Here is a listing of electronic publications authored by NAVBO members and of relevance to the entire Vascular Biology Community.
ePubs
Carbon monoxide-releasing molecule-3 (CORM-3) offers protection in an in vitro model of compartment syndrome
Authors:
Bihari A, Chung KA, Cepinskas G, Sanders D, Schemitsch E, Lawendy AR
Abstract:
OBJECTIVE: Limb compartment syndrome (CS), a complication of trauma, results in muscle necrosis and cell death; ischemia and inflammation contribute to microvascular dysfunction and parenchymal injury. Carbon monoxide-releasing molecule-3 (CORM-3) has been shown to protect microvascular perfusion and reduce inflammation in animal models of CS. The purpose of the study was to test the effect of CORM-3 in human in vitro CS model, allowing exploration of the mechanism(s) of CO protection and potential development of pharmacologic treatment.
METHODS: Confluent human vascular endothelial cells (HUVECs) were stimulated for 6 h with serum isolated from patients with CS. Intracellular oxidative stress (production of reactive oxygen species (ROS)) apoptosis, transendothelial resistance (TEER), polymorphonuclear leukocyte (PMN) activation and transmigration across the monolayer in response to the CS stimulus were assessed. All experiments were performed in the presence of CORM-3 (100 μM) or its inactive form, iCORM-3.
RESULTS: CS serum induced a significant increase in ROS, apoptosis and endothelial monolayer breakdown; it also increased PMN superoxide production, leukocyte rolling and adhesion/transmigration. CORM-3 completely prevented CS-induced ROS production, apoptosis, PMN adhesion, rolling and transmigration, while improving monolayer integrity.
CONCLUSION: CORM-3 offers potent anti-oxidant and anti-inflammatory effects, and may have a potential application to patients at risk of developing CS.
Citation:
Microcirculation. 2019 Jun 22:e12577. doi: 10.1111/micc.12577. [Epub ahead of print]
Regulation of CCL2 expression in human vascular endothelial cells by a neighboring divergently transcribed long noncoding RNA
Authors:
Khyzha N, Khor M, DiStefano PV, Wang L, Matic L, Hedin U, Wilson MD, Maegdefessel L, Fish JE
Abstract:
Atherosclerosis is a chronic inflammatory disease that is driven, in part, by activation of vascular endothelial cells (ECs). In response to inflammatory stimuli, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway orchestrates the expression of a network of EC genes that contribute to monocyte recruitment and diapedesis across the endothelium. Although many long noncoding RNAs (lncRNAs) are dysregulated in atherosclerosis, they remain poorly characterized, especially in the context of human vascular inflammation. Prior studies have illustrated that lncRNAs can regulate their neighboring protein-coding genes via interaction with protein complexes. We therefore identified and characterized neighboring interleukin-1β (IL-1β)-regulated messenger RNA (mRNA)-lncRNA pairs in ECs. We found these pairs to be highly correlated in expression, especially when located within the same chromatin territory. Additionally, these pairs were predominantly divergently transcribed and shared common gene regulatory elements, characterized by active histone marks and NF-κB binding. Further analysis was performed on lncRNA-CCL2, which is transcribed divergently to the gene, CCL2, encoding a proatherosclerotic chemokine. LncRNA-CCL2 and CCL2 showed coordinate up-regulation in response to inflammatory stimuli, and their expression was correlated in unstable symptomatic human atherosclerotic plaques. Knock-down experiments revealed that lncRNA-CCL2 positively regulated CCL2 mRNA levels in multiple primary ECs and EC cell lines. This regulation appeared to involve the interaction of lncRNA-CCL2 with RNA binding proteins, including HNRNPU and IGF2BP2. Hence, our approach has uncovered a network of neighboring mRNA-lncRNA pairs in the setting of inflammation and identified the function of an lncRNA, lncRNA-CCL2, which may contribute to atherogenesis in humans.
Citation:
Proc Natl Acad Sci U S A. 2019 Jul 26. pii: 201904108. doi: 10.1073/pnas.1904108116. [Epub ahead of print]
Augmented pulmonary vasoconstrictor reactivity following chronic hypoxia requires Src kinase and EGFR signaling
Authors:
Norton CE, Sheak JR, Yan S, Weise-Cross L, Jernigan NL, Walker BR, Resta TC
Abstract:
RATIONALE: Chronic hypoxia augments pressure- and agonist-induced pulmonary vasoconstriction through myofilament calcium sensitization. NADPH oxidases contribute to the development of pulmonary hypertension, and both epidermal growth factor receptor and Src kinases can regulate NADPH oxidase. OBJECTIVES: We tested the hypothesis that Src/epidermal growth factor receptor signaling mediates enhanced vasoconstrictor sensitivity following chronic hypoxia through NADPH oxidase-derived superoxide generation. METHODS: Protocols employed pharmacological inhibitors in isolated, pressurized rat pulmonary arteries to examine the contribution of a variety of signaling moieties to enhanced vascular tone following chronic hypoxia. Superoxide generation in pulmonary arterial smooth muscle cells was assessed using the fluorescent indicator dihydroethidium. Indices of pulmonary hypertension were measured in rats treated with the epidermal growth factor receptor inhibitor gefitinib. MEASUREMENTS AND RESULTS: Inhibition of NADPH oxidase, Rac1, and epidermal growth factor receptor abolished pressure-induced pulmonary arterial tone and endothelin-1-dependent calcium sensitization and vasoconstriction following chronic hypoxia. Consistently, chronic hypoxia augmented endothelin-1-induced superoxide production through epidermal growth factor receptor signaling, and rats treated chronically with gefitinib displayed reduced right ventricular pressure and diminished arterial remodeling. Src kinases were additionally activated by ET-1 following chronic hypoxia and contributed to enhanced basal arterial tone and vasoconstriction to ET-1. A role for matrix metalloproteinase 2 to mediate Src dependent-epidermal growth factor receptor activation is further supported by our findings. CONCLUSIONS: Our studies support a novel role for a Src kinase, epidermal growth factor receptor, NADPH oxidase signaling axis to mediate enhanced pulmonary vascular smooth muscle Ca2+ sensitization, vasoconstriction, and pulmonary hypertension following chronic hypoxia.
Citation:
Am J Respir Cell Mol Biol. 2019 Jul 2. doi: 10.1165/rcmb.2018-0106OC. [Epub ahead of print]
High‐Throughput Scaffold System for Studying the Effect of Local Geometry and Topology on the Development and Orientation of Sprouting Blood Vessels
Authors:
Szklanny, A. A., Debbi, L., Merdler, U., Neale, D., Muñiz, A., Kaplan, B., Guo, S., Lahann J. & Levenberg, S.
Abstract:
Live tissues require vascular networks for cell nourishing. Mimicking the complex structure of native vascular networks in vitro requires understanding the governing factors of early tubulogenesis. Current vascularization protocols allow for spontaneous formation of vascular networks; however, there is still a need to provide control over the defined network structure. Moreover, there is little understanding on sprouting decision and migration, especially within 3D environments. Here, tessellated polymer scaffolds with various compartment geometries and a novel two‐step seeding protocol are used to study vessel sprouting decisions. Endothelial cells first organize into hollow vessels tracing the shape contour with high fidelity. Subsequent sprouts emerge in specific directions, responding to compartment geometry. Time‐lapse imaging is used to track vessel migration, evidencing that sprouts frequently emerge from the side centers, mainly migrating toward opposing corners, where the density of support cells (SCs) is the highest, providing the highest levels of angiogenic factors. SCs distribution is quantified by smooth muscle actin expression, confirming the cells preference for curved compartment surfaces and corners. Displacements within the hydrogel correlate with SCs distribution during the initial tubulogenesis phase. This work provides new insight regarding vessel sprouting decisions that should be considered when designing scaffolds for vascularized engineered tissues.
Citation:
First published: 07 June 2019|https://doi.org/10.1002/adfm.201901335
https://onlinelibrary.wiley.com/doi/abs/10.1002/adfm.201901335
Single-cell transcriptome analyses reveal novel targets modulating cardiac neovascularization by resident endothelial cells following myocardial infarction
Authors:
Li Z, Solomonidis EG, Meloni M, Taylor RS, Duffin R, Dobie R, Magalhaes MS, Henderson BEP, Louwe PA, D'Amico G, Hodivala-Dilke KM, Shah AM, Mills NL, Simons BD, Gray GA, Henderson NC, Baker AH, Brittan M
Abstract:
AIMS: A better understanding of the pathways that regulate regeneration of the coronary vasculature is of fundamental importance for the advancement of strategies to treat patients with heart disease. Here, we aimed to investigate the origin and clonal dynamics of endothelial cells (ECs) associated with neovascularization in the adult mouse heart following myocardial infarction (MI). Furthermore, we sought to define murine cardiac endothelial heterogeneity and to characterize the transcriptional profiles of pro-angiogenic resident ECs in the adult mouse heart, at single-cell resolution. METHODS AND RESULTS: An EC-specific multispectral lineage-tracing mouse (Pdgfb-iCreERT2-R26R-Brainbow2.1) was used to demonstrate that structural integrity of adult cardiac endothelium following MI was maintained through clonal proliferation by resident ECs in the infarct border region, without significant contributions from bone marrow cells or endothelial-to-mesenchymal transition. Ten transcriptionally discrete heterogeneous EC states, as well as the pathways through which each endothelial state is likely to enhance neovasculogenesis and tissue regeneration following ischaemic injury were defined. Plasmalemma vesicle-associated protein (Plvap) was selected for further study, which showed an endothelial-specific and increased expression in both the ischaemic mouse and human heart, and played a direct role in regulating human endothelial proliferation in vitro. CONCLUSION: We present a single-cell gene expression atlas of cardiac specific resident ECs, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. These data provide a rich resource that could assist in the development of new therapeutic interventions to augment endogenous myocardial perfusion and enhance regeneration in the injured heart.
Citation:
Eur Heart J. 2019 Jun 4. pii: ehz305. doi: 10.1093/eurheartj/ehz305. [Epub ahead of print]
https://academic.oup.com/eurheartj/advance-article/doi/10.1093/eurheartj/ehz305/5510786
The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing
Authors:
Ishihara J, Ishihara A, Starke RD, Peghaire CR, Smith KE, McKinnon TA, Tabata Y, Sasaki K, White MJ, Fukunaga K, Laffan MA, Lutolf MP, Randi AM, Hubbell JA.
Abstract:
During wound healing, the distribution, availability and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GFs binding. The hemostatic protein von Willebrand factor (VWF), released by endothelial cells (ECs) in plasma and in the subendothelial matrix, has been shown to regulate angiogenesis; this function is relevant to patients where VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing, accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor (VEGF)-A isoforms and platelet derived growth factor (PDGF)-BB, mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and FGF-2 in human plasma and co-localizes with VEGF-A in EC. Incorporation of the VWF A1-HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1-HBD can function as a GFs reservoir, leading to effective angiogenesis and tissue regeneration.
Citation:
Blood. 2019 Apr 11. pii: blood.2019000510. doi: 10.1182/blood.2019000510. [Epub ahead of print]
Standardization of methods to quantify and culture endothelial colony-forming cells derived from peripheral blood: Position paper from the International Society on Thrombosis and Haemostasis SSC
Authors:
Smadja DM, Melero-Martin JM, Eikenboom J, Bowman M, Sabatier F, Randi AM
Citation:
J Thromb Haemost. 2019 Apr 25. doi: 10.1111/jth.14462. [Epub ahead of print]
A disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif 9 (ADAMTS9) regulates fibronectin fibrillogenesisand turnover
Authors:
Wang LW, Nandadasa S, Annis DS, Dubail J, Mosher DF, Willard BB, Apte SS
Abstract:
The secreted metalloprotease ADAMTS9 has dual roles in extracellular matrix (ECM) turnover and biogenesis of the primary ciliumduring mouse embryogenesis. Its gene locus is associated with several human traits and disorders, but ADAMTS9 has few knowninteracting partners or confirmed substrates. Here, using a yeast two-hybrid screen for proteins interacting with its C-terminalGon1 domain, we identified three putative ADAMTS9-binding regions in the ECM glycoprotein fibronectin. Using solid-phase bindingassays and surface plasmon resonance experiments with purified proteins, we demonstrate that ADAMTS9 and fibronectin interact.ADAMTS9 constructs, including those lacking Gon1, co-localized with fibronectin fibrils formed by cultured fibroblasts lackingfibrillin-1, which co-localizes with fibronectin and binds several ADAMTSs. We observed no fibrillar ADAMTS9 staining afterblockade of fibroblast fibronectin fibrillogenesis with a peptide based on the functional upstream domain of a Staphylococcus aureus adhesin. These findings indicate that ADAMTS9 binds fibronectin dimers and fibrils directly through multiple sites in bothmolecules. Proteolytically active ADAMTS9, but not a catalytically inactive variant, disrupted fibronectin fibril networksformed by fibroblasts in vitro, and ADAMTS9-deficient RPE1 cells assembled a robust fibronectin fibril network, unlike wildtype cells. Targeted LC–MS analysis of fibronectin digested by ADAMTS9-expressing cells identified a semi-tryptic peptidearising from cleavage at Gly-2196–Leu-2197. We noted that this scissile bond is in the linker between fibronectin modulesIII17 and I10, a region targeted also by other proteases. These findings, along with stronger fibronectin staining previouslyobserved in Adamts9 mutant embryos, suggest that ADAMTS9 contributes to fibronectin turnover during ECM remodeling.
Citation:
Journal of Biological Chemistry, First Published on May 13, 2019, doi: 10.1074/jbc.RA118.006479, jbc.RA118.006479
http://www.jbc.org/content/early/2019/05/13/jbc.RA118.006479.abstract
Type I Diabetes Delays Perfusion and Engraftment of 3D Constructs by Impinging on Angiogenesis; Which can be Rescued by Hepatocyte Growth Factor Supplementation
Authors:
Wafa Altalhi, Rupal Hatkar, James B. Hoying, Yasaman Aghazadeh, Sara S. Nunes
Abstract:
Introduction: The biggest bottleneck for cell-based regenerative therapy is the lack of a functional vasculature to support the grafts. This problem is exacerbated in diabetic patients, where vessel growth is inhibited. To address this issue, we aim to identify the causes of poor vascularization in 3D engineered tissues in diabetes and to reverse its negative effects. Methods: We used 3D vascularized constructs composed of microvessel fragments containing all cells present in the microcirculation, embedded in collagen type I hydrogels. Constructs were either cultured in vitro or implanted subcutaneously in non-diabetic or in a type I diabetic (streptozotocin-injected) mouse model. We used qPCR, ELISA, immunostaining, FACs and co-culture assays to characterize the effect of diabetes in engineered constructs. Results: We demonstrated in 3D vascularized constructs that perivascular cells secrete hepatocyte growth factor (HGF), driving microvessel sprouting. Blockage of HGF or HGF receptor signaling in 3D constructs prevented vessel sprouting. Moreover, HGF expression in 3D constructs in vivo is downregulated in diabetes; while no differences were found in HGF receptor, VEGF or VEGF receptor expression. Low HGF expression in diabetes delayed the inosculation of graft and host vessels, decreasing blood perfusion and preventing tissue engraftment. Supplementation of HGF in 3D constructs, restored vessel sprouting in a diabetic milieu. Conclusion: We show for the first time that diabetes affects HGF secretion in microvessels, which in turn prevents the engraftment of engineered tissues. Exogenous supplementation of HGF, restores angiogenic growth in 3D constructs showing promise for application in cell-based regenerative therapies.
Citation:
Cellular and Molecular Bioengineering, First Online: 21 May 2019
https://link.springer.com/article/10.1007%2Fs12195-019-00574-3
FAK and Pyk2 activity promote TNF-α and IL-1β-mediated pro-inflammatory gene expression and vascular inflammation
Authors:
James M. Murphy, Kyuho Jeong, Yelitza A. R. Rodriguez, Jung-Hyun Kim, Eun-Young Erin Ahn & Ssang-Taek Steve Lim
Abstract:
Protein tyrosine kinase (PTK) activity has been implicated in pro-inflammatory gene expression following tumor necrosis factor-α (TNF-α) or interkeukin-1β (IL-1β) stimulation. However, the identity of responsible PTK(s) in cytokine signaling have not been elucidated. To evaluate which PTK is critical to promote the cytokine-induced inflammatory cell adhesion molecule (CAM) expression including VCAM-1, ICAM-1, and E-selectin in human aortic endothelial cells (HAoECs), we have tested pharmacological inhibitors of major PTKs: Src and the focal adhesion kinase (FAK) family kinases - FAK and proline-rich tyrosine kinase (Pyk2). We found that a dual inhibitor of FAK/Pyk2 (PF-271) most effectively reduced all three CAMs upon TNF-α or IL-1β stimulation compared to FAK or Src specific inhibitors (PF-228 or Dasatinib), which inhibited only VCAM-1 expression. In vitro inflammation assays showed PF-271 reduced monocyte attachment and transmigration on HAoECs. Furthermore, FAK/Pyk2 activity was not limited to CAM expression but was also required for expression of various pro-inflammatory molecules including MCP-1 and IP-10. Both TNF-α and IL-1β signaling requires FAK/Pyk2 activity to activate ERK and JNK MAPKs leading to inflammatory gene expression. Knockdown of either FAK or Pyk2 reduced TNF-α-stimulated ERK and JNK activation and CAM expression, suggesting that activation of ERK or JNK is specific through FAK and Pyk2. Finally, FAK/Pyk2 activity is required for VCAM-1 expression and macrophage recruitment to the vessel wall in a carotid ligation model in ApoE−/− mice. Our findings define critical roles of FAK/Pyk2 in mediating inflammatory cytokine signaling and implicate FAK/Pyk2 inhibitors as potential therapeutic agents to treat vascular inflammatory disease such as atherosclerosis.
Citation:
Scientific Reports 9, Article number: 7617 (2019)
Nuclear Focal Adhesion Kinase Controls Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Through GATA4-mediated Cyclin D1 Transcription
Authors:
Kyuho Jeong, Jung-Hyun Kim, James M Murphy, Hyeonsoo Park, Su-Jeong Kim, Yelitza Rodriguez, Hyunkyung Kong, Chungsik Choi,Jun-Lin Guan,Joan M Taylor, William T Gerthoffer , Jun-Sub Kim, Eun-Young Erin Ahn, David D Schlaepfer, Ssang-Taek Steve Lim
Abstract:
Rationale: Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via focal adhesion kinase (FAK). Objective: To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia. Methods and Results: Using combined pharmacological FAK catalytic inhibition (VS-4718) and SMC-specific FAK kinase-dead (KD, Myh11-Cre-ERT2) mouse models, we report that FAK regulates SMC proliferation and neointimal hyperplasia in part by governing GATA4-cyclin D1 signaling. Inhibition of FAK catalytic activity facilitates FAK nuclear localization, which is required for proteasome-mediated GATA4 degradation in the cytoplasm. Chromatin immunoprecipitation identified GATA4 binding to the mouse cyclin D1 promoter and loss of GATA4-mediated cyclin D1 transcription diminished SMC proliferation. Stimulation with platelet-derived growth factor or serum activated FAK and redistributed FAK from the nucleus to cytoplasm, leading to concomitantly increased GATA4 protein and cyclin D1 expression. In a femoral artery wire injury model, increased neointimal hyperplasia was observed in parallel with elevated FAK activity, GATA4 and cyclin D1 expression following injury in control, but not in VS-4718-treated and SMC-specific FAK-KD mice. Finally, lentiviral shGATA4 knockdown in the femoral artery wire injury significantly reduced cyclin D1 expression, SMC proliferation, and neointimal hyperplasia compared to control mice. Conclusions: Nuclear enrichment of FAK by inhibition of FAK catalytic activity during vessel injury blocks SMC proliferation and neointimal hyperplasia through regulation of GATA4-mediated cyclin D1 transcription.
Citation:
Originally published, https://doi.org/10.1161/CIRCRESAHA.118.314344Circulation Research. ;0
https://www.ahajournals.org/doi/abs/10.1161/CIRCRESAHA.118.314344
Innate Immune Cells' Contribution to Systemic Lupus Erythematosus
Authors:
Andrés A. Herrada, Noelia Escobedo, Mirentxu Iruretagoyena, Rodrigo A. Valenzuela, Paula I. Burgos, Loreto Cuitino and Carolina Llanos
Abstract:
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of autoantibodies against nuclear antigens, immune complex deposition, and tissue damage in the kidneys, skin, heart and lung. Because of the pathogenic role of antinuclear antibodies and autoreactive T cells in SLE, extensive efforts have been made to demonstrate how B cells act as antibody-producing or as antigen-presenting cells that can prime autoreactive T cell activation. With the discovery of new innate immune cells and inflammatory mediators, innate immunity is emerging as a key player in disease pathologies. Recent work over the last decade has highlighted the importance of innate immune cells and molecules in promoting and potentiating SLE. In this review, we discuss recent evidence of the involvement of different innate immune cells and pathways in the pathogenesis of SLE. We also discuss new therapeutics targets directed against innate immune components as potential novel therapies in SLE.
Citation:
Front. Immunol., 15 April 2019| https://doi.org/10.3389/fimmu.2019.00772
https://www.frontiersin.org/articles/10.3389/fimmu.2019.00772/full
Disrupted inflammasome activation by CD39 in venous thrombosis
Authors:
Yadav V, Chi L, Zhao R, Tourdot BE, Yalavarthi S, Banka A, Jacobs BN, Liao H, Koonse S, Anyanwu AC, Visovatti SH, Holinstat M, Kahlenberg JM, Knight JS, Pinsky DJ, Kanthi Y
Abstract:
Deep vein thrombosis (DVT), caused by alterations in venous homeostasis is the third most common cause of cardiovascular mortality; however, key molecular determinants in venous thrombosis have not been fully elucidated. Several lines of evidence indicate that DVT occurs at the intersection of dysregulated inflammation and coagulation. The enzyme ectonucleoside tri(di)phosphohydrolase (ENTPD1, also known as CD39) is a vascular ecto-apyrase on the surface of leukocytes and the endothelium that inhibits intravascular inflammation and thrombosis by hydrolysis of phosphodiester bonds from nucleotides released by activated cells. Here, we evaluated the contribution of CD39 to venous thrombosis in a restricted-flow model of murine inferior vena cava stenosis. CD39-deficiency conferred a >2-fold increase in venous thrombogenesis, characterized by increased leukocyte engagement, neutrophil extracellular trap formation, fibrin, and local activation of tissue factor in the thrombotic milieu. This was orchestrated by increased phosphorylation of the p65 subunit of NFκB, activation of the NLRP3 inflammasome, and interleukin-1β (IL-1β) release in CD39-deficient mice. Substantiating these findings, an IL-1β-neutralizing antibody attenuated the thrombosis risk in CD39-deficient mice. These data demonstrate that IL-1β is a key accelerant of venous thrombo-inflammation, which can be suppressed by CD39. CD39 inhibits in vivo crosstalk between inflammation and coagulation pathways, and is a critical vascular checkpoint in venous thrombosis.
Citation:
J Clin Invest. https://doi.org/10.1172/JCI124804
Tuning the Thromboinflammatory Response to Venous Flow Interruption by the Ectonucleotidase CD39
Authors:
Anuli C. Anyanwu , Yogendra Kanthi , Keigo Fukase , Hui Liao , Tekashi Mimura , Karl C. Desch , Martin Gruca , Saabir Kaskar , Hussein Sheikh-Aden , Liguo Chi , Raymond Zhao , Vinita Yadav , Thomas W. Wakefield , Matthew C. Hyman , David J. Pinsky
Abstract:
Objective — Leukocyte flux contributes to thrombus formation in deep veins under pathological conditions, but mechanisms that inhibit venous thrombosis are incompletely understood. Ectonucleotide di(tri)phosphohydrolase 1 (ENTPD1 or Cd39), an ectoenzyme that catabolizes extracellular adenine nucleotides, is embedded on the surface of endothelial cells and leukocytes. We hypothesized that under venous stasis conditions, CD39 regulates inflammation at the vein:blood interface in a murine model of deep vein thrombosis. Approach and Results — CD39-null mice developed significantly larger venous thrombi under venous stasis, with more leukocyte recruitment compared with wild-type mice. Gene expression profiling of wild-type and Cd39-null mice revealed 76 differentially expressed inflammatory genes that were significantly upregulated in Cd39-deleted mice after venous thrombosis, and validation experiments confirmed high expression of several key inflammatory mediators. P-selectin, known to have proximal involvement in venous inflammatory and thrombotic events, was upregulated in Cd39-null mice. Inferior vena caval ligation resulted in thrombosis and a corresponding increase in both P-selectin and VWF (von Willebrand Factor) levels which were strikingly higher in mice lacking the Cd39 gene. These mice also manifest an increase in circulating platelet-leukocyte heteroaggregates suggesting heterotypic crosstalk between coagulation and inflammatory systems, which is amplified in the absence of CD39. Conclusions — These data suggest that CD39 mitigates the venous thromboinflammatory response to flow interruption.
Citation:
Originally published28 Feb 2019https://doi.org/10.1161/ATVBAHA.119.312407Arteriosclerosis, Thrombosis, and Vascular Biology. 2019;39:e118–e129
Genetic compensation triggered by mutant mRNA degradation
Authors:
Mohamed A. El-Brolosy, Zacharias Kontarakis, Andrea Rossi, Carsten Kuenne, Stefan Günther, Nana Fukuda, Khrievono Kikhi, Giulia L. M. Boezio, Carter M. Takacs, Shih-Lei Lai, Ryuichi Fukuda, Claudia Gerri, Antonio J. Giraldez & Didier Y. R. Stainier
Abstract:
Genetic robustness, or the ability of an organism to maintain fitness in the presence of harmful mutations, can be achieved via protein feedback loops. Previous work has suggested that organisms may also respond to mutations by transcriptional adaptation, a process by which related gene(s) are upregulated independently of protein feedback loops. However, the prevalence of transcriptional adaptation and its underlying molecular mechanisms are unknown. Here, by analysing several models of transcriptional adaptation in zebrafish and mouse, we uncover a requirement for mutant mRNA degradation. Alleles that fail to transcribe the mutated gene do not exhibit transcriptional adaptation, and these alleles give rise to more severe phenotypes than alleles displaying mutant mRNA decay. Transcriptome analysis in alleles displaying mutant mRNA decay reveals the upregulation of a substantial proportion of the genes that exhibit sequence similarity with the mutated gene's mRNA, suggesting a sequence-dependent mechanism. These findings have implications for our understanding of disease-causing mutations, and will help in the design of mutant alleles with minimal transcriptional adaptation-derived compensation.
Citation:
Nature(2019)
Loss of Endothelial Derived WNT5A is Associated with Reduced Pericyte Recruitment and Small Vessel Loss in Pulmonary Arterial Hypertension
Authors:
Yuan K, Shamskhou EA, Orcholski ME, Nathan A, Reddy S, Honda H, Mani V, Zeng Y, Ozen MO, Wang L, Demirci U, Tian W, Nicolls MR, de Jesus Perez VA
Abstract:
Background: Pulmonary arterial hypertension (PAH) is a life-threatening disorder of the pulmonary circulation associated with loss and impaired regeneration of microvessels. Reduced pericyte coverage of pulmonary microvessels is a pathological feature of PAH and is partly due to the inability of pericytes to respond to signaling cues from neighboring pulmonary microvascular endothelial cells (PMVECs). We have shown that activation of the Wnt/PCP pathway is required for pericyte recruitment but whether production and release of specific Wnt ligands by PMVECs is responsible for Wnt/PCP activation in pericytes is unknown. Methods: Isolation of pericytes and PMVECs from healthy donor and PAH lungs was carried out using 3G5 or CD31 antibody conjugated magnetic beads. Wnt expression profile of PMVECs was documented via qPCR using a Wnt primer library. Exosome purification from PMVEC media was carried out using the ExoTIC device. Hemodynamic profile, right ventricular function and pulmonary vascular morphometry were obtained in a conditional endothelial specific Wnt5a knockout (Wnt5aECKO) mouse model under normoxia, chronic hypoxia and hypoxia recovery. Results: Quantification of Wnt ligand expression in healthy PMVECs co-cultured with pericytes demonstrated a 35-fold increase in Wnt5a, a known Wnt/PCP ligand. This Wnt5a spike was not seen in PAH PMVECs, which correlated with inability to recruit pericytes in matrigel co-culture assays. Exosomes purified from media demonstrated an increase in Wnt5a content when healthy PMVECs were co-cultured with pericytes, a finding that was not observed in exosomes of PAH PMVECs. Furthermore, the addition of either recombinant Wnt5a or purified healthy PMVEC exosomes increased pericyte recruitment to PAH PMVECs in co-culture studies. While no differences were noted in normoxia and chronic hypoxia, Wnt5aECKO mice demonstrated persistent pulmonary hypertension and right ventricular failure four weeks after recovery from chronic hypoxia, which correlated with significant reduction, muscularization and decreased pericyte coverage of microvessels. Conclusions: We identify Wnt5a as a key mediator for the establishment of pulmonary endothelial-pericyte interactions and its loss could contribute to PAH by reducing the viability of newly formed vessels. We speculate that therapies that mimic or restore Wnt5a production could help prevent loss of small vessels in PAH.
Citation:
Originally published 11 Dec 2018 https://doi.org/10.1161/CIRCULATIONAHA.118.037642 Circulation. 2018
https://www.ahajournals.org/doi/abs/10.1161/CIRCULATIONAHA.118.037642
Endothelial miR-30c suppresses tumor growth via inhibition of TGF-β-induced Serpine1
Authors:
McCann JV, Xiao L, Kim DJ, Khan OF, Kowalski PS, Anderson DG, Pecot CV, Azam SH, Parker JS, Tsai YS, Wolberg AS, Turner SD, Tatsumi K, Mackman N, Dudley AC
Abstract:
In tumors, extravascular fibrin forms provisional scaffolds for endothelial cell (EC) growth and motility during angiogenesis. We report that fibrin-mediated angiogenesis was inhibited and tumor growth delayed following postnatal deletion of Tgfbr2 in the endothelium of Cdh5-CreERT2 Tgfbr2fl/fl mice (Tgfbr2iECKO mice). ECs from Tgfbr2iECKO mice failed to upregulate the fibrinolysis inhibitor plasminogen activator inhibitor 1 (Serpine1, also known as PAI-1), due in part to uncoupled TGF-β–mediated suppression of miR-30c. Bypassing TGF-β signaling with vascular tropic nanoparticles that deliver miR-30c antagomiRs promoted PAI-1–dependent tumor growth and increased fibrin abundance, whereas miR-30c mimics inhibited tumor growth and promoted vascular-directed fibrinolysis in vivo. Using single-cell RNA-Seq and a NanoString miRNA array, we also found that subtypes of ECs in tumors showed spectrums of Serpine1 and miR-30c expression levels, suggesting functional diversity in ECs at the level of individual cells; indeed, fresh EC isolates from lung and mammary tumor models had differential abilities to degrade fibrin and launch new vessel sprouts, a finding that was linked to their inverse expression patterns of miR-30c and Serpine1 (i.e., miR-30chi Serpine1lo ECs were poorly angiogenic and miR-30clo Serpine1hi ECs were highly angiogenic). Thus, by balancing Serpine1 expression in ECs downstream of TGF-β, miR-30c functions as a tumor suppressor in the tumor microenvironment through its ability to promote fibrin degradation and inhibit blood vessel formation.
Citation:
J Clin Invest. https://doi.org/10.1172/JCI123106
Effect of TRPA1 activator allyl isothiocyanate (AITC) on rat dural and pial arteries
Authors:
Anna Koldbro Hansted, Deepak Kumar Bhatt, Jes Olesen, Lars Jørn Jensen, Inger Jansen-Olesen
Abstract:
Background: Transient receptor potential ankyrin 1 (TRPA1) channels may have a role in migraine as some substances known to cause headache activate the channel. In the craniovascular system such activation causes a calcitonin gene-related peptide (CGRP)-dependent increase in meningeal blood flow. TRPA1 channels in the endothelium of cerebral arteries cause vasodilation when activated. The headache preventive substance feverfew inhibits activation of TRPA1 channels. In this study we aim to compare and characterize the effect of the TRPA1 agonist allyl isothiocyanate (AITC) on the diameter of rat dural and pial arteries in vivo. Material and methods: The genuine closed-cranial window technique in rats was used to examine changes in dural and pial artery diameter and mean arterial blood pressure (MABP) after intracarotid infusion of AITC. Blockade experiments were performed by intravenous infusion of olcegepant, HC-030031, sumatriptan or capsazepine immediately after infusion of AITC, in four different groups of rats. Results: AITC caused a significant dilation of dural arteries, which was inhibited by HC-030031, olcegepant and sumatriptan, but not by capsazepine. In pial arteries AITC caused a significant dilation, which was not inhibited by any of the pre-treatments, suggesting a poor penetration of the blood-brain barrier or autoregulation due to dimethyl sulfoxide (DMSO) mediated decrease in MABP during HC-030031 infusion. AITC did not cause a significant change in MABP. Conclusion: AITC causes dilation of dural arteries via a mechanism dependent on CGRP and TRPA1 that is sensitive to sumatriptan. AITC causes a small but significant dilation of pial arteries.
Citation:
Pharmacological Reports, Available online 21 February 2019
https://www.sciencedirect.com/science/article/abs/pii/S1734114018303463
Cardosin A Endocytosis Mediated by Integrin Leads to Lysosome Leakage and Apoptosis of Epithelial Cells
Authors:
Wu H, Castenheira P, Faro C, Tang J.
Abstract:
Cardosin A is an aspartic protease present in large amount in the pistils of cardoon flowers. This protease is known to contain an -Arg-Gly-Asp- (RGD) motif located on the molecular surface. In this study we found that isolated recombinant cardosin A attached to human epithelial cells A549, mediated by the binding of its RGD motif to cell surface integrins. The cell bound cardosin A was internalized to endosomes and lysosomes and triggered the permeability of lysosomal membrane leading to apoptosis of the epithelial cells. These events are identical to those observed for three RGD-containing aspartic proteases, Saps 4-6, secreted by Candida albicans.(1) Such a process, which has been called the Trojan Horse mechanism, is thought to benefit the invasion of C. albican into the epithelium of the host. The location of the RGD motifs of cardosin A and Saps 4-6 are on the opposite ends of the homologous three-dimensional structures, suggesting that the Trojan Horse mechanism is insensitive to the RGD position. Current finding also suggests that cardosin A may have a defensive function against the ingestion of cardoon flowers by human, insects, and other herbivores. This article is protected by copyright. All rights reserved.
Citation:
Proteins. 2019 Feb 20. doi: 10.1002/prot.25672. [Epub ahead of print]
Structural and functional analysis of single-nucleotide polymorphic variants of purine-rich element-binding protein B
Authors:
Lauren A. Ferris & Robert J. Kelm Jr.
Abstract:
Purine-rich element-binding protein B (Purβ) inhibits myofibroblast differentiation by repressing the expression of the smooth muscle α-actin gene (Acta2). Several reports have identified the structural domains in Purβ that enable its characteristic interaction with purine-rich single-stranded DNA (ssDNA) sequences in the Acta2 promoter. However, little is known about the physical and functional effects of single-nucleotide polymorphisms that alter individual amino acid residues in Purβ. This study evaluated seven rare single amino acid variants of human PURB engineered into the homologous mouse Purβ protein. Mapping the location of variant residues on a homology model of the Purβ homodimer suggested that most of the altered residues are remote from the predicted ssDNA-binding regions of the protein. The repressor activity of each Purβ variant was assessed in transfected fibroblasts and smooth muscle cells via Acta2 promoter-reporter assays. A Q64* nonsense variant was completely inactive while missense variants exhibited repressor activity that ranged from ~1.5-fold greater to ~2-fold less than wild-type Purβ. Lower activity variants P223L and R297Q were expressed in bacteria and purified to homogeneity. Each variant was physically indistinguishable from wild-type Purβ in terms of quaternary structure and thermostability. Results of DNA and protein-binding assays indicated that the P223L and R297Q variants retained high affinity and specificity for purine-rich ssDNA sequences but differed in their interaction with other Acta2 regulatory proteins. These findings suggest that the presence of certain variant residues affects the Acta2 repressor activity of Purβ by altering its interaction with other transcription factors but not with ssDNA.
Citation:
J Cell Biochem. 2019 Apr;120(4):5835-5851. doi: 10.1002/jcb.27869. Epub 2018 Nov 1.
Disturbed Flow Increases UBE2C (Ubiquitin E2 Ligase C) via Loss of miR-483-3p, Inducing Aortic Valve Calcification by the HIF-1α (Hypoxia-Inducible Factor-1α) Pathway in Endothelial Cells
Authors:
Esmerats JF, Villa-Roel N, Kumar S, Gu L, Salim MT, Ohh M, Taylor WR, Nerem RM, Yoganathan AP, Jo H
Abstract:
Objective- Calcific aortic valve (AV) disease, characterized by AV sclerosis and calcification, is a major cause of death in the aging population; however, there are no effective medical therapies other than valve replacement. AV calcification preferentially occurs on the fibrosa side, exposed to disturbed flow (d-flow), whereas the ventricularis side exposed to predominantly stable flow remains protected by unclear mechanisms. Here, we tested the role of novel flow-sensitive UBE2C (ubiquitin E2 ligase C) and microRNA-483-3p (miR-483) in flow-dependent AV endothelial function and AV calcification. Approach and Results- Human AV endothelial cells and fresh porcine AV leaflets were exposed to stable flow or d-flow. We found that UBE2C was upregulated by d-flow in human AV endothelial cells in the miR-483-dependent manner. UBE2C mediated OS-induced endothelial inflammation and endothelial-mesenchymal transition by increasing the HIF-1α (hypoxia-inducible factor-1α) level. UBE2C increased HIF-1α by ubiquitinating and degrading its upstream regulator pVHL (von Hippel-Lindau protein). These in vitro findings were corroborated by immunostaining studies using diseased human AV leaflets. In addition, we found that reduction of miR-483 by d-flow led to increased UBE2C expression in human AV endothelial cells. The miR-483 mimic protected against endothelial inflammation and endothelial-mesenchymal transition in human AV endothelial cells and calcification of porcine AV leaflets by downregulating UBE2C. Moreover, treatment with the HIF-1α inhibitor (PX478) significantly reduced porcine AV calcification in static and d-flow conditions. Conclusions- These results suggest that miR-483 and UBE2C are novel flow-sensitive anti- and pro-calcific AV disease molecules, respectively, that regulate the HIF-1α pathway in AV. The miR-483 mimic and HIF-1α pathway inhibitors may serve as potential therapeutics of calcific AV disease.
Citation:
Arterioscler Thromb Vasc Biol. 2019 Jan 3:ATVBAHA118312233. doi: 10.1161/ATVBAHA.118.312233. [Epub ahead of print]
miR-214 is Stretch-Sensitive in Aortic Valve and Inhibits Aortic Valve Calcification
Authors:
Salim MT, Esmerats JF, Arjunon S, Villa-Roel N, Nerem RM, Jo H, Yoganathan AP
Abstract:
miR-214 has been recently found to be significantly downregulated in calcified human aortic valves (AVs). ER stress, especially the ATF4-mediated pathway, has also been shown to be significantly upregulated in calcific AV disease. Since elevated cyclic stretch is one of the major mechanical stimuli for AV calcification and ATF4 is a validated target of miR-214, we investigated the effect of cyclic stretch on miR-214 expression as well as those of ATF4 and two downstream genes (CHOP and BCL2L1). Porcine aortic valve (PAV) leaflets were cyclically stretched at 15% for 48 h in regular medium and for 1 week in osteogenic medium to simulate the early remodeling and late calcification stages of stretch-induced AV disease, respectively. For both stages, 10% cyclic stretch served as the physiological counterpart. RT-qPCR revealed that miR-214 expression was significantly downregulated during the late calcification stage, whereas the mRNA expression of ATF4 and BCL2L1 was upregulated and downregulated, respectively, during both early remodeling and late calcification stages. When PAV leaflets were statically transfected with miR-214 mimic in osteogenic medium for 2 weeks, calcification was significantly reduced compared to the control mimic case. This implies that miR-214 may have a protective role in stretch-induced calcific AV disease.
Citation:
Ann Biomed Eng. 2019 Jan 22. doi: 10.1007/s10439-019-02206-3. [Epub ahead of print]
Staphylococcus aureus Leukocidins Target Endothelial DARC to Cause Lethality in Mice
Authors:
Lubkin A., Lee W.L., Alonzo F. 3rd, Wang C., Aligo J., Keller M., Girgis N. M., Reyes-Robles T., Chan R., O’Malley A., Buckley P., Vozhilla N., Vasquez M., Su J., Sugiyama M., Yeung S.T., Coffre M., Bajwa S., Chen E., Martin P., Kim S.Y., Loomis C., Worthen G.S., Shopsin B., Khanna K.M., Weinstock D., Lynch A.S., Koralov S.B., Loke P., Cadwell K., and Torres V.J.
Abstract:
The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytesand lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB),are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstratethat LukED and HlgAB cause vascular congestion and derangements in vascular fluiddistribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines(DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the criticaltarget for lethality. Consistent with this, LukED and HlgAB injure primary human endothelialcells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells areresistant to toxin-mediated lethality. During bloodstream infection in mice, DARCtargeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potentialfor S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulencefactors.
Citation:
Cell Host and Microbe, Published: February 21, 2019•DOI:https://doi.org/10.1016/j.chom.2019.01.015
https://www.cell.com/cell-host-microbe/fulltext/S1931-3128(19)30053-8
MicroRNA-dependent regulation of biomechanical genes establishes tissue stiffness homeostasis
Authors:
Moro A, Driscoll TP, Boraas LC, Armero W, Kasper DM, Baeyens N, Jouy C, Mallikarjun V, Swift J, Ahn SJ, Lee D, Zhang J, Gu M, Gerstein M, Schwartz M, Nicoli S
Abstract:
Vertebrate tissues exhibit mechanical homeostasis, showing stable stiffness and tension over time and recovery after changes in mechanical stress. However, the regulatory pathways that mediate these effects are unknown. A comprehensive identification of Argonaute 2-associated microRNAs and mRNAs in endothelial cells identified a network of 122 microRNA families that target 73 mRNAs encoding cytoskeletal, contractile, adhesive and extracellular matrix (CAM) proteins. The level of these microRNAs increased in cells plated on stiff versus soft substrates, consistent with homeostasis, and suppressed targets via microRNA recognition elements within the 3' untranslated regions of CAM mRNAs. Inhibition of DROSHA or Argonaute 2, or disruption of microRNA recognition elements within individual target mRNAs, such as connective tissue growth factor, induced hyper-adhesive, hyper-contractile phenotypes in endothelial and fibroblast cells in vitro, and increased tissue stiffness, contractility and extracellular matrix deposition in the zebrafish fin fold in vivo. Thus, a network of microRNAs buffers CAM expression to mediate tissue mechanical homeostasis.
Citation:
Nat Cell Biol. 2019 Feb 11. doi: 10.1038/s41556-019-0272-y. [Epub ahead of print]
Evolution of hemodynamic forces in the pulmonary tree with progressively worsening pulmonary arterial hypertension in pediatric patients
Authors:
Yang W, Dong M, Rabinovitch M, Chan FP, Marsden AL, Feinstein JA
Abstract:
Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling resulting in right ventricular (RV) dysfunction and ultimately RV failure. Mechanical stimuli acting on the vessel walls of the full pulmonary tree have not previously been comprehensively characterized. The goal of this study is to characterize wall shear stress (WSS) and strain in pediatric PAH patients at different stages of disease severity using computational patient-specific modeling. Computed tomography, magnetic resonance imaging and right heart catheterization data were collected and assimilated into pulmonary artery (PA) models for patients with and without PAH. Patients were grouped in three disease severity groups (control, moderate and severe) based on clinical evaluations. A finite element solver was employed to quantify hemodynamics and wall strains. To estimate WSS in the distal small PAs with diameters ranging from 50 to 500 [Formula: see text], a morphometric tree model was created, with inputs coming from outlets of the 3D model. WSS in the proximal PAs decreased with disease severity (control 20.5 vs. moderate 15.8 vs. severe 6.3 [Formula: see text], [Formula: see text]). Oscillatory shear index increased in the main pulmonary artery (MPA) with disease severity (0.13 vs. 0.13 vs. 0.2, [Formula: see text]). Wall strains measured by the first invariant of Green strain tensor decreased with disease severity (0.16 vs. 0.12 vs. 0.11, [Formula: see text]). Mean WSS for the distal PAs between 100 and 500 [Formula: see text] significantly increased with disease severity (20 vs. 52 vs. 116 [Formula: see text], [Formula: see text]). In conclusion, 3D flow simulations showed that WSS is significantly decreased in the MPA with disease while the mathematical morphometric model suggested increased WSS in the distal small vessels. Computational models can reveal mechanical stimuli acting on vessel walls that may inform patient risk stratification and flow shear experiments.
Citation:
Biomech Model Mechanobiol. 2019 Jan 12. doi: 10.1007/s10237-018-01114-0. [Epub ahead of print]
https://link.springer.com/article/10.1007/s10237-018-01114-0
Solid stress in brain tumours causes neuronal loss and neurological dysfunction and can be reversed by lithium
Authors:
Seano G, Nia HT, Emblem KE, Datta M, Ren J, Krishnan S, Kloepper J, Pinho M, Ho WW, Ghosh M, Askoxylakis V, Ferraro GB, Riedemann L, Gerstner ER, Batchelor TT, Wen PY, Lin NU, Grodzinsky AJ, Fukumura D, Huang P, Baish JW, Padera TP, Munn LL, Jain RK
Abstract:
The compression of brain tissue by a tumour mass is believed to be a major cause of the clinical symptoms seen in patients with brain cancer. However, the biological consequences of these physical stresses on brain tissue are unknown. Here, via imaging studies in patients and by using mouse models of human brain tumours, we show that a subgroup of primary and metastatic brain tumours, classified as nodular on the basis of their growth pattern, exert solid stress on the surrounding brain tissue, causing a decrease in local vascular perfusion as well as neuronal death and impaired function. We demonstrate a causal link between solid stress and neurological dysfunction by applying and removing cerebral compression, which respectively mimic the mechanics of tumour growth and of surgical resection. We also show that, in mice, treatment with lithium reduces solid-stress-induced neuronal death and improves motor coordination. Our findings indicate that brain-tumour-generated solid stress impairs neurological function in patients, and that lithium as a therapeutic intervention could counter these effects.
Citation:
Nature Biomedical Engineering(2019)
CD36 mediates albumin transcytosis by dermal but not lung microvascular endothelial cells - role in fatty acid delivery
Authors:
Raheel H, Ghaffari S, Khosraviani N, Mintsopoulos V, Auyeung D, Wang C, Kim YH, Mullen B, Sung HK, Ho M, Fairn G, Neculai D, Febbraio M, Heit B, Lee WL
Abstract:
In healthy blood vessels, albumin crosses the endothelium to leave the circulation by transcytosis. However, little is known about the regulation of albumin transcytosis or how it differs in different tissues; its physiological purpose is also unclear. Using total internal reflection fluorescence microscopy, we quantified transcytosis of albumin across primary human microvascular endothelial cells from both lung and skin. We then validated our in vitro findings using a tissue-specific knockout mouse model. We observed that albumin transcytosis was saturable in the skin but not the lung microvascular endothelial cells, implicating a receptor-mediated process. We identified the scavenger receptor CD36 as being both necessary and sufficient for albumin transcytosis across dermal microvascular endothelium, in contrast to the lung where macropinocytosis dominated. Mutations in the apical helical bundle of CD36 prevented albumin internalization by cells. Mice deficient in CD36 specifically in endothelial cells exhibited lower basal permeability to albumin and less basal tissue edema in the skin but not in the lung. Finally, these mice also exhibited a smaller subcutaneous fat layer despite having identical total body weights and circulating fatty acid levels as wild-type animals. In conclusion, CD36 mediates albumin transcytosis in the skin but not the lung. Albumin transcytosis may serve to regulate fatty acid delivery from the circulation to tissues.
Citation:
Am J Physiol Lung Cell Mol Physiol. 2019 Jan 31. doi: 10.1152/ajplung.00127.2018. [Epub ahead of print]
Mature vessel networks in engineered tissue promote graft–host anastomosis and prevent graft thrombosis
Authors:
Shahar Ben-Shaul, Shira Landau, Uri Merdler, and Shulamit Levenberg
Abstract:
Graft vascularization remains one of the most critical challenges facing tissue-engineering experts in their attempt to create thick transplantable tissues and organs. In vitro prevascularization of engineered tissues has been suggested to promote rapid anastomosis between the graft and host vasculatures; however, thrombotic events have been reported upon graft implantation. Here, we aimed to determine whether in vitro vessel maturation in transplantable grafts can accelerate vascular integration and graft perfusion and prevent thrombotic events in the grafts. To this end, endothelial cells and fibroblasts were cocultured on 3D scaffolds for 1, 7, or 14 d to form vasculature with different maturation degrees. Monitoring graft–host interactions postimplantation demonstrated that the 14-d in vitro-cultured grafts, bearing more mature and complex vessel networks as indicated by elongated and branched vessel structures, had increased graft–host vessel anastomosis; host vessel penetration into the graft increased approximately eightfold, and graft perfusion increased sixfold. The presence of developed vessel networks prevented clot accumulation in the grafts. Conversely, short-term cultured constructs demonstrated poor vascularization and increased thrombus formation. Elevated expression levels of coagulation factors, von Willebrand factor (vWF), and tissue factor (TF), were demonstrated in constructs bearing less mature vasculature. To conclude, these findings demonstrate the importance of establishing mature and complex vessel networks in engineered tissues before implantation to promote anastomosis with the host and accelerate graft perfusion.
Citation:
PNAS published ahead of print February 4, 2019 https://doi.org/10.1073/pnas.1814238116
AIBP-mediated cholesterol efflux instructs hematopoietic stem and progenitor cell fate
Authors:
Gu Q, Yang X, Lv J, Zhang J, Xia B, Kim JD, Wang R, Xiong F, Meng S, Clements TP, Tandon B, Wagner DS, Diaz MF, Wenzel PL, Miller YI, Traver D, Cooke JP, Li W, Zon LI, Chen K, Bai Y, Fang L
Abstract:
Hypercholesterolemia, the driving force of atherosclerosis, accelerates the expansion and mobilization of hematopoietic stem and progenitor cells (HSPCs). The molecular determinants connecting hypercholesterolemia with hematopoiesis are unclear. Here we report that a somite-derived pro-hematopoietic cue, AIBP, orchestrates HSPC emergence from the hemogenic endothelium, a type of specialized endothelium manifesting hematopoietic potential. Mechanistically, AIBP-mediated cholesterol efflux activates endothelial Srebp2, the master transcription factor for cholesterol biosynthesis, which in turn transactivates Notch and promotes HSPC emergence. Srebp2 inhibition impairs hypercholesterolemia-induced HSPC expansion. Srebp2 activation and Notch upregulation are associated with HSPC expansion in hypercholesterolemic human subjects. Genome-wide ChIP-seq, RNA-seq, and ATAC-seq indicate that Srebp2 trans-regulates Notch pathway genes required for hematopoiesis. Our studies outline an AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease.
Citation:
Science. 2019 Jan 31. pii: eaav1749. doi: 10.1126/science.aav1749. [Epub ahead of print]
http://science.sciencemag.org/content/early/2019/01/30/science.aav1749
Visualization and Quantification of Mitochondrial Structure in the Endothelium of Intact Arteries
Authors:
Durand MJ, Ait-Aissa K, Levchenko V, Staruschenko A, Gutterman DD, Beyer AM
Abstract:
Aims: To quantify the mitochondrial structure of ECs in intact arteries vs. cultured cells. Methods and Results: Cre-stop mito-Dendra2 mice, expressing the fluorescent protein Dendra2 in the mitochondrial matrix only, were used to label EC mitochondria using Cre-recombinase under the control of the VE-cadherin promoter. Conduit arteries, resistance arterioles and veins were fixed, mounted on glass slides and fluorescent images were obtained using a laser scanning confocal microscope (ex 488 nm; em 550 nm). ImageJ was used to calculate form factor (FF) and aspect ratio (AR) of the mitochondrial segments. Mitochondrial fragmentation count (MFC) was calculated by counting non-contiguous mitochondrial particles and dividing by the number of pixels which comprise the mitochondrial network. Primary aortic EC cultures (48 h on culture plates) were generated to compare the mitochondrial structure of cultured ECs vs. intact arteries. Aortic segments were also exposed to high glucose overnight (33 mM) ex vivo, and separate groups of mice were either infused with a high glucose saline solution (300 mM) via tail vein catheter for one hour or injected with Streptozotocin (STZ; 50 mg/kg) to cause hyperglycemia. Compared to cultured ECs, the mitochondria of ECs from the intact aorta were more fragmented (MFC: 6.4±2.5 vs. 18.6±9.4, respectively; p < 0.05). The mitochondrial segments of ECs within the aorta were more circular in shape (FF: 3.5±0.75 vs. 1.8±0.30, respectively; p < 0.05) and had less branching (AR: 2.9±0.60 vs. 2.0±0.25, respectively; p < 0.05) compared to cultured ECs. Ex vivo exposure of the intact aorta to high glucose overnight caused mitochondrial fission compared to normal glucose conditions (5 mM; MFC: 25.5±11.1 high glucose vs. 11.0±3.6 normal glucose; p < 0.05). Both one-hour infusion of high glucose saline (MFC: 22.4±4.3) and STZ treatment (MFC: 40.3±14.2) caused mitochondrial fission compared to freshly fixed aortas from control mice (MFC: 18.6±9.4; p < 0.05 vs. high glucose infusion and STZ treatment). Conclusions: Using a novel mouse model, we were able to, for the first time, obtain high resolution images of EC mitochondrial structure in intact arteries. We reveal the endothelial mitochondrial network is more fragmented in the intact aorta compared to cultured ECs, indicating that mitochondria assume a more elongated and branched phenotype in cell culture.
Citation:
Cardiovasc Res. 2018 Nov 22. doi: 10.1093/cvr/cvy294. [Epub ahead of print]
https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvy294/5198893
A dynamic and integrated epigenetic program at distal regions orchestrates transcriptional responses to VEGFA
Authors:
Wang S, Chen J, Garcia SP, Liang X, Zhang F, Yan P, Yu H, Wei W, Li Z, Wang J, Le H, Han Z, Luo X, Day DS, Stevens SM, Zhang Y, Park PJ, Liu ZJ, Sun K, Yuan GC, Pu WT, Zhang B
Abstract:
Cell behaviors are dictated by epigenetic and transcriptional programs. Little is known about how extracellular stimuli modulate these programs to reshape gene expression and control cell behavioral responses. Here, we interrogated the epigenetic and transcriptional response of endothelial cells to VEGFA treatment and found rapid chromatin changes that mediate broad transcriptomic alterations. VEGFA-responsive genes were associated with active promoters, but changes in promoter histone marks were not tightly linked to gene expression changes. VEGFA altered transcription factor occupancy and the distal epigenetic landscape, which profoundly contributed to VEGFA-dependent changes in gene expression. Integration of gene expression, dynamic enhancer, and transcription factor occupancy changes induced by VEGFA yielded a VEGFA-regulated transcriptional regulatory network, which revealed that the small MAF transcription factors are master regulators of the VEGFA transcriptional program and angiogenesis. Collectively these results revealed that extracellular stimuli rapidly reconfigure the chromatin landscape to coordinately regulate biological responses.
Citation:
Genome Res. 2019 Jan 22. doi: 10.1101/gr.239053.118. [Epub ahead of print]
Bola3 Deficiency Controls Endothelial Metabolism and Glycine Homeostasis in Pulmonary Hypertension
Authors:
Yu Q, Tai YY, Tang Y, Zhao J, Negi V, Culley MK, Pilli J, Sun W, Brugger K, Mayr J, Saggar R, Saggar R, Wallace WD, Ross DJ, Waxman AB, Wendell SG, Mullett SJ, Sembrat J, Rojas M, Khan OF, Dahlman J, Sugahara M, Kagiyama N, Satoh T, Zhang M, Feng N, Gorcsan J, Vargas SO, Haley KJ, Kumar R, Graham BB, Langer R, Anderson DG, Wang B, Shiva S, Bertero T, Chan SY
Abstract:
Background and Hypothesis: Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. The BolA Family Member 3 (BOLA3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. However, the exact mechanistic role of BOLA3 in PH remains undefined. Methods and Results: In cultured hypoxic PAECs as well as lung from human and multiple rodent models of PH, endothelial BOLA3 expression was down-regulated, which involved HIF-2α-dependent transcriptional repression via HDAC-mediated histone deacetylation. In vitro gain and loss of function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In addition, BOLA3 deficiency down-regulated the glycine cleavage system protein H (GCSH), thus bolstering intracellular glycine content. As a result of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction, while decreasing angiogenic potential. In vivo, pharmacologic knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 demonstrated that BOLA3 deficiency promotes histologic and hemodynamic manifestations of PH. Conclusion: In aggregate, BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic re-programming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, iron-sulfur biogenesis, and glycine biology, for diagnostic and therapeutic development in this disease.
Citation:
Originally published5 Nov 2018Circulation. 2018;138:A11422
https://www.ahajournals.org/doi/abs/10.1161/circ.138.suppl_1.11422
Ponatinib Combined With Rapamycin Causes Regression of Murine Venous Malformation
Authors:
Li X, Cai Y, Goines J, Pastura P, Brichta L, Lane A, Le Cras TD, Boscolo E
Abstract:
Objective- Venous malformations (VMs) arise from developmental defects of the vasculature and are characterized by massively enlarged and tortuous venous channels. VM grow commensurately leading to deformity, obstruction of vital structures, bleeding, and pain. Most VMs are associated with the activating mutation L914F in the endothelial cell (EC) tyrosine kinase receptor TIE2. Therapeutic options for VM are limited and ineffective while therapy with the mammalian target of rapamycin inhibitor rapamycin shows moderate efficacy. Here, we investigated novel therapeutic targets promoting VM regression. Approach and Results- We performed an unbiased screen of Food and Drug Administration-approved drugs in human umbilical vein ECs expressing the TIE2-L914F mutation (HUVEC-TIE2-L914F). Three ABL (Abelson) kinase inhibitors prevented cell proliferation of HUVEC-TIE2-L914F. Moreover, c-ABL, common target of these inhibitors, was highly phosphorylated in HUVEC-TIE2-L914F and VM patient-derived ECs with activating TIE2 mutations. Knockdown of c-ABL/ARG in HUVEC-TIE2-L914F reduced cell proliferation and vascularity of murine VM. Combination treatment with the ABL kinase inhibitor ponatinib and rapamycin caused VM regression in a xenograft model of VM based on injection of HUVEC-TIE2-L914F. A reduced dose of this drug combination was effective in this VM murine model with minimal side effects. The drug combination was antiproliferative, enhanced cell apoptosis, and vascular channel regression both in vivo and in a 3-dimensional fibrin gel assay. Conclusions- This is the first report of a combination therapy with ponatinib and rapamycin promoting regression of VM. Mechanistically, the drug combination enhanced AKT inhibition compared with single drug treatment and reduced PLCγ and ERK activity.
Citation:
Arterioscler Thromb Vasc Biol. 2019 Jan 10:ATVBAHA118312315. doi: 10.1161/ATVBAHA.118.312315. [Epub ahead of print]
Loss of estrogen-related receptor alpha facilitates angiogenesis in endothelial cells
Authors:
Likhite N, Yadav V, Milliman EJ, Sopariwala DH, Lorca S, Narayana NP, Sheth M, Reineke EL, Giguère V, Narkar V
Abstract:
Estrogen-related receptors (ERRs) have emerged as major metabolic regulators in various tissues. However, their expression and function in the vasculature remains unknown. Here we report the transcriptional program and cellular function of ERRα in endothelial cells (ECs), a cell type with a multi-faceted role in vasculature. Of the three ERR subtypes ECs exclusively express ERRα. Gene expression profiling of ECs lacking ERRα revealed that ERRα predominantly acts as a transcriptional repressor, targeting genes linked with angiogenesis, cell migration and cell adhesion. ERRα-deficient ECs exhibit decreased proliferation, but increased migration and tube formation. ERRα depletion increased basal, VEGFA and ANG1/2 stimulated angiogenic sprouting in endothelial spheroids. Moreover, retinal angiogenesis is enhanced in ERRα knockout mice compared to wild type mice. Surprisingly, ERRα is dispensable for the regulation of its classic targets such as metabolism, mitochondrial biogenesis and cellular respiration in the ECs. ERRα is enriched at the promoters of angiogenic, migratory and cell adhesion genes. Further, VEGFA increased ERRα recruitment to angiogenesis-associated genes and simultaneously decreased their expression. Despite increasing its gene occupancy, pro-angiogenic stimuli decrease ERRα expression in ECs. Our work shows that endothelial ERRα plays a repressive role in angiogenesis, and potentially fine-tunes growth factor-mediated angiogenesis.IMPORTANCE Impaired angiogenesis, the process of blood vessel formation, is central to various diseases including peripheral vascular disease, retinopathy, nephropathy, cardiac ischemia and cancer. While few angiogenic regulators have been described, full breadth of the cellular and molecular pathways that regulate this phenomenon are far from clear. Fundamental discoveries related to angiogenesis regulation could lead to new drugs that normalize blood vessels in the aforementioned diseases. Using a cell and molecular biology approach, our work highlights a gene regulator called ERRα in endothelial cells (the building blocks of the vasculature), which plays a role in limiting angiogenic activation by controlling endothelial gene expression. It can be envisioned that future pre-clinical and pharmaco/physiological studies could lead to advancement of ERRα as a therapeutic regulator of angiogenesis in vascular-linked diseases.
Citation:
Mol Cell Biol. 2019 Jan 2. pii: MCB.00411-18. doi: 10.1128/MCB.00411-18. [Epub ahead of print]
Induction of Brain Arteriovenous Malformation Through CRISPR/Cas9-Mediated Somatic Alk1 Gene Mutations in Adult Mice
Authors:
Zhu W, Saw D, Weiss M, Sun Z, Wei M, Shaligram S, Wang S, Su H
Abstract:
Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. The pathogenesis of bAVM has not been fully understood. Animal models are important tools for dissecting bAVM pathogenesis and testing new therapies. We have developed several mouse bAVM models using genetically modified mice. However, due to the body size, mouse bAVM models have some limitations. Recent studies identified somatic mutations in sporadic human bAVM. To develop a feasible tool to create sporadic bAVM in rodent and animals larger than rodent, we made tests using the CRISPR/Cas9 technique to induce somatic gene mutations in mouse brain in situ. Two sequence-specific guide RNAs (sgRNAs) targeting mouse Alk1 exons 4 and 5 were cloned into pAd-Alk1e4sgRNA + e5sgRNA-Cas9 plasmid. These sgRNAs were capable to generate mutations in Alk1 gene in mouse cell lines. After packaged into adenovirus, Ad-Alk1e4sgRNA + e5sgRNA-Cas9 was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brains of wild-type C57BL/6J mice. Eight weeks after viral injection, bAVMs were detected in 10 of 12 mice. Compared to the control (Ad-GFP/AAV-VEGF-injected) brain, 13% of Alk1 alleles were mutated and Alk1 expression was reduced by 26% in the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF-injected brains. Around the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF injected site, Alk1-null endothelial cells were detected. Our data demonstrated that CRISPR/Cas9 is a feasible tool for generating bAVM model in animals.
Citation:
Transl Stroke Res. 2018 Dec 3. doi: 10.1007/s12975-018-0676-1. [Epub ahead of print]
Partial Deletion of Tie2 Affects Microvascular Endothelial Responses to Critical Illness in a Vascular Bed and Organ-Specific Way
Authors:
Jongman RM, Zwiers PJ, van de Sluis B, van der Laan M, Moser J Zijlstra JG, Dekker D, Huijkman N, Moorlag HE, Popa ER, Molema G, van Meurs M
Abstract:
Tie2 is a tyrosine kinase receptor that is mainly expressed by endothelial cells. In animal models mimicking critical illness, Tie2 levels in organs are temporarily reduced. Functional consequences of these reduced Tie2 levels on microvascular endothelial behaviour are unknown. We investigated the effect of partial deletion of Tie2 on the inflammatory status of endothelial cells in different organs. Newly-generated heterozygous Tie2 knockout mice (exon 9 deletion, ΔE9/Tie2) exhibiting 50% reduction in Tie2 mRNA and protein, and wild type littermate controls (Tie2), were subjected to hemorrhagic shock and resuscitation (HS + R), or challenged with i.p. lipopolysaccharide (LPS). Kidney, liver, lung, heart, brain, and intestine were analyzed for mRNA levels of adhesion molecules E-selectin, VCAM-1, and ICAM-1, and CD45. Exposure to HS + R did not result in different expression responses of these molecules between organs from Tie2 or Tie2 mice and sham-operated mice. In contrast, the LPS-induced mRNA expression levels of E-selectin, VCAM-1, and ICAM-1, and CD45 in organs were attenuated in Tie2 mice when compared to Tie2 mice in kidney and liver, but not in the other organs studied. Furthermore, reduced expression of E-selectin and VCAM-1 protein, and reduced influx of CD45 cells upon LPS exposure, was visible in a microvascular bed-specific pattern in kidney and liver of Tie2 mice compared to controls. In contrast to the hypothesis that a dysbalance in the Ang/Tie2 system leads to increased microvascular inflammation, heterozygous deletion of Tie2 is associated with an organ-restricted, microvascular bed-specific attenuation of endothelial inflammatory response to LPS.
Citation:
Shock. 2018 Jul 30. doi: 10.1097/SHK.0000000000001226. [Epub ahead of print]
Early Heterogenic Response of Renal Microvasculature to Hemorrhagic Shock/Resuscitation and the Influence of NF-κB Pathway Blockade
Authors:
Yan R, van Meurs M, Popa ER, Li R, Zwiers PJ, Zijlstra JG, Molema G.
Abstract:
Hemorrhagic shock (HS) is associated with low blood pressure due to excessive loss of circulating blood and causes both macrocirculatory and microcirculatory dysfunction. Fluid resuscitation after HS is used in the clinic to restore tissue perfusion. The persistent microcirculatory damage caused by HS and/or resuscitation can result in multiple organ damage, with the kidney being one of the involved organs. The kidney microvasculature consists of different segments that possess a remarkable heterogeneity in functional properties. The aim of this study was to investigate the inflammatory responses of these different renal microvascular segments, i.e., arterioles, glomeruli, and postcapillary venules, to HS and resuscitation (HS/R) in mice and to explore the effects of intervention with an NF-κB inhibitor on these responses. We found that HS/R disturbed the balance of the Angiopoietin-Tie2 ligand-receptor system, especially in the glomeruli. Furthermore, endothelial adhesion molecules, pro-inflammatory cytokines, and chemokines were markedly upregulated by HS/R, with the strongest responses occurring in the glomerular and postcapillary venous segments. Blockade of NF-κB signaling during the resuscitation period only slightly inhibited HS/R induced inflammatory activation, possibly because NF-κB p65 nuclear translocation already occurred during the HS period. In summary, although all three renal microvascular segments were activated upon HS/R, responses of endothelial cells in glomeruli and postcapillary venules to HS/R, as well as to NF-κB inhibition were stronger than those in arterioles. NF-κB inhibition during the resuscitation phase does not effectively counteract NF-κB p65 nuclear translocation initiating inflammatory gene transcription.This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0.
Citation:
Shock. 2018 Feb 21. doi: 10.1097/SHK.0000000000001126. [Epub ahead of print]
Phenotypic characterization of murine models of cerebral cavernous malformations
Authors:
Zeineddine HA, Girard R, Saadat L, Shen L, Lightle R, Moore T, Cao Y, Hobson N, Shenkar R, Avner K, Chaudager K, Koskimäki J, Polster SP, Fam MD, Shi C, Lopez-Ramirez MA, Tang AT, Gallione C, Kahn ML, Ginsberg M, Marchuk DA, Awad IA
Abstract:
Cerebral cavernous malformations (CCMs) are clusters of dilated capillaries that affect around 0.5% of the population. CCMs exist in two forms, sporadic and familial. Mutations in three documented genes, KRIT1(CCM1), CCM2, and PDCD10(CCM3), cause the autosomal dominant form of the disease, and somatic mutations in these same genes underlie lesion development in the brain. Murine models with constitutive or induced loss of respective genes have been applied to study disease pathobiology and therapeutic manipulations. We aimed to analyze the phenotypic characteristic of two main groups of models, the chronic heterozygous models with sensitizers promoting genetic instability, and the acute neonatal induced homozygous knockout model. Acute model mice harbored a higher lesion burden than chronic models, more localized in the hindbrain, and largely lacking iron deposition and inflammatory cell infiltrate. The chronic model mice showed a lower lesion burden localized throughout the brain, with significantly greater perilesional iron deposition, immune B- and T-cell infiltration, and less frequent junctional protein immunopositive endothelial cells. Lesional endothelial cells in both models expressed similar phosphorylated myosin light chain immunopositivity indicating Rho-associated protein kinase activity. These data suggest that acute models are better suited to study the initial formation of the lesion, while the chronic models better reflect lesion maturation, hemorrhage, and inflammatory response, relevant pathobiologic features of the human disease.
Citation:
Lab Invest. 2018 Jun 26. doi: 10.1038/s41374-018-0030-y. [Epub ahead of print]
Cerebral cavernous malformations form an anticoagulant vascular domain
Authors:
Lopez-Ramirez MA, Pham A, Girard R, Wyseure T, Hale P, Yamashita A, Koskimäki J, Polster S, Saadat L, Romero IA, Esmon CT, Lagarrigue F, Awad IA, Mosnier LO, Ginsberg MH
Abstract:
Cerebral cavernous malformations (CCM) are common brain vascular dysplasias prone to acute and chronic hemorrhage with significant clinical sequelae. The pathogenesis of recurrent bleeding in CCM is incompletely understood. Here we show that central nervous system (CNS) hemorrhage in CCM is associated with locally elevated expression of the anticoagulant endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). TM levels are increased in human CCM lesions and in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of Krit1 (Krit1ECKO ) or Pdcd10 (Pdcd10ECKO ), which cause CCM formation, result in increased levels of vascular TM and EPCR, and in enhanced generation of activated protein C (APC) on endothelial cells. Increased TM expression is due to upregulation of transcription factors KLF2 and KLF4 consequent to the loss of KRIT1 or PDCD10 Increased TM expression contributes to CCM hemorrhage, because genetic inactivation of one or two copies of the Thbd gene decreases brain hemorrhage in Pdcd10ECKO mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in Pdcd10ECKO mice. Thus, a local increase in the endothelial co-factors that generate anticoagulant APC can contribute to bleeding in CCMs and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs.
Citation:
Blood. 2018 Nov 15. pii: blood-2018-06-856062. doi: 10.1182/blood-2018-06-856062. [Epub ahead of print]
Association of Somatic GNAQ Mutation With Capillary Malformations in a Case of Choroidal Hemangioma
Authors:
Colette A. Bichsel, Jeremy Goss, Mohammed Alomari , Sanda Alexandrescu, Richard Robb, Lois E. Smith , Marcelo Hochman, Arin Greene, Joyce Bischoff
Abstract:
Importance Choroidal hemangiomas are defined by a thickened choroid owing to vessel overgrowth, which may increase the intraocular pressure and lead to glaucoma. Choroidal hemangioma and glaucoma often co-occur in patients with Sturge-Weber syndrome, a rare neurocutaneous disorder characterized by capillary malformations. Objective To determine whether the mutation found in most capillary malformations, GNAQ R183Q (c.548G>A), was present in the choroidal hemangioma of a patient with Sturge-Weber syndrome. Design, Setting, and Participant Using laser-capture microdissection, choroidal blood vessels were isolated from paraffin-embedded tissue sections, and genomic DNA was extracted for mutational analysis. Choroidal sections were analyzed in parallel. A patient with choroidal hemangioma and Sturge-Weber syndrome who had undergone enucleation was analyzed in this study at Boston Children’s Hospital. Negative controls were choroidal tissue from an eye with retinoblastoma and unaffected lung tissue; brain tissue from a different patient with Sturge-Weber syndrome served as a positive control. Infantile hemangioma was analyzed as well. Data were analyzed in 2018. Main Outcomes and Measures The mutant allelic frequency of GNAQ R183 and GNAQ Q209L/H/P was determined by droplet digital polymerase chain reaction on isolated genomic DNA. The infantile hemangioma marker glucose transporter-1 was visualized by immunofluorescent staining of tissue sections. Results The GNAQ R183Q mutation was present in the patient’s choroidal vessels (21.1%) at a frequency similar to that found in brain tissue from a different patient with Sturge-Weber syndrome (25.1%). In contrast, choroidal vessels from a case of retinoblastoma were negative for the mutation (0.5%), as was lung tissue (0.2%). The patient’s choroidal tissue was negative for the 3 GNAQ mutations associated with congenital hemangioma and for the infantile hemangioma marker glucose transporter-1. Conclusions and Relevance The results suggest that a more accurate description for choroidal hemangioma in patients with Sturge-Weber syndrome is choroidal capillary malformation. This finding may explain why propranolol, used to treat infantile hemangiomas, has been largely ineffective in patients with choroidal hemangioma. Further studies are needed to corroborate this finding.
Citation:
JAMA Ophthalmol. Published online October 25, 2018. doi:10.1001/jamaophthalmol.2018.5141
HIV-Nef Protein Persists in the Lungs of Aviremic HIV Patients and Induces Endothelial Cell Death
Authors:
Chelvanambi S, Bogatcheva N, Bednorz M, Agarwal S, Maier B, Alves NJ, Li W, Syed F, Saber MM, Dahl N, Lu H, Day RB, Smith P, Jolicoeur P, Yu Q, Dhillon NK, Weissmann N, Twigg Iii HL, Clauss M
Abstract:
It remains a mystery why HIV-associated end-organ pathologies persist in the era of combined antiretroviral therapy (ART). One possible mechanism is the continued production of HIV encoded proteins in latently HIV-infected T cells and macrophages. The pro-apoptotic protein HIV-Nef persists in the blood of ART treated patients within extracellular vesicles (EV) and PBMC. Here we demonstrate that HIV-Nef is present in cells and extracellular vesicles (EV) isolated from bronchoalveolar lavage (BAL) of patients on ART. We hypothesize that HIV-Nef persistence in lung induces endothelial apoptosis leading to endothelial dysfunction and further pulmonary vascular pathologies. HIV-Nef presence in HIV patients correlates with the surface expression of the pro-apoptotic Endothelial-Monocyte Activating Polypeptide II (EMAPII), which was implicated in progression of pulmonary emphysema via mechanisms involving endothelial cell death. HIV-Nef protein induces EMAPII surface expression in HEK293T, T cells, and human and mouse lung endothelial cells. HIV-Nef packages itself into EV and increases the amount of EV secreted from Nef-expressing T cells and Nef-transfected HEK293T cells. EV from BAL of HIV-positive patients and Nef-transfected cells induce apoptosis in lung microvascular endothelial cells by upregulating EMAPII surface expression in a PAK2-dependent fashion. Transgenic expression of HIV-Nef in VE-Cadherin-positive endothelial cells leads to lung rarefaction, characterized by reduced alveoli and overall increase in lung inspiratory capacity. These changes occur concomitantly with lung endothelial cell apoptosis. Together, these data suggest that HIV-Nef induces endothelial cell apoptosis using an EMAPII dependent mechanism that is sufficient to cause pulmonary vascular pathologies even in the absence of inflammation.
Citation:
Am J Respir Cell Mol Biol. 2018 Oct 15. doi: 10.1165/rcmb.2018-0089OC. [Epub ahead of print]
Lysophosphatidic acid acts on LPA1 receptor to increase H2O2 during flow‐induced dilation in human adipose arterioles
Authors:
Chabowski DS, Kadlec AO, Ait-Aissa K, Hockenberry JC, Pearson PJ, Beyer AM, Gutterman DD
Abstract:
BACKGROUND AND PURPOSE: Nitric oxide (NO) produces arteriolar flow-induced dilation (FID) in healthy subjects but is replaced by mitochondria-derived hydrogen peroxide (mtH2 O2 ) in patients with coronary artery disease (CAD). Lysophosphatidic acid (LPA) is elevated in patients with risk factors for CAD but its functional effect in arterioles is unknown. We tested whether elevated LPA changes the mediator of FID from NO to mtH2 O2 in human visceral and subcutaneous adipose arterioles.
Citation:
Br J Pharmacol. 2018 Aug 28. doi: 10.1111/bph.14492. [Epub ahead of print]
https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14492
Cerebral Cavernous Malformations Develop Through Clonal Expansion of Mutant Endothelial Cells
Authors:
Matthew R Detter, Daniel A Snellings, and Douglas A Marchuk
Abstract:
Rationale: Vascular malformations arise in vessels throughout the entire body. Causative genetic mutations have been identified for many of these diseases; however, little is known about the mutant cell lineage within these malformations. Objective: We utilize an inducible mouse model of cerebral cavernous malformations (CCMs) coupled with a multi-color fluorescent reporter to visualize the contribution of mutant endothelial cells (ECs) to the malformation. Methods and Results: We combined a Ccm3 mouse model with the confetti fluorescent reporter to simultaneously delete Ccm3 and label the mutant EC with one of four possible colors. We acquired Z-series confocal images from serial brain sections and created 3D reconstructions of entire CCMs to visualize mutant ECs during CCM development. We observed a pronounced pattern of CCMs lined with mutant ECs labeled with a single confetti color (n=42). The close 3D distribution, as determined by the nearest neighbor analysis, of the clonally dominant ECs within the CCM was statistically different than the background confetti labeling of ECs in non-CCM control brain slices as well as a computer simulation (p<0.001). Many of the small (<100m diameter) CCMs consisted, almost exclusively, of the clonally dominant mutant ECs labeled with the same confetti color whereas the large (>100m diameter) CCMs contained both the clonally dominant mutant cells and wild type ECs. We propose of model of CCM development in which an EC acquires a second somatic mutation, undergoes clonal expansion to initiate CCM formation, and then incorporates neighboring wild type ECs to increase the size of the malformation. Conclusions: This is the first study to visualize, with single-cell resolution, the clonal expansion of mutant ECs within CCMs. The incorporation of wild type ECs into the growing malformation presents another series of cellular events whose elucidation would enhance our understanding of CCMs and may provide novel therapeutic opportunities.
Citation:
Originally published 18 Sep 2018 Circulation Research. 2018
https://www.ahajournals.org/doi/abs/10.1161/CIRCRESAHA.118.313970
Gene expression profiles of rat MMECs with different glucose levels and fgl2 gene silencing
Authors:
Zheng Z, Zhang F, Gao D, Wu Y, Wu H
Abstract:
BACKGROUND: Cardiac microvascular endothelial cells is one of the key factors in the process of diabetic cardiomyopathy, a common chronic complication of diabetes. Fibrinogen-like protein 2 (FGL2) is linked to apoptosis, angiogenesis, and inflammatory response, all of which also occur in diabetes. Thus, we investigate the role of FGL 2 and other genes in the pathology of diabetic cardiomyopathy.
Citation:
Diabetes Metab Res Rev. 2018 Aug 11:e3058. doi: 10.1002/dmrr.3058. [Epub ahead of print]
Epsin deficiency promotes lymphangiogenesis through regulation of VEGFR3 degradation in diabetes
Authors:
Wu H, Rahman HNA, Dong Y, Liu X, Lee Y, Wen A, To KH, Xiao L, Birsner AE, Bazinet L, Wong S, Song K, Brophy ML, Mahamud MR, Chang B, Cai X, Pasula S, Kwak S, Yang W, Bischoff J, Xu J, Bielenberg DR, Dixon JB, D'Amato RJ, Srinivasan RS, Chen H
Abstract:
Impaired lymphangiogenesis is a complication of chronic complex diseases, including diabetes. VEGF-C/VEGFR3 signaling promotes lymphangiogenesis, but how this pathway is affected in diabetes remains poorly understood. We previously demonstrated that loss of epsins 1 and 2 in lymphatic endothelial cells (LECs) prevented VEGF-C-induced VEGFR3 from endocytosis and degradation. Here, we report that diabetes attenuated VEGF-C-induced lymphangiogenesis in corneal micropocket and Matrigel plug assays in WT mice but not in mice with inducible lymphatic-specific deficiency of epsins 1 and 2 (LEC-iDKO). Consistently, LECs isolated from diabetic LEC-iDKO mice elevated in vitro proliferation, migration, and tube formation in response to VEGF-C over diabetic WT mice. Mechanistically, ROS produced in diabetes induced c-Src-dependent but VEGF-C-independent VEGFR3 phosphorylation, and upregulated epsins through the activation of transcription factor AP-1. Augmented epsins bound to and promoted degradation of newly synthesized VEGFR3 in the Golgi, resulting in reduced availability of VEGFR3 at the cell surface. Preclinically, the loss of lymphatic-specific epsins alleviated insufficient lymphangiogenesis and accelerated the resolution of tail edema in diabetic mice. Collectively, our studies indicate that inhibiting expression of epsins in diabetes protects VEGFR3 against degradation and ameliorates diabetes-triggered inhibition of lymphangiogenesis, thereby providing a novel potential therapeutic strategy to treat diabetic complications.
Citation:
J Clin Invest. 2018 Aug 13. pii: 96063. doi: 10.1172/JCI96063. [Epub ahead of print]
Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells
Authors:
Paik DT, Tian L, Lee J, Sayed N, Chen IY, Rhee S, Rhee JW, Kim Y, Wirka RC, Buikema JW, Wu SM, Red-Horse K, Quertermous T, Wu JC
Abstract:
Rationale: Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology.
Citation:
Circ Res. 2018 Jul 9. pii: CIRCRESAHA.118.312913. doi: 10.1161/CIRCRESAHA.118.312913. [Epub ahead of print]
https://www.ahajournals.org/doi/10.1161/CIRCRESAHA.118.312913
Estrogen Inhibits LDL (Low-Density Lipoprotein) Transcytosis by Human Coronary Artery Endothelial Cells via GPER (G-Protein–Coupled Estrogen Receptor) and SR-BI
Authors:
Ghaffari S, Naderi Nabi F, Sugiyama MG, Lee WL
Abstract:
Objective - The atheroprotective effects of estrogen are independent of circulating lipid levels. Whether estrogen regulates transcytosis of LDL (low-density lipoprotein) across the coronary endothelium is unknown. Approach and Results - Using total internal reflection fluorescence microscopy, we quantified transcytosis of LDL across human coronary artery endothelial cells from multiple donors. LDL transcytosis was significantly higher in cells from men compared with premenopausal women. Estrogen significantly attenuated LDL transcytosis by endothelial cells from male but not female donors; transcytosis of albumin was not affected. Estrogen caused downregulation of endothelial SR-BI, and overexpression of SR-BI was sufficient to restore LDL transcytosis. Similarly, depletion of SR-BI by siRNA attenuated endothelial LDL transcytosis and prevented any further effect of estrogen. In contrast, treatment with estrogen had no effect on SR-BI expression by liver cells. Inhibition of estrogen receptors α and β had no effect on estrogen-mediated attenuation of LDL transcytosis. However, estrogen’s effect on LDL transcytosis was blocked by depletion of the GPER (G-protein–coupled estrogen receptor). GPER was found to be enriched in endothelial cells compared with hepatocytes and is reported to signal via transactivation of the EGFR (epidermal growth factor receptor); inhibition of EGFR prevented the effect of estrogen on LDL transcytosis and SR-BI mRNA. Last, SR-BI expression was significantly higher in human coronary artery endothelial cells from male compared with premenopausal female donors. Conclusions - Estrogen significantly inhibits LDL transcytosis by downregulating endothelial SR-BI; this effect requires GPER.
Citation:
Arteriosclerosis, Thrombosis, and Vascular Biology. 2018;0:ATVBAHA.118.310792
https://www.ahajournals.org/doi/abs/10.1161/ATVBAHA.118.310792
Near‐Infrared IIb Fluorescence Imaging of Vascular Regeneration with Dynamic Tissue Perfusion Measurement and High Spatial Resolution
Authors:
Zhuoran Ma, Mingxi Zhang, Jingying Yue, Cynthia Alcazar, Yeteng Zhong, Timothy C. Doyle, Hongjie Dai Ngan F. Huang
Abstract:
Real‐time optical imaging is a promising approach for visualizing in vivo hemodynamics and vascular structure in mice with experimentally induced peripheral arterial disease (PAD). The application of a novel fluorescence‐based all‐optical imaging approach in the near‐infrared IIb (NIR‐IIb, 1500–1700 nm emission) window, for imaging hindlimb microvasculature and blood perfusion in a mouse model of PAD is reported. In phantom studies, lead sulfide/cadmium sulfide (PbS/CdS) quantum dots show better retention of image clarity, in comparison with single‐walled nanotube (SWNT) NIR‐IIa (1000–1400 nm) dye, at varying depths of penetration. When systemically injected to mice, PbS/CdS demonstrates improved clarity of the vasculature, compared to SWNTs, as well as higher spatial resolution than in vivo microscopic computed tomography. In a mouse model of PAD, NIR‐IIb imaging of the ischemic hindlimb vasculature shows significant improvement in blood perfusion over the course of 10 days (P < 0.05), as well as a significant increase in microvascular density over the first 7 days after induction of PAD. In conclusion, NIR‐IIb imaging of PbS/CdS vascular contrast agent is a useful multifunctional imaging approach for high spatial resolution imaging of the microvasculature and quantification of blood perfusion recovery.
Citation:
Advanced Functional Materials,First published: 23 July 2018, https://doi.org/10.1002/adfm.201803417
https://onlinelibrary.wiley.com/doi/abs/10.1002/adfm.201803417
Role of complement C5a and histones in septic cardiomyopathy
Authors:
Fattahi F, Frydrych LM, Bian G, Kalbitz M, Herron TJ, Malan EA, Delano MJ, Ward PA
Abstract:
Polymicrobial sepsis (after cecal ligation and puncture, CLP) causes robust complement activation with release of C5a. Many adverse events develop thereafter and will be discussed in this review article. Activation of complement system results in generation of C5a which interacts with its receptors (C5aR1, C5aR2). This leads to a series of harmful events, some of which are connected to the cardiomyopathy of sepsis, resulting in defective action potentials in cardiomyocytes (CMs), activation of the NLRP3 inflammasome in CMs and the appearance of extracellular histones, likely arising from activated neutrophils which form neutrophil extracellular traps (NETs). These events are associated with activation of mitogen-activated protein kinases (MAPKs) in CMs. The ensuing release of histones results in defective action potentials in CMs and reduced levels of [Ca2+]i-regulatory enzymes including sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) and Na+/Ca2+ exchanger (NCX) as well as Na+/K+-ATPase in CMs. There is also evidence that CLP causes release of IL-1β via activation of the NLRP3 inflammasome in CMs of septic hearts or in CMs incubated in vitro with C5a. Many of these events occur after in vivo or in vitro contact of CMs with histones. Together, these data emphasize the role of complement (C5a) and C5a receptors (C5aR1, C5aR2), as well as extracellular histones in events that lead to cardiac dysfunction of sepsis (septic cardiomyopathy).
Citation:
Mol Immunol. 2018 Jun 15. pii: S0161-5890(18)30194-9. doi: 10.1016/j.molimm.2018.06.006. [Epub ahead of print]
Endothelial cells in the innate response to allergens and initiation of atopic asthma
Authors:
Asosingh K, Weiss K, Queisser K, Wanner N, Yin M, Aronica M, Erzurum S
Abstract:
Protease-activated receptor 2 (PAR-2), an airway epithelial pattern recognition receptor (PRR), participates in the genesis of house dust mite-induced (HDM-induced) asthma. Here, we hypothesized that lung endothelial cells and proangiogenic hematopoietic progenitor cells (PACs) that express high levels of PAR-2 contribute to the initiation of atopic asthma. HDM extract (HDME) protease allergens were found deep in the airway mucosa and breaching the endothelial barrier. Lung endothelial cells and PACs released the Th2-promoting cytokines IL-1α and GM-CSF in response to HDME, and the endothelium had PAC-derived VEGF-C-dependent blood vessel sprouting. Blockade of the angiogenic response by inhibition of VEGF-C signaling lessened the development of inflammation and airway remodeling in the HDM model. Reconstitution of the bone marrow in WT mice with PAR-2-deficient bone marrow also reduced airway inflammation and remodeling. Adoptive transfer of PACs that had been exposed to HDME induced angiogenesis and Th2 inflammation with remodeling similar to that induced by allergen challenge. Our findings identify that lung endothelium and PACs in the airway sense allergen and elicit an angiogenic response that is central to the innate nonimmune origins of Th2 inflammation.
Citation:
J Clin Invest. 2018 Jun 18. pii: 97720. doi: 10.1172/JCI97720. [Epub ahead of print]
Oscillatory Strain Promotes Vessel Stabilization and Alignment through Fibroblast YAP‐Mediated Mechanosensitivity
Authors:
Shira Landau, Shahar Ben‐Shaul, Shulamit Levenberg
Abstract:
Endothelial cells form the interior layer of blood vessels and, as such, are constantly exposed to shear stress and mechanical strain. While the impact of shear stress on angiogenesis is widely studied, the role of mechanical strain is less understood. To this end, endothelial cells and fibroblasts are cocultured under oscillatory strain to create a vessel network. The two cell types show distinctly different sensitivities to the mechanical stimulation. The fibroblasts, sense the stress directly, and respond by increased alignment, proliferation, differentiation, and migration, facilitated by YAP translocation into the nucleus. In contrast, the endothelial cells form aligned vessels by tracking fibroblast alignment. YAP inhibition in constructs under mechanical strain results in vessel destruction whereas less damage is observed in the YAP‐inhibited static control. Moreover, the mechanical stimulation enhances vessel development and stabilization. Additionally, vessel orientation is preserved upon implantation into a mouse dorsal window chamber and promotes the invading host vessels to orient in the same manner. This study sheds light on the mechanisms by which mechanical strain affects the development of blood vessels within engineered tissues. This can be further utilized to engineer a more organized and stable vasculature suitable for transplantation of engineered grafts.
Citation:
Advanced Science, First published: 15 July 2018, https://doi.org/10.1002/advs.201800506
https://onlinelibrary.wiley.com/doi/abs/10.1002/advs.201800506
Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis
Authors:
Lino M, Wan MH, Rocca AS, Ngai D, Shobeiri N, Hou G, Ge C, Franceschi RT, Bendeck MP
Abstract:
OBJECTIVE: Vascular calcification is a common and severe complication in patients with atherosclerosis which is exacerbated by type 2 diabetes mellitus. Our laboratory recently reported that the collagen receptor discoidin domain receptor 1 (DDR1) mediates vascular calcification in atherosclerosis; however, the underlying mechanisms are unknown. During calcification, vascular smooth muscle cells transdifferentiate into osteoblast-like cells, in a process driven by the transcription factor RUNX2 (runt-related transcription factor 2). DDR1 signals via the phosphoinositide 3-kinase/Akt pathway, which is also central to insulin signaling, and upstream of RUNX2, and this led us to investigate whether DDR1 promotes vascular calcification in diabetes mellitus via this pathway. APPROACH AND RESULTS: Ddr1+/+; Ldlr-/- (single knock-out) and Ddr1-/-; Ldlr-/- (double knock-out) mice were placed on high-fat diet for 12 weeks to induce atherosclerosis and type 2 diabetes mellitus. Von Kossa staining revealed reduced vascular calcification in the aortic arch of double knock-out compared with single knock-out mice. Immunofluorescent staining for RUNX2 was present in calcified plaques of single knock-out but not double knock-out mice. Primary vascular smooth muscle cells obtained from Ddr1+/+ and Ddr1-/- mice were cultured in calcifying media. DDR1 deletion resulted in reduced calcification, a 74% reduction in p-Akt levels, and an 88% reduction in RUNX2 activity. Subcellular fractionation revealed a 77% reduction in nuclear RUNX2 levels in Ddr1-/- vascular smooth muscle cells. DDR1 associated with phosphoinositide 3-kinase, and treatment with the inhibitor wortmannin attenuated calcification. Finally, we show that DDR1 is important to maintain the microtubule cytoskeleton which is required for the nuclear localization of RUNX2. CONCLUSIONS: These novel findings demonstrate that DDR1 promotes RUNX2 activity and atherosclerotic vascular calcification in diabetes mellitus via phosphoinositide 3-kinase/Akt signaling.
Citation:
Arterioscler Thromb Vasc Biol. 2018 Jun 21. pii: ATVBAHA.118.311238. doi: 10.1161/ATVBAHA.118.311238. [Epub ahead of print]
http://atvb.ahajournals.org/content/early/2018/06/20/ATVBAHA.118.311238
Use of 4-phenylbutyrate to define therapeutic parameters for reducing intracerebral hemorrhage and myopathy in Col4a1 mutant mice
Authors:
Hayashi G, Labelle-Dumais C, Gould DB
Abstract:
Collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers that constitute a major component of nearly all basement membranes. COL4A1 and COL4A2 mutations cause a multisystem disorder that includes variable cerebrovascular and skeletal muscle manifestations. The pathogenicity of COL4A1 and COL4A2 mutations is generally attributed to impaired secretion into basement membranes. Sodium 4-phenylbutyrate (4PBA) is an FDA-approved drug that promotes mutant heterotrimer secretion in vitro and in vivo Here, we use different 4PBA treatment paradigms to define therapeutic parameters for preventing cerebrovascular and muscular pathologies in Col4a1 mutant mice. We show efficacy of long-term 4PBA treatment in reducing the severity of intracerebral hemorrhages (ICH) in Col4a1 mutant mice aged up to 8 months. In addition, we demonstrate that maximal efficacy of 4PBA on ICH and myopathy was achieved when treatment was initiated prenatally, whereby even transient 4PBA administration had lasting benefits after being discontinued. Importantly, postnatal treatment with 4PBA also reduced ICH and skeletal myopathy severities in Col4a1 mutant mice, which has significant clinical implications for patients with COL4A1 and COL4A2 mutations.
Citation:
Dis Model Mech. 2018 Jun 12. pii: dmm.034157. doi: 10.1242/dmm.034157. [Epub ahead of print]
http://dmm.biologists.org/content/early/2018/06/09/dmm.034157
EMAPII Monoclonal Antibody Ameliorates Influenza A Virus-Induced Lung Injury
Authors:
Hongyan Lu, Sarvesh Chelvanambi, Christophe Poirier, Jacob Saliba, Keith L. March, Matthias Clauss, Natalia V. Bogatcheva
Abstract:
Influenza A virus (IAV) remains a major worldwide health threat, especially to high-risk populations, including the young and elderly. There is an unmet clinical need for therapy that will protect the lungs from damage caused by lower respiratory infection. Here, we analyzed the role of EMAPII, a stress- and virus-induced pro-inflammatory and pro-apoptotic factor, in IAV-induced lung injury. First, we demonstrated that IAV induces EMAPII surface translocation, release, and apoptosis in cultured endothelial and epithelial cells. Next, we showed that IAV induces EMAPII surface translocation and release to bronchoalveolar lavage fluid (BALF) in mouse lungs, concomitant with increases in caspase 3 activity. Injection of monoclonal antibody (mAb) against EMAPII attenuated IAV-induced EMAPII levels, weight loss, reduction of blood oxygenation, lung edema, and increase of the pro-inflammatory cytokine TNF alpha. In accordance with the pro-apoptotic properties of EMAPII, levels of caspase 3 activity in BALF were also decreased by mAb treatment. Moreover, we detected EMAPII mAb-induced increase in lung levels of M2-like macrophage markers YM1 and CD206. All together, these data strongly suggest that EMAPII mAb ameliorates IAV-induced lung injury by limiting lung cell apoptosis and shifting the host inflammatory setting toward resolution of inflammation.
Citation:
Mol Ther. 2018 Jun 14. pii: S1525-0016(18)30220-X. doi: 10.1016/j.ymthe.2018.05.017. [Epub ahead of print]
https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(18)30220-X
Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells
Authors:
Ma N, Zhang J, Itzhaki I, Zhang SL, Chen H, Haddad F, Kitani T, Wilson KD, Tian L, Shrestha R, Wu H, Lam CK, Sayed N, Wu JC
Abstract:
Background -The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic "healthy" individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity. Methods -To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a "healthy" asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene MYL3, reported by the ClinVar database to be "likely pathogenic." Humaninduced pluripotent stem cells (iPSCs) were derived from the heterozygous VUSMYL3(170C>A) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected "healthy" control; (2) homozygous VUSMYL3(170C>A); (3) heterozygous frameshift mutation MYL3(170C>A/fs); and (4) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUSMYL3(170C>A), lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs). Results -The heterozygous VUSMYL3(170C>A)-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUSMYL3(170C>A)- and frameshift mutation MYL3(170C>A/fs)-iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular MYL3 variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic MYL3 mutation, MYL3(170C>G), and a carrier-specific iPSC-CMs line, carrying a MYBPC3(961G>A) HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments. Conclusions -Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrierspecific manner in a dish. As such, this platform represents a promising VUS riskassessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field.
Citation:
Circulation. 2018 Jun 18. pii: CIRCULATIONAHA.117.032273. doi: 10.1161/CIRCULATIONAHA.117.032273. [Epub ahead of print]
Progress, obstacles, and limitations in the use of stem cells in organ-on-a-chip models
Authors:
Wnorowski A, Yang H, Wu JC
Abstract:
In recent years, drug development costs have soared, primarily due to the failure of preclinical animal and cell culture models, which do not directly translate to human physiology. Organ-on-a-chip (OOC) is a burgeoning technology with the potential to revolutionize disease modeling, drug discovery, and toxicology research by strengthening the relevance of culture-based models while reducing costly animal studies. Although OOC models can incorporate a variety of tissue sources, the most robust and relevant OOC models going forward will include stem cells. In this review, we will highlight the benefits of stem cells as a tissue source while considering current limitations to their complete and effective implementation into OOC models.
Citation:
Adv Drug Deliv Rev. 2018 Jun 6. pii: S0169-409X(18)30132-7. doi: 10.1016/j.addr.2018.06.001. [Epub ahead of print]
Clinical factors that influence the cellular responses of saphenous veins used for arterial bypass
Authors:
Sobel M, Kikuchi S, Chen L, Tang GL, Wight TN, Kenagy RD
Abstract:
OBJECTIVE: When an autogenous vein is harvested and used for arterial bypass, it suffers physical and biologic injuries that may set in motion the cellular processes that lead to wall thickening, fibrosis, stenosis, and ultimately graft failure. Whereas the injurious effects of surgical preparation of the vein conduit have been extensively studied, little is known about the influence of the clinical environment of the donor leg from which the vein is obtained. METHODS: We studied the cellular responses of fresh saphenous vein samples obtained before implantation in 46 patients undergoing elective lower extremity bypass surgery. Using an ex vivo model of response to injury, we quantified the outgrowth of cells from explants of the adventitial and medial layers of the vein. We correlated this cellular outgrowth with the clinical characteristics of the patients, including the Wound, Ischemia, and foot Infection classification of the donor leg for ischemia, wounds, and infection as well as smoking and diabetes. RESULTS: Cellular outgrowth was significantly faster and more robust from the adventitial layer than from the medial layer. The factors of leg ischemia (P < .001), smoking (P = .042), and leg infection (P = .045) were associated with impaired overall outgrowth from the adventitial tissue on multivariable analysis. Only ischemia (P = .046) was associated with impaired outgrowth of smooth muscle cells (SMCs) from the medial tissue. Co-culture of adventitial cells and SMCs propagated from vein explants revealed that adventitial cells significantly inhibited the growth of SMCs, whereas SMCs promoted the growth of adventitial cells. The AA genotype of the -838C>A p27 polymorphism (previously associated with superior graft patency) enhanced these effects, whereas the factor of smoking attenuated adventitial cell inhibition of SMC growth. Comparing gene expression, the cells cultured from the media overexpress Kyoto Encyclopedia of Genes and Genomes pathways associated with inflammation and infection, whereas those from the adventitia overexpress gene families associated with development and stem/progenitor cell maintenance. CONCLUSIONS: The adverse clinical environment of the leg may influence the biologic behavior of the cells in the vein wall, especially the adventitial cells. Chronic ischemia was the most significant factor that retards adventitial cell outgrowth. The cells arising from the vein adventitia may be key players in determining a healthy adaptive or a pathologic response to the injuries associated with vein grafting.
Citation:
J Vasc Surg. 2018 Jun 15. pii: S0741-5214(18)31029-2. doi: 10.1016/j.jvs.2018.03.436. [Epub ahead of print]
Prolonged activation of cAMP signaling leads to endothelial barrier disruption via transcriptional repression of RRAS
Authors:
Carole Y. Perrot, Junko Sawada and Masanobu Komatsu
Abstract:
The increase in cAMP levels in endothelial cells triggers cellular signaling to alter vascular permeability. It is generally considered that cAMP signaling stabilizes the endothelial barrier function and reduces permeability. However, previous studies have only examined the permeability shortly after cAMP elevation and thus have only investigated acute responses. Because cAMP is a key regulator of gene expression, elevated cAMP may have a delayed but profound impact on the endothelial permeability by altering the expression of the genes that are vital for the vessel wall stability. The small guanosine triphosphate hydrolase Ras-related protein (R-Ras) stabilizes VE-cadherin clustering and enhances endothelial barrier function, thereby stabilizing the integrity of blood vessel wall. Here we show that cAMP controls endothelial permeability through RRAS gene regulation. The prolonged cAMP elevation transcriptionally repressed RRAS in endothelial cells via a cAMP response element–binding protein (CREB) 3–dependent mechanism and significantly disrupted the adherens junction. These effects resulted in a marked increase of endothelial permeability that was reversed by R-Ras transduction. Furthermore, cAMP elevation in the endothelium by prostaglandin E2 or phosphodiesterase type 4 inhibition caused plasma leakage from intact microvessels in mouse skin. Our study demonstrated that, contrary to the widely accepted notion, cAMP elevation in endothelial cells ultimately increases vascular permeability, and the cAMP-dependent RRAS repression critically contributes to this effect.—Perrot, C. Y., Sawada, J., Komatsu, M. Prolonged activation of cyclic AMP signaling leads to endothelial barrier disruption via transcriptional repression of RRAS.
Citation:
FASEB J. 2018 May 18:fj201700818RRR. doi: 10.1096/fj.201700818RRR. [Epub ahead of print]
https://www.fasebj.org/doi/abs/10.1096/fj.201700818RRR?journalCode=fasebj
Lack of Tone in Mouse Small Mesenteric Arteries Leads to Outward Remodeling, which can be Prevented by Prolonged Agonist-Induced Vasoconstriction
Authors:
Anika Klein, Philomeena Daphne Joseph, Vibeke Grøsfjeld Christensen, Lars Jørn Jensen, and Jens Christian Brings Jacobsen
Abstract:
Inward remodeling of resistance vessels is an independent risk factor for cardiovascular events. Thus far, the remodeling process remains incompletely elucidated, but the activation level of the vascular smooth muscle cell (VSMC) appears to play a central role. Accordingly, previous data suggest that an antagonistic and, supposedly beneficial, response - outward remodeling - may follow prolonged vasodilatation. This study aims to determine if 1) outward remodeling follows 3 days of vessel culture without tone, 2) a similar response can be elicited in a much shorter 4-hour time frame and finally 3) a 4-hour response can be prevented or reversed by the presence of vasoconstrictors in the medium. Cannulated mouse small mesenteric arteries (SMA) were organo-cultured for 3 days in the absence of tone, leading to outward remodeling that continued throughout the culture period. In more acute experiments in which cannulated SMAs were maintained in physiological saline without tone for 4 hours, we detected a similar outward remodeling that proceeded at a rate several times faster. In the 4-hour experimental setting, continuous vasoconstriction to approximately 50% tone by abluminal application of uridine 5'-triphosphate (UTP) or norepinephrine (NE) + neuropeptide Y (NPY) prevented outward remodeling but did not cause inward remodeling. Computational modelling was used to simulate and interpret these findings and to derive time constants of the remodeling processes. It is suggested that depriving resistance arteries of activation will lead to eutrophic outward remodeling, which can be prevented by VSMC activation induced by prolonged vasoconstrictor exposure.
Citation:
Am J Physiol Heart Circ Physiol. 2018 May 18. doi: 10.1152/ajpheart.00111.2018. [Epub ahead of print]
https://www.physiology.org/doi/abs/10.1152/ajpheart.00111.2018
Up-regulation of SFTPB expression and attenuation of acute lung injury by pulmonary epithelial cell-specific NAMPT knockdown
Authors:
Guangliang Bi, Lei Wu, Shamima Islam, Daniel P. Heruth, Li Qin Zhang, Ding-You Li, Venkatesh Sampath, Weimin Huang, Brett A. Simon, Ronald Blaine Easley, Joe G.N. Garcia and Shui Qing Ye
Abstract:
Although a deficiency of surfactant protein B (SFTPB) has been associated with lung injury, SFTPB expression has not yet been linked with nicotinamide phosphoribosyltransferase (NAMPT), a potential biomarker of acute lung injury (ALI). The effects of Nampt in the pulmonary epithelial cell on both SFTPB expression and lung inflammation were investigated in a LPS-induced ALI mouse model. Pulmonary epithelial cell-specific knockdown of Nampt gene expression, achieved by the crossing of Nampt gene exon 2 floxed mice with mice expressing epithelial-specific transgene Cre or by the use of epithelial-specific expression of anti-Nampt antibody cDNA, significantly attenuated LPS-induced ALI. Knockdown of Nampt expression was accompanied by lower levels of bronchoalveolar lavage (BAL) neutrophil infiltrates, total protein and TNF-α levels, as well as lower lung injury scores. Notably, Nampt knockdown was also associated with significantly increased BAL SFTPB levels relative to the wild-type control mice. Down-regulation of NAMPT increased the expression of SFTPB and rescued TNF-α-induced inhibition of SFTPB, whereas overexpression of NAMPT inhibited SFTPB expression in both H441 and A549 cells. Inhibition of NAMPT up-regulated SFTPB expression by enhancing histone acetylation to increase its transcription. Additional data indicated that these effects were mainly mediated by NAMPT nonenzymatic function via the JNK pathway. This study shows that pulmonary epithelial cell-specific knockdown of NAMPT expression attenuated ALI, in part, via up-regulation of SFTPB expression. Thus, epithelial cell-specific knockdown of Nampt may be a potential new and viable therapeutic modality to ALI.-Bi, G., Wu, L., Huang, P., Islam, S., Heruth, D. P., Zhang, L. Q., Li, D.-Y., Sampath, V., Huang, W., Simon, B. A., Easley, R. B., Ye, S. Q. Up-regulation of SFTPB expression and attenuation of acute lung injury by pulmonary epithelial cell-specific NAMPT knockdown.
Citation:
FASEB J. 2018 Feb 8:fj201701059R. doi: 10.1096/fj.201701059R. [Epub ahead of print]
Suppression of TGFβ-mediated conversion of endothelial cells and fibroblasts into cancer associated (myo)fibroblasts via HDAC inhibition
Authors:
Kim DJ, Dunleavey JM, Xiao L, Ollila DW, Troester MA, Otey CA, Li W, Barker TH, Dudley AC
Abstract:
BACKGROUND: Cancer-associated fibroblasts (CAFs) support tumour progression and invasion, and they secrete abundant extracellular matrix (ECM) that may shield tumour cells from immune checkpoint or kinase inhibitors. Targeting CAFs using drugs that revert their differentiation, or inhibit their tumour-supportive functions, has been considered as an anti-cancer strategy. METHODS: We have used human and murine cell culture models, atomic force microscopy (AFM), microarray analyses, CAF/tumour cell spheroid co-cultures and transgenic fibroblast reporter mice to study how targeting HDACs using small molecule inhibitors or siRNAs re-directs CAF differentiation and function in vitro and in vivo. RESULTS: From a small molecule screen, we identified Scriptaid, a selective inhibitor of HDACs 1/3/8, as a repressor of TGFβ-mediated CAF differentiation. Scriptaid inhibits ECM secretion, reduces cellular contraction and stiffness, and impairs collective cell invasion in CAF/tumour cell spheroid co-cultures. Scriptaid also reduces CAF abundance and delays tumour growth in vivo. CONCLUSIONS: Scriptaid is a well-tolerated and effective HDACi that reverses many of the functional and phenotypic properties of CAFs. Impeding or reversing CAF activation/function by altering the cellular epigenetic regulatory machinery could control tumour growth and invasion, and be beneficial in combination with additional therapies that target cancer cells or immune cells directly.
Citation:
Br J Cancer. 2018 Apr 26. doi: 10.1038/s41416-018-0072-3. [Epub ahead of print]
A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment
Authors:
Griveau A, Seano G, Shelton SJ, Kupp R, Jahangiri A, Obernier K, Krishnan S, Lindberg OR, Yuen TJ, Tien AC, Sabo JK, Wang N, Chen I, Kloepper J, Larrouquere L, Ghosh M, Tirosh I, Huillard E, Alvarez-Buylla A, Oldham MC, Persson AI, Weiss WA, Batchelor TT, Stemmer-Rachamimov A, Suvà ML, Phillips JJ, Aghi MK, Mehta S, Jain RK, Rowitch DH
Abstract:
Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.
Citation:
Cancer Cell. 2018 Apr 6. pii: S1535-6108(18)30125-9. doi: 10.1016/j.ccell.2018.03.020. [Epub ahead of print]
http://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30125-9
Thalidomide Reduces Hemorrhage of Brain Arteriovenous Malformations in a Mouse Model
Authors:
Zhu W, Chen W, Zou D, Wang L, Bao C, Zhan L, Saw D, Wang S, Winkler E, Li Z, Zhang M, Shen F, Shaligram S, Lawton M, Su H
Abstract:
BACKGROUND AND PURPOSE: Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current treatments for bAVM are all associated with considerable risks. There is no safe method to prevent bAVM hemorrhage. Thalidomide reduces nose bleeding in patients with hereditary hemorrhagic telangiectasia, an inherited disorder characterized by vascular malformations. In this study, we tested whether thalidomide and its less toxic analog, lenalidomide, reduce bAVM hemorrhage using a mouse model. METHODS: bAVMs were induced through induction of brain focal activin-like kinase 1 (Alk1, an AVM causative gene) gene deletion and angiogenesis in adult Alk1-floxed mice. Thalidomide was injected intraperitoneally twice per week for 6 weeks, starting either 2 or 8 weeks after AVM induction. Lenalidomide was injected intraperitoneally daily starting 8 weeks after AVM induction for 6 weeks. Brain samples were collected at the end of the treatments for morphology, mRNA, and protein analyses. The influence of Alk1 downregulation on PDGFB (platelet-derived growth factor B) expression was also studied on cultured human brain microvascular endothelial cells. The effect of PDGFB in mural cell recruitment in bAVM was explored by injection of a PDGFB overexpressing lentiviral vector to the mouse brain. RESULTS: Thalidomide or lenalidomide treatment reduced the number of dysplastic vessels and hemorrhage and increased mural cell (vascular smooth muscle cells and pericytes) coverage in the bAVM lesion. Thalidomide reduced the burden of CD68+ cells and the expression of inflammatory cytokines in the bAVM lesions. PDGFB expression was reduced in ALK1-knockdown human brain microvascular endothelial cells and in mouse bAVM lesion. Thalidomide increased Pdgfb expression in bAVM lesion. Overexpression of PDGFB mimicked the effect of thalidomide. CONCLUSIONS: Thalidomide and lenalidomide improve mural cell coverage of bAVM vessels and reduce bAVM hemorrhage, which is likely through upregulation of Pdgfb expression.
Citation:
Stroke. 2018 Mar 28. pii: STROKEAHA.117.020356. doi: 10.1161/STROKEAHA.117.020356. [Epub ahead of print]
Early Heterogenic Response of Renal Microvasculature to Hemorrhagic Shock/Resuscitation and the Influence of NF-κB Pathway Blockade
Authors:
Yan R, van Meurs M, Popa ER, Li R, Zwiers PJ, Zijlstra JG, Moser J, Molema G
Abstract:
Hemorrhagic shock (HS) is associated with low blood pressure due to excessive loss of circulating blood and causes both macrocirculatory and microcirculatory dysfunction. Fluid resuscitation after HS is used in the clinic to restore tissue perfusion. The persistent microcirculatory damage caused by HS and/or resuscitation can result in multiple organ damage, with the kidney being one of the involved organs. The kidney microvasculature consists of different segments that possess a remarkable heterogeneity in functional properties. The aim of this study was to investigate the inflammatory responses of these different renal microvascular segments, i.e., arterioles, glomeruli, and postcapillary venules, to HS and resuscitation (HS/R) in mice and to explore the effects of intervention with an NF-κB inhibitor on these responses. We found that HS/R disturbed the balance of the Angiopoietin-Tie2 ligand-receptor system, especially in the glomeruli. Furthermore, endothelial adhesion molecules, pro-inflammatory cytokines, and chemokines were markedly upregulated by HS/R, with the strongest responses occurring in the glomerular and postcapillary venous segments. Blockade of NF-κB signaling during the resuscitation period only slightly inhibited HS/R induced inflammatory activation, possibly because NF-κB p65 nuclear translocation already occurred during the HS period. In summary, although all three renal microvascular segments were activated upon HS/R, responses of endothelial cells in glomeruli and postcapillary venules to HS/R, as well as to NF-κB inhibition were stronger than those in arterioles. NF-κB inhibition during the resuscitation phase does not effectively counteract NF-κB p65 nuclear translocation initiating inflammatory gene transcription.This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0.
Citation:
Shock. 2018 Feb 21. doi: 10.1097/SHK.0000000000001126. [Epub ahead of print]
Mir-126 is a conserved modulator of lymphatic development
Authors:
Zacharias Kontarakis, Andrea Rossi, Sophie Ramas, Michael T. Dellinger, Didier Y.R. Stainier
Abstract:
Organ homeostasis relies upon cellular and molecular processes that restore tissue structure and function in a timely fashion. Lymphatic vessels help maintain fluid equilibrium by returning interstitial fluid that evades venous uptake back to the circulation. Despite its important role in tissue homeostasis, cancer metastasis, and close developmental origins with the blood vasculature, the number of molecular players known to control lymphatic system development is relatively low. Here we show, using genetic approaches in zebrafish and mice, that the endothelial specific microRNA mir-126, previously implicated in vascular integrity, regulates lymphatic development. In zebrafish, in contrast to mir-126 morphants, double mutants (mir-126a-/-; mir-126b-/-, hereafter mir-126-/-) do not exhibit defects in vascular integrity but develop lymphatic hypoplasia; mir-126-/- animals fail to develop complete trunk and facial lymphatics, display severe edema and die as larvae. Notably, following MIR-126 inhibition, human Lymphatic Endothelial Cells (hLECs) respond poorly to VEGFA and VEGFC. In this context, we identify a concomitant reduction in Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) and Vascular Endothelial Growth Factor Receptor-3 (VEGFR3, also known as FLT4) expression upon MIR-126 inhibition. In vivo, we further show that flt4 +/- zebrafish embryos exhibit lymphatic defects after mild miR-126 knockdown. Similarly, loss of Mir-126 in Flt4 +/- mice results in embryonic edema and lethality. Thus, our results indicate that miR-126 modulation of Vegfr signaling is essential for lymphatic system development in fish and mammals.
Citation:
Developmental Biology, Available online 15 March 2018
https://www.sciencedirect.com/science/article/pii/S0012160618301386
Cross-talk between blood vessels and neural progenitors in the developing brain
Authors:
Mathew Tata, Christiana Ruhrberg
Abstract:
The formation of the central nervous system (CNS) involves multiple cellular and molecular interactions between neural progenitor cells (NPCs) and blood vessels to establish extensive and complex neural networks and attract a vascular supply that support their function. In this review, we discuss studies that have performed genetic manipulations of chick, fish and mouse embryos to define the spatiotemporal roles of molecules that mediate the reciprocal regulation of NPCs and blood vessels. These experiments have highlighted core functions of NPC-expressed ligands in initiating vascular growth into and within the neural tube as well as establishing the blood–brain barrier. More recent findings have also revealed indispensable roles of blood vessels in regulating NPC expansion and eventual differentiation, and specific regional differences in the effect of angiocrine signals. Accordingly, NPCs initially stimulate blood vessel growth and maturation to nourish the brain, but blood vessels subsequently also regulate NPC behaviour to promote the formation of a sufficient number and diversity of neural cells. A greater understanding of the molecular cross-talk between NPCs and blood vessels will improve our knowledge of how the vertebrate nervous system forms and likely help in the design of novel therapies aimed at regenerating neurons and neural vasculature following CNS disease or injury.
Citation:
Neuronal Signaling, Mar 30, 2018, 2(1)NS20170139; DOI: 10.1042/NS20170139
Spatiotemporal Multi-omics Mapping Generates a Molecular Atlas of the Aortic Valve and Reveals Networks Driving Disease
Authors:
Schlotter F, Halu A, Goto S, Blaser MC, Body SC, Lee LH, Higashi H, DeLaughter DM, Hutcheson JD, Vyas P, Pham T, Rogers MA, Sharma A, Seidman CE, Loscalzo J, Seidman JG, Aikawa M, Singh SA, Aikawa E
Abstract:
Background -No pharmacological therapy exists for calcific aortic valve disease (CAVD), which confers a dismal prognosis without invasive valve replacement. The search for therapeutics and early diagnostics is challenging since CAVD presents in multiple pathological stages. Moreover, it occurs in the context of a complex, multi-layered tissue architecture, a rich and abundant extracellular matrix phenotype, and a unique, highly plastic and multipotent resident cell population. Methods -A total of 25 human stenotic aortic valves obtained from valve replacement surgeries were analyzed by multiple modalities, including transcriptomics and global unlabeled and label-based tandem-mass-tagged proteomics. Segmentation of valves into disease-stage-specific samples was guided by near infrared molecular imaging, and anatomical layer-specificity was facilitated by laser capture microdissection. Side-specific cell cultures were subjected to multiple calcifying stimuli, and their calcification potential and basal/stimulated proteomes were evaluated. Molecular (protein-protein) interaction networks were built and their central proteins and disease associations were identified. Results -Global transcriptional and protein expression signatures differed between the non-diseased, fibrotic, and calcific stages of CAVD. Anatomical aortic valve microlayers exhibited unique proteome profiles that were maintained throughout disease progression and identified glial fibrillary acidic protein (GFAP) as a specific marker of valvular interstitial cells (VICs) from the spongiosa layer. CAVD disease progression was marked by an emergence of smooth muscle cell activation, inflammation, and calcification-related pathways. Proteins overrepresented in the disease-prone fibrosa are functionally annotated to fibrosis and calcification pathways, and we found that in vitro, fibrosa-derived VICs demonstrated greater calcification potential than those from the ventricularis. These studies confirmed that the microlayer-specific proteome was preserved in cultured VICs, and that VICs exposed to ALPL-dependent and ALPL-independent calcifying stimuli had distinct proteome profiles, both of which overlapped with that of the whole tissue. Analysis of protein-protein interaction networks found a significant closeness to multiple inflammatory and fibrotic diseases. Conclusions -A spatially- and temporally-resolved multi-omics, and network and systems biology strategy identifies the first molecular regulatory networks in CAVD, a cardiac condition without a pharmacological cure, and describes a novel means of systematic disease ontology that is broadly applicable to comprehensive omics studies of cardiovascular diseases.
Citation:
Circulation. 2018 Mar 27. pii: CIRCULATIONAHA.117.032291. doi: 10.1161/CIRCULATIONAHA.117.032291. [Epub ahead of print]
http://circ.ahajournals.org/content/early/2018/03/26/CIRCULATIONAHA.117.032291
Minoxidil improves vascular compliance, restores cerebral blood flow and alters extracellular matrix gene expression in a model of chronic vascular stiffness
Authors:
Knutsen R, Beeman SC, Broekelmann TJ, Liu D, Tsang KM, Kovacs A, Ye L, Danback J, Watson A, Wardlaw A, Wagenseil J, Garbow JR, Shoykhet M, Kozel BA
Abstract:
Increased vascular stiffness correlates with higher risk of cardiovascular complications in aging adults. Elastin insufficiency, as observed in patients with Williams-Beuren syndrome or with familial supravalvular aortic stenosis, also increases vascular stiffness and leads to arterial narrowing. We used Eln+/- mice to test the hypothesis that pathologically increased vascular stiffness with concomitant arterial narrowing leads to decreased blood flow to end organs such as the brain. We also hypothesized that drugs which remodel arteries and increase lumen diameter would improve flow. To test these hypotheses, we compared carotid blood flow using ultrasound and cerebral blood flow using MRI-based arterial spin labeling in WT and Eln+/- mice. We then studied how minoxidil, a KATP channel opener and vasodilator, affects vessel mechanics, blood flow and gene expression. Both carotid and cerebral blood flows were lower in Eln+/- than in WT mice. Treatment of Eln+/- mice with minoxidil lowered blood pressure and reduced functional arterial stiffness to WT levels. Minoxidil also improved arterial diameter and restored carotid and cerebral blood flows in Eln+/- mice. Beneficial effects persisted for weeks after drug removal. RNA-Seq analysis revealed differential expression of 127 extracellular matrix-related genes among the treatment groups. These results indicate that elastin insufficiency impairs end organ perfusion, which may contribute to increased cardiovascular risk. Minoxidil, despite lowering blood pressure, improves end organ perfusion. Changes in matrix gene expression and persistence of treatment effects after drug withdrawal suggest arterial remodeling. Such remodeling may benefit patients with genetic or age-dependent elastin insufficiency.
Citation:
Am J Physiol Heart Circ Physiol. 2018 Mar 2. doi: 10.1152/ajpheart.00683.2017. [Epub ahead of print]
Elastin-driven genetic diseases
Authors:
Lasio MLD, Kozel BA
Abstract:
Elastic fibers provide recoil to tissues that undergo repeated deformation, such as blood vessels, lungs and skin. Composed of elastin and its accessory proteins, the fibers are produced within a restricted developmental window and are stable for decades. Their eventual breakdown is associated with a loss of tissue resiliency and aging. Rare alteration of the elastin (ELN) gene produces disease by impacting protein dosage (supravalvar aortic stenosis, Williams Beuren syndrome and Williams Beuren region duplication syndrome) and protein function (autosomal dominant cutis laxa). This review highlights aspects of the elastin molecule and its assembly process that contribute to human disease and also discusses potential therapies aimed at treating diseases of elastin insufficiency.
Citation:
Matrix Biol. 2018 Feb 28. pii: S0945-053X(17)30369-4. doi: 10.1016/j.matbio.2018.02.021. [Epub ahead of print]
Extracellular bone morphogenetic protein modulator BMPER and twisted gastrulation homolog 1 preserve arterial-venous specification in zebrafish blood vessel development and regulate Notch signaling in endothelial cells
Authors:
Esser JS, Steiner RE, Deckler M, Schmitt H, Engert B, Link S, Charlet A, Patterson C, Bode C, Zhou Q, Moser M
Abstract:
The bone morphogenetic protein (BMP) signaling pathway plays a central role during vasculature development. Mutations or dysregulation of the BMP pathway members have been linked to arteriovenous malformations. In the present study, we investigated the effect of the BMP modulators bone morphogenetic protein endothelial precursor-derived regulator (BMPER) and twisted gastrulation protein homolog 1 (TWSG1) on arteriovenous specification during zebrafish development and analyzed downstream Notch signaling pathway in human endothelial cells. Silencing of bmper and twsg1b in zebrafish embryos by morpholinos resulted in a pronounced enhancement of venous ephrinB4a marker expression and concomitant dysregulated arterial ephrinb2a marker expression detected by in situ hybridization. As arteriovenous specification was disturbed, we assessed the impact of BMPER and TWSG1 protein stimulation on the Notch signaling pathway on endothelial cells from different origin. Quantitative real-time PCR (qRT-PCR) and western blot analysis showed increased expression of Notch target gene hairy and enhancer of split, HEY1/2 and EPHRINB2. Consistently, silencing of BMPER in endothelial cells by siRNAs decreased Notch signaling and downstream effectors. BMP receptor antagonist DMH1 abolished BMPER and BMP4 induced Notch signaling pathway activation. In conclusion, we found that in endothelial cells, BMPER and TWSG1 are necessary for regular Notch signaling activity and in zebrafish embryos BMPER and TWSG1 preserve arteriovenous specification to prevent malformations.
Citation:
FEBS J. 2018 Feb 23. doi: 10.1111/febs.14414. [Epub ahead of print]
Peripheral post-ischemic vascular repair is impaired in a murine model of Alzheimer's disease
Authors:
Merkulova-Rainon T, Mantsounga CS, Broquères-You D, Pinto C, Vilar J, Cifuentes D, Bonnin P, Kubis N, Henrion D, Silvestre JS, Lévy BI
Abstract:
The pathophysiology of sporadic Alzheimer's disease (AD) remains uncertain. Along with brain amyloid-β (Aβ) deposits and neurofibrillary tangles, cerebrovascular dysfunction is increasingly recognized as fundamental to the pathogenesis of AD. Using an experimental model of limb ischemia in transgenic APPPS1 mice, a model of AD (AD mice), we showed that microvascular impairment also extends to the peripheral vasculature in AD. At D70 following femoral ligation, we evidenced a significant decrease in cutaneous blood flow (- 29%, P < 0.001), collateral recruitment (- 24%, P < 0.001), capillary density (- 22%; P < 0.01) and arteriole density (- 28%; P < 0.05) in hind limbs of AD mice compared to control WT littermates. The reactivity of large arteries was not affected in AD mice, as confirmed by unaltered size, and vasoactive responses to pharmacological stimuli of the femoral artery. We identified blood as the only source of Aβ in the hind limb; thus, circulating Aβ is likely responsible for the impairment of peripheral vasculature repair mechanisms. The levels of the majority of pro-angiogenic mediators were not significantly modified in AD mice compared to WT mice, except for TGF-β1 and PlGF-2, both of which are involved in vessel stabilization and decreased in AD mice (P = 0.025 and 0.019, respectively). Importantly, endothelin-1 levels were significantly increased, while those of nitric oxide were decreased in the hind limb of AD mice (P < 0.05). Our results suggest that vascular dysfunction is a systemic disorder in AD mice. Assessment of peripheral vascular function may therefore provide additional tools for early diagnosis and management of AD.
Citation:
Angiogenesis. 2018 Mar 7. doi: 10.1007/s10456-018-9608-7. [Epub ahead of print]
Postnatal development of lymphatic vasculature in the brain meninges
Authors:
Izen RM, Yamazaki T, Nishinaka-Arai Y, Hong YK, Mukouyama YS
Abstract:
BACKGROUND: Traditionally, the central nervous system (CNS) has been viewed as an immune-privileged environment with no lymphatic vessels. This view was partially overturned by the discovery of lymphatic vessels in the dural membrane that surrounds the brain, in contact with the interior surface of the skull. We here examine the distribution and developmental timing of these lymphatic vessels.
Citation:
Dev Dyn. 2018 Mar 1. doi: 10.1002/dvdy.24624. [Epub ahead of print]
Effects of teriparatide on morphology of aortic calcification in aged hyperlipidemic mice
Authors:
Hsu JJ, Lu J, Umar S, Lee JT, Kulkarni R, Ding Y, Chang CC, Hsiai TK, Hokugo A, Gkouveris I, Tetradis S, Nishimura I, Demer LL, Tintut Y
Abstract:
Calcific aortic vasculopathy correlates with bone loss in osteoporosis in an age-independent manner. Prior work suggests that teriparatide, the bone anabolic treatment for postmenopausal osteoporosis, may inhibit onset of aortic calcification. Whether teriparatide affects progression of pre-existing aortic calcification, widespread among this patient population, is unknown. Female apolipoprotein E-deficient mice were aged for over one year to induce aortic calcification, treated for 4.5 weeks with daily injections of control vehicle (PBS), teriparatide 40 µg/kg (PTH40) or 400 µg/kg (PTH400), and assayed for aortic calcification by micro-computed tomography (microCT) before and after treatment. In a follow-up cohort, aged female apolipoprotein E-deficient mice were treated with PBS or PTH400, and assayed for aortic calcification by serial microCT and micro-positron emission tomography (microPET). In both cohorts, aortic calcification detected by microCT progressed similarly in all groups. Mean aortic 18F-NaF incorporation, detected by serial microPET, increased in the PBS group (+14{plus minus}5%). In contrast, 18F-NaF incorporation decreased in the PTH400 group (-33{plus minus}20%; p=0.03). Quantitative histochemical analysis by Alizarin red staining revealed a lower mineral surface area index in the PTH400 group compared with the PBS group (p=0.04). Furthermore, Masson trichrome staining showed a significant increase in collagen deposition in the left ventricular myocardium of mice receiving PTH400 (2.1{plus minus}0.6% vs. controls, 0.5{plus minus}0.1%; p=0.02). In summary, while teriparatide may not affect the calcium mineral content of aortic calcification, it reduces 18F-NaF uptake in calcified lesions, suggesting the possibility that it may reduce mineral surface area with potential impact on plaque stability.
Citation:
Am J Physiol Heart Circ Physiol. 2018 Feb 16. doi: 10.1152/ajpheart.00718.2017. [Epub ahead of print]
Endothelial immune activation programs cell-fate decisions and angiogenesis by inducing DLL4 through TLR4-ERK-FOXC2 signalling
Authors:
Xia S, Menden HL, Korfhagen TR, Kume T, Sampath V
Abstract:
Endothelial cells (EC) mediate a specific and robust immune response to bacteria in sepsis through the activation of Toll-Like Receptor (TLR) signalling. The mechanisms by which bacterial ligands released during sepsis program endothelial cell (EC) specification and altered angiogenesis remain unclear. We postulated that the Forkhead box protein C2 (FOXC2) transcriptional factor directs EC cell-fate decisions and angiogenesis during TLR signalling. In human lung EC, LPS induced ERK phosphorylation, FOXC2, and DLL4 expression in a TLR4-dependent manner. LPS mediated ERK phosphorylation resulted in FOXC2-ERK protein ligation, ERK-dependent FOXC2 serine and threonine phosphorylation, and subsequent activation of DLL4 gene expression. Chemical inhibition of ERK or ERK-2 dominant negative transfection disrupted LPS-mediated FOXC2 phosphorylation and transcriptional activation of FOXC2. FOXC2-siRNA or ERK-inhibition attenuated LPS-induced DLL4 expression and angiogenic sprouting in vitro. In vivo, intraperitoneal LPS induced ERK and FOXC2 phosphorylation, FOXC2 binding to DLL4 promoter, and FOXC2/DLL4 expression in the lung. ERK-inhibition suppressed LPS-induced FOXC2 phosphorylation, FOXC2-DLL4 promoter binding, and induction of FOXC2 and DLL4 in mouse lung EC. LPS induced aberrant retinal angiogenesis and DLL4 expression in neonatal mice, which was attenuated with ERK-inhibition. FOXC2+/- mice treated with LPS showed a mitigated increase in FOXC2 and DLL4 compared to FOXC2+/+ mice. These data reveal a new mechanism (TLR4-ERK-FOXC2-DLL4) by which sepsis-induced EC TLR signalling programs EC specification and altered angiogenesis. This article is protected by copyright. All rights reserved.
Citation:
J Physiol. 2018 Jan 30. doi: 10.1113/JP275453. [Epub ahead of print]
RhoA increases ASIC1a plasma membrane localization and calcium influx in pulmonary arterial smooth muscle cells following chronic hypoxia
Authors:
Herbert, LM, Resta TC, Jernigan NL
Abstract:
Increases in pulmonary arterial smooth muscle cell (PASMC) intracellular Ca2+ levels and enhanced RhoA/Rho kinase-dependent Ca2+ sensitization are key determinants of PASMC contraction, migration, and proliferation accompanying the development of hypoxic pulmonary hypertension. We previously showed that acid-sensing ion channel 1a (ASIC1a)-mediated Ca2+ entry in PASMC is an important constituent of the active vasoconstriction, vascular remodeling, and right ventricular hypertrophy associated with hypoxic pulmonary hypertension. However, the enhanced ASIC1a-mediated store-operated Ca2+ entry in PASMC from pulmonary hypertensive animals is not dependent on an increase in ASIC1a protein expression, suggesting that chronic hypoxia (CH) stimulates ASIC1a function through other regulatory mechanism(s). RhoA is involved in ion channel trafficking, and levels of activated RhoA are increased following CH. Therefore, we hypothesize that activation of RhoA following CH increases ASIC1a-mediated Ca2+ entry by promoting ASIC1a plasma membrane localization. Consistent with our hypothesis, we found greater plasma membrane localization of ASIC1a following CH. Inhibition of RhoA decreased ASIC1a plasma membrane expression and largely diminished ASIC1a-mediated Ca2+ influx, whereas activation of RhoA had the opposite effect. A proximity ligation assay revealed that ASIC1a and RhoA colocalize in PASMC and that the activation state of RhoA modulates this interaction. Together, our findings show a novel interaction between RhoA and ASIC1a, such that activation of RhoA in PASMC, both pharmacologically and via CH, promotes ASIC1a plasma membrane localization and Ca2+ entry. In addition to enhanced RhoA-mediated Ca2+ sensitization following CH, RhoA can also activate a Ca2+ signal by facilitating ASIC1a plasma membrane localization and Ca2+ influx in pulmonary hypertension.
Citation:
Am J Physiol Cell Physiol. 2018 Feb 1;314(2):C166-C176. doi: 10.1152/ajpcell.00159.2017. Epub 2017 Oct 25.
http://www.physiology.org/doi/abs/10.1152/ajpcell.00159.2017?journalCode=ajpcell
Reduced membrane cholesterol after chronic hypoxia limits Orai1-mediated pulmonary endothelial Ca2+ entry
Authors:
Zhang B, Naik JS, Jernigan NL, Walker BR, Resta TC
Abstract:
Endothelial dysfunction in chronic hypoxia (CH)-induced pulmonary hypertension is characterized by reduced store-operated Ca2+ entry (SOCE) and diminished Ca2+-dependent production of endothelium-derived vasodilators. We recently reported that SOCE in pulmonary arterial endothelial cells (PAECs) is tightly regulated by membrane cholesterol and that decreased membrane cholesterol is responsible for impaired SOCE after CH. However, the ion channels involved in cholesterol-sensitive SOCE are unknown. We hypothesized that cholesterol facilitates SOCE in PAECs through the interaction of Orai1 and stromal interaction molecule 1 (STIM1). The role of cholesterol in Orai1-mediated SOCE was initially assessed using CH exposure in rats (4 wk, 380 mmHg) as a physiological stimulus to decrease PAEC cholesterol. The effects of Orai1 inhibition with AnCoA4 on SOCE were examined in isolated PAEC sheets from control and CH rats after cholesterol supplementation, substitution of endogenous cholesterol with epicholesterol (Epichol), or vehicle treatment. Whereas cholesterol restored endothelial SOCE in CH rats, both Epichol and AnCoA4 attenuated SOCE only in normoxic controls. The Orai1 inhibitor had no further effect in cells pretreated with Epichol. Using cultured pulmonary endothelial cells to allow better mechanistic analysis of the molecular components of cholesterol-regulated SOCE, we found that Epichol, AnCoA4, and Orai1 siRNA each inhibited SOCE compared with their respective controls. Epichol had no additional effect after knockdown of Orai1. Furthermore, Epichol substitution significantly reduced STIM1-Orai1 interactions as assessed by a proximity ligation assay. We conclude that membrane cholesterol is required for the STIM1-Orai1 interaction necessary to elicit endothelial SOCE. Furthermore, reduced PAEC membrane cholesterol after CH limits Orai1-mediated SOCE. NEW & NOTEWORTHY This research demonstrates a novel contribution of cholesterol to regulate the interaction of Orai1 and stromal interaction molecule 1 required for pulmonary endothelial store-operated Ca2+ entry. The results provide a mechanistic basis for impaired pulmonary endothelial Ca2+ influx after chronic hypoxia that may contribute to pulmonary hypertension.
Citation:
Am J Physiol Heart Circ Physiol. 2018 Feb 1;314(2):H359-H369. doi: 10.1152/ajpheart.00540.2017. Epub 2017 Nov 3.