Vascular biology
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.
Authors
Vinita Yadav, Liguo Chi, Raymond Zhao, Benjamin Tourdot, Srilakshmi Yalavarthi, Benjamin N. Jacobs, Alison Banka, Hui Liao, Sharon Koonse, Anuli C. Anyanwu, Scott Visovatti, Michael Holinstat, J. Michelle Kahlenberg, Jason S. Knight, David J. Pinsky, Yogendra Kanthi
Abstract
The lung is a specialized barrier organ that must tightly regulate interstitial fluid clearance and prevent infection in order to maintain effective gas exchange. Lymphatic vessels are important for these functions in other organs, but their roles in the lung have not been fully defined. In the present study, we addressed how the lymphatic vasculature participates in lung homeostasis. Studies using mice carrying a lymphatic reporter allele revealeded that, in contrast to other organs, lung lymphatic collecting vessels lack smooth muscle cells entirely, suggesting that forward lymph flow is highly dependent on movement and changes in pressure associated with respiration. Functional studies using CLEC2-deficient mice in which lymph flow is impaired due to loss of lympho-venous hemostasis or using inducible lung-specific ablation of lymphatic endothelial cells in a lung transplant model revealeded that loss of lymphatic function leads to an inflammatory state characterized by the formation of tertiary lymphoid organs (TLOs). In addition, impaired lymphatic flow in mice resulteds in hypoxia and features of lung injury that resemble emphysema. These findings reveal both a lung-specific mechanism of lymphatic physiology and a lung-specific consequence of lymphatic dysfunction that may contribute to chronic lung diseases that arise in association with TLO formation.
Authors
Hasina Outtz Reed, Liqing Wang, Jarrod Sonett, Mei Chen, Jisheng Yang, Larry Li, Petra Aradi, Zoltán Jakus, Jeanine M. D'Armiento, Wayne W. Hancock, Mark L. Kahn
Abstract
Mice selectively expressing PPARγ dominant negative mutation in vascular smooth muscle exhibit RhoBTB1-deficiency and hypertension. Our rationale was to employ genetic complementation to uncover the mechanism of action of RhoBTB1 in vascular smooth muscle. Inducible smooth muscle-specific restoration of RhoBTB1 fully corrected the hypertension and arterial stiffness by improving vasodilator function. Notably, the cardiovascular protection occurred despite preservation of increased agonist-mediated contraction and RhoA/Rho kinase activity, suggesting RhoBTB1 selectively controls vasodilation. RhoBTB1 augmented the cGMP response to nitric oxide by restraining the activity of phosphodiesterase 5 (PDE5) by acting as a substrate adaptor delivering PDE5 to the Cullin-3 E3 Ring ubiquitin ligase complex for ubiquitination inhibiting PDE5. Angiotensin-II infusion also caused RhoBTB1-deficiency and hypertension which was prevented by smooth muscle specific RhoBTB1 restoration. We conclude that RhoBTB1 protected from hypertension, vascular smooth muscle dysfunction, and arterial stiffness in at least two models of hypertension.
Authors
Masashi Mukohda, Shi Fang, Jing Wu, Larry N. Agbor, Anand R. Nair, Stella-Rita C. Ibeawuchi, Chunyan Hu, Xuebo Liu, Ko-Ting Lu, Deng-Fu Guo, Deborah R. Davis, Henry L. Keen, Frederick W. Quelle, Curt D. Sigmund
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.
Authors
James V. McCann, Lin Xiao, Dae Joong Kim, Omar F. Khan, Piotr S. Kowalski, Daniel G. Anderson, Chad V. Pecot, Salma H. Azam, Joel S. Parker, Yihsuan S. Tsai, Alisa S. Wolberg, Stephen D. Turner, Kohei Tatsumi, Nigel Mackman, Andrew C. Dudley
Abstract
Upon arterial injury, endothelial denudation leads to platelet activation, and delivery of multiple agents (e.g. TXA2, PDGF) promoting VSMC dedifferentiation, and proliferation, in injury repair (intimal hyperplasia). Resolution of vessel injury repair, and prevention of excessive repair (switching VSMC back to a differentiated quiescent state) is a poorly understood process. We now report that internalization of activated platelets by VSMCs promotes resolution of arterial injury by switching on VSMC quiescence. Ex vivo and in vivo studies using lineage tracing reporter mice (PF4-Cre x mTmG) demonstrated uptake of green platelets by red vascular smooth muscle cells upon arterial wire injury. Genome-wide miRNA sequencing of VSMCs co-cultured with activated platelets identified significant increases in platelet-derived miR-223. miR-223 appears to directly target PDGFRβ (in VSMCs) reversing the injury-induced dedifferentiation. Upon arterial injury platelet miR-223 knockout mice exhibit increased intimal hyperplasia, whereas miR-223 mimics reduced intimal hyperplasia. Diabetic mice with reduced expression of miR-223, exhibited enhanced VSMC dedifferentiation, proliferation, and increased intimal hyperplasia. Horizontal transfer of platelet-derived miRNAs into VSMCs provide a novel mechanism for regulating VSMC phenotypic switching. Platelets thus play a dual role in vascular injury repair, initiating an immediate repair process, and concurrently, a delayed process to prevent excessive repair.
Authors
Zhi Zeng, Luoxing Xia, Xuejiao Fan, Allison C. Ostriker, Timur Yarovinsky, Meiling Su, Yuan Zhang, Xiangwen Peng, Xie Yi, Lei Pi, Xiaoqiong Gu, Sookja Kim Chung, Kathleen A. Martin, Renjing Liu, John Hwa, Wai Ho Tang
Abstract
Local flow patterns determine the uneven distribution of atherosclerotic lesions. This research aims to elucidate the mechanism of regulation of nuclear translocation of Yes-associated protein (YAP) under oscillatory shear stress (OSS) in the atheroprone phenotype of endothelial cells (ECs). We report here that OSS led to tyrosine phosphorylation and strong, continuous nuclear translocation of YAP in ECs that is dependent on integrin α5β1 activation. YAP overexpression in ECs blunted the anti-atheroprone effect of an integrin-α5β1 blocking peptide (ATN161) in Apoe-/- mice. Activation of integrin α5β1 induced tyrosine, but not serine, phosphorylation of YAP in ECs. Blockage of integrin α5β1 with ATN161 abolished the phosphorylation of YAP at Y357 induced by OSS. Mechanistic studies showed that c-Abl inhibitor attenuated the integrin α5β1-induced YAP tyrosine phosphorylation. Furthermore, the phosphorylation of c-Abl and YAPY357 was significantly increased in ECs in atherosclerotic vessels of mice and in human plaques vs. normal vessels. Finally, bosutinib, a tyrosine kinase inhibitor, markedly reduced the level of YAPY357 and the development of atherosclerosis in Apoe-/- mice. The c-Abl/YAPY357 pathway serves as a mechanism for the activation of integrin α5β1 and the atherogenic phenotype of ECs in response to OSS, and provides a potential therapeutic strategy for atherogenesis.
Authors
Bochuan Li, Jinlong He, Huizhen Lv, Yajin Liu, Xue Lv, Chenghu Zhang, Yi Zhu, Ding Ai
Abstract
The aortic root is the predominant site for development of aneurysm caused by heterozygous loss-of-function mutations in positive effectors of the transforming growth factor-β (TGF-β) pathway. Using a mouse model of Loeys-Dietz syndrome (LDS) that carries a heterozygous kinase-inactivating mutation in TGF-β receptor I, we found that the effects of this mutation depend on the lineage of origin of vascular smooth muscle cells (VSMCs). Secondary heart field–derived (SHF-derived), but not neighboring cardiac neural crest–derived (CNC-derived), VSMCs showed impaired Smad2/3 activation in response to TGF-β, increased expression of angiotensin II (AngII) type 1 receptor (Agtr1a), enhanced responsiveness to AngII, and higher expression of TGF-β ligands. The preserved TGF-β signaling potential in CNC-derived VSMCs associated, in vivo, with increased Smad2/3 phosphorylation. CNC-, but not SHF-specific, deletion of Smad2 preserved aortic wall architecture and reduced aortic dilation in this mouse model of LDS. Taken together, these data suggest that aortic root aneurysm predisposition in this LDS mouse model depends both on defective Smad signaling in SHF-derived VSMCs and excessive Smad signaling in CNC-derived VSMCs. This work highlights the importance of considering the regional microenvironment and specifically lineage-dependent variation in the vulnerability to mutations in the development and testing of pathogenic models for aortic aneurysm.
Authors
Elena Gallo MacFarlane, Sarah J. Parker, Joseph Y. Shin, Shira G. Ziegler, Tyler J. Creamer, Rustam Bagirzadeh, Djahida Bedja, Yichun Chen, Juan F. Calderon, Katherine Weissler, Pamela A. Frischmeyer-Guerrerio, Mark E. Lindsay, Jennifer P. Habashi, Harry C. Dietz
Abstract
Non-coding RNAs are emerging as important players in gene regulation and disease pathogeneses. Here, we show that a previously uncharacterized long non-coding RNA, NEXN-AS1, modulates the expression of the actin-binding protein NEXN and that NEXN exerts a protective role against atherosclerosis. An expression microarray analysis showed that the expression of both NEXN-AS1 and NEXN were reduced in human atherosclerotic plaques. In vitro experiments revealed that NEXN-AS1 interacted with the chromatin remodeler BAZ1A and the 5′-flanking region of the NEXN gene, and upregulated NEXN expression. Augmentation of NEXN-AS1 expression inhibited toll-like receptor-4 oligomerization and NFκB activity, downregulated the expression of adhesion molecules and inflammatory cytokines by endothelial cells, and suppressed monocyte adhesion to endothelial cells. These inhibitory effects of NEXN-AS1 were abolished by knockdown of NEXN. In vivo experiments of ApoE knockout mice fed a Western high-fat diet demonstrated that NEXN deficiency promoted atherosclerosis and increased macrophage abundance in atherosclerotic lesions, with heightened expression of adhesion molecules and inflammatory cytokines, whereas augmented NEXN expression deterred atherosclerosis. A group of patients with coronary artery disease were found to have lower blood NEXN levels than healthy individuals. These results indicate that NEXN-AS1 and NEXN represent potential therapeutic targets in atherosclerosis related diseases.
Authors
Yan-Wei Hu, Feng-Xia Guo, Yuan-Jun Xu, Pan Li, Zhi-Feng Lu, David G. McVey, Lei Zheng, Qian Wang, John H. Ye, Chun-Min Kang, Shao-Guo Wu, Jing-Jing Zhao, Xin Ma, Zhen Yang, Fu-Chun Fang, Yu-Rong Qiu, Bang-Ming Xu, Lei Xiao, Qian Wu, Li-Mei Wu, Li Ding, Tom R. Webb, Nilesh J. Samani, Shu Ye
Abstract
BACKGROUND.l-Carnitine, an abundant nutrient in red meat, accelerates atherosclerosis in mice via gut microbiota–dependent formation of trimethylamine (TMA) and trimethylamine N-oxide (TMAO) via a multistep pathway involving an atherogenic intermediate, γ-butyrobetaine (γBB). The contribution of γBB in gut microbiota–dependent l-carnitine metabolism in humans is unknown. METHODS. Omnivores and vegans/vegetarians ingested deuterium-labeled l-carnitine (d3-l-carnitine) or γBB (d9-γBB), and both plasma metabolites and fecal polymicrobial transformations were examined at baseline, following oral antibiotics, or following chronic (≥2 months) l-carnitine supplementation. Human fecal commensals capable of performing each step of the l-carnitine→γBB→TMA transformation were identified. RESULTS. Studies with oral d3-l-carnitine or d9-γBB before versus after antibiotic exposure revealed gut microbiota contribution to the initial 2 steps in a metaorganismal l-carnitine→γBB→TMA→TMAO pathway in subjects. Moreover, a striking increase in d3-TMAO generation was observed in omnivores over vegans/vegetarians (>20-fold; P = 0.001) following oral d3-l-carnitine ingestion, whereas fasting endogenous plasma l-carnitine and γBB levels were similar in vegans/vegetarians (n = 32) versus omnivores (n = 40). Fecal metabolic transformation studies, and oral isotope tracer studies before versus after chronic l-carnitine supplementation, revealed that omnivores and vegans/vegetarians alike rapidly converted carnitine to γBB, whereas the second gut microbial transformation, γBB→TMA, was diet inducible (l-carnitine, omnivorous). Extensive anaerobic subculturing of human feces identified no single commensal capable of l-carnitine→TMA transformation, multiple community members that converted l-carnitine to γBB, and only 1 Clostridiales bacterium, Emergencia timonensis, that converted γBB to TMA. In coculture, E. timonensis promoted the complete l-carnitine→TMA transformation. CONCLUSION. In humans, dietary l-carnitine is converted into the atherosclerosis- and thrombosis-promoting metabolite TMAO via 2 sequential gut microbiota–dependent transformations: (a) initial rapid generation of the atherogenic intermediate γBB, followed by (b) transformation into TMA via low-abundance microbiota in omnivores, and to a markedly lower extent, in vegans/vegetarians. Gut microbiota γBB→TMA/TMAO transformation is induced by omnivorous dietary patterns and chronic l-carnitine exposure. TRIAL REGISTRATION. ClinicalTrials.gov NCT01731236. FUNDING. NIH and Office of Dietary Supplements grants HL103866, HL126827, and DK106000, and the Leducq Foundation.
Authors
Robert A. Koeth, Betzabe Rachel Lam-Galvez, Jennifer Kirsop, Zeneng Wang, Bruce S. Levison, Xiaodong Gu, Matthew F. Copeland, David Bartlett, David B. Cody, Hong J. Dai, Miranda K. Culley, Xinmin S. Li, Xiaoming Fu, Yuping Wu, Lin Li, Joseph A. DiDonato, W.H. Wilson Tang, Jose Carlos Garcia-Garcia, Stanley L. Hazen
Abstract
Activation of the type 1 angiotensin II receptor (AT1) triggers proinflammatory signaling through pathways independent of classical Gq signaling that regulate vascular homeostasis. Here, we report that the AT1 receptor preformed a heteromeric complex with the receptor for advanced glycation endproducts (RAGE). Activation of the AT1 receptor by angiotensin II (Ang II) triggered transactivation of the cytosolic tail of RAGE and NF-κB–driven proinflammatory gene expression independently of the liberation of RAGE ligands or the ligand-binding ectodomain of RAGE. The importance of this transactivation pathway was demonstrated by our finding that adverse proinflammatory signaling events induced by AT1 receptor activation were attenuated when RAGE was deleted or transactivation of its cytosolic tail was inhibited. At the same time, classical homeostatic Gq signaling pathways were unaffected by RAGE deletion or inhibition. These data position RAGE transactivation by the AT1 receptor as a target for vasculoprotective interventions. As proof of concept, we showed that treatment with the mutant RAGE peptide S391A-RAGE362–404 was able to inhibit transactivation of RAGE and attenuate Ang II–dependent inflammation and atherogenesis. Furthermore, treatment with WT RAGE362–404 restored Ang II–dependent atherogenesis in Ager/Apoe-KO mice, without restoring ligand-mediated signaling via RAGE, suggesting that the major effector of RAGE activation was its transactivation.
Authors
Raelene J. Pickering, Christos Tikellis, Carlos J. Rosado, Despina Tsorotes, Alexandra Dimitropoulos, Monique Smith, Olivier Huet, Ruth M. Seeber, Rekhati Abhayawardana, Elizabeth K.M. Johnstone, Jonathan Golledge, Yutang Wang, Karin A. Jandeleit-Dahm, Mark E. Cooper, Kevin D.G. Pfleger, Merlin C. Thomas
Copyright © 2019 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)