Vascular biology
Abstract
Vascular smooth muscle cells (SMCs) are heterogeneous, and their differential responses to vascular injury are not well understood. To address this question, we performed single-cell analysis of vascular cells to a ligation injury in mouse carotid arteries after 3 days. While endothelial cells had a homogeneous activation of mesenchymal genes, less than 30% of SMCs responded to the injury and generated 2 distinct clusters — i.e., proinflammatory SMCs and stress-responsive SMCs. Proinflammatory SMCs were enriched with high levels of inflammatory markers such as vascular cell adhesion molecule-1 while stress-responsive SMCs overexpressed heat shock proteins. Trajectory analysis suggested that proinflammatory SMCs were potentially derived from a specific subpopulation of SMCs. Ligand-receptor pair analysis showed that the interaction between macrophages and proinflammatory SMCs was the major cell-cell communication among all cell types in the injured arteries. In vitro coculture demonstrated that VCAM1+ SMCs had a stronger chemotactic effect on macrophage recruitment than VCAM1– SMCs. Consistently, the number of VCAM1+ SMCs significantly increased in injured arteries and atherosclerotic lesions of ApoE–/– mice and human arteries. These findings provide insights at the single-cell level on the distinct patterns of endothelial cells and SMC responses to vascular injury.
Authors
Xili Ding, Qin An, Weikang Zhao, Yang Song, Xiaokai Tang, Jing Wang, Chih-Chiang Chang, Gexin Zhao, Tzung Hsiai, Guoping Fan, Yubo Fan, Song Li
Abstract
Endothelial mitochondria play a pivotal role in maintaining endothelial cell (EC) homeostasis through constantly altering their size, shape, and intracellular localization. Studies show that the disruption of the basal mitochondrial network in EC, forming excess fragmented mitochondria, implicates cardiovascular disease. However, cellular consequences underlying the morphological changes in the endothelial mitochondria under distinctively different, but physiologically occurring, flow patterns (i.e., unidirectional flow [UF] versus disturbed flow [DF]) are largely unknown. The purpose of this study was to investigate the effect of different flow patterns on mitochondrial morphology and its implications in EC phenotypes. We show that mitochondrial fragmentation is increased at DF-exposed vessel regions, where elongated mitochondria are predominant in the endothelium of UF-exposed regions. DF increased dynamin-related protein 1 (Drp1), mitochondrial reactive oxygen species (mtROS), hypoxia-inducible factor 1, glycolysis, and EC activation. Inhibition of Drp1 significantly attenuated these phenotypes. Carotid artery ligation and microfluidics experiments further validate that the significant induction of mitochondrial fragmentation was associated with EC activation in a Drp1-dependent manner. Contrarily, UF in vitro or voluntary exercise in vivo significantly decreased mitochondrial fragmentation and enhanced fatty acid uptake and OXPHOS. Our data suggest that flow patterns profoundly change mitochondrial fusion/fission events, and this change contributes to the determination of proinflammatory and metabolic states of ECs.
Authors
Soon-Gook Hong, Junchul Shin, Soo Young Choi, Jeffery C. Powers, Benjamin M. Meister, Jacqueline Sayoc, Jun Seok Son, Ryan Tierney, Fabio A. Recchia, Michael D. Brown, Xiaofeng Yang, Joon-Young Park
Abstract
Loss of retinal blood flow autoregulation is an early feature of diabetes that precedes the development of clinically recognizable diabetic retinopathy (DR). Retinal blood flow autoregulation is mediated by the myogenic response of the retinal arterial vessels, a process that is initiated by the stretch‑dependent activation of TRPV2 channels on the retinal vascular smooth muscle cells (VSMCs). Here, we show that the impaired myogenic reaction of retinal arterioles from diabetic animals is associated with a complete loss of stretch‑dependent TRPV2 current activity on the retinal VSMCs. This effect could be attributed, in part, to TRPV2 channel downregulation, a phenomenon that was also evident in human retinal VSMCs from diabetic donors. We also demonstrate that TRPV2 heterozygous rats, a nondiabetic model of impaired myogenic reactivity and blood flow autoregulation in the retina, develop a range of microvascular, glial, and neuronal lesions resembling those observed in DR, including neovascular complexes. No overt kidney pathology was observed in these animals. Our data suggest that TRPV2 dysfunction underlies the loss of retinal blood flow autoregulation in diabetes and provide strong support for the hypothesis that autoregulatory deficits are involved in the pathogenesis of DR.
Authors
Michael O’Hare, Gema Esquiva, Mary K. McGahon, Jose Manuel Romero Hombrebueno, Josy Augustine, Paul Canning, Kevin S. Edgar, Peter Barabas, Thomas Friedel, Patrizia Cincolà, Jennifer Henry, Katie Mayne, Hannah Ferrin, Alan W. Stitt, Timothy J. Lyons, Derek P. Brazil, David J. Grieve, J. Graham McGeown, Tim M. Curtis
Abstract
A central feature of progressive vascular remodeling is altered smooth muscle cell (SMC) homeostasis; however, the understanding of how different cell populations contribute to this process is limited. Here, we utilized single cell RNA sequencing to provide insight into cellular composition changes within isolated pulmonary arteries (PA) from pulmonary arterial hypertension (PAH) and donor lungs. Our results revealed that remodeling skewed the balanced communication network between immune and structural cells, in particular SMC. Comparative analysis with murine PA showed that human PA harbor heterogeneous SMC populations with an abundant intermediary cluster displaying a gradient transition between SMC and adventitial fibroblasts. Transcriptionally distinct SMC populations were enriched in specific biological processes and could be distinguished into four major clusters: oxygen sensing (enriched in pericytes), contractile, synthetic and fibroblast-like. End-stage remodeling was associated with phenotypic shift of pre-existing SMC populations and accumulation of synthetic SMC in neointima. Distinctly regulated genes in clusters built non-redundant regulatory hubs encompassing stress response and differentiation regulators. The current study provides a blueprint of cellular and molecular changes on a single cell level that are defining pathological vascular remodeling process.
Authors
Slaven Crnkovic, Francesco Valzano, Elisabeth Fließer, Juergen Gindlhuber, Helene Thekkekara Puthenparampil, Maria C. Basil, Michael P. Morley, Jeremy Katzen, Elisabeth Gschwandtner, Walter Klepetko, Edward Cantu, Heimo Wolinski, Horst Olschewski, Jorg Lindenmann, You-Yang Zhao, Edward E. Morrisey, Leigh M. Marsh, Grazyna Kwapiszewska
Abstract
Lymphangiectasia, an anomalous dilation of lymphatic vessels first described in the 17th century, is frequently associated with chylous effusion, respiratory failure, and high mortality in young patients, yet the underlying molecular pathogenesis and effective treatments remain elusive. Here, we identify an unexpected causal link between MAPK activation and defective development of the lymphatic basement membrane that drives lymphangiectasia. Human pathological tissue samples from patients diagnosed with lymphangiectasia revealed sustained MAPK activation within lymphatic endothelial cells. Endothelial KRASG12D–mediated sustained MAPK activation in newborn mice caused severe pulmonary and intercostal lymphangiectasia, accumulation of chyle in the pleural space, and complete lethality. Pathological activation of MAPK in murine vasculature inhibited the Nfatc1-dependent genetic program required for laminin interactions, collagen crosslinking, and anchoring fibril formation, driving defective development of the lymphatic basement membrane. Treatment with ravoxertinib, a pharmacological inhibitor of MAPK, reverses nuclear-to-cytoplasmic localization of Nfatc1, basement membrane development defects, lymphangiectasia, and chyle accumulation, ultimately improving survival of endothelial KRAS mutant neonatal mice. These results reveal defective lymphatic basement membrane assembly and composition as major causes of thoracic lymphangiectasia and provide a potential treatment.
Authors
Harish P. Janardhan, Karen Dresser, Lloyd Hutchinson, Chinmay M. Trivedi
Abstract
The (Pro)renin receptor ((P)RR), also known as ATP6AP2, is a single-transmembrane protein that is implicated in a multitude of biological processes. However, the exact role of ATP6AP2 during blood vessel development remains largely undefined. Here, we use an inducible endothelial cell (EC)-specific Atp6ap2 knockout mouse model to investigate the role of ATP6AP2 during both physiological and pathological angiogenesis in vivo. We observed that postnatal deletion of Atp6ap2 in ECs results in cell migration defects, loss of tip cell polarity and subsequent impairment of retinal angiogenesis. In vitro, Atp6ap2 deficient ECs similarly displayed reduced cell migration, impaired sprouting, and defective cell polarity. Transcriptional profiling of ECs isolated from Atp6ap2 mutant mice further indicated regulatory roles in angiogenesis, cell migration and extracellular matrix composition. Mechanistically, we provided evidence that expression of various extracellular matrix components is controlled by ATP6AP2 via the extracellular-signal-regulated kinase (ERK) pathway. Furthermore, Atp6ap2 deficient retinas exhibited reduced revascularization in an oxygen induced retinopathy model. Collectively, our results demonstrated a critical role of ATP6AP2 as a regulator of developmental and pathological angiogenesis.
Authors
Nehal R. Patel, Rajan K C, Avery E. Blanks, Yisu Li, Minolfa C. Prieto, Stryder M. Meadows
Abstract
Angiopoietin-2 (Ang-2) is a key mediator of vascular disease during sepsis, and elevated plasma levels of Ang-2 are associated with organ injury scores and poor clinical outcomes. We have previously observed that biomarkers of endothelial glycocalyx (EG) damage correlate with plasma Ang-2 levels, suggesting a potential mechanistic linkage between EG injury and Ang-2 expression during states of systemic inflammation. However, the cell signaling mechanisms regulating Ang-2 expression following EG damage are unknown. In the current study, we determined the temporal associations between plasma heparan sulfate (HS) levels as a marker of EG erosion and plasma Ang-2 levels in children with sepsis and in mouse models of sepsis. Secondly, we evaluated the role of shear stress-mediated 5’-adenosine monophosphate-activated protein kinase (AMPK) signaling in Ang-2 expression following enzymatic HS cleavage from the surface of human primary lung microvascular endothelial cells (HLMVEC). We found that plasma HS levels peak prior to plasma Ang-2 levels in children and mice with sepsis. Further, we discovered that impaired AMPK signaling contributes to increased Ang-2 expression following HS cleavage from flow conditioned HLMVECs, establishing a novel paradigm by which Ang-2 may be upregulated during sepsis.
Authors
Robert P. Richter, Amit R. Ashtekar, Lei Zheng, Danielle Pretorius, Tripathi Kaushlendra, Ralph D. Sanderson, Amit Gaggar, Jillian R. Richter
Abstract
Blood clot formation initiates ischemic events, but coagulation roles during postischemic tissue repair are poorly understood. The endothelial protein C receptor (EPCR) regulates coagulation as well as immune and vascular signaling by protease activated receptors (PARs). Here, we show that endothelial EPCRPAR1 signaling supports reperfusion and neovascularization in hindlimb ischemia in mice. Whereas deletion of PAR2 or PAR4 did not impair angiogenesis, EPCR and PAR1 deficiency or PAR1 resistance to cleavage by activated protein C caused markedly reduced postischemic reperfusion in vivo and angiogenesis in vitro. These findings were corroborated by biased PAR1 agonism in isolated primary endothelial cells. Loss of EPCRPAR1 signaling upregulated hemoglobin expression and reduced endothelial nitric oxide (NO) bioavailability. Defective angiogenic sprouting was rescued by the NO donor DETA-NO, whereas NO scavenging increased hemoglobin and mesenchymal marker expression in human and mouse endothelial cells. Vascular specimens from patients with ischemic peripheral artery disease exhibited increased hemoglobin expression, and soluble EPCR and NO levels were reduced in plasma. Our data implicate endothelial EPCR−PAR1 signaling in the hypoxic response of endothelial cells and identify suppression of hemoglobin expression as an unexpected link between coagulation signaling, preservation of endothelial cell NO bioavailability, support of neovascularization, and prevention of fibrosis.
Authors
Magdalena L. Bochenek, Rajinikanth Gogiraju, Stefanie Großmann, Janina Krug, Jennifer Orth, Sabine Reyda, George S. Georgiadis, Henri Spronk, Stavros Konstantinides, Thomas Münzel, John H. Griffin, Philipp S. Wild, Christine Espinola-Klein, Wolfram Ruf, Katrin Schäfer
Abstract
Apolipoprotein C-III (apoC-III) is a critical regulator of triglyceride metabolism and correlates positively with hypertriglyceridemia and cardiovascular disease (CVD). ApoC-III also induces sterile inflammation via inflammasome activation, another CVD risk factor. It remains unclear if therapeutic apoC-III lowering reduces CVD risk, nor is it understood if the CVD correlation depends on the lipid-lowering or anti-inflammatory properties. Therefore, we determined the impact of interventional apoC-III lowering on atherogenesis via apoC-III antisense oligonucleotide (ASO) administration in two hypertriglyceridemic mouse models where the intervention lowers plasma triglycerides (Apoe-/-Ndst1f/fAlb-Cre+, Ldlr-/-Ndst1f/fAlb-Cre+) and in a third lipid-refractory model where the ASO cannot lower plasma triglycerides (Ldlr-/-Lrp1f/fAlb-Cre+). A high-cholesterol Western diet ApoC-III ASO treatment did not alter atherosclerotic lesion size but did significantly attenuate advanced and unstable plaque development in the two triglyceride responsive mouse models. In contrast, no lesion size or composition improvement was observed with apoC-III ASO in the lipid-refractory Ldlr-/-Lrp1f/fAlb-Cre+ mice. To circumvent confounding effects of continuous high cholesterol feeding, we tested the impact of interventional apoC-III lowering when switching to a cholesterol-poor diet after 12-weeks of Western diet. In this diet-switch regimen, ApoC-III ASO treatment significantly reduced plasma triglycerides, atherosclerotic lesion progression, and necrotic core area and increased fibrous cap thickness in Ldlr-/-Ndst1f/fAlb-Cre+ mice. Again, ApoC-III ASO treatment did not alter triglyceride levels, lesion development and lesion composition in Ldlr-/-Lrp1f/fAlb-Cre+ mice after the diet-switch. Thus, therapeutic apoC-III targeting increased fibrous cap thickness, and reduced necrotic core area and lesion size after diet intervention when triglyceride-lowering is achieved in murine models. Our findings suggest that interventional apoC-III lowering might be an effective strategy to reduce atherosclerosis lesion size and improve plaque stability.
Authors
Bastian Ramms, Sohan Patel, Xiaoli Sun, Ariane R. Pessentheiner, G. Michelle Ducasa, Adam E. Mullick, Richard G. Lee, Rosanne M. Crooke, Sotirios Tsimikas, Joseph L. Witztum, Philip L.S.M. Gordts
Abstract
Disruption of the neurovascular unit (NVU) underlies the pathophysiology of various CNS diseases.(1-3) One strategy to repair NVU dysfunction would use stem/progenitor cells to provide trophic support to the NVU’s functionally coupled and interdependent vasculature and surrounding CNS parenchyma.(4) A subset of endothelial progenitor cells, endothelial colony forming cells (ECFCs) with high expression of the CD44 hyaluronan receptor (CD44hi), provides such neurovasculotrophic support via a paracrine mechanism.(5) Here, we report that bioactive extracellular vesicles from CD44hi ECFCs (EVshi) are paracrine mediators, recapitulating the effects of intact cell therapy in murine models of ischemic/neurodegenerative retinopathy; vesicles from ECFCs with low expression levels of CD44 (EVslo) were ineffective. Small RNA sequencing comparing the microRNA (miR) cargo from EVshi and EVslo identified candidate miRs that contribute to these effects. EVshi may be used to repair NVU dysfunction through multiple mechanisms to stabilize hypoxic vasculature, promote vascular growth, and support neural cells.
Authors
Kyle V. Marra, Edith Aguilar, Wei Guoqin, Ayumi Usui-Ouchi, Yochiro Ideguchi, Susumu Sakimoto, Martin Friedlander
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