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 EPCR-PAR1 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 EPCR-PAR1 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.

Cardiovascular risk factors may act by modulating the composition and function of the adventitia. Here we examine how age affects perivascular adipose tissue (PVAT) and its paracrine activities during neointima formation. Aortic tissue and PVAT or primary aortic smooth muscle cells from male C57BL/6JRj mice aged 52 weeks (“middle-aged”) were compared to tissue or cells from mice aged 16 weeks (“adult”). Vascular injury was induced at the carotid artery using 10% ferric chloride. Carotid arteries from the middle-aged mice exhibited smooth muscle de-differentiation and elevated senescence marker expression, and vascular injury further aggravated media and adventitia thickening. Perivascular transplantation of PVAT had no effect on these parameters, but age-independently reduced neointima formation and lumen stenosis. Quantitative PCR analysis revealed a blunted increase in senescence-associated proinflammatory changes in perivascular tissue compared to visceral adipose tissue and higher expression of mediators attenuating neointima formation. Elevated levels of protein inhibitor of activated STAT1 (PIAS1) and lower expression of STAT1- or NFκB-regulated genes involved in adipocyte differentiation, inflammation, and apoptosis/senescence were present in mouse PVAT, whereas PIAS1 was reduced in the PVAT of patients with atherosclerotic vessel disease. Our findings suggest that age affects adipose tissue and its paracrine vascular activities in a depot-specific manner. PIAS1 may mediate the age-independent vasculoprotective effects of perivascular fat.

(1) Background: Endothelial dysfunction initiates cardiovascular pathologies, including peripheral artery disease (PAD). The pathophysiology of impaired new vessel formation in the presence of angiogenic stimuli, such as ischemia and inflammation, is unknown. We have recently shown in mice that reduced endothelial protein C receptor (EPCR) expression results in defective angiogenesis following experimental hindlimb ischemia. (2) Purpose: To determine soluble (s)EPCR levels in the plasma of patients with PAD and to compare them with the protein C activity and biomarkers of endothelial function, inflammation, and angiogenesis. (3) Methods and Results: Clinical tests of vascular function and immunoassays of plasma from patients with PAD stage II were compared to age- and sex-matched individuals with and without cardiovascular risk factors or PAD stage III/IV patients. sEPCR levels were significantly lower in PAD stage II patients compared to subjects with risk factors, but no PAD, and further decreased in PAD stage III/IV patients. Plasma protein C activity or levels of ADAM17, a mediator of EPCR shedding, did not differ. Significant associations between sEPCR and the ankle-brachial index (p = 0.0359), age (p = 0.0488), body mass index (p = 0.0110), and plasma sE-selectin levels (p = 0.0327) were observed. High-sensitive CRP levels and white blood cell counts were significantly elevated in PAD patients and associated with serum glucose levels, but not sEPCR. In contrast, plasma TNFα or IL1β levels did not differ. Circulating levels of VEGF were significantly elevated in PAD stage II patients (p = 0.0198), but not associated with molecular (sE-selectin) or functional (ankle-brachial index) markers of vascular health. (4) Conclusions: Our findings suggest that circulating sEPCR levels may be useful as biomarkers of endothelial dysfunction, including angiogenesis, in persons older than 35 years and that progressive loss of endothelial protein C receptors might be involved in the development and progression of PAD.

(1) Background: Vascular mural cells reside in the media and outer layers of the vessel wall. Their ability to proliferate and migrate or to change phenotype in response to external cues is a central feature of the vascular response to injury. Genetically engineered mice are used for loss- or gain-of-function analyses or lineage tracing in vivo, their primary cells for mechanistic studies in vitro. Whether and how cultivation conditions affect their phenotype and function is often overlooked. (2) Methods: Here, we systematically studied how the cultivation of primary mural cells isolated from the aorta of adult wild-type mice in either basal medium (DMEM) or special media formulated for the cultivation of fibroblasts or pericytes affects their phenotype and function. (3) Results: Medium composition did not alter cell viability, but the mRNA levels of differentiated smooth muscle cell markers were highest in vascular mural cells expanded in DMEM. Conversely, significantly higher numbers of proliferating and migrating cells were observed in cells expanded in Pericyte medium, and cytoskeletal rearrangements supported increased migratory capacities. Significantly reduced telomere lengths and metabolic reprogramming was observed in aortic mural cells cultured in Fibroblast medium. (4) Conclusions: Our findings underline the plasticity of primary aortic mural cells and highlight the importance of the culture media composition during their expansion, which could be exploited to interrogate their responsiveness to external stimuli or conditions observed in vivo or in patients.