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Objective Human monocytes can be divided into CD16− monocytes and CD16+ monocytes. Studies in mice suggested differential effects of monocyte subsets during new vessel formation. Methods The functional role of human monocyte subsets in neovascularization processes was investigated. For in vivo experiments, nude mice underwent unilateral hindlimb ischemia surgery before being injected with either total monocytes, CD16− monocytes or CD16+ monocytes isolated from healthy individuals. Results In vitro, cytokine array analysis demonstrated that monocytes release numerous angiogenic cytokines, some of which were differentially expressed in monocyte subsets. Sprout length was enhanced in EC spheroids being cultured in conditioned medium obtained from total monocytes and, to a lesser extent, also in supernatants of CD16− monocytes. Laser Doppler perfusion imaging up to day 28 after surgery revealed a trend toward improved revascularization in mice treated with monocytes, but no significant differences between monocyte subsets. Histological analyses four weeks after surgery showed an increased arteriole size in mice having received CD16+ monocytes, whereas the number of capillaries did not significantly differ between groups. Conclusions Our findings suggest additive and differential effects of monocyte subsets during neovascularization processes, possibly due to an altered secretion of angiogenic factors and their paracrine capacity to stimulate new vessel formation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Experimental and human autopsy studies have associated adventitial lymphangiogenesis with atherosclerosis. An analysis of perivascular lymphangiogenesis in patients with coronary artery disease is lacking. Here, we examined lymphangiogenesis and its potential regulators in perivascular adipose tissue (PVAT) surrounding the heart (C-PVAT) and compared it with PVAT of the internal mammary artery (IMA-PVAT). Forty-six patients undergoing coronary artery bypass graft surgery were included. Perioperatively collected C-PVAT and IMA-PVAT were analyzed using histology, immunohistochemistry, real time PCR, and PVAT-conditioned medium using cytokine arrays. C-PVAT exhibited increased PECAM-1 (platelet endothelial cell adhesion molecule 1)-positive vessel density. The number of lymphatic vessels expressing lymphatic vessel endothelial hyaluronan receptor-1 or podoplanin was also elevated in C-PVAT and associated with higher inflammatory cell numbers, increased intercellular adhesion molecule 1 (ICAM1) expression, and fibrosis. Significantly higher expression of regulators of lymphangiogenesis such as vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF receptor-3 was observed in C-PVAT compared to IMA-PVAT. Cytokine arrays identified angiopoietin-2 as more highly expressed in C-PVAT vs. IMA-PVAT. Findings were confirmed histologically and at the mRNA level. Stimulation of human lymphatic endothelial cells with recombinant angiopoietin-2 in combination with VEGF-C enhanced sprout formation. Our study shows that PVAT surrounding atherosclerotic arteries exhibits more extensive lymphangiogenesis, inflammation, and fibrosis compared to PVAT surrounding a non-diseased vessel, possibly due to local angiopoietin-2, VEGF-C, and VEGF-D overexpression. [ABSTRACT FROM AUTHOR]

Background: Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8 + T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8 + T cells and their effects on SMCs in established atherosclerosis., Methods: CD8 + T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient ( Ldlr -/- ) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8 + T cells were conducted., Results: Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8 + T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8 + T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8 + T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8 + T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1 , to be induced by CD8 + T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner., Conclusions: We here uncovered CD8 + T cells to control the SMC phenotype in atherosclerosis. CD8 + T cells promote SMC dedifferentiation and drive SMCs to adopt features of macrophage-like and osteoblast-like, procalcifying cell phenotypes. Given the critical role of SMCs in atherosclerotic plaque stability, CD8 + T cells could thus be explored as therapeutic target cells during lesion progression., Competing Interests: None.