ABSTRACT
BACKGROUND AND AIMS: Lysyl oxidase (LOX) catalyzes the crosslinking of collagen and elastin to maintain tensile strength and structural integrity of the vasculature. Excessive LOX activity increases vascular stiffness and the severity of occlusive diseases. Herein, we investigated the mechanisms by which LOX controls atherogenesis and osteogenic differentiation of vascular smooth muscle cells (SMC) in hyperlipidemic mice. METHODS: Gene inactivation of Lox in SMC was achieved in conditional knockout mice after tamoxifen injections. Atherosclerosis burden and vascular calcification were assessed in hyperlipidemic conditional [Loxf/fMyh11-CreERT2ApoE-/-] and sibling control mice [Loxwt/wtMyh11-CreERT2ApoE-/-]. Mechanistic studies were performed with primary aortic SMC from Lox mutant and wild type mice. RESULTS: Inactivation of Lox in SMCs decreased > 70 % its RNA expression and protein level in the aortic wall and significantly reduced LOX activity without compromising vascular structure and function. Moreover, LOX deficiency protected mice against atherosclerotic burden (13 ± 2 versus 23 ± 1 %, p < 0.01) and plaque calcification (5 ± 0.4 versus 11.8 ± 3 %, p < 0.05) compared to sibling controls. Interestingly, gene inactivation of Lox in SMCs preserved the contractile phenotype of vascular SMC under hyperlipidemic conditions as demonstrated by single-cell RNA sequencing and immunofluorescence. Mechanistically, the absence of LOX in SMC prevented excessive collagen crosslinking and the subsequent activation of the pro-osteogenic FAK/ß-catenin signaling axis. CONCLUSIONS: Lox inactivation in SMC protects mice against atherosclerosis and plaque calcification by reducing SMC modulation and FAK/ß-catenin signaling.
Subject(s)
Atherosclerosis , Disease Models, Animal , Hyperlipidemias , Mice, Knockout , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , Plaque, Atherosclerotic , Protein-Lysine 6-Oxidase , Vascular Calcification , Animals , Protein-Lysine 6-Oxidase/metabolism , Protein-Lysine 6-Oxidase/genetics , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/pathology , Myocytes, Smooth Muscle/metabolism , Atherosclerosis/genetics , Atherosclerosis/enzymology , Atherosclerosis/pathology , Atherosclerosis/metabolism , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/metabolism , Vascular Calcification/genetics , Vascular Calcification/pathology , Vascular Calcification/enzymology , Vascular Calcification/prevention & control , Vascular Calcification/metabolism , Hyperlipidemias/genetics , Hyperlipidemias/enzymology , Hyperlipidemias/complications , Hyperlipidemias/metabolism , Mice , Osteogenesis , Cells, Cultured , Aortic Diseases/pathology , Aortic Diseases/genetics , Aortic Diseases/enzymology , Aortic Diseases/prevention & control , Aortic Diseases/metabolism , Aorta/pathology , Aorta/enzymology , Aorta/metabolism , Male , Mice, Inbred C57BL , beta Catenin/metabolism , Signal Transduction , Extracellular Matrix ProteinsABSTRACT
The venous system has been historically understudied despite its critical roles in blood distribution, heart function, and systemic immunity. This study dissects the microanatomy of upper arm veins at the single cell level, and how it relates to wall structure, remodeling processes, and inflammatory responses to injury. We applied single-cell RNA sequencing to 4 non-diseased human veins (3 basilic, 1 cephalic) obtained from organ donors, followed by bioinformatic and histological analyses. Unsupervised clustering of 20,006 cells revealed a complex ecosystem of endothelial cell (EC) types, smooth muscle cell (SMCs) and pericytes, various types of fibroblasts, and immune cell populations. The venous endothelium showed significant upregulation of cell adhesion genes, with arteriovenous zonation EC phenotypes highlighting the heterogeneity of vasa vasorum (VV) microvessels. Venous SMCs had atypical contractile phenotypes and showed widespread localization in the intima and media. MYH11+DESlo SMCs were transcriptionally associated with negative regulation of contraction and pro-inflammatory gene expression. MYH11+DEShi SMCs showed significant upregulation of extracellular matrix genes and pro-migratory mediators. Venous fibroblasts ranging from secretory to myofibroblastic phenotypes were 4X more abundant than SMCs and widely distributed throughout the wall. Fibroblast-derived angiopoietin-like factors were identified as versatile signaling hubs to regulate angiogenesis and SMC proliferation. An abundant monocyte/macrophage population was detected and confirmed by histology, including pro-inflammatory and homeostatic phenotypes, with cell counts positively correlated with age. Ligand-receptor interactome networks identified the venous endothelium in the main lumen and the VV as a niche for monocyte recruitment and infiltration. This study underscores the transcriptional uniqueness of venous cells and their relevance for vascular inflammation and remodeling processes. Findings from this study may be relevant for molecular investigations of upper arm veins used for vascular access creation, where single-cell analyses of cell composition and phenotypes are currently lacking.