Vascular Smooth Muscle Cells Release Protease Nexin-1 in Cell Models of Atherosclerosis and Human Carotid Tissue

Abstract

Vascular smooth muscle cells (VSMCs) express protease nexin-1 (PN-1) in atherosclerotic lesions, but factors responsible for its release and its role in atherosclerosis remain unclear. PN-1 has been shown to induce collagen expression, a feature of plaque tissue and the synthetic VSMC phenotype. HDAC9 affects VSMC cellular phenotypic stability and has been implicated in large vessel ischemic stroke. We hypothesize that HDAC9 may influence PN-1 expression and be a target in atherosclerosis and stroke. Human primary VSMCs and control, asymptomatic and symptomatic (stroke or transient ischemic attack <7 days) carotid tissue from patients were examined. VSMCs were treated with cholesterol and oxidized phospholipids to induce the synthetic phenotype. Functional assays, qPCR, proteomic analysis and transwell co-culture with THP-1 monocytes, were used to assess phenotype characteristics and quantify protein and gene expression. Human VSMCs in atherogenic media have increased expression of PN-1, macrophage markers (LGALS3, CD68), and decreased expression of contractile genes (ACTA2). There was more PN-1 released from VSMCs grown in atherogenic media compared to control media. Monocytes exposed to atherogenic supernatant had higher rates of migration compared to control. Inhibition of HDAC9 by siRNA transfection decreased expression of PN-1, macrophage markers and restored VSMC contractile gene expression. Observations were replicated in human tissues. VSMCs from symptomatic plaque had increased expression of HDAC9, PN-1, macrophage markers and increased histone methylation, consistent with decreased contractile gene expression, compared to control and asymptomatic carotid tissues. HDAC9 and PN-1 have a previously unknown direct correlation, which is also related to VSMC synthetic phenotype. The mechanisms of this relationship and the role of PN-1 on plaque progression remains to be elucidated.