Oxidized Low-Density Lipoprotein-Induced Cyclophilin A Secretion Requires ROCK-Dependent Diphosphorylation of Myosin Light Chain

Abstract

Objective: Accumulation of cyclophilin A (CyPA) within atherosclerotic lesions is thought to be implicated in the progression of atherosclerosis. However, the source of CyPA within atherosclerotic lesions is still unknown. The aim of this study is to determine the role of oxidized low-density lipoproteins (ox-LDL) in vascular smooth muscle cell (VSMC)-derived CyPA secretion and the underlying mechanism. Methods and Results: Abundant CyPA and α-smooth muscle actin (α-SMA) expressed in atherosclerotic lesions was observed in apolipoprotein E-deficient mice. ox-LDL induced CyPA secretion from a primary culture of rat aortic smooth muscle cells in a dose- and time-dependent manner. Sulfosuccinimidyloleate, a CD36 inhibitor, prevented the ox-LDL-induced CyPA secretion. Pre-exposure to either the actin-depolymerizing agent cytochalasin D or the actin-polymerizing agent jasplakinolide inhibited CyPA secretion induced by ox-LDL. Gene silencing of vesicle-associated membrane protein 2 suppressed ox-LDL-induced CyPA secretion. ox-LDL caused the phosphorylation of myosin light chain (MLC). Inhibition of MLC by blebbistatin reversed the secretion of CyPA and the phosphorylation of MLC induced by ox-LDL. MLC kinase inhibitor ML-7 reduced the monophosphorylation of MLC but did not reduce CyPA secretion. Pretreatment with the rho-associated coiled-coil kinase (ROCK) inhibitor Y27632 blocked diphosphorylation of MLC and secretion of CyPA induced by ox-LDL. Conclusions: ox-LDL-induced CyPA secretion requires vesicle transportation, actin remodeling and ROCK-dependent diphosphorylation of MLC. VSMC-derived CyPA induced by ox-LDL may be associated with increased CyPA expression in atherosclerotic lesions.

Yes‐associated protein mediates angiotensin II‐induced vascular smooth muscle cell phenotypic modulation and hypertensive vascular remodelling

“…Proteins from cultured cells or rat carotid arteries were extracted and prepared following standard protocols and as previously described by our laboratory . Nuclear and cytoplasmic extracts were prepared using NE‐PER Nuclear and Cytoplasmic Extraction Reagents (#78833, ThermoFisher, Waltham, MA, USA), supplemented with protease inhibitor (#36978; ThermoFisher).…”

The Release of Cyclophilin A from Rapamycin-Stimulated Vascular Smooth Muscle Cells Mediated by Myosin II Activation: Involvement of Apoptosis but Not Autophagy

“…RASMCs were isolated from male Harlan Sprague-Dawley rats (200-250 g) and cultured in high-glucose (4.5 g/L) DMEM (Gibco) containing 10% foetal bovine serum (Gibco) at 37°C in a humidified incubator (5% CO 2 /95% air), as described previously [13]. Passages 4-10 at 70-80% confluence were used for experiments.…”

“…Conditioned medium (CM) was prepared as described previously [13]. Briefly, the culture medium was collected and centrifuged for 10 min at 800 g to remove cell debris.…”

“…Western blotting was performed as previously described [13]. Cells were lysed with radioimmunoprecipitation assay buffer supplemented with 1:100 Halt TM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific).…”

“…Autophagy is reportedly involved in the unconventional secretion of secretory proteins [10][11][12][13]. Therefore, we firstly explored the role of autophagy in the exocytosis of CyPA in RASMCs.…”