Vascular Smooth Muscle Cell Plasticity Research Articles

Abstract 18191: Mitochondrial Regulation of the Hyperproliferative Vascular Smooth Muscle Cell Phenotype

Background: During arterial restenosis and atherosclerosis, vascular smooth muscle cells (VSMCs) develop a highly proliferative and synthetic phenotype. Because such phenotypic changes are likely integrated with the energetic state of the cell, we hypothesized that changes in mitochondrial function regulate VSMC plasticity. Methods and Results: Exposure of VSMCs to platelet-derived growth factor (PDGF) promoted loss of the contractile proteins α-smooth muscle actin and calponin and was accompanied by remarkable changes in mitochondrial morphology. Confocal analysis showed that 97% of mitochondria in contractile VSMCs were filamentous; however, synthetic VSMCs showed a mitochondrial pool with >70% fragmented mitochondria (p<0.05). Mitochondrial membrane potential decreased by 86±1% with the attainment of the synthetic phenotype (p<0.01; n=3), but was not associated with mitophagy or decreases in the abundance of key proteins involved in oxidative phosphorylation. Compared with contractile VSMCs, synthetic VSMCs showed a 51±1.9% decrease in the abundance of mitofusin 2 (Mfn2; p<0.001; n=3) with no significant changes in Drp1, Fis1, or Opa1. Analysis of mitochondrial function in intact VSMCs showed a 20±3.7% decrease in glucose oxidation in synthetic cells, which was accompanied by a 17±2.6% increase in fatty acid oxidation (p<0.02; n=3). In permeabilized cells, complex II-mediated respiration was decreased by 20±2% in synthetic VSMCs (p<0.05; n=4). An inhibitor of mitochondrial fragmentation, mdivi1, decreased mitochondrial fragmentation by 50±6% (p<0.001; n=3), abolished the hyperproliferative response to PDGF (p<0.03; n=4), and reversed changes in mitochondrial respiration, yet it did not prevent PDGF-induced losses of contractile proteins. Conclusions: These results indicate that changes in mitochondrial morphology and bioenergetics contribute to the hyperproliferation of synthetic VSMCs, but do not affect the degradation of the contractile proteins. We propose that mitochondrial fragmentation that occurs during transition to the synthetic phenotype regulates mitochondrial substrate selection and bioenergetics and is a therapeutic target for vascular disorders characterized by VSMC hyperproliferation.

The epigenetic phenotypic switch of vascular smooth muscle cells involved in atherosclerosis

Atherosclerosis is a progressive and chronic inflammatory response to pathological fatty deposits in the arterial wall. The resulting disease spectrum ranges from angina pectoris, myocardial infarction, and sudden cardiac death to peripheral vascular disease and stroke. Vascular smooth muscle cells (VSMCs) are one of the major cell types contributing to neointimal formation of the affected arteries leading to atherosclerosis and in-stent restenosis. The severity of this disease has been linked to the cellular plasticity of VSMCs. At least two phenotypic states have been described: a contractile state, in which cells have increased cytoplasmic myofilaments and are involved in maintaining vascular tone, and a synthetic state, in which cells have relatively few contractile elements but in contrast upregulate the machinery required for protein synthesis and extracellular matrix secretion. In healthy vessels, VSMC can switch between these states, but regulation of this switch is disrupted in atherosclerosis and thought to contribute to the progression of disease.Our aim was to characterise the epigenetic signature of these VSMC phenotypes to better understand the pathophysiology of atherosclerosis. We used primary mouse VSMC from aorta and characterised the phenotypic changes occurring in response to transforming growth factor β, which promote the contractile phenotype, and platelet-derived growth factor that is involved in VSMC proliferation and migration. First we showed that VSMC develop into a contractile or synthetic phenotype in response to growth factor stimulation. We then characterised the gene expression profile and pattern of epigenetic histone modifications of VSMC selective genes. This identified novel histone modification proteins which could be involved in promoting the atherosclerotic phenotype. We have also shown that key VSMC selective genes are regulated through different epigenetic gene regulation mechanisms. This cell model will be used to investigate the molecular pathways involved in VSMC phenotypic regulation and identify novel candidates for future therapeutic intervention. FundingUK National Health Service and British Heart Foundation.

Mitochondrial fission induced by platelet-derived growth factor regulates vascular smooth muscle cell bioenergetics and cell proliferation

Vascular smooth muscle cells (VSMCs) develop a highly proliferative and synthetic phenotype in arterial diseases. Because such phenotypic changes are likely integrated with the energetic state of the cell, we hypothesized that changes in cellular metabolism regulate VSMC plasticity. VSMCs were exposed to platelet-derived growth factor-BB (PDGF) and changes in mitochondrial morphology, proliferation, contractile protein expression, and mitochondrial metabolism were examined. Exposure of VSMCs to PDGF resulted in mitochondrial fragmentation and a 50% decrease in the abundance of mitofusin 2. Synthetic VSMCs demonstrated a 20% decrease in glucose oxidation, which was accompanied by an increase in fatty acid oxidation. Results of mitochondrial function assays in permeabilized cells showed few changes due to PDGF treatment in mitochondrial respiratory chain capacity and coupling. Treatment of VSMCs with Mdivi-1—an inhibitor of mitochondrial fission—inhibited PDGF-induced mitochondrial fragmentation by 50% and abolished increases in cell proliferation; however, it failed to prevent PDGF-mediated activation of autophagy and removal of contractile proteins. In addition, treatment with Mdivi-1 reversed changes in fatty acid and glucose oxidation associated with the synthetic phenotype. These results suggest that changes in mitochondrial morphology and bioenergetics underlie the hyperproliferative features of the synthetic VSMC phenotype, but do not affect the degradation of contractile proteins. Mitochondrial fragmentation occurring during the transition to the synthetic phenotype could be a therapeutic target for hyperproliferative vascular disorders.

CGMP-Dependent Protein Kinase I and Smooth Muscle Relaxation

See related article, pages 1096–1103 The maintenance of vascular tone is central to the regulation of blood pressure and tissue perfusion and plays a role in the pathogenesis of hypertension and atherosclerosis. Vascular tone is determined by the balance of vasodilator and vasoconstrictor stimuli. After several decades of research, the NO/cGMP/cGMP-dependent protein kinase (cGK) pathway is now recognized as an important mediator of vasodilation. However, the mechanisms by which cGK causes smooth muscle relaxation continue to be an important question. Smooth muscle contraction and relaxation are tightly coupled to the phosphorylation and dephosphorylation, respectively, of the regulatory myosin light chain.1 Myosin light chain phosphorylation state is determined by the relative activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). MLCK phosphorylates MLC leading to contraction,2 and MLCP dephosphorylates MLC, leading to relaxation3 (Figure). Both MLCK and MLCP activities are highly regulated. MLCK activity is activated by the binding of calcium/calmodulin and thus is the primary mechanism linking intracellular calcium concentration to smooth muscle contractility.4 MLCP activity is regulated by both vasodilator and vasoconstrictor stimuli, and is therefore responsible for much of the calcium-independent regulation of contractility (reviewed in5). Figure. MLC phosphorylation determines smooth muscle contractility. Contractile agonists lead to inositol 1,4,5 triphosphate (IP3) production or activation of RhoA (RhoA-GTP). IP3 binding to its receptor in the sarcoplasmic reticulum leads to release of Ca2+. Ca2+/calmodulin binds to and activates MLCK, which in turn phosphorylates MLC (calcium-dependent contraction). Activated RhoA binds to and activates ROCK, leading to phosphorylation and inhibition of MLCP, inhibiting MLC dephosphorylation (calcium-independent contraction). cGKI mediates relaxation by inhibiting both calcium-dependent and -independent contraction. cGKIα activates MLCP by a direct interaction and by inhibition of RhoA, and activates RGS2 to inhibit Gαq …