Proteomic and Metabolomic Analysis of Vascular Smooth Muscle Cells

Abstract

Abstract-Recent developments of proteomic and metabolomic techniques provide powerful tools for studying molecular mechanisms of cell function. Previously, we demonstrated that neointima formation was markedly increased in vein grafts of PKC␦-deficient mice compared with wild-type controls. To clarify the underlying mechanism, we performed a proteomic and metabolomic analysis of cultured vascular smooth muscle cells (SMCs) derived from PKC␦ ϩ/ϩ and PKC␦ Ϫ/Ϫ mice. Using 2-dimensional electrophoresis and mass spectrometry, we identified Ͼ30 protein species that were altered in PKC␦ Ϫ/Ϫ SMCs, including enzymes related to glucose and lipid metabolism, glutathione recycling, chaperones, and cytoskeletal proteins. Interestingly, nuclear magnetic resonance spectroscopy confirmed marked changes in glucose metabolism in PKC␦ Ϫ/Ϫ SMCs, which were associated with a significant increase in cellular glutathione levels resulting in resistance to cell death induced by oxidative stress. Furthermore, PKC␦ Ϫ/Ϫ SMCs overexpressed RhoGDI␣, an endogenous inhibitor of Rho signaling pathways. Inhibition of Rho signaling was associated with a loss of stress fiber formation and decreased expression of SMC differentiation markers. Thus, we performed the first combined proteomic and metabolomic study in vascular SMCs and demonstrate that PKC␦ is crucial in regulating glucose and lipid metabolism, controlling the cellular redox state, and maintaining SMC differentiation. (Circ Res. 2004;94:e87-e96.)Key Words: proteomics Ⅲ metabolomics Ⅲ smooth muscle cells Ⅲ PKC Ⅲ signal transduction P roteomic and metabolomic techniques are ideal for clarifying quantitative protein and metabolite changes in physiological and diseased conditions, respectively. [1][2][3][4][5] In vascular research, however, proteomics and metabolomics are still in their infancies 6 -8 and no studies have been performed so far comparing proteomic and metabolomic profiles in vascular smooth muscle cells (SMCs).PKC␦ represents a novel PKC isoform as characterized on the basis of its structure and maximal activation by diacylglycerol in the absence of calcium. 9,10 We recently developed knockout mice lacking PKC␦ and studied its effect on neointima formation in vein grafts. 11 We demonstrated that loss of PKC␦ markedly accelerated neointima formation, resulting in complete occlusion of the vessel lumen in one-third of the vein grafts. As with p53-deficient mice, 12 neointimal lesions in PKC␦ Ϫ/Ϫ vein grafts contained twice as many SMCs as wild-type controls and showed significantly lower numbers of apoptotic SMCs. 11 In vitro experiments revealed that SMCs derived from PKC␦ Ϫ/Ϫ mice were less sensitive to various apoptotic stimuli, including cytokine treatment. Their apoptotic resistance appeared to involve a loss of free radical generation as evidenced by redoxsensitive fluorescent dyes. 11 Besides modulating apoptosis, PKC␦ was found to be important for cytoskeleton rearrangement and cell migration. 13 However, the molecular mechanisms of resistance to apoptosis and cyt...

Isozyme-specific Interaction of Protein Kinase Cδ with Mitochondria Dissected Using Live Cell Fluorescence Imaging

“…In particular, phorbol esters have been observed to induce the translocation of endogenous and exogenous PKC␦ to mitochondria in multiple cell lines, with prolonged phorbol ester treatment leading to apoptosis (22,23). PKC␦ knock-out mice also exhibit cardiac phenotypes, which tend to show that PKC␦ is cardioprotective (24,25), and proteomic analysis of cardiac tissue from PKC␦ knock-out mice shows changes in the levels of various metabolic proteins (24,26). Importantly, PKC␦ has been implicated in the regulation of the pyruvate dehydrogenase complex (PDHC), which controls the flux of fuel entering the citric acid cycle by converting pyruvate to acetyl CoA in the mitochondrial matrix, thereby regulating mitochondrial respiration.…”

Determination of PKC isoform-specific protein expression in pulmonary arteries of rats with chronic hypoxia-induced pulmonary hypertension

“…These results are consistent with previous observations from in vivo cerebral ischemia and primary neuronal cultures damaged by glutamate [33,34], which suggest that decreased cPKCβII activation probably provides a negative feedback mechanism in cell hypoxic growth, and the down-regulation of cPKC isoform-specific protein expression might be linked to the augmented growth capacity of pulmonary artery SMC. Mayr et al also reported that nPKCζ is crucial in maintaining SMC differentiation [35]. The decrease of cPKCα, βI and βII protein expressions may be the result of enhanced proteolytic degradation by Ca 2+ -activated proteinases during chronic hypoxia [36].…”

Metabolic homeostasis is maintained in myocardial hibernation by adaptive changes in the transcriptome and proteome

“…For in vivo inhibition of K(ATP) channels, glibenclamide (0.3 mg/kg bolus i.p. ; Sigma Chemical Corporation) was administered as a single dose to 2-week-old mice and RNA was harvested after 24 h. Key techniques involved adaptations of previously published protocols, including those for difference in-gel electrophoresis (DIGE) [10], liquid chromatography tandem mass spectrometry (LC-MS/MS) [10], proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) [13], immunoblotting [10], real-time PCR (qPCR) [7] and hypoxyprobe™ staining [7]. The Affymetrix Genechip mRNA expression analysis data were previously described by May et al [7].…”