Vascular smooth muscle cell (VSMC) proliferation is an important etiological factor in vascular proliferative diseases such as primary atherosclerosis, hypertension, arterial and in-stent restenosis, and transplant vasculopathy. Our studies established that butyrate, a bacterial fermentation product of dietary fiber and a chromatin modulator, is a potent inhibitor of VSMC proliferation. The cardiovascular health benefits of a high-fiber diet, the principle source of butyrate in the body, have been known for a long time, however, very little is known about the antiatherogenic potential of butyrate. Because oxidative stress plays an important role in the pathogenesis of atherosclerosis, we examined involvement of the glutathione/glutathione S-transferase (GST) antioxidant system in butyrate's inhibition of VSMC proliferation. Treatment of proliferating VSMCs with butyrate leads to the induction of several GSTs. Interestingly, our study also demonstrated the nuclear localization of GST-P1 (GST-7-7), which is considered to be a cytosolic protein; this was demonstrated using immunostaining and was corroborated by western blotting. Also, the butyrate-induced antiproliferative action, and the induction of GST-P1 and its nuclear localization are downregulated when butyrate is withdrawn. Furthermore, assessment of intracellular glutathione levels reveals their augmentation by butyrate. Conversely, butyrate treatment reduces the levels of reactive oxygen species in VSMCs. Collectively, the butyrate-treatment-related increase in glutathione content, the reduction in reactive oxygen species, the upregulation of GST and the nuclear localization of GST-P1 in growth-arrested VSMCs imply that butyrate's antiproliferative action involves modulation of the cellular redox state. Thus, induction of the glutathione/GST antioxidant system appears to have other regulatory role(s) besides detoxification and regulation of the cellular redox state, for example, cell-cycle control and cell proliferation, which are both critical to atherogenesis.
Vascular aging is characterized by endothelial dysfunction that is primarily attributed to increased superoxide production, the exact source of which remains ambiguous. This study compared the NAD(P)H and xanthine oxidase (XO) systems as sources of superoxide and impaired vascular function in aging. Male Sprague Dawley rats, 4-months-old (young) and 18-months-old (Aging), were used. Systolic blood pressure was higher (36 +/- 3%) in the aging group compared with young rats, and this was accompanied by reduced acetylcholine-induced renal vasodilatation. Urinary excretion of nitrite was lower in the aging rats (P < 0.05), and this was associated with reduced nitric oxide synthase (NOS) activity and reduced eNOS and iNOS protein expression in the aorta. Aged rats showed a n approximately twofold increase in free radical generation, as evident by increased plasma 8-isoprostane level, and an approximately fourfold increase in proteinuria compared with the young rats. Vascular NADP(H) oxidase was unchanged between both groups, as was the expression of p67phox or p47phox components of NAD(P)H oxidase. However, XO activity was increased (19 +/- 1%; P < 0.05) as well as XO expression in the aorta of aging rats. These results suggest that increased free radical generation-associated increase in SBP in aging rats is XO but not NAD(P)H oxidase-dependent.
Vascular remodeling is a characteristic feature of cardiovascular diseases. Altered cellular processes of vascular smooth muscle cells (VSMCs) is a crucial component in vascular remodeling. Histone deacetylase inhibitor (HDACI), butyrate, arrests VSMC proliferation and promotes cell growth. The objective of the study is to determine the mechanism of butyrate-induced VSMC growth. Using proliferating VSMCs exposed to 5 mM butyrate, immunoblotting studies are performed to determine whether PI3K/Akt pathway that regulates different cellular effects is a target of butyrate-induced VSMC growth. Butyrate inhibits phosphorylation-dependent activation of PI3K, PDK1, and Akt, eliciting differential effects on downstream targets of Akt. Along with previously reported Ser9 phosphorylation-mediated GSK3 inactivation leading to stability, increased expression and accumulation of cyclin D1, and epigenetic histone modifications, inactivation of Akt by butyrate results in: transcriptional activation of FOXO1 and FOXO3 promoting G1 arrest through p21Cip1/Waf1 and p15INK4B upregulation; inactivation of mTOR inhibiting activation of its targets p70S6K and 4E-BP1 impeding protein synthesis; inhibition of caspase 3 cleavage and downregulation of PARP preventing apoptosis. Our findings imply butyrate abrogates Akt activation, causing differential effects on Akt targets promoting convergence of cross-talk between their complimentary actions leading to VSMC growth by arresting proliferation and inhibiting apoptosis through its effect on dual targets, HDAC activity and PI3K/Akt pathway network.
Excessive proliferation of vascular smooth muscle cells (VSMCs) is a critical element in the development of several vascular pathologies, particularly in atherosclerosis and in restenosis due to angioplasty. We have shown that butyrate, a powerful antiproliferative agent, a strong promoter of cell differentiation and an inducer of apoptosis inhibits VSMC proliferation at physiological concentrations with no cytotoxicity. In the present study, we have used cDNA array technology to unravel the molecular basis of the antiproliferative effect of butyrate on VSMCs. To assess the involvement of gene expression in butyrate-inhibited VSMC proliferation, proliferating VSMCs were exposed to 5 mmol/l butyrate 1 through 5 days after plating. Expression profiles of 1.176 genes representing different functional classes in untreated control and butyrate treated VSMCs were compared. A total of 111 genes exhibiting moderate (2.0-5.0 fold) to strong (> 5.0 fold) differential expression were identified. Analysis of these genes indicates that butyrate treatment mainly alters the expression of four different functional classes of genes, which include: 43 genes implicated in cell growth and differentiation, 13 genes related to stress response, 11 genes associated with vascular function and 8 genes normally present in neuronal cells. Examination of differentially expressed cell growth and differentiation related genes indicate that butyrate-inhibited VSMC proliferation appears to involve down-regulation of genes that encode several positive regulators of cell growth and up-regulation of some negative regulators of growth or differentiation inducers. Some of the down-regulated genes include proliferating cell nuclear antigen (PCNA), retinoblastoma susceptibility related protein p130 (pRb), cell division control protein 2 homolog (cdc2), cyclin B1, cell division control protein 20 homolog (p55cdc), high mobility group (HMG) 1 and 2 and several others. Whereas the up-regulated genes include cyclin D1, p21WAF1, p141NK4B/p15INK5B, Clusterin, inhibitor of DNA binding 1 (ID1) and others. On the other hand, butyrate-responsive stress-related genes include some of the members of heat shock protein (HSP), glutathione-s-transferase (GST), glutathione peroxidase (GSH-PXs) and cytochrome P450 (CYP) families. Additionally, several genes related to vascular and neuronal function are also responsive to butyrate treatment. Although involvement of genes that encode stress response, vascular and neuronal functional proteins in cell proliferation is not clear, cDNA expression array data appear to suggest that they may play a role in the regulation of cell proliferation. However, cDNA expression profiles indicate that butyrate-inhibited VSMC proliferation involves combined action of a proportionally large number of both positive and negative regulators of growth, which ultimately causes growth arrest of VSMCs. Furthermore, these butyrate-induced differential gene expression changes are not only consistent with the antiproliferative effect of butyrate but a...
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