Search

Genetic mutations and aberrant epigenetic alterations are the triggers for carcinogenesis. The emergence of the drugs targeting epigenetic aberrations has provided a better outlook for cancer treatment. Histone deacetylases (HDACs) are epigenetic modifiers playing critical roles in numerous key biological functions. Inappropriate expression of HDACs and dysregulation of PI3K signaling pathway are common aberrations observed in human diseases, particularly in cancers. Histone deacetylase inhibitors (HDACIs) are a class of epigenetic small-molecular therapeutics exhibiting promising applications in the treatment of hematological and solid malignancies, and in non-neoplastic diseases. Although HDACIs as single agents exhibit synergy by inhibiting HDAC and the PI3K pathway, resistance to HDACIs is frequently encountered due to activation of compensatory survival pathway. Targeted simultaneous inhibition of both HDACs and PI3Ks with their respective inhibitors in combination displayed synergistic therapeutic efficacy and encouraged the development of a single HDAC-PI3K hybrid molecule via polypharmacology strategy. This review provides an overview of HDACs and the evolution of HDACs-based epigenetic therapeutic approaches targeting the PI3K pathway.

Oxidative stress and inflammation are major contributors to acrolein toxicity. Peroxisome proliferator activated receptor gamma (PPARγ) has antioxidant and anti-inflammatory effects. We investigated the contribution of PPARγ ligand GW1929 to the attenuation of oxidative stress in acrolein-induced insult. Male gp91phox knock-out (KO) mice were treated with acrolein (0.5 mg·(kg body mass)–1 by intraperitoneal injection for 7 days) with or without GW1929 (GW; 0.5 mg·(kg body mass)–1·day–1, orally, for 10 days). The livers were processed for further analyses. Acrolein significantly increased 8-isoprostane and reduced PPARγ activity (P < 0.05) in the wild type (WT) and KO mice. GW1929 reduced 8-isoprostane (by 32% and 40% in WT and KO mice, respectively) and increased PPARγ activity (by 81% and 92% in WT and KO, respectively). Chemokine activity was increased (by 63%) in acrolein-treated WT mice, and was reduced by GW1929 (by 65%). KO mice exhibited higher xanthine oxidase (XO). Acrolein increased XO and COX in WT mice and XO in KO mice. GW1929 significantly reduced COX in WT and KO mice and reduced XO in KO mice. Acrolein significantly reduced the total antioxidant status in WT and KO mice (P < 0.05), which was improved by GW1929 (by 75% and 74%). The levels of NF-κB were higher in acrolein-treated WT mice. GW1929 reduced NF-κB levels (by 51%) in KO mice. Acrolein increased CD36 in KO mice (by 43%), which was blunted with GW1929. Data confirms that the generation of free radicals by acrolein is mainly through NAD(P)H, but other oxygenates play a role too. GW1929 may alleviate the toxicity of acrolein by attenuating NF-κB, COX, and CD36.

Virtual screening has become one of the important tools in the discovery of novel hits for the given target. The present study reports the successful application of ligand-based virtual screening method for the discovery of novel vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors. We generated a ligand query model with pharmacophore features from the reported VEGFR-2 inhibitors using vROCS tool and performed virtual screening. Among the 2.4 million lead like molecules of ZINC database screened, nineteen prioritized compounds were purchased from Enamine and ChemBridge and tested for VEGFR-2 inhibitory activity using Promega's ADP-Glo™ kinase assay. Experimental validation led to the discovery of four compounds 3, 7, 10, and 13. Compound 10 exhibited moderate inhibitory activity with the IC50 value of 19.3 μM. Molecular docking was performed for these compounds and the predicted binding modes reported in this paper may further guide to explore the possible structural changes to get more potent VEGFR-2 inhibitors.

It is known that moderate exercise can prevent the development of cardiovascular diseases, but the exact molecular mechanisms mediating cardioprotective effect of exercise remain unknown. Emerging evidence suggests that exercise has great impact on the biogenesis of exosomes, which have been found in both interstitial fluid and circulation, and play important roles in cellular communication. Exosomes carry functional molecules such as mRNAs, microRNA, and specific proteins, which can be used in the early diagnosis and targeted therapy of a variety of diseases. Our review focus on the current knowledge on exosome production, secretion, uptake and how exercise influence exosome content. We also highlight recent research development in exosome based approach for cardiac repair.

Use of sterile maggot larvae in the removal of infected tissue has been practiced for centuries in different cultures The practice was declined once antibiotics were introduced Today due to antibiotic resistant development or when conventional treatment is not possible use of maggot therapy in management of infection in different conditions has been shown to be effective Maggot therapy has been reported to be especially effective in treating diabetic wounds once it starts early in the process nbsp Here we are reporting a case study where maggot therapy was applied by a family member at home to an elderly patient when two months treatment of a wound in a diabetic foot with conventional antibiotics showed no improvement in progression of healing However addition of maggot therapy at home was effective in complete healing of the wound in three months Our observations indicate a decrease in exudation and edema a week after the application of maggot therapy significant improvement three weeks after and a complete healing after three months of maggot therapy nbsp Based on these observations we are concluding that maggot therapy can be safely applied by an untrained person at home setting This is a low cost and convenient alternative to antibiotic treatment that can be used at home for treating diabetic wounds without the need of a professional nbsp

Small Interfering RNAs (siRNAs) are one of the valuable tools to investigate the functions of genes, and are also used for gene silencing. It has a wide scope in drug discovery through in vivo target validation. siRNA therapeutics are not optimal drug-like molecules due to poor bioavailability, immunogenic and off-target effects. In order to overcome the challenges associated with siRNA therapeutics, identification of appropriate chemical modifications that improves the stability, specificity and potency of siRNA is essential. This review focuses on the various chemical modifications and their implications in siRNA therapy.

Oxidative stress and inflammation are major contributors to acrolein toxicity. Peroxisome proliferator activated receptor gamma (PPARγ) has antioxidant and anti-inflammatory effects. We investigated the contribution of PPARγ ligand GW1929 to the attenuation of oxidative stress in acrolein-induced insult. Male gp91

It is known that moderate exercise can prevent the development of cardiovascular diseases, but the exact molecular mechanisms mediating cardioprotective effect of exercise remain unknown. Emerging evidence suggests that exercise has great impact on the biogenesis of exosomes, which have been found in both interstitial fluid and circulation, and play important roles in cellular communication. Exosomes carry functional molecules such as mRNAs, microRNA, and specific proteins, which can be used in the early diagnosis and targeted therapy of a variety of diseases. Our review focus on the current knowledge on exosome production, secretion, uptake and how exercise influence exosome content. We also highlight recent research development in exosome based approach for cardiac repair.

Epigenetic mechanisms by altering the expression and, in turn, functions of target genes have potential to modify cellular processes that are characteristics of atherosclerosis, including inflammation, proliferation, migration and apoptosis/cell death. Butyrate, a natural epigenetic modifier and a histone deacetylase inhibitor (HDACi), is an inhibitor of vascular smooth muscle cell (VSMC) proliferation, a critical event in atherogenesis. Here, we examined whether glutathione peroxidases (GPxs), a family of antioxidant enzymes, are modulated by butyrate, contributing to its antiproliferation action on VSMC through the regulation of the inflammatory response by using western blotting, immunostaining methods and activity assay. Treatment of VSMC with butyrate not only upregulates glutathione peroxidase (GPx) 3 and GPx4, but also increases the overall catalytic activity of GPx supporting involvement of antioxidant effect in butyrate arrested VSMC proliferation. Moreover, analysis of the redox-sensitive NF-κB transcription factor system, the target of GPx, reveals that butyrate causes downregulation of IKKα, IKKβ, IkBα and NF-κBp65 expression and prevents NF-κBp65 phosphorylation at serine536 causing inhibition of the expression NF-κB target inflammatory genes, including inducible nitric oxide synthase, VCAM-1 and cyclooxygenase-2. Overall, these observations suggest a link between the antioxidant effect and anti-inflammatory response in butyrate-arrested VSMC proliferation, accentuating the atheroprotective and therapeutic potential of natural products, like butyrate, in vascular proliferative diseases.

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.

The histone deacetylase (HDAC) inhibitors, butyrate and trichostatin A (TSA), are epigenetic histone modifiers and proliferation inhibitors by downregulating cyclin D1, a positive cell cycle regulator, and upregulating p21Cip1 and INK family of proteins, negative cell cycle regulators. Our recent study indicated cyclin D1 upregulation in vascular smooth muscle cells (VSMC) that are proliferation-arrested by butyrate. Here we investigate whether cyclin D1 upregulation is a unique response of VSMC to butyrate or a general response to HDAC inhibitors (HDACi) by evaluating the effects of butyrate and TSA on VSMC. While butyrate and TSA inhibit VSMC proliferation via cytostatic and cytotoxic effects, respectively, they downregulate cdk4, cdk6, and cdk2, and upregulate cyclin D3, p21Cip1 and p15INK4B, and cause similar effects on key histone H3 posttranslational modifications. Conversely, cyclin D1 is upregulated by butyrate and inhibited by TSA. Assessment of glycogen synthase 3-dependent phosphorylation, subcellular localization and transcription of cyclin D1 indicates that differential effects of butyrate and TSA on cyclin D1 levels are linked to disparity in cyclin D1 gene expression. Disparity in butyrate- and TSA-induced cyclin D1 may influence transcriptional regulation of genes that are associated with changes in cellular morphology/cellular effects that these HDACi confer on VSMC, as a transcriptional modulator.

HDACs and HATs regulate histone acetylation, an epigenetic modification that controls chromatin structure and through it, gene expression. Butyrate, a dietary HDAC inhibitor, inhibits VSMC proliferation, a crucial factor in atherogenesis, and the principle mechanism in arterial and in-stent restenosis. Here, the link between antiproliferation action of butyrate and the portraits of global covalent modifications of histone H3 that it induces are characterized to understand the mechanics of butyrate-arrested VSMC proliferation. Analysis of histone H3 modifications specific to butyrate arrested VSMC proliferation display induction of histone H3-Lysine9 acetylation, inhibition of histone H3-Serine10 phosphorylation, reduction of histone H3-Lysine9 dimethylation and stimulation of histone H3-Lysine4 di-methylation, which is linked to transcriptional activation, cell cycle/mitosis, transcriptional suppression and activation, respectively. Conversely, untreated VSMCs exhibit inhibition of H3-Lysine9 acetylation, induction of H3-Serine10 phosphorylation, stimulation of H3-Lysine9 di-methylation and reduction in H3-Lysine4 di-methylation. Butyrate's cooperative effects on H3-Lysine9 acetylation and H3-Serine10 phosphorylation, and contrasting effects on di-methylation of H3-Lysine9 and H3-Lysine4 suggests that the interplay between these site-specific modifications cause distinct chromatin alterations that allow cyclin D1 and D3 induction, G1-specific cdk4, cdk6 and cdk2 downregulation, and upregulation of cdk inhibitors, p15INK4b and p21Cip1. Regardless of butyrate's effect on D-type cyclins, downregulation of G1-specific cdks and upregulation of cdk inhibitors by butyrate prevents cell cycle progression by failing to inactivate Rb. Overall, through chromatin remodeling, butyrate appears to differentially alter G1-specific cell cycle proteins to ensure proliferation arrest of VSMCs, a crucial cellular component of blood vessel wall.

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.

The metabotropic glutamate receptors (mGlu receptors) have emerged as attractive targets for number of neurological and psychiatric disorders. Recently, mGluR5 negative allosteric modulators (NAMs) have gained considerable attention in pharmacological research. Comparative Molecular Field Analysis (CoMFA) was performed on 73 analogues of aryl ether which were reported as mGluR5 NAMs. The study produced a statistically significant model with high correlation coefficient and good predictive abilities.

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/1 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, p14INK4B/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), and 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 are also in agreement with the roles that these gene products play in cell proliferation.

Acrolein, a major component of cigarette smoke, an environmental pollutant and an endogenous lipid peroxidation product, has been implicated in the development of atherosclerosis. Although a link between vascular injury and acrolein has been indicated, the exact molecular mechanism of acrolein-induced toxicity to vasculature is unknown. In an effort to elucidate the molecular basis of acrolein-induced vascular toxicity, the possibility of the intracellular signaling system as one of the targets of acrolein-induced toxicity is investigated in the present study. Exposure of cultured rat vascular smooth muscle cells (VSMCs) to different doses of acrolein not only causes cytotoxicity but also alters cellular morphology in a concentration and time-dependent manner. VSMCs exhibit cytotoxicity to a narrow concentration range of 5–10 μg/ml and display no toxicity to 2 μg/ml acrolein even after 24 h of exposure. Furthermore, exposure to acrolein results in activation of members of the mitogen-activated protein kinase (MAPK) family and protein tyrosine kinases. The extracellular signal-regulated kinases 1 and 2 (ERK1/2), stress-activated protein kinases/c-jun NH2-terminal kinases (SAPK/JNK) and p38MAPK are effectively and transiently activated by acrolein in a concentration and time-dependent fashion. While all three MAPKs exhibit significant activation within 5 min of exposure to acrolein, maximum activation (ERK1/2 and p38MAPK) or close to maximum activation (SAPK/JNK) occurs on exposure to 5 μg/ml acrolein for 2 h. Acrolein-induced activation of MAPKs is further substantiated by the activation of transcription factors, c-jun and activator transcription factor-2 (ATF-2), by acrolein-activated SAPK/JNK and p38MAPK, respectively. Additionally several cellular proteins exhibit spectacular protein tyrosine phosphorylation, particularly in response to 2 and 5 μg/ml of acrolein. Interestingly, the acrolein-induced activation of MAPKs precedes acrolein-stimulated protein tyrosine phosphorylation, which occurs after 2 h of exposure to acrolein. However, the time course of maximum protein tyrosine phosphorylation profile corresponds to the peak activation profile of MAPKs. The activation of MAPKs and protein tyrosine phosphorylation by acrolein appears to be independent of acrolein-induced toxicity. VSMCs exposed to 2 μg/ml acrolein exhibit no toxicity but stimulates significant activation of MAPKs and protein tyrosine phosphorylation. Although acrolein-induced VSMC toxicity is not blocked by MAPK inhibitors, PD98059, an inhibitor of MAPK kinase and SB203580, an inhibitor of p38MAPK, either alone or in combination, each MAPK responds differently to the inhibitors. Most prominently, although SB203580, an inhibitor of both SAPK/JNK and p38MAPK, significantly inhibited acrolein-induced activation of p38MAPK, it also stimulated SAPK/JNK activation by acrolein alone and in combination with PD98059. These results provide the first evidence that the activation of both growth-regulated (ERK1/2) and stress-regulated (SAPK/JNK and p38MAPK) MAPKs as well as tyrosine kinases are involved in the mediation of acrolein-induced effects on VSMC, which may play a crucial role in vascular pathogenesis due to environmentally and endogenously produced acrolein.

Epigenetic mechanisms by altering the expression and in turn, functions of target genes, without changing their primary structure have potential to modify cellular processes that are characteristic...

Arterial injury-induced vascular smooth muscle cell (VSMC) proliferation in intima is the important etiologic factor in vascular proliferative disorders such as atherosclerosis, hypertension and restenosis after balloon angioplasty. Butyrate, a naturally occurring short chain fatty acid, is produced by bacterial fermentation of dietary fiber and by mammary glands of certain mammals. Studies have shown that butyrate at millimolar concentrations, which are physiological, induces growth arrest, differentiation and apoptosis. We examined the effect of physiological concentrations of butyrate on rat VSMC proliferation and proliferation-induced PCNA expression to determine anti-atherogenic potential of butyrate. Butyrate concentrations, closer to physiological range, exhibited antiproliferative effects on both serum-induced proliferation of serum-starved quiescent VSMCs and actively proliferating non-confluent VSMCs. Treatment of serum-starved quiescent VSMCs with 1-8 mmol/l concentration of butyrate caused a concentration-dependent decrease in serum-induced VSMC proliferation and cell proliferation-associated increase in total cellular proteins and RNA levels. Similarly, exposure of actively growing VSMCs to 5 mmol/l butyrate resulted in the inhibition of cell proliferation and proliferation-induced increase in cellular proteins and RNA levels. Furthermore, cellular morphology was significantly altered. Analysis of cell cycle regulatory proteins indicated that levels of PCNA, an excellent marker for cell proliferation, was significantly altered by butyrate both in actively proliferating and serum-induced quiescent VSMCs. These observations suggest that butyrate exhibits potential antiatherogenic capability by inhibiting VSMC proliferation and proliferation-associated increase in PCNA expression and thus merits further investigations regarding therapeutic significance of butyrate in vascular proliferative disorders.

Abstract Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key regulatory enzyme of glycolysis, which exists in nuclei and functions as a DNA-binding protein as well as a nuclear protein, appears to be modulated by cellular activities. Exposure of quiescent rat smooth muscle cells (SMCs) to platelet-derived growth factor BB (PDGF-BB), which stimulates SMCs proliferation, caused a time-dependent increase in mRNA for GAPDH and its catalytic activity. Treatment of quiescent SMCs with sodium butyrate (SB), which is shown to inhibit PDGF-BB-induced SMC proliferation, caused a time- and concentration-dependent decrease in PDGF-BB-induced GAPDH mRNA expression and its catalytic activity. Nuclear run-on studies revealed that the PDGF-BB-induced rate of GAPDH gene transcription was reduced by about 50% in the presence of 5 mmol/L SB. The protein synthesis inhibitor, cycloheximide, failed to abolish the SB-inhibited PDGF-BB-induced rate of transcription of GAPDH, suggesting that SB is not dependent on ongoing protein synthesis to exert its effects on PDGF-BB-induced GAPDH transcription. Furthermore, measurement of GAPDH mRNA stability at various times after the inhibition of transcription with actinomycin D indicated that 5 mmol/L SB has no significant effect on the half-life of PDGF-BB-induced mRNA. The reduction in PDGF-BB-induced GAPDH expression by SB is probably caused by a cycloheximide-insensitive transcriptional mechanism. Thus, the inhibition of PDGF-BB-induced expression of GAPDH by SB suggests a link between SMC proliferation, energy consumption, and GAPDH gene upregulation.

Although until recently it is presumed that the greater portion of the genome has no biological role, the current advances in genome research and RNA biology have provided evidence indicating that a large section of the human and most eukaryotic genome is transcribed as nonprotein-coding RNAs or non-coding RNAs (ncRNAs)[1,2]. Only about 2% of the eukaryotic genome sequence codes for protein encoding genes and the remaining so called “junk” DNA are thought to have no functional significance [3, 4]. Based on large scale studies of human and other eukaryotic genomes it is estimated that about 98 % of the transcriptional output of their genomes is RNA that does not encode protein implying that the genomes are gorged with either inept RNA transcripts or with ncRNA transcripts that exhibit unanticipated functions in eukaryotic biology. However, recent development of new technologies in molecular biology and human genetics such as genome tiling [4,5], microarrays, and next generation RNAsequencing (RNA-Seq) [6,7] have enabled the discovery of different types of ncRNAs that do not code for protein product [8-10]. Even though ncRNAs do not encode proteins, they play pivotal roles in the complex networks that are necessary to regulate cellular functions via transcriptional and translational regulation of protein coding genes that are crucial to normal development and physiology, and to disease [11]. Moreover, many of the ncRNAs are highly conserved and susceptible to epigenetic and genetic defects that affect normal development and disease process significantly [12-15].

Atherosclerosis and restenosis, complex pathologies of blood vessels, are multifactorial diseases triggered by the inflammatory response to injury of endothelium. Remodeling of the injured vessel, proliferation and migration of vascular smooth muscle cells (VSMC) and elaboration and accumulation of extracellular matrix proteins are main traits of these diseases (Dzau et al., 2002; Libby, 2002; Pons et al., 2009; Ranganna et al., 2006; Ross, 1995;). Despite the substantial progress in understanding the etiology and the clinical management of atherosclerosis and restenosis, they are still life threatening diseases. Precise reasons are not still fully transparent. Different cell types; distinct cellular pathways and processes; and multiple genes within each participating cell types that are vulnerable to both genetic and environmental risk factors participate in the pathogenesis of atherosclerosis and restenosis. Recently, it is recognized that besides the genetic control epigenetic mechanisms regulate development and maintenance of organisms or their interaction with surrounding environment through the coordination of a set of reversible modifications that turn parts of the genome ‘off’ and ‘on’ at strategic times and at specific sites causing changes in gene expression with no changes in DNA sequences (Ekstrom, 2009; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). The two well-known epigenetic mechanisms, DNA methylation and histone modifications change the chromatin structure and dynamics that alter gene functions by influencing gene expressions. Dysregulation of epigenetic processes has been linked to human diseases, which influences many aspects of cell biology including cell growth, cell cycle control, proliferation, differentiation, and cell death. Reversing the dysregulation of epigenetic mechanisms may offer effective treatment strategy for many diseases including cardiovascular disease due to atherosclerosis and restenosis. This review presents the current advancement in the epigenetics of VSMC proliferation and potential use of histone epigenetic modifiers in the intervention of atherosclerosis and restenosis.

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.

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 are also in agreement with the roles that these gene products play in cell proliferation.

Acrolein, a major component of cigarette smoke, an environmental pollutant and an endogenous lipid peroxidation product, has been implicated in the development of atherosclerosis. Although a link between vascular injury and acrolein has been indicated, the exact molecular mechanism of acrolein-induced toxicity to vasculature is unknown. In an effort to elucidate the molecular basis of acrolein-induced vascular toxicity, the possibility of the intracellular signaling system as one of the targets of acrolein-induced toxicity is investigated in the present study. Exposure of cultured rat vascular smooth muscle cells (VSMCs) to different doses of acrolein not only causes cytotoxicity but also alters cellular morphology in a concentration and time-dependent manner. VSMCs exhibit cytotoxicity to a narrow concentration range of 5-10 microg/ml and display no toxicity to 2 microg/ml acrolein even after 24 h of exposure. Furthermore, exposure to acrolein results in activation of members of the mitogen-activated protein kinase (MAPK) family and protein tyrosine kinases. The extracellular signal-regulated kinases 1 and 2 (ERK1/2), stress-activated protein kinases/c-jun NH2-terminal kinases (SAPK/JNK) and p38MAPK are effectively and transiently activated by acrolein in a concentration and time-dependent fashion. While all three MAPKs exhibit significant activation within 5 min of exposure to acrolein, maximum activation (ERK1/2 and p38MAPK) or close to maximum activation (SAPK/JNK) occurs on exposure to 5 microg/ml acrolein for 2 h. Acrolein-induced activation of MAPKs is further substantiated by the activation of transcription factors, c-jun and activator transcription factor-2 (ATF-2), by acrolein-activated SAPK/JNK and p38MAPK, respectively. Additionally several cellular proteins exhibit spectacular protein tyrosine phosphorylation, particularly in response to 2 and 5 microg/ml of acrolein. Interestingly, the acrolein-induced activation of MAPKs precedes acrolein-stimulated protein tyrosine phosphorylation, which occurs after 2 h of exposure to acrolein. However, the time course of maximum protein tyrosine phosphorylation profile corresponds to the peak activation profile of MAPKs. The activation of MAPKs and protein tyrosine phosphorylation by acrolein appears to be independent of acrolein-induced toxicity. VSMCs exposed to 2 microg/ml acrolein exhibit no toxicity but stimulates significant activation of MAPKs and protein tyrosine phosphorylation. Although acrolein-induced VSMC toxicity is not blocked by MAPK inhibitors, PD98059, an inhibitor of MAPK kinase and SB203580, an inhibitor of p38MAPK, eitheralone or in combination, each MAPK responds differently to the inhibitors. Most prominently, although SB203580, an inhibitor of both SAPK/JNK and p38MAPK, significantly inhibited acrolein-induced activation of p38MAPK, it also stimulated SAPK/JNK activation by acrolein alone and in combination with PD98059. These results provide the first evidence that the activation of both growth-regulated (ERK1/2) and stress-regulated (SAPK/JNK and p38MAPK) MAPKs as well as tyrosine kinases are involved in the mediation of acrolein-induced effects on VSMC, which may play a crucial role in vascular pathogenesis due to environmentally and endogenously produced acrolein.

Abstract Sodium butyrate (SB), a naturally occurring short-chain fatty acid, was investigated for its therapeutic value as an antiproliferative agent for vascular smooth muscle cells (SMCs). At 5-mmol/L concentration, SB had no significant effect on rat SMC proliferation. However, at the same concentration, SB inhibited platelet-derived growth factor (PDGF)-AA–, -AB–, and -BB–induced proliferation of SMCs. Exposure of SMCs to PDGF-BB resulted in activation of receptor intrinsic tyrosine kinase activity and autophosphorylation of β-PDGF–receptor (β-PDGFR). The activated β-PDGFR physically associated and phosphorylated signaling molecules such as ras -GTPase activating protein (GAP) and phospholipase Cγ (PLCγ). SB, in the absence of PDGF-BB, caused neither β-PDGFR tyrosine phosphorylation nor phosphorylation and association of GAP and PLCγ with β-PDGFR. PDGF-BB–enhanced activation of receptor intrinsic tyrosine kinase activity and autophosphorylation of tyrosine residues of β-PDGFR were unaffected by SB irrespective of whether SMCs were preincubated with SB before exposure to PDGF-BB plus SB or incubated concomitantly with PDGF-BB plus SB. Likewise, phosphorylation and association of GAP and PLCγ with PDGF-BB–activated β-PDGFR were unaffected. In addition, SB did not block PDGF-BB–stimulated, PLCγ-mediated production of inositol triphosphate. Similarly, PDGF-BB–induced β-PDGFR degradation was unaffected when SMCs were exposed to PDGF-BB plus SB, and SB by itself had no influence on β-PDGFR degradation. Unlike β-PDGFR kinase activity, mitogen-activated protein kinase (MAP-kinase) activity was stimulated by SB by about 2.7-fold. Exposure of SMCs to PDGF-BB caused an ≈11.4-fold increase in MAP-kinase activity and this increase in activity was not significantly affected when cells were coincubated with PDGF-BB and SB (10.3-fold). However, pretreatment of SMCs with SB for 30 minutes and subsequent incubation in PDGF-BB plus SB abolished most of the PDGF-BB–induced MAP-kinase activity (4.6-fold). Transcription of growth response genes such as c- fos , c- jun , and c- myc were induced by PDGF-BB, and their induction was suppressed, particularly c- myc , by incubating SMCs with PDGF-BB plus SB. Similarly, preincubation of cells with SB for 30 minutes and subsequent incubation in PDGF-BB plus SB diminished PDGF-BB–induced transcription of c- fos , c- jun , and c- myc . However, SB by itself had no significant effect on c- fos , c- jun , and c- myc transcription. Our data suggest that the inhibition of PDGF-BB–induced proliferation of SMCs by SB involves MAP-kinase–regulated events as well as transcription of growth-response genes.

Nitric oxide (NO) regulates renal proximal tubular (PT) Na+ handling through modulation of Na+-K+ ATPase. Peroxisome Proliferator Activated Receptorα (PPARα), a nuclear transcription factor, is expressed in PTs and has been reported to influence NO generation/activity in renal tissues. This study tested the hypothesis that PPARα interacts with NO and thereby affects renal tubular Na+ transport. Urinary excretion of nitrite (UNOXV) and Na+ (UNaV) and PT Na+ transport (Na+-K+ ATPase activity) were determined in rats treated with clofibrate (250 mg/kg i.p) or WY14643 (45 mg/kg; i.p.), a PPARα ligand, 2% NaCl (orally), clofibrate/NaCl, L-NAME, an inhibitor of NO production (100 mg/kg; orally), L-NAME/Clofibrate. Clofibrate or WY14643 increased PPARα expression by 106 ± 7% (p < 0.05) and 113 ± 8% (p < 0.05), respectively. Similarly, clofibrate and WY14643 increased expression of MCAD, a downstream target protein of PPARα by 123 ± 8% (p < 0.05) and 143 ± 8% (p < 0.05), respectively. L-NAME attenuated clofibrate-induced increase in PPARα expression by 27 ± 2% (p < 0.05) but did not affect MCAD expression. UNOXV excretion increased 3–4 fold in rats treated with clofibrate, WY14643 or NaCl from 44 ± 7 to 170 ± 15, 144 ± 18 or 132 ± 11 nmol/24 hr, respectively (p < 0.05). Similarly, clofibrate, WY14643 or NaCl elicited a 2–5 fold increase in UNaV. L-NAME significantly reduced basal UNOXV and UNaV and abolished the clofibrate-induced increase. Clofibrate, WY14643, NaCl or clofibrate + NaCl treatment reduced Na+-K+-ATPase activity in the PT by 89 ± 23, 62 ± 10, 43 ± 9 and 82 ± 15% (p < 0.05), respectively. On the contrary, L-NAME or ODQ, inhibitor of sGC, abolished the inhibition of Na+-K+-ATPase activity by clofibrate (p < 0.05). Clofibrate either alone or with NaCl elicited ~2-fold increase in the expression of the α1 subunit of Na+-K+ ATPase in the PT while L-NAME abolished clofibrate-induced increase in Na+-K+ ATPase expression. These data suggest that PPARα activation, through increased NO generation promotes renal excretion of Na+ through reduced Na+-K+ ATPase activity in the PT probably via post translational modification of Na+-K+-ATPase.