Kasturi Ranganna

Relationship between PPARα activation and NO on proximal tubular Na+ transport in the rat

BackgroundNitric 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.ResultsClofibrate 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.ConclusionThese 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.

Acrolein activates mitogen-activated protein kinase signal transduction pathways in rat vascular smooth muscle cells.

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.

Butyrate inhibits proliferation-induced Proliferating Cell Nuclear Antigen expression (PCNA) in rat vascular smooth muscle cells

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.

Gene expression profile of butyrate-inhibited vascular smooth muscle cell proliferation

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.

Involvement of glutathione/glutathione S‐transferase antioxidant system in butyrate‐inhibited vascular smooth muscle cell proliferation

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 butyrates 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 butyrates 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.

Differential Cellular and Molecular Effects of Butyrate and Trichostatin A on Vascular Smooth Muscle Cells

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.

Involvement of the Antioxidant Effect and Anti-inflammatory Response in Butyrate-Inhibited Vascular Smooth Muscle Cell Proliferation

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.

Therapeutic potential of chemically modified siRNA: Recent trends

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 and immunogenic and off‐target effects. 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.

MicroRNAome of Vascular Smooth Muscle Cells: Potential for MicroRNA-Based Vascular Therapies

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].

Emerging Epigenetic Therapy for Vascular Proliferative Diseases

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.