Inkyeom Kim

Tubastatin A suppresses renal fibrosis via regulation of epigenetic histone modification and Smad3-dependent fibrotic genes.

Inflammation and fibrosis are implicated in the pathogenesis of hypertensive kidney damage. We previously demonstrated that a nonspecific histone deacetylase (HDAC) inhibitor attenuates cardiac fibrosis in deoxycorticosterone acetate-salt hypertensive rats, which induces HDAC6 protein and enzymatic activity. However, the HDAC inhibitors effect and mechanism have not yet been demonstrated. We sought to determine whether an HDAC6-selective inhibitor could treat hypertension and kidney damage in angiotensin II-infused mice. Hypertension was induced by infusion of ANG in mice. Tubastatin A, an HDAC6 selective inhibitor, did not regulate blood pressure. Hypertensive stimuli enhanced the expression of HDAC6 in vivo and in vitro. We showed that the inhibition of HDAC6 prevents fibrosis and inflammation as determined by quantitative real-time PCR, western blot, and immunohistochemistry. Small interfering RNA (siRNA) against HDAC6 or Smad3 attenuated hypertensive stimuli-induced fibrosis and inflammation, whereas Smad2 siRNA failed to inhibit fibrosis. Interestingly, the combination of the HDAC6 inhibitor and Smad3 knockdown synergistically blocked transforming growth factor β (TGF-β) or ANG-induced fibrosis. We also demonstrated for the first time, to our knowledge, that acetylation of collagen type I can be regulated by HDAC6/p300 acetyltransferase. The chromatin immunoprecipitation assay revealed that the HDAC6 inhibitor suppressed TGF-β-induced acetylated histone H4 or phospho-Smad2/3 to Smad3 binding elements in the fibrosis-associated gene promoters including collagen type I. These results suggest that HDAC6 may be a valuable therapeutic target for the treatment of hypertension-induced kidney fibrosis and inflammation.

Histone Deacetylase Inhibition Attenuates Cardiac Hypertrophy and Fibrosis through Acetylation of Mineralocorticoid Receptor in Spontaneously Hypertensive Rats

Inhibition of histone deacetylases (HDACs) by valproic acid (VPA) attenuates inflammatory, hypertrophic, and fibrotic responses in the hearts of spontaneously hypertensive rats (SHRs); however, the molecular mechanism is still unclear. We hypothesized that HDAC inhibition (HDACi) attenuates cardiac hypertrophy and fibrosis through acetylation of mineralocorticoid receptor (MR) in SHRs. Seven-week-old SHRs and Wistar-Kyoto rats were treated with an HDAC class I inhibitor (0.71% w/v in drinking water; VPA) for 11 weeks. Sections of heart were visualized after trichrome stain as well as H&E stain. Histone modifications, such as acetylation (H3Ac [acetylated histone 3]) and fourth lysine trimethylation (H3K4me3) of histone 3, and recruitment of MR and RNA polymerase II (Pol II) into promoters of target genes were measured by quantitative real-time polymerase chain reaction after chromatin immunoprecipitation assay. MR acetylation was determined by Western blot with anti–acetyl-lysine antibody after immunoprecipitation with anti-MR antibody. Treatment with VPA attenuated cardiac hypertrophy and fibrosis. Although treatment with VPA increased H3Ac and H3K4me3 on promoter regions of MR target genes, expression of MR target genes as well as recruitment of MR and Pol II on promoters of target genes were decreased. Although HDACi did not affect MR expression, it increased MR acetylation. These results indicate that HDACi attenuates cardiac hypertrophy and fibrosis through acetylation of MR in spontaneously hypertensive rats.

Effect of heat shock on the vascular contractility in isolated rat aorta.

Stresses such as heat shock, ischemia, and irradiation have been known to induce heat shock proteins in various tissues. We investigated the effects of heat shock on the vascular contractility by using isolated rat aorta. Rat thoracic aortic rings were mounted in an organ bath maintained at 37 degrees C. For heat shock, aortic ring preparations were exposed to 42 degrees C for either 15 or 45 min (heat shock group), whereas the control group was left at 37 degrees C. Eight hours after heat shock, aortic ring preparations were subjected to contractions with high K(+) membrane-depolarizing solution. After functional study, tissues were frozen for measurement of heat shock protein 70 (HSP70). Heat shock not only increased the expression of HSP70 in the rat aorta, but also augmented contractions to KCl whether endothelium was present or denuded. Short exposure of tissues to 42 degrees C for 15 min did not work either. These results suggest that heat shock increases vascular contractility in isolated aortic strips.

Histone Deacetylase 3 and 4 Complex Stimulates the Transcriptional Activity of the Mineralocorticoid Receptor.

Histone deacetylases (HDACs) act as corepressors in gene transcription by altering the acetylation of histones, resulting in epigenetic gene silencing. We previously reported that HDAC3 acts as a coactivator of the mineralocorticoid receptor (MR). Although HDAC3 forms complexes with class II HDACs, their potential role in the transcriptional activity of MR is unclear. We hypothesized that HDAC4 of the class II family stimulates the transcriptional activity of MR. The expression of MR target genes was measured by quantitative real-time PCR. MR and RNA polymerase II recruitment to promoters of MR target genes was analyzed by chromatin immunoprecipitation. The association of MR with HDACs was investigated by co-immunoprecipitation. MR acetylation was determined with an anti-acetyl-lysine antibody after immunoprecipitation with an anti-MR antibody. Among the class II HDACs, HDAC4 interacted with both MR and HDAC3 after aldosterone stimulation. The nuclear translocation of HDAC4 was mediated by protein kinase A (PKA) and protein phosphatases (PP). The transcriptional activity of MR was significantly decreased by inhibitors of PKA (H89), PP1/2 (calyculin A), class I HDACs (MS-275), but not class II HDACs (MC1568). MR acetylation was increased by H89, calyculin A, and MS-275, but not by MC1568. Interaction between MR and HDAC3 was significantly decreased by H89, calyculin A, and HDAC4 siRNA. A non-genomic effect of MR via PKA and PP1/2 induced nuclear translocation of HDAC4 to facilitate the interaction between MR and HDAC3. Thus, we have uncovered a crucial role for a class II HDAC in the activation of MR-dependent transcription.

Effects of 3',4'-dihydroxyflavonol on vascular contractions of rat aortic rings

1. 3′,4′‐Dihydroxyflavonol (DiOHF) is an effective vasodilator with anti‐oxidant activity. The aim of the present study was to elucidate the effects of DiOHF on vascular contractions.

Glucosamine increases vascular contraction through activation of RhoA/Rho kinase pathway in isolated rat aorta

Diabetes is a well-known independent risk factor for vascular disease. However, its underlying mechanism remains unclear. It has been reported that increased influx of the hexosamine biosynthesis pathway (HBP) induces O-GlcNAcylation of proteins, leading to insulin resistance. In this study, we determined whether or not O-GlcNAc modification of proteins could increase vessel contraction. Using an endothelium-denuded aortic ring, we observed that glucosamine induced OGlcNAcylation of proteins and augmented vessel contraction stimulated by U46619, a thromboxane A(2) agonist, via augmentation of the phosphorylation of MLC(20), MYPT1(Thr855), and CPI17, but not phenylephrine. Pretreatment with OGT inhibitor significantly ameliorated glucosamine-induced vessel constriction. Glucosamine treatment also increased RhoA activity, which was also attenuated by OGT inhibitor. In conclusion, glucosamine, a product of glucose influx via the HBP in a diabetic state, increases vascular contraction, at least in part, through activation of the RhoA/Rho kinase pathway, which may be due to O-GlcNAcylation.

HEAT SHOCK‐INDUCED AUGMENTATION OF VASCULAR CONTRACTILITY IS INDEPENDENT OF RHO‐KINASE

1 In a previous study, we demonstrated that heat shock augments the contractility of vascular smooth muscle through the stress response. 2 In the present study, we investigated whether Rho‐kinases play a role in heat shock‐induced augmentation of vascular contractility in rat isolated aorta. 3 Rat aortic strips were mounted in organ baths, exposed to 42C for 45 min and subjected to contractile or relaxant agents 5 h later. 4 The level of expression of Rho‐kinases in heat shock‐exposed tissues was no different to that of control tissues, whereas heat shock induced heat shock protein (Hsp) 72 at 3 and 5 h. Heat shock resulted in an increase in vascular contractility in response to phenylephrine 5 h later. 5 The Rho‐kinase inhibitors Y27632 (30 nmol/L‐10 mmol/L) or HA 1077 (10 nmol/L‐10 mmol/L) relaxed 1.0 mmol/L phenylephrine‐precontracted vascular strips in a concentration‐dependent manner; these effects were attenuated in heat shock‐exposed strips. Pretreatment with Y27632 resulted in greater inhibition of the maximum contraction in control strips compared with those in heat shock‐exposed strips. 6 The results of the present study suggest that Rho‐kinases are unlikely to be involved in heat shock‐induced augmentation of vascular contractility.

Improvement of oral contraceptive-induced glucose dysregulation and dyslipidemia by valproic acid is independent of circulating corticosterone

Abstract Context: Cardiometabolic disorders are rapidly becoming major public health challenges. Valproic acid (VPA) is a widely prescribed anticonvulsant drug. Objective: We hypothesized that treatment with VPA would improve the regulation of glucose and atherogenic dyslipidemia through reduction in circulating corticosterone. Methods: Female Wistar rats recieved (p.o.) combined oral contraceptive (COC) containing 1.0 µg ethinylestradiol plus 5.0 µg levonorgestrel and valproic acid (VPA; 20 mg) for 8 weeks. Results: Treatment with COC led to elevated fasting blood glucose, insulin, corticosterone, triglycerides (TG), TG/HDL-cholesterol ratio, insulin resistance (IR) and impaired glucose tolerance. VPA significantly attenuated the alterations induced by COC treatment, but did not affect the corticosterone level. However, VPA treatment led to significant increases in plasma insulin, corticosterone, atherogenic lipids and impaired glucose tolerance in rats not treated with COC. Conclusion: The findings in this study suggest that VPA mitigates against the development of COC-induced insulin resistance and dyslipidemia independent of elevated circulating corticosterone.

Enrichment of (pro)renin receptor promoter with activating histone codes in the kidneys of spontaneously hypertensive rats

Background: The (pro)renin receptor [(P)RR] non-proteolytically, through conformational change, activates prorenin which can convert angiotensinogen to angiotensin I in addition to the classic conversion of angiotensinogen to angiotensin I by circulating renin. Since renal (P)RR is upregulated in hypertension and implicated in the pathogenesis of malignant hypertension, we hypothesized that (pro)renin receptor promoter is enriched with activating histone codes in the kidney of spontaneously hypertensive rats (SHR). Methods: The mRNA and protein expression levels were measured by real-time polymerase chain reaction (PCR) and western blot, respectively. The DNA methylation status of (P)RR promoter region was analyzed by bisulfite sequencing. The histone modifications were determined by chromatin immunoprecipitation followed by real-time PCR. Results: The (P)RR mRNA expression in the kidney was about six times greater in SHR than in Wistar–Kyoto (WKY) rats. The (P)RR promoter was little methylated in the kidneys of both WKY and SHR. Acetylated histone H3 (H3Ac) and di-methylated histone H3 at lysine 4 (H3K4me2), activating histone codes, were about 25 and three times higher in SHR than in WKY, respectively. On the other hand, di-methylated histone H3 at lysine 9 (H3K9me2), a suppressive histone code, was 50 times lower in SHR than in WKY. Conclusion: These results suggest that the (P)RR promoter is enriched with activating histone codes in the kidneys of SHR.

Enhanced contractility of vascular smooth muscle after brief exposure to arsenate.

Epidemiological studies indicate that arsenic exposure induces hypertension. We hypothesized that arsenate exposure modulates the contractility of vascular smooth muscle through the stress response. Intraperitoneal injection of sodium arsenate (15mg/kg) 16h before increased not only the blood pressure of rats but also the pressor response to preganglionic nerve stimulation (2 and 16Hz) or to bolus injection of vasopressin or phenylephrine in pithed rats as compared with the control rats. Exposure of rat aortic rings to 4mM sodium arsenate for 60min enhanced the contractile responses to KCl or phenylephrine as well as the HSP 70 expression 8h later, but did not affect the relaxation responses to acetylcholine, histamine, or sodium nitroprusside. These results suggest that brief exposure to arsenate is associated with enhanced contractility of vascular smooth muscle through the stress response.