The Quantitative Trait Loci Mapping of Rice Plant and the Components of Its Extract Confirmed the Anti-Inflammatory and Platelet Aggregation Effects In Vitro and In Vivo.

Unpredictable climate change might cause serious lack of food in the world. Therefore, in the present world, it is urgent to prepare countermeasures to solve problems in terms of human survival. In this research, quantitative trait loci (QTLs) were analyzed when rice attacked by white backed planthopper (WBPH) were analyzed using 120 Cheongcheong/Nagdong double haploid lines. Moreover, from the detected QTLs, WBPH resistance-related genes were screened in large candidate genes. Among them, OsCM, a major gene in the synthesis of Cochlioquinone-9 (cq-9), was screened. OsCM has high homology with the sequence of chorismate mutase, and exists in various functional and structural forms in plants that produce aromatic amino acids. It also induces resistance to biotic stress through the synthesis of secondary metabolites in plants. The WBPH resistance was improved in rice overexpressed through map-based cloning of the WBPH resistance-related gene OsCM, which was finally detected by QTL mapping. In addition, cq-9 increased the survival rate of caecal ligation puncture (CLP)-surgery mice by 60%. Moreover, the aorta of rat treated with cq-9 was effective in vasodilation response and significantly reduced the aggregation of rat platelets induced by collagen treatment. A cq-9, which is strongly associated with resistance to WBPH in rice, is also associated with positive effect of CLP surgery mice survival rate, vasodilation, and significantly reduced rat platelet aggregation induced by collagen treatment. Therefore, cq-9 presents research possibilities as a substance in a new paradigm that can act on both Plant-Insect in response to the present unpredictable future.

HDAC5 inhibition reduces angiotensin II-induced vascular contraction, hypertrophy, and oxidative stress in a mouse model.

Non-specific histone deacetylase (HDAC) inhibition reduces high blood pressure in essential hypertensive animal models. However, the exact HDAC isoforms that play a critical role in controlling hypertension are not known. Here, we investigated the role of HDAC5 in vascular contraction, hypertrophy, and oxidative stress in the context of angiotensin II (Ang II)-induced hypertension. Genetic deletion of HDAC5 and treatment with class IIa HDAC inhibitors (TMP269 and TMP195) prevented Ang II-induced increases in blood pressure and arterial wall thickness. Hdac5-knockout mice were also resistant to the thromboxane A2 agonist (U46619)-induced vascular contractile response. Furthermore, the expression of Rho-associated protein kinase (ROCK) 2 was downregulated in the aortas of Ang II-treated Hdac5-knockout mice. Knockdown of HDAC5, RhoA, or ROCK2 reduced collagen gel contraction, whereas silencing of ROCK1 increased it. VSMC hypertrophy reduced on knocking down HDAC5, ROCK1, and ROCK2. Here we showed that genetic deletion of HDAC5 and pharmacological inhibition of class IIa HDACs ameliorated Ang II-induced ROS generation. Moreover, ROCK1 and ROCK2, the downstream targets of HDAC5, influenced ROS generation. The relative protein levels of HDAC5, ROCK1, and ROCK2 were increased both in the cytoplasm and nuclear fraction in response to Ang II stimulation in vascular smooth muscle cells. Inhibition of HDAC5 expression or activity reduced vascular hypertrophy, vasoconstriction, and oxidative stress in the Ang II-induced hypertension model. These findings indicate that HDAC5 may serve as a potential target in the treatment of hypertension.

Mountain ginseng inhibits skeletal muscle atrophy by decreasing muscle RING finger protein-1 and atrogin1 through forkhead box O3 in L6 myotubes.

ETHNOPHARMACOLOGICAL RELEVANCE: Mountain ginseng (Panax ginseng C.A. Meyer) is a medicinal herb with immune effects, muscle damage protection and energy metabolism effects. However, the pharmacological role of mountain ginseng in dexamethasone (DEXA)-induced muscle atrophy through the forkhead box O (FOXO) family is not understood. Therefore, we hypothesized that mountain ginseng inhibits skeletal muscle atrophy by decreasing muscle RING finger protein-1 (MuRF1) and atrogin1 through FOXO3 in L6 myotubes. METHODS: Rat myoblast (L6) cells or Sprague-Dawley (SD) rats were exposed to DEXA and mountain ginseng. The expressions of muscle atrophy targets such as MuRF1, atrogin1, MyHC (myosin heavy chain), HSP90, p-Akt, Akt, p-ERK1/2, ERK, FOXO3a, FOXO1, myostatin, and follistatin were analyzed by using Western blot analysis or real-time PCR. The diameter of myotubes was measured. Recruitment of glucocorticoid receptor (GR) or FOXO3a was analyzed by performing a chromatin immunoprecipitation (ChIP) assay. RESULTS: Mountain ginseng treatment reduced muscle weight loss and collagen deposition in DEXA-induced rats. Mountain ginseng treatment led to decreases in MuRF1, atrogin1, p-ERK1/2, FOXO3a, FOXO1, and myostatin. Also, mountain ginseng treatment led to increases in the diameter of myotubes, MyHC, HSP90, p-Akt, and follistatin. Treatment with mountain ginseng reduced enrichment of GR, FOXO3a, and RNA polymerase II on the promoters. CONCLUSIONS: These results suggest that mountain ginseng inhibits skeletal muscle atrophy by decreasing MuRF1 and atrogin1 through FOXO3a in L6 myotubes.

Anticancer Effect of Mountain Ginseng on Human Breast Cancer: Comparison with Farm-Cultivated Ginseng.

Mountain ginseng has been used generally as a pharmacopuncture for cancer therapy in clinical practice in Northeast Asia. Nonetheless, there have been few scientific reports for the anticancer action of mountain ginseng. In this study, we investigated whether mountain ginseng extract (MGE) could inhibit the growth of breast cancer in in vitro and in vivo models. MGE showed stronger cytotoxicity than farm-cultivated ginseng extract (FGE) through promoting ROS generation. Also MGE dose-dependently brought about mitochondrial dysfunction in MCF-7 cells. In addition, MGE induced apoptosis through enhancing the activities of caspase-3/7 by regulation of expression of Bcl-2, Bax, cytochrome c, and cleaved caspase-3 in the MCF-7 cells. Consistent with the in vitro results, MGE significantly reduced tumor weights compared with FGE in mice transplanted with MCF-7 cells, and it regulated the expression of apoptosis-related proteins, such as Bcl-2, Bax, cytochrome c, cleaved caspase-3, and cleaved PARP, in the tumor tissues. Additionally, MGE included higher total ginsenoside contents than FGE. In conclusion, MGE, which is richer in ginsenosides, exerts a stronger anticancer action than FGE in breast cancer. The anticancer action of MGE may be closely correlated with caspase-mediated apoptosis through upregulating ROS generation. Therefore, these findings may be helpful for a clinical understanding of the anticancer mechanism of MGE for breast cancer patients.

Selective inhibition of histone deacetylase 8 improves vascular hypertrophy, relaxation, and inflammation in angiotensin II hypertensive mice.

BACKGROUND: The dysregulation of histone deacetylase (HDAC) protein expression or its enzyme activity is implicated in a variety of diseases. Cardiac HDAC6 and HDAC8 enzyme activity induced by deoxycorticosterone acetate (DOCA) hypertension was attenuated by sodium valproate, a pan-HDAC inhibitor. However, the HDAC6-selective inhibitor, tubastatin A, did not attenuate angiotensin II-induced hypertension. The purpose of this study was to investigate whether PCI34051, an HDAC8-selective inhibitor, can modulate angiotensin II-induced hypertension and its regulatory mechanism. METHODS: An angiotensin II-regulated mouse model was used in this study. Animals received vehicle or PCI34051 (3 mg·kg - 1·day- 1) via intraperitoneal injection. Systolic blood pressure was measured by the tail-cuff method. Blood vessel thickness was measured following hematoxylin and eosin staining, VCAM-1 immunohistochemistry was performed in the aortas, and mRNA expression of renin-angiotensin system components, inflammation markers, and NADPH oxidase (Nox) was determined by RT-PCR. The effect of PCI34051 on vasorelaxation was studied in rat aortic rings, and its effect on nitric oxide (NO) production was determined using DAF-FM DA, a fluorescent dye, in human umbilical vascular endothelial cells (HUVECs). RESULTS: PCI34051 administration reduced systolic blood pressure via downregulation of angiotensin II receptor type 1 (AT1) mRNA expression. PCI34051 treatment attenuated vascular hypertrophy by decreasing E2F3 and GATA6 mRNA expression. Vascular relaxation after PCI34051 treatment was more dependent on vascular endothelial cells and it was blocked by an NO synthase (NOS) inhibitor. In addition, NO production increased in HUVECs after PCI34051 treatment; this was decreased by the NOS inhibitor. The expression of inflammatory molecules and adhesion molecules VCAM-1 and ICAM-1 decreased in the aortas of angiotensin II-infused mice after PCI34051 administration. However, PCI34051 did not affect Nox or its regulatory subunits. CONCLUSIONS: PCI34051 lowered high blood pressure through modulation of arterial remodeling, vasoconstriction, and inflammation in an angiotensin II-induced hypertension model. We suggest that HDAC8 could be a potential therapeutic target for hypertension.

Class I histone deacetylase inhibitor MS-275 attenuates vasoconstriction and inflammation in angiotensin II-induced hypertension.

OBJECTIVE: Non-selective histone deacetylase (HDAC) inhibitors are known to improve hypertension. Here, we investigated the therapeutic effect and regulatory mechanism of the class I HDAC selective inhibitors, MS-275 and RGFP966, in angiotensin (Ang) II-induced hypertensive mice. METHODS AND RESULTS: MS-275 inhibited the activity of HDAC1, HDAC2, and HDAC3, while RGFP966 weakly inhibited that of HDAC3 in a cell-free system. MS-275 and RGFP966 treatment reduced systolic blood pressure and thickness of the aorta wall in Ang II-induced hypertensive mice. MS-275 treatment reduced aorta collagen deposition, as determined by Masson's trichrome staining. MS-275 decreased the components of the renin angiotensin system and increased vascular relaxation of rat aortic rings via the nitric oxide (NO) pathway. NO levels reduced by Ang II were restored by MS-275 treatment in vascular smooth muscle cells (VSMCs). However, MS-275 dose (3 mg·kg-1·day-1) was not enough to induce NO production in vivo. In addition, MS-275 did not prevent endothelial nitric oxide synthase (eNOS) uncoupling in the aorta of Ang II-induced mice. Treatment with MS-275 failed to inhibit Ang II-induced expression of NADPH oxidase (Nox)1, Nox2, and p47phox. MS-275 treatment reduced proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and monocyte chemoattractant protein (MCP)-1, as well as adhesion molecules. Histological analysis showed that Ang II-induced macrophage infiltration was reduced by MS-275 and RGFP966 administration. CONCLUSIONS: Our results indicate that class I HDAC selective inhibitors may be good therapeutic agents for the treatment of hypertension through the regulation of vascular remodeling and vasoconstriction, as well as inflammation.

Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension.

Here, we report that LMK235, a class I and histone deacetylase (HDAC6)-preferential HDAC inhibitor, reduces hypertension via inhibition of vascular contraction and vessel hypertrophy. Angiotensin II-infusion mice and spontaneously hypertensive rats (SHRs) were used to test the anti-hypertensive effect of LMK235. Daily injection of LMK235 lowered angiotensin II-induced systolic blood pressure (BP). A reduction in systolic BP in SHRs was observed on the second day when SHRs were treated with 3 mg/kg LMK235 every 3 days. However, LMK235 treatment did not affect angiotensin-converting enzyme 1 and angiotensin II receptor mRNA expression in either hypertensive model. LMK235, acting via the nitric oxide pathway, facilitated the relaxing of vascular contractions induced by a thromboxane A2 agonist in the rat aortic and mesenteric artery ring test. In addition, LMK235 increased nitric oxide production in HUVECs and inhibited the increasing of aortic wall thickness in both animal hypertensive models. LMK235 decreased the enhanced cell cycle-related genes cyclin D1 and E2F3 in angiotensin II-infusion mice and restored the decreased p21 expression. In addition, LMK235 suppressed calcium calmodulin-dependent protein kinase II (CaMKII) α, which is related to vascular smooth muscle cell proliferation. Inhibition or knockdown of HDAC5 blocked the CaMKIIα-induced cell cycle gene expression. Immunoprecipitation demonstrated that class I HDACs were involved in the inhibition of CaMKII α-induced HDAC4/5 by LMK235. We suggest that LMK235 should be further investigated for its use in the development of new therapeutic options to treat hypertension via reducing vascular hyperplasia or vasoconstriction.

Repression of Transcriptional Activity of Forkhead Box O1 by Histone Deacetylase Inhibitors Ameliorates Hyperglycemia in Type 2 Diabetic Rats.

Type 2 diabetes mellitus (T2DM) is a chronic disease manifested by hyperglycemia. It is essential to effectively control hyperglycemia to prevent complications of T2DM. Here, we hypothesize that repression of transcriptional activity of forkhead box O1 (FoxO1) via histone deacetylase inhibitors (HDACi) ameliorates hyperglycemia in T2DM rats. METHODS: Male Long-Evans Tokushima Otsuka (LETO) and Otsuka Long-Evans Tokushima Fatty (OLETF) rats aged 14 weeks were administered sodium valproate (VPA, 0.71% w/v) dissolved in water for 20 weeks. Electrophoretic mobility shift assay (EMSA) and luciferase assay were performed for elucidation of transcriptional regulation through acetylation of FoxO1 by HDACi. RESULTS: VPA attenuated blood glucose levels in accordance with a decrease in the expression of gluconeogenic genes in hyperglycemic OLETF rats. It has been shown that HDAC class I-specific and HDAC class IIa-specific inhibitors, as well as pan-HDAC inhibitors decrease FoxO1 enrichment at the cis-element of target gene promoters. Mutations in FoxO1 prevent its acetylation, thereby increasing its transcriptional activity. HDAC3 and HDAC4 interact with FoxO1, and knockdown of HDAC3, HDAC4, or their combination increases FoxO1 acetylation, thereby decreasing the expression of gluconeogenic genes. CONCLUSIONS: These results indicate that HDACi attenuates the transcriptional activity of FoxO1 by impeding deacetylation, thereby ameliorating hyperglycemia in T2DM rats.

Gallic Acid Reduces Blood Pressure and Attenuates Oxidative Stress and Cardiac Hypertrophy in Spontaneously Hypertensive Rats.

Gallic acid (GA) has been reported to have beneficial effects on cancer, vascular calcification, and diabetes-induced myocardial dysfunction. We hypothesized that GA controls hypertension via oxidative stress response regulation in an animal model for essential hypertension. Spontaneously hypertensive rats (SHRs) were administered GA for 16 weeks. GA treatment lowered elevated systolic blood pressure in SHRs through the inhibition of vascular contractility and components of the renin-angiotensin II system. In addition, GA administration reduced aortic wall thickness and body weight in SHRs. In SHRs, GA attenuated left ventricular hypertrophy and reduced the expression of cardiac-specific transcription factors. NADPH oxidase 2 (Nox2) and GATA4 mRNA expression was induced in SHR hearts and angiotensin II-treated H9c2 cells; this expression was downregulated by GA treatment. Nox2 promoter activity was increased by the synergistic action of GATA4 and Nkx2-5. GA seems to regulate oxidative stress by inhibiting the DNA binding activity of GATA4 in the rat Nox2 promoter. GA reduced the GATA4-induced Nox activity in SHRs and angiotensin II-treated H9c2 cells. GA administration reduced the elevation of malondialdehyde levels in heart tissue obtained from SHRs. These findings suggest that GA is a potential therapeutic agent for treating cardiac hypertrophy and oxidative stress in SHRs.

Activation of cardiac renin-angiotensin system and plasminogen activator inhibitor-1 gene expressions in oral contraceptive-induced cardiometabolic disorder.

CONTEXT: Clinical studies have shown that combined oral contraceptive (COC) use is associated with cardiometabolic disturbances. Elevated renin-angiotensin system (RAS) and plasminogen activator inhibitor-1 (PAI-1) have also been implicated in the development of cardiometabolic events. OBJECTIVE: To determine the effect of COC treatment on cardiac RAS and PAI-1 gene expressions, and whether the effect is circulating aldosterone or corticosterone dependent. METHODS: Female rats were treated (p.o.) with olive oil (vehicle) or COC (1.0 µg ethinylestradiol and 10.0 µg norgestrel) daily for six weeks. RESULTS: COC treatment led to increases in blood pressure, HOMA-IR, Ace1 mRNA, Atr1 mRNA, Pai1 mRNA, cardiac PAI-1, plasma PAI-1, C-reactive protein, uric acid, insulin and corticosterone. COC treatment also led to dyslipidemia, decreased glucose tolerance and plasma 17ß-estradiol. CONCLUSION: These results demonstrates that hypertension and insulin resistance induced by COC is associated with increased cardiac RAS and PAI-1 gene expression, which is likely to be through corticosterone-dependent but not aldosterone-dependent mechanism.

Combined oral contraceptive-induced hypertension is accompanied by endothelial dysfunction and upregulated intrarenal angiotensin II type 1 receptor gene expression.

Combined oral contraceptive (COC) use is associated with increased risk of developing hypertension. Activation of the intrarenal renin-angiotensin system (RAS) and endothelial dysfunction play an important role in the development of hypertension. We tested the hypothesis that COC causes hypertension that is associated with endothelial dysfunction and upregulation of intrarenal angiotensin-converting enzyme 1 (Ace1) and angiotensin II type 1 receptor (At1r). Female Sprague-Dawley rats aged 12 weeks received (p.o.) olive oil (control) and a combination of 0.1 µg ethinylestradiol and 1.0 µg norgestrel (low COC) or 1.0 µg ethinylestradiol and 10.0 µg norgestrel (high COC) daily for 6 weeks. Blood pressure was recorded by tail cuff plethysmography. Expression of genes in kidney cortex was determined by quantitative real-time polymerase chain reaction. COC treatment led to increased blood pressure, circulating uric acid, C-reactive protein and plasminogen activator inhibitor-1, renal uric acid, and expression of renal Ace1 and At1r. COC treatment resulted in increased contractile responses to phenylephrine in endothelium-denuded aortic rings. Endothelium-dependent relaxation responses to acetylcholine, but not endothelium-independent relaxation responses to nitric oxide (NO) donation by sodium nitroprusside, were attenuated in COC-exposed rings. Impaired relaxation responses to acetylcholine were masked by the presence of NO synthase inhibitor (L-NAME) in the COC-exposed rings, whereas the responses to acetylcholine in the presence of selective cyclooxygenase-2 inhibitor (NS-398) were enhanced. These findings indicate that COC induces hypertension that is accompanied by endothelial dysfunction, upregulated intrarenal Ace1 and At1r expression, and elevated proinflammatory biomarkers.

Lysine deacetylase inhibition attenuates hypertension and is accompanied by acetylation of mineralocorticoid receptor instead of histone acetylation in spontaneously hypertensive rats.

Inhibition of lysine deacetylase (KDAC) attenuated development of hypertension in spontaneously hypertensive rats (SHRs). We hypothesized that KDAC inhibition attenuates hypertension and is accompanied by acetylation of mineralocorticoid receptors (MR) instead of histone acetylation in SHRs. Valproate (VPA, 0.71 % wt/vol), an inhibitor of class I KDACs, was administered in drinking water to 7-week-old SHRs and Wistar Kyoto rats for 11 weeks. MR acetylation was determined by immunoprecipitation with anti-MR antibody followed by western blot with anti-acetyl-lysine antibody. Expression levels of acetylated histone H3, KDACs, MR target genes, or MR corepressors in the kidney cortex were measured by using western blot analysis or real-time PCR. Recruitment of MR and RNA polymerase II (Pol II) and histone modifications on promoters of target genes were analyzed by performing a chromatin immunoprecipitation (ChIP) assay. Treatment of SHR with VPA increased MR acetylation without affecting MR expression, which attenuated development of hypertension in SHR VPA decreased expression of KDAC class I but globally increased acetylated histone H3. Although VPA treatment increased histone 3 acetylation (H3Ac) and trimethylation of the fourth lysine (H3K4me3) in the promoter regions of MR target genes, it decreased the expression of target genes as well as recruitment of MR and Pol II. These results suggest that KDAC inhibition attenuates the development of hypertension in SHRs and is accompanied by acetylation of MR that is independent of histone acetylation.

Combined oral contraceptive synergistically activates mineralocorticoid receptor through histone code modifications.

Clinical studies have shown that the use of combined oral contraceptive in pre-menopausal women is associated with fluid retention. However, the molecular mechanism is still elusive. We hypothesized that combined oral contraceptive (COC) ethinyl estradiol (EE) and norgestrel (N) synergistically activates mineralocorticoid receptor (MR) through histone code modifications. Twelve-week-old female Sprague-Dawley rats were treated with olive oil (control), a combination of 0.1µg EE and 1.0µg N (low COC) or 1.0µg EE and 10.0µg N (high COC) as well as 0.1 or 1.0µg EE and 1.0 or 10.0µg N daily for 6 weeks. Expression of MR target genes in kidney cortex was determined by quantitative real-time polymerase chain reaction. MR was quantified by western blot. Recruitment of MR and RNA polymerase II (Pol II) on promoters of target genes as well as histone code modifications was analyzed by chromatin immunoprecipitation assay. Treatment with COC increased renal cortical expression of MR target genes such as serum and glucocorticoid-regulated kinase 1 (Sgk-1), glucocorticoid-induced leucine zipper (Gilz), epithelial Na(+)channel (Enac) and Na(+)-K(+)-ATPase subunit α1 (Atp1a1). Although COC increased neither serum aldosterone nor MR expression in kidney cortex, it increased recruitment of MR and Pol II in parallel with increased H3Ac and H3K4me3 on the promoter regions of MR target genes. However, treatment with EE or N alone did not affect renal cortical expression of Sgk-1, Gilz, Enac or Atp1a1. These results indicate that COC synergistically activates MR through histone code modifications.

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.

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.

Lysine acetyltransferases cyclic adenosine monophosphate response element-binding binding protein and acetyltransferase p300 attenuate transcriptional activity of the mineralocorticoid receptor through its acetylation.

Acetylation of the mineralocorticoid receptor (MR) by inhibition of lysine deacetylases attenuates MR's transcriptional activity. However, the specific lysine acetyltransferases that are responsible for acetylation of the MR remain unknown. We hypothesized that the acetyltransferases cyclic adenosine monophosphate response element-binding binding protein (CBP) and acetyltransferase p300 (p300) attenuate transcriptional activity of the MR through its acetylation. Expression of MR target genes was measured by quantitative real-time polymerase chain reaction. Recruitment of MR and RNA polymerase II (Pol II) on promoters of target genes was analysed by chromatin immunoprecipitation. Acetylation of the MR was determined by western blot with an anti-acetyl-lysine antibody after immunoprecipitation with an anti-MR antibody. In human embryonic kidney (HEK) 293 cells, overexpression of CBP or p300, but not p300/CBP-associated factor, increased MR acetylation and decreased expression of MR target genes. The downregulation of target genes coincided with a decrease in the recruitment of MR and Pol II to specific hormone response elements. These results demonstrate that overexpression of CBP or p300 attenuates the transcriptional activity of the MR through its acetylation in HEK 293 cells. Our data provide strong evidence identifying CBP and p300 as lysine acetyltransferases responsible for the regulation of MR that may provide new therapeutic targets for the treatment of hypertension.

GPER-1 agonist G1 induces vasorelaxation through activation of epidermal growth factor receptor-dependent signalling pathway.

OBJECTIVES: The G protein-coupled oestrogen receptor-1 (GPER-1) agonist G1 induces endothelium-dependent relaxation. Activation of the epidermal growth factor (EGF) receptor leads to transduction of signals from the plasma membrane for the release of nitric oxide. We tested the hypothesis that G1 induces endothelium-dependent vasorelaxation through activation of the EGF receptor. METHODS: Rat aortic rings were mounted in organ baths. After pretreatment with various inhibitors, aortic rings contracted with 11,9-epoxymethano-prostaglandin F2α or KCl were subjected to relaxation by G1. KEY FINDINGS: G1 induced endothelium-dependent vasorelaxation, which was attenuated by pretreatment with either L -N(ω) -nitroarginine methyl ester (L -NAME), an inhibitor of nitric oxide synthase, or (3aS,4R,9bR)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline HB-EGF, heparin-binding EGF-like growth factor, a GPER-1 antagonist. Neither a general oestrogen receptor antagonist, ICI 182 780, nor a selective oestrogen receptor-α antagonist, methyl-piperidino-pyrazole dihydrochloride (MPP), had an effect on G1-induced vasorelaxation. However, pretreatment with EGF receptor blockers, AG1478 or DAPH, resulted in attenuated G1-induced vasorelaxation. In addition, pretreatment with Src inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or Akt inhibitor VIII also resulted in attenuated vascular relaxation induced by the cumulative addition of G1. However, neither phosphatidylinositol-3 kinase inhibitors LY294002 and wortmannin nor an extracellular signal-regulated kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene monoethanolate had effect on vascular relaxation induced by the cumulative addition of G1. CONCLUSIONS: G1 induces endothelium-dependent vasorelaxation through Src-mediated activation of the EGF receptor and the Akt pathway in rat aorta.

3',4'-Dimethoxythioflavone induces endothelium-dependent vasorelaxation through activation of epidermal growth factor receptor.

It is of interest to investigate whether synthetic thioflavonoids have vasorelaxant actions as natural flavonoids. We tested the hypothesis that 3',4'-dimethoxythioflavone induces endothelium-dependent vasorelaxation through activation of epidermal growth factor (EGF) receptor. Rat aortic rings were mounted in organ baths and subjected to relaxation upon contraction. 3',4'-Dimethoxythioflavone induced endothelium-dependent vasorelaxation, which was attenuated by pretreatment with either L-N (ω)-nitroarginine methyl ester, an inhibitor of nitric oxide synthase, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. 3',4'-Dimethoxythioflavone-induced vasorelaxation was not affected by pretreatment with a general estrogen receptor antagonist ICI 182,780, a selective estrogen receptor-α antagonist methyl-piperidino-pyrazole dihydrochloride, or a G protein-coupled receptor 30 antagonist G15. However, pretreatment with EGF receptor blockers AG1478 or DAPH, but not with a phosphatidylinositol-3 kinase inhibitor LY294002 or an Akt1/2 kinase inhibitor Akt inhibitor VIII, attenuated 3',4'-dimethoxythioflavone-induced vasorelaxation. In addition, pretreatment with a Src inhibitor PP2 or an ERK inhibitor U0126 also attenuated vascular relaxation induced by the cumulative addition of 3',4'-dimethoxythioflavone. However, neither a mitochondrial electron transport inhibitor rotenone, an NADPH oxidase inhibitor apocynin, nor a superoxide dismutase mimetic MnTMPyP affected the vascular relaxation induced by the cumulative addition of 3',4'-dimethoxythioflavone. In conclusion, 3',4'-dimethoxythioflavone induces endothelium-dependent vasorelaxation through activation of EGF receptor and Src/ERK pathway in rat aorta.

17ß-Estradiol induces vasorelaxation in a G-protein-coupled receptor 30-independent manner.

17ß-Estradiol (E2) exerts rapid non-genomic vascular effects through activation of its plasma membrane receptors. We tested the hypothesis that E2 induces vasorelaxation through activation of the G-protein-coupled receptor 30 (GPR30) in rat aorta. Rat aortic rings were mounted in organ baths and subjected to contraction followed by relaxation. Whether endothelium was intact or denuded, both E2 and G1, a GPR30 agonist, induced vasorelaxation in concentration-dependent manners. Although G15, a specific GPR30 antagonist, blocked G1-induced vasorelaxation, it did not block E2-induced vasorelaxation. In conclusion, 17ß-estradiol induces vasorelaxation in a GPR30-independent manner in rat aorta.

Upregulation of the Na(+)-K(+)-2Cl(-) cotransporter 1 via histone modification in the aortas of angiotensin II-induced hypertensive rats.

The Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) is upregulated in diverse models of hypertension. We hypothesized that NKCC1 is upregulated via histone modification in the aortas of angiotensin II (Ang II)-induced hypertensive rats. An osmotic mini-pump containing Ang II was implanted in the subcutaneous tissues of the backs of Sprague-Dawley (SD) rats for 7 days. The systolic blood pressure was recorded every day by the tail-cuff method. On days 3 and 7, the mesenteric arteries were excised, cut into rings, mounted in organ baths and subjected to vascular contraction. The levels of Nkcc1 mRNA and protein in the aortas were measured using real-time PCR and Western blotting, respectively. The histone modifications and recruited proteins at the Nkcc1 promoter were determined by chromatin immunoprecipitation. The inhibition of concentration-response curves to phenylephrine by bumetanide, an inhibitor of NKCCs, was greater in Ang II-infused rats than in sham-operated (sham) rats . The levels of Nkcc1 mRNA and protein in the aortas increased gradually as Ang II was infused into the rats. Acetylated histone H3 (H3Ac), an activating histone code, was increased but trimethylated histone H3 at lysine 27 (H3K27me3), a repressive histone code, was greatly decreased in Ang II-infused rats compared with sham. RNA polymerase II was recruited to the Nkcc1 promoter with increased KDM6b. We conclude that the NKCC1 is upregulated via histone modification in the aortas of Ang II-induced hypertensive rats. Thus, we suggest that this ion transporter is epigenetically upregulated by histone modification or DNA demethylation upon the development of hypertension.