Hydrogen Sulfide Mitigates Cardiac Remodeling During Myocardial Infarction via Improvement of Angiogenesis

Exogenous hydrogen sulfide (H2S) leads to down-regulation of inflammatory responses and provides myocardial protection during acute ischemia/reperfusion injury; however its role during chronic heart failure (CHF) due to myocardial infarction (MI) is yet to be unveiled. We previously reported that H2S inhibits antiangiogenic factors such, as endostatin and angiostatin, but a little is known about its effect on parstatin (a fragment of proteinase-activated receptor-1, PAR-1). We hypothesize that H2S inhibits parstatin formation and promotes VEGF activation, thus promoting angiogenesis and significantly limiting the extent of MI injury. To verify this hypothesis MI was created in 12 week-old male mice by ligation of left anterior descending artery (LAD). Sham surgery was performed except LAD ligation. After the surgery mice were treated with sodium hydrogen sulfide (30 μmol/l NaHS, a donor for H2S, in drinking water) for 4 weeks. The LV tissue was analyzed for VEGF, flk-1 and flt-1, endostatin, angiostatin and parstatin. The expression of VEGF, flk-1 and flt-1 were significantly increased in treated mice while the level of endostatin, angiostatin and parstatin were decreased compared to in untreated mice. The echocardiography in mice treated with H2S showed the improvement of heart function compared to in untreated mice. The X-ray and Doppler blood flow measurements showed enhancement of cardiac-angiogenesis in mice treated with H2S. This observed cytoprotection was associated with an inhibition of anti-angiogenic proteins and stimulation of angiogenic factors. We established that administration of H2S at the time of MI ameliorated infarct size and preserved LV function during development of MI in mice. These results suggest that H2S is cytoprotective and angioprotective during evolution of MI

Mitochondrial division/mitophagy inhibitor (Mdivi) Ameliorates Pressure Overload Induced Heart Failure

Background: We have previously reported the role of anti-angiogenic factors in inducing the transition from compensatory cardiac hypertrophy to heart failure and the significance of MMP-9 and TIMP-3 in promoting this process during pressure overload hemodynamic stress. Several studies reported the evidence of cardiac autophagy, involving removal of cellular organelles like mitochondria (mitophagy), peroxisomes etc., in the pathogenesis of heart failure. However, little is known regarding the therapeutic role of mitochondrial division inhibitor (Mdivi) in the pressure overload induced heart failure. We hypothesize that treatment with mitochondrial division inhibitor (Mdivi) inhibits abnormal mitophagy in a pressure overload heart and thus ameliorates heart failure condition. Materials and Methods: To verify this, ascending aortic banding was done in wild type mice to create pressure overload induced heart failure and then treated with Mdivi and compared with vehicle treated controls. Results: Expression of MMP-2, vascular endothelial growth factor, CD31, was increased, while expression of anti angiogenic factors like endostatin and angiostatin along with MMP-9, TIMP-3 was reduced in Mdivi treated AB 8 weeks mice compared to vehicle treated controls. Expression of mitophagy markers like LC3 and p62 was decreased in Mdivi treated mice compared to controls. Cardiac functional status assessed by echocardiography showed improvement and there is also a decrease in the deposition of fibrosis in Mdivi treated mice compared to controls

Folic acid mitigates angiotensin-II-induced blood pressure and renal remodeling.

Clinical data suggests an association between systolic hypertension, renal function and hyperhomocysteinemia (HHcy). HHcy is a state of elevated plasma homocysteine (Hcy) levels and is known to cause vascular complications. In this study, we tested the hypothesis whether Ang II-induced hypertension increases plasma Hcy levels and contributes to renovascular remodeling. We also tested whether folic acid (FA) treatment reduces plasma Hcy levels by enhancing Hcy remethylation and thus mitigating renal remodeling. Hypertension was induced in WT mice by infusing Ang II using Alzet mini osmotic pumps. Blood pressure, Hcy level, renal vascular density, oxidative stress, inflammation and fibrosis markers, and angiogenic- and anti-angiogenic factors were measured. Ang II hypertension increased plasma Hcy levels and reduced renal cortical blood flow and microvascular density. Elevated Hcy in Ang II hypertension was associated with decreased 4, 5-Diaminofluorescein (DAF-2DA) staining suggesting impaired endothelial function. Increased expression of Nox-2, -4 and dihydroethidium stain revealed oxidative stress. Excess collagen IV deposition in the peri-glomerular area and increased MMP-2, and -9 expression and activity indicated renal remodeling. The mRNA and protein expression of asymmetric dimethylarginine (ADMA) was increased and eNOS protein was decreased suggesting the involvement of this pathway in Hcy mediated hypertension. Decreased expressions of VEGF and increased anti-angiogenic factors, angiostatin and endostatin indicated impaired vasculogenesis. FA treatment partially reduced hypertension by mitigating HHcy in Ang II-treated animals and alleviated pro-inflammatory, pro-fibrotic and anti-angiogenic factors. These results suggest that renovascular remodeling in Ang II-induced hypertension is, in part, due to HHcy

Folic acid (FA) reduced blood pressure and plasma Hcy levels in Ang II-induced hypertension.

<p>(<b>A</b>) Ang II was infused using alzet mini pump (1000 ng/kg/min) for 4 weeks and blood pressure was measured by radiotelemetry as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083813#s2" target="_blank">Materials and Methods</a>. Folic acid (0.015 g/L) was given in drinking water 14 days after pump insertion and continued till the end of the experiment; n = 8 animals/group. * p<0.05 between Ang II + FA vs. Ang II. (<b>B</b>) Plasma Hcy was measured by high performance liquid chromatography (HPLC) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083813#s2" target="_blank">Materials and Methods</a>. Data was first analyzed with ANOVA and pairwise comparison was performed using Bonferroni method. Data is presented as mean ± SEM, n = 6 mice/group. * p<0.05 vs. vehicle (saline) and, ^† p<0.05 vs. Ang II (1000 ng/kg/min), ^‡ p = 0.05 vs. Vehicle.</p

Folic acid treatment increased renal cortical blood flow and vascular density in Ang II infused kidney.

<p>(<b>A</b>) Renal cortical blood flow was measured at end-point using Speckle contrast Imager (MoorFLPI, Wilmington, DE). Animals were anesthetized with TBE (Tribromoethanol, 240 mg/kg b.w. i.p.) and the left kidney exposed. All measurements were done under standard conditions of light and temperature control. (<b>B</b>) Data was first analyzed with ANOVA and pairwise comparison was performed using Bonferroni method. Summarized bar diagram represents mean ± SEM, n = 5–6 animals/group. * p<0.05 vs. vehicle and ^†p<0.05 vs. Ang II. (<b>C</b>) Mice were infused with Barium sulfate (100 mg/ml, at pH 5.0) through PE10 catheter (ID -0.28 MM, Franklin Lakes, NJ) inserted in the carotid artery directed towards the aorta and a constant rate of 200 µL/min was injected. Two minute X-ray images were captured with Kodak 4000 MM image station (Molecular Imaging System; Carestream Health Inc., Rochester, NY). Image analyses were done by ImagePro software (a representative analysis image is shown at the bottom right). Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. Bar diagram indicates percent change of vascular density against the background using vehicle treatment as control, n = 3 mice/group. * p<0.05 vs. vehicle; ^† p<0.05 vs. Ang II.</p

Hydrogen Sulfide Promotes Bone Homeostasis by Balancing Inflammatory Cytokine Signaling in CBS-Deficient Mice through an Epigenetic Mechanism

Abstract Previously, we have shown hyperhomocysteinemia (HHcy) to have a detrimental effect on bone remodeling, which is associated with osteoporosis. During transsulfuration, Hcy is metabolized into hydrogen sulfide (H2S), a gasotransmitter molecule known to regulate bone formation. Therefore, in the present study, we examined whether H2S ameliorates HHcy induced epigenetic and molecular alterations leading to osteoporotic bone loss. To test this mechanism, we employed cystathionine-beta-synthase heterozygote knockout mice, fed with a methionine rich diet (CBS+/− +Met), supplemented with H2S-donor NaHS for 8 weeks. Treatment with NaHS, normalizes plasma H2S, and completely prevents trabecular bone loss in CBS+/− mice. Our data showed that HHcy caused inhibition of HDAC3 activity and subsequent inflammation by imbalancing redox homeostasis. The mechanistic study revealed that inflammatory cytokines (IL-6, TNF-α) are transcriptionally activated by an acetylated lysine residue in histone (H3K27ac) of chromatin by binding to its promoter and subsequently regulating gene expression. A blockade of HDAC3 inhibition in CBS+/− mice by HDAC activator ITSA-1, led to the remodeling of histone landscapes in the genome and thereby attenuated histone acetylation-dependent inflammatory signaling. We also confirmed that RUNX2 was sulfhydrated by administration of NaHS. Collectively, restoration of H2S may provide a novel treatment for CBS-deficiency induced metabolic osteoporosis

FA mitigated glomerulosclerosis and collagen IV expression in Ang II hypertension.

<p>(<b>A</b>) Histological kidney sections (5 µm thick) were stained with Masson's trichrome. Collagen is shown as deep blue color (yellow arrows); 200× magnification. (<b>B</b>) Collagen IV was detected in the cortical tissue extracted protein by Western blot. (<b>C</b>) Bar diagram represents densitometric analysis of collagen IV expression as fold change against vehicle group, n = 5–6 group. Statistical analyses were performed by Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. * p<0.05 vs. vehicle and, ^† p<0.05 vs. Ang II.</p

FA treatment reduces ROS production in Ang II hypertension by decreasing Nox-2 and Nox-4 isoforms.

<p>(<b>A</b>) Equal amount of protein from each group was separated on a SDS-PAGE and immunoblotted with anti-Nox-2, and -4 antibodies. (<b>B</b>) The pixel densities of bands from n = 6/group were quantified using ImageJ software (National Institute of Health, NIH) and presented as fold change using β-actin as control. Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. * p<0.05 vs. vehicle; p<0.05 vs. Ang II. (<b>C</b>) Superoxide was detected in the glomerulus by dihydroethidium (DHE) staining as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083813#s2" target="_blank">Materials and Methods</a>. Scale 20 µm.</p

mRNA and protein expression of CBS/CSE and MTHFR is decreased in Ang II induced hypertension

<p>Effect of vehicle, Ang II and FA on the mRNA and protein expression of CBS (<b>A</b>); CSE (<b>C</b>) and MTHFR (<b>E</b>) as determined by semiquantitative RT-PCR and Western blotting. Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. Bar diagrams represent fold change from n = 6 experiments using GAPDH as control. * p<0.05 vs. vehicle; ^† p<0.05 vs. Ang II, ^‡ p<0.05 vs. vehicle.</p