Chronic kidney disease activates the HDAC6-inflammatory axis in the heart and contributes to myocardial remodeling in mice: inhibition of HDAC6 alleviates chronic kidney disease-induced myocardial remodeling.

Chronic kidney disease (CKD) adversely affects the heart. The underlying mechanism and the interplay between the kidney and the heart are still obscure. We examined the cardiac effect using the unilateral ureteral obstruction (UUO)-induced CKD pre-clinical model in mice. Echocardiography, histopathology of the heart, myocardial mRNA expression of ANP and BNP, the extent of fibrotic (TGF-ß, α-SMA, and collagen I) and epigenetic (histone deacetylases, namely HDAC3, HDAC4, and HDAC6) proteins, and myocardial inflammatory response were assessed. Six weeks of post-UUO surgery, we observed a compromised left-ventricular wall thickness and signs of cardiac hypertrophy, accumulation of fibrosis associated, and inflammatory proteins in the heart. In addition, we observed a perturbation of epigenetic proteins, especially HDAC3, HDAC4, and HDAC6, in the heart. Pharmacological inhibition of HDAC6 using ricolinostat (RIC) lessened cardiac damage and improved left-ventricular wall thickness. The RIC treatment substantially restored the serum cardiac injury markers, namely creatine kinase-MB and lactate dehydrogenase (LDH) activities, ANP and BNP mRNA expression, and heart histological changes. The extent of myocardial fibrotic proteins, phospho-NF-κB (p65), and pro-inflammatory cytokines (TNF-α, IL-18, and IL-1ß) were significantly decreased in the RIC treatment group. Further findings revealed the CKD-induced infiltration of CD3, CD8a, CD11c, and F4/80 positive inflammatory cells in the heart. Treatment with RIC substantially reduced the myocardial infiltration of these inflammatory cells. From these findings, we believe that CKD-induced myocardial HDAC6 perturbation has a deteriorative effect on the heart, and inhibition of HDAC6 can be a promising approach to alleviate CKD-induced myocardial remodeling.

Intranasal Delivery of Carvedilol- and Quercetin-Encapsulated Cationic Nanoliposomes for Cardiovascular Targeting: Formulation and In Vitro and Ex Vivo Studies.

Carvedilol (CVD), an adrenoreceptor blocker, is a hydrophobic Biopharmaceutics Classification System class II drug with poor oral bioavailability due to which frequent dosing is essential to attain pharmacological effects. Quercetin (QC), a polyphenolic compound, is a potent natural antioxidant, but its oral dosing is restricted due to poor aqueous solubility and low oral bioavailability. To overcome the common limitations of both drugs and to attain synergistic cardioprotective effects, we formulated CVD- and QC-encapsulated cationic nanoliposomes (NLPs) in situ gel (CVD/QC-L.O.F.) for intranasal administration. We designed CVD- and QC-loaded cationic nanoliposomal (NLPs) in situ gel (CVD/QC-L.O.F.) for intranasal administration. In vitro drug release studies of CVD/QC-L.O.F. (16.25%) exhibited 18.78 ± 0.57% of QC release and 91.38 ± 0.93% of CVD release for 120 h. Ex vivo nasal permeation studies of CVD/QC-L.O.F. demonstrated better permeation of QC (within 96 h), i.e., 75.09% compared to in vitro drug release, whereas CVD permeates within 48 h, indicating the better interaction between cationic NLPs and the negatively charged biological membrane. The developed nasal gel showed a sufficient mucoadhesive property, good spreadability, higher firmness, consistency, and cohesiveness, indicating suitability for membrane application and intranasal administration. CVD-NLPs, QC-NLPs, and CVD/QC-NLPs were evaluated for in vitro cytotoxicity, in vitro ROS-induced cell viability assessment, and a cellular uptake study using H9c2 rat cardiomyocytes. The highest in vitro cellular uptake of CVD/QC-cationic NLPs by H9c2 cells implies the benefit of QC loading within the CVD nanoliposomal carrier system and gives evidence for better interaction of NLPs carrying positive charges with the negatively charged biological cells. The in vitro H2O2-induced oxidative stress cell viability assessment of H9c2 cells established the intracellular antioxidant activity and cardioprotective effect of CVD/QC-cationic NLPs with low cytotoxicity. These findings suggest the potential of cationic NLPs as a suitable drug delivery carrier for CVD and QC combination for the intranasal route in the treatment of various cardiovascular diseases like hypertension, angina pectoris, etc. and for treating neurodegenerative disorders.

Total Synthesis of Diterpenoid Quinone Methide Tumor Inhibitor, (+)-Taxodione.

An asymmetric polyene cyclization (92% ee) strategy has been successfully applied for the first asymmetric total synthesis of oxidized abietane, anticancer agent, taxodione (1) sharing a trans-decalin system. Additionally, the total syntheses of pomiferin B (2) and gaultheric acid (3) (a nor-abietane) were achieved utilizing this unified approach.

Description of an In Vitro Platelet Function Analyzer-PFA-100™.

This manuscript represents a republication of a manuscript originally published in STH in 1995. This republication is to help celebrate 50 years of publishing for STH. The original abstract follows.A new in vitro system for the detection of platelet dysfunction, PFA-100®, has been developed. It provides a quantitative measure of platelet function in anticoagulated whole blood. The system comprises a microprocessor-controlled instrument and a disposable test cartridge containing a biologically active membrane. The instrument aspirates a blood sample under constant vacuum from the sample reservoir through a capillary and a microscopic aperture cut into the membrane. The membrane is coated with collagen and epinephrine or adenosine 5'-diphosphate. The presence of these biochemical stimuli, and the high shear rates generated under the standardized flow conditions, result in platelet attachment, activation, and aggregation, slowly building a stable platelet plug at the aperture. The time required to obtain full occlusion of the aperture is reported as the "closure time." We have found that impairment of von Willebrand factor, or inhibition of platelet receptors glycoprotein Ib or IIb/IIIa with monoclonal antibodies or peptides, resulted in abnormal closure times. An antifibrinogen antibody, in contrast, failed to show any effect. The test appears to be sensitive to platelet adherence and aggregation abnormalities. The PFA-100® system has potential applications in routine evaluation of platelet function in the clinical setting because of its accuracy, case of operation, and rapid turnaround of results.

Anti-inflammatory activity of carvacrol protects the heart from lipopolysaccharide-induced cardiac dysfunction by inhibiting pyroptosis via NLRP3/Caspase1/Gasdermin D signaling axis.

AIMS: Lipopolysaccharide (LPS) is a well-known agent to induce septic conditions. Sepsis-induced cardiomyopathy has an overwhelming death rate. Carvacrol (CVL), a monoterpene phenol, has anti-inflammatory and antioxidant properties. This research aimed to investigate the effect of CVL on LPS-induced dysfunction in the heart. In this study, we evaluated the effect of CVL in LPS-stimulated H9c2 cardiomyoblast cells and Balb/C mice. MAIN METHODS: LPS was used to induce septic conditions in H9c2 cardiomyoblast cells in vitro and in Balb/C mice. A survival study was conducted to assess the survival rate of mice after LPS and/or CVL treatment. KEY FINDINGS: In vitro studies indicated that CVL inhibits reactive oxygen species (ROS) generation and abates pyroptosis mediated by NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in H9c2 cells. In mice, CVL intervention improved the survival rate in septic conditions. The CVL administration markedly improved the echocardiographic parameters and alleviated the LPS-induced reduction in the ejection fraction (%) and fraction shortening (%). The CVL intervention restored the myocardial antioxidants and histopathological alterations and decreased the pro-inflammatory cytokine contents in the heart. Further findings disclosed that CVL reduced the protein levels of NLRP3, apoptosis-associated speck-like protein (ASC), caspase 1, interleukin (IL)-18, IL-1ß, and the pyroptosis-indicative protein, gasdermin-D (GSDMD) in the heart. The autophagy-indicative proteins, beclin 1 and p62 in the heart were also restored in the CVL-treated group. SIGNIFICANCE: Altogether, our findings demonstrated that CVL has a beneficial effect and can be a potential molecule against sepsis-induced myocardial dysfunction.

Alternanthera brasiliana L. extract alleviates carbon tetrachloride-induced liver injury and fibrotic changes in mice: Role of matrix metalloproteinases and TGF-ß/Smad axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Alternanthera brasiliana L. is a flowering plant belonging to the family Amaranthaceae and is popularly known as "penicillin". It is used in folk medicine to treat infections, coughs, wound healing, and inflammatory diseases. AIM OF THE STUDY: We investigated the effect of Alternanthera brasiliana L. leaves hydroalcoholic extract (AB) against oxidative stress, inflammation, and fibrotic changes in an experimental model of carbon tetrachloride (CCl4)-induced liver injury and fibrosis in mice. MATERIALS AND METHODS: Thirty-six male Balb/C mice were randomized into five groups: normal control, AB control, CCl4 control, CCl4 + AB-200 mg/kg, and CCl4 + AB-400 mg/kg. In mice, liver injury was induced by intraperitoneal injection of CCl4 (20% in corn oil, 5 ml/kg body weight) thrice a week for six consecutive weeks. AB extract at two doses (200 mg/kg and 400 mg/kg body weight) was administered orally for six consecutive weeks. Liver injury-related serum markers (ALT, AST, ALP), antioxidants (GSH, GST, SOD, and vitamin C), pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-18, ultrasonographic and histological alterations, proteins of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase-1 (TIMP-1), nuclear factor-κB (p65) (NF-κB), nod-like receptor protein 3 (NLRP3), and TGF-ß/Smad signaling were accessed. LC-Q-TOF-MS/MS analysis of AB was performed. RESULTS: AB treatment significantly decreased the CCl4-induced rise in serum ALT, AST, and ALP activities and improved the histological alterations. Compared with the CCl4-treated group, treatment with AB significantly restored the hepatic antioxidants and reduced the pro-inflammatory cytokines in the liver. The antioxidant activity of AB may be attributed to its terpenoid constituents, which was confirmed by LC-Q-TOF-MS/MS analysis. The CCl4-induced rise in expression of MMP-2 and MMP-9 and decrease in TIMP-1 were markedly restored in the AB-treated groups. Further findings revealed a significant reduction in the protein levels of phospho-NF-κB (p65), NLRP3, TGF-ß, pSmad2/3, collagen I, and α-smooth muscle actin (α-SMA) in the AB treatment groups. CONCLUSIONS: The hepatoprotective effect of AB may be attributed to the high content of terpenoid compounds and alleviates liver injury and associated fibrotic changes through modulating MMPs, NF-κB (p65), and the TGF-ß/Smad axis.

Nuciferine alleviates intestinal inflammation by inhibiting MAPK/NF-κB and NLRP3/Caspase 1 pathways in vivo and in vitro.

Nuciferine (NCF) is an aporphine alkaloid and a principal bioactive constituent in the lotus plant. Herewith, we investigated the potential anti-inflammatory effect and underlying mechanisms of NCF employing dextran sulfate sodium (DSS)-induced ulcerative colitis in mice, a predominant intestinal inflammatory disease, and mouse RAW 264.7 cells in vitro. Lipopolysaccharide (LPS) was used to generate an inflammatory response in the RAW 264.7 cells. The disease activity index (DAI), colon morphology, colonoscopy, and colon histopathology were performed to assess experimental colitis. The biochemical assays, enzyme-linked immunosorbent assay (ELISA), and immunoblot analysis were performed to understand the underlying mechanisms. In RAW 264.7 cells, NCF pretreatment significantly decreased the expression of inducible nitric oxide synthase (iNOS), the expression and release of pro-inflammatory cytokines including interleukin (IL)-1ß, IL-18, and tumor necrosis factor-α (TNF-α) and interfered with the activation of mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and NOD-like family pyrin domain containing 3 (NLRP3) signaling pathways. The oral treatment of NCF substantially alleviated the DSS-induced DAI, increased colon length, and restored colon morphology and histology. Compared to the DSS-induced mice, the proteins involved in the activation of MAPK/NF-κB/NLRP3 pathways and the cytokines were markedly decreased in the NCF-treated mice. Moreover, the tight junction architecture of the colon was well-maintained in NCF treatment groups by regulating the expression of claudin-1 and zonula occludens-1 (ZO-1) proteins. All these findings suggest that NCF can be a promising molecule to modulate ulcerative colitis.

Biological Activities, Pharmacokinetics and Toxicity of Nootkatone: A Review.

Plant-based drugs have a significant impact on modern therapeutics due to their vast array of pharmacological activities. The integration of herbal plants in the current healthcare system has emerged as a new field of research. It can be used for the identification of novel lead compound candidates for future drug development. Nootkatone is a sesquiterpene derivative and an isolate of grapefruit. Shreds of evidence illustrate that nootkatone targets few molecular mechanisms to exhibit its pharmacological activity and yet needs more exploration. The current review is related to nootkatone, drafted through a literature search using research articles and books from different sources, including Science Direct, Google Scholar, Elsevier, PubMed, and Scopus. It has been reported to possess a wide range of pharmacological activities such as anti-inflammatory, anticancer, antibacterial, hepatoprotective, neuroprotective, and cardioprotective. Although preclinical studies in experimental animal models suggest that nootkatone has therapeutic potential, it is further warranted to evaluate its toxicity and pharmacokinetic parameters before being applied to humans. Hence, in the present review, we have summarized the scientific knowledge on nootkatone with a particular emphasis on its pharmacological properties to encourage researchers for further exploration in preclinical and clinical settings.

Exogenous hydrogen sulfide and miR-21 antagonism attenuates macrophage-mediated inflammation in ischemia reperfusion injury of the aged kidney.

Ischemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in the aging population. A reduction of hydrogen sulfide (H2S) production in the old kidney and renal IRI contribute to renal pathology and injury. Recent studies suggest that microRNAs (miRs) play an important role in the pathophysiology of AKI and a significant crosstalk exists between H2S and miRs. Among the miRs, miR-21 is highly expressed in AKI and is reported to have both pathological and protective role. In the present study, we sought to determine the effects of age-induced reduction in H2S and mir-21 antagonism in AKI. Wild type (WT, C57BL/6J) mice aged 12-14 weeks and 75-78 weeks underwent bilateral renal ischemia (27 min) and reperfusion for 7 days and were treated with H2S donor, GYY4137 (GYY, 0.25 mg/kg/day, ip) or locked nucleic acid anti-miR-21 (20 mg/kg b.w., ip) for 7 days. Following IRI, old kidney showed increased macrophage polarization toward M1 inflammatory phenotype, cytokine upregulation, endothelial-mesenchymal transition, and fibrosis compared to young kidney. Treatment with GYY or anti-miR-21 reversed the changes and improved renal vascular density, blood flow, and renal function in the old kidney. Anti-miR-21 treatment in mouse glomerular endothelial cells showed upregulation of H2S-producing enzymes, cystathionine ß-synthase (CBS), and cystathionineγ-lyase (CSE), and reduction of matrix metalloproteinase-9 and collagen IV expression. In conclusion, exogenous H2S and inhibition of miR-21 rescued the old kidney dysfunction due to IRI by increasing H2S levels, reduction of macrophage-mediated injury, and promoting reparative process suggesting a viable approach for aged patients sustaining AKI.

Hydrogen Sulfide Protects Hyperhomocysteinemia-Induced Renal Damage by Modulation of Caveolin and eNOS Interaction.

The accumulation of homocysteine (Hcy) during chronic kidney failure (CKD) can exert toxic effects on the glomeruli and tubulo-interstitial region. Among the potential mechanisms, the formation of highly reactive metabolite, Hcy thiolactone, is known to modify proteins by N-homocysteinylation, leading to protein degradation, stress and impaired function. Previous studies documented impaired nitric oxide production and altered caveolin expression in hyperhomocysteinemia (HHcy), leading to endothelial dysfunction. The aim of this study was to determine whether Hhcy homocysteinylates endothelial nitric oxide synthase (eNOS) and alters caveolin-1 expression to decrease nitric oxide bioavailability, causing hypertension and renal dysfunction. We also examined whether hydrogen sulfide (H2S) could dehomocysteinylate eNOS to protect the kidney. WT and Cystathionine ß-Synthase deficient (CBS+/-) mice representing HHcy were treated without or with sodium hydrogen sulfide (NaHS), a H2S donor (30 µM), in drinking water for 8 weeks. Hhcy mice (CBS+/-) showed low levels of plasma H2S, elevated systolic blood pressure (SBP) and renal dysfunction. H2S treatment reduced SBP and improved renal function. Hhcy was associated with homocysteinylation of eNOS, reduced enzyme activity and upregulation of caveolin-1 expression. Further, Hhcy increased extracellular matrix (ECM) protein deposition and disruption of gap junction proteins, connexins. H2S treatment reversed the changes above and transfection of triple genes producing H2S (CBS, CSE and 3MST) showed reduction of vascular smooth muscle cell proliferation. We conclude that during Hhcy, homocysteinylation of eNOS and disruption of caveolin-mediated regulation leads to ECM remodeling and hypertension, and H2S treatment attenuates renovascular damage.

Estrogen-regulated expression of P450arom genes in the brain and ovaries of adult female Indian climbing perch, Anabas testudineus.

Cytochrome P450arom (CYP19), a product of cyp19a1 gene, catalyzes the conversion of androgens to estrogens and is essential for regulation of reproductive function in vertebrates. In the present study, we isolated partial cDNA encoding the ovarian (cyp19a1a) and brain (cyp19a1b) P450arom genes from adult female perch, Anabas testudineus and investigated their regulation by estrogen in vivo. Results demonstrated that cyp19a1a and cyp19a1b predominate in ovary and brain respectively, with quantity of both attuned to reproductive cycle. To elucidate estrogen-regulated expression of cyp19a1b in brain and cyp19a1a in ovary, dose- and time-dependent studies were conducted with estrogen in vitellogenic-stage fish in the presence or absence of specific aromatase inhibitor fadrozole. Results demonstrated that treatment of fish with 17ß-estradiol (E2; 1.0 µM)) for 6 days caused significant upregulation of cyp19a1b transcripts, aromatase B protein, and aromatase activity in brain in a dose- and time-dependent manner. Ovarian cyp19a1a mRNA, aromatase protein, and aromatase activity, however, was less responsive to E2 than brain. Treatment of fish with an aromatase inhibitor fadrozole for 6 days attenuated both brain and ovarian cyp19a1 mRNAs expression and stimulatory effects of E2 was also significantly reduced. These results indicate that expression of cyp19a1b in brain and cyp19a1a in ovary of adult female A. testudineus was closely associated to plasma E2 levels and seasonal reproductive cycle. Results further show apparent differential regulation of cyp19a1a and cyp19a1b expression by E2/fadrozole manipulation.

Toll-like Receptor 4 Deficiency Reduces Oxidative Stress and Macrophage Mediated Inflammation in Hypertensive Kidney.

Oxidative stress and inflammation are integral to hypertension-induced renal injury. A unifying feature for the two components is Toll-like receptors (TLR), which are key regulators of the innate immune system. Recent studies implicate TLR4 activation and oxidative stress in cardiovascular diseases and also as a link between inflammation and hypertension. However, its role in hypertension induced renal injury remains unexplored. In the present study, we investigated whether TLR-4 deficiency reduces Ang-II-induced renal injury and fibrosis by attenuating reactive oxygen species (ROS) production and inflammation. C3H/HeOuJ mice with normal TLR-4 and C3H/HeJ Lps-d with dysfunctional TLR4 (TLR4 deficiency) were treated without or with Ang-II. In response to Ang-II, TLR4 deficient mice had reduced renal resistive index and increased renal cortical blood flow compared to mice with normal TLR4. Further, TLR4 deficiency reduced oxidative stress and increased antioxidant capacity (MnSOD, CuSOD and Catalase activity). TLR4 deficiency was also associated with reduced inflammation (MCP-1, MIP-2, TNF-α, IL-6 and CD68), decreased accumulation of bone marrow-derived fibroblasts and TGF-ß expression. Our data suggests that in C3H/HeJ Lps-d mice, deficiency of functional TLR4 reduces oxidative stress and macrophage activation to decrease TGF-ß-induced extracellular matrix protein deposition in the kidney in Ang-II induced hypertension.

Moderate intensity exercise prevents diabetic cardiomyopathy associated contractile dysfunction through restoration of mitochondrial function and connexin 43 levels in db/db mice.

AIMS: Although the cardiovascular benefits of exercise are well known, exercise induced effects and mechanisms in prevention of cardiomyopathy are less clear during obesity associated type-2 diabetes. The current study assessed the impact of moderate intensity exercise on diabetic cardiomyopathy by examining cardiac function and structure and mitochondrial function. METHODS: Obese-diabetic (db/db), and lean control (db/+) mice, were subjected to a 5 week, 300 m run on a tread-mill for 5 days/week at the speeds of 10-11 m/min. Various physiological parameters were recorded and the heart function was evaluated with M-mode echocardiography. Contraction parameters and calcium transits were examined on isolated cardiomyocytes. At the molecular level: connexin 43 and 37 (Cx43 and 37) levels, mitochondrial biogenesis regulators: Mfn2 and Drp-1 levels, mitochondrial trans-membrane potential and cytochrome c leakage were assessed through western blotting immunohistochemistry and flow cytometry. Ability of exercise to reverse oxygen consumption rate (OCR), tissue ATP levels, and cardiac fibrosis were also determined. RESULTS: The exercise regimen was able to prevent diabetic cardiac functional deficiencies: ejection fraction (EF) and fractional shortening (FS). Improvements in contraction velocity and contraction maximum were noted with the isolated cardiomyocytes. Restoration of interstitial and micro-vessels associated Cx43 levels and improved gap junction intercellular communication (GJIC) were observed. The decline in the Mfn2/Drp-1 ratio in the db/db mice hearts was prevented after exercise. The exercise regimen further attenuated transmembrane potential decline and cytochrome c leakage. These corrections further led to improvements in OCR and tissue ATP levels and reduction in cardiac fibrosis. CONCLUSIONS: Moderate intensity exercise produced significant cardiovascular benefits by improving mitochondrial function through restoration of Cx43 networks and mitochondrial trans-membrane potential and prevention of excessive mitochondrial fission.

Hyperhomocysteinemia: a missing link to dysfunctional HDL via paraoxanase-1.

Paraoxanase-1 (PON1) is an HDL-associated enzyme that contributes to the antioxidant and antiatherosclerotic properties of HDL. Lack of PON1 results in dysfunctional HDL. HHcy is a risk factor for cardiovascular disorders, and instigates vascular dysfunction and ECM remodeling. Although studies have reported HHcy during atherosclerosis, the exact mechanism is unclear. Here, we hypothesize that dysfunctional HDL due to lack of PON1 contributes to endothelial impairment and atherogenesis through HHcy-induced ECM re-modeling. To verify this hypothesis, we used C57BL6/J and PON1 knockout mice (KO) and fed them an atherogenic diet. The expression of Akt, ADMA, and DDAH, as well as endothelial gap junction proteins such as Cx-37 and Cx-40 and eNOS was measured for vascular dysfunction and inflammation. We observed that cardiac function was decreased and plasma Hcy levels were increased in PON1 KO mice fed the atherogenic diet compared with the controls. Expression of Akt, eNOS, DDAH, Cx-37, and Cx-40 was decreased, and the expression of MMP-9 and ADMA was increased in PON1 KO mice fed an atherogenic diet compared with the controls. Our results suggest that HHcy plays an intricate role in dysfunctional HDL, owing to the lack of PON1. This contributes to vascular endothelial impairment and atherosclerosis through MMP-9-induced vascular remodeling.

DNA hypermethylation in hyperhomocysteinemia contributes to abnormal extracellular matrix metabolism in the kidney.

Hyperhomocysteinemia (HHcy) is prevalent in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD). Emerging studies suggest that epigenetic mechanisms contribute to the development and progression of fibrosis in CKD. HHcy and its intermediates are known to alter the DNA methylation pattern, which is a critical regulator of epigenetic information. In this study, we hypothesized that HHcy causes renovascular remodeling by DNA hypermethylation, leading to glomerulosclerosis. We also evaluated whether the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-Aza) could modulate extracellular matrix (ECM) metabolism and reduce renovascular fibrosis. C57BL/6J (wild-type) and cystathionine-ß-synthase (CBS(+/-)) mice, treated without or with 5-Aza (0.5 mg/kg body weight, i.p.), were used. CBS(+/-) mice showed high plasma Hcy levels, hypertension, and significant glomerular and arteriolar injury. 5-Aza treatment normalized blood pressure and reversed renal injury. CBS(+/-) mice showed global hypermethylation and up-regulation of DNA methyltransferase-1 and -3a. Methylation-specific PCR showed an imbalance between matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and -2 and also increased collagen and galectin-3 expression. 5-Aza reduced abnormal DNA methylation and restored the MMP-9/TIMP-1, -2 balance. In conclusion, our data suggest that during HHcy, abnormal DNA methylation and an imbalance between MMP-9 and TIMP-1 and -2 lead to ECM remodeling and renal fibrosis.

MMP-9- and NMDA receptor-mediated mechanism of diabetic renovascular remodeling and kidney dysfunction: hydrogen sulfide is a key modulator.

Previously we reported that matrix metalloproteinase-9 (MMP-9) plays an important role in extracellular matrix (ECM) remodeling in diabetic kidney. Induction of NMDA-R and dysregulation of connexins (Cxs) were also observed. We concluded that this was due to decreased H2S production by downregulation of CBS and CSE enzymes. However, the potential role of H2S to mitigate ECM dysregulation and renal dysfunction was not clearly understood. The present study was undertaken to determine whether H2S supplementation reduces MMP-9-induced ECM remodeling and dysfunction in diabetic kidney. Wild type (C57BL/6J), diabetic (Akita, C57BL/6J-Ins2(Akita)), MMP-9 knockout (MMP-9(-/-), M9KO) and double KO of Akita/MMP-9(-/-) (DKO) mice were treated without or with 0.005 g/l of NaHS (as a source of H2S) in drinking water for 30 days. Decreased tissue production and plasma content of H2S in Akita mice were ameliorated with H2S supplementation. Dysregulated expression of MMP-9, CBS, CSE, NMDA-R1 and Cxs-40, -43 was also normalized in Akita mice treated with H2S. In addition, increased renovascular resistive index (RI), ECM deposition, plasma creatinine, and diminished renal vascular density and cortical blood flow in Akita mice were normalized with H2S treatment. We conclude that diminished H2S production in renal tissue and plasma levels in diabetes mediates adverse renal remodeling, and H2S therapy improves renal function through MMP-9- and NMDA-R1-mediated pathway.

Hydrogen sulfide mitigates hyperglycemic remodeling via liver kinase B1-adenosine monophosphate-activated protein kinase signaling.

Hyperglycemia (HG) reduces AMPK activation leading to impaired autophagy and matrix accumulation. Hydrogen sulfide (H2S) treatment improves HG-induced renovascular remodeling however, its mechanism remains unclear. Activation of LKB1 by the formation of heterotrimeric complex with STRAD and MO25 is known to activate AMPK. We hypothesized that in HG; H2S induces autophagy and modulates matrix synthesis through AMPK-dependent LKB1/STRAD/MO25 complex formation. To address this hypothesis, mouse glomerular endothelial cells were treated with normal and high glucose in the absence or presence of sodium hydrogen sulfide (NaHS), an H2S donor. HG decreased the expression of H2S regulating enzymes CBS and CSE, and autophagy markers Atg5, Atg7, Atg3 and LC3B/A ratio. HG increased galectin-3 and periostin, markers of matrix accumulation. Treatment with NaHS to HG cells increased LKB1/STRAD/MO25 formation and AMPK phosphorylation. Silencing the encoded genes confirmed complex formation under normoglycemia. H2S-mediated AMPK activation in HG was associated with upregulation of autophagy and diminished matrix accumulation. We conclude that H2S mitigates adverse remodeling in HG by induction of autophagy and regulation of matrix metabolism through LKB1/STRAD/MO25 dependent pathway.

Endothelial dysfunction: the link between homocysteine and hydrogen sulfide.

High level of homocysteine (hyperhomocysteinemia, HHcy) is associated with increased risk for vascular disease. Evidence for this emerges from epidemiological studies which show that HHcy is associated with premature peripheral, coronary artery and cerebrovascular disease independent of other risk factors. Possible mechanisms by which homocysteine causes vascular injury include endothelial injury, DNA dysfunction, proliferation of smooth muscle cells, increased oxidative stress, reduced activity of glutathione peroxidase and promoting inflammation. HHcy has been shown to cause direct damage to endothelial cells both in vitro and in vivo. Clinically, this manifests as impaired flow-mediated vasodilation and is mainly due to a reduction in nitric oxide synthesis and bioavailability. The effect of impaired nitric oxide release can in turn trigger and potentiate atherothrombogenesis and oxidative stress. Endothelial damage is a crucial aspect of atherosclerosis and precedes overt manifestation of disease. In addition, endothelial dysfunction is also associated with hypertension, diabetes, ischemia reperfusion injury and neurodegenerative diseases. Homocysteine is a precursor of hydrogen sulfide (H2S) which is formed by transulfuration process catalyzed by the enzymes, cystathionine ß-synthase and cystathionine γ-lyase. H2S is a gasotransmitter that has emerged recently as a novel mediator in cardiovascular homeostasis. As a potent vasodilator, it plays several roles which include regulation of vessel diameter, protection of endothelium from redox stress, ischemia reperfusion injury and chronic inflammation. However, the precise mechanism by which it mediates these beneficial effects is complex and still remains unclear. Current evidence indicates H2S modulates cellular functions by a variety of intracellular signaling processes. In this review, we summarize the mechanisms of HHcy-induced endothelial dysfunction and the metabolism and physiological functions of H2S as a protective agent.

Epigenetic regulation of aortic remodeling in hyperhomocysteinemia.

Hyperhomocysteinemia (HHcy) is prevalent in patients with hypertension and is an independent risk factor for aortic pathologies. HHcy is known to cause an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), leading to the accumulation of collagen in the aorta and resulting in stiffness and development of hypertension. Although the exact mechanism of extracellular matrix (ECM) remodeling is unclear, emerging evidence implicates epigenetic regulation involving DNA methylation. Our purpose was to investigate whether 5-aza-2'-deoxycytidine (Aza), a DNA methyltransferase (DNMT1) inhibitor, reduces high blood pressure (BP) by regulating aortic ECM remodeling in HHcy. Wild-type and cystathionine ß-synthase (CBS)(+/-) HHcy mice were treated with Aza (0.5 mg/kg body weight). In HHcy mice, Aza treatment normalized the plasma homocysteine (Hcy) level and BP. Thoracic and abdominal aorta ultrasound revealed a reduction in the resistive index and wall-to-lumen ratio. Vascular response to phenylephrine, acetylcholine, and sodium nitroprusside improved after Aza in HHcy mice. Histology showed a marked reduction in collagen deposition in the aorta. Aza treatment decreased the expression of DNMT1, MMP9, TIMP1, and S-adenosyl homocysteine hydrolase (SAHH) and upregulated methylene tetrahydrofolate reductase (MTHFR). We conclude that reduction of DNA methylation by Aza in HHcy reduces adverse aortic remodeling to mitigate hypertension.

Hydrogen sulfide deficiency and diabetic renal remodeling: role of matrix metalloproteinase-9.

Matrix metalloproteinase-9 (MMP-9) causes adverse remodeling, whereas hydrogen sulfide (H2S) rescues organs in vascular diseases. The involvement of MMP-9 and H2S in diabetic renovascular remodeling is, however, not well characterized. We determined whether MMP-9 regulates H2S generation and whether H2S modulates connexin through N-methyl-d-aspartate receptor (NMDA-R)-mediated pathway in the diabetic kidney. Wild-type (WT, C57BL/6J), diabetic (Akita, C57BL/6J-Ins2(Akita)), MMP-9(-/-) (M9KO), double knockout (DKO) of Akita/MMP-9(-/-) mice and in vitro cell culture were used in our study. Hyperglycemic Akita mice exhibited increased level of MMP-9 and decreased production of H2S. H2S-synthesizing enzymes cystathionine-ß-synthase and cystathionine-γ-lyase were also diminished. In addition, increased expressions of NMDA-R1 and connexin-40 and -43 were observed in diabetic kidney. As expected, MMP-9 mRNA was not detected in M9KO kidneys. However, very thin protein expression and activity were detected. No other changes were noticed in M9KO kidney. In DKO mice, all the above molecules showed a trend toward baseline despite hyperglycemia. In vitro, glomerular endothelial cells treated with high glucose showed induction of MMP-9, attenuated H2S production, NMDA-R1 induction, and dysregulated conexin-40 and -43 expressions. Silencing MMP-9 by siRNA or inhibition of NMDA-R1 by MK801 or H2S treatment preserved connexin-40 and -43. We conclude that in diabetic renovascular remodeling MMP-9 plays a major role and that H2S has therapeutic potential to prevent adverse diabetic renal remodeling.