Although matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) play vital role in tumor angiogenesis and TIMP-3 caused apoptosis, their role in cardiac angiogenesis is unknown. Interestingly, a disruption of coordinated cardiac hypertrophy and angiogenesis contributed to the transition to heart failure, however, the proteolytic and anti-angiogenic mechanisms of transition from compensatory hypertrophy to decompensatory heart failure were unclear. We hypothesized that after an aortic stenosis MMP-2 released angiogenic factors during compensatory hypertrophy and MMP-9/TIMP-3 released antiangiogenic factors causing decompensatory heart failure. To verify this hypothesis, wild type (WT) mice were studied 3 and 8 wks after aortic stenosis, created by banding the ascending aorta in WT and MMP-9−/− (MMP-9KO) mice. Cardiac function (echo, PV loops) was decreased at 8 wks after stenosis. The levels of MMP-2 (western blot) increased at 3 wks and returned to control level at 8 wks, MMP-9 increased only at 8 wks. TIMP-2 and −4 decreased at 3 and even more at 8 wks. The angiogenic VEGF increased at 3 wks and decreased at 8 wks, the antiangiogenic endostatin and angiostatin increased only at 8 wks. CD-31 positive endothelial cells were more intensely labeled at 3 wks than in sham operated or in 8 wks banded mice. Vascularization, as estimated by x-ray angiography, was increased at 3 wks and decreased at 8 wks post-banding. Although vast majority of studies were performed on control WT mice only, interestingly, MMP9-KO mice seemed to have increased vascular density 8 wks after banding. These results suggested that there was increase in MMP-2, decrease in TIMP-2 and −4, increase in angiogenic factors and vascularization in compensatory hearts. However, in decompensatory hearts there was increase in MMP-9, TIMP-3, endostatin, angiostatin and vascular rarefaction.

We reported previously that although there is disruption of coordinated cardiac hypertrophy and angiogenesis in transition to heart failure, matrix metalloproteinase (MMP)-9 induced antiangiogenic factors play a vital role in this process. Previous studies have shown the cardioprotective role of hydrogen sulfide (H₂S) in various cardiac diseases, but its role during transition from compensatory hypertrophy to heart failure is yet to be unveiled. We hypothesize that H₂S induces MMP-2 activation and inhibits MMP-9 activation, thus promoting angiogenesis, and mitigates transition from compensatory cardiac hypertrophy to heart failure. To verify this, aortic banding (AB) was created to mimic pressure overload in wild-type (WT) mice, which were treated with sodium hydrosulfide (NaHS, H₂S donor) in drinking water and compared with untreated control mice. Mice were studied at 3 and 8 wk. In the NaHS-treated AB 8 wk group, the expression of MMP-2, CD31, and VEGF was increased while the expression of MMP-9, endostatin, angiostatin, and tissue inhibitor of matrix metalloproteinase (TIMP)-3 was decreased compared with untreated control mice. There was significant reduction in fibrosis in NaHS-treated groups. Echocardiograph and pressure-volume data revealed improvement of cardiac function in NaHS-treated groups over untreated controls. These results show that H₂S by inducing MMP-2 promotes VEGF synthesis and angiogenesis while it suppresses MMP-9 and TIMP-3 levels, inhibits antiangiogenic factors, reduces intracardiac fibrosis, and mitigates transition from compensatory hypertrophy to heart failure.

High levels of homocysteine (Hcy) known as hyperhomocysteine-mia (HHcy), contribute to autophagy and ischemia/reperfusion injury (I/R). Previous studies have shown that I/R injury and HHcy cause increased cerebrovascular permeability; however, the associated mechanism remains obscure. Interestingly, during HHcy, cytochome-c becomes homocysteinylated (Hcy-cyto-c). Cytochrome-c (cyto-c) transports electrons and facilitates bioenergetics in the system. However, its role in autophagy during ischemia/reperfusion injury is unclear. Tetrahydrocurcumin (THC) is a major herbal antioxidant and anti-inflammatory agent. Therefore, the objective of this study was to determine whether THC ameliorates autophagy during ischemia/reperfusion injury by reducing homocysteinylation of cyto-c in hyperhomocysteinemia pathological condition. To test this hypothesis we employed 8–10 weeks old male cystathionine-beta-synthase heterozygote knockout (CBS+/−) mice (genetically hyperhomocystemic mice). Experimental group was: CBS+/−, CBS+/−+THC (25mg/kg in 0.1%DMSO dose); CBS (+/−)/I/R and CBS (+/−)/I/R+THC(25mg/kg in 0.1%DMSO dose). Ischemia was performed for 30 min and reperfusion for 72 hours. THC was injected intra-peritoneally (I.P.) once daily for a period of 3 days after 30 of ischemia. The infarct area was measured using 2,3,5-triphenyltetrazolium chloride staining. Permeability was determined by brain edema and Evans Blue extravasation. The brain tissues were analyzed for oxidative stress, matrix metalloproteinase-9 (MMP-9), damage-regulated autophagy modulator (DRAM), and microtubule-associated protein 1 light chain 3 (LC3) by Western blot. The mRNA levels of S-Adenosyl-L-homocysteine hydrolases (SAHH) and Methylenetetrahydrofolate reductase (MTHFR) genes were measured by Quantitative real-time polymerase chain reaction. Co-immunoprecipitation was used to determine the homocysteinylation of cyto-c. We found that brain edema and Evans Blue leakage were reduced in I/R+THC treated groups as compared to sham operated groups along with reduced brain infarct size. THC also decreased oxidativedamage and ameliorated the homocysteinylation of cyto-c in-part by MMP-9 activation which leads to autophagy in I/R groups as compared to sham operated groups. This study suggests a potential therapeutic role of dietary THC in cerebral ischemia.

Hyperhomocysteinemia (HHcy) is a risk factor for neuroinflammatory and neurodegenerative diseases. Homocysteine (Hcy) induces redox stress, in part, by activating matrix metalloproteinase-9 (MMP-9), which degrades the matrix and leads to blood–brain barrier dysfunction. Hcy competitively binds to γ-aminbutyric acid (GABA) receptors, which are excitatory neurotransmitter receptors. However, the role of GABA-A receptor in Hcy-induced cerebrovascular remodeling is not clear. We hypothesized that Hcy causes cerebrovascular remodeling by increasing redox stress and MMP-9 activity via the extracellular signal-regulated kinase (ERK) signaling pathway and by inhibition of GABA-A receptors, thus behaving as an inhibitory neurotransmitter. Hcy-induced reactive oxygen species production was detected using the fluorescent probe, 2′–7′-dichlorodihydrofluorescein diacetate. Hcy increased nicotinamide adenine dinucleotide phosphate-oxidase-4 concomitantly suppressing thioredoxin. Hcy caused activation of MMP-9, measured by gelatin zymography. The GABA-A receptor agonist, muscimol ameliorated the Hcy-mediated MMP-9 activation. In parallel, Hcy caused phosphorylation of ERK and selectively decreased levels of tissue inhibitors of metalloproteinase-4 (TIMP-4). Treatment of the endothelial cell with muscimol restored the levels of TIMP-4 to the levels in control group. Hcy induced expression of iNOS and decreased eNOS expression, which lead to a decreased NO bioavailability. Furthermore muscimol attenuated Hcy-induced MMP-9 via ERK signaling pathway. These results suggest that Hcy competes with GABA-A receptors, inducing the oxidative stress transduction pathway and leading to ERK activation.

Autophagy is an important process in the pathogenesis of cardiovascular diseases; however, the proximal triggers for mitochondrial autophagy were unknown. The N-methyl-D-aspartate receptor 1 (NMDA-R1) is a receptor for homocysteine (Hcy) and plays a key role in cardiac dysfunction. Cardiac-specific deletion of NMDA-R1 has been shown to ameliorate Hcy-induced myocyte contractility. Hcy activates mitochondrial matrix metalloproteinase-9 (mtMMP-9) and induces translocation of connexin-43 (Cxn-43) to the mitochondria (mtCxn-43). We sought to show cardiac-specific deletion of NMDA-R1 mitigates Hcy-induced mtCxn-43 translocation, mtMMP-9-mediated mtCxn-43 degradation, leading to mitophagy, in part, by decreasing mitochondrial permeability (MPT). Cardiac-specific knockout (KO) of NAMDA-R1 was generated using the cre/lox approach. The myocyte mitochondria were isolated from wild type (WT), WT + Hcy (1.8 g of DL-Hcy/L in the drinking water for 6 weeks), NMDA-R1 KO + Hcy, and NR1 fl/fl /Cre (NR1 fl/fl ) genetic control mice. Mitochondrial respiratory capacity and MPT were measured by fluorescence-dye methods. The mitochondrial superoxide and peroxinitrite levels were detected by confocal microscopy using Mito-SOX and dihydrorhodamine-123. The mtMMP-9 activity and expression were detected by zymography and RT-PCR analyses. The mtCxn-43 translocation was detected by confocal microscopy. The degradation of mtCxn-43 and LC3-I/II (a marker of autophagy) were detected by Western blot. These results suggested that Hcy enhanced intramitochondrial nitrosative stress in myocytes. There was a robust increase in mtMMP-9 activity. An increase in translocation and degradation of mtCxn-43 was also noted. These increases led to mitophagy. The effects were ameliorated by cardiac-specific deletion of NMDA-R1. We concluded that HHcy increased mitochondrial nitrosative stress, thereby activating mtMMP-9 and inciting the degradation of mtCxn-43. This led to mitophagy, in part, by activating NMDA-R1. The findings of this study will lead to therapeutic ramifications for mitigating cardiovascular diseases by inhibiting the mitochondrial mitophagy and NMDA-R1 receptor. KeywordsMitochondrial MMP; mitochondrial connexin-43 and microtubule-associated protein light chain 3; nitrosative stress