Elevated blood level of Fibrinogen (Fg) is commonly associated with vascular dysfunction. We tested the hypothesis that at pathologically high levels, Fg increases cerebrovascular permeability by activating matrix metalloproteinases (MMPs). Fibrinogen (4 mg/mL blood concentration) or equal volume of phosphate-buffered saline (PBS) was infused into male wild-type (WT; C57BL/6J) or MMP-9 gene knockout (MMP9À/À) mice. Pial venular leakage of fluorescein isothiocyanate-bovine serum albumin to Fg or PBS alone and to topically applied histamine (10 À5 mol/L) were assessed. Intravital fluorescence microscopy and image analysis were used to assess cerebrovascular protein leakage. Pial venular macromolecular leakage increased more after Fg infusion than after infusion of PBS in both (WT and MMP9À/À) mice but was more pronounced in WT compared with MMP9À/À mice. Expression of vascular endothelial cadherin (VE-cadherin) was less and plasmalemmal vesicle-associated protein-1 (PV-1) was greater in Fg-infused than in PBS-infused both mice groups. However, in MMP9À/À mice, VE-cadherin expression was greater and PV-1 expression was less than in WT mice. These data indicate that at higher levels, Fg compromises microvascular integrity through activation of MMP-9 and downregulation of VE-cadherin and upregulation of PV-1. Our results suggest that elevated blood level of Fg could have a significant role in cerebrovascular dysfunction and remodeling.

Background/Aims: In this study we tested the hypothesis that H₂S regulates collagen deposition, matrix metalloproteinases (MMP) and inflammatory molecules during hyperhomocysteinemia (HHcy) resulting in attenuation of glomerulosclerosis and improved renal function. Materials and Methods: A genetic model of HHcy, cystathionine β-synthase heterozygous (CBS+/–) and wild-type (WT) 2-kidney (2K) mice were used in this study and supplemented with or without NaHS (30 µmol/l, H₂S donor) in drinking water for 8 weeks. To expedite the renal damage associated with HHcy, uninephrectomized (1K) mice of similar groups were also used. Results: Results demonstrated that NAD(P)H oxidase (p47^phoxsubunit) and blood pressure were upregulated in WT 1K, CBS+/– 2K and CBS+/– 1K mice with downregulation of H₂S production and reduced glomerular filtration rate. These changes were normalized with H₂S supplementation. Both pro- and active MMP-2 and -9 and collagen protein expressions and glomerular depositions were also upregulated in WT 1K, CBS+/– 2K and CBS+/– 1K mice. Increased expressions of inflammatory molecules, intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1, as well as increased macrophage infiltration, were detected in WT 1K, CBS+/– 2K and CBS+/– 1K mice. These changes were ameliorated with H₂S supplementation. Conclusion: Together, these results suggest that increased oxidative stress and decreased H₂S in HHcy causes matrix remodeling and inflammation resulting in glomerulosclerosis and reduced renal function.

Hydrogen sulfide (H2S) has recently been identified as a regulator of various physiological events, including vasodilation, angiogenesis, antiapoptotic, and cellular signaling. Endogenously, H2S is produced as a metabolite of homocysteine (Hcy) by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Although Hcy is recognized as vascular risk factor at an elevated level [hyperhomocysteinemia (HHcy)] and contributes to vascular injury leading to renovascular dysfunction, the exact mechanism is unclear. The goal of the current study was to investigate whether conversion of Hcy to H2S improves renovascular function. Ex vivo renal artery culture with CBS, CSE, and 3MST triple gene therapy generated more H2S in the presence of Hcy, and these arteries were more responsive to endothelial-dependent vasodilation compared with nontransfected arteries treated with high Hcy. Cross section of triple gene-delivered renal arteries immunostaining suggested increased expression of CD31 and VEGF and diminished expression of the antiangiogenic factor endostatin. In vitro endothelial cell culture demonstrated increased mitophagy during high levels of Hcy and was mitigated by triple gene delivery. Also, dephosphorylated Akt and phosphorylated FoxO3 in HHcy were reversed by H2S or triple gene delivery. Upregulated matrix metalloproteinases-13 and downregulated tissue inhibitor of metalloproteinase-1 in HHcy were normalized by overexpression of triple genes. Together, these results suggest that H2S plays a key role in renovasculopathy during HHcy and is mediated through Akt/FoxO3 pathways. We conclude that conversion of Hcy to H2S by CBS, CSE, or 3MST triple gene therapy improves renovascular function in HHcy.

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.

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.