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Aortic valve (AV) disease involves stiffening of the AV cusp with progression characterized by inflammation, fibrosis, and calcification. Here, we examine the relationship between biomechanical valve function and proteomic changes before and after the development of AV pathology in the Emilin1−/− mouse model of latent AV disease. Biomechanical studies were performed to quantify tissue stiffness at the macro (micropipette) and micro (atomic force microscopy (AFM)) levels. Micropipette studies showed that the Emilin1−/− AV annulus and cusp regions demonstrated increased stiffness only after the onset of AV disease. AFM studies showed that the Emilin1−/− cusp stiffens before the onset of AV disease and worsens with the onset of disease. Proteomes from AV cusps were investigated to identify protein functions, pathways, and interaction network alterations that occur with age- and genotype-related valve stiffening. Protein alterations due to Emilin1 deficiency, including changes in pathways and functions, preceded biomechanical aberrations, resulting in marked depletion of extracellular matrix (ECM) proteins interacting with TGFB1, including latent transforming growth factor beta 3 (LTBP3), fibulin 5 (FBLN5), and cartilage intermediate layer protein 1 (CILP1). This study identifies proteomic dysregulation is associated with biomechanical dysfunction as early pathogenic processes in the Emilin1−/− model of AV disease.

Aortic valve disease is a burgeoning public health problem associated with significant mortality. Loss of function mutations in NOTCH1 cause bicuspid aortic valve (BAV) and calcific aortic valve disease. Because calcific nodules manifest on the fibrosa side of the cusp in low fluidic oscillatory shear stress (OSS), elucidating pathogenesis requires approaches that consider both molecular and mechanical factors. Therefore, we examined the relationship between NOTCH loss of function (LOF) and biomechanical indices in healthy and diseased human aortic valve interstitial cells (AVICs). An orbital shaker system was used to apply cyclic OSS, which mimics the cardiac cycle and hemodynamics experienced by AVICs in vivo. NOTCH LOF blocked OSS-induced cell alignment in human umbilical vein endothelial cells (HUVECs), whereas AVICs did not align when subjected to OSS under any conditions. In healthy AVICs, OSS resulted in decreased elastin (ELN) and α-SMA (ACTA2). NOTCH LOF was associated with similar changes, but in diseased AVICs, NOTCH LOF combined with OSS was associated with increased α-SMA expression. Interestingly, AVICs showed relatively higher expression of NOTCH2 compared to NOTCH1. Biomechanical interactions between endothelial and interstitial cells involve complex NOTCH signaling that contributes to matrix homeostasis in health and disorganization in disease.

Overexposure of the kidneys to cisplatin [Cisplatin (cis- diaminedichloroplatinum II) (CDDP)] and cisplatin complexes cause degradation of the renal cells, and eventually lead to renal failure. Agents like antioxidants, modulators of nitric oxide (NO) and anti-apoptotic molecules are reported to reduce nephrotoxicity. In the present study we tested the hypothesis that supplementation of antioxidant spirulina and vitamin c reduces cisplatin induced nephrotoxicity. The study was carried out on female Balb /C mice of the following five groups: control (i); cisplatin (ii); vitamin c (iii); spirulina (iv); and vitamin c + spirulina + cisplatin (v). Plasma urea and creatinine levels were estimated 3 and 6 days after the injection of cisplatin and uptake studies with 99m Technician-ethylene dicysteine was performed to assess the renal clearance rate. Kidneys were isolated and processed for electron microscopy and light microscopy. The activity and concentration of antioxidant enzymes such as glutathione, catalase, superoxide dismutase and GST was also determined. Dual supplementation of spirulina and vitamin c significantly protected cisplatin-induced increased levels of serum urea, creatinine, improved renal clearance rate, the declined renal antioxidant enzymes and increased LPO levels. Hence, we conclude that supplementation of spirulina and vitamin c had a beneficial effect on cisplatin induced nephrotoxicity in mice.

Elevated levels of plasma homocysteine (Hcy) called hyperhomocysteinemia (HHcy) have been implicated in inflammation and remodeling in intestinal vasculature, and HHcy is also known to aggravate the pathogenesis of inflammatory bowel disease (IBD). Interestingly, colon is the pivotal site that regulates Hcy levels in the plasma. We hypothesize that HHcy decreases intestinal motility through matrix metalloproteinase-9 (MMP-9)-induced intestinal remodeling leading to constipation. To verify this hypothesis, we used C57BL/6J or wild-type (WT), cystathionine β-synthase (CBS+/−), MMP-9−/−, and MMP-9−/− + Hcy mice. Intestinal motility was assessed by barium meal studies and daily feces output. Plasma Hcy levels were measured by HPLC. Expression of ICAM-1, inducible nitric oxide synthase, MMP-9, and tissue inhibitors of MMPs was studied by Western blot and immunohistochemistry. Reactive oxygen species (ROS) including super oxide were measured by the Invitrogen molecular probe method. Tissue nitric oxide levels were assessed by a commercially available kit. Plasma Hcy levels in the treated MMP-9 group mice were comparable to CBS+/− mice. Barium meal studies suggest that intestinal motility is significantly decreased in CBS+/− mice compared with other groups. Fecal output-to-body weight ratio was significantly reduced in CBS+/− mice compared with other groups. There was significant upregulation of MMP-9, iNOS, and ICAM-1 expression in the colon from CBS+/− mice compared with WT mice. Levels of ROS, superoxide, and inducible nitric oxide were elevated in the CBS+/− mice compared with other groups. Results suggest that HHcy decreases intestinal motility due to MMP-9-induced intestinal remodeling leading to constipation.

More than 29 million adults in the United States have been diagnosed with hearing loss. Interestingly, elevated homocysteine (Hcy) levels, known as hyperhomocysteinemia (HHcy), are also associated with impaired hearing. However, the associated mechanism remains obscure. The collagen receptor such as discoidin domain receptor 1 and matrix metalloproteinase (MMP) play a significant role in inner ear structure and function. We hypothesize that HHcy increases hearing thresholds by compromise in inner ear vasculature resulted from impaired Hcy metabolism, increased oxidative stress, collagen IVa and collagen Ia turnover. The treatment with folic acid (FA) protects elevated hearing thresholds and prevents reduction in vessel density by lowering abundant collagen deposition and oxidative stress in inner ear. To test this hypothesis we employed 8 weeks old male wild type (WT), cystathionine-beta-synthase heterozygote knockout (CBS+/-) mice, WT + FA (0.0057 mu g/g/day, equivalent to a 400 mu g/70 kg/day human dose in drinking water); and CBS(+/-) +FA. The mice were treated for four weeks. The hearing thresholds were determined by recording the auditory brainstem responses. Integrity of vessels was analyzed by perfusion of horseradish peroxidase (HRP) tracer. Endothelial permeability was assessed, which indicated restoration of HRP leakage by FA treatment. A total Hcy level was increased in stria vascularis (SV) and spiral ligament (SL) of CBS+/- mice which was lowered by FA. Interestingly, FA treatment lowered Col IVa Immunostaining by affecting its turnover. The levels of MMP-2, -9, methylenetetrahydrofolate reductase (MTHFR) and cystathione gamma lyase (CSE) were measured by Western blot analysis. The oxidative stress was high in SV and SL of CBS+/- compared to WT however the treatment with FA lowered oxidative stress in CBS+/- mice. These data suggested that hearing loss in CBS+/- mice was primarily due to leakage in inner ear circulation, also partly by induced collagen imbalance, increase in Hcy and oxidative stress in inner ear.

In the present study we tested the hypothesis whether hyperhomocysteinemia, an elevated homocysteine level, induces venous phenotype in artery. To test our hypothesis, we employed wild type (WT) and cystathionine β-synthase heterozygous (+/-) (CBS+/-) mice treatment with or without folic acid (FA). Aortic blood flow and velocity were significantly lower in CBS+/-mice compared to WT. Aortic lumen diameter was significantly decreased in CBS+/-mice, whereas FA treatment normalized it. Medial thickness and collagen were significantly increased in CBS+/-aorta, whereas elastin/collagen ratio was significantly decreased. Superoxide and gelatinase activity was significantly high in CBS+/-aorta vs WT. Western blot showed significant increase in MMP-2, -9,-12, TIMP-2 and decrease in TIMP-4 in aorta. RT-PCR revealed significant increase of vena cava marker EphB4, MMP-13 and TIMP-3 in aorta. We summarize that chronic HHcy causes vascular remodelling that transduces changes in vascular wall in a way that artery expresses vein phenotype.

Deficiencies in folate lead to increased serum concentrations of homocysteine (Hcy), which is known as hyperhomocysteinemia (HHcy), is associated with bone disorders. Although, homocysteine (Hcy) accumulates collagen in bone and contribute to decrease in bone strength. The mechanism of Hcy induced bone loss and remodeling is unclear. Therefore, the present study was aimed to determine the role of folic acid in genetically HHcy associated decrease in bone blood flow and remodeling. Wildtype (WT) and cystathionine-β-synthase heterozygous (CBS+/−) mice were used in this study and supplemented with or without folic acid (FA 300 mg/kg, Hcy reducing agent) in drinking water for 6 weeks. The tibial bone blood flow was measured by laser Doppler and ultrasonic flow probe method. The tibial bone density was assessed by dual energy X-ray absorptiometry. The bone homogenates were analyzed for oxidative stress,NOX-4 as oxidative marker and thioredoxin-1 (Trx-1) as anti-oxidant marker, bone remodeling (MMP-9) and bio-availability of nitric oxide (eNOS/iNOS/NO) by Western blot method. The results suggested that there was decrease in tibial blood flow in CBS+/− mice. The bone density was also reduced in CBS+/− mice. There was an increase in NOX-4, iNOS, MMP -9 protein as well as MMP-9 activity in CBS+/− mice and decrease in Trx-1, eNOS protein levels, in part by decreasing NO bio-availability in CBS+/− mice. Interestingly, these effects were ameliorated by folic acid and suggested that folic acid supplementation may have therapeutic potential against genetically HHcy induced bone loss.

Although protease activated receptor-1 (PAR-1) and matrix metalloproteinase-9 (MMP-9) play significant role in vascular remodelling in hyperhomocysteinemia (HHcy due to cystathionine beta synthase deficiency, CBS−/+) and diabetes, mechanism remains nebulous. We hypothesized that differential vascular density and remodelling in different vascular beds in HHcy and diabetes were responsible for an adaptive metabolic homeostasis during the pathogenesis. To test this hypothesis, vascular density in mice lacking PAR-1, MMP-9, CBS and Insulin-2 gene mutant (Ins2−/+, Akita) was measured and compared with wild type (WT, C57BL/6J) mice. The vascular density was detected by x-ray angiography using KODAK 4000 MM image station, using barium sulphate as contrasting agent. The % vascular density in the hearts of WT, CBS−/+ (HHcy), MMP-9−/−, PAR-1−/+ and Ins2−/+ (type-1 diabetes) was 100 ± 2.8, 85 ± 3.3, 90 ± 3.3, 95 ± 3.8 and 73 ± 1.7, respectively. The vascular density in CBS−/+ and Akita hearts decreased while it was increased in lungs of CBS−/+ and MMP-9−/−. There was decreased vascular density in liver and kidney of Akita mice. Vascular density in brain, kidney and mesentery was decreased in CBS−/+ mice. These findings support the notation that metabolic derangement in diabetes and HHcy causes the chronic decline and/or rarefaction in vascular density.

Aortic valve disease (AVD) is a common condition with a progressive natural history, and presently, there are no pharmacologic treatment strategies. Elastic fiber fragmentation (EFF) is a hallmark of AVD, and increasing evidence implicates developmental elastic fiber assembly defects. Emilin1 is a glycoprotein necessary for elastic fiber assembly that is present in both developing and mature human and mouse aortic valves. The Emilin1‐ deficient mouse ( Emilin1 −/− ) is a model of latent AVD, characterized by activated TGFβ/MEK/p‐Erk signaling and upregulated elastase activity. Emilin1 −/− aortic valves demonstrate early EFF and aberrant angiogenesis followed by late neovascularization and fibrosis. The objective of this study was to test the effectiveness of three different targeted therapies. Aged (12–14 months) Emilin1 −/− mice were treated with refametinib (RDEA‐119, MEK1/2 inhibitor), doxycycline (elastase inhibitor), or G6‐31 (anti‐VEGF‐A mouse antibody) for 4 weeks. Refametinib‐ and doxycycline‐treated Emilin1 −/− mice markedly reduced MEK/p‐Erk activation in valve tissue. Furthermore, both refametinib and doxycycline attenuated elastolytic cathepsin K, L, MMP‐2, and MMP‐9 activation, and abrogated macrophage and neutrophil infiltration in Emilin1 −/− aortic valves. RNAseq analysis was performed in aortic valve tissue from adult (4 months) and aged (14 months) Emilin1 −/− and age‐matched wild‐type control mice, and demonstrated upregulation of genes associated with MAPK/MEK/p‐Erk signaling and elastases at the adult stage and inflammatory pathways at the aged stage controlling for age. These results suggest that Erk1/2 signaling is an important modulator of early elastase activation, and pharmacological inhibition using refametinib may be a promising treatment to halt AVD progression

Aortic valve disease (AVD) is characterized by elastic fiber fragmentation (EFF), fibrosis and aberrant angiogenesis. Emilin1 is an elastin-binding glycoprotein that regulates elastogenesis and inhibits TGF-β signaling, but the role of Emilin1 in valve tissue is unknown. We tested the hypothesis that Emilin1 deficiency results in AVD, mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β dysregulation. Using histology, immunohistochemistry, electron microscopy, quantitative gene expression analysis, immunoblotting and echocardiography, we examined the effects of Emilin1 deficiency (Emilin1−/−) in mouse aortic valve tissue. Emilin1 deficiency results in early postnatal cell-matrix defects in aortic valve tissue, including EFF, that progress to latent AVD and premature death. The Emilin1−/− aortic valve displays early aberrant provisional angiogenesis and late neovascularization. In addition, Emilin1−/− aortic valves are characterized by early valve interstitial cell activation and proliferation and late myofibroblast-like cell activation and fibrosis. Interestingly, canonical TGF-β signaling (phosphorylated Smad2 and Smad3) is upregulated constitutively from birth to senescence, whereas non-canonical TGF-β signaling (phosphorylated Erk1 and Erk2) progressively increases over time. Emilin1 deficiency recapitulates human fibrotic AVD, and advanced disease is mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β activation. The early manifestation of EFF and aberrant angiogenesis suggests that these processes are crucial intermediate factors involved in disease progression and therefore might provide new therapeutic targets for human AVD.

Introduction Heart valve disease affects over 2% of the population, leading to at least 20,000 deaths and $1 billion in direct costs in the US each year. Calcific aortic valve disease (CAVD) is linked to loss of function mutations in NOTCH1 that result in osteogenic gene induction. In vitro animal studies have established that multiple osteogenic targets are mediated through induction of p-ERK 1/2 signaling; however, the relationship between NOTCH1 and p-ERK has not been examined in primary human aortic valvar interstitial cells (AVICs). Hypothesis We tested the hypothesis that chemically-induced NOTCH1 suppression results in mineralization due to p-ERK 1/2 activation in human AVICs. Methods Human AVICs were isolated from CAVD specimens at the time of valve replacement. AVICs were cultured for two weeks in complete media containing DMEM and 10% FBS, and treated with calcific media (10mM b-glycerol phosphate; 60μM ascorbic acid-2-phosphate; 1μM dexamethasone), which has been shown to suppress NOTCH1 signaling. Standard real-time RT-PCR, western blot, and immunocytochemistry were used to compare groups. Results Treatment with calcific media resulted in a 2.3 fold decrease in the level of NOTCH1 expression and a 3 fold increase in p-ERK 1/2 expression, assessed with both western blots and immunocytochemistry. Interestingly, calcific media also induced a 3.5 fold decrease in VEGF, consistent with early osteogenic differentiation. Additionally, the AVICs treated with calcific media demonstrated overt calcification by Alizarin Red staining. Conclusions Human AVICs demonstrated calcification associated with p-ERK 1/2 activation and reduced NOTCH1 levels, suggesting p-ERK inhibition may mitigate the effects of NOTCH loss of function, thereby representing a potential pharmacologic therapy for CAVD. Further investigation of the molecular mechanisms linking NOTCH1 and p-ERK are warranted, including studies that specifically inhibit NOTCH1 signaling and define the impact of mechanical forces on molecular pathways, thereby identifying mechanisms underlying CAVD progression.

Although elevated levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is associated with inflammatory bowel disease, the mechanism of Hcy action is unclear. In the present study we tested the hypothesis that HHcy activates matrix metalloproteinase-9 (MMP-9), which in turn enhances permeability of human intestinal microvascular endothelial cell (HIMEC) layer by decreasing expression of endothelial junction proteins and increasing caveolae formation. HIMECs were grown in Transwells and treated with 500 μM Hcy in the presence or absence of MMP-9 activity inhibitor. Hcy-induced permeability to FITC-conjugated bovine serum albumin (FITC-BSA) was assessed by measuring fluorescence intensity of solutes in the Transwells’ lower chambers. The cell-cell interaction and cell barrier function was estimated by measuring trans-endothelial electrical impedance. Confocal microscopy and flow cytometry were used to study cell junction protein expressions. Hcy-induced changes in transcellular transport of HIMECs were estimated by observing formation of functional caveolae defined as caveolae labeled by cholera toxin and antibody against caveolin-1 and one that have taken up FITC-BSA. Hcy instigated HIMEC monolayer permeability through activation of MMP-9. The increased paracellular permeability was associated with degradation of vascular endothelial cadherin and zona occludin-1 and transcellular permeability through increased caveolae formation in HIMECs. Elevation of Hcy content increases permeability of HIMEC layer affecting both paracellular and transcellular transport pathways, and this increased permeability was alleviated by inhibition of MMP-9 activity. These findings contribute to clarification of mechanisms of inflammatory bowel disease development.

High levels of homocysteine (Hcy) known as hyperhomocysteinemia (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-week-old male cystathionine-beta-synthase heterozygote knockout (CBS+/−) mice (genetically hyperhomocystemic mice). Experimental group was: CBS+/−, CBS+/− + THC (25 mg/kg in 0.1% DMSO dose); CBS (+/−)/I/R, and CBS (+/−)/I/R + THC (25 mg/kg in 0.1% DMSO dose). Ischemia was performed for 30 min and reperfusion for 72 h. THC was injected intra-peritoneally (I.P.) once daily for a period of 3 days after 30 min 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 oxidative damage 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.

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. Conclusion Above results suggest that Mdivi inhibits the abnormal cardiac mitophagy response during sustained pressure overload stress and propose the novel therapeutic role of Mdivi in ameliorating heart failure.

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 (MMP9-/-) mice. Pial venular leakage of fluorescein isothiocyanate-bovine serum albumin to Fg or PBS alone and to topically applied histamine (105mol/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 MMP9-/-) mice but was more pronounced in WT compared with MMP9-/-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 MMP9-/-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.

Cervical cancer is caused by human papilloma virus (HPV) expressing E6 and E7 oncoproteins, which are known to inactivate tumor suppressor proteins p53 and pRb, respectively. Repression of HPV oncoproteins would therefore result in reactivation of tumor suppressor pathways and cause apoptosis in cancer cells. Withaferin A (WA), the active component of the medicinal plant Withania Somnifera, has exhibited inhibitory effects against several different cancers. We examined the activity of WA on human cervical cancer cells in vitro and in vivo. WA potently inhibited proliferation of the cervical cancer cells, CaSki (IC(50) 0.45 ± 0.05 μM). Mechanistically, WA was found to (i) downregulate expression of HPV E6 and E7 oncoproteins, (ii) induce accumulation of p53, (iii) increase levels of p21(cip1/waf1) and its interaction with proliferating cell nuclear antigen (PCNA), (iv) cause G(2)/M cell cycle arrest, associated with modulation of cyclin B1, p34(cdc2) and PCNA levels, (v) decrease the levels of STAT3 and its phosphorylation at Tyr(705) and Ser(727) and (vi) alter expression levels of p53-mediated apoptotic markers-Bcl2, Bax, caspase-3 and cleaved PARP. In vivo, WA resulted in reduction of nearly 70% of the tumor volume in athymic nude mice with essentially similar trend in the modulation of molecular markers as in vitro. This is the first demonstration indicating that WA significantly downregulates expression of HPV E6/E7 oncogenes and restores the p53 pathway, resulting in apoptosis of cervical cancer cells. Together, our data suggest that WA can be exploited as a potent therapeutic agent for the treatment and prevention of cervical cancer without deleterious effects.

Many inflammatory diseases are associated with elevated blood concentration of fibrinogen (Fg) leading to vascular dysfunction. We showed that pathologically high (4 mg/ml) content of Fg disrupts integrity of endothelial cell (EC) layer and causes macromolecular leakage affecting tight junction proteins. However, role of adherence junction proteins, particularly vascular endothelial cadherin (VE-cadherin) and matrix metalloproteinase-9 (MMP-9) in this process is not clear. We tested the hypothesis that at high levels Fg affects integrity of mouse brain endothelial cell (MBEC) monolayer through activation of MMP-9 and downregulation of VE-cadherin expression and in part its translocation to the cytosol. The effect of Fg on cultured MBEC layer integrity was assessed by measuring transendothelial electrical resistance. Cellular expression and translocation of VE-cadherin were assessed by Western blot and immunohistochemical analyses, (respectively). Our results suggest that high content of Fg decreased VE-cadherin expression at protein and mRNA levels. Fg induced translocation of VE-cadherin to cytosol, which led to disruption of cell-to-cell interaction and cell to subendothelial matrix attachment. Fg-induced alterations in cell layer integrity and their attachment were diminished during inhibition of MMP-9 activity. Thus Fg compromises EC layer integrity causing downregulation and translocation of VE-cadherin and through MMP-9 activation. These results suggest that increased level of Fg could play a significant role in vascular dysfunction and remodeling.

Although matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) play a vital role in tumour angiogenesis and TIMP-3 caused apoptosis, their role in cardiac angiogenesis is unknown. Interestingly, a disruption of co-ordinated 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 anti-angiogenic factors causing decompensatory heart failure. To verify this hypothesis, wild type (WT) mice were studied 3 and 8 weeks 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 weeks after stenosis. The levels of MMP-2 (western blot) increased at 3 weeks and returned to control level at 8 weeks, MMP-9 increased only at 8 weeks. TIMP-2 and -4 decreased at 3 and even more at 8 weeks. The angiogenic VEGF increased at 3 weeks and decreased at 8 weeks, the anti-angiogenic endostatin and angiostatin increased only at 8 weeks. CD-31 positive endothelial cells were more intensely labelled at 3 weeks than in sham operated or in 8 weeks banded mice. Vascularization, as estimated by x-ray angiography, was increased at 3 weeks and decreased at 8 weeks post-banding. Although the vast majority of studies were performed on control WT mice only, interestingly, MMP9-KO mice seemed to have increased vascular density 8 weeks 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.

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 (p47phoxsubunit) 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.