Biomedical subjects

S C Tyagi

Publications and source records attributed to S C Tyagi.

At least 19 recordsLinked to original sources

PubMed2004

Proteomic analysis of homocysteine inhibition of microvascular endothelial cell angiogenesis.

Although homocysteine (Hcy) inhibits angiogenesis in vivo and in vitro, the mechanism(s) underlying this phenomenon are largely unclear. The hypothesis of the present work is that Hcy, while inducing the expression of antiangiogenic factors, inhibits the production of angiogenic factors. Mouse brain microvascular endothelial cells (MVEC) were cultured in the presence and absence of 20 microM Hcy for 24 hr in serum-free medium. Cell homogenates were incubated with Trans-Signal Angiogenesis Antibody Array containing antibodies to angiogenic activators (ANG, HGF, leptin, VEGF, IL-6, IL-8, PIGF, FGF-alpha/beta, TNF-alpha and TGF-alpha) and inhibitors (IFN-gamma, IL-12, IP-10, TIMP-1 and -2). The array membranes were scanned and normalized with positive controls. Angiogenesis and formation of capillaries were measured by culturing the MVEC in Matrigels. The capillary-like structures were identified by transmission microscopy. Hcy decreased the expression of leptin, IL-6, -8, PIGF, FGF-alpha and VEGF, while the levels of anti-angiogenic IL-12, IP-10 (chemokine) and TIMP-1 were increased by Hcy. The vascular tube-like structures by MVEC were decreased by increased Hcy. However, the addition of VEGF to Hcy-treated MVEC ameliorated the decreased Hcy-mediated capillary formation. The results suggest that Hcy inhibits angiogenesis, in part, by decreasing VEGF and increasing TIMP-1.

Animals↗

PubMed2003

Gelatinase B(MMP-9) an apoptotic factor in diabetic transgenic mice.

AIMS/HYPOTHESIS: Although matrix metalloproteinase-9 (MMP-9) is specifically induced and apoptosis of endothelial cells is evidenced in diabetes mellitus, the mechanism of endocardial endothelial dysfunction in diabetes mellitus is not clear. The increase in MMP-9 activity is associated with endocardial endothelial apoptosis and dysfunction in diabetes mellitus. METHODS: Diabetes was created by injecting 65 mg/kg alloxan in tail vein of MMP-9 knockout (-/-) and wild-type (WT, C57BL/J6) mice. At 8 weeks mice were grouped: (i) WT+saline; (ii) WT+alloxan; (iii) MMP+saline; (iv) MMP+alloxan. The MMP-9 genotype was determined by observing single PCR product of different mobility than the PCR product from wild-type in blood from tail vein. RESULTS: MMP-9 activity, measured by zymography, increased in plasma and in the left ventricle of alloxan-induced diabetic wild-type mice. The concentrations of cardiac inhibitor of metalloproteinase, that blocks MMP-9 activity, were decreased in diabetic MMP-9 knockouts as well as in wild-type mice. Diabetes induced apoptosis, detected by TUNEL assays, in wild-type but not in MMP-9 knockouts. Endocardial endothelial function was severely impaired in diabetic wild-type mice compared with normoglycaemic animals, while non-diabetic MMP-9 knockout mice showed partial endocardial endothelial dysfunction which was not further exacerbated by the developments of diabetes. CONCLUSION/INTERPRETATION: The results suggest an association between increased MMP-9 activity and endocardial endothelial apoptosis in diabetic mice, while genetic ablation of MMP-9 correlated with amelioration of endocardial endothelial dysfunction and apoptosis.

Animals↗

PubMed2002

Cardiac remodelling in end stage heart failure: upregulation of matrix metalloproteinase (MMP) irrespective of the underlying disease, and evidence for a direct inhibitory effect of ACE inhibitors on MMP.

OBJECTIVE: To investigate matrix metalloproteinases (MMP-2 and MMP-9) in heart failure caused by ischaemic and idiopathic dilated cardiomyopathy, and the impact of angiotensin converting enzyme (ACE) inhibition on MMP. DESIGN AND MAIN OUTCOME MEASURES: MMP were extracted from myocardium of patients with heart failure (coronary artery disease, n = 13; idiopathic dilated cardiomyopathy (IDCM), n = 16) and from controls (n = 6). The active form of MMP-2 and MMP-9 was measured by enzyme linked immunosorbent assay; activity of MMPs by zymography; mRNA expression of MMPs by reverse transcriptase polymerase chain reaction. RESULTS: Active MMP-9 was significantly increased in coronary artery disease (mean (SD) 1.6 (0.35) ng/ml) and IDCM (2.11 (0.54) ng/ml) in comparison with controls (0.53 (0.15) ng/ml). Increased MMP-2 was only found in IDCM (3.68 (0.41) ng/ml). There were corresponding increases in MMP activity but no upregulation of mRNA expression was found. The ACE inhibitors captopril and ramiprilate inhibited MMP-2 and MMP-9 activity in vitro (inhibitory capacity (IC50), in mmol/l: MMP-2: captopril 2.0 (0.16), ramiprilate 2.1 (0.3); MMP-9: captopril 1.65 (0.18), ramiprilate 2.0 (0.3)). Lisinopril inhibited MMP-9 significantly but did not inhibit MMP-2 in vitro (IC50 MMP-2: 7.4 (0.88); MMP-9: 7.86 (2.23)). Inhibition of MMP activity by ACE inhibitors was blunted by zinc excess. CONCLUSIONS: Upregulation of MMP-9 activity is common in the failing myocardium, independent of the underlying disease. Missing upregulation of transcription suggests that activation of latent forms of MMP is the source of increased MMP activity, rather than increased de novo synthesis. Some ACE inhibitors may influence MMP activity by a direct effect.

Adult↗

PubMed2001

Tissue inhibitor of metalloproteinase-4 instigates apoptosis in transformed cardiac fibroblasts.

Tumor cells become malignant, in part, because of their activation of matrix metalloproteinases (MMPs) and inactivation of tissue inhibitor of metalloproteinases (TIMPs). Myocardial tumors are rarely malignant. This raises the possibility that the MMPs and TIMPs are differentially regulated in the heart compared to other tissues. Therefore, we hypothesized that a tissue specific tumor suppressor exists in the heart. To test this hypothesis we prepared cardiac tissue extracts from normal (n = 4), ischemic cardiomypathic (ICM) [n = 5], and dilated cardiomyopathic (DCM) [n = 8] human heart end-stage explants. The level of cardiospecific TIMP-4 was determined by SDS-PAGE and Western-blot analysis. The results suggested reduced levels of TIMP-4 in ICM and DCM as compared to normal heart. TIMP-4 was purified by reverse phase HPLC and gelatin-sepharose affinity chromatography. Collagenase inhibitory activity of chromatographic peaks was determined using fluorescein-conjugated collagen as substrate and fluorescence spectroscopy. The activity of TIMP-4 (27 kDa) was characterized by reverse zymography. The role of TIMP-4 in cardiac fibroblast cell migration was examined using Boyden chamber analysis. The results suggested that TIMP-4 inhibited cardiac fibroblast cells migration and collagen gel invasion. To test whether TIMP-4 induces apoptosis, we cultured cardiac normal and polyomavirus transformed fibroblast cells in the presence and absence of TIMP-4. The number of cells were measured and DNA laddering was determined. The results suggested that TIMP-4 controlled normal cardiac fibroblast transformation and induced apoptosis in transformed cells. Cardiospecific TIMP-4 plays a significant role in regulating the normal cell phenotype. The reduced levels of TIMP-4 elicit cellular transformation and may lead to adverse extracellular matrix degradation (remodeling), cardiac hypertrophy and failure. This study suggests a possible protective role of TIMP-4 in other organs which are susceptible to malignancy.

Apoptosis↗

PubMed2001

Induction of oxidative stress by homocyst(e)ine impairs endothelial function.

Previous studies have demonstrated a relationship between hyperhomocysteinemia and endothelial dysfunction, reduced bioavailability of nitric oxide, elastinolysis and, vascular muscle cell proliferation. In vivo decreased nitric oxide production is associated with increased matrix metalloproteinase (MMP) activity and formation of nitrotyrosine. To test the hypothesis that homocysteine neutralizes vascular endothelial nitric oxide, activates metalloproteinase, causes elastinolysis and vascular hypertrophy, we isolated aortas from normotensive Wistar rats and cultured them in medium containing homocysteine, and calf serum for 14 days. Homocysteine-mediated impairment of endothelial-dependent vasodilatation was reversed by co-incubation of homocysteine with nicotinamide (an inhibitor of peroxinitrite and nitrotyrosine), suggesting a role of homocysteine in redox-mediating endothelial dysfunction and nitrotyrosine formation. The Western blot analysis, using anti-nitrotyrosine antibody, on aortic tissue homogeneates demonstrated decreased nitrotyrosine in hyperhomocysteinemic vessels treated with nicotinamide. Zymographic analysis revealed increased elastinolytic gelatinase A and B (MMP-2, -9) in homocysteine treated vessels and the treatment with nicotinamide decreases the homocysteine-induced MMP activation. Morphometric analyses revealed significant medial hypertrophic thickening (1.4 +/- 0.2-fold of control, P = 0.03) and elastin disruption in homocysteine-treated vessels as compared to control. To determine whether homocysteine causes endothelial cell injury, cross-sections of aortas were analyzed for caspase activity by incubating with Ac-YVAD-AMC (substrate for apoptotic enzyme, caspase). The endothelium of homocysteine treated vessels, and endothelial cells treated with homocysteine, showed marked labeling for caspase. The length-tension relationship of homocysteine treated aortas was shifted to the left as compared to untreated aortas, indicating reduced vascular elastic compliance in homocysteine-treated vessels. Co-incubation of homocysteine and inhibitors of MMP, tissue inhibitor of metalloproteinase-4 (TIMP-4), and caspase, YVAD-CHO, improved vascular function. The results suggest that alteration in vascular elastin/collagen ratio and activation of MMP-2 are associated with decreased NO production in hyperhomocysteinemia.

Acetylcholine↗

PubMed2001

Activation of matrix metalloproteinase dilates and decreases cardiac tensile strength.

Previous studies demonstrated that transition from compensatory pressure overload hypertrophy to decompensatory volume overload heart failure is associated with decreased cardiac tensile strength and activation of matrix metalloproteinase (MMP) in spontaneously hypertensive rat (SHR). To test the hypothesis that in the absence of nitric oxide activation of MMP during cardiac failure causes disruption in the organization of extracellular matrix (ECM) and leads to decrease systolic and diastolic cardiac tensile strength, we employed SHR of 24--32 weeks, which demonstrates significant cardiac hypertrophy and fibrosis. The normotensive Wistar rats (NWR) were used as control. To determine whether cardiac hypertrophy is associated with increased elastinolytic matrix metalloproteinase-2 (MMP-2) activity; quantitative elastin-zymography was performed on cardiac tissue homogenates. The MMP-2 activity was normalized by the levels of actin. The MMP-2/actin ratio was 2.0+/-0.5 in left ventricle (LV) and 1.5+/-0.25 in right ventricle (RV) of SHR(32wks); and 0.5+/-0.25 in LV and 0.25+/-0.12 in RV of NWR(32wks) (P<0.02 when SHR compared with NWR). To measure passive diastolic cardiac function, rings from LV as well as RV through transmyocardial wall from male SHR and NWR of 6--8 weeks and 24--36 weeks were prepared. The LV wall thickness from endocardium to epicardium was 3.75+/-0.25 mm in SHR(32wks) as compared to 2.25+/-0.50 mm in NWR(32wks) (P<0.01). The ring was placed in tissue myobath and length--tension relationships were assessed. The pressure--length relationship was shifted to left in SHR as compared to NWR. The amounts of cardiac elastin and collagen were determined spectrophotometrically by measuring desmosine--isodesmosine and hydroxyproline contents, respectively. A negative correlation between elastic tensile strength and elastin/collagen ratio was elucidated. To create situation analogous to heart failure and MMP activation, we treated cardiac rings with active MMP-2 and length--tension relation was measured. The relationship was shifted to right in both SHR and NWR when compared to their respective untreated groups. The results suggested that activation of MMP led to decreased cardiac tissue tensile strength and may cause systolic and diastolic dysfunction.

Analysis of Variance↗

PubMed2001

"A" is for amylin and amyloid in type 2 diabetes mellitus.

Amyloid deposits within the islet of the pancreas have been known for a century. In 1987, the islet amyloid precursor polypeptide (IAPP) amylin (a 37 amino acid) was discovered. Recently there has been an explosion of amylin's importance in the development of type 2 diabetes mellitus (T2DM). This review is intended to share what is understood about amylin derived amyloid and the role it plays in T2DM. Whether islet amyloid is an epiphenomenona, a tombstone, or a trigger it leaves an indelible footprint in greater that 70% of the patients with T2DM. There is current data supporting the damaging role of intermediate sized toxic amyloid particles to the beta cell resulting in a beta cell defect which contributes to a relative deficiency or loss of insulin secretion. Within the islet there is an intense redox stress which may be associated with the unfolding of amylin's native secondary structure compounding its amyloidogenic properties. In addition to the beta cell defect there may be an absorptive defect as a result of amyloid deposition in the basement membranes which form an envelope around the inta-islet capillary endothelium. We have an opportunity to change our current treatment modalities with newer medications and we should attempt to diagnose T2DM earlier and use these newer treatment strategies in combination to decrease glucotoxicity without elevating endogenous insulin and amylin. In the 21st century our goal should be to prevent remodeling, save the pancreatic islet, conquer islet amyloid, and amyloid diabetes.

Amino Acid Sequence↗

PubMed2000

Homocyst(e)ine induces calcium second messenger in vascular smooth muscle cells.

Homocysteine found in the plasma of patients with coronary heart disease, induces vascular smooth muscle cell (VSMC) proliferation and increases deposition of extracellular matrix (ECM) components. Yet, the mechanism by which homocysteine mediates this effect and its role in vascular disease is largely unknown. We hypothesized that homocysteine induces ECM production via intracellular calcium release in VSMC. To test this hypothesis, aortic VSMC from Sprague-Dawley rats were isolated and characterized by positive labeling for vascular smooth muscle alpha-actin. Early passage cells (p2-3) were grown in monolayer on coverslips. Calcium transients were quantified with fura2/AM spectrofluorometry. Homocysteine induced intracellular calcium [Ca(2+)](i) transients with an EC(50) of 60 +/- 5 nM. The EC(50) for glutathione and cysteine were 10 and 100-fold lower, respectively. Depleting extracellular calcium did not alter the homocysteine effect on intracellular calcium; however, thapsigargin pretreatment, which depletes intracellular Ca(2+) stores, abolished the homocysteine effect, demonstrating its dependence on intracellular Ca(2+) stores. Extracellular sodium depletion significantly (P < 0.05) increased [Ca(2+)](i) also suggesting a possible role of sodium-calcium exchange in the process. To begin to elucidate the intracellular pathways by which homocysteine might act, VSMC were pretreated with specific inhibitors and stimulators prior to homocysteine stimulation. Staurosporine and phorbol myrisate acetate (PMA), potent simulators of protein kinase C, augmented the release of Ca(2+) by homocysteine. Interestingly, pretreatment with the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) greatly exacerbated the sensitivity of VSMC to homocysteine. In contrast, pretreatment with either the phospholipase A(2) activator neomycin, the antioxidant and hepatic hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase inhibitor, pravastatin, the tyrosine kinase inhibitor genestein, or the calcium channel blocker, felodipine completely inhibited the homocysteine-induced Ca(2+) signal in VSMC. This suggests the role of multiple signaling pathways in the homocysteine effect on VSMC Ca(2+). Effects of homocysteine on collagen production, as ascertained by immunoblot analysis, correlated with its effect in intracellular calcium. Regardless of the signaling pathways involved, homocysteine, by virtue of its role on VSMC proliferation and ECM deposition, has the potential to affect vascular reactivity. To determine the effect of homocysteine on the ability of VSMC to react to potent agonist such as angiotensin II, VSMC were pretreated with homocysteine and exposed to a range of angiotensin II concentrations which normally have no effect on intracellular Ca(2+). After homocysteine pretreatment, VSMC were extremely responsive to angiotensin II at concentrations well below the physiologic range. These data taken together suggested that an initial effect of homocysteine is to induce release of intracellular Ca(2+) in VSMC and may induce vascular reactivity. The transient in Ca(2+) correlates with the effect on ECM associated with homocysteine.

Angiotensin II↗

PubMed2000

Hyperhomocyst(e)inemia induces multiorgan damage.

Hyperhomocyst(e)inemia has been associated with the development of hypertension, stroke, and cardiovascular, cerebral/neuronal, renal, and liver diseases. To test the hypothesis that homocyst(e)ine plays an integrated role in multiorgan injury in hypertension, we employed: (1) spontaneously hypertensive rats (SHR) in which endogenous homocyst(e)ine levels are moderately high (18.1 +/- ().5 microM); (2) control age- and sex-matched Wistar Kyoto (WKY) rats in which homocyst(e)ine levels are normal (3.7 +/- 0.3 microM). To create the pathophysiological condition of hyperhomocyst(e)inemia, 20 mg/day homocyst(e)ine was administered for 12 weeks in (3) SHR (SHR-H) and in (4) WKY (WKY-H) rats. (5) Endogenous homocyst(e)ine levels were reduced slightly but not significantly from 18.1 +/- 0.5 microM to 12.5 +/- 0.7 microM in SHR by folic acid administration (SHR-F). Plasma and tissue levels of homocyst(e)ine were determined by HPLC and spectrophotometric methods. Plasma and sympathetic ganglion (neuronal) matrix metalloproteinase (MMP) activity was measured by zymography. Activity of neuronal MMP was increased in hyperhomocyst(e)inemic rats as compared with controls. Mean arterial pressure (mmHg) was 95 +/- 5, 126 +/- 8,157 +/- 10, 188 +/- 5, and 165 +/- 12 in WKY, WKY-H, SHR, SHR-H, and SHR-F, respectively. Urinary protein (mg/day) was 0.11 +/- 0.03, 0.88 +/- 0.22, 0.47 +/- 0.10, 0.89 +/- 0.21, and 0.81 +/- 0.21 in WKY, WKY-H. SHR, SHR-H, and SHR-F, respectively, as measured by the Bio-Rad dye binding assay. The relationships between increased arterial pressure, plasma homocyst(e)ine, and urinary protein were delineated. Plasma and neuronal creatinine phosphokinase (CK) isoenzymes were measured by agarose gel electrophoresis. All three CK isoenzymes, i.e., MM, MB, and BB, specific for skeletal, cardiac, and nerve tissue, respectively, were induced following 12 weeks' hyperhomocyst(e)inemia, suggesting multiorgan injury by homocyst(e)ine. Homocyst(e)ine induces endocardial endothelial cell (capillary) apoptosis and may reduce capillary cell density. Structural damage to aorta, myocardium, kidney, and renalureter was analyzed by histology. Results suggested an integrated physiological role of homocyst(e)ine in injury to the endothelial/epithelial cell lining in the respective organs.

Animals↗

PubMed2000

Remodeling of the endocrine pancreas: the central role of amylin and insulin resistance.

BACKGROUND: Remodeling of the endocrine pancreas, caused by the deleterious effects of amylin as it is co-synthesized, co-packaged, and co-secreted with insulin, gives clinicians and researchers cause to ponder. METHODS: A literature search was done, and relevant publications and texts on amylin and islet amyloid polypeptide (IAPP) were reviewed. RESULTS: The mechanisms and clinical consequences attributed to the remodeling of the endocrine pancreas, along with proposals for reevaluating the methods of treating patients who have type 2 diabetes are illustrated and discussed. CONCLUSIONS: In addition to controlling the devastating effects ofglucotoxicity, lipotoxicity, and hypertension, we should consider the newer hypoglycemic agents with regard to their effects on the remodeling of the endocrine pancreas. This remodeling results in structural and subsequent functional changes, causing continued elevations of hemoglobin A1C. Studies are indicated to determine whether amylin (IAPP) may be implicated in the remodeling of the arterial vessel wall, the glomerulus of the kidney, and the cardiac interstitium.

Administration, Oral↗

PubMed1999

Responses of vascular smooth muscle cell to extracellular matrix degradation.

Vessels remodel to compensate for increases in blood flow/pressure. The chronic exposure of blood vessels to increased flow and circulatory redox-homocysteine may injure vascular endothelium and disrupt elastic laminae. In order to understand the role of extracellular matrix (ECM) degradation in vascular structure and function, we isolated human vascular smooth muscle cells (VSMC) from normal and injured coronary arteries. The apparently normal vessels were isolated from explanted human hearts. The vessels were injured by inserting a blade into the lumen of the vessel, which damages the inner elastic laminae in the vessel wall and polarizes the VSMC by producing a pseudopodial phenotypic shift in VSMC. This shift is characteristic of migratory, invasive, and contractile nature of VSMC. We measured extracellular matrix metalloproteinases (MMPs), tissue plasminogen activator (tPA), tissue inhibitor of metalloproteinase (TIMP), and collagen I expression in VSMC by specific substrate zymography and Northern blot analyses. The injured and elastin peptide, val-gly-val-ala-pro-gly, treated VSMC synthesized active MMPs and reduced expression of TIMP. The level of tPA and collagen type I was induced in the injured, invasive VSMC and in the val-gly-val-ala-pro-gly treated cells. To demonstrate the angiogenic role of elastin peptide to VSMC we performed in vitro organ culture with rings from normal coronary artery. After 3 days in culture the vascular rings in the collagen gel containing elastin peptide elaborated MMP activity and sprouted and grew. The results suggest that val-gly-val-ala-pro-gly peptide generated at the site of proteolysis during vascular injury may have angiogenic activity.

Amino Acid Sequence↗

PubMed1999

Temporal regulation of extracellular matrix components in transition from compensatory hypertrophy to decompensatory heart failure.

OBJECTIVE: Extracellular matrix, particularly type I fibrillar collagen, provides tensile strength that allows cardiac muscle to perform systolic and diastolic functions. Collagen is induced during the transition from compensatory hypertrophy to heart failure. We hypothesized that cardiac stiffness during decompensatory hypertrophy is partly due to a decreased elastin:collagen ratio. MATERIALS AND METHODS: We prepared left ventricular tissue homogenates from spontaneously hypertensive rats (SHR) aged 30-36 weeks, which had compensatory hypertrophy with no heart failure, and from SHR aged 70-92 weeks, which had decompensatory hypertrophy with heart failure. Age- and sex-matched Wistar-Kyoto (WKY) rats were used as normotensive controls. In both SHR groups, increased levels of collagen were detected by immuno-blot analysis using type I collagen antibody. Elastin and collagen were quantitated by measuring desmosine/isodesmosine and hydroxyproline spectrophometrically, respectively. To determine whether the decrease in elastin content was due to increased elastinolytic activity of matrix metalloproteinase-2, we performed gelatin and elastin zymography on left ventricular tissue homogenates from control rats, SHR with compensatory hypertrophy and SHR with heart failure. RESULTS: The elastin:collagen ratio was 0.242 +/- 0.008 in hearts from WKY rats. In SHR without heart failure, the ratio was decreased to 0.073 +/- 0.003 and in decompensatory hypertrophy with heart failure, the ratio decreased to 0.012 +/- 0.005. Matrix metalloproteinase-2 activity was increased significantly in SHR with heart failure compared with controls (P < 0.001). The level of tissue inhibitor of metalloproteinase-4 was increased in compensatory hypertrophy and markedly reduced in heart failure. Decorin was strongly reduced in decompensatory heart failure compared with control hearts. CONCLUSIONS: Since collagen was induced in SHR with heart failure, decorin and elastin were decreased and the ratios of gelatinase A and elastase to tissue inhibitor of metalloproteinase-4 were increased, we conclude that heart failure is associated with adverse extracellular matrix remodeling.

Animals↗

PubMed1999

Homocyst(e)ine and heart disease: pathophysiology of extracellular matrix.

Occlusive coronary artery disease is an important factor of cardiovascular morbidity and mortality. The rupture of the thin fibrous cap of the atheroma may be one of the causes of acute coronary syndrome, however, the mechanism of formation of fibrous plaque are poorly understood. Elevation of plasma homocysteine, hyperhomocystinemia, H(e), has emerged as an independent risk factor for hypertension and fibrotic heart disease. The extracellular matrix (ECM) components, particularly fibrillar collagen, are elevated in the atherosclerotic lesions and are the essential integral element in holding the oxidized low density lipoproteins (LDL), homocystine, macrophage and foam cells in milieu, constituting the primary atherosclerotic and secondary restenotic lesions. In vivo and in vitro physiological, morphological, cellular, biochemical and molecular experiments have suggested the role of tissue homocystine in cardiovascular fibrosis and adverse ECM remodeling following H(e). The tissue homocystine induces cardiovascular fibrosis and may lead to heart failure via the redox-receptor pathway. The underlying cause and mechanism of cardiovascular fibrosis associated with arteriosclerosis, atherosclerosis, hypertension and coronary heart disease, involve changes in the levels of tissue redox state.

Animals↗

PubMed1999

Homocyst(e)ine impairs endocardial endothelial function.

Homocyst(e)ine injured vascular endothelium and modulated endothelial-dependent vascular function. Endothelium plays an analogous role in both the vessel and the endocardium. Therefore, we hypothesized that homocyst(e)ine modulated endocardial endothelium (EE) dependent cardiac function. The ex vivo cardiac rings from normal male Wistar-Kyoto rats were prepared. The contractile responses of left and right ventricular rings were measured in an isometric myobath, using different concentrations of CaCl2. The response was higher in the left ventricle than right ventricle and was elevated in endocardium without endothelium. The half effective concentration (EC50) and maximum tension generated by homocyst(e)ine were 10(6) and 5-fold lower than endothelin (ET) and angiotensin II (AII), respectively. However, in endothelial-denuded endocardium, homocyst(e)ine response was significantly increased (p<0.005, compared with intact endothelium) and equal to the response to ET and AII. To determine the physiological significance of ET, AII, homocyst(e)ine, and endothelial nitric oxide in EE function, cardiac rings were pretreated with AII (10(-10) M) or ET (10(-13) M) and then treated with homocyst(e)ine (10(-8) M). Results suggested that at these concentrations AII, ET, or homocyst(e)ine alone had no effect on cardiac contraction. However, in the presence of 10(-10) M AII or 10(-13) M ET, the cardiac contraction to homocyst(e)ine (10(-8) M) was significantly enhanced (p<0.01, compared with without pretreatment) and further increased in the endocardium without endothelium. The pretreatment of cardiac ring with the inhibitor of nitric oxide, Nomega-nitro-L-arginine methyl ester (L-NAME), increased contractile response to homocyst(e)ine. These results suggested that homocyst(e)ine impaired EE-dependent cardiac function and acted synergistically with AII and ET in enhancing the cardiac contraction.

Angiotensin II↗

PubMed1998

Activation of extracellular matrix metalloproteinases in equine laminitis.

Samples of connective tissue obtained from the hoof of six laminitic and eight non-laminitic adult horses were analysed zymographically to investigate whether connective tissue matrix metalloproteinases are activated or induced during laminitis. The activity or matrix metalloproteinases was substantially greater in the tissues from the laminitic horses than in the tissues from the non-laminitic horses. A comparison of the collagenolytic activity in the laminitic and control tissues showed that collagenolytic activities corresponding to the 92 kDa (P < 0.001), 72 kDa (P < 0.01) and 66 kDa (P < 0.01) bands were induced in the laminitic tissues.

Animals↗

PubMed1998

Extracellular matrix dynamics in heart failure: a prospect for gene therapy.

In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is impairment of myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs), which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation and heart failure. There is no known program that has declared its aim the investigation of the role of ECM gene therapy in heart failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or downregulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor-beta1 (TGF-beta), decorin, and collagen in cardiomyopathy and heart failure.

Extracellular Matrix↗

PubMed1998

Stretch-induced membrane type matrix metalloproteinase and tissue plasminogen activator in cardiac fibroblast cells.

In the normal heart, cardiomyocytes are surrounded by extracellular matrix (ECM) and latent matrix metalloproteinases (MMPs), which are produced primarily by cardiac fibroblasts. An activator of latent MMPs might be induced by ischemic conditions or pressure-induced stretching. To test the hypothesis that an activator of latent MMP is induced in the ischemic heart during transformation of a compensatory hypertrophic response to a decompensatory failing response in cardiac fibroblast cells, we stretched the human cardiac fibroblasts at 25 cycles/min in serum-free or 5% serum culture condition. The membrane type (MT)-MMP activity in stretched cells was measured by zymography and immuno-blot analyses using MT-MMP-2 antibody. The MT-MMP activity was further characterized by transverse-urea gradient (TUG)-zymography. The results suggested that stretch induced a membrane MMP in the fibroblasts that was similar to the MT-MMP induced in ischemic heart. Furthermore, we observed that membrane MMP has distinct mobility in TUG-zymography. To localize the MT-MMP and tissue plasminogen activator (tPA) of latent MMPs, the membrane and cytosol were separated by a method employing a detergent and sedimentation. The MT-MMP and tPA activities of cytosol and membrane fractions were measured by gelatin- and plasminogen-zymography, respectively. Differential-display mRNA analysis was performed on control and stretched cells. In situ immuno-labelling was performed to localize the MT-MMP. The results indicate that induction of MT-MMP occurred in the membrane fractions. The secretion of tPA was elevated in the stretched cells. The MT-MMP activity was inhibited by prior incubation with an antibody generated to membrane MMP. The tPA activity was inhibited by using tPA antibody. These results suggest that, under stretched conditions, neutral transmembrane matrix proteinases are induced in the cardiac fibroblasts. This may lead to activation of adverse ECM remodeling, cardiac dilatation, and failure.

Blotting, Northern↗

PubMed1998

Pouch tissue and angiotensin peptide generation.

Myofibroblasts and their potential to generate angiotensin (Ang) II and transforming growth factor beta 1 (TGF-beta 1) at sites of infarction in the rat heart have been implicated in tissue repair. These cells likewise contribute to repair in a subcutaneous pouch model of fibrous tissue formation. Their appearance in pouch tissue coincides with high density ACE and Ang II receptor binding, suggesting a role for Ang II in tissue repair. Using pouch tissue studied at different time points of repair, the present study examined the expression of requisite mRNA for Ang peptide generation: angiotensinogen, Ao; an aspartyl protease, either cathepsin-D, Cat-D, or renin: and angiotensin converting enzyme, ACE, TGF-beta 1 and type I collagen mRNA expression was also addressed. Unlike pouch studied on day 2 and 4, at 7, 14 and 21 days, we found: (a) expression of Ao, Cat-D but not renin, ACE and TGF-beta 1 mRNA; (b) Ang I and Ang II peptides in pouch tissue and exudate; (c) the presence of Cat-D activity but no renin activity; (d) an increase in type I collagen mRNA with time; (e) upregulation of pouch tissue ACE mRNA expression by lisinopril treatment, whereas AT1 and AT2 receptor antagonists (losartan and PD 123177, respectively) downregulated the expression of mRNA for ACE, when compared to untreated controls; (f) downregulation of TGF-beta 1 mRNA expression by lisinopril and losartan compared to untreated controls; and (g) PD 123177 had no effect, whereas lisinopril and losartan treatment significantly (P < 0.05) reduced type I collagen mRNA expression. Thus, in this model of fibrous tissue formation, we found expression of component genes involved in Ang peptide (I and II) and TGF-beta 1 generation and Ang II upregulation of TGF-beta 1 expression, suggesting Ang II and/or TGF-beta 1 may upregulate type I collagen expression during tissue repair. Pharmacologic intervention studies with lisinopril or losartan indicate Ang II plays a role in the reciprocal regulation of ACE mRNA expression, which modulates Ang II levels at sites of repair.

Angiotensin I↗