S100A8/A9 in Myocardial Infarction.

S100A8/A9 represents a novel biomarker and therapeutic target in sterile inflammatory diseases. Among the various S100 proteins, S100A8 and S100A9 have been shown to be the most important of all the damage-associated molecular pattern (DAMP) proteins in sterile inflammatory conditions such as diabetes, cardiovascular disease, autoimmune disorders, etc. We present here methods to quantify S100A8/A9 expression in various tissues in mouse models of myocardial infarction (MI) using flow cytometry (FC), immunofluorescence, quantitative real-time polymerase chain reaction (q-RT-PCR), and enzyme-linked immunosorbent assays (ELISA).

GPCR agonist-induced transactivation of the EGFR upregulates MLC II expression and promotes hypertension in insulin-resistant rats.

AIMS: The presence of metabolic abnormalities such as insulin resistance and elevated levels of various vasoconstrictor G-protein-coupled receptor (GPCR) agonists contributes to the development of hypertension. Recent studies have suggested a link between disease progression and activation of growth factor receptor signalling pathways such as the epidermal growth factor receptor (EGFR) by matrix metalloproteinases (MMPs). We hypothesized that excessive stimulation of GPCRs such as alpha(1)-adrenergic receptors activates MMP-dependent EGFR transactivation and contributes to the development of hypertension by promoting increased synthesis of contractile proteins in vascular smooth muscle (VSM). METHODS AND RESULTS: We tested this concept in experiments using insulin-resistant VSM cells (VSMCs) and fructose hypertensive rats (FHRs), a model of acquired systolic hypertension and insulin resistance. We found that insulin resistance and agonist stimulation increased the expression and activity of MMPs (MMP-2 and MMP-7), the EGFR, contractile proteins such as myosin light chain kinase and MLC II, and their transcriptional activators including P90 ribosomal kinase (P90RSK) and serum response factor, possibly via the activation of extracellular signal-regulated kinase (ERK1/2) in VSMCs. Further, in insulin-resistant VSMCs and arteries from FHRs, disruption of MMP-EGFR signalling either by a pharmacological or small interfering RNA approach normalized the increased expression and activity of contractile proteins and their transcriptional activators and prevented the development of hypertension in FHRs. CONCLUSION: Our data suggest that the MMP-EGFR pathway could be a potential target in the treatment of hypertension in insulin resistance and/or hyperglycaemic conditions such as type 2 diabetes.

Selective inhibition of protein kinase C beta(2) attenuates inducible nitric oxide synthase-mediated cardiovascular abnormalities in streptozotocin-induced diabetic rats.

OBJECTIVE: Impaired cardiovascular function in diabetes is partially attributed to pathological overexpression of inducible nitric oxide synthase (iNOS) in cardiovascular tissues. We examined whether the hyperglycemia-induced increased expression of iNOS is protein kinase C-beta(2) (PKCbeta(2)) dependent and whether selective inhibition of PKCbeta reduces iNOS expression and corrects abnormal hemodynamic function in streptozotocin (STZ)-induced diabetic rats. RESEARCH DESIGN AND METHODS: Cardiomyocytes and aortic vascular smooth muscle cells (VSMC) from nondiabetic rats were cultured in low (5.5 mmol/l) or high (25 mmol/l) glucose or mannitol (19.5 mmol/l mannitol + 5.5 mmol/l glucose) conditions in the presence of a selective PKCbeta inhibitor, LY333531 (20 nmol/l). Further, the in vivo effects of PKCbeta inhibition on iNOS-mediated cardiovascular abnormalities were tested in STZ-induced diabetic rats. RESULTS: Exposure of cardiomyocytes to high glucose activated PKCbeta(2) and increased iNOS expression that was prevented by LY333531. Similarly, treatment of VSMC with LY333531 prevented high glucose-induced activation of nuclear factor kappaB, extracellular signal-related kinase, and iNOS overexpression. Suppression of PKCbeta(2) expression by small interference RNA decreased high-glucose-induced nuclear factor kappaB and extracellular signal-related kinase activation and iNOS expression in VSMC. Administration of LY333531 (1 mg/kg/day) decreased iNOS expression and formation of peroxynitrite in the heart and superior mesenteric arteries and corrected the cardiovascular abnormalities in STZ-induced diabetic rats, an action that was also observed with a selective iNOS inhibitor, L-NIL. CONCLUSIONS: Collectively, these results suggest that inhibition of PKCbeta(2) may be a useful approach for correcting abnormal hemodynamics in diabetes by preventing iNOS mediated nitrosative stress.

Maintenance of adrenergic vascular tone by MMP transactivation of the EGFR requires PI3K and mitochondrial ATP synthesis.

AIMS: G-protein-coupled receptors (GPCRs) modulate vascular tone, at least in part, via matrix metalloproteinase (MMP) transactivation of the epidermal growth factor receptor (EGFR). We previously have identified novel signalling pathways downstream of the EGFR suggestive of mitogen-activated protein kinase and mitochondrial redox control of vascular tone. In the present study, we examined whether MMP modulation of vascular tone involves phosphoinositide 3-kinase (PI3K) and mitochondrial ATP synthesis. METHODS AND RESULTS: To determine whether PI3K is required for the maintenance of adrenergic vascular tone, we first constricted rat small mesenteric arteries with phenylephrine (PE) and then perfused with PI3K inhibitors, LY294002 and wortmannin, both of which produced a dose-dependent vasodilatation. Next, to investigate whether MMPs modulate PI3K activity, we cultured rat aortic vascular smooth muscle cells (VSMCs) and stimulated them with GPCR agonists such as PE and angiotensin II. Inhibition of MMPs (by GM6001) or EGFR (by AG1478) or suppressing the expression of MMP-2 or MMP-7 or the EGFR by small interfering RNA blunted the PI3K phosphorylation of Akt induced by PE. Further, in VSMCs, PI3K inhibitors reduced the PE-induced increase in ATP synthesis and glucose transporter-4 translocation, an effect that was also observed with MMP and the EGFR inhibitors. Further, the PE-induced increase in ATP synthesis activated MMP-7 by mechanisms involving purinergic (P2X) receptors and calcium. CONCLUSION: These data suggest that the maintenance of adrenergic vascular tone by the MMP-EGFR pathway requires PI3K activation and ATP synthesis. Further, our data support the view that elevated levels of GPCR agonists exaggerate the MMP transactivation of EGFR response and contribute to enhanced vascular tone and development of cardiovascular disease such as hypertension.

Chronic inhibition of inducible nitric oxide synthase ameliorates cardiovascular abnormalities in streptozotocin diabetic rats.

Previous studies from our lab have demonstrated cardiovascular abnormalities such as depressed mean arterial blood pressure and heart rate, endothelial dysfunction and attenuated pressor responses to vasoactive agents in streptozotocin diabetic rats. We investigated whether these abnormalities are due to diabetes-associated chronic activation of inducible nitric oxide synthase (iNOS). Control and streptozotocin (60 mg/kg, iv) diabetic rats were treated with either vehicle or N6-(1-Iminoethyl)-L-lysine dihydrochloride (L-NIL, 3 mg/kg/day, p.o), a specific inhibitor of iNOS for 8 weeks. At the end of treatment, the mean arterial blood pressure and heart rate were measured in freely moving conscious rats. Further, pressor responses to bolus doses of methoxamine were determined. Endothelial nitric oxide synthase (eNOS) and iNOS expression as well as nitrotyrosine (NT) levels were assessed in the heart and superior mesenteric arteries by western blot and immunohistochemistry. Untreated diabetic rats showed depressed mean arterial blood pressure and heart rate and exhibited vascular hyporeactivity that were significantly improved by treatment with L-NIL. Further, decreased eNOS expression and increased iNOS expression and activity were associated with increased NT levels in the heart and superior mesenteric arteries of untreated diabetic rats. L-NIL treatment of diabetic rats normalized the expression of eNOS and NT levels without any effect on iNOS expression in the heart and superior mesenteric arteries. The results of our study suggest that induction of iNOS in cardiovascular tissues contributes significantly to the depressed mean arterial blood pressure, heart rate and pressor responses to vasoactive agents. Chronic inhibition of iNOS in diabetes may prove beneficial in the treatment of cardiovascular abnormalities.

Gonadectomy prevents endothelial dysfunction in fructose-fed male rats, a factor contributing to the development of hypertension.

Insulin resistance has been shown to be associated with increased blood pressure (BP). The sex hormones estrogen and testosterone have opposing effects in the development of increased BP. Since testosterone has been implicated in increased BP following insulin resistance, we have tried to dissect out the effects of insulin resistance on endothelium-dependent vasorelaxation in the presence and absence of testosterone. Both gonadectomized and sham-operated male Wistar rats fed with a high-fructose diet developed insulin resistance, but BP increased only in the sham-operated rats. Reintroduction of testosterone in vivo restored the increase in BP, thereby abolishing the protective effects of gonadectomy. Fructose feeding did not affect plasma testosterone levels. Insulin resistance induced endothelial dysfunction in the mesenteric arteries of sham-operated rats, which was prevented by gonadectomy, thus suggesting a key role for testosterone in the pathogenesis of secondary vascular complications. Subsequent to blocking the actions of endothelium-dependent hyperpolarizing factor (EDHF), relaxation to acetylcholine (ACh) was lower in sham-operated fructose-fed rats compared with other groups, suggesting the involvement of nitric oxide (NO) in vasorelaxation. Inhibition of NO synthesis nearly abolished the ACh-evoked relaxation in both fructose-fed groups, thus suggesting a testosterone-independent impairment of EDHF-mediated relaxation. The improvement in endothelial function following gonadectomy could be ascribed to a NO component, although plasma nitrite and nitrate levels were unchanged. In summary, testosterone is essential in vivo for the development of endothelial dysfunction and hypertension secondary to insulin resistance, suggesting a facilitatory role for testosterone in increasing BP in fructose-fed male rats.

N-acetylcysteine prevents nitrosative stress-associated depression of blood pressure and heart rate in streptozotocin diabetic rats.

Previous studies have indicated that cardiovascular abnormalities such as depressed blood pressure and heart rate occur in streptozotocin (STZ) diabetic rats. Chronic diabetes, which is associated with increased expression of inducible nitric oxide synthase (iNOS) and oxidative stress, may produce peroxynitrite/nitrotyrosine and cause nitrosative stress. We hypothesized that nitrosative stress causes cardiovascular depression in STZ diabetic rats and therefore can be corrected by reducing its formation. Control and STZ diabetic rats were treated orally for 9 weeks with N-acetylcysteine (NAC), an antioxidant and inhibitor of iNOS. At termination, the mean arterial blood pressure (MABP) and heart rate (HR) were measured in conscious rats. Nitrotyrosine and endothelial nitric oxide synthase (eNOS) and iNOS expression were assessed in the heart and mesenteric arteries by immunohistochemistry and Western blot experiments. Untreated diabetic rats showed depressed MABP and HR that was prevented by treatment with NAC. In untreated diabetic rats, levels of 15-F(2t)-isoprostane, an indicator of lipid peroxidation increased, whereas plasma nitric oxide and antioxidant concentrations decreased. Furthermore, decreased eNOS and increased iNOS expression were associated with elevated nitrosative stress in blood vessel and heart tissue of untreated diabetic rats. N-acetylcysteine treatment of diabetic rats not only restored the antioxidant capacity but also reduced the expression of iNOS and nitrotyrosine and normalized the expression of eNOS to that of control rats in heart and superior mesenteric arteries. The results suggest that nitrosative stress depress MABP and HR following diabetes. Further studies are required to elucidate the mechanisms involved in nitrosative stress mediated depression of blood pressure and heart rate.

Withania somnifera improves bone calcification in calcium-deficient ovariectomized rats.

Osteoporosis, characterized by reduction in bone density, is a significant source of mortality among the elderly, particularly in oestrogen-deficient women. We studied the effect of Withania somnifera (WS) root extract (ethanolic), which contains oestrogen-like withanolides for anti-osteoporotic activity. Female Sprague-Dawley rats were either sham operated (n = 12) or ovariectomized (n = 12) and treated with WS/vehicle (65 mg kg(-1)), orally for 16 weeks (n = 12). All rats were allowed free access to a calcium-deficient diet (0.04% Ca) and distilled water. At termination, urinary excretion of calcium (Ca) and phosphorus (P) and serum levels of Ca, P and alkaline phosphatase (ALP) were measured. Femur and tibia bones were processed for histological (histology), morphological (scanning electron microscopy, SEM), biomechanical strength (impact test) and mineral composition (ash) analysis. Ovariectomized (OVX) rats showed a significant increase in serum ALP levels and urinary Ca and P excretion. Histological findings revealed narrowed, and disappearance of, trabeculae with widened medullary spaces in the OVX group. Ash analysis showed a reduction in ash weight, percent ash, ash Ca, ash P and ash magnesium levels in the OVX group. Further, SEM examination revealed metaphyseal bone loss in femurs and impact test showed a reduction in biomechanical strength of tibias in OVX rats. WS treatment markedly prevented the above changes in OVX rats and thus may be a potential agent in the treatment of osteoporosis.

Increased expression of iNOS is associated with endothelial dysfunction and impaired pressor responsiveness in streptozotocin-induced diabetes.

Studies in streptozotocin (STZ)-induced diabetic rats have demonstrated cardiovascular abnormalities such as depressed mean arterial blood pressure (MABP) and heart rate (HR), endothelial dysfunction, and attenuated pressor responses to vasoactive agents. We investigated whether these abnormalities are due to diabetes-associated activation of inducible nitric oxide synthase (iNOS). In addition, the effect of the duration of diabetes on these abnormalities was also evaluated. Diabetes was induced by administration of 60 mg/kg STZ via the tail vein. One, 3, 9, or 12 wk after STZ injection, MABP, HR, and endothelial function were measured in conscious unrestrained rats. Pressor response curves to bolus doses of methoxamine (MTX) and angiotensin II (ANG II) were constructed in the presence of N-[3(aminomethyl)benzyl]-acetamidine, dihydrochloride (1400W), a specific inhibitor of iNOS. Depressed MABP and HR and impairment of endothelial function were observed as early as 3 wk after induction of diabetes. Acute inhibition of iNOS with 1400W (3 mg/kg i.v.) restored the attenuated pressor responses to both MTX and ANG II without affecting the basal MABP and HR. Immunohistochemical and Western analysis blot studies in cardiovascular tissues revealed decreased expression of endothelial nitric oxide synthase (eNOS) concomitant with increased expression of iNOS and nitrotyrosine with the progression of diabetes. Our findings suggest that induction of iNOS in cardiovascular tissues is dependent on the duration of diabetes and contributes significantly to the depressed pressor responses to vasoactive agents and potentially to endothelial dysfunction.

Oral administration of sodium tungstate improves cardiac performance in streptozotocin-induced diabetic rats.

Normalization of hyperglycemia and hyperlipidemia is an important objective in preventing diabetes-induced cardiac dysfunction. Our study investigated the effects of sodium tungstate on cardiac performance in streptozotocin-induced (STZ) diabetic rats based on its potential antidiabetic and antioxidant activity. Male Wistar rats were made STZ-diabetic and then treated with tungstate in their drinking water for 9 weeks. Body mass, food and fluid intake, plasma glucose, insulin, triglyceride, and free fatty acids levels were measured. At the termination of the study period, an oral glucose tolerance test (OGTT) was performed, and cardiac performance was evaluated using an isolated working heart apparatus. Tungstate-treated STZ-diabetic rats showed a significant reduction in fluid and food intake, plasma glucose, triglycerides, and free fatty acid levels, and improved tolerance to glucose in OGTT, owing to tungstate-mediated enhancement of insulin activity rather than increased insulin levels. Left ventricular pressure development, the rate of contraction (+dP/dT), and the rate of relaxation (-dP/dT) were significantly improved in tungstate-treated diabetic rats. Apart from a decreased rate of body mass gain, no other signs of toxicity or hypoglycemic episodes were observed in tungstate-treated rats. This study extends previous observations on the antidiabetic activities of tungstate, and also reports for the first time the salutary effects in preventing diabetic cardiomyopathy.