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EL-MINIA MED. BULL. VOL. 19, NO. 2, JUNE, 2008 Ibrahim et al NITRIC OXIDE AND FIBRONECTIN IN CARDIAC TISSUE OF DIABETIC HYPERTENSIVE RATS; EFFECT OF CAPTOPRIL By Mohamed Abdellah Ibrahim*, Tetsuto Kanzaki**, Adel Hussian Saad***, Ahmed Mohmed Mahmoud**** and Shiro Ueda*****. Departments of *Pharmacology, Minia University, Minia City, Egypt. **Clinical Medicine, Faculty of Pharmacy, Chiba Institute of Science, Shiomi-cho, Choshi, Chiba, JAPAN, ***Physiology, Minia University, Minia City, Egypt, ****Drug Information and Communication, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana, Chuo-ku, Chiba City, Japan and *****Biochemistry, Minia University, Minia City, Egypt. ABSTRACT: Diabetes and hypertension are interrelated diseases that represent major risk factors for developing cardiovascular complications such as cardiomyopathy. The mechanism of developing diabetic cardiomyopathy is not well documented and may include modulation of nitric oxide (NO) and extracellular matrix, fibronectin. We studied effect of experimentally induced diabetes on cardiac NO metabolites (NOx) and fibronectin in a model of spontaneous hypertensive rats (SHR); and possible modulation by administration of captopril. Diabetes caused significant decrease in NOx and increase in fibronectin expression in cardiac tissue of SHR. Four weeks treatment with captopril prevented cardiac hypertrophy, and decreased fibronectin expression but not affecting NOx in cardiac tissues of SHR. KEYWORDS: Nnitric oxide Diabetic cardiomyoathy Fibronectin Captopril multifactorial, including modulation of nitric oxide (NO) and expression of extracellular matrix, fibronectin (Sowers et al. 1993; Chen et al. 2003). INTRODUCTION: Hypertension and diabetes are interrelated diseases that represent major risk factors for cardiovascular diseases (Epstein and Sowers 1992; Charles and Lee 1995). Both diabetes and hypertension are associated with cardiomyopathy (Grossman and Messerli 1996; Sowers and Epstein 2001). Although the clinical and morphologic features of hypertensivediabetic cardiomyopathy were descrybed more than 2 decades ago (Factor et al. 1980), the mechanism of developing hypertensive or diabetic cardiomyopathy is not well documented (Lip et al. 2000). It has been reported that the mechanisms by which diabetes produces cardiomyopathy are Nitric oxide, which is synthesized from L-arginine by nitric oxide synthase (NOS), plays an important role in regulating cardiac functions. Long-term treatment of rats with the NOS inhibitor NG-nitro-Larginine methylester (L-NAME) results in development of arterial hypertension, bradycardia and left ventricular hypertrophy (Johnson and FRREMAN 1992; Bernatova et al. 1996) decrease of cardiac output (Hampl et al. 1993), and increased cardiac fibrosis (Babal et al. 1997; 328 EL-MINIA MED. BULL. VOL. 19, NO. 2, JUNE, 2008 Bernatova 1999). The level of NO in diabetes is somewhat controversial, with studies providing for an increase (Graier et al. 1996; Cosentino et al. 1997) a decrease [Balon and Nadler 1997; Pieper GM 1998), or no change (Smits et al. 1993; Schmetterer et al. 1997). Fibronectin is an extracellular matrix glycoprotein, and its overexpression may contribute to vascular diseases (Sowers et al. 1993). It plays a central role in regulating morphogenesis and functional maturation in developing tissues through its effects on cell adhesion, differenttiation, and migration (Pichika and Homandberg, 2004). Both NO and fibronectin play a role in the development of vascular tissues and the NOS inhibitor L-NGmonomethylarginine (LNMMA) decreased fibronectin synthesis, whereas the NO donor, Snitroso-N-acetylpenicillamine (SNAP), increased fibronectin synthesis (Catherine et al. 1999; Pichika and Homandberg, 2004). Diabetes causes overproduction of cardiac extracellular matrix (ECM), which contributes to diastolic dysfunction (Chen et al. 2000). Ibrahim et al strating that ACE-inhibition was associated with antitrophic effects in diabetes and hypertension (Lassila et al. 2003). Mechanisms that account for the effects of ACE inhibitors on diabetes-induced cardiovascular dysfunctions are not clear but may be related to effects on insulin sensitivity (Torlone et al. 1993), inhibition of oxidative stress (Rajagoplan and Harrison 1996) and/or modulation of NO pathway (Yayuz et al. 2003). In the present investigation, we report the effect of diabetes on cardiac tissue NO and fibronectin in spontaneous hypertensive rats (SHR) and the modulation of these effects by the administration of captopril. MATERIALS AND METHODS: Chemicals: The sources of materials were as follows: streptozotocin (STZ), and bovine serum albumin from Sigma Chemical Co. (St. Louis, MO, USA); Captopril, Sankyo Co. (Tokyo, Japan); Nitrite/nitrate colorimetric assay kit from Caymen Chemical Co. (Ann Arbor, MI, USA); DC protein assay kit from Bio Rad Lab (Hercules, CA, USA); Western blot ECL immunodetection kit from GE healthcare UK limited (Buckinghamshire, UK). Currently, one of the most important therapeutic approaches to prevent diabetic cardiaovasculr complications is rigorous blood pressure control (Cooper and Johnston 2000). Angiotensin converting enzyme (ACE) inhibitors, because of their protective effects on the cardiovascular system and kidney, are regarded as the firstline antihypertensive therapy for patients with diabetes (World Health Organization 1999). It has been reported that reninangiotensin system may be involved in the pathogenesis of diabetic cardiomyopathy, since captopril can improve and reverse the cardiomyopathy of diabetic rats (Rösen et al. 1995). lassila M, et al. demon- Antibody: Antibody against fibronectin was a generous gift from Dr. Tetsuto Kanzaki (Kanzaki et al. 1997). Animal protocol: Male SHR (Nippon SLC Co. Ltd. Shizuoka, Japan) aged 10 weeks, weighing 240-290 g were acclimatized for one week to handling and blood pressure measurement by tail-cuff method. All experimental procedures were done in accordance with 329 EL-MINIA MED. BULL. VOL. 19, NO. 2, JUNE, 2008 institutional ethical guidelines for animal research (Chiba University).” Ibrahim et al reaction using a commercial colorimetric assay kit. Briefly, in the first step, nitrate was converted to nitrite utilizing nitrate reductase. In the second step, the addition of Griess reagents converted nitrite into a deep purple azo compound. Photometric measurements by the microplate reader (Titertek Multiskan MCC/340, Dainippon seiyaku, Osaka, Japan) at 540 nm were used to determine the NOx levels. Induction of diabetes: Diabetes was induced by a single intraperitoneal injection of 65 mg/kg STZ (Ibrahim et al. 2005). Rats with blood glucose ≥ 16.7mmol/L were considered diabetic and used in the experiments. The rats were allocated into 3 groups: control group 1, (number (n) = 7) non-diabetic SHR group; group 2, (n = 7) diabetic SHR group; group 3, (n = 8) diabetic SHR group receiving captopril for 4 weeks at 80 mg/kg in drinking water, a dose previously reported to reduce blood pressure in both diabetic and nondiabetic SHR (Sharma and Kesavarao 2002). Immunoblotting of fibronectin in cardiac homogenates: The frozen samples were homogenized in lysis buffer (1% SDS, 300 mM NaCl, 10 mM Tris-HCl, PH 7.4, protease inhibitor Cocktail (Sigma Co., Japan) 1/100). The homogenates were centrifuged at 15,000g for 5 min at 4 C, and the supernatant served as tissue extracts. Total protein of tissue extracts was assayed using DC protein assay kit (Bio RAD Co., Japan). Equal amounts of tissue proteins were separated on SDS-PAGE (7.5% polyacrylamide gels) and electrically transferred to a nitrocellulose membrane (GE healthcare UK Limited). Western blot analysis was done using polyclonal antibody against rat fibronectin (a gift from Dr. Kanzaki) after blocking non-specific binding sites with 5% bovine serum albumin (Sigma). For immunodetection, we used an ECL kit (GE healthcare UK Limited), according to manufacturer protocol. Chemiluminescence signals were quantified using the Las-1000 plus. Measurement of blood pressure: Body weight and mean blood pressure (MBP) were determined once a week during the course of the experiment. MBP was measured by the tail-cuff method in conscious, lightly restrained rats (Zartz 1990). At least 3 determinations were made at every session, and the mean of the three values was used to obtain MBP. Blood and organ collections: Rats were anesthetized with diethyl ether and blood samples were collected from abdominal aortas. Plasma samples were stored at –70oC until assayed. Determination of cardiac hypertrophy: The heart was rapidly excised, dried, weighed and divided by body weight to determine cardiac index. The heart was stored at – 70oC for further assays. Statistical analysis: Data were expressed as mean ± SD. Statistical significance of differences between groups was evaluated by using ANOVA, unpaired Student’s t test and Mann whitney test. Detection of the level of NO: Nitrite/nitrate, as marker of NO level, was determined by Griess 330 EL-MINIA MED. BULL. VOL. 19, NO. 2, JUNE, 2008 Ibrahim et al mm Hg) compared with either group 1 (165 ± 5.85 mm Hg) or group 2 (160 ± 12.5 mm Hg) (Table 1). RESULTS: 1- Blood glucose, blood pressure, and cardiac index Blood glucose levels, four weeks after STZ injection were: group 1 (8.8±1.0 mmol/L); group 2, (20.6±5.1 mmol/L); group 3 (26.9±6.9 mmol/L). There was no significant difference in blood glucose between groups 2 and 3 (Table 1). MBP was significantly lower in the captopril-treated group 3 (120 ± 7.21 Cardiac index (heart weight (mg)/body weight (g)) was signifycantly lower in the captopril-treated group 3 (3.4 ± 0.14 mg/g) compared with either group 1 (3.8 ± 0.15 mg/g) or group 2 (3.8 ± 0.22 mg/g) (Table 1). Table 1: Animal conditions and cardiac index (at 0 week) Body Blood weight glucose (g) (mm/L) at sacrifice (4 weeks) Body Blood weight glucose (g) (mg/dL) Mean Mean Cardiac blood blood index pressure pressure (mg/g) (mmHg) (mmHg) Group1 264 7.2 143 329 8.8 165 3.8 (n = 7) ± 21 ± 0.9 ± 6.55 ± 15 ± 1.0 ±6 ± 0.15 Group2 278 18.1 139 278* 20.6 160 3.8 (n = 7) ±4 ± 1.0 ± 10.96 ± 23 ± 5.1 ± 13 ± 0.22 Group3 278 19.9 137 258* 26.9 120* ☼ 3.4 (n = 8) ± 13 ± 3.6 ± 11.66 ± 37 ± 6.9 ±7 ± 0.14* The results were expressed as means ± S.D., and analyzed by unpaired student t- test. Values of P < 0.05 were considered statistically significant. * Significant difference compared with group 1. ☼ Significant difference compared with group 2. Cardiac NOx level was significantly decreased in groups 2 and 3 compared with control group 1. Captopril treatment did not affect the cardiac NOx level 2- NO in cardiac tissues Cardiac NOx levels were 43 ± 18 nmol/mg protein, 22 ± 9 nmol/mg protein and 24 ± 6 nmol/mg protein in groups 1, 2 and 3, respectively (Fig. 1). . 331 EL-MINIA MED. BULL. VOL. 19, NO. 2, JUNE, 2008 Ibrahim et al Fig. 1. Changes of cardiac nitrite and nitrate NOx in cardiac tiisues NOx Con. (nmol/mg protein) 70 60 50 40 *** *** 30 20 10 0 Group 1 Group 2 Group 3 *** Significant difference compared with group 1 Cardiac nitrite and nitrate were assayed as described in Materials & Methods. Values are expressed as mean+SD (n=7). diabetic group compared with control group 1 (Fig. 2). The band intensities of group 3 was significantly weaker than group2, indicating that diabetes increased fibronectin expression in aortic tissue of SHR but captopril administration decreased fibronectin expression in aortic tissue of diabetic SHR. 3Immunoblot analysis of fibronectin in aortic tissue Fibronectin expressions were examined by immunoblot analysis. The bands detected by using antibody to fibronectin, but not by non-immune serum, were specific to fibronectin (data not shown). The intensities of this band were significantly denser in Fig. 2. Immunoblot analysis of cardiac fibronectin A) 1 2 3 332 EL-MINIA MED. BULL. VOL. 19, NO. 2, JUNE, 2008 Ibrahim et al Fibronectin in cardiac tissue B) Relative fibronectin density 1.6 * 1.4 1.2 ** 1 0.8 0.6 0.4 0.2 0 1 2 3 Immunoblot analysis of cardiac fibronectin was performed as described in Materials and Methods. Fig. 2-A shows the typical band of non-diabetic SHR (1), diabetic SHR (2) and diabetic SHR receiving captopril (3). Densities of the bands were calculated, and the relative densities are shown as mean ± SD of four different experiments (Fig. 2-B). *Significant difference compared with group 1. **Significant difference compared with group 2. pathway in endothelium (Shen and Zheng 1999), and a deficiency in tetrahydrobiopterin (BH4), a cofactor necessary for NOS activity (Meininger et al. 2000). DISCUSSION: Our findings showed that diabetes caused a significant decrease in NO metabolites, nitrite/nitrate (NOx), in cardiac tissues of SHR compared with non-diabetic SHR. It has been reported that diabetes decreased vascular NO formation, leading to dysfunction of the vascular endothelium and contributing to the development of vascular diseases (Oyadomari et al. 2001; Shafiei et al. 2003). Ibrahim, et al., reported that NOS-3 expression and NOx decreased in rat aorta after STZ injection (Ibrahim et al. 2005). The reduction in NO availability in diabetes has been attributed to either enhanced degradation of nitric oxide by excessive production of superoxide anion (Hink et al. 2001), or impairment of NO formation due to several factors including a defect in substrate supply for NO synthesis such as a defect in the utilization of L-arginine by NOS (Pieper and Dondlinger 1997), impaired capacitative Ca2+ entry The molecular mechanism of endothelial NO regulation in diabetes is complex and not fully understood, but the following mechanisms have been suggested (Artwohl et al. 2003). First, hyperglycemia can markedly activate the beta II isoform of protein kinase (PKC) in endothelial cells by promoting de novo synthesis of diacylglycerol (DAG) and increasing mitochondrial superoxide production. Then, activated PKC can suppress NOS-3 transcription. Second, hyperglycemia, by increasing free radical production, can lead to a decrease in NO synthesis and/or availability. Third, chronic hyperglycemia increases aldose reductase activity, leading to an increase in glucose metabolism through the polyol pathway that consumes NADPH. Because NADPH 333 EL-MINIA MED. BULL. VOL. 19, NO. 2, JUNE, 2008 is also an essential cofactor for NOS in the synthesis of NO, its depletion as a result of hyperglycemia could lead to a reduction in NO production. Fourth, accumulation of advanced glycation end products as a result of sustained high plasma glucose concentrations has been shown to reduce NO production by directly reacting with NO or triggering apoptosis in vascular endothelial cells. In addition, the lack of insulin in diabetes may contribute to the effect of decreased NOS-3, because physiological concentrations of insulin can increase NOS-3 gene expression, protein and activity (Kuboki et al. 2000). Ibrahim et al finding is consistent with previous reports. (Lindsay et al. 1997; Vural et al. 2002). Captopril reduced blood pressure without affecting NOx concentrations in cardiac tissue of diabetic SHR. In support of this finding, Bernatova et al. reported that captopril prevented hypertension and left ventricular hypertrophy without affecting NOS activity in rats (Bernatova et al. 1996). Similarily, Kawabata et al. reported that protection of the myocardium against ischemia reperfusion injury with ACE inhibitor, temocaprilat, was not abolished by nitric oxide synthase inhibitor in rabbit hearts (Kawabata et al. 2001). Trauernich et al. reported that enalapril prevented cerebrovascular dysfunction in diabetic rats without altering the expression of NOS-3 protein in cerebral microvessels (Trauernich et al. 2003). In contrast, other studies reported the enhanced effect of ACE inhibitors on NOS expression and activity (Linz et al., 1997; Bosc et al. 2000). Although the reason for these conflicting data is unclear, differences in the duration of drug administration and/or doses administered might provide some explanations. Indeed, it has been reported that administration of ACE inhibitors, captopril or enalapril for 1 or 3 months respecttively, did not improve NO production or NOS expression (Bernatova et al. 1996; Trauernich et al. 2003). While treatment with the ACE inhibitors, enalapril or ramipril for 6 months or 2 years, respectively, induced a significant increase in NOS activity and expression [(Linz et al., 1997; Bosc et al. 2000). Scribner et al. reported that captopril at only high concentrations (above 30 μM) increased NO synthesis in aortic endothelial cells as evidenced by the However, there is no clear consensus regarding NO production and the relation between NO production and cardiaovascular dysfunction in diabetes (Pieper 1999). This diversity of opinion is probably based on differences in the type of the vessel examined, duration of diabetes, degree of hyperglycemia, insulin concentration, presence or absence of diabetic complications, and the experimental model (Pieper 1999). StockklauserFarber et al. reported that the myocardial NOS activity of diabetic rats reached 2.5 times as high as controls after 4 weeks of diabetes, declined to the control level after 15 weeks, and decreased to lower than control after 46 weeks (StockklauserFarber et al. 2000). Diabetic SHR treated with captopril for 4 weeks showed a significant reduction in MBP. The blood pressure-lowering effect of captopril has been well documented (Silberbauer et al. 1982). The MBP values of diabetic rats were similar to those of non-diabetic rats, indicating that STZ-induced diabetes had no significant effect on blood pressure during the period of this study. This 334 EL-MINIA MED. BULL. VOL. 19, NO. 2, JUNE, 2008 increased production of cyclic GMP (Scribner et al. 2003). Ibrahim et al decrease in NOx in cardiac tissue, our findings raise a question about the possible link between NO and fibrenectin in diabetic cardimyopathy. It has been reported that Both NO and fibronectin play a role in the development of vascular tissues and the NOS inhibitor L-NMMA decreased fibronectin synthesis, whereas the NO donor, SNAP, increased fibronectin synthesis (Catherine et al.1999; Pichika and Homandberg, 2004). Further investigation is needed to confirm such a relation. Our results showed that diabetes significantly increased fibronectin expression in hearts of SHR. Although, the pathogenesis of diabetic cardiovascular disease is multifactorial, excessive accumulation of ECM is one of the main pathological hallmarks (Evans et al. 2000). It has been reported that hyperglycemia increased fibronectin protein and mRNA expression in embryonic hearts (Smoak 2004). A study by Argano M et al. reported that diabetic cardiomyopathy is characterized by myocyte loss and showed that oxidative stress, induced by hyperglycemia, caused myocardial fibrosis and impaired contractile function in the left ventricle of diabetic rats (Aragno et al. 2008). In summary, diabetes decreased NO level and increased fibronectin expression in hearts of SHR rats. Antihypertensive and anti-hypertrophic effects of captopril are associated with decrease in fibronectin expression in cardiac tissue without significant changes in cardiac NO. Administration of captopril in diabetic SHR prevented cardiac hypertrophy and decreased fibronectin expression in cardiac tissue compared with non-treated rats. It has been reported that captopril, prevented cardiomyopathy in experimental chronic aortic valve regurgitation and these effects might be related to decrease of fibronectin expression in left ventricles (Plante et al. 2004). REFERENCES: 1. 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VOL. 19, NO. 2, JUNE, 2008 مستوي اكسيد النيتريك والفيبرونيكتين في نسيج عضلة القلب في الجرزان المصابة بمرض السكر المصاحب لضغط الدم المرتفع .تأثير عقار الكابتوبريل ً إن مرض البول السكري وارتفاع ضغط الدم من االمراض المتداخلة والتي تمثل واحدة من أخطر العوامل المؤدية الي مشكالت وأمراض القلب والشرايين مثل ضعف عضلة القلب. ان ألية حدوث ضعف عضلة القلب في حاالت مرض البول السكري غير مؤكدة حتي االن والتي قد تشمل بعض التعديل في مستوي اكسيد النيتريك و القلب الخلوى الخارجى والفيبرونيكتين .قد تم دراسة بدراسة تأثير مرض البول السكري علي مستوى اكسيد النيتريك والفيبرونيكتين في عضلة القلب في نموذج الجرزان ذات ضغط الدم المرتفع التلقائي وكذلك احتمالية تعديلها باستخدام عقار الكابتوبرل .لقد أثبتت الدراسة أن مرض السكر سبب نقص ذات داللة احصائية ملحوظة في مستوي اكسيد النيتريك ومعدل افراز الفيبرونيكتين في عضلة القلب للجرذان المرتفعة ضغط الدم التلقائي مقارنة بالمجموعة الضابطة .كما أن أربعة أسابيع من العالج بواسطة الكابتوبرل خفضت ضغط الدم ومنعت تضخم عضلة القلب وقللت من معدل افراز الفيبرونيكتين ولكنها لم تكن ذا تأثير ملحوظ علي مستوي اكسيد النيتريك. 339