Blockade Of AngiotensinⅡType 1 Receptor Alters ACE2 Activity, ENOS Expression And CD44-hyaluronan Interaction In Rats With Hypertension And Myocardial Fibrosis

Hypertension could induce myocardial injury which is characterized by the progressive perivascular fibrosis and interstitial fibrosis that contribute to an increase in cardiac muscle stiffness and development of heart failure. Multiple triggers have been proposed in induction of hypertension and cardiac fibrosis, including inflammatory cytokines, oxidative stress, endothelial dysfunction, and so on. Although the current therapeutic drugs by targeting these mediators have been shown to be effective to decrease blood pressure and attenuate cardiac fibrosis, clinical outcomes are still unsatisfactory, suggesting that some other unknown factors may also be involved. Therefore, clarification of mechanisms of action underlying development of hypertension induced cardiac fibrosis may help to further improve the treatment and prophylaxis in patient with heart failure.Angiotensin II(Ang II) has been demonstrated to play a major role in the development of hypertension and cardiac fibrosis through stimulating Ang II type 1 receptor(AT1). Ang II induces vascular constriction, systemic inflammatory response, tissue macrophage production/fibroblast proliferation and interstitial collagen deposition, eventually resulting in hypertension and tissue fibrosis. In contract, angiotensin converting enzyme 2(ACE2), a homolog of angiotensin converting enzyme, functions biologically to oppose cardiac fibrosis, proliferation, inflammation and oxidation by converting Ang II to Ang(1-7). It has been demonstrated that the Ang(1-7) can activate the endothelial nitric oxide synthase(e NOS) to synthesize nitric oxide(NO). NO exerts systemic vasodilation, reduces the preload and afterload of heart and inhibits the pathological process of tissue fibrosis. However, whether ACE2 and e NOS co-participate in inhibition of hypertension and cardiac hypertrophy induced by Ang II and the potential role of AT1 receptor in modulation of ACE2 and e NOS have not fully been illustrated.Abnormal deposition of extracellular collagens in peri-vascular and interstitial regions is the main feature of cardiac fibrosis. Recently, hyaluronic acid(HA) accumulation has been demonstrated to be associated with development of tissue fibrosis. HA can be degraded to low molecular weight fragments(LMW-HA) by hyaluronidases(Hayl). LMW-HA can then combine its receptor CD44, a transmembrane adhesion molecule, to regulate cellular growth, adhesion molecule expression and inflammatory cell recruitment. Increasing evidence has suggested that CD44-HA interaction can also promote tissue fibrosis in lung, liver and kidney. However, the role of CD44-HA involvement in pathogenesis of Ang II mediated myocardial fibrosis remains elusive.Therefore, the present study was designed to determine whether ACE2, e NOS and CD44-HA interaction co-participate in Ang II-induced hypertension and cardiac fibrosis following activation of the AT1 receptor. Three specific aims are as follows:1. To demonstrate whether Ang II induced hypertension and cardiac hypertrophy is mediated by reducing vascular ACE2 activity and e NOS expression following stimulation of the AT1 receptor.2. To illustrate whether Ang II induced cardiac fibrosis is related to upregulated CD44 expression and enhanced hyaluronidase activity, which are potentially stimulated by AT1 receptor.3. To confirm whether Ang II stimulated proliferation of cardiac fibroblasts is associated with enhanced expression of CD44, TGF-β1, p Smad2, Smad4 and collagen I, and to provide cellular evidence for induction of cardiac fibrosis.Study part 1: Blockade of angiotensin II type 1 receptor reduces blood pressure and attenuates cardiac hypertrophy by altering ACE2 activity and e NOS expressionObjective: To demonstrate whether downregulation the expression of ACE2 and e NOS in vascular endothelium co-participates in the pathological process of Ang II elevated blood pressure and cardiac hypertrophy.Groups and methods: 1. Male Sprague-Dawley rats were subjected to Ang II infusion using Alzet osmotic minipumps. Two study protocols(i.e., 2 and 4 weeks of Ang II infusion) were selected for all experimental groups. Rats were randomized into one of three groups(n=6/each group/observational period).(1) Ang II infusion: rats received subcutaneous Ang II infusion at a rate of 500 ng/kg/min;(2) Ang II plus telmisartan: AT1 receptor blocker, telmisartan(Telmi) was administered by gastric gavage at a dose of 10 mg/kg/day during the periods of Ang II infusion;(3) Sham plus telmisartan: rats were infused with a saline pump and telmisartan given by gastric gavage at same dose in the telmisartan group. 2. Systemic blood pressure was determined in conscious rats using a noninvasive blood pressure measuring system. 3. The heart weight/body weight(HW/BW) ratio was calculated. Cardiomyocyte cross-sectional area was conducted using HE staining and the protein level was analyzed by Western blot assay. 4. ACE2 activity was measured using a fluorescence assay. 5. Localization of AT1, AT2, ACE2 and e NOS in the myocardium was observed using immunohistochemistry staining and the protein level was analyzed by Western blot assay.Results:1. During 4 weeks of Ang II infusion, mean arterial pressure continued to rise and increased by 2.26 times compared with the Sham group at week 4. Telmi intervention significantly reduced blood pressure back to the baseline values as compared with Ang II infusion. Telmi treatment had no effect on blood pressure in the sham group. 2. Myocardial hypertrophy determined by cardiomyocyte cross-sectional and HW/BW ratio was found in the Ang II group at the end of week 2 but more obviously at week 4. 3. Ang II infusion caused a significant upregulation in expression of AT1 receptor and downregulation of AT2 receptor. Along with these changes, ACE2 activity and e NOS expression were reduced as demonstrated using immunohistochemistry and Western blot assay. Inhibition of AT1 receptor expression by Telmi, reversed changes in Ang II infusion induced ACE2 and e NOS.Conclusion: These results suggested that downregulation of ACE2 and e NOS by Ang II is largely caused by stimulation AT1 receptor. Enhancement in ACE2 and e NOS with Telmi is assoacited with reduction in blood pressure and cardiac hypertrophy, suggesting their roles in regulation of blood pressure and normal morphology.Study part 2: Blockade of angiotensin II type 1 receptor inhibits cardiac fibrosis by altering CD44-hyaluronan interactionObjective: To illustrate whether CD44 and HA interaction is involved in Ang II induced cardiac fibrosis.Groups and methods: 1. The rats were randomly divided into three groups, i.e., Sham control, Ang II infusion and Telmi treatment plus Ang II infusion as described in Study part 1. 2. The expression of CD44 was detected by immunhistochemical staining and Western blot. 3. Hyaluronidase activity was measured by zymography. 4. Immunhistochemical staining and Masson’s trichrome method were used to detect macrophage infiltration, myofibroblast proliferation and cardiac fibrosis. 5. The protein level of TGF-β1, Smad2, Smad3, Smad4, Smad7 and Collagen I was analyzed by Western blot assay.Results: 1. The level of CD44 and hyaluronidase activity was significantly increased in the Ang II group at the end of week 2, and maintained at a higher level until week 4 relative to the sham group. However, CD44 expression and hyaluronidase activity were significantly reduced by treatment with Telmi. 2. In the Ang II group, the vascular accumulation of monocytes was enhanced at week 2, but accumulation of macrophages and proliferation of myofibroblasts were significantly incressed at week 4, which were inhibited by 4 weeks of Telmi treatment. 3. At the end of 4 weeks of Ang II infusion, the levels of TGF-β1, Smad2, Smad3, Smad4 was up-regulated and Smad7 was down-regulated, which all were reversed with Telmi. These results were consistent with an inhibition of cardiac fibrosis at this time point.Conclusion: Blockade of the AT1 receptor by Telmi downregulated CD44 expression, reduced hyaluronidase activity and attenuated cardiac fibrosis. These changes were primarily associated with an inhibition in extravasation of macrophages and proliferation of myofibroblasts-initiated TGF-β1/Smads singaling pathways.Study part 3: Mechanisms of action underlying angiotensin II induced proliferation of cardiac fibroblastsObjective: To demonstrate whether Ang II stimulated proliferation of cardiac fibroblasts is associated with enhanced expression of CD44, TGF-β1, p Smad2, Smad4 and collagen I.Groups and methods: 1. Primary cultures of neonatal rat cardiac fibroblasts(CFs) were obtained from the ventricles of 1 to 3-day old Sprague-Dawley rats using the differential attachment method and identified by immunofluorescence staining. 2. Dose-response of Ang II in induction of cell proliferation was assessed by cell counting kit-8 assay. 3. After Ang II stimulation, the expression level of CD44, TGF-β1, p Smad2, Smad4 and Collagen I were detected by Western blot and Real-time PCR. In order to determine whether AT1 receptor was involved in the cell proliferation, the AT1 receptor blocker Telmisartan at a dose of 100 n M was co-applied with 50 n M of Ang II. So, study groups included in this aim were the Control, Ang II group(Ang II 50 n M) and Telmi group(Ang II 50 n M + Telmi 100 n M).Results: 1. The first passage of cultured neonatal rat CFs showed the typical morphological characteristics of fibroblasts, and the purity of cells was more than 95% identified by immunofluorescence staining.2. Coincubation of Ang II with fibroblasts at different concentrations for 24 hr significantly increased proliferative capability of fibroblasts with a marxiaml effect at 50 n M. 3. The expression of CD44 was significantly upregulated by Ang II stimulation at 50 n M, in accordance with increased expression of TGF-β1, p Smad2, Smad4 and Collagen I. Pretreatment with Telmi at 100 n M reversed Ang II induced expression of these proteins.Conclusion: Blockade of the AT1 receptor inhibited Ang II activated CD44 expression and proliferation of cardiac fibroblasts, largely mediated by inhibiting TGF-β1/Smads meadited production of collagen.Research Prospect This study shows that ACE2 activity, e NOS expression and the interaction between CD44/HA are involved in Ang II inducted hypertension and cardiac fibrosis. Reduction of blood pressure by dietary telmisartan may be associated with reduced AT1/AT2 receptor ratio and augmented expression of both ACE2 and e NOS. Mechanisms underlying prevention of Ang II initiated myocardial fibrosis may be associated with inhibition of CD44/HA interaction and TGF-β1/Smads singnaling pathways. These data suggest that pharmacologically selective activation of ACE2/e NOS or inhibition of CD44/HA interaction might be considered as alternative therapeutic options to be combined with other existing agents in treatment with hypertension and cardiac fibrosis derived heart failure.