| Background:Myocardial fibrosis can occur as a result of hypertension, ischemic injury or valvular heart disease, and is a major cause of left ventricular diastolic dysfunction that accounts for40% to 50% of heart failure patients. Pathologically, fibrosis is characterized by the accumulation of fibrillar collagens and it develops from the perivascular area to the intermyocardium with a more diffuse distribution pattern. The lack of resolution of this excessive collagen deposition in tissue is associated with reduction of cardiac muscle compliance, filling impairment, and ultimately heart failure.Many basic studies and clinical trials have shown that angiotensin II(Ang II) derived from the renin-angiotensin system plays an important role in development of hypertension,maladaptive cardiac repair after myocardial infarction, myocardial fibrosis and heart failure. Through understanding in deleterious effects of Ang II on cardiovascular injury and development in anti-Ang II therapy, it has well been reorganized that Ang II plays a major role in pathogenesis of a variety of cardiovascular diseases. As is known, pharmacological interventions using angiotensin converting enzyme(ACE) inhibitors to reduce the conversion of angiotensin(Ang) I to Ang II or Ang II type 1 receptor(AT1) antagonists to block the action of Ang II have become one of the most successful therapeutic approaches in treatment of different cardiovascular disease. However, data from clinical observations have also revealed that the use of ACE inhibitors is associated with higher rate of renin sensitivity, dry cough, angioedema and limited usage in renal stenosis, while hypotensive symptoms and different personal response are often identified with AT1 receptor antagonists. Therefore, adjunctive therapies to avoid these unfavorable actions and reduce morbidity and mortality of cardiovascular diseases by inhibiting Ang II/AT1 systemreserve significance of translational medicine in hospital.Curcumin, the yellow pigment extracted from the rhizomes of the plant curcuma longa, has previously been demonstrated to exhibit a variety of potent beneficial effects,such as anti-oxidant, anti-inflammation and anti-fibrosis. Therefore, curcumin is a great concern of people. We have previously reported that dietary administration of curcumin reduces infarct size and improves cardiac function after ischemia. However, we do not know whether these protective effects of curcumin are associated with modulation in expression of ACE and Ang II receptors. In the rat model of ischemia/reperfusion-induced heart failure, therefore, we tested the hypothesis that curcumin inhibits fibrotic process and promotes cardiac function through altering expression of ACE and Ang II receptors.Furthermore, we selected a well-established rat model of Ang II infusion-induced tissue fibrosis to examine the direct protective effect of curcumin on myocardial fibrosis induced by angiotensin II. In this model, we demonstrated the characteristics of cardiac injury induced by angiotensin II, and the protective effect of curcumin after injury, and clarified its mechanism. The study was divided into two parts as follows:PART 1Protective effects of curcumin on myocardial fibrosis and cardiac function by inhibiting angiotensin converting enzyme and blocking angiotensin II AT1 receptorObjective:To demonstrate the changes of angiotensin converting enzyme and angiotensin II receptors in the period of myocardial fibrosis after myocardial infarction. To investigate the anti-fibrosis with curcumin and its possible mechanism.Study hypothesis:It has been shown that angiotensin II plays a major role in pathogenesis of a variety of cardiovascular diseases, such as myocardial fibrosis and heart failure. As is well known,anti-fibrosis is one of important effects of curcumin. Our study group has found that curcumin reduces infarct size after myocardial ischemia and improves cardiac function in heart failure patients. Taken together, the protective effect of curcumin on the maladaptive myocardial repair after myocardial infarction may be related to the expression of angiotensin converting enzyme and angiotensin II receptors. These results provide a basis for the further study of the effect of curcumin on the heart protection. Therefore, we tested the hypothesis that the protective effect of curcumin in inhibiting the fibrosis process was achieved by altering the expression of angiotensin converting enzyme and angiotensin II receptors.Methods:Male Sprague-Dawley rats were anesthetized with an initial intraperitoneal injection of a mixture of ketamine(90 mg/kg) and zylaxine(10 mg/kg). After pericardiotomy, a 6-0proline ligature was placed under the left coronary artery and the ends of the tie were threaded through a small plastic tube to form a snare for reversible coronary occlusion.After ischemia, the chest was closed and the proline ligature was kept in the chest for re-ligation at the end of the study.The rats were randomly divided into four groups: 1) Control(n=8): rats were subjected to 45 min of coronary occlusion followed by 7 and 42 days of reperfusion,respectively, and no treatment was given during the experiment; 2) Curcumin treatment(n=8): curcumin(Sigma-Aldrich, St. Louis, MO) was administered by gastric gavage at a dose of 150 mg/kg/day only during reperfusion as we reported recently; 3) Sham(n=6): no coronary occlusion was conducted; 4) Sham plus curcumin(n=6): no coronary occlusion was conducted and curcumin was given by gastric gavage at same dose in the curcumin group.At the end of 7 or 42 days of reperfusion, two-dimensional(2D) guided M-mode ultrasound systemwas used to assess left ventricular(LV) systolic and diastolic function.The hearts were isolated and tissue blocks were saved for histological analysis. Masson’s trichrome staining was used for detection of tissue fibrosis; Western blot assay was used to measure tissue protein levels; immunohistochemical staining was selected to detect expression of enzyme and receptors. The purchased H9c2 cells were stimulated using hydrogen peroxide(H2O2, 400 μM) for 5 min. The viability of H9c2 cells was evaluated by performing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and propidium iodide(PI) staining in 96-well plates, respectively.Results:Echocardiography showed the wall thickness of the infarcted anterior septum in the curcumin group was significantly greater than that in the control group. Cardiac contractile function was improved in the curcumin treated animals as measured by fraction shortening and ejection fraction. As evidenced by less collagen deposition in fibrotic myocardium,curcumin also reduced the extent of collagen-rich scar and increased mass of viable myocardium detected by Masson’s trichrome staining. Relative to the control animals,dietary treatment with curcumin significantly reduced levels of ACE and AT1 receptor protein as determined by Western blot assay, coincident with less locally-expressed ACE and AT1 receptor in myocardium and coronary vessels as identified by immunohistochemistry. Along with this inhibition, curcumin significantly increased protein level of AT2 receptor and its expression compared with the control. In cultured cardiac muscle cells, curcumin inhibited oxidant-induced AT1 receptor expression and promoted cell survival.Conclusion:These results suggest that a daily oral administration of curcumin inhibits expression of ACE and AT1 receptor during reperfusion. Along with this ablation, expression of AT2 receptor was up-regulated, as evidenced by a reduced AT1/AT2 receptor ratio. In accord with these findings, the collagen deposition and fibrotic tissue in ischemic/reperfusedventricular wall were attenuated, which was further corroborated by increased ventricular wall thickness. Furthermore, the fraction shortening and ejection fraction were increased,suggesting an improvement in cardiac contractile function. In vitro data showed that curcumin inhibits H2O2-stimulated AT1 receptor expression in H9c2 cells and enhances cell survival, which were comparable to those when these cells were treated using losartan,suggesting that curcumin is capable of protecting cells against oxidant-induced cell injury through modulating AT1 receptor.PART 2Attenuation of myocardialfibrosis with curcumin is mediated by modulating angiotensin II receptors/TGFβ1/Smad signaling and expression of angiotensin converting enzyme 2Objective:To demonstrate mechanisms of action underlying Ang II induced myocardial fibrosis and cardiac protection with curcumin.Study hypothesis:In study part 1, we found that Ang II induced myocardial fibrosis can be reversed by curcumin. Based on our previous studies, curcumin is known to improve cardiac function by balancing degradation and synthesis of collagens after myocardial infarction.TGFβ1/Smads signaling pathways are involved in induction of myocardial fibrosis, but we do know whether they also participate in signaling curcumin inhibited tissue fibrosis. In response to Ang II stimulation, activation of AT1 receptor induces vascular constriction,systemic inflammatory response, interstitial collagen deposition and tissue fibrosis.However, activation of AT2 receptor may evoke cardioprotective effects by countervailing over-all deleterious effects of AT1 receptor. On the other hand, angiotensin convertingenzyme 2(ACE2) is a homolog of ACE, sharing 42% sequence identity and 61% sequence similarity with catalytic domain of ACE, but not inhibited by ACE inhibitors. An increase in ACE2 activity is associated with a decrease in tissue level of Ang II through degradation of Ang II to the Ang(1-7), hence functioning effectively as a negative regulator of Ang II-mediated fibrosis. In the following proposed study, we tested the hypothesis that curcumin could modulate expression of AT1/AT2 receptors and ACE2. Furthermore,treatment with curcumin reduces myocardial fibrosis.Methods:Male Sprague-Dawley rats were used in this study. An intraperitoneal injection of a mixture of ketamine(90 mg/kg) and zylaxine(10 mg/kg) was selected to anesthetize the rats on the day for a minipump implantation. The skin was swabbed with povidone-iodine and alcohol after shaving. An incision between the scapulae was made and a small pocket was produced using a hemostat to spread the subcutaneous connective tissue. An osmotic minipump was inserted into the pocket and the incision was closed with sutures. The osmotic minipumps were used to perfuse the Ang II. These pumps were preincubated in the tubes with 0.9% sterile saline only or saline containing Ang II at 37o C for overnight prior to implantation.A non-invasive blood pressure(NIBP) measuring system was selected to detect the NIBP change from all animals using a pulse transducer. This system operates by occluding blood flow to the tail with a specialized cuff. The transducer intermittently measures blood pressure based on the periodic occlusion of blood flow in the tail. The measurement of NIBP was considered valid only when three consecutive readings did not differ by more than 10 mm Hg. The change of blood pressure on the day 0,7,14,28 after Ang II infusion was recorded.Two observational points(i.e., 2 and 4 weeks of Ang II infusion) were selected for all experimental groups. Animals were randomized into one of four groups(n=6/each observational period): 1) Control: rats were received Ang II infusion only at a rate of 500ng/kg/min; 2) Ang II plus curcumin: curcumin(Sigma-Aldrich, St. Louis, MO, USA) wasadministered by gastric gavage at a dose of 150 mg/kg/day during Ang II infusion; 3)Sham: rats were infused with a saline pump; 4) Sham plus curcumin(normal): rats were infused with a saline pump and curcumin was given by gastric gavage at same dose in the curcumin group.At the end of the experimental period in each group, the rat was euthanized and the heart was removed. The transversal myocardial slices were fixed in 10%phosphate-buffered formalin solution for 24 h, and embedded in paraffin wax for histological analysis. Masson’s trichrome staining was used for detection of tissue fibrosis;Western blot assay was used to detect expression of AT1, AT2, TGFβ1, p-Smad2,p-Smad3, collagen I and ACE2; immunohistochemical staining was selected to detect expression of receptors.Results:We found that arterial blood pressure was significantly increased over time with Ang II infusion relative to the Sham control, reaching to a peak at 21 days. Curcumin reduced blood pressure(i.e., systolic, diastolic and mean pressure) during entire period of study.There were significant changes in myocardial fibrosis at the end of 4 weeks of Ang II infusion, evidenced as expanded peri-vascular fibrotic space and inter-myocardium fibrotic area; enhanced accumulation of macrophages and α-SMA expressing myofibroblasts;increased deposition of type I collagen. Relative to these changes in the control animals,treatment with curcumin for 4 weeks significantly reduces fibrotic tissue formation,inflammatory cell accumulation and collagen deposition. We also found that there are no significant statistical differences in all parameters measured at 2 weeks of Ang II infusion.However, at the end of 4 weeks, relative to the sham control, Ang II caused significant changes in all parameters analyzed, as manifested as enhanced expression of AT1 receptor and reduced expression of AT2 receptor, which were further confirmed by immunohistochemical staining at peri-vascular space and inter-myocardium area.Consistent with these changes, expression of TGFβ1, p-Smad2 and Smad3 was increased.Furthermore, expression of ACE2 were attenuated. Treatment with curcumin for 4 weeksreversed all parameters measured, evidenced by inhibited expression of TGFβ1, p-Smad2 and p-Smad3. On the other hand, curcumin enhanced ACE2 expression which were confirmed by immunohistochemical staining at inter-myocardium area.Conclusion:These results suggest that Ang II has significant effects on fibrotic tissue formation and macrophages/myofibroblast accumulation that are largely associated with collagen deposition seen at 4 weeks of Ang II infusion. Curcumin reduces myocardial fibrosis. The results also suggest that Ang II induces myocardial fibrosis through Ang II AT1receptor/TGFβ1/Smads mediated signaling pathways. We demonstrated that curcumin reduces AT1 receptor protein level, up-regulates AT2 receptor, and therefore, re-balances expression between AT1 and AT2 receptors, as evidenced by a reduced ratio of AT1 to AT2 receptor. It also activated ACE2 and increased ACE2 expression in myocardium. In accord with these findings, the production of macrophages and expression of TGFβ1/Smads were attenuated and proliferation of myofibroblasts was inhibited.Consistent with the modulatory effects of curcumin on Ang II receptors and TGFβ1/Smads signaling pathways, collagen synthesis and cardiac fibrosis were attenuated. These results were consistent with its protective effect on cardiovascular system as shown in previous studies and provide evidence showing a direct effect on the heart after Ang II stimulation.Study prospect Our studies demonstrated for the first time that anti-fibrosis effect of curcumin has been associated with expressions of angiotensin II receptors and angiotensin converting enzyme. We observed that curcumin can also reduced the hypertension induced by angiotensin II. Our data provided a direct experimental evidence to explain the cardiovascular protection of curcumin. Curcumin, as a kind of plant extracts, is a daily consumption seasoning in India. Preclinical studies and clinical trials have addressed its pharmacokinetics, safety, efficacy and therapeutic potential. Currently, since there is nounique therapy for cardiovascular diseases to improve final outcome in patient with fibrosis-induced heart failure, curcumin might be selected as an adjunctive therapeutic compound with other commonly used cardiovascular drugs, such as ACE inhibitor, AT1 receptor blocker or beta receptor blocker, to help lowing blood pressure and better protect the heart against cardiac dysfunction. |
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