[show abstract][hide abstract] ABSTRACT: The present study evaluated the reversal of diabetes-mediated impairment of angiogenesis in a myocardial infarction model of type 1 diabetic rats by intramyocardial administration of an adenoviral vector encoding thioredoxin-1 (Ad.Trx1). Various studies have linked diabetes-mediated impairment of angiogenesis to dysfunctional antioxidant systems in which thioredoxin-1 plays a central role.
Ad.Trx1 was administered intramyocardially in nondiabetic and diabetic rats immediately after myocardial infarction. Ad.LacZ was similarly administered to the respective control groups. The hearts were excised for molecular and immunohistochemical analysis at predetermined time points. Myocardial function was measured by echocardiography 30 days after the intervention. The Ad.Trx1-administered group exhibited reduced fibrosis, oxidative stress, and cardiomyocyte and endothelial cell apoptosis compared with the diabetic myocardial infarction group, along with increased capillary and arteriolar density. Western blot and immunohistochemical analysis demonstrated myocardial overexpression of thioredoxin-1, heme oxygenase-1, vascular endothelial growth factor, and p38 mitogen-activated protein kinase-beta, as well as decreased phosphorylated JNK and p38 mitogen-activated protein kinase-alpha, in the Ad.Trx1-treated diabetic group. Conversely, we observed a significant reduction in the expression of vascular endothelial growth factor in nondiabetic and diabetic animals treated with tin protoporphyrin (SnPP, a heme oxygenase-1 enzyme inhibitor), even after Ad.Trx1 therapy. Echocardiographic analysis after 4 weeks of myocardial infarction revealed significant improvement in myocardial functional parameters such as ejection fraction, fractional shortening, and E/A ratio in the Ad.Trx1-administered group compared with the diabetic myocardial infarction group.
This study demonstrates for the first time that impairment of angiogenesis and myocardial dysfunction can be regulated by Ad.Trx1 gene therapy in streptozotocin-induced diabetic rats subjected to infarction.
[show abstract][hide abstract] ABSTRACT: Hypertension, the major risk factor for many cardiovascular diseases, is a result of multiple causes along with excessive generation of reactive oxygen species (ROS) resulting in imbalance of redox status.
In this study, we investigated the therapeutic potential of Adenoviral-Thioredoxin-1 (Adeno-Trx-1) in spontaneous hypertensive rats (SHRs) at a dosage of 1 x 10(9) pfu. The rats were assigned as normotensive Wistar-Kyoto (WKY), SHR, SHR + Adeno-Lac-Z (SHRLac-Z), and SHR + Adeno-Trx-1 (SHRTrx-1). Echo-guided injection of adeno virus was done 48 h before permanent myocardial infarction (MI) by left anterior descending coronary artery (LAD) occlusion.
Decreased infarct size (52 +/- 4.1% vs. 67 +/- 6.1%), number of apoptotic cardiomyocytes (161 +/- 14.8 vs. 240 +/- 22.2), left ventricular inner diameter (7 +/- 0.33 vs. 9 +/- 0.46 mm), increased ejection fraction (52 +/- 6.3 vs. 42 +/- 3.3%), and fractional shortening (28 +/- 1.8 vs. 22 +/- 2.04 %) was observed in the SHRTrx-1 compared to SHR. Western Blot and immunohistochemical analysis demonstrated increased expression of Trx-1, HO-1, and Bcl-2 in the SHRTrx-1 compared to SHR. In addition, increased HO-1 activity was also observed in SHRTrx-1 as compared to SHR and SHRLac-Z groups.
Our study demonstrates that the cardioprotective effect of Adeno-Trx-1 gene therapy in SHR is Trx-1/HO-1/Bcl-2 mediated and may represent future target to develop therapy against hypertension associated cardiac failure.
American Journal of Hypertension 01/2009; 22(2):183-90. · 3.67 Impact Factor
[show abstract][hide abstract] ABSTRACT: Endothelial dysfunction and impaired angiogenesis constitute a hallmark of hypercholesterolemia. This study was designed to examine the effects of resveratrol, an antioxidant with lipid-lowering properties similar to those of statins, on neovascularization along with caveolar interaction with proangiogenic molecules in hypercholesterolemic rats. Animals were divided into: rats maintained on a normal diet (control group); rats maintained on a 5% high-cholesterol diet for 8 weeks (HC group); and rats maintained on a 5% high-cholesterol diet for 8 weeks and administered resveratrol (20 mg/kg) orally for 2 weeks (HCR group). Myocardial infarction was induced by ligating the left anterior descending artery. Herein we examined a novel method for stimulating myocardial angiogenesis by pharmacological preconditioning with resveratrol at both the capillary and arteriolar levels and the potential role of hemeoxygenase-1, endothelial nitric oxide synthase and caveolin-1 in mediating such a response. We also investigated the functional relevance of such treatment by assessing whether the induced neovascularization can help preserve left ventricle-contractile functional reserve in the setting of a chronic hypercholesterolemic condition. Four weeks after sham surgery and left anterior descending artery occlusion, rats underwent echocardiographic evaluation, which revealed improvement in ejection fraction and fractional shortening in the HCR group compared with the HC group. Left ventricular tissue sections displayed increased capillary and arteriolar density in the HCR group compared with the HC group. Western blot analysis revealed downregulation of vascular endothelial growth factor and hemeoxygenase-1 and increased association of caveolin-1 eNOS in the HC group, decreasing the availability of eNOS to the system; which was reversed with resveratrol treatment in the HCR group. This study was further validated in cardiac-specific hemeoxygenase-1-overexpressed mice assuming molecular cross-talk between the targets. Hence, our data identified potential regulators that primarily attenuate endothelial dysfunction by resveratrol therapy in hypercholesterolemic myocardium.
Free Radical Biology and Medicine 08/2008; 45(7):1027-34. · 5.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sildenafil citrate (SC), a drug for erectile dysfunction, is now emerging as a cardiopulmonary drug. Our study aimed to determine a novel role of sildenafil on cardioprotection through stimulating angiogenesis during ischaemia (I) reperfusion (R) at both capillary and arteriolar levels and to examine the role of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in this mechanistic effect. Rats were divided into: control sham (CS), sildenafil sham (SS), control+IR (CIR) and sildenafil+IR (SIR). Rats were given 0.7 mg/kg, (i.v) of SC or saline 30 min. before occlusion of left anterior descending artery followed by reperfusion (R). Sildenafil treatment increased capillary and arteriolar density followed by increased blood flow (2-fold) compared to control. Treatment with sildenafil demonstrated increased VEGF and Ang-1 mRNA after early reperfusion. PCR data were validated by Western blot analysis. Significant reduction in infarct size, cardiomyocyte and endothelial apoptosis were observed in SC-treated rats. Increased phosphorylation of Akt, eNOS and expression of anti-apoptotic protein Bcl-2, and thioredoxin, hemeoxygenase-1 were observed in SC-treated rats. Echocardiography demonstrated increased fractional shortening and ejection fraction following 45 days of reperfusion in the treatment group. Stress testing with dobutamine infusion and echocardiogram revealed increased contractile reserve in the treatment group. Our study demonstrated for the first time a strong additional therapeutic potential of sildenafil by up-regulating VEGF and Ang-1 system, probably by stimulating a cascade of events leading to neovascularization and conferring myocardial protection in in vivo I/R rat model.
Journal of Cellular and Molecular Medicine 04/2008; 12(6B):2651-64. · 4.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: This study examined the hypothesis that the ubiquitin proteasome system (UPS) degrades proteins damaged by exposure to hyperglycemia. Experimental hyperglycemia was induced in male rats by treatment with streptozotocin. After 30 days, echocardiography confirmed the presence of cardiomyopathy as ejection fraction, fractional shortening, and diastolic function (E/A ratio) were decreased, and chamber diameter was increased in hyperglycemic animals. Proteasome non-ATP-dependent chymotryptic activity was increased over 2-fold in hyperglycemic hearts, but the ATP-dependent activity was decreased and levels of ubiquitinated proteins were increased. Protein levels of the PA28alpha of the 11S-activator ring were increased by 128% and the PA28beta subunit increased by 58% in the hyperglycemic hearts. The alpha3 subunit of the 20S-proteasome was increased by 82% while the catalytic beta5 subunit was increased by 68% in hyperglycemic hearts. Protein oxidation as indicated by protein carbonyls was significantly higher in hyperglycemic hearts. These studies support the conclusion that the UPS becomes dysfunctional during long term hyperglycemia. However, 11S-activated proteasome was increased suggesting a response to oxidative protein damage and a potential role for this form of the proteasome in a cardiac pathophysiology.
Journal of Molecular and Cellular Cardiology 04/2008; 44(3):618-21. · 5.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hypercholesterolemia (HC) induced endothelial cell dysfunction and decreased endothelial nitric oxide formation results in impaired angiogenesis and subsequent cardiovascular disorders. Therapeutic angiogenesis is known to be a novel strategy for treatment of patients with ischemic heart disease. We have shown that secoisolariciresinol diglucoside (SDG) is angiogenic as well as cardioprotective against myocardial ischemia. In the present study, we examined the efficacy of SDG in a hypercholesterolemic myocardial infarction (MI) model. The rats were maintained on a normal and high cholesterol diet (2%) for 8 weeks followed by oral administration of SDG (20 mg/kg) for 2 weeks. The rats were divided into four groups (n=24 in each): Control (C); SDG control (SDG); HC; and HC+SDG (HSDG). Isolated hearts subjected to 30 min of global ischemia followed by 120 min of reperfusion were used to measure the cardiac functions, infarct size and to examine the protein expression profile. After treatment, MI was induced by ligating the left anterior descending artery. Echocardiographic parameters were examined 30 days after MI. Significant reduction in total cholesterol, LDL-cholesterol, triglycerides and an increase in HDL-cholesterol levels were observed in HSDG as compared to the HC. Decreased infarct size was observed in the HSDG group (43%) compared to the HC (54%). Increased phosphorylation of endothelial nitric oxide synthase (p-eNOS) (3.1-fold), vascular endothelial growth factor (1.9-fold) and heme oxygenase-1 (2.3-fold) was observed in the HSDG group as compared to the HC group. Significant improvement in left ventricular functions was also observed in the HSDG group as evidenced by increased ejection fraction (55% vs. 45%), fractional shortening (28% vs. 22%) and decreased left ventricular inner diameter in systole (8 vs. 6 mm) in HSDG compared to HC. Moreover, MI model has shown increased capillary density (2531 vs. 1901) and arteriolar density (2.6 vs. 1.8) in SDG-treated rats as compared to the HC. The increased capillary and arteriolar density along with increased left ventricular functions on SDG treatment might be due to increased HO-1, VEGF and p-eNOS expression. In conclusion, our study demonstrates for the first time that SDG treatment reduces ventricular remodeling by neovascularization of the infarcted HC myocardium.
Journal of Molecular and Cellular Cardiology 02/2008; 44(1):170-9. · 5.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Excessive oxidative stress has been implicated in the pathology and complications of diabetes, which leads to myocardial ischemia reperfusion injury. The present study was designed to examine whether resveratrol (trans-3,5,4'-trihydroxystilbene), a polyphenolic compound present in red wine has a direct cardioprotective effect on diabetic myocardium. Resveratrol (2.5 mg/kg body wt/day) and L-NAME (25 mg/kg body wt/day) were administered orally for 15 days to streptozotocin (65 mg/kg)-induced diabetic rats. Sprague Dawley rats were divided into 5 groups: (i) control, (ii) diabetic, (iii) diabetic+resveratrol, (iv) diabetic+resveratrol+L-NAME (nitric oxide synthase inhibitor), and (v) diabetic+L-NAME. In our present study resveratrol demonstrated significant reduction in glucose level in diabetic rats. After the treatment, the hearts were excised and subjected to 30 min of global ischemia followed by 2 h of reperfusion. Resveratrol-treated diabetic rats demonstrated significant reduction in glucose levels as compared to the nontreated diabetic animals, and improved left ventricular function throughout reperfusion compared to the diabetic or L-NAME-treated animals (dp/dt(max) 1457+/-51 vs 999+/-44 mm Hg/s at 120 min reperfusion). Cardioprotection from ischemic injury in resveratrol-treated diabetic rats showed decreased infarct size (42% vs 51%) and cardiomyocyte apoptosis (35% vs 40%) as compared with diabetic animals. Resveratrol produced significant induction of p-AKT, p-eNOS, Trx-1, HO-1, and VEGF in addition to increased activation of MnSOD activity in diabetic animals compared to nondiabetic animals. However treatment with L-NAME in resveratrol-treated and nontreated diabetic animals demonstrated significant downregulation of the above-noted protein expression profile and MnSOD activity. In the present study we found that the mechanism(s) responsible for the cardioprotective effect of resveratrol in the diabetic myocardium include upregulation of Trx-1, NO/HO-1, and VEGF in addition to increased MnSOD activity and reduced blood glucose level. Thus this study shows a novel mechanism of pharmacological preconditioning with resveratrol in the diabetic myocardium.
Free Radical Biology and Medicine 10/2007; 43(5):720-9. · 5.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: Reactive oxygen species (ROS) generated during ischemia-reperfusion (I/R) enhance myocardial injury, but brief periods of myocardial ischemia followed by reperfusion [ischemic preconditioning (IP)] induce cardioprotection. Ischemia is reported to stimulate glucose uptake through the translocation of GLUT-4 from the intracellular vesicles to the sarcolemma. In the present study we demonstrated involvement of ROS in IP-mediated GLUT-4 translocation along with increased expression of caveolin (Cav)-3, phospho (p)-endothelial nitric oxide synthase (eNOS), p-Akt, and decreased expression of Cav-1. The rats were divided into the following groups: 1) control sham, 2) N-acetyl-L-cysteine (NAC, free radical scavenger) sham (NS), 3) I/R, 4) IP + I/R (IP), and 5) NAC + IP (IPN). IP was performed by four cycles of 4 min of ischemia and 4 min of reperfusion followed by 30 min of ischemia and 3, 24, 48 h of reperfusion, depending on the protocol. Increased mRNA expression of GLUT-4 and Cav-3 was observed after 3 h of reperfusion in the IP group compared with other groups. IP increased expression of GLUT-4, Cav-3, and p-AKT and p-eNOS compared with I/R. Coimmunoprecipitation demonstrated decreased association of Cav-1/eNOS in the IP group compared with the I/R group. Significant GLUT-4 and Cav-3 association was also observed in the IP group. This association was disrupted when NAC was used in conjunction with IP. It clearly documents a significant role of ROS signaling in Akt/eNOS/Cav-3-mediated GLUT-4 translocation and association in IP myocardium. In conclusion, we demonstrated a novel redox mechanism in IP-induced eNOS and GLUT-4 translocation and the role of caveolar paradox in making the heart euglycemic during the process of ischemia, leading to myocardial protection in a clinically relevant rat ischemic model.
[show abstract][hide abstract] ABSTRACT: Hypercholesterolemia (HC) is a common health problem that significantly increases risk of cardiovascular disease. Both statin (S) and resveratrol (R) demonstrated cardioprotection through nitric oxide-dependent mechanism. Therefore, the present study was undertaken to determine whether combination therapy with statin and resveratrol is more cardioprotective than individual treatment groups in ischemic rat heart model. The rats were fed with 2% high cholesterol diet and after 8 weeks of high cholesterol diet the animals were treated with statin (1 mg/kg bw/day) and resveratrol (20 mg/kg bw/day) for 2 weeks. The rats were assigned to: (1) Control (C), (2) HC, (3) HCR, (4) HCS and (5) HCRS. The hearts, subjected to 30-min global ischemia followed by 120-min reperfusion were used as experimental model. The left ventricular functional recovery (+dp/dt(max)) was found to be significantly better in the HCRS (1926+/-43), HCR (1556+/-65) and HCS (1635+/-40) compared to HC group (1127+/-16). The infarct sizes in the HCRS, HCS and HCR groups were 37+/-3.6, 43+/-3.3 and 44+/-4.2 respectively compared to 53+/-4.6 in HC. The lipid level was found to be decreased in all the treatment groups when compared to HC more significantly in HCS and HCRS groups when compared to HCR. Increased phosphorylation of Akt and eNOS was also observed in all the treatment groups resulting in decreased extent of cardiomyocyte apoptosis but the extent of reduction in apoptosis was more significant in HCRS group compared to all other groups. In vivo rat myocardial infarction (MI) model subjected to 1 week of permanent left descending coronary artery (LAD) occlusion documented increased capillary density in HCR and HCRS treated group when compared to HCS treatment group. We also documented increased beta-catenin translocation and increased VEGF mRNA expression in all treatment groups. Thus, we conclude that the acute as well as chronic protection afforded by combination treatment with statin and resveratrol may be due to pro-angiogenic, anti-hyperlipidemic and anti-apoptotic effects and long-term effects may be caused by increased neo-vascularization of the MI zone leading to less ventricular remodeling.
Journal of Molecular and Cellular Cardiology 04/2007; 42(3):508-16. · 5.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Therapeutic angiogenesis represents a novel approach for the prevention and treatment of ischemic heart disease. This study examined a novel method of stimulating myocardial angiogenesis using secoisolariciresinol diglucoside (SDG), a plant lignan isolated from flaxseed. SDG has been shown to decrease serum cholesterol and reduce the extent of atherosclerosis. In the present study, the angiogenic properties of SDG were investigated in three different models. First, in the in vitro model, human coronary arteriolar endothelial cells (HCAEC) treated with SDG (50 and 100 microM) showed a significant increase in tubular morphogenesis compared with control. Western blot analysis indicated an increased expression of vascular endothelial growth factor (VEGF), kinase insert domain-containing receptor (KDR), Flt-1, angiopoietin-1 (Ang-1), Tie-1, and phosphorylated endothelial nitric oxide synthase (p-eNOS) in the SDG-treated cells. Second, in the ex vivo ischemia/reperfusion model, SDG-treated rats (20 mg/kg b.wt./day for 2 weeks orally) showed an increased level of aortic flow and functional recovery after 2 h of reperfusion following 30 min of ischemia compared with the control group [dP/dt (mm Hg/s) of 2110 +/- 35 versus 1752 +/- 62]. SDG reduced infarct size compared with the control group by 32% (38 versus 26%) and also decreased cardiomyocyte apoptosis. Increased protein expression of VEGF, Ang-1, and p-eNOS was also observed in the SDG-treated group. Third, in the in vivo myocardial infarction model, SDG increased capillary density and myocardial function as evidenced by increased fractional shortening and ejection fraction. In conclusion, these results suggest that SDG has potent angiogenic and antiapoptotic properties that may contribute to its cardioprotective effect in ischemic models.
Journal of Pharmacology and Experimental Therapeutics 03/2007; 320(2):951-9. · 3.89 Impact Factor