Suresh Varma Penumathsa

Thioredoxin-1 Gene Therapy Enhances Angiogenic Signaling and Reduces Ventricular Remodeling in Infarcted Myocardium of Diabetic Rats

Background— 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. Methods and Results— 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-β, as well as decreased phosphorylated JNK and p38 mitogen-activated protein kinase-α, 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. Conclusions— 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.

Akt/FOXO3a/SIRT1-Mediated Cardioprotection by n-Tyrosol against Ischemic Stress in Rat in Vivo Model of Myocardial Infarction : Switching Gears toward Survival and Longevity

Moderate consumption of wine has been associated with decreased risk of cardiovascular events. Recently we have shown that white wine is equally as cardioprotective as red wine. However, unlike resveratrol (polyphenol in red wine), the white wine component, n-tyrosol [2-(4-hydroxyphenyl)ethanol] has not been explored for its cardioprotective effect and mechanism of action. Therefore, the present study was designed to evaluate the effect of tyrosol treatment (5 mg/kg/day for 30 days) on myocardial ischemic stress in a rat in vivo model of Myocardial Infarction (MI) and to identify key molecular targets involved in this mechanism. MI was induced by Left Anterior Descending (LAD) coronary artery ligation. Reduced infarct size (32.42 vs 48.03%) and cardiomyocyte apoptosis (171 vs 256 counts/100 HPF) were observed along with improvement in the myocardial functional parameters such as LVIDs (5.89 vs 6.58 mm), ejection fraction (51.91 vs 45.09%), and fractional shortening (28.46 vs 23.52%) as assessed by echocardiography in the tyrosol-treated animals when compared to the nontreated controls. We have also observed significant increase in the phosphorylation of Akt (1.4-fold), eNOS (3-fold) and FOXO3a (2.6-fold). In addition, tyrosol induced the expression of longevity protein SIRT1 (3.2-fold) in the MI group as compared to the non-treated MI control. Therefore tyrosols SIRT1, Akt and eNOS activating power adds another dimension to the white wine research, because it adds a great link to the French paradox. In conclusion these findings suggest that tyrosol induces myocardial protection against ischemia related stress by inducing survival and longevity proteins that may be considered as anti-aging therapy for the heart. However, human intervention studies would be necessary before establishing any recommendations about dietary habits for tyrosol intake or administration of dietary supplements containing tyrosol.

Resveratrol: a promising agent in promoting cardioprotection against coronary heart disease.

The inverse association between alcohol intake and coronary heart disease has been consistently reported in cross-culture, case-control, and cohort studies. Over the past couple of decades, however, many studies have explained promising health benefits associated with wine consumption. Some studies suggest that red wine is more cardioprotective than white wine, possibly due to the increased content of flavanoid antioxidants found in red wine. Several experimental studies, including ours, support the evidence that these beneficial effects are due to resveratrol, the polyphenolic compound present in red wine. Many studies have provided evidence that resveratrol possesses antioxidant and antiapoptotic effects apart from activation of longevity proteins (such as SIRT-1). We have recently reported the angiogenic, antihypercholesterolemic, and antidiabetic effects of resveratrol and the mechanisms involved in reduced ventricular remodeling and increased cardiac functions. We have also shown different strategic target molecules involved in resveratrol-mediated cardioprotection. Therefore, this review discusses the potential effect of resveratrol and the mechanisms involved in resveratrol-mediated cardioprotection during myocardial infarction, hypercholesterolemia, and diabetes rendering its beneficial effects during health and disease.

Sildenafil-mediated neovascularization and protection against myocardial ischaemia reperfusion injury in rats: role of VEGF/angiopoietin-1

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.

Strategic targets to induce neovascularization by resveratrol in hypercholesterolemic rat myocardium: Role of caveolin-1, endothelial nitric oxide synthase, hemeoxygenase-1, and vascular endothelial growth factor

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.

Niacin bound chromium treatment induces myocardial Glut-4 translocation and caveolar interaction via Akt, AMPK and eNOS phosphorylation in streptozotocin induced diabetic rats after ischemia-reperfusion injury.

Diabetes, one of the major risk factors of metabolic syndrome culminates in the development of Ischemic Heart Disease (IHD). Refined diets that lack micronutrients, mainly trivalent chromium (Cr(3+)) have been identified as the contributor in the rising incidence of diabetes. We investigated the effect of niacin-bound chromium (NBC) during ischemia/reperfusion (IR) injury in streptozotocin induced diabetic rats. Rats were randomized into: Control (Con); Diabetic (Dia) and Diabetic rats fed with NBC (Dia+NBC). After 30 days of treatment, the isolated hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. NBC treatment demonstrated significant increase in left ventricular functions and significant reduction in infarct size and cardiomyocyte apoptosis in Dia+NBC compared with Dia. Increased Glut-4 translocation to the lipid raft fractions was also observed in Dia+NBC compared to Dia. Reduced Cav-1 and increased Cav-3 expression along with phosphorylation of Akt, eNOS and AMPK might have resulted in increased Glut-4 translocation in Dia+NBC. Our results indicate that the cardioprotective effect of NBC is mediated by increased activation of AMPK, Akt and eNOS resulting in increased translocation of Glut-4 to the caveolar raft fractions thereby alleviating the effects of IR injury in the diabetic myocardium.

Secoisolariciresinol Diglucoside: Relevance to Angiogenesis and Cardioprotection against Ischemia-Reperfusion Injury

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 μM) 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.

Bromelain induces cardioprotection against ischemia-reperfusion injury through Akt/FOXO pathway in rat myocardium.

Bromelain (Br), a proteolytic enzyme extracted from the stem of the pineapple, is known to possess anti-inflammatory activity and has been shown to reduce blood viscosity, prevent the aggregation of blood platelets, and improve ischemia-reperfusion (I/R) injury in a skeletal muscle model. We investigated the capacity of Br to limit myocardial injury in a global I/R model. Adult male Sprague-Dawley rats were divided into two groups: control (PBS) and Br at 10 mg/kg in PBS administered via intraperitoneal injection (twice/day) for 15 consecutive days. On day 16, the hearts were excised and subjected to 30 min of global ischemia followed by 2 h of reperfusion. Br treatment showed higher left ventricular functional recovery throughout reperfusion compared with the controls [maximum rate of rise in intraventricular pressure (dP/dt max), 2,225 vs. 1,578 mmHg/s at 2 h reperfusion]. Aortic flow was also found to be increased in Br treatment when compared with that in untreated rats (11 vs. 1 ml). Furthermore, Br treatment reduced both the infarct size (34% vs. 43%) and the degree of apoptosis (28% vs. 37%) compared with the control animals. Western blot analysis showed an increased phosphorylation of both Akt and FOXO3A in the treatment group compared with the control. These results demonstrated for the first time that Br triggers an Akt-dependent survival pathway in the heart, revealing a novel mechanism of cardioprotective action and a potential therapeutic target against I/R injury.

Adeno-sh-β-Catenin Abolishes Ischemic Preconditioning–Mediated Cardioprotection by Downregulation of Its Target Genes VEGF, Bcl-2, and Survivin in Ischemic Rat Myocardium

beta-Catenin, the downstream target of glycogen synthase kinase-3beta (GSK-3beta), plays a vital role in ischemic preconditioning (IP)-mediated cardioprotection. In the present study, we investigated the mechanism of IP-mediated cardioprotection through suppression of beta-catenin expression by intramyocardial injection of adeno-sh-RNA against beta-catenin (BCT) (4 x 10(8) pfu). Adeno-LacZ (LZ) was used as control. The rats were randomized into (a) LZ + ischemia-reperfusion (IR); (b) LZIPIR; (c) BCTIR; and (d) BCTIPIR. Isolated hearts from each group were subjected to 30 min of I followed by 2 h of R. Both IPIR group hearts were subjected to IP (5 min I + 10 min R; four cycles) before IR. Significant reduction in left ventricular functional recovery (78 vs. 88 mm Hg), dp/dt(max) (1,802 vs. 2,189 mm Hg/sec), and aortic flow (4 vs. 9 ml/min) was observed in BCTIPIR compared with LZIPIR at 120 min of reperfusion. Increased infarct size (42 vs. 24%) and apoptotic cardiomyocytes (122 vs. 58 counts/60 HPF) were observed in BCTIPIR compared with LZIPIR. Realtime PCR and Western blot analysis showed significant downregulation in mRNA and protein expression of VEGF, Bcl-2, and survivin in BCTIPIR compared with LZIPIR. These findings indicated for the first time that silencing beta-catenin abolished IP-mediated cardioprotection, probably through inhibition of VEGF-Bcl-2 and survivin.

Thioredoxin-1 Gene Delivery Induces Heme Oxygenase-1 Mediated Myocardial Preservation After Chronic Infarction in Hypertensive Rats

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSION 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.