Vaithinathan Selvaraju

Diabetes, oxidative stress, molecular mechanism, and cardiovascular disease – an overview

In recent years, diabetes and its associated complications have come to represent a major public health concern. It is a complex disease characterized by multiple metabolic derangements and is known to impair cardiac function by disrupting the balance between pro-oxidants and antioxidants at the cellular level. The subsequent generation of reactive oxygen species (ROS) and accompanying oxidative stress are hallmarks of the molecular mechanisms responsible for cardiovascular disease. Among several oxidative stress-mediated mechanisms that have been proposed, ROS-mediated oxidative stress has received the most attention. ROS have been shown to interact with proteins, lipids, and DNA, causing damage to the cellular macromolecules and subsequently, deterioration of cellular function. Induction of thioredoxin-1 (Trx1) gene expression has been demonstrated to protect the diabetic myocardium from dysfunction by reducing oxidative stress and enhancing the expression of heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF). The failure of antioxidants to consistently demonstrate clinical benefit necessitates further investigation of the role of oxidative stress in diabetes-mediated cardiovascular disease.

Conundrum of pathogenesis of diabetic cardiomyopathy: role of vascular endothelial dysfunction, reactive oxygen species, and mitochondria

Diabetic cardiomyopathy and heart failure have been recognized as the leading causes of mortality among diabetics. Diabetic cardiomyopathy has been characterized primarily by the manifestation of left ventricular dysfunction that is independent of coronary artery disease and hypertension among the patients affected by diabetes mellitus. A complex array of contributing factors including the hypertrophy of left ventricle, alterations of metabolism, microvascular pathology, insulin resistance, fibrosis, apoptotic cell death, and oxidative stress have been implicated in the pathogenesis of diabetic cardiomyopathy. Nevertheless, the exact mechanisms underlying the pathogenesis of diabetic cardiomyopathy are yet to be established. The critical involvement of multifarious factors including the vascular endothelial dysfunction, microangiopathy, reactive oxygen species (ROS), oxidative stress, mitochondrial dysfunction has been identified in the mechanism of pathogenesis of diabetic cardiomyopathy. Although it is difficult to establish how each factor contributes to disease, the involvement of ROS and mitochondrial dysfunction are emerging as front-runners in the mechanism of pathogenesis of diabetic cardiomyopathy. This review highlights the role of vascular endothelial dysfunction, ROS, oxidative stress, and mitochondriopathy in the pathogenesis of diabetic cardiomyopathy. Furthermore, the review emphasizes that the puzzle has to be solved to firmly establish the mitochondrial and/or ROS mechanism(s) by identifying their most critical molecular players involved at both spatial and temporal levels in diabetic cardiomyopathy as targets for specific and effective pharmacological/therapeutic interventions.

Thioredoxin-1 (Trx1) engineered mesenchymal stem cell therapy increased pro-angiogenic factors, reduced fibrosis and improved heart function in the infarcted rat myocardium

INTRODUCTION Engraftment of mesenchymal stem cells (MSCs) has emerged as a powerful candidate for mediating myocardial repair. In this study, we genetically modified MSCs with an adenovector encoding thioredoxin-1 (Ad.Trx1). Trx1 has been described as a growth regulator, a transcription factor regulator, a cofactor, and a powerful antioxidant. We explored whether engineered MSCs, when transplanted, are capable of improving cardiac function and angiogenesis in a rat model of myocardial infarction (MI). METHODS Rat MSCs were cultured and divided into MSC, MSC+Ad.LacZ, and MSC+Ad.Trx1 groups. The cells were assayed for proliferation, and differentiation potential. In addition, rats were divided into control-sham (CS), control-MI (CMI), MSC+Ad.LacZ-MI (MLZMI), and MSC+Ad.Trx1-MI (MTrxMI) groups. MI was induced by left anterior descending coronary artery (LAD) ligation, and MSCs preconditioned with either Ad.LacZ or Ad.Trx1 were immediately administered to four sites in the peri-infarct zone. RESULTS The MSC+Ad.Trx1 cells increased the proliferation capacity and maintained pluripotency, allowing them to divide into cardiomyocytes, smooth muscle, and endothelial cells. Western blot analysis, 4 days after treatment showed increased vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1), and C-X-C chemokine receptor type 4 (CXCR4). Also capillary density along with myocardial function as examined by echocardiography was found to be increased. Fibrosis was reduced in the MTrxMI group compared to MLZMI and CMI. Visualization of Connexin-43 by immunohistochemistry confirmed increased intercellular connections in the MTrxMI rats compared to MLZMI. CONCLUSION Engineering MSCs to express Trx1 may prove to be a strategic therapeutic modality in the treatment of cardiac failure.

Glutaredoxin-1 Overexpression Enhances Neovascularization and Diminishes Ventricular Remodeling in Chronic Myocardial Infarction

Oxidative stress plays a critical role in the pathophysiology of cardiac failure, including the modulation of neovascularization following myocardial infarction (MI). Redox molecules thioredoxin (Trx) and glutaredoxin (Grx) superfamilies actively maintain intracellular thiol-redox homeostasis by scavenging reactive oxygen species. Among these two superfamilies, the pro-angiogenic function of Trx-1 has been reported in chronic MI model whereas similar role of Grx-1 remains uncertain. The present study attempts to establish the role of Grx-1 in neovascularization and ventricular remodeling following MI. Wild-type (WT) and Grx-1 transgenic (Grx-1Tg/+) mice were randomized into wild-type sham (WTS), Grx-1Tg/+ Sham (Grx-1Tg/+S), WTMI, Grx-1Tg/+MI. MI was induced by permanent occlusion of the LAD coronary artery. Sham groups underwent identical time-matched surgical procedures without LAD ligation. Significant increase in arteriolar density was observed 7 days (d) after surgical intervention in the Grx-1Tg/+MI group as compared to the WTMI animals. Further, improvement in myocardial functional parameters 30 d after MI was observed including decreased LVIDs, LVIDd, increased ejection fraction and, fractional shortening was also observed in the Grx-1Tg/+MI group as compared to the WTMI animals. Moreover, attenuation of oxidative stress and apoptotic cardiomyocytes was observed in the Grx-1Tg/+MI group as compared to the WTMI animals. Increased expression of p-Akt, VEGF, Ang-1, Bcl-2, survivin and DNA binding activity of NF-κB were observed in the Grx-1Tg/+MI group when compared to WTMI animals as revealed by Western blot analysis and Gel-shift analysis, respectively. These results are the first to demonstrate that Grx-1 induces angiogenesis and diminishes ventricular remodeling apparently through neovascularization mediated by Akt, VEGF, Ang-1 and NF-κB as well as Bcl-2 and survivin-mediated anti-apoptotic pathway in the infarcted myocardium.

Simvastatin treatment inhibits hypoxia inducible factor 1-alpha-(HIF-1alpha)-prolyl-4-hydroxylase 3 (PHD-3) and increases angiogenesis after myocardial infarction in streptozotocin-induced diabetic rat

BACKGROUND Statins (HMG-CoA reductase inhibitors), are known to improve cardiac function in diabetes-induced cardiovascular disease. We investigated the mechanism by which statins ameliorate cardiac function after myocardial infarction (MI). Simvastatin (S) increased tube formation and migration of HUVEC in vitro. We examined the role of simvastatin on cardiac function in streptozotocin (STZ) induced diabetic rats subjected to MI. METHODS Rats were randomly assigned to 1) Control (non-diabetic) Sham (CS); 2) Control (non-diabetic) MI (CMI); 3) Control Statin treated Sham (CSS); 4) Control Statin treated MI (CSMI); 5) Diabetic Sham (DS); 6) Diabetic MI (DMI); 7) Diabetic Statin treated Sham (DSS); 8) Diabetic Statin treated MI (DSMI). Two weeks after STZ/saline injection Simvastatin (1mg/kg.b.wt) was gavaged for 15 days (d). MI was induced 30 d after treatment by permanent LAD ligation. RESULTS The S treated MI groups exhibited increased arteriolar density (23 ± 0.6 vs. 14.8 ± 0.4 counts/mm(2), DSMI vs. DMI) and reduced fibrosis at 30 d post-MI. VEGF measurement by ELISA after 4d post-MI showed increased expression in DSMI group compared to DMI group. Western blot analysis showed decreased Prolyl-4-Hydroxylase 3 (PHD-3) in DSMI group as compared to DMI group. Echocardiographic analysis 4 weeks after post-MI showed significant improvement in ejection fraction (50.11 ± 1.83 vs. 32.46 ± 2.19%; DSMI vs. DMI) and fractional shortening (26.77 ± 1.12 vs.16.36 ± 1.22%; DSMI vs. DMI) in both statin-treated MI groups regardless of diabetic status. CONCLUSION These results suggest that statin therapy mitigates impairment of angiogenesis and myocardial dysfunction following MI in the diabetic rat through PHD3 inhibition.

Deletion of prolyl hydroxylase domain proteins (PHD1, PHD3) stabilizes hypoxia inducible factor-1 alpha, promotes neovascularization, and improves perfusion in a murine model of hind-limb ischemia

BACKGROUND There is an emerging focus on investigating innovative therapeutic molecules that can potentially augment neovascularization in order to treat peripheral arterial disease (PAD). Although prolyl hydroxylase domain proteins 1 and 3 (PHD1 and PHD3) may modulate angiogenesis via regulation of hypoxia inducible factor-1α (HIF-1α), there has been no study directly addressing their roles in ischemia-induced vascular growth. We hypothesize that PHD1(-/-) or PHD3(-/-) deficiency might promote angiogenesis in the murine hind-limb ischemia (HLI) model. STUDY DESIGN Wild type (WT), PHD1(-/-) and PHD3(-/-) male mice aged 8-12weeks underwent right femoral artery ligation. Post-procedurally, motor function assessment and laser Doppler imaging were periodically performed. The mice were euthanized after 28days and muscles were harvested. Immunohistochemical analysis was performed to determine the extent of angiogenesis by measuring capillary and arteriolar density. VEGF expression was quantified by enzyme-linked immunosorbent assay (ELISA). Bcl-2 and HIF-1α were analyzed by immunofluorescence. Fibrosis was measured by picrosirius red staining. RESULTS PHD1(-/-) and PHD3(-/-) mice showed significantly improved recovery of perfusion and motor function score when compared to WT after femoral artery ligation. These mice also exhibited increased capillary and arteriolar density, capillary/myocyte ratio along with decreased fibrosis compared to WT. VEGF, Bcl-2 and HIF-1α expression increased in PHD1(-/-) and PHD3(-/-) mice compared to WT. CONCLUSIONS Taken together these results suggest that PHD1 and PHD3 deletions promote angiogenesis in ischemia-injured tissue, and may present a promising therapeutic strategy in treating PAD.

Targeted Gene Deletion of Prolyl Hydroxylase Domain Protein 3 Triggers Angiogenesis and Preserves Cardiac Function by Stabilizing Hypoxia Inducible Factor 1 Alpha Following Myocardial Infarction

The key oxygen sensing molecules, Prolyl-hydroxylase domain 1-3 enzymes (PHD1-3), regulate hypoxia-inducible factor (HIF) under hypoxia. In the settings of cardiomyopathy and ischemia-reperfusion injury, PHD3 expression is elevated, resulting in decreased HIF activation. The role of PHD3 in myocardial injury is poorly understood. Hence, we aimed to determine the effects of PHD3 deletion in mice on HIF-1α and other related pathways following myocardial infarction (MI). Left coronary artery (LAD) in both wild type and prolyl hydroxylase 3 knock out (PHD3⁻/⁻) mice was ligated to induce myocardial infarction. Electrophoretic mobility shift analysis showed significant increase in DNA-binding activity of HIF-1α in PHD3⁻/⁻ mice as compared to wild type (WT) mice post MI. The PHD3⁻/⁻MI group also showed decreased fibrosis. Seven days after MI, enhanced capillary/arteriolar density was observed compared to WTMI group. PHD3⁻/⁻ mice subjected to MI also showed improved cardiac functions (Ejection fraction and Fractional shortening), as assessed by echocardiogram, compared to WT. Western blot analysis showed increased VEGF, Ang-1 & Bcl-2 expression in PHD3⁻/⁻MI group. In conclusion, ablation of the PHD3 gene resulted in increased angiogenesis and cardiac function after infarction thereby offering a potential target for pharmacological management of ischemic myocardial disease.

Increased survivability of ischemic skin flap tissue in Flk-1+/− mice by Pellino-1 intervention

Reduced skin flap survival due to ischemia is a serious concern during reconstructive cosmetic surgery. The absence of VEGF and its receptors during ischemia may lead to flap failure. We identified Peli1, a 46‐kDa protein, as a proangiogenic molecule and is directly regulated by VEGF. Therefore, we hypothesized that Peli1 acts downstream of Flk‐1/VEGFR2 and aids in skin flap survival during ischemia.

Overexpression of Thioredoxin1 enhances functional recovery in a mouse model of hind limb ischemia

BACKGROUND There is keen interest in finding nonsurgical treatments for peripheral vascular disease (PVD). Previously, we demonstrated that selective activation of Thioredoxin1 (Trx1), a 12-kDa cytosolic protein, initiates redox-dependent signaling and promotes neovascularization after ischemic heart disease. Therefore, Trx1 might possess immense potential to not only treat murine hind limb ischemia (HLI) through effective angiogenesis but also provide PVD patients with nonsurgical therapy to enhance neovascularization and improve blood perfusion. METHODS To determine whether activation of Trx1 increases blood perfusion in HLI, two different strategies were used-gene therapy and transgenic model system. In adenoviral-mediated gene therapy, 8- to 12-wk-old mice were divided into two groups: (1) control Adeno-LacZ (Ad-LacZ) and (2) Adeno-Thiroedoxin1 (Ad-Trx1). The mice underwent surgical intervention to induce right HLI followed by injection with Ad-LacZ or Ad-Trx1, respectively. In the second strategy, we used wild-type and transgenic mice overexpressing Trx1 (Trx1Tg/+). All the animals underwent Doppler imaging for the assessment of limb perfusion followed by immunohistochemistry and Western blot analysis. RESULTS Significant increases in perfusion ratio were observed in all the Trx1 overexpressed groups compared with their corresponding controls. Expressions of heme oxygenase-1, vascular endothelial growth factor, and the vascular endothelial growth factor receptors Flk-1 and Flt-1 were increased in Trx1 overexpressed mice compared with their respective controls. Blood perfusion in the ischemic limb gradually improved and significantly recovered in Trx1Tg/+ and Ad-Trx1 groups compared with their corresponding controls. The capillary and arteriolar density in the ischemic zone were found to be higher in Trx1Tg/+ group compared with wild type. CONCLUSIONS The overall outcomes of our study demonstrate that Trx1 enhances blood perfusion and increases angiogenic protein expression in a rodent HLI model. These results suggest that Trx1 is a potential target for clinical trials and drug therapy for the treatment of PVD.

Thioredoxin-1 augments wound healing and promote angiogenesis in a murine ischemic full-thickness wound model

Background Nonhealing wounds are a continuing health problem in the United States. Overproduction of reactive oxygen species is a major causative factor behind delayed wound healing. Previously we reported that thioredoxin‐1 treatment could alleviate oxidative stress under ischemic conditions, such as myocardial infarction and hindlimb ischemia. In this study, we explored the potential for thioredoxin‐1 gene therapy to effectively aid wound healing through improved angiogenesis in a murine ischemic wound model. Methods Full‐thickness, cutaneous, ischemic wounds were created in the dorsum skin flap of 8‐ to 12‐week‐old CD1 mice. Nonischemic wounds created lateral to the ischemic skin flap served as internal controls. Mice with both ischemic wounds and nonischemic wounds were treated with Adeno‐LacZ (1 × 109 pfu) or Adeno‐thioredoxin‐1 (1 × 109 pfu), injected intradermally around the wound. Digital imaging was performed on days 0, 3, 6, and 9 to assess the rate of wound closure. Tissue samples collected at predetermined time intervals were processed for immunohistochemical analysis. Results No significant differences in wound closure were identified among the nonischemic wounds control, nonischemic wounds‐LacZ, and nonischemic wounds–thioredoxin‐1 groups. Hence, only mice with ischemic wounds were further analyzed. The ischemic wounds–thioredoxin‐1 group had significant improvement in wound closure on days 6 and 9 after surgery compared with the ischemic wounds control and ischemic wounds‐LacZ groups. Immunohistochemical analysis indicated increased thioredoxin‐1, vascular endothelial cell growth factor, and &bgr;‐catenin levels in the ischemic wounds–thioredoxin‐1 group compared with the ischemic wounds control and ischemic wounds‐LacZ groups, as well as increased capillary density and cell proliferation, as represented by Ki‐67 staining. Conclusion Taken together, thioredoxin‐1 gene therapy promotes vascular endothelial cell growth factor signaling and re‐epithelialization and activates wound closure in mice with ischemic wounds.