Sanjiv Dhingra

A concise description of cardioprotective strategies in doxorubicin-induced cardiotoxicity.

Doxorubicin (Dox) is frequently used as a frontline chemotherapeutic agent against a variety of cancers. Tremendous progress has been made on its optimal usage over the last 40 years. However, cardiotoxicity still remains a major concern. The great promise in this matter is that the mechanisms leading to antitumor activity appear to be different from those leading to Dox-induced cardiomyopathy. In this regard, various cardioprotective agents have been discussed. Attention should be drawn to probucol, a lipid-lowering agent with potent antioxidant properties, which provides complete protection against Dox-induced cardiomyopathy in rats without interfering with the antitumor properties of Dox in an experimental setting. Clinical trials employing Dox therapy in combination with probucol are needed to determine whether the outstanding findings in animal experiments can be extrapolated to clinical results. We have much further to go before the establishment of cancer therapies without any risk of cardiac side effects.

Preserving Prostaglandin E2 Level Prevents Rejection of Implanted Allogeneic Mesenchymal Stem Cells and Restores Postinfarction Ventricular Function

Background— Allogeneic mesenchymal stem cells (MSCs) were immunoprivileged early after cardiac implantation and improved heart function in preclinical and clinical studies. However, long-term preclinical studies demonstrated that allogeneic MSCs lost their immunoprivilege and were rejected in the injured myocardium, resulting in recurrent ventricular dysfunction. This study identifies some of the mechanisms responsible for the immune switch in MSCs and suggests a new treatment to maintain immunoprivilege and preserve heart function. Methods and Results— Rat MSC immunoprivilege was mediated by prostaglandin E2 (PGE2)–induced secretion of 2 critical chemokines, CCL12 and CCL5. These chemokines stimulated the chemoattraction of T cells toward MSCs, suppressed cytotoxic T-cell proliferation, and induced the production of T regulatory cells. MSCs treated with 5-azacytidine for 24 hours differentiated into myogenic cells after 2 weeks, which was associated with decreased PGE2 and chemokine production and the loss of immunoprivilege. Treatment of differentiated MSCs with PGE2 restored chemokine levels and preserved MSC immunoprivilege. In a rat myocardial infarction model, allogeneic MSCs (3×106 cells/rat) were injected into the infarct region with or without a biodegradable hydrogel that slowly released PGE2. Five weeks later, the transplanted MSCs expressed myogenic lineage markers and were rejected in the control group, but in the PGE2-treated group, the transplanted cells survived and heart function improved. Conclusions— Allogeneic MSCs maintained immunoprivilege by PGE2-induced secretion of chemokines CCL12 and CCL5. Differentiation of MSCs decreased PGE2 levels, and immunoprivilege was lost. Maintaining PGE2 levels preserved immunoprivilege after differentiation, prevented rejection of implanted MSCs, and restored cardiac function.

Interleukin-6 downregulation with mesenchymal stem cell differentiation results in loss of immunoprivilege.

Allogeneic mesenchymal stem cell (MSC) transplantation improves cardiac function, but cellular differentiation results in loss of immunoprivilege and rejection. To explore the mechanism involved in this immune rejection, we investigated the influence of interleukin‐6 (IL‐6), a factor secreted by MSCs, on immune privilege after myogenic, endothelial and smooth muscle cell differentiation induced by 5‐azacytidine, VEGF, and transforming growth factor‐β (TGF‐β), respectively. Both RT‐PCR and ELISA showed that myogenic differentiation of MSCs was associated with significant downregulation of IL‐6 expression (P < 0.01), which was also observed following endothelial (P < 0.01) and smooth muscle cell differentiation (P < 0.05), indicating that IL‐6 downregulation was dependent on differentiation but not cell phenotype. Flow cytometry demonstrated that IL‐6 downregulation as a result of myogenic differentiation was associated with increased leucocyte‐mediated cell death in an allogeneic leucocyte co‐culture study (P < 0.01). The allogeneic reactivity associated with IL‐6 downregulation was also observed following MSC differentiation to endothelial and smooth muscle cells (P < 0.01), demonstrating that leucocyte‐mediated cytotoxicity was also dependent on differentiation but not cell phenotype. Restoration of IL‐6 partially rescued the differentiated cells from leucocyte‐mediated cell death. These findings suggest that rejection of allogeneic MSCs after implantation may be because of a reduction in cellular IL‐6 levels, and restoration of IL‐6 may be a new target to retain MSC immunoprivilege.

Erythropoietin protects against doxorubicin-induced heart failure

The hormone erythropoietin (EPO) has been demonstrated to have cardioprotective properties. The present study investigates the role of EPO to prevent heart failure following cancer treatment with doxorubicin [adriamycin (AD)]. Male Wistar rats (150 ± 10 g) were treated with saline (vehicle control group); with EPO, subcutaneously at 1,000 IU/kg body wt, three times per week for 4 wk (EPO group); with adriamycin, intraperitoneally at 2.5 mg/kg body wt, three times per week for 2 wk (AD group); and with adriamycin and EPO (EPO-AD group). Echocardiographic measurements showed that EPO-AD treatment prevented the AD-induced decline in cardiac function. Each of the hearts was then exposed to ischemia and reperfusion during Langendorff perfusion. The percentage of recovery after ischemia-reperfusion was significantly greater in EPO-AD than the AD-treated group for left ventricular developed pressure, maximal increase in pressure, and rate pressure product. The level of oxidative stress was significantly higher in AD (5 μM for 24 h)-exposed isolated cardiomyocytes; EPO (5 U/ml for 48 h) treatment prevented this. EPO treatment also decreased AD-induced cardiomyocyte apoptosis, which was associated with the decrease in the Bax-to-Bcl2 ratio and caspase-3 activation. Immunostaining of myocardial tissue for CD31 showed a significant decrease in the number of capillaries in AD-treated animals. EPO-AD treatment restored the number of capillaries. In conclusion, EPO treatment effectively prevented AD-induced heart failure. The protective effect of EPO was associated with a decreased level of oxidative stress and apoptosis in cardiomyocytes as well as improved myocardial angiogenesis.

Challenges in allogeneic mesenchymal stem cell-mediated cardiac repair.

Autologous mesenchymal stem cells (MSCs) have been proven safe in phase I and II clinical trials in patients who have suffered a myocardial infarction. However, their potential for proliferation and differentiation decreases with age, which limits their efficacy in elderly patients. Allogeneic MSCs offer several key advantages over autologous MSCs, including a high regenerative potential and availability for clinical use without the delay required for expansion. It was believed that allogeneic MSCs were immune privileged and thus able to escape the recipients immune system. In several preclinical studies, allogeneic MSCs were successful in regenerating the myocardium, and the transplanted MSCs improved heart function early after implantation. However, the long-term ability of allogeneic MSCs to preserve heart function is limited because of a transition from an immune privileged to an immunogenic phenotype after the cells differentiate. The initial phase I/II clinical study using allogeneic MSCs in patients with acute myocardial infarction was safe, and no side effects were observed. However, the long-term safety and efficacy of allogeneic MSCs remain to be established. In this review, we discuss the challenges of using allogeneic MSCs for cardiac repair and present strategies to prevent the immune rejection of allogeneic MSCs to increase their potential for use in cardiac patients.

Expression of CNPY2 in Mouse Tissues: Quantification and Localization

Canopy FGF signaling regulator 2 (CNPY2) is a FGF21-modulated protein containing a saposin B-type domain. In vitro studies have shown CNPY2 is able to enhance neurite outgrowth in neurons and stabilize the expression of low density lipoprotein receptor in macrophages and hepatocytes. However, no in vivo data are available on the normal expression of CNPY2 and information is lacking on which cell types express this protein in tissues. To address this, the present study examined CNPY2 expression at the mRNA and protein levels. Quantitative PCR and ELISA examination of mouse tissues showed that CNPY2 varies between organs, with the highest expression in the heart, lung and liver. Immunohistochemistry detected CNPY2 in a variety of cell types including skeletal, cardiac and smooth muscle myocytes, endothelial cells and epithelial cells. CNPY2 was also detectable in mouse blood and human and mouse uteri. These data demonstrate CNPY2 is widely distributed in tissues and suggest the protein has biological functions that have yet to be identified. Using these new observations we discuss possible functions of the protein.

Prophylactic supplementation of resveratrol is more effective than its therapeutic use against doxorubicin induced cardiotoxicity

Resveratrol (RSV), a polyphenolic compound and naturally occurring phytoalexin, has been reported to exert cardio-protective effects in several animal studies. However, the outcome of initial clinical trials with RSV was less effective compared to pre-clinical studies. Therefore, RSV treatment protocols need to be optimized. In this study we evaluated prophylactic versus therapeutic effect of resveratrol (RSV) in mitigating doxorubicin (Dox)-induced cardiac toxicity in rats. To investigate prophylactic effects, RSV was supplemented for 2 weeks along with Dox administration. After 2 weeks, Dox treatment was stopped and RSV was continued for another 4 weeks. To study therapeutic effects, RSV treatment was initiated after 2 weeks of Dox administration and continued for 4 weeks. Both prophylactic and therapeutic use of RSV mitigated Dox induced deterioration of cardiac function as assessed by echocardiography. Also RSV treatment (prophylactic and therapeutic) prevented Dox induced myocardial damage as measured by cardiac enzymes (LDH and CK-MB) in serum. Which was associated with decrease in Dox induced myocardial apoptosis and fibrosis. Interestingly our study also reveals that prophylactic use of RSV was more effective than its therapeutic use in mitigating Dox induced apoptosis and fibrosis in the myocardium. Therefore, prophylactic use of resveratrol may be projected as a possible future adjuvant therapy to minimize cardiotoxic side effects of doxorubicin in cancer patients.

Modulation of Alloimmune Responses by Interleukin-10 Prevents Rejection of Implanted Allogeneic Smooth Muscle Cells and Restores Postinfarction Ventricular Function.

Interleukin-10 (IL-10) gene transduction into allogeneic smooth muscle cells (SMCs) was evaluated to improve the long-term benefits of allogeneic cell transplantation into infarcted myocardium. Allogeneic cells, including SMCs, have been demonstrated to restore cardiac function and repair the infarcted myocardium, but late rejection of the transplanted cells by the host immune system may reverse the benefits of cell therapy. In a rat myocardial infarction model, three groups of rats were injected with either unmodified autologous, unmodified allogeneic, or allogeneic + IL-10 SMCs into the infarct region. Three weeks later, most of the allogeneic cells were rejected, whereas autologous cells were engrafted in the myocardium. IL-10 gene transduction of the allogeneic SMCs significantly improved the cell survival. To understand the mechanism of this improved survival, we evaluated the host immune responses against the SMCs. Allogeneic SMCs expressing IL-10 decreased leukocyte-mediated cytotoxicity in coculture, decreased the number of cytotoxic CD8+ T-cells, and increased the number of CD4+CD25+ regulatory T-cells in vitro and in vivo. Furthermore, IL-10 prevented the production of antidonor antibodies by the recipients against the allogeneic SMCs. Transplantation of unmodified autologous SMCs, but not unmodified allogeneic SMCs, significantly improved fractional shortening and left ventricular dimensions compared to the media-injected control group. However, IL-10 gene-enhanced allogeneic SMCs improved ventricular function, increased wall thickness, and decreased scar length in association with their enhanced survival. We conclude that IL-10 gene-enhanced cell therapy with allogeneic SMCs prevents detrimental alloimmune responses in the recipient, thereby increasing the survival of transplanted allogeneic SMCs and more effectively restoring cardiac function.

Stem cell therapy for cardiac regeneration: hits and misses.

Cardiac injury and loss of cardiomyocytes is a causative as well as a resultant condition of cardiovascular disorders, which are the leading cause of death throughout the world. This loss of cardiomyocytes cannot be completely addressed through the currently available drugs being administered, which mainly function only in relieving the symptoms. There is a huge potential being investigated for regenerative and cell replacement therapies through recruiting stem cells of various origins namely embryonic, reprogramming/induction, and adult tissue. These sources are being actively studied for translation to clinical scenarios. In this review, we attempt to discuss some of these promising scenarios, including the clinical trials and the obstacles that need to be overcome, and hope to address the direction in which stem cell therapy is heading.

Graphene Oxide-Gold Nanosheets Containing Chitosan Scaffold Improves Ventricular Contractility and Function After Implantation into Infarcted Heart

Abnormal conduction and improper electrical impulse propagation are common in heart after myocardial infarction (MI). The scar tissue is non-conductive therefore the electrical communication between adjacent cardiomyocytes is disrupted. In the current study, we synthesized and characterized a conductive biodegradable scaffold by incorporating graphene oxide gold nanosheets (GO-Au) into a clinically approved natural polymer chitosan (CS). Inclusion of GO-Au nanosheets in CS scaffold displayed two fold increase in electrical conductivity. The scaffold exhibited excellent porous architecture with desired swelling and controlled degradation properties. It also supported cell attachment and growth with no signs of discrete cytotoxicity. In a rat model of MI, in vivo as well as in isolated heart, the scaffold after 5 weeks of implantation showed a significant improvement in QRS interval which was associated with enhanced conduction velocity and contractility in the infarct zone by increasing connexin 43 levels. These results corroborate that implantation of novel conductive polymeric scaffold in the infarcted heart improved the cardiac contractility and restored ventricular function. Therefore, our approach may be useful in planning future strategies to construct clinically relevant conductive polymer patches for cardiac patients with conduction defects.