Saher Hamed

Nitric oxide: a key factor behind the dysfunctionality of endothelial progenitor cells in diabetes mellitus type-2

Diabetes mellitus type-2 (DM-2) contributes to atherogenesis by inducing endothelial cell injury and dysfunction. Endothelial progenitor cells (EPCs) are essential to blood vessel formation, can differentiate into mature endothelial cells, and promote the repair of damaged endothelium. In DM-2, the circulating EPC count is low and their functionality is impaired. The mechanisms that underlie this reduced count and impaired functionality are poorly understood. Nitric oxide (NO) is a short-lived signalling molecule that is produced by vascular endothelial cells and participates in the maintenance of vascular tone. NO is also known to participate in other physiological processes, such as cell survival, proliferation, and migration. The bioavailability of NO is reduced in EPCs from DM-2 patients. Interestingly, an inverse relationship exists between the reduction in NO bioavailability in EPCs and the patients plasma glucose and glycated haemoglobin levels. In addition, NO bioavailability in EPCs correlates with plasma oxidized low-density lipoprotein levels in DM-2. Although this reduction in NO bioavailability could be attributed to oxidative stress in DM-2 patients, it also may be due to impairment of one or more members of the protein signalling cascades that are responsible for NO production. The stimulation of NO production or its signalling cascades in EPCs may increase their numbers and improve their function, thus attenuating endothelium damage, independent of the vasodilatory effects of NO. This review summarizes the metabolic alterations that underlie the molecular mechanisms that may be responsible for EPC decrease and dysfunction in DM-2 with emphasis on the involvement of the NO system.

Nitric oxide and superoxide dismutase modulate endothelial progenitor cell function in type 2 diabetes mellitus

BackgroundThe function of endothelial progenitor cells (EPCs), which are key cells in vascular repair, is impaired in diabetes mellitus. Nitric oxide (NO) and reactive oxygen species can regulate EPC functions. EPCs tolerate oxidative stress by upregulating superoxide dismutase (SOD), the enzyme that neutralizes superoxide anion (O2-). Therefore, we investigated the roles of NO and SOD in glucose-stressed EPCs.MethodsThe functions of circulating EPCs from patients with type 2 diabetes were compared to those from healthy individuals. Healthy EPCs were glucose-stressed, and then treated with insulin and/or SOD. We assessed O2- generation, NO production, SOD activity, and their ability to form colonies.ResultsEPCs from diabetic patients generated more O2-, had higher NAD(P)H oxidase and SOD activity, but lower NO bioavailability, and expressed higher mRNA and protein levels of p22-phox, and manganese SOD and copper/zinc SOD than those from the healthy individuals. Plasma glucose and HbA1c levels in the diabetic patients were correlated negatively with the NO production from their EPCs. SOD treatment of glucose-stressed EPCs attenuated O2- generation, restored NO production, and partially restored their ability to form colonies. Insulin treatment of glucose-stressed EPCs increased NO production, but did not change O2- generation and their ability to form colonies. However, their ability to produce NO and to form colonies was fully restored after combined SOD and insulin treatment.ConclusionOur data provide evidence that SOD may play an essential role in EPCs, and emphasize the important role of antioxidant therapy in type 2 diabetic patients.

Topical Erythropoietin Promotes Wound Repair in Diabetic Rats

Wound healing in diabetic patients is slower than in healthy individuals. Erythropoietin (EPO) has non-hemopoietic targets in the skin, and systemically administered EPO promotes wound healing in experimental animals. This study investigated the effect of topical EPO treatment on defective wound repair in the skin of diabetic rats. Full-thickness excisional skin wounds were made in 38 rats, of which 30 had diabetes. The wounds were then treated topically with a cream that contained either vehicle, 600 IU ml(-1) EPO (low dose), or 3,000 IU ml(-1) (high dose) EPO. We assessed the rate of wound closure during the 12-day treatment period, and microvascular density (MVD), vascular endothelial growth factor (VEGF), and hydroxyproline (HP) contents, and the extent of apoptosis in wound tissues at the end of the 12-day treatment period. Topical EPO treatment significantly reduced the time to final wound closure. This increased rate of closure of the two EPO-treated wounds in diabetic rats was associated with increased MVD, VEGF, and HP contents, and a reduced extent of apoptosis. In light of our finding that topical EPO treatment promotes skin wound repair in diabetic rats, we propose that topical EPO treatment is a therapeutically beneficial method of treating chronic diabetic wounds.

Erythropoietin Improves the Survival of Fat Tissue after Its Transplantation in Nude Mice

Background Autologous transplanted fat has a high resorption rate, providing a clinical challenge for the means to reduce it. Erythropoietin (EPO) has non-hematopoietic targets, and we hypothesized that EPO may improve long-term fat graft survival because it has both pro-angiogenic and anti-apoptotic properties. We aimed to determine the effect of EPO on the survival of human fat tissue after its transplantation in nude mice. Methodology/Principal Findings Human fat tissue was injected subcutaneously into immunologically-compromised nude mice, and the grafts were then treated with either 20 IU or 100 IU EPO. At the end of the 15-week study period, the extent of angiogenesis, apoptosis, and histology were assessed in the fat grafts. The results were compared to vascular endothelial growth factor (VEGF)-treated and phosphate-buffered saline (PBS)-treated fat grafts. The weight and volume of the EPO-treated grafts were higher than those of the PBS-treated grafts, whose weights and volumes were not different from those of the VEGF-treated grafts. EPO treatment also increased the expression of angiogenic factors and microvascular density, and reduced inflammation and apoptosis in a dose-dependent manner in the fat grafts. Conclusions/Significance Our data suggest that stimulation of angiogenesis by a cluster of angiogenic factors and decreased fat cell apoptosis account for potential mechanisms that underlie the improved long-term survival of fat transplants following EPO treatment.

Fibronectin Potentiates Topical Erythropoietin-Induced Wound Repair in Diabetic Mice

Diabetes mellitus disrupts all phases of the wound repair cascade and leads to development of chronic wounds. We previously showed that topical erythropoietin (EPO) can promote wound repair in diabetic rats. Fibronectin (FN) has a critical role throughout the process of wound healing, yet it is deficient in wound tissues of diabetic patients. Therefore, we investigated the effect of topical treatment of both EPO and FN (EPO/FN) on wound repair in diabetic mice. Full-thickness excisional skin wounds in diabetic and nondiabetic mice were treated with a cream containing vehicle, EPO, FN, or EPO/FN. We assessed the rate of wound closure, angiogenesis, apoptosis, and expression of inflammatory cytokines, endothelial nitric oxide synthase (eNOS) and β1-integrin, in the wound tissues. We also investigated the effect of EPO, FN, and EPO/FN on human dermal microvascular endothelial cells and fibroblasts cultured on fibrin-coated plates, or in high glucose concentrations. EPO/FN treatment significantly increased the rate of wound closure and this effect was associated with increased angiogenesis, increased eNOS and β1-integrin expression, and reduced expression of inflammatory cytokines and apoptosis. Our findings show that EPO and FN have an additive effect on wound repair in diabetic mice.

Erythropoietin, a novel repurposed drug: An innovative treatment for wound healing in patients with diabetes mellitus

Developing a new drug is expensive: the cost of going from bench to bedside is about

Treating fat grafts with human endothelial progenitor cells promotes their vascularization and improves their survival in diabetes mellitus.

US1 billion. Therefore, the repurposing of an approved drug is potentially rewarding because it expands the drugs existing therapeutic profile and preempts additional development costs. As the safety profile of a repurposed drug is already well known, any new investigations could then focus on its efficacy and other therapeutic benefits. Recombinant erythropoietin (EPO) is a potential candidate for repurposing because the results of numerous studies have shown that systemic and topical EPO is therapeutically beneficial when it is administered to healthy and diabetic animals with acute and chronic skin wounds and burns. Moreover, the molecular mechanisms of EPOs actions have been elucidated: EPO acts on those nonhematopoietic cells which are involved in the innate immune response where it promotes cellular proliferation and differentiation, exerts its cytoprotective actions, and inhibits apoptosis. In this review, the mechanism of EPOs action in skin wound healing is reviewed, and its potential for treating acute and chronic skin wounds and stimulating tissue regeneration in diabetic patients is discussed.

The chemokine stromal cell-derived factor-1α promotes endothelial progenitor cell-mediated neovascularization of human transplanted fat tissue in diabetic immunocompromised mice.

Background: Bone marrow–derived endothelial progenitor cells are required for vascularization of a fat graft to form a functional microvasculature within the graft and to facilitate its integration into the surrounding tissues. Organ transplantation carries a high risk of graft loss and rejection in patients with diabetes mellitus because endothelial progenitor cell function is impaired. The authors investigated the influence of endothelial progenitor cell treatment on the phenotype and survival of human fat grafts in immunocompromised mice with experimentally induced diabetes mellitus. Methods: The authors injected 1 ml of human fat tissue into the scalps of 14 nondiabetic and 28 diabetic immunocompromised mice, and then treated some of the grafts with endothelial progenitor cells that were isolated from the blood of a human donor. The phenotype of the endothelial progenitor cell–treated fat grafts from the 14 diabetic mice was compared with that of the untreated fat grafts from 14 nondiabetic and 14 diabetic mice, 18 days and 15 weeks after fat transplantation. Determination of graft phenotype included measurements of weight and volume, vascular endothelial growth factor levels, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase, and caspase 3 expression levels, and histologic analysis of the extent of vascularization. Results: The untreated grafts from the diabetic mice were fully resorbed 15 weeks after fat transplantation. The phenotype of endothelial progenitor cell–treated fat grafts from the diabetic mice was similar to that of the untreated fat grafts from the nondiabetic mice. Conclusion: Endothelial progenitor cell treatment of transplanted fat can increase the survival of a fat graft by inducing its vascularization and decreasing the extent of apoptosis.

Topical Erythropoietin Treatment Accelerates the Healing of Cutaneous Burn Wounds in Diabetic Pigs Through an Aquaporin-3-Dependent Mechanism

Background: Stromal cell–derived factor-1&agr; is a chemokine and mediates endothelial progenitor cell–induced neovascularization. Because vascularization of a graft is crucial for its survival, the authors investigated whether stromal cell–derived factor-1&agr; could improve fat graft survival by inducing endothelial progenitor cell–mediated neovascularization and preventing its resorption. Methods: The authors injected 1 ml of human fat tissue into the scalps of 30 diabetic and 10 nondiabetic immunocompromised mice. The fat grafts were treated with phosphate-buffered saline or stromal cell–derived factor-1&agr;. Determination of graft phenotype included measurements of their weights and volumes, vascular endothelial growth factor (VEGF) levels, the stromal cell–derived factor-1&agr; receptor CXCR4, VEGF receptor 2, endothelial nitric oxide synthase, serine/threonine-specific protein kinase (protein kinase B), caspase 3, and cytochrome c expression levels, and the extent of vascularization. Results: Eighteen days after transplantation, stromal cell–derived factor-1&agr; treatment of the grafts in the diabetic mice (1) increased plasma VEGF levels; (2) raised VEGF receptor 2, CXCR4, endothelial nitric oxide synthase, and protein kinase B expression levels; and (3) reduced caspase 3 and cytochrome c expression levels in the fat grafts. Fifteen weeks after transplantation, stromal cell–derived factor-1&agr; treatment of the grafts prevented their resorption and increased the extent of their vascularization. Conclusion: Locally delivered stromal cell–derived factor-1&agr; increases fat graft survival by stimulating neovascularization and reducing fat cell apoptosis through an endothelial progenitor cell–mediated mechanism.

Methods of improving transplantation using sdf-1alpha

We have previously reported that the topical application of erythropoietin (EPO) to cutaneous wounds in rats and mice with experimentally induced diabetes accelerates their healing by stimulating angiogenesis, reepithelialization, and collagen deposition, and by suppressing the inflammatory response and apoptosis. Aquaporins (AQPs) are integral membrane proteins whose function is to regulate intracellular fluid hemostasis by enabling the transport of water and glycerol. AQP3 is the AQP that is expressed in the skin where it facilitates cell migration and proliferation and re-epithelialization during wound healing. In this report, we provide the results of an investigation that examined the contribution of AQP3 to the mechanism of EPO action on the healing of burn wounds in the skin of pigs with experimentally induced type 1 diabetes. We found that topical EPO treatment of the burns accelerated their healing through an AQP3-dependent mechanism that activates angiogenesis, triggers collagen and hyaluronic acid synthesis and the formation of the extracellular matrix (ECM), and stimulates reepithelialization by keratinocytes. We also found that incorporating fibronectin, a crucial constituent of the ECM, into the topical EPO-containing gel, can potentiate the accelerating action of EPO on the healing of the burn injury.