Erythropoietin-induced upregulation of endothelial nitric oxide synthase but not vascular endothelial growth factor prevents musculocutaneous tissue from ischemic damage

Abstract

Recent findings have attested the protective effects of erythropoietin (EPO) in ischemically challenged organs. We therefore aimed at elaborating the underlying mechanism of EPO-mediated protection in musculocutaneous tissue undergoing persistent ischemia after acute injury. Mice were assigned to five experimental groups equipped with a randomly perfused flap fixed in a dorsal skinfold chamber, whereas the sixth group did not undergo flap preparation: EPO, L-Name, EPO and L-Name, EPO and bevacizumab, untreated flap, and nonischemic chamber (control). Intravital fluorescence microscopic analysis of microhemodynamics, apoptotic cell death, macromolecular leakage and angiogenesis was carried out over a 10-day period. Further, immunohistochemical analysis was used to study the protein expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF). Increased expression of eNOS in EPO-administered mice correlated with significant arteriolar dilation and thus increased blood flow resulting in a maintained functional capillary density (FCD) at day 10. In addition, EPO induced a VEGF upregulation, which was associated with newly formed capillaries. In addition, EPO was able to reduce ischemia-induced apoptotic cell death and finally to significantly reduce flap necrosis. In contrast, coadministration of L-Name abolished EPO-mediated tissue protection by abrogating the dilatory effect resulting in reduced FCD and tissue survival, without counteracting angiogenesis and apoptotic cell death, whereas additional administration of bevacizumab did not influence the beneficial effect of EPO on flap survival despite abrogating angiogenesis. Macromolecular leakage was found to be increased in all treatment groups. This study shows that EPO administration prevents musculocutaneous tissue from ischemic necrosis as a consequence of an eNOS-dependent arteriolar hyperperfusion maintaining capillary perfusion, thus representing a promising approach to pharmacologically protect ischemically challenged tissue.

Erythropoietin Action in Stress Response, Tissue Maintenance and Metabolism

“…In a mouse model for cell therapy for muscular dystrophy, overexpression of EpoR in myoblasts and/or EPO treatment at the time of transplant was suggested to increase donor cell survival and increase the number of fibers expressing dystrophin without change in hematocrit [139]. The contribution of endothelial EPO response to skeletal muscle injury was suggested by reports of EPO treatment in rodents that protect musculocutaneous tissue from ischemic necrosis via eNOS activation and maintaining capillary perfusion [144], and in a hind limb ischemia injury model in mice that enhances blood flow recovery [145]; EPO was also observed to increase regeneration of skeletal muscle tissue after severe trauma and to improve microcirculation in muscle tissue in rat [146]. …”

Systemic Treatment with Erythropoietin Protects the Neurovascular Unit in a Rat Model of Retinal Neurodegeneration

“…This leads to an increase in endothelial cell and pericytes number by exclusion of highly damaged fields in PKD retinae. One published mechanism by which EPO can mediate its vasoprotective effect is an increase in synthesis of nitric oxide (NO) by endothelial NO-synthase and an increase in VEGF-production [18]–[20]. The vasculoprotective effect of EPO can also be mediated by Tie-1 (Tyrosine kinase with immunoglobulin-like and EGF-like domains 1), Angiopoietin-2 and bFGF (basic fibroblast growth factor) [21].…”

Isoflurane Preconditioning Increases Survival of Rat Skin Random-Pattern Flaps by Induction of HIF-1a Expression

“…In this study, we just demonstrated induction of VEGF and HO-1 expression. Other genes may also have participated in isoflurane-induced survival of the random-pattern skin flap, e.g., isoflurane preconditioning was able to induce erythropoietin (EPO) expression [14], which is also capable to prevent musculocutaneous tissue from ischemic necrosis [37]. In addition, bone marrow-derived cells including hematopoietic stem cells, mesenchymal stem cells, endothelial progenitor cells and their generations improve wound healing by mobilization, differentiation and angiogenesis [38,39,40].…”