ERYTHROPOIETIN REQUIRES ENDOTHELIAL NITRIC OXIDE SYNTHASE TO COUNTERACT TNF-α-INDUCED MICROCIRCULATORY DYSFUNCTION IN MURINE STRIATED MUSCLE

In the present study, we aimed to evaluate whether erythropoietin (EPO) treatment may exert nonhematopoietic endothelial protection against TNF-alpha-induced microvascular inflammation and to determine the involvement of the nitric oxide (NO)-producing enzyme isoforms endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). Murine dorsal skinfold chambers of wild-type (WT) animals were topically stimulated with TNF-alpha after pretreatment with epoetin beta (1,000 IU/kg body weight i.p.) or physiological saline. Leukocyte behavior, microvascular perfusion, and apoptosis were assessed by in vivo fluorescence microscopy. To study the involvement of NO, we compared eNOS-deficient (eNOS(-/-)) and iNOS-deficient (iNOS(-/-)) mice with WT animals. TNF-alpha-associated leukocyte activation, perfusion failure, and apoptosis were substantially attenuated in EPO-pretreated WT mice, which was accompanied by marked reduction of perivascular infiltration with F4/80-stained macrophages. The anti-inflammatory protective effects of EPO were abolished in eNOS(-/-), but not in iNOS(-/-) mice, both with unaffected intercellular adhesion molecule 1 expression. However, the antiapoptotic effect of EPO was maintained in both eNOS(-/-) and iNOS(-/-) mice, indicating that this mechanism might rather be independent of NO. We conclude that EPO treatment elicits protection against TNF-alpha-induced microcirculatory dysfunction, depending on NO derived from endothelial cells, but not on the inducible isoform.