Erythropoietin requires endothelial nitric oxide synthase to counteract TNF-[alpha]-induced microcirculatory...

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.