Role of Endothelial NO Synthase Phosphorylation in Cerebrovascular Protective Effect of Recombinant Erythropoietin During Subarachnoid Hemorrhage– Induced Cerebral Vasospasm

Abstract

Background and Purpose-In the present study, the effect of subarachnoid hemorrhage (SAH) on the phosphorylation of endothelial NO synthase (eNOS) and the ability of recombinant erythropoietin (Epo) to augment this vasodilator mechanism in the spastic arteries were studied. Methods-Recombinant adenoviral vectors (10 9 plaque-forming units per animal) encoding genes for human Epo (AdEpo), and ␤-galactosidase were injected immediately after injection of autologous arterial blood into the cisterna magna (day 0) of rabbits. Cerebral angiography was performed on day 0 and day 2, and basilar arteries were harvested for Western blots, measurement of cGMP levels, and analysis of vasomotor functions. Results-Injection of autologous arterial blood into cisterna magna resulted in significant vasospasm of the basilar arteries.Despite the narrowing of arterial diameter and reduced expression of eNOS, expressions of phosphorylated protein kinase B (Akt) and phosphorylated eNOS were significantly increased in spastic arteries. Gene transfer of AdEpo reversed the vasospasm. AdEpo-transduced basilar arteries demonstrated significant augmentation of the endotheliumdependent relaxations to acetylcholine, whereas the relaxations to an NO donor, 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt, were not affected. Transduction with AdEpo further increased the expression of phosphorylated Akt and eNOS and elevated basal levels of cGMP in the spastic arteries. Conclusions-Phosphorylation

In vivo stimulatory effect of erythropoietin on endothelial nitric oxide synthase in cerebral arteries

“…In an earlier study, we reported that the cerebral vascular protective effect of recombinant Epo during subarachnoid hemorrhage-induced cerebral vasospasm is mediated, in part, by augmentation of endothelium-dependent relaxations, increased phosphorylation of eNOS, and elevation of cGMP levels in the cerebral arteries (39). On the basis of these observations, we first examined the effect of Epo on contractions and relaxations in AdEpo-transduced arteries.…”

“…Hence, divergent effects of Epo on the vasculature have been reported to date. In a recent study, we observed that Epo increased the phosphorylation of eNOS and reversed cerebral vasospasm rabbits subjected to subarachnoid hemorrhage (39). From these the findings, we hypothesized that in the cerebral vasculature, Epo increases the expression and activation of eNOS and augments vasodilatation.…”

“…Epo levels in the CSF and plasma were measured by a two-site chemiluminescence immunoassay using the Nichols Erythropoietin Immunoassay kit (Nichols Institute Diagnostics, San Clemente, CA) as described elsewhere (39).…”

“…Soluble proteins were extracted by mincing and homogenizing tissues in lysis buffer as described earlier (39). Briefly, 50 g protein were separated by electrophoresis and transferred onto nitrocellulose membrane.…”

Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting NF-κB and upregulating endothelial nitric oxide synthase

“…The discovery that EpoRs are expressed in glomerular, mesangial, and tubular epithelial cells in human, rat, and mouse kidneys have prompted studies of the effects of EPO in small-rodent models of AKI (41,44). Such studies have shown that EPO not only stimulates erythropoiesis but also has antiinflammatory properties, as well as exerting the following effects (7,19,21,38): reducing apoptosis, oxidative stress, and lipid peroxidation; promoting renal tubular cell regeneration, vascular regeneration, and neoangiogenesis; mobilizing endothelial progenitor cells; and upregulating eNOS expression. It is also known that EPO mediates neuroprotection against axonopathy and apoptosis in the peripheral nervous system by…”