Prazosin Can Prevent Glucocorticoid Mediated Capillary Rarefaction.

Glucocorticoids (GC) elicit skeletal muscle capillary rarefaction, which can subsequently impair blood distribution and muscle function; however, the mechanisms have not been established. We hypothesized that CORT would inhibit endothelial cell survival signals but that treatment with the alpha-1 adrenergic receptor inhibitor prazosin, which leads to angiogenesis in skeletal muscle of healthy rats, would reverse these effects and induce angiogenesis within the skeletal muscle of corticosterone (CORT)-treated rats. Male Sprague Dawley rats were implanted subcutaneously with CORT pellets (400 mg/rat), with or without concurrent prazosin treatment (50mg/L in drinking water), for 1 or 2 weeks. Skeletal muscle capillary rarefaction, as indicated by a significant reduction in capillary-to-fiber ratio (C:F), occurred after 2 weeks of CORT treatment. Concurrent prazosin administration prevented this capillary rarefaction in CORT-treated animals but did not induce angiogenesis or arteriogenesis as was observed with prazosin treatment in control rats. CORT treatment reduced the mRNA level of Angiopoietin-1 (Ang-1), which was partially offset in the muscles of rats that received 2 weeks of co-treatment with prazosin. In 2W CORT animals, prazosin treatment elicited a significant increase in vascular endothelial growth factor-A (VEGF-A) mRNA and protein. Conversely prazosin did not rescue CORT-induced reductions in transforming growth factor beta-1 (TGFß1 and matrix metalloproteinase-2 (MMP-2) mRNA. To determine if CORT impaired shear stress dependent signaling, cultured rat skeletal muscle endothelial cells were pre-treated with CORT (600nM) for 48 hours, then exposed to 15 dynes/cm2 shear stress or maintained with no flow. CORT blunted the shear stress-induced increase in pSer473 Akt, while pThr308 Akt, ERK1/2 and p38 phosphorylation and nitric oxide (NO) production were unaffected. This study demonstrates that GC-mediated capillary rarefaction is associated with a reduction in Ang-1 mRNA within the skeletal muscle microenvironment and that concurrent prazosin treatment effectively increases VEGF-A levels and prevents capillary loss.

Identification of a mechanism underlying regulation of the anti-angiogenic forkhead transcription factor FoxO1 in cultured endothelial cells and ischemic muscle.

Chronic limb ischemia, a complication commonly observed in conjunction with cardiovascular disease, is characterized by insufficient neovascularization despite the up-regulation of pro-angiogenic mediators. One hypothesis is that ischemia induces inhibitory signals that circumvent the normal capillary growth response. FoxO transcription factors exert anti-proliferative and pro-apoptotic effects on many cell types. We studied the regulation of FoxO1 protein in ischemic rat skeletal muscle following iliac artery ligation and in cultured endothelial cells. We found that FoxO1 expression was increased in capillaries within ischemic muscles compared with those from rats that underwent a sham operation. This finding correlated with increased expression of p27(Kip1) and reduced expression of Cyclin D1. Phosphorylated Akt was reduced concurrently with the increase in FoxO1 protein. In skeletal muscle endothelial cells, nutrient stress as well as lack of shear stress stabilized FoxO1 protein, whereas shear stress induced FoxO1 degradation. Endogenous FoxO1 co-precipitated with the E3 ubiquitin ligase murine double minute-2 (Mdm2) in endothelial cells, and this interaction varied in direct relation to the extent of Akt and Mdm2 phosphorylation. Moreover, ischemic muscles had a decreased level of Mdm2 phosphorylation and a reduced interaction between Mdm2 and FoxO1. Our results provide novel evidence that the Akt-Mdm2 pathway acts to regulate endothelial cell FoxO1 expression and illustrate a potential mechanism underlying the pathophysiological up-regulation of FoxO1 under ischemic conditions.