Two tandem Netrin genes in Drosophila are expressed at the midline of the developing CNS and in different subsets of neurons, muscles, and epidermal patches. In embryos carrying a small deficiency that deletes both genes, CNS axon commissures are partially missing or thinner. This phenotype is rescued by expressing either Netrin gene at the midline. Pan-neural expression of either gene causes disruption of commissural and longitudinal tracts, indicating that the pattern of Netrin expression is crucial and that Netrins function as instructive cues. The double mutant also shows defects in motor axon projections. Expression of either Netrin gene in all muscles also results in aberrant motor projections. Thus, Drosophila Netrins are required for the guidance of commissural axons at the midline, and of motor axons to their target muscles.

Angiogenesis, which is essential for the physiological adaptation of skeletal muscle to exercise, occurs in response to the mechanical forces of elevated capillary shear stress and cell stretch. Increased production of VEGF is a characteristic of endothelial cells undergoing either stretch-or shear-stress-induced angiogenesis. Because VEGF production is regulated by hypoxia inducible factors (HIFs), we examined whether HIFs play a significant role in the angiogenic process initiated by these mechanical forces. Rat extensor digitorum longus (EDL) muscles were overloaded to induce stretch, or exposed to the dilator prazosin to elevate capillary shear stress, and capillaries from these muscles were isolated by laser capture microdissection for RNA analysis. HIF-1α and HIF-2α transcript levels increased after 4 and 7 days of stretch, whereas a transient early induction of HIF-1α and HIF-2α transcripts was detected in capillaries from prazosin-treated muscles. Skeletal muscle microvascular endothelial cells exposed to 10% stretch in vitro showed an elevation in HIF-1α and HIF-2α mRNA, which was preceded by increases in HIF-binding activity. Conversely, HIF-1α and HIF-2α mRNA were reduced significantly, and HIF-α proteins were undetectable, after 24 h exposure to elevated shear stress (16 dyn cm −2 (16 ×10 −5 N cm −2 ). Given the disparate regulation of HIFs in response to these mechanical stimuli, we tested the requirement of HIF-α proteins in stretch-and shear-stress-induced angiogenesis by impeding HIF accumulation through use of the geldanamycin derivative 17-DMAG. Treatment with 17-DMAG significantly impaired stretch-induced, but not shear-stress-induced, angiogenesis. Together, these results illustrate that activation of HIF-1α and HIF-2α contributes significantly to stretch-but not to shear-stress-induced capillary growth.

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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.