Previous studies showed that targeted endothelial nitric oxide synthase (eNOS) disruption in mice with femoral artery occlusion does not impede and transgenic eNOS overexpression does not stimulate collateral artery growth after femoral artery occlusion, suggesting that nitric oxide from eNOS does not play a role in arteriogenesis. However, pharmacologic nitric oxide synthase inhibition with L-NAME markedly blocks arteriogenesis, suggestive of an important role of nitric oxide. To solve the paradox, we studied targeted deletion of eNOS and of inducible nitric oxide synthase (iNOS) in mice and found that only iNOS knockout could partially inhibit arteriogenesis. However, the combination of eNOS knockout and treatment with the iNOS inhibitor L-NIL completely abolished arteriogenesis. mRNA transcription studies (reverse transcriptase-polymerase chain reaction) performed on collateral arteries of rats showed that eNOS and especially iNOS (but not neural nitric oxide synthase) become upregulated in shear stress-stimulated collateral vessels, which supports the hypothesis that nitric oxide is necessary for arteriogenesis but that iNOS plays an important part. This was strengthened by the observation that the nitric oxide donor DETA NONOate strongly stimulated collateral artery growth, activated perivascular monocytes, and increased proliferation markers. Shear stress-induced nitric oxide may activate the innate immune system and activate iNOS. In conclusion, arteriogenesis is completely dependent on the presence of nitric oxide, a large part of it coming from mononuclear cells.

Objective— Arteriogenesis, the development of a collateral circulation, is important for tissue survival but remains functionally defective because of early normalization of fluid shear stress (FSS). Using a surgical model of chronically elevated FSS we showed that rabbits exhibited normal blood flow reserve after femoral artery ligature (FAL). Inhibition of the Rho pathway by Fasudil completely blocked the beneficial effect of FSS. In a genome-wide gene profiling we identified actin-binding Rho activating protein ( Abra ), which was highly upregulated in growing collaterals. Methods and Results— qRT-PCR and Western blot confirmed highly increased FSS-dependent expression of Abra in growing collaterals. NO blockage by L-NAME abolished FSS-generated Abra expression as well as the whole arteriogenic process. Cell culture studies demonstrated an Abra-triggered proliferation of smooth muscle cells through a mechanism that requires Rho signaling. Local intracollateral adenoviral overexpression of Abra improved collateral conductance by 60% in rabbits compared to the natural response after FAL. In contrast, targeted deletion of Abra in CL57BL/6 mice led to impaired arteriogenesis. Conclusions— FSS-induced Abra expression during arteriogenesis is triggered by NO and leads to stimulation of collateral growth by smooth muscle cell proliferation.

Trpv4 showed significantly increased expression in FSS-stimulated cerebral collaterals. Pharmacological Trpv4 activation enhanced cerebral arteriogenesis, pinpointing Trpv4 as a possible candidate for the development of new therapeutic concepts.