| Background and Objective: Cerebrovascular disease is the second most common cause of death after ischemic heart disease, and one of the leading causes of disability worldwide. Due to the unique properties of cerebral vasculature and limited reparative capability of central nervous system, it has been difficult to devise effective neuroprotective therapies after stroke. Angiogenesis is one of important early events after cerebral focal ischemia. Accelerated neovessels formation seems to be essential for enhancing endogenous neural function restoration, suggesting that proangiogenic treatment could be a promising approach in stroke treatment. RGMa(Repulsive guidance molecule a) was initially revealed to be chemorepulsion in guiding cell interactions in the nervous system, has now emerged as a molecule with pleiotropic roles involving repulsion, adhesion, migration and differentiation in nervous system. Neuvous system share similar signaling pathways with vascular system that mediate differentiation, maturity, and migration, have similar parallel branching structures which might interact with each other. Neogenin, the receptor of RGMa, was identified to be involved in angiogenesis regulated by Netrins. It was reported RGMa inhibited tube formation through FAK dephosphorylation downstream of Neogenin in human umbilical artery endothelial cells(HUAEC) and suppresses angiogenesis in Matrigel plug assay. However, to the best of our knowledge, the effect of RGMa on angiogenesis in the adult brain remains to be clarified. VEGF is the most prototypical mediator among the growth factors which involved in angiogenesis. Besides, angiopoietin(Ang)–Tie signaling system was identified as a vascular-specific receptor tyrosine kinase pathway that is essential for vessel development. Signaling by Tie receptors appears to complement the VEGF pathway by contributing to later stages of vascular development. Ang-1 plays a prominent role in angiogenesis as well as vascular stability. While Ang2 acts as an antagonist for Ang1, inhibits Ang1-promoted Tie2 signaling and decreases blood vessel maturation and stabilization. BDNF is a member of the neurotrophin family which is crucial in protection and proliferation of neuronal, glial, and endothelial cells. Besides, BDNF has been reported to have angiogenic effects and plays an important role in recovery after stroke. Our previous findings observed that recombinant adenovirus rAd-shRGMa could suppress the expression of RGMa via RNA interference in rats MCAO/Reperfusion model. The extracellular portion of Neogenin contains four Immunoglobulin like(4Ig) and six Fibronectin type III(6FNIII) domains. It has been reported that 6FNIII interacts with full-length RGMa. To examine the angiogenic response in the peri-ischemic cortex, we observed angiogenesis in MCAO/Reperfusion rats after injected rAd-shRGMa. The present study had further performed 6FNIII peptide injection to assess whether Neogenin is necessary for the inhibitory effect of RGMa on angiogenesis.Methods:1. Twenty-five male SD rats were randomly divided into sham operated group(S), ischemia/reperfusion(I/R) group(2d and 7d), I/R+rAd-shRGMa and I/R+rAd-HK(n=5 in each group), to observe the location of RGMa/Negonein expression after ischemia/reperfusion injury and the change of angiogenesis after treated with RNA interference by using rAd-sh RGMa. The rats were received middle cerebral artery occlusion(MCAO) immediately after treated with rAd-shRGMa to determine the effect of RGMa on angiogenesis after stroke. Poststroke angiogenesis was assessed by CD31 immunohistochemistry and Ki67/CD31 immunofluorescence.2. Thirty male SD rats were randomly divided into six groups to determine the optimal concentration of 6FNⅢ: sham operated group(S), ischemia/reperfusion(I/R) group, I/R+6FNⅢ(H)(1μg/μl), I/R+medium concentration 6FNⅢ(M)(0.5μg/μl), I/R+low concentration 6FNⅢ groups(L)(0.25μg/μl)and I/R+normal saline(NS) group(n=5 in each group). In addition, ninety rats were randomly divided into six groups(n=5 in each group), including sham operated group(S), ischemia/reperfusion(I/R) group, I/R+rAd-sh RGMa, I/R+6FNⅢ, I/R+rAd-HK, and I/R+NS control, to determine the expression of VEGF, Ang1, Ang2 and BDNF 2 days,7 days and 14 days after reperfusion.Results:1. Both RGMa and its receptor Neogenin were expressed in neurons and epithelial cells after MCAO/Reperfusion. Angiogenesis was enhanced after RNA interference against RGMa, and neurological deficits were greatly improved.2. 6FNⅢ could effectively decrease the RGMa protein in the ipsilateral cortex. Medium concentration group showed higher inhibition efficiency than the low concentration group, and did almost the same thing as high concentration, it was the optimal concentration for treatment. VEGF, Ang1,Ang2 and BDNF expression were significantly increased after intervened with rAd-shRGMa or 6FNⅢ.Conclusions:1. Suppression of RGMa via RNA interference induced angiogenesis in the peri-ischemic cortex and provided a favorable environment for neuronal recovery.2. RGMa/Neogenin signal could be blocked by peptide 6FNⅢ by intra-ventricular injection after MCAO/Reperfusion in rats. RGMa might suppress angiogenesis via VEGF, Ang1, Ang2 and BDNF by binding to Neogenin after cerebral ischemia/reperfusion injury. |
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