Beside activation of adenosine receptors A a A
Beside activation of adenosine receptors (A2a.1, A2a.2, A2b) by FSK treatment, the expression of adenosine receptors was also upregulated, when embryos were exposed to inhibitors (SU5416 and DAPT) alone and also combined with FSK. Our data imply that adenosine receptor was associated with a positive regulation on angiogenesis signaling pathways, while modulating VEGF and Notch signaling with exposure to time. Recently, it was shown that under normoxia, inhibition of Notch leads to capillary endothelial tip sprouting without stimulating the formation of new Aliskiren Hemifumarate . Moreover, Notch interacts molecularly at several levels and has been shown to underlie arteriovenous specification in zebrafish as VEGF-induced Notch and Notch ligand expression . A feedback loop comprising VEGF and Notch signaling would greatly aid such a scheme and it has been supported by increased filopodia and branching with DLL4 expression in zebrafish retinal study [31,32]. Similar to expression of FSK, embryos when treated with NECA revealed upregulated adenosine receptor (A1, A2a.1, A2a.2, A2b), HIF1a, VEGFA, VEFG R2, NRP1a, NOTCH1a, DLL4 gene expression significantly, proposing hypoxic condition in zebrafish embryos. Additionally, embryos treated with NECA showed increased hear beat similar to hypoxic condition . Whilst combined treatments were carried out with FSK and NECA, resulted in dramatically increased gene expression, might be due to additive effect on adenosine and its receptors. Consistent with previous studies, our data also suggest that increased adenosine signaling might be accountable for increased VEGF and NOTCH gene respective to angiogenic signaling. It is clear that the amount of VEGF expression, affects NOTCH expression and hence influencing angiogenic signaling in zebrafish embryos . In addition, VEGF and Notch was proposed to be act side by side with one other [, , ]. Recently, Fedeles and associates reported that VEGF and NOTCH expresses in gradient dependent manner with time dependent fashion . Notably HIF1a expression was also increased during VEGF and NOTCH gene upregulation, representing HIF1a might also be involved in regulating angiogenesis . Although no direct interaction has been reported between NOTCH and NRP1a, NRP1a is found to be significantly altered during VEGF and NOTCH expression thorough unknown mechanism. Results from our study, indicates that FSK could be a potent inducer of adenosine signaling pathway [3,4,23] which could play a key role in modulating VEGF and NOTCH expression in zebrafish embryos. Although involvement of adenosine in VEGF and Notch signaling pathways were relatively unclear, Notch was known to alter the angiogenic targets by upregulating adenosine receptors during angiogenesis under hypoxia . And also known that proliferation and expression of VEGF in endothelial cells based on local adenosine concentration. Recent study on interaction of VEGF-Notch expression in cell line  and model  suggest it as potential important determinant in development and progression of blood vessels towards the angiogenic stimuli  also in proper neuronal development , cardiac development and brain [21,22]. VEGF requires Notch signaling to inhibit surplus sprouting, differentiation and also Notch signaling mediates VEGF induced tumour cell angiogenesis, according to adenosine and adenosine receptor balance . Recently Nedvetsky et al. , reported that protein kinase A (PKA) was essential for stabilizing vascular development in Notch independent manner, demonstrating that PKA and Notch might independently regulates angiogenesis in endothelial cells. Indeed, molecular mechanism behind regulation of PKA activation has to be explained whether PKA actively regulates either at tip cell or stalk cell. While other reports [31,32,36] remarkably suggested that co-ordinated balance between VEGF and Notch gradient leads to successful complete vessel formation and stabilization, at tip cell via Notch and stalk cell via VEGF. Although the underlying mechanisms remain uncharacterized, it could be possible that inhibition of the adenosine pathway might lead to alter angiogenesis in endothelial cell tip and stalk cell formation. Suggesting, that adenosine could play an important role during angiogenesis, along with VEGF and Notch signaling. These interesting findings warrant further mechanistic investigation. Nevertheless, our findings provide important clues regarding VEGF and Notch signaling, that might be altered due to the involvement of adenosine receptors. Thus adenosine receptors might be involved as key regulator in hypoxia induced angiogenesis and providing new insight for targeting angiogenesis via adenosine receptor which might gain therapeutic significance.