Adenosine receptors are proposed to play

Adenosine receptors are proposed to play proangiogenic role in vascular and immune gsk-3 within microenvironment of hypoxic tissues to maintain tissue oxygenation in chronic ischemic condition [9]. Adenosine also stimulates the production of angiopoetin-1, VEGF and Interleukin-6 (IL6) via adenosine receptor on vascular cells that may contribute to angiogenesis [3,10,11]. It is also fairly well established that other growth factors such as tissue growth factor-β (TGF-β) [12], epidermal growth factor (EGF) [13] and platelet-derived growth factor BB (PDGF-BB) activates VEGF expression [14] to stimulate angiogenesis to supply oxygen and nutrition for wound healing, developing tumour and tissue regeneration. However, the specific function of adenosine receptor and its isoforms are not known [4,15]. Numerous studies have found that 5′-N ethylcarboxamidoadenosine (NECA) promotes a response similar to hypoxia by inducing adenosine receptor, an agonist that significantly increases intracellular cAMP levels and VEGF [16,17]. Also plant extract diterpene Forskolin (FSK), a potent and unique activator of adenylyl cyclase, enhanced various endothelial events, including angiogenesis by elevating the intracellular cAMP level and adenosine receptors. These processes were mediated by modulating adenosine receptor and VEGF expression in angiogenic pathways [18]. Hence we have used FSK to induce adenosine and its receptor to study role of VEGF and Notch signal pathways during adenosine induced angiogenesis using zebrafish model. Zebrafish (Danio rerio), Indian teleost has excellent utility as a human disease model system. It offers several advantages, including ease of experimentation, drug administration, rapid development, optical transparency and amenability to in-vivo genetic manipulation indeed when compared to other vertebrate model system [19,20]. This is an excellent model for studying angiogenesis, since genetic studies have revealed conservation of the molecular pathways between teleost and mammals [20]. The pattern of angiogenesis is simple and at molecular level angiogenesis in zebrafish is similar to other vertebrates [21]. Recently, zebrafish embryonic models have been developed to dissect the detailed events of hypoxia-induced tumour cell invasion and metastasis in association with angiogenesis under normoxia or hypoxic conditions [22,23]. The results are found to have positive impacts on embryos by altering the angiogenesis patterns during hypoxic condition suggesting that zebrafish is a predictive model for testing angiogenesis modulators.
Materials and methods
Results
Discussion and conclusion Adenosine is known to stimulate endothelial cell migration, proliferation and tube formation for the formation of new capillary networks in case of hypoxic urge or any-other pathological condition [34,35]. Recently it is estimated that adenosine can contribute up to 50%–70% of the angiogenic response in ischemic condition. Adenosine receptor, agonists or modulator of adenosine and its metabolism has been established in in-vivo and in-vitro models of angiogenesis, which causes release of VEGF [31,34]. This stimulates both proliferation and expression of VEGF in endothelial cells based on local adenosine concentration. In addition, Notch has been suggested as potentially important determinant in development and progression of blood vessels, towards the angiogenic stimuli [36]. However, little is known on the role of adenosine in link between VEGF and Notch. In this study, we have treated zebrafish embryos with FSK, inducer of adenosine [3] to explore the role of adenosine signaling in VEGF and NOTCH expression. Results from our study indicate that FSK was potentially involved in upregulating adenosine receptors, VEGF and NOTCH. It also enhanced hatching and growth of embryos. When embryos were treated with FSK at higher concentration (50 μM, 100 μM and 150 μM) caused severe morphological defects including curling and shunt growth leading to “whirling phenotype” with sever pericardial edema. Embryos also displayed hyper pigmentation and sensitivity to light.