Role of Adenosine Receptors in the Regulation of Angiogenic Factors and Neovascularization in Hypoxia

Abstract

Because hypoxia increases extracellular adenosine levels and stimulates angiogenesis, we evaluated the relative roles of reduced oxygen concentrations and adenosine receptor activation in the production of angiogenic factors. In vitro, we analyzed the effects of hypoxia and adenosine on the secretion of angiogenic factors from human microvascular endothelial cells (HMEC-1). To study the effects of hypoxia alone, we scavenged adenosine from the hypoxic medium with adenosine deaminase, and we used the stable adenosine analog 5Ј-N-ethylcarboxamidoadenosine (NECA) to study the effects of stimulation of adenosine receptors. In the absence of adenosine, hypoxia stimulated vascular endothelial growth factor (VEGF) but not interleukin-8 (IL-8) secretion from HMEC-1. In contrast, NECA stimulated both VEGF and IL-8 secretion. VEGF secretion was increased 1.9 Ϯ 0.04-fold with NECA (10 M) and 1.7 Ϯ 0.1-fold with hypoxia (5% O 2 ) but 3.8 Ϯ 0.1-fold when these two stimuli were combined. Thus, adenosine receptors act in a cooperative fashion with hypoxia to stimulate VEGF and induce IL-8 secretion not stimulated by hypoxia alone. In vivo, antagonism of adenosine receptors with caffeine abrogated VEGF up-regulation induced by local injection of NECA into the mouse hind limb and produced a 46% reduction of neovascularization in a mouse ischemic hind limb model. Our study suggests that adenosine actions are not redundant but rather are complementary to the direct effects of hypoxia. Stimulation of adenosine receptors not only contributes to the overall effect of hypoxia but also has additional actions in the regulation of angiogenic factors. Thus, adenosine receptors represent a potential therapeutic target for regulation of neovascularization.Hypoxia elicits a number of compensatory homeostatic mechanisms aimed to restore oxygen supply. Acutely, it induces local vasodilation, resulting in reactive hyperemia. Chronically, hypoxia promotes growth of new blood vessels in a process known as angiogenesis. Angiogenesis is regulated by a delicate balance of multiple pro-and antiangiogenic factors. Hypoxia promotes angiogenesis by up-regulating the expression of several angiogenic factors including vascular endothelial growth factor (VEGF). It is commonly accepted that VEGF production is regulated by the oxygen-sensitive hypoxia-inducible factor-1 (HIF-1). Accumulating evidence, however, suggests that hypoxia-induced secretion of angiogenic factors involves diverse mechanisms. Several studies implicate interleukin-8 (IL-8) as contributing to hypoxiainduced angiogenesis through mechanisms independent of HIF-1 (Xie, 2001;Mizukami et al., 2005). It is possible therefore, that other alternative oxygen-sensitive mechanisms could be involved in the overall effect of hypoxia. Adenosine is known to be released in hypoxic tissues and could be an ideal candidate to contribute to hypoxia-induced angiogenesis.Adenosine is an intermediate product of adenine nucleotide metabolism. It is generated as ATP is catabolized when energy demands inc...

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