Increase of blood capillary density at the interface between normal and ischemic tissue after acute myocardial infarction reduces infarct size and improves cardiac function. Cardiac injury triggers the production of the matricellular component thrombospondin-1 (TSP-1) and adenosine. Adenosine is thought to be involved in cardiac repair and is known to stimulate angiogenesis. The role of TSP-1 in angiogenesis is less clear, since both anti- and pro-angiogenic activities have been reported. We hypothesized that adenosine controls angiogenesis through modulation of TSP-1 production.Methods
Primary human macrophages were obtained by differentiation of peripheral blood monocytes from healthy volunteers, and were treated with adenosine (0.1-50 μmol/L) under ischemic conditions (hypoxia and starvation) or stimulation by cytokines (IL-1β and TNF-α). A rat aortic ring assay was implemented to evaluate angiogenesis.Results
Adenosine dose-dependently increased the production of TSP-1 by macrophages, reaching a 4-fold increase at 10 μmol/L (n = 11, P < 0.001). A 13-fold induction of TSP-1 mRNA expression was measured. These effects were observed both under basal conditions, during ischemia, and after stimulation with cytokines. Use of agonists and antagonist of adenosine receptors, coupled to RNA interference experiments suggested that the A2A and A2B receptors mediate the effect of adenosine on TSP-1. This effect was reproduced by cholera toxin (Gs protein activator) and forskolin (adenylate cyclase activator), and blocked by the PKA inhibitor H89. Low doses of purified TSP-1 ( < 5μg/mL) increased microvessel outgrowth from rat aortic rings, whereas high doses ( > 5μg/mL) decreased this outgrowth. Conditioned medium from adenosine-treated macrophages (containing approximately 0.2ng/mL of TSP-1) enhanced microvessel outgrowth. Addition of anti-TSP-1 antibodies to conditioned medium blocked angiogenesis in this model.Conclusions
We show for the first time that adenosine up-regulates TSP-1 production by macrophages. This effect involves the A2-type adenosine receptors and is mediated through the cAMP/PKA pathway. In our model, adenosine induced low levels of TSP-1 which were pro-angiogenic. However, higher levels of adenosine and TSP-1, which are anti-angiogenic, may be reached in the ischemic myocardium. This is an important information for the design of adenosine-based cardiac therapies.