Adenosine inhibits the proliferation of lymphatic endothelial cells

Background: The presence of a lymphatic system in the human heart has been demonstrated several decades ago. After myocardial infarction, lymphatic vessel density increases, predominantly in the myocardium, but also in the epicardium and endocardium of both ventricles and atria. The biological relevance of this increase is poorly known. Recent studies suggest that it may be a protective element in the response to myocardial injury, through efficient removal of cellular debris produced by cardiomyocyte death. Adenosine has the potential to stimulate cardiac repair, partly through activation of angiogenesis. However, whether adenosine affects the lymphatic system is unknown. Methods: Human adult dermal microvascular lymphatic endothelial cells (HMVEC) were used to study the direct effect of adenosine on the lymphatic system. Expression of adenosine receptors was investigated using quantitative PCR. Proliferation of lymphatic cells was measured using CyQUANT and Ki-67, migration of the cells was assessed using a wound healing assay and a Boyden chamber assay, and tube formation was evaluated using a tubulogenesis assay in collagen and spheroids matrix. Cytotoxicity was evaluated using the Live/Dead viability/cytotoxicity assay. Lymphangiogenesis was characterized ex vivo using mouse lymphatic ring explants. To identify potential paracrine effects of adenosine on lymphangiogenesis, the murine cardiomyocyte cell line HL-1 and the human fibroblast cell line 1226 KI were used. Results: HMVEC expressed A2A and A2B, but not A1 and A3, adenosine receptors. Adenosine dose-dependently decreased the proliferation of HMVEC (-40% for 10 μmol/L adenosine), and inhibited their migration and tube formation. Adenosine also decreased microvessel outgrowth from lymphatic rings explanted from mouse lymphatic thoracic duct. A potential cytotoxic effect of adenosine was ruled out. Adenosine decreased the production of vascular endothelial growth factor C (VEGFC), the main growth factor of lymphatic endothelial cells, by fibroblasts. Conditioned medium from fibroblasts treated with adenosine inhibited the proliferation of HMVEC. Adenosine did not alter the production of VEGFC by cardiomyocytes. Conclusion: We have shown for the first time that adenosine down-regulates lymphangiogenesis. This observation may be important for the design of adenosine-based therapies to stimulate cardiac repair.