Background: Angiogenesis is a fundamental process in which new blood vessels are formed. It is critical for survival and in the regenerative response to tissue hypoxia and injury. However, an imbalance in its regulation causes excessive pathological angiogenesis that accelerates inflammatory diseases such as cancer and atherosclerosis. Increasing evidence implicates the CC chemokine class in the promotion of inflammation driven pathological angiogenesis. The chemokine binding protein ‘35K’ uniquely inhibits the CC-chemokine class and does not affect chemokines from other classes, including CXCL12, that play key roles in physiological hypoxia-driven angiogenesis.
Aim: To determine if CC-chemokine inhibition with 35K suppresses inflammation-induced pathological angiogenesis, but not hypoxia driven physiological angiogenesis in key in vitro angiogenic functional assays with human coronary artery endothelial cells (ECs).
Results: Incubation with conditioned media (CM) from stimulated human macrophages, significantly increased EC tubule formation (55.0%, p<0.05) in a matrix gel tubulogenesis assay, when compared to control cells. The addition of 35K protein strikingly inhibited CM-stimulated tubule formation reaching almost complete inhibition at 50nM (95.8%, p<0.001). Using the EdU ClickItTM assay, we found that inhibition of CM-induced EC proliferation required higher concentrations of 35K. At 200nM, 35K reduced proliferation by 22.5% and at 400nM by 53.6% (p<0.05). When ECs were subjected to hypoxia EC tubulogenesis increased (23.0%). However, 50nM of 35K was less effective at reducing hypoxia-induced tubule formation (77.7%, p<0.01), than in the context of inflammation. Inhibition of hypoxia induced EC proliferation by 35K was also less effective, where 200nM had no effect and 400nM reduced proliferation by 43.8% (p<0.05).
Conclusion: Broad-spectrum chemokine inhibition by 35K blocks inflammation-induced angiogenic processes, with a less striking effect on hypoxia-driven angiogenesis. CC-chemokine inhibition may present as a therapeutic strategy to reduce pathological angiogenic disease, without the severe side effects of other angiogenic inhibitors that block angiogenesis in all pathophysiological contexts.