by Giovanni Ligresti, Alfred C. Aplin, Bruce E. Dunn, Ann Morishita, Roberto F. Nicosia
Tissues respond to injury by releasing acute phase reaction (APR) proteins which regulate inflammation and angiogenesis. Among the genes upregulated in wounded tissues are tumor necrosis factor-alpha (TNFa) and the acute phase reactant orosomucoid-1 (ORM1). ORM1 has been shown to modulate the response of immune cells to TNFa, but its role on injury- and TNFa-induced angiogenesis has not been investigated. This study was designed to characterize the role of ORM1 in the angiogenic response to injury and TNFa. Methods and Results
Angiogenesis was studied with in vitro, ex vivo, and in vivo angiogenesis assays. Injured rat aortic rings cultured in collagen gels produced an angiogenic response driven by macrophage-derived TNFa. Microarray analysis and qRT-PCR showed that TNFa and ORM1 were upregulated prior to angiogenic sprouting. Exogenous ORM1 delayed the angiogenic response to injury and inhibited the proangiogenic effect of TNFa in cultures of aortic rings or isolated endothelial cells, but stimulated aortic angiogenesis over time while promoting VEGF production and activity. ORM1 inhibited injury- and TNFa-induced phosphorylation of MEK1/2 and p38 MAPK in aortic rings, but not of NF?B. This effect was injury/TNFa-specific since ORM1 did not inhibit VEGF-induced signaling, and cell-specific since ORM1 inhibited TNFa-induced phosphorylation of MEK1/2 and p38 MAPK in macrophages and endothelial cells, but not mural cells. Experiments with specific inhibitors demonstrated that the MEK/ERK pathway was required for angiogenesis. ORM1 inhibited angiogenesis in a subcutaneous in vivo assay of aortic ring-induced angiogenesis, but stimulated developmental angiogenesis in the chorioallantoic membrane (CAM) assay. Conclusion
ORM1 regulates injury-induced angiogenesis in a time- and context-dependent manner by sequentially dampening the initial TNFa-induced angiogenic response and promoting the downstream stimulation of the angiogenic process by VEGF. The context-dependent nature of ORM1 angioregulatory function is further demonstrated in the CAM assay where ORM1 stimulates developmental angiogenesis without exerting any inhibitory activity.