Multimerin2 Influences Vascularization Through Modulation of the Extracellular Matrix

Background: Vascular development is crucial in normal physiology and pathophysiology, and is in part orchestrated by a dynamic balance between extracellular matrix (ECM) and endothelial cells. The molecular mechanisms that govern this balance and as such play a role in the formation of new blood vessels remain largely unknown. In order to identify new genes involved in blood vessel formation, we performed a genome-wide gene expression array analysis in Flk1+ angioblasts during mouse development using a commercial platform (Affymetrix). Multimerin2 (Mmrn2) expression was found to be upregulated in angioblasts and endothelial cells and in the developing vasculature of zebrafish and mice. Methods & Results: Subsequent in vitro studies using an adenoviral construct to induce Mmrn2 overexpression showed a negative effect on angiogenesis. Mmrn2 overexpression resulted in a reduction of 2D tube formation capacity of human aortic endothelial cells (HAECs), as seen in a decrease in the number of junctions and tubules (P<0.05, N=3). Another observed effect after Mmrn2 overexpression was a G0/G1 cell cycle arrest in HAECs (P<0.001, N=8) and decreased migration of the cells in a Boyden chamber assay (P<0.05, N=10). Further in vitro experiments, using immunofluorescence, showed Mmrn2 was detected outside of HAECs, were it co-localized with the extracellular matrix components perlecan, laminin and fibronectin. Direct interaction with these proteins was confirmed using a combination of co-immunoprecipitation followed by proteomics (LC-MS), and western blot to identify possible binding partners. Interestingly, overexpression of Mmrn2 also resulted in enhanced expression of these extracellular components, suggesting that Mmrn2 changes the phenotype from a more proliferative endothelial cell towards a more productive endothelial cell. Conclusions: These findings suggest that Mmrn2 is an abundant and important extracellular matrix protein, secreted by endothelial cells, which influences the expression of other ECM proteins such as fibronectin, laminin and perlecan. We hypothesize that Mmrn2 switches the phenotype of the endothelial cell and subsequently diminishes endothelial cell proliferation, migration and overall tube formation.