A subpopulation of peritoneal macrophages form capillary-like lumens and branching patterns in vitro

Abstract

Objective: We have previously shown that monocytes/macrophages (MC/Mph) influence neovascularization by extracellular matrix degradation, and by direct incorporation into growing microvessels. To date, neither the phenotype of these cells, nor the stages of their capillary-like conversion were sufficiently characterized. Methods: We isolated mouse peritoneal Mph from transgenic mice expressing fluorescent proteins either ubiquitously, or specifically in the myelocytic lineage. These Mph were embedded in Matrigel which contained fluorescent protease substrates, exposed to an MCP-1 chemotactic gradient, and then examined by confocal microscopy after various intervals. Results: Within 3 hrs after gel embedding, we detected TIMP-1 and MMP-12 dependent proteolysis of the matrix surrounding Mph, mostly in the direction of high concentrations of MCP-1. After 2 days, Mph developed intracellular vacuoles containing degradation product. At 5 days these vacuoles were enlarged and/or fused to generate trans-cellular lumens in approximately 10% of cells or more (depending on animal’s genetic background). At this stage, Mph became tubular, and occasionally organized in three-dimensional structures resembling branched microvessels. Conclusion: Isolated mouse peritoneal Mph penetrate Matrigel and form tunnels via a metalloprotease-driven proteolysis and phagocytosis. Following a morphological adjustment driven by occurrence, enlargement and/or fusion process of intracellular vacuoles, similar to that described in bona fide endothelium, a subpopulation of these cells end up by lining a capillary-like lumen in vitro. Thus we show that adult Mph, not only the more primitive ‘endothelial progenitors’, have functional properties until now considered defining of the endothelial phenotype.