Abstract

Abstract 3043

Poster Board II-1019

Angiogenic sprouting needs to navigate through tissues to establish a vascular branching pattern. Such angiogenic guidance is mainly inferred by nonrandom angiogenic sprouting in the developing central nervous system (CNS). In mammalian retina, a vascular plexus initially forms, superimposed on a preexisting astrocyte-plexus, and retinal astrocytes begin to express vascular endothelial growth factor (VEGF-A), which stimulates endothelial cells to sprout radially from the optic nerve head into the retinal periphery. However, there has been little investigation of the role of astrocyte-plexus in new vessel formation of other ischemic sites besides retina. We recently found that infiltrated CD11b+ cells in ischemic muscles differentiate into endothelial-like cells in vitro, and that direct-injection of muscle-derived CD11b+ cells enhances recovery of blood perfusion in ischemic hind-limbs of C57BL/6 mice. To study if the astrocytes are related to the neovascularization process in ischemic limbs, infiltrated CD11b+ myeloid-cells in ischemic muscles were isolated 4 days after femoral artery dissection, and then seeded at 0.5×105/cm2 onto Matrigel and cultured in EGM-2 medium. Under these conditions, we tested the following hypotheses: (1) cultured cells initially form an astrocyte-network in vitro, and (2) astrocytes are involved in the formation of vascular structure. Seven days after the cells were embedded in Matrigel, spindle shaped cells grew, spread-out radially from cell clusters to the periphery, and established a mesh-like network expressing glial fibrillary acidic protein (GFAP), a marker for astrocyte. These spindle shaped cells were also positive for pericyte markers: desmin, NG-2, and platelet-derived growth factor receptor (PDGFR)-β. Interestingly, proliferating endothelial cells were closely associated with astrocytes by extension of endothelial cell filopodia on astrocytes. This observation is consistent that astrocyte scaffold guides extension of endothelial cell filopodia. Two weeks after cells were embedded in Matrigel, CD11b+ cells expanded and were layered in the same way to form vessel wall-like structures consisting of hundreds of cells. Spindle shaped GFAP-positive cells gradually expressed smooth muscle-actin at the bottom of the culture plate, migrated to the vessel wall-like structures, and covered the surface of the walls like pericytes. Taken together with the novel finding of astrocyte-like cell differentiation from CD11b+ cells from Matrigel culture, mobilization of CD11b+ myeloid-cells to regions of muscle ischemia would appear to play an important role in neovascularization after ischemic injury.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.