Acute lung injury (ALI) results in extensive destruction of the alveolar epithelium and microcirculatory endothelium, in addition to interstitial edema. Accompanying the loss of microcirculation and influx of inflammatory cells, the wound space becomes hypoxic, and microvessel endothelial cells (EC) not destroyed by the injury process are in close proximity to a microenvironment which may be particularly conducive to the elaboration of new capillary networks. EC synthesize a potent angiogenic factor Basic Fibroblast Growth Factor (bFGF), which is also stored in extracellular matrices beneath them, providing the opportunity for autocrine stimulation of new vessel growth. The hypoxic environment of the wound space could have a profound effect on the availability/activity of this growth factor, by increasing production and/or release of this factor from cytosolic and matrix pools. Recent advances in cell culture technology now permit the study of new vessel formation (angiogenesis) in vitro. It has been demonstrated that EC themselves possess all the genetic information necessary to form capillaries with tight junctions and correct polarity, similar to those formed in situ. This proposal will utilize cultured pulmonary microvessel endothelial cells (PMVEC) to study the direct effect of hypoxia on the process of angiogenesis. Preliminary experiments have demonstrated that exposure of MPVEC to 5% 02 causes marked stimulation of capillary network formation.
Specific Aim : 1) To investigate the quantity and distribution of bFGF in confluent PMVEC relative to the onset of hypoxia-induced capillary network formation, using biochemical and morphological techniques. 2) TO characterize the structural/biochemical changes which occur in close temporal proximity to changes in growth factor availability/activity, addressing changes in actin cytoarchitecture, protease activity, and matrix protein remodeling, 3) To probe the role of bFGF in hypoxia-induced angiogenesis by addition of exogenous bFGF to normoxia cultures and by neutralization of bFGF in cultures expose to hypoxia. It is essential to study the capillary forming potential of EC exposed to a hypoxic environment, since these cells are central to the elaboration of new vessels and may have evolved unique mechanisms for responding to decreased oxygen tensions. Information about the mechanisms vessel-forming cells use to function in a hypoxic environment may eventually assist in the formulation of therapeutic strategies to modulate the process of angiogenesis in clinical settings involving hypoxia/ischemia.
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