Pulmonary hypertension is an important clinical problem, either as a primary event or secondary to other injury. The walls of injured blood vessels rapidly thicken, especially those of the microvessels, where lumen restriction increases resistance to blood flow and arterial pressure. As cells proliferate in the microvessels, and as contractile cells develop from (a) interstitial fibroblasts recruited to the vessel wall or (b) vascular intermediate cells, matrix components organize these cells into intimal and medial layers. The endothelium adjacent to the developing contractile cells is an important source of agents that modify their function and proliferation. Our structural data demonstrate that basement membranes and a primary (1 degrees) elastic lamina form de novo to incorporate the fibroblasts into the vessel wall, and a secondary (2 degrees) elastic lamina forms to separate these cells from endothelium; following injury, basement membranes reform around intermediate cells, the pre-existing 1 degrees lamina thickens, and a 2 degrees lamina forms in a similar way. The endothelial cell mediators expressed in the hypertensive lung, the constituents and assembly of the laminae, and of basement membranes modulating cell movement, attachment and proliferation, are unknown. We propose that (i) the expression of endothelial-derived vasoactive mediators shifts to favor hyperplasia and contraction of the developing cells, (ii) elastin synthesis is accompanied by the differential synthesis of molecules associated with lamina formation and (iii) that the ability of cells to organize within the wall reflects expression of specific regulatory molecules in the basement membrane and extracellular matrix. As the microvessels (vessels <100 micromED) remodel, and as the walls of normally muscular vessels thicken (resistance vessels 200microm to 2000micromED) we will assess these changes by high resolution immunogold studies. We will analyze endothelial mediators, e.g., endothelin-1 and nitric oxide synthetase (AIM 1), the constituents and assembly of elastic laminae by expression of microfibrillar proteins (e.g., 31kDa microfibrillin-associated glycoprotein, a 35kDa protein and 350kDa fibrillin), tropoelastin and elastin (AIM 2), and the constituents of basement membranes (e.g., collagen type IV, laminin, tenascin, perlecan, chondroitin sulfate proteoglycan and fibronectin, AIM 3). This new data will significantly increase our understanding of the basis of cell organization in the vessel wall during the development of critical lesions in pulmonary hypertension - ones that are the structural basis of a maintained rise in pressure.
