The vascular endothelium performs critical functions in the physiology of mammals, men included. It mediates vast, continuous exchanges of water and hydrophilic and hydrophobic solutes (small as well as large molecules) between the blood-plasma and interstitial fluids. The survival of all cells of the tissues and organs of the body depends on these continuous exchanges. It participates in the control of coagulant and anticoagulant processes affecting the blood. It plays an essential role in the initial phases of inflammatory reactions. And it is involved in normal and pathological neovascularization (angiogenesis), including tumor vascularization on which depends tumor growth and metastasis. On these accounts, endothelial malfunction is the starting baseline for a large number of human pathological conditions. Work done under this grant has already provided useful information on cellular and molecular interactions involved in each of the critical functions listed above. It has also opened new vistas which the Principal Investigator proposes to explore during the continuation of the grant. He believes that increased understanding of these functions will help prevent, mitigate and if possible cure endothelial malfunctions at some time in the future. The studies are proposed under five Specific Aims. I. In the first specific aim, experiments utilizing alpha1-acidic glycoprotein (orosomucoid) as a tracer are proposed to reinforce the concept/process of transcytosis in the handling of macromolecules in continuous endothelia bounded by intercellular junctions. Morphological and cell fractionation methods will be employed to study the entrance and exit of the tracer via transendothelial channels formed by the fusion of Pvs or caveolae. Attempts will be made to identify two populations of caveolae, i.e., small and large ones, accommodating 40 angstrom and 250 angstrom dinitrophenylated probes, respectively. The studies will be extended also to identify the plasmalemmal protein(s) which anchor(s) the alpha1-acidic glycoproteins and constitute the fiber matrix. Further experiments to identify the factor(s) responsible for the fusion of the caveolae are also proposed. In these experiments, fusion proteins would be generated utilizing vectors containing cDNA for NEM sensitive factor (NSF) and SNAPs; their corresponding polyclonal antibodies will be generated, and used to study the distribution in the endothelial compartments. The distribution would be confirmed by using other approaches, such as, with the employment of specific monoclonal antibodies. II. In the second specific aim, the studies proposed will focus on fenestrated endothelia with or without diaphragms, and utilize the methodologies as described in specific aim # I. III. The third specific aim relates to the studies of structural modulations of caveolae induced by platelet activating factor (PAF) and vascular endothelial growth factor (VEGF). A particular attention will be paid to study the changes in the paracaveolar glycoprotein assembly, defined by the PI as """"""""corset"""""""", upon perfusion of PAF and VEGF into the vasculature. Since their actions are mediated via their receptors, the experiments are outlined to study the receptor phosphorylation under the influence of these growth factors. The studies will be extended to investigate the influence of negative and possible positive modulators of transcytosis, and they include NEM, Na-nitroprusside, ANP and Ca++ ionophores. In these experiments, alterations, if any, in the interendothelial junctions will be investigated. IV.
The specific aim # 4 deals with the biochemical characterization of glycoproteins enriched in the caveolar fractions isolated from pulmonary endothelia, utilizing cationized silica procedures. Their isolation will be facilitated by the use of various monoclonal antibodies. These studies would be extended to characterize the multiplicity of the functions of caveolae. V.
In specific aim # 5, the characterization of fenestrated endothelia from diaphragmatic capillaries, e.g., peritubular capillaries will be carried out by utilizing the techniques described in above specific aims.
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