The objective of this project has been the study or matrix protein synthesis by endothelial (EC) and smooth muscle cells (SM) under normal conditions and after in vitro injury. In this revised renewal application, the proposed research forms a logical extension of ongoing studies that aim at understanding mechanisms of vascular injury and tissue repair. EC and SM will be isolated from umbilical vein and artery, respectively, and from bovine aorta and subjected to viral injury by: a) viral infection and b) viral transformation. Viruses known to cause lytic and persistent infections in vivo and in vitro will be used, including herpes simplex (types 1 and 2), measles and coxsackie-B4. The experiments are designed to test the hypothesis that viral infection leads to viral replication, EC injury and loss, followed by alteration of the subendothelium and subsequent interference with normal healing leading to a chronic lesion.
The specific aims are to: 1) Examine the effect of acute viral infection on the synthesis of matrix proteins, including collagen types I, III and IV, fibronectin, laminin and thrombospondin. This will be contrasted with the effect on intracellular proteins such as actin, tubulin and histones. An attempt will be made to answer the following questions. a) How do different viruses affect matrix protein synthesis? b) Is inhibition of protein synthesis occurring at the transcriptional or translational level? c) Is there polysome dissociation and/or mRNA degradation? d) Are these events virus dose and/or virus synthesis dependent? 2) Examine the effect of viral infection on the protein composition of the extracellular substratum. The questions to be answered include a) How does multiplicity of infection affect the ratio of host-cell to viral protein in the matrix? b) What is the nature of the viral proteins in the matrix? c) What is the effect of the altered substratum on cell adhesion, morphology, replication and phenotypic expression? 3) Examine the effect of in vitro persistent viral infection of EC and SM on cellular, and in particular, matrix protein synthesis. Cell protein synthesis will be investigated when viral replication is periodic as with herpes simplex and when it is continuous, as with Coxsackie-B4. A variety of approaches, including isotopic labeling, gel electrophoresis, immunofluorescence and immunoelectron microscopy, will be used. Recombinant DNA clones will be used to measure levels of mRNAs encoding for matrix and non-matrix cell proteins.
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