Anastomotic intimal hyperplasia remains as the most common cause of delayed prothetic arterial graft failure. Our hypothesis is that various genes are either up or down-regulated at various time intervals during graft healing, thereby contributing to the formation of hyperplasia.
The specific aims of this study are to: 1) determine differential gene expression at various time intervals following graft implantation and 2) elucidate the role of these genes in hyperplasia formation. Using a canine model of arterial reconstruction, mRNA differential display will be employed to screen both normal arteries (control) and hyperplasia lesions in order to determine altered gene expression. These candidates will then be amplified via RT: PCR and radiolabeled probes will be synthesized. These radiolabeled probes will then be used to confirm altered gene expression using Northern Blot analysis. These candidates will be sequenced and evaluated in Genbank to determine the relation of the identified sequence to known gene sequences. Selective digoxigenin-labeled riboprobes and antibodies from these clones will be created in order to determine cellular location of the gene products. Novel clones will be hybridized to known cDNA/RNA libraries to either identify the gene itself or evaluate expression in other tissues. Gene or gene products from the differential display studies will be evaluated in tissue culture. Interferon gamma upregulated protein or PA2 (an activator of the 20s proteasome), which was found by our group to have altered expression at 90 days in hyperplasia tissue as compared to control artery, will be initially evaluated. The presence or absence of PA28-beta, MECL-1, LMP7 an dLMP2 proteasome subunits in hyperplasia tissue will be examined in order to determine if PA28 is regulated independently of other known gamma interferon-expressed proteins. Endothelial and smooth muscle cell cultures will be employed to assess PA28 and proteasome function in cellular migration and proliferation, MAP Kinase regulation, production of nitric oxide and regulation of nuclear factor kappa-B. Through these assays, the importance of PA28 function in vascular cells and anastomotic intimal hyperplasia will be elucidated. Other genes and gene products from our differential display studies such as human retinoblastoma susceptibility gene, apolipoprotein J, creatine kinase, alpha-1 protease inhibitor and type III collagen will selectively be assessed in similar fashion. This ongoing study of gene expression following prosthetic arterial grafting will continue to yield new insight into the mechanism(s) of anastomotic intimal hyperplasia and provide new avenues for therapeutic intervention.
Showing the most recent 10 out of 41 publications
