Adenoviral delivery of a leukocyte-type 12 lipoxygenase ribozyme inhibits effects of glucose and platelet-derived growth factor in vascular endothelial and smooth muscle cells

The lipoxygenase (LO) pathway has been implicated as an important mediator of chronic glucose and platelet-derived growth factor (PDGF)-induced effect s in the vascular system. Endothelial cells treated with 12LO products or c ultured in high glucose showed enhanced monocyte adhesion, an important ste p in atherogenesis, We have previously reported that PDGF increased HETE le vels in porcine aortic smooth muscle cells. Although several pharmacologica l inhibitors to the LO pathway are available, most lack specificity and may harbor undesirable side effects, Therefore, we developed a recombinant ade novirus expressing a hammerhead ribozyme (AdRZ) targeted against the porcin e leukocyte-type 12LO mRNA to investigate the involvement of LO in glucose- and PDGF-mediated effects in vascular cells. Infection of porcine aortic e ndothelial cells with AdRZ reduced the level of glucose-enhanced 12LO mRNA expression as determined by quantitative, real-time reverse transcriptase-p olymerase chain reaction. Reverse phase HPLC and RIA analysis also revealed a corresponding decrease in glucose-stimulated I2HETE production in both t he cellular and supernatant fractions. In the ribozyme-treated porcine aort ic endothelial cells, there was marked inhibition of high glucose-stimulate d monocyte adhesion. Infection with AdRZ also reduced PDGF-induced porcine aortic smooth muscle cell migration by approximately 50%. These studies dem onstrate the efficacy of recombinant adenovirus expressing 12LO ribozyme in studying the effects of 12LO in vascular wall cells. They document an impo rtant role for the 12LO pathway in regulating inflammatory changes in endot helial cells and smooth muscle cells.