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 effects in the vascular system. Endothelial cells treated with 12LO products or cultured in high glucose showed enhanced monocyte adhesion, an important step in atherogenesis. We have previously reported that PDGF increased HETE levels in porcine aortic smooth muscle cells. Although several pharmacological inhibitors to the LO pathway are available, most lack specificity and may harbor undesirable side effects. Therefore, we developed a recombinant adenovirus expressing a hammerhead ribozyme (AdRZ) targeted against the porcine leukocyte-type 12LO mRNA to investigate the involvement of LO in glucose- and PDGF-mediated effects in vascular cells. Infection of porcine aortic endothelial cells with AdRZ reduced the level of glucose-enhanced 12LO mRNA expression as determined by quantitative, real-time reverse transcriptase-polymerase chain reaction. Reverse-phase HPLC and RIA analysis also revealed a corresponding decrease in glucose-stimulated 12HETE production in both the cellular and supernatant fractions. In the ribozyme-treated porcine aortic endothelial cells, there was marked inhibition of high glucose-stimulated monocyte adhesion. Infection with AdRZ also reduced PDGF-induced porcine aortic smooth muscle cell migration by approximately 50%. These studies demonstrate the efficacy of recombinant adenovirus expressing 12LO ribozyme in studying the effects of 12LO in vascular wall cells. They document an important role for the 12LO pathway in regulating inflammatory changes in endothelial cells and smooth muscle cells.