Role of calcium-independent phospholipase a₂β in high glucose-induced activation of RhoA, Rho Kinase, and CPI-17 in cultured vascular smooth muscle cells and vascular smooth muscle hypercontractility in diabetic animals

Previous studies suggest that high glucose-induced RhoA/ Rho kinase/CPI-17 activation is involved in diabetes-associated vascular smooth muscle hypercontractility. However, the upstream signaling that links high glucose and RhoA/Rho kinase/ CPI-17 activation is unknown. Here we report that calcium-independent phospholipase A₂β (iPLA₂β) is required for high glucose-induced RhoA/Rho kinase/CPI-17 activation and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility. We demonstrate that high glucose increases iPLA ₂β mRNA, protein, and iPLA₂ activity in a time-dependent manner. Protein kinase C is involved in high glucose-induced iPLA₂ βprotein up-regulation. Inhibiting iPLA₂β activity with bromoenol lactone or preventing its expression by genetic deletion abolishes high glucose-induced RhoA/Rho kinase/CPI-17 activation, and restoring expression of iPLA₂β in iPLA₂β-deficient cells also restores high glucose-induced CPI-17 phosphorylation. Pharmacological and genetic inhibition of 12/15-lipoxygenases has effects on high glucose-induced CPI-17 phosphorylation similar to iPLA₂β inhibition. Moreover, increases in iPLA₂ activity and iPLA₂β protein expression are also observed in both type 1 and type 2 diabetic vasculature. Pharmacological and genetic inhibition of iPLA₂β, but not iPLA₂γ, diminishes diabetes-associated vascular smooth muscle hypercontractility. In summary, our results reveal a novel mechanism by which high glucose-induced, protein kinase C-mediated iPLA₂β up-regulation activates the RhoA/Rho kinase/CPI-17 via 12/15-lipoxygenases and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility.