JNK2 Gene Silencing for Elastic Matrix Regenerative Repair.

Elastic fibers do not naturally regenerate in many proteolytic disorders, such as in abdominal aortic aneurysms (AAAs), and prevent restoration of tissue homeostasis. We have shown drug-based attenuation of the stress-activated protein kinase, JNK-2 to stimulate elastic matrix neoassembly and to attenuate cellular proteolytic activity. We now investigate if JNK2 gene knockdown with siRNA provides greater specificity of action and improved regenerative/anti-proteolytic outcomes in a proteolytic injury culture model of rat aneurysmal smooth muscle cell (EaRASMCs). An siRNA dose of 12.5nM delivered with a transfection reagent significantly enhanced downstream elastic fiber assembly and maturation vs untreated EaRASMC cultures. The optimal siRNA dose was also delivered as a complex with a polymeric transfection vector, polyethyleneimine (PEI) in preparation for future in vivo delivery. Linear 25kDa PEI-siRNA (5:1 molar ratio of amine to phosphate) and linear 40kDa PEI-siRNA (2.5:1 ratio) were effective in downregulating the JNK2 gene, and significantly increasing expression of elastic fiber assembly proteins, and decreases in elastolytic matrix metalloprotease (MMP)-2 vs treatment controls to significantly increase mature elastic fiber assembly. The current work has identified siRNA dosing and siRNA-PEI complexing conditions that are safe and efficient in stimulating processes contributing to improved elastic matrix neoassembly via JNK2 gene knockdown. The results represent a mechanistic basis of a broader therapeutic approach to reverse elastic matrix pathophysiology in tissue disorders involving aberrations of elastic matrix homeostasis, such as in aortic aneurysms.