Nanocarrier-driven dual targeting VCAM-1/Collagen IV enables RNA interference-mediated silencing of Smad3 and Runx2 to mitigate aortic valve disease

BACKGROUNDCalcific aortic valve disease (CAVD) is a common malady with few treatment options other than valve replacement by surgery or transcatheter aortic valve implantation (TAVI). Endothelial-to-mesenchymal transition (EndMT) of valvular endothelial cells and osteogenic differentiation of valvular interstitial cells are crucial processes of CAVD. Smad3 and Runx2 are key transcription factors (TFs) that drive these processes by regulating gene expression and cellular functions. We hypothesize that downregulation of these TFs with nanoparticle-mediated RNA interference could mitigate aortic valve stenosis and calcification. METHODSWe engineered dual-targeted lipid-polymer hybrid nanocarriers (lipopolyplexes, LPP) to deliver short-hairpin RNA (shRNA) for gene silencing in pathologically remodeled aortic valve. The nanocarriers simultaneously target vascular cell adhesion molecule-1 (VCAM-1) and collagen IV, enhancing specificity toward inflamed and fibrotic valvular tissue. Encapsulated shRNA constructs were designed to silence either Smad3 or Runx2 (yielding formulations V/Cp-LPP/shSmad3 and V/Cp-LPP/shRunx2). Therapeutic efficacy was evaluated in a mouse model of atherosclerosis aggravated by diabetes, mimicking the pathological environment of CAVD. RESULTSThe dual-targeted lipopolyplexes effectively facilitated gene delivery to the aortic valve, ensuring efficient transfection. Treatment with V/Cp-LPP/shSmad3 and V/Cp-LPP/shRunx2 resulted in marked silencing of Smad3 and Runx2, accompanied by significant suppression of osteogenic markers, including osteopontin, alkaline phosphatase, and osteocalcin, as well as reduced SMA expression in valve tissue. Our data further identify Runx2 as a novel upstream modulator of Smad3 expression, unveiling a previously unrecognized Runx2-Smad3 regulatory axis with important implications for valvular pathology and targeted therapy. Beyond localized effects, systemic administration of these lipopolyplexes led to reduced plasma concentrations of alkaline phosphatase, cholesterol, and triglycerides, while maintaining hepatic and renal function, suggesting additional benefits on systemic metabolic homeostasis. CONCLUSIONSThese findings highlight the pivotal role of Smad3 and Runx2 downregulation in mitigating aortic valve calcification, unveiling both molecules as compelling therapeutic targets in CAVD. HighlightsO_LILipopolyplexes dual-targeted to VCAM-1 and Collagen IV successfully transfect valvular cells and drive plasmid expression in the valve leaflets of a CAVD mouse model. C_LIO_LITherapeutic delivery of shRNA specific for Smad3 and Runx2 led to significant downregulation of osteogenic markers (osteopontin, alkaline phosphatase, osteocalcin) and reduced SMA in the aortic valve. C_LIO_LIThe study establishes Runx2 as an upstream regulator of Smad3 expression with implications for valve disease intervention. C_LIO_LIThe engineered lipopolyplexes demonstrated a favorable safety profile and systemic benefits: decreased plasma levels of alkaline phosphatase, cholesterol, and triglycerides. C_LIO_LIThe study validates the lipopolyplexes-based strategy as a safe and targeted platform for localized molecular reprogramming in calcific aortic valve disease. C_LI