Abstract
PYCARD (PYD and CARD domain containing), a pivotal adaptor protein in inflammasome assembly and activation, contributes to innate immunity, and plays an essential role in the pathogenesis of atherosclerosis and restenosis. However, its roles in microRNA biogenesis remain unknown. Therefore, this study aimed to investigate the roles of PYCARD in miRNA biogenesis and neointima formation using pycard knockout (pycard-/-) mice. Deficiency of Pycard reduced circulating miRNA profile and inhibited Mir17 seed family maturation. The systemic pycard knockout also selectively reduced the expression of AGO2 (argonaute RISC catalytic subunit 2), an important enzyme in regulating miRNA biogenesis, by promoting chaperone-mediated autophagy (CMA)-mediated degradation of AGO2, specifically in adipose tissue. Mechanistically, pycard knockout increased PRMT8 (protein arginine N-methyltransferase 8) expression in adipose tissue, which enhanced AGO2 methylation, and subsequently promoted its binding to HSPA8 (heat shock protein family A (Hsp70) member 8) that targeted AGO2 for lysosome degradation through chaperone-mediated autophagy. Finally, the reduction of AGO2 and Mir17 family expression prevented vascular injury-induced neointima formation in Pycard-deficient conditions. Overexpression of AGO2 or administration of mimic of Mir106b (a major member of the Mir17 family) prevented Pycard deficiency-mediated inhibition of neointima formation in response to vascular injury. These data demonstrate that PYCARD inhibits CMA-mediated degradation of AGO2, which promotes microRNA maturation, thereby playing a critical role in regulating neointima formation in response to vascular injury independently of inflammasome activity and suggest that modulating PYCARD expression and function may represent a powerful therapeutic strategy for neointima formation.Abbreviations: 6-AN: 6-aminonicotinamide; ACTB: actin, beta; aDMA: asymmetric dimethylarginine; AGO2: argonaute RISC catalytic subunit 2; CAL: carotid artery ligation; CALCOCO2: calcium binding and coiled-coil domain 2; CMA: chaperone-mediated autophagy; CTSB: cathepsin B; CTSD: cathepsin D; DGCR8: DGCR8 microprocessor complex subunit; DOCK2: dedicator of cyto-kinesis 2; EpiAdi: epididymal adipose tissue; HSPA8: heat shock protein family A (Hsp70) member 8; IHC: immunohistochemical; ISR: in-stent restenosis; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; miRNA: microRNA; NLRP3: NLR family pyrin domain containing 3; N/L: ammonium chloride combined with leupeptin; PRMT: protein arginine methyltransferase; PVAT: peri-vascular adipose tissues; PYCARD: PYD and CARD domain containing; sDMA: symmetric dimethylarginine; ULK1: unc-51 like kinase 1; VSMCs: vascular smooth muscle cells; WT: wild-type.