Nidogen-2 is a Novel Endogenous Ligand of LGR4 to Inhibit Vascular Calcification

Background:Vascular calcification is closely related to the all-cause mortality of cardiovascular events. Basement membrane protein nidogen-2 is a key component of the vascular extracellular matrix microenvironment and we recently found it is pivotal for the maintenance of contractile phenotype in vascular smooth muscle cells (VSMCs). However, whether nidogen-2 is involved in VSMCs osteochondrogenic transition and vascular calcification remains unclear. Methods:VSMCs was treated with high-phosphate to study VSMC calcification in vitro. Three different mice models (5/6 nephrectomy-induced chronic renal failure, cholecalciferol-overload, and periadventitially administered with CaCl2) were used to study vascular calcification in vivo. Membrane protein interactome, coimmunoprecipitation, flow cytometric binding assay, surface plasmon resonance, G protein signaling, VSMCs calcium assays were performed to clarify the phenotype and elucidate the molecular mechanisms. Results:Nidogen-2 protein levels were significantly reduced in calcified VSMCs and aortas from mice in different vascular calcification model. Nidogen-2 deficiency exacerbated high-phosphate-induced VSMC calcification, whereas the addition of purified nidogen-2 protein markedly alleviated VSMC calcification in vitro. Nidogen-2(-/-) mice exhibited aggravated aorta calcification compared to wild-type (WT) mice in response to 5/6 nephrectomy, cholecalciferol-overload, and CaCl2 administration. Further unbiased coimmunoprecipitation and interactome analysis of purified nidogen-2 and membrane protein in VSMCs revealed that nidogen-2 directly binds to LGR4 (leucine-rich repeat G-protein-coupled receptor 4) with K-D value 26.77 nM. LGR4 deficiency in VSMCs in vitro or in vivo abolished the protective effect of nidogen-2 on vascular calcification. Of interest, nidogen-2 biased activated LGR4-G alpha q-PKC alpha (protein kinase C alpha)-AMPK alpha 1 (AMP-activated protein kinase alpha 1) signaling to counteract VSMCs osteogenic transition and mineralization. Conclusions:Nidogen-2 is a novel endogenous ligand of LGR4 that biased activated G alpha q- PKC alpha-AMPK alpha 1 signaling and inhibited vascular calcification.