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

Circ Res. 2022 Dec 2;131(12):1037-1054. doi: 10.1161/CIRCRESAHA.122.321614. Epub 2022 Nov 10.

Abstract

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 KD 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-Gαq-PKCα (protein kinase Cα)-AMPKα1 (AMP-activated protein kinase α1) signaling to counteract VSMCs osteogenic transition and mineralization.

Conclusions: Nidogen-2 is a novel endogenous ligand of LGR4 that biased activated Gαq- PKCα-AMPKα1 signaling and inhibited vascular calcification.

Keywords: LGR4; extracellular matrix; nidogen-2; smooth muscle cells; vascular calcification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium Chloride
  • Cells, Cultured
  • Cholecalciferol / metabolism
  • Cholecalciferol / pharmacology
  • Ligands
  • Membrane Glycoproteins* / metabolism
  • Mice
  • Muscle, Smooth, Vascular* / metabolism
  • Myocytes, Smooth Muscle / metabolism
  • Phosphates / adverse effects
  • Protein Kinase C-alpha / metabolism
  • Receptors, G-Protein-Coupled* / metabolism
  • Vascular Calcification* / genetics
  • Vascular Calcification* / prevention & control

Substances

  • Calcium Chloride
  • Cholecalciferol
  • LGR4 protein, mouse
  • Ligands
  • Membrane Glycoproteins
  • Phosphates
  • Protein Kinase C-alpha
  • Receptors, G-Protein-Coupled
  • Nid2 protein, mouse