Mono-allelic and bi-allelic ENPP1 deficiency promote post-injury neointimal hyperplasia associated with increased C/EBP homologous protein expression

Atherosclerosis. 2014 Apr;233(2):493-502. doi: 10.1016/j.atherosclerosis.2014.01.003. Epub 2014 Jan 21.

Abstract

Objective: Bi-allelic function-inactivating ENPP1 mutations cause artery media calcification (AMC) with associated severe myointimal hyperplasia in generalized arterial calcification of infancy (GACI), whereas mono-allelic ENPP1 deficiency is phenotypically normal. Here, we tested if ENPP1 deficiency promotes abnormal vascular smooth muscle cell (VSMC)-driven responses to injury, with or without calcification. The ER stress mediator C/EBP homologous protein (CHOP) affects neointimal hyperplasia and atherosclerosis, and has paradoxical effects on bone formation. Hence, we assessed relationships between ENPP1 and CHOP in VSMCs.

Methods: We studied ENPP1-deficient mice and control littermates subjected to left carotid artery ligation, and isolated and studied VSMCs from these and Chop-/- mice, or with CHOP siRNA treatment.

Results: Normal Enpp1-/+ mice, in addition to Enpp1-/- mice prior to AMC development, had accelerated neointimal hyperplasia in response to carotid artery ligation at 7-8 weeks age. Neointimal hyperplasia was linked with robust artery media CHOP expression in situ, but with marked AMC only in injured Enpp1-/- arteries. Cultured, ENPP1-deficient and CHOP-deficient VSMCs had increased migration and proliferation to PDGF. Cultured Chop-/- VSMCs demonstrated increased Pi donor-induced calcification. CHOP was significantly increased in Pi donor treated Enpp1-/- and Enpp1-/+ cultured VSMCs. CHOP siRNA treatment of Enpp1-/- VSMCs increased calcification, associated with elevated expression of tissue nonspecific alkaline phosphatase and the master osteoblastic transcription factor RUNX2.

Conclusions: Both mono-allelic and bi-allelic ENPP1 deficiency promote dysregulated VSMC function, with robust lesion CHOP expression and enhanced neointimal hyperplasia after injury in vivo, but marked post-injury calcification limited to Enpp1-/- mice. Intimal hyperplasia in GACI appears regulated by biologic effects of ENPP1 deficiency other than calcification, including ER stress. VSMC CHOP excess in ENPP1 deficiency may primarily function to limit VSMC calcification.

Keywords: Artery media calcification; CHOP; ER stress; Generalized arterial calcification of infancy; Unfolded protein response.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alkaline Phosphatase / biosynthesis
  • Alkaline Phosphatase / genetics
  • Alleles
  • Animals
  • Carotid Stenosis / metabolism
  • Carotid Stenosis / pathology*
  • Cell Differentiation
  • Core Binding Factor Alpha 1 Subunit / biosynthesis
  • Core Binding Factor Alpha 1 Subunit / genetics
  • Disease Models, Animal
  • Genotype
  • Hyperplasia
  • Ligation
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Myocytes, Smooth Muscle / metabolism
  • Myocytes, Smooth Muscle / pathology*
  • Neointima / physiopathology*
  • Phosphoric Diester Hydrolases / deficiency
  • Phosphoric Diester Hydrolases / genetics
  • Phosphoric Diester Hydrolases / physiology*
  • Pyrophosphatases / deficiency
  • Pyrophosphatases / genetics
  • Pyrophosphatases / physiology*
  • Transcription Factor CHOP / biosynthesis*
  • Transcription Factor CHOP / deficiency
  • Transcription Factor CHOP / genetics
  • Tunica Intima / injuries*
  • Tunica Intima / pathology
  • Tunica Media / pathology
  • Unfolded Protein Response / physiology
  • Vascular Calcification / etiology
  • Vascular Calcification / genetics
  • Vascular Calcification / metabolism
  • Vascular Calcification / pathology
  • Vascular Calcification / physiopathology

Substances

  • Core Binding Factor Alpha 1 Subunit
  • Runx2 protein, mouse
  • Transcription Factor CHOP
  • ALPL protein, mouse
  • Alkaline Phosphatase
  • Phosphoric Diester Hydrolases
  • ectonucleotide pyrophosphatase phosphodiesterase 1
  • Pyrophosphatases

Supplementary concepts

  • Arterial calcification of infancy