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

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.