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

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 Iinked 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.

ENPP1-Fc prevents neointima formation in generalized arterial calcification of infancy through the generation of AMP

“…However, in our study, injury-triggered myointimal proliferation was significantly increased in ENPP1-deficient ttw/ttw mice when compared to WT mice within 14 days after carotid artery ligation. Serrano et al 25 demonstrated that the increased proliferation observed during ENPP1 deficiency is associated with dysregulated VSMC function and ER stress. The authors assumed that there were biologic effects of ENPP1 deficiency on VSMCs other than osteochondral differentiation and calcification.…”

“…Neither of the mouse models, ttw/ttw and Enpp1 − / − , show myointimal proliferation per se. However, Enpp1 − / − mice show marked intimal vascular smooth muscle cell (VSMC) proliferation in response to arterial injury in vivo 25 . Thus far, it is not known whether injury-triggered intimal proliferation is also increased in ttw/ttw mice.…”

Enpp1 inhibits ectopic joint calcification and maintains articular chondrocytes by repressing hedgehog signaling

“…The mouse lines have been described previously: Enpp1 −/− mice (C57BL/6-Enpp1tm1) (Serrano et al, 2014); Gnas f/f (Chen et al, 2005), Ptch1 +/− (Mak et al, 2008a), Gli2 +/− (Bai and Joyner, 2001) and Osx-GFP::Cre mice (Rodda and McMahon, 2006). Genotyping was carried out using tail genomic DNA by standard PCR-based procedures.…”

Endoplasmic Reticulum Stress in Arterial Smooth Muscle Cells: A Novel Regulator of Vascular Disease

“…In mouse ASMCs lacking the ENPP1 enzyme, which synthesizes pyrophosphate, a natural calcification inhibitor, the absence of CHOP increased mineralisation [137]. …”