Serrano Ramon L, Yu Weifang, Terkeltaub Robert
San Diego VA Healthcare System and Department of Medicine, University of California, 111K, 3350 La Jolla Village Dr., San Diego, CA 92161, USA.
San Diego VA Healthcare System and Department of Medicine, University of California, 111K, 3350 La Jolla Village Dr., San Diego, CA 92161, USA.
Atherosclerosis. 2014 Apr;233(2):493-502. doi: 10.1016/j.atherosclerosis.2014.01.003. Epub 2014 Jan 21.
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.
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.
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.
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.
双等位基因功能失活的ENPP1突变会导致婴儿期全身性动脉钙化（GACI）中的动脉中层钙化（AMC）以及相关的严重肌内膜增生，而单等位基因ENPP1缺陷在表型上是正常的。在此，我们测试了ENPP1缺陷是否会促进异常血管平滑肌细胞（VSMC）对损伤的反应，无论有无钙化。内质网应激介质C/EBP同源蛋白（CHOP）会影响内膜增生和动脉粥样硬化，并且对骨形成有矛盾的影响。因此，我们评估了VSMC中ENPP1与CHOP之间的关系。
我们研究了接受左颈动脉结扎的ENPP1缺陷小鼠和对照同窝小鼠，并从这些小鼠以及Chop-/-小鼠中分离并研究了VSMC，或对其进行CHOP siRNA处理。
正常的Enpp1+/-小鼠，除了AMC发生前的Enpp1-/-小鼠外，在7-8周龄时对颈动脉结扎的反应中内膜增生加速。内膜增生与动脉中层原位CHOP的强烈表达有关，但仅在受伤的Enpp1-/-动脉中有明显的AMC。在培养中，ENPP1缺陷和CHOP缺陷的VSMC对血小板衍生生长因子（PDGF）的迁移和增殖增加。培养的Chop-/-VSMC显示出无机磷供体诱导的钙化增加。在无机磷供体处理的Enpp1-/-和Enpp1+/-培养的VSMC中，CHOP显著增加。对Enpp1-/-VSMC进行CHOP siRNA处理会增加钙化，这与组织非特异性碱性磷酸酶和主要成骨转录因子RUNX2的表达升高有关。
单等位基因和双等位基因ENPP1缺陷均会促进VSMC功能失调，体内损伤后病变中CHOP表达强烈且内膜增生增强，但损伤后明显的钙化仅限于Enpp1-/-小鼠。GACI中的内膜增生似乎受ENPP1缺陷的生物学效应调控，而非钙化，包括内质网应激。ENPP1缺陷时VSMC中CHOP过量可能主要起到限制VSMC钙化的作用。
