Saturated phosphatidic acids mediate saturated fatty acid–induced vascular calcification and lipotoxicity
Masashi Masuda, … , Makoto Kuro-o, Makoto Miyazaki
Masashi Masuda, … , Makoto Kuro-o, Makoto Miyazaki
Published December 1, 2015; First published October 26, 2015
Citation Information: J Clin Invest. 2015;125(12):4544-4558. https://doi.org/10.1172/JCI82871.
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Research Article Vascular biology

Saturated phosphatidic acids mediate saturated fatty acid–induced vascular calcification and lipotoxicity

Abstract

Recent evidence indicates that saturated fatty acid–induced (SFA-induced) lipotoxicity contributes to the pathogenesis of cardiovascular and metabolic diseases; however, the molecular mechanisms that underlie SFA-induced lipotoxicity remain unclear. Here, we have shown that repression of stearoyl-CoA desaturase (SCD) enzymes, which regulate the intracellular balance of SFAs and unsaturated FAs, and the subsequent accumulation of SFAs in vascular smooth muscle cells (VSMCs), are characteristic events in the development of vascular calcification. We evaluated whether SMC-specific inhibition of SCD and the resulting SFA accumulation plays a causative role in the pathogenesis of vascular calcification and generated mice with SMC-specific deletion of both Scd1 and Scd2. Mice lacking both SCD1 and SCD2 in SMCs displayed severe vascular calcification with increased ER stress. Moreover, we employed shRNA library screening and radiolabeling approaches, as well as in vitro and in vivo lipidomic analysis, and determined that fully saturated phosphatidic acids such as 1,2-distearoyl-PA (18:0/18:0-PA) mediate SFA-induced lipotoxicity and vascular calcification. Together, these results identify a key lipogenic pathway in SMCs that mediates vascular calcification.

Authors

Masashi Masuda, Shinobu Miyazaki-Anzai, Audrey L. Keenan, Kayo Okamura, Jessica Kendrick, Michel Chonchol, Stefan Offermanns, James M. Ntambi, Makoto Kuro-o, Makoto Miyazaki

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Figure 6

UFA cotreatment normalizes mineralization, osteogenic differentiation, ER stress, and PA accumulation.

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UFA cotreatment normalizes mineralization, osteogenic differentiation, E...
(A–C) UFAs blocked SCDi-induced mineralization (A and B) and osteogenic differentiation (C) of VSMCs. Human VSMCs were treated with 200 μM UFAs in the presence of 300 nM SCDi for 7 days in the presence of 2.0 mM inorganic phosphate. (A) Seven days after the treatments, the cells were stained with Alizarin red to identify calcium deposits. (B) Calcium content was analyzed using a colorimetric assay. (C) Alp mRNA levels were analyzed by qPCR. (D and E) Calcitriol-induced mineralization is blocked by 18:1n-9 supplementation. Human VSMCs were treated with 100 nM calcitriol in the presence of 200 μM 18:1n-9 for 7 days. (F–H) UFAs such as 18:1n-9 completely blocked ER stress induced by SCDi. VSMCs were treated with SCDi acid for 24 hours. Total protein extracts were subjected to immunoblot analysis with ATF4- and CHOP-specific antibodies. Atf4 and Chop mRNA levels were quantified by qPCR. (I) Autoradiography and (J) quantification of 14C-18:0 incorporation into the lipid fraction. Human VSMCs were treated with 200 μM 18:1n-9 in the presence of 300 nM SCDi and 14C-18:0 (1 μCi). The black line in I indicates that the image was derived from noncontiguous lanes of the same plate. Lipids were separated on a boric acid–coated TLC. (K) Absolute levels of PA species in VSMCs cotreated with SCDi and 18:1n-9. Human VSMCs were treated with 200 μM 18:1n-9 in the presence of 300 nM SCDi for 24 hours. Each PA content was analyzed with LC-MS/MS. NL, neutral lipids. n = 4, *P < 0.01 vs. vehicle (Veh) and #P < 0.01 vs. SCDi (n = 3–6, one-way ANOVA).