Despite the established role of alveolar type II epithelial cells for the maintenance of pulmonary function, little is known about the deregulation of lipid composition in the pathogenesis of pulmonary fibrosis. The elongation of long-chain fatty acids family member 6 (Elovl6) is a rate-limiting enzyme catalysing the elongation of saturated and monounsaturated fatty acids. Here we show that Elovl6 expression is significantly downregulated after an intratracheal instillation of bleomycin (BLM) and in human lung with idiopathic pulmonary fibrosis. Elovl6-deficient (Elovl6 À / À ) mice treated with BLM exhibit severe fibroproliferative response and derangement of fatty acid profile compared with wild-type mice. Furthermore, Elovl6 knockdown induces a change in fatty acid composition similar to that in Elovl6 À / À mice, resulting in induction of apoptosis, TGF-b1 expression and reactive oxygen species generation. Our findings demonstrate a previously unappreciated role for Elovl6 in the regulation of lung homeostasis, and in pathogenesis and exacerbation of BLM-induced pulmonary fibrosis.
BackgroundFatty acids constitute the critical components of cell structure and function, and dysregulation of fatty acid composition may exert diverging vascular effects including proliferation, migration, and differentiation of vascular smooth muscle cells (VSMCs). However, direct evidence for this hypothesis has been lacking. We investigated the role of elongation of long‐chain fatty acid member 6 (Elovl6), a rate‐limiting enzyme catalyzing the elongation of saturated and monounsaturated long‐chain fatty acid, in the regulation of phenotypic switching of VSMC.Methods and ResultsNeointima formation following wire injury was markedly inhibited in Elovl6‐null (Elovl6−/−) mice, and cultured VSMCs with siRNA‐mediated knockdown of Elovl6 was barely responsive to PDGF‐BB. Elovl6 inhibition induced cell cycle suppressors p53 and p21 and reduced the mammalian targets of rapamycin (mTOR) phosphorylation and VSMC marker expression. These changes are ascribed to increased palmitate levels and reduced oleate levels, changes that lead to reactive oxygen species (ROS) production and resulting AMP‐activated protein kinase (AMPK) activation. Notably, Elovl6 inhibition robustly induced the pluripotency gene Krüppel‐like factor 4 (KLF4) expression in VSMC, and KLF4 knockdown significantly attenuated AMPK‐induced phenotypic switching of VSMC, indicating that KLF4 is a bona fide target of AMPK.ConclusionsWe demonstrate for the first time that dysregulation of Elovl6‐driven long‐chain fatty acid metabolism induces phenotypic switching of VSMC via ROS production and AMPK/KLF4 signaling that leads to growth arrest and downregulation of VSMC marker expression. The modulation of Elovl6‐mediated cellular processes may provide an intriguing approach for tackling atherosclerosis and postangioplasty restenosis.
Introduction:
Elovl6, the elongase of long chain fatty acids 6, is a rate-limiting enzyme catalyzing the elongation of saturated and monounsaturated fatty acids with 12, 14 and 16 carbons. Our recent study showed that Elovl6 is abundantly expressed in vascular smooth muscle cells (VSMC) and is dramatically induced in neointima in rat.
Hypothesis:
In this study, we tested the hypothesis that changes of fatty acid (FA) composition by Elovl6 affects the proliferation of VSMC and contributes to neointimal formation in vivo.
Methods and Results:
Abundant Elovl6 expression was observed in mice femoral artery at 2 weeks after wire-injury and in intimal thickening lesion of human coronary artery. Furthermore, Elovl6 mRNA expression in cultured human aortic SMC (HASMC) was significantly increased by platelet-derived growth factor-BB (2.4-fold, p<0.05) or hypoxic stress (6.7-fold, p<0.01) in a dose- or time-dependent manner. Furthermore, knockdown of Elovl6 expression in HASMC markedly suppressed cell proliferation (16%, relative to control, p<0.01) and migration, concomitantly induced the expression of p21 and phospholyration of AMP-activated protein kinase (p-AMPK) and suppressed mTOR expression. Consistent with in vitro data, Elovl6 deficient (Elovl6 -/-) mice at 2 weeks after injury showed markedly suppressed neointimal formation compared with wild-type (WT) mice (intima/media ratio: WT, 1.4 ± 0.6; Elovl6 -/-, 0.5 ± 0.2; Ki67-positive cells: 0.2 fold relative to WT mice; N=6-7, p<0.05). Of an importance, analysis of FA composition in SMC isolated from Elovl6 -/- mice showed that high levels of palmitic acid and low levels of oleic acid were detected as compared with that from WT mice. In accordance with these results, exogenous treatment of palmitic acid in SMC substantially suppressed cell proliferation (42%, relative to control, p<0.01) and migration, induced p21 and p-AMPK expressions. Conversely, these effects were blunted by adenovirus-mediated Elovl6-overexpression or exogenous oleic acid treatment.
Conclusions:
Collectively, our study demonstrates that proliferation of VSMC is tightly regulated by FA composition modulated by Elvlo6, offering a novel therapeutic target for arterial proliferative disease in which VSMC plays a key role.
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