锌指Krüppel样因子4抑制平滑肌内源性脂合成的新功能及其分子机制研究

Enhanced and disordered in situ lipogenesis in vascular smooth muscle cells(VSMCs) maybe the earliest and the primary event in the development of atheroma. Krüppel-like factor 4(KLF4) is a zinc-finger transcription factor that plays a critical role in the regulation of cellular proliferation, differentiation and oncogenesis. KLF4 has been newly identified as a key regulator controlling lipid metabolism in blood vessels in our study.Indeed,a strong association between KLF4 expression and de novo lipogenesis has been implied in our experiments, conferring an advantage to VSMCs. Our previous studies have demonstrated that silencing of KLF4 by small interfering RNA in endothelial cells permits a decrease in pulsatile laminar shear stress-stimulated insig1 expression. Furthermore, our preliminary data have also revealed that KLF4 substantially inhibited the interleukin-1(IL-1)-induced lipogenic enzymes expression and intracellular accumulation of neutral lipids, which are storage products of fatty acid and cholesterol in VSMCs.Although remarkable progress has been achieved, much less is known concerning whether KLF4 directly modulates the de novo lipogenesis in VSMCs,and the molecular mechanisms by which this occurs have not yet been defined. Therefore, it is essential to elucidate the novel role that KLF4 exerts in de novo lipogenesis in VSMCs and the mechanisms involved... In this study, we hypothesize for the first time that KLF4 may exert a novel function in inhibiting the lipogenic enzymes- mediated de novo lipogenesis in primary coronary artery VSMCs under inflammatory stress, thereby decreasing the massive accumulation of neutral lipids during atheroma.To confirm this hypothesis,a strategy for the integration of molecular biology,cellular biology,bioinformatics and ApoE-/- mice,is proposed. The present study was undertaken : 1) to clarify the direct effect of KLF4 on the profiles of specific sets of lipogenic genes and its putative mechanisms; 2)to investigate whether KLF4 rescues cytokine-induced the endogenous lipid synthesis through the regulation of insig; 3)to determine the phenotypic outcomes ascending from the novel function of KLF4 in animal model.Proceeding along the above scheme,our findings may provide novel insights into the patho-physiological mechanisms of the anti-atherosclerosis properties of KLF4, and the unexpected lipogenesis-inhibitory function of KLF4 in VSMCs may provide a new paradigm for linking de novo lipogenesis and atherom.

平滑肌内源性(endogenous)脂质合成异常是动脉粥样硬化(AS)早期首要病理环节。锌指Krüppel样因子4(KLF4)是我们前期研究中新近发现的一种调节血管脂代谢的关键因子，前期和预实验结果表明：KLF4介导了脉冲式层流诱导的内皮insig1表达；KLF4可显著降低IL-1诱导的平滑肌内源性脂质积聚。然而,KLF4是否直接调控平滑肌内源性脂质合成及其机制尚不明确。. 本研究以KLF4抑制平滑肌内源性脂质合成(de novo lipogenesis)的新功能及机制为靶点，将分子细胞生物学、生物信息学与动物模型相交叉、整合，首次1）阐明KLF4对内源性脂合成的影响及调控机制；2）探讨KLF4对炎症诱导的平滑肌脂质合成异常增加的影响；3）揭示KLF4新功能的表型转归。循分子-细胞-整体水平阐释KLF4抗平滑肌内源性脂质合成的分子病理生理学机制及其意义,藉此为AS机理研究开辟新途经。

平滑肌内源性脂质合成异常是动脉粥样硬化早期首要病理环节。KLF4是我们前期研究中新近发现的一种调节血管脂代谢的关键因子。肌源性假说认为平滑肌通过清道夫受体摄入ox-LDL，脂质摄取与外流之间平衡被破坏导致泡沫细胞形成。如果平滑肌内源性脂质合成增加，则可引起内皮细胞增殖和血管新生，导致斑块破裂。然而，内源性脂质合成途径在肌源性泡沫细胞形成中的始动作用机理尚不明确。本项目旨在探讨锌指KLF4对平滑肌内源性脂质合成的影响及其机制。我们应用小鼠模型及原代平滑肌细胞，深入研究KLF4对平滑肌内源性脂质合成的分子病理生理学机制。结果表明，平滑肌KLF4水平升高可调控大量参与脂合成的分子亚群（如FAS、ACC、GPAT、SREBPs以及insig-1）。在insig-1的promoter区存在多个KLF4结合位点，KLF4可显著上调insig-1，下调FAS、ACC、GPAT表达水平。ChIP assay表明KLF4可结合于insig-1启动子区-2167/-2161bp。转染siRNA-insig-1，KLF4抑制脂质合成基因的作用减弱。Oil red O染色检测到KLF4可抑制平滑肌内源性脂质合成增加。平滑肌转染insig-1 siRNA，KLF4抑制平滑肌内源性脂质异常增加的作用减弱。在体实验中将ApoE-/-小鼠高脂喂养，尾静脉注射AdKLF4，横向切片染色观察了KLF4与粥样硬化损害的相关性。免疫组化染色检测了insigs、SREBPs在KLF4处理组和对照组中病变局部的表达差异，在KLF4高表达的主动脉弓大弯侧，insig-1有较高表达，SREBP-1表达很低,而在KLF4低表达的主动脉弓小弯侧，insig-1表达不明显，而SREBP-1水平很高，表明KLF4调节内源性脂合成的新功能与insigs-SREBPs分子直接相关。以上问题的阐明将在肌源性泡沫细胞形成的始动环节上发现KLF4抑制脂质合成的重要地位，为阐明KLF4与治疗动脉粥样硬化的关系提供实验证据。

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