病理剪切力介导的内皮细胞Dickkopf1的调控在内皮-平滑肌细胞间通讯中的作用及对动脉粥样硬化的影响

Pathological shear stress is one of the most important factors leading to atherosclerosis (AS) development, involving endothelial intracellular and intercellular signaling abnormalities. DKK1 is a secreted factor which plays important roles in many physiological and pathological processes in an autocrine and/or paracrine way. Studies have shown that DKK1 is closely related to atherosclerosis (AS), but the underlying mechanism is not clear. Our previous studies firstly confirmed that atheroprone oscillatory shear stress (OSS) upregulated endothelial DKK1 expression and in turn promoted AS; DKK1 mediated the dysfunction of endothelial cells and smooth muscle cells. However, the mechanisms of endothelial DKK1 upregulation by OSS and the roles of endothelial secreted DKK1 in smooth muscle cells need to be further explored. We propose the following hypothesis: OSS increases the expression and secretion of DKK1 in endothelial cells via activating the transcription factor c-Ets, secreted DKK1 are further recognized by surface receptors of smooth muscle cells and then play roles in the development of AS. Our study will construct endothelial DKK1 gene knockout mice model and take advantage of cellular biology and proteomics techniques to fully test our hypothesis both in vitro and in vivo and verify the mechanism of OSS-induced endothelial DKK1 expression and the role of DKK1-mediated endothelial cell-smooth muscle cell communication in the process of AS. This study will expand our information of the biological function of DKK1 and provide new insight into the potential therapeutic targets of AS. Besides, our study might establish theoretical basis for the understanding of cardiovascular response induced by DKK1 intervention in cancers.

病理剪切力是致动脉粥样硬化（AS）的重要原因，涉及内皮细胞及细胞间异常。DKK1是一种具有自分泌和旁分泌作用的糖蛋白，是重要的肿瘤治疗靶点，并与AS密切相关，但其机制尚未明了。我们首次证实致AS的震荡剪切力（OSS）上调内皮细胞DKK1表达，引起内皮细胞和平滑肌细胞功能异常，促进AS发生。但OSS上调内皮细胞DKK1表达机制及分泌的DKK1如何影响平滑肌细胞仍不明了。我们提出如下假说：OSS刺激内皮细胞经c-Ets上调DKK1，分泌后被平滑肌细胞表面受体识别，促进AS发生发展。本课题将通过构建内皮细胞DKK1基因条件性敲除小鼠，利用分子生物学、蛋白质组学等技术，在体内外研究验证该假说，明确OSS引起内皮细胞DKK1上调及其介导的内皮-平滑肌细胞间通讯致AS的作用与机制。本研究将拓展对 DKK1生物学功能的新认识，为AS的防治提供新的靶点；同时为明确肿瘤干预治疗潜在的心血管反应提供理论基础。

病理剪切力干预下，血管内皮细胞分泌的多种炎症因子及血管活性物质可通过旁分泌参与对平滑肌细胞功能的调节，从而导致血管重构。DKK1与肿瘤疾病密切相关，但在心血管疾病中的作用尚未明了。本课题研究：（1）体外实验发现病理剪切力促进内皮细胞DKK1的表达，c-Ets/CBP与c-jun共同介导HUVECs中病理性低剪切力对DKK1的上调；（2）病理性低剪切力干预下内皮细胞DKK1 通过旁分泌作用对共培养的平滑肌细胞的增殖具有促进作用；通过构建内皮条件性DKK1敲除的小鼠并构建颈动脉结扎模型，体内实验验证内皮细胞分泌的DKK1能够促进平滑肌细胞的增殖；（3）对照组及敲除组小鼠行血管组织全基因组测序分析(RNA-seq)，测序结果显示基因COX2及转录因子MYB的表达水平有明显差异，COX2是c-myb的靶基因，DKK1通过c-myb/COX2促进平滑肌细胞的增殖；体外实验证实DKK1主要通过与平滑肌细胞表面的CKAP4受体结合，通过PI3K/AKT通路促进平滑肌细胞的增殖；（4）内皮细胞分泌的DKK1，一方面通过上调平滑肌细胞脂质受体 SR-A1的表达促进胆固醇沉积，另一方面通过下调平滑肌细胞脂质输出分子ABCA1的表达抑制胆固醇流出，从而促进平滑肌细胞内胆固醇沉积进而形成泡沫化细胞；（5）通过CRISPR/Cas9技术构建Tek-Cre-ERT2/+/DKK1fl/flApopE-/-小鼠模型，进一步验证了DKK1对病理剪切力作用区域斑块形成的影响。本研究拓展了对 DKK1生物学功能的新认识，为AS的防治提供新的理论基础；DKK1作为肿瘤药物治疗靶点，本研究也为DKK1将来应用于肿瘤心血管疾病的治疗提供了理论基础。

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