Background: Dickkopf-related protein 3 (DKK3) is a secreted protein that is involved in the regulation of cardiac remodeling and vascular smooth muscle cell differentiation, but little is known about its role in atherosclerosis.

Methods: We tested the hypothesis that DKK3 is atheroprotective using both epidemiological and experimental approaches. Blood DKK3 levels were measured in the Bruneck Study in 2000 (n=684) and then in 2005 (n=574). DKK3-deficient mice were crossed with apolipoprotein E^-/- mice to evaluate atherosclerosis development and vessel injury-induced neointimal formation. Endothelial cell migration and the underlying mechanisms were studied using in vitro cell culture models.

Results: In the prospective population-based Bruneck Study, the level of plasma DKK3 was inversely related to carotid artery intima-media thickness and 5-year progression of carotid atherosclerosis independently from standard risk factors for atherosclerosis. Experimentally, we analyzed the area of atherosclerotic lesions, femoral artery injury-induced reendothelialization, and neointima formation in both DKK3^-/-/apolipoprotein E^-/- and DKK3^+/+/apolipoprotein E^-/- mice. It was demonstrated that DKK3 deficiency accelerated atherosclerosis and delayed reendothelialization with consequently exacerbated neointima formation. To explore the underlying mechanisms, we performed transwell and scratch migration assays using cultured human endothelial cells, which exhibited a significant induction in cell migration in response to DKK3 stimulation. This DKK3-induced migration activated ROR2 and DVL1, activated Rac1 GTPases, and upregulated JNK and c-jun phosphorylation in endothelial cells. Knockdown of the ROR2 receptor using specific siRNA or transfection of a dominant-negative form of Rac1 in endothelial cells markedly inhibited cell migration and downstream JNK and c-jun phosphorylation.

Conclusions: This study provides the evidence for a role of DKK3 in the protection against atherosclerosis involving endothelial migration and repair, with great therapeutic potential implications against atherosclerosis.

背景： Dickkopf 相关蛋白 3 (DKK3) 是一种分泌蛋白，参与调节心脏重塑和血管平滑肌细胞分化，但对其在动脉粥样硬化中的作用知之甚少。

方法：我们使用流行病学和实验方法检验了 DKK3 具有动脉粥样硬化保护作用的假设。2000 年（n=684）和 2005 年（n=574）在 Bruneck 研究中测量了血液 DKK3 水平。DKK3缺陷小鼠与载脂蛋白E ^-/-小鼠杂交以评估动脉粥样硬化发展和血管损伤诱导的新内膜形成。使用体外细胞培养模型研究内皮细胞迁移和潜在机制。

结果：在基于人群的前瞻性 Bruneck 研究中，血浆 DKK3 水平与颈动脉内膜中层厚度和颈动脉粥样硬化 5 年进展呈负相关，独立于动脉粥样硬化的标准危险因素。在实验上，我们分析了DKK3 ^-/- /apolipoprotein E ^-/-和DKK3 ^+/+ /apolipoprotein E ^-/-的动脉粥样硬化病变面积、股动脉损伤诱导的再内皮化和新内膜形成老鼠。已证明 DKK3 缺乏会加速动脉粥样硬化并延迟再内皮化，从而加剧新内膜的形成。为了探索潜在的机制，我们使用培养的人内皮细胞进行了 transwell 和划痕迁移测定，这些细胞在响应 DKK3 刺激时表现出显着的细胞迁移诱导。这种 DKK3 诱导的迁移激活了 ROR2 和 DVL1，激活了 Rac1 GTPases，并上调了内皮细胞中的 JNK 和 c-jun 磷酸化。使用特异性 siRNA 敲低 ROR2 受体或转染内皮细胞中显性失活形式的 Rac1 可显着抑制细胞迁移和下游 JNK 和 c-jun 磷酸化。

结论：本研究为 DKK3 在预防涉及内皮迁移和修复的动脉粥样硬化中的作用提供了证据，对动脉粥样硬化具有巨大的治疗潜力。