Abstract
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. 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. 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. 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.
Highlights
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
We found that the plasma level of dickkopf-related protein 3 (DKK3), a member of the dickkopf family, is negatively correlated with atherosclerosis in human subjects
We demonstrated that DKK3 promotes reendothelialization in murine models of atherosclerosis and wire-induced femoral artery injury, revealing its atheroprotective role
Summary
An expanded Methods is available in the online-only Data Supplement. Study PopulationPopulation recruitment was performed as part of the prospective community-based Bruneck Study.[28,29] The survey area was located in the north of Italy (Bolzano Province). An expanded Methods is available in the online-only Data Supplement. Population recruitment was performed as part of the prospective community-based Bruneck Study.[28,29] The survey area was located in the north of Italy (Bolzano Province). Special features of the study design and protocol have been described previously in detail[28,29,30] and are provided in the online-only Data Supplement. The current study focused on the evaluation in 2000 (n=684) and follow-up between 2000 and 2005. The appropriate ethics committees approved the study protocol, and all study subjects gave their written informed consent before entering the study
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