Dedicator of Cytokinesis 2 in smooth muscle phenotype modulation

描述（由申请人提供）：本提案的总体目标是建立一种新的机制，通过该机制，细胞分裂2奉献体（DOCK2）调节血管平滑肌细胞（SMC）的表型调节。SMC从分化表型到去分化状态的转变伴随着新内膜形成/血管重塑，在动脉粥样硬化、血管成形术或搭桥术后再狭窄、糖尿病血管并发症、移植动脉病变、哮喘和癌症的发生中起着关键作用。然而，调节SMC表型调节和新内膜形成的机制和因素尚不清楚。在生理条件下，DOCK2仅在造血细胞中表达，通过激活Rac来调节肌动蛋白细胞骨架，从而控制淋巴细胞的迁移和活化。我们令人兴奋的初步数据表明，血小板衍生生长因子(PDGF)-BB，一种SMC表型调节剂，可诱导SMC中DOCK2的表达。敲低DOCK2阻断pdgf - bb诱导的SMC表型调节、增殖和迁移。体内动物研究表明，DOCK2在正常大鼠颈动脉的SMC中检测不到，但在球囊导管诱导的血管损伤后，DOCK2首先在中膜SMC中被诱导，随后在新内膜SMC中被诱导。重要的是，敲低DOCK2可显著抑制损伤诱导的新生内膜形成。DOCK2依赖性血管重构/新生内膜形成的最确凿证据是，敲除DOCK2 （DOCK2-/-）可显著阻断动脉结扎诱导的小鼠颈动脉新生内膜增生。有趣的是，DOCK2定位于SMC的细胞膜和细胞核中，这表明DOCK2除了在细胞骨架/细胞迁移中起作用外，还可能参与SMC基因的转录。事实上，DOCK2过表达阻断了SMC基因mRNA的表达和心肌素诱导的平滑肌-肌动蛋白启动子的激活。因此，中心假设是DOCK2通过抑制SMC基因转录和刺激SMC迁移/增殖来调节SMC表型调节，从而导致新内膜形成/血管重塑。采用SMC原代培养、大鼠球囊损伤和小鼠丝损伤模型，结合分子、细胞和组织学方法，研究DOCK2通过调控SMC基因转录调控SMC表型的分子机制；2)研究DOCK2是否通过激活Rac/RhoA/ Cdc42诱导SMC迁移；3)确定DOCK2在SMC表型调节和血管重塑中的重要作用。该项目的完成将揭示SMC表型调节的新机制，并为DOCK2是否是对抗常见疾病（包括糖尿病、再狭窄、动脉粥样硬化和癌症）相关血管损伤的潜在治疗靶点提供新的见解。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to establish a novel mechanism by which dedicator of cytokinesis 2 (DOCK2) regulates the phenotypic modulation of vascular smooth muscle cells (SMC). Transition of SMC from a differentiated phenotype to a dedifferentiated state accompanied by neointima formation/vascular remodeling plays a critical role in the development of atherosclerosis, restenosis after angioplasty or bypass, diabetic vascular complications, transplantation arteriopathy, asthma, and cancer. The mechanisms and factors that regulate SMC phenotypic modulation and neointima formation, however, are poorly understood. Under physiological conditions, DOCK2 is only expressed in hematopoietic cells and controls lymphocyte migration and activation by regulating actin cytoskeleton through Rac activation. Our exciting preliminary data demonstrate that platelet derived growth factor (PDGF)-BB, a SMC phenotype modulator, induced DOCK2 expression in SMC. Knockdown of DOCK2 blocked PDGF-BB-induced SMC phenotypic modulation, proliferation, and migration. In vivo animal studies showed that DOCK2 was undetectable in SMC of normal rat carotid arteries, but was induced in the media layer SMC initially and neointimal SMC subsequently following balloon catheter-induced vascular injury. Importantly, knockdown of DOCK2 dramatically inhibited the injury- induced neointima formation. The most conclusive evidence for DOCK2-dependent vascular remodeling/ neointima formation was that knockout of DOCK2 (DOCK2-/-) dramatically blocked artery ligation-induced neointima hyperplasia in mouse carotid artery. Interestingly, DOCK2 is localized in both cell membrane and nuclei of SMC, suggesting that in addition to its role in cytoskeleton/cell migration, DOCK2 may be involved in SMC gene transcription. Indeed, DOCK2 overexpression blocked both SMC gene mRNA expression and myocardin-induced activation of smooth muscle ¿-actin promoter. Thus, the central hypothesis is that DOCK2 regulates SMC phenotypic modulation by suppressing SMC gene transcription and stimulating SMC migration/ proliferation, leading to neointima formation/vascular remodeling. Using primary culture of SMC, in vivo rat balloon injury and mouse wire injury models combining with molecular, cellular and histological approaches, we will 1) study the molecular mechanisms by which DOCK2 modulates SMC phenotype through regulating SMC gene transcription; 2) investigate if DOCK2 induces SMC migration through activation of Rac/RhoA/ Cdc42; and 3) determine the essential role of DOCK2 in SMC phenotypic modulation and vascular remodeling in vivo. The completion of this project will unravel a novel mechanism regulating SMC phenotypic modulation and provide novel insights into whether DOCK2 is a potential therapeutic target for countering vascular damage associated with common diseases including diabetes, restenosis, atherosclerosis, and cancer.