SIGNAL TRANSDUCTION PATHWAYS INVOLVED IN VASCULAR SMOOTH MUSCLE CELL MIGRATION

血管的机械损伤导致血管平滑肌细胞（VSMCs）相对缓慢地聚集在管腔界面形成新内膜(N)，这一过程模拟了移植动脉粥样硬化和血管再狭窄的实验模型。血管平滑肌细胞通常位于内侧(M)细胞层。我们之前已经证明，钙/钙调素依赖性蛋白激酶II （CamKII）的激活是调节pdgf引导的血管平滑肌细胞（VSMC）向外迁移的关键细胞内信号事件。CamKII整合整合素（如alphav/beta3）、整合相关蛋白（如IAP）和细胞外基质分子（如血栓反应蛋白（TSP））的信号，协调趋化剂诱导的细胞向新生内膜的运动。最近，我们研究了细胞凋亡在新生内膜形成中的作用。正常血管不表达被称为ARC的肌肉特异性细胞凋亡抑制因子。然而，血管损伤后，ARC选择性地在新生内膜中积累。与分离的M-VSMCs相比，从损伤部位分离的N-VSMCs对凋亡的抵抗力增强。在m - vsmc中，强迫表达与N-VMSCs相当水平的ARC，而不是其card缺陷突变体，增加了它们对细胞凋亡的抵抗力。因此，响应血管损伤的ARC上调可能赋予N-VSMCs对凋亡的不同抗性，从而促进新内膜细胞的积累。识别。因此，针对TSP、IAP或alphav/beta3的抗体都能阻断VSMC的迁移和CamKII的激活。强制表达组成型激活的CamKII恢复向抗体处理的细胞以及从IAP-/-小鼠分离的细胞的迁移。在其他细胞类型中，我们已经表明，从β 3整合素复合物到其他整合素的外向信号传导（整合素串扰）也由CamKII及其对肌球蛋白轻链激酶（MLCK）的影响介导，后者在非肌肉细胞中促进应激纤维的形成。在这些细胞类型以及迁移的VSMCs中，MLCK的药理学抑制剂模拟CamKII激活的作用，促进迁移，抑制应激纤维的形成，同时促进皮质肌动蛋白沉积。CamKII对MLCK活性的抑制作用可能是直接通过酶的磷酸化或间接通过中间分子的磷酸化来实现的。一个可能的中介是TIAM，一种rac-GEF，最近被证明是CamKII磷酸化的底物。CamKII磷酸化TIAM导致rac活性增加，皮质肌动蛋白形成，并通过rac相关激酶PAK抑制MCLK。我们已经证明TIAM的显性阴性突变体抑制pdgf导向的迁移，这种抑制作用可以被rac的组成型激活突变体覆盖，但CamKII却不能。这些结果表明，rac的调控是CamKII调控VSMC迁移和细胞内不同整合素间串扰的关键下游效应因子。

SUMMARY OF WORK Mechanical injury to blood vessels results in the relatively slow accumulation of vascular smothh muscle cells (VSMCs), which normally reside in the medial (M) cell layer, at the luminal interface to form a neointima (N), a process that mimics and is an experimental model for transplant atherosclerosis and vessel restenosis. We have previously shown that activation of calcium/calmodulin-dependent protein kinase II (CamKII) is a key intracellular signaling event regulating PDGF-directed vascular smooth muscle cell (VSMC) migration out of the media. CamKII integrates signals from integrins, such as alphav/beta3, integrated associated proteins (e.g., IAP),and extracellular matirx molecules, such as thrombospondin (TSP)to coordinate chemoattractant-induced cell movement into the neointima. More recently, we have investigated the role of apoptosis in neointima formation. Normal blood vessels do not express the muscle-specific repressor of apoptosis, known as ARC. However, ARC selectively accumulates in the developing neointima after vessel injury. N-VSMCs isolated from the site of injury show increased resistance to apoptosis compared to isolated M-VSMCs. Forced expression of ARC, but not its CARD-defective mutant, in M-VSMCs to levels comparable to that in N-VMSCs increases their resistance to apoptosis. Upregulation of ARC in respond to vessel injury, therefore, may confer on N-VSMCs a differntial resistant to apoptosis that contributes to neointimal cell accumulation. recognition. Accordingly, antibodies to TSP, IAP, or alphav/beta3 all block VSMC migration as well as CamKII activation. Forced expression of constitutively activated CamKII restores migration to antibody-treated cells as well as to cells isolated from IAP-/- mice. In other cell types, we have shown that outside-in signaling from the beta3 integrin complex to other integrins (integrin crosstalk) is also mediated by CamKII and its effects on myosin light chain kinase (MLCK), which in nonmuscle cells promotes stress fiber formation. In these cell types as well as in migrating VSMCs, pharmacological inhibitors of MLCK mimic the effect of CamKII activation, promote migration, and inhibit stress fiber formation while promoting cortical actin deposition. The inhibitory effect of CamKII on MLCK activity may be direct via phosphorylation of the enzyme or indirect through phosphorylation of intermediary molecules. One possible intermediary is TIAM, a rac-GEF that has recently been shown to be a substrate for CamKII phosphorylation. Phosphorylation of TIAM by CamKII results in increased rac activity, cortical actin formation, and MCLK inhibition through the rac-associated kinase, PAK. We have shown that a dominant negative mutant of TIAM inhibits PDGF-directed migration and this inhibition can be overriden by constitutuvely activated mutants of rac, but not of CamKII. These results demonstrate that the regulation of rac is a critical downstream effector of CamKII in the regulation of VSMC migration and the crosstalk between different integrins in the cell.