Endothelial Cell Regulation of Smooth Muscle Phenotype

血管平滑肌细胞去分化为合成表型在广泛的血管疾病过程中具有重要意义。SMC表型调控是控制再狭窄、动脉粥样硬化、血管生成和侧支血管形成的基本过程。调控SMC表型的细胞内信号转导过程目前还不完全清楚。已有研究表明，内皮细胞可引导血管平滑肌细胞向更分化的表型分化。我们实验室的初步数据表明，这种情况发生的机制是通过PI3-激酶/AKT途径。利用共培养系统，内皮细胞控制的细胞内信号通路将被定义为调节平滑肌细胞表型的信号通路。Western blotts和实时定量聚合酶链式反应将被用来证明EC促进分化的平滑肌表型，这是由分化蛋白标记物的表达决定的。平滑肌细胞的分化也将通过细胞形态和细胞大小来评估。为了确定这是否通过IRS-1/PI3-Kinase/AKT途径介导，我们将在单独培养和与内皮细胞共同培养的平滑肌细胞中对这些蛋白及其下游靶点进行Western blotting。这些实验将使用PI3-激酶抑制剂Wortmannin、LY294002、显性阴性AKT腺病毒和肉豆蔻酰化的成分活性AKT腺病毒重复进行。AKT的下游靶点是GSK3B，它是GATA转录因子的已知调节因子。GATA-6已被证明能促进平滑肌细胞分化。为了确定内皮细胞是否通过AKT激活转录因子GATA-6来调节SMC的分化，将使用分化蛋白标志物平滑肌肌球蛋白重链和α-1整合素的GATA-6结合区产生的探针进行电迁移率漂移分析。启动子报告分析将使用来自相同分化蛋白标记的启动子区域进行。将对已知与GATA-6协同作用的转录因子和共激活剂进行进一步的研究。从这些研究中获得的数据将为控制平滑肌细胞表型的细胞内信号过程提供新的见解，从而可能加强对广泛血管疾病的治疗。

Dedifferentiation of vascular smooth muscle cells to the synthetic phenotype is of central importance in a broad range of vascular disease processes. SMC phenotype regulation is a basic process that controls the development of restenosis, atherosclerosis, angiogenesis, and collateral blood vessel formation. The intracellular signaling processes that regulate SMC phenotype are at present, incompletely understood. Endothelial cells have been shown to direct vascular smooth muscle cells toward a more differentiated phenotype. Preliminary data from our laboratory suggests that the mechanism by which this occurs is through the PI3-kinase / AKT pathway. Using a co-culture system endothelial cell controlled intracellular signaling pathways that regulate smooth muscle cell phenotype will be defined. Western blots and real time PCR will be used to demonstrate that EC promote a differentiated smooth muscle phenotype as determined by differentiation protein marker expression. Smooth muscle cell differentiation will also be evaluated by cell morphology and cell size. To determine if this is mediated through the IRS-1/PI3-kinase/AKT pathway western blots will be performed for these proteins as well as their down stream targets in smooth muscle cells cultured alone and co-cultured with endothelial cells. These experiments will be repeated using the PI3-kinase inhibitors wortmannin, LY294002, a dominant negative AKT adenovirus, and a myristoylated constitutively active AKT adenovirus. A downstream target of AKT is GSK3B, a known regulator of the GATA transcription factors. GATA-6 has been shown to promote smooth muscle cell differentiation. To determine if endothelial cells regulate SMC differentiation through AKT activation of the transcription factor GATA-6, electromobility shift assays will be performed using probes generated from the GATA-6 binding region of the differentiation protein markers smooth muscle myosin heavy chain and alpha-1 integrin. Promoter reporter assays will be performed using promoter regions from the same differentiation protein markers. Additional studies will be performed for transcription factors and co-activators known to act synergistically with GATA-6. Data obtained from these studies will provide new insight into the intracellular signaling processes that control smooth muscle cell phenotype, allowing for potential enhanced treatment of a wide range of vascular diseases.