Elucidation of the mechanisms regulating the vascular tone and proliferation : Development of a novel technique to introduce protein into the intact cells and its applications

通过使用细胞穿透肽，这是在人类免疫缺陷病毒转录因子Tat中发现的，由11个氨基酸组成，我开发了一种快速，可逆和定量地将蛋白质引入血管组织和细胞的新技术。利用细菌表达系统制备了与细胞穿透肽融合的重组蛋白。我已经开发了表达式向量。利用这种蛋白质转导的新技术，我对血管生物学做出了以下贡献。(1)首次发现肌球蛋白磷酸酶调节亚基MYPT1的n端区域在完整动脉中对血管张力的调节具有生理作用。(2)通过以时间特异性的方式引入小G蛋白RhoA和Rac1的抑制剂蛋白，我首次发现在血管内皮细胞中，Rho蛋白的活性需要在G1后期ph…更多的酶才能使细胞周期从G1期进入S期。(3)我发现，用RhoA抑制剂蛋白处理动脉组织24小时后，动脉收缩力以依赖于内皮NO生成的方式减弱。而Rac1抑制剂蛋白则无此作用。因此，这一发现首次提供了直接证据，表明RhoA在原位内皮细胞中NO的产生中起生理作用。(4)我发现用Rac1抑制剂蛋白处理培养的平滑肌细胞24 h后，凝血酶受体PAR1的细胞表面表达降低。而RhoA抑制剂蛋白则没有这种作用。这一观察结果表明，Rac1在血管平滑肌细胞中对PAR1的表达起关键作用。(5)雌激素抑制TNF-α-诱导的血管内皮细胞凋亡。然而，Akt显性负突变体的引入抑制了雌激素的抗凋亡作用。这一发现为Akt在血管内皮细胞雌激素诱导的抗凋亡信号传导中发挥关键作用提供了第一个直接证据。少

By using a cell-penetrating peptide, which is found in the human immunodeficiency viral transcription factor Tat and is composed of 11 amino acids, I have developed a novel technique to rapidly, reversibly and quantitatively introduce proteins into the vascular tissues and cells. The cargo protein to be introduced into the cells was prepared as a recombinant protein fused to the cell-penetrating peptide using a bacterial expression system. I have developed the expression vectors. Using this new technique of the protein transduction, I have made the following contribution to the vascular biology.(1)I have for the first time found that the N-terminal region of the regulatory subunit of myosin phosphatase, MYPT1, plays a physiological role in regulating the vascular tone in an intact artery.(2)By introducing the inhibitor proteins of the small G proteins RhoA and Rac1 in a time-specific manner, I have for the first time found that the activity of Rho proteins is required at the late G1 ph … More ase for the cell cycle to progress from the G1 phase to the S phase in the vascular endothelial cells.(3)I found that the 24 h treatment of the arterial tissue with the RhoA inhibitor protein attenuated the arterial contractility in a manner dependent on the endothelial NO production. However, Rac1 inhibitor protein had no such effect. This finding thus for the first time present direct evidence that RhoA plays a physiological role in the production of NO in in situ endothelial cells.(4)I found that the 24 h treatment of the cultured smooth muscle cells with the Rac1 inhibitor protein reduced the cell surface expression of the thrombin receptor PAR1. However, the RhoA inhibitor protein had no such effect. This observation thus suggests that Rac1 plays a critical role in determining the expression of PAR1 in the vascular smooth musclecells.(5)Estrogen inhibited the TNF-α-induced apoptosis in the vascular endothelial cells. However, the introduction of the dominant negative mutant of Akt inhibited the anti-apoptotic effect of estrogen. This finding thus provide the first direct evidence that Akt plays a critical role in the anti-apoptotic signaling elicited by estrogen in the vascular endothelial cells. Less