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

通过使用在人免疫缺陷病毒转录因子达特中发现的由11个氨基酸组成的细胞穿透肽，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。使用细菌表达系统将待引入细胞的货物蛋白制备为与细胞穿透肽融合的重组蛋白。我已经开发了表达载体。利用这种新的蛋白转导技术，我对血管生物学做出了以下贡献。(1)I首次发现肌球蛋白磷酸酶MYPT 1调节亚基的N-末端区域在调节完整动脉的血管张力中起生理作用。(2)By通过以时间特异性的方式引入小G蛋白RhoA和Rac 1的抑制蛋白，我首次发现Rho蛋白的活性在G1晚期pH是必需的。 ...更多信息 使血管内皮细胞的细胞周期从G1期进入S期。(3)I发现用RhoA抑制剂蛋白处理动脉组织24小时以依赖于内皮NO产生的方式减弱动脉收缩性。然而，Rac 1抑制剂蛋白没有这种作用。因此，这一发现第一次提出了直接的证据表明，RhoA在原位内皮细胞中NO的产生中起着生理作用。(4)I发现用Rac 1抑制剂蛋白处理培养的平滑肌细胞24小时降低了凝血酶受体PAR 1的细胞表面表达。然而，RhoA抑制剂蛋白没有这种作用。因此，这一观察结果表明，Rac 1在决定血管平滑肌细胞中PAR 1的表达中起着关键作用。（5）雌激素可抑制TNF-α诱导的血管内皮细胞凋亡。然而，Akt的显性负突变体的引入抑制了雌激素的抗凋亡作用。因此，这一发现提供了第一个直接证据，Akt在血管内皮细胞中雌激素诱导的抗凋亡信号转导中起关键作用。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