Regulation of Signaling Molecules in Endothelial Cells by Steady Flow and Pulsatile Flow

血液流動所產生的剪力可以調節血管內皮細胞的結構與功能，因而在血管生理及動脈粥狀硬化過程中扮演十分重要的角色。血液在血管內以脈動方式流動，其作用在血管內皮細胞的剪力大小會隨著時間做週期性改變。本研究分別以蠕動幫浦和往復式針筒幫浦產生兩種不同型態的流動：亦即穩定流 (steaty flow) 和脈動流 (pulsatile flow)，以這兩種流動方式刺激牛主動脈內皮細胞 (BAECs)，探討並比較其對於細胞內訊息傳導分子如：PKCα、PKCε、ERK1/2、β-catenin、Akt、eNOS 以及活性氧族群 (ROS) 的影響。 蠕動幫浦和往復式針筒幫浦所建構的流動實驗裝置，經過測量可產生剪力大小12 dyn/cm2 的穩定流和12±4 dyn/cm2 的脈動流，並選定此接近正常生理範圍的流動進行實驗。兩種流動型態對於 PKCα、PKCε 之磷酸化並無顯著影響；PKC 的下游 ERK1/2 之磷酸化程度在受到剪力刺激後會大幅增加，造成快速而短暫的活化，兩種流動型態也都會誘導 Akt 之磷酸化在10分鐘達到最高值，並持續達30分鐘，2小時後完全回到基礎狀態。至於 β-catenin 的穩定性則受 Axin complex、PKCα 對 β-catenin 的 serine/threonine 殘基之磷酸化影響，以兩種流動型態刺激細胞後，並未發現 β-catenin 磷酸化之改變，而使用共軛焦顯微鏡觀察 β-catenin 的位置則發現它持續分佈在 adherens junctions，分布情形未有明顯變化。已知 eNOS 的活性會受 Akt 調控，而在兩種流動型態的剪力刺激下皆可活化 Akt，一如預期也均可觀察到 eNOS 分子上 Ser1179 位置磷酸化程度約增加為靜態基礎值的4倍，而 Ser635 位置磷酸化程度約增加為靜態基礎值的3至5倍，且此位置之磷酸化較持久；另外，關於細胞內 ROS 的含量，在兩種流動型態的剪力刺激30分鐘後，胞內 ROS 量均增加為靜態下的2.5至3倍。 本研究中12 dyn/cm2 的穩定流和12±4 dyn/cm2 的脈動流造成的剪力刺激，對內皮細胞之生理功能均有類似之調控：兩種流動型態可能以磷酸化以外的方式來活化PKCα、PKCε，而活化 ERK1/2、Akt 有助於細胞存活、抗凋亡，兩者也都活化 eNOS 產生 NO，藉此可維持細胞內 ROS 含量不至於過高，並能達到保護心血管的目的，而少量的 ROS 含量增加具有細胞內訊息傳導的作用。穩定流和脈動流均被視為對內皮細胞具有保護效果的流動型態，而本實驗也在正常生理範圍的流動下進行，因而均一致得觀察到對細胞無害的現象。Shear stress generated by blood flow regulates vascular endothelial cell structure and function, thus playing a crucial role in the cardiovascular physiology and atherosclerosis process. Since the blood flow in blood vessels is pulsatile, the magnitude of shear stress applied on the vessel wall alters periodically. We used the roller pump and the syringe pump to generate steady flow and pulsatile flow, respectively. We investigated and compared the influence of the two types of flow on the signaling molecules, including protein kinase Cα (PKCα), PKCε, ERK1/2, β-catenin, Akt, eNOS and reactive oxygen species (ROS) in bovine arotic endothelial cells (BAECs). The flow chamber system, which consisted of roller pump and syringe pump, was able to produce steady flow and pulsatile flow with shear stress magnitude of 12 dyn/cm2 and 12±4 dyn/cm2, respectively. The two types of flow did not have significant effects upon the phosphorylation of PKCα and PKCε. The phosphorylation of ERK1/2, as the downstream of PKC, was transiently induced by the stimulation of the flow. Both steady flow and pulsatile flow activated Akt with the maximum activation at 10 minutes and maintained for 30 minutes after BAECs subjected to the flow. The stability of β-catenin was regulated by its phosphorylation at serine/threonine residues by Axin complex and PKCα. After BAECs were exposed to the two types of flow, there were no significant changes in the phosphorylation of β-catenin. We also used the confocal microscopy to identify the location of β-catenin in BAECs and observed that β-catenin continuously appeared at adherens junctions. Known that eNOS activity was regulated by Akt, and Akt was activated under the both types of flow, the phosphorylation of eNOS at Ser1179 increased 4 times as compared with basal level, as expected. The phosphorylation of eNOS at Ser635 increased 3 to 5 times as compared with basal level, and the phosphorylation sustained longer. After BAECs were stimulated by the two types of flow for 30 minutes, the ROS levels in BAECs increased 2.5 to 3 times as compared with basal level. In summary, the steady flow and the pulsatile flow might activate PKCα and PKCε by the way other than phosphorylation. Both types of flow activated ERK1/2 and Akt, which promoted cell survival and prevented cell apoptosis. Both types of flow also activated eNOS to counteract the elevated ROS levels and providing protective effects for cardiovascular system. Slightly increased ROS levels might function as signaling molecules to mediate cellular signal transduction