High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells

Laminar shear stress produced by high fluid flow across endothelial cells causes the cells to produce vasodilatory nitric oxide (NO) and to elongate from polygonal to ellipsoid such that their long axes become parallel with direction of the flow (Nerem et al. 1981, Dewey et al. 1981, reviewed in Tamargo et al. 2023). Nitric oxide produced by endothelial cells modulates soluble guanylyl cyclase and cGMP-dependent kinase in surrounding smooth muscle cells to cause vasodilation (reviewed in Feletou et al. 2008, 2012). By optimizing blood flow without inflammation, the response to laminar shear stress is atheroprotective.<br>Laminar shear stress on endothelial cells is detected by the glycocalyx, caveolae, cilia, the mechanosensitive ion channel PIEZO1 located on the apex of the cell, and the PECAM1:CDH5:KDR (PECAM1:VE-cadherin:VEGFR2) complex located on the lateral surfaces between adjacent cells (reviewed in Tanaka et al. 2021). The active molecular components, mechanisms of activation, and downstream events related to the glycocalyx, caveolae, and cilia are incompletely characterized so the annotation here focuses more on PIEZO1 and the PECAM1:CDH5:KDR complex.<br>The force of the flow on the membrane of the endothelial cell activates the mechanosensitive ion channel PIEZO1 and, indirectly, the ion channel TRPV4 to transport cations, notably calcium, from the extracellular region to the cytosol (reviewed in Li et al. 2014, Ranade et al. 2014, Fang et al. 2021, Xiao et al. 2023). Cytosolic calcium activates the protease complex Calpain2 to cleave the cytoskeletal proteins TALIN1 and VINCULIN, resulting in changes to the cytoskeleton that alter the shape of the endothelial cell (Miyazaki et al. 2007).<br>Flow-sensitive potassium channels (which may include Kir2.1 and TREK1) and chloride channels (which may include LRRC8A) are also observed to open, however their mechanisms of activation and downstream events are incompletely characterized (reviewed in Tanaka et al. 2021).<br>Cytosolic calcium activates Pannexin channels to release ATP (Wang et al. 2016), which binds the P2RY2 (P2Y2) receptor on the cell surface in an autocrine and paracrine manner and thereby activates Galpha(q/11)-PI3K-AKT1 signaling. Both signaling by P2RY2 and signaling by a mechanosensitive complex containing PECAM1 and KDR (VEGFR2) (inferred from mouse homologs in Tzima et al. 2005) produce phosphatidylinositol 3,4,5-trisphosphate (PIP3), which binds AKT1 and enhances the phosphorylation of AKT1 on serine-475 by the mTORC2 complex.<br>Through a PI3K-independent mechanism, P2RY2 signaling and cytosolic calcium activate the kinase PDPK1, which phosphorylates the kinase PKN2 (PRK2) (Jin et al. 2021). Phospho-PKN2 then phosphorylates AKT1 on threonine-308 (Jin et al. 2021). Phospho-T308,S475-AKT1 phosphorylates serine-1177 of NOS3 (eNOS) while phospho-PKN2 also phosphorylates serine-1179 of NOS3 (Jin et al. 2021), causing increased nitric oxide production (reviewed in Cabou and Martinez 2022).<br>Laminar shear stress increases secretion of Adrenomedullin (ADM), a vasodilator, by endothelial cells through an uncharacterized mechanism (Iring et al. 2019). ADM binds the AM1 receptor and signals through G-alpha(s), adenylate cyclase, and resultant cAMP to activate protein kinase A (PKA) to phosphorylate serine-633 of NOS3, further increasing nitric oxide production (Iring et al. 2019).<br>The sphingosine 1-phosphate receptor S1PR1, which couples to Galpha(i1) and Galpha(i3), contributes in a ligand-independent manner to activation of AKT and NOS3, however the intermediate steps are incompletely characterized (reviewed in Tanaka et al. 2021). Other GPCRs such as GPR68 also become activated, possibly through flow-induced deformation of the extracellular domain (reviewed in Tanaka et al. 2021).

层流切应力由高速流体流经内皮细胞产生，导致细胞分泌血管舒张因子一氧化氮（NO），并从多边形延长至椭球形，使细胞的长轴与流体流动方向平行（参见 Nerem 等，1981 年，Dewey 等，1981 年，Tamargo 等，2023 年综述）。内皮细胞产生的一氧化氮调节周围平滑肌细胞中的可溶性鸟苷酸环化酶和cGMP依赖性激酶，以引起血管舒张（参见 Feletou 等，2008 年，2012 年综述）。通过优化血流而不引起炎症，层流切应力的反应具有抗动脉粥样硬化的作用。<br>内皮细胞上的层流切应力由糖萼、小窝、纤毛、位于细胞顶端的机械敏感性离子通道PIEZO1，以及位于相邻细胞之间的侧面上的PECAM1:CDH5:KDR（PECAM1:VE-钙粘蛋白:VEGFR2）复合物检测到（参见 Tanaka 等，2021 年综述）。关于糖萼、小窝和纤毛的活性分子成分、激活机制和下游事件的表征尚不完全，因此此处注释更侧重于PIEZO1和PECAM1:CDH5:KDR复合物。<br>流体对内皮细胞膜的压力激活了机械敏感性离子通道PIEZO1，间接地激活了离子通道TRPV4，以将阳离子，尤其是钙离子，从细胞外区域运输到细胞质中（参见 Li 等，2014 年，Ranade 等，2014 年，Fang 等，2021 年，Xiao 等，2023 年综述）。细胞质中的钙离子激活了蛋白酶复合物Calpain2，裂解细胞骨架蛋白TALIN1和VINCULIN，从而导致细胞骨架的变化，改变内皮细胞的形状（参见 Miyazaki 等，2007 年）。<br>观察到的流动敏感钾通道（可能包括Kir2.1和TREK1）和氯通道（可能包括LRRC8A）也被观察到开启，但其激活机制和下游事件表征尚不完全（参见 Tanaka 等，2021 年综述）。<br>细胞质中的钙离子激活Pannexin通道释放ATP（参见 Wang 等，2016 年），ATP通过自分泌和旁分泌的方式与细胞表面上的P2RY2（P2Y2）受体结合，从而激活Galpha(q/11)-PI3K-AKT1信号通路。P2RY2信号通路和含有PECAM1和KDR（VEGFR2）的机械敏感性复合物（从Tzima等，2005年的小鼠同源物推断）产生的磷脂酰肌醇-3,4,5-三磷酸（PIP3）与AKT1结合，并通过mTORC2复合物增强AKT1在丝氨酸-475位的磷酸化。<br>通过PI3K非依赖性机制，P2RY2信号通路和细胞质中的钙离子激活激酶PDPK1，PDPK1磷酸化激酶PKN2（PRK2）（参见 Jin 等，2021 年）。磷酸化的PKN2随后磷酸化AKT1的苏氨酸-308位（参见 Jin 等，2021 年）。磷酸化T308,S475-AKT1磷酸化NOS3（eNOS）的丝氨酸-1177位，而磷酸化的PKN2也磷酸化NOS3的丝氨酸-1179位（参见 Jin 等，2021 年），导致一氧化氮产生增加（参见 Cabou 和 Martinez，2022 年综述）。<br>层流切应力通过未知的机制增加内皮细胞分泌肾上腺髓质素（ADM），一种血管舒张剂（参见 Iring 等，2019 年）。ADM与AM1受体结合，通过G-alpha(s)、腺苷酸环化酶和生成的cAMP信号通路激活蛋白激酶A（PKA），PKA磷酸化NOS3的丝氨酸-633位，进一步增加一氧化氮的产生（参见 Iring 等，2019 年）。<br>与Galpha(i1)和Galpha(i3)偶联的鞘氨醇-1-磷酸受体S1PR1，以配体非依赖性方式参与AKT和NOS3的激活，然而中间步骤表征尚不完全（参见 Tanaka 等，2021 年综述）。其他GPCR，如GPR68，也可能被激活，可能是通过流体诱导的细胞外结构域变形（参见 Tanaka 等，2021 年综述）。