Mechanosensation is perhaps the last sensory modality not understood at the molecular level. Ion channels that sense mechanical force are postulated to play critical roles in a variety of biological processes including sensing touch/pain (somatosensation), sound (hearing), and shear stress (cardiovascular physiology); however, the identity of these ion channels has remained elusive. We previously identified Piezo1 and Piezo2 as mechanically activated cation channels that are expressed in many mechanosensitive cell types. Here, we show that Piezo1 is expressed in endothelial cells of developing blood vessels in mice. Piezo1-deficient embryos die at midgestation with defects in vascular remodeling, a process critically influenced by blood flow. We demonstrate that Piezo1 is activated by shear stress, the major type of mechanical force experienced by endothelial cells in response to blood flow. Furthermore, loss of Piezo1 in endothelial cells leads to deficits in stress fiber and cellular orientation in response to shear stress, linking Piezo1 mechanotransduction to regulation of cell morphology. These findings highlight an essential role of mammalian Piezo1 in vascular development during embryonic development.

The focal nature of atherosclerotic lesions suggests an important role of local hemodynamic environment. Recent studies have demonstrated significant roles of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in mediating mechanotransduction and vascular homeostasis. The objective of this study is to investigate the functional role of YAP/TAZ in the flow regulation of atheroprone endothelial phenotypes and the consequential development of atherosclerotic lesions. We found that exposure of cultured endothelial cells (ECs) to the atheroprone disturbed flow resulted in YAP/TAZ activation and translocation into EC nucleus to up-regulate the target genes, including cysteine-rich angiogenic inducer 61 (CYR61), connective tissue growth factor (CTGF), and ankyrin repeat domain 1 (ANKRD1). In contrast, the atheroprotective laminar flow suppressed YAP/TAZ activities. En face analysis of mouse arteries demonstrated an increased nuclear localization of YAP/TAZ and elevated levels of the target genes in the endothelium in atheroprone areas compared with athero-protective areas. YAP/TAZ knockdown significantly attenuated the disturbed flow induction of EC proliferative and proinflammatory phenotypes, whereas overexpression of constitutively active YAP was sufficient to promote EC proliferation and inflammation. In addition, treatment with statin, an antiatherosclerotic drug, inhibited YAP/TAZ activities to diminish the disturbed flow-induced proliferation and inflammation. In vivo blockade of YAP/TAZ translation by morpholino oligos significantly reduced endothelial inflammation and the size of atherosclerotic lesions. Our results demonstrate a critical role of the activation of YAP/TAZ by disturbed flow in promoting atheroprone phenotypes and atherosclerotic lesion development. Therefore, inhibition of YAP/TAZ activation is a promising athero-protective therapeutic strategy.atherogenesis | disturbed flow | endothelial cells | mechanotransduction

Endothelial cells (ECs) respond to changes in mechanical forces, leading to the modulation of signaling networks and cell function; an example is the inhibition of EC proliferation by steady laminar flow. MicroRNAs (miRs) are short noncoding 20-22 nucleotide RNAs that negatively regulate the expression of target genes at the posttranscriptional level. This study demonstrates that miRs are involved in the flow regulation of gene expression in ECs. With the use of microRNA chip array, we found that laminar shear stress (12 dyn/cm 2 , 12 h) regulated the EC expression of many miRs, including miR-19a. We further showed that stable transfection of miR-19a significantly decreased the expression of a reporter gene controlled by a conserved 3′-untranslated region of the cyclinD1 gene and also the protein level of cyclin D1, leading to an arrest of cell cycle at G1/S transition. Laminar flow suppressed cyclin D1 protein level, and this suppressive effect was diminished when the endogenous miR-19a was inhibited. In conclusion, we demonstrated that miR-19a plays an important role in the flow regulation of cyclin D1 expression. These results revealed a mechanism by which mechanical forces modulate endothelial gene expression.

Interstitial flow in and around bone tissue is oscillatory in nature and affects the mechanical microenvironment for bone cell growth and formation. We investigated the role of oscillatory shear stress (OSS) in modulating the proliferation of human osteoblast-like MG63 cells and its underlying mechanisms. Application of OSS (0.5 ؎ 4 dynes/cm 2 ) to MG63 cells induced sustained activation of phosphatidylinositol 3-kinase (PI3K)/ Akt/mTOR/p70S6K (p70S6 kinase) signaling cascades and hence cell proliferation, which was accompanied by increased expression of cyclins A and D1, cyclin-dependent protein kinases-2, -4, and -6, and bone formation-related genes (c-fos, Egr-1, and Cox-2) and decreased expression of p21 CIP1 and p27 KIP1. OSS-induced activation of PI3K/Akt/mTOR/p70S6K and cell proliferation were inhibited by specific antibodies or small interference RNAs of ␣ v ␤ 3 and ␤ 1 integrins and by dominant-negative mutants of Shc (Shc-SH2) and focal adhesion kinase (FAK) (FAK(F397Y)). Co-immunoprecipitation assay showed that OSS induces sustained increases in association of Shc and FAK with ␣ v ␤ 3 and ␤ 1 integrins and PI3K subunit p85, which were abolished by transfecting the cells with FAK(F397Y) or Shc-SH2. OSS also induced sustained activation of ERK, which was inhibited by the specific PI3K inhibitor LY294002 and was required for OSS-induced activation of mTOR/p70S6K and proliferation in MG63 cells. Our findings provide insights into the mechanisms by which OSS induces osteoblast-like cell proliferation through activation of ␣ v ␤ 3 and ␤ 1 integrins and synergistic interactions of FAK and Shc with PI3K, leading to the modulation of downstream ERK and Akt/ mTOR/p70S6K pathways.Mechanical loading is critical for the formation of new bone (1-3). During dynamic and periodic loading of intact bone, the reciprocating flow of interstitial fluid through the canaliculi generates oscillatory shear stress (OSS), 3 which is detected by osteocytes in the canaliculi and osteoblasts lining the endosteal and periosteal surfaces of bone (4, 5). Stimulation of osteocytes by fluid shear stress induces their release of osteoblastic factors, which are transferred via gap junctions of the osteocyte-interconnecting network to induce osteoblast recruitment and hence bone growth (4, 6). There is increasing evidence that fluid shear stress regulates signaling, gene expression, and differentiation in osteocytes and osteoblasts (4 -9). Recent studies using flow channels have demonstrated that application of steady fluid shear stress to osteoblasts induces cell proliferation (10, 11) and the expression of many genes, including c-fos (8, 12), Egr-1 (early growth response-1) (8, 13), and Cox-2 (cyclooxygenase-2) (8, 12), all of which have been shown to play a role in bone formation in vivo (14 -17).The signaling molecules that have been shown to regulate mechanically induced proliferation in osteoblasts include NO (10,[18][19][20], prostaglandin E 2 , prostacyclin (10, 18 -20), and ERK (10,11,18). Kapur et al. (11) demonstrated that ERK1...