Rapid flow-induced activation of Gαq/11 is independent of Piezo1 activation.

Endothelial cell (EC) mechanochemical transduction is the process by which mechanical stimuli are sensed by ECs and transduced into biochemical signals and ultimately into physiological responses. Identifying the mechanosensor/mechanochemical transducer(s) and describing the mechanism(s) by which they receive and transmit the signals has remained a central focus within the field. The heterotrimeric G protein, Gαq/11, is proposed to be part of a macromolecular complex together with PECAM-1 at EC junctions and may constitute the mechanochemical transducer as it is rapidly activated within seconds of flow onset. The mechanically activated cation channel Piezo1 has recently been implicated due to its involvement in mediating early responses, such as calcium and ATP release. Here, we investigate the role of Piezo1 in rapid shear stress-induced Gαq/11 activation. We show that flow-induced dissociation of Gαq/11 from PECAM-1 in ECs at 15 s is abrogated by BIM-46187, a selective inhibitor of Gαq/11 activation, suggesting that Gαq/11 activation is required for PECAM-1/Gαq/11 dissociation. Although siRNA knockdown of Piezo1 caused a dramatic decrease in PECAM-1/Gαq/11 association in the basal condition, it had no effect on flow-induced dissociation. Interestingly, siRNA knockdown of Piezo1 caused a marked decrease in PECAM-1 expression. Additionally, selective blockade of Piezo1 with ion channel inhibitors had no effect on flow-induced PECAM-1/Gαq/11 dissociations. Lastly, flow onset caused increased association of Gß1 with Piezo1 as well as with the p101 subunit of phosphoinositide 3-kinase, which were both blocked by the Gßγ inhibitor gallein. Together, our results indicate that flow-induced activation of Piezo1 is not upstream of G protein activation.

Yoda1-induced phosphorylation of Akt and ERK1/2 does not require Piezo1 activation.

Piezo1 is a mechanosensitive cation channel that is activated by shear stress in endothelial cells (ECs). It has been shown to mediate shear-induced EC responses, including increased calcium influx, and vascular functions, such as vascular tone and blood pressure. Yoda1, a selective Piezo1 activator, has been shown to mimic shear-induced responses in ECs. Since shear-induced calcium influx causes Akt and ERK1/2 activation in ECs, we examined the effects of Yoda1 and the role of Piezo1 on their activation. Here, we show that Yoda1 robustly activates Akt and ERK1/2 in ECs. Additionally, the Piezo1 antagonists, gadolinium and ruthenium red, but not GsMTx4, effectively blocks Yoda1-induced Akt activation. Our results suggest that Yoda1-induced Akt and ERK1/2 activation is not dependent on Piezo1.

Shear stress induces Gαq/11 activation independently of G protein-coupled receptor activation in endothelial cells.

Mechanochemical signal transduction occurs when mechanical forces, such as fluid shear stress, are converted into biochemical responses within the cell. The molecular mechanisms by which endothelial cells (ECs) sense/transduce shear stress into biological signals, including the nature of the mechanosensor, are still unclear. G proteins and G protein-coupled receptors (GPCRs) have been postulated independently to mediate mechanotransduction. In this study, we used in situ proximity ligation assay (PLA) to investigate the role of a specific GPCR/Gαq/11 pair in EC shear stress-induced mechanotransduction. We demonstrated that sphingosine 1-phosphate (S1P) stimulation causes a rapid dissociation at 0.5 min of Gαq/11 from its receptor S1P3, followed by an increased association within 2 min of GPCR kinase-2 (GRK2) and ß-arrestin-1/2 with S1P3 in human coronary artery ECs, which are consistent with GPCR/Gαq/11 activation and receptor desensitization/internalization. The G protein activator AlF4 resulted in increased dissociation of Gαq/11 from S1P3, but no increase in association between S1P3 and either GRK2 or ß-arrestin-1/2. The G protein inhibitor guanosine 5'-(ß-thio) diphosphate (GDP-ß-S) and the S1P3 antagonist VPC23019 both prevented S1P-induced activation. Shear stress also caused the rapid activation within 7 s of S1P3/Gαq/11 There were no increased associations between S1P3 and GRK2 or S1P3 and ß-arrestin-1/2 until 5 min. GDP-ß-S, but not VPC23019, prevented dissociation of Gαq/11 from S1P3 in response to shear stress. Shear stress did not induce rapid dephosphorylation of ß-arrestin-1 or rapid internalization of S1P3, indicating no GPCR activation. These findings suggest that Gαq/11 participates in the sensing/transducing of shear stress independently of GPCR activation in ECs.

Fluid shear stress induces upregulation of COX-2 and PGI2 release in endothelial cells via a pathway involving PECAM-1, PI3K, FAK, and p38.

Vascular endothelial cells play an important role in the regulation of vascular function in response to mechanical stimuli in both healthy and diseased states. Prostaglandin I2 (PGI2) is an important antiatherogenic prostanoid and vasodilator produced in endothelial cells through the action of the cyclooxygenase (COX) isoenzymes COX-1 and COX-2. However, the mechanisms involved in sustained, shear-induced production of COX-2 and PGI2 have not been elucidated but are determined in the present study. We used cultured endothelial cells exposed to steady fluid shear stress (FSS) of 10 dyn/cm2 for 5 h to examine shear stress-induced induction of COX-2/PGI2 Our results demonstrate the relationship between the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1) and the intracellular mechanoresponsive molecules phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and mitogen-activated protein kinase p38 in the FSS induction of COX-2 expression and PGI2 release. Knockdown of PECAM-1 (small interference RNA) expression inhibited FSS-induced activation of α5ß1-integrin, upregulation of COX-2, and release of PGI2 in both bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs). Furthermore, inhibition of the PI3K pathway (LY294002) substantially inhibited FSS activation of α5ß1-integrin, upregulation of COX-2 gene and protein expression, and release of PGI2 in BAECs. Inhibition of integrin-associated FAK (PF573228) and MAPK p38 (SB203580) also inhibited the shear-induced upregulation of COX-2. Finally, a PECAM-1-/- mouse model was characterized by reduced COX-2 immunostaining in the aorta and reduced plasma PGI2 levels compared with wild-type mice, as well as complete inhibition of acute flow-induced PGI2 release compared with wild-type animals.NEW & NOTEWORTHY In this study we determined the major mechanotransduction pathway by which blood flow-driven shear stress activates cyclooxygenase-2 (COX-2) and prostaglandin I2 (PGI2) release in endothelial cells. Our work has demonstrated for the first time that COX-2/PGI2 mechanotransduction is mediated by the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1).

Endomucin prevents leukocyte-endothelial cell adhesion and has a critical role under resting and inflammatory conditions.

Endomucin is a membrane-bound glycoprotein expressed luminally by endothelial cells that line postcapillary venules, a primary site of leukocyte recruitment during inflammation. Here we show that endomucin abrogation on quiescent endothelial cells enables neutrophils to adhere firmly, via LFA-1-mediated binding to ICAM-1 constitutively expressed by endothelial cells. Moreover, TNF-α stimulation downregulates cell surface expression of endomucin concurrent with increased expression of adhesion molecules. Adenovirus-mediated expression of endomucin under inflammatory conditions prevents neutrophil adhesion in vitro and reduces the infiltration of CD45(+) and NIMP-R14(+) cells in vivo. These results indicate that endomucin prevents leukocyte contact with adhesion molecules in non-inflamed tissues and that downregulation of endomucin is critical to facilitate adhesion of leukocytes into inflamed tissues.

Heparan sulfates mediate the interaction between platelet endothelial cell adhesion molecule-1 (PECAM-1) and the Gαq/11 subunits of heterotrimeric G proteins.

The endothelial cell-cell junction has emerged as a major cell signaling structure that responds to shear stress by eliciting the activation of signaling pathways. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and heterotrimeric G protein subunits Gαq and 11 (Gαq/11) are junctional proteins that have been independently proposed as mechanosensors. Our previous findings suggest that they form a mechanosensitive junctional complex that discriminates between different flow profiles. The nature of the PECAM-1·Gαq/11 interaction is still unclear although it is likely an indirect association. Here, we investigated the role of heparan sulfates (HS) in mediating this interaction and in regulating downstream signaling in response to flow. Co-immunoprecipitation studies show that PECAM-1·Gαq/11 binding is dramatically decreased by competitive inhibition with heparin, pharmacological inhibition with the HS antagonist surfen, and enzymatic removal of HS chains with heparinase III treatment as well as by site-directed mutagenesis of basic residues within the extracellular domain of PECAM-1. Using an in situ proximity ligation assay, we show that endogenous PECAM-1·Gαq/11 interactions in endothelial cells are disrupted by both competitive inhibition and HS degradation. Furthermore, we identified the heparan sulfate proteoglycan syndecan-1 in complexes with PECAM-1 that are rapidly decreased in response to flow. Finally, we demonstrate that flow-induced Akt activation is attenuated in endothelial cells in which PECAM-1 was knocked down and reconstituted with a binding mutant. Taken together, our results indicate that the PECAM-1·Gαq/11 mechanosensitive complex contains an endogenous heparan sulfate proteoglycan with HS chains that is critical for junctional complex assembly and regulating the flow response.

The role of shear-induced transforming growth factor-ß signaling in the endothelium.

OBJECTIVE: Vascular endothelial cells (ECs) are continuously exposed to blood flow that contributes to the maintenance of vessel structure and function; however, the effect of hemodynamic forces on transforming growth factor-ß (TGF-ß) signaling in the endothelium is poorly described. We examined the potential role of TGF-ß signaling in mediating the protective effects of shear stress on ECs. APPROACH AND RESULTS: Human umbilical vein ECs (HUVECs) exposed to shear stress were compared with cells grown under static conditions. Signaling through the TGF-ß receptor ALK5 was inhibited with SB525334. Cells were examined for morphological changes and harvested for analysis by real-time polymerase chain reaction, Western blot analysis, apoptosis, proliferation, and immunocytochemistry. Shear stress resulted in ALK5-dependent alignment of HUVECs as well as attenuation of apoptosis and proliferation compared with static controls. Shear stress led to an ALK5-dependent increase in TGF-ß3 and Krüppel-like factor 2, phosphorylation of endothelial NO synthase, and NO release. Addition of the NO donor S-nitroso-N-acetylpenicillamine rescued the cells from apoptosis attributable to ALK5 inhibition under shear stress. Knockdown of TGF-ß3, but not TGF-ß1, disrupted the HUVEC monolayer and prevented the induction of Krüppel-like factor 2 by shear. CONCLUSIONS: Shear stress of HUVECs induces TGF-ß3 signaling and subsequent activation of Krüppel-like factor 2 and NO, and represents a novel role for TGF-ß3 in the maintenance of HUVEC homeostasis in a hemodynamic environment.

Early VEGFR2 activation in response to flow is VEGF-dependent and mediated by MMP activity.

Although several potential mechanosensors/mechanotransducers have been proposed, the precise mechanisms by which ECs sense and respond to mechanical forces and translate them into biochemical signals remains unclear. Here, we report that two major ligand-dependent tyrosine autophosphorylation sites of VEGFR2, Y1175 and Y1214, are rapidly activated by shear stress in human coronary artery endothelial cells (HCAECs). Neutralizing antibody against VEGFR2 not only abrogates flow-induced phosphorylation of these tyrosine residues, but also has a marked inhibitory effect on downstream eNOS activation. In situ proximity ligation assay revealed that VEGF and VEGFR2 are closely associated in HCAECs, and more importantly, this association is increased with flow. Finally, we show that flow-induced VEGFR2 activation is attenuated in the presence of the broad spectrum matrix metalloproteinase (MMP) inhibitor, GM6001. Taken together, our results suggest that a ligand-dependent mechanism involving the activity of MMPs plays a key role in the early, shear stress-induced activation of VEGFR2.

Role of shear-stress-induced VEGF expression in endothelial cell survival.

Vascular endothelial growth factor (VEGF) plays a crucial role in developmental and pathological angiogenesis. Expression of VEGF in quiescent adult tissue suggests a potential role in the maintenance of mature blood vessels. We demonstrate, using a Vegf-lacZ reporter mouse model, that VEGF is expressed by arterial but not by venous or capillary endothelial cells (ECs) in vivo. Using an in vitro model, we show that arterial shear stress of human umbilical vein ECs (HUVECs) decreases apoptosis and increases VEGF expression, which is mediated by the induction of Krüppel-like factor 2 (KLF2). Additionally, shear stress stimulates the expression of VEGF receptor 2 (VEGFR2) and is associated with its activation. Knockdown of VEGF in shear stressed HUVECs blocks the protective effect of shear stress, resulting in EC apoptosis equivalent to that in control ECs cultured under static conditions. Similarly, treatment of ECs subjected to arterial shear stress with the VEGF receptor tyrosine kinase inhibitor SU1498, or VEGFR2 neutralizing antiserum, led to increased apoptosis, demonstrating that the mechanoprotection from increased shear is mediated by VEGFR2. Taken together, these studies suggest that arterial flow induces VEGF-VEGFR2 autocrine-juxtacrine signaling, which is a previously unidentified mechanism for vascular EC survival in adult arterial blood vessels.

Arterial versus venous endothelial cells.

Vascular endothelial cells (ECs) form the inner lining of all blood vessels from the largest artery and veins, viz., the aorta and venae cavae, respectively, to the capillaries that connect the arterial and venous systems. Because these two major conducting systems of the cardiovasculature differ functionally, it is not surprising that the physical makeup of arteries and veins, including the ECs that line their lumina, are also distinct. Although few would argue that the local environment contributes to the differences between arteries and veins, recent evidence has shown that the specification of arterial and venous identity is largely genetically determined.

Regulation of NF-kappaB-dependent gene expression by the POU domain transcription factor Oct-1.

Maintenance of the cells of the vessel wall in a quiescent state is an important aspect of normal vascular physiology. Transcriptional repressors are widely believed to regulate this process, yet the exact factors involved and the mechanism of repression are not known. Here, we report that the POU domain transcription factor Oct-1 represses the expression of E-selectin and vascular cell adhesion molecule (VCAM-1), two cytokine-inducible, NF-kappaB-dependent endothelial-leukocyte adhesion molecules that participate in the leukocyte recruitment phase of the inflammatory response. Co-transfection and microinjection studies demonstrate that Oct-1 blocks tumor necrosis factor alpha-stimulated E-selectin and VCAM-1 expression. Gene expression arrays indicate that control of tumor necrosis factor alpha-induced, NF-kappaB-dependent gene expression by Oct-1 is promoter-specific. A DNA-binding mutant of Oct-1 represses NF-kappaB-dependent reporter gene expression. Biochemically, Oct-1 interacts with p65, suggesting that Oct-1 is involved in the regulation of NF-kappaB transactivation function. NF-kappaB-dependent gene expression is more pronounced in Oct-1-deficient than in wild-type murine embryonic fibroblasts, and reintroduction of human Oct-1 abolishes these differences. Finally, the cytokine interleukin-6 induces Oct-1 gene expression, providing a biologically relevant means by which NF-kappaB-dependent gene expression can be selectively reverted by Oct-1 to quiescent levels.