Acute mechanical stretch promotes eNOS activation in venous endothelial cells mainly via PKA and Akt pathways.

In the vasculature, physiological levels of nitric oxide (NO) protect against various stressors, including mechanical stretch. While endothelial NO production in response to various stimuli has been studied extensively, the precise mechanism underlying stretch-induced NO production in venous endothelial cells remains incompletely understood. Using a model of continuous cellular stretch, we found that stretch promoted phosphorylation of endothelial NO synthase (eNOS) at Ser¹¹77, Ser6³³ and Ser6¹5 and NO production in human umbilical vein endothelial cells. Although stretch activated the kinases AMPKα, PKA, Akt, and ERK1/2, stretch-induced eNOS activation was only inhibited by kinase-specific inhibitors of PKA and PI3K/Akt, but not of AMPKα and Erk1/2. Similar results were obtained with knockdown by shRNAs targeting the PKA and Akt genes. Furthermore, inhibition of PKA preferentially attenuated eNOS activation in the early phase, while inhibition of the PI3K/Akt pathway reduced eNOS activation in the late phase, suggesting that the PKA and PI3K/Akt pathways play distinct roles in a time-dependent manner. Finally, we investigated the role of these pathways in stretch-induced endothelial exocytosis and leukocyte adhesion. Interestingly, we found that inhibition of the PI3K/Akt pathway increased stretch-induced Weibel-Palade body exocytosis and leukocyte adhesion, while inhibition of the PKA pathway had the opposite effects, suggesting that the exocytosis-promoting effect of PKA overwhelms the inhibitory effect of PKA-mediated NO production. Taken together, the results suggest that PKA and Akt are important regulators of eNOS activation in venous endothelial cells under mechanical stretch, while playing different roles in the regulation of stretch-induced endothelial exocytosis and leukocyte adhesion.

Hypertensive stretch regulates endothelial exocytosis of Weibel-Palade bodies through VEGF receptor 2 signaling pathways.

Regulated endothelial exocytosis of Weibel-Palade bodies (WPBs), the first stage in leukocyte trafficking, plays a pivotal role in inflammation and injury. Acute mechanical stretch has been closely associated with vascular inflammation, although the precise mechanism is unknown. Here, we show that hypertensive stretch regulates the exocytosis of WPBs of endothelial cells (ECs) through VEGF receptor 2 (VEGFR2) signaling pathways. Stretch triggers a rapid release (within minutes) of von Willebrand factor and interleukin-8 from WPBs in cultured human ECs, promoting the interaction between leukocytes and ECs through the translocation of P-selectin to the cell membrane. We further show that hypertensive stretch significantly induces P-selectin translocation of intact ECs and enhances leukocyte adhesion both ex vivo and in vivo. Stretch-induced endothelial exocytosis is mediated via a VEGFR2/PLCγ1/calcium pathway. Interestingly, stretch also induces a negative feedback via a VEGFR2/Akt/nitric oxide pathway. Such dual effects are confirmed using pharmacological and genetic approaches in carotid artery segments, as well as in acute hypertensive mouse models. These studies reveal mechanical stretch as a potent agonist for endothelial exocytosis, which is modulated by VEGFR2 signaling. Thus, VEGFR2 signaling pathways may represent novel therapeutic targets in limiting hypertensive stretch-related inflammation.

Vascular endothelial growth factor (VEGF) receptor-2 tyrosine 1175 signaling controls VEGF-induced von Willebrand factor release from endothelial cells via phospholipase C-gamma 1- and protein kinase A-dependent pathways.

There is increasing evidence that vascular endothelial growth factor (VEGF) contributes to inflammation independent of its angiogenic functions. Targeting some of the components in endothelial Weibel-Palade bodies (WPBs) effectively inhibits VEGF-induced inflammation, but little is known about how VEGF regulates WPB exocytosis. In this study, we showed that VEGF receptor-2 (VEGFR2), but not VEGFR1, is responsible for VEGF-induced release of von Willebrand factor (vWF), a major marker of WPBs. This is in good contrast to VEGF-stimulated interleukin-6 release from endothelium, which is selectively mediated through VEGFR1. We further demonstrated that VEGFR2-initiated phospholipase C-gamma1 (PLCgamma1)/calcium signaling is important but insufficient for full vWF release, suggesting the possible participation of another effector pathway. We found that cAMP/protein kinase A (PKA) signaling is required for full vWF release. Importantly, a single mutation of Tyr(1175) in the C terminus of VEGFR2, a tyrosine residue crucial for embryonic vasculogenesis, abolished vWF release, concomitant with defective activations of both PLCgamma1 and PKA. These data suggest that Tyr(1175) mediates both PLCgamma1-dependent and PKA-dependent signaling pathways. Taken together, our results not only reveal a novel Tyr(1175)-mediated signaling pathway but also highlight a potentially new therapeutic target for the management of vascular inflammation.