Shear stress augments mitochondrial ATP generation that triggers ATP release and Ca2+ signaling in vascular endothelial cells

Vascular endothelial cells (ECs) sense and transduce hemodynamic shear stress into intracellular biochemical signals, and Ca2+ signaling plays a critical role in this mechanotransduction, i.e., ECs release ATP in the caveolae in response to shear stress and, in turn, the released ATP activates P2 purinoceptors, which results in an influx into the cells of extracellular Ca2+. However, the mechanism by which the shear stress evokes ATP release remains unclear. Here, we demonstrated that cellular mitochondria play a critical role in this process. Cultured human pulmonary artery ECs were exposed to controlled levels of shear stress in a flow-loading device, and changes in the mitochondrial ATP levels were examined by real-time imaging using a fluorescence resonance energy transfer-based ATP biosensor. Immediately upon exposure of the cells to flow, mitochondrial ATP levels increased, which was both reversible and dependent on the intensity of shear stress. Inhibitors of the mitochondrial electron transport chain and ATP synthase as well as knockdown of caveolin-1, a major structural protein of the caveolae, abolished the shear stress-induced mitochondrial ATP generation, resulting in the loss of ATP release and influx of Ca2+ into the cells. These results suggest the novel role of mitochondria in transducing shear stress into ATP generation: ATP generation leads to ATP release in the caveolae, triggering purinergic Ca2+ signaling. Thus, exposure of ECs to shear stress seems to activate mitochondrial ATP generation through caveola- or caveolin-1-mediated mechanisms. NEW & NOTEWORTHY The mechanism of how vascular endothelial cells sense shear stress generated by blood flow and transduce it into functional responses remains unclear. Real-time imaging of mitochondrial ATP demonstrated the novel role of endothelial mitochondria as mechanosignaling organelles that are able to transduce shear stress into ATP generation, triggering ATP release and purinoceptor-mediated Ca2+ signaling within the cells.

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Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01385.2006 ◽

2007 ◽

Vol 293 (3) ◽

pp. H1646-H1653 ◽

Cited By ~ 71

Author(s):

Kimiko Yamamoto ◽

Nobutaka Shimizu ◽

Syotaro Obi ◽

Shinichiro Kumagaya ◽

Yutaka Taketani ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Cell Surface ◽

Lipid Rafts ◽

Atp Synthase ◽

Vascular Endothelial Cells ◽

Atp Release ◽

Dependent Manner ◽

Caveolin 1 ◽

Atp Generation

Endothelial cells (ECs) release ATP in response to shear stress, a mechanical force generated by blood flow, and the ATP released modulates EC functions through activation of purinoceptors. The molecular mechanism of the shear stress-induced ATP release, however, has not been fully elucidated. In this study, we have demonstrated that cell surface ATP synthase is involved in shear stress-induced ATP release. Immunofluorescence staining of human pulmonary arterial ECs (HPAECs) showed that cell surface ATP synthase is distributed in lipid rafts and co-localized with caveolin-1, a marker protein of caveolae. Immunoprecipitation indicated that cell surface ATP synthase and caveolin-1 are physically associated. Measurement of the extracellular metabolism of [3H]ADP confirmed that cell surface ATP synthase is active in ATP generation. When exposed to shear stress, HPAECs released ATP in a dose-dependent manner, and the ATP release was markedly suppressed by the membrane-impermeable ATP synthase inhibitors angiostatin and piceatannol and by an anti-ATP synthase antibody. Depletion of plasma membrane cholesterol with methyl-β-cyclodextrin (MβCD) disrupted lipid rafts and abolished co-localization of ATP synthase with caveolin-1, which resulted in a marked reduction in shear stress-induced ATP release. Pretreatment of the cells with cholesterol prevented these effects of MβCD. Downregulation of caveolin-1 expression by transfection of caveolin-1 siRNA also markedly suppressed ATP-releasing responses to shear stress. Neither MβCD, MβCD plus cholesterol, nor caveolin-1 siRNA had any effect on the amount of cell surface ATP synthase. These results suggest that the localization and targeting of ATP synthase to caveolae/lipid rafts is critical for shear stress-induced ATP release by HPAECs.

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Lowering Caveolin-1 Expression in Human Vascular Endothelial Cells Inhibits Signal Transduction in Response to Shear Stress

International Journal of Cell Biology ◽

10.1155/2009/532432 ◽

2009 ◽

Vol 2009 ◽

pp. 1-9 ◽

Cited By ~ 6

Author(s):

A. D. van der Meer ◽

M. M. J. Kamphuis ◽

A. A. Poot ◽

J. Feijen ◽

I. Vermes

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Shear Stress ◽

Vascular Endothelial Cells ◽

Vascular Endothelial ◽

Human Endothelial Cells ◽

Caveolin 1 ◽

Low Levels ◽

Endothelial Growth Factor

Vascular endothelial cells have an extensive response to physiological levels of shear stress. There is evidence that the protein caveolin-1 is involved in the early phase of this response. In this study, caveolin-1 was downregulated in human endothelial cells by RNAi. When these cells were subjected to a shear stress of 15 dyn/cm2for 10 minutes, activation of Akt and ERK1/2 was significantly lower than in control cells. Moreover, activation of Akt and ERK1/2 in response to vascular endothelial growth factor was significantly lower in cells with low levels of caveolin-1. However, activation of integrin-mediated signaling during cell adhesion onto fibronectin was not hampered by lowered caveolin-1 levels. In conclusion, caveolin-1 is an essential component in the response of endothelial cells to shear stress. Furthermore, the results suggest that the role of caveolin-1 in this process lies in facilitating efficient VEGFR2-mediated signaling.

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Low Shear Stress Upregulates CX3CR1 Expression by Inducing VCAM-1 via the NF-κB Pathway in Vascular Endothelial Cells

Cell Biochemistry and Biophysics ◽

10.1007/s12013-020-00931-4 ◽

2020 ◽

Vol 78 (3) ◽

pp. 383-389 ◽

Cited By ~ 1

Author(s):

Yiwei Zhao ◽

Peile Ren ◽

Qiufang Li ◽

Shafiu Adam Umar ◽

Tan Yang ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Protein Expression ◽

Vascular Endothelial Cells ◽

Critical Role ◽

Mrna Levels ◽

Vascular Endothelial ◽

Low Shear Stress ◽

Low Shear ◽

Cx3cr1 Expression

Abstract Atherosclerosis is a significant cause of mortality and morbidity. Studies suggest that the chemokine receptor CX3CR1 plays a critical role in atherogenesis. Shear stress is an important mechanical force that affects blood vessel function. In this study, we investigated the effect of shear stress on CX3CR1 expression in vascular endothelial cells (VECs). First, cells were exposed to different shear stress and then CX3CR1 mRNA and protein were measured by quantitative RT-PCR and western blot analysis, respectively. CX3CR1 gene silencing was used to analyze the molecular mechanisms underlying shear stress-mediated effects on CX3CR1 expression. CX3CR1 mRNA and protein expression were significantly increased with 4.14 dyne/cm2 of shear stress compared with other tested levels of shear stress. We observed a significant increase in CX3CR1 mRNA levels at 2 h and CX3CR1 protein expression at 4 h. CX3CR1-induced VCAM-1 expression in response to low shear stress by activating NF-κB signaling pathway in VECs. Our findings demonstrate that low shear stress increases CX3CR1 expression, which increases VCAM-1 expression due to elevated NF-κB activation. The current study provides evidence of the correlation between shear stress and atherosclerosis mediated by CX3CR1.

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