scholarly journals PI3K, Rho, and ROCK play a key role in hypoxia-induced ATP release and ATP-stimulated angiogenic responses in pulmonary artery vasa vasorum endothelial cells

2009 ◽  
Vol 297 (5) ◽  
pp. L954-L964 ◽  
Author(s):  
Heather N. Woodward ◽  
Adil Anwar ◽  
Suzette Riddle ◽  
Laimute Taraseviciene-Stewart ◽  
Miguel Fragoso ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Atp Release ◽  
Brefeldin A ◽  
Extracellular Atp ◽  
Vasa Vasorum ◽  
Angiogenic Factor ◽  
Dependent Manner

We recently reported that vasa vasorum expansion occurs in the pulmonary artery (PA) adventitia of chronically hypoxic animals and that extracellular ATP is a pro-angiogenic factor for isolated vasa vasorum endothelial cells (VVEC). However, the sources of extracellular ATP in the PA vascular wall, as well as the molecular mechanisms underlying its release, remain elusive. Studies were undertaken to explore whether VVEC release ATP in response to hypoxia and to determine signaling pathways involved in this process. We found that hypoxia (1–3% O2) resulted in time- and O2-dependent ATP release from VVEC. Preincubation with the inhibitors of vesicular transport (monensin, brefeldin A, and N-ethylmaleimide) significantly decreased ATP accumulation in the VVEC conditioned media, suggesting that hypoxia-induced ATP release occurs through vesicular exocytosis. Additionally, both hypoxia and exogenously added ATP resulted in the activation of PI3K and accumulation of GTP-bound RhoA in a time-dependent manner. Pharmacological inhibition of PI3K and ROCK or knockout of RhoA by small interfering RNA significantly abolished hypoxia-induced ATP release from VVEC. Moreover, RhoA and ROCK play a critical role in ATP-induced increases in VVEC DNA synthesis, migration, and tube formation, indicating a functional contribution of PI3K, Rho, and ROCK to both the autocrine mechanism of ATP release and ATP-mediated angiogenic activation of VVEC. Taken together, our findings provide novel evidence for the signaling mechanisms that link hypoxia-induced increases in extracellular ATP and vasa vasorum expansion.

scholarly journals c-Jun, Foxo3a, and c-Myc Transcription Factors are Key Regulators of ATP-Mediated Angiogenic Responses in Pulmonary Artery Vasa Vasorum Endothelial Cells †

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 416
Author(s):  
Derek Strassheim ◽  
Vijaya Karoor ◽  
Hala Nijmeh ◽  
Philip Weston ◽  
Martin Lapel ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factors ◽  
Pulmonary Artery ◽  
Molecular Mechanisms ◽  
Cell Cycle Control ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Vasa Vasorum ◽  
Angiogenic Factor ◽  
Angiogenic Genes

Angiogenic vasa vasorum (VV) expansion plays an essential role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH), a cardiovascular disease. We previously showed that extracellular ATP released under hypoxic conditions is an autocrine/paracrine, the angiogenic factor for pulmonary artery (PA) VV endothelial cells (VVECs), acting via P2Y purinergic receptors (P2YR) and the Phosphoinositide 3-kinase (PI3K)-Akt-Mammalian Target of Rapamycin (mTOR) signaling. To further elucidate the molecular mechanisms of ATP-mediated VV angiogenesis, we determined the profile of ATP-inducible transcription factors (TFs) in VVECs using a TranSignal protein/DNA array. C-Jun, c-Myc, and Foxo3 were found to be upregulated in most VVEC populations and formed nodes connecting several signaling networks. siRNA-mediated knockdown (KD) of these TFs revealed their critical role in ATP-induced VVEC angiogenic responses and the regulation of downstream targets involved in tissue remodeling, cell cycle control, expression of endothelial markers, cell adhesion, and junction proteins. Our results showed that c-Jun was required for the expression of ATP-stimulated angiogenic genes, c-Myc was repressive to anti-angiogenic genes, and Foxo3a predominantly controlled the expression of anti-apoptotic and junctional proteins. The findings from our study suggest that pharmacological targeting of the components of P2YR-PI3K-Akt-mTOR axis and specific TFs reduced ATP-mediated VVEC angiogenic response and may have a potential translational significance in attenuating pathological vascular remodeling.

scholarly journals Extracellular ATP is a pro-angiogenic factor for pulmonary artery vasa vasorum endothelial cells

Angiogenesis ◽  
2007 ◽  
Vol 11 (2) ◽  
pp. 169-182 ◽  
Author(s):  
Evgenia V. Gerasimovskaya ◽  
Heather N. Woodward ◽  
Doug A. Tucker ◽  
Kurt R. Stenmark
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Extracellular Atp ◽  
Vasa Vasorum ◽  
Angiogenic Factor

scholarly journals P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells

2011 ◽  
Vol 300 (2) ◽  
pp. C266-C275 ◽  
Author(s):  
Taras Lyubchenko ◽  
Heather Woodward ◽  
Kristopher D. Veo ◽  
Nana Burns ◽  
Hala Nijmeh ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Purinergic Receptors ◽  
Purinergic Receptor ◽  
Extracellular Atp ◽  
Vasa Vasorum ◽  
Receptor Subtypes ◽  
Extracellular Nucleotide ◽  
Selective Antagonist ◽  
Intracellular Ca

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca2+ concentration ([Ca2+]i) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca2+ signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca2+]i via Ca2+ influx through plasma membrane channels as well as Ca2+ mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca2+ responses in both cytosolic and nuclear compartments. An increase in [Ca2+]i was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPβS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αβMeATP, and βγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca2+ responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.

scholarly journals Mechanosensation and Mechanotransduction by Lymphatic Endothelial Cells Act as Important Regulators of Lymphatic Development and Function

2021 ◽  
Vol 22 (8) ◽  
pp. 3955
Author(s):  
László Bálint ◽  
Zoltán Jakus
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Fate ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Mechanical Forces ◽  
Lymphatic Endothelial Cells ◽  
Lymphatic Development ◽  
And Function ◽  
The Impact

Our understanding of the function and development of the lymphatic system is expanding rapidly due to the identification of specific molecular markers and the availability of novel genetic approaches. In connection, it has been demonstrated that mechanical forces contribute to the endothelial cell fate commitment and play a critical role in influencing lymphatic endothelial cell shape and alignment by promoting sprouting, development, maturation of the lymphatic network, and coordinating lymphatic valve morphogenesis and the stabilization of lymphatic valves. However, the mechanosignaling and mechanotransduction pathways involved in these processes are poorly understood. Here, we provide an overview of the impact of mechanical forces on lymphatics and summarize the current understanding of the molecular mechanisms involved in the mechanosensation and mechanotransduction by lymphatic endothelial cells. We also discuss how these mechanosensitive pathways affect endothelial cell fate and regulate lymphatic development and function. A better understanding of these mechanisms may provide a deeper insight into the pathophysiology of various diseases associated with impaired lymphatic function, such as lymphedema and may eventually lead to the discovery of novel therapeutic targets for these conditions.

Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells

1992 ◽  
Vol 1134 (1) ◽  
pp. 61-72 ◽  
Author(s):  
Kevin D. Lustig ◽  
Laurie Erb ◽  
David M. Landis ◽  
Cathy S. Hicks-Taylor ◽  
Xiaoke Zhang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Extracellular Atp

Abstract 3380: Acetylation-dependent Regulation Of Notch1 Signaling In Endothelial Cells

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Virginia Guarani ◽  
Franck Dequiedt ◽  
Andreas M Zeiher ◽  
Stefanie Dimmeler ◽  
Michael Potente
Keyword(s):  
Endothelial Cells ◽  
Notch Signaling ◽  
Cell Fate ◽  
Molecular Mechanisms ◽  
Trichostatin A ◽  
Notch Pathway ◽  
Dependent Manner ◽  
Class Iii ◽  
Cell Fate Decisions ◽  
Knock Down

The Notch signaling pathway is a versatile regulator of cell fate decisions and plays an essential role for embryonic and postnatal vascular development. As only modest differences in Notch pathway activity suffice to determine dramatic differences in blood vessel development, this pathway is tightly regulated by a variety of molecular mechanisms. Reversible acetylation has emerged as an important post-translational modification of several non-histone proteins, which are targeted by histone deacetylases (HDACs). Here, we report that specifically the Notch1 intracellular domain (NICD) is itself an acetylated protein and that its acetylation level is tightly regulated by the SIRT1 deacetylase, which we have previously identified as a key regulator of endothelial angiogenic functions during vascular growth. Coexpression of NICD with histone acetyltransferases such as p300 or PCAF induced a dose- and time-dependent acetylation of NICD. Blocking HDAC activity using the class III HDAC inhibitor nicotinamid (NAM), but not the class I/II HDAC inhibior trichostatin A, resulted in a significant increase of NICD acetylation suggesting that NICD is targetd by class III HDACs for deacetylation. Among the class III HDACs with deacetylase activity (SIRT1, 2, 3, 5), knock down of specifically SIRT1 resulted in enhanced acetylation of NICD. Moreover, wild type SIRT1, but not a catalytically inactive mutant catalyzed the deacetylation of NICD in a nicotinamid-dependent manner. SIRT1, but SIRT2, SIRT3 or SIRT5, associated with NICD through its catalytic domain demonstrating that SIRT1 is a direct NICD deacetylase. Enhancing NICD acetylation by either overexpression of p300 or inhibition of SIRT1 activity using NAM or RNAi-mediated knock down resulted in enhanced NICD protein stability by blocking its ubiquitin-mediated degradation. Consistent with these results, loss of SIRT1 amplified Notch target gene expression in endothelial cells in response to NICD overexpression or treatment with the Notch ligand Dll4. In summary, our results identify reversible acetylation of NICD as a novel molecular mechanism to control Notch signaling and suggest that deacetylation of NICD by SIRT1 plays a key role in the dynamic regulation of Notch signaling in endothelial cells.

P2X7 receptor cross-talk regulates ATP-induced pannexin 1 internalization

2017 ◽  
Vol 474 (13) ◽  
pp. 2133-2144 ◽  
Author(s):  
Andrew K.J. Boyce ◽  
Leigh Anne Swayne
Keyword(s):  
Nervous System ◽  
Cross Talk ◽  
Purinergic Receptors ◽  
Atp Release ◽  
Extracellular Atp ◽  
Signalling Pathways ◽  
Physical Interaction ◽  
Cell Periphery ◽  
Dependent Manner ◽  
Pannexin 1

In the nervous system, extracellular ATP levels transiently increase in physiological and pathophysiological circumstances, effecting key signalling pathways in plasticity and inflammation through purinergic receptors. Pannexin 1 (Panx1) forms ion- and metabolite-permeable channels that mediate ATP release and are particularly enriched in the nervous system. Our recent study demonstrated that elevation of extracellular ATP triggers Panx1 internalization in a concentration- and time-dependent manner. Notably, this effect was sensitive to inhibition of ionotropic P2X7 purinergic receptors (P2X7Rs). Here, we report our novel findings from the detailed investigation of the mechanism underlying P2X7R–Panx1 cross-talk in ATP-stimulated internalization. We demonstrate that extracellular ATP triggers and is required for the clustering of P2X7Rs and Panx1 on Neuro2a cells through an extracellular physical interaction with the Panx1 first extracellular loop (EL1). Importantly, disruption of P2X7R–Panx1 clustering by mutation of tryptophan 74 within the Panx1 EL1 inhibits Panx1 internalization. Notably, P2X7R–Panx1 clustering and internalization are independent of P2X7R-associated intracellular signalling pathways (Ca2+ influx and Src activation). Further analysis revealed that cholesterol is required for ATP-stimulated P2X7R–Panx1 clustering at the cell periphery. Taken together, our data suggest that extracellular ATP induces and is required for Panx1 EL1-mediated, cholesterol-dependent P2X7R–Panx1 clustering and endocytosis. These findings have important implications for understanding the role of Panx1 in the nervous system and provide important new insights into Panx1–P2X7R cross-talk.

scholarly journals Toll-Like Receptor-Triggered Calcium Mobilization Protects Mice against Bacterial Infection through Extracellular ATP Release

2014 ◽  
Vol 82 (12) ◽  
pp. 5076-5085 ◽  
Author(s):  
Hua Ren ◽  
Yunfei Teng ◽  
Binghe Tan ◽  
Xiaoyu Zhang ◽  
Wei Jiang ◽  
...  
Keyword(s):  
Immune Responses ◽  
Pyridoxal Phosphate ◽  
Atp Release ◽  
Extracellular Atp ◽  
Calcium Mobilization ◽  
Innate Immune ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Innate Immune Responses ◽  
Toll Like Receptor

ABSTRACTExtracellular ATP (eATP), released as a “danger signal” by injured or stressed cells, plays an important role in the regulation of immune responses, but the relationship between ATP release and innate immune responses is still uncertain. In this study, we demonstrated that ATP was released through Toll-like receptor (TLR)-associated signaling in bothEscherichia coli-infected mice and lipopolysaccharide (LPS)- or Pam3CSK4-treated macrophages. This ATP release could be blocked completely only byN-ethylmaleimide (NEM), not by carbenoxolone (CBX), flufenamic acid (FFA), or probenecid, suggesting the key role of exocytosis in this process. Furthermore, LPS-induced ATP release could also be reduced dramatically through suppressing calcium mobilization by use of U73122, caffeine, and thapsigargin (TG). In addition, the secretion of interleukin-1β (IL-1β) and CCL-2 was enhanced significantly by ATP, in a time- and dose-dependent manner. Meanwhile, macrophage-mediated phagocytosis of bacteria was also promoted significantly by ATP stimulation. Furthermore, extracellular ATP reduced the number of invading bacteria and protected mice from peritonitis by activating purinergic receptors. Mechanistically, phosphorylation of AKT and ERK was overtly increased by ATP in antibacterial immune responses. Accordingly, if we blocked the P2X- and P2Y-associated signaling pathway by using suramin and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid), tetrasodium salt (PPADS), the ATP-enhanced immune response was restrained significantly. Taken together, our findings reveal an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of calcium mobilization-mediated ATP release in infectious diseases.

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

2018 ◽  
Vol 315 (5) ◽  
pp. H1477-H1485 ◽  
Author(s):  
Kimiko Yamamoto ◽  
Hiromi Imamura ◽  
Joji Ando
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Vascular Endothelial Cells ◽  
Critical Role ◽  
Atp Release ◽  
Vascular Endothelial ◽  
The Novel ◽  
Caveolin 1 ◽  
Atp Generation

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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