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

Gerasimovskaya EV, Woodward HN, Tucker DA, Stenmark KR - Angiogenesis (2007)

Bottom Line: Using pharmacological inhibitors, Western blot analysis, and Phosphatidylinositol-3 kinase (PI3K) in vitro kinase assays, we found that PI3K/Akt/mTOR and ERK1/2 play a critical role in mediating the extracellular ATP-induced mitogenic and migratory responses in VVEC.However, PI3K/Akt and mTOR/p70S6K do not significantly contribute to extracellular ATP-induced tube formation on Matrigel.Our studies indicate that VVEC, isolated from the sites of active angiogenesis, exhibit distinct functional responses to ATP, compared to endothelial cells derived from large pulmonary or systemic vessels.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Colorado at Denver and Health Sciences Center, B131, 4200 East 9th Ave, Denver, CO 80262, USA. Evgenia.Gerasimovskaya@UCHSC.edu

ABSTRACT
Expansion of the vasa vasorum network has been observed in a variety of systemic and pulmonary vascular diseases. We recently reported that a marked expansion of the vasa vasorum network occurs in the pulmonary artery adventitia of chronically hypoxic calves. Since hypoxia has been shown to stimulate ATP release from both vascular resident as well as circulatory blood cells, these studies were undertaken to determine if extracellular ATP exerts angiogenic effects on isolated vasa vasorum endothelial cells (VVEC) and/or if it augments the effects of other angiogenic factors (VEGF and basic FGF) known to be present in the hypoxic microenvironment. We found that extracellular ATP dramatically increases DNA synthesis, migration, and rearrangement into tube-like networks on Matrigel in VVEC, but not in pulmonary artery (MPAEC) or aortic (AOEC) endothelial cells obtained from the same animals. Extracellular ATP potentiated the effects of both VEGF and bFGF to stimulate DNA synthesis in VVEC but not in MPAEC and AOEC. Analysis of purine and pyrimidine nucleotides revealed that ATP, ADP and MeSADP were the most potent in stimulating mitogenic responses in VVEC, indicating the involvement of the family of P2Y1-like purinergic receptors. Using pharmacological inhibitors, Western blot analysis, and Phosphatidylinositol-3 kinase (PI3K) in vitro kinase assays, we found that PI3K/Akt/mTOR and ERK1/2 play a critical role in mediating the extracellular ATP-induced mitogenic and migratory responses in VVEC. However, PI3K/Akt and mTOR/p70S6K do not significantly contribute to extracellular ATP-induced tube formation on Matrigel. Our studies indicate that VVEC, isolated from the sites of active angiogenesis, exhibit distinct functional responses to ATP, compared to endothelial cells derived from large pulmonary or systemic vessels. Collectively, our data support the idea that extracellular ATP participates in the expansion of the vasa vasorum that can be observed in hypoxic conditions.

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PI3K/Akt, ERK1/2, and mTOR/p70S6K pathways play a critical role in extracellular ATP-induced VVEC migration. Growth arrested VVEC (100,000 cells/well) were plated on top of inserts in serum free DMEM. Cells were preincubated with LY294002 (20 μM, 60 min) rapamycin (10 nM, 60 min), wortmannin (100 nM, 120 min), U0126 (10 μM, 60 min), or vechicle for 60 min. Cell migration was stimulated by adding ATP (100 μM) in the lower transwell compartment. At the end of incubation, cells on the bottom of filter were fixed and counted in three fields at 10X magnification. Quantitative data for each experimental condition represent the means ± SE Similar results were reproduced in at least three experiments on four distinct cell populations; *P < 0.05 vs. nonstimulated control; #P < 0.05 vs. ATP-stimulated cells
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Fig8: PI3K/Akt, ERK1/2, and mTOR/p70S6K pathways play a critical role in extracellular ATP-induced VVEC migration. Growth arrested VVEC (100,000 cells/well) were plated on top of inserts in serum free DMEM. Cells were preincubated with LY294002 (20 μM, 60 min) rapamycin (10 nM, 60 min), wortmannin (100 nM, 120 min), U0126 (10 μM, 60 min), or vechicle for 60 min. Cell migration was stimulated by adding ATP (100 μM) in the lower transwell compartment. At the end of incubation, cells on the bottom of filter were fixed and counted in three fields at 10X magnification. Quantitative data for each experimental condition represent the means ± SE Similar results were reproduced in at least three experiments on four distinct cell populations; *P < 0.05 vs. nonstimulated control; #P < 0.05 vs. ATP-stimulated cells

Mentions: Similarly, pre-incubation of VVEC with LY294002, wortmannin, and U0126 significantly diminished ATP-induced migration. Rapamycin had a partial, but significant inhibitory effect on VVEC migration (Fig. 8). Consistent with the role of PI3K/mTOR and ERK1/2 in VVEC proliferation, these data demonstrate that activation of these pathways is also critically important to extracellular ATP-induced VVEC migration.Fig. 8


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

Gerasimovskaya EV, Woodward HN, Tucker DA, Stenmark KR - Angiogenesis (2007)

PI3K/Akt, ERK1/2, and mTOR/p70S6K pathways play a critical role in extracellular ATP-induced VVEC migration. Growth arrested VVEC (100,000 cells/well) were plated on top of inserts in serum free DMEM. Cells were preincubated with LY294002 (20 μM, 60 min) rapamycin (10 nM, 60 min), wortmannin (100 nM, 120 min), U0126 (10 μM, 60 min), or vechicle for 60 min. Cell migration was stimulated by adding ATP (100 μM) in the lower transwell compartment. At the end of incubation, cells on the bottom of filter were fixed and counted in three fields at 10X magnification. Quantitative data for each experimental condition represent the means ± SE Similar results were reproduced in at least three experiments on four distinct cell populations; *P < 0.05 vs. nonstimulated control; #P < 0.05 vs. ATP-stimulated cells
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2480488&req=5

Fig8: PI3K/Akt, ERK1/2, and mTOR/p70S6K pathways play a critical role in extracellular ATP-induced VVEC migration. Growth arrested VVEC (100,000 cells/well) were plated on top of inserts in serum free DMEM. Cells were preincubated with LY294002 (20 μM, 60 min) rapamycin (10 nM, 60 min), wortmannin (100 nM, 120 min), U0126 (10 μM, 60 min), or vechicle for 60 min. Cell migration was stimulated by adding ATP (100 μM) in the lower transwell compartment. At the end of incubation, cells on the bottom of filter were fixed and counted in three fields at 10X magnification. Quantitative data for each experimental condition represent the means ± SE Similar results were reproduced in at least three experiments on four distinct cell populations; *P < 0.05 vs. nonstimulated control; #P < 0.05 vs. ATP-stimulated cells
Mentions: Similarly, pre-incubation of VVEC with LY294002, wortmannin, and U0126 significantly diminished ATP-induced migration. Rapamycin had a partial, but significant inhibitory effect on VVEC migration (Fig. 8). Consistent with the role of PI3K/mTOR and ERK1/2 in VVEC proliferation, these data demonstrate that activation of these pathways is also critically important to extracellular ATP-induced VVEC migration.Fig. 8

Bottom Line: Using pharmacological inhibitors, Western blot analysis, and Phosphatidylinositol-3 kinase (PI3K) in vitro kinase assays, we found that PI3K/Akt/mTOR and ERK1/2 play a critical role in mediating the extracellular ATP-induced mitogenic and migratory responses in VVEC.However, PI3K/Akt and mTOR/p70S6K do not significantly contribute to extracellular ATP-induced tube formation on Matrigel.Our studies indicate that VVEC, isolated from the sites of active angiogenesis, exhibit distinct functional responses to ATP, compared to endothelial cells derived from large pulmonary or systemic vessels.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Colorado at Denver and Health Sciences Center, B131, 4200 East 9th Ave, Denver, CO 80262, USA. Evgenia.Gerasimovskaya@UCHSC.edu

ABSTRACT
Expansion of the vasa vasorum network has been observed in a variety of systemic and pulmonary vascular diseases. We recently reported that a marked expansion of the vasa vasorum network occurs in the pulmonary artery adventitia of chronically hypoxic calves. Since hypoxia has been shown to stimulate ATP release from both vascular resident as well as circulatory blood cells, these studies were undertaken to determine if extracellular ATP exerts angiogenic effects on isolated vasa vasorum endothelial cells (VVEC) and/or if it augments the effects of other angiogenic factors (VEGF and basic FGF) known to be present in the hypoxic microenvironment. We found that extracellular ATP dramatically increases DNA synthesis, migration, and rearrangement into tube-like networks on Matrigel in VVEC, but not in pulmonary artery (MPAEC) or aortic (AOEC) endothelial cells obtained from the same animals. Extracellular ATP potentiated the effects of both VEGF and bFGF to stimulate DNA synthesis in VVEC but not in MPAEC and AOEC. Analysis of purine and pyrimidine nucleotides revealed that ATP, ADP and MeSADP were the most potent in stimulating mitogenic responses in VVEC, indicating the involvement of the family of P2Y1-like purinergic receptors. Using pharmacological inhibitors, Western blot analysis, and Phosphatidylinositol-3 kinase (PI3K) in vitro kinase assays, we found that PI3K/Akt/mTOR and ERK1/2 play a critical role in mediating the extracellular ATP-induced mitogenic and migratory responses in VVEC. However, PI3K/Akt and mTOR/p70S6K do not significantly contribute to extracellular ATP-induced tube formation on Matrigel. Our studies indicate that VVEC, isolated from the sites of active angiogenesis, exhibit distinct functional responses to ATP, compared to endothelial cells derived from large pulmonary or systemic vessels. Collectively, our data support the idea that extracellular ATP participates in the expansion of the vasa vasorum that can be observed in hypoxic conditions.

Show MeSH
Related in: MedlinePlus