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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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Extracellular ATP induces VVEC network formation through activation of ERK1/2, and not of PI3K/mTOR pathways. Panels a–d: Growth-arrested VVEC (125,000 cells/well) were plated on growthfactor-reduced Matrigel 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. Formation of tube-like networks was stimulated by the addition ATP (100 μM). At the end of incubation, images were captured in three fields using AxioVision program. Quantitative data for total and average tube (cord) lengths and area of cell aggregates represent the means ± SE. Similar results were reproduced in at least three experiments on three distinct cell populations; *P < 0.05 vs. nonstimulated control; #P < 0.05 vs. ATP stimulated-cells
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Fig9: Extracellular ATP induces VVEC network formation through activation of ERK1/2, and not of PI3K/mTOR pathways. Panels a–d: Growth-arrested VVEC (125,000 cells/well) were plated on growthfactor-reduced Matrigel 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. Formation of tube-like networks was stimulated by the addition ATP (100 μM). At the end of incubation, images were captured in three fields using AxioVision program. Quantitative data for total and average tube (cord) lengths and area of cell aggregates represent the means ± SE. Similar results were reproduced in at least three experiments on three distinct cell populations; *P < 0.05 vs. nonstimulated control; #P < 0.05 vs. ATP stimulated-cells

Mentions: Because cell rearrangement in the tube-like networks requires a proper cell migration, we sought to determine if PI3K/mTOR and ERK1/2 pathways, are important to this response. We found that pre-incubation of VVEC with PI3K and mTOR pathway-specific inhibitors (LY294002, wortmannin, rapamycin) had only minor effects in ATP-stimulated cells. (Fig. 9). Calculation of the areas occupied by the tube-like structures (total tube length and total cell aggregate diameter) indicated that there were no statistically significant contributions of PI3K and mTOR/p70S6K pathways to ATP-induced formation of VVEC networks. (Fig 9b–e). A small, but statistically significant effect on the total and average tube length was observed in U0126-treated cell pointing out a role for ERK1/2 in this response (Fig 9a, b). These findings suggest that extracellular ATP-induced VVEC rearrangements in the tube-like networks requires additional, as yet unidentified intracellular signaling pathways.Fig. 9


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

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

Extracellular ATP induces VVEC network formation through activation of ERK1/2, and not of PI3K/mTOR pathways. Panels a–d: Growth-arrested VVEC (125,000 cells/well) were plated on growthfactor-reduced Matrigel 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. Formation of tube-like networks was stimulated by the addition ATP (100 μM). At the end of incubation, images were captured in three fields using AxioVision program. Quantitative data for total and average tube (cord) lengths and area of cell aggregates represent the means ± SE. Similar results were reproduced in at least three experiments on three 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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Fig9: Extracellular ATP induces VVEC network formation through activation of ERK1/2, and not of PI3K/mTOR pathways. Panels a–d: Growth-arrested VVEC (125,000 cells/well) were plated on growthfactor-reduced Matrigel 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. Formation of tube-like networks was stimulated by the addition ATP (100 μM). At the end of incubation, images were captured in three fields using AxioVision program. Quantitative data for total and average tube (cord) lengths and area of cell aggregates represent the means ± SE. Similar results were reproduced in at least three experiments on three distinct cell populations; *P < 0.05 vs. nonstimulated control; #P < 0.05 vs. ATP stimulated-cells
Mentions: Because cell rearrangement in the tube-like networks requires a proper cell migration, we sought to determine if PI3K/mTOR and ERK1/2 pathways, are important to this response. We found that pre-incubation of VVEC with PI3K and mTOR pathway-specific inhibitors (LY294002, wortmannin, rapamycin) had only minor effects in ATP-stimulated cells. (Fig. 9). Calculation of the areas occupied by the tube-like structures (total tube length and total cell aggregate diameter) indicated that there were no statistically significant contributions of PI3K and mTOR/p70S6K pathways to ATP-induced formation of VVEC networks. (Fig 9b–e). A small, but statistically significant effect on the total and average tube length was observed in U0126-treated cell pointing out a role for ERK1/2 in this response (Fig 9a, b). These findings suggest that extracellular ATP-induced VVEC rearrangements in the tube-like networks requires additional, as yet unidentified intracellular signaling pathways.Fig. 9

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