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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 robustly activates ERK1/2, PI3K/Akt, and mTO/p70S6K signaling pathways in VVEC but only modestly in AOEC and MPAEC. Growth- arrested cells were stimulated with ATP (100 μM) for the indicated times. Equivalent amounts of total cell protein (25–40 μg) were subjected to Western blot analysis with antibodies against phospho-ERK1/2 (Thr202/Tyr204), phospho-Akt (Ser 473), phospho-mTOR (Thr 2448), and phospho-p70S6K (Thr421/Ser424). Data shown on each panel illustrate representative experiments for each cell type. Similar results were reproduced in at least three independent experiments. The maximal phosphorylation responses observed within the indicated time frame, are discussed in the text
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Fig5: Extracellular ATP robustly activates ERK1/2, PI3K/Akt, and mTO/p70S6K signaling pathways in VVEC but only modestly in AOEC and MPAEC. Growth- arrested cells were stimulated with ATP (100 μM) for the indicated times. Equivalent amounts of total cell protein (25–40 μg) were subjected to Western blot analysis with antibodies against phospho-ERK1/2 (Thr202/Tyr204), phospho-Akt (Ser 473), phospho-mTOR (Thr 2448), and phospho-p70S6K (Thr421/Ser424). Data shown on each panel illustrate representative experiments for each cell type. Similar results were reproduced in at least three independent experiments. The maximal phosphorylation responses observed within the indicated time frame, are discussed in the text

Mentions: Studies in vivo and in vitro have shown the importance of ERK1/2 and PI3K/mTOR pathways in controlling angiogenesis-related events and metastatic tumor growth [36–40]. Because of the differences in ATP-induced proliferative and migratory responses in VVEC, AOEC, and MPAEC, we examined the effects of ATP on activation of ERK1/2 (as measured by Thr202/Tyr204 phosphorylation), Akt (as measured by Ser473 phosphorylation), mTOR (as measured by Ser2448 phosphorylation), and p70S6K (as measured by Thr421/Ser424 phosphorylation) in the different endothelial cell types (Fig. 5). We found that ATP induced a greater stimulation of all examined signaling pathways in VVEC than in AOEC or MPAEC (Fig. 5). In particular, ATP exerted greater (maximum 10.8 fold increase) and more prolonged increase in phosphorylation of ERK1/2 in VVEC than in AOEC (maximum 4.2-fold) and in MPAEC (maximum 5.0-fold). ATP-induced phosphorylation of ERK1/2 was consistently observed in VVEC at 120 min and was not observed at this time point in AOEC or MPAEC. ATP-induced phosphorylation of Akt occurred earlier and was greater in VVEC than in AOEC and MPAEC (maximal fold increase 5.3, 3.2, and 2.4, respectively). ATP-induced phosphorylation of mTOR was greater in VVEC than in AOEC and MPAEC (maximal fold increase 4.8, 1.8, and 2.2, respectively). Maximal phosphorylation of p70S6K was also greater and more prolonged in VVEC than in AOEC and MPAEC (maximal fold increase 8.9, 2.0, and 2.4, respectively) Thus, in aggreement with the robust effect of extracellular ATP on DNA synthesis, these data support the idea that in adventitial VVEC, ERK1/2, PI3K/Akt, and mTOR/p70S6K pathways contribute to the ATP-dependent pro-angiogenic phenotype.Fig. 5


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 robustly activates ERK1/2, PI3K/Akt, and mTO/p70S6K signaling pathways in VVEC but only modestly in AOEC and MPAEC. Growth- arrested cells were stimulated with ATP (100 μM) for the indicated times. Equivalent amounts of total cell protein (25–40 μg) were subjected to Western blot analysis with antibodies against phospho-ERK1/2 (Thr202/Tyr204), phospho-Akt (Ser 473), phospho-mTOR (Thr 2448), and phospho-p70S6K (Thr421/Ser424). Data shown on each panel illustrate representative experiments for each cell type. Similar results were reproduced in at least three independent experiments. The maximal phosphorylation responses observed within the indicated time frame, are discussed in the text
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2480488&req=5

Fig5: Extracellular ATP robustly activates ERK1/2, PI3K/Akt, and mTO/p70S6K signaling pathways in VVEC but only modestly in AOEC and MPAEC. Growth- arrested cells were stimulated with ATP (100 μM) for the indicated times. Equivalent amounts of total cell protein (25–40 μg) were subjected to Western blot analysis with antibodies against phospho-ERK1/2 (Thr202/Tyr204), phospho-Akt (Ser 473), phospho-mTOR (Thr 2448), and phospho-p70S6K (Thr421/Ser424). Data shown on each panel illustrate representative experiments for each cell type. Similar results were reproduced in at least three independent experiments. The maximal phosphorylation responses observed within the indicated time frame, are discussed in the text
Mentions: Studies in vivo and in vitro have shown the importance of ERK1/2 and PI3K/mTOR pathways in controlling angiogenesis-related events and metastatic tumor growth [36–40]. Because of the differences in ATP-induced proliferative and migratory responses in VVEC, AOEC, and MPAEC, we examined the effects of ATP on activation of ERK1/2 (as measured by Thr202/Tyr204 phosphorylation), Akt (as measured by Ser473 phosphorylation), mTOR (as measured by Ser2448 phosphorylation), and p70S6K (as measured by Thr421/Ser424 phosphorylation) in the different endothelial cell types (Fig. 5). We found that ATP induced a greater stimulation of all examined signaling pathways in VVEC than in AOEC or MPAEC (Fig. 5). In particular, ATP exerted greater (maximum 10.8 fold increase) and more prolonged increase in phosphorylation of ERK1/2 in VVEC than in AOEC (maximum 4.2-fold) and in MPAEC (maximum 5.0-fold). ATP-induced phosphorylation of ERK1/2 was consistently observed in VVEC at 120 min and was not observed at this time point in AOEC or MPAEC. ATP-induced phosphorylation of Akt occurred earlier and was greater in VVEC than in AOEC and MPAEC (maximal fold increase 5.3, 3.2, and 2.4, respectively). ATP-induced phosphorylation of mTOR was greater in VVEC than in AOEC and MPAEC (maximal fold increase 4.8, 1.8, and 2.2, respectively). Maximal phosphorylation of p70S6K was also greater and more prolonged in VVEC than in AOEC and MPAEC (maximal fold increase 8.9, 2.0, and 2.4, respectively) Thus, in aggreement with the robust effect of extracellular ATP on DNA synthesis, these data support the idea that in adventitial VVEC, ERK1/2, PI3K/Akt, and mTOR/p70S6K pathways contribute to the ATP-dependent pro-angiogenic phenotype.Fig. 5

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