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

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

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

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

Adenosine-induced AKT phosphorylation in VVEC is mediated via Gαi.

<p>To dissect a role of Gi proteins in Akt activation, VVEC-Co (<b>A</b>) and VVEC-Hyp (<b>C</b>) were pre-treated with PTx (100 ng/ml, 18 h) and stimulated with 100 μM adenosine (Ado) or 10 nM CCPA for the indicated periods of time. To determine the role of adenosine A1R in Akt activation, VVEC-Co (<b>B</b>) and VVEC-Hyp (<b>D</b>) were pre-treated with 10 nM PSB 36 (30 min), a specific A1R antagonist, followed by stimulation with 100 µM adenosine (Ado) or 10 nM CCPA for the indicated periods of time. Data are representative from at least three independent experiments.</p

A1R is involved in adenosine-induced VVEC barrier function. Effect of A1R siRNA on CCPA-induced increase in TER in VVEC.

<p>(<b>A, B</b>) VVEC were incubated with A1R specific siRNA or non-specific siRNA for 48 h and then cells were stimulated with CCPA (1 nM) in TER measurement assay. The depletion of A1R mRNA and protein was confirmed by RT-PCR (<b>C</b>) and the Western blot analysis with anti-A1R antibody. (<b>D</b>). Results are presented as mean ± SE from three independent experiments.</p

The qRT-PCR analysis of adenosine receptors expression in VVEC.

<p>The fold change in mRNA expression of each adenosine receptor relative to internal housekeeping gene (ß-actin) was calculated. (<b>A</b>) Adenosine receptor mRNA levels were normalized versus VVEC-Co A3R, whose expression level was arbitrarily established as 1. The results are shown as mean ± SEM from at least three distinct VVEC populations. Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test for VVEC-Co and VVEC-Hyp separately. (<b>B</b>) Adenosine receptor mRNA levels in VVEC-Hyp were normalized to VVEC-Co for each gene. Data were analyzed by Student's t-test. **p<0.01, ***p<0.001.</p