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* From University of Colorado Health Sciences Center (Drs. Gerasimovskaya, Davie, and Stenmark, and Mr. Tucker), Denver; and National Jewish Medical and Research Center (Drs. Ahmad and White), Denver, CO.
Correspondence to: Evgenia V. Gerasimovskaya, PhD, University of Colorado Health Sciences Center, 4200 E 9th Ave, B-131, Denver, CO 80262; e-mail: Evgenia.Gerasimovskaya{at}UCHSC.edu
Long-term exposure (persistent or intermittent) of man and most mammalian species to hypoxia results in the development of pulmonary hypertension. Significant changes in the structure of pulmonary vessels accompany the hypertensive process and include marked thickening of both the media and adventitia with particularly striking fibroproliferative changes observed in adventitia of chronically hypoxic neonatal animals. The cellular and molecular mechanisms contributing to these changes remain unclear. In this context, it is important to note that the adventitial compartment of at least elastic and large muscular arteries includes within its borders a blood supply, the vasa vasorum. This microcirculatory network has traditionally been assigned a passive role in maintaining vessel integrity through supply of oxygen and nutrients to the outer part of the vessel wall. Data123 have emerged, however, suggesting that the vasa network may contribute to the initiation and progression of vascular diseases in the systemic circulation, including atherosclerosis, restenosis, and vasculitis. We have reported4 marked neovascularization of the vasa vasorum at all points along the longitudinal axis of the pulmonary circulation in neonatal calves exposed to long-term hypoxia. These observations raise questions as to how this neovascularization occurs and ultimately how this process affects structural remodeling of the pulmonary circulation under hypoxic conditions.
Extracellular nucleotides (both purines and pyrimidines) have been proposed to be important in angiogenic responses. Specifically, extracellular adenosine triphosphate (ATP) and uridine triphosphate have been demonstrated to stimulate DNA synthesis in endothelial cells, smooth-muscle cells, and adventitial fibroblasts.567 In addition, extracellular nucleotides appear to directly affect migration of vascular as well as nonvascular cells including monocytes. It has been suggested that some of these effects may be mediated through a direct effect of ATP on vascular cell adhesion molecule-1 expression in endothelial cells, which is mediated by nucleotide receptor (P2Y2) transactivation of a receptor tyrosine kinase-2.8 It is also known that extracellular nucleotides are released in response to mechanical forces and other environmental stresses such as osmotic shock, acidosis, hyperoxia and hypoxia by cells of the vasculature, airways, and gut and play a key role in transducing cellular responses via activation of purinergic receptors in these organs.91011 In fact, hypoxia-induced release of ATP has been shown to play a critical role in modulating proliferative and secretory functions of endothelial cells and fibroblasts.711 We have previously shown712 that hypoxia induces release of ATP from adventitial fibroblasts, and that this ATP acts in an autocrine manner to stimulate adventitial fibroblast proliferation, a response previously shown to be critical in the vascular remodeling observed in response to hypoxia. We also found that extracellular ATP acts synergistically with cytokines and peptide mitogens known to be released by vascular cells under hypoxic conditions (eg, platelet-derived growth factor, epidermal growth factor, insulin-like growth factor) to stimulate growth, emphasizing the important role of ATP in hypoxia-induced vascular remodeling.7 These observations stimulated us to evalulate the possibility that the endothelium of newly formed adventitial vasa vasorum represents another, as yet unidentified, source of extracellular ATP, and that extracellular ATP would act to contribute to vasa vasorum neovascularization.
To test this hypothesis, we first had to isolate vasa vasorum endothelial cells (VECs) from the adventitia of pulmonary arteries. Using explant techniques followed by cloning ring-enhanced cell purification, VECs were successfully isolated from pulmonary artery adventitia of chronically hypoxic calves. These cells expressed CD31, Flk-1, Tie-1, von Willebrand factor, and endothelial nitric oxide synthase, as did pulmonary artery endothelial cells cultured from the same vessel. In addition, VECs expressed ephrin B2, Ac133, c-kit, and the lectin Lycopersicon esculentum, suggesting that these VECs represent an activated microvascular endothelial phenotype.
We evaluated the effect of hypoxic exposure (3% O2; PO2 of 45 to 50 mm Hg; 30 to 60 min) on ATP release from VECs. Extracellular ATP concentration increased from 0.02 nmol (basal) to 1.95 to 2.12 nmol in the media of hypoxia-exposed VECs. We also sought to determine if extracellular ATP would stimulate VEC proliferation and thus raising the possibility that ATP could act as an autocrine growth regulator of VECs. We found that extracellular ATP dose-dependently increased DNA synthesis (up to 15-fold) in VECs.
We next sought to define the intracellular pathways involved in ATP-induced mitogenic responses and hypoxia-induced ATP release. We found that ATP (100 µmol, 60 min), as well as hypoxia (3% O2, 60 min) stimulated PI3K activity measured in vitro in phosphotyrosine and PI3K regulatory subunit p85-immunoprecipitates in VEC. In addition, extracellular ATP induced phosphorylation of ERK1/2, Akt, and p70S6K, all known to be associated with the proliferative responses. We found that the PI3K inhibitors wortmannin (1 µmol) and LY294002 (20 µmol) blocked hypoxia-induced ATP release by 67% and 63%, respectively, and that the Rho kinase inhibitor Y27632 (10 µmol) decreased the release by 52%. These findings indicate that similar molecular pathways are involved in hypoxia-induced ATP release as well as in ATP-induced mitogenic responses in VEC. In summary, our data support the idea that ATP released by VECs and fibroblasts into the local adventitial environment may serve as a potent autocrine/paracrine factor contributing to vasa vasorum neovascularization in chronic hypoxic pulmonary hypertension (Fig 1 ).
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This work has been supported by National Institutes of Health Specialized Center of Research grant No. 5-P50 HL57144–08 and PPG# 2P01 HL014985–31.
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