Role of purinergic signaling in pathological pulmonary vascular remodeling

Irreversible vascular remodeling has a central role in a variety of pathophysiological conditions including pulmonary arterial hypertension (PAH). Hypoxia and inflammation are prominent features in PAH, along with hyperplasia and hypertrophy of vessel wall layers. Although, endothelial cell (EC) dysfunction is thought to drive the multiple forms of vascular remodeling, the origins of this phenomenon are poorly understood. Extracellular ATP and its metabolites are important regulators of vascular tone, permeability, and homeostasis. Yet little is known about their role in pathological vascular remodeling. By using chronic hypoxia and PAH animal models as well as human PAH patient samples, this study was undertaken to evaluate the catalytic activities and expression levels of nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, otherwise known as CD39) and other purine-converting ectoenzymes with a primary focus on vascular EC. For this purpose we employed thin-layer chromatographic enzyme assays with H-labelled nucleotide substrates, in combination with various immunoassays and qPCR. In addition we have developed a highly sensitive assay for simultaneous sensing of extracellular ATP and its metabolites and also a novel method for measuring CD39 activity in modeled to in vivo conditions. In functional assays, cells or animals were stimulated through purine signaling pathways and proliferation, apoptosis, permeability, and DNA damage were assayed. Our results clearly demonstrated that the activity of CD39 was downregulated in chronic hypoxia, monocrotaline induced animal models of PAH and in human PAH patients. Attenuated enzyme activities could create a niche in the vasculature where ATP levels were increased and adenosine levels were decreased. Even a small increase in ATP concentration was enough to induce an apoptosis-resistant, hyper-proliferative, and DNA-damageresistant phenotype in ECs of pulmonary origin. The observed effects were at least partly dependent on P2Y11 receptor activation. In addition, we found that low ATP concentrations could induce pulmonary smooth muscle cell proliferation and migration. Interestingly, we found that small apelin peptide could directly restore the downregulated CD39 activity. This study implies that purinergic signaling, ATP mediated cell activation in particular, plays a truly significant role in pathological vascular remodeling, and that it could be used as a therapeutic target. Moreover, purinergic signaling pathways could be used before vascular injury to precondition EC against irradiation or chemotherapy induced DNA damage.