Objective— Inflammation and dysregulated angiogenesis are features of endothelial dysfunction in pulmonary hypertension. Neutrophil extracellular traps (NETs), produced by dying neutrophils, contribute to pathogenesis of numerous vascular disorders but their role in pulmonary hypertension has not been studied. We sought evidence of (NETs) formation in pulmonary hypertension and investigated the effect of NETs on endothelial function. Approach and Results— Plasma and lung tissues of patients with pulmonary hypertension were analyzed for NET markers. The effects of NETs on endothelial function were studied in vitro and in vivo. Patients with chronic thromboembolic pulmonary hypertension and idiopathic pulmonary hypertension showed elevated plasma levels of DNA, neutrophil elastase, and myeloperoxidase. NET-forming neutrophils and extensive areas of NETosis were found in the occlusive plexiform lesions and vascularized intrapulmonary thrombi. NETs induced nuclear factor κB–dependent endothelial angiogenesis in vitro and increased vascularization of matrigel plugs in vivo. Angiogenic responses were associated with increased release of matrix metalloproteinase-9, heparin-binding epidermal growth factor–like growth factor, latency-associated peptide of the transforming growth factor β1, and urokinase-type plasminogen activator, accompanied by increased endothelial permeability and cell motility. NETs-induced responses depended on myeloperoxidase/H 2 O 2 -dependent activation of Toll-like receptor 4/nuclear factor κB signaling. NETs stimulated the release of endothelin-1 in HPAECs (human pulmonary artery endothelial cells) and stimulated pulmonary smooth muscle cell proliferation in vitro. Conclusions— We are the first to implicate NETs in angiogenesis and provide a functional link between NETs and inflammatory angiogenesis in vitro and in vivo. We demonstrate the potential pathological relevance of this in 2 diseases of disordered vascular homeostasis, pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension.

AimsRhoB plays a key role in the pathogenesis of hypoxia-induced pulmonary hypertension. Farnesylated RhoB promotes growth responses in cancer cells and we investigated whether inhibition of protein farnesylation will have a protective effect.Methods and resultsThe analysis of lung tissues from rodent models and pulmonary hypertensive patients showed increased levels of protein farnesylation. Oral farnesyltransferase inhibitor tipifarnib prevented development of hypoxia-induced pulmonary hypertension in mice. Tipifarnib reduced hypoxia-induced vascular cell proliferation, increased endothelium-dependent vasodilatation and reduced vasoconstriction of intrapulmonary arteries without affecting cell viability. Protective effects of tipifarnib were associated with inhibition of Ras and RhoB, actin depolymerization and increased eNOS expression in vitro and in vivo. Farnesylated-only RhoB (F-RhoB) increased proliferative responses in cultured pulmonary vascular cells, mimicking the effects of hypoxia, while both geranylgeranylated-only RhoB (GG-RhoB), and tipifarnib had an inhibitory effect. Label-free proteomics linked F-RhoB with cell survival, activation of cell cycle and mitochondrial biogenesis. Hypoxia increased and tipifarnib reduced the levels of F-RhoB-regulated proteins in the lung, reinforcing the importance of RhoB as a signalling mediator. Unlike simvastatin, tipifarnib did not increase the expression levels of Rho proteins.ConclusionsOur study demonstrates the importance of protein farnesylation in pulmonary vascular remodelling and provides a rationale for selective targeting of this pathway in pulmonary hypertension.

Study objectives: Small GTPase RhoB contributes to pulmonary vascular remodelling in pulmonary hypertension. Farnesylation of RhoB augments cell proliferation while geranylgeranylation of RhoB is believed to be pro-apoptotic. We hypothesized that farnesyltransferase inhibitors may prevent vascular pathology in pulmonary hypertension. We studied the effects of farnesyltransferase inhibitor Tipifarnib on pulmonary vascular remodelling and vasoreactivity in chronically hypoxic pulmonary hypertensive mice and cultured human pulmonary artery endothelial cells (HPAECs). Chemical proteomics was used to characterise Tipifarnib-induced changes in protein prenylation while label- free quantitative proteomics was used to characterise changes induced by overexpression of RhoB prenylation mutants: farnesylated-only or geranlygeranylated-only RhoB in HPAECs. The effects of Tipifarnib on Ras and Rho GTPases expression and activity, filamentous and globular actin levels and eNOS pathway, were also studied. Results: Oral administration of Tipifarnib significantly attenuated chronic hypoxia-induced pulmonary hypertension in mice. This protective effect was associated with a marked improvement of endothelium-dependent vasodilatation, reduction in pulmonary vascular muscularization and a decrease in the right ventricular systolic pressure. Tipifarnib reduced farnesylation and increased geranyl-geranylation of several proteins, including Rho GTPases and Ras and reduced their activity in vitro and in vivo. Tipifarnib-induced changes in cultured cells were associated with a decrease in cell proliferation, decreased polymerization of actin and increased eNOS mRNA and protein levels. Proteomic analysis of HPAECs overexpressing prenylation mutants of RhoB suggests that protective effects of Tipifarnib may result, at least in part, from inhibition of RhoB farnesylation. Conclusions: We demonstrate protective effects of farnesyltransferase inhibitor Tipifarnib in chronic hypoxia-induced pulmonary hypertension. The effects of Tipifarnib may result from inhibition of small GTPases activity and increase in NO signalling.