Pulmonary vascular remodeling is key to the pathogenesis of idiopathic pulmonary arterial hypertension (IPAH). We recently reported that fibroblast growth factor (FGF)2 is markedly overproduced by pulmonary endothelial cells (P-ECs) in IPAH and contributes significantly to smooth muscle hyperplasia and disease progression. Excessive FGF2 expression in malignancy exerts pathologic effects on tumor cells by paracrine and autocrine mechanisms.We hypothesized that FGF2 overproduction contributes in an autocrine manner to the abnormal phenotype of P-ECs, characteristic of IPAH. In distal pulmonary arteries (PAs) of patients with IPAH, we found increased numbers of proliferating ECs and decreased numbers of apoptotic ECs, accompanied with stronger immunoreactivity for the antiapoptotic molecules, B-cell lymphoma (BCL)2, and BCL extra long (BCL-xL) compared with PAs from control patients. These in situ observations were replicated in vitro, with cultured P-ECs from patients IPAH exhibiting increased proliferation and diminished sensitivity to apoptotic induction with marked increases in the antiapoptotic factors BCL2 and BCL-xL and levels of phosphorylated extracellular signal-regulated (ERK)1/2 compared with control P-ECs. IPAH P-ECs also exhibited increased FGF2 expression and an accentuated proliferative and survival response to conditioned P-EC media or exogenous FGF2 treatment. Decreasing FGF2 signaling by RNA interference normalized sensitivity to apoptosis and proliferative potential in the IPAH P-ECs. Our findings suggest that excessive autocrine release of endothelial-derived FGF2 in IPAH contributes to the acquisition and maintenance of an abnormal EC phenotype, enhancing proliferation through constitutive activation of ERK1/2 and decreasing apoptosis by increasing BCL2 and BCL-xL.

Mutations affecting transforming growth factor-beta (TGF-β) superfamily receptors, activin receptor-like kinase (ALK)-1, and endoglin (ENG) occur in patients with pulmonary arterial hypertension (PAH). To determine whether the TGF-β/ALK1/ENG pathway was involved in PAH, we investigated pulmonary TGF-β, ALK1, ALK5, and ENG expressions in human lung tissue and cultured pulmonary-artery smooth-muscle-cells (PA-SMCs) and pulmonary endothelial cells (PECs) from 14 patients with idiopathic PAH (iPAH) and 15 controls. Seeing that ENG was highly expressed in PEC, we assessed the effects of TGF-β on Smad1/5/8 and Smad2/3 activation and on growth factor production by the cells. Finally, we studied the consequence of ENG deficiency on the chronic hypoxic-PH development by measuring right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary arteriolar remodeling in ENG-deficient (Eng+/−) and wild-type (Eng+/+) mice. We also evaluated the pulmonary blood vessel density, macrophage infiltration, and cytokine expression in the lungs of the animals. Compared to controls, iPAH patients had higher serum and pulmonary TGF-β levels and increased ALK1 and ENG expressions in lung tissue, predominantly in PECs. Incubation of the cells with TGF-β led to Smad1/5/8 phosphorylation and to a production of FGF2, PDGFb and endothelin-inducing PA-SMC growth. Endoglin deficiency protected mice from hypoxic PH. As compared to wild-type, Eng+/− mice had a lower pulmonary vessel density, and no change in macrophage infiltration after exposure to chronic hypoxia despite the higher pulmonary expressions of interleukin-6 and monocyte chemoattractant protein-1. The TGF-β/ALK1/ENG signaling pathway plays a key role in iPAH and experimental hypoxic PH via a direct effect on PECs leading to production of growth factors and inflammatory cytokines involved in the pathogenesis of PAH.

Idiopathic pulmonary arterial hypertension (IPAH) is a fatal disease of unknown etiology characterized by a progressive and Introduction: sustained increase in pulmonary arterial pressure and vascular remodeling that eventually obliterates the small pulmonary arteries. We have shown previously that endothelial-derived fibroblast growth factor-2 (FGF-2) is overproduced in IPAH and contributes to smooth muscle cell hyperplasia and to the progression of the disease in humans and rodents. We have now investigated the possible autocrine participation of the endothelial-derived FGF-2 in the hyperproliferative, apoptosis-resistant phenotype of pulmonary microvascular endothelial cells (PMECs) in IPAH.Lung specimens were collected during lung transplantation in 10 patients with IPAH and during lobectomy or pneumonectomy Methods: for localized lung cancer in 10 controls. The incidence of PMEC apoptosis / proliferation was investigated in distal pulmonary in situ arteries by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and proliferating cell nuclear antigen (PCNA) immunostaining. Then, primary cultures of PMECs were generated from these lung specimens and cultured before evaluation of the balance proliferation/cell death using flow cytometric and cell count analyses in basal conditions or following induction of apoptosis by different methods: serum starvation, treatment with hydrogen peroxide or cycloheximide. Expression levels of pro-(Bax) and anti-(Bcl2, Bcl-xL) apoptotic were assessed using quantitative real time-PCR and western blot analyses. We conducted parallel evaluation of the balance proliferation/cell death and pro-and anti-apoptotic protein levels with or without siRNA-mediated gene FGF2 silencing in CTR-PMECs vs. IPAH-PMECs.Compared with controls, we found in lung specimens from patients with IPAH small numbers of apoptotic PMECs and high Results: numbers of proliferating PMECs in walls of distal pulmonary arteries., we found that IPAH-PMECs are hyperproliferative and In vitro exhibited a diminished sensitivity to the induction of apoptosis in response to serum starvation, exposure to hydrogen peroxide or cycloheximide when compared to CTR-PMECs. In addition, IPAH-PMECs showed a marked decrease in the Bax/Bcl-2 and Bax/Bcl-xL ratios compared with control cells, phenomena partially mediated by an autocrine FGF2 production.Taken together our results strongly suggest that the endothelial-FGF-2 overproduction in IPAH contributes to many Conclusions: components of IPAH including maintenance of the apoptosis resistant phenotype via an autocrine loop.

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