Study On The Mechanism Of FGF - 2 And Its Downstream Signaling Pathway In The Pathogenesis Of Pulmonary Hypertension

Part I:The changes of FGF-2 and its downstream signaling pathways in pulmonary arterial hypertensionBackground:Pulmonary Arterial Hypertension (PAH) is a progressive disease induced by multiple factors, and its pathophysiologic mechanisms have not been fully understood. Fibroblast growth factor-2 (FGF-2), a member of a family of heparin binding growth factors, is a potent mitogen for a large number of cell types. It exerts its biological activity by binding to high-affinity tyrosine kinase FGF receptors (FGFRs), such as FGFR-1, expressed on the surface of vascular cells. This study aimed to investigate the time-course changes of FGF-2 and its downstream signaling pathways in PAH.Methods:The PAH model was established by intraperitoneal injection of 60mg/kg monocrotaline (MCT). Pulmonary hemodynamics were evaluated through right heart catheterization and pulmonary arteriole morphological changes were analyzed after staining with hematoxylin-eosin and modified weigert elastic stain. Western blot and immunohistochemical staining were used to investigate the changes of FGF-2/FGFR-1 and its downstream signaling pathways in lung tissues from PAH patients and rats. Fresh blood samples from 66 idiopathic PAH patients were collected, and plasma FGF-2 and BMP-2 levels were assessed with commercially available enzyme-linked immunosorbent assays (ELISA).Results:PAH rats showed an increase in pulmonary arterial pressure after monocrotaline injection, and they were characterized by pulmonary vascular media hypertrophy and mononuclear cell infiltration in morphological analysis. The expression of FGF-2 and FGFR-1 was significantly up-regulated in lung tissues from idiopathic PAH patients and MCT rats. Immunohistochemical analyses revealed that FGF-2 was predominantly located in the adventitial of pulmonary arterioles and lung mesenchyma in MCT rats, while FGFR-1 was predominantly located in the intimal layer of pulmonary arterioles. We observed an increase of ERK1/2 activity and an decrease of Akt activity in lung tissues from MCT rats. Besides, PCNA, a-SMA and cleaved caspase-3 was significantly increased after MCT injection. Conversely, the ratio of Bcl-2 to Bax, an antiapoptotic index, was lower in MCT rats. We also found that the expression of BMPR-II and p-Smad 1/5/8 were significantly decreased after MCT injection. Moreover, plasma FGF-2 and BMP-2 levels were significantly higher in patients with idiopathic PAH than in control subjects, but they did not show any correlation with clinical parameters.Conclusion:FGFR-1 and its downstream ERK1/2 signaling pathways were activated in MCT rats, and it was followed by increased smooth muscle cell proliferation and endothelial cell apoptosis.Part Ⅱ:Inhibition of FGFR signaling with PD173074 ameliorates monocrotaline-induced pulmonary arterial hypertensionBackground:Pulmonary Arterial Hypertension (PAH) is a progressive disease characterized by intima proliferation and media thickening, and the prognosis is poor. Numerous studies have demonstrated that fibroblast growth factor-2 (FGF-2) signaling may play a pivotal role in the development of pulmonary arterial hypertension. PD 173074 is a potent FGF receptor 1 (FGFR-1) inhibitor that displays high activity and selectivity. The aim of this study was to investigate the effects of PD 173074 on monocrotaline-induced PAH and FGF-2 induced pulmonary artery smooth muscle cells (PASMCs) proliferation.Methods:27 rats randomly divided into 3 groups (n=9):control, MCT+Vehicle and MCT+PD173074. PAH model was established by a single intraperitoneal injection of monocrotaline. And then a daily intraperitoneal injection of PD 173074 (20 mg/kg) was administered from day 14 to day 28. Hemodynamic parameters, right ventricular hypertrophy index and morphometry were evaluated at day 28. Western blot and immunohistochemical analysis were used to determine the expression of FGF-2 and BMP signaling in lung tissues. Rat PASMCs were prepared and cultured, MTT and western blot were used to detect the effects of PD 173074 on FGF-2 induced PASMCs proliferation and its downstream activation of ERK1/2 and Akt.Results:PD 173074 treatment partly ameliorated PAH and vascular remodeling. It decreased ERK1/2 activation and rescued total Akt expression, leading to a reduction in both proliferation and apoptosis in the lung. Besides, the expression of BMPR-Ⅱ and p-Smad 1/5/8 were significantly decreased after MCT injection, and treatment with PD173074 reduced the down-regulation of BMPR-Ⅱ and p-Smad 1/5/8. In vitro studies suggested that FGF-2 treatment promoted PASMCs proliferation. ERK1/2 and Akt were activated by FGF-2, whereas inhibition of ERK1/2 by PD98059 as well as inhibition of Akt by LY294002 ameliorated FGF-2 induced PASMCs proliferation. Furthermore, PD173074 inhibited FGF-2 induced proliferation in PASMCs through ERK1/2 and Akt inhibition.Conclusion:These results suggested that FGFR-1 inhibition with PD 173074 ameliorated PAH and vascular remodeling, and rescued BMPR-Ⅱ expression. PD 173074 inhibited FGF-2 induced proliferation in PASMCs through ERK1/2 and Akt inhibition in vitro. Thus, PD173074 may be an effective novel option for the treatment of PAH.Part Ⅲ:Plasma soluble ST2 levels correlate with disease severity and predict clinical worsening in patients with pulmonary arterial hypertensionBackground:Soluble suppression of tumorgenicity (sST2) has been proposed to be a marker for biomechanical strain and a possible predictor of mortality in patients with chronic heart failure. The use of sST2 in pulmonary arterial hypertension (PAH) has not been well defined. We Hypothesize that plasma sST2 levels may correlate with the disease severity and predict clinical worsening in PAH.Methods:We performed a cohort study of 40 IPAH patients with data on demographics, exercise capacity, echocardiographic parameters, laboratory tests, hemodynamics, and medications. Plasma sST2 was assessed with the high-sensitivity ST2 ELISA kit at diagnostic catheterization. All patients were followed-up since the date of blood sampling. The endpoint was clinical worsening.Results:sST2 was significantly elevated in patients with IPAH compared with control subjects (28.9±13.9 vs.20.7 ± 7.5 ng/ml, P=0.003). Pearson’s correlation analysis revealed that sST2 levels correlated with cardiac index (r=-0.534, p=0.000) and pulmonary vascular resistance (r=0.350, p=0.027), and could reflect disease severity of PAH. After a mean follow-up of 14±5 months, twelve patients showed clinical worsening. ROC analysis suggested that sST2 levels higher than 31.4ng/ml discriminated clinical worsening with a sensitivity and specificity of 83.3% and 78.6%, respectively. Kaplan-Meier analysis showed that higher sST2 levels (>31.4 ng/ml) were associated with poor clinical outcomes (P=0.008). Multivariate Cox regression analysis showed that sST2 was an independent predictor of clinical worsening (HR=6.067; 95% CI 1.317-27.948; P=0.021).Conclusions:sST2 correlates with disease severity and is a significant predictor of clinical worsening in patients with PAH.