Autocrine fibroblast growth factor-2 signaling contributes to altered endothelial phenotype in pulmonary hypertension

Abstract

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.

Figure 1. Representative photomicrographs of distal-pulmonary arteries (PAs) in lung sections from IPAH patients and controls

Co-immunolocalization of (A) vWF factor and PCNA, (B) vWF and TUNEL, (C) vWF and BCL2, (D) vWF and BCL-xL. The percentage of PCNA- and TUNEL-positive ECs were obtained from the analysis of 15–20 different distal pulmonary arteries selected randomly in each patient. [PCNA- or TUNEL-positive P-ECs (arrows); PCNA- or TUNEL-positive cells (*)]. Scale bar=50 μm. Values are mean±SEM (n=3–5). **P<0.01, ***P<0.001 compared to control-PAs.

Figure 2. In vitro proliferation and apoptosis analysis of human pulmonary endothelial cells (P-ECs) isolated from IPAH- and control-lung biopsies

Proliferation of control- and IPAH-P-ECs was assessed by (A) BrdU incorporation and (B) viable cell counts under basal conditions, in presence of FCS or after apoptosis induction by serum deprivation (24h). (C) Representative two-color FACS analysis of the annexinV-FITC assays and quantification of the percentage of annexinV⁺/PI⁻ P-EC under basal conditions, in presence of FCS and after apoptosis induction by serum deprivation. (D) Evaluation of sensitivity to apoptosis induced by H₂O₂ and cycloheximide. *P<0.05, **P<0.01, ***P<0.001 compared to control-P-ECs under basal conditions. §P<0.05, §§P<0.01, §§§P<0.001 between control- and IPAH-P-ECs subjected to the same treatment.

Figure 3. Evaluation of the expression of pro-apoptotic (Bax), anti-apoptotic (BCL2 and BCL-xL) factors, and ERK1/2 phosphorylation status in cultured human control- and IPAH-P-ECs and role in the apoptosis-resistance and the proliferative potential

(A) Relative levels of mRNAs encoding Bax, BCL2, BCL-xL, and values of the Bax:BCL2 and Bax:BCL-xL ratios. (B) Representative western immunoblots of Bax, BCL2, BCL-xL, and β-actin loading control and quantification of the Bax:BCL2 and Bax:BCL-xL ratios. (C) Representative western immunoblots of Bax, BCL2, BCL-xL, and β-actin loading control in IPAH-P-ECs 72h after transfection with lipofectamine alone, scrambled-sequence, BCL2-siRNA, BCL-xL-siRNA. (D) Quantification of the percentage of annexinV⁺/PI⁻ cells in the IPAH-P-EC population with 10%-FCS or after apoptosis induction by serum deprivation (24h), 48h after transfection with BCL2-siRNA and/or BCL-xL-siRNA or scrambled-sequence. (E) Representative western immunoblots of phosphoERK1/2 and ERK2 and quantification of the phosphoERK1/2:ERK2 ratio. (F) Proliferation of control- and IPAH-P-ECs assessed by BrdU incorporation under basal condition in presence of PD98059 or vehicle. Values are mean±SEM (n=5–10). *P<0.05, **P<0.01, ***P<0.001 between control- and IPAH-P-ECs or compared to IPAH-P-ECs transfected with the scrambled-sequence. §P<0.05 between control- and IPAH-P-ECs subjected to the same treatment.

Figure 4. Analysis of mRNA levels of FGF2 and its receptors in cultured human control- and IPAH-P-ECs and evaluation of their responsiveness to FGF2 under serum deprivation

(A) Relative levels of mRNAs encoding FGF2, and FGF-Rs in control- and IPAH-P-ECs. (B) Representative western immunoblots of FGF-R2, and β-actin loading control and quantification of the FGF-R2:β-actin ratio. (C) Evaluation of the balance between proliferation/apoptosis of quiescent control- and IPAH-P-ECs under serum deprivation with or without conditioned media from IPAH- or control-P-ECs (D) or rHu-FGF2. Bars represent mean±SEM (n=4–8). *P<0.05, **P<0.01, ***P<0.001 compared to control-P-ECs. §P<0.05, §§P<0.01 between control- and IPAH-P-ECs subjected to the same treatment.

Figure 5. Effect of FGF2 on the expression of Bax, BCL2, BCL-xL, on the ERK1/2 phosphorylation status, and its autocrine contribution to protection against serum-deprivation-induced apoptosis and to the excessive proliferation

(A) Representative western immunoblots of Bax, BCL2, BCL-xL, and β-actin loading control and quantification of the Bax:BCL2, and Bax:BCL-xL ratios. (B) Representative western immunoblots of phosphoERK1/2, ERK2 and quantification of the phosphoERK1/2:ERK2 ratio. (C) FGF2 protein levels in conditioned media from control- and IPAH-P-ECs with or without FGF2-siRNA or scrambled sequence. (D) Representative two-color FACS analysis of the annexinV-FITC assays and quantification of the percentage of annexinV⁺/PI⁻ cells in control-P-EC and IPAH-P-EC population after apoptosis induction by serum deprivation (24h), 48h after transfection with either FGF2-siRNA or scrambled-sequence with or without rHu-FGF2 (10ng/mL). (E) Representative western immunoblots of Bax, BCL2, and BCL-xL and quantification of the Bax:BCL2, and Bax:BCL-xL ratios. (F) Proliferation of control- and IPAH-P-ECs 48h after transfection with either FGF2-siRNA or scrambled-sequence assessed by BrdU incorporation. (G) Representative western immunoblots of phosphoERK1/2 and ERK2 and quantification of the phosphoERK1/2:ERK2 ratio. Values are mean±SEM (n=3–7). *P<0.05, **P<0.01 ***P<0.001 compared to 0ng/mL of rHu-FGF2 or to control-P-ECs transfected with the scrambled sequence. §P<0.05 between control- and IPAH-P-ECs subjected to the same treatment.