Hypoxia induces pulmonary arterial fibroblast proliferation, migration, differentiation and vascular remodeling via the PI3K/Akt/p70S6K signaling pathway

Abstract

The present study was designed to examine whether hypoxia induces the proliferation, migration and differentiation of pulmonary arterial fibroblasts (PAFs) via the PI3K/Akt/p70S6K signaling pathway. PAFs were subjected to normoxia (21% O2) or hypoxia (1% O2). The proliferation, migration, differentiation and cellular p110α, p‑Akt, and p‑p70S6K expression levels of the PAFs were examined in vitro. In addition, rats were maintained under hypoxic conditions, and the right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI) and right ventricular weight/body weight ratio (RV/BW) were examined. The expression levels of p110α, p‑Akt, p70S6K, fibronectin and α‑SMA in the rat pulmonary vessels were also examined. Hypoxia significantly elevated the proliferation, migration and differentiation of rat PAFs. It also strongly elevated the expression of p110α, p‑Akt and p‑p70S6K in PAFs in vitro. NVP‑BEZ235 was revealed to significantly reduce the hypoxia‑induced proliferation, migration and differentiation. In vivo experiments demonstrated that hypoxia significantly induced the elevation of RVSP, RVHI, RV/BW, medial thickening, adventitious thickening, and fibronectin and collagen deposition around pulmonary artery walls. The expression of p110α, p‑Akt and p70S6K was evident in the pulmonary arteries of the hypoxic rats. NVP‑BEZ235 significantly reduced the hypoxia‑induced hypoxic pulmonary vascular remodeling, as well as fibronectin and collagen deposition in the pulmonary arteries. Therefore, hypoxia was demonstrated to induce the proliferation, migration and differentiation of PAFs and the hypoxic pulmonary vascular remodeling of rats via the PI3K/Akt/p70S6K signaling pathway.

Figure 1

Hypoxia-induces the proliferation of PAFs in vitro. (A) The proliferation of PAFs was measured using a cell counting kit-8 assay following treatment as indicated for 24 h (n=8). (B) Western blot analysis of PCNA expression in all PAF groups following treatment for 24 h (n=3). (C) PCNA expression analysis of the western blot analyses. N, normoxia; H, hypoxia; HB, hypoxia with NVP-BEZ235; B, NVP-BEZ235; PAFs, pulmonary arterial fibroblasts; PCNA, proliferating cell nuclear antigen. ^**P<0.01 and ***P<0.001.

Figure 2

Hypoxia induces the migration of PAFs in vitro. (A) Results of the scratch assay in PAFs following 12 h of treatment (n=3). (B) Migration area results of the scratch assay (n=6). (C) Cell numbers in the Transwell migration assay determined by cell counting following staining (n=6). PAFs, pulmonary arterial fibroblasts; N, normoxia; H, hypoxia; HB, hypoxia with NVP-BEZ235; HR, hypoxia with rapamycin; HL, hypoxia with LY294002; B, NVP-BEZ235; R, rapamycin; L, LY294002. ^**P<0.01 and ^***P<0.001.

Figure 3

Hypoxia-induced differentiation of PAFs requires activation of the PI3K/Akt/p70S6K signaling pathway. (A) Cellular morphology of PAFs under normoxic and hypoxic conditions. The images were captured using an optical microscope (magnification, main images, ×200, enlarged images, ×450). A portion of the photographs was enlarged and displayed at the bottom left corner. (B) PAFs were treated as indicated for 24 h, then subjected to immunofluorescence staining. α-SMA was stained green, GAPDH was stained red and the nuclei were stained blue by DAPI in the merged images. (C) The expression of α-SMA in the immunofluorescence photographs was examined (n=3). (D) Western blot analysis was performed to evaluate the expression of α-SMA and GAPDH in the PAFs. (E) The expression of α-SMA in the western blot was quantified. PAFs, pulmonary arterial fibroblasts; N, normoxia; H, hypoxia; HB, hypoxia with NVP-BEZ235; HR, hypoxia with rapamycin; HL, hypoxia with LY294002; α-SMA, α-smooth muscle actin. ^**P<0.01 and ^***P<0.001.

Figure 4

Hypoxia upregulates the PI3K/Akt/p70S6K signaling pathway in PAFs. (A) Western blot analysis of the indicated proteins in PAFs following treatment with NVP-BEZ235 and hypoxia as indicated for 24 h. Quantification of (B) p110α, (C) p-Akt and (D) p-p70S6K expression in the western blot assay (n=3). PAFs, pulmonary arterial fibroblasts; PI3K, phosphatidylinositol-3-kinase; Akt, protein kinase B; p70S6K, p70 ribosomal protein S6 kinase; p, phosphorylated; N, normoxia; H, hypoxia; HB, hypoxia with NVP-BEZ235; B, NVP-BEZ235. ^*P<0.05 and ^**P<0.01.

Figure 5

NVP-BEZ235 inhibits the PI3K/Akt/p70S6K signaling pathway in rat PAFs. (A) Western blot analysis of the dose response of PAFs treated with NVP-BEZ235 for 24 h. Quantitative analysis of (B) p110α, (C) p-Akt and (D) p-p70S6K expression in the western blot assay (n=3). (E) Western blot analysis of the response of PAFs treated with NVP-BEZ235 (10 nM) for increasing time periods. Quantitative analysis of (F) p110α, (G) p-Akt and (H) p-p70S6K expression in the western blot assay (n=3). PAFs, pulmonary arterial fibroblasts; PI3K, phosphatidylinositol-3-kinase; Akt, protein kinase B; p70S6K, p70 ribosomal protein S6 kinase; p, phosphorylated. ^*P<0.05, ^**P<0.01 and ^***P<0.001.

Figure 6

NVP-BEZ235 inhibits the hypoxia-induced proliferation, migration and differentiation of rat PAFs in a dose- and time-dependent manner. (A) The proliferation results of PAFs treated with increasing concentrations of NVP-BEZ235 as measured by a CCK-8 assay (n=8). (B) The proliferation results of PAFs treated with 10 nM NVP-BEZ235 for increasing periods of time as measured by a CCK-8 assay (n=8). The cell migration results of PAFs treated with (C) increasing concentrations of NVP-BEZ235 and (D) 10 nM NVP-BEZ235 for increasing time periods as measured by a Transwell assay (n=8). (E) Western blot analysis of the expression of α-SMA and β-actin in PAFs treated with increasing concentrations of NVP-BEZ235 for 24 h. (F) Quantitative analysis of the α-SMA expression observed in the western blot analyses (n=3). (G) Western blot analysis of the expression of α-SMA and GAPDH in PAFs treated with 10 nM NVP-BEZ235 for increasing time periods. (H) Quantitative analysis of the α-SMA expression observed in the western blot analyses (n=3). PAFs, pulmonary arterial fibroblasts; CCK-8, cell counting kit-8; α-SMA, α-smooth muscle actin. ^*P<0.05, ^**P<0.01 and ^***P<0.001.

Figure 7

The PI3K/Akt/p70S6K signaling pathway contributes to the physiological process of hypoxia in rats. (A) RVSP, (B) RVHI, (C) HCT and (D) RV/BW results from rats in different groups. RVSP, right ventricular systolic pressure; RVHI, right ventricular hypertrophy index; HCT, hematocrit; RV/BW, right ventricular/body weight ratio; N, normoxia; H, hypoxia; HB, hypoxia plus NVP-BEZ235. ^**P<0.01 and ^***P<0.001.

Figure 8

The PI3K/Akt/p70S6K signaling pathway contributes to the pulmonary vascular remodeling of hypoxic rats. (A) H&E, Russell Movat, Masson and immunohistochemical staining of FN and α-SMA in lung tissue isolated from rats in the different groups. The black arrows indicate the pulmonary vessels. The (B) medial thickness, (C) medial cross-sectional area, (D) adventitial thickness and (E) collagen deposition area were calculated. A total of 3 rats were randomly selected from each group and at least five pulmonary arterials of each rat were measured. Scale bar, 100 µm. N, normoxia; H, hypoxia; HB, hypoxia with NVP-BEZ235; FN, fibronectin; H&E, hematoxylin and eosin. ^**P<0.01 and ^***P<0.001.

Figure 9

The PI3K/Akt/p70S6K signaling pathway is upregulated in the pulmonary vessels of rats following hypoxia. Immunohistochemistry results of p110α, p-Akt and p70S6K in the pulmonary vessels of rats. The black arrows identify the positively stained areas. Scale bar, 100 µm. N, normoxia; H, hypoxia; HB, hypoxia with NVP-BEZ235; Akt, protein kinase B; p, phosphorylated; p70S6K, p70 ribosomal protein S6 kinase.