Sildenafil attenuates hypoxic pulmonary remodelling by inhibiting bone marrow progenitor cells

Abstract

The recruitment of bone marrow (BM)-derived progenitor cells to the lung is related to pulmonary remodelling and the pathogenesis of pulmonary hypertension (PH). Although sildenafil is a known target in PH treatment, the underlying molecular mechanism is still elusive. To test the hypothesis that the therapeutic effect of sildenafil is linked to the reduced recruitment of BM-derived progenitor cells, we induced pulmonary remodelling in rats by two-week exposure to chronic hypoxia (CH, 10% oxygen), a trigger of BM-derived progenitor cells. Rats were treated with either placebo (saline) or sildenafil (1.4 mg/kg/day ip) during CH. Control rats were kept in room air (21% oxygen) with no treatment. As expected, sildenafil attenuated the CH-induced increase in right ventricular systolic pressure and right ventricular hypertrophy. However, sildenafil suppressed the CH-induced increase in c-kit⁺ cells in the adventitia of pulmonary arteries. Moreover, sildenafil reduced the number of c-kit⁺ cells that colocalize with tyrosine kinase receptor 2 (VEGF-R2) and CD68 (a marker for macrophages), indicating a positive effect on moderating hypoxia-induced smooth muscle cell proliferation and inflammation without affecting the pulmonary levels of hypoxia-inducible factor (HIF)-1α. Furthermore, sildenafil depressed the number of CXCR4⁺ cells. Collectively, these findings indicate that the improvement in pulmonary haemodynamic by sildenafil is linked to decreased recruitment of BM-derived c-kit⁺ cells in the pulmonary tissue. The attenuation of the recruitment of BM-derived c-kit⁺ cells by sildenafil may provide novel therapeutic insights into the control of pulmonary remodelling.

Keywords: CXCR4 receptor; bone marrow progenitor cells; c-kit cells; chronic hypoxia; pulmonary hypertension; sildenafil.

Figure 1

Sildenafil preserves survival pathways in hypoxia. (A) Left panel. Representative Western blots of whole pulmonary tissue homogenates for phospho‐eNOS (P‐eNOS), eNOS, phospho‐Akt (P‐Akt) and Akt. GAPDH was used as a loading control. Right panel. Densitometric analysis, calculated as fold increase versus control. (B) Left panel. Representative confocal immunofluorescence images of pulmonary tissue stained with antibody against Ki‐67 (red) with nuclei counterstained with HOECHST (blue). Right panel. Percentage of cells with a positive nuclear staining for Ki‐67 with respect to all nuclei. Scale bars = 20 μm. Data are presented as mean±SEM (n = 5/group); *P < 0.05 versus control, ^#P < 0.05 versus chronic hypoxia (CH) (one‐way anova with Bonferroni post‐test).

Figure 2

Sildenafil reduces hypoxia‐induced increase in c‐kit⁺ cell recruitment. (A) Representative confocal immunofluorescence staining for c‐kit⁺ cells (red). Nuclei were counterstained with HOECHST (blue). Scale bar = 20 μm. The plot (B) shows the quantification of the number of c‐kit⁺ cells/vessel (5 fields per section). Same details as in Fig. 1.

Figure 3

Sildenafil reduced hypoxia‐induced increase in c‐kit⁺/VEGF‐R2⁺g. Representative confocal immunofluorescence costainings for c‐kit⁺ (red) and VEGF‐R2 (green). Nuclei were counterstained with HOECHST (blue). Scale bars = 50 μm. The plot shows the quantification of the number of c‐kit⁺/VEGF‐R2⁺ cells (5 fields per section). Same details as in Fig. 1.

Figure 4

Sildenafil reduced hypoxia‐induced increase in c‐kit⁺/CD68⁺. Representative confocal immunofluorescence costainings for c‐kit⁺ (red) and CD‐68 (green). Nuclei were counterstained with HOECHST (blue). Scale bars = 50 μm. The plot shows the quantification of the number of c‐kit⁺/CD68 cells (5 fields per section). Same details as in Fig. 1.

Figure 5

Effects of chronic hypoxia (CH) and sildenafil on muscularization and inflammation. (A) Left panel. Representative confocal images of vessels stained with α‐SMA (red) and PECAM‐1 (green). The plot (right panel) shows the index of muscularization of vessels calculated as per cent of α‐SMA staining on PECAM‐1‐positive vessels. Same details as in Fig. 1. (B) Representative confocal immunofluorescence staining of pulmonary sections for α‐SMA (green) and Ki‐67 (red). Nuclei were counterstained with HOECHST (blue). Scale bars = 20 μm. (C) Representative confocal immunofluorescence staining of pulmonary sections for CD68 (red) and Ki‐67 (green). Nuclei were counterstained with HOECHST (blue). Scale bars = 20 μm.

Figure 6

Sildenafil regulates mobilization and recruitment of CXCR4⁺ cells. (A) Left panel. mRNA quantification of HIF‐1α. Same details as in Fig. 1. Right panel. Immunohistochemical staining (brown) of HIF‐1α in haematoxylin pulmonary sections. (B) Left panel. Representative confocal microscopy images for CXCR4 (red) in pulmonary tissue. Nuclei were counterstained with HOECHST (blue). Scale bar = 50 μm. The right panel shows the number of CXCR4⁺ cells per vessel (5 fields per section). Same details as in Fig. 1.

Figure 7

Outcome of altered recruitment of c‐kit⁺ cells. (A) Quantification in 10 fields/sample of the number of vessels with diameter <50 μm. (B) Morphometric analysis performed on 20 pulmonary vessels (<50 μm diameter) to assess the percentage of medial wall thickness of pulmonary arteries. (C) Right ventricular hypertrophy, calculated as the weight of RV/left ventricle + septum. Right panel. (D) Left and the right ventricular pressures. Same details as in Fig. 1.