Endothelial Progenitor Cells Attenuate Ventilator-Induced Lung Injury with Large-Volume Ventilation

Abstract

Ventilator-induced lung injury (VILI) is a common complication that results from treatment with mechanical ventilation (MV) in acute respiratory distress syndrome (ARDS) patients. The present study investigated the effect of endothelial progenitor cell (EPC) transplantation on VILI. Wistar rats were divided into three groups (n = 8): sham (S), VILI model (V) induced by tidal volume ventilation (17 mL/kg), and VILI plus EPC transplantation (VE) groups. The lung PaO₂/FiO₂ ratio, pulmonary wet-to-dry (W/D) weight ratio, number of neutrophils, total protein, neutrophil elastase level, and inflammatory cytokines in bronchoalveolar lavage fluid (BALF) and serum were examined. Furthermore, the histological and apoptotic analysis, and lung tissue protein expression analysis of Bax, Bcl-2, cleaved caspase-3, matrix metalloproteinase (MMP)-9, total nuclear factor kappa B (total-NF-κB), phosphorylated NF-κB (phospho-NF-κB) and myosin light chain (MLC) were performed. The ventilation-induced decrease in PaO₂/FiO₂ ratio, and the increase in W/D ratio and total protein concentration were prevented by the EPC transplantation. The EPC transplantation (VE group) significantly attenuated the VILI-induced increased expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8, MMP-9, phospho-NF-κB and MLC, neutrophil elastase levels and neutrophil counts in BALF. In addition, the anti-inflammatory factor IL-10 increased in the VE group. Furthermore, pulmonary histological injury and apoptosis (TUNEL-positive cells, increase in Bax and cleaved caspase-3) were considerably diminished by the EPC transplantation. The EPC transplantation ameliorated the VILI. The mechanism may be primarily through the improvement of epithelial permeability, inhibition of local and systemic inflammation, and reduction in apoptosis.

Keywords: acute lung injury; endothelial progenitor cell; mechanical ventilation; ventilator.

Figure 1.

Characterization of EPCs. Rat peripheral blood mononuclear cells were isolated by density gradient centrifugation, as described in the Materials and Methods. The endothelial characteristics were identified through the positive staining of both DiI-acetyl-LDL (A, red) and FITC-UEA-1 (B, green). The merged image of DiI-acetyl-LDL and FITC-UEA-1 is presented in C (yellow). Furthermore, EPCs were recognized through the cytoplasmic positive signals of both VEGFR-2 (D, 400 × magnification, red) and CD34 (E, 400 × magnification, green). The merged image of VEGFR2 and CD34 is presented in F (yellow). In addition, EPCs were identified with VEGFR-2 (G, 400 × magnification, red) and CD133 (H, 400 × magnification, green). The merged image of VEGFR2 and CD34 is presented in I (yellow). From the tenth day after isolation, these cells were characteristic of a monolayer (J, 400 × magnification). Abbreviations: EPCs, endothelial progenitor cells; LDL, low density lipoprotein; UEA-1, ulexeuropaeus agglutinin-1; VEGFR2, vascular endothelial growth factor receptor 2.

Figure 2.

Tracking EPCs following the transplantation in lung tissue. EPCs transplanted to lung tissues at 24 h after injection were detected and identified by acetyl-LDL immunofluorescence staining. The positive acetyl-LDL stain presented as red (A), and the nuclei was visualized by DAPI (blue, B). The merged image is presented in C (200 × magnification). The DAPI stain in the tissue of rats in the V group is presented in D. Abbreviations: DAPI, 4-,6-diamidino-2-phenylindole; EPCs, endothelial progenitor cells; LDL, low density lipoprotein. Each testing group contained eight rats.

Figure 3.

The lung transplantation of EPCs improved the ventilation-induced PaO₂/FiO₂ ratio, W/D weight ratio and protein concentration. The PaO₂/FiO₂ ratio (A), lung W/D weight ratio (B), and protein concentration in the BALF (C) were analyzed in the samples obtained from rats in the S group. The VILI model and EPC transplanted rats before (left) and after 24-h ventilation (right) are presented. Each testing group contained eight rats. *p < 0.05 vs. the S group; ^# p < 0.05 vs. the V group. Abbreviations: BALF, bronchoalveolar lavage fluid; EPCs, endothelial progenitor cells; PaO₂/FiO₂, partial pressure of O₂ to fraction inspiratory O₂; S group, sham; VILI, ventilator-induced lung injury; W/D, pulmonary wet/dry weight. The levels of TNF-α (D), IL-1β (E), IL-8 (F), IL-10 (G), neutrophil count (H), and neutrophil elastase (I) in the BALF were examined in the S, V, and VE groups by ELISA assay. *p < 0.05 vs. the S group; ^# p < 0.05 vs. the V group. The serum levels of TNF-α (J), IL-1β (K), and IL-8 (L), IL-10 (M), ICAM-1 (N) and MIP-2 (O) in the S group (), V group () and VE group () were detected using the corresponding ELISA kits before (left panels) and after (right panels) ventilation. *p < 0.05, when compared with the S group; ^# p < 0.05, when compared with the V group. Abbreviations: ELISA, enzyme-linked immunosorbent assay; EPCs, endothelial progenitor cells; ICAM, intercellular adhesion molecule; IL, interleukin; MIP, macrophage inflammatory protein; S group, sham; TNF, tumor necrosis factor; V group, VILI group; VE group, VILI with EPC transplantation; VILI, ventilator-induced lung injury. Each testing group contained eight rats.

Figure 4.

EPC transplantation decreased the ventilation-induced increase of MMP-9, phosphorylated NF-κB and phosphorylated MLC in the rat VILI model. The representative results of the expression of MMP-9 (upper panel of A and B), total-NF-κB (secondary panel of A and C), phosphorylated NF-κB (third panel of A and D), and phosphorylated MLC (forth panel of A and E) in lung tissues in the S group (left bands of A and of B–E), V group (middle bands of A and of B–E) and VE group (right bands of A and of B–E) were detected by Western blot. The corresponding protein expression was evaluated by densitometry and presented in B (MMP-9), C (total-NF-κB), D (phosphorylated NF-κB), and E (phosphorylated MCL). Data from each group were calculated from three independent experiments. *p < 0.05, when compared with the S group; ^# p < 0.05, when compared with the V group. Abbreviations: EPCs, endothelial progenitor cells; MLC, myosin light chain; MMP, matrix metalloprotease; NF-κB, nuclear factor kappa B; S group, sham; V group, VILI; VE group, VILI with EPC transplantation; VILI, ventilator-induced lung injury.

Figure 5.

EPC transplantation attenuated histological lung injury in the rat VILI model. The histopathological alteration of lung tissue in the S (A and D), V (B and E), and VE (C and F) groups were analyzed by H&E staining. A-C: 200 × magnification; D-F: 400 × magnification. The histological change in the V group (B and E) revealed a thickened alveolar wall, pulmonary edema, broken alveoli and hemorrhage. EPC transplantation (VE group) clearly mitigated these alterations (C and E). Abbreviations: EPCs, endothelial progenitor cells; H&E, hematoxylin and eosin; S group, sham; V group, VILI; VE group, VILI with EPC transplantation; VILI, ventilator-induced lung injury.

Figure 6.

EPC transplantation mitigated the ventilation-induced apoptotic damage in lung tissue. The representative results of apoptosis in lung tissue in the S group (Aa), V group (Ab), and EPC transplantation (VE group; Ac) were detected by TUNEL staining using fluorescence microscopy (A, 400 × magnification). TUNEL-positive cells were stained green and the nucleus was stained blue with DAPI. The representative results of the protein expression of Bax (first panel of Ba and Bb), Bcl-2 (second panel of Ba and Bc), native caspase-3 (third panel of Ba and Bd), and cleaved caspase-3 (fourth panel of Ba and Be) in lung tissue in the S group (left bands of Ba, ), V group (middle bands of Ba; ) and VE group (right bands of Ba; ) were detected by Western blot. The expression of the corresponding proteins was evaluated by densitometry analysis, and presented in Bb (Bax), Bc (Bcl-2), Bd (native caspase 3), and Be (cleaved caspase 3). Data in each group were calculated from three independent tests. *p < 0.05 vs. the S group; ^# p < 0.05 vs. the V group. Abbreviations: EPCs, endothelial progenitor cells; S group, sham; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; V group, VILI; VE group, VILI with EPC transplantation; VILI, ventilator-induced lung injury.