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

Isolation and Culture of EPCs

The present study was approved by the Ethics Committee of Harbin Medical University. All treatments were performed according to the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Harbin Medical University and national animal treatment guidelines.

Peripheral blood was withdrawn from the caudal vein of Wistar rats (6–8 weeks, 140–160 g), and mononuclear cell isolation was achieved using density gradient centrifugation with Ficoll-Plaque Plus (Amersham Pharmacia Biotech AB, Uppsala, Sweden). After washing with phosphate-buffered saline (PBS), the isolated mononuclear cells were incubated in complete endothelial growth medium (EGM)-2 (Lonza Corp., Basel, Switzerland) on six-well plates pre-coated with human fibronectin at 37°C with 5% CO₂. The culture medium was changed daily. After 10 days of culture, the EPCs were lifted with 0.025% trypsin containing 0.02% EDTA and harvested for further analysis or transplantation.

Characterization of EPCs

The EPCs isolated from rats were identified using a methodology from a previous study ^17,18 . Mononuclear cells were incubated with Dil-acetyl-low density lipoprotein (LDL, 10 μg/mL; Invitrogen, Carlsbad, CA, USA) and fluorescein isothiocyanate-ulexeuropaeus agglutinin-1 (UEA-1, 5 μg/mL; Sigma-Aldrich, Saint Louis, MO, USA). EPCs stained with DiI-acetyl-LDL (absorption wavelength: 555 nm) and UEA-1 (absorption wavelength: 495 nm) were identified using a confocal microscope. These EPCs were also stained with vascular endothelial growth factor receptor (VEGFR)-2 (Abcam, Cambridge, UK), CD34, and CD133 (Santa Cruz Biotechnology, Santa Cruz, California, USA), as described in a previous study ^17,19 . Furthermore, in order to characterize the subtype of EPCs, cells with antibodies against FITC-CD14 (Santa Cruz Biotechnology) and PE-CD45 (Biolegend, San Diego, CA, USA) were analyzed using a flow cytometer. CD14^-/CD45^- cells were considered as advanced EPCs (endothelial colony-forming cells), while CD14⁺/CD45⁺ cells were considered as early EPCs ²⁰ .

VILI Model and EPC Transplantation

All male Wistar rats (250–280 g) were fed with water and standard diet ad libitum before the study. Twenty-four rats were randomly divided into three groups (n = 8/group): sham group (S group), ventilation group (V group), and ventilation/EPC transplantation group (VE group). Rats in the S group were only exposed to anesthesia, while rats in the V and VE groups received MV, with a tidal volume of ²¹ 17 mL/kg (inspiratory gas: 50% O₂ + 50% N₂; 50% respiratory rate: 50/min; inspiratory to expiratory ratio: 1:1) for 4 h. Then, all rats were anesthetized with 3% pentobarbital sodium (a total volume of 30 mg/kg) via intraperitoneal (IP) injection. Following local analgesia with lidocaine, peripheral blood was collected through cannulation of the caudal vein and artery for arterial blood gas analysis. In addition, saline infusion was achieved through cannulation. After additional anesthesia with a rocuronium injection (0.6 mg/kg), rats in the V and VE groups were intubated with a 14 G tube and exposed to MV (a tidal volume of 17 mL/kg for 4 h). Rats in the VE group were intravenously given approximately 10⁶ of EPCs (in 1 mL of PBS) immediately at the end of MV ²² . PBS alone was intravenously given to rats in the V group as controls. The anesthesia was maintained with 3% pentobarbital sodium (10 mg/kg) and rocuronium (0.6 mg/kg) over 1-h intervals. Once spontaneous breath recovered after 4 h of ventilation, the rats were extubated. All rats were anesthetized and cannulated. After the collection of blood samples, all rats were sacrificed with an overdose of anesthetics at 24 h following ventilation ²³ .

Arterial Blood Gas Analysis

In order to observe the effect of EPC transplantation on pulmonary gas exchange function, arterial blood gases were analyzed at baseline and at 24 h after ventilation using a Bayer Rapid lab 348 ventilator (Bayer Diagnostics, Leverkusen, Germany). The ratio of oxygen partial pressure to fraction inspiratory oxygen (PaO₂/FiO₂) was calculated.

Alveolar–Capillary Permeability

After sacrifice at 24 h after ventilation, the right upper lung lobes of rats were harvested and weighed while wet. Then, these were dried at 60°C for 48 h and weighed again. Afterwards, the wet/dry weight (W/D) ratio was calculated to indirectly determine the effect of the EPC transplantation on alveolar–capillary permeability ^24,25 .

Inflammatory Cytokine Detection in Bronchoalveolar Lavage Fluid and Serum

The right bronchus was blocked using an artery clamp. Then, sterile saline (4°C, 15 mL/kg) was injected into the left lung and withdrawn from the pulmonary airway for five times. Next, bronchoalveolar lavage fluid (BALF) was collected and centrifuged (1,000 × g, 15 min, 4°C). The cytokines in the supernatant of the BALF, including interleukin (IL)-1β, IL-8, IL-10, tumor necrosis factor (TNF)-α, and neutrophil elastase, were detected using the corresponding ELISA kit (Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), according to product instructions. The protein concentration in the supernatant of the BALF and neutrophil count in the BALF pellet were also measured.

Peripheral femoral venous blood was collected at baseline and at 24 h after ventilation, and centrifuged (1,500 × g, 4°C, 10 min). Then, the serum (supernatant) was transferred into a separate Eppendorf tube and stored at –80°C. The levels of IL-1β, IL-8, IL-10, TNF-α, macrophage inflammatory protein (MIP)-2, and intercellular adhesion molecule (ICAM)-1 were detected using the corresponding ELISA kits above.

Histopathologic Injury Examination

The lung tissue harvested from the lower right lobe was used to examine the histological alterations. The tissue was fixed with 4% paraformaldehyde, embedded in paraffin, cut in 4-μm-thick sections, and placed onto a glass slide. Then, the treated lung tissue on the slide was stained with hematoxylin and eosin (H&E). Two blinded independent pathologists evaluated the extent of lung injury under a light microscope. The pathological alterations that were analyzed included edema, alveolar congestion, hemorrhage, airspace/vessel wall neutrophil infiltration, alveolar wall thickness, and hyaline membrane formation. The histological score was evaluated from 0 to 4 (0, normal histological presentation; 1, light infiltration of inflammatory cells; 2, severe perivascular infiltration of inflammatory cells; 3, infiltration of inflammatory cells in the alveolar septum/space; 4, diffused infiltration of mononuclear cells in the perivascular, interstitial and airspace).

Tracking of Transplanted EPCs in Lung Tissue

In order to identify the distribution of EPCs in lung tissue and distinguish the EPCs injected from EPCs derived from rats, EPCs with acetyl-LDL (37°C for 2 h) were pre-labeled and injected into another eight rats. Then, the transplanted acetyl-LDL-labeled EPCs were detected in rat pulmonary tissue using a fluorescent microscope. Briefly, after 24 h, the harvested pulmonary tissue was fixed with paraformaldehyde and embedded in paraffin. The sectioned pulmonary tissue on a glass slide was deparaffinized with xylene and rehydrated with decreasing alcohol gradients. The slide was stained with 4,6-diamidino-2-phenylindole (DAPI; 1 μg/mL) for 30 min to visualize the nuclei. The distribution of EPCs in lung tissue was finally detected using a confocal microscope (absorption wavelength: 555 nm for acetyl-LDL).

Apoptosis Assay

Pulmonary tissues from the right middle lobe was collected to detect the apoptosis by TUNEL staining using an apoptosis assay kit (Roche, Mannheim, Germany). Briefly, the sectioned pulmonary tissue on a slide was immersed with proteinase K at 37°C for 30 min. After washing with PBS twice, the slide was immersed in the TUNEL reaction mixture (TdT and fluorochrome-conjugated dUTP) at 37°C for 60 min in a dark chamber, incubated with DAPI (1 μg/mL) for 30 min, and visualized using a confocal microscope (absorption wavelength: 490 nm).

Western Blot

Protein was extracted from the right lung, and the protein concentration was determined using the Bradford assay. Aliquot amounts of the protein for each sample were loaded onto a SDS-polyacrylamide gel, and transferred onto a polyvinylidene fluoride (PVDF) membrane. Then, the membrane was blocked with 5% milk for 30 min, and incubated with a primary antibody overnight at 4°C. The primary antibodies included the following: Bax, Bcl-2, matrix metalloproteinase (MMP)-9, cleaved caspase-3 (Sigma-Aldrich), phosphorylated myosin light chain (phospho-MLC; Sigma-Aldrich), and total nuclear factor kappa B (total-NF-κB) and phosphorylated NF-κB (phospho-NF-κB) (Santa Cruz Biotechnology). After washing with TBS-tween buffer (3 × 5 min), the membrane was incubated with horseradish peroxidase (HRP)-linked secondary antibodies (Santa Cruz Biotechnology) for 1 h, and the probed-bands on the membrane were visualized by enhanced chemiluminescence.

Statistical Analysis

Normally distributed data were presented as mean ± standard deviation (SD), and statistically analyzed by repeated measures analysis of variance (ANOVA) (with Bonferroni post-hoc test) using the SPSS 11.0 software (SPSS, Chicago, IL, USA). p < 0.05 was considered statistically significant.

Characterization of EPCs

Rat peripheral blood mononuclear cells were isolated by density gradient centrifugation. After 10 days of incubation, approximately 8–10×10⁶ cells proliferated, and were analyzed. Endothelial cells expressed DiI-acetyl-LDL (Fig. 1A) and isothiocyanate-UEA-1 (Fig. 1B). The merged image of the anti-UEA-1 and DiI-ac-LDL staining is presented in Fig. 1C. The expression of both (Figs 1C) was detected using fluorescent antibodies in the isolated monolayer of cells. These results demonstrate that the monolayer of cells were endothelial cells. The biological characteristics of EPCs were further identified by the positive staining of VEGFR-2 (Fig. 1D) and CD34 (Fig. 1E). The merged image of the anti-VEGFR-2 and CD34 staining is presented in Fig. 1F. VEGFR-2 (Fig. 1G) and CD133 (Fig. 1H) were also detected. The merged image of the VEGFR-2 and CD133 staining is presented in Fig. 1I. These data indicate that the isolated monolayer of cells were EPCs. Furthermore, all analyzed EPCs presented as spindle-shaped (Fig. 1J). The subtype of EPCs was also characterized with FITC-CD14 and PE-CD45 using a flow cytometer. The percentage of CD14⁺/CD45⁺ cells was 14.6 ± 3.2, while the percentage of CD14^-/CD45^- cells was 40.5 ± 4.1 (Fig. 1K and 1L). Furthermore, some cells were also found with CD14⁺/CD45^- and CD14^-/CD45⁺. At present, no study has named or classified these cells. Hence, it was hypothesized that these cells may be special cells between early and advanced EPCs. However, this hypothesis needs further studies to be confirmed.

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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.

Detection of Transplanted EPCs

The EPCs transplanted in rat lungs in the VE group were observed by immunofluorescent staining using an acetyl-LDL (Fig. 2A) antibody and DAPI (Fig. 2B) with fluorescence microscopy. It was found that these EPCs were successfully transplanted and survived in rat lungs in the VE group (Fig. 2C). In contrast, samples obtained from rats in the V group did not show any acetyl-LDL staining (Fig. 2D).

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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.

EPCs Improved the Gas Exchange Index and Alveolar–Capillary Permeability

In the present study, the PaO₂/FiO₂ index significantly decreased at post-ventilation 24 h in the V and VE groups, when compared with the S group (p < 0.05). The EPC transplantation (VE group) dramatically increased the PaO₂/FiO₂ ratio, when compared with the V group (Fig. 3A, p < 0.05). In addition, the pulmonary W/D weight ratio and total protein in the BALF were analyzed to detect the effect of EPCs on alveolar–capillary permeability. Interestingly, the pulmonary W/D weight ratio (Fig. 3B) and total protein level in the BALF (Fig. 3C) were significantly higher in the V and VE groups, when compared with the S group (p < 0.05). However, this increase significantly declined after EPC transplantation (VE group, p < 0.05). These results clearly demonstrate that the transplantation of EPCs significantly attenuated the VILI in the rat model.

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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.

Local and Systemic Inhibition of Inflammation by Transplanted EPCs

The concentration of various inflammatory cytokines, including TNF-α (Fig. 3D), IL-1β (Fig. 3E), IL-8 (Fig. 3F), and IL-10 (Fig. 3G), as well as the neutrophil count (Fig. 3H) and neutrophil elastase (Fig. 3I), in the BALF was measured to explore the effect of EPCs on local inflammation in the rat VILI model. The levels of TNF-α, IL-1β, IL-8, and neutrophil elastase, and neutrophil count in the BALF were significantly higher in the V and VE groups, when compared with the S group (p < 0.05). However, these elevations significantly decreased after the EPC transplantation (VE group, p < 0.05). In contrast, the level of IL-10 was significantly upregulated by the EPC transplantation (VE group), when compared with the V group (p < 0.05).

Next, the serum levels of TNF-α (Fig. 3J), IL-1β (Fig. 3K), IL-8 (Fig. 3L), IL-10 (Fig. 3M), ICAM-1 (Fig. 3N), and MIP-2 (Fig. 3O) were analyzed to explore the effect of EPCs on systemic inflammation in the rat VILI model. All cytokines in serum were upregulated by ventilation (V and VE groups), when compared with the S group (p < 0.05). Similar to the responses in the BALF, the levels of TNF-α, IL-1β, IL-8, ICAM-1, and MIP-2 significantly decreased after EPC transplantation (VE group), when compared with the V group (p < 0.05). However, the level of IL-10 was significantly upregulated after the EPC transplantation (VE group, p < 0.05; Fig. 3M).

Compared with the S group, MMP-9, total-NF-κB, phospho-NF-κB, and phospho-MLC were significantly upregulated following ventilation (V and VE groups, p < 0.05). However, these upregulations were considerably attenuated by the lung transplantation of EPCs (VE group), when compared with the V group (Fig. 4).

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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.

EPC Transplantation Attenuated Ventilation-Induced Histological Injury in the Rat VILI Model

The effect of EPC transplantation on histological changes in the rat VILI model was evaluated by H&E staining. Compared with the S group (Fig. 5A), typical pathological changes, including edema, alveolar wall thickening, the formation of a hyaline membrane, and hemorrhage, were observed in the V (Fig. 5B) and VE groups (Fig. 5C). However, these alterations were milder in the EPC transplantation (VE group) samples than those in the V group (Fig. 5D).

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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.

EPC Transplantation Reduced Ventilation-Induced Apoptosis in the Rat VILI Models

Compared with the S group (Fig. 6Aa), an increased number of TUNEL-positive cells (green) was observed in the V group (Fig. 6Ab). However, the EPC transplantation (Fig. 6Ac) reduced the number of TUNEL-positive cells in the VE group, when compared with the VILI model (V group). In addition, the expression of Bax (Fig. 6Ba and 6Bb) and both cleaved (Fig. 6Ba and 6Be) and uncleaved (Figs 6Ba and 6 Bd) caspase-3, which are critical factors to regulate the apoptotic process, was significantly higher in the V group, and this increase could be partially prevented by EPC transplantation (VE group). In contrast, the expression of Bcl-2 (Fig. 6Ba and 6Bc), which is a critical anti-apoptotic factor, significantly decreased in the rat VILI model (V group), and the decrease in Bcl-2 was partially attenuated by EPCs.

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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.