Background
Acute lung injury (ALI) is a common clinical syndrome characterized by high mortality [
1]. ALI pathogenesis can be caused by biological and abiotic pathogens. Biological pathogens include bacteria, viruses, fungi, atypical pathogens, and malignant tumors, while abiotic pathogens include acidic substances, drugs, toxic gas inhalation, and mechanical ventilation-related injuries [
2]. Initially, endotoxins and other harmful substances in the blood circulation injure pulmonary capillary endothelial cells (PCEC), causing an increase in permeability in endothelial cell monolayers, as well as endothelial cell shrinkage and death. Approximately two hours post-injury, pulmonary interstitial edema can occur, and alveolar edema occurs approximately 12–24 h post-injury [
3].
Endothelial cell dysfunction and inflammation play important roles in ALI initiation and development [
3]. In ALI, an improved patient survival correlates with a higher colony count of endothelial progenitor cell (EPC) [
4]. EPC engraftment maintains the integrity of pulmonary alveolar-capillary barrier, reestablishes endothelial function in vessels, and ameliorates the inflammatory state in LPS-induced ALI in rat models [
5]. Therefore, EPC contributes to the repair of endothelial impairment and prevention of ALI development. However, patients with ALI have increased levels of pro-inflammatory mediators (e.g., ox-LDL, IL-6, TNF-α), which impair EPC function, including proliferation, migration, and tube formation. These impaired EPC are unable to restore the damaged endothelial system in patients with ALI [
6]. Therefore, increasing the number of EPC and alleviating EPC dysfunction induced by pro-inflammatory mediators may suppress the development of ALI.
High-density lipoprotein (HDL) regulates EPC mobilization from bone marrow, prevents EPC apoptosis, and promotes differentiation of EPC into endothelial cells [
7]. Apolipoprotein A-I (apoA-I), an important functional component of HDL, has 243 amino acids, and improves survival in septic rats [
8]. Because the lipid-binding properties of apoA-I are largely related to its class A amphipathic helices, designed mimetic peptides, which contain four phenylalanine residues on the hydrophobic face (4F; F being the biochemical symbol for phenylalanine) and mimic the class A amphipathic helices, have no sequence homology with apoA-I [
9]. The 4F residues have anti-inflammatory and antioxidant activities, and improve survival in septic rats [
8]. Kruger et al. have shown that the mimetic peptide D-4F can prevent the loss of certain EPC functions by increasing endothelial nitric oxide synthase (eNOS) in EPC [
10]. Our previous studies revealed that reverse D-4F (Rev-D4F) acts via a PI3K/AKT/eNOS-dependent mechanism to significantly improve EPC proliferation, migration, and tube formation [
11]. Thus, HDL, apoA-I, and its mimetic peptides are new drugs for the treatment of lung injury [
12]. However, the precise mechanisms of the protective effects of HDL and HDL mimetic peptides Rev-D4F on EPC damage induced by LPS have not been entirely clarified. In this study, we investigated the effect of Rev-D4F on EPC numbers and function in LPS-induced ALI mice.
Methods
Animals
Male C57 mice (6 weeks old) were obtained from the Vital River Company (Beijing, China) and randomly divided into four groups with six mice in each group: the control group (PBS), Rev-D4F group (Rev-D4F 10 mg/Kg and PBS), LPS group (PBS and LPS 10 mg/Kg), and Rev-D4F treatment group (Rev-D4F 10 mg/Kg and LPS 10 mg/Kg). Either vehicle or Rev-D4F (10 mg/kg) was administered by intraperitoneal injection.
All animal procedures complied with the Guide for the Care and Use of Laboratory Animals published by Weifang Medical University, and the protocol was approved by the Committee on the Ethics of Animal Experiments of Weifang Medical University. Mice were anesthetized by intraperitoneal injection of 400 mg/kg chloral hydrate.
Rev-D4F synthesis
The Reverse D4F mimetic peptide synthesis method was used to obtain inverse chirality with D-amino acids only and reverse order of D4F (Ac-DWFKAFYDKVAEKFKEAF-NH2) via a practical approach of Fmoc solid-phase peptide synthesis by the Scilight-Peptide INC (Beijing, BJ, China). The structure and purity (98%) were determined by GC/MS.
Analysis of wet: dry weight ratio
The left lung was taken, and lung surface liquid was absorbed with an absorbent paper. Weight of the left lung was considered the wet weight. The lung was dried at 60 °C for 48 h, and then reweighed to obtain the dry weight. The degree of lung edema was evaluated by calculating the wet: dry weight ratio.
Measurement of serum cytokines
ELISA kits (Blue Gene, Shanghai, China) were used to measure TNF-α and ET-1 levels in mice plasma according to the manufacturer’s instructions.
Histopathologic evaluations and Immunohistochemical staining
The right lobes of the lungs were fixed in 10% formalin (Sigma-Aldrich, St Louis, MO, USA), embedded in paraffin, cut to 6 μm sections, and then stained with Hematoxylin-eosin (Sigma-Aldrich, St Louis, MO, USA). Immunohistochemistry (IHC) was performed with specific antibodies against Sca-1 (Abcam, Cambridge, UK), VCAM-1, ICAM-1, eNOS, iNOS, or vWF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and the samples were then counterstained with Hematoxylin.
Red and white blood cells in the lung parenchyma were counted. Red and white blood cell counts, as well as quantitative alveolar wall thickness measurements were performed based on the segmented morphology in 10 randomly selected fields in each section (n = 6/group) using high power fields (× 200).
Flow cytometric analysis of EPC
Red blood cell-lysing buffer (BD Biosciences, San Jose, CA, USA) was used to lyse peripheral blood erythrocytes. Then, other cells were labeled with the FITC-conjugated antibodies against CD34, PE-conjugated antibodies against FLK-1, and APC-conjugated antibodies against CD133 (BD Biosciences, San Jose, CA, USA). A gating strategy and isotype-identical antibodies were used to exclude debris and nonspecific fluorescent signals.
EPC isolation
In vitro isolation of mice bone marrow-derived EPC were performed as described previously [
11]. The mononuclear cells (MNCs) were obtained from the femurs of 4-week-old male C57 mice by density gradient centrifugation (Sigma-Aldrich, St Louis, MO, USA), and plated on fibronectin-coated 6 well plates in EGM-2MV (Endothelial cell basal medium-2, plus FBS, VEGF, R-IGF-1, rhEGF, rhFGF-B, GA-1000, hydrocortisone and ascorbic acid) (Lonza, Basel, Switzerland). After 3 days, non-adherent cells were removed, and fresh medium was applied every 3 days.
Characterization of EPC
MNCs from mice bone marrow were cultured for 10 days, incubated with 2.5 mg/L DiI-ac-LDL (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 2 h at 37 °C, and 10 mg/L FITC-UEA (Sigma-Aldrich, St Louis, MO, USA) for 1 h at 37 °C, and then fixed for 5 min with 1–2% paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA). Double-positive cells and total cell numbers were counted in five random fields under a fluorescence microscope (× 100). The positive cells (%) represent the numbers of double-positive cells compared with the total number of cells.
EPC treatment
MNCs obtained from mice bone marrow were induced for 21 days by EGM-2MV and differentiated into late outgrowth EPC. Before experiment, the cell medium was changed to basic medium (M199 + 5% FBS), and cells were separated into 5 groups and treated as follows: Control (M199 + 5% FBS), Rev-D4F (M199 + 5% FBS + 50 μg/ml reverse D-4F), LPS (M199 + 5% FBS + 30 μg/ml LPS), LPS + Rev-D4F (M199 + 5% FBS + 50 μg/ml reverse D-4F + 30 μg/ml LPS), LPS + Rev-D4F + LY294002 (M199 + 5% FBS + 50 μg/ml reverse D-4F + 30 μg/ml LPS + 30 μM LY294002). After pretreatment with the PI3-kinase inhibitor LY294002 (30 μM) (Sigma-Aldrich, St Louis, MO, USA) for 2 h, EPC were incubated with Rev-D4F (50 μg/ml) for 6 h, and then treated with LPS for 36 h to induce EPC damage without removal of LY294002 and Rev-D4F.
EPC viability and proliferation
EPC viability was determined by MTT assay. After different treatments, cells were incubated with 20 μl 5 mg/ml MTT (Sigma-Aldrich, St Louis, MO, USA) for 4 h at 37 °C. The supernatant was aspirated, and 150 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, St Louis, MO, USA) was added to each well and shaken for 10 min. OD values were measured at 490 nm.
EPC proliferation was evaluated by flow cytometry analysis. After treatments, cells were digested with 0.25% trypsin (Sigma-Aldrich, St Louis, MO, USA), fixed with 75% ethanol, and stained with FITC-conjugated antibodies against Ki67 (BD Biosciences, San Jose, CA, USA). A gating strategy and isotype-identical antibodies were used to exclude debris and nonspecific fluorescent signals.
EPC migration
EPC migration was measured using an 8 μm pore 24-well Cell Migration Assay kit (BD Biosciences, San Jose, CA, USA). After treatments, cells were digested with 0.25% trypsin, and 1.2 × 104 cells in M199 were placed in the upper chamber. EGM-2MV medium was placed in the lower compartment. After 24 h incubation at 37 °C, the upper cells were removed with a cotton wool swab, and the transwell filters were fixed and stained with DAPI (Sigma-Aldrich, St Louis, MO, USA). The migratory cells were counted in five random microscopic fields (× 100).
EPC adhesion
Mice bone marrow-derived MNCs were cultured on fibronectin-coated 6 well plates at 106 cells per cm2. After 3 days, non-adherent cells were removed, and adherent cells were cultured for additional 4 days. Then, we randomly selected five fields of view and counted the number of adherent cells under a microscope (× 100).
After different treatments, cells were digested with 0.25% trypsin and 1.0 × 104 cells in M199 plus 10% FBS were placed on matrigel (BD Biosciences, San Jose, CA, USA) in a 96 wells plate. After 18 h incubation, the average total length of complete tubes formed by cells was measured under a microscope (× 40) in five random microscopic fields using computer software, Image-Pro Plus.
Western blot analysis
Total protein was extracted with RIPA lysis buffer and quantified using the BCA method. In total, 30 μg of protein was electrophoresed on a 10% denaturing polyacrylamide gel, and transferred onto PVDF membranes. After blocking with 5% dried skimmed milk, the membranes were incubated with antibodies against β-actin (1: 20000, Sigma-Aldrich, St Louis, MO, USA), AKT (1: 2000, Cell Signaling Technologies, Beverly, MA, USA), phosphor-AKT (1: 1000, Cell Signaling Technologies, Beverly, MA, USA), eNOS (1: 500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphor-eNOS (1: 1000, Cell Signaling Technologies, Beverly, MA, USA), CD133 (1: 500, Abcam, Cambridge, UK), FLK-1 (1: 500,Abcam, Cambridge, UK) or Sca-1 (1: 500, Abcam, Cambridge, UK) for 3 h, and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1: 3000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The signals were detected using the Phototope-HRP Western Detection Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).
Immunofluorescence staining
MNCs were cultured and differentiated into relatively mature endothelial cells. The lung tissues were used to make frozen sections. Next, cells and frozen sections were blocked with goat serum at room temperature for 30 min, and then incubated with anti-mouse CD31 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), vWF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD133 (Abcam, Cambridge, UK) or FLK-1 (Abcam, Cambridge, UK) antibodies for 1 h at 37 °C. Finally, cells and tissues were incubated with secondary antibodies conjugated with Cy3 or FITC, and staining was assessed under fluorescence microscopy (× 200).
Statistical analysis
All data are presented as mean ± SD. Variables between groups were assessed by one-way ANOVA using the software program SPSS11.5. Values of P<0.05 were considered significant.
Discussion
EPC were first isolated from peripheral blood using magnetic microbeads [
15]. EPC contributes to physiological neovascularization, wound healing in vessels, tissue regeneration in ischemia [
16], and repair of lung injury [
17]. Our present findings demonstrate that Rev-D4F inhibits LPS-induced pulmonary edema, decreases plasma levels of TNF-αand ET-1, inhibits infiltration of red and white blood cells into the interstitial space, reduces injury-induced lung inflammation, and restores injured PCEC. Our previous study indicated that Rev-D4F improved EPC proliferation, migration, tube formation, and NO-releasing function, which was partially dependent on the PI3K/AKT/eNOS/NO pathway [
11]. Our present results demonstrate that Rev-D4F improves the function of EPC impaired by LPS both in vivo and in vitro, and that Rev-D4F increases EPC numbers to repair injured PCEC.
The protective effects of apoA-I mimetic peptide 4F on vascular function in endotoxemic rats have been demonstrated. The anti-inflammatory effects of apolipoprotein A-I mimetic peptide (L-4F) in ARDS are due to the direct inhibition of endotoxin activity and increase of HDL antioxidant activity [
18‐
20]. Similarly, our results demonstrate that another apoA-I mimetic peptide, Rev-D4F, repairs LPS-induced lung damage in mice. In addition, our results show that Rev-D4F increases numbers of EPC, both in peripheral blood and in lung tissues, and improves EPC function in LPS-treated mice.
Chronic inflammation decreases numbers of circulating EPC and suppresses their activity, resulting in suppressed restoration of vessel injury and continuous inflammation [
21]. Reduced numbers of circulating EPC are associated with hypercholesterolemia [
22], inflammation [
23], obesity [
24], and diabetes [
25]. HDL, active in anti-inflammation and anti-oxidation, increases numbers of circulating EPC and promotes re-endothelialization in wound healing via the PI3K/Akt/cyclin D1 signaling pathway. In addition, HDL slows down EPC senescence by increasing NO and promoting telomerase activity via the PI3K/Akt signaling pathway [
26]. ApoA-I, the major structural apolipoprotein of HDL, increases numbers of circulating EPC, promotes endothelial regeneration, and attenuates neointima formation in a murine model of transplant arteriosclerosis [
27]. ApoA-I mimetic peptide D-4F, mimicking ApoA-I class A amphipathic helices, promotes EPC proliferation, migration, and adhesion [
28]. Our previous study has indicated that reverse D-4F, a novel ApoA-I mimetic peptide, improves EPC proliferation, migration, tube formation, and NO-releasing function, partially through the PI3K/AKT/eNOS/NO pathway [
11]. Few studies have investigated whether an ApoA-I mimetic peptide D-4F, especially reverse D-4F, exerts protective effects against EPC injury induced by LPS, and little is known about the potential mechanism.
In this study, we have found that LPS significantly impairs EPC functions, such as proliferation, migration, and tube formation, and Rev-D4F inhibits the LPS-induced EPC dysfunction. Patruno et al. have found that LPS reduces the phosphorylation levels of AKT in H9c2 cells, and that AKT is an important determinant of eNOS phosphorylation at Ser1177 [
29]. PI3K/Akt activation plays an essential role in HDL-induced EPC proliferation, migration, and angiogenesis [
30]. Our western blot results show that LPS reduces levels of eNOS, Ser1177-phosphor-eNOS, and phosphor-AKT in EPC, and that Rev-D4F restores the LPS-attenuated levels of these proteins. In addition, our results demonstrate that the PI3K/AKT inhibitor LY294002 inhibits the Rev-D4F-restorative effects on phosphor-AKT and phosphor-eNOS levels, indicating that the Rev-D4F-mediated restoration of EPC function in LPS-treated cells is mediated by PI3K/AKT /eNOS signaling pathway.
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