Correlation between NADPH oxidase-mediated oxidative stress and dysfunction of endothelial progenitor cell in hyperlipidemic patients

Study subjects

Patients with hyperlipidemia (n = 30) and healthy volunteers (n = 30) from Xiangya Hospital (Changsha, China) were enrolled for this study. All patients were newly diagnosed with hyperlipidemia based on the results of total cholesterol (TC) and/or total triglycerides (TG) examinations. Exclusion criteria included cardiomyopathy, congenital heart disease, liver or renal diseases, bleeding disorders, and malignant diseases. Age- and sex-matched controls were selected from community-based inhabitants, free of any hyperlipidemia following the same exclusion criteria as mentioned above. The relevant characteristics of hyperlipidemic patients and controls are summarized in Table 1.

Table 1.

Body weight and plasma lipids

Isolation and characterization of EPCs

Peripheral venous blood samples (15 mL) from the enrolled patients or controls were collected in a sterile tube containing the anticoagulant heparin for plasma or EPCs isolation. EPCs were isolated and cultured according to instructions provided by commercial kits. Briefly, EPCs were obtained by density gradient centrifugation with Ficoll-Paque PREMIUM (GE-Healthcare, Freiburg, Germany). After washing, 10⁷ cells were seeded on culture plates coated with human fibronectin (Millipore, Temecula, CA, USA) and maintained in endothelial basal medium-2 (Lonza, Walkersville, MD, USA) containing endothelial growth medium-2 (EGM-2) SingleQuots (Lonza). At the 7th day of culture, EPCs were identified by immunofluorescence staining to detect surface markers and dual binding characteristics of acetylated LDL incorporation and lectin.

Measurement of plasma lipids

For the measurement of the levels of total TC, TG, LDL and high density lipoprotein in plasma, commercial diagnostic kits were used and the measurements were carried out according to the instructions provided by the manufacturer (Jiancheng Institute of Biotechnology, Nanjing, China). The levels of ox-LDL in plasma were assayed using a commercial ELISA (enzyme-linked immunosorbent assay) kit (R&D Systems, Minneapolis, MN, USA).

Immunofluorescence staining

EPCs were verified by immunofluorescence staining with primary antibody against CD34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), fetal liver kinase 1 (Flk-1, Abcam, Cambridge, MA, USA), CD144 (Santa Cruz Biotechnology), CD31 (Abcam), endothelial nitric oxide synthase (eNOS, Life Science, Shanghai, China), followed by a secondary antibody of goat anti-rabbit Cy3-conjugated immunoglobulin G (IgG, Beyotime, Jiangsu, China) or goat anti-mouse DyLight 488-conjugated IgG (Beyotime). The cell nuclei were labeled with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime). Images were taken under a fluorescence microscope (Olympus IX71, Olympus, Tokyo, Japan). For the negative control, PBS was utilized to replace the primary antibodies.

Functional analysis of EPCs

The functions of EPCs were assessed through evaluating the abilities of migration, adhesion and tube formation. For migration assay, 1 × 10⁵ EPCs were put in the upper chamber of a 24-well Transwell (Corning, NY, USA), and 600 μL EGM-2 media containing 50 ng/mL of vascular endothelial growth factor were placed to the lower compartment of the chamber. After 6 hours incubation at 37°C, media in the upper and lower chamber were aspirated and EPCs on the top membrane were gently wiped off with a cotton swab. The Transwell membrane was washed with PBS and then fixed in 4% paraformaldehyde for 15 minutes at room temperature. For quantitation, 0.1% crystal violet solution (Beyotime) was used to stain cells, and cells migrating into the lower chamber were counted manually in ten random fields under ×200 magnification.

For adhesion assay, 5 × 10⁴ EPCs were seeded in 12-well plates coated with 10 μg/mL human plasma fibronectin (Millipore). After 30 minutes incubation, plates were washed three times with PBS to remove non-adherent cells, and then incubated in 4% paraformaldehyde. Cells were stained with DAPI (Beyotime) and counted in five random fields under ×100 magnification.

For tube formation assay, Matrigel basement membrane matrix (ECMatriX, BD Biosciences, Franklin Lakes, NJ, USA) was thawed at 4°C overnight. Then 60 μL matrigel was added in a 96-well plate for 1 hour at 37°C. Ten thousand EPCs were added on the top of the solidified matrix containing EGM-2. After 16 hours incubation, tube morphology was imaged and 10 random fields were counted for the tube numbers under ×40 magnification.

Measurement of ROS production in EPCs

Intracellular ROS levels were measured based on the fluorescent signal of 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), a cell-permeable ROS indicator (Beyotime). Non-fluorescent DCFH-DA is oxidized to fluorescent DCF by intracellular ROS once the acetate groups are removed. Briefly, EPCs were washed with PBS and then incubated with 10 μM DCFH-DA at 37°C for 20 minutes. Then, the ROS-mediated fluorescence was monitored under a fluorescent microscope with excitation at 502 nm and emission at 523 nm. Arbitrary fluorescent units were normalized to the control and presented as fold changes.

Measurement of H₂O₂ production in EPCs

To measure the H₂O₂ generation in EPCs, mixtures of 50 μL of EPCs lysate and 100 μL of work solution (0.25 mM ammonium ferrous II sulfate, 100 mM sorbitol, 25 mM H2SO4, and 125 mM xylenol orange) were incubated at room temperature for 20 minutes. The change of absorbance at 560 nm was observed, and the level of H₂O₂ was computed according to a standard curve made from the standard solutions offered by the supplier (Beyotime).

Measurement of NOX activity

The NOX activities were measured by analyzing the NADPH-dependent superoxide dismutase (SOD)-inhibited cytochrome c reduction (Genmed, Shanghai, China). Briefly, the reaction buffer (900 μL) with the NOX substrate (NADPH) and the oxidized cytochrome c was incubated in a quartz cuvette at 30°C for 3 minutes, and then an aliquot (100 μL) of the supernatant from EPCs lysate was incubated with the reaction mixture at 30°C for 15 minutes. The change of absorbance at 550 nm was detected in a spectrophotometer. SOD-inhibited cytochrome c reduction in buffer blank was monitored and subtracted from each sample. The NOX activity was evaluated as SOD-inhibited cytochrome c reduction and expressed as O^2– in nmol/mg/min.

Real-time polymerase chain reaction

Real-time polymerase chain reaction (PCR) was performed to determine mRNA levels of NOX2 and NOX4 in EPCs, and β-actin served as a loading control. The primers for NOX2, NOX4, and β-actin were listed in Table 2. Real-time PCR was conducted following standard protocols. Briefly, 2 μg of total RNA from each sample was subjected to reverse transcription reaction by Reverse Transcription System (Takara, Kyoto, Japan) to obtain the cDNAs. Then, 10 μL of reaction mixtures containing 5 μL SYBR Master mix, 2 μL cDNA template, 2.4 μL H₂O, 0.20 μL ROX, and 0.20 μL of each primer were amplified following the procedures: denaturing at 95°C for 10 minutes and 45 cycles of the amplification steps (denaturation at 95°C for 15 seconds, annealing and extension at 60°C for 1 minute). Data analysis was carried out by comparative cycle threshold method using 7300 System SDS Software (Applied Biosystems, Foster City, CA, USA). Results were expressed as the ratio of NOX2 or NOX4 mRNA to β-actin mRNA.

Table 2.

Primers for real-time polymerase chain reaction

Western blot analysis

EPCs were homogenized in ice-cool lysis buffer, sonicated for 1 minute, and then centrifuged at 15, 000 ×g for 15 minutes. The protein concentration in supernatants was determined by a BCA Protein Assay kit (Beyotime). Samples containing ∼40 μg of protein were loaded to 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel and then transferred to polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies against NOX2, or NOX4, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. The signals of bands were examined using Luminata Crescendo Western HRP substrate through Molecular Imager ChemiDoc XRS System (Bio-Rad, Philadelphia, PA, USA). The densitometric quantification was performed with Image J 1.43 (NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/). Antibody against β-actin (Beyotime) was used as loading control.

Ethics

This study has been performed in accordance with the Declaration of Helsinki (2013) of the World Medical Association, and was approved by the local Ethics Committee at Xiangya Hospital of Central South University (Changsha, China). Written informed consent was obtained from all individuals at the time of enrollment. All investigators were qualified to undertake this study.

Statistics

The statistical analysis was conducted using the SPSS version 20.0 (IBM Corp., Armonk, NY, USA). Data were presented as mean ± standard error of the mean (SEM). Differences in measured values between two groups were assessed by Student t test. For categorical variables, the chi-square test was used. The correlations between functions of EPCs and NOX activity were calculated using Pearson correlation coefficient. Differences were considered as significant when p < 0.05.

Phenotypic characterization of human EPCs

The isolated mononuclear cells from human peripheral blood samples were cultured for 7 days. At the 7th day, EPCs were identified as the attached cells, showing functions of acetylated LDL uptake (incorporation) and lectin binding (Fig. 1A), as well as expressing the stem cell marker (CD34) and endothelial cell markers (eNOS, CD144 Flk-1, and CD31) (Fig. 1B).

Figure 1.

Characterization of human endothelial progenitor cells (EPCs) from peripheral blood. (A) Three-color fluorescent imaging shows Dil-acetylated low density lipoprotein (Dil-acLDL) incorporation (red) and FITC lectin (FITC-UEA-1) binding (green) by EPCs. Nuclei appear dark blue (DAPI). (B) Immunof luorescence staining indicates the expression of CD34 (the stem cell marker), Flk-1, CD31, CD144, and eNOS (the endothelial cell markers) in EPCs. Nuclei were counterstained with DAPI (blue).

Dysfunction of circulating EPCs in hyperlipidemic patients

The functions of EPCs including the abilities of migration, adhesion, and tube formation were evaluated. Compared to the controls, EPCs migration was dramatically reduced in the hyperlipidemia patients (Fig. 2A), concomitant with decreases in adhesive EPCs numbers (Fig. 2B) and tube numbers (Fig. 2C).

Figure 2.

Dysfunctions of the circulating endothelial progenitor cells (EPCs) in hyperlipidemic patients. (A) Left panel, representative images for EPCs migration (Transwell) assay; right panel, the migrated EPC numbers. (B) Left panel, representative images for EPCs adhesion assay; right panel, the adhesive EPC numbers. (C) Left panel, representative images for EPCs tube formation assay; right panel, the tube numbers. All values are presented as mean ± SEM (7 individual samples per group for migration, adhesion and tube formation analysis). ^ap < 0.01 vs. control.

Up-regulation of NOX in EPCs from hyperlipidemic patients

To investigate whether NOX, a major source of ROS in cardiovascular system, involved in the dysfunction of EPCs in hyperlipidemic patients, we measured the expression of NOX (NOX2 and NOX4) and analyzed the correlation between NOX activity and EPC functions. Compared to the controls, mRNA and protein levels of NOX2 and NOX4 in the EPCs of the hyperlipidemic patients were significantly up-regulated (Fig. 3). Consistently, NOX activity was dramatically increased concomitant with an elevation in plasma levels of H₂O₂ and the intracellular ROS levels (Fig. 4). The correlation analysis showed that NOX activity in EPCs was inversely correlated with the EPC functions including the abilities of migration and adhesion (Fig. 5).

Figure 3.

Up-regulation of NOX2 and NOX4 in endothelial progenitor cells (EPCs) of hyperlipidemic patients. (A, B) NOX2 or NOX4 mRNA expression. (C, D) NOX2 or NOX4 protein expression. Left, representative images of Western blot results for NOX2 or NOX4 and β-actin; right, relative changes in optical density for NOX2 or NOX4. All values are presented as mean ± SEM (6 individual samples per group for mRNA and protein analysis, respectively). NOX, NADPH oxidase. ^ap < 0.01 vs. control.

Figure 4.

Increase of NADPH oxidase (NOX) activity and reactive oxygen species (ROS) production in endothelial progenitor cells (EPCs) of hyperlipidemic patients. (A) NOX activity in EPCs. (B) H₂O₂ levels in plasma. (C) Representative image of fluorescent signal of 2’,7’-dichlorodihydrof luorescein diacetate (DCFHDA) for total ROS in EPCs. (D) Statistic value for f luorescent density. All values are presented as mean ± SEM (7 individual samples per group for NOX activity and ROS level analysis). ^ap < 0.01 vs. control.

Figure 5.

Negative correlation between NADPH oxidase (NOX) activity and endothelial progenitor cells (EPCs) migration (A) or adhesion (B).