Human endothelial progenitor cells were isolated from human mammary arteries/saphenous vein as described earlier (Ranjan et al., 2009). Briefly, 2–4 cm long pieces of human mammary artery/saphenous vein were collected as de-identified surgical waste following written informed consent from 50 to 70 years old individuals undergoing cardiac surgery. Mammary artery/saphenous vein samples were collected in M199 medium containing antibiotics (penicillin 25 unit/ml, streptomycin 25 μg/ml, Ciprofloxacin 30 μg/ml, GIBCO-BRL, Burlington, ON), then washed with PBS. Collection of such material was approved by the NCCS research ethics committee (Ethics No. NCCS06/198) National Center for Cell Science, Pune, India. EPCs were detached from the luminal wall of arteries using collagenase (1 mg/ml, Sigma, St. Louis, MO) for 15 min at 37°C. Sterile PBS were used to flush out the detached EPCs from the lumen, and collected in 15 ml Falcon tubes. EPCs were washed twice with 10 ml of PBS and then cell seeded in 10% human AB sera and antibiotics (5 unit/ml penicillin, 5 μg/ml streptomycin, 6 μg/ml Ciprofloxacin, GIBCO-BRL, Burlington, ON) containing endothelial growth medium mix or EGMM, which was made by mixing 50% M199 medium (GIBCO-BRL, Burlington, ON) with EBM-2 medium (Lonza, Walkersville, MD). Isolated EPCs adhere to culture plates within 6–8 h, after which, all non-adherent cells were removed. Adherent EPCs were expanded following procedures described earlier (Ranjan et al., 2009) and used for the present study.

EPCs were harvested, washed in Ca⁺⁺ and Mg⁺⁺ free PBS before blocking for non-specific antigens using 4% normal donkey serum for 20 min. CD133 one step staining was carried out using phycoerythrin(PE)-tagged mouse anti-human CD133 antibody (1:50; Miltenyi Biotec, Germany) and incubation at 4°C for 45 min. Later, EPCs were washed with 5 ml PBS for three times. Cells were then re-suspended in 300 ml of PBS. Each sample was assessed for at least 10,000 events on a FACS Canto II system (BD, Franklin Lakes, NJ) and then analyzed using the FACS Diva software as described earlier (Ranjan et al., 2009).

EPCs were cultured on 1% fibronectin coated dishes in EGM-2 Bullet Kit with 15% fetal bovine serum (FBS) known as EGM-2 growth medium (Lonza, VIC, Australia). EPCs were cultured under standard cell culture conditions using a 5% CO2 incubator at 37°C. Tumor necrosis factor (TNF-α) is well known to induce Nox isoforms and cell death. In some conditions, cells were treated with human TNF-α (20 ng/ml; Sigma, St. Louis, MO) for 6 or 48 h prior to cell harvest.

We silenced the expression of NOX4 gene using adenoviral vectors carrying small interfering RNA targeting nucleotides 418–436 from the start codon of human NOX4 (Ad-NOX4i). Briefly, 5 × 10⁴ EPCs were cultured in a 10 cm² plate 1 day before infecting them with Quiet-U6 vector (Welgen) expressing the sequence against human NOX4 (nucleotides 418–436 from the start codon). The construct was ligated into a linearized adenoviral genome for subsequent generation of adenoviral vector (Ad-NOX4i) (Chen et al., 2008). Adenovirus containing green fluorescent protein gene (Adv-GFP) was used as a control and optimized multiplicity of infection as shown in Supplementary Figure S1. EPCs were infected with 1,000 MOI of Ad-GFP or Ad-NOX4 RNAi for 24 h in Opti-MEM medium (Life Technologies, Victoria, Australia), then allowed to recover for another 24 h using EGM-2 growth medium. All experiments were performed 48 h after infection.

EPCs (10³ cells/well) were seeded on 1% fibronectin coated 24-well plates in EGM-2 Bullet Kit with 5% FBS (EGM-2 growth media, Lonza, Victoria, Australia) and allowed to attach following overnight incubation. For LDL uptake assay, cultured medium was replaced with 10 μg/ml of acetylated low density lipoprotein (Ac-LDL), labeled with 1,1′-dioctadecyl– 3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL, Biomedical Technologies), then cells were incubated for additional 4 h. After the end of the incubation period, the solution was aspirated and fresh growth medium was added before capturing the images using fluorescence microscopy (Zeiss AxioImager.2 microscope).

Cells were fixed in 4% fresh paraformaldehyde, permeabilized with chilled 50% methanol, blocked with 4% normal donkey serum and then incubated with specific antisera. Primary antibodies; mouse anti- PECAM-1 antibody (Chemicon, Temecula, CA, 1:100), mouse anti-VE-cadherin (Chemicon, Temecula, CA, 1:100), rabbit anti-eNOS mouse antibody (Chemicon, Temecula, CA, 1:100) and UEA1 (Ulex europeous agglutinin; Sigma, St. Louis, MO; 1:100), were incubated overnight at 4°C, washed with PBS and then incubated with the secondary antibodies; Alexa-Fluor 488, Alexa-Fluor 546 (Molecular Probes, Carlsbad, CA, 1:200) at 37°C for 1 h. We used 4′, 6-diamidino-2-phenylindole (DAPI) or propidium iodide to visualize nuclei. Slides were washed extensively with generous volumes of PBS and mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Confocal images for CD31 and CD144 were obtained using a Zeiss LSM 510 laser scanning microscope. All other images obtained using Zeiss AxioImager-2 microscope fluorescence microscopy. Results presented are representative fields confirmed from at least three different biological samples.

Cells (10⁵ cells/well) were seeded on 6-well plates. Serum deprived cells were treated with tumor necrosis factor; TNFα (20 ng/ml) for 6 or 48 h. Total RNA from treated cells was extracted with the TRI reagent according to manufacturer's instructions (Ambion, Austin, TX, USA) and reverse-transcribed to cDNA using TaqMan high performance reverse transcription reagents (Applied Biosystems, Life Technologies, Victoria, Australia) at 25°C for 10 min, 37°C for 2 h followed by 85°C for 5 s in a Thermal cycler (BioRad-DNA Engine, Bio-Rad, New South Wales, Australia). Real-time PCR reactions were performed in a 7,300 real-time quantitative PCR system (Applied Biosystems, Life Technologies) using TaqMan Universal PCR master mix and pre-designed (off the shelf) gene specific probes and primer sets for NOX2 (Hs00166163_m1) and NOX4 (Hs01558199_m1) The cycle threshold (CT) values from all real-time PCR experiments were analyzed using ^ΔΔCT method. Data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; human 4326317E) and expressed as fold changes over that in the control treatment group.

Extracellular hydrogen peroxide (H₂O₂) levels were detected using Amplex® Red assay kit (Molecular Probes, Life Technologies, Victoria, Australia) according to manufacturer's instructions. EPCs (10⁴ cells/well) were seeded on 1% fibronectin coated 24-well plates. Serum-deprived cells were trypsinized and suspended in Krebs-HEPES buffer (HBSS, in mM: NaCl 98.0, KCl 4.7, NaHCO₃ 25.0, MgSO₄ 1.2, 137 KH₂PO₄ 1.2, CaCl₂ 2.5, d-glucose 11.1 and Hepes-Na 20.0) containing Amplex® Red reagent (10 μM) and horseradish peroxidase (0.1 U/ml). Fluorescence was then measured with excitation and emission at 550 nm and 590 nm respectively, using a polarstar microplate reader (BMG Labtech, Germany) at 37°C. Fluorescence values were normalized to cell numbers determined by Alamar® Blue cell viability assays as according to manufacturer's instructions (Life Technologies, Victoria, Australia).

EPCs (5 × 10³ cells/well) were seeded on 24-well plates for 24 h before serum starvation. Serum starved-cells were induced to proliferate by adding EGM-2 growth medium with 1% FBS. After 48 h, cells were trypsinized and numbers were analyzed by using trypan blue (Life Technologies, Victoria, Australia) and counted using a haemocytometer.

The transwell migration assay was performed using 24-well transwell permeable inserts containing polycarbonate membranes (6.5 mm diameter, 8 μm pore size, and 0.3 cm³ bottom area, CLS3422; Sigma-Aldrich). Inserts were incubated with 0.1% gelatin and dried. Then, 600 μl of the chemo attractant (EGM-2 growth medium with 1% FBS) was dispensed into each well of the 24-well transwell plates and incubated at 37°C for 1 h. The transwell inserts were placed in the bottom of wells containing pre-warmed chemo attractant. EPC cells were trypsinized and resuspended in EGM-2 growth medium with 1% FBS. 1 × 10⁴ cells (600 μl from the cell suspension) were added to each well. The transwell plates were then incubated at 37°C. The number of migrated cells was counted after 16 h. The medium in the inserts was next removed, and the membranes were washed twice in PBS. Transwell membranes were then fixed in cold methanol for 1 min, air dried and washed three times before staining for nuclei using DAPI for 1 min. Transwell membranes were then washed at least three times with generous amounts of PBS to remove excess stain. The non-migrated cells on the top side of the membranes were gently wiped off using wet cotton swabs. The membranes were left to air dry and carefully peeled off from the inserts and placed on microscopic slides, with the migrated cells facing up down, then mounted in DEPEX mounting medium. Quantification was carried out by imaging 10 random X10 high-power fields per membrane using an Olympus inverted light/fluorescent microscope (Model No. IX81) and the number of migrated cells was counted using Image J software.

Cells (2.5 × 10⁵ cells/well) were cultured in 6-well plates, and protein was extracted as previously described. Equal amounts of protein were then separated by electrophoresis using gradient SDS-PAGE gel, and transferred to nitrocellulose membranes (Amersham). After blocking with 5% non-fat milk in a buffer containing Tris–HCl (20 mM, pH 7.5), NaCl (100 mM) and Tween 20 (0.1%), respective membranes were incubated at 4°C overnight with primary rabbit monoclonal anti-NOX4 (1:1,000, Abcam, Australia) and mouse monoclonal anti-GAPDH (1:1,000, Merck Millipore) antibodies. Proteins were detected using an enhanced chemiluminescence detection kit (GE Healthcare, New South Wales, Australia) with horseradish peroxidase conjugated to appropriate secondary antibodies (Bio-Rad, New South Wales, Australia). The image was captured and processed using CanoScan 8800F/PhotoStudio 5.5 software.

Data are expressed as mean ± standard error of the mean (SEM). The mean data were analyzed with Student's t-test or One-way analysis of the variance (ANOVA) followed by post-hoc Tukey or Dunnett analysis. A value of P < 0.05 was regarded as statistically significant.

EPCs have been successfully isolated and expanded in culture from different sources (Takahashi et al., 1999; Kalka et al., 2000; Yamaguchi et al., 2003; Ingram et al., 2004; Li et al., 2006). Here, EPCs were isolated from the luminal wall of human mammary artery/saphenous vein and expanded in vitro under low cell density culture conditions (10³ cells/cm²). EPCs formed colonies within 14 days (Figure 1A) and immunophenotyping showed high expression of hematopoietic marker CD133 at low density as compared to isotype controls (Figures 1B,C). Adherent EPCs stained positive for Dil-Ac-LDL (Figure 1D) and UEA-1 lectin (Figure 1E). Moreover, these cells stained positive for expression of the endothelial marker protein endothelial nitric oxide synthase (eNOS; Figure 1F).

Early and late EPCs can be distinguished by their proliferative property and life span in culture (Marui et al., 1993; Asahara et al., 1997; Hur et al., 2004). We examined proliferative potential of our expanded progenitor cells from three different patients. Cells were plated on day 0, and then serially passaged to maintain cells under proliferative phase for 60 days. Remarkably, colonies derived from EPCs started to appear from 20 days (Figure 2), suggesting that these cells are late EPCs. Every 10 days, the number of EPCs expanded until the induction reached around 3,500-fold within 60 days. This finding suggests that late EPCs can be expanded for a long time in vitro.

EPCs can be readily differentiated to endothelial cells. We examined the differentiation capacity of late EPCs which were plated at a high cell density (8 × 10⁴ cells/cm²) onto tissue culture treated chambers. Within 8 days late EPC had matured into endothelial cells which showed cobblestone morphology, as well as expressing endothelial markers PECAM-1 (Figure 3A) and VE-cadherin (Figure 3B).

To demonstrate the importance of NOX4 function in EPCs, adenovirus expressing RNA interference targeting human NOX4 (Ad-NOX4i) was used to suppress NOX4 gene expression. Inhibition of NOX4 using Ad-NOX4i (1,000 MOI) markedly reduced both NOX4 mRNA (Figure 4A) and protein (Figure 4B) expression compared with the control group (Ad-GFP). Moreover, Ad-NOX4i suppressed H₂O₂ production in late EPCs (Figure 4C), confirming that NOX4 promoted H₂O₂ generation in late EPCs.

NOX4 has been also reported to mediate vascular cells proliferation and migration (Petry et al., 2006; Sturrock et al., 2006; Datla et al., 2007; Ismail et al., 2009; Peshavariya et al., 2009, 2014a,b). As shown in Figure 5A, EPC proliferation was clearly reduced after knocking down NOX4 using Ad-NOX4i when compared with control (Ad-GFP). We also examined the contribution of Nox4 on EPC migration using transwell migration assay. For this purpose, 1 × 10⁴ of Ad-GFP and Ad-NOX4i EPCs were seeded on the upper chamber of the transwell inserts and stimulated with EGM-2 growth medium + 1% FBS to pass to the lower chamber. The number of migratory cells was significantly decreased after inhibition of NOX4 gene when compared with the control (Ad-GFP) (Figures 5B,C). Thus NOX4 is required for proliferation and migration of EPCs.

Pro-inflammatory cytokines such as TNFα, induce to oxidative stress via escalating ROS production (Kim et al., 2010) particularly from NADPH oxidase (Chandel et al., 2001). TNFα up-regulated the expression of NOX2 over 6 h, which was further induced in NOX4- deficient cells (Figure 6A). In contrast, NOX4 expression decreased after TNFα treatment. There was no significant difference in NOX4 expression before and after TNFα treatment in NOX4 deficient cells (Figure 6B), indicating that TNFα selectively modulates NOX isoforms in EPCs.

Ad-GFP carrying EPCs treated with TNFα (20 ng/ml) for 48 h significantly increased cell death. Suppression of NOX4 gene using NOX4 RNAi in the absence of TNFα also increased cell death. Interestingly, the effect of TNFα on cell death was further enhanced in NOX4 deficient cells (Figure 7), indicating that NOX4 helps to protect EPCs.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.