Role of endothelial colony forming cells (ECFCs) Tetrahydrobiopterin (BH4) in determining ECFCs functionality in coronary artery disease (CAD) patients

Nitric oxide (NO.) is critical for functionality of endothelial colony forming cells (ECFCs). Dimerization of endothelial nitric oxide synthase (eNOS) is must to produce NO. and tetrahydrobiopterin (BH4) plays a crucial role in stabilizing this state. We investigated BH4 level in ECFCs and its effect on ECFCs functionality in CAD patients. Intracellular biopterin levels and ECFCs functionality in terms of cell viability, adhesion, proliferation, in vitro wound healing and angiogenesis were assessed. Guanosine Triphosphate Cyclohydrolase-1 (GTPCH-1) expression was studied in ECFCs. Serum total reactive oxygen/nitrogen species was measured and effect of nitrosative stress on ECFC’s biopterins level and functionality were evaluated by treating with 3-morpholino sydnonimine (SIN-1). BH4 level was significantly lower in ECFCs from CAD patients. Cell proliferation, wound closure reflecting cellular migration as well as in vitro angiogenesis were impaired in ECFCs from CAD patients. Wound healing capacity and angiogenesis were positively correlated with ECFC’s BH4. A negative effect of nitrosative stress on biopterins level and cell functionality was observed in SIN-1 treated ECFCs. ECFCs from CAD exhibited impaired functionality and lower BH4 level. Association of BH4 with wound healing capacity and angiogenesis suggest its role in maintaining ECFC’s functionality. Oxidative stress may be a determinant of intracellular biopterin levels.

Coronary artery disease (CAD) is the most prevalent type of cardiovascular diseases (CVDs) and leading cause of death worldwide. Damage of vascular endothelium is the first step which initiates a cascade leading to the development of CAD 1 . The repair and maintenance of vascular endothelium is critical and is not only dependent on proliferation of local endothelial cells (ECs) but also on bone marrow derived endothelial progenitor cells (EPCs) 2 . EPCs have been shown to inversely associate with coronary artery disease severity and is a strong predictor of multivessel CAD after age 3 . Circulating EPC levels also predicts severe endothelial dysfunction in CAD patients independent of classical cardiovascular risk factors 4 . Endothelial nitric oxide synthase (eNOS) derived NO . plays a critical role in mobilization EPCs from bone marrow and their recruitment at the site of vascular injury 5 . In vascular disease state, oxidative stress increases and eNOS derived endothelial nitric oxide (NO . ) bioactivity diminishes resulting in endothelial dysfunction. Tetrahydrobiopterin (BH4) the cofactor of eNOS plays a crucial role in maintaining the enzymatic "coupling" state of eNOS as well as biosynthesis of NO . . The balance between NO . and superoxide production by eNOS seems to be dependent on bioavailability of BH4, whereas dihydrobiopterin (BH2), the oxidized form of BH4 does not act as cofactor of eNOS, rather competes with BH4 for eNOS binding which further leads to enzyme uncoupling and superoxide production 6 .
In vitro and in vivo studies on diabetic subjects have shown that depletion of BH4 levels either by decreased expression of guanosine triphosphate cyclohydrolase-1 (GTPCH-1), the rate limiting enzyme of BH4 biosynthesis, or oxidative loss of BH4 leads to increased production of superoxide and peroxynitrite, which in turn affect ECFCs characterization. Flow cytometry phenotypic characterization. Phenotypic characterization of ECFCs were carried out at passage 2, in 05 representative samples from both CAD patients and healthy subjects, by flowcytometry. The expression of endothelial antigens CD31, KDR and CD146, progenitor antigens CD34, CD117, leucocytic antigen CD45 and monocytic antigen CD14 were assessed. Briefly, the adherent ECFCs were harvested by treating with Accutase cell detachment reagent (Cat. 561,527; Becton Dickinson) and then stained with 0.5 µL of FITC-conjugated mouse anti-human CD31 (Cat. 303,103; BioLegend), 0.5 µL of CD146 (Cat. 361,011; BioLegend), 1µL of PE-conjugated mouse anti-human KDR (Cat. FAB357P; R&D Systems) and 1 µL of CD117 (Cat. FAB332P; R&D Systems), 1 µL of APC-conjugated mouse anti-human CD34 (Cat. FAB7227A; R&D Systems), 0.5 µL of PE-Cy7-conjugated mouse anti-human CD14 (Cat. 557,742; BD Biosciences) and 2 µL of PECy5.5-conjugated mouse anti-human CD45 (Cat. 555,484; BD Biosciences). Cells were incubated with fluorophore conjugated antibodies at 4 °C for 30 min and stained cells were detected with Gallios flowcytometer (Beckman Coulter) and results were analysed with Kaluza software (Kaluza Analysis Software; version 2.1). ECFC characterization was carried out in representative samples of both CAD and control subjects. Cultured ECFCs were considered as negative for the leucocyte and monocyte marker CD45 and CD14, positive for the stem cell marker CD117, CD34 and positive for endothelial marker CD31, KDR, CD146 respectively.
Immunofluorescence characterization. Cultured ECFCs were assessed for KDR and CD34 expression in 05 representative samples from each CAD patients and healthy subjects. Briefly, cells at passage 2 with approximately 60-70% confluency were fixed with 4% paraformaldehyde (PFA) solution and blocked with 1% BSA. Cells were then incubated with mouse monoclonal anti-human KDR primary antibody (20 µg/ml) (Cat. MAB3571; R&D Systems) for 1 h followed by rhodamine conjugated goat antimouse secondary antibody (1:100 dil) (Cat. SR131, G-Biosciences) in dark for 1 h. Thereafter, cells were incubated with Alexa Fluor 488 conjugated mouse anti-human CD34 antibody (10 µg/ml) (Cat. FAB7227G; R&D Systems) for 1 h in dark. Counter staining was achieved by incubating cells with DAPI and images were obtained using upright Epi fluorescence microscope (Nikon Eclipse Ni with DSRi-2 colour camera).
In 05 representative ECFC samples from both CAD patients and healthy subjects, ability of cells to uptake acetylated low-density lipoprotein; Ac-LDL and Ulex-lectin binding was assessed. In brief, ECFCs at passage 2 were incubated with 1,1'-dioctadecyl-3,3,3' ,3'-tetramethylindo-carbocyanine labelled Ac-LDL (Dil-Acy-LDL; 5 µg/ml) ( Quantification of intracellular ECFC's tetrahydrobiopterin (BH4) and dihydrobiopterin (BH2). ECFC's tetrahydrobiopterin, dihydrobiopterin and biopterin were quantified as described by González et al. 12 with minor modifications. Briefly adherent cells at passage 3 for both CAD subjects and healthy controls were harvested and 1 × 10 6 cells were used for biopterin estimation using high performance liquid chromatography (HPLC) (Agilent 1260 Infinity II) with fluorescence detector. The harvested cells were washed with ice cold phosphate buffer saline (PBS) in amber vial and then cell pellet was resuspended in 300 µl of ice-cold lysis buffer (composition: 50 Tris-HCl, 2 DTT, 1 EDTA mmol/L) and lysed by sonication using probe sonicator (4 cycles; 10 s each cycle at 100 W) on ice. The cell lysate was then centrifuged and supernatant was collected. Next, 25 µl of supernatant was used for protein estimation by Bicinchoninic Acid Assay (BCA) method. HClO4: H3PO4 was added to residual supernatant and vortexed vigorously for 5 s followed by 30 min incubation in dark at 4 °C for denaturation of protein in cell lysate. The cell lysate was then centrifuged at 13500xg for 5 min at 4 °C to pellet down the protein content and supernatant was collected for acid and alkali oxidation. For acid oxidation, 10 µl of acidic I 2 /KI (1% I 2 /2% KI Solution in 1 M HCl) was added to 90 µl of supernatant whereas 10 µl of 1 M NaOH and 10 µl of alkali I 2 /KI (1% I 2 /2% KI Solution in 1 M NaOH) were added to 80 µl of supernatant and then incubated for 1 h in dark. The alkali oxidized sample was then acidified with 20 µl of 1 M H 3 PO 4 and both acid and alkali oxidized samples were then neutralized by adding 5 µl of 20 mg/ml ascorbic acid. After neutralizing, samples were filtered through 0.2 µm filter with 4 mm diameter under centrifugal force at 13500xg for 5 min at 23 °C. The filtered samples (50 µl) were then injected into C18 reverse phase analytical column. The total run time was 10 min with flow rate of 1 ml/minute of 5% methanol solvent system. Detection of biopterin was carried out at excitation and emission wavelengths of 350 and 450 nm respectively. Biopterins were quantified against standard curve of L-biopterin with 06 different concentrations (20-0.625 [picomoles/ml) was prepared by serial dilutions and % recovery of biopterin from BH4 and BH2 after chemical derivatization was assessed using standard BH4 and BH2 compounds. The chromatogram was obtained and peak area was analysed using Chemstation software, Agilent Technologies. The quantification of BH4 in cell lysate was carried out from the difference between peak areas of acid and alkali oxidation, whereas BH2 was determined as the difference between the peak areas of alkali oxidation and samples without any oxidation process. The values of BH4, BH2 and biopterin levels were expressed in picomol per milligram of protein after normalization with the level of intracellular total protein.

GTPCH mRNA expression by quantitative real-time PCR (qRT-PCR). ECFCs at passage 4 in both
CAD and control subjects were dissolved in 1.0 ml of TRIzol reagent (Ref. No.:15596018; Life Technologies, USA) and total RNA was isolated as per manufacturer instruction. The isolated total RNA was further dissolved in nuclease free water and quantified at 260 nm and 280 nm using NanoDrop (Thermo Scientific). Next for the cDNA synthesis, 500 ng of total RNA was reverse transcribed into cDNA using iScript cDNA Synthesis Kit (Cat. No: 1708891; BioRad). cDNA synthesis was carried out in Agilent Mastercycler (SureCycler 8800). For qRT-PCR, 3.0 µL cDNA, 15 µL master mix (Cat. No: 1725121; iTaq Universal SYBR Green Supermix; BioRad), 1.5 µL primers and 10.5 µL nuclease free water were pipetted into the appropriate strip tubes. Real-time PCR was performed on an Agilent AriaMx (G8830A AriaMx Real-Time PCR) with the following protocol; step 1: 1 cycle: for 30 s at 95 °C, step 2: 40 cycle: for 5 s at 95 °C and 15 s at 60 °C followed by continuous melt curve and melt peak analysis to ensure accuracy of amplified product. For each sample, runs were performed in triplicates. The primer sequences used to determine mRNA levels of GTPCH and housekeeping gene β Actin were: Forward 5' CGA GGA GGA TAA CGA GCT GA3' , Reverse 5'CTG GTA GCC CTT GGT GAA GA3' , Forward 5' AGA AAA TCT GGC ACC ACA CC3' , Reverse 5'GGG GTG TTG AAG GTC TCA AA3' .
The relative quantification of gene expression was calculated by ∆∆Ct method and the expression of target gene (GTPCH) was normalized with housekeeping gene; β Actin.
ECFCs functional parameters. Functional assays including cell proliferation, adhesion, viability, in vitro wound healing and angiogenesis were carried out in duplicate in all samples from CAD patients and control subjects in which ECFC appeared, that is 27 CAD patients and 09 healthy control subjects. Angiogenesis assay was carried out in triplicates in a representative sample, that is 17 CAD patients and 09 healthy subjects.
Population doubling time. In vitro ECFCs proliferation was assessed by population doubling time (PDT) at passage 3. 1 × 10 5 cells were seeded on collagen coated 35 mm culture dishes in duplicate and incubated at 37 °C with 5% CO 2 . After 72 h of incubation, cells were harvested and counted by neubauer chamber under phase contrast microscope. PDT of ECFCs was assessed using the formula: PDT = T ln 2 /ln (Xe/Xb), where T is time period (h), Xb is cell number at the beginning of incubation time and Xe is number of cells at the end of incubation time.
Expression of cell nuclear antigen Ki67. ECFCs proliferation was assessed through % expression of cell nuclear antigen Ki67 by flowcytometry. We investigated Ki67 expression in ECFCs of both CAD and control subjects at passage 3. Briefly, 1 × 10 5 cells/well were seeded on a 6 well plate and incubated for 72 h at 37 °C, 5% CO 2 . After incubation, cells were harvested and permeabilized by treating with pre-chilled 70% ethanol and incubated for 1 h at − 20 °C. Thereafter, cells were resuspended into staining buffer and stained with PE conjugated Ki67 (Cat. 350,503, BioLegend) antibody and incubated for 1 h at room temperature. Cells were acquired in FACS LSR-II www.nature.com/scientificreports/ (Becton Dickinson). The unstained cell sample was used as negative control and Ki67-PE signal in logarithmic mode was recorded and data expressed as % expression of Ki67 in histogram.
Cell viability. Viability of ECFCs was monitored up to 7 days at 48 h interval post-seeding by performing the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Briefly at passage 3, 1 × 10 4 cells/well were seeded on collagen coated 96 well plate and incubated at 37 °C with 5% CO 2 . At 24 h and subsequent days with 48 h interval, cultures were incubated with MTT solution (12 mM) for 4 h at 37 °C. Then, the MTT solution was aspirated out and formazan crystals were solubilized with dimethyl sulfoxide. Absorbance of the dye was measured at a wavelength of 570 nm and recorded using a microplate reader (Synergy-2, Biotek).
Cell adhesion. ECFCs adhesion ability was assessed at passage 4, using method as described by Oren M. Tepper et al. 13 with minor modifications. In brief, 1 × 10 5 ECFCs/well were first seeded on collagen coated (5 µg/ cm 2 surface area) 24 well plate and incubated with EGM-2 for 1 h at 37 °C in 5% CO 2 . Adherent cells were then fixed with 4% PFA followed by staining with 0.1% crystal violet for 10 min, rinsing excess stain with water and addition of 200 µl of 10% acetic acid for elution of the stain. The absorbance of the eluted stain was measured at a wavelength of 600 nM with a microtiter plate reader (Synergy-2, Biotek).
Wound healing assay. In vitro wound healing assays were carried out using culture inserts as per the manufacturer's instruction (IBIDI, Germany). Briefly, ECFCs at passage 4 were seeded at 2 × 10 4 per well, in order to obtain confluent cell monolayer in 12 h, in two wells of the insert separated by a wall. Then culture inserts were removed creating a reproducible cell free gap of about 500 µm for each condition. The wound area filling was captured immediately, at 4 h and at 8 h by phase contrast microscope at 10 × magnification. The experiment was carried out in duplicate for all the samples and the microscopic images were analysed by T-Scratch software (TScratch version 1.0; Martin Schulz ETH Zurich) and data was expressed as percent of wound area at 0 h, 4 h and 8 h. The percentage of wound closure was calculated by using the formula as described by Bronson I. Pinto 14 ; % of wound closure (X) = (i-f/i) × 100, where i = initial width of cell free gap at 0 h, f = final width of cell free gap.
Vasculogenesis assay. In vitro vasculogenesis or angiogenesis was assessed at passage 4 by plating 1 × 10 4 cells on 10 µl of growth factor reduced matrigel matrix (Cat. 354,230; Corning) in µ-slides for angiogenesis (IBIDI, Germany). Cells were imaged using phase contrast microscope and 10 × objective after 12 h incubation at 37 °C and 5% CO 2 . This assay was performed in triplicate and number of tube junctions, number of branches of capillary tube and tube length were analysed using Image J (1.51 J8; National Institute of Health, USA) software with angiogenesis analyser plugin (http:// rsb. info. nih. gov/ ij/ macros/ tools ets/ Angio genes is% 20Ana lyzer. txt) in 6 images per participant obtained from random selected microscopic field.

Estimation of circulatory reactive oxygen and nitrogen species (ROS/RNS). Estimation of total
ROS/RNS in serum samples of both CAD and control subjects were carried out using OxiSelect in vitro ROS/ RNS assay kit (Cell Biolab) as per the manufacturer instruction.
In vitro treatment of SIN-1, a peroxynitrite generator, on ECFC viability assessed by MTT assay. At passage 2, ECFCs from healthy subjects (n = 3) were selected for the experiment. In brief, cells (1 × 10 4 /well) in 96 well plate were divided into control and treatment group. As described by Yu Dong et al. 15 with minor modification, the cells in the five treatment groups were supplemented with 5 different concentrations of SIN-1 (00,221-1; Biotium) (200, 400, 600, 800, 1000 µM) with EGM-2 containing 1% FBS for 24 h. The effective concentration for subsequent experiments was determined by MTT. After, 24 h of treatment, MTT (12 mM) was added to each well and incubated for 4 h at 37 °C and the formazan crystals was dissolved in DMSO. The absorbance was measured at 570 nm and the cell viability was expressed as optical density with reference to the control.

The in vitro effect of SIN-1 on ECFC's intracellular biopterin levels. Estimation of intracellular
ECFCs biopterin levels was performed after treatment with SIN-1. In brief, at passage 2, 1 × 10 5 cells/ well were seeded in 6 well plate and incubated for 24 h at 37 °C, 5% CO 2 . Cells were then supplemented with starvation media containing EGM-2 with 1% FBS and incubated for overnight. In the treated group, SIN-1 was added in starvation media whereas for control, only EGM-2 with 1% FBS was added and incubated for 24 h. After treatment, estimation of biopterins were carried out by HPLC as described previously.

ECFCs functionality after SIN-1 treatment in vitro.
ECFCs functionalities in vitro were assessed in terms of cell proliferation, migration, in vitro wound healing and angiogenesis after treating the cells with SIN-1 for 24 h.
ECFCs proliferation as expression of cell nuclear antigen Ki67. The proliferation of ECFCs were assessed in terms of % expression of cell nuclear antigen Ki67 after SIN-1 treatment. Briefly, 1 × 10 5 cells/well were seeded on 6 well plate. Cells underwent SIN-1 treatment for 24 h. After treatment, cells were stained with PE-conjugated Ki67 as described before and expression of proliferation marker Ki67 in SIN-1 treated and untreated samples was assessed by flowcytometry. www.nature.com/scientificreports/ ECFCs migration. ECFCs migratory function was assessed in transwell migration assay as described by Tsai et al. 2 with minor modifications. Briefly, EGM-2 medium supplemented with 10% FBS was added into the lower chamber, while 5 × 10 4 cells in endothelial basal media (EBM-2) were seeded onto upper chamber of transwell permeable supports (8 µm pore size, polycarbonate membrane, SPL Life Sciences). Thereafter, cells were incubated for 4 h at 37 °C, 5% CO2. Suspension cells remaining in the upper chamber were removed and the migrated cells present in the lower side of the transwell insert were fixed with 4% paraformaldehyde. The migrated cells were next stained with DAPI and images from random selected fields and counted number of migrated cells using inverted fluorescence microscope (Nikon ECLIPSE Ts2RFL) and number of migrated cells were quantified and evaluated by Image J software (version 1.51 J8; National Institute of Health, USA).
The effect of SIN-1 treatment on in vitro wound healing by ECFCs. In vitro wound healing by ECFCs was assessed as described earlier after treating the cells with SIN-1 for 24 h. The experiment was carried out in duplicate and microscopic images at 0, 04 and 08 h were analysed using T-Scratch software (TScratch version 1.0; Martin Schulz ETH Zurich) and data was represented as percentage of wound closure at two different time points (0 to 04 h and 04 to 08 h).

Consent for publication.
All the authors has given consent for the publication of this article.

Results
Baseline characteristics. Demographic, clinical characteristics and baseline biochemical parameters of CAD and controls are summarized in Table 1. The mean age of CAD patients was 52.7 years and that of control subjects, 49.0 years. The mean BMI (body mass index) was significantly different between CAD and control groups. A significant proportion of CAD patients (87%) were on lipid lowering drugs and consequently cholesterol, triglyceride, HDL, LDL, VLDL were significantly lower in them compared to control subjects. Table 2 shows baseline characteristics of CAD case (n = 27) and controls (n = 9) in which successful ECFC cultures were established in vitro. Other than fasting glucose, which was significantly lower in controls, no significant differences were noted any of the baseline characteristics between CAD cases and controls.

In vitro ECFC culture and their expansion in CAD and control subjects. ECFCs appeared in cul-
ture with typical cobble stone morphology (Supplemental Fig. 1) similar to that of native endothelial cells. ECFC appeared in culture in 30 out of 53 patients with CAD (56%) and fourteen out of 42 control subjects (33%). Out of the 30 cases in which ECFCs appeared in culture, 03 did not proliferate adequately and no confluency was attained. Whereas similar limited proliferation was seen in 5 control samples.

GTPCH mRNA expression.
No significant difference in GTPCH-1 mRNA expression was observed.
ECFCs functionalities. ECFC proliferation. Figure 3 shows the population doubling time (PDT) of ECF-Cs isolated from CAD subjects and healthy controls. PDT of ECFCs from CAD subjects was significantly prolonged compared to ECFCs from healthy control during 72 h of culture (49.8 ± 4.3; 34.1 ± 1.0; p = 0.049). The % expression of Ki67 as cell proliferation marker was significantly (p = 0.002) lower in ECFCs cultured from CAD patients as compared to control group (37.6 ± 4.2; 65.3 ± 6.9) (Fig. 4). Flowcytometry data acquisition for determination of ECFC's Ki67 expression has been shown in supplemental figure 5.
Cell viability. Cell viability was carried out by MTT assay and was found to increase from day 1 to day 7 in ECFCs from both CAD and control subjects with no significant difference between groups (Fig. 5).
Cell adhesion. Cell adhesion ability of ECFCs did not differ between ECFCs from CAD and control subjects as shown in Fig. 6B. The microscopic image of ECFCs with crystal violet staining has shown in Fig. 6A.  Fig. 6A).
In vitro angiogenesis. Matrigel tubule assay was performed to investigate capacity of ECFCs to differentiate into tubule like structures. The number of branches of tube, length of branches and number of tube junctions were assessed in 17 and 09 representative ECFC samples from CAD and healthy control group respectively. All three parameters of in vitro capillary tube formation were significantly (p < 0.001) reduced in CAD patients compared to control group (Fig. 8A-E). In vitro angiogenesis by ECFCs by means of both number of capillary tube junctions and branch length of capillary tube were positively correlated with intracellular BH4 level (r = 0.54, p = 0.022; r = 0.51, p = 0.03) (Supplemental Fig. s6B & C).

Circulatory total reactive oxygen and nitrogen species (ROS/RNS). Since the ROS/RNS is very
unstable and highly susceptible to deterioration outside living system, the estimation was carried out only in few representative serum samples (CAD; n = 22, control; n = 15) stored at − 80 °C for not more than 60 days. The concentration of circulatory total ROS/RNS was measured in terms of µM H2O2 and was found to be significantly higher in CAD patients as compared to control subjects (p = 0.009; Fig. 9).  Fig. 7), so SIN-1 at this concentration was used to assess the effect of oxidative stress particularly nitrosative stress on intracellular biopterin levels and ECFCs functionalities including cell proliferation, migration, in vitro wound healing, angiogenesis.   Fig. 10). However, there was no significant difference in BH4/BH2 ratio between the groups. After treatment with SIN-1 for 24 h the ECFC's when observed under bright field microscope showed a changed morphology with elongated shape whereas untreated cells showed the typical cobble stone morphology (Supplementary figure 8).
The effect of SIN-1 on proliferative potential of ECFCs, assessed in terms of expression of cell nuclear antigen Ki67, was found to be significantly p = 0.02) lower in SIN-1 treated ECFCs compare to untreated cells (Fig. 11).
After 24 h of SIN-1 treatment significantly lesser number of ECFCs migrated to the lower transwell chamber as compared to the untreated ECFCs (67.8 ± 3.8 vs 160 ± 20; p = 0.012) (Fig. 12). In the histogram, Y axis represent PDT in hours and X axis represent the study groups (CAD and control group). Student t-test was used to assess the significance difference. Data is represented as mean ± SE. Error bar represent standard error of mean; dots represent average values of the parameters obtained from experimental duplicates; p ≤ 0.05 considered statistically significant. www.nature.com/scientificreports/ In the in vitro scratch wound assay, the % wound regression in SIN-1 treated ECFCs was significantly lower as compared to that in untreated ECFCs (35.6 ± 1.2 vs 47.2 ± 2.6; p = 0.016) at 4 h. However, at 08 h the % wound regression in treated and untreated ECFCs were comparable (Fig. 13).
In the in vitro 2D matrigel assay capillary tube formation was measured in terms of number of junction, number of branches and branch length of capillary tubes SIN-1 treated and untreated ECFCs. All three measurements of capillary tube formation were significantly reduced in case of SIN-1 treated ECFCs as compared to untreated. Data is shown in Fig. 14.

Discussion
In the current study we looked at the correlation of ECFC tetrahydrobiopterin levels with its functionality in CAD patients and healthy controls. We observed that ECFC from CAD patients were less proliferative as evidenced by lower Ki67 expression and prolonged population doubling time, and showed impairment in migration as seen by delayed wound healing capacity in vitro and significant decline in angiogenesis potential as compared to ECFCs from healthy controls. A significant association between ECFC BH4 levels and its wound healing capacity and angiogenesis potential was observed. An elevated circulatory ROS/RNS levels was evidenced in CAD patients pointing to peripheral oxidative stress. Oxidative stress induced in ECFC in vitro with SIN-1 was associated with reduction in BH4 levels and decreased proliferation, migration and angiogenesis. A concise graphical abstract of the present study findings has been illustrated in Fig. 15.
An impairment in early EPC functionality in terms of their adhesion and migration capacity has been reported in diabetic and hypertensive patients 13,[16][17][18] . Studies on functional capacity of late EPCs or ECFC, which are the true functional EPCs is limited. Kevin Tan et al. found that ECFCs from patients with proliferative diabetic retinopathy (PDR) were impaired in their ability to migrate towards SDF-1 as well as human sera. Also, ECFCs from patients with PDR showed impaired ability to incorporate into and form vascular tubes with human retinal endothelial cells 19 . In contrary, John D. Stroncek et al. reported no significant difference in population doubling time of late outgrowth EPCs in CAD and healthy controls, PDT was however assessed after thawing of cryopreserved cells. The authors also reported no significant difference in matrigel tube formation by late out growth EPCs between CAD and healthy control groups. The study was however limited by very small sample size 20 . In another study on ischemic cardiomyopathy patients no difference in angiogenic potential of blood outgrowth endothelial cells was reported between patients and healthy subjects 21 however the patient group comprised of stable ischemic cadiomyopathy as compared to patients in our study who had coronary artery disease. To the best of our knowledge, no study has comprehensively looked at all the four functional assays that is in vitro adhesion, migration, proliferation and angiogenesis assays in ECFCs in CAD patients. Assessment of all functional aspects of ECFC, that is proliferation or the ability of EPC to expand after vascular damage or tissue ischemia, adhesion of ECFCs to endothelium for re-endothelization and angiogenesis, migration of ECFC through the extracellular matrix for growth of new vessels and finally the ability of ECFC to form vascular structure, is important to understand the global function of ECFC's in a disease state 22 .
Our finding of a reduced ECFC BH4 in CAD patients as compared to healthy controls point to either an oxidative reduction of BH4 to BH2 levels due to reactive oxygen and nitrogen species in patients or impaired biosynthesis of BH4. We did not find any significant difference in the GTPCH-1 expression between CAD patients and controls. We did find an increase in circulating ROS/RNS in CAD patients compared to controls. The effect of oxidative stress particularly nitrosative stress was confirmed by treating ECFCs with SIN-1, a peroxynitrite generator, which resulted in a reduced BH4 further confirming the role of nitrosative stress on determining intracellular biopterin levels. The reduced BH4 level and imbalance in the ratio of reduced to oxidized biopterin in the present study may lead to uncoupling of eNOS enzyme in ECFCs of CAD patients. In vivo, in vitro and clinical study have shown the transformation of eNOS from protective enzyme to a contributor of oxidative stress 23 . In line with our finding of increased peripheral oxidative stress , da Cunha et al. have postulated that,  15 . We also observed a reduced BH2 levels with SIN-1 treatment which possibly reflects impaired BH4 biosynthesis. Both oxidative loss of BH4 and reduced biosynthesis of BH4 may be acting to determine intracellular biopterin levels.  www.nature.com/scientificreports/ The strength of our study is that we have studied all functional aspects of ECFCs which is important to understand global function. Secondly, we have done our study in ECFCs which are true cells of endothelial lineage unlike early EPCs on which most studies have been conducted. Thirdly we have also confirmed the role of nitrosative stress on biopterin levels in ECFCs in vitro. We also note some limitations. We did not measure the catalytic active state of eNOS to conclusively state that the reduced BH4 levels are causing uncoupling of eNOS. Further we have also not demonstrated the direct effect of augmenting BH4 levels on ECFCs functionality.
In conclusion, present study suggests the role of intracellular ECFC's tetrahydrobiopterin as well as reduced to oxidized biopterin ratio in maintaining the ECFC functionality in CAD patients. Although we have looked at ECFC's GTPCH-1 expression, the rate limiting enzyme of BH4 biosynthetic pathway in CAD patients, it is also imperative to look at expression of DHFR which is the rate limiting enzyme of the salvage pathway for regeneration of BH4. This will help to understand whether the reduced level of BH4 is contributed by oxidative  www.nature.com/scientificreports/ degradation, by reduced biosynthesis or impaired regeneration through salvage pathway. Future studies should also be done to demonstrate the involvement of eNOS activity with lower BH4 levels in ECFC functionality. Since BH4 regulatory system itself offer multiple pharmacological targets for therapeutic prevention of endothelial dysfunction and the progression of cardiovascular diseases a combinatorial approach of antioxidant therapy and BH4 supplementation might be useful in treatment of cardiovascular diseases 25 Figure 11. Effect of SIN-1 on ECFC's Ki67 expression. Dot plot represents Ki67 expression of ECFCs in terms of median fluorescence intensity (MFI) in untreated and SIN-1 treated groups with individual data points and error bars (n = 3 for each group). Significance was assessed by student ttest and data is represented as mean ± SE. Error bar represent standard error of mean; dots represent average values of the parameter obtained from experimental duplicates p < 0.05 was considered statistically significant. Significance was assessed by student ttest and data is represented as mean ± SE. Error bar represent standard error of mean; dots represent average values of the parameter obtained from experimental duplicates p < 0.05 was considered statistically significant.