Race-Specific Differences in Endothelial Function: Predisposition of African Americans to Vascular Diseases

Human umbilical vein endothelial cells (HUVECs) were isolated into primary cultures from 12 white and 12 black female donors by Clonetics and purchased as proliferating cells. All cell culture donors were healthy, and none had pregnancy or perinatal complications. The clinical characteristics of the donors are reported in Table 1. None of the donors took any drugs regularly, and all were nonsmokers and consumed regular caloric/content diet. Before selection for the study, donors of each group were screened by clinical history, physical examination, routine chemical analyses, and ECG. Exclusion criteria were history or evidence of present or past hypertension, diabetes mellitus, renal disease, cardiac disease, peripheral vascular disease, vasculitis, coagulopathy, or any other disease predisposing the donors to vascular complications. The local Research Ethics Committee approved collection of tissue specimens, and all donors gave written informed consent.

The HUVEC culture was incubated in 95% air/5% CO₂ at 37°C and passaged by an enzymatic (trypsin) procedure.¹⁰ The confluent cells (4×10⁵ to 5×10⁵ cells/35-mm dish) were placed with minimum essential medium containing 3 mmol/L l-arginine and 0.1 mmol/L H₄B [(6R)-5,6,7,8-tetrahydrobiopterin]. Before the experiments, the cells (from the second or third passage) were rinsed twice with Tyrode’s solution–HEPES buffer with 1.8 mmol/L CaCl₂. All experiments were blinded to the race of the endothelial cell donors and study treatment.

Concurrent measurements of NO, O₂⁻, and ONOO⁻ were performed with electrochemical microsensors^7–9 combined into 1 working unit with a total diameter of 3 to 4 μm. Their design was based on previously developed and well-characterized chemically modified carbon-fiber technology. Each of the sensors was made by depositing a sensing material on the tip of carbon fiber (length, 4 to 5 μm; diameter, 0.5 μm). The fibers were sealed with nonconductive epoxy and electrically connected to copper wires with conductive silver epoxy. We used conductive film of polymeric nickel(II)tetrakis(3-methoxy-4-hydroxyphenyl)porphyrin for the NO sensor,^9,11 an immobilized polypyrrole/horseradish peroxidase (PPy/HRP) for the O₂⁻ sensor,⁸ and polymeric film of Mn(III)-[2,2]paracyclophenylporphyrin for the ONOO⁻ sensor for the ONOO⁻ sensor.^7,12

The tandem NO/O₂⁻/ONOO⁻ nanosensors with a platinum wire (0.1 mm) counterelectrode and saturated calomel reference electrode were applied. Differential pulse voltametry (DPV) and amperometry were performed with a computer-based Gamry VFP600 multichannel potentiostat. DPV was used to measure the basal NO, O₂⁻, and ONOO⁻ concentrations, and amperometry was used to measure changes in NO, O₂⁻, and ONOO⁻ concentrations from its basal level with time (detection limit of 1 nmol/L and resolution time <50 ms for each sensor). The DPV current at the peak potential characteristic for NO (0.65 V) oxidation and ONOO⁻ (−0.45 V) or O₂⁻ (−0.23 V) reduction was directly proportional to the local concentrations of these compounds in the immediate vicinity of the sensor. Linear calibration curves were constructed for each sensor from 5 nmol/L to 3 μmol/L before and after measurements with aliquots of NO, O₂⁻, and ONOO⁻ standard solutions, respectively. The tandem system of NO/O₂⁻/ONOO⁻ nanosensors was lowered with the help of a computer-controlled micromanipulator until it reached the surface of the cell membrane (a small piezoelectric signal, 6 to 8 pA, of 1 to 3 ms duration was observed at this point). The sensors were slowly raised 4±1 μm from the surface of a single endothelial cell. The eNOS agonists calcium ionophore A23187 (CaI) and acetylcholine were then injected with a nanoinjector that was also positioned by a computer-controlled micromanipulator. In the experiments with eNOS agonist stimulation, endothelial cells were pretreated for 3 hours with 0.3 mmol/L N^G-nitro-l-arginine methyl ester (L-NAME), an eNOS inhibitor; 0.5 mmol/L 3-morpho-linosydnonimine-N-ethylcarbamide (SIN-1),¹³ a releaser of both NO and O₂⁻; 0.01 mmol/L Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP) or 1.0 mmol/L tempol (4-hydroxytetramethylpiperidine-1-oxyl), both cell-permeable superoxide dismutase (SOD) mimetics, or 0.01 mmol/L 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride (FeTPPS) or 0.1 mmol/L uric acid, both ONOO⁻ scavengers. Immediately before the measurements of eNOS agonist–stimulated NO, O₂⁻, and ONOO⁻ release, the incubation was stopped by washing the cells twice with the buffer free of the test substances. In the experiments for determination of basal NO, O₂⁻, and ONOO⁻, the cells were preincubated for 3 hours before and during the measurements with various oxidase inhibitors (mmol/L): 3.0 apocynin,³ 0.05 6,8-diallyl 5,7-dihydroxy 2-(2-allyl 3-hydroxy 4-methoxyphenyl)1-H benzo(b)pyran-4-one (S17834) (Servier),¹⁴ 0.1 oxypurinol, 0.1 rotenone, 0.01 meclofenamate, or 0.3 L-NAME.

Endothelial O₂⁻ production was also measured by SOD-inhibitable ferricytochrome c reduction assay as described previously.¹⁵ Briefly, equal protein samples of endothelial cell homogenate were incubated in 1 mL of buffer containing ferricytochrome c (80 μmol/L) in the presence of NAD(P)H or NADH (100 μmol/L) at 37°C for 45 minutes, and then absorbance was measured at 550 nm. All experiments were performed with or without SOD (400 U/mL). Superoxide production was calculated as the portion of ferricytochrome c reduction inhibited by SOD.

Samples of endothelial cell homogenate, equalized for protein content, were separated by SDS-PAGE (5% gels) and transferred to PVDF membranes. NADPH-oxidase components were detected with goat polyclonal antibodies against p67phox or p47phox or p22phox, and eNOS was detected with polyclonal anti-eNOS antibody (Santa Cruz Biotechnology).¹⁶ To compare the NADPH-oxidase subunits and eNOS expressions with the expression of another protein, we analyzed the expression of β-actin by Western blot using a monoclonal anti-β-actin antibody. Bands were detected by horseradish peroxidase–conjugated secondary antibodies and visualized by chemiluminescence.

All chemicals were purchased from Sigma-Aldrich, unless otherwise noted.

When applicable (comparison between 2 values), statistical analysis was done with Student’s t test. For multiple comparisons, results were analyzed by ANOVA followed by Bonferroni’s and Dunn’s correction.¹⁷ Data are presented as mean±SEM. Means were considered significantly different at P<0.05.

Compared with whites, endothelial cells from blacks elicited reduced release of biologically active (diffusible) NO with an accompanying increase in the release of both O₂⁻ and ONOO⁻ (Table 2). This suggests that in blacks, a decrease in NO bioavailability is not a result of a decrease of NO synthesis but rather of an increase of NO consumption by excess of O₂⁻. To investigate the endothelial enzymatic sources of O₂⁻ production in both racial groups, we measured O₂⁻ release with concurrent measurements of NO and ONOO⁻ in response to a range of potential oxidase inhibitors. In both blacks and whites, O₂⁻ production was inhibited by apocynin and S17834, NAD(P)H-oxidase inhibitors. Also, both apocynin and S17834 completely abolished differences between whites and blacks in release of the detected molecules. When the cells were treated with oxypurinol, meclofenamate, or rotenone, only meclofenamate appreciably suppressed O₂⁻ release in both racial groups. However, the differences in NO, O₂⁻, and ONOO⁻ release from HUVECs between whites and blacks were still maintained in the presence of each of the 3 inhibitors. The presence of L-NAME resulted in a significant increase of O₂⁻ release in both whites and blacks because of the loss of O₂⁻ scavenging by NO. Intriguingly, the proportionally greater increase in O₂⁻ release from HUVECs in the presence of L-NAME in whites than blacks (16.2±1.5 versus 5.2±1.8 nmol/L; P<0.01) suggests that the net effect of NOS activity in blacks is associated with diminishing O₂⁻ scavenging by eNOS-derived NO. In both groups, NO and ONOO⁻ release in the presence of L-NAME were suppressed below the detection limits.

To ascertain whether the increased O₂⁻ release from endothelial cells in blacks is caused by an increase in NAD(P)H activity, we compared NADH- and NAD(P)H-dependent O₂⁻ production in the endothelial cells from whites and blacks. As expected, in endothelial cells, NADH stimulated O₂⁻ production with greater potency than NAD(P)H in both racial groups (Figure 1). NADH/NAD(P)H-stimulated O₂⁻ production from HUVECs was significantly greater in blacks than whites. In both racial groups, NADH/NAD(P)H-stimulated O₂⁻ production was inhibited by apocynin or S17834 but not by oxypurinol, rotenone, meclofenamate, or L-NAME. We also investigated the relative abundance of NAD(P)H-oxidase protein subunits in the endothelial cells. Relative quantification of protein bands, normalized to β-actin, revealed increased levels of the p22phox membrane-bound subunit and the p67phox and p47phox cytosolic subunits in HUVECs from blacks compared with whites (Figure 2).

As in NAD(P)H-oxidase protein subunits, Western blot analysis revealed a parallel increase in eNOS expression in HUVECs from blacks (Figure 2). Also, the NOS activity was significantly greater in HUVEC lysates in blacks than whites when we used an NOS activity assay¹⁸ based on conversion of ³H-l-arginine to ³H-l-citrulline (data not shown). This suggested that endothelial cells in blacks compared with whites overproduce not only O₂⁻ but also NO, which finally leads to the formation of ONOO⁻. To investigate further whether the racial difference in the endothelial capabilities of O₂⁻ and NO production results in a disparity in NO bioavailability, we studied the eNOS-stimulated kinetics of free NO release with simultaneous detection of the kinetics of O₂⁻ and ONOO⁻ release by the tandem NO/O₂⁻/ONOO⁻ nanosensors. In both racial groups, the stimulated (by CaI) NO release from a single endothelial cell was associated with concurrent release of O₂⁻ and ONOO⁻ (Figure 3). In a HUVEC from whites, the pattern of ONOO⁻ release was similar to that for NO release, but it was delayed with a time-shift of ≈1 second. The sharp peaks of NO and ONOO⁻ concentrations were reached at 0.9 and 1.8 seconds after CaI stimulation, respectively. In contrast, the peak value of ONOO⁻ concentration recorded in a HUVEC from blacks was ≈2 seconds before reaching the maximum for NO concentration, and it was at the time of the peak NO concentration in a HUVEC from whites. The blunted peaks of O₂⁻ concentrations were observed in the same time span (3 to 4 seconds) in HUVECs from both racial groups. There were significant differences in the kinetics of the initial rates of NO, O₂⁻, and ONOO⁻ release between blacks and whites from a single HUVEC. The rate of NO release was ≈5 times slower in blacks than whites (94 versus 505 nmol/L per second), whereas the rates of release were ≈2 times faster for O₂⁻ and ≈4 times faster for ONOO⁻ in blacks than whites (9.4 versus 22.1 nmol/L per second for O₂⁻ and 209 versus 810 nmol/L per second for ONOO⁻).

We confirmed that the release of NO, O₂⁻, and ONOO⁻ was caused by eNOS activation by the finding that eNOS inhibition by L-NAME prevented CaI-stimulated release of the detected molecules in both racial groups (Figure 4). In contrast to L-NAME, pretreatment with the specific NAD(P)H-oxidase inhibitors in concentrations that significantly inhibited NAD(P)H-dependent O₂⁻ production in the endothelial cells (Figure 2), apocynin 3 mmol/L or S17834 0.05 mmol/L, abolished the difference in the eNOS-dependent release of the detected molecules from HUVECs between whites and blacks (Figure 4). The slow continuous generation of ONOO⁻ by SIN-1 (0.5 mmol/L) during pretreatment with apocynin or S17834 caused the differences between whites and blacks to be maintained. This suggested that the either apocynin or S17834 was effective in abolishing the difference in the eNOS function in HUVECs between both groups by preventing O₂⁻ formation that eventually reacted with NO to form ONOO⁻. Accordingly, pretreatment of the cells with either MnTMPyP (0.01 mmol/L), a specific O₂⁻ scavenger, or FeTPPS (0.01 mmol/L), a specific ONOO⁻ scavenger, abolished the differences in eNOS-dependent NO, O₂⁻, and ONOO⁻ release. The effect of abolishing racial differences in NO, O₂⁻, and ONOO⁻ release was also observed when the cells were pretreated in the same way with other specific scavengers of O₂⁻ and ONOO⁻, tempol (1 mmol/L), or uric acid (0.1 mmol/L), respectively (data not shown). As in the studies with CaI, the differences between whites and blacks in the peak NO, O₂⁻, and ONOO⁻ concentrations were also revealed in HUVECs in response to acetylcholine (1 μmol/L), a receptor-independent eNOS agonist (data with acetylcholine are not shown). Likewise, the racial differences were abolished by pretreatment with the NAD(P)H-oxidase inhibitors (SIN-1 inhibited this effect) and the scavengers of O₂⁻ or ONOO⁻.

Of note, the racial differences in NO/O₂⁻/ONOO⁻ balance and both eNOS and NAD(P)H regulation were revealed even in prolonged cell cultures when the cells from the eighth passages were investigated.