Abstract
Background
African American (AA) subjects with essential hypertension (EH) have greater inflammation and cardiovascular complications than Caucasian EH. An impaired endogenous cellular repair system may exacerbate vascular injury in hypertension, yet whether these differ between AA EH and Caucasian EH remains unknown. Vascular repair by circulating endothelial progenitor cells (EPCs) is controlled by regulators of EPC mobilization, homing, adhesion and new vessel formation, but can be hindered by various cytokines. We hypothesized that EPC levels and function would be impaired in AA EH compared with Caucasian EH, in association with increased levels of inflammatory mediators and EPC regulators.
Methods
CD34+/KDR+ EPCs were isolated from inferior vena cava and renal vein blood samples of AA EH and Caucasian EH patients (n = 18 each) and from peripheral veins of 17 healthy volunteers (HVs) and enumerated using fluorescence-activated cell sorting. Angiogenic function of late-outgrowth endothelial cells expanded from these samples for 3 weeks was tested in vitro. Levels of inflammatory mediators, angiogenic factors and EPC regulators were measured by Luminex.
Results
EPC levels were decreased in both AA and Caucasian EH compared with HVs, whereas their late-outgrowth endothelial cell angiogenic function was comparable. Levels of several inflammatory mediators were elevated in AA EH compared with Caucasian EH and HVs. Contrarily, vascular endothelial growth factor and its receptor-2 were lower. EPC levels inversely correlated with blood pressure in all hypertensive patients and estimated glomerular filtration rate with inflammatory mediators only in AA EH.
Conclusions
Despite lower EPC numbers, decreased vascular endothelial growth factor signaling and inflammation, EPC function is preserved in AA EH compared with Caucasian EH and HVs, suggesting compensatory mechanisms for vascular repair.
Keywords: African American, endothelial progenitor cells, hypertension
INTRODUCTION
Endothelial progenitor cells (EPCs) that patrol the systemic circulation are necessary for repair and continued function of the vascular endothelium, and their depletion is considered a marker of ongoing vascular damage [1]. EPCs may also undergo functional changes during exposure to cardiovascular risk factors [2], leading to an impaired ability to repair endothelial damage [3]. Late-outgrowth endothelial cells (LOECs), putative representatives of cultured EPCs, appear after 17–23 days in culture [4]. LOECs are highly proliferative, directly participate in endothelial tubulogenesis, differentiate into vascular endothelial cells and form vascular networks invitro and invivo [5–7] and thus are considered useful surrogates of EPCs invitro. For example, impaired invitro function of LOECs obtained from patients with diabetes correlates with low microvessel density invivo in murine of hindlimb ischemia [8]. Furthermore, a previous study in patients with end-stage renal disease showed that LOEC characteristics invitro reflect their capability for vessel network formation invivo [6]. The number of circulating EPCs is lower in medically treated Caucasian essential hypertensive (EH) patients compared with healthy individuals [9], yet their function invitro is overall preserved [10]. However, the potential effects of racial background on EPC function in treated hypertension remain to be elucidated.
Inflammatory factors not only contribute to the etiology of hypertension and lead to endothelial dysfunction [11], but also serve as injury and homing cues for EPCs. EPCs released into the peripheral circulation after stimulation by an inflammatory response home in on the injured site and participate in tissue repair [12]. Growth factors and cytokines promote EPC migration to these sites [12], adhesive molecules promote their local adhesion to the endothelial monolayer [13] and various inflammatory and growth factors regulate EPC angiogenic function [12]. Contrarily, some inflammatory mediators may diminish the number and function of EPCs in patients [14]. Therefore, the overall inflammatory milieu may dictate the reparative potency of vascular repair by EPCs.
African Americans (AA) have a higher risk of hypertension and cardiovascular end-organ damage compared with Caucasians [15–17]. The pathogenesis of enhanced hypertension in AA patients has been postulated to involve increased vascular injury and impairment of endothelial function [18], which are often consequences of inadequate mechanisms to repair vascular damage [19]. However, it remains unknown whether the endogenous vascular repair system is impaired in hypertensive AA subjects.
Thus the purpose of this study was to test the hypothesis that EPC levels and function would be reduced in AA compared with Caucasian hypertensive patients and healthy volunteers (HVs), in association with increased levels of inflammatory mediators.
MATERIALS AND METHODS
Nondiabetic patients with EH (18 Caucasian and 18 AA) were prospectively enrolled. We excluded patients with secondary hypertension, uncontrolled hypertension [systolic blood pressure (SBP) >180 mmHg], cardiovascular events within the past 6 months (myocardial infarction, stroke or congestive heart failure) and serum creatinine levels >1.6 mg/dL. Caucasian HVs (n = 17) without cardiovascular risk factors were recruited through the Mayo Clinic Biobank. The Mayo Clinic institutional review board approved this study and informed consent was obtained. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki.
A 3-day inpatient protocol was performed in all hypertensive patients as part of another study [20]. All hypertensive patients were taking angiotensin-converting enzyme inhibitors (ACEis) or angiotensin receptor blockers (ARBs) and continued with this regimen during the study. During the protocol, dietary sodium intake was controlled at 150 mEq/day; 24-h urine protein and serum creatinine were measured on the first day. Blood pressure was measured three times daily using an automated oscillometric device. Estimated glomerular filtration rate (eGFR) was calculated by the Chronic Kidney Disease Epidemiology Collaboration formula. On the third day, the common femoral vein was cannulated and blood samples selectively collected from the inferior vena cava (IVC) and the right and left renal veins (RVs) with a 5F pigtail Cobra catheter (Cook Inc., Bloomington, IN, USA). HV recruitment was initiated in parallel with and completed a few months after the inpatient protocol in hypertensive patients. In HVs, blood samples from a peripheral (antecubital) vein and a urine sample were collected during a single visit, and dietary sodium intake was not regulated.
Mononuclear cells were immediately isolated from blood samples, characterized for surface markers and the number of CD34+/KDR+ EPCs determined using fluorescence-activated cell sorting (FACS). In addition, mononuclear cells were cultured for 3 weeks and angiogenic function (migration, proliferation and tube formation) studied invitro in the resultant LOECs [2, 10]. Inflammatory mediator, EPC mobilizing and homing factor levels were measured using Luminex. In hypertensive patients, values measured in the right and left RVs were averaged and a cross-renal gradient (RV-IVC) for each marker was estimated.
EPC isolation and characterization
Shortly after sampling, mononuclear cells were obtained using the density-gradient centrifugation of heparinized blood with Histopaque 1077 (Sigma, St. Louis, MO, USA) at 1600 rpm for 25 min (Beckman Coulter, Minneapolis, MN, USA), as described previously [10, 21]. These cells were incubated with primary antibodies against CD34 (R&D Systems, Minneapolis, MN, USA; catalog no. AF3890, NS0-derived rpCD34) and KDR (Santa Cruz Biotechnology, Dallas, TX, USA; catalog no. sc-504, Clone: C-1158) for 1 h at room temperature. CD34+/KDR+ EPCs were sorted using FACS and counted by CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA) [9]. Results were described as percent per 100 000 cell counts.
LOEC function test in vitro
Mononuclear cells were resuspended in EGM-2 Singlequot medium (Lonza, Walkersville, MD, USA) and plated on six-well plates (Corning, Corning, NY, USA) coated with fibronectin (1 µg/m2; Sigma). After 3 weeks of culture, LOECs emerged, which exhibit mature endothelial cell characteristic and angiogenic capabilities [5]. LOEC cultures were examined by light microscopy and samples were assayed for function.
Migration
QCM Haptotaxis cell migration kit was used for LOEC migration test (Millipore, Billerica, MA, USA) [2, 10]. LOECs (1 × 106 cells/mL) were set in fibronectin-coated or BSA-coated (negative control) wells, incubated for 24 h and stained. After stained LOECs were solubilized with extraction buffer, 100-μL of buffer was transferred to a 96-well plate. Migration was measured in a plate reader at absorbance 562 nm.
Proliferation
The MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (Promega, Madison, WI, USA) was used to study proliferation [2, 10]. LOECs (3 × 103 cells/well) were placed in 96-well, flat-bottom plates with EGM-2 medium containing 2% fetal calf serum (FCS) and allowed to adhere for 24 h. The MTS solution was added at 20 μL/well and the conversion of MTS to formazan after 4 h of culture was measured in a plate reader at 490 nm.
Tube formation
Matrigel tube formation assay was performed using a BD BioCoat angiogenesis system (BD Biosciences, Bedford, MA, USA) [2, 10]. Matrigel was dispensed onto 24-well plates and polymerized for 15 min at 37°C. LOECs (1 × 104 cells) were labeled with DiI (Molecular Probes, Eugene, OR, USA), mixed with human umbilical vein endothelial cells (PromoCell, Heidelberg, Germany) (4 × 104 cells) in 24-well plates and incubated at 37°C for 24 h with EGM-2 culture medium. The lengths of formed tubes were quantified using Meta-Morph image analysis software (Meta Imaging Series 6.3.2, Allentown, PA, USA).
Inflammatory mediators and EPC regulating factors
Blood samples were centrifuged and assayed by Luminex for levels of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1), VEGFR-2, granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), stromal cell–derived factor 1 (SDF-1), soluble E-selectin (sE-selectin), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), macrophage inflammatory protein-1δ (MIP-1δ), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interlukin-6 (IL-6), IL-10, matrix metalloproteinase-9 (MMP-9), myeloperoxidase (MPO) and plasminogen activator inhibitor-1 (PAI-1) (Millipore, catalog nos. HCYTOMAG-60K, HCYP2MAG-62K, HSCR-32K). Signals were measured by the Bio Plex 200 system (Bio-Rad, Hercules, CA, USA) [10].
Statistical analysis
Statistical analysis was performed using JMP software version 10.0 (SAS Institute Cary, NC, USA). Results are presented as median (interquartile range) since most data did not show a normal distribution. Nonparametric (Wilcoxon and Kruskal–Wallis) tests were used for comparisons. Multivariate models were applied to account for age, body mass index (BMI), mean arterial pressure (MAP), eGFR, statin use and racial effects on EPCs and inflammatory mediators. Regressions were calculated by Spearman rank correlation coefficient. P-values ≤0.05 was considered statistically significant.
RESULTS
Patients’ characteristics
The characteristics of participants are summarized in Table 1. AA EH patients were younger and their BMIs were higher than Caucasian EH patients (both P = 0.006), but had similar durations of hypertension. Treated diastolic blood pressure (DBP) was higher in AA EH (P = 0.001) and Caucasian EH (P = 0.04) compared with HVs, as was MAP (P = 0.001 and P = 0.05, respectively). SBP was higher only in AA EH compared with HVs (P = 0.02). Serum creatinine and urine protein levels did not differ among the groups, but eGFR was higher in AA EH compared with the two other groups (P = 0.01 versus HVs, P = 0.02 versus Caucasian EH). Lipid profile showed no difference among the three groups. Antihypertensive medications were similar between hypertensive patients, all of who were taking ACEis and/or ARBs. Caucasian EH was more frequently treated with statins than AA EH (P = 0.05).
Table 1.
Characteristics of HVs, Caucasian and AA patients with EH
| Caucasian HVs (n = 17) | Caucasian EH (n = 18) | AA EH (n = 18) | |
|---|---|---|---|
| Age (years) | 58 (52.75–68) | 66.5 (51–71.75) | 50 (46–52.75)*,† |
| Sex (men:women) | 5:12 | 5:13 | 5:13 |
| Weight (kg) | 78.7 (60.2–91.8) | 75.1 (65.4–87.9) | 90.8 (80.2-103.2)*,† |
| BMI (kg/m2) | 25.4 (22.9–29.3) | 25.8 (23.3–31.2) | 31.8 (28.6–39.7)*,† |
| SBP (mmHg) | 124 (114.5–128.5) | 129 (121.3–145) | 133 (120–145)* |
| DBP (mmHg) | 71 (62.5–73.5) | 73.5 (69–80.8)* | 79.5 (72.3–86)* |
| MAP (mmHg) | 86.7 (80.7–91.8) | 91.7 (85.7–100.9)* | 97.2 (87.5–103.5)* |
| Duration of hypertension | 0 | 14 (5.8–16)* | 8 (5–15.5)* |
| Serum creatinine (mg/dL) | 0.8 (0.8–1.0) | 0.9 (0.8–1.1) | 0.8 (0.7–1.2) |
| eGFR (mL/min/1.73 m2) | 77.1 (69.55–87.1) | 83.6 (59.7–94.65) | 97.3 (81–116.8)*,† |
| Urine protein (mg/mL) | 70 (33–109) | 47 (37.5–105.5) | 48 (30–69) |
| Total cholesterol (mg/dL) | 176 (146.5–204.5) | 182 (161–198) | 181 (164–230) |
| High-density lipoprotein (mg/dL) | 58 (47.5–74) | 53 (43–61) | 55 (43–64.8) |
| Low-density lipoprotein (mg/dL) | 95.8 (66.6–116.9) | 107 (81–120) | 108 (90–143.8) |
| Triglycerides (mg/dL) | 113 (82.5–145) | 103 (91–173) | 91.5 (77.3–136.8) |
| ARB | 0 | 8/18 | 8/18 |
| Angiotensin-converting enzyme inhibitor | 0 | 10/18 | 10/18 |
| Calcium-channel blocker | 0 | 5/18 | 6/18 |
| Diuretics | 0 | 13/18 | 15/18 |
| α-Blockers | 0 | 1/18 | 3/18 |
| Statins | 0 | 8/18 | 3/18† |
Data are median (interquartile range).
*P ≤ 0.05 versus HVs,
P ≤ 0.05 versus Caucasian EH.
EPC numbers and LOECs’ function
IVC CD34+/KDR+ EPC levels were lower in both Caucasian EH (P = 0.003) and AA EH (P = 0.04) than peripheral vein levels in HVs (Figure 1A). There was no measurable difference in IVC and RV CD34+/KDR+ EPC levels between Caucasian EH and AA EH, and no meaningful RV-IVC of EPCs in either Caucasian EH or AA EH (Figure 1A). Invitro, migration, proliferation and tube formation capacity of systemic (IVC or peripheral) and RV LOECs were similar among the three groups (Figure 1B–D). There was no change in EPC numbers and function after adjustment for age, BMI, MAP, eGFR and statin use.
FIGURE 1.
Levels and function of EPCs. (A) Systemic (IVC or peripheral) and renal vein levels of EPCs (per 100 000 cell counts). IVC CD34+/KDR+ EPC levels were lower in both Caucasian and AA patients with EH than peripheral levels in HVs. Cross-renal gradient of EPCs was very small in both Caucasian and AA EH. (B–D) There was no measurable difference in (B) LOEC migration (C) proliferation and (D) tube formation capacity among the three groups. *P ≤ 0.05 versus HVs.
Inflammatory mediators and EPC regulating factors
EPC mobilizing and homing factors
Systemic VEGF levels were lower in AA EH than in both Caucasian EH and HVs (Figure 2A; all P = 0.01). Systemic VEGFR-1 levels were lower in Caucasian EH than in HVs (all P = 0.02), whereas systemic VEGFR-2 levels were decreased in AA EH compared with both Caucasian EH (P = 0.005) and HVs (P = 0.02). RV VEGF, VEGFR-1 and VEGFR-2 levels were all lower in AA EH than Caucasian EH (Figure 2B; P = 0.03, 0.001 and 0.005, respectively) and AA EH had a negative VEGFR-1 gradient across the kidney (Figure 2C). Systemic SCF levels were markedly elevated in AA EH compared with HVs (Table 2; P < 0.001) and Caucasian EH (P < 0.001) and SDF-1 levels compared with HVs (P = 0.02). Systemic G-CSF levels were not significantly different among the three groups. RV SCF and SDF levels were higher in AA EH than Caucasian EH (P < 0.001), whereas RV G-CSF levels were similar in AA EH and Caucasian EH.
FIGURE 2.
VEGF and VEGFR levels. (A) VEGF and VEGFR levels in systemic (peripheral or IVC) blood. VEGF levels decreased in AA patients with EH compared with HVs and Caucasian patients with EH. VEGFR-1 levels were lower in Caucasian EH patients than HVs, whereas VEGFR-2 levels were lower in AA EH patient than HVs and Caucasian EH patients. (B) VEGF, VEGFR-1 and VEGFR-2 levels in RV blood were lower in AA EH than Caucasian EH. (C) RV-IVC of VEGF, VEGFR-1 and VEGFR-2. In AA EH patient kidneys, RV levels of VEGFR-1 were significantly lower than their IVC levels, resulting in negative gradients. *P ≤ 0.05 versus HV, †P ≤ 0.05 versus Caucasian EH.
Table 2.
Plasma levels of inflammatory mediators and EPC regulators in HVs and Caucasian and AA patients with EH
| Inflammatory mediators | Peripheral vein | Systemic |
Renal vein |
||
|---|---|---|---|---|---|
| HVs | Caucasian EH | AA EH | Caucasian EH | AA EH | |
| EPC mobilizing and homing factors | |||||
| G-CSF | 11.8 (7.5–16.8) | 12.3 (8.8–32.4) | 15.9 (11.7–28.2) | 13.2 (8.9–30.9) | 18.0 (11.3–30.7) |
| SCF | 6.4 (1.3–11.4) | 14.2 (2.0–22.2) | 113.2 (108.8–169.6)**,†† | 15.8 (2.0–23.4) | 117.2 (101–177.1)‡‡ |
| SDF-1 | 1468.2 (955.3–1716.0) | 2042.7 (433.1–3472.1) | 3212.9 (582.7–5372.8) | 1480.7 (188.8–2985.3) | 2669.3 (504.1–4863.1)‡‡ |
| Adhesion molecules | |||||
| sE-selectin (ng/mL) | 12.8 (7.9–21.0) | 26.4 (15.3–43.8)* | 29.9 (18.9–72.0)** | 23.5 (13.6–37.7) | 30.7 (20.1–72.5) |
| sVCAM-1 | 614.4 (451.5–829.0) | 1110.0 (771.9–278.0)** | 792.3 (621.6–3447.6)* | 1034.2 (722.1–1188.2) | 912.1 (593.4–2931.7) |
| sICAM-1 | 162.4 (123.4–222.4) | 143.9 (88.3–256.4) | 139.1 (112.8–2539.1) | 134.4 (91.0–250.3) | 140.3 (110.8–2652.9) |
| Chemokines | |||||
| MIP-1δ | 2906.3 (1487.4–6187.8) | 3842.3 (2681.2–521.9) | 6891.3 (4043.3–20945.5) | 3297.6 (2023.8–5864.6) | 5604.0 (3160.7–21510.1) |
| MCP-1 | 99.5 (96.2–138.2) | 137.2 (108.8–171.9) | 156.0 (136.3–205.8)** | 128.9 (82.1–154.8) | 139.6 (129.1–184.2) |
| TNF and interleukins | |||||
| TNF-α | 3.5 (2.5–5.8) | 4.0 (3.0–7.4) | 3.6 (2.6–5.2) | 3.5 (2.8–5.6) | 3.15 (2.62–4.72) |
| IL-6 | 1.6 (1.2–6.3) | 3.7 (1.3–13.8) | 2.0 (0–11.6) | 1.9 (1.5–8.3) | 2.7 (0–9.89) |
| IL-10 | 2.8 (2.8–6.6) | 2.2 (0.5–15.0) | 0 (0–8.8) | 1.8 (1.1–12.1) | 0 (0–8.33) |
| Other biomarkers | |||||
| MMP-9 | 71.0 (47.7–86.9) | 61.3 (50.8–99.1) | 85.1 (46.1–3012.2) | 78.9 (37.3–107.4) | 85.0 (54.2–2459.9) |
| MPO | 15.7 (11.6–20.6) | 13.5 (9.1–24.3) | 24.5 (17.8–53.0)*,† | 10.9 (8.0–22.7) | 20.6 (14.1–45.7)‡ |
| PAI-1 | 16.7 (8.3–28.6) | 20.3 (8.4–57.3) | 31.7 (12.9–539.1) | 24.3 (9.5–44.3) | 33.9 (13.5–579.6) |
Data are median (interquartile range) in pg/mL, except where noted.
P ≤ 0.01 and
P ≤ 0.001 versus HV,
P ≤ 0.01 and
P ≤ 0.001 versus systemic levels of Caucasian EH,
P ≤ 0.01 and
P ≤ 0.001 versus renal vein levels of Caucasian EH.
Adhesion molecules
Systemic sE-selectin and sVCAM-1 levels were elevated in AA EH (Table 2; P < 0.001 and P = 0.01, respectively) and Caucasian EH (P = 0.003 and P < 0.001, respectively) compared with HVs. In contrast, systemic sICAM-1 showed no difference among the three groups. RV adhesion molecule levels were similar between AA EH and Caucasian EH.
Chemokines
Systemic MIP-1δ and MCP-1 levels were higher in AA EH than HVs (Table 2; P = 0.02 and P < 0.001, respectively) and Caucasian EH (all P = 0.05), whereas their RV levels were similar.
Cytokines
Systemic and renal TNF-α levels were similar among the three groups. Systemic IL-10 levels decreased in AA EH compared with HVs (Table 2; P = 0.02) and Caucasian EH (P = 0.05) and RV IL-10 levels tended to be lower than in Caucasian EH (P = 0.07). Levels of IL-6 were similar among the groups.
Other biomarkers
Systemic MMP-9 levels were similar among the three groups. Systemic MPO levels were elevated in AA EH compared with HVs (Table 2; P = 0.006) and Caucasian EH (P = 0.01), and PAI-1 levels compared with HVs (P = 0.03). RV MPO levels were higher in AA EH than in Caucasian EH (P = 0.01), while RV MMP-9 and PAI-1 levels were not different between them.
Multivariate analysis
After adjustment for age, BMI, MAP, eGFR and statin use, systemic levels of SCF, adhesion molecules, chemokines and other biomarkers remained higher in AA EH than in HVs, while VEGFR-2 remained lower. In Caucasian EH, only systemic sVCAM-1 levels were higher than HVs. Compared with Caucasian EH, IVC and RV levels of VEGF and VEGFR-2 were decreased in AA EH, whereas levels of SCF, sICAM-1, all chemokines and other biomarkers remained elevated at both sites, except for IVC MCP-1 and RV PAI-1 levels, which that showed only a trend toward elevation (Table 3).
Table 3.
Multivariate analysis of inflammatory mediator levels among HVs and Caucasian and AA patients with EH
| HVs versus AA EH |
HVs versus Caucasian EH |
Caucasian EH versus AA EH |
||||||
|---|---|---|---|---|---|---|---|---|
| IVC |
RVs |
|||||||
| Parameter estimate | P-value | Parameter estimate | P-value | Parameter estimate | P-value | Parameter estimate | P-value | |
| EPC mobilizing and homing factors | ||||||||
| VEGF | 29.88 | 0.55 | −94.14 | 0.51 | 474.90 | 0.001 | 478.19 | 0.001 |
| VEGFR-1 | 197.52 | 0.28 | 231.45 | 0.13 | −143.44 | 0.12 | 202.53 | 0.07 |
| VEGFR-2 | 3555.26 | 0.008 | 2.36 | 0.99 | 2887.55 | 0.006 | 2795.76 | 0.005 |
| G-CSF | −6.03 | 0.44 | −14.91 | 0.40 | 13.46 | 0.35 | 6.75 | 0.28 |
| SCF | −70.99 | <0.001 | 0.58 | 0.87 | −53.72 | <0.001 | −59.44 | <0.001 |
| SDF-1 | −694.62 | 0.12 | 39.41 | 0.89 | −591.23 | 0.14 | −415.87 | 0.22 |
| Adhesion molecules | ||||||||
| sE-selectin | −25.33 | 0.003 | −4.77 | 0.14 | −9.99 | 0.12 | −10.74 | 0.09 |
| sVCAM-1 | −1092.25 | 0.006 | −211.42 | 0.009 | −451.00 | 0.11 | −547.64 | 0.05 |
| sICAM-1 | −830.40 | 0.009 | −8.64 | 0.73 | −486.93 | 0.03 | −498.52 | 0.04 |
| Chemokines | ||||||||
| MIP-1δ | −6121.803 | 0.003 | −115.57 | 0.88 | −3409.37 | 0.02 | −3283.21 | 0.03 |
| MCP-1 | −51.73 | 0.006 | −0.28 | 0.97 | −28.07 | 0.06 | −30.67 | 0.03 |
| TNF and interleukins | ||||||||
| TNF-α | −0.79 | 0.16 | 0.27 | 0.58 | 0.41 | 0.44 | 0.49 | 0.29 |
| IL-6 | −8.36 | 0.07 | −3.01 | 0.55 | 2.00 | 0.70 | −0.65 | 0.89 |
| IL-10 | 1.29 | 0.68 | −5.20 | 0.56 | 8.97 | 0.21 | 4.44 | 0.19 |
| Other biomarkers | ||||||||
| MMP-9 | −1962.54 | 0.007 | 13.84 | 0.23 | −1065.77 | 0.05 | −906.68 | 0.03 |
| MPO | −29.53 | 0.003 | −1.00 | 0.82 | −19.89 | 0.01 | −20.10 | <0.001 |
| PAI-1 | −252.29 | 0.01 | −6.50 | 0.26 | −155.36 | 0.03 | −135.78 | 0.06 |
Models were adjusted for age, BMI, MAP, eGFR and statin use.
RV-IVC gradients
In AA EH kidneys, RV levels of VEGFR-1 were significantly lower than their IVC levels both before (P = 0.009) and after (P < 0.001) eGFR adjustment, resulting in negative gradients significantly different from Caucasian EH (Figure 2C; P < 0.001). RV-IVCs of no other markers were significantly different between Caucasian EH and AA EH.
IVC EPC levels were inversely correlated with SBP and MAP in AA EH and Caucasian EH (MAP in AA EH showed only a trend) (Figure 3). In AA EH, eGFR correlated inversely with IVC levels of MPO and MMP-9 and with RV levels of MPO, MMP-9, TNF-α, MCP-1 and G-CSF (Figure 4).
FIGURE 3.
Correlation of EPC number with blood pressure in AA and Caucasian patients with EH. (A) IVC EPC levels were inversely correlated with SBP and showed a trend for inverse correlation with MAP in AA EH. (B) SBP and MAP correlated inversely with IVC EPC levels in Caucasian EH.
FIGURE 4.
Correlation of eGFR with inflammatory mediators, EPC mobilizing and homing factors in AA patients with EH. (A and B) eGFR correlated inversely with IVC and (A) RV and (B) IVC levels of MMP-9. (C–E) eGFR correlated inversely with RV levels of MCP-1, TNF-α and G-CSF.
DISCUSSION
This study shows that the number of circulating EPCs are decreased in both AA EH and Caucasian EH compared with healthy controls, yet the angiogenic activity of LOECS invitro is preserved. Interestingly, EPC number and function were similar in AA EH and Caucasian EH, despite the observation that VEGF signaling was attenuated, and the levels of many inflammatory cytokines were elevated in AA EH compared with Caucasian EH even after multivariate adjustment. Possibly, elevated levels of SCF, adhesion molecules and chemokines enhanced mobilization of EPCs and their retention and angiogenic activity in hypertension target organs such as the kidney.
In this study, CD34+/KDR+ EPC levels were similarly lower in AA EH and Caucasian EH than in HV, and blood pressure correlated inversely with their systemic levels. Several studies have shown a decreased number of EPCs in hypertension [22, 23], including medically treated EH patients [9, 24]. Notably, some studies have suggested that antihypertensive medications can improve EPC number in addition to function. Valsartan stimulates EPC colony formation [25], and a 2.5-fold increase in EPC count was observed after 4 weeks of ramipril treatment [26]. Yet, our hypertensive patients treated with blockers of the renin-angiotensin-aldosterone system (RAAS) showed decreased levels of circulating EPCs. These divergent observations may result from differences in the observation period in those latter studies, the variability of EPC definition, methods of isolation, underlying disease, comorbidities or medications used.
Previous studies have shown that EPCs become dysfunctional during aging, hypertension, vascular disease, diabetes mellitus or chronic kidney disease [27–32]. The balance between nitric oxide (NO) availability and increased oxidative stress is important in the regulation of EPC mobilization and function [33], which might be impaired in hypertension. Angiotension II causes EPC senescence through increasing oxidative stress and changing telomerase activity, and senescence may lead to EPC dysfunction [34]. ARBs inhibit nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase, a major source of reactive oxygen species, stimulate EPCs through the peroxisome proliferator–activated receptor gamma pathway [35] and improve EPC function in hypertensive patients [28]. ACE inhibition lessens oxidative stress [36] and improves the proliferation, adhesion and migration capacity of EPCs in hypertension [29]. Indeed, EPC angiogenic activity is preserved in renovascular and EH patients treated with ARBs or ACEis [10]. However, the effect of racial background on EPC function remained unknown. In the current study, we observed that LOEC function invitro was preserved in both AA EH and Caucasian EH, possibly because all patients were treated with ARBs or ACEis. These results support the use of autologous EPC therapy in medically treated hypertensive patient regardless of race.
The EPC vascular repair system is controlled by multiple factors through a complex sequence including EPC mobilization from the bone marrow, circulation, homing, adhesion to the impaired sites and new blood vessel formation [12]. VEGF is an important stimulus that mobilizes EPCs from the bone marrow and regulates their angiogenic properties [12]. The current study suggests that VEGF signaling in hypertensive patients might be race dependent. We found that systemic and RV levels of VEGF and its primary receptor VEGFR-2 were both lower in AA EH than in Caucasian EH and remained significantly lower after adjustment for age, BMI, MAP, eGFR and statin use. Furthermore, VEGFR-1, a decoy receptor that negatively sequesters VEGF, was downregulated in Caucasian EH, but not in AA EH, compared with HVs. These results imply greater VEGF pathway availability in Caucasian EH than in AA EH. Importantly, low VEGF levels have been linked to cardiovascular disease [37], and VEGF signaling pathway inhibitors often induce hypertension [38], possibly due to a decrease in NO bioavailability [39]. Given that AAs a priori have lower bioavailability of endothelial nitric oxide (NO) than Caucasians [19], they might be more vulnerable to decreases in levels of VEGF. In addition, VEGF genetic polymorphisms show racial differences [40] and impacts susceptibility to hypertension [41], hypertensive complications [42] and antihypertensive responses [43]. Further studies are required to establish race dependency of VEGF signaling pathways, which may be relevant for development of therapeutic targets in AA EH patients.
Many modulating factors also affect EPC recruitment and mobilization from the stem cell niche [44], and adhesion molecules play a role in their local retention in injured tissues [45]. In the current study, while AA EH patients showed decreased VEGF levels, they exhibited elevated levels of SCF, adhesion molecules, chemokines, MMP-9, MPO and PAI-1 levels compared with HVs, which remained significant after multivariate adjustment. Only sVCAM-1 remained higher in Caucasian EH compared with HVs. Conceivably, elevated levels of homing factors, adhesion molecules and chemokines may serve to compensate for decreased VEGF signaling and ensure preserved EPC recruitment and function in AA EH.
We found higher inflammatory mediators levels in AA EH than in Caucasian EH despite similar regimens with ACEis or ARBs, representing the complex pathogenesis of AA EH. Several studies have demonstrated that the blood pressure–lowering efficacy of inhibitors of the renin–angiotensin system is attenuated in AA EH [16, 46]. Future studies will need to determine the effect of renin–angiotensin system blockers on inflammation in AA EH.
The kidneys are important target organs in hypertension, and renal damage is more common in AA EH than in Caucasian EH [16]. In the current study, RV levels of most inflammatory cytokines (except VEGFR-1) were not significantly different from their systemic levels in either AA or Caucasian EH. These data argue against the kidney being the source of these inflammatory mediators in hypertensive patients with relatively preserved renal function. Interestingly, cross-renal VEGFR-1 gradients were negative in AA EH, suggesting sequestration or urinary excretion of the decoy receptor in AA EH kidney. RV levels of inflammatory mediators were higher in the AA EH compared with Caucasian EH despite elevated eGFR. However, the inverse correlation of eGFR with several mediators in AA EH argues against renal hyperfiltration as a trigger for release of injury signals in AA EH.
Limitations of our study include its cross-sectional nature and a greater proportion of women, so that findings cannot be extrapolated to the general population. This study has A relatively small sample size, given the invasive nature of RV and IVC sampling, hence, for ethical reasons, blood was collected from a peripheral vein in HVs. Notwithstanding, this study afforded evaluation of the RV levels of EPCs and inflammatory mediators in hypertensive patients. Furthermore, preliminary comparisons of cytokine measurements between a peripheral vein and IVC disclosed very similar levels (unpublished data), and there is no reason to believe that EPC numbers would differ between these vascular beds. AA EH patients were younger, more obese and had higher eGFRs and less usage of statins than Caucasian EH patients and HVs. Adjustments for these variables failed to abolish the differences among the groups. Yet, we cannot rule out some uncorrected effects of these factors and cannot fully exclude the possibility that the younger age of AA EH patients contributed to preserved EPC numbers. Several subjects were also treated with additional drugs other than ACEis or ARBs, which might affect EPC and inflammatory mediators. Additionally, the HV group included only Caucasian subjects, due to practical difficulties in enrollment.
Our study demonstrates that circulating EPC numbers were similarly decreased, and their function similarly preserved, in medically treated AA and Caucasian hypertensive patients compared with HVs. Levels of a spectrum of inflammatory cytokines, injury signals and regulators of EPC recruitment were significantly higher in AA than in Caucasian hypertensive patients, which may account for preserved vascular repair capacity at an early stage of EH. On the other hand, VEGF signaling is deficient in the AA group. These observations may help direct race-selective management strategies in hypertensive subjects. Further studies are needed to determine mechanisms of vascular injury in AA EH.
FUNDING
This study was partly supported by National Institutes of Health grant numbers DK100081, DK73608, HL123160 and DK102325.
CONFLICT OF INTEREST STATEMENT
None declared.
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