Endothelial Nitric Oxide Synthase Uncoupling Impairs Endothelial Progenitor Cell Mobilization and Function in Diabetes

The study conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (publ. no. 85-23, revised 1996). Clinical study approval from the ethics committee of the University of Würzburg was obtained, as was informed written consent from patients.

We isolated peripheral blood mononuclear cells (PBMCs) derived from leucophoresis material of healthy volunteers or from whole blood of type 2 diabetic (n = 5, aged 70.6 ± 1.7 years) and nondiabetic (n = 5, aged 70.0 ± 2.9 years) patients, as described (11). Glucose concentrations and A1C values were determined in the morning after an overnight fasting period. Detailed patient characteristics are shown in Table 1. As diabetes had not been diagnosed previously in these patients, they were not on insulin or any oral antidiabetes medication at the time of blood withdrawal. Rat PBMCs were isolated by Ficoll density centrifugation (20). Hollow bones of rat legs were prepared by standard surgical procedures, and whole bone marrow was harvested by flushing marrow with 500 μl PBS using a syringe with a 20-gauge needle as described (20). Bone marrow was pelleted and shock frozen before further analysis.

We used an adhesion-related selection method for isolation of monocytic EPCs, as described (11,15,20,21). PBMCs (2 × 10⁵) were cultured on fibronectin-precoated eight-well chamber slides (Lab-Tek; Nunc, Wiesbaden, Germany) in endothelial basal medium (EBM)-2 culture medium supplemented with EBM SingleQuots (Cambrex, Verviers, Belgium) and 20% fetal calf serum for 3 days. To exclude contamination with mature circulating endothelial cells, we carefully removed culture supernatant 2 h after initial seeding and placed it on new fibronectin-precoated chamber slides. EPCs were characterized by cellular uptake of acetylated LDL (dil-acLDL; Molecular Probes, Eugene, Oregon), binding of fluorescein isothiocyanate–conjugated lectin from Ulex europeus (UEA-1; Sigma, Deisenhofen, Germany), expression of vascular endothelial growth factor receptor-2 and eNOS, capacity for integration during endothelial tube formation, and cellular migration and colony forming unit (CFU) capacity, as described (20). Additionally, EPCs were stained with an anti–von Willebrand factor antibody (Sigma).

Diabetes was induced by a single intravenous injection of streptozotocin (50 mg/kg) in male Wistars rats (180–200 g) obtained from Harlan-Winkelmann (Borchen, Germany). After 12 weeks hyperglycemia was confirmed by a blood glucose monitoring system in the morning after an overnight fasting period (Ascensia Elite; Bayer, Leverkusen, Germany). Only streptozotocin-induced diabetic rats with blood glucose levels >300 mg/dl were included in the study.

Westen blotting was performed as described (22). In brief, cell extracts were mixed with sample loading buffer and separated under reducing conditions on 12% SDS-polyacrylamide gel. Proteins were electro-transferred onto polyvinylidine fluoride membranes (Immun-Blot 0.2 μm; Bio-Rad), incubated for 2 h in Tris-buffered saline with Tween (TBS-T) with 5% blocking agent (Amersham), and then incubated overnight at 4°C with primary antibodies. The bands were detected using a chemiluminescence assay (ECL+Plus; Amersham). Primary antibodies used included mouse anti-eNOS and anti–phospho-eNOS (Transduction Laboratories, BD Biosciences, Heidelberg, Germany) and mouse anti-GAPDH (Abcam, Acris, Germany).

Cell extracts were mixed with 3× SDS sample buffer (187.5 mmol/l Tris-HCl [pH 6.8], 6% wt/vol SDS, 30% glycerol, 0.03% wt/vol bromophenol blue, and 15% vol/vol 2-mercaptoethanol) at 0°C. Samples were loaded on 7.5% polyacrylamide gels and subjected to electrophoresis. Gels and buffers were cooled to 4°C before electrophoresis and the buffer tank placed in an ice bath during electrophoresis. eNOS dimer/monomer protein was detected by Western blot analysis.

A variety of different techniques were used to measure reactive oxygen species (ROS) production, as described below.

Malondialdehyde-thiobarbituric acid adducts were measured by high-performance liquid chromatography (HPLC) as described (23). Bone marrow supernatants were separated, and fractionation of the protein-free extract was performed using a C18 column (Micro Bondapak; Waters, Milford, MA) in an HPLC system (Pharmacia LKB, Freiburg, Germany). We used a flow rate of 1 ml/min at a pressure of 1,800 psi at 35°C. Exact quantification was achieved using 1,13,3-tetraethoxypropane standards.

Superoxide anion formation was measured by lucigenin-enhanced chemiluminescence. Cell extracts were transferred into scintillation vials containing lucigenin and Krebs/HEPES buffer (final composition: 0.005 mmol/l lucigenin, 99.01 mmol/l NaCl, 4.69 mmol/l KCl, 2.5 mmol/l CaCl₂, 1.2 mmol/l MgSO₄, 1.03 mmol/l KH₂PO₄, 25 mmol/l NaHCO₃, 20 mmol/l Na-HEPES, and 5.6 mmol/l glucose [pH 7.4]). Signals were assessed over 20 min in a luminometer (Wallac, Freiburg, Germany) at 30-s intervals. To address the effects of NOS inhibition on superoxide production, aliquots of bone marrow extracts were incubated with N^G-nitro-l-arginine (l-NNA, 1 mmol/l) for 20 min.

The redox-sensitive, cell-permeable fluorophore dihydroethidium becomes oxidized in the presence of O₂⁻ to yield fluorescent ethidium. Thus, dye oxidation is an indirect measure of the presence of reactive oxygen intermediates. Cultured EPCs were incubated with dihydroethidium (2.5 μmol/l) for 30 min. After washing mean channel fluorescence, each sample was measured during fluorescence-microscopic evaluation of at least four different visual fields. Intracellular ROS in human EPCs were additionally detected using a FACSCalibur flow cytometer (Becton Dickinson, San Juan, CA). Fluorescence was measured using an FL-3 filter (670 nm), and histograms of 10,000 events were analyzed per experiment. The degree of fluorescence in EPCs was then evaluated using Cell Quest software (Becton Dickinson).

Peroxynitrite reacts quickly with a variety of different biomolecules to produce 3-nitrotyrosine, which therefore is a good biomarker for reactive nitrogen species (24). Human EPCs were treated with glucose (30 mmol/l, 24 h), and then cell lysates were prepared on ice using cell lysis buffer (1 mol/l Tris-HCl [pH 7.6], 0.5 mol/l EDTA, and 1.25 mol/l dithiothreitol). 3-Nitrotyrosine from cell lysates was then isolated by HPLC and subjected to a sensitive gas chromatography–tandem mass spectrometry method, as recently described by us (24), in the electron capture negative-ion chemical ionization mode on a triple-stage quadrupole mass spectrometer model ThermoQuest TSQ 7,000 (Finnigan MAT, San Jose, CA) directly interfaced with a Trace 2000 series gas chromatograph equipped with an autosampler AS 2000 (CE Instruments, Austin, TX) (24).

Measurements of biopterin content in EPCs were performed using HPLC analysis and a differential oxidation method as previously described (25). The amount of BH₄ was determined from the difference between total (BH₄ plus BH₂ plus biopterin) and alkaline-stable oxidized (BH₂ plus biopterin) biopterin. A Nucleosil C-18 column (4.6 × 250 mm, 5 μm) was used with 5% methanol/95% water as a solvent at a flow rate of 1.0 ml/min. The fluorescence detector was set at 350 nm for excitation and 450 nm for emission.

Endothelial CFUs from isolated PBMCs of diabetic and nondiabetic patients were determined using the EndoCult system (StemCell Technologies) as described (11).

Migratory capacity of EPCs was investigated using the modified Boyden chamber assay as previously described (11,20). After treatment, 5 × 10⁴ EPCs were cultured in inlets (Falcon HTS Fluoro Blok insert, 8-μm pore size), which were placed in 24-well culture dishes containing EBM (Clonetics) and 50 ng/ml vascular endothelial growth factor, and 100 ng/ml stromal cell–derived factor-1. After 24 h, migrated cells were stained with dil-acLDL and manually counted by fluorescence-based microscopic evaluation of the bottom side of the membrane.

Data are expressed as means ± SEM. Statistical analysis was performed by one-way ANOVA followed by multiple comparisons using Fisher’s protected least significant difference test. Statistical analysis was performed using StatView 5.0 statistic program (Abacus Concepts, Berkley, CA). Statistical significance was assumed at P < 0.05.

Diabetic patients were characterized by increased plasma glucose concentration (294.0 ± 32.0 vs. 100.0 ± 6.4 mg/dl) and increased A1C values (8.8 ± 0.6 vs 5.4 ± 0.1%) compared with nondiabetic age-matched subjects. Patient characteristics are shown in Table 1.

Diabetic patients had reduced formation of endothelial CFUs compared with nondiabetic subjects (9.8 ± 1.9 vs. 22.6 ± 1.7 CFUs, P = 0.001; Fig. 1A and B). A detailed description of human EPC characterization has been previously published (11) (Fig. 1). EPCs from diabetic patients displayed increased ROS formation (11.7 ± 0.7 vs. 6.2 ± 1.1 arbitrary units, P < 0.001; Fig. 2A and B) but impaired migratory capacity compared with EPCs from nondiabetic age-matched subjects (40.4 ± 4.6 vs. 72.7 ± 6.2 migrated cells, P < 0.0001; Fig. 2C). After NOS inhibition with l-NNA in EPCs from diabetic patients, ROS production was attenuated and cellular migration improved by 34.6 ± 5.6% (P < 0.05; Fig. 2A–C). Incubation of diabetic EPCs with pegylated superoxide dismutase completely inhibited ROS formation and improved migration by 60.1 ± 11.3% (P < 0.001; Fig. 2A–C).

To test whether observations from the clinical study can be related to the increased glucose concentration in patients, we carried out further in vitro studies. Glucose treatment increased ROS production up to threefold and impaired migratory capacity of cultured EPCs by 55.7 ± 8.2% (P < 0.001), whereas treatment with mannitol (30 mmol/l) as osmotic control had no effect (Fig. 3A and B). Increased concentrations of O₂⁻ react with NO in a reaction forming peroxynitrite, the latter being a powerful toxicant to a wide range of cells (26). Peroxynitrite is difficult to measure and reacts quickly with a variety of different biomolecules to produce 3-nitrotyrosine. In glucose-challenged EPCs, 3-nitrotyrosine concentration was substantially higher than in control subjects, whereas NOS and to a lesser extent NADPH oxidase inhibition attenuated exaggerated 3-nitrotyrosine production (Fig. 3C). Combined NOS and NADPH oxidase inhibition completely inhibited 3-nitrotyrosine production in glucose-challenged EPCs (Fig. 3C).

The glucose-mediated impairment of EPC migration was diminished when cells were concomitantly treated with pegylated supoxide dismutase (Fig. 3B). Inhibition of NOS with l-NNA attenuated the detrimental effects of glucose (Fig. 3A and B).

Because PKC is involved in vascular O₂⁻ in diabetic vessels (4), we analyzed its importance in glucose-mediated ROS production in EPCs. Inhibition of PKC by chelerythrine attenuated O₂⁻ production after treatment of EPCs with glucose. In parallel, EPC function was improved (Fig. 3A and B). PKC may also activate NADPH oxidases (27); therefore, we additionally tested the contribution of NADPH oxidases in glucose-mediated impairment of EPCs. Inhibition of NADPH oxidases resulted in a slight reduction of intracellular O₂⁻ levels after glucose challenge, whereas combined inhibition of NOS and NADPH oxidases strongly lowered O₂⁻ production. A trend for improved migratory capacity was also observed after concomitant treatment with glucose and DPI, although this was not statistically significant. However, synergistic effects on the improvement of EPC migration after glucose challenge were observed after combined treatment with l-NNA and DPI (Fig. 3).

In addition, a reduction of the essential eNOS cofactor BH₄ has been described to be mechanistically involved in eNOS uncoupling (4,7). Intracellular levels of BH₄ and total and oxidized biopterin levels were measured in lysates from glucose-challenged and control EPCs. Glucose treatment significantly reduced the concentration of intracellular BH₄ by 59%, whereas oxidized biopterin levels raised by 36% (P < 0.05). Total biopterin levels were basically unchanged (Fig. 4A and B). To test whether reversal of the glucose-mediated reduction in intracellular BH₄ levels would rescue EPC function, we performed further studies. Exogenous treatment of glucose-challenged EPCs with BH₄ increased their intracellular availability by fivefold compared with untreated controls. As a result, glucose-mediated exaggerated O₂⁻ production was attenuated and EPC migratory capacity significantly improved (Fig. 4C and D).

To understand whether reduced levels of EPCs in diabetes are potentially mediated by changes of EPC-mobilizing pathways in bone marrow, we used rats with streptozotocin-induced diabetes. Diabetic animals had higher serum glucose levels compared with control animals (459 ± 39 vs. 151 ± 15 mg/dl, P < 0.0001). In contrast, levels of circulating EPCs were reduced to 39 ± 5% compared with nondiabetic animals (P < 0.05) (Fig. 5A). A detailed rat EPC characterization has been previously published (20).

Diabetes was associated with 4.9-fold increased malondialdehyde-thiobarbituric acid adduct formation, an index of ROS generation (data not shown), as well as with increased O₂⁻ production in bone marrow (Fig. 5B and C). eNOS protein expression was increased in bone marrow extracts of diabetic rats (Fig. 5D), whereas eNOS phosphorylation (Fig. 5E), as well as eNOS dimer-to-monomer ratio, was reduced (Fig. 5F). NOS inhibition by l-NNA increased O₂⁻-mediated chemiluminescence in bone marrow extracts from control subjects but decreased O₂⁻ levels in diabetic bone marrow, indicating uncoupling of eNOS to be involved in exaggerated O₂⁻ production (Fig. 5C).

Diabetic patients are at high risk for endothelial lesions and arteriosclerosis. EPCs essentially contribute to vascular lesion repair (8). In the present study, we demonstrate for the first time uncoupling of the eNOS in EPCs from diabetic patients, in glucose-treated EPCs, and in bone marrow from diabetic rats. eNOS uncoupling explains at least in part reduced levels and impaired function of EPCs observed in diabetes and provides a valuable pharmacological target.

Under physiological conditions, eNOS confers anti-arteriosclerotic vascular protection (28). Indeed, eNOS-deficient mice display enhanced onset and rapid progression of arteriosclerosis (29). Exogenous NO may reverse the migratory defect of EPCs associated with diabetes (30). In general, increased eNOS expression is considered to be beneficial. However, under certain pathophysiological conditions, upregulation of eNOS expression is associated with reduced endothelium-dependent vasodilatation (31–34) explained by the so-called “eNOS uncoupling” (rev. in 7,33,34). Under this condition eNOS itself can be a source of O₂⁻, instead of NO (7,31–34). eNOS uncoupling was shown in a variety of experimental and clinical vascular disease states, especially in diabetes (6,7,33,35). Diabetes-associated eNOS uncoupling has been described in the heart, vessels, and the kidney (4,34–39), resulting in decreased NO bioavailibility, increased superoxide production, and disrupted eNOS dimer formation within the vascular wall, while eNOS mRNA and/or protein levels are maintained or even increased (36–38). A reduction of the essential eNOS cofactor BH₄ has been described to be mechanistically involved in eNOS uncoupling (7,31,35,37,40).

In our present study, EPCs from diabetic patients had increased intracellular ROS levels, reduced endothelial CFU capacity, and impaired migratory function, which were improved after NOS inhibition. In vitro, high glucose concentration led to increased ROS production and 3-nitrotyrosine levels and impairment of EPC function in EPCs. While EPCs are equipped with powerful antioxidative enzyme systems that may reduce the damaging effects of ROS (41), a direct correlation between functional capacity of EPCs and intracellular ROS production recently has been proposed (42,43). Glutathione peroxidase-1–deficient mice had increased ROS production and suffered from impaired migratory capacity of EPCs and reduced angiogenesis in vitro (42). The protective role of eNOS may only be maintained under a normoglycemic environment, as under hyperglycemic conditions eNOS has been shown to become uncoupled to produce O₂⁻ (4–7). Addition of superoxide dismutase, which highly efficiently detoxifies O₂⁻, completely normalized ROS production and EPC function. As NOS inhibition only partly attenuated O₂⁻ production, other systems may also be involved in ROS production and EPC dysfunction in a diabetic environment, such as excessive mitochondrial production of ROS (42) or increased activation of the p38 MAP kinase in EPCs (44). However, eNOS uncoupling in EPCs as shown in the present study seems to play an important and dominant role in glucose-mediated EPC dysfunction.

The underlying molecular mechanisms by which increased ROS may disturb EPC function are unclear, but reduced bioavailability of NO or the eNOS cofactor BH₄ may be involved (7,17,20,29,40). Indeed, intracellular BH₄ levels depend by large on their degradation due to excessive oxidation (rev. in 7). Reduced BH₄ levels have been reported in diabetic eNOS uncoupling (7,31,35), and restoration of BH₄ levels can “recouple” eNOS and enhance its regular enzymatic activity (7,40). In the present study we demonstrate for the first time a critical role for intracellular BH₄ levels for coupling and uncoupling of eNOS in EPCs. Indeed, we observed a significant reduction of intracellular BH₄ levels after challenging EPCs with high concentrations of glucose, whereas oxidized biopterin levels were significantly increased. This switch from BH₄ levels to oxidized biopterin strengthens our results that during glucose challenge eNOS in EPCs becomes uncoupled. Strikingly, addition of exogenous BH₄ resulted in reduction of intracellular O₂⁻ levels and complete rescue of EPC function during high-dose glucose treatment.

PKC is involved in the vascular O₂⁻ production in diabetic vessels (4). Its inhibition restores vascular NO bioavailability and endothelial function (4), but a role in EPCs has not been investigated so far. We demonstrate PKC to be essential in translation of the detrimental effects of high levels of glucose to EPCs. PKC inhibition resulted in reduced glucose-mediated O₂⁻ production and improvement of EPC function, suggesting a potential therapeutic role of PKC inhibitors in dysfunctional EPCs.

While reduced circulating EPC levels in diabetes have been observed in several human studies (13–14,45,46), alterations in EPC mobilizing pathways within diabetic bone marrow have not been identified so far. A pivotal role for eNOS in the regulation of EPC mobilization was shown with eNOS-deficient mice, which show impaired capacity to mobilize EPCs and impaired function of isolated EPCs (17). In the bone marrow, eNOS is the uniquely expressed NOS isoform, whereas iNOS or neuronal NOS are not detected (47). We observed enhanced eNOS expression in the bone marrow of diabetic rats, whereas eNOS monomerization and superoxide anion production were increased. O₂⁻ production was blocked upon NOS inhibition. Our data therefore provide evidence for eNOS uncoupling within bone marrow in diabetes. The uncoupling of eNOS has previously been linked to its monomerization upon treatment of endothelial cells with peroxynitrite or glucose (37,48). In diabetic bone marrow, eNOS dimer-to-monomer ratio was shifted to monomerization. Changes in the dimer-to-monomer ratio are not directly linked to functional uncoupling of eNOS, as only the dimeric form appears to be biochemically active and is able to generate either NO or O₂⁻. However, the eNOS monomer can be viewed as a marker for eNOS uncoupling (5). eNOS uncoupling in bone marrow lowers NO bioavailability and thus likely contributed to the observed reduced levels of circulating EPCs.

We cannot rule out that other O₂⁻-producing systems play an additional role in the observed effects. For instance, we also observed a minor contribution of NADPH oxidases in glucose-mediated increase in ROS formation and subsequent reduction in EPC function. However, a stronger attenuation of O₂⁻ formation was observed after eNOS inhibition, indicating a major role for uncoupled eNOS in superoxide production in diabetes.

In conclusion, eNOS uncoupling impairs EPC number and function in diabetes, thus contributing to the pathogenesis of vascular disease.

This work was supported in part by the IZKF Würzburg (D22 to J.B.; E-31 Nachwuchsgruppe Cardiac Wounding and Healing to T.T.), the Novartis Foundation (to T.T. and J.B.), and the Ernst und Berta Grimmke-Stiftung (to T.T.). J.D.W. was supported by the Deutsche Akademie der Naturforscher Leopoldina (BMBF-LPD 9901/8-97).

The authors thank Helga Wagner (University of Würzburg) and Anja Mitschke (Medical School Hannover) for expert technical assistance.