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From the Cardiovascular Research Center,* INSERM Lariboisière U689, Université Paris, Paris, France; the Cardiovascular Division, King’s College, London, United Kingdom; the Departments of Vascular Surgery and Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands; and the Institut fur Kardiovaskulare Physiologie, Universitat Frankfurt, Frankfurt, Germany
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Abstract |
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1.5-fold greater than untreated diabetic BM-MNCs (P < 0.05). Thus, inhibition of NADPH oxidase-derived ROS overproduction improves the angiogenic and vasculogenic processes and restores postischemic neovascularization in type 1 diabetic mice.
Reactive oxygen species (ROS) including superoxide, hydrogen peroxide, hydroxyl radical, and reactive nitrogen species, such as nitric oxide and peroxynitrite, are biologically active species that are increasingly recognized to play major roles in vascular biology through redox signaling.7-9 Each of these species derives from specific enzymatic or chemical reactions. A predominant source of ROS in the normal vessel is thought to be a family of membrane-associated NADPH oxidases.7,8,10 The NADPH oxidase, as found in neutrophils and endothelial cells,7,8,11 consists of a membrane-localized cytochrome b558 comprised of two subunits, gp91phox (or Nox 2) and p22phox, as well as cytosolic components p47phox and p67phox. The small GTPase Rac1 is also necessary for full NADPH oxidase activity. ROS have been suggested as important mediators of angiogenesis. H2O2 stimulates cell migration and proliferation in endothelial cells,12 and ROS directly modulate VEGF-A expression and vascular smooth muscle cell proliferation.13 Previous reports also suggest that both the gp91phox-containing NADPH oxidase and Rac1 play a major role in VEGF-A-induced endothelial cell proliferation.14 Similarly, mice lacking gp91phox displayed impaired postischemic neovascularization.15 Taken together, these observations suggest that both gp91phox and Rac1 play a significant role in NADPH oxidase-induced angiogenesis.
Diabetes is associated with increased NADPH oxidase activation leading to ROS overproduction.16,17 A link between ROS and diabetes has been evidenced in experimental models of type 1 and 2 diabetes, as well as in human patients with type 2 diabetes.7,8,16,17 Furthermore, increased ROS production may contribute to diabetes-induced vascular complications. In support of this view, ROS overproduction has been shown to participate in endothelial dysfunction in both rodent models and patients with type 2 diabetes.16,18
The aim of our study was to analyze the role of diabetes-induced ROS overproduction in postischemic neovascularization in a mouse model of unilateral hindlimb ischemia. We hypothesized that oxidative stress is one of the key events that triggers pathways leading to vascular complications in diabetes. In particular, we sought to investigate the involvement of NADPH oxidase-derived ROS in diabetes-induced alterations of VEGF-A expression and postnatal vasculogenesis.
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Materials and Methods |
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All of the experiments were performed in accordance with the European Community guidelines for the care and use of laboratory animals (no. 07430). To induce diabetes, C57BL/6 wild-type mice and C57BL/6 gp91phox-deficient mice were injected intraperitoneally with 60 mg/kg streptozotocin in sodium citrate buffer (0.05 mol/L, pH 4.5) daily for 5 days. Three days after the fifth injection, blood glucose levels were measured. Mice with glucose levels <300 mg/dl were excluded from the study, as previously described.3,5 In control groups, mice were injected intraperitoneally with sodium citrate buffer (0.05 mol/L, pH 4.5). After 2 months of monitored diabetes, mice underwent surgical ligation of the proximal part of the right femoral artery, just above the origin of the circomflexa femoris lateralis, as previously described.19,20 Diabetic and nondiabetic mice were then treated with or without the anti-oxidant N-acetyl-L-cysteine (NAC; 200 mg/kg/day, i.p.) and in vivo electrotransfer of a pCAG expression plasmid encoding for dominant-negative mutant Rac1 (N17Rac1, 50 µg injected in the gastrocnemius muscle). An additional group of mice also received 50 µg of empty pCAG expression plasmid as a negative control. To further analyze the role of VEGF-A up-regulation in NAC-induced restoration of neovascularization, NAC-treated diabetic mice also received an intraperitoneal injection of neutralizing antibody directed against VEGF receptor 2 (anti-Flk-1, 100 µg/day, clone DC101; ImClone Systems Inc., New York, NY21 ).
Quantification of Neovascularization in the Hindlimb
Three weeks after the onset of ischemia, vessel density was evaluated by three different methods, as previously described19,20 : 1) high-definition microangiography using barium sulfate (1 g/ml) injected in the abdominal aorta, followed by image acquisition with a digital X-ray transducer and computerized quantification of vessel density expressed as a percentage of pixels per image occupied by vessels in the quantification area; 2) assessment of capillary density in gastrocnemius muscle by immunostaining with a rabbit polyclonal antibody directed against total fibronectin (dilution 1/50; Chemicon Int., Temecula, CA) and morphometric quantification using Histolab software (Microvisions, Paris, France); and 3) laser Doppler perfusion imaging to assess in vivo tissue foot perfusion.
Determination of Protein Expression
Gastrocnemius muscles from ischemic and nonischemic hindlimbs were thawed and homogenized in 300 µl of buffer (200 mmol/L sucrose, 20 mmol/L HEPES, pH 7.4) with protease inhibitors. Protein extracts from BM-MNCs were obtained by lysing cells in 200 µl of buffer (20% sodium dodecyl sulfate, 100 mmol/L sodium orthovanadate, 0.5 mol/L Tris, pH 7.4) with protease inhibitors. Proteins were separated in 12% denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose sheets (Hybond ECL; Amersham, Orsay, France). Antibodies against VEGF-A (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA), gp91phox (1:500, BD Transduction, Franklin Lakes, NJ), p47phox (1:2000, BD Transduction), p67phox (1:2000, BD Transduction), Rac1 (1:1000, Cell Signaling, Danvers, MA), phospho-p38 mitogen-activated protein kinase (p38MAPK) (1:1000, Cell Signaling) and pan-p38MAPK (1:1000, Santa Cruz Biotechnology) were used for immunoblotting. As a protein loading control, membranes were stripped, incubated with a goat polyclonal antibody directed against total actin (dilution 1/1000, Santa Cruz Biotechnology), and specific chemiluminescent signal was detected as previously described.19,20
Proangiogenic Effect of BM-MNCs
BM-MNCs were obtained by flushing tibia and femur of wild-type and gp91phox-deficient control and diabetic mice treated with or without NAC. Low-density mononuclear cells were then isolated by centrifugation on a Ficoll gradient, as previously described.5 Five hours after hindlimb ischemia, control animals received intravenous injections of 1 x 106 BM-MNCs. Animals were euthanized at day 10 after ischemia, and the neovascularization reaction was assessed as described above.
BM-MNC Differentiation into EPCs
BM-MNCs (1.5 x 106) per ml were plated on 11-mm cell-culture dishes coated with rat plasma vitronectin (Sigma, St. Quentin Fallavier, France) and gelatin (0.1%) and maintained in endothelial basal medium (EGM2; BioWhittaker, Walkersville, MD). BM-MNCs were treated with or without apocynin (200 µmol/L), NAC (3 mmol/L), or a p38MAPK inhibitor (LY333351, 10 µmol/L) for 7 days. Nonadherent cells were then removed and adherent cells underwent im-munochemical analysis. To detect the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (AcLDL-Dil), cells were incubated in medium containing AcLDL-Dil (Tebu, Le Perray en Yvelines, France) at 37°C for 1 hour. Cells were then fixed with 2% paraformaldehyde and incubated with fluorescein isothiocyanate-labeled BS-1 lectin (Sigma). Dual-positive staining for both AcLDL-Dil and BS-1 lectin characterized EPCs. EPC numbers were counted and expressed in cells per well, as previously described.5 Five replicates were counted for each experimental condition. Three independent investigators evaluated the number of EPCs per well by counting three randomly selected high-power fields under epifluorescence microscopy. Results are expressed as percentages of total cell numbers.
BM-MNC Apoptosis Measurement
BM-MNCs were incubated with tumor necrosis factor-
(10 ng/ml) plus cycloheximide (10 µmol/L) or C2-ceramide (100 µmol/L) for 8 hours. BM-MNCs were fixed with paraformaldehyde for 12 hours at 4°C, stained with 4,6-diamidino-2-phenylindole (Calbiochem, Paris, France) for 30 minutes for examination of nuclear morphology by fluorescence microscopy. Total and apoptotic nuclei showing condensed chromatin and fragmented nuclear bodies (karyorrhexis, pyknosis) were counted in five random fields per treatment, totaling a minimum of 150 cells in each of three independent experiments. Results are presented as percentage of apoptotic cells. Alternatively, cell death was assessed by propidium iodide DNA staining and fluorescence-activated cell sorting analysis. Fractions of the cell population in the apoptotic G0/G1 and G2/M cell cycle phases were evaluated and expressed in percentages.
Immunohistochemistry
Frozen tissue sections (7 µm) were incubated with rabbit polyclonal anti-nitrotyrosine antibody (dilution of 1/50) to evaluate 3-nitrotyrosine formation as a biomarker for in vivo nitration. To account for variables including heterogeneity of ROS levels, three different sections were performed at three different levels of gastrocnemius muscle for each animal. In addition, we compared the number of nitrotyrosine-specific stainings between the three different levels for each animal. Using one-way analysis of variance, we did not observe any statistical differences in the experimental group. Immunostains were visualized by fluorescence microscopy and then analyzed in randomly chosen fields (five fields per section) of a defined area with Histolab software.
Luminescence Assay
Tissue and cellular ROS levels, reflecting a balance between oxidant production and removal by endogenous antioxidants, were also quantified using L-012 as described recently.22,23 Gastrocnemius muscle and BM-MNCs were lysed in 50 mmol/L Tris buffer (pH 7.5) containing protease inhibitors (Boehringer-Mannheim, Mannheim, Germany) and centrifuged at 10,000 x g for 15 minutes at 4°C. Supernatants were then incubated with L-012 100 µmol/L (Wako, Neuss, Germany) with or without diphenylene iodonium (DPI; 30 µmol/L) or apocynin (1 mmol/L). The same part of the gastrocnemius muscle was used in the different treated groups. Luminescence was counted (Topcount NXT; Perkin Elmer) during 20 seconds after a 10-minute interval, allowing for the plates to become dark-adapted.
Statistical Analysis
Results were expressed as mean ± SEM. One-way analysis of variance was used to compare each parameter. Comparisons between groups were performed using post hoc Bonferroni t-test when the analysis of variance test was statistically significant. P < 0.05 was considered significant.
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Results |
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In control animals, 7 days of ischemia raised 3-nitrotyrosine-positive staining in hindlimb muscle (Figure 1a)
. These results were substantiated by L-012 luminescence demonstrating that ischemia enhanced ROS levels in control animals (Figure 1b)
. Treatment with DPI or apocynin impaired ischemia-induced increase in ROS levels (P < 0.01 and P < 0.05 for DPI and apocynin treatment, respectively; Figure 1b
). Although the experiments with DPI and apocynin are suggestive, we used gp91phox-deficient mice to obtain more definitive evidence and showed that ischemia-induced up-regulation of ROS levels was abrogated in this setting (Figure 1b)
. Taken together, these results suggest that ischemia activates NADPH oxidase-dependent ROS synthesis. In diabetic mice, ROS accumulation in ischemic tissue was further up-regulated by 1.8-fold compared to control animals (P < 0.01 versus ischemic control; Figure 1b
). Treatment with NAC and dominant-negative Rac-1 or gp91phox deficiency abrogated ischemia- and diabetes-induced increases in ROS (Figure 1, a and b)
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. In diabetic mice, gp91phox and Rac1 protein levels were further increased by 1.4- and 1.3-fold in diabetic ischemic hindlimbs compared to ischemic controls (P < 0.05; Figure 2
). In contrast, p47phox and p67phox protein content was decreased in diabetic ischemic muscles in reference to ischemic control animals (P < 0.05; Figure 2
). Taken together, these results indicate an up-regulation of gp91phox and Rac1 protein content and increased ROS levels in diabetic ischemic muscles.
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We next assessed the role of ROS and NADPH oxidase activity in postischemic vessel growth 21 days after ischemia. In control animals, neovascularization was impaired by NAC administration, or the absence of gp91phox, as revealed by the 40% decrease in angiographic scores, the 43% decrease in capillary density, and the 40% reduction in foot Doppler perfusion scores (Figure 3)
. In diabetic animals, angiography scores, capillary density, and foot perfusion were decreased by 40%, 60%, and 43%, respectively, compared to control animals (Figure 3)
. The scavenging of ROS by NAC administration or the inhibition of ROS generation by gp91phox deficiency or administration of dominant-negative Rac1 restored postischemic neovascularization in diabetic animals to control levels (Figure 3)
. Hence, reduction in ROS levels restored vessel growth in diabetic ischemic leg whereas it impaired neovascularization in control ischemic tissue.
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We sought to determine the molecular and cellular pathways affected by diabetes-induced oxidative stress. VEGF-A signaling represents a critical rate-limiting step in both physiological and pathological angiogenesis. In control mice, VEGF-A protein levels were decreased by 25% in NAC-treated animals and gp91phox-deficient mice compared to control mice (P < 0.05; Figure 4
). In diabetic mice, VEGF-A levels were reduced by 60% in ischemic tissues compared to ischemic control hindlimbs (P < 0.001; Figure 4
). Interestingly, VEGF-A protein contents were significantly increased by 2.5-, 2.0-, and 2.7-fold in diabetic animals treated with NAC or dominant-negative Rac1 and in gp91phox-deficient diabetic mice, respectively, compared to untreated diabetic mice (Figure 4)
. In addition, co-treatment with anti-Flk-1 abrogated NAC-related effects suggesting that VEGF-A up-regulation participates in the restoration of vessel growth in NAC-treated diabetic mice (Figure 3)
.
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We finally evaluated the effects of diabetes-induced increased ROS on the ability of BM-MNCs to stimulate neovascularization. Protein expression of NADPH oxidase subunits was detected in control BM-MNCs. Their levels were up-regulated in diabetic versus control BM-MNCs (Figure 5a)
. ROS levels were also increased by 2.3-fold in diabetic BM-MNCs compared to control BM-MNCs (P < 0.01; Figure 5b
). Treatment with NAC or deficiency in gp91phox reduced ROS levels in both control and diabetic cells.
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The proangiogenic potential of BM-MNCs isolated from control mice treated with NAC or gp91phox-deficient mice was lower than that observed with BM-MNCs isolated from control mice. However, this difference did not reach statistical significance (Figure 6)
. In contrast, transplantation of BM-MNCs isolated from diabetic mice treated with NAC improved the angiography score by 1.6-fold, capillary numbers by 1.4-fold, and foot perfusion by 1.5-fold greater than BM-MNCs isolated from untreated diabetic animals (Figure 6)
. Similarly, injection of BM-MNCs isolated from gp91phox-deficient diabetic mice improved the angiography score by 1.7-fold, capillary numbers by 1.4-fold, and foot perfusion by 1.6-fold, compared to BM-MNCs isolated from untreated diabetic animals (Figure 6)
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We could not detect any significant difference in percentages of apoptotic cells in control and diabetic animals, suggesting that major alterations in BM-MNC apoptosis were not a significant cause of diabetes-induced BM-MNC dysfunction (Figure 7)
. Treatment of BM-MNCs isolated from control animals with NAC and apocynin reduced the number of DilLDL/BS1 lectin double-positive cells (Figure 8a)
suggesting that low levels of ROS may facilitate BM-MNC differentiation into EPCs. The ability of diabetic BM-MNCs to differentiate into EPCs was markedly reduced, as revealed by the 66% reduction in numbers of DilLDL/BS1 lectin-positive cells (Figure 8a)
. Treatment with NAC and apocynin restored the percentage of diabetic double-positive cells to control BM-MNC levels (Figure 8a)
. This effect is likely related to changes in p38MAPK phosphorylation. Indeed, p38MAPK plays a pivotal role in the signal transduction pathways that regulate numbers of EPCs ex vivo.24
Diabetes increased p38MAPK phosphorylation by twofold in BM-MNCs (P < 0.01). NAC treatment reduced p38MAPK phosphorylation by 44% in diabetic cells. p38MAPK phosphorylation was also decreased by 45% in diabetic gp91phox-deficient cells (Figure 8c)
. Furthermore, p38MAPK inhibition with LY333351 improved the number of diabetic DilLDL/BS1 lectin-positive cells (Figure 8a)
, suggesting that the decreased differentiation of diabetic BM-MNCs into EPCs is mediated by enhanced p38MAPK phosphorylation resulting from increased ROS levels.
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Discussion |
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We found that ROS levels were higher in diabetes, in association with the up-regulation of gp91phox and Rac1 expression. However, down-regulation of p47phox and p67phox is puzzling and does not fit with the increase in ROS levels in ischemic diabetic tissue. One may speculate that the time course of ischemia-induced p47phox and p67phox protein regulation may differ from that observed for gp91phox or Rac-1. Alternatively, the down-regulation of p67phox and p47phox may counterbalance the rise in gp91phox and Rac-1 protein levels and limit ROS overproduction in diabetic tissue. Nevertheless, previous studies show evidence that NADPH oxidase activity and expression are significantly increased in diabetic tissue.16,18 Antioxidant defense capacity is reduced in animal models of diabetes, and this can also contribute to diabetes-induced oxidative stress.25 We showed here that blockade of NADPH oxidase activity or the scavenging of ROS restores postischemic neovascularization in diabetes. This effect is related to an up-regulation of major proangiogenic factors such as VEGF-A.3,19,26 We also demonstrated that diabetes-induced increases in ROS enhanced p38MAPK phosphorylation in BM-MNCs, reduced BM-MNC differentiation into EPCs in vitro, and impaired their proangiogenic potential in vivo. Similarly, diabetes has been shown to activate p38MAPK in vascular cells via PKC-dependent and -independent pathways.27 Furthermore, p38MAPK activation is known to down-regulate EPC proliferation and differentiation.24 Taken together, our results suggest that increased oxidative stress constitutes an underlying pathogenic mechanism that affects both angiogenesis and vasculogenesis in the setting of diabetes. In addition, this study supports the concept that the effects of ROS on vascular function depend critically on the amounts present. In low amounts under physiological conditions, ROS can act as intracellular second messengers, modulating proangiogenic pathways such as VEGF-A signaling and postnatal vasculogenesis. Conversely, higher amounts of ROS can bear negatively on vessel growth.7,8,28,29 Nevertheless, because gp91phox and Rac-1 may have multiple roles in nonphagocytic cells, one cannot exclude the possibility that gp91phox and Rac-1 may modulate neovascularization through ROS-independent related pathways.
Because ROS are involved in numerous signaling pathways, the mechanisms by which they may affect neovascularization are potentially diverse. A considerable body of evidence implicates oxidative stress as an important pathogenic element in diabetic endothelial dysfunction.30 The chronic treatment of diabetic animals with NAC improved or normalized endothelium-dependent responses.31 Alternatively, increased superoxide production by NADPH oxidase likely reduces NO bioactivity by scavenging or through uncoupling of endothelial nitric oxide synthase and may also lead to the formation of other signaling species such as peroxynitrite.16,18 In keeping with this, we found increased 3-nitrotyrosine staining in ischemic muscles, consistent with increased levels of nitrating species. Changes in NO signaling might contribute to alterations in postischemic neovascularization because NO is known to mediate BM-MNC mobilization and differentiation, as well as basal neovascularization reaction.32,33
In conclusion, this study reports for the first time that diabetes-induced increases in NADPH oxidase-derived ROS reduces postischemic neovascularization through the inhibition of proangiogenic pathways and impaired postnatal vasculogenesis. Our results also highlight the concept that inhibition of this oxidative stress might be a potential therapeutic avenue to promote neovascularization in the setting of diabetes.
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Acknowledgements |
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Footnotes |
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Supported by the British Heart Foundation (Chair of Cardiology at King’s College London to A.M.S.); Agence Nationale de la Recherche (young investigator grant JC05-45445 and 2005 Cardiovascular, Obesity, and Diabetes grant ANR-05-PCOD-028-01 to J.-S.S.); The Netherlands Organization for Health Research and Development (Agiko stipend 920-03-291 to B.M.); the Prof. Michaël-van Vloten Fund; and the European Commission (INSERM U689 is partner of the European Vascular Genomics Network, a Network of Excellence granted by contract no. LSHM-CT-2003-503254).
Accepted for publication May 2, 2006.
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P < 0.05, 
