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. Author manuscript; available in PMC: 2006 May 31.
Published in final edited form as: Circ Res. 2005 Dec 22;98(2):254–261. doi: 10.1161/01.RES.0000200740.57764.52

Impaired Angiogenesis in Glutathione Peroxidase-1– Deficient Mice Is Associated With Endothelial Progenitor Cell Dysfunction

Gennaro Galasso 1,*, Stephan Schiekofer 1,*, Kaori Sato 1, Rei Shibata 1, Diane E Handy 1, Noriyuki Ouchi 1, Jane A Leopold 1, Joseph Loscalzo 1, Kenneth Walsh 1
PMCID: PMC1472658  NIHMSID: NIHMS9810  PMID: 16373599

Abstract

Several vascular disease are characterized by elevated levels of reactive oxygen species (ROS). Vascular endothelium is protected from oxidant stress by expressing enzymes such as glutathione peroxidase type 1 (GPx-1). In this study, we investigated the effect of vascular oxidant stress on ischemia-induced neovascularization in a murine model of homozygous deficiency of GPx-1. GPx-1– deficient mice showed impaired revascularization following hindlimb ischemic surgery based on laser Doppler measurements of blood flow and capillary density in adductor muscle. GPx-1– deficient mice also showed an impaired ability to increase endothelial progenitor cell (EPC) levels in response to ischemic injury or subcutaneous administration of vascular endothelial growth factor protein. EPCs isolated from GPx-1– deficient mice showed a reduced ability to neutralize oxidative stress in vitro, which was associated with impaired migration toward vascular endothelial growth factor and increased sensitivity to ROS-induced apoptosis. EPCs isolated from GPx-1– deficient mice were impaired in their ability to promote angiogenesis in wild-type mice, whereas wild-type EPCs were effective in stimulating angiogenesis in GPx-1– deficient mice. These data suggest that EPC dysfunction is a mechanism by which elevated levels of ROS can contribute to vascular disease.

Keywords: glutathione peroxidase-1, hindlimb ischemia, endothelial progenitor cells, angiogenesis

Several vascular diseases, including hypertension, diabetes, and atherosclerosis, are characterized by elevated levels of reactive oxygen species (ROS).1,2 ROS-mediated apoptosis of endothelial cells has been previously demonstrated,3,4 and an increase in ROS, leading to increased oxidant stress in the vasculature, promotes endothelial dysfunction.5 The vascular endothelium is protected from oxidant stress by expressing enzymes with antioxidant properties such as the selenocysteine-containing protein glutathione peroxidase type 1 (GPx-1). GPx-1 diminishes oxidant stress by using glutathione (GSH) to reduce H2O2 and lipid peroxides to their corresponding alcohols.6,7 The glutathione peroxidases exist in several isoforms, and the most abundant intracellular isoform is cellular GPx, or GPx-. Previous studies have supported a relationship between decreased GPx-1 activity and vascular injury. Indeed, GPx-1 activity is decreased in atherosclerotic plaque excised from carotid artery.8 Moreover, hyperhomocysteinemia, an independent risk factor for coronary artery disease, is associated with a significant reduction in GPx-1 expression,9,10 whereas over-expression of GPx-1 restores the normal endothelial phenotype in cultured endothelial cells exposed to elevated homocysteine concentrations.11 Taken together, these data indicate that decreases in the expression of this enzyme may contribute to vascular oxidant stress and the progression of atherothrombosis. Consistent with this hypothesis, mice with GPx-1 deficiency have endothelial dysfunction and significant structural vascular and cardiac abnormalities.12,13

Recent evidence suggests that the regeneration of injured endothelium involves the participation of cells from adult bone marrow. A number of laboratories have reported that bone marrow– derived endothelial progenitor cells (EPCs) are present in the systemic circulation and that they home to sites of ischemic injury where they function to promote neovascularization.14–17 EPCs express endothelial cell marker proteins, and autologous delivery of these cells can improve wound healing responses in animal models of ischemia. Circulating EPCs are also indicators of overall cardiovascular health. Vasa et al18 initially showed that levels of circulating EPCs (CD34/KDR-positive) are higher in healthy volunteers than in patients with coronary artery disease. Similarly, Hill et al19 analyzed “colony-forming units” of EPCs and found that this measurement negatively correlated with Framing-ham risk factor score. These investigators also found that a reduction in EPC colonies was a good predictor of impairment in flow-mediated brachial-artery reactivity. In a related study, it was reported that EPCs isolated from patients with type 2 diabetes mellitus display impaired proliferation and reduced incorporation into tube-like structures on Matrigel.20 Conversely, statin (HMG-CoA reductase inhibitor) therapy increases circulating EPC levels in patients with coronary artery disease.21 Lastly, an age-dependent decrease in EPC mobilization has been reported.22 All of these risk factors are also associated with elevated markers of ROS stress.23

Stem cells express high levels of genes that are associated with DNA repair and protection from stress.24,25 Recently, it has been shown that EPCs have lower levels of basal and stress-induced intracellular ROS than primary endothelial cells because they express higher levels of catalase, manganese superoxide dismutase (MnSOD) and GPx-1.26,27 It has also been shown that the collective inhibition of catalase, MnSOD, and GPx-1 increases ROS levels in EPCs and that this inhibition impairs EPC survival and migration26; however, these in vitro findings have yet to be validated in a mouse model of oxidant stress. Because GPx-1 plays a central role in protecting the vasculature from oxidant stress, we studied the effect of increased ROS flux on angiogenesis by using a murine model that is deficient in GPx-1 gene expression. Here, we show that ischemia-induced angiogenesis is impaired in GPx-1– deficient mice. These mice also did not display robust increases in EPC levels in response to ischemic injury or vascular endothelial growth factor (VEGF) administration, and EPCs from GPx-1– deficient mice were functionally deficient in promoting angiogenesis. These data suggest oxidant stress can promote EPC dysfunction and contribute to vascular disease.

Materials and Methods

Animals

Homozygously glutathione peroxidase-1 deficient (GPx-1 KO) and wild-type (WT) mice in a C57/BL6 background were used for this study. See the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org.

Mouse Model of Angiogenesis

GPx-1 KO and WT mice were subjected to unilateral hindlimb surgery under anesthesia with sodium pentobarbital (50 mg/kg intraperitoneally). In this model, the entire left femoral artery and vein were excised surgically.28 (See also the expanded Materials and Methods section.)

EPC Isolation and Characterization

Circulating mouse EPCs were harvested 5 days after ischemic hindlimb surgery and cultured according to previously described techniques.29,30 Proliferation, apoptosis, migration, and differentiation were assessed as described in the expanded Materials and Methods section.

Dichlorofluorescein Fluorescence

Cellular ROS accumulation was determined in a microplate fluorometer (SpectraMax Gemini, Molecular Devices) as described31,32 (see also the expanded Materials and Methods section).

Flow Cytometry

Fluorescence-activated cell sorter (FACS) analysis was used to detect the cell surface expression of the endothelial cell antigen Flk-1 on cultured EPCs from mice (see also the expanded Materials and Methods section).

Statistical Analysis

Results from at least 3 independent experiments are expressed as mean±SD. Comparison between groups was performed by Student’s paired 2-tailed t test or ANOVA for experiments with more than 2 subgroups. Post hoc analysis and pairwise multiple comparisons were performed using the 2-sided t test with Bonferroni adjustment. Probability values <0.05 were considered statistically significant. All analyses were performed with SPSS 11.5 software (SPSS Inc).

Results

Impaired Ischemia-Induced Angiogenesis in GPx-1–Deficient Mice

All mice survived after surgical induction of unilateral left hindlimb ischemia and appeared to be healthy during the follow-up period. Body weight did not differ between the 2 groups (WT 30.1±1g versus GPx-1 29.3±1.2; P=NS). Immediately after left femoral artery and vein resection, the ratio of blood flow between the ischemic and nonischemic hindlimbs decreased to 0.23±0.05 in WT and 0.24±0.06 in GPx-1 KO mice, indicating that the severity of the induced ischemia was similar in both groups. Figure 1A shows representative laser Doppler blood flowmetry (LDBF) images of hindlimb blood flow before, immediately post (day 0) and 28 days after surgery in the WT and GPx-1 KO. In WT mice, hindlimb blood flow perfusion increased to 50% to 60% of the nonischemic limb by day 7 and ultimately returned to ≈80% of the nonischemic limb by day 28 (Figure 1B). In contrast to WT mice, flow recovery in GPx-1 KO mice was impaired. Flow in GPx-1 KO mice was significantly less than WT by day 14 after surgery, and the flow difference persisted up to 28 days after surgery. Because the laser Doppler procedure is limited to analysis of blood flow near the limb surface, these data were corroborated by a quantitative analysis of capillary density in ischemic adductor muscle was measured in histologic sections harvested from the WT or GPx-1 KO mice on postoperative day 28. Figure 2A shows representative photomicrographs of tissue immunostained with CD31. Quantitative analysis of CD31-positive cells revealed that the capillary density was significantly diminished in the ischemic adductor muscle of GPx-1 KO mice compared with WT mice (Figure 2B).

Figure 1.

Figure 1.

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Impaired ischemia-induced angiogenesis response in the hindlimbs of GPx-1– deficient mice. A, Low-perfusion signal (dark blue) was observed in the ischemic hindlimb of GPx-1 KO mice, whereas a higher-perfusion signal (white to red) was detected in WT mice on postoperative day 28. B, Quantitative analysis of the ischemic/nonischemic LDBF ratio in WT (square) and GPx-1 KO (circle) mice (n=8 in each group). *P<0.01, **P<0.001 WT vs GPx-1 KO mice.

Figure 2.

Figure 2.

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Reduced capillary density in ischemic hindlimbs of GPx-1– deficient mice. A, Representative immunostaining of ischemic tissues from WT and GPx-1 KO mice with anti-CD31 monoclonal antibody (brown) on postoperative day 14. B, Quantitative analysis of capillary density in ischemic adductor muscle of WT and GPx-1 KO mice on postoperative day 14 (n=8 in each group). Capillary density was expressed as the number of capillaries per high-power field (hpf) (×400) (top) and capillaries per muscle fiber (bottom). #P<0.05 vs WT mice.

EPC Levels in GPx-1–Deficient Mice

As we have shown that GPx-1 KO mice are compromised in hindlimb reperfusion after ischemia, we investigated EPC levels after culture in WT and GPx-1 KO mice. EPCs were generated from peripheral blood mononuclear cells.29,30 Culturing MNCs for 5 days resulted in an adherent population of acetylated LDL (Ac-LDL)– and lectin-positive cells that were also positive for expression of the endothelial transcripts VEGF receptor-2 and endothelial NO synthase (eNOS) (data not shown), consistent with an EPC phenotype. As shown in Figure 3A, no differences were found in the level of EPCs characterized before ischemia between WT and GPx-1 KO mice. Five days after the induction of ischemia, the number of EPCs derived from WT mice increased significantly, whereas GPx-1 KO mice showed little or no increase in EPC levels in response to ischemic surgery. To investigate EPC behavior further, changes in EPC levels in response to subcutaneous VEGF protein administration was evaluated. After VEGF165 (500 μg per kilogram per day) subcutaneous administration for 3 days, EPCs were cultured and stained as described above. Whereas the number of EPCs significantly increased after VEGF administration in WT mice, the increase in EPC levels in GPx-1 KO mice was of markedly lower magnitude (Figure 3B). To corroborate these findings, EPCs cultured from WT and GPx-1 KO mice were subjected to FACS analysis of Flk-1 expression as described by others26,30 (Figure 3C). The numbers of Flk-1–positive cells were similar in WT and GPx-1 KO mice under basal conditions, but the marked increase in Flk-1–positive cells observed in response to ischemia surgery or VEGF administration in WT were not observed in similarly treated GPx-1 KO mice (Figure 3D). Taken together, these data show that the increase in EPC levels in response to mobilizing stimuli are impaired in GPx-1 KO mice.

Figure 3.

Figure 3.

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Suppression of EPC levels in GPx-1– deficient mice. A, Quantitative analysis of EPC levels before and after induction of ischemia in WT and GPx-1 KO mice. Peripheral blood monocytes were collected before surgery and at 5 days postsurgery. After 5 days of cultivation, attached cells were stained for the uptake of DiI-Ac-LDL and lectin. Cells were quantified by examining 15 random microscopic fields, and double-positive cells were counted as EPCs. Values are expressed as mean±SD. *P<0.01 vs baseline in WT mice (n=5); #P<0.05 vs ischemia in GPx-1 KO mice. hpf indicates high-power field. B, EPC levels after treatment with VEGF. Mice received subcutaneous injections of 500 μg of VEGF per kg/d or saline for a period of 3 days. EPCs were isolated and quantified as described above. Values are expressed as mean±SD. *P<0.01 vs baseline in WT mice, P<0.01 vs VEGF administration in GPx-1 KO mice. hpf indicates high-power field. C, Expression of Flk-1 assessed by fluorescence FACS. EPCs were isolated from mice 5 days following ischemic hindlimb surgery and cultured for 5 days before analysis. Alternatively, mice were administered VEGF daily before EPC isolation and analysis. D, The results of 3 experiments are shown. *P<0.01 relative to KO mice.

ROS Accumulation in GPx-1–Deficient EPCs

To investigate EPC resistance to oxidative stress, EPCs isolated from GPx-1 KO and WT mice were incubated with dichlorofluorescein for 30 minutes and ROS accumulation was determined using a fluorometer (Figure 4). Baseline ROS accumulation was higher in the EPCs of GPx-1 KO then WT mice. To test whether EPCs of GPx-1 null mice display an inability to handle free radicals, we treated GPx-1 KO and WT EPCs with H2O2. Addition of H2O2 to cells for 4 hours resulted in a much larger increase in intracellular ROS levels in GPx-1 KO than in WT EPCs (Figure 4A). Consistent with the results of Dernbach et al,26 H2O2-induced ROS accumulation was lower in WT EPCs than in cultured HUVECs. However, ROS accumulation in response to H2O2 occurred to a greater extent in GPx-1– deficient EPCs than HUVECs. To exclude that ROS accumulation in GPx-1– deficient EPCs might be delayed in response to H2O2, we performed a time-course analysis and found that EPCs manifest the higher sensitivity to oxidative stress up to 12 hours following incubation with H2O2 (data not shown). Finally, these results were corroborated by independent experiments analyzing ROS accumulation in EPCs by FACS analysis (Figure 4B). Intracellular ROS was elevated in GPx-1 KO EPCs compared with EPCs from WT mice, and treatment with H2O2 led to marked elevation of ROS in EPCs from GPx-1 KO but not WT mice. Collectively, these data show that GPx-1 KO EPCs have a reduced capacity to neutralize hydrogen peroxide, suggesting that a GPx-1 deficit impairs the defense of EPCs against oxidative stress.

Figure 4.

Figure 4.

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Increased ROS in GPx-1– deficient EPCs. A, Intracellular ROS levels were measured in EPCs harvested from WT and GPx-1– deficient mice 5 days after hindlimb ischemia. After 5 days in culture, EPCs were loaded with DCF and analyzed in a microplate fluorometer. Control experiments were performed using HUVECs. Values are expressed are mean±SD of DCF fluorescence. **P<0.001 KO plus H2O2 vs WT without H2O2, vs WT plus H2O2, vs KO without H2O2, and vs HUVECs with or without H2O2. **P<0.001 KO without H2O2 vs WT and HUVEC without H2O2. B, Detection of ROS accumulation by flow cytometry in EPCs isolated from WT on GPx-1 KO mice that had undergone hindlimb ischemia surgery.

ROS-Induced EPC Apoptosis

Previous reports indicate that a reduction in GPx-1 may exacerbate the level of neuronal cell death under conditions of increased ROS following ischemia-reperfusion injury.33,34 Thus, having shown that GPx-1– deficient EPCs exhibit higher intracellular ROS levels compared with WT EPCs, we tested the resistance of these cells to ROS-induced apoptosis. TUNEL-positive cells were analyzed after incubation with 500 μmol/L H2O2 for 12 hours. Apoptosis induced by H2O2 was significantly enhanced in GPx-1–impaired EPCs as compared with WT EPCs (Figure 5A). To further explore the effect of ROS on apoptosis, we analyzed Annexin V staining by FACS (Figure 5B). This analysis also revealed a significant increase in oxidative stress–induced apoptosis in the GPx-1 KO EPCs as compared with WT after addition of H2O2.

Figure 5.

Figure 5.

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Increased apoptosis in GPx-1 KO EPCs in response to oxidative stress. A, Quantitative analysis of TUNEL staining in cultured EPCs. EPCs from GPx-1 KO and WT mice were incubated 12 hours in the presence or absence of 500 μmol/L H2O2. Data are shown as mean±SD. **P<0.001 vs WT plus H2O2, vs KO without H2O2; n=5. B, Quantitative analysis of Annexin V staining. After ischemic surgery, EPCs were isolated and cultured in the presence or absence of H2O2. Annexin V staining was detected by FACS analysis. Data are reported as mean±SD. KO EPCs after addition of H2O2 are significantly more sensitive to apoptosis compared with WT EPCs (**P<0.001 vs WT plus H2O2, n=5).

Effect of EPCs on Endothelial Cell Differentiation

EPCs exhibit the ability to promote the formation of vascular networks in vitro.20 The coculture of HUVECs and DiI-Ac– labeled EPCs on a Matrigel matrix revealed that significantly more WT EPCs were physically associated with the developing network structure compared with the EPCs derived from GPx-1– deficient mice (Figure 6A and 6B). Furthermore, the total number of network projections per microscopic field was significantly higher when WT-derived EPCs were cocultured with HUVECs compared with GPx-1– derived EPC (Figure 6C). Because vascular network formation requires cellular migration,29 we investigated the ability of EPCs to migrate toward VEGF in a transwell assay (Figure 6D). This assay showed that GPx-1– derived EPCs were impaired in their migratory activity relative to WT EPCs. In contrast, proliferative activity assessed by MTS assay30 showed no significant difference between WT- and GPx-1– derived EPCs (data not shown). Collectively, these data suggested that EPCs derived from GPx-1– deficient mice have an impaired angiogenic capacity in vitro.

Figure 6.

Figure 6.

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Vascular network formation in vitro. A, Representative superimposed phase contrast and fluorescent photomicrographs are shown of HUVECs cocultured with EPCs isolated from WT or GPx-1– deficient mice. EPCs harvested from WT or GPx-1– deficient mice following ischemic hindlimb surgery were tagged with DiI-Ac-LDL to assess physical association with the HUVEC network. B, Fluorescence microscopy revealed that fewer GPx-1 KO EPCs were associated with endothelial cell networks when compared with WT EPCs (*P<0.01). C, Numbers of network projections formed in each group after 24 hours of incubation. Data are presented as mean±SD. *P<0.01 vs WT. D, EPC migration assay was performed using a modified Boyden chamber. Cell migration was performed by manually counting cells in five random high-power field (hpf). Values are expressed as mean±SD. *P<0.01 vs KO (n=5).

Effect of EPCs on Neovascularization

To assess whether the impaired mobilization of EPCs contributes to the reduced neovascularization in GPx-1 KO mice, “rescue” experiments were performed comparing WT and GPx-1 KO EPCs. Both GPx-1 KO and WT mice underwent unilateral hindlimb ischemia surgery. On the day of surgery, mice received an intravenous infusion of either GPx-1 KO or WT EPCs (2×105), and revascularization was assessed by LDBF analysis 14 days after surgery. Interestingly, the impairment in limb reperfusion in the GPx-1 KO mice was rescued by intravenous infusion of WT EPCs (Figure 7A and 7B) but not GPx-1 KO EPCs. As expected, flow recovery of WT mice was more robust than that observed in GPx-1 KO mice. Delivery of WT EPCs increased flow recovery in WT mice, but GPx-1 EPCs failed to do so. Quantitative histologic analysis of CD31-positive cells revealed that capillary density was significantly increased in the ischemic adductor muscle of both WT and GPx-1 KO recipient mice when injected with WT-EPCs but not with GPx-1 KO EPCs (Figure 7C). Taken together, these data suggest that impaired limb perfusion in GPx-1 KO mice can be rescued by intravenous infusion of WT EPCs and that GPx-1 KO EPCs are functionally impaired in their ability to promote neovascularization in vivo.

Figure 7.

Figure 7.

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Rescue of impaired angiogenic phenotype in GPx-1 KO mice by intravenous injection of WT EPCs. A, Representative LDBF images showing improved perfusion of ischemic limbs in WT and GPx-1 KO mice by IV injection of WT but not GPx-1 KO ex vivo cultured EPCs on the day of surgery. LDBF measurements were made 14 days after the induction of ischemia. B, Quantitative analysis of ischemic/nonischemic LDBF ratio in GPx-1 KO and WT mice on postoperative day 14 with or without IV injection of ex vivo cultured EPCs from WT or GPx-1 KO mice. *P<0.01 vs control (no EPCs) and GPx-1 KO– derived EPCs for the WT recipient; *P<0.01 vs control and GPx-1 KO– derived EPC for the GPx-1 KO recipient mice (n=4). C, Quantitative analysis of capillary density in ischemic adductor muscle of WT and GPx-1 KO mice on postoperative day 14. Capillary density is expressed as the number of capillaries per high-power field (hpf) (×400); #P<0.05 vs control and GPx-1 KO– derived EPCs for the WT recipient. *P<0.01 vs control and GPx-1 KO– derived EPCs for the GPx KO recipient.

Discussion

The present study provides the first evidence that a deficiency of GPx-1 results in impaired neovascularization following hindlimb ischemia. It is also shown that EPC levels are suppressed in GPx-1 mice that are challenged with ischemic injury or exogenous VEGF treatment. EPCs from GPx-1– deficient mice exhibit impaired angiogenic capacity both in vitro and in vivo and increased susceptibility to oxidative stress and ROS-induced cell death in vitro. Collectively, these data suggest that an imbalance in ROS can contribute to EPC dysfunction and their lack of angiogenic function.

Stem cells exhibit resistance to environmental stress as well as self-renewing capabilities and the capacity to differentiate into multiple cell lineages.24,25,35 A recent study provided evidence that human circulating EPCs are enriched for the expression of genes encoding for antioxidative proteins, resulting in low baseline ROS levels and a reduced sensitivity toward ROS-induced cell death when compared with HUVECs.26 As ischemic tissue is characterized by high levels of inflammatory cytokines, which activate ROS production,36 it has been proposed that high levels of ROS metabolizing enzymes in EPCs are essential to maintain their survival during tissue regeneration under conditions of injury. GPx-1 is a key enzyme for the cellular defense against ROS in endothelial cells,6 and mice heterozygous for this enzyme exhibit endothelial dysfunction.12 In this regard, we found that EPCs harvested from GPx-1 KO mice are ineffective in promoting neovascularization in ischemic tissue when administered systemically. Thus, the impairment in reperfusion in mice lacking GPx-1 may be related to EPC dysfunction and their inability to augment the neovascularization process. Consistent with this hypothesis, we find that GPx-1 KO EPCs are impaired in their abilities to migrate toward VEGF in vitro and stimulate network formation when they are cocultured with endothelial cells on a Matrigel matrix. Our data also suggest that the reduced angiogenic capacity in GPx-1 KO mice may also be related to a reduction in EPC mobilization from bone marrow following ischemia. Furthermore, because VEGF administration also failed to efficiently increase EPC levels in GPx-1 KO mice, the impairment is likely to result from intrinsic differences between WT and GPx-1 KO EPC rather than potential differences in tissue injury between the 2 strains.

Although our study shows that EPCs from GPx-1 KO mice are dysfunctional, we rarely detected DiI-Ac-LDL–positive, CD31-positive cells from either wild-type or GPx-1 KO, incorporated into the vascular structure following the intravenous administration of cells (data not shown). Although it is widely believed that EPCs facilitate angiogenesis, there is considerable controversy regarding the incorporation of these cells into the endothelium of growing vessels. Others have reported that these cells do not incorporate into the growing adult vasculature.37 In the latter case, investigators have proposed that bone marrow– derived cells deliver “software” that facilitates the neovascularization process.38 Overall, our findings are consistent with the prevailing view is that the incorporation of EPCs with an endothelial cell phenotype into the vasculature is generally very low.39

Because GPx-1 plays a central role in protecting cells from ROS, its deficiency may lead to an increase of oxidant stress in the cell.12 Accordingly, we found that EPCs lacking GPx-1 display an increase in ROS levels and a reduced ability to maintain viability when incubated with H2O2 in vitro. GPx-1 expression is normally increased by oxidant exposure40,41 and lack of GPx-1 results in enhanced sensitivity to exogenous oxidants in cultured cells and in vitro.40 Similarly, generation of endogenous ROS, such as in models of cardiac or neuronal ischemia-reperfusion injury, results in enhanced injury in GPx-1– deficient mice.13,33 Furthermore, in vitro and in vivo studies indicate that GPx-1 overexpression is protective against exogenous or endogenously generated ROS.11,42–44 Taken together, these data illustrate the central role of GPx1 in preventing injury and death in response to oxidant stress in differentiated tissues as well as progenitor cells.

We have previously demonstrated that GPx-1 deficiency is associated with a decrease in bioavailable endothelium-derived NO, which is not caused by decreased expression of eNOS protein.12 Interestingly, Aicher et al reported reduced EPC mobilization caused by a reduction in NO bioavailability in eNOS-deficient mice.45 Thus, we can infer that impaired EPC mobilization in GPx-1 KO mice may be attributable, at least in part, to a depletion of bioavailable NO that occurs after induction of ischemia and increased ROS flux in GPx-deficient mice.

The results of the present study may have implications for cardiovascular disease progression. It is well established that several cardiovascular risk factors, including hypertension, hypercholesterolemia, and diabetes are associated with oxidative stress.23 Numerous studies support the notion that modulation of oxidant and antioxidant enzymes plays an important role in the pathogenesis of vascular disease, and low levels of GPx-1 activity were recently shown to be an independent risk factor for cardiovascular events in patients with coronary artery disease.46 Consequently, our data support the hypothesis that the defective mobilization and function of EPCs caused by oxidative stress may impair neovascularization, thereby contributing to the pathogenesis and the progression of vascular disease.

Acknowledgments

This work was supported by National Heart, Lung, and Blood Institute grant NO1-HV-28178 from the NIH; NIH grants HL81587, AR40197, HL77774, and AG15052 (to K.W.); and NIH grants HL81587, HL58976, and HL61795 (to J.L.).

Footnotes

Correspondence to Dr Kenneth Walsh, PhD, Molecular Cardiology/Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany St, W611, Boston, Massachusetts 02118. E-mail kxwalsh@bu.edu

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