Essential Role of Extracellular SOD in Reparative Neovascularization Induced by Hindlimb Ischemia
Neovascularization is an important physiological repair mechanism in response to ischemic injury, and its process is dependent on reactive oxygen species (ROS). Overproduction of superoxide anion (O2·−) rather contributes to various cardiovascular diseases. The extracellular superoxide dismutase (ecSOD) is one of the major antioxidant enzymes against O2·− in blood vessels; however, its role in neovascularization induced by tissue ischemia is unknown. Here we show that hindlimb ischemia of mice stimulates a significant increase in ecSOD activity in ischemic tissues where ecSOD protein is highly expressed at arterioles. In mice lacking ecSOD, ischemia-induced increase in blood flow recovery, collateral vessel formation, and capillary density are significantly inhibited. Impaired neovascularization in ecSOD−/− mice is associated with enhanced O2·− production, TUNEL-positive apoptotic cells and decreased levels of NO2−/NO3− and cGMP in ischemic tissues as compared with wild-type mice, and it is rescued by infusion of the SOD mimetic tempol. Recruitment of inflammatory cells into ischemic tissues as well as numbers of inflammatory cells and endothelial progenitor cells (c-kit+/CD31+ cells) in both peripheral blood and bone marrow (BM) are significantly reduced in these knockout mice. Of note, ecSOD expression is markedly increased in BM after ischemia. NO2−/NO3− and cGMP levels are decreased in ecSOD−/− BM. Transplantation of wild-type BM into ecSOD−/− mice rescues the defective neovascularization. Thus, ecSOD in BM and ischemic tissues induced by hindlimb ischemia may represent an important compensatory mechanism that blunts the overproduction of O2·−, which may contribute to reparative neovascularization in response to ischemic injury.
- superoxide dismutase
- reactive oxygen species
- bone marrow
- endothelial progenitor cells
Neovascularization is important repair mechanism to preserve tissue integrity in response to ischemic injury. It is a key process involved in normal development and wound repair as well as in the various pathophysiologies such as ischemic heart and limb diseases. New blood vessels grow postnatally via angiogenesis and arteriogenesis.1,2 Moreover, circulating endothelial progenitor cells (EPCs) mobilized from bone marrow (BM) also contribute to the formation of new blood vessels (vasculogenesis) after tissue ischemia.3,4 Inflammation is an early key event required for ischemia-induced neovascularization.5,6 Reactive oxygen species (ROS) such as superoxide anion (O2·−) and hydrogen peroxide (H2O2) are involved in physiological and pathophysiological responses. Excess amounts of ROS contribute to the pathogenesis of many cardiovascular diseases by inducing apoptosis and cell death.7 In contrast, physiological levels of ROS produced by growth factor and tissue ischemia are involved in proliferation and migration of endothelial cells (ECs), thereby contributing angiogenesis in vivo.7 Indeed, we previously demonstrated that hindlimb ischemia stimulates ROS production via gp91phox (Nox2)-based NADPH oxidase in ischemic tissues, and that blood flow recovery are markedly impaired in Nox2−/− mice in which ischemia-induced ROS production is abolished.8 In contrast, overproduction of Nox2-derived ROS rather contributes to impairment of postischemic neovascularization in pathological conditions such as diabetes.9 Thus, function of ROS during new blood vessel formation is dependent on its concentration.
Levels of ROS are tightly controlled by the balance of activity of pro- and antioxidative enzymes. The extracellular superoxide dismutase (ecSOD) is the major antioxidant enzyme against O2·− in the extracellular space where ROS are accumulated.10,11 It is synthesized and secreted by vascular smooth muscle cells and fibroblasts, and anchored to the extracellular matrix and endothelial cell surface through binding to the heparan sulfate proteoglycan, collagen, and fibulin-5.12,13 Because of its location, ecSOD plays an important role in regulating endothelial function by modulating the levels of O2·− and bioavailability of nitric oxide (NO).14–16 We and others reported that ecSOD expression is influenced by multiple stimuli, including nitric oxide and proinflammatory cytokines.17,18 Previous studies using ecSOD−/− mice demonstrate that ecSOD functions to prevent oxidative stress–dependent pathological states such as ischemia-reperfusion injury and lung injury induced by hyperoxia.12,17 In some of these models, protective effects of ecSOD are associated with a decrease in recruitment of inflammatory cells to the injury sites.19,20 Moreover, adenovirus-mediated delivery of ecSOD inhibits growth of B16 melanoma tumors by blocking neovascularization in mice.21
We performed the present study to determine whether endogenous ecSOD is involved in neovascularization in response to hindlimb ischemia by modulating the levels of O2·− using ecSOD−/− mice. We demonstrate that hindlimb ischemia of mice increases expression of ecSOD in ischemic tissues and BM. Mice lacking ecSOD show enhanced ischemia-induced O2·− production, decreased NO2−/NO3− and cyclic GMP levels, increased apoptosis in ischemic tissues, which may contribute to impairment of neovascularization. Numbers of inflammatory cells and EPCs in both peripheral blood and BM are reduced in ecSOD−/− mice, which is associated with decrease in BM cells differentiation into EPCs as well as levels of NO2−/NO3− and cyclic GMP in BM. Defective neovascularization in ecSOD−/− mice is rescued by SOD mimetic infusion as well as by transplantation of BM from WT mice. These findings suggest that ecSOD plays an essential role in reparative neovascularization in response to ischemic injury by protecting ischemic tissues and BM from overproduction of ROS.
Materials and Methods
Mouse Ischemia Hindlimb Model
The ecSOD-deficient mice in a C57Blk/6 background and C57Blk/6 (wild-type) mice were used for this study.15 Surgical procedure are performed as previously described (see the online data supplement, available at http://circres.ahajournals.org).8
Laser Doppler Blood Flow Analysis
We measured hindlimb blood flow using a laser Doppler blood flow (LDBF) analyzer (PIM III, Perimed) as previously described (see the online data supplement).8
Blood Flow Measurement by Microsphere
Fluorescent microspheres (15-μm diameter, 5×105 beads, Molecular Probe) were used to analyze the formation of conductance collateral vessels as previously described (see the online data supplement).8
Staining for capillary, proliferating arterioles, inflammatory leukocytes, and macrophages are performed as previously described (see also the online data supplement).8
Microcomputed Tomography (micro-CT) Analysis
Tissues were prepared, and analyzed as previously described (see the online data supplement).22
Measurement of ecSOD Activity
The ecSOD activity was measured by cytochrome c reduction assay after separation of ecSOD fraction with ConA-Sepharose column as described in the online data supplement.23
Measurements of Superoxide (O2·−) Production in Hindlimb Tissues
O2·− production was measured with 5 μmol/L lucigenin-enhanced chemiluminescence, as previously described.8 As a second approach to measure O2·− in hindlimb tissues in situ, dihydroethidium (DHE) were used as previously described.13
Measurement of NO2−/NO3− and cGMP Production
Levels of the nitrite [NO2−] plus nitrate [NO3−] were measured by the Griess method (Cayman), according to manufacturer’s protocol. Tissue cGMP was measured using a cGMP enzyme-immunoassay system (Amersham Life Science).
Fluorescence-activated cell sorter (FACS) analysis was used to quantify inflammatory cells and EPC-like mononuclear cells (MNCs) in both peripheral blood (PB) and bone marrow (BM) from WT and ecSOD−/− mice as described in the online data supplement.
BM Culture Assay
Isolation of BM-MNCs and Fluorescent chemical detection of EPCs were performed as previously described (see the online data supplement).24
BM transplantation was performed as described in the online data supplement.
All values were expressed as mean±SE. Blood flow recovery in the ischemic hindlimb was compared between 2 groups by 2-way repeated measures ANOVA, followed by Bonferroni post hoc analysis. Comparison between 2 mean values was evaluated by an unpaired Student 2-tailed t test, and between 3 or more groups was evaluated by 1-way ANOVA followed by Bonferroni post hoc analysis. Statistical significance was accepted at P<0.05.
Ischemia-Induced Neovascularization Is Impaired in ecSOD−/− Mice
All mice survived after induction of unilateral hindlimb ischemia, appeared to be healthy, and showed no significant change of blood pressure and heart rate during the follow-up period. To determine the role of ecSOD in ischemia-induced neovascularization, we measured blood flow recovery in ischemic and nonischemic limbs after ligation of femoral artery in WT and ecSOD−/− mice. Figure 1A using LDBF analysis shows that in WT mice, hindlimb blood flow was markedly decreased immediately after surgery, partially restored on day 3, and recovered to the level of that of the nonischemic limb by day 7. In ecSOD−/− mice, ischemia-induced blood flow recovery was delayed, and the LDBF ratio at 7 days after ischemia was significantly decreased as compared with that in WT mice. Similar precipitous reduction in hindlimb flow occurred in WT and ecSOD−/− mice, indicating that the severity of ischemia was similar in both groups.
Neovascularization induced by tissue ischemia is mediated through arteriogenesis and angiogenesis.1 We thus examined the role of ecSOD in ischemia-induced arteriogenesis using microspheres of 15 μm diameter to analyze the formation of conductance collateral vessels.8 Figure 1B shows that the recovery of collateral blood flow at 7 days after ischemia was significantly reduced in ecSOD−/− mice compared with WT mice. Consistent with this result, immunofluorescence analysis in collateral vessels revealed that the number of BrdU positive arterioles was decreased in ischemic hindlimbs in ecSOD−/− mice (Figure 1C). To confirm further, we performed micro-CT analysis and found that collateral formation was markedly reduced in ecSOD−/− mice compared with WT mice (supplemental Figure I). Taken together, arteriogenesis is impaired in ecSOD−/− mice.
To determine the role of ecSOD in ischemia-induced angiogenesis, we measured capillary density by staining ischemic tissue with Griffonia simplicifolia lectin which detects ECs with high efficiency in our system.8 Figure 1D shows that lectin-positive capillary density was significantly reduced in the ischemic adductor muscle of ecSOD−/− mice compared with WT mice.
ecSOD Activity and Protein Expression in Ischemic Tissues Are Increased in Response to Hindlimb Ischemia
We next examined the ecSOD activity and protein expression after hindlimb ischemia. Figure 2A shows that ecSOD activity in ischemic tissues as measured by cytochrome c reduction assay was significantly increased after femoral artery ligation. Immunocytochemistry with double staining for ecSOD and α-actin or lectin shows that ecSOD protein was highly expressed at α-actin positive arterioles, not at lectin-positive capillary-like ECs, in ischemic tissues (Figure 2B). We also found that ecSOD is expressed in inflammatory cells such as macrophage (Mac3 positive) (supplemental Figure II).
Ischemia-Induced O2·− Production Is Enhanced in ecSOD−/− Mice
Because ecSOD is one of the major antioxidant enzymes against O2·− in the vasculature, we examined O2·− levels in ischemic and nonischemic limbs in WT- and ecSOD−/− mice. Figure 3A using lucigenin assays demonstrate that hindlimb ischemia stimulates O2·− production in ischemic tissues at 7 days after operation in WT mice. Mice deficient in ecSOD show enhanced ischemia-induced, but not basal, O2·− production as compared with WT mice, suggesting a protective role of ecSOD from overproduction of ROS induced by ischemic injury. To confirm this result further, we performed DHE staining which is specific for O2·−25 in ischemic and nonischemic hindlimb tissue sections. Figure 3B shows an increase of DHE fluorescence in ischemic tissue in WT mice, which was further enhanced in ecSOD−/− mice. Of note, an increase in DHE staining was almost completely abolished by coincubation with SOD, demonstrating ischemia-induced increase in O2·− in WT mice and its augmentation in ecSOD−/−mice.
Chronic Tempol Infusion Rescues Impairment of Ischemia-Induced Neovascularization in ecSOD−/− Mice
To determine whether overproduction of ROS contributes to impairment of postischemic neovascularization in ecSOD−/− mice, we infused the SOD mimetic tempol in WT and ecSOD−/− mice, and measured O2·− production and blood flow recovery. Tempol treatment into WT mice significantly reduced the ischemia-induced flow recovery and O2·− level in ischemic tissues (Figure 4A) at 7 days after operation (Figure 4B). In ecSOD−/− mice, treatment with tempol rather rescued the impairment of ischemia-induced blood flow recovery (Figure 4B) by reducing the overproduction of O2·− to the levels observed in WT mice without tempol after hindlimb ischemia (Figure 4A). These results are consistent with the notion that optimal levels of ROS are required but excess amount of ROS are inhibitory for neovascularization induced by tissue ischemia.
Decrease in NO2−/NO3− and cGMP Levels and Increase in Apoptosis in ecSOD−/− Mice
It has been shown that ecSOD preserves bioavailable NO by preventing the reaction of NO with excess amount of O2·−.14–16 NO also plays an important role in postnatal neovascularization.24,26 To assess the mechanisms by which overproduction of O2·− by ecSOD deficiency inhibits neovascularization, we measured NO2−/NO3− and cGMP levels in ischemic tissues from WT and ecSOD−/− mice. Figure 4C shows that NO2−/NO3− and cGMP levels in ischemic tissue were markedly decreased in ecSOD−/− mice as compared with WT mice. Of note, their reduction in ecSOD−/− mice was restored by tempol treatment (data not shown). Moreover, we found that the numbers of apoptotic cells as measured by TUNEL staining were markedly increased in ischemic tissues in ecSOD−/− mice as compared with WT mice (supplemental Figure III). These results suggest that overproduction of O2·− results in decrease in NO/cGMP levels and increased apoptosis, which may contribute to defective neovascularization in ecSOD−/− mice.
Numbers of Inflammatory Cells in Ischemic Sites as Well as in Peripheral Blood and Bone Marrow Are Decreased in ecSOD−/− Mice
Because inflammation plays a key role in ischemia-induced neovascularization, we examined the number of inflammatory cells infiltrated into the ischemic hindlimbs in WT and ecSOD−/− mice. Figure 5 using immunocytochemial analysis shows that the numbers of infiltrated CD45 positive leukocytes and Mac3-positive macrophages were significantly decreased in ischemic tissue from ecSOD−/− mice at 3 days after ischemia. FACS analysis of peripheral blood (PB) and bone marrow (BM) reveals that there was significant decrease in the numbers of CD45 positive neutrophils and monocytic cells in PB (Figure 6A) as well as CD45 positive myeloid cells in BM (Figure 6B) in ecSOD−/− mice compared with WT mice.
EPC-Like Cells in Peripheral Blood and Bone Marrow Are Decreased in ecSOD−/− Mice
Because postnatal neovascularization is also dependent on vasculogenesis,3,4 we examined the number of c-kit+/CD31+ EPC-like cells in PB and BM in WT and ecSOD−/− mice. FACS analysis shows a significant reduction in the number of c-kit+/CD31+ cells in both PB (Figure 7A) and BM (Figure 7B) at 3 days after ischemia in ecSOD−/− mice compared with WT mice.
NO2−/NO3− and cGMP Levels in BM as Well as BM-MNC Differentiation Into EPCs are Decreased in ecSOD−/− Mice
Above results suggest that BM function is impaired in ecSOD−/− mice. To assess underlying mechanisms, we examined NO2−/NO3− and cGMP levels in BM cells as well as capacity of BM-MNC differentiation into EPCs in WT- and ecSOD−/− mice. Figure 7C shows that both NO2−/NO3− and cGMP levels were significantly lower in BM from ecSOD−/− mice than that from WT mice. As shown in Figure 7D, BM culture assay reveals that differentiation of BM-MNCs into EPCs, as detected by DiI-acLDL and BS lectin double positive cells, was markedly decreased in ecSOD−/− mice.
ecSOD+/+ Bone Marrow Rescues Impairment of Neovascularization in ecSOD−/− Mice
To assess the role of ecSOD in BM function in vivo, we examined the ecSOD protein expression in BM in response to hindlimb ischemia and performed BM transplantation between WT and ecSOD−/− mice. Western analysis shows that ecSOD protein expression was markedly increased in the BM at 3 and 7 days after ischemia while Cu/Zn SOD protein was not changed (Figure 8A). Figure 8B shows that WT, but not ecSOD−/−, BM transplantation to the irradiated ecSOD−/− mice rescued the impairment of blood flow recovery after hindlimb ischemia in ecSOD−/− mice. Moreover, WT mice transplanted with BM from ecSOD−/−, but not WT, mice showed the significant decrease in blood flow recovery after ischemia. Taken together, these results suggest that ecSOD induced in BM after tissue ischemia plays an important role for BM function, thereby regulating postnatal neovascularization.
The present study provides novel evidence that endogenous ecSOD plays an essential role for postischemic neovascularization. We found that: (1) Hindlimb ischemia of mice stimulates a significant increase in ecSOD activity in ischemic tissues where ecSOD protein is highly expressed at arterioles and in part at inflammatory cells; (2) Mice lacking ecSOD show overproduction of O2·− in ischemic tissues, which is associated with decreased NO2−/NO3− and cGMP levels as well as increased apoptosis, thereby contributing to impairment of neovascularization; (3) Recruitment of inflammatory cells into ischemic tissues as well as number of inflammatory cells and EPCs in both PB and BM are significantly decreased in ecSOD−/− mice; (4) ecSOD−/− BM is dysfunctional because NO2−/NO3− and cGMP levels in BM as well as BM-MNC differentiation into EPCs are decreased in ecSOD−/− mice; (5) Ischemia increases ecSOD expression in BM, and BM transplantation of WT-BM into ecSOD−/− mice rescues the defective blood flow recovery.
We demonstrate that ecSOD activity is significantly increased in the ischemic tissues after hindlimb ischemia, which is associated with an increase in ROS production and neovascularization. Immunocytochemial analysis reveals that ecSOD protein is predominantly expressed at α-actin positive arterioles and in part at mac3-positive inflammatory cells, but not at lectin-positive capillary-like ECs. This is consistent with the previous reports that ecSOD is highly expressed in vascular smooth muscle and inflammatory cells, but not in endothelial cells.11,27,28,29 Functional role of upregulation of ecSOD in neovascularization is demonstrated by the observation that ischemia-induced increase in blood flow recovery and capillary density (angiogenesis) are significantly inhibited in ecSOD−/− mice. Moreover, formation of collateral artery (arteriogenesis) as measured by microsphere (15 μm diameter), number of BrdU-positive arterioles, and micro-CT are significantly impaired in ecSOD−/− mice. These results suggest that endogenous ecSOD plays an important role in ischemia-induced neovascularization. In contrast, Wheeler et al reported that overexpression of ecSOD using adenovirus inhibits tumor angiogenesis.21 This discrepancy may be attributable to the difference of angiogenesis model system; ie, hindlimb ischemia and B16 melanoma.
Lucigenin assay and DHE fluorescence analysis demonstrate that ischemia-induced O2·− production in ischemic tissue is further enhanced in ecSOD−/− mice compared with WT mice. Infusion of the SOD mimetic tempol into ecSOD−/− mice rescues the impairment of ischemia-induced blood flow recovery by reducing the overproduction of O2·− to the levels observed in WT mice. These results suggest that overproduction of ROS attributable to ecSOD deficiency contributes to the impairment of ischemia-induced neovascularization. Consistent with this, Ebrahimian et al reported that excess amount of ROS impairs postischemic neovascularization in pathological conditions such as type1 diabetes.9 By contrast, WT mice treated with tempol inhibits neovascularization with concomitant decrease in O2·− production, suggesting that optimal levels of ROS are required for neovascularization. Indeed, we and others previously demonstrated that ROS are essential for angiogenesis signaling in cultured endothelial cells and postnatal neovascularization in vivo.8,30–32 Thus, ischemia-induced upregulation of ecSOD expression may represent an important compensatory mechanism that protects ischemic tissues from overproduction of O2·−, thereby preserving appropriate level of ROS required for new blood vessel formation. Moreover, our findings are consistent with the notion that optimal low levels of ROS are required but excess amounts of ROS are inhibitory for neovascularization induced by tissue ischemia.
We and others previously demonstrated that ecSOD deficiency is associated with a decrease in NO bioavailability and endothelial dysfunction caused by a rapid reaction of O2·− and NO.15,15,16 NO also plays an important role in postnatal neovascularization.24,26 The present study demonstrates that NO2−/NO3− and cGMP levels in ischemic tissue are markedly decreased in ecSOD−/− mice, which are restored by tempol treatment. Moreover, TUNEL-positive apoptotic cells are markedly increased in ischemic tissues in ecSOD−/− mice as compared with WT mice. Consistent with this, high concentrations of ROS have been shown to cause apoptosis and cell death.7 Taken together, these results suggest that overproduction of O2·− induced by ecSOD knockout results in decreased available NO and increased apoptosis, thereby contributing to impairment of neovascularization. Moreover, it is possible that ecSOD deficiency may cause formation of peroxynitrite17 which causes endothelial NO synthase uncoupling to produce excess amount of O2·−.33 The detailed relationships between ecSOD and NO or associated reactive nitrogen species in postnatal neovascularization require further investigation.
Inflammation is a key early process responsible for ischemia-induced neovascularization.5,6,34,35 The present study demonstrates that ecSOD deficiency inhibits infiltration of inflammatory leukocytes (CD45 positive) and macrophage (Mac3 positive) into ischemic limbs at 3 days after tissue ischemia. Given that infiltrated inflammatory leukocytes release cytokines and angiogenic factors including VEGF, it is conceivable that reduced inflammatory responses in ecSOD−/− mice may contribute to impairment of neovascularization. This is in contrast to the previous reports suggesting an antiinflammatory function of ecSOD in other models including lung injury induced by various stimulants such as LPS and hyperoxia.12,19,20 This discrepancy may be attributable to the difference of mechanism of inflammation between ischemia-induced angiogenesis and lung injury. Importantly, the present study demonstrates that the number of inflammatory cells in both PB and BM after hindlimb ischemia is significantly reduced in ecSOD−/− mice. Thus, the decrease of inflammatory cells in ischemic tissue induced by ecSOD deficiency may be at least in part attributable to the impairment of BM function which is important for maintenance and mobilization of inflammatory cells.
BM is one of the major reservoirs of EPCs. In response to ischemic injury, EPCs are mobilized from BM and homes to sites of injury, thereby contributing to neovascularization.3,4 Flow cytometry analysis shows that the numbers of EPC-like c-kit+/CD31+ cells in both PB and BM are significantly decreased in ecSOD−/− mice. To assess the mechanism by which the numbers of EPCs and inflammatory cells are decreased in ecSOD−/− BM, we examined NO2−/NO3− and cGMP levels in BM cells in WT- and ecSOD−/− mice. Both NO2−/NO3− and cGMP levels are significantly lower in ecSOD−/− BM compared with those in WT-BM. Aicher et al reported that eNOS deficiency in the BM microenvironment impairs the mobilization of stem and progenitor cells from the BM.24 It has been shown that the local BM microenvironment, so-called stem cell niche, which includes macrophages, fibroblasts, endothelial cells, and extracellular matrixes is important for hematopoiesis and EPC differentiation.3,36 In the present study, BM culture assay reveals that differentiation of BM-MNCs into EPCs, as detected by Dil-acLDL and BS lectin double positive cells, is markedly decreased in ecSOD−/− mice. In line with our finding, previous studies show that EPCs are enriched in antioxidant enzymes which are protective against oxidative stress to maintain their function required for postischemic neovascularization,37,38 and that overproduction of ROS in the setting of diabetes impairs BM-MNCs differentiation into EPCs.9 Thus, ecSOD may play an important role in BM function by preserving NO availability in the BM microenvironment from excess amounts of ROS.
To assess the role of ecSOD in BM function, we examined the ecSOD protein expression in BM in response to hindlimb ischemia and performed BM transplantation between WT and ecSOD−/− mice. We demonstrate that ecSOD protein expression is markedly increased in the BM after ischemia whereas Cu/ZnSOD protein is not changed. Moreover, impaired blood flow recovery in ecSOD−/− mice is rescued by reception of WT-BM. WT mice transplanted with ecSOD−/− BM show significant decrease in ischemia-induced restoration of blood flow. These data strongly suggest that ischemia-induced upregulation of ecSOD in BM is involved in preserving BM function, thereby promoting neovascularization. Understanding the role of ecSOD in BM cells in neovascularization is the subject of future investigation.
In summary, the present study demonstrates that ecSOD plays a critical role in neovascularization in response to ischemic injury. Ischemia-induced upregulation of ecSOD in BM and ischemic hindlimbs may represent a novel compensatory mechanism that protects against overproduction of ROS, thereby maintaining ROS levels which are required for reparative neovascularization induced by tissue ischemia. These findings provide novel insight into ecSOD as a potential therapeutic target for ischemic cardiovascular diseases. Moreover, our present study using ecSOD−/− mice and previous studies using Nox2−/− mice8,9 support the concept for a double-edged role of ROS in postischemic neovascularization; optimal low levels of ROS are required but excess amounts of ROS are inhibitory for new blood vessel formation.
We are extremely grateful to Dr Rodney J. Folz for providing us with ecSOD-deficient mice.
Sources of Funding
This research was supported by NIH R01 HL70187 (to T.F.), AHA Grant-In-Aid 0455242B (to T.F.), NIH grant HL 077524 (to M.U.-F.), and AHA Grant-in-Aid 0555308B and 0755805Z (to M.U.-F.).
↵*These authors contributed equally to this study.
Original received April 8, 2007; revision received June 7, 2007; accepted June 20, 2007.
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