GTP Cyclohydrolase I/BH4 Pathway Protects EPCs via Suppressing Oxidative Stress and Thrombospondin-1 in Salt-Sensitive Hypertension
Endothelial progenitor cells (EPCs) are both reduced and dysfunctional in hypertension that correlates inversely with its mortality, but the mechanisms are poorly understood. Endothelial nitric oxide synthase (eNOS) critically regulates EPC mobilization and function but is uncoupled in salt-sensitive hypertension because of the reduced cofactor tetrahydrobiopterin (BH4). We tested the hypothesis that GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 de novo synthesis, protects EPCs and its function in deoxycorticosterone acetate (DOCA)-salt mice. EPCs were isolated from peripheral blood and bone marrow of wild-type (WT), WT DOCA-salt, endothelial-specific GTPCH transgenic (Tg-GCH), GTPCH transgenic DOCA-salt, and BH4-deficient hph-1 mice. In WT DOCA-salt and hph-1 mice, EPCs were significantly decreased with impaired angiogenesis and adhesion, which were restored in Tg-GCH DOCA-salt mice. Superoxide (O2−) and nitric oxide (NO) levels in EPCs were elevated and reduced, respectively, in WT DOCA-salt and hph-1 mice; both were rescued in Tg-GCH DOCA-salt mice. eNOS−/−/GCH+/− hybrid mice demonstrated that GTPCH preserved the circulating EPC number, reduced intracellular O2− in EPCs, and ameliorated EPC dysfunction independent of eNOS in DOCA-salt hypertension. Secreted thrombospondin-1 (TSP-1; a potent angiogenesis inhibitor) from EPCs was elevated in WT DOCA-salt and hph-1 but not DOCA-salt Tg-GCH mice. In vitro treatment with BH4, polyethylene glycol-superoxide dismutase (PEG-SOD), or Nomega-nitro-L-arginine (L-NNA) significantly augmented NO and reduced TSP-1 and O2− levels from EPCs of WT DOCA-salt mice. These results demonstrated, for the first time, that the GTPCH/BH4 pathway critically regulates EPC number and function in DOCA-salt hypertensive mice, at least in part, via suppressing TSP-1 expression and oxidative stress.
- endothelial progenitor cell
- GTP cyclohydrolase
- nitric oxide synthase
Although the standard nomenclature of endothelial progenitor cells (EPCs) is still lacking, putative EPCs are a circulating, bone marrow-derived cell population that participates in vasculogenesis1 by differentiating into endothelial cells and have been used to successfully enhance angiogenesis under ischemia.2 The integrity and function of the endothelium plays a key role in the prevention of hypertension.3 Recent clinical studies indicate that the number of circulating EPCs may serve as a surrogate marker for cardiovascular risks, affecting the progression of cardiovascular diseases including hypertension.4,5 In addition, hypertension has been identified as a major independent predictor for impaired EPC function.6 Treatment with antihypertensive drugs, such as angiotensin-converting enzyme inhibitors, can increase the number of circulating EPCs in patients with cardiovascular risk factors.7 However, the mechanisms underlying EPC dysfunction in hypertension are poorly understood.
Endothelial nitric oxide synthase (eNOS) regulates EPC mobilization and function, and nitric oxide (NO)-mediated signaling pathways are essential for EPC mobilization.8 However, eNOS is uncoupled in deoxycorticosterone acetate (DOCA)-salt hypertension because of the reduced level of its essential cofactor tetrahydrobiopterin (BH4).9–11 When the BH4 level is decreased, the enzymatic reduction of molecular oxygen by eNOS is no longer coupled to L-arginine oxidation, resulting in generation of superoxide anion (O2−) rather than NO, thus exacerbating oxidative stress.12 Recent studies have shown that eNOS uncoupling impairs EPC number and function in diabetes.13 In the present study, we tested the hypothesis that BH4 overexpression, which recouples eNOS, might preserve EPCs and its function in DOCA-salt mice. Since the effects of systemic BH4 supplementation may be mediated in part by nonspecific antioxidant effects of high-dose BH4, here we investigated the influence of endogenous BH4 on EPC dysfunction using a transgenic mouse model of endothelial-specific overexpression of GTPCH I (Tg-GCH), the rate-limiting enzyme of de novo BH4 synthesis.11,14
Thrombospondin-1 (TSP-1) is a key inhibitor of EPC function,15 and decreased NO production has been shown to induce TSP-1 expression in cultured endothelial cells.16,17 Previous studies also demonstrated that high glucose upregulates TSP-1 expression in rat mesangial cells, and this effect is reversed by BH4.18 On the basis of these findings, we also examined the role of TSP-1 on EPCs from DOCA-salt hypertension. We demonstrate, for the first time, that the GTPCH/BH4 pathway critically regulates EPC number and function in DOCA-salt hypertensive mice, at least in part, via suppressing TSP-1 expression and oxidative stress.
Materials and Methods
Wild-type (WT) C57BL/6 male mice (10 to 12 weeks, 20 to 25 g) were obtained from Charles River Breeding Laboratories (Portage, Mich.). Tg-GCH and BH4-deficient hph-1 mice of the C57BL/6 background were bred in house.11,19 Positive expression of transgenic GTPCH was confirmed by polymerase chain reaction (PCR). Tg-GCH (GCH+/−) and eNOS knockout (eNOS−/−) mice were crossbred to produce the offspring with the genotype of eNOS+/−/GCH+/−, which were further crossbred with eNOS knockout mice to develop a new strain of hybrid (HY) mice with the genotype of eNOS−/−/GCH+/−. Potential HY mice were screened by PCR of genomic DNA from tail tips. The primer sequences were as follows: GTPCH I (5′-GGGAAGTCGCAAAGTTGTGAGTT-3′ and 5′-GAACCCATTGCTGCACCTGG-3′); eNOS (5′-TGGCTACCCGTGATATTGCT-3′, 5′-ATTTCCTGTCCCCTGCCTTC-3′, and 5′-GGCCAGTCTCAGAGCCATAC-3′). Both 150-bp (GCH+/−) and 500-bp (eNOS−/−) PCR products were identified in the HY mice (Figure S1, available at http://hyper.ahajournals.org). All animal procedures were performed according to the guidelines of the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC).
Isolation of EPCs and Characterizations
Mouse circulating and bone marrow-derived EPCs were isolated and cultured according to our described technique.20,21
All other methods are described in the online supplemental material.
Values were expressed as mean±SEM. Statistical significance of difference between groups was performed using the Student’s 2-tailed unpaired t test. When more than 2 groups were compared, 1-way ANOVA was used. A value of P<0.05 was considered statistically significant.
Effect of GTPCH I on Blood Pressure
Five groups of mice (WT sham, WT DOCA-salt, Tg-GCH, Tg-GCH DOCA-salt, and BH4-deficient hph-1 mice) were used in the present studies, and the mean systolic blood pressure (BP) levels of the animals were observed. There were no significant differences in baseline BP (day 0) among all the groups (data not shown). Compared to WT sham mice, BP was significantly increased in WT DOCA-salt mice after a 3-week period (107±1.6 versus 139±4.8 mm Hg on day 21, P<0.01, n=6). BP in Tg-GCH DOCA-salt mice was significantly lower than that of WT DOCA-salt mice on day 21 (123±2.8 mm Hg, P<0.01, n=6 versus WT-DOCA). BH4-deficient hph-1 mice showed a slightly higher BP level than WT sham and Tg-GCH mice, but the difference did not reach statistical significance (115±4.5 versus 107±1.6 and 106±3.4 mm Hg, P>0.05, n=6 to 7).
GTPCH I Overexpression Preserves the Number of Circulating EPCs in DOCA-Salt Mice
The number of circulating stem cell antigen-1/fetal liver kinase-1 (also known as vascular endothelial growth factor receptor 2) (Sca-1/Flk-1) double-positive cells was significantly lower in WT DOCA-salt mice compared to WT sham mice (1.52±0.13% versus 2.63±0.14%, n=6 to 12, P<0.01), and was preserved in Tg-GCH DOCA-salt mice (2.41±0.27%, n=8, P<0.05 versus WT-DOCA). EPC number was also reduced in BH4-deficient hph-1 mice (1.56±0.11%, n=8, P<0.01 versus WT sham), which was increased in Tg-GCH mice (2.30±0.21%, n=5, P<0.05 versus hph-1) (Figure 1A and 1B). No difference was found between WT sham and Tg-GCH mice. These results were also confirmed by counting Dil-acLDL and lectin double-positive adherent cells under a fluorescence microscope (Figure 1C and 1D). These results demonstrate that GTPCH I overexpression preserved the level of circulating EPCs in DOCA-salt hypertensive mice.
GTPCH I Overexpression Maintains Intracellular BH4 and NO Levels in EPCs of DOCA-Salt Mice
The intracellular BH4 level in EPCs was significantly decreased in WT DOCA-salt (4.82±0.47 pmol/mg protein, n=6, P<0.05) and hph-1 (1.61±0.59 pmol/mg protein, n=6, P<0.01) mice compared to sham controls (7.04±0.48 pmol/mg protein, n=7). There was an ∼2-fold elevation of intracellular BH4 in EPCs of Tg-GCH mice over sham mice under control condition (14.0±1.29 pmol/mg protein, n=5, P<0.01 versus WT sham), which was maintained in Tg-GCH DOCA-salt mice (8.18±0.85 pmol/mg protein, n=6, P>0.05 versus WT sham) (Figure 2A).
The NO level in bone marrow-derived EPCs was reduced in BH4-deficient hph-1 mice (0.41±0.09, n=6, P<0.05 versus WT sham), which was increased in Tg-GCH mice (1.45±0.32, n=6, P<0.01 versus hph-1). No difference was found between WT sham and Tg-GCH mice (Figure 2B). Triple-staining flow cytometry (Sca-1+/Flk-1+/DAF-FM+) showed that the NO level in circulating EPCs from WT DOCA-salt mice was significantly lower than that from WT sham mice (0.52±0.09 versus 1.0±0.06, n=5 to 8, P<0.01), which was augmented in Tg-GCH DOCA-salt mice (0.86±0.11, n=5, P<0.05 versus WT-DOCA) (Figure S2).
GTPCH I Overexpression Decreases Intracellular O2− Level in EPCs of DOCA-Salt Mice
The O2− level in bone marrow-derived EPCs was elevated in BH4-deficient hph-1 mice (2.83±0.28, n=8, P<0.01 versus WT sham), which was reduced in Tg-GCH mice (1.13±0.14, n=5, P<0.01 versus hph-1). No difference was found between WT sham and Tg-GCH mice (Figure 3A and 3B). Data from triple-staining flow cytometry (Sca-1+/Flk-1+/DHE+) showed that the O2− level in circulating EPCs from WT DOCA-salt mice was significantly higher compared to WT sham mice (2.74±0.27 versus 1.0±0.19, n=8, P<0.01), which was reduced in Tg-GCH DOCA-salt mice (1.26±0.40, n=5, P<0.01 versus WT-DOCA) (Figure S2).
GTPCH I Overexpression Preserves Circulating EPCs and Decreases Their Intracellular O2− Level Independent of eNOS in DOCA-Salt Mice
To further elucidate the role of eNOS in the effects of GTPCH I on EPCs, a new strain of HY mice was developed that possesses the genotype of eNOS−/−/GCH+/−. Circulating Sca-1/Flk-1 double-positive cells in the HY mice were similar to that in WT sham mice (2.0±0.66% versus 2.38±0.18%, n=4 to 15, P>0.05), which was preserved in HY DOCA-salt mice (2.10±0.79%, n=4 to 15, P>0.05 versus WT sham) (Figure 4A). Flow cytometry data revealed that the O2− level of circulating Sca-1/Flk-1 double-positive cells from WT DOCA-salt mice was significantly higher than that from WT sham mice (2.74±0.27 versus 1.0±0.19, n=8, P<0.01), which was reduced in HY DOCA-salt mice (1.74±0.10, n=4 to 8, P<0.05 versus WT-DOCA). No difference was found between WT sham and HY mice (Figure 4B). These results suggest that GTPCH I overexpression preserves circulating EPCs and reduces their intracellular O2− level independent of eNOS in DOCA-salt hypertensive mice.
GTPCH I Overexpression Protects EPC Functions in DOCA-Salt Mice
Both tube formation and adhesion functions in EPCs from WT DOCA-salt mice were significantly reduced compared to that from WT sham mice (24.8±4.5 versus 65.8±5.2, n=5 to 8, P<0.01; and 43.7±4.7 versus 75.0±3.9, n=7, P<0.01, respectively), which were enhanced in Tg-GCH DOCA-salt mice (61.0±3.8, n=7, P<0.01; and 69.8±4.1, n=6, P<0.01, respectively, versus WT-DOCA). Tube formation and adhesion functions in EPCs were also decreased in BH4-deficient hph-1 mice (20.7±2.5, n=5, P<0.01; and 30.3±4.3, n=8, P<0.01, respectively, versus WT sham), which was ameliorated in Tg-GCH mice (66.2±4.2, n=7, P<0.01; and 89.9±2.3, n=5, P<0.01, respectively, versus hph-1). No difference was found between WT sham and Tg-GCH mice (Figure 5A through 5D).
To further elucidate the role of eNOS in the effects of GTPCH I on EPC functions, EPCs from the HY and HY DOCA-salt mice were also assessed. Both tube formation and adhesion functions in EPCs were considerably enhanced in HY and HY DOCA-salt mice compared to WT DOCA-salt mice, and no difference was found between HY and HY DOCA-salt mice, suggesting that GTPCH I ameliorates EPC function independent of eNOS in DOCA-salt hypertensive mice (Figure 5E and 5F). However, tube formation in EPCs was significantly lower in HY and HY DOCA-salt mice than that in WT sham, Tg-GCH, and Tg-GCH DOCA-salt mice, suggesting an important role of eNOS in the angiogenic function of EPCs (Figure 5E).
GTPCH I Overexpression Reduces Secreted TSP-1 in EPCs of DOCA-Salt Mice
Since TSP-1 is a secreted protein and its level in EPCs was too low to be detected (data not shown), we examined the TSP-1 level in EPC media using Western blot analysis. The TSP-1 level in EPC media from WT DOCA-salt mice was increased ≈3-fold compared to WT sham mice (2.92±0.27 versus 1.0±0.22, n=12, P<0.01), which was reduced in Tg-GCH DOCA-salt mice (1.61±0.31, n=9, P<0.01 versus WT-DOCA). The TSP-1 level in EPC media was also significantly enhanced in BH4-deficient hph-1 mice (2.87±0.40, n=8, P<0.01 versus WT sham), which was decreased in Tg-GCH mice (1.05±0.22, n=7, P<0.01 versus hph-1). No difference was found between WT sham and Tg-GCH mice (Figure 6).
In Vitro Treatment of EPCs With BH4 Increases Their Intracellular BH4 and Reduces TSP-1 in DOCA-Salt Mice
In vitro BH4 treatment was performed to determine the direct effects of BH4 on TSP-1 expression in EPCs, excluding the influence of other factors (eg, BP). The intracellular BH4 level was increased ≈6-fold following in vitro treatment with BH4 (10 μmol/L) of EPCs from WT DOCA-salt mice (Figure S3A). In vitro treatment with BH4 (5 to 20 μmol/L) concentration-dependently reduced the secreted TSP-1 level from EPCs of WT DOCA-salt mice (Figure S3B).
In Vitro Treatment With BH4, Polyethylene Glycol-Superoxide Dismutase, or Nomega-Nitro-L-Arginine Reduces TSP-1 and O2− Levels in EPCs of DOCA-Salt Mice
In vitro pharmacological interventions were also performed to verify the roles of intracellular O2− and NO in the effect of BH4 on TSP-1 expression in EPCs. The secreted TSP-1 level and intracellular O2− level in EPCs from WT DOCA-salt mice were significantly increased compared to those from WT sham mice (1.0±0.22 versus 2.92±0.27, n=12, P<0.01; and 1.0±0.12 versus 2.17±0.13, n=5 to 7, P<0.01, respectively), which was reduced following in vitro treatment with BH4 (10 μmol/L), polyethylene glycol-superoxide dismutase (PEG-SOD) (100 U/L), or Nomega-nitro-L-arginine (L-NNA) (0.8 mmol/L) (Figure 7A and 7B). In addition, the intracellular NO level in EPCs from WT DOCA-salt mice was significantly decreased compared to those from WT sham mice (1.0±0.05 versus 0.53±0.08, n=7 to 12, P<0.01), which was enhanced following in vitro treatment with BH4 (10 μmol/L) and PEG-SOD (100 U/L) but not L-NNA (0.8 mmol/L) (Figure 7C). These results suggest that restoration of intracellular O2− and NO levels contributes to the effects of BH4 on TSP-1 expression in EPCs.
The major new findings of the present study are as follows: (1) endothelium-specific GTPCH I overexpression ameliorates the decreased intracellular BH4 level and protects EPCs and its functions in DOCA-salt hypertensive mice; (2) BH4 attenuates the increased O2− level and augments the decreased NO level in EPCs of DOCA-salt mice both in vitro and in vivo; (3) BH4 reduces the O2− level and preserves EPC number and functions independent of eNOS in DOCA-salt mice; (4) TSP-1 expression in EPCs is upregulated in DOCA-salt hypertension, which is reduced by BH4 in vitro and in vivo; (5) in vitro treatment with PEG-SOD or L-NNA reduces the secreted TSP-1 level from EPCs of DOCA-salt mice; and (6) eNOS is uncoupled in EPCs from DOCA-salt hypertension.
Although clinical studies have shown that EPCs are both reduced and dysfunctional in hypertension that correlates inversely with its mortality,6 limited knowledge exists regarding the underlying causes. In this study, we demonstrate that the sca-1/Flk-1 double-positive cells in peripheral blood were significantly decreased in DOCA-salt mice (a salt-sensitive hypertension model with low plasma renin) compared with the normotensive controls, which was in parallel with the results of Dil-acLDL/lectin double-staining assessment. In addition, both angiogenic and adhesion functions of EPCs were impaired in DOCA-salt mice compared to sham mice.
eNOS is uncoupled in DOCA-salt hypertension because of its reduced essential cofactor BH4,9–11 whereby electrons no longer flow to L-arginine to form NO but instead reduce molecular oxygen to generate O2−, resulting in exacerbated oxidative stress.12 It has been shown that eNOS critically regulates normal EPC mobilization and function.8 However, the functional state of eNOS in EPCs under hypertension was unclear. In the present study, NOS inhibitor L-NNA reduced the intracellular O2− level, while it did not increase the NO level, suggesting eNOS uncoupling in EPCs from DOCA-salt mice. In addition, recent findings including ours indicate that reactive oxygen species is increased in salt-sensitive hypertension.9–11,22 The increased oxidative stress observed in both animal and clinical hypertensives may affect the survival of EPCs.23 Thus, eNOS uncoupling and the subsequent increase in intracellular O2− and reduction in intracellular NO, as observed in the present study, may represent a major mechanism underlying EPC dysfunction in DOCA-salt mice (Figure 4S). Accordingly, it is reasonable to expect that the supplement of BH4, which could recouple eNOS, may rescue EPC dysfunction in this setting. Considering the fact that BH4 is highly unstable as a potent reducing molecule, we used transgenic mice overexpressing endothelial-specific GTP cyclohydrolase I (Tg-GCH). GTPCH I overexpression markedly elevated intracellular BH4 and NO levels, reduced the intracellular O2− level, and preserved both EPC number and function in DOCA-salt Tg-GCH mice. These results demonstrate that BH4 ameliorates oxidative stress-induced EPC dysfunction through reducing O2− and enhancing NO levels in DOCA-salt mice. Restoration of intracellular O2− and NO levels by BH4 may be attributable to the recoupling of eNOS in EPCs (Figure 4S).
However, we suspected whether the observed effects of BH4 on EPCs were completely eNOS dependent, or whether BH4 could produce these effects partly via a non-eNOS recoupling mechanism. To address this issue, we used a new strain of HY mice with the genotype of eNOS−/−/GCH+/−. It was demonstrated that GTPCH I could reduce the intracellular O2− level in EPCs and thus preserve EPC number and function independent of eNOS in DOCA-salt mice, suggesting that BH4 may exert a direct effect on EPCs in addition to recoupling eNOS. The augmented BH4 level caused by GTPCH I overexpression may decrease the O2− level by chemically reacting with O2−, which may help to explain the BH4 non-eNOS recoupling effects on EPCs. In addition, angiogenic function of EPCs was significantly lower in these HY mice compared to the WT sham and Tg-GCH mice, demonstrating the importance of eNOS for EPC-mediated angiogenesis.
It has been demonstrated that TSP-1 inhibits angiogenesis and modulates endothelial cell adhesion, motility, and growth.24 Furthermore, a study has proposed that TSP-1 acts as a key inhibitor of EPC adhesion.15 However, no direct evidence for TSP-1 upregulation has been shown in hypertensive EPCs to date. Our data demonstrate the upregulation of TSP-1 in EPCs under hypertensive conditions for the first time. In addition, we observed an inverse relationship between TSP-1 level and EPC number and function, suggesting that TSP-1 upregulation might, in part, contribute to EPC dysfunction in DOCA-salt hypertension. Consistent with this notion, our data also revealed that a downregulation of TSP-1 by BH4 in vivo was accompanied by an amelioration of EPC dysfunction in this model, indicating a negative correlation of EPC function and TSP-1 level. Collectively, our experimental observations provide the first evidence that BH4 supplement reduces TSP-1 expression in EPCs both in vivo and in vitro and enhanced EPC function in vivo in DOCA-salt hypertension. Hence, in addition to reducing the intracellular O2− level and enhancing the NO level, BH4 could also ameliorate oxidative stress-induced EPC dysfunction by suppressing TSP-1 expression in EPCs of DOCA-salt mice (Figure 4S).
Studies have shown that decreased NO production induces TSP-1 expression in cultured endothelial cells.16,17 Augmented oxidative stress can reduce NO bioavailability by increasing NO scavenging. Since our data showed that secreted TSP-1 and intracellular superoxide levels were enhanced and the intracellular NO level was reduced in EPCs of DOCA-salt mice (both ameliorated by increasing BH4 in vivo), we then tested the hypothesis that BH4 exerts its effects on TSP-1 expression in EPCs through reducing superoxide and enhancing the NO level, in a series of in vitro studies. Besides BH4, PEG-SOD and NOS inhibitor L-NNA reduced the superoxide level and TSP-1 expression in EPCs of DOCA-salt mice, suggesting that the decreased superoxide level was involved in the mechanisms underlying the effect of BH4 on TSP-1 expression in EPCs. In addition, because the intracellular NO level in EPCs of WT DOCA-salt mice was upregulated following BH4 or PEG-SOD treatment in vitro, the enhanced NO level might also contribute to the effect of BH4 on TSP-1 expression (Figure 4S).
As Tg-GCH DOCA-salt mice exhibited a significantly lower BP than WT DOCA-salt mice, possibility exists that BP reduction might also contribute to the beneficial effect of BH4 on EPCs in Tg-GCH DOCA-salt mice, considering that these in vivo studies could not exclude the effect of BP from that of BH4. However, in vitro studies possess the advantage that allowed us to determine the direct effects of BH4, PEG-SOD, and L-NNA on EPCs, which do not involve BP and other possible influences from our in vivo studies. In addition, EPCs used in the present study are heterogenous. It is possible that some of the changes detected in the present study are a consequence of alterations in cell populations induced by hypertension.
The present study demonstrates, for the first time, that the GTPCH/BH4 pathway critically regulates EPC number and function in DOCA-salt hypertension, at least in part, via suppressing TSP-1 expression and reducing oxidative stress. Because BH4 is highly unstable and easily oxidized (thus not suitable for chronic oral administration), our findings on how the GTPCH/BH4 pathway regulates EPC function may provide a mechanistic basis of augmenting endogenous BH4 levels by targeting GTPCH as a new rational therapeutic strategy to recouple eNOS and combat EPC and endothelial dysfunction in cardiovascular diseases, including hypertension.
Sources of Funding
This work was supported by National Institute of General Medical Science/National Institutes of Health grant R01GM077352, American Heart Association Grant 0855601G, International Collaborative grant 30728021 from the Natural Sciences Foundation of China (NSFC) (to A.F.C.), and NSFC grants 30700307 and 30971158 (to H.-H.X.). D.D.C. is the recipient of an American Heart Association postdoctoral fellowship award (0720114Z).
- Received August 2, 2010.
- Revision received August 26, 2010.
- Accepted October 5, 2010.
Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964–967.
Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003; 13: 593–600.
Bahlmann FH, de Groot K, Mueller O, Hertel B, Haller H, Fliser D. Stimulation of endothelial progenitor cells: a new putative therapeutic effect of angiotensin II receptor antagonists. Hypertension. 2005; 45: 526–529.
Zheng JS, Yang XQ, Lookingland KJ, Fink GD, Hesslinger C, Kapatos G, Kovesdi I, Chen AF. Gene transfer of human GTP cyclohydrolase I restores vascular tetrahydrobiopterin level and endothelial dysfunction in low renin hypertension. Circulation. 2003; 108: 1238–1245.
Du YH, Guan YY, Alp NJ, Channon KM, Chen AF. Endothelium-specific GTP cyclohydrolase I overexpression attenuates blood pressure progression in salt-sensitive low-renin hypertension. Circulation. 2008; 117: 1045–1054.
Katusic ZS. Vascular endothelial dysfunction: does tetrahydrobiopterin play a role? Am J Physiol Heart Circ Physiol Heart Circ Physiol. 2001; 281: H981–H986.
Thum T, Fraccarollo D, Schultheiss M, Froese S, Galuppo P, Widder JD, Tsikas D, Ertl G, Bauersachs J. Endothelial nitric oxide synthase uncoupling impairs endothelial progenitor cell mobilization and function in diabetes. Diabetes. 2007; 56: 666–674.
Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, Goh N, Rockett KA, Channon KM. Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I overexpression. J Clin Invest. 2003; 112: 725–735.
Ii M, Takenaka H, Asai J, Ibusuki K, Mizukami Y, Maruyama K, Yoon YS, Wecker A, Luedemann C, Eaton E, Silver M, Thorne T, Losordo DW. Endothelial progenitor thrombospondin-1 mediates diabetes-induced delay in reendothelialization following arterial injury. Circ Res. 2006; 17: 98: 697–704.
Wang S, Shiva S, Poczatek MH, Darley-Usmar V, Murphy-Ullrich JE. Nitric oxide and cGMP-dependent protein kinase regulation of glucose-mediated thrombospondin 1-dependent transforming growth factor-beta activation in mesangial cells. J Biol Chem. 2002; 277: 9880–9888.
Marrotte E, DD Chen, Hakim JS, Chen AF. Restoration of endothelial progenitor cell function with manganese superoxide dismutase accelerates wound healing in diabetic mice. J Clin Invest. 2010;120:In press.
Zhao T, Li J, Chen AF. MicroRNA-34a induces endothelial progenitor cell senescence and impedes its angiogenesis via suppressing silent information regulator 1. Am J Physiol Endocrinol Metab. 2010; 299: E110–E116.
Li L, Fink GD, Watts SW, Northcott CA, Galligan JJ, Pagano PJ, Chen AF. Endothelin-1 increases vascular superoxide via endothelin(A)-NADPH oxidase pathway in low-renin hypertension. Circulation. 2003; 107: 1053–1058.
Guo N, Krutzsch HC, Inman JK, Roberts DD. Thrombospondin 1 and type I repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells. Cancer Res. 1997; 57: 1735–1742.