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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 17, 2001 as doi:10.1096/fj.01-0338fje. |
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Departments of Medicine and Molecular Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 01125, USA; and
* Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
2Correspondence: Molecular Medicine, Beth Israel Deaconess Medical Center, RW-663, 330 Brookline Ave., Boston MA 02215, USA. E-mail: waird{at}caregroup.harvard.edu
SPECIFIC AIM
The aim of this study was to test the hypothesis that vascular endothelial growth factor (VEGF) signaling in the endothelium is coupled to the redox state of the cell. We demonstrate that 1) VEGF induces the expression of the manganese-superoxide dismutase (MnSOD) gene in cultured human endothelial cells and 2) this effect is dependent on NADPH oxidase activity.
PRINCIPAL FINDINGS
1. VEGF induces MnSOD mRNA and protein in primary human endothelial cells
VEGF treatment of primary human endothelial cells resulted in a time-and dose-dependent increase in MnSOD mRNA and protein levels, with maximal levels occurring at 4 and 24 h, respectively (Fig. 1
). In contrast, endothelial nitric oxide synthase (eNOS) mRNA was not induced in either cell type by administration of VEGF (Fig. 1)
. VEGF-mediated induction of MnSOD mRNA was abolished by actinomycin D, indicating that MnSOD induction by VEGF requires de novo mRNA synthesis.
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2. VEGF-mediated induction of MnSOD is inhibited by chemical inhibitors of NADPH oxidase and by adenoviral-mediated overexpression of catalase, Cu, Zn-SOD, and Rac1N17
VEGF-mediated induction of MnSOD at 4 h was inhibited in a dose-dependent manner by reactive oxygen species (ROS) scavengers (anthrone and DMSO) and by NADPH oxidase inhibitors [diphenyleneiodonium (DPI) and 2-aminoethyl-benzenesulfonyl fluoride (AEBSF)]. In contrast, VEGF-mediated induction of MnSOD was not affected by pretreatment with inhibitors of cytochrome P450 (methoxsalen and troleandomycin), xanthine oxidase (allopurinol), nitric oxide synthase (N-monomethyl-L-arginine, or L-NAME), or mitochondrial site I electron transport (rotenone). Taken together, the above studies suggest that VEGF induces MnSOD by an NADPH oxidase-dependent mechanism.
To further test our hypothesis, we used an adenoviral gene transfer system to infect primary human coronary artery endothelial cells (HCAEC) with control virus (Adßgal) or virus containing the cDNA of catalase (AdCat), Cu, Zn-SOD (AdSOD), or a dominant negative form of the small GTPase component of NADPH oxidase, Rac1 (AdRac1N17) at 50 multiplicity of infection (MOI). As shown in Fig. 2
, VEGF-mediated induction of MnSOD mRNA was abrogated by overexpression of catalase or Cu, Zn-SOD and significantly inhibited by expression of Rac1N17 compared with control. Together with the results of the chemical inhibitor studies, these findings suggest that the VEGF-mediated response of MnSOD depends critically on the redox state of the cell and is mediated in part by a Rac1-dependent oxidase.
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3. Basal levels ROS in endothelial cells are generated by NADPH oxidase
To determine the relative contributions of the ROS-generating enzymes in endothelial cells, HCAEC were labeled with cell-permeable 2',7'-dichlorofluorescein diacetate and oxidation of intracellular DCF was measured by fluorescence-activated cell sorting analysis. Baseline oxidation of the fluorophore was inhibited in a dose-dependent manner by DPI and AEBSF, whereas allopurinol had no such effect. Addition of L-NAME resulted in a slight but significant increase in ROS generation, suggesting that NOS inhibition has a net pro-oxidant effect. Preincubation of HCAEC with 300 µM anthrone significantly reduced ROS levels. Taken together, these results suggest that the NADPH oxidase enzyme complex is responsible for generating ambient levels of ROS in HCAEC.
4. VEGF produces a transient increase in Rac1 oxidase-dependent ROS in primary human endothelial cells
Confocal laser scanning microscopy was used to examine DCF fluorescence in HCAEC between 5 and 30 min after addition of VEGF. VEGF resulted in a significant transient increase in ROS at 20 min (47% and 56% in uninfected and control Adßgal-infected HCAEC, respectively). This effect was completely abrogated by adenoviral-mediated expression of AdRac1N17 compared with control Adßgal. Based on these findings, we conclude that VEGF produces a transient increase in Rac1 oxidase-dependent ROS.
CONCLUSIONS AND SIGNIFICANCE
Whereas ROS have traditionally been viewed as cytotoxic molecules, it is now recognized they play a critical role in signal transduction and transcriptional regulation within the vascular tree. There is increasing evidence for an important role of NADPH oxidase in generating ROS within endothelial cells. The NADPH oxidase complex consists of a membrane component comprising two subunits, gp91phox and p22phox, and several cytosolic components including p40phox, p47phox, p67phox, and the small GTPase Rac (Rac1 or Rac2). Various components of the leukocyte NADPH oxidase complex have been identified in endothelial cells, including gp91phox, p47phox, and p22phox. Moreover, some studies have provided evidence for the role of NADPH oxidase as the primary determinant of basal ROS generation in the endothelium. Finally, temporal changes in NADPH oxidase activity and secondary increases in ROS production have been reported in studies of endothelial cells that were exposed to oscillatory and steady state shear stress, cyclical strain, ischemia, T 78 F
, or high concentrations of K+.
In the present study, we have shown that the addition of VEGF to two different primary endothelial cell types (HCAEC and HPAEC) resulted in a significant induction of MnSOD mRNA and protein. This effect was completely blocked by the intracellular overexpression of the ROS scavenger enzymes catalase or Cu, Zn-SOD, suggesting that both O2- and H2O2 are involved as signaling intermediates. We also demonstrated that VEGF transiently increased ROS levels in endothelial cells and that this effect was abrogated by overexpression of a dominant negative form of the small GTPase component of the NADPH oxidase complex, Rac1 (Rac1N17).
VEGF induction of MnSOD was dependent on both inducible and basal levels of ROS. Overexpression of RacN17, which prevented an increase in ROS but did not decrease basal levels of ROS, only partially blunted the MnSOD response. In contrast, the addition of chemical inhibitors that decreased inducible and basal levels of ROS completely abrogated MnSOD induction. Together, our findings suggest that VEGF/ROS-dependent increases in MnSOD expression are mediated, at least in part, by a Rac1-dependent oxidase and that VEGF signals through a pathway that is sensitive to both basal and inducible levels of NADPH oxidase-derived ROS.
Our finding that VEGF signaling induces MnSOD expression has important biological implications. First, ROS have been shown to induce mitochondrial damage and dysfunction, leading to impaired function of Krebs’ cycle and activation of apoptotic pathways. Since MnSOD catalyzes the removal of O2-, the enzyme has the potential to enhance cell survival. Indeed, it is tempting to speculate that VEGF-mediated induction of MnSOD represents an important mechanism by which the growth factor exerts its anti-apoptotic effects. Second, VEGF-mediated increases in MnSOD activity would serve to enhance the local production of H2O2. Since H2O2 readily diffuses between cellular compartments, newly generated H2O2 would be free to interact with cytosolic signal transduction pathways. In other words, the mitochondria might function as signaling modules in VEGF-treated endothelial cells. Third, increased levels of MnSOD may protect the mitochondria from the toxic effects of peroxynitrite (ONOO-). VEGF has been shown to induce NO activity in endothelial cells. Newly generated NO may react with O2- to produce ONOO-, leading to endothelial cell dysfunction and mitochondrial damage. SOD competes with NO for scavenging of O2-, thereby inhibiting the production of ONOO- and increasing the bioavailability of NO. Based on the results of the present study, we propose that VEGF-mediated induction of MnSOD may offset the ROS-generating capacity of elevated NO. The coregulation of SOD expression and NO activity by VEGF may serve to reduce the pro-oxidant potential of NO and thus to divert NO to biologically important functions. It would follow that VEGF-mediated changes in MnSOD may serve to attenuate further ROS production and protect against cytotoxicity (Fig. 3
).
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In summary, we have demonstrated that VEGF induces MnSOD expression and that VEGF signaling requires a threshold level of NADPH oxidase-generated ROS. The study raises interesting questions that require further investigation: 1) How does the redox state influence other functions of VEGF? 2) What is the net effect of VEGF on the redox potential of the various subcellular compartments? 3) To what extent does VEGF alter the expression of other antioxidants in endothelial cells such as Cu, Zn-SOD, catalase, or glutathione? 4) Does the redox state and its role in VEGF signaling vary between different types of endothelial cells? The answers to these questions should provide important insights into the role of VEGF and ROS in endothelial cell biology.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0338fje; to cite this article, use FASEB J. (September 17, 2001) 10.1096/fj.01-0338fje 
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