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Dephosphorylation of endothelial nitric oxide synthase contributes to the anti-angiogenic effects of endostatin¹

CARMEN URBICH, AGNES REISSNER, EMMANOUIL CHAVAKIS, ELISABETH DERNBACH, JUDITH HAENDELER, INGRID FLEMING^*, ANDREAS M. ZEIHER, MARIETTA KASZKIN and STEFANIE DIMMELER²

Division of Molecular Cardiology, Department of Internal Medicine IV,
^* Institute for Cardiovascular Physiology and
Pharmazentrum Frankfurt, University of Frankfurt, 60590 Frankfurt, Germany

²Correspondence: Division of Molecular Cardiology, Dept. of Internal Medicine IV, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail: Dimmeler{at}em.uni-frankfurt.de

SPECIFIC AIMS

Regulation of blood vessel growth by promotion or inhibition of angiogenesis is a therapeutic option to treat coronary artery disease or cancer. Angiogenesis is counteracted by the anti-angiogenic molecule endostatin. Here we investigated the molecular intracellular mechanisms by which endostatin affects endothelial cell function. Because Akt-dependent phosphorylation of the endothelial NO synthase (eNOS) at Ser 1177 plays a key role in angiogenesis signaling, we investigated the effect of endostatin on eNOS phosphorylation and activation.

PRINCIPAL FINDINGS

1. Endostatin inhibits VEGF-induced endothelial cell migration and NO synthesis
Human recombinant endostatin dose-dependently blocked VEGF-induced endothelial cell migration. Although VEGF-induced endothelial migration was completely blocked at 100 ng/ml endostatin, endothelial cells were not rendered apoptotic. To investigate the effects of endostatin on the downstream signaling pathways induced by VEGF, we measured endothelial NO production and the formation of prostanoids, which have both been implicated in angiogenesis signaling. VEGF-stimulated NO synthesis was abolished by endostatin. In contrast, VEGF-induced formation of prostacyclin was not significantly affected by endostatin.

2. Endostatin acts downstream of Akt
Having demonstrated that endostatin blocks VEGF-induced synthesis of NO, we addressed the pathways that lead to eNOS activation. VEGF was shown to stimulate eNOS activity via the Akt-dependent phosphorylation of Ser 1177. We transfected endothelial cells with either Akt or a phospho-mimetic eNOS construct, which exhibits enhanced activity at basal levels of intracellular Ca²⁺, and determined the influence of endostatin. The Akt and eNOS constructs both increased cell migration and NO synthesis (Fig. 1 A, B). However, whereas the Akt-mediated stimulation of endothelial cell migration and eNOS activation was blocked by endostatin, cells transfected with the phospho-mimetic eNOS construct were unaffected by endostatin (Fig. 1) . Treatment with endostatin did not affect endothelial cell migration stimulated by the exogenous NO donor SNAP (Fig. 1C ). These results were confirmed in a second experimental model by measuring tube formation in a human angiogenesis assay. Endostatin inhibited VEGF-induced tube formation but did not affect that induced by SNAP. Moreover, the NO donor reversed the inhibitory effect of endostatin on VEGF-induced tube formation and EC migration. These data suggest that endostatin interferes with the activation of eNOS downstream of Akt.

View larger version (26K): [in this window] [in a new window]   Figure 1. Endostatin acts downstream of Akt. A, B) HUVEC were transfected with the phospho-mimetic constructs of Akt (Akt T308D/S473D) or eNOS (eNOS S1177D) and incubated with VEGF (10 ng/ml) in the presence or absence of endostatin (10 ng/ml). A) Cell migration was detected after 24 h using a ‘scratched wound assay’. Data are mean ± SE, n = 6, *P < 0.01 vs. vector+VEGF; #P < 0.05 vs. Akt. B) Cells were stained with the fluorescent indicator DAF-2 DA. Data are mean ± SE, n = 5, *P < 0.05 vs. vector+VEGF; #P < 0.05 vs. Akt. C) HUVEC were incubated with VEGF (10 ng/ml) or SNAP in the presence or absence of endostatin (10 ng/ml) for 24 h. Data are mean ± SE, n = 4, *P < 0.05 vs. VEGF; #P < 0.05 VEGF+endostatin. D) HUVEC were exposed to laminar shear stress (15 dynes/cm2) in the presence or absence of endostatin (10 ng/ml) for 24 h. Data are mean ± SE, n = 4.

3. Endostatin specifically inhibits phosphorylation of eNOS at Ser 1177
To test whether endostatin interferes with the phosphorylation of eNOS, VEGF-stimulated phosphorylation of Ser 1177 was determined by Western blot analysis using a phospho-specific antibody. As described previously, VEGF enhanced Ser 1177 phosphorylation. Coincubation with endostatin inhibited the VEGF-induced phosphorylation of Ser 1177 (Fig. 2 A, B). In contrast, VEGF-induced Akt phosphorylation was not affected by endostatin (Fig. 2) . To investigate the role of the serine/threonine protein phosphatase 2A (PP2A), which is known to dephosphorylate eNOS on Ser 1177, HUVEC were transfected with the catalytic PP2A subunit PP2Ac or an inactive, dominant negative PP2Ac construct (L199P). Endostatin inhibited VEGF-induced EC migration in vector-transfected cells and in cells transfected with wild-type PP2A. In contrast, the inhibitory effect of endostatin was reduced in cells transfected with the dominant negative PP2A construct (Fig. 2C ). Moreover, endostatin stimulated PP2A activity in the presence or absence of VEGF (Fig. 2D ).

View larger version (27K): [in this window] [in a new window]   Figure 2. Endostatin reduces VEGF-induced phosphorylation of eNOS. A) HUVEC were incubated in serum-free medium for 6 h and incubated with VEGF (10 ng/ml) in the presence or absence of endostatin (10 ng/ml) for 1 h, and phosphorylation of eNOS or Akt was determined by Western blot analysis. Total eNOS or Akt serves as loading control. Representative blots of 5 independent experiments are shown. B) Blots were scanned and quantified by densitometric analysis. Data are mean ± SE, n = 5, *P < 0.05 vs. VEGF. C) HUVEC were transfected with wild-type PP2A (PP2A wt) or PP2A mutant (PP2A L199P) and incubated with VEGF (10 ng/ml) in the presence or absence of endostatin (10 ng/ml) for 24 h. Data are mean ± SE, n = 4, *P < 0.05 vs. vector+VEGF; #P < 0.05 vs. PP2A wt+VEGF. D) PP2A activity was measured after incubation of HUVEC with endostatin (100 ng/ml) and/or VEGF (10 ng/ml) for 1 h. Data are mean ± SE, n = 4.

CONCLUSIONS

The results of the present study demonstrate that endostatin blocks VEGF-induced NO synthesis and prevents VEGF-induced EC migration and tube formation. However, endostatin did not affect EC migration induced by a NO donor or by overexpression of a nondephosphorylatable and constitutively active eNOS construct. Similarly, tube formation induced by NO donors was resistant to endostatin, suggesting that endostatin interferes with the signals leading to eNOS activation. Indeed, endostatin prevented VEGF-induced phosphorylation of eNOS on Ser 1177. VEGF-induced phosphorylation of Akt was not impaired by endostatin. In addition, endostatin blocked Akt-stimulated EC migration. These data implicate that endostatin engages downstream of Akt but upstream of NO formation via dephosphorylation of eNOS at Ser 1177.

Two recent studies suggest that PP2A dephosphorylates eNOS at Ser 1177. Therefore, we addressed a potential contribution of PP2A in endostatin-induced eNOS dephosphorylation. Since pharmacological inhibitors such as okadaic acid showed marked toxicity and cell detachment after incubation for several hours, we transfected the cells with a dominant-negative inactive construct of the catalytic subunit of PP2A (PP2Ac-L199P) that has been characterized. Overexpression of PP2Ac-L199P rendered the cells resistant to endostatin-induced inhibition of cell migration, suggesting that PP2A plays a central role in mediating the cellular effects of endostatin. However, it is unclear why Akt, which is known to be prone to PP2A-dependent dephosphorylation, was unaffected. This apparent discrepancy may be because the individual subunits of PP2A are encoded by a heterogeneous group of genes that give rise to a multitude of different PP2A holoenzyme complexes with diverse substrate specificities. Although the molecular identity of the PP2A holoenzyme is not yet clear, the data of the present study clearly indicate that endostatin induces the dephosphorylation of Ser 1177, thus functionally antagonizing the Akt-dependent activation of the eNOS. Endothelial NO synthesis is essential for EC migration, endothelial cell survival, and angiogenesis in vivo. Therefore, blockade of this central signaling pathway may essentially contribute to the anti-angiogenic effects of endostatin seen in various studies.

Taken together, endostatin appears to specifically interfere with eNOS phosphorylation at Ser 1177 whereas the upstream signaling pathway, including VEGF-induced Akt activation, is not affected. We also demonstrated that VEGF-induced prostacyclin synthesis is not reduced by endostatin treatment, implicating a specific interference of endostatin with the NO pathway. The present data further suggest that endostatin—by a yet undefined signaling pathway—activates the serine/threonine phosphatase PP2A, which in turn reduces eNOS phosphorylation and prevents VEGF-induced EC migration.

View larger version (37K): [in this window] [in a new window]   Figure 3. Schematic illustration of the findings.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/doi/10.1096/fj.01-0637fje; to cite this article, use FASEB J. (March 12, 2002)10.1096/fj.01-0637fje.

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