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VEGF differentially activates STAT3 in microvascular endothelial cells ¹

MANUELA BARTOLI^*,2, DAN PLATT, TAHIRA LEMTALSI, XIAOLIN GU^¶, STEVEN E. BROOKS^#, MARIO B. MARRERO and RUTH B. CALDWELL^¶

Vascular Biology Center,
^* Department of Pathology,
^¶ Department of Cellular Biology and Anatomy,
Department of Pharmacology and Toxicology, and the
^# Department of Ophthalmology, Medical College of Georgia, Augusta, Georgia, USA

²Correspondence: Vascular Biology Center, CB 3209, Medical College of Georgia, 1459 Laney Walker Blvd., Augusta, GA 30912, USA. E-mail: mbartoli{at}mail.mcg.edu

SPECIFIC AIM

Vascular endothelial growth factor (VEGF) expression is up-regulated during normal and pathological angiogenesis. Recent studies have indicated that the transcription factor signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in normal and pathological angiogenesis by up-regulating VEGF expression.

In this study we determined the role of STAT3 in VEGF autocrine expression in endothelial cells. We have previously shown that in aortic macrovascular endothelial cells STAT3 is tyrosine phosphorylated in response to VEGF, but is not translocated to the nucleus and thus lacks transcriptional activity. Since angiogenesis primarily targets microcapillary structures and little is known about how angiogenic factors can selectively exert their proangiogenic effects on microvascular endothelium, we wanted to determine whether STAT3 could be differentially activated by VEGF in microvascular endothelial cells and if this event could result in different patterns of VEGF expression.

PRINCIPAL FINDINGS

1. VEGF induces STAT3 nuclear translocation in microvascular endothelial cells and this effect is mediated by VEGFR2
Bovine retinal microvascular endothelial cells (BREC) were cultured to 85% confluency, then switched to serum-free medium. BREC were challenged with 10 ng/mL VEGF for different times and STAT3 tyrosine phosphorylation was assessed by Western blot analysis. VEGF-stimulated STAT3 tyrosine phosphorylation in BREC was different from what we had previously shown in bovine aortic endothelial cells (BAEC). The pattern of phosphorylation started earlier and was more prolonged.

We have previously shown that in BAEC, VEGF induced STAT3 tyrosine phosphorylation but not nuclear translocation; therefore, we determined STAT3 mobilization in the nuclear compartment of VEGF-stimulated BREC. Western blot and confocal immunocytochemistry analyses revealed STAT3 immunoreactivity in the nuclei of VEGF-stimulated BREC (Fig. 1 b–d) and not in control unstimulated cells (Fig. 1a ). Maximal STAT3 nuclear translocation was detected 15 min after VEGF treatment (Fig. 1c ). These data indicate that unlike aortic macrovascular endothelial cells, VEGF stimulation of BREC results in full STAT3 activation and transcriptional ability.

View larger version (139K): [in this window] [in a new window]   Figure 1. STAT3 activation in BREC. Confocal immunocytochemistry of STAT3 (green) nuclear mobilization in BREC stimulated with 10 ng/mL VEGF for different times in comparison with control unstimulated BREC. The nuclei are counterstained with propidium iodide (red). Confocal sections along the axis yz and xz have been determined to identify STAT3 immunoreactivity within the nuclear compartment. Overlapping of the green (STAT3) and the red (nucleus) colors (=yellow) indicates STAT3's presence in the nuclei of BREC. a) BREC not stimulated (control); b) BREC stimulated for 5 min; c) BREC stimulated for 15 min; d) BREC stimulated for 30 min with 10 ng/mL VEGF.

STAT protein nuclear translocation is an active process that requires interaction with shuttle proteins bearing a nuclear localization signal (NLS). VEGF receptor 2 (VEGFR2) possesses a NLS and, upon ligand interaction, has been shown to migrate into the nucleus of the activated cells within specialized intracellular structures called caveolae. STAT3 mobilization to caveolar vesicles has been shown in different cell types. Based on this evidence we performed coimmunoprecipitation analysis to identify STAT3/VEGFR2 complexes in BREC. We found that VEGFR2 and STAT3 coprecipitated, indicating that these proteins form a complex. In BAEC, where VEGF did not induce STAT3 nuclear translocation, we failed to detect VEGFR2/STAT3 complex formation.

2. VEGF induces its own expression in a STAT3-dependent fashion in microvascular endothelial cells but not in macrovascular endothelial cells
To determine whether differential activation of STAT3 in BAEC and BREC resulted in different patterns of VEGF autocrine gene expression, we performed real-time quantitative reverse transcription polymerase chain reaction analysis and measured VEGF-specific RNA formation in treated cells. BREC showed a significant increase in VEGF transcription that peaked 1 h after VEGF treatment, whereas BAEC showed no response. We assessed VEGF autocrine gene expression in human dermal microvascular endothelial cells (HDMEC) to determine whether the observed effect was limited to BREC or was also represented in other microvascular endothelial cells. We found that HDMEC activated VEGF autocrine production and that VEGF-induced STAT3 activation followed a pattern similar to that observed in BREC.

Finally, to determine whether VEGF expression in microvascular endothelial cells is a STAT3-dependent transcriptional event, we tested the effect of STAT3 lack of function, obtained with antisense transfection, on VEGF autocrine RNA production in BREC. Antisense-STAT3 transfection significantly inhibited VEGF expression in BREC in response to 1 h VEGF stimulation whereas in BREC treated with the sense construct, the response remained unchanged (Fig. 2 ).

View larger version (9K): [in this window] [in a new window]   Figure 2. STAT3 lack of function effects on VEGF expression in BREC. The effects of VEGF treatment (25 ng/mL) on VEGF expression were assessed by quantitative RT-PCR in controls (C+V) and sense- (S+V) or antisense-transfected (As+V) BREC. The results are expressed as a percentage increase vs. control unstimulated BREC (C) and represent mean values ± SE. *P < 0.001, **P< 0.005 vs. control (C), n = 3. #P < 0.05 vs. control not transfected cells stimulated with VEGF (C+V), n= 3.

CONCLUSIONS AND SIGNIFICANCE

STAT3 has been shown to be involved in angiogenesis by up-regulating VEGF expression. STAT3 binding sites have been identified in the VEGF promoter and mutations of these regions can block specific RNA transcription. Its constitutive activation has been correlated with an increased rate of VEGF expression and angiogenesis in myocardial tissue and in tumoral cells and human cancers.

Our results show that in VEGF-stimulated BREC, STAT3 is rapidly tyrosine-phosphorylated and translocated to the nucleus. Moreover, STAT3 is associated with VEGFR2 in BREC but not in BAEC. Because STAT3 is nuclear translocated only in BREC, where it is associated with VEGFR2, our data strongly suggest that VEGFR2 mediates STAT3 nuclear translocation. This hypothesis is also supported by the observation that in cytokine signaling STAT3 localizes within the caveolar compartment and this subcellular localization appears to determine its transcriptional activity. Furthermore, VEGF’s ability to stimulate its own production is significantly stimulated in BREC and in human dermal microvascular endothelial cells (HDMEC) but not in BAEC. Antisense-mediated reduction of STAT3 bioavailability results in significant inhibition of VEGF autocrine gene expression.

Our studies are indicating for the first time that VEGF can differentially induce the pattern of STAT3 activation in micro- vs. macrovascular endothelial cells and that this effect is linked to VEGFR2/STAT3 complex formation and correlates with VEGF’s ability to induce its own expression in an autocrine fashion in microvascular endothelial cells. The relevance of our findings, summarized in Fig. 3 , is the identification of STAT3 as a specific mediator of microvascular endothelial cell’s biological responses and as a major player of VEGF autocrine production. The first aspect applies to those pathological situations in which systemic anti-angiogenic therapies may negatively affect macrovascular structures. This is the case with diabetic vasculopathy, which affects macro- and microvascular districts in opposing ways. The last aspect finds its application in tumor biology, where VEGF autocrine activity affects tumor angiogenesis and tumor progression. Finally, in light of what is discussed above, our study provides further support to the importance of STAT3 as a pharmacological target for the therapeutic management of pathological angiogenesis.

View larger version (13K): [in this window] [in a new window]   Figure 3. Schematic diagram of the principal findings. VEGF-induced STAT3 activation is different in BAEC and BREC (see full paper on-line). In VEGF-stimulated BREC, STAT3 is nuclear translocated and possess transcriptional activity, whereas in BAEC STAT3 nuclear translocation does not occur. STAT3 is associated with VEGFR2 only in BREC and not in BAEC (see Fig. 3, full-text paper). VEGF autocrine production in these cells is different and strongly correlates with STAT3's ability to be associated with VEGFR2 and to be nuclear translocated. Indeed, only BREC respond to VEGF-stimulated autocrine production and this effect is STAT3 dependent.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1084fje; doi: 10.1096/fj.02-1084fje

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