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. 2008 Dec;7(12):1994-2003.
doi: 10.4161/cbt.7.12.6967. Epub 2008 Dec 11.

Activated STAT3 is a mediator and biomarker of VEGF endothelial activation

Shao-Hua Chen  1 , Danielle A Murphy, Wiem Lassoued, Gavin Thurston, Michael D Feldman, William M F Lee
Affiliations

Activated STAT3 is a mediator and biomarker of VEGF endothelial activation

Shao-Hua Chen et al. Cancer Biol Ther. 2008 Dec.

Abstract

STAT3 plays important roles in cell proliferation and survival signaling and is often constitutively activated in transformed cells. In this study, we examined STAT3 activation in endothelial cells (EC) during angiogenic activation and therapeutic angiogenesis inhibition. VEGF stimulation of cultured EC induced STAT3 phosphorylation by a VEGFR2- and Src-dependent mechanism. FGF2 but not PlGF also induced EC STAT3 activation in vitro. Activated STAT3 mediated VEGF induction of EC Bcl-2 and contributed to VEGF protection of EC from apoptosis. In vivo, p-STAT3 was absent by immunohistological staining in the vascular EC of most normal mouse organs but was present in the vessels of mouse and human tumors. Tumor vascular p-STAT3 increased as tumors were induced to overexpress VEGF, indicating that VEGF is an activator of EC p-STAT3 in vivo. Tumor vascular p-STAT3 decreased during angiogenesis inhibition by antagonists of VEGF-VEGFR signaling, VEGF Trap and SU5416, indicating that VEGF contributed to the EC STAT3 activation seen in the tumors prior to treatment and that p-STAT3 may be used to monitor therapy. These studies show that p-STAT3 is a mediator and biomarker of endothelial activation that reports VEGF-VEGFR2 activity and may be useful for studying the pharmacodynamics of targeted angiogenesis inhibitors.

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Figures

Figure 1
Figure 1
STAT3 is activated in endothelial cells of tumors but not in endothelial cells of most murine normal organs. Thin sections of paraffin-embedded K1735 (A–D) and RENCA (E and F) murine tumors, normal mouse organs, kidney (G and H), liver (I and J) and lung (M and N), and human colorectal cancer (O and P) were stained with anti-p-STAT3 antibody using DAB chromogen (brown) and with anti-CD34 antibody using SG blue chromogen (blue in A, B, G, H–J, O and P) or by immunofluorescence (green in C–F, M and N). Regions of 400X fields of view (A, E, G, I, M, O) outlined by red boxes are shown at higher magnification in the ensuing panels (B, F, H, J, N and P) to reveal detail. Examples of endothelial cell (EC) nuclei staining for p-STAT3 are pointed out by red arrows; examples of EC nuclei not staining for p-STAT3 in tumors are pointed out by black arrows in (D) and are abundant in normal organs (H and J) except the lung. Note that a large fraction of tumor cell nuclei stain positively for p-STAT3 (B–D and F). Normal mouse liver (K and higher magnification, L) was stained with anti-STAT3 antibody using DAB chromogen and with anti-CD34 antibody using SG blue chromogen.
Figure 2
Figure 2
VEGF and FGF2 activate STAT3 in endothelial cells. HUVEC and MS1 endothelial cells were cultured in medium containing 0.5% serum for 24 hours and then cultured in medium containing 10 ng/ml VEGF (A and B) or 25 ng/ml FGF2 (C) for different durations, or 2–200 ng/ml PlGF (D) in HUVEC cell for 10 minutes. Western blots of cell lysates were probed for p-STAT3, stripped and reprobed for STAT3 (A, C and D). HUVEC cells were fixed and stained using anti-p-STAT3 antibody. DAPI counterstaining of nuclei is shown (B). All experiments were performed twice with similar results.
Figure 3
Figure 3
VEGF induces endothelial cell VEGFR2, Src and STAT3 activation and intermolecular association. HUVEC and MS1 endothelial cells were cultured in medium containing 0.5% serum for 24 hours and then cultured in medium containing 10 ng/ml VEGF for different durations. Western blots of the lysates were probed with antibody specific for phospho-VEGFR2, stripped and reprobed for VEGFR2 (A), and probed with antibody specific for phospho-Src, stripped and reprobed for Src (B). HUVEC cells were pretreated with anti-human VEGFR2 inhibitory antibody (1 μg/ml) or IgG (1 μg/ml) for one hour and were stimulated with 10 ng/ml VEGF for 10 min. Western blots of the lysates were probed with antibody specific for phospho-VEGFR2, phospho-Src, phospho-STAT3, stripped and reprobed for VEGFR2, Src and STAT3 (C). HUVEC lysates were immunoprecipitated with anti-STAT3 antibody and Western blots of the precipitate were probed with antibody to VEGFR2, stripped and reprobed with antibody to STAT3 (D). HUVEC lysates were immunoprecipitated with anti-Src antibody and Western blots of the precipitate were probed sequentially with antibodies to VEGFR2, STAT3, STAT5 and Src (E). All experiments were performed twice with similar results.
Figure 4
Figure 4
VEGFR2 or Src inhibition abrogates STAT3 activation by VEGF. HUVEC were cultured in medium containing 0.5% serum for 24 hours and exposed to different concentrations of SU5416, PP1 or PP2 inhibitor for one hour. VEGF (10 ng/mg) was added, and cell lysates were prepared 10 minutes later. Western blots of lysates from cells treated with SU5416, a VEGFR2 inhibitor, were probed with antibodies to p-VEGFR2, p-Src and p-STAT3; the last blot was stripped and reprobed with antibody to STAT3 (A). Blots of lysates from cells treated with c-Src inhibitors PP1 (B) or PP2 (C) were probed with antibodies to p-Src and p-STAT3; these blots were stripped and reprobed with antibodies to Src and STAT3, respectively. All experiments were performed twice with similar results.
Figure 5
Figure 5
VEGF induces Bcl-2 and protects endothelial cells from death by a STAT3-dependent mechanism. HUVEC were placed in serum-free medium overnight, stimulated with medium containing 0.5% serum + VEGF (100 ng/ml) for 48 or 72 hours. Blots of the lysates were probed with antibody to Bcl-2, stripped and reprobed with antibody to GAPDH (A). HUVEC were transfected with STAT3 siRNA (siRNA; 100 nM) for 48, 72 or 96 hours, transfected with negative control siRNA (NC; 100 nM) for 96 hours, mock transfected (Mock) or untransfected (UT). Blots were probed sequentially with antibodies to STAT3, VEGFR2 and GAPDH (B). HUVEC were transfected with STAT3 siRNA (siRNA; 100 nM) or negative control siRNA (NC; 100 nM) for 72 hours. The transfected cells were cultured in serum-free medium overnight and then placed in medium containing 0.5% serum + VEGF (100 ng/ml). Cell death was assayed after 24 hours and cell lysates were prepared after 48 hours. Blots were probed with antibodies to Bcl-2 and STAT3, stripped and reprobed with antibody to GAPDH (C). Cell death was assayed by TUNEL staining (D). This experiment was performed a total of three times with similar results. HUVEC were treated with 20 μM p-STAT3 inhibitory peptide linked to a membrane translocating sequence (MTS-SIP) or with unlinked SIP at 20 μM in serum-free medium for 16 hours. They were then stimulated with VEGF (100 ng/ml) for 10 minutes. Cells were fixed and stained using anti-p-STAT3 antibody. DAPI counterstaining of nuclei is shown (E). HUVEC were treated with 0.2, 2 or 20 μM p-STAT3 MTS-SIP or with unlinked SIP at 20 μM in serum-free medium for 16 hours. They were then cultured in medium containing 0.5% serum + VEGF (100 ng/ml) and the peptides. Cell death was assayed after 24 hours and cell lysates were prepared after 48 hours. Blots were probed with antibodies to Bcl-2, stripped and reprobed with antibody to GAPDH (F). Cell death was assayed by TUNEL staining (G). All Western blot experiments were performed twice and the other experiments were performed a total of three times with similar results.
Figure 6
Figure 6
Tumor endothelial cell p-STAT3 is induced by VEGF and decreased by inhibitors of VEGF and VEGFR2. Tumors were raised in syngeneic mice by subcutaneous inoculation of cultured tumor cells. When tumors reached 5 mm in diameter, hosts were treated with the agents described and the tumors harvested at various times after start of treatment. Vascular p-STAT3 expression was examined immunohistochemically by dual staining with anti-p-STAT3 antibody (DAB chromogen, brown chromogen) and anti-CD34 antibody (SG blue, blue chromogen). C3H/HeN mice bearing K1735.VI4 tumors (engineered to express murine VEGF165 under doxycycline, Dox, induction) were given Dox (2 mg/ml) in their drinking water or not for two days (number of tumors analyzed for each group indicated within histogram) (A). Mice bearing K1735 tumors were treated with control Fc or VEGF Trap (25 mg/kg given subcutaneously twice a week) and their tumors excised 3, 7 or 14 days after start of treatment (B). BALB/c mice bearing RENCA tumors were treated with control Fc or VEGF Trap and their tumors analyzed as described (C). Mice bearing K1735 tumors were treated with vehicle or SU5416 (20 mg/kg daily) (D) and their tumors analyzed after seven days of treatment. Differences were analyzed by Student’s test and p value was shown.
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