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. 2017 Feb;20(1):85-96.
doi: 10.1007/s10456-016-9530-9. Epub 2016 Nov 2.

Tissue factor is an angiogenic-specific receptor for factor VII-targeted immunotherapy and photodynamic therapy

Zhiwei Hu  1   2 , Jijun Cheng  3   4 , Jie Xu  3   5 , Wolfram Ruf  6 , Charles J Lockwood  7
Affiliations

Tissue factor is an angiogenic-specific receptor for factor VII-targeted immunotherapy and photodynamic therapy

Zhiwei Hu et al. Angiogenesis. 2017 Feb.

Abstract

Identification of target molecules specific for angiogenic vascular endothelial cells (VEC), the inner layer of pathological neovasculature, is critical for discovery and development of neovascular-targeting therapy for angiogenesis-dependent human diseases, notably cancer, macular degeneration and endometriosis, in which vascular endothelial growth factor (VEGF) plays a central pathophysiological role. Using VEGF-stimulated vascular endothelial cells (VECs) isolated from microvessels, venous and arterial blood vessels as in vitro angiogenic models and unstimulated VECs as a quiescent VEC model, we examined the expression of tissue factor (TF), a membrane-bound receptor on the angiogenic VEC models compared with quiescent VEC controls. We found that TF is specifically expressed on angiogenic VECs in a time-dependent manner in microvessels, venous and arterial vessels. TF-targeted therapeutic agents, including factor VII (fVII)-IgG1 Fc and fVII-conjugated photosensitizer, can selectively bind angiogenic VECs, but not the quiescent VECs. Moreover, fVII-targeted photodynamic therapy can selectively and completely eradicate angiogenic VECs. We conclude that TF is an angiogenic-specific receptor and the target molecule for fVII-targeted therapeutics. This study supports clinical trials of TF-targeted therapeutics for the treatment of angiogenesis-dependent diseases such as cancer, macular degeneration and endometriosis.

Keywords: Angiogenesis; Factor VII-targeted therapy; Tissue factor; VEGF; Vascular endothelial cells.

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Conflict of interest statement

Z.H. is co-inventor of US patents on neovascular-targeted immunoconjugates (ICON) and is co-inventor of two US patent applications on “Factor VII conjugates for selectively treating neovascularization disorders.” W.R. is currently serving as a consultant for Iconic Therapeutics, a startup biotech based on ICON patents. Other authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
TF is an angiogenic-specific receptor on endothelial cells. a–c. Representative imaging of Western blots and expression fold changes (normalized to GAPDH) for TF in HMVEC (a), HUVEC (b) and HAEC (c) before (0 h) and after VEGF stimulation (2–24 h). d Representative confocal imaging of TF (green) and endothelial marker CD31 (red) expression on HMVEC before (0 h) and after VEGF stimulation (2–24 h). Cell nuclei were counterstained by DAPI (blue). Scale bars: 20 μm. p values were calculated by 2-way ANOVA with multiple comparisons test. Data in a–c are presented as Mean ± SD and representative of two independent experiments. (Color figure online)
Fig. 2
Fig. 2
Selective binding of fVII agents to in vitro angiogenic VEC models. a–f Representative confocal imaging of different expression patterns of TF on in vitro angiogenic and quiescent VEC models. HMVEC (a, b), HUVEC (c, d) and HAEC (e, f). g–l Selective binding of fVII agents (mfVII/hIgG1 Fc and mfVII peptides) and anti-TF antibody to angiogenic VECs (g, i, k), but not to quiescent VECs (h, j, l). HMVEC (g, h), HUVEC (i, j) and HAEC (k, l). Scale bars in a–f: 20 μm. p values were calculated by 2-way ANOVA with multiple comparisons test. Data in g–l are presented as Mean ± SD and representative of two independent experiments
Fig. 3
Fig. 3
Selective binding of fVII agents to angiogenic VECs is mediated by TF. a–b. Choice of anti-TF antibodies for inhibiting the binding of fVII agent (mfVII/hIgG1 Fc) to TF. MDA-MB-231 is a human breast cancer line with high level of TF expression and is used here as a TF-expressing cell model. Two antihuman TF antibodies, goat anti-HTF and mouse anti-HTF (Clone HTF1), display similar binding to the MDA-MB-231 cells (a) but goat anti-TF antibody shows stronger inhibitory effect than monoclonal HTF1 in blocking mfVII/hIgG1 Fc binding to cancer cell TF (b). c–e Selective binding of mfVII/hIgG1 Fc to angiogenic HMVEC (c), HUVEC (d) and HAEC (e) can be completely blocked by goat anti-HTF, but not by control antibody. f Representative Western blots using mfVII/hIgG1 Fc and hfVII/hIgG1 Fc to immune-precipitate their cognate receptor TF (f). The negative control was the untransfected CHO-K1 cells. Human IgG was an isotype control. p values were calculated by 2-way ANOVA with multiple comparisons test. Data in a–e are presented as Mean ± SD and representative of two independent experiments
Fig. 4
Fig. 4
fVII-tPDT is selective and effective in eradicating angiogenic VEC. a–c fVII-SnCe6 conjugates retains the binding activity and selectivity to angiogenic VECs (HMVEC). Goat anti-TF was a positive control (a), SnCe6 was separately conjugated with mfVII/Sp (b) and mfVII/NLS (c) d Representative imaging of crystal violet-stained VEGF-stimulated and unstimulated HMVECs right after being treated with fVII-tPDT or ntPDT (2 μM and 635 nm laser light at 36 J/cm2). Control HMVECs include an untreated control and a maximal killing control (completely lysed by 1% Triton X-100). Original magnification: 400× phase contrast. e Complete eradication (no colonies formed) of angiogenic VEC (HMVEC) by fVII-tPDT using fVII/NLS-SnCe6 or fVII/Sp-SnCe6 (p < 0.001 vs. untreated control), whereas ntPDT has no therapeutic effect in killing angiogenic VEC under the same condition as for fVII-tPDT (2 μM SnCe6 and 635 nm laser light at 36 J/cm2). f The fVII-tPDT is effective and selective in killing angiogenic VEC (angiogenic HMVEC) with an EC50 of 0.031 μM SnCe6 in fVII/NLS-SnCe6, whereas it has no side effects to quiescent VEC (HMVEC). The fVII-tPDT conditions were as follows: 635 nm laser light at 36 J/cm2 and the SnCe6 concentrations in the fVII/NLS-SnCe6 conjugate (x axis) were 0.0 (buffer only), 0.5, 1 and 2 μM, respectively. Note that the VEC cells without fVII/NLS-SnCe6 (0.0 μM) also served as the light only control as they were also irradiated with 635 nm laser light (36 J/cm2). g The underlining mechanism of fVII/NLS-tPDT involves rapid induction of apoptosis and necrosis. SnCe6 concentration was reduced to 1 μM (36 J/cm2) so that not all of the treated cells undergo necrosis immediately after fVII-tPDT (See the stained HMVEC in d). Annexin V-FITC (green) stains for apoptotic cell membrane (green arrow) and propidium iodide (PI, red) stains for the nuclei of dead cells (red arrows). Original magnification: 400×. p values were calculated by 2-way ANOVA with multiple comparisons test. Data are presented as Mean ± SD and representative of two or three independent experiments. (Color figure online)
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