Faricimab Reverts VEGF-A165-Induced Impairment of the Barrier Formed by Retinal Endothelial Cells
Abstract
1. Introduction
2. Results
2.1. General Information
2.2. Faricimab Strongly Suppressed Higher Secretion of Ang-2 by VEGF-A165-Treated iBREC
2.3. Faricimab Efficiently Reverted VEGF-A165-Induced Barrier Impairment
2.4. Faricimab Reverted VEGF-A165-Induced Changes in the Expression of Claudin-1 and PLVAP
3. Discussion
4. Materials and Methods
4.1. Antibodies and Reagents
4.2. Cultivation of iBREC
4.3. Cell Index Measurements
4.4. Measurement of VGEF-A165, Ang-2 and IL-6 by ELISA
4.5. Western Blot Analyses of Protein Extracts
4.6. Statistical Analyses
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Note
Abbreviations
AJ | adherens junction |
Ang-2 | angiopoietin-2 |
CI | cell index |
EC | endothelial cells |
ECGM | endothelial cell growth medium |
FBS | fetal bovine serum |
hEGF | human epidermal growth factor |
HRP | horseradish peroxidase |
HuREC | human retinal endothelial cells |
(i)BREC | (immortalized) bovine retinal endothelial cells |
PLVAP | plasma lemma vesicle associated protein |
REC | retinal endothelial cells |
TJ | tight junction |
VEcadherin | vascular endothelial cadherin |
VEGF-A | vascular endothelial growth factor-A |
VEGFR | vascular endothelial growth factor receptor |
WB | Western blot analyses |
References
- Antonetti, D.A.; Barber, A.J.; Hollinger, L.A.; Wolpert, E.B.; Gardner, T.W. Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occludens 1. J. Biol. Chem. 1999, 274, 23463–23467. [Google Scholar] [CrossRef] [PubMed]
- Deissler, H.; Deissler, H.; Lang, G.E. Inhibition of VEGF is sufficient to completely restore barrier malfunction induced by growth factors in microvascular retinal endothelial cells. Br. J. Ophthalmol. 2011, 95, 1151–1156. [Google Scholar] [CrossRef] [PubMed]
- Qaum, T.; Xu, Q.; Joussen, A.M.; Clemens, M.W.; Qin, W.; Miyamoto, K.; Hassessian, H.; Wiegand, S.J.; Rudge, J.; Yancopoulos, G.D.; et al. VEGF-initiated blood-retinal barrier breakdown in early diabetes. Investig. Ophthalmol. Vis. Sci. 2001, 42, 2408–2413. [Google Scholar]
- Eichmann, A.; Simons, M. VEGF signaling inside vascular endothelial cells and beyond. Curr. Opin. Cell Biol. 2012, 24, 188–193. [Google Scholar] [CrossRef]
- Deissler, H.L.; Stutzer, J.-N.; Lang, G.K.; Grisanti, S.; Lang, G.E.; Ranjbar, M. VEGF receptor 2 inhibitor nintedanib completely reverts VEGF-A165-induced disturbances of barriers formed by retinal endothelial cells or long-term cultivated ARPE-19 cells. Exp. Eye Res. 2020, 194, 108004. [Google Scholar] [CrossRef]
- Suarez, S.; McCollum, G.W.; Bretz, C.A.; Yang, R.; Capozzi, M.E.; Penn, J.S. Modulation of VEGF-induced retinal vascular permeability by peroxisome proliferator-activated receptor-β/δ. Investig. Ophthalmol. Vis. Sci. 2014, 55, 8232–8240. [Google Scholar] [CrossRef]
- Wisniewska-Kruk, J.; Hoeben, K.A.; Vogels, I.M.; Gaillard, P.J.; Van Noorden, C.J.; Schlingemann, R.O.; Klaassen, I. A novel co-culture model of the blood-retinal barrier based on primary retinal endothelial cells, pericytes and astrocytes. Exp. Eye Res. 2012, 96, 181–190. [Google Scholar] [CrossRef]
- Sun, M.; Fu, H.; Cheng, H.; Cao, Q.; Zhao, Y.; Mou, X.; Zhang, X.; Liu, X.; Ke, Y. A dynamic real-time method for monitoring epithelial barrier function in vitro. Anal. Biochem. 2012, 425, 96–103. [Google Scholar] [CrossRef]
- Bischoff, I.; Hornburger, M.C.; Mayer, B.A.; Beyerle, A.; Wegener, J.; Fürst, R. Pitfalls in assessing microvascular endothelial barrier function: Impedance-based devices versus the classic macromolecular tracer assay. Sci. Rep. 2016, 6, 23671. [Google Scholar] [CrossRef]
- Deissler, H.L.; Lang, G.K.; Lang, G.E. Inhibition of single routes of intracellular signaling is not sufficient to neutralize the biphasic disturbance of a retinal endothelial cell barrier induced by VEGF-A165. Cell. Physiol. Biochem. 2017, 42, 1493–1513. [Google Scholar] [CrossRef]
- Deissler, H.; Deissler, H.; Lang, G.K.; Lang, G.E. Generation and characterization of iBREC: Novel hTERT-immortalized bovine retinal endothelial cells. Int. J. Mol. Med. 2005, 15, 65–70. [Google Scholar] [CrossRef]
- Deissler, H.L.; Rehak, M.; Busch, C.; Wolf, A. Blocking of VEGF-A is not sufficient to completely revert its long-term effects on the barrier formed by retinal endothelial cells. Exp. Eye Res. 2022, 216, 108945. [Google Scholar] [CrossRef] [PubMed]
- Deissler, H.L.; Rehak, M.; Wolf, A. Impairment of the retinal endothelial cell barrier induced by long-term treatment with VEGF-A165 no longer depends on the growth factor’s presence. Biomolecules 2022, 12, 734. [Google Scholar] [CrossRef] [PubMed]
- McCann, M.; Li, Y.; Baccouche, B.; Kazlauskas, A. VEGF induces expression of genes that either promote or limit relaxation of the retinal endothelial barrier. Int. J. Mol. Sci. 2023, 24, 6402. [Google Scholar] [CrossRef]
- Ferrara, N.; Damico, L.; Shams, N.; Lowman, H.; Kim, R. Development of ranibizumab, an anti-vascular endothelial growth factor antigen binding fragment, as therapy for neovascular age-related macular degeneration. Retina 2006, 26, 859–870. [Google Scholar] [CrossRef]
- Gaudreault, J.; Gunde, T.; Floyd, H.S.; Ellis, J.; Tietz, J.; Binggeli, D.; Keller, B.; Schmidt, A.; Escher, A. Preclinical pharmacology and safety of ESBA1008, a single-chain antibody fragment, investigated as potential treatment for age related macular degeneration. Investig. Ophthalmol. Vis. Sci. 2012, 53, 3025. [Google Scholar]
- Deissler, H.L.; Rehak, M.; Lytvynchuk, L. VEGF-A165a and angiopoietin-2 differently affect the barrier formed by retinal endothelial cells. Exp. Eye Res. 2024, 247, 110062. [Google Scholar] [CrossRef]
- Rangasamy, S.; Srinivasan, R.; Maestas, J.; McGuire, P.G.; Das, A. A potential role for angiopoietin 2 in the regulation of the blood–retinal barrier in diabetic retinopathy. Investig. Ophthalmol. Vis. Sci. 2011, 52, 3784–3791. [Google Scholar] [CrossRef]
- Eyre, J.J.; Williams, R.L.; Levis, H.J. A human retinal microvascular endothelial-pericyte co-culture model to study diabetic retinopathy in vitro. Exp. Eye Res. 2020, 201, 108293. [Google Scholar] [CrossRef]
- Regula, J.T.; Lundh von Leithner, P.; Foxton, R.; Barathi, V.A.; Cheung, C.M.; Bo Tun, S.B.; Wey, Y.S.; Iwata, D.; Dostalek, M.; Moelleken, J.; et al. Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases. EMBO Mol. Med. 2016, 8, 1265–1288. [Google Scholar] [CrossRef]
- Holash, J.; Davis, S.; Papadopoulos, N.; Croll, S.D.; Ho, L.; Russell, M.; Boland, P.; Leidich, R.; Hylton, D.; Burova, E.; et al. VEGF-Trap: A VEGF blocker with potent antitumor effects. Proc. Natl. Acad. Sci. USA 2002, 99, 11393–11398. [Google Scholar] [CrossRef] [PubMed]
- Lange, C.; Tetzner, R.; Strunz, T.; Rittenhouse, K.D. Aflibercept suppression of angiopoietin-2 in a rabbit retinal vascular hyperpermeability model. Transl. Vis. Sci. Technol. 2023, 12, 5. [Google Scholar] [CrossRef] [PubMed]
- Bosma, E.K.; van Noorden, C.J.F.; Schlingemann, R.O.; Klaassen, I. The role of plasmalemma vesicle-associated protein in pathological breakdown of blood–brain and blood–retinal barriers: Potential novel therapeutic target for cerebral edema and diabetic macular edema. Fluids Barriers CNS 2018, 15, 24. [Google Scholar] [CrossRef] [PubMed]
- Bosma, E.K.; Darwesh, S.; Habani, Y.I.; Cammeraat, M.; Serrano Martinez, P.; van Breest Smallenburg, M.E.; Zheng, J.Y.; Vogels, I.M.C.; van Noorden, C.J.F.; Schlingemann, R.O.; et al. Differential roles of eNOS in late effects of VEGF-A on hyperpermeability in different types of endothelial cells. Sci. Rep. 2023, 13, 21436. [Google Scholar] [CrossRef]
- Wisniewska-Kruk, J.; van der Wijk, A.E.; van Veen, H.A.; Gorgels, T.G.; Vogels, I.M.; Versteeg, D.; Van Noorden, C.J.; Schlingemann, R.O.; Klaassen, I. Plasmalemma vesicle-associated protein has a key role in blood-retinal barrier loss. Am. J. Pathol. 2016, 186, 1044–1054. [Google Scholar] [CrossRef]
- Kluger, M.S.; Clark, P.R.; Tellides, G.; Gerke, V.; Pober, J.S. Claudin-5 controls intercellular barriers of human dermal microvascular but not human umbilical vein endothelial cells. Arterioscler. Thromb. Vasc. Biol. 2014, 33, 489–500. [Google Scholar] [CrossRef]
- Busch, C.; Rehak, M.; Hollborn, M.; Wiedemann, P.; Lang, G.K.; Lang, G.E.; Wolf, A.; Deissler, H.L. Type of culture medium determines properties of cultivated retinal endothelial cells: Induction of substantial phenotypic conversion by standard DMEM. Heliyon 2021, 7, e06037. [Google Scholar] [CrossRef]
- Wong, T.Y.; Haskova, Z.; Asik, K.; Baumal, C.R.; Csaky, K.G.; Eter, N.; Ives, J.A.; Jaffe, G.J.; Korobelnik, J.F.; Lin, H.; et al. YOSEMITE and RHINE investigators. Faricimab treat-and-extend for diabetic macular edema: 2-year results from the randomized phase 3 YOSEMITE and RHINE trials. Ophthalmology 2024, 131, 708–723. [Google Scholar] [CrossRef]
- Strohl, L.L.; Zang, J.B.; Ding, W.; Manni, M.; Zhou, X.K.; Granstein, R.D. Norepinephrine and adenosine-5′-triphosphate synergize in inducing IL-6 production by human dermal microvascular endothelial cells. Cytokine 2013, 64, 605–612. [Google Scholar] [CrossRef]
- Lytvynchuk, L.; Nolte, K.L.; Deissler, H.L. Novel needle for intravitreal injections maintains activity of VEGF-binding proteins. In Proceedings of the 2025 ARVO Annual Meeting, Salt Lake City, UT, USA, 4–8 May 2025. [Google Scholar]
- Dejana, E.; Tournier-Lasserve, E.; Weinstein, B.M. The control of vascular integrity by endothelial cell junctions: Molecular basis and pathological implications. Dev. Cell 2009, 16, 209–221. [Google Scholar] [CrossRef]
- Wolf, H.N.; Guempelein, L.; Schikora, J.; Pauly, D. Inter-tissue differences in oxidative stress susceptibility reveal a less stable endothelial barrier in the brain than in the retina. Exp. Neurol. 2024, 380, 114919. [Google Scholar] [CrossRef] [PubMed]
- Eigenmann, D.E.; Xue, G.; Kim, K.S.; Moses, A.V.; Hamburger, M.; Oufir, M. Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood–brain barrier model for drug permeability studies. Fluids Barriers CNS 2013, 10, 33. [Google Scholar] [CrossRef] [PubMed]
- Barton, W.A.; Tzvetkova-Robev, D.; Miranda, E.P.; Kolev, M.V.; Rajashankar, K.R.; Himanen, J.P.; Nikolov, D.B. Crystal structures of the Tie2 receptor ectodomain and the angiopoietin-2-Tie2 complex. Nat. Struct. Mol. Biol. 2006, 13, 524–532. [Google Scholar] [CrossRef] [PubMed]
- Maisonpierre, P.C.; Suri, C.; Jones, P.F.; Bartunkova, S.; Wiegand, S.J.; Radziejewski, C.; Compton, D.; McClain, J.; Aldrich, T.H.; Papadopoulos, N.; et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 1997, 277, 55–60. [Google Scholar] [CrossRef]
- Scharpfenecker, M.; Fiedler, U.; Reiss, Y.; Augustin, H.G. The Tie-2 ligand angiopoietin-2 destabilizes quiescent endothelium through an internal autocrine loop mechanism. J. Cell Sci. 2005, 118, 771–780. [Google Scholar] [CrossRef]
- Diack, C.; Avery, R.L.; Cheung, C.M.G.; Csaky, K.G.; Gibiansky, L.; Jaminion, F.; Gibiansky, E.; Sickert, D.; Stoilov, I.; Cosson, V.; et al. Ocular pharmacodynamics of intravitreal faricimab in patients with neovascular age-related macular degeneration or diabetic macular edema. Transl. Vis. Sci. Technol. 2024, 13, 13. [Google Scholar] [CrossRef]
- Deissler, H.L.; Busch, C.; Wolf, A.; Rehak, M. Beovu, but not Lucentis impairs the function of the barrier formed by retinal endothelial cells in vitro. Sci. Rep. 2022, 12, 12493. [Google Scholar] [CrossRef]
- Martos, A.; Koch, W.; Jiskoot, W.; Wuchner, K.; Winter, G.; Friess, W.; Hawe, A. Trends on analytical characterization of polysorbates and their degradation products in biopharmaceutical formulations. J. Pharm. Sci. 2017, 106, 1722–1735. [Google Scholar] [CrossRef]
- Bruns, A.F.; Shane, P.H.; Odell, A.F.; Jopling, H.M.; Hooper, N.M.; Zachary, I.C.; Walker, J.H.; Ponnambalam, S. Ligand-stimulated VEGFR2 signaling is regulated by co-ordinated trafficking and proteolysis. Traffic 2010, 11, 161–174. [Google Scholar] [CrossRef]
- Guo, L.; Zhang, H.; Hou, Y.; Wie, T.; Liu, J. Plasmalemma vesicle-associated protein: A crucial component of vascular homeostasis. Exp. Ther. Med. 2016, 12, 1639–1644. [Google Scholar] [CrossRef]
- Hamilton, B.J.; Tse, D.; Stan, R.V. Phorbol esters induce PLVAP expression via VEGF and additional secreted molecules in MEK1-dependent and p38, JNK and PI3K/Akt-independent manner. J. Cell. Mol. Med. 2019, 23, 920–933. [Google Scholar] [CrossRef] [PubMed]
- Aiello, L.P.; Avery, R.L.; Arrigg, P.G.; Keyt, B.A.; Jampel, H.D.; Shah, S.T.; Pasquale, L.R.; Thieme, H.; Iwamoto, M.A.; Park, J.E.; et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N. Engl. J. Med. 1994, 331, 1480–1487. [Google Scholar] [CrossRef] [PubMed]
- Peters, S.; Cree, I.A.; Alexander, R.; Turowski, P.; Ockrim, Z.; Patel, J.; Boyd, S.R.; Joussen, A.M.; Ziemssen, F.; Hykin, P.G.; et al. Angiopoietin modulation of vascular endothelial growth factor: Effects on retinal endothelial cell permeability. Cytokine 2007, 40, 144–150. [Google Scholar] [CrossRef] [PubMed]
- Watanabe, D.; Suzuma, K.; Suzuma, I.; Ohashi, H.; Ojima, T.; Kurimoto, M.; Murakami, T.; Kimura, T.; Takagi, H. Vitreous levels of angiopoietin 2 and vascular endothelial growth factor in patients with proliferative diabetic retinopathy. Am. J. Ophthalmol. 2005, 139, 476–481. [Google Scholar] [CrossRef]
- Klaassen, I.; Avery, P.; Schlingemann, R.O.; Steel, D.H.W. Vitreous protein networks around ANG2 and VEGF in proliferative diabetic retinopathy and the different effects of aflibercept versus bevacizumab pre-treatment. Sci. Rep. 2022, 12, 21062. [Google Scholar] [CrossRef]
- Hammes, H.P.; Lin, J.; Wagner, P.; Feng, Y.; Vom Hagen, F.; Krzizok, T.; Renner, O.; Breier, G.; Brownlee, M.; Deutsch, U. Angiopoietin-2 causes pericyte dropout in the normal retina: Evidence for involvement in diabetic retinopathy. Diabetes 2004, 53, 1104–1110. [Google Scholar] [CrossRef]
Target | Host, Type and Conjugate | Source a | Working Concentration |
---|---|---|---|
actin | mouse, monoclonal | clone 5J11, Novus Biologicals, #NBP2-25142 | 700 ng/mL |
β-actin | mouse, monoclonal | clone BA3R, Invitrogen, #MA5-15739 | 100 ng/mL |
claudin-1 | rabbit, polyclonal | Invitrogen, #51-9000 | 250 ng/mL |
claudin-5 | rabbit, polyclonal | Invitrogen, #34-1600 | 100 ng/mL |
PLVAP b | rabbit, polyclonal | Invitrogen, #PA5-110183 | 2 µg/mL |
VEcadherin | rabbit, polyclonal | Cell Signaling Technology B.V., #2158 | 1:2000 |
whole rabbit IgG | goat, polyclonal, coupled to HRP | Biorad, #170-5046 | 1:15,000 |
whole mouse IgG | goat, polyclonal, coupled to HRP | Biorad, #170-5047 | 1:30,000 |
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Jung, D.M.; Fuezy, I.; Lytvynchuk, L.; Deissler, H.L. Faricimab Reverts VEGF-A165-Induced Impairment of the Barrier Formed by Retinal Endothelial Cells. Int. J. Mol. Sci. 2025, 26, 4318. https://doi.org/10.3390/ijms26094318
Jung DM, Fuezy I, Lytvynchuk L, Deissler HL. Faricimab Reverts VEGF-A165-Induced Impairment of the Barrier Formed by Retinal Endothelial Cells. International Journal of Molecular Sciences. 2025; 26(9):4318. https://doi.org/10.3390/ijms26094318
Chicago/Turabian StyleJung, Dominik M., Isabell Fuezy, Lyubomyr Lytvynchuk, and Heidrun L. Deissler. 2025. "Faricimab Reverts VEGF-A165-Induced Impairment of the Barrier Formed by Retinal Endothelial Cells" International Journal of Molecular Sciences 26, no. 9: 4318. https://doi.org/10.3390/ijms26094318
APA StyleJung, D. M., Fuezy, I., Lytvynchuk, L., & Deissler, H. L. (2025). Faricimab Reverts VEGF-A165-Induced Impairment of the Barrier Formed by Retinal Endothelial Cells. International Journal of Molecular Sciences, 26(9), 4318. https://doi.org/10.3390/ijms26094318