Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
권호 표지
DOI QR코드

DOI QR Code

Celafolin A-1 Ameliorates Subretinal Fibrosis via Inhibition of Crystallin Alpha B in a Laser-Induced Choroidal Neovascularization Mouse Model

  • Eunhye Yu (College of Pharmacy, Chungbuk National University) ;
  • Sun Mi Gu (College of Pharmacy, Chungbuk National University) ;
  • Haechan Kim (College of Pharmacy, Chungbuk National University) ;
  • Jun Gu Kim (College of Pharmacy, Chungbuk National University) ;
  • Bang Yeon Hwang (College of Pharmacy, Chungbuk National University) ;
  • Jung Kee Min (Department of Ophthalmology, Ulsan University Hospital, University of Ulsan, College of Medicine) ;
  • Jaesuk Yun (College of Pharmacy, Chungbuk National University)
  • Received : 2025.05.28
  • Accepted : 2026.01.10
  • Published : 2026.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

Age-related macular degeneration (AMD) is a leading cause of blindness in people over 65 years old. Anti-vascular endothelial growth factor (VEGF) therapy is the first-line treatment for neovascular AMD (nAMD); however, fibrosis remains an unmet medical need due to the lack of effective medications. This study evaluated eight natural products from Celastrus orbiculatus, which has been reported to have anti-inflammatory effects, for their effects on the fibrotic pathological process as well as choroidal neovascularization (CNV). We investigated the half-maximal inhibitory concentration (IC50) values of these compounds on VEGF and alphasmooth muscle actin (α-SMA, a fibrosis marker) expression-induced by human acute monocytic cell (THP-1) conditioned media in retinal pigment epithelium cells (ARPE-19). Four compounds reduced both VEGF and α-SMA at 10 µM, with three-Celafolin A-1, COFH5645, and COFH543435-showing the highest potency. Intravitreal injections of the compound in a mouse model of CNV induced by laser photocoagulation confirmed its efficacy. Celafolin A-1 significantly reduced α-SMA and VEGF expression and decreased hyper-reflective lesions and CNV areas. Binding affinity measurements using biolayer interferometry identified an interaction between Celafolin A-1 and crystallin alpha B (Cryab), a protein involved in stress responses and fibrosis. Celafolin A-1 reduced the expression levels of Cryab as well as its phosphorylated form at Ser45, indicating that its mechanism involves the regulation of Cryab phosphorylation. Taken together, Celafolin A-1 exhibits dual inhibitory effects on VEGF and fibrosis, suggesting it as a candidate for the treatment of nAMD.

Keywords

Acknowledgement

This work was supported by the Bio&Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korea government (MSIT) (No. RS-2024-00440787), Basic Science Research Program through the NRF funded by the Ministry of Education (RS-2024-00460411), the NRF of Korea grant funded by the Korea government (MSIT) (No. RS-2025-02273102, No. NRF-2022R1F1A1069431), the research grant by an Ulsan University Hospital Research Grant (UUH-2020-07), and a funding for the academic research program of Chungbuk National University in 2025. This work was conducted during the research year of Chungbuk National University in 2025.

References

  1. Abdalhameid, E., Abd El-Haleim, E. A., Abdelsalam, R. M., Georgy, G. S., Fawzy, H. M. and Kenawy, S. A. (2024) Cinnamic acid mitigates methotrexate-induced lung fibrosis in rats: comparative study with pirfenidone. Naunyn Schmiedebergs Arch. Pharmacol. 397, 1071-1079. https://doi.org/10.1007/s00210-023-02652-w
  2. Abdiche, Y., Malashock, D., Pinkerton, A. and Pons, J. (2008) Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet. Anal. Biochem. 377, 209-217. https://doi.org/10.1016/j.ab.2008.03.035
  3. Adisakwattana, S. (2017) Cinnamic acid and its derivatives: mechanisms for prevention and management of diabetes and its complications. Nutrients 9, 163. https://doi.org/10.3390/nu9020163
  4. Andley, U. P. (2007) Crystallins in the eye: function and pathology. Prog. Retin. Eye Res. 26, 78-98. https://doi.org/10.1016/j.preteyeres.2006.10.003
  5. Bai, Y., Tan, D., Deng, Q., Miao, L., Wang, Y., Zhou, Y., Yang, Y., Wang, S., Vong, C. T. and Cheang, W. S. (2025) Cinnamic acid alleviates endothelial dysfunction and oxidative stress by targeting PPARδ in obesity and diabetes. Chin. Med. 20, 13.
  6. Bellaye, P. S., Wettstein, G., Burgy, O., Besnard, V., Joannes, A., Colas, J., Causse, S., Marchal-Somme, J., Fabre, A., Crestani, B., Kolb, M., Gauldie, J., Camus, P., Garrido, C. and Bonniaud, P. (2014) The small heat-shock protein αB-crystallin is essential for the nuclear localization of Smad4: impact on pulmonary fibrosis. J. Pathol. 232, 458-472. https://doi.org/10.1002/path.2014.232.issue-4
  7. Bouma, B. E., de Boer, J. F., Huang, D., Jang, I. K., Yonetsu, T., Leggett, C. L., Leitgeb, R., Sampson, D. D., Suter, M., Vakoc, B., Villiger, M. and Wojtkowski, M. (2022) Optical coherence tomography. Nat. Rev. Methods Primers 2, 79. https://doi.org/10.1038/s43586-022-00162-2
  8. Concepcion, J., Witte, K., Wartchow, C., Choo, S., Yao, D., Persson, H., Wei, J., Li, P., Heidecker, B., Ma, W., Varma, R., Zhao, L. S., Perillat, D., Carricato, G., Recknor, M., Du, K., Ho, H., Ellis, T., Gamez, J., Howes, M., Phi-Wilson, J., Lockard, S., Zuk, R. and Tan, H. (2009) Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization. Comb. Chem. High Throughput Screen. 12, 791-800. https://doi.org/10.2174/138620709789104915
  9. Daniel, E., Toth, C. A., Grunwald, J. E., Jaffe, G. J., Martin, D. F., Fine, S. L., Huang, J., Ying, G. S., Hagstrom, S. A., Winter, K. and Maguire, M. G. (2014) Risk of scar in the comparison of age-related macular degeneration treatments trials. Ophthalmology 121, 656-666. https://doi.org/10.1016/j.ophtha.2013.10.019
  10. De, S., Rabin, D. M., Salero, E., Lederman, P. L., Temple, S. and Stern, J. H. (2007) Human retinal pigment epithelium cell changes and expression of αB-crystallin: a biomarker for retinal pigment epithelium cell change in age-related macular degeneration. Arch. Ophthalmol. 125, 641-645. https://doi.org/10.1001/archopht.125.5.641
  11. Dimberg, A., Rylova, S., Dieterich, L. C., Olsson, A. K., Schiller, P., Wikner, C., Bohman, S., Botling, J., Lukinius, A., Wawrousek, E. F. and Claesson-Welsh, L. (2008) alphaB-crystallin promotes tumor angiogenesis by increasing vascular survival during tube morphogenesis. Blood 111, 2015-2023. https://doi.org/10.1182/blood-2007-04-087841
  12. Ecroyd, H., Meehan, S., Horwitz, J., Aquilina, J. A., Benesch, J. L., Robinson, C. V., Macphee, C. E. and Carver, J. A. (2007) Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity. Biochem. J. 401, 129-141. https://doi.org/10.1042/BJ20060981
  13. Evans, J. R. (2001) Risk factors for age-related macular degeneration. Prog. Retin. Eye Res. 20, 227-253. https://doi.org/10.1016/S1350-9462(00)00023-9
  14. Fichna, J. P., Potulska-Chromik, A., Miszta, P., Redowicz, M. J., Kaminska, A. M., Zekanowski, C. and Filipek, S. (2017) A novel dominant D109A CRYAB mutation in a family with myofibrillar myopathy affects αB-crystallin structure. BBA Clin. 7, 1-7. https://doi.org/10.1016/j.bbacli.2016.11.004
  15. Flores, R., Carneiro, Â., Vieira, M., Tenreiro, S. and Seabra, M. C. (2021) Age-related macular degeneration: pathophysiology, management, and future perspectives. Ophthalmologica 244, 495-511. https://doi.org/10.1159/000517520
  16. Guo, J., Yan, L., Yang, Q., Li, H., Tian, Y., Yang, J., Xie, J., Zhang, F. and Hao, E. (2025) Multiple strategies confirm the anti hepatocellular carcinoma effect of cinnamic acid based on the PI3k-AKT pathway. Pharmaceuticals 18, 1205. https://doi.org/10.3390/ph18081205
  17. Guo, Y. S., Liang, P. Z., Lu, S. Z., Chen, R., Yin, Y. Q. and Zhou, J. W. (2019) Extracellular αB-crystallin modulates the inflammatory responses. Biochem. Biophys. Res. Commun. 508, 282-288. https://doi.org/10.1016/j.bbrc.2018.11.024
  18. Hayashi, J. and Carver, J. A. (2020) The multifaceted nature of αBcrystallin. Cell Stress Chaperones 25, 639-654. https://doi.org/10.1007/s12192-020-01098-w
  19. Ishikawa, K., Kannan, R. and Hinton, D. R. (2016) Molecular mechanisms of subretinal fibrosis in age-related macular degeneration. Exp. Eye Res. 142, 19-25. https://doi.org/10.1016/j.exer.2015.03.009
  20. Ito, H., Okamoto, K., Nakayama, H., Isobe, T. and Kato, K. (1997) Phosphorylation of alphaB-crystallin in response to various types of stress. J. Biol. Chem. 272, 29934-29941. https://doi.org/10.1074/jbc.272.47.29934
  21. Jin, H. Z., Hwang, B. Y., Kim, H. S., Lee, J. H., Kim, Y. H. and Lee, J. J. (2002) Antiinflammatory constituents of Celastrus orbiculatus inhibit the NF-kappaB activation and NO production. J. Nat. Prod. 65, 89-91. https://doi.org/10.1021/np010428r
  22. Kase, S., He, S., Sonoda, S., Kitamura, M., Spee, C., Wawrousek, E., Ryan, S. J., Kannan, R. and Hinton, D. R. (2010) alphaB-crystallin regulation of angiogenesis by modulation of VEGF. Blood 115, 3398-3406. https://doi.org/10.1182/blood-2009-01-197095
  23. Kiriakidis, S., Andreakos, E., Monaco, C., Foxwell, B., Feldmann, M. and Paleolog, E. (2003) VEGF expression in human macrophages is NF-kappaB-dependent: studies using adenoviruses expressing the endogenous NF-kappaB inhibitor IkappaBalpha and a kinasedefective form of the IkappaB kinase 2. J. Cell Sci. 116, 665-674. https://doi.org/10.1242/jcs.00286
  24. Klaver, C. C., Wolfs, R. C., Vingerling, J. R., Hofman, A. and de Jong, P. T. (1998) Age-specific prevalence and causes of blindness and visual impairment in an older population: the Rotterdam Study. Arch. Ophthalmol. 116, 653-658. https://doi.org/10.1001/archopht.116.5.653
  25. Lambert, N. G., ElShelmani, H., Singh, M. K., Mansergh, F. C., Wride, M. A., Padilla, M., Keegan, D., Hogg, R. E. and Ambati, B. K. (2016) Risk factors and biomarkers of age-related macular degeneration. Prog. Retin. Eye Res. 54, 64-102. https://doi.org/10.1016/j.preteyeres.2016.04.003
  26. Lambert, V., Lecomte, J., Hansen, S., Blacher, S., Gonzalez, M.-L. A., Struman, I., Sounni, N. E., Rozet, E., de Tullio, P., Foidart, J. M., Rakic, J.-M. and Noel, A. (2013) Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice. Nat. Protoc. 8, 2197-2211. https://doi.org/10.1038/nprot.2013.135
  27. Léger, H., Santana, E., Beltran, W. A. and Luca, F. C. (2019) Preparation of mouse retinal Cryo-sections for immunohistochemistry. J. Vis. Exp. 149, e59683.
  28. Little, K., Ma, J. H., Yang, N., Chen, M. and Xu, H. (2018) Myofibroblasts in macular fibrosis secondary to neovascular age-related macular degeneration - the potential sources and molecular cues for their recruitment and activation. EBioMedicine 38, 283-291. https://doi.org/10.1016/j.ebiom.2018.11.029
  29. Malik, D., Tarek, M., Caceres del Carpio, J., Ramirez, C., Boyer, D., Kenney, M. C. and Kuppermann, B. D. (2014) Safety profiles of anti-VEGF drugs: bevacizumab, ranibizumab, aflibercept and zivaflibercept on human retinal pigment epithelium cells in culture. Br. J. Ophthalmol. 98 Suppl 1, i11-i16. https://doi.org/10.1136/bjophthalmol-2014-305302
  30. Nair, A., Preetha Rani, M. R., Salin Raj, P., Ranjit, S., Rajankutty, K. and Raghu, K. G. (2022) Cinnamic acid is beneficial to diabetic cardiomyopathy via its cardioprotective, anti-inflammatory, antidyslipidemia, and antidiabetic properties. J. Biochem. Mol. Toxicol. 36, e23215.
  31. Peschek, J., Braun, N., Rohrberg, J., Back, K. C., Kriehuber, T., Kastenmüller, A., Weinkauf, S. and Buchner, J. (2013) Regulated structural transitions unleash the chaperone activity of αB-crystallin. Proc. Natl. Acad. Sci. U. S. A . 110, E3780-E3789. https://doi.org/10.1073/pnas.1308898110
  32. Qian, Y. Y., Zhang, H., Hou, Y., Yuan, L., Li, G. Q., Guo, S. Y., Hisamits, T. and Liu, Y. Q. (2012) Celastrus orbiculatus extract inhibits tumor angiogenesis by targeting vascular endothelial growth factor signaling pathway and shows potent antitumor activity in hepatocarcinomas in Vitro and in Vivo. Chin. J. Integr. Med. 18, 752-760. https://doi.org/10.1007/s11655-011-0819-7
  33. Rosenfeld, P. J., Brown, D. M., Heier, J. S., Boyer, D. S., Kaiser, P. K., Chung, C. Y. and Kim, R. Y. (2006) Ranibizumab for neovascular age-related macular degeneration. N. Engl. J. Med. 355, 1419-1431. https://doi.org/10.1056/NEJMoa054481
  34. Ryan, S. J. (1979) The development of an experimental model of subretinal neovascularization in disciform macular degenera tion. Trans. Am. Ophthalmol. Soc. 77, 707-745.
  35. Schwesinger, C., Yee, C., Rohan, R. M., Joussen, A. M., Fernandez, A., Meyer, T. N., Poulaki, V., Ma, J. J., Redmond, T. M., Liu, S., Adamis, A. P. and D'Amato, R. J. (2001) Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial growth factor in the retinal pigment epithelium. Am. J. Pathol. 158, 1161-1172. https://doi.org/10.1016/S0002-9440(10)64063-1
  36. Sherman, W., Day, T., Jacobson, M. P., Friesner, R. A. and Farid, R. (2006) Novel procedure for modeling ligand/receptor induced fit effects. J. Med. Chem. 49, 534-553. https://doi.org/10.1021/jm050540c
  37. Sprague-Piercy, M. A., Rocha, M. A., Kwok, A. O. and Martin, R. W. (2021) α-Crystallins in the vertebrate eye lens: complex oligomers and molecular chaperones. Annu. Rev. Phys. Chem. 72, 143-163. https://doi.org/10.1146/physchem.2021.72.issue-1
  38. Tamiya, S., Liu, L. and Kaplan, H. J. (2010) Epithelial-mesenchymal transition and proliferation of retinal pigment epithelial cells initiated upon loss of cell-cell contact. Invest. Ophthalmol. Vis. Sci. 51, 2755-2763. https://doi.org/10.1167/iovs.09-4725
  39. Wang, H., Luo, Y., Ou, S., Ni, T., Chu, Z., Feng, X., Dai, X., Zhang, X. and Liu, Y. (2023) Celastrus orbiculatus Thunb. extract inhibits EMT and metastasis of gastric cancer by regulating actin cytoskeleton remodeling. J. Ethnopharmacol. 301, 115737. https://doi.org/10.1016/j.jep.2022.115737
  40. Wang, J. J., Rochtchina, E., Lee, A. J., Chia, E. M., Smith, W., Cumming, R. G. and Mitchell, P. (2007) Ten-year incidence and progression of age-related maculopathy: the blue Mountains Eye Study. Ophthalmology 114, 92-98. https://doi.org/10.1016/j.ophtha.2006.07.017
  41. Yu, E., Kim, H., Park, H., Hong, J. H., Jin, J., Song, Y., Woo, J. M., Min, J. K. and Yun, J. (2024) Targeting the VEGFR2 signaling pathway for angiogenesis and fibrosis regulation in neovascular age-related macular degeneration. Sci. Rep. 14, 25682.
  42. Yu, E., Song, Y., Gu, S. M., Jo, Y. H., Yeon, S. W., Han, K. J., Lee, M. K., Min, J. K. and Yun, J. (2022) Alpinumisoflavone ameliorates choroidal neovascularisation and fibrosis in age-related macular degeneration in in vitro and in vivo models. Sci. Rep. 12, 14316.
  43. Yun, J. and Min, J. K. (2020) Elevated vitreous αB-crystallin in patients with rhegmatogenous retinal detachment and association with proliferative vitreoretinopathy and retinal detachment area. Int. Ophthalmol. 40, 2461-2467. https://doi.org/10.1007/s10792-020-01425-1