Glycosaminoglycans compositional analysis of Urodele axolotl (Ambystoma mexicanum) and Porcine Retina

  • So Young Kim
  • Joydip Kundu
  • Asher Williams
  • Anastasia S. Yandulskaya
  • James R. Monaghan
  • Rebecca L. CarrierEmail author
  • Robert J. LinhardtEmail author
Original Article


Retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are major causes of blindness worldwide. Humans cannot regenerate retina, however, axolotl (Ambystoma mexicanum), a laboratory-bred salamander, can regenerate retinal tissue throughout adulthood. Classic signaling pathways, including fibroblast growth factor (FGF), are involved in axolotl regeneration. Glycosaminoglycan (GAG) interaction with FGF is required for signal transduction in this pathway. GAGs are anionic polysaccharides in extracellular matrix (ECM) that have been implicated in limb and lens regeneration of amphibians, however, GAGs have not been investigated in the context of retinal regeneration. GAG composition is characterized native and decellularized axolotl and porcine retina using liquid chromatography mass spectrometry. Pig was used as a mammalian vertebrate model without the ability to regenerate retina. Chondroitin sulfate (CS) was the main retinal GAG, followed by heparan sulfate (HS), hyaluronic acid, and keratan sulfate in both native and decellularized axolotl and porcine retina. Axolotl retina exhibited a distinctive GAG composition pattern in comparison with porcine retina, including a higher content of hyaluronic acid. In CS, higher levels of 4- and 6- O-sulfation were observed in axolotl retina. The HS composition was greater in decellularized tissues in both axolotl and porcine retina by 7.1% and 15.4%, respectively, and different sulfation patterns were detected in axolotl. Our findings suggest a distinctive GAG composition profile of the axolotl retina set foundation for role of GAGs in homeostatic and regenerative conditions of the axolotl retina and may further our understanding of retinal regenerative models.


Amphibian Axolotl Glycosaminoglycans Regeneration Retina 



This research was funded by the NIH in the form of grants DK111958, CA231074, HL125371 (to RJL) and by grant NSF-CBET #1606128 (to RLC).

Compliance with ethical standards

Conflicts of interest

The authors declare to have no conflicts of interest.

Ethical approval

Animals in this study were approved by Institutional Animal Care and Use Committee (IACUC).


  1. 1.
    Wong, W.L., Su, X., Li, X., Cheung, C.M., Klein, R., Cheng, C.Y., Wong, T.Y.: Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Glob. Health. 2, e106–e116 (2014)CrossRefGoogle Scholar
  2. 2.
    Barbosa-Sabanero, K., Hoffmann, A., Judge, C., Lightcap, N., Tsonis, P.A., Del Rio-Tsonis, K.: Lens and retina regeneration: new perspectives from model organisms. Biochem. J. 447, 321–334 (2012)CrossRefGoogle Scholar
  3. 3.
    Del Rio-Tsonis, K., Tsonis, P.A.: Eye regeneration at the molecular age. Dev. Dyn. 226, 211–224 (2003)CrossRefGoogle Scholar
  4. 4.
    Haynes, T., Del Rio-Tsonis, K.: Retina repair, stem cells and beyond. Curr. Neurovasc. Res. 1, 231–239 (2004)CrossRefGoogle Scholar
  5. 5.
    Hayashi, T., Mizuno, N., Ueda, Y., Okamoto, M., Kondoh, H.: FGF2 triggers iris-derived lens regeneration in newt eye. Mech. Dev. 121, 519–526 (2004)CrossRefGoogle Scholar
  6. 6.
    Spence, J.R., Aycinena, J.C., Del Rio-Tsonis, K.: Fibroblast growth factor-hedgehog interdependence during retina regeneration. Dev. Dyn. 236, 1161–1174 (2007)CrossRefGoogle Scholar
  7. 7.
    Susaki, K., Chiba, C.: MEK mediates in vitro neural transdifferentiation of the adult newt retinal pigment epithelium cells: is FGF2 an induction factor? Pigment Cell Res. 20, 364–379 (2007)CrossRefGoogle Scholar
  8. 8.
    Spence, J.R., Madhavan, M., Aycinena, J.C., Del Rio-Tsonis, K.: Retina regeneration in the chick embryo is not induced by spontaneous Mitf downregulation but requires FGF/FGFR/MEK/Erk dependent upregulation of Pax6. Mol. Vis. 13, 57–65 (2007)Google Scholar
  9. 9.
    Schlessinger, J., Plotnikov, A.N., Ibrahimi, O.A., Eliseenkova, A.V., Yeh, B.K., Yayon, A., Linhardt, R.J., Mohammadi, M.: Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol. Cell. 6, 743–750 (2000)CrossRefGoogle Scholar
  10. 10.
    Sterner, E., Meli, L., Kwon, S.J., Dordick, J.S., Linhardt, R.J.: FGF–FGFR signaling mediated through glycosaminoglycans in microtiter plate and cell-based microarray platforms. Biochemistry. 50, 9009–9019 (2013)CrossRefGoogle Scholar
  11. 11.
    Linhardt, R.J., Toida, T.: Role of glycosaminoglycans in cellular communication. Acc. Chem. Res. 7, 431–438 (2004)CrossRefGoogle Scholar
  12. 12.
    Kim, S.Y., Zhao, J., Liu, X., Fraser, K., Lin, L., Zhang, X., Zhang, F., Dordick, J.S., Linhardt, R.J.: Interaction of Zika virus envelope protein with glycosaminoglycans. Biochemistry. 56, 1151–1162 (2017)CrossRefGoogle Scholar
  13. 13.
    Kim, S.Y., Zhang, F., Gong, W., Chen, K., Xia, K., Liu, F., Gross, R.A., Wang, J.M., Linhardt, R.J., Cotton, M.L.: Copper regulates the interactions of antimicrobial piscidin peptides from fish mast cells with formyl peptide receptors and heparin. J. Biol. Chem. 293, 15381–15396 (2018)CrossRefGoogle Scholar
  14. 14.
    Kaprinis, K., Symeonidis, C., Papakonstantinou, E., Tsinopoulos, I., Dimitrakos, S.A.: Decreased hyaluronan concentration during primary rhegmatogenous retinal detachment. Eur. J. Ophthalmol. 26, 633–638 (2016)CrossRefGoogle Scholar
  15. 15.
    Park, P.J., Shukla, D.: Role of heparan sulfate in ocular diseases. Exp. Eye Res. 110, 1–9 (2013)CrossRefGoogle Scholar
  16. 16.
    McManus, L.M., Mitchell, R.N.: Pathobiology of human disease: a dynamic encyclopedia of disease mechanisms. Elsevier. (2014)Google Scholar
  17. 17.
    Dreyfuss, J.L., Regatieri, C.V., Lima, M.A., Paredes-Gamero, E.J., Brito, A.S., Chavante, S.F., Belfort Jr., R., Farah, M.E., Nader, H.B.: A heparin mimetic isolated from a marine shrimp suppresses neovascularization. J. Thromb. Haemost. 8, 1828–1837 (2010)CrossRefGoogle Scholar
  18. 18.
    Jiang, X., Couchman, J.R.: Perlecan and tumor angiogenesis. J. Histochem. Cytochem. 51, 1393–1410 (2003)CrossRefGoogle Scholar
  19. 19.
    Regatieri, C.V., Dreyfuss, J.L., Melo, G.B., Lavinsky, D., Hossaka, S.K., Rodrigues, E.B., Farah, M.E., Maia, M., Nader, H.B.: Quantitative evaluation of experimental choroidal neovascularization by confocal scanning laser ophthalmoscopy: fluorescein angiogram parallels heparan sulfate proteoglycan expression. Braz. J. Med. Biol. Res. 43, 627–633 (2010)CrossRefGoogle Scholar
  20. 20.
    Clark, S.J., Bishop, P.N., Day, A.J.: Complement factor H and age-related macular degeneration: the role of glycosaminoglycan recognition in disease pathology. Biochem. Soc. Trans. 38, 1342–1348 (2010)CrossRefGoogle Scholar
  21. 21.
    Clark, S.J., Perveen, R., Hakobyan, S., Morgan, B.P., Sim, R.B., Bishop, P.N., Day, A.J.: Impaired binding of the age-related macular degeneration-associated complement factor H 402H allotype to Bruch’s membrane in human retina. J. Biol. Chem. 285, 192–202 (2010)Google Scholar
  22. 22.
    Kelly, U., Yu, L., Kumar, P., Ding, J.D., Jiang, H., Hageman, G.S., Arshavsky, V.Y., Frank, M.M., Hauser, M.A., Rickman, C.B.: Heparan sulfate, including that in Bruch’s membrane, inhibits the complement alternative pathway: implications for age-related macular degeneration. J. Immunol. 185, 5486–5494 (2010)CrossRefGoogle Scholar
  23. 23.
    Alibardi, L.: Hyaluronic acid in the tail and limb of amphibians and lizards recreates permissive embryonic conditions for regeneration due to its hygroscopic and immunosuppressive properties. J Exp Zool B Mol Dev Evol. 328, 760–771 (2017)CrossRefGoogle Scholar
  24. 24.
    Ouyang, X., Panetta, N.J., Talbott, M.D., Payumo, A.Y., Halluin, C., Longaker, M.T., Chen, J.K.: Hyaluronic acid synthesis is required for zebrafish tail fin regeneration. PLoS One. 12, e0171898 (2017)CrossRefGoogle Scholar
  25. 25.
    Kulyk, W.M., Zalik, S.E., Dimitrov, E.: Hyaluronic acid production and hyaluronidase activity in the newt iris during lens regeneration. Exp. Cell Res. 172, 180–191 (1987)CrossRefGoogle Scholar
  26. 26.
    Gardiner, D.M.: Regulation of regeneration by heparan sulfate proteoglycans in the extracellular matrix. Regen Eng Transl Med. 3, 192–198 (2017)CrossRefGoogle Scholar
  27. 27.
    Phan, A.Q., Lee, J., Oei, M., Flath, C., Hwe, C., Mariano, R., Vu, T., Shu, C., Dinh, A., Simkin, J., Muneoka, K., Bryant, S.V., Gardiner, D.M.: Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl (Ambystoma mexicanum). Regeneration. 2, 182–201 (2015)CrossRefGoogle Scholar
  28. 28.
    Ramachandra, R., Namburi, R.B., Dupont, S.T., Ortega-Martinez, O., van Kuppevelt, T.H., Lindahl, U., Spillmann, D.: A potential role for chondroitin sulfate/dermatan sulfate in arm regeneration in Amphiura filiformis. Glycobiology. 27, 438–449 (2017)Google Scholar
  29. 29.
    Becker, C.G, Becker, T.: Repellent guidance of regenerating optic axons by chondroitin sulfate glycosaminoglycans in zebrafish. J. Neurosci. 22, 842–853 (2002)Google Scholar
  30. 30.
    Rauvala, H., Paveliev, M., Kuja-Panula, J., Kulesskaya, N.: Inhibition and enhancement of neural regeneration by chondroitin sulfate proteoglycans. Neural Regen. Res. 12, 687–691 (2017)CrossRefGoogle Scholar
  31. 31.
    Inatani, M., Tanihara, H.: Proteoglycans in retina. Prog. Retin. Eye Res. 21, 429–447 (2002)CrossRefGoogle Scholar
  32. 32.
    Joven, A., Simon, A.: Homeostatic and regenerative neurogenesis in salamanders. Prog. Neurobiol. 170, 81–98 (2018)CrossRefGoogle Scholar
  33. 33.
    Roy, S., Levesque, M.: Limb regeneration in axolotl: is it superhealing? Sci. World J. 6, 12–25 (2006)CrossRefGoogle Scholar
  34. 34.
    Alunni, A., Bally-Cuif, L.: A comparative view of regenerative neurogenesis in vertebrates. Development. 143, 741–753 (2016)CrossRefGoogle Scholar
  35. 35.
    Sun, Y.B., Xiong, Z.J., Xiang, X.Y., Liu, S.P., Zhou, W.W., Tu, X.L., Zhong, L., Wang, L., Wu, D.D., Zhang, B.L., Zhu, C.L.: Whole-genome sequence of the Tibetan frog Nanorana parkeri and the comparative evolution of tetrapod genomes. Proc. Natl. Acad. Sci. 112, 1257–1262 (2015)CrossRefGoogle Scholar
  36. 36.
    Nowoshilow, S., Schloissnig, S., Fei, J.F., Dahl, A., Pang, A.W., Pippel, M., Winkler, S., Hastie, A.R., Young, G., Roscito, J.G., Falcon, F.: The axolotl genome and the evolution of key tissue formation regulators. Nature. 554, 50–55 (2018)CrossRefGoogle Scholar
  37. 37.
    Svistunov, S.A., Mitashov, V.I.: Proliferative activity of the pigment epithelium and regenerating retinal cells in Ambystoma mexicanum. Ontogenez. 14, 597–606 (1983)Google Scholar
  38. 38.
    Voss, S.R., Epperlein, H.H., Tanaka, E.M.: Ambystoma mexicanum, the axolotl: a versatile amphibian model for regeneration, development, and evolution studies. Cold Spring Harb Protoc. 8, pdb.emo128, (2009)Google Scholar
  39. 39.
    Linhardt, R.J., Turnbull, J.E., Wang, H.M., Loganathan, D., Gallagher, J.T.: Examination of the substrate specificity of heparin and heparan sulfate lyases. Biochemistry. 29, 2611–2617 (1990)CrossRefGoogle Scholar
  40. 40.
    Wang, H., He, W., Jiang, P., Yu, Y., Lin, L., Sun, X., Koffas, M., Zhang, F., Linhardt, R.J.: Construction and functional characterization of truncated versions of recombinant keratanase II from Bacillus circulans. Glycoconj. J. 34, 643–649 (2017)CrossRefGoogle Scholar
  41. 41.
    Kundu, J., Michaelson, A., Talbot, K., Baranov, P., Young, M.J., Carrier, R.L.: Decellularized retinal matrix: natural platforms for human retinal progenitor cell culture. Acta Biomater. 31, 61–70 (2016)CrossRefGoogle Scholar
  42. 42.
    Wright, A.F., Chakarova, C.F., El-Aziz, M.M., Bhattacharya, S.S.: Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait. Nat Rev Genet. 11, 273–284 (2010)CrossRefGoogle Scholar
  43. 43.
    Grzybowski, A.: General structure and function of the retina. Acta Ophthalmol. 94, (2016)Google Scholar
  44. 44.
    Sanchez, I., Martin, R., Ussa, F., Fernandez-Bueno, I.: The parameters of the porcine eyeball. Graefes Arch. Clin. Exp. Ophthalmol. 249, 475–482 (2011)CrossRefGoogle Scholar
  45. 45.
    Porrello, K., Lavail, M.M.: Immunocytochemical localization of chondroitin sulfates in the interphotoreceptor matrix of the normal and dystrophic rat retina. Curr. Eye Res. 5, 981–993 (1986)CrossRefGoogle Scholar
  46. 46.
    Singhal, S., Lawrence, J.M., Bhatia, B., Ellis, J.S., Kwan, A.S., MacNeil, A., Luthert, P.J., Fawcett, J.W., Perez, M.T., Khaw, P.T., Limb, G.A.: Chondroitin sulfate proteoglycans and microglia prevent migration and integration of grafted Müller stem cells into degenerating retina. Stem Cells. 26, 1074–1082 (2008)CrossRefGoogle Scholar
  47. 47.
    Suzuki, T., Akimoto, M., Imai, H., Ueda, Y., Mandai, M., Yoshimura, N., Swaroop, A., Takahashi, M.: Chondroitinase ABC treatment enhances synaptogenesis between transplant and host neurons in model of retinal degeneration. Cell Transplant. 16, 493–503 (2007)CrossRefGoogle Scholar
  48. 48.
    Ichijo, H., Kawabata, I.: Roles of the telencephalic cells and their chondroitin sulfate proteoglycans in delimiting an anterior border of the retinal pathway. J. Neurosci. 21, 9304–9314 (2001)CrossRefGoogle Scholar
  49. 49.
    Brittis, P.A., Canning, D.R., Silver, J.: Chondroitin sulfate as a regulator of neuronal patterning in the retina. Science. 255, 733–736 (1992)CrossRefGoogle Scholar
  50. 50.
    Bakalash, S., Rolls, A., Lider, O., Schwartz, M.: Chondroitin sulfate-derived disaccharide protects retinal cells from elevated intraocular pressure in aged and immunocompromised rats. Invest. Ophthalmol. Vis. Sci. 48, 1181–1190 (2007)CrossRefGoogle Scholar
  51. 51.
    Tate, D.J., Oliver, P.D., Miceli, M.V., Stern, R., Shuster, S., Newsome, D.A.: Age-dependent change in the hyaluronic acid content of the human chorioretinal complex. Arch. Ophthalmol. 111, 963–967 (1993)CrossRefGoogle Scholar
  52. 52.
    Hollyfield, J.G., Rayborn, M.E., Tammi, M., Tammi, R.: Hyaluronan in the interphotoreceptor matrix of the eye: species differences in content, distribution, ligand binding and degradation. Exp. Eye Res. 66, 241–248 (1998)CrossRefGoogle Scholar
  53. 53.
    Inoue, Y., Yoneda, M., Miyaishi, O., Iwaki, M., Zako, M.: Hyaluronan dynamics during retinal development. Brain Res. 1256, 55–60 (2009)CrossRefGoogle Scholar
  54. 54.
    Solursh, M., Vaerewyck, S.A., Reiter, R.S.: Depression by hyaluronic acid of glycosaminoglycan synthesis by cultured chick embryo chondrocytes. Dev. Biol. 41, 233–244 (1974)CrossRefGoogle Scholar
  55. 55.
    Munaim, S.I., Klagsbrun, M., Toole, B.P.: Hyaluronan-dependent pericellular coats of chick embryo limb mesodermal cells: induction by basic fibroblast growth factor. Dev. Biol. 143, 297–302 (1991)CrossRefGoogle Scholar
  56. 56.
    Vatne, H.O., Syrdalen, P.: The use of sodium hyaluronate (Healon) in the treatment of complicated cases of retinal detachment. Acta Ophthalmol. 64(169–172), (1986)Google Scholar
  57. 57.
    Lipton, S.A., Wagner, J.A., Madison, R.D., D’Amore, P.A.: Acidic fibroblast growth factor enhances regeneration of processes by postnatal mammalian retinal ganglion cells in culture. Proc. Natl. Acad. Sci. U. S. A. 85, 2388–2392 (1988)CrossRefGoogle Scholar
  58. 58.
    Nagy, T., Reh, T.A.: Inhibition of retinal regeneration in larval Rana by an antibody directed against a laminin–heparan sulfate proteoglycan. Brain Res. Dev. 81, 131–134 (1994)CrossRefGoogle Scholar
  59. 59.
    Schubert, D., LaCorbiere, M.: Isolation of a cell-surface receptor for chick neural retina adherons. J. Cell Biol. 100, 56–63 (1985)CrossRefGoogle Scholar
  60. 60.
    Carri, N.G., Perris, R., Johansson, S., Ebendal, T.: Differential outgrowth of retinal neurites on purified extracellular matrix molecules. J. Neurosci. Res. 19, 428–439 (1988)CrossRefGoogle Scholar
  61. 61.
    Chernousov, M.A., Carey, D.J.: N-syndecan (Syndecan 3) from neonatal rat brain binds basic fibroblast growth factor. J. Biol. Chem. 268, 16810–16814 (1993)Google Scholar
  62. 62.
    Ornitz, D.M., Itoh, N.: The fibroblast growth factor signaling pathway. Wiley Interdiscip. Rev. Dev. Biol. 4, 215–266 (2015)CrossRefGoogle Scholar
  63. 63.
    Keenan, T.D., Pickford, C.E., Holley, R.J., Clark, S.J., Lin, W., Dowsey, A.W., Merry, C.L., Day, A.J., Bishop, P.N.: Age-dependent changes in heparan sulfate in human Bruch's membrane: implications for age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 55, 5370–5379 (2014)CrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Biochemistry and Biophysics Graduate Program, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  2. 2.Department of Chemical EngineeringNortheastern UniversityBostonUSA
  3. 3.Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  4. 4.Department of BiologyNortheastern UniversityBostonUSA
  5. 5.Department of Biological Science, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  6. 6.Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  7. 7.Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA

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