Molecular and Cellular Biochemistry

, Volume 402, Issue 1–2, pp 123–139 | Cite as

Limbal epithelial stem-microenvironmental alteration leads to pterygium development

  • Prosun Das
  • Arjun Gokani
  • Ketaki Bagchi
  • Gautam Bhaduri
  • Samaresh Chaudhuri
  • Sujata Law


Maintenance of tissue homeostasis relies on the accurate regulation of tissue specific stem cell activity which is governed by the dynamic interaction between the positive and negative feedback modulating mechanism of stem cell microenvironmental niche. Alteration or deregulation of the “stem-microenvironmental networking” provokes disease development. Limbal epithelial stem cells (LESC) are the initiator hierarchy that maintains corneal integrity. Compartmentalization of LESC within the limbal vicinity provides an opportunity to understand the stem-microenvironmental relationship. The purpose of this study was to determine the microenvironmental alteration associated with LESCs fate in pterygium condition in comparison with healthy state. Clinical observations evaluated the ocular surface disorder with respect to corneal vascularization, tear film abnormality, and thickening of limbal area in pterygium patients. Structural alteration of limbal stem/progenitor cells and its neighboring niche components were observed using histology and scanning electron microscopy. Receptor overexpression of TGFβ-R1, EGF-R1, and IL6-Rα and alteration of IL2-Rα expression pointed toward aberration of “stem-microenvironmental networking” in the limbal vicinity during disease development. Increased cell proliferation index along with TERT, Cyclin-D1, and PCNA over-expression in limbal part of pterygium epithelial cells indicated increased cellular proliferation and disturbed homeostatic equilibrium. We postulate that pterygium is associated with limbal microenvironmental anomaly where the resident epithelial cells became hyperproliferative.


Pterygium Stem-microenvironmental networks Cytology Scanning electron microscopy Explant culture Growth factor Cell cycle 



Limbal epithelial stem cells


Ultra violet-B


Transforming growth factor beta-receptor 1


Epidermal growth factor-receptor 1


Interleukin 6- Receptor alpha


Telomerase reverse transcriptase


Proliferating cell nuclear antigen


Anterior segment optical coherence tomography


Peripheral corneal thickness


Central corneal thickness


Scanning electron microscopy


Proliferation index


Limbal stem cell deficiency diseases


Epithelial–mesenchymal transition


Mitogen activated protein kinase


Glycoprotein 130


Fetal bovine serum



We are thankful to the Indian Council of Medical Research (ICMR) for their sponsorship, The Director, Calcutta School of Tropical Medicine (CSTM), The Director, Regional Institute of Ophthalmology (RIO) Kolkata and The West Bengal University of Health Sciences (WBUHS) for the facilities provided. This work was supported by Indian Council of Medical Research (80/10/2008/BMS/Stem cell).

Conflict of interest

Authors indicate no potential conflicts of interest.


  1. 1.
    Hime GR, Somers WG (2009) Micro-RNA mediated regulation of proliferation, self-renewal and differentiation of mammalian stem cells. Cell Adh Migr 3(4):425–432CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Biteau B, Hochmuth CE, Jasper H (2011) Maintaining tissue homeostasis: dynamic control of somatic stem cell activity. Cell Stem Cell 9(5):402–411CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Wong VW, Stange DE, Page ME, Buczacki S, Wabik A, Itami S, van de Wetering M, Poulsom R, Wright NA, Trotter MW, Watt FM, Winton DJ, Clevers H, Jensen KB (2012) Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signaling. Nat Cell Biol 14(4):401–408CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Jones DL, Wagers AJ (2008) No place like home: anatomy and function of the stem cell niche. Nat Rev Mol Cell Biol 9(1):11–21CrossRefPubMedGoogle Scholar
  5. 5.
    Scadden DT (2006) The stem-cell niche as an entity of action. Nature 441(7097):1075–1079CrossRefPubMedGoogle Scholar
  6. 6.
    Rizo A, Vellenga E, de Haan G, Schuringa JJ (2006) Signaling pathways in self-renewing hematopoietic and leukemic stem cells: do all stem cells need a niche? Hum Mol Genet 15(2):210–219CrossRefGoogle Scholar
  7. 7.
    Keung AJ, Kumar S, Schaffer DV (2010) Presentation counts: microenvironmental regulation of stem cells by biophysical and material cues. Annu Rev Cell Dev Biol 26:533–556CrossRefPubMedGoogle Scholar
  8. 8.
    Das P, Chatterjee S, Basak P, Das M, Pereira JA, Dutta R, Chaklader M, Bagchi K, Bhaduri G, Chaudhuri S, Law S (2010) Sca-1 expression pattern in the mouse limbal epithelium and its association with cell cycle. J Stem Cells 5(2):65–74PubMedGoogle Scholar
  9. 9.
    Shortt AJ, Secker GA, Munro PM, Khaw PT, Tuft SJ, Daniels JT (2007) Characterization of the limbal epithelial stem cell niche: novel imaging techniques permit in vivo observation and targeted biopsy of limbal epithelial stem cells. Stem Cells 25(6):1402–1409CrossRefPubMedGoogle Scholar
  10. 10.
    Lathrop KL, Gupta D, Kagemann L, Schuman JS, Sundarraj N (2012) Optical coherence tomography as a rapid, accurate, noncontact method of visualizing the palisades of Vogt. Invest Ophthalmol Vis Sci 53(3):1381–1387CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Pajoohesh-Ganji A, Stepp MA (2005) In search of markers for the stem cells of the corneal epithelium. Biol Cell 97(4):265–276CrossRefPubMedGoogle Scholar
  12. 12.
    Crane AM, Hua HU, Coggin AD, Gugiu G, Lam BL, Bhattacharya SK (2012) Mass spectrometric analyses of phosphatidylcholines in alkali-exposed corneal tissue. Invest Ophthalmol Vis Sci 53(11):7122–7130CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Norval M, Lucas RM, Cullen AP, de Gruijl R, Longstreth J, Takizawa Y, van der Leun JC (2011) The human health effects of ozone depletion and interactions with climate change. Photochem Photobiol Sci 10(2):199–225CrossRefPubMedGoogle Scholar
  14. 14.
    Singh J, Singh RP, Dubey AK (2012) Effects of ultraviolet-B (UV-B) radiation on two cryptogamic plants pigments growing at high altitude of central Himalayan region, India. African J Env Sci Tech 6(1):9–16Google Scholar
  15. 15.
    Wang F, Gao Q, Hu L, Gao N, Ge T, Yu J, Liu Y (2012) Risk of eye damage from the wavelength-dependent biologically effective UVB spectrum irradiances. PLoS ONE 7(12):e52259CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Moran DJ, Hollows FC (1984) Pterygium and ultraviolet radiation: a positive correlation. Br J Ophthalmol 68(5):343–346CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Ayanniyi AA, Badmos KB, Olatunji FO, Owoeye JF, Sanni TO (2011) Blindness caused by Pterygium. Sierra Leone J Biomed Res 3(1):60–62CrossRefGoogle Scholar
  18. 18.
    Shiroma H, Higa A, Sawaguchi S, Iwase A, Tomidokoro A, Amano S, Araie M (2009) Prevalence and risk factors of pterygium in a southwestern island of Japan: the Kumejima Study. Am J Ophthalmol 148(5):766–771CrossRefPubMedGoogle Scholar
  19. 19.
    Youngson RM (1972) Recurrence of pterygium after excision. Br J Ophthalmol 56(2):120–125CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Sebastiá R, Ventura MP, Solari HP, Antecka E, Orellana ME, Burnier MN (2013) Immunohistochemical detection of Hsp90 and Ki-67 in pterygium. Diagn Pathol 8(32):1–7Google Scholar
  21. 21.
    Dushku N, John MK, Schultz GS, Reid TW (2001) Pterygia pathogenesis: corneal invasion by matrix metalloproteinase expressing altered limbal epithelial basal cells. Arch Ophthalmol 119(5):695–706CrossRefPubMedGoogle Scholar
  22. 22.
    Menzel-Severing J, Polisetti N, Schlötzer-Schrehardt U, Kruse F (2012) Limbal stem cells and their niche: implications for bioengineered tissue constructs. Klin Monbl Augenheilkd 229(12):1191–1197CrossRefPubMedGoogle Scholar
  23. 23.
    Ordonez P, Di Girolamo N (2012) Limbal epithelial stem cells: role of the niche microenvironment. Stem Cells 30(2):100–107CrossRefPubMedGoogle Scholar
  24. 24.
    Marc B & Taub OD (2004) Ocular effects of ultraviolet radiation. UV special, UV special. Optometry Today pp 34–38Google Scholar
  25. 25.
    Dohlman CH (1971) The function of the corneal epithelium in health and disease. The Jonas S. Friedenwald Memorial Lecture. Invest Ophthalmol 10(6):383–407PubMedGoogle Scholar
  26. 26.
    Patwardhan AA, Khan M, Mollan SP, Haigh P (2008) The importance of central corneal thickness measurements and decision making in general ophthalmology clinics: a masked observational study. BMC Ophthalmol 8:1–5CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Bechmann M, Thiel M, Roesen B, Ullrich S, Ulbig MW, Ludwig K (2000) Central corneal thickness determined with optical coherence tomography in various types of glaucoma. Br J Ophthalmol 84(11):1233–1237CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Wang J, Abou SM, Perez VL, Karp CL, Yoo SH, Shen M, Cui L, Hurmeric V, Du C, Zhu D, Chen Q, Li M (2011) Ultra-high resolution optical coherence tomography for imaging the anterior segment of the eye. Ophthalmic Surg Lasers Imag 42(Suppl):S15–S27CrossRefGoogle Scholar
  29. 29.
    Kieval JZ, Karp CL, Abou SM, Galor A, Hoffman RA, Dubovy SR, Wang J (2012) Ultra-high resolution optical coherence tomography for differentiation of ocular surface squamous neoplasia and pterygia. Ophthalmol 119(3):481–486CrossRefGoogle Scholar
  30. 30.
    Soliman W, Mohamed TA (2012) Spectral domain anterior segment optical coherence tomography assessment of pterygium and pinguecula. Acta Ophthalmol 90:461–465CrossRefPubMedGoogle Scholar
  31. 31.
    Buchwald HJ, Muller A, Kampmeier J, Lang GK (2003) Optical coherence tomography versus ultrasound biomicroscopy of conjunctival and eyelid lesions. Klin Monbl Augenheilkd 220:822–829CrossRefPubMedGoogle Scholar
  32. 32.
    Welch MN, Reilly CD, Kalwerisky K, Johnson A, Waller SG (2011) Pterygia measurements are more accurate with anterior segment optical coherence tomography—a pilot study. Nep. J. Ophthalmol. 3(5):9–12Google Scholar
  33. 33.
    Sangwan VS (2001) Limbal stem cells in health and disease. Biosci Rep 21:385–405CrossRefPubMedGoogle Scholar
  34. 34.
    Egbert PR, Lauber S, Maurice DM (1977) A simple conjunctival biopsy. Am J Ophthalmol 84:798–801CrossRefPubMedGoogle Scholar
  35. 35.
    Tseng SCG, Chen JJY, Huang AJW, Kruse FE, Maskin SL, Tsai RJF (1990) Classification of conjunctival surgeries for corneal disease based on stem cell concept. Ophthalmol Clin N Am 3:595–610Google Scholar
  36. 36.
    Puangsricharern V, Tseng SC (1995) Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology 102(10):1476–1485CrossRefPubMedGoogle Scholar
  37. 37.
    Anderson DF, Ellies P, Pires RT, Tseng SC (2001) Amniotic membrane in partial limbal stem cell deficiency. Br J Ophthalmol 85:567–575CrossRefPubMedCentralPubMedGoogle Scholar
  38. 38.
    Fatima A, Iftekhar G, Sangwan VS, Vemuganti GK (2008) Ocular surface changes in limbal stem cell deficiency caused by chemical injury: a histologic study of excised pannus from recipients of cultured corneal epithelium. Eye 22:1161–1167CrossRefPubMedGoogle Scholar
  39. 39.
    Sejpal K, Bakhtiari P, Deng SX (2013) Presentation, diagnosis and management of limbal stem cell deficiency. Middle East Afr J Ophthalmol 20(1):5–10CrossRefPubMedCentralPubMedGoogle Scholar
  40. 40.
    Ahmad S (2012) Concise review: limbal stem cell deficiency, dysfunction, and distress. Stem Cells Transl Med 1(2):110–115CrossRefPubMedCentralPubMedGoogle Scholar
  41. 41.
    Felix N, Marina K (2013) The bone marrow microenvironment as niche retreats for hematopoietic and leukemic stem cells. Adv Hematol 2013:953–982Google Scholar
  42. 42.
    Raaijmakers MH, Scadden DT (2008) Evolving concepts on the microenvironmental niche for hematopoietic stem cells. Curr Opin Hematol 15(4):301–306CrossRefPubMedGoogle Scholar
  43. 43.
    Bron A (2001) The architecture of the corneal stroma. Br J Ophthalmol 85(4):379–381CrossRefPubMedCentralPubMedGoogle Scholar
  44. 44.
    Chan CM, Liu YP, Tan DT (2002) Ocular surface changes in pterygium. Cornea 21(1):38–42CrossRefPubMedGoogle Scholar
  45. 45.
    Bandyopadhyay R, Nag D, Mondal SK, Gangopadhyay S, Bagchi K, Bhaduri G (2010) Ocular surface disorder in pterygium: role of conjunctival impression cytology. Indian J Pathol Microbiol 53:692–695CrossRefPubMedGoogle Scholar
  46. 46.
    Shimazaki J, Yang H, Tsubata K (1997) Amniotic membrane transplantation for ocular surface reconstruction in patients with chemical and thermal burns. Ophthalmol 104:2068–2076CrossRefGoogle Scholar
  47. 47.
    Nelson DJ (1988) Impression cytology. Cornea 7:71–81CrossRefPubMedGoogle Scholar
  48. 48.
    Das P, Pereira JA, Chaklader M, Law A, Bagchi K, Bhaduri G, Chaudhuri S, Law S (2013) Phenotypic alteration of limbal niche-associated limbal epithelial stem cell deficiency by ultraviolet-B exposure–induced phototoxicity in mice. Biochem Cell Biol 91(3):165–175CrossRefPubMedGoogle Scholar
  49. 49.
    Joseph A, Powell-Richards AO, Shanmuganathan VA, Dua HS (2004) Epithelial cell characteristics of cultured human limbal explants. Br J Ophthalmol 88(3):393–398CrossRefPubMedCentralPubMedGoogle Scholar
  50. 50.
    Kato N, Shimmura S, Kawakita T, Miyashita H, Ogawa Y, Yoshida S, Higa K, Okano H, Tsubota K (2007) Beta-catenin activation and epithelial-mesenchymal transition in the pathogenesis of pterygium. Invest Ophthalmol Vis Sci 48(4):1511–1517CrossRefPubMedGoogle Scholar
  51. 51.
    Willis BC, Borok Z (2007) TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol 293(3):L525–L534CrossRefPubMedGoogle Scholar
  52. 52.
    Nishimura T, Toda S, Mitsumoto T, Oono S, Sugihara H (1998) Effects of hepatocyte growth factor, transforming growth factor-beta1 and epidermal growth factor on bovine corneal epithelial cells under epithelial-keratocyte interaction in reconstruction culture. Exp Eye Res 66(1):105–116CrossRefPubMedGoogle Scholar
  53. 53.
    Wilson SE, He YG, Lloyd SA (1992) EGF, EGF receptor, basic FGF, TGF beta-1, and IL-1 alpha mRNA in human corneal epithelial cells and stromal fibroblasts. Invest Ophthalmol Vis Sci 33(5):1756–1765PubMedGoogle Scholar
  54. 54.
    Di Girolamo N, Coroneo M, Wakefield D (2005) Epidermal growth factor receptor signaling is partially responsible for the increased matrix metalloproteinase-1 expression in ocular epithelial cells after UVB radiation. Am J Pathol 167(2):489–503CrossRefPubMedCentralPubMedGoogle Scholar
  55. 55.
    Sullivan NJ, Sasser AK, Axel AE, Vesuna F, Raman V, Ramirez N, Oberyszyn TM, Hall BM (2009) Interleukin-6 induces an epithelial-mesenchymal transition phenotype in human breast cancer cells. Oncogene 28(33):2940–2947CrossRefPubMedGoogle Scholar
  56. 56.
    Somers W, Stahl M, Seehra JS (1997) 1.9 A crystal structure of interleukin 6: implications for a novel mode of receptor dimerization and signaling. EMBO J 16(5):989–997CrossRefPubMedCentralPubMedGoogle Scholar
  57. 57.
    Jager MJ, Gregerson DS, Streilein JW (1995) Regulators of immunological responses in the cornea and the anterior chamber of the eye 9(Pt 2):241–246Google Scholar
  58. 58.
    Akpek EK, Gottsch JD (2003) Immune defense at the ocular surface. Eye (Lond) 17(8):949–956CrossRefGoogle Scholar
  59. 59.
    Waldmann TA (2006) The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design. Nat Rev Immunol 6(8):595–601CrossRefPubMedGoogle Scholar
  60. 60.
    Taniguchi T, Minami Y (1993) The IL-2/IL-2 receptor system: a current overview. Cell 73(1):5–8CrossRefPubMedGoogle Scholar
  61. 61.
    De Paiva CS, Yoon KC, Pangelinan SB, Pham S, Puthenparambil LM, Chuang EY, Farley WJ, Stern ME, Li DQ, Pflugfelder SC (2009) Cleavage of functional IL-2 receptor alpha chain (CD25) from murine corneal and conjunctival epithelia by MMP-9. J Inflamm (Lond) 6:31CrossRefGoogle Scholar
  62. 62.
    Cotsarelis G, Cheng G, Dong G, Sun TT, Lavker RM (1989) Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell 57:201–209CrossRefPubMedGoogle Scholar
  63. 63.
    Lehrer MS, Sun TT, Lavker RM (1998) Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation. J Cell Sci 111(Pt 19):2867–2875PubMedGoogle Scholar
  64. 64.
    Chung EH, Hutcheon AE, Joyce NC, Zieske JD (1999) Synchronization of the G1/S transition in response to corneal debridement. Invest Ophthalmol Vis Sci 40(9):1952–1958PubMedGoogle Scholar
  65. 65.
    Joyce NC, Meklir B, Joyce SJ, Zieske JD (1996) Cell cycle protein expression and proliferative status in human corneal cells. Invest Ophthalmol Vis Sci 37(4):645–655PubMedGoogle Scholar
  66. 66.
    Sandell LL, Zakian VA (1993) Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell 75(4):729–739CrossRefPubMedGoogle Scholar
  67. 67.
    Schiestl RH (1989) DNA-damaging agents show different kinetics in induction of heterothallic mating-type switching during growth after treatment in yeast. Mutat Res 227(4):269–274CrossRefPubMedGoogle Scholar
  68. 68.
    Colitz CM, Sugimoto Y, Lu P, Barden CA, Thomas-Ahner J, Chandler HL (2009) ERalpha increases expression and interacts with TERT in cataractous canine lens epithelial cells. Mol Vis 15:2259–2267PubMedCentralPubMedGoogle Scholar
  69. 69.
    Lamy E, Herz C, Lutz-Bonengel S, Hertrampf A, Márton MR, Mersch-Sundermann V (2013) The MAPK pathway signals telomerase modulation in response to isothiocyanate-induced DNA damage of human liver cancer cells. PLoS ONE 8(1):e53240CrossRefPubMedCentralPubMedGoogle Scholar
  70. 70.
    Holt SE, Wright WE, Shay JW (1996) Regulation of telomerase activity in immortal cell lines. Mol Cell Biol 16(6):2932–2939PubMedCentralPubMedGoogle Scholar
  71. 71.
    Engelhardt M, Kumar R, Albanell J, Pettengell R, Han W, Moore MA (1997) Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells. Blood 90(1):182–193PubMedGoogle Scholar
  72. 72.
    Ueda M, Ouhtit A, Bito T, Nakazawa K, Lübbe J, Ichihashi M, Yamasaki H, Nakazawa H (1997) Evidence for UV-associated activation of telomerase in human skin. Cancer Res 57(3):370–374PubMedGoogle Scholar
  73. 73.
    Sherr CJ (1993) Mammalian G1 cyclins. Cell 73(6):1059–1065CrossRefPubMedGoogle Scholar
  74. 74.
    Yang K, Hitomi M, Stacey DW (2006) Variations in cyclin D1 levels through the cell cycle determine the proliferative fate of a cell. Cell Div 1:32CrossRefPubMedCentralPubMedGoogle Scholar
  75. 75.
    Kubben FJ, Peeters-Haesevoets A, Engels LG, Baeten CG, Schutte B, Arends JW, Stockbrügger RW, Blijham GH (1994) Proliferating cell nuclear antigen (PCNA): a new marker to study human colonic cell proliferation. Gut 35(4):530–535CrossRefPubMedCentralPubMedGoogle Scholar
  76. 76.
    Shivji KK, Kenny MK, Wood RD (1992) Proliferating cell nuclear antigen is required for DNA excision repair. Cell 69(2):367–374CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Prosun Das
    • 1
  • Arjun Gokani
    • 1
  • Ketaki Bagchi
    • 1
  • Gautam Bhaduri
    • 1
  • Samaresh Chaudhuri
    • 1
  • Sujata Law
    • 1
  1. 1.Stem Cell Research and Application Unit, Department of Biochemistry and Medical BiotechnologyCalcutta School of Tropical MedicineKolkataIndia

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