Effect of elevated extracellular glucose on adhesion and migration of sv40 transformed human corneal epithelial cells

A. M. McDermott, T. S. Kern, Christopher J Murphy

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Abstract

Purpose Corneal epithelial wound healing requires orchestrated cell proliferation, migration and adhesion. Disturbances in any or all of these events may underlie impaired corneal wound healing in diabetes. To separate these components, we have begun to characterize the effects of elevated extracellular glucose concentration on SV40 transformed human corneal epithelial cells (Araki-Sasaki, et ai IOVS, 1995, 36 614-621) Methods Cells were maintained in DMEM/F12 (1 1) or in this media containing 38mM glucose Adhesion Cells were allowed to adhere for 1 hour at 37°C to 96 well plates coated with extracellular matrix proteins. Non-adherent cells were removed by washing, then attached cells quantitated by using the fluorescent dye Calcein AM Migration: Cell migration in response to 10% fetal bovine serum was determined in blind well migration chambers. Results Adhesion Collagen 1 was the preferred substrate, then fibronectin = vitronectin > laminin (ECso 0 15+/-004, 3 95+/-1 9, 6 1+/-2 1 and >100ug/ml respectively, n=2or3). Pre-incubation of the cells with a monoclonal antibody to βl-integrin inhibited adhesion, indicating involvement of this protein in adhesion. Western blotting with an anti-βl polyclonal antibody confirmed the presence of this integrin in these cells. Exposure of the cells to 38mM glucose for i-5 weeks had no significant effect on adhesion. In contrast, cell migration was attenuated by up to 50% in cells that had been maintained in 38mM glucose Conclusions Supplementation of the growth media with glucose did not affect the adherent capabilities of SV40 transformed human corneal epithelial cells, but did lead to attenuated cell migration.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume38
Issue number4
StatePublished - 1997
Externally publishedYes

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Epithelial Cells
Glucose
Cell Movement
Cell Adhesion
Integrins
Wound Healing
Vitronectin
Extracellular Matrix Proteins
Laminin
Fluorescent Dyes
Fibronectins
Collagen
Western Blotting
Monoclonal Antibodies
Cell Proliferation
Antibodies
Growth
Serum
Proteins

ASJC Scopus subject areas

  • Ophthalmology

Cite this

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title = "Effect of elevated extracellular glucose on adhesion and migration of sv40 transformed human corneal epithelial cells",
abstract = "Purpose Corneal epithelial wound healing requires orchestrated cell proliferation, migration and adhesion. Disturbances in any or all of these events may underlie impaired corneal wound healing in diabetes. To separate these components, we have begun to characterize the effects of elevated extracellular glucose concentration on SV40 transformed human corneal epithelial cells (Araki-Sasaki, et ai IOVS, 1995, 36 614-621) Methods Cells were maintained in DMEM/F12 (1 1) or in this media containing 38mM glucose Adhesion Cells were allowed to adhere for 1 hour at 37°C to 96 well plates coated with extracellular matrix proteins. Non-adherent cells were removed by washing, then attached cells quantitated by using the fluorescent dye Calcein AM Migration: Cell migration in response to 10{\%} fetal bovine serum was determined in blind well migration chambers. Results Adhesion Collagen 1 was the preferred substrate, then fibronectin = vitronectin > laminin (ECso 0 15+/-004, 3 95+/-1 9, 6 1+/-2 1 and >100ug/ml respectively, n=2or3). Pre-incubation of the cells with a monoclonal antibody to βl-integrin inhibited adhesion, indicating involvement of this protein in adhesion. Western blotting with an anti-βl polyclonal antibody confirmed the presence of this integrin in these cells. Exposure of the cells to 38mM glucose for i-5 weeks had no significant effect on adhesion. In contrast, cell migration was attenuated by up to 50{\%} in cells that had been maintained in 38mM glucose Conclusions Supplementation of the growth media with glucose did not affect the adherent capabilities of SV40 transformed human corneal epithelial cells, but did lead to attenuated cell migration.",
author = "McDermott, {A. M.} and Kern, {T. S.} and Murphy, {Christopher J}",
year = "1997",
language = "English (US)",
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issn = "0146-0404",
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TY - JOUR

T1 - Effect of elevated extracellular glucose on adhesion and migration of sv40 transformed human corneal epithelial cells

AU - McDermott, A. M.

AU - Kern, T. S.

AU - Murphy, Christopher J

PY - 1997

Y1 - 1997

N2 - Purpose Corneal epithelial wound healing requires orchestrated cell proliferation, migration and adhesion. Disturbances in any or all of these events may underlie impaired corneal wound healing in diabetes. To separate these components, we have begun to characterize the effects of elevated extracellular glucose concentration on SV40 transformed human corneal epithelial cells (Araki-Sasaki, et ai IOVS, 1995, 36 614-621) Methods Cells were maintained in DMEM/F12 (1 1) or in this media containing 38mM glucose Adhesion Cells were allowed to adhere for 1 hour at 37°C to 96 well plates coated with extracellular matrix proteins. Non-adherent cells were removed by washing, then attached cells quantitated by using the fluorescent dye Calcein AM Migration: Cell migration in response to 10% fetal bovine serum was determined in blind well migration chambers. Results Adhesion Collagen 1 was the preferred substrate, then fibronectin = vitronectin > laminin (ECso 0 15+/-004, 3 95+/-1 9, 6 1+/-2 1 and >100ug/ml respectively, n=2or3). Pre-incubation of the cells with a monoclonal antibody to βl-integrin inhibited adhesion, indicating involvement of this protein in adhesion. Western blotting with an anti-βl polyclonal antibody confirmed the presence of this integrin in these cells. Exposure of the cells to 38mM glucose for i-5 weeks had no significant effect on adhesion. In contrast, cell migration was attenuated by up to 50% in cells that had been maintained in 38mM glucose Conclusions Supplementation of the growth media with glucose did not affect the adherent capabilities of SV40 transformed human corneal epithelial cells, but did lead to attenuated cell migration.

AB - Purpose Corneal epithelial wound healing requires orchestrated cell proliferation, migration and adhesion. Disturbances in any or all of these events may underlie impaired corneal wound healing in diabetes. To separate these components, we have begun to characterize the effects of elevated extracellular glucose concentration on SV40 transformed human corneal epithelial cells (Araki-Sasaki, et ai IOVS, 1995, 36 614-621) Methods Cells were maintained in DMEM/F12 (1 1) or in this media containing 38mM glucose Adhesion Cells were allowed to adhere for 1 hour at 37°C to 96 well plates coated with extracellular matrix proteins. Non-adherent cells were removed by washing, then attached cells quantitated by using the fluorescent dye Calcein AM Migration: Cell migration in response to 10% fetal bovine serum was determined in blind well migration chambers. Results Adhesion Collagen 1 was the preferred substrate, then fibronectin = vitronectin > laminin (ECso 0 15+/-004, 3 95+/-1 9, 6 1+/-2 1 and >100ug/ml respectively, n=2or3). Pre-incubation of the cells with a monoclonal antibody to βl-integrin inhibited adhesion, indicating involvement of this protein in adhesion. Western blotting with an anti-βl polyclonal antibody confirmed the presence of this integrin in these cells. Exposure of the cells to 38mM glucose for i-5 weeks had no significant effect on adhesion. In contrast, cell migration was attenuated by up to 50% in cells that had been maintained in 38mM glucose Conclusions Supplementation of the growth media with glucose did not affect the adherent capabilities of SV40 transformed human corneal epithelial cells, but did lead to attenuated cell migration.

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