VEGF rescues cigarette smoking-induced human RPE cell death by increasing autophagic flux: Implications of the role of autophagy in advanced age-related macular degeneration

Young Kwang Chu, Sungchul Lee, Suk Ho Byeon

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11 Citations (Scopus)

Abstract

Purpose. Cigarette smoking (CS) is the most consistent risk factor for advanced age-related macular degeneration (AMD). To verify the molecular basis for CS-induced RPE alterations, RPE cell survival levels after being exposed to CS in relation with VEGF expression and autophagic flux were evaluated. Methods. Cigarette smoking extract (CSE) was added to ARPE-19 cells and hydrogen peroxide (HP) was used as a pure oxidant control. Cell survival was measured by flow cytometry with annexin V-fluorescein isothiocyanate. Cell survival analysis was performed after pretreatment with anti-VEGF or recombinant VEGF. The expression of VEGF-A, VEGF-R1/R2, and soluble VEGF-R1 was determined by semiquantitative RT-PCR. LC3B-I (microtubule-associated protein-1 inhibitors), LC3B-II, and phosphorylation of Akt or Erk were measured with Western blot. Autophagic flux was determined by increasing LC3B-II levels with inhibitors of lysosomal proteases. Results. Incubation with 5% CSE for 16 hours induced approximately 30% cell death, which was similar to cell death levels when exposed to concentrations of 200 μM HP. Pretreatment with anti-VEGF did not decrease cell survival under CSE, unlike the decrease in cell survival shown with HP. However, supplementation with VEGF rescued CSE-induced RPE cell death. Interestingly, CSE caused an increase in autophagic flux, which was augmented with VEGF pretreatment. Cigarette smoking extract also degraded the total amounts of Akt levels, and VEGF blunted CSE-induced phosphorylation of Erk. Conclusions. Cigarette smoking extract, similar to HP, affects cell viability and induces expression of VEGF and its receptors. Increased autophagic flux accelerated by treatment of exogenous VEGF may have a role in rescuing CSE-induced RPE cell death.

Original languageEnglish
Pages (from-to)7329-7337
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume54
Issue number12
DOIs
Publication statusPublished - 2013 Oct 1

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Autophagy
Macular Degeneration
Vascular Endothelial Growth Factor A
Smoking
Cell Survival
Hydrogen Peroxide
Cell Death
Phosphorylation
Vascular Endothelial Growth Factor Receptor
Microtubule-Associated Proteins
Annexin A5
Survival Analysis
Protease Inhibitors
Fluorescein
Oxidants
Flow Cytometry
Western Blotting

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

@article{1ab4ce7282d74a0da933bf7e28768611,
title = "VEGF rescues cigarette smoking-induced human RPE cell death by increasing autophagic flux: Implications of the role of autophagy in advanced age-related macular degeneration",
abstract = "Purpose. Cigarette smoking (CS) is the most consistent risk factor for advanced age-related macular degeneration (AMD). To verify the molecular basis for CS-induced RPE alterations, RPE cell survival levels after being exposed to CS in relation with VEGF expression and autophagic flux were evaluated. Methods. Cigarette smoking extract (CSE) was added to ARPE-19 cells and hydrogen peroxide (HP) was used as a pure oxidant control. Cell survival was measured by flow cytometry with annexin V-fluorescein isothiocyanate. Cell survival analysis was performed after pretreatment with anti-VEGF or recombinant VEGF. The expression of VEGF-A, VEGF-R1/R2, and soluble VEGF-R1 was determined by semiquantitative RT-PCR. LC3B-I (microtubule-associated protein-1 inhibitors), LC3B-II, and phosphorylation of Akt or Erk were measured with Western blot. Autophagic flux was determined by increasing LC3B-II levels with inhibitors of lysosomal proteases. Results. Incubation with 5{\%} CSE for 16 hours induced approximately 30{\%} cell death, which was similar to cell death levels when exposed to concentrations of 200 μM HP. Pretreatment with anti-VEGF did not decrease cell survival under CSE, unlike the decrease in cell survival shown with HP. However, supplementation with VEGF rescued CSE-induced RPE cell death. Interestingly, CSE caused an increase in autophagic flux, which was augmented with VEGF pretreatment. Cigarette smoking extract also degraded the total amounts of Akt levels, and VEGF blunted CSE-induced phosphorylation of Erk. Conclusions. Cigarette smoking extract, similar to HP, affects cell viability and induces expression of VEGF and its receptors. Increased autophagic flux accelerated by treatment of exogenous VEGF may have a role in rescuing CSE-induced RPE cell death.",
author = "Chu, {Young Kwang} and Sungchul Lee and Byeon, {Suk Ho}",
year = "2013",
month = "10",
day = "1",
doi = "10.1167/iovs.13-12149",
language = "English",
volume = "54",
pages = "7329--7337",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "12",

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TY - JOUR

T1 - VEGF rescues cigarette smoking-induced human RPE cell death by increasing autophagic flux

T2 - Implications of the role of autophagy in advanced age-related macular degeneration

AU - Chu, Young Kwang

AU - Lee, Sungchul

AU - Byeon, Suk Ho

PY - 2013/10/1

Y1 - 2013/10/1

N2 - Purpose. Cigarette smoking (CS) is the most consistent risk factor for advanced age-related macular degeneration (AMD). To verify the molecular basis for CS-induced RPE alterations, RPE cell survival levels after being exposed to CS in relation with VEGF expression and autophagic flux were evaluated. Methods. Cigarette smoking extract (CSE) was added to ARPE-19 cells and hydrogen peroxide (HP) was used as a pure oxidant control. Cell survival was measured by flow cytometry with annexin V-fluorescein isothiocyanate. Cell survival analysis was performed after pretreatment with anti-VEGF or recombinant VEGF. The expression of VEGF-A, VEGF-R1/R2, and soluble VEGF-R1 was determined by semiquantitative RT-PCR. LC3B-I (microtubule-associated protein-1 inhibitors), LC3B-II, and phosphorylation of Akt or Erk were measured with Western blot. Autophagic flux was determined by increasing LC3B-II levels with inhibitors of lysosomal proteases. Results. Incubation with 5% CSE for 16 hours induced approximately 30% cell death, which was similar to cell death levels when exposed to concentrations of 200 μM HP. Pretreatment with anti-VEGF did not decrease cell survival under CSE, unlike the decrease in cell survival shown with HP. However, supplementation with VEGF rescued CSE-induced RPE cell death. Interestingly, CSE caused an increase in autophagic flux, which was augmented with VEGF pretreatment. Cigarette smoking extract also degraded the total amounts of Akt levels, and VEGF blunted CSE-induced phosphorylation of Erk. Conclusions. Cigarette smoking extract, similar to HP, affects cell viability and induces expression of VEGF and its receptors. Increased autophagic flux accelerated by treatment of exogenous VEGF may have a role in rescuing CSE-induced RPE cell death.

AB - Purpose. Cigarette smoking (CS) is the most consistent risk factor for advanced age-related macular degeneration (AMD). To verify the molecular basis for CS-induced RPE alterations, RPE cell survival levels after being exposed to CS in relation with VEGF expression and autophagic flux were evaluated. Methods. Cigarette smoking extract (CSE) was added to ARPE-19 cells and hydrogen peroxide (HP) was used as a pure oxidant control. Cell survival was measured by flow cytometry with annexin V-fluorescein isothiocyanate. Cell survival analysis was performed after pretreatment with anti-VEGF or recombinant VEGF. The expression of VEGF-A, VEGF-R1/R2, and soluble VEGF-R1 was determined by semiquantitative RT-PCR. LC3B-I (microtubule-associated protein-1 inhibitors), LC3B-II, and phosphorylation of Akt or Erk were measured with Western blot. Autophagic flux was determined by increasing LC3B-II levels with inhibitors of lysosomal proteases. Results. Incubation with 5% CSE for 16 hours induced approximately 30% cell death, which was similar to cell death levels when exposed to concentrations of 200 μM HP. Pretreatment with anti-VEGF did not decrease cell survival under CSE, unlike the decrease in cell survival shown with HP. However, supplementation with VEGF rescued CSE-induced RPE cell death. Interestingly, CSE caused an increase in autophagic flux, which was augmented with VEGF pretreatment. Cigarette smoking extract also degraded the total amounts of Akt levels, and VEGF blunted CSE-induced phosphorylation of Erk. Conclusions. Cigarette smoking extract, similar to HP, affects cell viability and induces expression of VEGF and its receptors. Increased autophagic flux accelerated by treatment of exogenous VEGF may have a role in rescuing CSE-induced RPE cell death.

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U2 - 10.1167/iovs.13-12149

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