Limits...
Cigarette smoke-related hydroquinone dysregulates MCP-1, VEGF and PEDF expression in retinal pigment epithelium in vitro and in vivo.

Pons M, Marin-Castaño ME - PLoS ONE (2011)

Bottom Line: Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls.VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio.Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Miller School of Medicine, Bascom Palmer Eye Institute, University of Miami, Miami, Florida, United States of America.

ABSTRACT

Background: Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly population. Debris (termed drusen) below the retinal pigment epithelium (RPE) have been recognized as a risk factor for dry AMD and its progression to wet AMD, which is characterized by choroidal neovascularization (CNV). The underlying mechanism of how drusen might elicit CNV remains undefined. Cigarette smoking, oxidative damage to the RPE and inflammation are postulated to be involved in the pathophysiology of the disease. To better understand the cellular mechanism(s) linking oxidative stress and inflammation to AMD, we examined the expression of pro-inflammatory monocyte chemoattractant protein-1 (MCP-1), pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial derived factor (PEDF) in RPE from smoker patients with AMD. We also evaluated the effects of hydroquinone (HQ), a major pro-oxidant in cigarette smoke on MCP-1, VEGF and PEDF expression in cultured ARPE-19 cells and RPE/choroids from C57BL/6 mice.

Principal findings: MCP-1, VEGF and PEDF expression was examined by real-time PCR, Western blot, and ELISA. Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls. Both MCP-1 mRNA and protein were downregulated in ARPE-19 cells and RPE/choroids from C57BL/6 mice after 5 days and 3 weeks of exposure to HQ-induced oxidative injury. VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio. Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo.

Conclusion: We propose that impaired RPE-derived MCP-1-mediated scavenging macrophages recruitment and phagocytosis might lead to incomplete clearance of proinflammatory debris and infiltration of proangiogenic macrophages which along with increased VEGF/PEDF ratio favoring angiogenesis might promote drusen accumulation and progression to CNV in smoker patients with dry AMD.

Show MeSH

Related in: MedlinePlus

Sustained and repetitive HQ-induced oxidative injury decreases on MCP-1 expression in human RPE cells.Confluent serum-starved ARPE-19 cells were treated with (A, B) 10 µM HQ every 24 hours for 5 consecutive days or (C, D) 50 µM HQ every 4 days for 24 hours for 3 consecutive weeks in phenol red-free 0.1% FBS medium. Total RNA was extracted to assess MCP-1 mRNA expression by real-time PCR (A, C). GAPDH was used as internal control. Supernatants were collected to assess MCP-1 protein concentration by ELISA (B, D). Data are mean± SE and represent the average results of 3 independent experiments run in duplicate. * is p<0.05, ** is p<0.01 and *** is p<0.001 versus control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3046136&req=5

pone-0016722-g002: Sustained and repetitive HQ-induced oxidative injury decreases on MCP-1 expression in human RPE cells.Confluent serum-starved ARPE-19 cells were treated with (A, B) 10 µM HQ every 24 hours for 5 consecutive days or (C, D) 50 µM HQ every 4 days for 24 hours for 3 consecutive weeks in phenol red-free 0.1% FBS medium. Total RNA was extracted to assess MCP-1 mRNA expression by real-time PCR (A, C). GAPDH was used as internal control. Supernatants were collected to assess MCP-1 protein concentration by ELISA (B, D). Data are mean± SE and represent the average results of 3 independent experiments run in duplicate. * is p<0.05, ** is p<0.01 and *** is p<0.001 versus control.

Mentions: Prolonged oxidative injury has been suggested as one of the causes of a number of retinal pathologic conditions, including AMD. We have previously investigated the effects of repetitive acute (6 hours every 3 days for 4 weeks) and transient (6 hours followed by a recovery phase, every 5 days for 6 weeks) exposure of ARPE-19 cells to HQ on matrix metalloproteinase-2 activity and extracellular matrix turnover relevant to the pathogenesis of dry AMD [26]. Regulation of RPE-derived MCP-1 expression following HQ-mediated oxidative injury has not been investigated. Here, we found that sustained exposure of ARPE-19 cells to HQ 10 µM every 24 hours for 5 consecutive days decreased MCP-1 mRNA expression by 24% compared with control cells (0.76±0.02 versus 1.0±0.03, p<0.001) (Figure 2A) with a concomitant moderate ∼12% decrease in MCP-1 protein released in the supernatants as measured by ELISA (88.27±1.8 versus 100.0±2.1%, p<0.01) (Figure 2B) without causing any cell death (94.6±6.1% of cells survived after treatment with NT versus 100.0±3.5% in control conditions, p>0.05). We also tested the effect of repetitive long-term exposure to sub-lethal oxidative stress by treating ARPE-19 cells with HQ 50 µM every 4 days for 24 hours for 3 consecutive weeks. We observed a ∼20% decline in MCP-1 mRNA expression (0.80±0.9 versus 1.0±0.01, p<0.05) (Figure 2C) with a concomitant ∼27% decrease in protein secretion (72.95±12.27 versus 100.0±2.0% p<0.05) (Figure 2D) relative to control cells without any cell death (95.4±5.9% of cells survived after treatment with NT versus 100.0±1.3% in control conditions, p>0.05). These observations suggest that cigarette smoke-related HQ-induced downregulation of RPE-derived MCP-1 expression may play an important role in the pathogenesis of AMD.


Cigarette smoke-related hydroquinone dysregulates MCP-1, VEGF and PEDF expression in retinal pigment epithelium in vitro and in vivo.

Pons M, Marin-Castaño ME - PLoS ONE (2011)

Sustained and repetitive HQ-induced oxidative injury decreases on MCP-1 expression in human RPE cells.Confluent serum-starved ARPE-19 cells were treated with (A, B) 10 µM HQ every 24 hours for 5 consecutive days or (C, D) 50 µM HQ every 4 days for 24 hours for 3 consecutive weeks in phenol red-free 0.1% FBS medium. Total RNA was extracted to assess MCP-1 mRNA expression by real-time PCR (A, C). GAPDH was used as internal control. Supernatants were collected to assess MCP-1 protein concentration by ELISA (B, D). Data are mean± SE and represent the average results of 3 independent experiments run in duplicate. * is p<0.05, ** is p<0.01 and *** is p<0.001 versus control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046136&req=5

pone-0016722-g002: Sustained and repetitive HQ-induced oxidative injury decreases on MCP-1 expression in human RPE cells.Confluent serum-starved ARPE-19 cells were treated with (A, B) 10 µM HQ every 24 hours for 5 consecutive days or (C, D) 50 µM HQ every 4 days for 24 hours for 3 consecutive weeks in phenol red-free 0.1% FBS medium. Total RNA was extracted to assess MCP-1 mRNA expression by real-time PCR (A, C). GAPDH was used as internal control. Supernatants were collected to assess MCP-1 protein concentration by ELISA (B, D). Data are mean± SE and represent the average results of 3 independent experiments run in duplicate. * is p<0.05, ** is p<0.01 and *** is p<0.001 versus control.
Mentions: Prolonged oxidative injury has been suggested as one of the causes of a number of retinal pathologic conditions, including AMD. We have previously investigated the effects of repetitive acute (6 hours every 3 days for 4 weeks) and transient (6 hours followed by a recovery phase, every 5 days for 6 weeks) exposure of ARPE-19 cells to HQ on matrix metalloproteinase-2 activity and extracellular matrix turnover relevant to the pathogenesis of dry AMD [26]. Regulation of RPE-derived MCP-1 expression following HQ-mediated oxidative injury has not been investigated. Here, we found that sustained exposure of ARPE-19 cells to HQ 10 µM every 24 hours for 5 consecutive days decreased MCP-1 mRNA expression by 24% compared with control cells (0.76±0.02 versus 1.0±0.03, p<0.001) (Figure 2A) with a concomitant moderate ∼12% decrease in MCP-1 protein released in the supernatants as measured by ELISA (88.27±1.8 versus 100.0±2.1%, p<0.01) (Figure 2B) without causing any cell death (94.6±6.1% of cells survived after treatment with NT versus 100.0±3.5% in control conditions, p>0.05). We also tested the effect of repetitive long-term exposure to sub-lethal oxidative stress by treating ARPE-19 cells with HQ 50 µM every 4 days for 24 hours for 3 consecutive weeks. We observed a ∼20% decline in MCP-1 mRNA expression (0.80±0.9 versus 1.0±0.01, p<0.05) (Figure 2C) with a concomitant ∼27% decrease in protein secretion (72.95±12.27 versus 100.0±2.0% p<0.05) (Figure 2D) relative to control cells without any cell death (95.4±5.9% of cells survived after treatment with NT versus 100.0±1.3% in control conditions, p>0.05). These observations suggest that cigarette smoke-related HQ-induced downregulation of RPE-derived MCP-1 expression may play an important role in the pathogenesis of AMD.

Bottom Line: Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls.VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio.Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Miller School of Medicine, Bascom Palmer Eye Institute, University of Miami, Miami, Florida, United States of America.

ABSTRACT

Background: Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly population. Debris (termed drusen) below the retinal pigment epithelium (RPE) have been recognized as a risk factor for dry AMD and its progression to wet AMD, which is characterized by choroidal neovascularization (CNV). The underlying mechanism of how drusen might elicit CNV remains undefined. Cigarette smoking, oxidative damage to the RPE and inflammation are postulated to be involved in the pathophysiology of the disease. To better understand the cellular mechanism(s) linking oxidative stress and inflammation to AMD, we examined the expression of pro-inflammatory monocyte chemoattractant protein-1 (MCP-1), pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial derived factor (PEDF) in RPE from smoker patients with AMD. We also evaluated the effects of hydroquinone (HQ), a major pro-oxidant in cigarette smoke on MCP-1, VEGF and PEDF expression in cultured ARPE-19 cells and RPE/choroids from C57BL/6 mice.

Principal findings: MCP-1, VEGF and PEDF expression was examined by real-time PCR, Western blot, and ELISA. Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls. Both MCP-1 mRNA and protein were downregulated in ARPE-19 cells and RPE/choroids from C57BL/6 mice after 5 days and 3 weeks of exposure to HQ-induced oxidative injury. VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio. Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo.

Conclusion: We propose that impaired RPE-derived MCP-1-mediated scavenging macrophages recruitment and phagocytosis might lead to incomplete clearance of proinflammatory debris and infiltration of proangiogenic macrophages which along with increased VEGF/PEDF ratio favoring angiogenesis might promote drusen accumulation and progression to CNV in smoker patients with dry AMD.

Show MeSH
Related in: MedlinePlus