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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.

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MCP-1 expression is decreased in RPE/choroids from mice exposed to HQ.(A) MCP-1 mRNA expression was downregulated in response to HQ-induced oxidative injury. Total RNA was extracted from microdissected RPE/choroid complexes after 5 days and 3 weeks of exposure to HQ in drinking water (0.8%). MCP-1 mRNA expression was measured by real-time PCR. GAPDH was used as internal control (n = 5 eyes per group). (B) MCP-1 protein expression was downregulated in response to HQ-induced oxidative injury. Total protein was extracted from microdissected RPE/choroid complexes after 5 days and 3 weeks of exposure to HQ in drinking water (0.8%). Equivalent amounts of protein from 5 eyes per group were pooled for each lane. MCP-1 protein expression was evaluated by Western blot and normalized to GAPDH. Top: representative Western blot gel. The numbers to the left are molecular weights in kilodaltons (KDa). Bottom: average densitometry results. Data are expressed as percentage of control and are means ± SE. * is p<0.05 and **p<0.01 versus control.
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pone-0016722-g003: MCP-1 expression is decreased in RPE/choroids from mice exposed to HQ.(A) MCP-1 mRNA expression was downregulated in response to HQ-induced oxidative injury. Total RNA was extracted from microdissected RPE/choroid complexes after 5 days and 3 weeks of exposure to HQ in drinking water (0.8%). MCP-1 mRNA expression was measured by real-time PCR. GAPDH was used as internal control (n = 5 eyes per group). (B) MCP-1 protein expression was downregulated in response to HQ-induced oxidative injury. Total protein was extracted from microdissected RPE/choroid complexes after 5 days and 3 weeks of exposure to HQ in drinking water (0.8%). Equivalent amounts of protein from 5 eyes per group were pooled for each lane. MCP-1 protein expression was evaluated by Western blot and normalized to GAPDH. Top: representative Western blot gel. The numbers to the left are molecular weights in kilodaltons (KDa). Bottom: average densitometry results. Data are expressed as percentage of control and are means ± SE. * is p<0.05 and **p<0.01 versus control.

Mentions: Based on the above observations obtained in vitro on ARPE-19 cells, we hypothesized that pro-oxidant HQ might also regulate MCP-1 expression in mice treated with 0.8% HQ in drinking water. A longer exposure for 5 days almost completely abolished MCP-1 mRNA expression compared with control mice (0.04±0.006 versus 1.0±0.12, p<0.001) (Figure 3A) but the expressed protein was only ∼46% downregulated (54.1±6.2 versus 100.0±0.01%, p<0.01) (Figure 3B). After 3 consecutive weeks of exposure to HQ, MCP-1 mRNA expression in RPE/choroids was dramatically downregulated compared with a 5 day-treatment but remained 39% lower compared with control mice (0.61±0.13 versus 1.0±0.12, p<0.05) (Figure 3A). At 3 weeks, MCP-1 expression was decreased by ∼30% at the translational level (70.5±4.3 versus 100.0±0.01%, p<0.01) (Figure 3B). These observations are consistent with aforementioned data on ARPE-19 cells at 5 days and 3 weeks indicating that cumulative cigarette-smoke related HQ-induced oxidative injury might decline RPE-derived MCP-1expression therefore inhibiting the recruitment of scavenging macrophages therefore playing a crucial role in the progression 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)

MCP-1 expression is decreased in RPE/choroids from mice exposed to HQ.(A) MCP-1 mRNA expression was downregulated in response to HQ-induced oxidative injury. Total RNA was extracted from microdissected RPE/choroid complexes after 5 days and 3 weeks of exposure to HQ in drinking water (0.8%). MCP-1 mRNA expression was measured by real-time PCR. GAPDH was used as internal control (n = 5 eyes per group). (B) MCP-1 protein expression was downregulated in response to HQ-induced oxidative injury. Total protein was extracted from microdissected RPE/choroid complexes after 5 days and 3 weeks of exposure to HQ in drinking water (0.8%). Equivalent amounts of protein from 5 eyes per group were pooled for each lane. MCP-1 protein expression was evaluated by Western blot and normalized to GAPDH. Top: representative Western blot gel. The numbers to the left are molecular weights in kilodaltons (KDa). Bottom: average densitometry results. Data are expressed as percentage of control and are means ± SE. * is p<0.05 and **p<0.01 versus control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046136&req=5

pone-0016722-g003: MCP-1 expression is decreased in RPE/choroids from mice exposed to HQ.(A) MCP-1 mRNA expression was downregulated in response to HQ-induced oxidative injury. Total RNA was extracted from microdissected RPE/choroid complexes after 5 days and 3 weeks of exposure to HQ in drinking water (0.8%). MCP-1 mRNA expression was measured by real-time PCR. GAPDH was used as internal control (n = 5 eyes per group). (B) MCP-1 protein expression was downregulated in response to HQ-induced oxidative injury. Total protein was extracted from microdissected RPE/choroid complexes after 5 days and 3 weeks of exposure to HQ in drinking water (0.8%). Equivalent amounts of protein from 5 eyes per group were pooled for each lane. MCP-1 protein expression was evaluated by Western blot and normalized to GAPDH. Top: representative Western blot gel. The numbers to the left are molecular weights in kilodaltons (KDa). Bottom: average densitometry results. Data are expressed as percentage of control and are means ± SE. * is p<0.05 and **p<0.01 versus control.
Mentions: Based on the above observations obtained in vitro on ARPE-19 cells, we hypothesized that pro-oxidant HQ might also regulate MCP-1 expression in mice treated with 0.8% HQ in drinking water. A longer exposure for 5 days almost completely abolished MCP-1 mRNA expression compared with control mice (0.04±0.006 versus 1.0±0.12, p<0.001) (Figure 3A) but the expressed protein was only ∼46% downregulated (54.1±6.2 versus 100.0±0.01%, p<0.01) (Figure 3B). After 3 consecutive weeks of exposure to HQ, MCP-1 mRNA expression in RPE/choroids was dramatically downregulated compared with a 5 day-treatment but remained 39% lower compared with control mice (0.61±0.13 versus 1.0±0.12, p<0.05) (Figure 3A). At 3 weeks, MCP-1 expression was decreased by ∼30% at the translational level (70.5±4.3 versus 100.0±0.01%, p<0.01) (Figure 3B). These observations are consistent with aforementioned data on ARPE-19 cells at 5 days and 3 weeks indicating that cumulative cigarette-smoke related HQ-induced oxidative injury might decline RPE-derived MCP-1expression therefore inhibiting the recruitment of scavenging macrophages therefore playing a crucial role in the progression 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