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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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VEGF-PEDF balance is altered in RPE/choroids from mice exposed to HQ.(A) VEGF and (B) PEDF mRNA expression 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%). VEGF and PEDF mRNA expression was measured by real-time PCR. GAPDH was used as internal control (n = 5 eyes per group). (C) VEGF and PEDF protein expression in response to HQ-induced oxidative injury. (D) VEGF-to-PEDF protein ratio. 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. VEGF and PEDF protein expression was evaluated by Western blot and normalized to GAPDH. Top: representative Western blots of the indicated proteins. 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-g007: VEGF-PEDF balance is altered in RPE/choroids from mice exposed to HQ.(A) VEGF and (B) PEDF mRNA expression 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%). VEGF and PEDF mRNA expression was measured by real-time PCR. GAPDH was used as internal control (n = 5 eyes per group). (C) VEGF and PEDF protein expression in response to HQ-induced oxidative injury. (D) VEGF-to-PEDF protein ratio. 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. VEGF and PEDF protein expression was evaluated by Western blot and normalized to GAPDH. Top: representative Western blots of the indicated proteins. 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: The observations reported above led us to investigate the impact of HQ-induced oxidative damage on VEGF and PEDF expression in RPE/choroids from C57BL/6 mice treated with 0.8% HQ in drinking water for different periods of time. Our data show that VEGF mRNA levels were ∼40% higher than in control mice after exposure to HQ for 5 days (1.40±0.13 versus 1.02±0.05%, p<0.01) (Fig. 7A) whereas the expressed protein was upregulated by ∼109% (208.0±12.1 versus 100.0±0.02%, p<0.01) (Fig. 7C). However, VEGF mRNA expression was not modified after 3 consecutive weeks of oxidative stress (0.87±0.03 versus 1.02±0.05, p>0.05) (Fig. 7A) whereas VEGF protein expression was increased by ∼60% relative to control mice (160.3±6.4 versus 100.0±0.02%, p<0.05) (Fig. 7C). In addition, 5 days of exposure to HQ led to ∼80% increase in PEDF mRNA expression relative to control (1.80±0.11 versus 1.00±0.07%, p<0.01) (Fig. 7B) while the protein levels were increased by ∼50% (149.7±1.5 versus 100.0±0.03%, p<0.05) (Fig. 7C). After 3 weeks of oxidative stress with HQ, PEDF mRNA expression was not modified in RPE/choroids from HQ-treated mice (1.25±0.1 versus 1.00±0.07, p>0.05) (Fig. 7B) and PEDF protein expression was increased by ∼25% compared to control (124.8±0.06 versus 100.0±0.03%, p<0.05) (Fig. 7C). Overall, exposure to HQ for 5 days and 3 weeks enhanced the VEGF-to-PEDF ratio in mice RPE/choroid complexes by ∼45% (1.45±0.04 versus 1.0±0.01, p<0.05) and 30% (1.33±0.06 versus 1.0±0.01, p<0.05) respectively (Fig. 7D). Our data demonstrate changes in the levels of VEGF and PEDF in RPE/choroids from mice following HQ-induced oxidative injury revealing a disturbance of the angiogenic homeostatic balance which may play a role in the development of CNV.


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)

VEGF-PEDF balance is altered in RPE/choroids from mice exposed to HQ.(A) VEGF and (B) PEDF mRNA expression 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%). VEGF and PEDF mRNA expression was measured by real-time PCR. GAPDH was used as internal control (n = 5 eyes per group). (C) VEGF and PEDF protein expression in response to HQ-induced oxidative injury. (D) VEGF-to-PEDF protein ratio. 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. VEGF and PEDF protein expression was evaluated by Western blot and normalized to GAPDH. Top: representative Western blots of the indicated proteins. 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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Related In: Results  -  Collection

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pone-0016722-g007: VEGF-PEDF balance is altered in RPE/choroids from mice exposed to HQ.(A) VEGF and (B) PEDF mRNA expression 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%). VEGF and PEDF mRNA expression was measured by real-time PCR. GAPDH was used as internal control (n = 5 eyes per group). (C) VEGF and PEDF protein expression in response to HQ-induced oxidative injury. (D) VEGF-to-PEDF protein ratio. 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. VEGF and PEDF protein expression was evaluated by Western blot and normalized to GAPDH. Top: representative Western blots of the indicated proteins. 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: The observations reported above led us to investigate the impact of HQ-induced oxidative damage on VEGF and PEDF expression in RPE/choroids from C57BL/6 mice treated with 0.8% HQ in drinking water for different periods of time. Our data show that VEGF mRNA levels were ∼40% higher than in control mice after exposure to HQ for 5 days (1.40±0.13 versus 1.02±0.05%, p<0.01) (Fig. 7A) whereas the expressed protein was upregulated by ∼109% (208.0±12.1 versus 100.0±0.02%, p<0.01) (Fig. 7C). However, VEGF mRNA expression was not modified after 3 consecutive weeks of oxidative stress (0.87±0.03 versus 1.02±0.05, p>0.05) (Fig. 7A) whereas VEGF protein expression was increased by ∼60% relative to control mice (160.3±6.4 versus 100.0±0.02%, p<0.05) (Fig. 7C). In addition, 5 days of exposure to HQ led to ∼80% increase in PEDF mRNA expression relative to control (1.80±0.11 versus 1.00±0.07%, p<0.01) (Fig. 7B) while the protein levels were increased by ∼50% (149.7±1.5 versus 100.0±0.03%, p<0.05) (Fig. 7C). After 3 weeks of oxidative stress with HQ, PEDF mRNA expression was not modified in RPE/choroids from HQ-treated mice (1.25±0.1 versus 1.00±0.07, p>0.05) (Fig. 7B) and PEDF protein expression was increased by ∼25% compared to control (124.8±0.06 versus 100.0±0.03%, p<0.05) (Fig. 7C). Overall, exposure to HQ for 5 days and 3 weeks enhanced the VEGF-to-PEDF ratio in mice RPE/choroid complexes by ∼45% (1.45±0.04 versus 1.0±0.01, p<0.05) and 30% (1.33±0.06 versus 1.0±0.01, p<0.05) respectively (Fig. 7D). Our data demonstrate changes in the levels of VEGF and PEDF in RPE/choroids from mice following HQ-induced oxidative injury revealing a disturbance of the angiogenic homeostatic balance which may play a role in the development of CNV.

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