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The objective of the present study was to investigate the effect of alcohol and nicotine consumption on the pathogenesis of choroidal neovascularization (CNV) in rats after laser-photocoagulation. Confocal microscopic analysis demonstrated an increase in CNV complex size in rats fed with alcohol (2.3-fold), nicotine (1.9-fold), and the combination of alcohol and nicotine (2.7-fold) compared with the control groups.
Immunohistochemical analysis revealed that alcohol and nicotine consumption increased MAC deposition and VEGF expression in laser spots. Expression of CD59 by RT-PCR and Western blot was drastically reduced in the animals that were fed with alcohol, nicotine and alcohol and nicotine compared to those fed with water alone and this was associated with exacerbation of CNV.
Age-related macular degeneration (AMD) is a complex disease that has been associated with multiple genetic and environmental risk factors [1–8]. Identification of risk factors is one of the first steps toward preventing and designing new strategies for AMD treatment. Smoking and chronic alcoholism are two environmental risk factors that have been strongly associated with human AMD severity and incidence. [7,8]. Cigarette smoking has been associated with a 2- to 3-fold increased in incidence of neovascular AMD [5–8]. Recent reports have indicated that a heavy alcohol intake may be associated with an increased risk of exudative AMD [9,10].
Choroidal neovascularization (CNV) is the creation of new blood vessels in the choroid layer of the eye, which is a common symptom of the degenerative maculopathy wet AMD. Many reports, including those from our laboratory, have demonstrated that the presence and activation of complement system is crucial for CNV development [11–14]. Several independent and unrelated studies have indicated that smoking and alcohol consumption can activate the complement system [15–20]. However, the complement system's exact role in alcohol and nicotine-induced CNV exacerbation is not known.
In our previous publications, we demonstrated that the alternative pathway of complement activation is critical for CNV development . We have also established that membrane attack complex (MAC) formation and its regulation by CD59 plays a critical role in CNV pathogenesis . CD59 is a complement regulatory protein that regulates the assembly and the activity of MAC .
In this study, we investigated the effect of both nicotine and alcohol consumption on laser-induced CNV in rats. Laser-induced CNV model is an accelerated model of wet type AMD and generates acute inflammation compared to subtle inflammation seen in human AMD. We have also investigated CD59's role in CNV exacerbation in alcohol or nicotine-fed rats. For this study, we have only focused on CD59 because we have already established its role  in the development and pathogenesis CNV or wet AMD.
Male Brown Norway rats (4–6 weeks old) were purchased from Harlan Sprague Dawley (Indianapolis, IN). This study was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Arkansas for Medical Sciences, Little Rock, AR.
Photocoagulation, using Argon red laser (50 μm spot size, 0.05 s duration, 350 mW) induced CNV and three laser spots were placed in each eye area surrounding the optic nerve as previously described [11–13,22].
After laser the rats were randomly divided into five groups. Group 1 was fed alcohol (8 g/kg body weight), group 2 was fed nicotine (200 μg/ml) and group 3 was fed with combination of alcohol (8 g/kg body weight) and nicotine (200 μg/ml). Group 4, fed with water alone, served as controls and group 5, fed with glucose in water, served as pair-fed controls. All group animals were fed regular diet and were killed after 4 weeks. Alcohol and nicotine were mixed with water and the bottles were changed daily. One rat was housed per cage. The daily alcohol and nicotine consumption was measured as follows: alcohol and water mixture contained 20 ml of ethyl alcohol (100% proof, density = 0.78) and 180 ml of water. Each rat drank 31–35 ml of the mixture daily. Assuming that 10% of alcohol evaporated from the mixture daily, each rat, weighing approximately 300 g, received 8 g/kg. Dehydration was controlled by feeding the rats in alcohol group with water only for 2 h daily before the next alcohol feeding. This 4 week dose of alcohol feeding produced serum alcohol levels similar to those found in alcoholics . The nicotine and water mixture contained 200 μg/ml of (−)- nicotine-hemisulfate (Sigma–Aldrich, St. Louis, MO). Each rat drank 15–20 ml of the mixture daily and consumed 3 mg/day (200 μg/ml /day). This 4 week dose of nicotine feeding produced serum nicotine levels similar to those found in chronic, moderate smokers [23,24].
Four weeks after the laser treatment, including alcohol and nicotine feeding, all animals were anesthetized (ketamine/xylazine mixture, 1:1), perfused with 1 ml of PBS containing 50 mg/ml fluorescein-labeled dextran (FITC-dextran; average molecular mass, 2 × 106; Sigma–Aldrich, St. Louis, MO) and sacrificed. Eyes were harvested and fixed in 10% phosphate-buffered formalin for 1 h, and retinal pigment epithelium (RPE)–choroid–scleral flat mounts were prepared as previously described [11–13,22]. RPE–choroid–scleral flat mounts were stained for elastin using a monoclonal antibody specific for elastin (1.0 mg/ml; 1/200 dilution; Sigma–Aldrich) followed by a Cy3-labeled secondary antibody (1.0 mg/ml; 1/200 dilution; Sigma–Aldrich). CNV incidence and size were determined by confocal microscopy (Zeiss LSM510). The CNV complex size was graded by morphometric analysis of the images (Image Pro 5.0 software; Media Cybernetics Inc., Silver Spring, MD) obtained from confocal microscopy [11–13].
Eyes were fixed with normal buffered formalin and embedded in paraffin. Sections (4 μm) were stained with hematoxylin and eosin. Stained section images were captured with Q Imaging GO-5 digital camera and Olympus microscope. Capillary amounts were counted and CNV area was measured using ImagePro program. Number of capillaries per 1mm2 of CNV area was calculated.
The paraffin embedded tissue sections (4 μm) were immunostained for MAC and vascular endothelial growth factor (VEGF). For MAC detection, polyclonal rabbit anti-rat C9 (1:500) was used as the primary antibody. This antibody was kindly provided by Prof. B.P. Morgan (School of Medicine, Cardiff University, Cardiff, UK). AlexaFluore 594 conjugated goat anti-rabbit IgG (invitrogen) were used as the secondary antibody. Control stains were performed with non-relevant antibodies (IgG whole molecule from rabbit serum) at concentrations similar to those of the primary antibodies. Additional controls consisted of staining by omission of the primary or secondary antibody. Sections were examined under fluorescence microscope (Olympus, Center Valley, PA). Rabbit polyclonal anti-rat VEGF164 antibody (1:1600; R&D Systems, Minneapolis, MN), secondary goat biotinylated anti-rabbit IgG (H + L) (1:800; VectorLab, Burlingame, CA), Vectastain ABC Elite KIT (VectorLab) and Vector VIP substrate kit for peroxidase (VectorLab) were used to detect VEGF expression. Nuclei were counterstained with Vector Methyl Green (VectorLab). For negative controls, slides were incubated with 1% BSA in TBS instead of primary antibodies. After immunohistochemical labeling, sections were mounted in Permount (Fisher, Fair Lawn, NJ) and were examined under bright field microscope (Olympus). Semiquantitative scoring of positive signal was performed in the choroid. Staining intensity was graded from 0 to 3 (0 – no staining; 1 – faint; 2 – moderate; 3 – intense). Score mean value was calculated for each laser-injured area.
After 30 days , animals from each group (n = 5/group) were killed; RPE–choroid–scleral tissues harvested from the enucleated eyes were pooled separately for each group and total RNA was prepared using the RNA Isolation kit (Qiagen, Germantown, MD). Equal amounts of the total RNA (0.1 μg) were converted to cDNA, and then used to detect the mRNA levels of β-actin, and CD59 by semiquantitative RT-PCR using reagents (Applied Biosystems, Foster City, CA). Sense and antisense oligonucleotide primers were synthesized at Integrated DNA Technologies, and 30 cycles were used for PCR. Negative controls consisted of RNA omission or reverse transcriptase from the reaction mixture. Primer sequences including the predicted sizes of amplified cDNA are as follows: β-actin (318 bp) forward: 5′-GTTTGAGACCTTCAACACC-3′, reverse: 5′-GTGGCCATCTCTTGCTCGAAGTC-3′; CD59 (302 bp) forward: 5′-CTGCTTCTGGCTGTCCTCTG-3′, reverse: 5′-ACGCTGTCTTCCCCAATAGG-3′.
PCR products analyzed on a 1% agarose gel were examined by using Quantity One (Bio-Rad, Hercules, CA). All reactions were normalized to β-actin expression. Experiments were repeated three times with similar results.
RPE–choroid–scleral tissues harvested from all animal groups (as described above for RT-PCR) were pooled separately. Pooled tissue was homogenized and solubilized in ice-cold PBS containing protease inhibitors and total protein concentration was determined [11–13]. After SDS–PAGE on 12% linear slab gel, separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were incubated with mouse anti-rat CD59 (AbD Serotec, Raleigh, NC), or monoclonal anti-β-actin (mouse IgG1; Sigma–Aldrich). After washing and incubation with HRP-conjugated secondary antibody, blots were developed using the ECL Western blotting detection system “ECL Plus” (Amersham Biosciences, Piscataway, NJ). Quantitation of CD59 and β-actin was determined by analyzing band intensity using Quantity One 4.2.0 (Bio-Rad). Experiments were repeated three times with similar results.
Data were expressed as the means ± S.D. and were analyzed and compared using Student's t-test, and differences were considered statistically significant with P < 0.05.
All animals were divided into five groups and CNV was induced by laser-photocoagulation as described in Section 2. After laser treatment, group 1 was fed alcohol (8 g/kg body weight), group 2 was fed nicotine (200 μg/ml) and group 3 was fed a combination of alcohol (8 g/kg body weight) and nicotine (200 μg/ml). Water-fed animals were placed in group 4 (no nicotine or alcohol) and served as controls. Group 5 animals were fed with glucose mixed in water and served as pair-fed control. All animals were monitored daily for alcohol/nicotine consumption and change in body weight. After initial weight loss, rate of weight gain in all groups was similar to that in controls (data not shown).
Animals were killed after 4 weeks of treatments, perfused with FITC-dextran, and eyes were enucleated. RPE–choroid– scleral flat mounts were stained with goat anti-elastin antibody (primary antibody). CNV incidence and size was determined by confocal microcopy. Fig. 1 shows representative confocal micrographs of the RPE–choroid–scleral flat mounts from each group. Red color in the micrographs represents the exposed Bruch's membrane due to laser treatment and green fluorescence represents the new vessels formed after the laser treatment. CNV complex size (i.e. area) was measured using Image Pro-Plus software in micron unit. Confocal analyses demonstrated a significant increase in CNV complex size in alcohol-fed rats (Fig. 1C), nicotine-fed rats (Fig. 1D) and combination of alcohol and nicotine-fed rats (Fig. 1E) compared to control rats that were fed with water alone (no nicotine or alcohol; Fig. 1A) or water with glucose (Fig. 1B). CNV size comparison, using ImagePro software, revealed that alcohol-fed rats had an increased CNV size compared to controls (~125% increase; Fig. 1F). Nicotine-fed rats also had an increased CNV size compared to controls (~86% increase; Fig. 1F). Combination nicotine- and alcohol-fed rats had a drastic increase in CNV size as compared to controls (~170% increase; Fig. 1F). Furthermore, CNV incidence was also found to be highest in alcohol-fed rats (92%), nicotine-fed (89%) and combination alcohol- and nicotine-fed (93%; Table 1) as compared to controls (77% and 80%; Table 1). Since there was no difference in CNV complex size between water-fed rats (Fig. 1A) and glucose and water-fed rats, (Fig. 1B), water-fed rats were used only as controls in our subsequent experiments.
Eyes were harvested and fixed in formalin for paraffin embedding. Four micrometer sections of the paraffin embedded eyes were stained with hematoxylin and eosin. Histological analysis of hematoxylin and eosin stained sections revealed several blood vessels in the laser injury area (shown as red arrow heads) in control group (Fig. 2A). The number and sizes of blood vessels in the laser-injured sites were found to be significantly (P < 0.05) higher in alcohol-fed animals (Fig. 2B and E), nicotine-fed (Fig. 2C and E) and combination of alcohol and nicotine-fed (Fig. 2D and E).
Previously, we reported that increased MAC deposition in CNV complex is critical for CNV development and MAC plays an important role in CNV immunopathogenesis [11–13]. Therefore, in the present study, we investigated if the CNV exacerbation in alcohol- and nicotine-fed animals is associated with increased MAC deposition. Paraffin embedded (4 μm) sections of harvested eyes from alcohol-fed nicotine-fed or combination of alcohol and nicotine-fed rats and controls were stained with anti-rat C9 (neoepitope). A strong red fluorescence in the choroid of controls (Fig. 3A) revealed MAC deposition after laser, thus, confirming our previous observations [11,12]. Increased MAC deposition was detected in the choroid of alcohol (Fig. 3B), nicotine (Fig. 3C) and a combination of alcohol and nicotine (Fig. 3D) fed animals. Negative control, stained with nonspecific isotype control antibody, did not show any fluorescence (data not shown).
Previously, we reported that MAC deposition plays an important role in the secretion/release of growth factors during laser-induced CNV [11–13]. In the present study, we observed an increased MAC deposition in the choroid of rats fed with both nicotine and alcohol, their VEGF expression was also investigated. Using immunohistochemical staining, VEGF was localized in choroidal vessels (Fig. 4). Increased VEGF expression was observed in the choroidal vessels of alcohol-fed (Fig. 4C), nicotine-fed (Fig. 4D) or combination of alcohol and nicotine-fed rats (Fig. 4E) compared to water-fed rats (Fig. 4B). Fig. 4A is negative control (NC) without primary antibody, showed no VEGF staining (Fig. 4F).
Increased MAC deposition in the choroid of alcohol and nicotine-fed rats observed in our study could be due to reduced expression/activity of MAC regulator CD59. To investigate alcohol and nicotine consumption effect on CD59 expression, rats were divided into four groups and were fed with alcohol, nicotine, a combination of alcohol and nicotine, or water (control) for 4 weeks. After 4 weeks, animals were killed and the RPE–choroid–scleral tissue was harvested and processed for RNA or protein extraction as described in Section 2. CD59 expression was assessed by RT-PCR (mRNA) and Western blot (protein) analysis. Using semiquantitative RT-PCR analysis, we detected a strong band representing CD59 transcript in water-fed rats (control group; Fig. 5A and B). CD59 mRNA levels were reduced in the alcohol-fed, nicotine-fed, and combination of alcohol and nicotine-fed rats. (Fig. 5A and B). Lowest levels of CD59 mRNA were detected in the combination of alcohol and nicotine-fed rats. (Fig. 5A and B). CD59 protein expression in RPE–choroid–scleral tissue was studied by semiquantitative Western blot analysis. Compared to controls, a decrease in CD59 protein levels was detected in the alcohol-fed, nicotine-fed and combination of alcohol and nicotine-fed animals (Fig. 5C and D).
In the present study, we investigated the effect of alcohol and nicotine on CNV and observed that consumption of both alcohol and nicotine leads to exacerbation of laser-induced CNV. The underlying mechanisms leading to CNV exacerbation by alcohol and nicotine up-take were also explored.
We used the rat model of laser-induced CNV to study the effect of alcohol and nicotine consumption on CNV. Our results clearly demonstrated that alcohol feeding results in 2.3-fold increase in CNV complex size compared to water-fed and pair-fed controls. Furthermore, our results demonstrated that there was a 1.9-fold increase in CNV size in rats treated with nicotine as compared to controls while the combination of alcohol and nicotine consumption resulted in 2.7-fold increase in CNV size. Also, in alcohol- and nicotine-fed animals, increased blood vessel density was also noted in the laser spots. Taken together, these results demonstrated that the alcohol and nicotine consumption increased CNV severity in rats. It is well documented that nicotine increases the proliferation of choroidal endothelial cells and vascular smooth muscle cells [23–28] and accelerates the growth of tumor and atheroma in association with increased neovascularization [25–28]. Alcohol has also been reported to contribute to angiogenic stimulations [29–34] including proliferation of endothelial cells [35,36].
We have documented that the complement system plays a central role in CNV pathogenesis and local MAC deposition is critical for the up-regulation of growth factors, including VEGF, required for the neovascularization [11–13]. In the present study, we investigated local MAC deposition and VEGF expression in alcohol and nicotine-fed animals. Our results show that there is an increased deposition of MAC in alcohol, nicotine, or combination of alcohol and nicotine-fed animals compared to control animals. This indicates that the alcohol and nicotine consumption increased the activation of the complement system in these animals.
Several studies have shown that cigarette smoke can result in activation of the complement system [15–17]. Similarly, many investigators have reported that chronic alcohol consumption can up-regulate the expression of many complement components and increases the complement activity as observed by increased deposition of complement activation products [18–20]. Using the immunohistochemistry of paraffin embedded tissues; we detected increased VEGF expression 4 weeks after laser treatment in the choroid of nicotine-or alcohol-fed rats. Furthermore, VEGF expression was found to be highest in combination alcohol- and nicotine-fed rats. This may be due to the cumulative effect of alcohol and nicotine consumption. Increased MAC deposition, as observed in the present study, could be due to reduced/loss of complement regulation as a result of decreased expression of complement regulatory proteins.
Järveläinen et al. previously reported that ethanol treatment leads to the reduction in levels of complement regulatory proteins (CD59 and Crry) in the liver and that this may increase the susceptibility of tissues to complement-mediated damage in alcohol-fed rats . Another independent study demonstrated that decay accelerating factor plays a critical role in ethanol-induced fatty liver by regulating the complement system . In the present study, we observed a drastic reduction in CD59 expression at both mRNA and protein levels in alcohol-fed, nicotine-fed, or combination of alcohol and nicotine-fed rats compared to controls. This reduction of CD59 expression may be due to the direct effect of nicotine and alcohol on CD59 gene transcription.
It has been reported that nicotine treatment inhibits the expression of several genes including IL-1 and IL-8 by modulating the activity of the transcription factor NF-κB . This modulation of NF-κB has been directly linked to the nicotine-induced accumulation of inhibitory protein IκB. Nicotine has also been shown to directly inhibit NF-κB expression in endothelial cells . Similar mechanisms may also be responsible for the reduction in CD59 expression observed in our present study. However, further investigations are required to elucidate the exact mechanism leading to CD59 reduction as a result of nicotine and alcohol up-take.
Previous studies, from our laboratory and from other investigators demonstrated that various components of the complement system are synthesized locally and the local complement activation and regulation is critical for CNV development [11–13]. Therefore, we investigated the mRNA level in retina/choroid. We also measured the complement activity (using hemolysis assay) in the serum samples of these animals and observed no significant difference between the groups (data not shown). In the present study, we used the hemolytic assay to measure the complement activation . The hemolytic assay is a measure of terminal complement activation in the serum. Although ethanol increases the expression of various complement components, it also suppresses the expression of complement components at the terminal end of complement activation [18,19].
We have also observed significant decrease in the CD59 expression in the eye (Fig. 5) which is a terminal component of the complement pathway. This may be the reason that we do not see any significant increase in the systemic complement activity even though we see an increased deposition of MAC in the eye. Thus, we believe that the local complement activation plays an important role in CNV development [11–13].
Taken together, our results indicate that alcohol and nicotine consumption may decrease CD59 levels which may lead to increased MAC deposition and exacerbation of laser-induced CNV. Our results may have important clinical implications. Although smoking and alcoholism are reversible risk factor of AMD, the intervention in the complement system using recombinant complement regulatory proteins may represent a promising strategy to treat/manage AMD patients with a history of smoking and/or alcoholism.
We thank Dr. Richard Kurten (Digital and Confocal Microscopy Laboratory, University of Arkansas for Medical Sciences, Little Rock, AR) for his help with the use of confocal microscope, Dr. Ammar Safar for the help with the use of argon laser and Ms. Cynthia Bond for editing the manuscript. This study was supported in part by NIH Grants EY 014623, EY 016205 and the Pat and Willard Walker Eye Research Center, Jones Eye Institute, University of Arkansas for Medical Sciences (Little Rock, AR). Laboratory supported by NIH Grant 2 P20 RR 16460 (PI: Larry Cornett, INBRE, Partnerships for Biomedical Research in Arkansas) and NIH/NCRR Grant 1 S10 RR 19395 (PI: Richard Kurten, “Zeiss LSM 510 META Confocal Microscope System”).