Báo cáo khoa học: Alcohol linked to enhanced angiogenesis in rat model of choroidal neovascularization pot

Alcohol linked to enhanced angiogenesis in rat model of choroidal neovascularization Puran S Bora*, Sankaranarayanan Kaliappan*, Qin Xu*, Shalesh Kumar*, Yali Wang*, Henry J Kaplan and Nalini S Bora* Department of Ophthalmology and Visual Science, Kentucky Lions Eye Center, University of Louisville, KY, USA Keywords adiponectin; angiogenesis; choroids; macular degeneration; neovascularization Correspondence P S Bora, Department of Ophthalmology, Jones Eye Institute, University of Arkansas for Medical Sciences, 4301 West Markham, # 523 Little Rock, AR 72205, USA Fax: +1 501 686 7037 Tel: +1 501 686 5150 E-mail: pbora@uams.edu *Present address Jones Eye Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA (Received 17 January 2006, accepted February 2006) doi:10.1111/j.1742-4658.2006.05163.x One of the pathologic complications of exudative (i.e wet-type) age-related macular degeneration (AMD) is choroidal neovascularization (CNV) The aim of this study was to investigate whether chronic and heavy alcohol consumption influenced the development of CNV in a rat model The oxidative metabolism of alcohol is minimal or absent in the eye, so that ethanol is metabolized via a nonoxidative pathway to form fatty acid ethyl esters (FAEE) Fatty acid ethyl ester synthase (FAEES) was purified from the choroid of Brown Norway (BN) rats The purified protein was 60 kDa in size and the antibody raised against this protein showed a single band on western blot BN rats on a regular diet were fed alcohol for 10 weeks Control rats were fed water with a regular diet and pair-fed control rats were fed regular diet, water and glucose We found that FAEES activity was increased 4.0-fold in the choroid of alcohol-treated rats compared with controls The amount of ethyl esters produced in the choroid of 10 week alcohol-fed rats was 7.4-fold more than rats fed alcohol for week The increased accumulation of ethyl esters was associated with a 3.0-fold increased expression of cyclin E and cyclin E ⁄ CDK2; however, the level of the cyclin kinase inhibitor, p27Kip, did not change The increased accumulation of ethyl esters was also associated with 3.0-fold decreased expression of APN in the choroid We also found that the size of CNV increased by 28% in alcohol-fed rats Thus, our study showed that chronic, heavy alcohol intake was associated with both an increased accumulation of ethyl esters in the choroid and an exacerbation of the CNV induced by laser treatment These results may provide insight into the link between heavy alcohol consumption and exudative AMD Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world in people aged over 55 Exudative (i.e wet-type) AMD is characterized by the development of new vessels within the choroid of the eye (i.e choroidal neovascularization; CNV) [1] This neovasculature penetrates Bruch’s membrane and grows beneath the retinal pigment epithelium and neurosensory retina [2–7] Many different growth factors have been implicated in the development of choroidal angiogenesis [8–18]; including vascular endothelial growth factor (VEGF) [9,19,20] More recently, an important role for various cell populations and cellular proteins – macrophages, hematopoietic stem cells and complement proteins [21–23] has been identified Several studies have shown that the nonoxidative ethanol metabolites, fatty acid ethyl esters (FAEE), are synthesized by the esterification of free fatty acids The reaction is catalyzed by FAEE synthase (FAEES) Abbreviations AMD, age-related macular degeneration; APN, adiponectin; BN rat, brown Norway rat; CDK2, cyclin E-dependent kinase 2; CNV, choroidal neovascularization; FAEE, fatty acid ethyl ester; FAEES, fatty acid ethyl ester synthase; PEDF, pigment epithelium-derived factor; RPE, retinal pigment epithelium; VEGF, vascular endothelial growth factor FEBS Journal 273 (2006) 1403–1414 ª 2006 The Authors Journal compilation ª 2006 FEBS 1403 Alcohol and choroidal neovascularization P S Bora et al [24–32] In addition to heart, brain, liver and pancreas, nonoxidative alcohol metabolism also exists in the eye [24,25] The nonoxidative ethanol metabolism pathway and its metabolites such as FAEE may play an important role in the development and ⁄ or enhancement of CNV Cell proliferation is governed by regulatory cellcycle proteins, such as cyclin E and cyclin E ⁄ CDK2, Li et al [33], showed that hepatic stellate cells treated with FAEE (linoelic acid ethyl ester) have increased cyclin E expression and cyclin E ⁄ CDK2 activity, suggesting that FAEE may have promitogenic effects [34] Adiponectin (APN) is an antiangiogenesis protein and circulating concentrations of APN decreased significantly following chronic consumption of alcohol [35,36] The balance between angiogenic factors and inhibitors is important for the angiogenesis process and has not previously been studied in the choroid of alcoholic rats Although there have been numerous studies using laser-induced CNV as a model of choroidal angiogenesis [2–7,21–23], none has studied the effect of chronic and heavy alcohol consumption on the neovasculature We investigated the effect of chronic and heavy alcohol feeding on alcohol metabolism in the choroid and on the development of CNV in a rat model We used ethyl alcohol (100% proof mixed with water) to feed the rats Because it is easy to feed alcohol to rats and they are a good source of choroidal tissue from which to purify FAEES enzyme, we used a rat model to induce CNV in this study Results Purification of FAEES FAEES was purified to homogeneity from rat choroid using a previously described method [29] The enzyme was purified 9000-fold to homogeneity on a hydroxylapatite column with 30% yield; SDS ⁄ PAGE showed a single band of molecular mass 60 kDa (Fig 1A) The N-terminal sequence of the purified protein matched 100% with rat adipose tissue FAEES protein (data not shown) [29] Purified FAEES was transferred to a nitrocellulose membrane and the purified protein reacted with an antibody raised in the rabbit against FAEES; a single 60 kDa band was observed on the western blot (Fig 1B) These results suggested that FAEES, a 60 kDa protein, is present in the choroid of the rat eye A B 94 kDa – 67 kDa – 60 kDa 60 kDa 43 kDa – 29 kDa – 20 kDa – 14 kDa – 2 Fig (A) SDS ⁄ PAGE analysis of purified FAEES from rat choroid Purification was performed as described in the Experimental procedures Lane 1, molecular mass markers; lane 2, purified FAEES, 60 kDa (B) Western blot analysis of purified FAEES Antibody (raised in rabbit) against FAEES was used to react with pure FAEES protein Lane shows no band (control lane) Rat albumin was used as control protein Lane shows a clear band at 60 kDa activity No significant increase in FAEES activity was observed in the cornea, iris and ciliary body, lens and retina of either group However, a 4.0-fold increase in FAEES activity was observed in the choroid of alcoholfed rats compared with regular controls (Fig 2A) Choroidal FAEES activity in regular and pair-fed controls was the same (data not shown) The kinetic constants of the enzyme purified from the choroid had similar Km values compared with the heart enzyme for both oleic acid and ethanol; however, the Vmax values were fourfold lower for both substrates compared with the heart enzyme (Table 1) The calculated Km and Vmax values of the choroidal enzyme for oleic acid were 0.24 mm and 1020 nmolỈmg)1Ỉh)1, respectively, and the calculated Km and Vmax for ethanol were 0.41 m and 930 nmolỈ mg)1Ỉh)1, respectively (Table 1) Immunohistochemistry studies Paraffin sections of the posterior segment of eyes were immunostained for FAEES using anti-FAEES serum (raised in rabbits) The eyes of rats fed alcohol for 10 weeks showed increased staining for FAEES compared with control rats (Fig 2B) FAEES activity The cornea, iris and ciliary body, lens, retina and choroid were collected separately from the eyes of control rats and rats fed alcohol for 10 weeks to assay synthase 1404 FAEE production The induction of FAEES activity produced 7.2- and 7.4-fold more ethyl esters in the choroid of rats fed FEBS Journal 273 (2006) 1403–1414 ª 2006 The Authors Journal compilation ª 2006 FEBS P S Bora et al Alcohol and choroidal neovascularization FAEES ACTIVITY (nmol/mg protein/hr) A 10 control alcohol fed cornea iris&cb lens retina Choroid B a b Fig (A) fatty acid ethyl ester synthase (FAEES) activity was assayed using the method described previously [28–31] Cornea, iris and ciliary body, lens, retina and choroid were separated from the eyes of control rats (no alcohol) and rats fed alcohol for 10 weeks to assay synthase activity No significant increase in FAEES activity was seen in the cornea, iris and ciliary body, lens and retina However, a 4.0-fold increase in FAEES activity was observed in the choroid of alcohol-fed rats compared with controls (B) Immunohistological analysis of FAEES in the posterior region of the rat eye Immunohistology was performed using FAEES antibody and immunoperoxidase staining kit (A) Control rats showed mild staining (brown color in the choroid) (B) Rats fed alcohol for 10 weeks showed several fold increase in staining (brown staining is marked by black arrows in the choroid) compared with controls Cell layers are marked as shown here: SC, sclera; CH, choroids; RPE, retinal pigment epithelium; OLM, outer limiting membrane; ONL, outer nuclear layer; OSL, outer synaptic layer; INL, inner nuclear layer; ISL, inner synaptic layer Table Kinetic constants of FAEES, purified from rat choroid Oleic acid and ethanol were used as substrates for FAEES activity Enzyme Substrate FAEES (Choroid) FAEES (Heart)a Oleic acid Ethanol Oleic acid Ethanol 0.24 0.41 0.20 0.30 Vmax (nmolỈmg protein)1Ỉh)1) Km ± ± ± ± 0.07 mM 0.05 M 0.02 mM 0.04 M 1020 930 3950 3700 ± ± ± ± 5.24 4.35 9.85 8.22 a See Bora et al [29] alcohol for and 10 weeks, respectively, compared with rats fed alcohol for week (Table 2) Ethyl esters production increased as the number of weeks of alcohol feeding increased (Table 2) However, no Table Ethyl esters produced in the choroid of alcohol-fed rats A 7.2- and 7.4-fold increase in the production of ethyl esters was observed in 9- and 10-week alcohol-fed rats, respectively, compared with 1-week alcohol-fed rats in the choroid However, no difference in ethyl ester production was observed in 4, 8, and 10 weeks of alcohol feeding compared with 1-week alcohol-fed (control) rats in the cornea, iris and ciliary body, lens and retina Weeks of alcohol feeding Ethyl ester formation (nmolỈg)1 wet weight) 10 2.30 6.50 10.10 16.60 17.10 FEBS Journal 273 (2006) 1403–1414 ª 2006 The Authors Journal compilation ª 2006 FEBS ± ± ± ± ± 0.80 0.92 0.75 1.10 1.22 1405 Alcohol and choroidal neovascularization P S Bora et al 18 CNV Size (ì 103 àm) 16 14 12 10 Control Pair-fed Alcohol-fed Fig Comparison of CNV complex size (i.e area) of regular control, pair-fed control and 10-week alcohol-fed rats CNV complex size was measured using IMAGE PRO-PLUS software in micron unit There was a 28% increase (P < 0.005) in the CNV complex size in alcohol-fed rats compared with both the controls rats (regular control and pair-fed control) The values in each group were averaged from 80 laser spots The incidence of CNV in each group was 92% significant changes were seen in ethyl esters production in the cornea, iris and ciliary body, lens and retina of 9- and 10-week alcohol-fed rats (Table 2) Size of the CNV complex Ten days after laser treatment (on day 70) alcohol-fed, regular and pair-fed control rats were killed to evaluate the effect of alcohol on the size of CNV There was a 28% increase (P < 0.005) in CNV complex size in alcohol-fed rats compared with regular and pair-fed controls (Fig 3) However, there was no difference in CNV size between regular and pair-fed controls (Fig 3) The values in each group were averaged from 80 laser spots Confocal microcopy images of flat mounts (RPE– choroid–sclera) are shown in Fig 4Aa–d There was a significant increase in the size of the CNV complex in alcohol-fed rats (Fig 4Ab) compared with controls (Fig 4Aa) Interestingly, we did not observe any significant difference in CNV complex size in rats fed alcohol for weeks (Fig 4Ac,d) The CNV size in pair-fed controls (Fig 4Ba) was the same as in regular controls (Fig 4Bb) However, alcohol-fed rats (Fig 4Bd) had a significantly higher CNV size compared with pair-fed rats (Fig 4B) These results indicated that chronic and heavy consumption of alcohol for 10 weeks increased the size of the CNV in the laser-induced rat model Cyclin E and cyclin E ⁄ CDK2 In order to explore the mechanism of the alcohol-associated increase in CNV size we measured the levels of 1406 cyclin E and cyclin E ⁄ CDK2 by western blot in alcohol-fed rats We found a 3.0-fold increase in cyclin E and cyclin E ⁄ CDK2 levels in rats fed alcohol for 10 weeks compared with controls (nonalcohol and 1-week alcohol-fed rats) (Fig 5A) Laser photocoagulation alone had no effect on the expression of these proteins (Fig 5A) Interestingly, we saw no change in cyclin E and cyclin E ⁄ CDK2 expression levels in rats fed alcohol for weeks (Fig 5B) These rats also showed no change in CNV size after laser induction Expression of the cyclin kinase inhibitor, p27Kip, was not changed in any of these groups (Fig 5A,B) APN study Immunohistochemistry showed that APN was expressed in the choroid of the rat eye No other tissue was stained in the rat eye (Fig 6A) RT-PCR data showed that expression of APN mRNA in the rat choroid was significantly decreased in the choroids of rats fed alcohol for 10 weeks compared with controls (Fig 6B, upper) Similarly, western blot showed a significantly decreased level of protein amount in the choroid of alcoholic rats compared with regular controls (Fig 6B, middle) In an in vitro study when rat choroidal endothelial cells were treated with 50 lm ethyl esters, there was significantly decreased expression of APN mRNA compared with control cells (not treated with ethyl esters) (Fig 6B, lower) Discussion AMD is the most common cause of permanent visual impairment among the elderly, and several million new cases are diagnosed each year around the world [37,38] It consists of two major forms, nonexudative (dry-type) and exudative (wet-type) AMD Approximately 10–12% of patients have the wet form, which is characterized by the presence of CNV and is responsible for 90% of severe central vision loss from AMD [1,37–39] There is no treatment that will reliably recover lost central vision, although there are currently several clinical trials in phase II ⁄ III testing using antiangiogenic pharmaceuticals Although there is no perfect animal model to study CNV that resembles humans, the laser-induced model may be the best available animal model to study CNV CNV can be induced by laser in mouse, rat, pig, rabbit and monkey In this study we used rats to study CNV because rats are easy to feed alcohol and it is economical to get enough choroidal tissue to purify FAEES enzyme There are currently 12 million alcoholics in the USA and over 80 million worldwide [26] The epidemiologic FEBS Journal 273 (2006) 1403–1414 ª 2006 The Authors Journal compilation ª 2006 FEBS P S Bora et al Alcohol and choroidal neovascularization A a b c d B a Fig (A, (a–d)) Confocal microcopy images of flat mounts (RPE–choroid–sclera) from rats perfused with fluorescein–dextran (green color) and stained with elastin (red color) Elastin stains Bruchs membrane (red), the CNV complex is green There was a significant increase in the size of the CNV complex in alcohol-fed (10 weeks) rats (A, b; 2500·), compared with control rats (A, a; 2500·) However, no significant increase in the CNV complex size was observed in rats fed alcohol for weeks (A, d; 2500·) compared with control rats (A, c; 2500·) (B, (a–d)) Confocal microcopy images of flat mounts (RPE–choroid–sclera) from rats perfused with fluorescein–dextran (green) and stained with elastin (red) Elastin stains Bruchs membrane (red), the CNV complex is green There was a significant increase in size of the CNV complex in alcohol-fed (10 weeks) rats (b, d; 2500·), compared with pair-fed controls (B, c; 2500·) However, there was no difference in the CNV complex size of pair-fed controls (B, a; 2500·) compared with regular controls (B, bx; 2500 ·) b c d FEBS Journal 273 (2006) 1403–1414 ª 2006 The Authors Journal compilation ª 2006 FEBS 1407 Alcohol and choroidal neovascularization P S Bora et al A Cyclin E Cyclin E/CDK2 p27 kip B Cyclin E Cyclin E/CDK2 p27 kip Fig (A, B) Western blots of cyclin E, cyclin E ⁄ CDK2 and p27Kip Rat cyclin E and p27Kip antibodies were used to blot these proteins Immunoprecipitation of cyclin E ⁄ CDK2 was performed using rat cyclin E antibody Choroid was removed from the following groups of rats, homogenized in solublizing buffer and the supernatant were used to run the gels (A) Expression of cyclin E, cyclin E ⁄ CDK2 was increased threefold in rats fed alcohol for weeks compared with rats fed alcohol for week and rats not fed alcohol (lane 1, no alcohol control; lane 2, week of alcohol; lane 3, weeks of alcohol; lane 4, weeks of alcohol and laser photocoagulated) However, no change was observed in the expression of p27Kip in these rats (B) No change in the expression of cyclin E, cyclin E ⁄ CDK2 and p27Kip was observed in the rats fed alcohol for weeks compared with those fed alcohol for week (lane 1, week of alcohol; lane 2, weeks of alcohol) The amount of protein used to run the gel was (A) cyclin E, lane 1–4, 6.0 lg; cyclin E ⁄ CDK2, lane 1–4, 6.0 lg; p27Kip, lane 1–4, 10.0 lg (B) Cyclin E, lane 1–2, 6.5 lg; cyclin E ⁄ CDK2, lane 1–2, 10.0 lg; p27 Kip, lane 1–2, 8.5 lg association between alcohol consumption and AMD is not clear The Beaver Dam eye studies and studies by Azen et al [40–43] suggested that a heavy alcohol intake is associated with an increased risk of exudative AMD [43] 1408 Alcohol is metabolized in the eye mainly via a nonoxidative pathway using the enzyme FAEES [24,25] Although ADH4 is present in eye tissue, it does not metabolize alcohol because of the high Km value for alcohol Retinol oxidation is the main function of the enzyme in the eye [44–47] Purified rat choroidal FAEES is 60 kDa in size The Km values for two substrates, oleic acid and ethanol, were similar to the value for the enzyme purified from the human heart; however, the Vmax values were fourfold lower than that of human heart FAEES This may be due to a species difference [29,44,45] In this study, we showed that feeding a rat alcohol (8 gỈkg)1) for 10 weeks increased FAEES activity in the choroid by 4.0-fold No change in FAEES activity was noted in the cornea, iris and ciliary body, lens or retina Immunohistological staining showed a qualitative increase in FAEES in the choroid of alcohol-treated rats compared with controls The increased FAEES activity resulted in increased production and accumulation of ethyl esters in the choroid of alcoholfed rats, with the size of the increase related to the number of weeks of alcohol feeding The increased concentration of ethyl esters within the choroid corresponded to a statistically significant increase in the size of CNV in alcohol-fed rats (10 weeks) compared with regular and pair-fed control rats There was no difference in the CNV size of regular controls compared with pair-fed controls The data suggest that either one or both of the controls may be used for the study We believe that this is the first report to show an effect of alcohol consumption on experimental choroidal angiogenesis We also observed increased expression of cell-cycle proteins, cyclin E and cyclin E ⁄ CDK2, in the choroid after 10 weeks of alcohol feeding These proteins are known to play an important role in the proliferation of cells, including endothelial cells [33,34,48,49] The change in expression of these cell-cycle proteins may be related to the amount of ethyl ester in the choroid Previous studies have shown that ethyl esters have promitogenic activity and can increase the expression of cyclin E and cyclin E ⁄ CDK2 [33] As shown in Table 2, the accumulation of ethyl esters increased with the number of weeks of alcohol feeding The amount (17.10 nmolỈg)1 wet weight) of ethyl ester accumulated after 10 weeks of alcohol feeding may be enough (just above the threshold level) to increase the expression of cyclin E and cyclin E ⁄ CDK2 by 3.0-fold, which may have contributed to the 28% increase in the size of CNV Because laser treatment in rats on a normal diet did not change cyclin E and cyclin E ⁄ CDK2 expression in the choroid, we believe that chronic and heavy alcohol FEBS Journal 273 (2006) 1403–1414 ª 2006 The Authors Journal compilation ª 2006 FEBS P S Bora et al Fig (A) Immunohistological analysis of choroidal APN Immunohistology was performed using adiponectin antibody (primary antibody) in paraffin-embedded rat eyes with CY3-labeled goat (antimouse IgG) serum (secondary antibody), mounting with AquaMount (Lerner Laboratories, PA) Sections were examined under fluorescence microscope (Zeiss) Only choroids showed staining (red in the choroid) No other tissue was stained in the eye APN is expressed specifically in the choroid (B) (Upper) Adiponectin (APN) mRNA expression in alcohol-fed (lane 3) and control (lane 4) BN rat choroids Lanes and are GAPDH lanes The figure shows ethidium bromide-stained bands for PCR product after UV exposure Equal amounts of total RNA (0.2 lg) were used to detect the mRNA levels of GAPDH and APN APN expression in 10-weeks alcoholic rat choroid was significantly decreased compared with control choroid (Middle) Western blot of rat choroid APN antibody was used to blot choroidal proteins Choroids were removed from the alcoholic and control rats, homogenized and the supernatant was used to run the gel to blot with APN antibody APN protein was significantly decreased in 10-week alcoholic rats (lane 1) compared with controls (lane 2) The amount of protein loaded in each lane was lg (Lower) APN mRNA expression in rat choroidal endothelial cell Lane 3, 50 lM ethyl ester-treated cells; lane 4, control cells, not treated with ethyl esters Lanes and are GAPDH lanes The figure shows ethidium bromide-stained bands for PCR product after UV exposure Equal amounts of the total RNA (0.2 lg) were used to detect the mRNA levels of GAPDH and APN APN expression in 50 lM ethyl ester-treated cells was significantly decreased compared with control cells Alcohol and choroidal neovascularization A B Rat choroid RT-PCR Rat choroid western blot intake was important in the increased expression of these proteins Our hypothesis that a certain level of ethyl esters was needed to increase the size of CNV complex was further supported by our observation that cyclin E and cyclin E ⁄ CDK2 expression did not change in the choroid of alcohol-fed rats after weeks and we found no significant difference in the size of the CNV complex after weeks of alcohol consumption Cyclin E and cyclin E ⁄ CDKs can be regulated by growth-promoting signals on cyclin synthesis and on the assembly of cyclin ⁄ CDK complexes The complexes can be further regulated by cyclin kinase inhibitors [33,48,49] Although we observed a 3.0-fold increase in the expression of cyclin E and cyclin E ⁄ CDK2 in the choroid of alcohol-fed rats at 10 weeks, we did not see a change in the expression of p27Kip, a cyclin kinase inhibitor Thus, the increased expression of cyclin E and cyclin E ⁄ CDK2 appeared to be the result of increased production and not decreased cyclin kinase inhibition Studies have shown that actively growing healthy or pathological tissues express high levels of angiogenic factors It is also known that high levels of angiogenic factors may not be sufficient to induce angiogenesis, i.e VEGF is expressed at high levels in Rat choroidal endothelial cell RT-PCR several quiescent adult tissues that lack active angiogenesis Thus, downregulation of antiangiogenic factors may help to enhance angiogenesis In a mouse tumor model, Brakenhielm et al [35] showed that APN was a direct inhibitor of angiogenesis Xu et al [36] showed that circulating APN was decreased in alcoholics We have also shown that choroidal APN expression and protein levels were decreased in 10-week alcoholic rats compared with controls Thus, downregulation of APN may be another factor that helped to enhance the choroidal angiogenesis in 10-week alcoholic rats We did not see any affect of alcohol feeding in the expression of pigment epithelium-derived factor (PEDF, an angiogenesis inhibitor) in the laser spots of alcohol-fed rats compared with controls (data not shown) There is no evidence in the literature to show the affect of alcohol in the expression of other endogenous angiogenesis inhibitors FEBS Journal 273 (2006) 1403–1414 ª 2006 The Authors Journal compilation ª 2006 FEBS 1409 Alcohol and choroidal neovascularization P S Bora et al In conclusion, in an experimental rat model of laserinduced CNV, we have shown that heavy and prolonged alcohol consumption was associated with increased FAEES expression and increased ethyl ester concentration in the choroid Furthermore, the increased cell-cycle regulatory proteins, cyclin E and cyclin E ⁄ CDK2 and decreased APN mRNA and protein in the choroid correlated with an increased CNV size after 10 weeks of alcohol feeding We have experimental evidence that heavy and chronic alcohol feeding in rats enhances choroidal angiogenesis with implications for the prognosis of AMD, and possibly, tumors horseradish peroxidase-conjugated secondary antibody (1 : 1000 dilution), blots were developed using the enhanced chemiluminescence western blotting detection system ‘ECL + Plus’ (Amersham Pharmacia Biotech, Arlington Heights, IL) Immunoprecipitation and western blots for cyclin E, cyclin E ⁄ CDK2 and p27Kip performed according to the methods described by Li et al [33] or as described above Antibodies for cyclin E and p27Kip were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) Antibodies for APN was purchased from BioVision, Inc (Mountain View, CA) Alcohol feeding Male BN rats (4–6 weeks old) were killed, the eyes were enucleated and the choroids (n ¼ 118) collected in phosphate buffer solution, minced and homogenized These procedures were performed at °C Homogenate was centrifuged and the resulting supernatant was collected Protein was estimated using the Bradford method with gamma-globin as standard The enzyme, FAEES, was purified using the method described previously The molecular mass and purity of the enzyme were determined by SDS ⁄ PAGE SDS ⁄ PAGE gels were run at 150 V (stacking) and 200 V (separating), after which they were fixed and stained with Coomassie Brilliant Blue Molecular mass was calculated using polypeptide standards of known molecular mass [29,30] Animals were divided in three groups The control group was fed regular diet and water for 10 weeks, the pair-fed group was fed regular diet, water and glucose for 10 weeks Alcohol-fed rats received gỈkg)1 alcohol and regular diet for 10 weeks Alcohol was mixed with water and the bottles were changed everyday One rat was housed per cage The amount of alcohol consumed was measured everyday [50,51] The alcohol and water mixture contained 20 mL of ethyl alcohol (100% proof, density ¼ 0.78) and 180 mL of water Rats were drinking 31–35 mL of the mixture everyday, assuming 10% of alcohol was evaporated everyday from the mixture, the amount of alcohol received by each rat (300 g) was gỈkg)1 Dehydration was controlled by feeding the alcohol group rats with only water for h everyday before next round of alcohol and water mixture was started The comparable amount of alcohol in humans will be about six glasses of whiskey or rum We included a pairfed group as a second control group Each rat from alcohol fed group was taking total 68 kcalỈday)1, 56 kcalỈday)1 were coming from alcohol and the rest from the regular diet Pair-fed rats also received total of 68 kcalỈday)1 by feeding regular diet (12 kcal) and glucose (56 kcal) The calorific value of glucose is kcalỈg)1 and the calorific value of alcohol is 7.0 kcalỈg)1 (ethanol, 100% proof) In a regular water bottle, 14 g of glucose was dissolved in 100 mL of water and one bottle was supplied to each cage After the glucose water was consumed by the rats the bottle was replaced by a regular water bottle Western blot analysis Rates of FAEE synthesis After SDS ⁄ PAGE on 12% linear slab gel, under reducing conditions, separated proteins were transferred to a poly(vinylidene difluoride) membrane using Trans-Blot semidry electrophoretic transfer cell (Bio-Rad, Richmond, CA) After electroblotting the gels were stained with Coomassie Brilliant Blue to ensure equal loading and equal transfer Blots were stained at room temperature with appropriate antibody (1 : 1000 dilution) for h or overnight at °C Control blots were treated with the same dilution of normal goat or rabbit serum After washing and incubating with The animals in both groups were sacrificed after 1, 4, 8, 9, and 10 weeks of alcohol feeding and the eyes were enucleated to remove cornea, iris and ciliary body, lens, retina and choroid to perform enzyme assay Rates of FAEE synthesis was determined by incubating samples containing enzyme with 0.4 mm [14C] oleic acid (20 000 dpmỈnmol)1) and 200 mm ethanol in 60 mm sodium phosphate buffer, pH 7.2, in a total volume of 0.17 mL in capped vials at 37 °C At the end of the incubation interval, the reaction was terminated by the Experimental procedures Animals Male Brown Norway (BN) rats (4–6 weeks old, 250–300 g) were purchased from Harlan (Indianapolis, IN) The animals were maintained in accord with guidelines established by the Committee on Animals at the University of Louisville Medical School and University of Arkansas for Medical Sciences Purification of FAEES 1410 FEBS Journal 273 (2006) 1403–1414 ª 2006 The Authors Journal compilation ª 2006 FEBS P S Bora et al addition of mL of cold acetone containing a known amount of ethyl [3H] oleate and 0.6 lmol of carrier ethyl oleate Volumes were reduced by evaporation under stream of nitrogen at 37 °C and residual lipids in acetone were chromatographed on silica OF plates, developed with petroleum ether ⁄ diethyl ether ⁄ acetic acid (75 : 25 : v ⁄ v ⁄ v) After visualization of lipids with iodine vapor, fatty acid ethyl ester spots were scrapped, and the lipid was eluted with acetone and counted for radioactivity The 14C counts were adjusted for yield as determined by recovery of 3H, and after subtraction of blanks, results were expressed as nmoles of fatty acid ethyl ester formed per milliliter per hour [29–31] Measurement of ethyl ester production Accumulated ethyl esters were measured using a method described previously [26] Briefly, homogenates of cornea, iris and ciliary body, lens, retina and choroid were incubated with 14C ethanol and phosphate buffer for 60 At the end of the incubation interval, the reaction was terminated by the addition of mL of cold acetone containing a known amount of ethyl [3H] oleate and 0.6 lmol of carrier ethyl oleate Volumes were reduced by evaporation under stream of nitrogen at 37 °C and residual lipids in acetone were chromatographed on silica OF plates, developed with petroleum ether ⁄ diethyl ether ⁄ acetic acid (75 : 25 : v ⁄ v ⁄ v) After visualization of lipids with iodine vapor, fatty acid ethyl ester spots were scrapped, and the lipid was eluted with acetone and counted The 14C counts were adjusted for yield as determined by recovery of 3H, and after subtraction of blanks, the results were expressed as nmolỈg)1 wet weight [26] Immunohistochemistry studies Paraffin embedded sections of the eyes from 10-week alcohol-fed and control rats were used for immunostaining of FAEES Briefly, slides were deparaffinized twice for in xylene, twice for in absolute ethanol, in 95% ethanol, in 70% ethanol and 15 in NaCl ⁄ Pi Fixed sections were immersed in a 0.3% hydrogen peroxide ⁄ methanol solution for 20 to inactivate endogenous peroxidase and then rinsed well in NaCl ⁄ Pi The slides were rinsed and incubated for 20 with serum from the same species as the origin of secondary antibody for blocking nonspecific binding After rinsing, they were incubated with rabbit antirat FAEES (1 : 200 dilution) for 60 at room temperature The slides were stained using anti-(rabbit IgG) immunoperoxidase staining kit (Vector, Burlingame, CA) according to the manufacturer’s instructions The sections were treated with DAB for 10 min, counterstained with Mayer’s hematoxylin for 10 min, washed thoroughly in cold tap water, and covered with coverslip with a mounting media for viewing by light microscopy For APN immunohistochemistry, APN antibody (1: 200 dilutions) was used as primary antibody and CY3-labeled anti-(goat IgG) serum was Alcohol and choroidal neovascularization used as secondary antibody RT-PCR of APN for mRNA expression was performed according to the methods used elsewhere [23] The following primers (GAPDH and APN) were used for RT-PCR GAPDH (forward 5¢-TGAAGGTCGGTGTGAACG GATTTGGC-3¢, reverse 5¢-CATGTAGGCCATGAGGTC CACCAC-3¢); APN (forward 5¢-ATGGGCTATGGGTA GTTGCAGTCA-3¢, reverse 5Â-TAGCTTCATGCTTTGG GTCCTCCA-3Â Laser photocoagulation Control (n ẳ 10), pair-fed (n ¼ 10) and alcohol-fed (n ¼ 10) animals were laser photocoagulated on day 60 by anesthetizing with a mixture of ketamine ⁄ xylazine (1 : v ⁄ v), and the pupils were dilated with a single drop of 1% tropicamide Krypton red laser photocoagulation (50 lm spot size, 0.05 s duration, 350 mW) was used to generate four laser spots in each eye surrounding the optic nerve The animals were killed on day 70 to evaluate the presence of CNV, the eyes were removed and choroid–scleral flat mounts were stained for elastin using a monoclonal antibody specific for elastin (Sigma, St Louis, MO) followed by a Cy-3-labeled secondary antibody (Sigma) The size of the CNV was determined by confocal microcopy [23,32] Measuring neovascularization Rats were anesthetized (ketamine ⁄ xylazine mixture, : 1) and perfused through the heart with mL NaCl ⁄ Pi containing 50 mgỈmL)1 fluorescein-labeled dextran (2 · 106 Da average molecular mass, Sigma) The eyes were removed and fixed for h in 10% phosphate-buffered formalin The cornea and lens were removed and the neurosensory retina was carefully dissected from the eyecup Five radial cuts were made from the edge of the eyecup to the equator; the sclera–choroid–RPE complex was flat-mounted, with the sclera facing down, on a glass slide in Aqua mount Flat mounts were stained with a monoclonal antibody against elastin (Sigma) and a CY3-conjugated secondary antibody (Sigma) and examined using a confocal microscope (Zeiss LSM510, Carl Zeiss AG, Jena, Germany) The CNV stained green, whereas the elastin in the Bruch’s membrane stained red The incidence of the CNV complex was determined by confocal microscopy The size of the neovascular complex was measured by using image pro-plus software [23,32] Primary culture of rat choroidal endothelial cells Rat (BN, weeks old) eyes were removed, cleaned from connective tissue and blood vessels and were dipped in 70% alcohol for After washing with NaCl ⁄ Pi, cornea, lens, RPE and sclera were removed Choroid was stored in HBSS buffer with out Ca and Mg Enzyme diges- FEBS Journal 273 (2006) 1403–1414 ª 2006 The Authors Journal compilation ª 2006 FEBS 1411 Alcohol and choroidal neovascularization P S Bora et al tion was performed using collagenase ⁄ dispase, mgỈmL)1 and DNase I, 17.3 lgỈmL)1 The supernatant was removed and the cells resuspended in HBSS (with out Ca and Mg) After washing five times with same buffer the cells were separated by 40 lm strainer and cultured in fibronectin coated plates After washing with HBSS, Dulbecco’s modified Eagle’s medium was added to the plates The culture was kept at 37 °C in a humidified atmosphere (5% CO2 and 95% air) Cultures were checked for the purity of the endothelial cells by positive labeling with CD31 [52] The medium was changed 24 h after plating and every 48 h thereafter until reached to confluence Methods described by Penfold et al [50] and Li et al [33] were followed for further growth of the cells Linoleic acid ethyl ester (Sigma) was dissolved in dimethylsulfoxide and 50 lm was added to the cells Control cells were treated with dimethylsulfoxide only Statistical analysis 10 11 Differences between groups were evaluated by Student’s t-test 12 Acknowledgements We thank Drs Douglas Borchman, Sean Kuntz and Purushottam Jha for critical review of the manuscript and Guirong Liu and Dr Jose M.C Cruz for helping in APN staining This study was supported by Commonwealth of Kentucky Research Challenge Trust Fund; Research to Prevent Blindness, Inc., New York and grants from NEI and NEI core grant, 1R24EY015636-01 14 15 References Ferris FL III, Fine SL & Hyman LA (1984) Age related macular degeneration and blindness due to neovascular maculopathy Arch Ophthalmol 102, 1640–1642 Grossniklaus HE, Hutchinson AK, Capone A Jr, Woolfson J & Lambert HM (1994) Clinicopathologic features of surgically-excised choroidal neovascular membranes Ophthalmology 101, 1099–1111 Grossniklaus HE & Gass JDM (1998) Clinicopathologic correlation of surgically-excised type and type choroidal neovascular membranes Am J Ophthalmol 126, 59–69 Hans E, Grossniklaus HE & Green WR (2004) Choroidal neovascularization Am J Ophthalmol 137, 496–503 Frank G, Holz D, Pauleikhoff RK & Bird AC (2004) Pathogenesis of lesions in late age-related macular disease Am J Ophthalmol 137, 504–510 Kliffen M, Sharma HS, Mooy CM, Kerkvliet S & Jong PT (1997) Increased expression of angiogenic growth 1412 13 16 17 18 19 factors in age-related maculopathy Br J Ophthalmol 81, 154–162 Frank RN (1997) Growth factors in age-related macular degeneration: pathogenic and therapeutic implications Ophthalmic Res 29, 341–353 Abraham JA, Mergia A, Whang JL, Tumolo A, Friedman J, Hjerrild KA, Gospodarowicz D & Fiddes JC (1986) Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor Science 1, 545–548 Amin R, Puklin JE & Frank RN (1994) Growth factor localization in choroidal neovascular membranes of agerelated macular degeneration Invest Ophthalmol Vis Sci 35, 3178–3188 Antoinette C, Lambooij KHM, van Wely DJ, Lindenbergh-Kortleve RWAM, Kuijpers MK & Cornelia MM (2003) Insulin-like growth factor-I and its receptor in neovascular age-related macular degeneration Invest Ophthalmol Vis Sci 44, 2192–2198 Smith LE, Kopchick JJ, Chen W, Knapp J, Kinose F, Daley D, Foley E, Smith RG & Schaeffer JM (1997) Essential role of growth hormone in ischemia-induced retinal neovascularization Science 276, 1706–1709 Jin M., Chen Y, He S, Ryan SJ & Hinton DR (2004) Hepatocyte growth factor and its role in the pathogenesis of retinal detachment Invest Ophthalmol Vis Sci 45, 323–329 Laterra J, Nam M, Rosen E, Rao JS, Lamszus K, Goldberg ID & Johnston P (1997) Scatter factor ⁄ hepatocyte growth factor gene transfer enhances glioma growth and angiogenesis in vivo Lab Invest 76, 565–577 Leibovich SJ, Polverini PJ, Shepard HM, Wiseman DM, Shively V & Nuseir N (1987) Macrophage-induced angiogenesis is mediated by tumour necrosis factoralpha Nature 329, 630–632 Oh H, Takagi H, Takagi C, Suzuma K, Otani A, Ishida K, Matsumura M, Ogura Y & Honda Y (1999) The potential angiogenic role of macrophages in the formation of choroidal neovascular membranes Invest Ophthalmol Vis Sci 40, 1891–1898 Tsutsumi C, Sonoda K-H, Egashira K, Qiao H, Hisatomi T, Nakao S, Ishibashi M, Charo IF, Sakamoto T, Murata T et al (2003) The critical role of ocularinfiltrating macrophages in the development of choroidal neovascularization J Leukoc Biol 74, 25–32 Killingsworth MC, Sarks JP & Sarks SH (1990) Macrophages related to Bruch’s membrane in age-related macular degeneration Eye 4, 613–621 Dastgheib K & Green WR (1994) Granulomatous reaction to Bruch’s membrane in age-related macular degeneration Arch Ophthalmol 112, 813–818 Shikun He, Man LJ, Worpel V & Hinton DR (2003) A role for connective tissue growth factor in the pathogenesis of choroidal neovascularization Arch Ophthalmol 121, 1283–1288 FEBS Journal 273 (2006) 1403–1414 ª 2006 The Authors Journal compilation ª 2006 FEBS P S Bora et al 20 Madri JA, Reidy MA, Kocher O & Bell L (1989) Endothelial cell behavior after denudation injury is modulated by transforming growth factor-beta1 and fibronectin Lab Invest 60, 755–765 21 Kaplan HJ, Leibole MA, Tezel TH & Ferguson TA (1999) Fas-ligand (CD95 ligand) controls angiogenesis beneath retina Nat Med 5, 292–297 22 Grant MB, May WS & Caballero S (2002) Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization Nat Med 6, 607–612 23 Bora PS, Sohn SG, Jha P, Kaplan HJ & Bora NS (2005) Role of complement and complement membrane attack complex in laser-induced choroidal neovascularization J Immunol 174, 491–497 24 Bora PS, Guruge BL & Bora NS (2003) Molecular characterization of human eye and heart fatty acid ethyl ester synthase ⁄ carboxylesterase by site-directed mutagenesis Biochem Biophys Res Commun 26, 1094–1098 25 Bora PS, Bora NS, Wu X, Kaplan HJ & Lange LG (1994) Molecular cloning, sequencing and characterization of smooth muscle myosin alkali light chain from human eye cDNA: homology with myocardial fatty acid ethyl ester synthase-III cDNA Genomics 19, 186–188 26 Lange LG & Sobel BE (1983) Myocardial metabolites of ethanol Circ Res 52, 479–482 27 Bora PS, Spilburg CA & Lange LG (1989) Purification to homogeneity and characterization of major fatty acid ethyl ester synthase from human myocardium FEBS Lett 258, 236–239 28 Bora PS, Spilburg CA & Lange LG (1989) Metabolism of ethanol and carcinogens by glutathione transferases Proc Natl Acad Sci USA 86 (4470), 4473 29 Bora PS, Guruge DG, Miller DD, Chaitman BR & Ruyle MS (1996) Purification and characterization of human heart fatty acid ethyl ester synthase ⁄ carboxylesterase J Mol Cell Cardiol 28, 2027–2032 30 Bora PS, Wu X, Spilburg CA & Lange LG (1992) Purification and characterization of fatty acid ethyl ester synthase-II from human myocardium J Biol Chem 267, 13217–13221 31 Mogelson S & Lange LG (1984) Nonoxidative ethanol metabolism in rabbit myocardium: purification to homogeneity of fatty acyl ethyl ester synthase Biochemistry 23, 4075–4081 32 Bora PS, Hu Z, Tezel TH, Sohn JH, Kang SG, Cruz JM, Bora NS, Garen A & Kaplan HJ (2003) Immunotherapy for choroidal neovascularization in a laser-induced mouse model simulating exudative (wet) macular degeneration Proc Natl Acad Sci USA 100, 2679–2684 33 Li JJ, Hu W, Baldassare JJ, Bora PS, Chen S, Britton RS & Bacon BR (2003) The ethanol metabolite, linolenic acid sthyl ester, stimulates mitogen activated protein Alcohol and choroidal neovascularization 34 35 36 37 38 39 40 41 42 43 44 45 46 kinase and cyclin signaling in hepatic stellate cells Life Sci 73, 1083–1096 Shankland SJ, Hugo C, Pippin J, Roberts JM, Couser WG & Johnson RJ (1996) Changes in cell cycle protein expression during experimental mesangeal proliferative glomerulonephritis Kidney Int 50, 1230–1239 Brakenhielm E, Veitonmaki N, Cao R, Kihara S, Matsuzawa Y, Zhivotovsky B, Funahashi T & Cao Y (2004) Adiponectin-induced antiangiogenesis and antitumor activity involve caspase-mediated endothelial cell apoptosis Proc Natl Acad Sci USA 101, 2476–2481 Xu A, Wang Y, Keshaw H, Xu LY, Lam KL & Cooper GS (2003) The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice J Clin Invest 112, 91–100 Cohen SY, Laroche A, Leguen Y, Soubrane G & Coscas GJ (1996) Etiology of choroidal neovascularization in young patients Ophthalmology 103, 1241–1244 Singerman LJ (1988) Current management of choroidal neovascularization Ann Ophthalmol 20, 415–420 Atebara NH, Thomas MA, Holekamp NM, Mandell BA & Del Priore LV (1998) Surgical removal of extensive peripapillary choroidal neovascularization associated with presumed ocular histoplasmosis syndrome Ophthalmology 105, 1598–1605 Smith W & Mitchell P (1996) Alcohol intake and agerelated maculopathy Am J Ophthalmol 122, 743–745 Moss SE, Klein R, Klein BE, Jensen SC & Meuer SM (1998) Alcohol consumption and the 5-year incidence of age-related maculopathy: the Beaver Dam eye study Ophthalmology 105, 789–794 Ritter LL, Klein R, Klein BE, Mares-Perlman JA & Jensen SC (1995) Alcohol use and age-related maculopathy in the Beaver Dam eye study Am J Ophthalmol 120, 190–196 Azen SP, fraser-Bell S, Klein R & Varma R (2004) Smoking, alcohol and risk of advanced age-related macular degeneration Los Angeles Latino Eye Study ARVO 3057, 126 Zgombic-Knight M, Ang HL, Foglio MH & Duester G (1995) Cloning of the mouse class IV alcohol dehydrogenase (retinol dehydrogenase) cDNA and tissue-specific expression patterns of the murine ADH gene family J Biol Chem 270, 10868–10877 Zgombic-Knight M, Foglio MH & Duester G (1995) Genomic structure and expression of the ADH7 gene encoding human class IV alcohol dehydrogenase, the form most efficient for retinol metabolism in vitro J Biol Chem 270, 4305–4311 Martras S, Alvarez R, Martinez SE, Torres D, Gallego O, Duester G, Farres J, DeLera AR & Pares X (2004) The specificity of alcohol dehydrogenase with cis-retinoids: activity with 11-cis-retinol and localization in retina Eur J Biochem 271, 1660–1670 FEBS Journal 273 (2006) 1403–1414 ª 2006 The Authors Journal compilation ª 2006 FEBS 1413 Alcohol and choroidal neovascularization P S Bora et al 47 Julia P, Farres J & Pares X (1986) Ocular alcohol dehydrogenase in the rat: regional distribution and kinetics of the ADH-1 isoenzyme with retinol and retinal Exp Eye Res 42, 305–314 48 Winston J, Dong F & Pledger WJ (1998) Differential modulation of G1 cyclins and the CDK inhibitor p27Kip by platelet derived growth factor and plasma factors in density arrested fibroblasts J Biol Chem 271, 11253–11260 49 Shankland SJ, Pippen J, Flanagan M, Coats SR, Couser WG & Johnson RJ (1997) Mesangial proliferation mediated by PDGF and bFGF is determinedby levels of the cyclin kinase inhibitor p27Kip Kidney Intl 51, 1088– 1099 1414 50 Port EA & Gomez Dumm CL (1968) A new experimental approach in the study of chronic alcoholism Laboratory Invest 18, 352–364 51 Edes I, Toszegi A, Csanady M & Bozoky B (1986) Myocardial lipid peroxidation in rats after chronic alcohol ingestion and the effects of different antioxidants Cardiovas Res 20, 542–548 52 Penfold PL, Wen L, King NJC & Provis JM (2002) Modulation of permeability and adhesion molecule expression by human choroidal endothelial cells Invest Ophthalmol Vis Sci 43, 3125–3130 FEBS Journal 273 (2006) 1403–1414 ª 2006 The Authors Journal compilation ª 2006 FEBS ... of cyclin E and cyclin E ⁄ CDK2 in the choroid of alcohol- fed rats at 10 weeks, we did not see a change in the expression of p27Kip, a cyclin kinase inhibitor Thus, the increased expression of. .. increased FAEES activity resulted in increased production and accumulation of ethyl esters in the choroid of alcoholfed rats, with the size of the increase related to the number of weeks of alcohol. .. immunoperoxidase staining kit (A) Control rats showed mild staining (brown color in the choroid) (B) Rats fed alcohol for 10 weeks showed several fold increase in staining (brown staining is marked