The role of omega-3 long-chain polyunsaturated fatty acids in health and disease of the retina
Progress in Retinal and Eye Research 24(2005)87–138
The role of omega-3long-chain polyunsaturated fatty acids in health
and disease of the retina
John Paul SanGiovanni ?,Emily Y.Chew
Division of Epidemiology and Clinical Research,National Eye Insitute,National Institutes of Health,31Center Drive,Building 31,Room 6A52,
MSC 2510,Bethesda,MD 20892-2510,USA
Abstract
In this work we advance the hypothesis that omega-3(o -3)long-chain polyunsaturated fatty acids (LCPUFAs)exhibit cytoprotective and cytotherapeutic actions contributing to a number of anti-angiogenic and neuroprotective mechanisms within the retina.o -3LCPUFAs may modulate metabolic processes and attenuate effects of environmental exposures that activate molecules implicated in pathogenesis of vasoproliferative and neurodegenerative retinal diseases.These processes and exposures include ischemia,chronic light exposure,oxidative stress,in?ammation,cellular signaling mechanisms,and aging.A number of bioactive molecules within the retina affect,and are effected by such conditions.These molecules operate within complex systems and include compounds classi?ed as eicosanoids,angiogenic factors,matrix metalloproteinases,reactive oxygen species,cyclic nucleotides,neurotransmitters and neuromodulators,pro-in?ammatory and immunoregulatory cytokines,and in?ammatory phospholipids.We discuss the relationship of LCPUFAs with these bioactivators and bioactive compounds in the context of three blinding retinal diseases of public health signi?cance that exhibit both vascular and neural pathology.
How is o -3LCPUFA status related to retinal structure and function?Docosahexaenoic acid (DHA),a major dietary o -3LCPUFA,is also a major structural lipid of retinal photoreceptor outer segment membranes.Biophysical and biochemical properties of DHA may affect photoreceptor membrane function by altering permeability,?uidity,thickness,and lipid phase properties.Tissue DHA status affects retinal cell signaling mechanisms involved in phototransduction.DHA may operate in signaling cascades to enhance activation of membrane-bound retinal proteins and may also be involved in rhodopsin regeneration.Tissue DHA insuf?ciency is associated with alterations in retinal function.Visual processing de?cits have been ameliorated with DHA supplementation in some cases.
What evidence exists to suggest that LCPUFAs modulate factors and processes implicated in diseases of the vascular and neural retina?Tissue status of LCPUFAs is modi?able by and dependent upon dietary intake.Certain LCPUFAs are selectively accreted and ef?ciently conserved within the neural retina.On the most basic level,o -3LCPUFAs in?uence retinal cell gene expression,cellular differentiation,and cellular survival.DHA activates a number of nuclear hormone receptors that operate as transcription factors for molecules that modulate reduction-oxidation-sensitive and proin?ammatory genes;these include the peroxisome proliferator-activated receptor-a (PPAR-a )and the retinoid X receptor.In the case of PPAR-a ,this action is thought to prevent endothelial cell dysfunction and vascular remodeling through inhibition of:vascular smooth muscle cell proliferation,inducible
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Abbreviations:A2E,N -retinylidene-N -retinylethanolamine;AA,arachidonic acid (20:4o -6);AMD,age-related macular degeneration;ARM,age-related maculopathy;COX,cyclooxygenase;DHA,docosahexaenoic acid (22:6o -3);DPA,docosapentaenoic acid;DR,diabetic retinopathy;EFA,essential fatty acid;EPA,eicosapentaenoic acid (20:5o -3);GA,geographic atrophy;HETE,hydroxyeicosatetraenoic acid;HPETE,
hydroperoxyeicosatetraenoic acid;HUVEC,human umbilical vein endothelial cell;ICAM,intracellular cell adhesion molecule;IFN,interferon;Ig,immunoglobulin;IL,interleukin;IPM,interphotoreceptor matrix;LA,linoleic acid (18:2o -6);LCPUFA,long-chain polyunsaturated fatty acid;LOX,lipoxygenase;LT,leukotriene;NF k B,nuclear-factor kappa B;NPDR,non-proliferative diabetic retinopathy;NV,neovascular;PAF,platelet-activating factor;PC,phosphatidylcholine;PDR,proliferative diabetic retinopathy;PEA,phosphatidylethanolamine;PG,prostaglandin;PI,
phosphatidylinositol;PKC,protein kinase C;PLA 2,phospholipase A 2;PPAR,peroxisome proliferator-activated receptor;PS,phosphatidylserine;redox,oxidation-reduction;ROP,retinopathy of prematurity;RPE,retinal pigment epithelium;RXR,retinoid X receptor;TNF,tumor necrosis factor;TX,thromboxane;uPA,urokinase-type plasminogen activator;VEGF,vascular endothelial growth factor;a -LLNA,a -linolenic acid (18:3o -3)?Corresponding author.Tel.:+1-301-496-1331;fax:+1-301-496-2297.E-mail address:jpsangio@https://www.wendangku.net/doc/2512175054.html, (J.P.SanGiovanni).
nitric oxide synthase production,interleukin-1induced cyclooxygenase (COX)-2production,and thrombin-induced endothelin 1production.
Research on model systems demonstrates that o -3LCPUFAs also have the capacity to affect production and activation of angiogenic growth factors,arachidonic acid (AA)-based vasoregulatory eicosanoids,and MMPs.Eicosapentaenoic acid (EPA),a substrate for DHA,is the parent fatty acid for a family of eicosanoids that have the potential to affect AA-derived eicosanoids implicated in abnormal retinal neovascularization,vascular permeability,and in?ammation.EPA depresses vascular endothelial growth factor (VEGF)—speci?c tyrosine kinase receptor activation and expression.VEGF plays an essential role in induction of:endothelial cell migration and proliferation,microvascular permeability,endothelial cell release of metalloproteinases and interstitial collagenases,and endothelial cell tube formation.The mechanism of VEGF receptor down-regulation is believed to occur at the tyrosine kinase nuclear factor-kappa B (NF k B).NF k B is a nuclear transcription factor that up-regulates COX-2expression,intracellular adhesion molecule,thrombin,and nitric oxide synthase.All four factors are associated with vascular instability.COX-2drives conversion of AA to a number angiogenic and proin?ammatory eicosanoids.Our general conclusion is that there is consistent evidence to suggest that o -3LCPUFAs may act in a protective role against ischemia-,light-,oxygen-,in?ammatory-,and age-associated pathology of the vascular and neural retina.Published by Elsevier Ltd.
Contents 1.Introduction ..............................................................................892.
LCPUFAs:general descriptions,functions,actions,and associations ......................................902.1.DHA,EPA,and AA are LCPUFAs ........................................................902.2.DHA,EPA,and AA are fatty acids of physiological signi?cance ....................................913.
Metabolism,transport,accretion,and intake of EFAs and LCPUFAs .....................................923.1.LCPUFAs are obtained through diet or biosynthesized from EFAs ..................................923.2.Transport and accretion of LCPUFAs.....................
..................................923.2.1.LCPUFAs in RPE and photoreceptors ................................................943.2.2.LCPUFAs in the vascular retina .....................................................953.3.EFA and LCPUFA intake .............................
..................................954.
Role of LCPUFAs in structure and function of sensory retina...........................................964.1.DHA is an essential structural component of retinal membranes ....................................964.2.DHA tissue status is associated with alterations in retinal and visual function.
..........................964.2.1.Inherited retinal degenerations ......................................................974.2.2.Metabolic insuf?ciency............................................................974.2.3.Dietary insuf?ciency..............................................................974.3.DHA affects retinal cell signaling mechanisms in phototransduction..................................974.4.LCPUFAs in?uence retinal cell gene expression,differentiation,and survival .
..........................984.4.1.Gene expression.................................................................984.4.2.Cellular differentiation ............................................................984.4.3.Survival.............................................
(98)
5.
Metabolic and environmental bioactivators ........................................................985.1.Role of PLA 2in LCPUFA hydrolysis .......................................................995.2.Role of COX in eicosanoid biosynthesis......................................................995.3.Role of LOX in eicosanoid biosynthesis......................................................995.4.Retinal ischemia..................................................................
.....995.4.1.Vascular networks in the retina.....................................................1005.4.2.LCPUFAs affect factors and processes implicated retinal ischemia:vasoregulatory eicosanoids and
vascular response...........................................................
....1005.4.3.Lipoprotein metabolism ..........................................................1015.4.4.LCPUFAs affect energy production,regulation,and metabolism .............................1015.5.Light exposure...................................................................
....1025.5.1.LCPUFAs affect factors and processes implicated in retinal light damage.......................1025.6.Oxidation-reduction balance .........................................................
....1025.6.1.Reactive oxygen species and free radicals ..............................................1025.6.2.Metabolic and environmental bioactivators affect redox balance .........................
(103)
J.P.SanGiovanni,E.Y.Chew /Progress in Retinal and Eye Research 24(2005)87–138
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1.Introduction
Long-chain polyunsaturated fatty acids(LCPUFAs) demonstrate anti-angiogenic,anti-vasoproliferative,and neuroprotective actions on factors and processes im-plicated in the pathogenesis of proliferative and degenerative retinal diseases.Many retinal diseases of public health signi?cance manifest tissue and cellular dysfunction in the forms of abnormal angiogenesis, proliferative neovascularization,excessive vascular per-meability,immunoregulatory dysfunction,alterations in physiologic reduction-oxidation(redox)balance,or neuronal/retinal pigment epithelial(RPE)cell degenera-tion.A number of bioactive molecules within the eye
5.6.3.LCPUFAs affect factors and processes implicated in maintaining redox balance (104)
5.7.In?ammation (104)
5.7.1.Eicosanoid metabolism (105)
5.7.2.LCPUFAs affect factors and processes implicated in ocular in?ammation (105)
5.7.3.LCPUFAs are ligands for nuclear hormone receptors involved in signaling pathways (108)
5.8.Neuroactive compounds and cell signaling pathways (108)
5.8.1.Cyclic nucleotides (109)
5.8.2.Endocannabinoids (109)
5.8.3.Glutamate and PAF (109)
5.8.4.Dopamine (110)
5.8.5.Ionic transport and channel dynamics (110)
5.9.Developmental processes (111)
5.9.1.Aging and the chororiocapillaris–Bruch’s membrane–RPE—photoreceptor complex (111)
5.9.2.Aging and oxidative stress (112)
5.9.3.Lipofuscin (113)
5.9.4.Astrocytes (113)
5.9.5.LCPUFAs affect factors and processes implicated in aging (113)
6.Role of LCPUFAs in structure and function of vascular retina (114)
6.1.LCPUFAs affect pathogenic processes implicated in neovasculaization (114)
6.1.1.Production and release of angiogenic factors (114)
6.1.2.Growth factors bind to receptors on endothelial cells (118)
6.1.3.Endothelial cells are activated and send signals to the nucleus for production of signaling molecules
and enzymes (118)
6.1.4.Enzymes digest the basement membrane (118)
6.1.5.Endothelial cells divide and migrate through basement membrane toward growth factors (118)
6.1.6.Adhesion molecules,or integrins(a v b3,a v b5)help pull the blood vessel sprout forward (118)
6.1.
7.MMP are produced to dissolve the tissue in front of the sprouting vessel tip in order to accommodate it.
As the vessel extends,the tissue is remolded around the vessel (119)
6.1.8.Sprouting endothelial cells form tubes (119)
7.Retinal diseases of public health signi?cance (119)
7.1.Diabetic retinopathy (119)
7.1.1.DR and the vascular retina (120)
7.1.2.DR and the neural retina (120)
7.1.3.Putative role of o-3LCPUFAs in modulating factors and processes implicated in pathogenesis of DR..120
7.2.Age-related macular degeneration (121)
7.2.1.Advanced AMD and CNV (121)
7.2.2.Advanced AMD and degeneration of RPE and neural retina (122)
7.2.3.Putative role of o-3LCPUFAs in modulating factors and processes implicated in pathogenesis of AMD122
7.3.Retinopathy of prematurity (123)
7.3.1.ROP and the vascular retina (123)
7.3.2.ROP and the neural retina (125)
7.3.3.Putative role of o-3LCPUFAs in modulating factors and processes implicated in pathogenesis of ROP.125
8.Summary and future directions (125)
Acknowledgements (126)
References (126)
J.P.SanGiovanni,E.Y.Chew/Progress in Retinal and Eye Research24(2005)87–13889
affect,and are effected by,such conditions.These molecules are activated in response to ischemia,light exposure,oxygen/energy metabolism and oxidative stress,apoptosis,cell signaling pathways,in?ammation, and developmental processes associated with aging. They operate within complex systems and include eicosanoids,angiogenic factors,matrix metalloprotei-nases(MMPs),reactive oxygen species,cyclic nucleo-tides,neurotransmitters and neuromodulators,pro-in?ammatory and immunoregulatory cytokines,and in?ammatory phospholipids.Effects and actions of metabolic and environmental bioactivators and bioac-tive molecules include activation of phospholipase A2 (PLA2),cyclooxgenase(COX),and lipoxygenase (LOX).Activation of this enzyme system yields a pool of LCPUFAs and bioactive eicosanoids.
Omega-3(o-3)LCPUFAs demonstrate the capacity to modulate production,activation,and potency of bioactive molecules.In some cases these LCPUFAs operate as lipid–protein complexes via signaling cas-cades in nuclear and cytosolic compartments.In others, they affect substrate pools or availability of biosynthetic enzymes.They in?uence gene expression as ligands to a number of transcription factors and act as endocanna-binoid autocoids.Docosahexaenoic acid(DHA, C22:6o-3),a major dietary o-3LCPUFA,is also a major structural lipid in sensory and vascular retina. Metabolic and dietary DHA insuf?ciency is associated with alterations in visual system structure and function. DHA and its substrate,eicosapentaenoic acid(EPA, C20:5o-3),in?uence eicosanoid metabolism by reducing o-6LCPUFA levels(mainly arachidonic acid(C20:4o-6,AA))and competing for enzymes(COX and LOX) used to produce AA-based angiogenic and proin?am-matory series2-and4-eicosanoids.
In this work we present the body evidence implicating LCPUFAs as key modulators of processes in?uencing retinal health and disease.Section2contains a general overview of properties,functions,and actions of LCPUFAs;a more detailed treatment of the issue appears in Chow(2000).Section3contains an overview of LCPUFA metabolism,intake,transport,and accre-tion to the retina;additional information exists in Neuringer(1993),Salem et al.(2001),and Bazan et al. (1993).In Section4we consider actions of LCPUFAs on biochemical and biophysical processes that de?ne properties of retinal membranes and signaling systems. Section5contains information on metabolic and environmental factors and processes that activate molecules driving retinal neovascularization and neural cell death.These bioactivating factors include ischemia, chronic light exposure,cellular redox balance,cell death, in?ammation,neuroactive signaling molecules,and the aging process.In Section6we consider the role of LCPUFAs in the structure and function of the vascular retina.In Section7we consider the means by which o-3LCPUFAs may operate as protective factors in retinal diseases that manifest vascular and neural pathology;we present three examples:diabetic retinopathy(DR),age-related macular degeneration(AMD),and retinopathy of prematurity(ROP).These diseases were selected on the basis of life-span risk,the burden they exert on society,and the coexistence of vascular and neural degenerative pathologies.Our general conclusion is that there is consistent evidence to suggest that o-3 LCPUFAs may act in a protective role against ischemia-,light-,oxygen-,in?ammatory-,or age-asso-ciated retinal diseases.Section conclusions are displayed in Table1.
2.LCPUFAs:general descriptions,functions,actions, and associations
2.1.DHA,EPA,and AA are LCPUFAs
Fatty acids are compounds synthesized through condensation of malonyl coenzyme A units by a fatty acid synthase complex.Two families of essential fatty acids(EFAs)exist in nature;o-3and o-6.o-3and o-6 LCPUFAs contain a carboxyl head group and an even numbered carbon chain(X18carbons)with two-or-more methylene-interrupted double(unsaturated) bonds.EFAs and LCPUFAs are structurally classi?ed by the number of carbons,double bonds,and proximity of the?rst double bond to the methyl(omega)terminal of the fatty acid acyl chain.Fatty acids of the o-3family contain a double bond at the third carbon;those of the o-6family contain a double bond at the sixth carbon. The chemical structure of fatty acids is commonly abbreviated by a listing of the number of carbons,the number of double bonds,and the location of the?rst double bond from the methyl terminal.For example, DHA is represented as C22:6o-3,indicating carbon chain length of22with6double bonds;the?rst unsaturated bond is inserted at carbon3.Body stores of LCPUFAs exist mainly as esteri?ed complexes in the sn-2position of gylcerophosphates(also known as glycer-ophospholipids or phospholipids)or trihydric glycerols (also known as triacylglycerols or triglycerides).Within the neural retina,phospholipids represent the predomi-nant LCPUFA-rich lipid class;these compounds are stored mainly as structural elements of membranes. Phosphatidylcholine(PC)composes40–50%of retinal phospholipids and is localized mainly in the outer lea?et of the membrane.Phosphatidylethanolamine(PEA)and phosphatidylserine(PS)represent30–35%and5–10% of retinal phospholipids,respectively;both species tend to orient within the cytoplasmic lea?et.Phosphatidyli-nositol(PI)composes3–6%of retinal phospholipids and may be a constituent of membrane domains acting in signaling cascades(Gordon and Bazan,1997).
J.P.SanGiovanni,E.Y.Chew/Progress in Retinal and Eye Research24(2005)87–138 90
2.2.DHA,EPA,and AA are fatty acids of physiological signi?cance
DHA(D4,7,10,13,16,19-DHA;C22H32O2)is an o-3 LCPUFA with a molecular weight of328.488.Highest body concentrations of DHA per unit weight are found in phospholipids of retinal photoreceptor outer seg-ments;DHA is also found in substantial amounts within retinal vascular tissue and glia.PEA and PS are the dominant retinal DHA-containing phospholipid species. EPA(D5,8,11,14,17-EPA;C20H30O2)is the other major dietary o-3LCPUFA.This compound contains5 double bonds and has a molecular weight of302.451. EPA is present in blood components,but is not accreted to tissue in great amounts as it is quickly used in DHA or eicosanoid biosynthesis(reviewed in Nelson,2000). AA(D5,8,11,14-eicosatetraenoic acid;C20H32O2)is an o-6LCPUFA with4double bonds and a molecular weight of304.467.AA is a major fatty acid of neural and vascular tissue of the retina and brain.Highest concentrations of AA in human retina are found in PC, and then PEA.
Table1
Section conclusions of this report
DESCRIPTIONS,FUNCTIONS,ACTIONS,AND ASSOCIATIONS OF LCPUFAs
DHA is a o-3LCPUFA.It has22carbons and6methylene-interupted double bonds.
EPA is a o-3LCPUFA.It has20carbons and5methylene-interupted double bonds.
AA is a o-6LCPUFA.It has20carbons and4methylene-interupted double bonds.
DHA,EPA,and AA are LCPUFAs of physiologic signi?cance,as they act as constituents of lipid–protein complexes,substrates for bioactive eicosanoids or endocannabinoids,and natural ligands to nuclear transcription factors.
LCPUFA METABOLISM,INTAKE,TRANSPORT,AND ACCRETION
LCPUFAs may be of dietary or cellular origin.The body does not have the enzymatic capacity to meet tissue needs for LCPUFA through biosynthesis.Tissue status is modi?able and dependent on intake.
DHA is selectively accreted and ef?ciently retained in photoreceptors.
The hepatocyte is the major site of LCPUFA biosynthesis.
LCPUFAs are esteri?ed into triglycerides and phospholipids,integrated with chylomicrons or very low-density lipoproteins before transport to the choriocapillaris.
LCPUFA-rich phospholipids are hydrolyzed and taken up by a high af?nity,receptor-mediated process at the choroid-RPE.They are then transported through the interphotoreceptor matrix to the photoreceptor inner segment.Esteri?ed DHA-phospholipid compounds are then hydrolyzed,actively transferred to the cytosol of the inner segment and re-esteri?ed into phospholipids.These moieties are then incorporated into photoreceptor disk membranes and transferred to the outer segment.Disks migrate to the apical tip of the photoreceptor with time,they are shed and phagocytized by RPE cells.DHA is then stored within oil droplets in the RPE and ef?ciently recycled to the inner segment via a receptor mediated process.
LCPUFAs of cellular origin may also be biosynthesized on neural(astrocytes,photoreceptor)and vascular retinal endoplasmic reticulum and peroxisomes.
o-3LCPUFA-rich foods are limited and less frequently consumed than other foods in Western diets
LCPUFAs IN RETINAL STRUCTURE AND FUNCTION OF THE SENSORY RETINA
DHA is a major structural component of retinal membranes
DHA tissue status insuf?ciency is associated with reduced visual processing capacity.
DHA affects retinal cell signaling mechanisms involved in phototransduction.
LCPUFAs in?uence retinal cell gene expression,differentiation,and survival.
METABOLIC AND ENVIRONMENTAL ACTIVATORS
PLA
2
hydrolyzes LCPUFAs from their esteri?ed form within membranes and lipoproteins to a free form capable of acting as a substrate for eicosanoid synthesis.PLA2is activated by ischemia,light exposure,oxidative stress,apoptosis,in?ammation,cell signaling molecules,and aging.Retinal diseases of public health signi?cance are associated both with PLA2activity and with these metabolic and environmental factors.
COX and LOX catalyze conversion of LCPUFAs from free forms to eicosanoids.COX and LOX are activated by ischemia,light exposure, oxidative stress,apoptosis,in?ammation,cell signaling molecules,and aging.Retinal diseases of public health signi?cance are associated both with COX/LOX activity and with these metabolic and environmental factors.
LCPUFAs IN RETINAL STRUCTURE AND FUNCTION OF THE VASCULAR RETINA
Long-chain polyunsaturated fatty acids(LCPUFAs)demonstrate anti-angiogenic,anti-vasoproliferative,and neuroprotective actions on factors and processes implicated in the pathogenesis of proliferative and degenerative retinal diseases.
These actions affect eicosanoids,angiogenic factors,matrix metalloproteinases,reactive oxygen species,cyclic nucleotides,neurotransmitters and neuromodulators,pro-in?ammatory and immunoregulatory cytokines,and in?ammatory phospholipids.
LCPUFAs AND RETINAL DISEASES OF PUBLIC HEALTH SIGNIFICANCE
LCPUFAs have the capacity to affect pathogenic factors and processes implicated in retinal neovascularization
LCPUFAs have the capacity to affect pathogenic factors and processes implicated in retinal neural degeneration
J.P.SanGiovanni,E.Y.Chew/Progress in Retinal and Eye Research24(2005)87–13891
The chemical structures for DHA,EPA,and AA are represented in Fig.1.These compounds act as:
Key structural constituents of phospholipid membranes.DHA and AA are major fatty acids of neural and vascular retinal tissue.
Ligands to transcription factors for genes in?uencing:(a)cellular differentiation and growth;(b)lipid,protein,and carbohydrate metabolism.DHA,EPA,and AA affect gene expression through regulation of transcription factor activity and concentration within the nucleus.Transcription factors containing an LCPUFA binding domain include peroxisome pro-liferator-activated receptor (PPAR),retinoid X re-ceptor (RXR),nuclear-factor kappa B (NF k B),and sterol regulatory element binding proteins (SREBPs).In some cases,metabolites of the LCPUFAs also act as ligands.
Effectors of signal transduction pathways regulating gene transcription .These pathways include enzyme-based LOX,COX,protein kinase C (PKC),and sphingomyelinase.LCPUFAs may also regulate path-ways affecting tyrosine kinase-linked-and G-protein receptors.
Substrates for eicosanoid or endocannabinoid autocoids involved in inter-and intracellular signaling cascades that in?uence vascular,neural,and immune function.
3.Metabolism,transport,accretion,and intake of EFAs and LCPUFAs
3.1.LCPUFAs are obtained through diet or biosynthesized from EFAs
Humans do not have capacity for de novo biosynth-esis of EFAs (a -linolenic and linoleic acid,LA),due to a
natural absence of D -15and-12desaturase enzymes.We are thus dependent on dietary sources of these compounds.LCPUFAs may be obtained directly through the diet or formed from 18-carbon EFAs.Enzymatic reactions yielding LCPUFAs do not satisfy the body’s requirements.
After EFAs are obtained through the diet they are desaturated (by insertion of double bonds)and elon-gated (by addition of 2-carbon units)to LCPUFAs on the hepatic or retinal endoplasmic reticulum (ER).a -linolenic acid (a -LLNA,C18:3o -3)is the dietary precursor to EPA and DHA.Linolenic acid (LA,C18:2o -6)is the dietary precursor to AA.Conversion from 24to 22carbon LCPUFAs requires b -oxidation in the peroxisome.Fig.2displays biosynthetic pathways for the o -3and o -6families.Because both EFA families compete for the same biosynthetic enzymes,dietary lipid balance and composition will affect production and tissue accretion of these nutrients.Although biosynth-esis of LCPUFAs from EFAs is possible,the ef?ciency of tissue accretion is highest when they are ingested in the preformed state (Su et al.,1999).3.2.Transport and accretion of LCPUFAs
Gordon and Bazan (1997)and Rodriguez de Turco et al.(1999)discuss pathways by which LCPUFAs may be accreted to the retina (see Fig.3).EFAs and LCPUFAs exist mainly in esteri?ed forms as triacylglycerols (TG,triacylglycerol)within foods.During early phases of dietary lipid absorption,free fatty acids are cleaved (hydrolyzed)from the sn-1and sn-3positions of triglyceride s by pancreatic lipase within the intestine.DHA appears to predominantly occupy the sn-2position of the resulting 2-monoglyceride.EPA may occupy the sn-3,and to a lesser extent,the sn-1position (Nettleton,1995).Free LCPUFA and
COOH
22
20
19
17
16
14 13
11 10
8
7
5 4
2
21
18 15
12
9
6
3
COOH 20
18
17
15
14
12 11
9 8
6
5
3
COOH
20
18
16
13
10
7
4
2
19
17
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19 14 13107 4
2
Eicosapentaenoic Acid (C 20H 30O 2, 20:5ω-3, MW: 302.451)
Docosahexaenoic Acid (C 22H 32O 2, 22:6ω-3, MW: 328.448)
Arachidonic Acid (C 20H 32O 2, 20:4ω-6, MW: 304.467)
Fig.1.Chemical structures of DHA (D 4,7,10,13,16,19-docosahexaenoic acid),EPA (D 5,8,11,14,17-eicosapentaenoic acid),and AA (D 5,8,11,14-eicosatetraenoic acid).Molecules are oriented with methyl (omega)terminal on the reader’s left.
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LCPUFA-monoacylglycerol complexes are then re-esteri?ed to triglycerides and phospholipids within enterocytes of the intestinal epithelium.Triglycerides and phospholipids are then integrated to chylomicrons and very low density lipoproteins (VLDLs),secreted into the lymphatic system,and circulated from the thoracic duct via blood to the liver.DHA of cellular origin is synthesized mainly from a -LLNA within the liver (Scott and Bazan,1989).a -LLNA enters the hepatocyte through a receptor-mediated process and is activated by coenzyme A (CoA).The complex then enters the smooth ER where it is elongated and desaturated to DHA–CoA.DHA–CoA enters the rough ER where it is esteri?ed to phospholipids and forms a
Peroxisome
-9
22:5ω -622:4ω -6
22:6ω -3-oxidation (-2C)
-oxidation (-2C)
-oxidation (-2C)
Fig.2.Biosynthetic pathways of o -3,-6,and -9fatty acids.Fatty acid notation represents total number of carbons:number of double bonds,
position of the ?rst double bond relative to the methyl terminal of the hydrocarbon chain.For example,22:6o -3indicates that the fatty acid chain is 22carbons long with the ?rst of 6double bonds inserted between the third and fourth carbons from the methyl terminal.a -LLNA=a -linolenic acid,EPA=eicosapentaenoic acid,DHA=docosahexaenoic acid,LA=linoleic acid,AA=arachidonic acid,DPA=docosapentaenoic acid.A detailed representation of this pathway with chemical structures is presented in Bazan (1990).
Fig.3.Pathway of DHA transport from the liver to the choriocapillaris to the RPE to the photoreceptor.DHA is carried from the liver on phospholipids and triacyclglycerols synthesized in the liver and packaged into lipoproteins.At the basal surface of the RPE DHA is taken up via a receptor-mediated process s (e.g.highly speci?c fatty acid binding protein+lipoprotein lipase).This process may occur simultaneously or subsequently via receptor mediated uptake at the myoid region of the inner segment.Transport through the IPM may be mediated by interphotoreceptor binding protein.DHA is then packaged into disk membranes and transported to the outer segment.As the apical membranes of the outer segment are shed and phagocytized by RPE cells,DHA-rich phospholipids from degraded phagosomes are immediately reintroduced to the pathway.This leads to a very ef?cient local conservation of DHA (from Bazan,NG.In:LaVail MM,Anderson RE,Holly?eld JG,eds.Inherited and environmentally induced retinal degenerations;Copyright r 1989,Alan Liss,New York.This material is used by permission of John Wiley &Sons,Inc.).
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complex with apoproteins.These complexes are trans-ported in vesicles to the Golgi bodies where they are assembled into lipoproteins and secreted(Bazan,1990). DHA of cellular origin is then transported with dietary DHA via VLDL lipoproteins to the choriocapillaris. Lipoprotein lipase hydrolyzes chylomicrons remnants and VLDL within the choriocapillaris.
The bulk of lymph-borne esteri?ed LCPUFAs are carried in the triglyceride class of the chylomicron and VLDL fractions;they exist to a lesser extent as free fatty acids and within other lipid pools(PC,cholesterol ester, monoglycerides,and diglycerides).DHA is accreted mainly to phospholipids species composing membranes (PEA,PC,PS)in the retina.Within the circulation LCPUFAs on chylomicron-bound triglycerides are hydrolyzed to their free forms by capillary-endothelial-cell-derived lipoprotein lipase.These free fatty acids may subsequently form loose(non-covalent)bonds with albumin in blood plasma for delivery to tissues.
The capacity of photoreceptors to synthesize DHA is limited(Wang and Anderson,1993;Wetzel et al.,1991). RPE(Wang and Anderson,1993),retinal endothelium (Delton-Vandenbroucke et al.,1997),and brain astro-cytes(Moore,2001)are able to synthesize DHA.Retinal biosynthesis of DHA is slow(Wetzel et al.,1991)and may be insuf?cient to support the needs of photorecep-tors.Gordon and Bazan(1989)and Li et al.(2001)have demonstrated that the liver is a key site for LCPUFA biosynthesis.Availability and distribution of LCPUFAs in plasma lipids and lipoproteins are driven by liver biosynthesis,lipoprotein assembly,and tissue uptake (Gordon and Bazan,1997).Transport via the chorioca-pillaris to the RPE and inner segments appears to be mediated by a high af?nity receptor mediated uptake. Hepatectomized rats demonstrate rapid accretion of LCPUFAs in neural tissue(Anderson and Connor, 1988),suggesting that transport mechanisms and speci?c binding proteins for these compounds operate effectively within the nervous system.DHA is trans-ported from the choriocapillaris via the RPE cells and interphotoreceptor matrix(IPM,an extracellular region between the RPE and outer limiting membrane).The hydrophobic nature of fatty acids requires specialized cytoplasmic transport systems,speci?c binding proteins, and receptors to transfer LCPUFAs to the photorecep-tors.As there is no direct contact between photorecep-tors and the choroidal circulation,adjacent cell types (RPE cells,astrocytes,and Mu ller cells)must aid in the process.
3.2.1.LCPUFAs in RPE and photoreceptors
Gordon and Bazan(1989)have traced the fate of radiolabeled a-LLNA(injected intraperitoneally)from the liver to the retina in the rat.Li et al.(2001)have used similar techniques to trace orally ingested compounds. Study results were concordant on a number of factors.Both reports identify the liver as the key site for LCPUFA metabolism,observe that retinal accretion of radiolabeled a-LLNA-derived DHA was negligible,and suggest that delivery of LCPUFAs is in part regulated at the choroid-RPE interface.Li et al.(2001)state that dietary LCPUFAs are?rst esteri?ed to triglycerides within enterocytes of the intestinal epithelium.Trigly-cerides are then integrated with chylomicrons,secreted to the lymph,and enter the circulation via the thoracic duct.At4h post-ingestion,and thereafter,labeled compounds appeared predominantly in the phospholi-pid classes of the liver.These authors suggest such a pro?le may indicate lipolytic actions on the chylomicron triglycerides as a?rst step in biosynthesis of phospho-lipids.Speci?c activity of DHA peaked in liver at4h post-ingestion and had dropped by more than half by 24h.Radiolabeled DHA was detected in rod outer segments at1h post-ingestion,increased rapidly from2 to4h(approximately eight-fold),peaked at24h,and remained at levels at least seven-fold higher than those measured at the2-hour sample for the duration of the study(96h).
In vivo tracer studies on amphibians have demon-strated a higher degree of LCPUFA labeling in RPE than in photoreceptors.As in mammalian species,RPE labeling preceded that of photoreceptors and the time course of transport from initial exposure to the tracer was substantially shorter.Authors of these studies speculate that RPE plays an important role in regulation and release of DHA from plasma to the IPM(reviewed in Gordon and Bazan,1997).In amphibians there was a selective accretion of LCPUFAs with carbon-chain lengths greater than20to the neural retina(Chen and Anderson,1993).The RPE contained20-and22-carbon LCPUFAs;the bulk of20-carbon species were AA.In a tracer study on rats,level of AA in plasma was equivalent to that in RPE after14weeks of feeding (Wang and Anderson,1992).Relative to RPE,the speci?c activity of AA in rod outer segments was4–16 times lower.The amount of22-carbon LCPUFAs (DHA and docosapentaenoic acid(DPA)(22:5o-6)) in photoreceptors was3–5times greater than that in RPE.These studies support the notion of a selec-tive accretion of22-carbon LCPUFAs in the photo-receptors.
How is DHA delivered to subcellular membrane systems in the photoreceptor?LCPUFA-containing phospholipids enter the RPE or photoreceptor inner segment via a receptor mediated transport process. Gordon and Bazan(1997)suggest operation of a high af?nity fatty acid binding protein with a lipoprotein lipase.LCPUFAs enter the inner segment in a smooth ER-dense area adjacent to the base of the outer segment (the myoid)that exhibits a preferential uptake of DHA. After enzymatic degradation of the DHA-triglyceride in the inner segment,activation of fatty acid co-enzyme A
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leads to re-esteri?cation of DHA to phosphatidic acid.
A process of de novo phospholipid,di-and triglyceride biosynthesis then occurs.Tracer studies indicate DHA-containing phospholipids are then integrated as struc-tural constituents of photoreceptor disk membranes and are retained in proximity to rhodopsin molecules across the life-span of these organelles.There are effective and ef?cient mechanisms of repair to oxidized DHA that allow it to remain within disks.These properties of repair and selective retention are unique among photo-receptor lipids.As disks migrate to the outer segment-RPE interface,are shed,and phagocytized the photo-receptor DHA content remains unchanged.Phagosomes are degraded in the RPE to form large oil droplets containing DHA-rich triglycerides.Triglycerides are then and transported back to the myoid for re-uptake. It is interesting to note that RPE cytosol remains virtually free of DHA-containing lipid and lipoprotein species.This condition may have important conse-quences for disease prevention,as discussed in later sections.
3.2.2.LCPUFAs in the vascular retina
Lecomte et al.(1996)determined the fatty acid composition of isolated bovine retinal microvessels and con?uent endothelial cell/pericyte monolayers. DHA and AA each represented approximately10%of total fatty acids in puri?ed intact vessels.In primary cultures the value for DHA was reduced by approxi-mately2%and the value for AA did not change.DHA levels in cultured endothelial cells and pericytes were restored with10m M supplementation of unesteri?ed DHA.In the case of endothelial cells,supplementation did not alter AA concentration;in the case of pericytes, AA concentration was reduced.Levels of EPA in all preparations were more than10times less than those of DHA and AA.Although substantial variation existed across tissue types,the mol%of EPA in retinal microvessels was5times higher than that in non-vascular retina(0.5%vs.0.1%).In human serum, retroconversion of DHA to EPA is estimated at9–11% (Conquer and Holub,1996,1997).Likewise,endothelial cultures from bovine macrovascular networks(aorta) exhibit considerable retroconversion.In the Lecomte et al.(1996)report,retroconversion was negligible,in-dicating a speci?city of fatty acid metabolism,based upon the origin of vascular tissue.
Delton-Vandenbroucke et al.(1997)examined capa-city of cultured bovine retinal endothelial cells to produce DHA and concluded that,in this model system it is possible via desaturation of DPA(C22:5)of the o-3 family.While EPA was the major metabolite of DPA o-3desaturation,DPA has been shown to constitute 2mol%of isolated and puri?ed bovine microvessels (Lecomte et al.,1996).3.3.EFA and LCPUFA intake
Typical intake of total o-3fatty acids is1.6g/d in the US(approximately0.7%of total energy intake)(Kris-Etherton et al.,2000).Most is in the form of a-LLNA. EPA and DHA usually compose6–12%of this value (0.1–0.2g/d).The main sources of a-LLNA are vege-table oils;of common types,linseed,canola,and soybean oils have highest levels.EPA and DHA are concentrated in fatty?sh and marine mammals and these are the main sources in the Western diet. Approximately10%of DHA is typically derived from eggs.o-3LCPUFAs are also commercially available as dietary supplements in the form of oil and capsules. Capsules typically contain120mg DHA and180mg EPA(Kris-Etherton et al.,2002).The main source of EPA for these products is?sh oil.DHA may be derived from?sh oil or single-celled organisms.A list of commercially available supplements containing DHA and/or EPA,the nutrient composition of these supple-ments,and the supplement manufacturers exists at The Natural Medicines Comprehensive Database(http:// https://www.wendangku.net/doc/2512175054.html,).As this chapter went to press,this database listed121products.The main dietary sources of LA are sun?ower,saf?ower,corn and soybean oils.Intake of o-6fatty acids is12–16g/d in the US(approximately6.0%of total energy intake). AA consumption is approximately0.1g/d.Major diet-ary sources of AA are terrestrial animal meats,organ meats,and egg yolk.
Because DHA and EPA are concentrated only in a small number of less-frequently consumed marine-based foods,these nutrients may show merit as modi?able factors in diet-or nutrient-based interventions designed to reduce the risk of vascular and degenerative retinal diseases.A scienti?c statement issued by the American Heart Association(AHA)on o-3fatty acids and cardiovascular disease reviews safety of o-3fatty acids and?sh(Kris-Etherton et al.,2002).The AHA statement cites formal population-based dietary intake recommendations of0.3–0.5g/d of EPA+DHA from The World Health Organization,North Atlantic Treaty Organization,and National Health ministries of Aus-tralia,Canada,Japan,Sweden,and the United King-dom.The US Food and Drug Administration(FDA, 1997)has formally stated that consumption of up to3g/ d of marine-based o-3fatty acids is generally regarded as safe(GRAS).The FDA(2002)has also approved a health claim for DHA and EPA in supplement form. Governmental regulatory bodies have issued state-ments concerning the potential for hemorrhagic risk with intake of o-3LCPUFAs43g/d(2002)(discussed in Kris-Etherton et al.,2002).The anti-thrombotic and anti-haemostatic effects of o-3LCPUFAs operate within physiologic limits at intakes between 1.0and 3.0g/d(Dyerberg and Bang,1979;Levine et al.,1989;
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Li and Steiner,1991;Vericel et al.,1999;von Schacky et al.,1985);at these levels hemorrhagic risk is not considered a major issue.
4.Role of LCPUFAs in structure and function of sensory retina
The contents of this section describe the role of LCPUFAs in the structure and function of the sensory retina.Neuringer et al.(forthcoming publication in Progress in Retinal and Eye Research)review these issues in detail.
4.1.DHA is an essential structural component of retinal membranes
DHA is the major fatty acid in structural lipids of retinal photoreceptor outer segment disc membranes (Fliesler and Anderson,1983).Outer segment discs contain rhodopsin,the photopigment necessary for initiating visual sensation;DHA is ef?ciently incorpo-rated and selectively retained in disc membranes(Bazan et al.,1993).Highest body concentrations of DHA per unit area are found in the disc membranes and the overall percent of DHA(30%of total retinal fatty acids) is50mol%greater than in the next most concentrated tissue(Neuringer,1993).
Composition of retinal photoreceptor outer segments is unique in that80–90%of structural lipids are glycerophospholipids and8–10%are neutral lipids (Daemen,1973;Fliesler and Anderson,1983).Neutral lipid species are mainly cholesterol,with a lower concentration of free fatty acids.A phospholipid is a polar molecule with a hydrophilic phosphate head group and two hydrophobic fatty acid tails on a glycerol backbone.Retinal phospholipids are unique because many are polyenoic in nature.Polyenoic phospholipids contain polyunsaturated fatty acids(PUFAs)in the C1 (sn-1)or C2(sn-2)positions of the molecule’s glycerol backbone.The majority of phospholipid species in the outer segments are dipolyenoic(Aveldano and Sprecher, 1987;Choe and Anderson,1990;Wiegand et al.,1991). Dipolyenoic species are known to increase the rate of rhodopsin activation(metarhodopsin II formation) in model membrane systems(Litman and Mitchell, 1996);this is an essential event in the process of phototransduction.
Fliesler and Anderson(1983)provide a detailed review on chemistry and metabolism of lipids in the vertebrate retina.Retinal phospholipid species include PEA as$40%of outer segment lipids,PS as$12%,and PC as$10%of total outer segment lipids.PC,PE,PS, PI represent$48%,32%,9%,and4%of retinal phospholipids,respectively.DHA composes approxi-mately20%of the fatty acids for outer segment PC,and $30%for each of PEA and PS(Anderson,1970;Fliesler and Anderson,1983).Half of all PC fatty acids are saturated($30%palmitic acid and$20%stearic acid); in PE these values are$10%and36%,respectively. Thirty percent of PS fatty acids are saturated,with the greatest proportion being stearic acid($28%).
What is the functional signi?cance of the unique fatty acid composition in retinal outer segment photoreceptor disc membranes?The biophysical and biochemical properties of DHA affect membrane function by altering permeability,?uidity,thickness,lipid phase properties,and the activation of membrane-bound proteins(Clandinin et al.,1994;Jumpsen and Clandinin, 1997).DHA-rich membranes impart properties to outer segment discs that in?uence the dynamic of the inter-and intracellular communication(Litman and Mitchell, 1996;Litman et al.,2001;Mitchell et al.,2001;Niu et al.,2001,2002;Treen et al.,1992).The stereochemical structure of DHA with its22carbons and6double bonds allows an ef?cient conformational change of the transmembrane protein rhodopsin,in response to light absorption(photon capture).Membranes highly con-centrated with PUFAs exhibit less rigid global proper-ties than membranes concentrated in sterol esters or saturated fatty acids,because the multiple unsaturated bonds in PUFAs do not allow dense packing of the hydrophobic fatty acid components.LCPUFAs,with their long-chain nature,also contribute to a less-dense structure.A more?uid membrane allows a faster response to stimulation.For DHA,the position of the ?rst unsaturated bond at the o-3(between D-20and D-19)carbon provides an advantage in ef?ciency of membrane dynamics over that observed in an otherwise structurally identical fatty acid with the?rst double bond at the o-6carbon(Mitchell et al.,2003). Biochemical characteristics of DHA may also explain why it is concentrated in the metabolically active retinal outer segment.Fatty acids in membrane phos-pholipids are a primary source of signaling mole-cules that modulate intercellular communication and autocrine signaling from the plasma membrane.These processes in?uence nuclear control of gene expres-sion(de Urquiza et al.,2000;Doucet et al.,1990;Lin et al.,1999a;Miles and Calder,1998;Yaqoob,1998). Although esteri?ed AA is more ef?ciently released from membrane stores than DHA(Salem et al.,2001), retinal astrocytes probably provide a readily mobilized source of DHA for such purposes(Kim and Edsall, 1999).
4.2.DHA tissue status is associated with alterations in retinal and visual function
DHA de?ciency is associated with structural and functional abnormalities in the visual system(Uauy et al.,2000,2001).Evidence to support this concept
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exists for genetic,metabolic,and behavioral factors that in?uence DHA tissue status and dietary intake.Three examples are provided below.
4.2.1.Inherited retinal degenerations
Retinitis pigmentosa(RP)is the term used to describe a family inherited retinal diseases de?ned by photo-receptor atrophy,progressive night blindness,and loss of peripheral visual?elds(Hoffman et al.,2001). Photoreceptor outer segment lipid DHA concentration was reduced in canine progressive rod-cone degenera-tion(pcrd)(Aguirre et al.,1997)and rodent transgenic models of RP(Anderson et al.,2001).A study of subjects with X-linked RP demonstrated a30–40% lower concentration of erythrocyte(RBC)o-3LCPU-FAs than that observed in a normally sighted compar-ison group(Hoffman and Birch,1995;Schaefer et al., 1995).When people with non-X-linked RP were compared with a normally sighted group,their plasma and RBC DHA were lower.Similar results for circulat-ing DHA were observed in canine models of pcrd (Anderson et al.,1999,1991).Hoffman et al.(2001) have suggested that these differences may be due to decreased biosynthesis of DHA in people with RP. ELOVL4is a photoreceptor-speci?c gene responsible for two dominant forms of macular dystrophy(Star-gardt-like macular dystrophy and autosomal dominant macular dystrophy).This gene has a homology to a family of yeast proteins(ELO)that operate within the ER to elongate fatty acids.Zhang et al.(2001)suggest that ELOVL4may be an essential factor in the biosythesis of DHA.
4.2.2.Metabolic insuf?ciency
Human populations with certain peroxisomal disor-ders(Zellweger syndrome,neonatal adrenal leukody-strophy,and infantile Refsum disease)exhibit abnormalities in metabolism of LCPUFAs.In these disorders,tissue de?ciency of DHA is present in retina, brain,liver,kidney,and blood(Martinez,1989,1990, 1992).People with these diseases demonstrate gross visual processing de?cits that can be ameliorated with o-3LCPUFA supplementation(Martinez et al.,2000).
4.2.3.Dietary insuf?ciency
Feeding studies in mice,rats,rabbits,and non-human primates have demonstrated electroretinogram(ERG) wave form differences that vary on the basis of dietary o-3EFA/LCPUFA intake(reviewed in(Jeffrey et al., 2001).Studies on primates demonstrate alterations in visual resolution acuity is associated with dietary LCPUFA composition and tissue markers of retinal LCPUFAs.Clinical trials in preterm infants have demonstrated transient differences in visual resolution acuity at2-and4-months of age,favoring DHA supplementation over DHA-free formulas(reviewed in (SanGiovanni et al.,2000b).Studies on full-term infants demonstrate differences at2-months-of-age(reviewed in (SanGiovanni et al.,2000a).
4.3.DHA affects retinal cell signaling mechanisms in phototransduction
Photoreceptor outer segment phospholipid fatty acid composition affects the ef?ciency of intercellular signal-ing in the visual transduction pathway.The leading portion of the ERG a-wave is associated with the phototransduction pathway;o-3de?cient animals produce wave forms that are both delayed and of reduced amplitude when compared to those of o-3 replete animals(Connor and Neuringer,1988;Pawlosky et al.,1997;Weisinger et al.,1996).
Litman et al.have investigated mechanisms by which membrane composition may affect aspects of photo-transduction(Litman and Mitchell,1996;Litman et al., 2001;Niu et al.,2001).Interpreting this body of evidence requires a basic understanding of the photo-transduction process.Phototransduction is the process through which the retina processes light energy and converts it to a pattern of neuronal activity.In a dark-adapted state,retinal photoreceptors generate a depo-larizing‘dark current’that is mediated by the effect of high cytosolic concentrations of30,50-cyclic guanosine monophosphate(cGMP)that open Na+/Ca2+chan-nels.Phototransduction is initiated with the capture of a photon by rhodopsin.Rhodopsin is then transformed to metarhodpsin II(M(II)).M(II)binds to and activates the a subunit of the trimeric G-protein transducin.The M(II)–transducin complex binds to and activates tetra-meric cGMP phosphodiesterase(PDE)through removal of one of its inhibitory g subunits.Activated PDE hydrolyzes cGMP to GMP,which results in membrane hyperpolarization due to dissociation of cGMP from Na+/Ca2+ion channels.The hyperpolarized state of the photoreceptor leads to a graded decrease of in release of the neurotransmitter glutamate into the photoreceptor synapses on horizontal and bipolar cells.Bipolar cells form synapses with retinal ganglion cells;axons of the retinal ganglion cells constitute the optic nerve.The process is deactivated when rhodopsin is phosphory-lated by rhodopsin kinase and then bound with visual arrestin;this process inhibits formation of the M(II)–transducin complex.
How does DHA tissue status alter dynamics of the phototransduction cascade?:M(II)formation to an activated membrane-bound receptor state is higher in DHA-containing model membrane systems than in those containing the AA and cholesterol(Litman and Mitchell,1996)DHA also enhances production of M(II).Activation of the M(II)–transducin complex is more than two times greater in DHA containing systems than it is in those concentrated with saturated and
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monounsaturated fatty acid species(Salem et al.,2001). This indicates that ef?ciency of interaction in M(II)–transducin coupling is enhanced for DHA-rich mem-branes.Similar relationships have been observed for PDE activity(Litman et al.,2001).
How may these?ndings relate to alterations in the timing and magnitude of the ERG a-wave?Salem et al. (2001)suggest that displacement of the leading edge of the ERG a-wave may be constrained by formation of the M(II)–transducin complex and that the reduced a-wave amplitude may be related to a reduced activation of the formed complex(Salem et al.,2001).Others have suggested that a rate limiting step may also exist downstream from activation of the M(II)–transducin complex,in the process of maintaining an adequate supply of photopigment.To this end,DHA may have a role in the regeneration of rhodopsin,as it affects the transport of11-cis-retinal(a component of rhodopsin) by interphotoreceptor retinal binding protein(IRBP) from the RPE(Chen et al.,1996).
4.4.LCPUFAs in?uence retinal cell gene expression, differentiation,and survival
4.4.1.Gene expression
DHA is a ligand to the nuclear hormone receptors peroxisome PPAR(Lin et al.,1999)and RXR(de Urquiza et al.,2000).DHA binds to speci?c DNA motifs present on cis-regulatory elements in promoter regions of target genes.This event modulates activation of the PPAR and RXR receptors that subsequently operate as transcription factors(Gottlicher et al.,1993). Isoforms of PPAR receptors affected by DHA include a, b,and g(Dreyer et al.,1993;Gottlicher et al.,1993;Yu et al.,1995).DHA also may act directly in transcription, as it is highly concentrated in PS,a negatively charged aminophospholipid known to activate protein kinases involved in gene expression(Salem et al.,2001).DHA may operate at the posttranscriptional level by acting as a ligand to induce changes of phosphorylation events in native mRNA processing,mRNA transport and stabi-lization,and mRNA degradation rates(Uauy et al., 2001).
4.4.2.Cellular differentiation
DHA operates as a trophic molecule in photoreceptor development,differentiation,and growth.It has been shown to increase opsin expression and apical process differentiation in developing rat photoreceptors in vitro (Politi et al.,2001;Rotstein et al.,1997,1998).The protein opsin combines with the11-cis-retinal to form rhodopsin;the relevance of this issue for retinal health is that expression of the opsin gene may be required for assembly of photoreceptor disc membranes(Uauy et al., 2001).4.4.3.Survival
DHA prolongs survival of rat photoreceptors in vitro (Politi et al.,2001;Rotstein et al.,1996,1997,1998).The number photoreceptors supplemented with DHA that survived for11days in vitro was approximately twice of that observed within a culture existing on DHA-free media.The proportion DHA-fed cells expressing opsin was signi?cantly higher than in those from a DHA-free culture.Measures of apoptosis(fragmented photore-ceptor nuclei and dysfunctional mitochondria)sug-gested a protective effect of DHA at post-plating days 7and11.Serum-starved PC-12and Neuro-2A cells preincubated in DHA and vitamin E for at least24h had lower amounts of genomic DNA fragmentation than cultures fed DHA-free media(Kim et al.,2001). Caspase-3is an enzyme that mediates mammalian apoptosis.In serum-starved,DHA-enriched cultures, the activity of caspase-3was maintained at the levels of adequately fed control cultures.DHA-feeding also reduced the expression of caspase-3mRNA.This was not the case of serum-starved cultures incubated with DHA-free media(Kim et al.,2001).
5.Metabolic and environmental bioactivators
A number of metabolic and environmental factors and processes serve as bioactivators of molecules associated with abnormal angiogenesis,proliferative neovascularization,excessive vascular permeability,im-munoregulatory dysfunction,alterations in physiologic redox balance,and neuronal/RPE cell degeneration. Key factors and processes affecting the retina include ischemia,light exposure,oxidative stress,apoptosis, in?ammation,neuroactive cell signaling molecules,and developmental processes associated with aging.In addition to affecting molecules associated with the pathogenesis of retinal disease,such factors and processes also modulate:(1)release of unesteri?ed LCPUFAs from phospholipid membranes by PLA2; and(2)activation of COXs and LOXs that catalyze eicosanoid synthesis.It is important at this point to acknowledge the dominant role of diet in affecting the LCPUFA substrate pool.Metabolic and environmental factors and processes affect fatty acid cleavage-and biosynthetic enzymes.As the concentration and compo-sition of o-3LCPUFAs stored in phospholipid mem-branes is modi?able by and dependent upon dietary intake,the balance of free LCPUFAs and their metabolites is thus affected after activation of PLA2, COX,and LOX.
In this section we discuss seven major metabolic and environmental factors and processes associated with activation or generation of eicosanoids,angiogenic growth factors,MMPs,reactive oxygen species,cyclic nucleotides,neurotransmitters and neuromodulators,
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pro-in?ammatory and immunoregulatory cytokines, and in?ammatory phospholipids operating in vascular and degenerative retinal diseases.The metabolic and environmental bioactivators presented here work in an interrelated and recursive system as a complex of aetiologic agents.Considering the role of factors capable of altering the concentrations of free LCPUFAs and the activity of key fatty acid cleaveage and biosynthetic enzymes is essential when investigating potential actions of o-3LCPUFAs in the retina.As such,we?rst review basic concepts related to PLA2,COX,and LOX in the context of the retinal metabolic and environmental exposures.
5.1.Role of PLA2in LCPUFA hydrolysis
PLA2s catalyze hydrolysis of fatty acids from the ester bond at the sn-2position of phospholipids to yield free LCPUFAs and lysophospholipids.At least19groups of PLA2s have been identi?ed and they are generally classi?ed into cytosolic(cPLA2),secretory(sPLA2),and calcium-independent(iPLA2)isoforms(Phillis and O’Regan,2003).cPLA2s are high molecular weight and preferentially cleave AA;there are Ca2+-dependent and independent forms.Intracellular sPLA2s are low molecular weight;while these enzymes do not show speci?city for particular fatty acids,Han et al.(2003) demonstrated that group IIa and V sPLA2s can regulate cPLA2a activity to affect AA release.cPLA2a has an N-terminal calcium-dependent phospholipid domain that may allow post-translational regulation by calcium or phosphorylation via mitogen-activated protein kinase (MAPK)and PKC(Geijsen et al.,2000;Kramer et al., 1996;Lin et al.,1993;Nemenoff et al.,1993;Qiu and Leslie,1994).Extracellular signal-regulated kinases (ERKs)are implicated in communication between cPLA2a and sPLA2s(Balsinde and Dennis,1996; Hernandez et al.,1998).DHA has been shown to decrease PLA2activity in nerve growth cones of nerve growth factor-differentiated PC12cells,with a predo-minant effect on sPLA2in calcium-independent path-ways(Martin,1998).PLA2is activated in response to ischemia(Kolko et al.,2002;Phillis and O’Regan,2003), light exposure(Jung and Reme,1994),oxidative stress (Martinez and Moreno,2001;Goldman et al.,1997), apoptosis(Goldman et al.,1997),in?ammation(Bazan et al.,2002),cell signaling molecules(Hayakawa et al., 1996;Schalkwijk et al.,1996),and developmental processes associated with aging(Balazy and Nigam, 2003).
5.2.Role of COX in eicosanoid biosynthesis
COX(prostaglandin endoperoxide synthase)is a protein complex that?rst converts20-carbon LCPUFA substrates from o-6(AA)and o-3(EPA)families to G-PG endoperoxides via hydrogen subtraction(at carbon11)and subsequent addition of2molecules of oxygen.A hydroperoxidase(HOX)then uses glu-tathione to convert the G-PGs to H-PGs.FitzGerald (2003)reviews basic aspects of COX production, structure,and metabolite actions.The constitutive form (COX-1)is found in most cell types(mainly in the gastric mucosa,kidney,and platelets)and operates primarily in the role of haemostatic regulation.The inducible form(COX-2)is found constitutively in the central nervous system,is activated by cytokines and mitogens,and acts in formation of PGs in in?ammatory response.Ringbom et al.(2001)have demonstrated that DHA and EPA are effective in inhibiting COX-1and COX-2catalyzed PG biosynthesis an in vitro assay. There was a higher potency of inhibition for COX-2. Corey et al.(1983)discuss potential for DHA to operate as a competitive inhibitor of COX.COXs are activated in response to PLA2activation and free LCPUFA concentration.As such,COX-2activation is associated with ischemia(Ju et al.,2003;Candelario-Jalil,2003), light exposure(Hendrickx et al.,2003),oxidative stress (Kiritoshi et al.,2003;Feng et al.,1995),cell death (Bizik et al.,2004),in?ammation(Sennlaub et al.,2003; Bazan et al.,1997;Dubois et al.,1998),neuroactive cell signaling molecules(Nakamichi et al.,2003;Hurst and Bazan,1995),and developmental processes associated with aging(reviewed in Han et al.,2004).
5.3.Role of LOX in eicosanoid biosynthesis
5-Lipoxygenase(5-LOX)converts AA or EPA to hydroperoxides(hydroperoxyeicosatetraenoic acids, HPETE)via removal of hydrogen at carbon7and insertion of moleular oxygen at carbon5.HPETE is used in leukotriene(LT)biosynthesis;it may also be reduced to hydroxyeicosatetraenoic acid(HETE).Acti-vation of5-LOX is modulated by calcium,adenosine triphosphate(ATP),and5-LOX activating protein (FLAP).Upon activation5-LOX is translocated to the nuclear membrane.5-LOX metabolites operate in immunoregulation within the in?ammatory response (reviewed in Romano and Claria,2003).LOXs are activated in response to PLA2activation and free LCPUFA concentration.As such,LOXs activation is associated with ischemia(Phillis and O’Regan,2003), light exposure(Naveh et al.,2000),oxidative stress (Werz et al.,2000)and in?ammation(Flamand et al., 2002).12-and15-LOX are other LOX enzymes that catalyze bioconversion of20-carbon chain LCPUFAs to compounds of physiological signi?cance.
5.4.Retinal ischemia
Retinal ischemia activates PLA2and in?uences processes implicated the pathogenesis of DR(Frank,
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2004),AMD(Ambati et al.,2003),and ROP(Kohner and Chibber,2001).Retinal ischemia is a state wherein the blood supply is insuf?cient to meet the metabolic needs of the retina;it is associated with alterations in oxygen delivery and regulation.Osborne et al.(2004) provide a comprehensive review on the subject.DHA and AA are major fatty acids within intraretinal (Lecomte et al.,1996)and choroidal(Kulkarni et al., 2002)capillary networks of vertebrate species.
5.4.1.Vascular networks in the retina
The metabolic demand for oxygen in retina exceeds that of most other tissues(reviewed in Beatty et al., 2000;Wangsa-Wirawan and Linsenmeier,2003).Since storage of oxygen within retina tissue is not possible,it must be provided continuously by the vascular system (Vanderkooi et al.,1991).The central retinal(ophthal-mic)artery and choriocapillaris(from the posterior ciliary arteries)supply blood to the mammalian retina. Two-thirds to85%of retinal blood?ow travels to the choroid.The majority of oxygen carried via this pathway supports photoreceptor metabolism(Wangsa-Wirawan and Linsenmeier,2003).There are three vascular networks within the choroidal system:(1)the inner layer is adjacent to Bruch’s membrane and the RPE;(2)the outer layer is adjacent to the sclera;
(3)the medial layer is positioned between these two. Vascular endothelium of the choriocapillaris is thin and fenestrated in the area adjacent to the RPE.It is interesting to note that VEGF is secreted constitutively from the basal surface of RPE cells.Vascular endothe-lium is thick and non-fenestrated in the area adjacent to the choroid(Mancini et al.,1986).
Twenty to33%of retinal blood supply enters at the optic nerve head from the ophthalmic artery to form an endartery(often called intraretinal)network supporting the inner retina.There are three major intraretinal capillary networks:(1)radial peripapillary capillaries (RPCs);(2)the inner capillary layer;and(3)the outer capillary layer.The RPCs occupy the inner area of the nerve?ber layer.In the healthy retina,astrocytes are present exclusively in the nerve?ber layer,were their processes cover the vascular tissue(Gardner et al., 2002).The inner capillary layer occupies the ganglion cell layer.The outer layer projects from the inner-to outer-plexiform layers.Vascular endothelium in the intraretinal vascular network is characterized by tight junctions and is impermeable to macromolecules (Gardner et al.,2002;Antonetti et al.,1999).
The intraretinal microvascular system does not receive autonomic input.In order to permit transmis-sion of light to the photoreceptors,intraretinal blood vessels are sparsely distributed at intercapillary distances more of than50m m.At the same time,this vascular network must deliver energy substrates(oxygen,glu-cose,and lipids),enzymatic substrates(nutrients),and remove waste products from this tissue of high meta-bolic activity.Because retinal neurons are not able to sustain prolonged ischemic insult and the nature of the tightly sealed capillaries of the blood-retina barrier,the system must operate ef?ciently in controlling perfusion. One suggested pathway has been through modulation of pericyte activity.Pericytes encase vascular endothelial cells and demonstrate contractile properties in vitro (Kawamura et al.,2003;Kelley et al.,1987;Matsugi et al.,1997;Wu et al.,2003).Pericyte loss is a prominent feature in vascular forms of DR.
The unique characteristics of systems modulating retinal oxygenation and their effects on pathogenesis and treatment of ischemia-induced retinopathies are the subject of a recent review(Wangsa-Wirawan and Linsenmeier,2003).These characteristics include the dual circulatory systems discussed above—with absence of metabolic oxygen regulation in one(choroid)and autoregulation in the other(intraretinal),and dense concentrations of mitochondria within photoreceptor inner segments.
In retinal or choroidal vascular disease,blood?ow may be affected by capillary occlusion or increases in platelet activity and aggregation.Occlusion may lead to infarction and concomitant structural alterations,ne-crosis,and functional loss in vascular tissue.Diseases affecting blood or vessel walls often manifest neural comorbidity.Modulators of retinal ischemia operate within blood and on vascular membranes.Eicosanoids, reactive oxygen species,and cytokines are potent modulators.Actions of these compounds are discussed in detail in following sections.As such o-3-derived compounds may show some advantage in modulating vasoregulatory processes.
5.4.2.LCPUFAs affect factors and processes implicated retinal ischemia:vasoregulatory eicosanoids and vascular response
How may retinal blood?ow and oxygen regulation be affected by LCPUFA intake and status?One means is by providing a substrate for eicosanoids that act as auto-or paracrine effectors of vascular membrane response and alter properties of blood constituents. Another is by altering lipoprotein metabolism.Mem-brane composition and concentration of LCPUFAs will determine the nature of the resultant free fatty acid pool that serves as substrates for eicosanoid biosynthesis. Vasoregulatory eicosanoids vary in bioactivity and structure on the basis of their LCPUFA substrate. EPA serves as the substrate for series-3compounds and AA is the precursor to series-2compounds.Eicosanoids are produced in diverse cell types.Thromboxanes(TX) and PG are the main eicosanoids produced within platelets and vascular endothelial cells that are asso-ciated with haemostasis and vasomotility.TXA2is a potent vasoconstrictor and induces platelet aggregation;
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the o-3-based analog,TXA3,is approximately ten-fold less prothrombotic.Prostacyclins(PGI)of the2-and3-series have equivalent potency in vasodilatation and platelet anti-aggregation.
5.4.2.1.Blood pressure.Results of two meta-analysis suggest reduction in blood pressure among persons with hypertension who consumed o-3LCPUFAs(Appel et al.,1993;Morris et al.,1993).Results have been observed for untreated hypertensive subjects consuming 43g/d of o-3LCPUFAs(Appel et al.,1993).The effect of the anti-hypertensive beta-blocker propranolol is enhanced with?sh oil intake(Singer et al.,1990).Bazan and Rodriquez de Turco comment that interventions with amphiphilic cationic drugs(such as propranolol) demonstrate active esteri?cation of DHA into mem-brane lipids through endogenous biosynthesis(Bazan and Rodriguez de Turco,1994).
5.4.2.2.Blood?ow,vasomotility,thrombosis,platelet activity and aggregation.Knapp(1997)has reviewed the role of fatty acids in modulating blood?ow, haemostasis,and thrombosis.A number of studies demonstrate anti-aggregant effects of o-3LCPUFAs (Dyerberg and Bang,1979;Levine et al.,1989;Vericel et al.,1999;von Schacky et al.,1985).Bayon et al.(1995) have observed that since DHA is less likely to be hydrolyzed to a free fatty acid form by PLA2than either EPA or AA,that it may prevent platelet aggregation, while in a membrane-bound esteri?ed form(as opposed to serving as a mobilizeable source of EPA).This group has demonstrated that when DHA was esteri?ed in into PC of platelet membranes,af?nity of eicosanoids to platelet TXA2/PGH2receptor was reduced.Platelet aggregation is reduced with increased consumption of o-3LCPUFAs(Agren et al.,1997;Knapp,1997;Mori et al.,1997).Effects of LCPUFAs on thrombosis are equivocal,as measured by alteration in the coagulation factors?brinogen(Barcelli et al.,1985;Marckmann et al.,1997;Shahar et al.,1993),Factor VIII(Archer et al.,1998;Marckmann et al.,1997;Shahar et al., 1993),von Willebrand factor(Archer et al.,1998; Marckmann et al.,1997;Shahar et al.,1993),and thrombomodulin(Johansen et al.,1999).
5.4.3.Lipoprotein metabolism
Lipoprotein metabolism and composition affects ischemia and oxygen regulation.Harris(1997)has reviewed data on the relationship of dietary o-3 LCPUFAs with serum lipoprotein response.The rela-tionship of o-3LCPUFA intake with triglyceride lowering follows a dose–response pattern.Reductions were observed with consumption of o2g/d(Roche and Gibney,1996).Consumption of approximately4g/d of o-3LCPUFAs from?sh oil was associated with a 25–30%reduction in serum triglycerides.LCPUFAs exert this effect mainly by reducing triglyceride synthesis in the liver and release of VLDL into the circulation (Bordin et al.,1998;Nenseter et al.,1992;Nestel,2000; Vasandani et al.,2002).The mechanism driving these events is believed to occur via o-3LCPUFA binding to nuclear transcription factors involved in fatty acid and triglyceride regulation.o-3LCPUFAs act as ligands to PPAR genes;PPARs form a heterodimer with RXR in binding with DNA in promoter regions of genes involved in fatty acid transport,fatty acid binding, and PUFA desatuarion(Jump,2004).LCPUFAs operate in a inhibitory feedback mechanism to reduce the nuclear abundance of SREBPs;this process occurs via modulation of proteolytic SREBP processing or SREBP-1c transcription(Jump,2002).SREBPs are involved in fatty acid biosynthesis and triglyceride metabolism(Horton et al.,2002).SREBP-1a increases transcription of all SREBP-responsive genes and is thus involved in cholesterol,triglyceride,and fatty acid biosynthesis.SREBP-1c activates transcription of genes for acetyl CoA carboxylase,fatty acid synthase,stearoyl CoA desaturase-1;it is associated with de novo fatty acid synthesis and desaturation.SREBP-2activates genes involved in the inhibition of triglyceride biosynth-esis(HMG-CoA reductase,HMG-CoA synthase,far-neysyl diphosphate synthase,squaline synthase)(Price et al.,2000).The reasoning is that increase in fatty acid catabolism and decrease in fatty acid biosythesis reduces the pool of materials essential for triglyceride synthesis. This subsequently in?uences triglyceride release into the circulation as well as the rate of VLDL production (Harris et al.,1990;Roche and Gibney,2000).Harris (1997)also reports o-3LCPUFA-intake-related in-creases in serum high-density lipoprotein(HDL)of 1–3%and in LDL of5–10%.
5.4.4.LCPUFAs affect energy production,regulation, and metabolism
The metabolically active neural retina supports its energy requirements in the form of ATP that is produced from oxygen and nutrient-based substrates (fatty acids and glucose,pyruvate,and lactate)within mitochondria of the photoreceptor inner segments. Mitochondria consume90%of oxygen used by the body.o-3LCPUFAs may in?uence ef?ciency of energy production within the retina.After ischemic challenge, recovery of mitochondrial function in cardiac tissue of rats fed a?sh oil diet was better than that observed in a group consuming an o-3LCPUFA-free diet(Demaison et al.,1994).Increased ef?ciency of ATP production and energy use within mitochondrial membranes in cardiac tissue of animals with higher levels of phospholipid o-3 LCPUFAs has also been observed(Grynberg and Demaison,1996).These results suggest that o-3 LCPUFAs enhance processes of energy metabolism with minimal cost of energy substrate expenditure.The
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mitochondria are a major site of reactive oxygen species generation and gains in energy processing ef?ciency are believed to lower production rates and volume of these compounds.
5.5.Light exposure
Light exposure induces PLA2activation and libera-tion of LCPUFAs bound to the sn-2position of membrane phospholipids(Jung and Reme,1994; Reinboth et al.,1996).Photic damage in?uences processes implicated the pathogenesis of AMD.Under normal physiological conditions,retinal photic damage is unlikely to occur,despite the high density of photosensitive compounds(chromophores)and the chronic nature of light exposure.Boulton et al.(2001) discuss3processes by which light damage may occur. Light-induced ionization is a mechanical process in-itiated by high irradiance,high frequency exposures;the retina is less susceptible to this form of damage than others.The destructive physical effect of ionization on ocular tissue is caused by periodic‘shock waves.’Light-induced thermal photocoagulation is the result of energy capture,retention,and non-radioactive decay within photothermal chromophores.Thermal damage is asso-ciated with cellular temperature increases that occur as chromophores are reduced from activated to ground states.Light-induced photochemical retinal damage is a process initiated after an activated chromophore shifts from a singlet to a more stable triplet energy state.In this triplet form,the activated chromophore may exist long enough to transfer energy to substrates of reactive oxygen species;the result is production of singlet oxygen and free radicals.Photochemical damage is assumed to occur under exposure to ambient light and,depending on spectral sensitivity of chromophores,may affect rod outer segments or rod outer segments and RPE cells (rhodopsin-based,green action spectrum peak),or RPE cells(non-rhodopsin-based,blue action spectrum peak).
5.5.1.LCPUFAs affect factors and processes implicated in retinal light damage
How may o-3LCPUFA intake and status facilitate cytoprotective mechanisms in response to light damage? Cellular response to chronic light exposure involves regulation of rhodopsin and membrane lipid concentra-tion.The purpose of this process is to allow the sensory retina to maintain a stable capacity for photon capture that is independent of stimulus intensity.DHA may contribute to this process as it binds to transport proteins implicated in regulation of photopigment regeneration.Boulton et al.(2001)discuss the potential for photosensitized chromophore-oxidation products to form cytotoxic compounds.All-trans-retinaldehyde(vi-tamin A aldehyde)is hydrolyzed from opsin during the isomerization of11-cis-retinaldehyde.All-trans-retinal-dehyde is subsequently reduced to all-trans-retinol by all-trans-retinol dehydrogenase.All-trans-retinol is car-ried to the RPE where it undergoes oxidation and isomerization to11-cis-retinaldehyde.Because all-trans-retinaldehyde exhibits a peak absorption spectrum in the range of high-energy short-wavelength light,increased concentrations of this compound may increase the potential for photic damage.All-trans-retinol exhibits membranolytic characteristics(reviewed in Boulton et al.,2001);the fact that this compound is concentrated both in photoreceptor outer segments and in RPE cells indicates that accumulation may have pervasive effects on retinal structure and function.
Accumulation of all-trans-retinol,and some other retinoids of the visual cycle,is modulated by retinoid binding proteins that travel across the IPM.IRBP is a 140-kDa glycoprotein that constitutes the major soluble protein fraction of the IPM(Chen et al.,1996).IRBP contains2retinoid binding sites and exhibits highest af?nity for11-cis-retinaldehyde and all-trans-retinol. IRBP also demonstrates af?nity for LCPUFAs with highest speci?city for DHA.In bovine retina DHA rapidly and speci?cally displaced11-cis-retinaldehyde from IRBP(Chen et al.,1996).On the basis of these ?ndings,and information suggesting a steep gradient of DHA between RPE(3.5%of total lipids as DHA)and photoreceptor cells(20%of total lipids as DHA),Chen et al.proposed a model by which lipids and retinoids may interact with IRBP in the regeneration of visual photopigment.The model posits that when IRBP is in the proximity of the RPE,the hydrophilic retinoid-binding site is occupied by11-cis-retinal(this compound has a higher speci?city to the receptor than RPE-associated lipids).As the protein comes in contact with the DHA-rich photoreceptor,the11-cis retinoid is released,and the site is occupied by DHA.All-trans-retinol also exhibits a high af?nity to the receptor and may thus bind to the complex as it approaches the outer segments in transit to the RPE.
Light adaptation is linked to reduction of oxidative stress,as it associated with a decrease in photoreceptor oxygen consumption(Wangsa-Wirawan and Linsenme-ier,2003).Evidence to suggest that light damage and photopigment concentration affect oxidative processes is based on the observation that heme oxygenase,an oxygen sensitive stress protein,is upregulated by retinal photic injury and rhodopsin loss(Organisciak et al., 1998).
5.6.Oxidation-reduction balance
5.6.1.Reactive oxygen species and free radicals Reactive oxygen species activate PLA2.Alterations in cellular redox balance are implicated in the pathogenesis of AMD(Beatty et al.,2000),DR(Cai and Boulton, 2002)and ROP(Hutcheson,2003).As part of a
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comprehensive review on the role of oxidative stress in AMD,Beatty et al.(2000)discuss the basic biochemistry of oxidative processes,as well as the generation and speci?c actions of reactive oxygen species in the retina. Oxidation involves the removal of electrons from an atom or ion and results in an electropositive state. Reactive oxygen species include free radicals(e.g.lipid peroxyl(ROO d),hydroperoxyl(HO2d),hydroxyl(HO d), nitric oxide(d NO)and superoxide anion(O2àd)),singlet oxygen(1O2),and hydrogen peroxide(H2O2).Free radicals are unstable molecules characterized by one or more unpaired electrons in their outer bond orbitals; they accept electrons(hydrogen)from other molecules to attain a balanced electric state.The molecule operating as the electron donor in this electron transfer reaction then becomes unstable and,in the process,acts to extract electrons from an adjacent molecule.This may result in an oxidative cascade.Reactivity and half-life in?uence the effects of these compounds.Most free radicals have a half life of a few seconds to$10à6s.Free radicals react with bases in nucleic acids,amino acid side chains in proteins,and unsaturated bonds in fatty acids. Singlet oxygen is formed when molecular oxygen is energized to redistribute both electrons in the outer-shell octet(that exist in separate pi*2p orbitals)to a single pi*2orbital.The outer bond orbit of singlet oxygen is complete,but the molecule exhibits a higher energy state than molecular oxygen since the2electrons in its outer shell orbit in opposite trajectories.As singlet oxgyen degrades to molecular oxygen,energy is released—this can damage adjacent molecules.
Mitochondria are the main site for superoxide genera-tion;since this molecule is highly reactive,it is unlikely to exist far from the cytosolic regions containing mitochon-dria.The superoxide anion is yielded via addition of an electron to molecular oxygen.Superoxide reacts with nitric oxide to form peroxynitrate(ONOOà).The hydroxyl radical is the product of peroxynitrate degrada-tion.Superoxide may also be enzymatically converted to to hydrogen peroxide.Hydrogen peroxide has a relatively long half-life and that allows it to travel to the nuclear domain;hydrogen peroxide oxidizes–SH groups of resident proteins.Hydrogen peroxide also reacts with divalent metal catalysts(released from injury or haemo-lysis),and via single electron transfer,yields highly reactive hydroxyl radicals.The hydroxyl radical is the most reactive oxygen species present in the body.While the half-life of of the hydroxyl radical is relatively short ($10à8s),it has a relatively high oxidation potential. This radical may be formed in the nucleus and lead to covalent cross-linking of nucleic acid bases.The hydroxyl radical also reacts with membrane-bound lipids to yield lipid radicals.Lipid radicals combine with oxygen to yield highly reactive lipid peroxyl and hydroperoxyl radicals. Lipid peroxyl and hydroperoxyl radicals exist mainly in biological membranes rich in PUFA.
Phospholipid bilayers that constitute cell membranes are rich sources of electrons in the case that the acyl chains of their fatty acids contain unsaturated double bonds.Balazy and Nigam(2003)review the multiple aspects of lipid peroxidation.Free radicals extract hydrogen from these unsaturated bonds,yielding lipid peroxyl radicals and lipid peroxides.Adjacent fatty acids are subsequently oxidized in the attempt to reduce the peroxyl radical to a stable compound.
Beatty et al.(2000)discuss retinal characteristics that facilitate imbalance of cellular redox balance to favor oxidation.These are:the high volume of oxygen consumption necessary to support the metabolic needs of the photoreceptors,the high concentration of photosensitizing compounds in the photoreceptors and RPE,the high concentration of unsaturated fatty acids in photoreceptors,and active phagocytosis of photo-receptor outer segments by the RPE.These character-istics are discussed in the following section within the context of metabolic,environmental,and developmental bioactivators.
5.6.2.Metabolic and environmental bioactivators affect redox balance
Redox balance may be altered by natural and pathologic metabolic processes.Cellular concentrations of reactive oxygen intermediates may be associated with energy metabolism;as nutrient-based energy substrates (carbohydrates,lipids,and proteins)are oxidized to CO2 and H2O they yield hydrogen atoms that are stored within reduced nicotinamide adenine dinucleotide (NADH)and?avin adenine dinucleotide(FAD(2H)). These coenzymes donate electrons to oxygen within the electron transport chain to yield energy that drives the oxidative phosphorylation of adenosine50-diphosphate (ADP)to adenosine50-triphosphate(ATP)by ATP synthase within the mitochondria.Most free radicals are by-products of mitochondrial respiration.As a means of supporting the metabolic needs of the cell,photorecep-tor inner segments are densely packed with these with these organelles.
Retinal oxygen delivery occurs mainly via the choriocapillaris and factors affecting blood?ow(blood or vascular tissue)and oxygen saturation will alter rates of energy production;these conditions affect rates of reactive oxygen intermediate generation.As partial pressure of oxygen increases there is a concomitant rise in reactive oxygen intermediates.It is important to note that oxygen regulation does not occur within chorioca-pillaris.Likewise,the effects of hyperoxia on vasoo-bliteration seen in the intraretinal capillary beds are not observed in the choroid.The in?ammatory response is associated with increases in reactive oxygen species production and thus affects redox balance.
Cellular redox balance may be altered by environ-mental exposures.Chronic and intense acute retinal
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irradiation increase production of free radicals and H2O2.Cigarette smoke,O3,and NO2are other environmentally based factors that increase production of reactive oxygen species.Cellular redox balance may be altered in response to the aging process(Balazy and Nigam,2003)under conditions of chronic exposure.The cellular structure and metabolic ef?ciency of the retina changes across developmental periods(see Section 5.9.1).Mitochondrial structure and function are affected with age;this may favor increased production of reactive oxygen species.In addition to the potential effects of age-related cumulative oxygen load,there is a concomitant alteration in tissue status of aqueous and lipid soluble vitamins with anti-oxidant properties (reviewed in Boulton et al.,2001).
5.6.3.LCPUFAs affect factors and processes implicated in maintaining redox balance
The biochemical nature of DHA and AA and the accretion of these compounds to metabolically active neural retinal tissue would appear to facilitate formation of lipid radicals,lipid peroxyl radicals,and lipid peroxides.Considering the high concentration of DHA in retinal photoreceptor outer segments(areas chroni-cally exposed to high levels of irradiation)the selective tissue distribution of these compounds is perplexing. The importance of LCPUFAs in the retina is indicated by the ef?cient conservation and use of these‘easily oxidized’lipids in areas highly susceptible to oxidative stress and under conditions that facilitate production of reactive oxygen species(Gordon and Bazan,1997). There is evidence to suggest that foveal regions exposed to highest intensity light have lower concentration of LCPUFAs(van Kuijk and Buck,1992);in age-related (chronic)retinal disease the fovea is often spared until late stages of disease.
In vitro studies on model membranes and liposomes have generally reported reactive LCPUFA peroxidation in response to energy or oxygen exposure.This has not been the case for most in vivo studies.Muggli(2003)) reviews studies examining the relationship of o-3 LCPUFA or?sh intake with reactive oxygen species-mediated events,effects on reactive oxygen species biomarkers,and effects on anti-oxidant defense systems. Free radical-induced haemolysis(Mabile et al.,2001) and in vitro LDL oxidation(Wander et al.,1998)were both reduced in samples from people consuming?sh oil. Urinary F2-isoprostanes are in vivo markers of lipid peroxidation and oxidant stress.Non-smoking,treated-hypertensive,type2diabetic subjects consuming4g/d of puri?ed EPA and DHA had lower levels of this biomarker than subjects sharing these characteristics, but consuming an olive oil supplement(Mori et al., 2000).In human tissue,?sh oil exposure is also associated with reduction in superoxide anion genera-tion(Chen et al.,1994;Luostarinen and Saldeen,1996).In some cases,in vivo oxidation of LDL was not altered as a function of LCPUFA intake(Higgins et al.,2001; Brude et al.,1997;Bonanome et al.,1996;Higdon et al., 2000;Frankel et al.,1994);in others it was decreased (Ando et al.,1999).In aged subjects o-3LCPUFA intake at low doses(180mg/d)was associated with decreases in oxidative stress within platelets(Vericel et al.,1999).At higher doses(50m mol/L)DHA operated as a pro-oxidant(Vericel et al.,2003).
An in vitro study on human retina reported an age-and area-related susceptibility to peroxidation,with the posterior pole oxidation increased among tissue from the oldest subjects(De La Paz and Anderson,1992).The oxidative damage of peripheral retina did not vary with age.Rotstein et al.(2003)applied and in vitro model of oxidative stress on pure rat retina neurons to elucidate a mechanism by which DHA may operate as a neuropro-tective factor.After cells were exposed to an environ-mental oxidant(paraquat)that generates the superoxide anion,they were observed to die by apoptosis;loss of mitochondrial membrane integrity was seen a key factor in this event.Addition of DHA to the cultures protected photoreceptors from oxidative stress induced apoptosis. Authors speculate that DHA operates to preserve mitochondrial membrane structure and function by reducing Bax and increasing bcl-2expression.In rats, lower DHA tissue status is associated with lower susceptibility to light damage from acute exposure of 700–800lux followed by90min of darkness(Bush et al., 1991).After exposure to intense green light using intermittent or hyperthermic light treatments rats fed a depleted o-3diet exhibited better structural outcomes than rats fed an linolenic acid-enriched diet from ?axseed(Organisciak et al.,1996).
5.7.In?ammation
In?ammation activates PLA2and in?uences processes implicated the pathogenesis of AMD(Penfold et al., 2001)and DR(Gardner et al.,2000;Frank,2004). In?ammation is an immediate biologic response to injury or infection;it is the result of increased capillary permeability and blood?ow.Increased capillary perme-ability allows regulatory proteins(antibodies,comple-ment,and cytokines)and leukocytes(monocytes, macrophages,natural killer lymphocytes,and granulo-cytes)to pass from the bloodstream across the vascular endothelial wall.Integration of this innate immune response with an acquired one then occurs as activated macrophages and monocytes present antigen to cyto-toxic(CD8+)and helper(CD4+)T lymphocytes. Helper T-lymphocytes express CD4+receptors that recognize cell surface peptide fragments bound in class II major histocompatibility complex(MHCII).These peptides are derived from extracellular pathogens that have either been phagocytosed by macrophages or
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endocytosed by antigen-presenting cells(macrophages, dendritic cells,B lymphocytes).
With the cell-mediated response to the antigen,T lymphocytes secrete cytokines that:modulate B and T lymphocyte proliferation;induce B lymphocyte anti-body production;and regulate monocyte,macrophage, and natural killer lymphocyte activity.Activity within innate immune system monocytes and macrophages leads to production of proin?ammatory cytokines (tumor necrosis factor(TNF)-a,interleukin(IL)-1,and IL-6)that modulate many aspects of innate and acquired immune response.
Helper T lymphocytes are key factors in the produc-tion of immunoregulatory cytokines;they originate from a common progenitor(Th0)and are functionally classi?ed by the effects of the cytokines they produce. Th1cells differentiate from Th0after exposure to IL-12 or interferon(IFN)-g.These cells produce IL-2and IFN-g to activate cytotoxic T lymphocytes,natural killer lymphocytes,macrophages,and monocytes.Th2cells differentiate after exposure to IL-4.These cells produce IL-4(to induce IgE production by B lymphocytes and suppress the Th1response),IL-5(to activate eosino-phils),and IL-10(to suppress the Th1response).
The eye is highly susceptible to attack by systemic autoimmune diseases since it contains cells originating from each of the three embryonic layers.In their diverse origin,these cells express cell surface and intracellular proteins that exist within many systems of the body. Under pathologic conditions,these proteins may be target sites for the immune system.Eicosanoids affect the activities of such factors.Eicosanoids are derived from tissue stores and circulating20carbon LCPUFAs; most20carbon LCPUFA species in the human body are of dietary origin.We discuss LCPUFA–eicosanoid–cy-tokine relationships in Section5.7.1.
5.7.1.Eicosanoid metabolism
Eicosanoids are lipid-based molecules that operate as mediators of in?ammation and immunity.As discussed in Section5.4.2,EPA is the precursor for series-5LTs and series-3PGs and TXs.AA is the substrate for series-4LTs and series-2PGs and TXs.Section6.1.1.1 presents details on eicosanoids as they relate to vascular pathology.AA-derived eicosanoids have the common effect of increasing vascular permeability and activating cells that produce proin?ammatory cytokines(key processes in the in?ammatory response).Autoimmune uveitis is a chronic in?ammatory disease ocular struc-tures in the uvea;certain types of uveitis manifest forms of retinal vasculitis.Autoimmune diseases commonly exhibit a dysregulated Th-1type response(alterations in IL-1,TNF-a production)and enhanced production of AA-derived eicosanoids(particularly PGE2and LTB4). The role of in?ammatory mediators in DR,AMD,and ROP is discussed in Section5.7.2.Fig.4represents the relationship of o-3LCPUFAs with AA-derived eicosa-noid metabolism and neovascularization,vascular per-meability,and in?ammation.
The potential for o-3LCPUFAs to modulate production of AA-derived eicosanoids is important for a number of reasons.First LTB4is associated with TNF-a production(Wallace et al.,2000).TNF-a mediates production of a number of potent proin?am-matory and immunoregulatory cytokines(Calder,2001, see Fig.5).Also,eicosanoids may operate directly on factors in the immune system or via a number soluble mediators,the in?ammatory phospholipids platelet-activating factor(PAF),nitric oxide(NO),and tyrosine and serine/threonine kinases.PGE2decreases T-cell proliferation,lymphocyte migration,and secretion of IL-1and IL-2.PGI2blocks leukocyte aggregation,T-cell proliferation,and lymphocyte migration and secre-tion of IL-1and IL-2.TXA2increases lymphocyte proliferation.LTB4increases leukocyte chemotaxis and aggregation,T-cell proliferation,and the release of TNF-a,IFN-g,IL-1,and IL-2.While AA-derived eicosanoids play different roles the in?ammatory process,they are all associated with vascular leakage.
5.7.2.LCPUFAs affect factors and processes implicated in ocular in?ammation
What evidence exists to implicate o-3LCPUFAs in alteration of the in?ammatory response?Table2
Fig.4.The relationship of o-3LCPUFAs with AA-derived eicosanoids,neovascularization,vascular permeability,and in?ammation.a-LNA=a-linolenic acid;AA=arachidonic acid;DHA=docosahexaenoic acid;EPA=eicosapentaenoic acis;LA=linoleic acid.
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presents three lines of evidence to support the mod-ulatory role of o -3LCPUFAs in immune and in?am-matory https://www.wendangku.net/doc/2512175054.html,rmation was extracted from a comprehensive review by Calder (2001).
LCPUFAs affect both innate and acquired immune systems.In vitro studies on human cell lines incubated with o -3LCPUFAs have demonstrated decreased:(1)monocyte cell surface antigen presentation (Hughes et al.,1996),TNF-a and IL-1b expression (Baldie et al.,1993);(2)neutrophil superoxide presentation (Chen et al.,1994);(3)natural killer lymphocyte activation (Purasiri et al.,1997;Yamashita et al.,1986);(4)lymphocyte proliferation (Brouard and Pascaud,1993;Calder et al.,1992;Calder and Newsholme,1992;Khalfoun et al.,1996;Purasiri et al.,1997;Santoli et al.,1990;Virella et al.,1991),antigen expression (Tappia et al.,1995),and IL-2production (Calder and Newsholme,1992;Purasiri et al.,1997).De Caterina et al.(2000)have added DHA to adult saphenous vein endothelial cell cultures activated by cytokines.The result was reduced expression of IL-6and IL-8.
Animal feeding studies have demonstrated differences in immune system factors between animals receiving o -3LCPUFA-rich diets and those receiving o -3LCPUFA-free diets;animals consuming o -3LCPUFAs show decreased:
1.Macrophage reactive oxygen species production (D’Ambola et al.,1991;Eicher and McVey,1995;Hubbard et al.,1991;Joe and Lokesh,1994),cell surface antigen presentation (Huang et al.,1992;Sanderson et al.,1997),TNF-a expression (Billiar
et al.,1988;Renier et al.,1993;Wallace et al.,2000;Yaqoob and Calder,1995a ),IL-1b expression (Billiar et al.,1988;Renier et al.,1993;Wallace et al.,2000;Yaqoob and Calder,1995a ),IL-6expression (Billiar et al.,1988;Renier et al.,1993;Wallace et al.,2000;Yaqoob and Calder,1995a ),and IFN-g receptor expression (Feng et al.,1999).
2.Monocyte TNF-a and IL-1b expression (Grimm et al.,1994).
3.
Natural Killer cell activation (Meydani et al.,1988;Peterson et al.,1998;Sanderson et al.,1995;Yaqoob et al.,1994c ).
4.Cytotoxic T lymphocyte activation (Fritsche and Cassity,1992).
5.
Lymphocyte proliferation (Alexander and Smythe,1988;Fritsche and Cassity,1992;Fritsche et al.,1991;Jolly et al.,1997;Kelley et al.,1988;Kuratko,2000;Peterson et al.,1998;Sanderson et al.,2000;Wallace et al.,2001;Yaqoob and Calder,1995b ;Yaqoob et al.,1994a,b )and production of IL-2and IFN-g (Wallace,2001).
Human feeding studies demonstrate similar results to the animal studies at high doses of o -3LCPUFAs.These studies also highlight the importance of consider-ing the balance of o -3/o -6LCPUFAs.Three (Endres et al.,1989;Schmidt et al.,1989,1992)of four studies showed decreased monocyte chemotaxis in populations consuming o -3LCPUFA-rich diets;subjects in the study that did not demonstrate a difference between dietary groups (Schmidt et al.,1996)received a relatively lower amount of o -3LCPUFAs.Monocyte surface
Vascular Leakage
Inhibition/Inactivation Enhancement/Production
Fig.5.The relationship of o -3LCPUFAs with leukotriene B 4,cytokines,and actions of cytokines.AA=arachidonic acid;IL=interleukin;PAF=platelet-activating factor.PKC=protein kinase C;PLA 2=phospholipase A 2;TNF-a =tumor necrosis factor-a .
J.P.SanGiovanni,E.Y.Chew /Progress in Retinal and Eye Research 24(2005)87–138
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