20051028, Curcio-2 1

Mechanism of Cholesterol Deposition in Bruch’s Membrane

Christine A. Curcio, Ph.D.

Department of Ophthalmology

School of Medicine

University of Alabama

20051028, Curcio-2 2

Outline

• Conclusion• Intro to apoB/MTP system; role in atherosclerosis• Evidence for an intra-ocular apoB lipoprotein• Conclusion & models

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ReferencesCurcio CA, Millican CL, Bailey T, Kruth HS: Accumulation of cholesterol with age in human Bruch's

membrane, Invest. Ophthalmol. Vis. Sci. 2001, 42:265-274

Malek G, Li C-M, Guidry C, Medeiros NE, Curcio CA: Apolipoprotein B in cholesterol-containing drusen and basal deposits in eyes with age-related maculopathy, Am. J. Pathol. 2003, 162:413-425

Ruberti JW, Curcio CA, Millican CL, Menco BP, Huang JD, Johnson M: Quick-freeze/deep-etch visualization of age-related lipid accumulation in Bruch's membrane, Invest Ophthalmol Vis Sci 2003, 44:1753-1759.

Li C-M, Presley JB, Zhang X, Dashti N, Chung BH, Medeiros NE, Guidry C, Curcio CA: Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy, J. Lipid Res. 2005, 46:628 - 640

Curcio CA, Presley JB, Millican CL, Medeiros NE: Basal deposits and drusen in eyes with age-related maculopathy: evidence for solid lipid particles, Exp Eye Res 2005, 80:761-775

Li CM, Chung BH, Presley JB, Malek G, Zhang X, Dashti N, Li L, Chen J, Bradley K, Kruth HS, Curcio CA: Lipoprotein-like particles and cholesteryl esters in human Bruch's membrane: initial characterization, Invest Ophthalmol Vis Sci 2005, 46:2576-2586

Curcio CA, Presley JB, Medeiros NE, Malek G, Avery DV, Kruth HS: Esterified and unesterified cholesterol in drusen and basal deposits of eyes with age-related maculopathy, Exp Eye Res 2005, in press:

20051028, Curcio-2 4

Hypothesis• Age-related maculopathy, like coronary artery disease, involves the

Response-to-Retention of apolipoprotein B-containing lipoprotein particles in a vascular intima, with the twist that the apo B-containing lipoprotein comes largely from the RPE rather than plasma.

BiologicalProcess

ApoB-Lipoprotein

Source

ApoB-Lipoprotein

Particle

Retention inIntima/ BrM

LipoproteinModification

CellularResponse

ThreateningComplications

Age-related Maculopathy

Coronary Artery Disease

Fuel transport Liver,Intestine

VLDL/ LDL,Chylomicrons/

remnants

Oxidative,Non-oxidative

Macrophages,Smooth muscle

Endothelium

Neovascularization,Rupture, thrombosis

RPE?MacrophagesEndothelium Neovascularization

Bindingof apo B

OS lipiddisposal? RPE? RPE lipoprotein?

Binding,Molecular sieve?

Oxidative,Non-oxidative

20051028, Curcio-2 5

Lipids in Bruch’s Membrane

Holz, Sheraidah, Pauleikhoff, & Bird (1994) Arch. Ophthalmol. 112: 402.

Sheraidah, Steinmetz, Maguire, Pauleikhoff, Marshall, & Bird (1993). Ophthalmology 100: 47.

Oil red O stain 35 years 74 years

Pauleikhoff, Harper, Marshall, & Bird (1990) Ophthalmology 97:171

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Coronary Artery Disease:a Model for ARM

• We should seek not only molecules of interest but a testable hypothesis that accounts for the overall trajectory of ARM

• Principal lesions (drusen and basal linear deposit) are in a vessel wall outside the blood-retina barrier

• A disease featuring extracellular lipid deposition in a vessel wall should be guided by research on coronary artery disease

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Lipoprotein Classes

Vance et al, Biochemistry of Lipids, Lipoproteins and Membranes 2002;

nm nmnmnmnm

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Cholesterol• Free (unesterified, UC)

– A component of all eukaryotic membranes

• Not metabolized by cells but turned over and released to circulating lipoproteins for hepatic clearance via bile salts

– Reverse cholesterol transport

• Bound by an ester linkage to a fatty acid at 3- OH group (esterified, EC)

– Intracellular storage – Transport

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Lipoprotein Metabolism

Lusis et al. Circulation.

2004;110:1868

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ApoB• 1 gene/ 2 proteins/ 1 molecule per particle

– mRNA editing; stop-codon at 2153 for apo B-48

– ApoB-100/ 4536 a.a./ liver/ VLDL– ApoB-48/ 2153 a.a. / intestine/ chylomicron

• 5 domains in apoB-100– -amphipathic, -sheet, globular

• Structurally related to vitellogenin, an egg yolk protein

• Lipidation required for secretion– Degraded via ubiquitin-proteosome– Insoluble when delipidated

• Function: delivery of TG (fuel)• Unique

– Only non-exchangeable apolipoprotein– In mice, only apolipoprotein whose absence

or dysfunction is fatal

Segrest, Jones, De Loof, Dashti J Lipid Res 2001, 42:1346

20051028, Curcio-2 11Segrest et al., J Lipid Res 1999, 40:1401

MTP and Lipoprotein Assembly

• Microsomal triglyceride transfer protein

– Cells that express apo B without MTP cannot secrete lipoproteins

– All cells expressing MTP secrete lipoproteins

• Functional heterodimer in ER– Large (97KD)– Small (55KD, protein disulfide

isomerase)

• Transfers neutral lipid (TG, EC)• Required for initial lipidation of apo B• Role in subsequent steps under study• Loss of functional MTP protein:

abetalipoproteinemia

ER

Nascent apo B

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Response to Retention HypothesisAccumulated cholesterol

Chylomicrons VLDL Apo B-48 Apo B-100

Remnants LDL

Binding & retention

PG Chemotaxis,stimulation

Adhesionmoleculeexpression

Monocyteattachment

Foam cell formation

Uptake of modified lipoproteins Smooth

musclecellsExtravasation

NeovascularizationRuptureHemorrhage

Williams & Tabas. ATVB 1995, 15:551

Figure after Proctor et al. Curr Opin Lipidol 2002, 13:461.

Transcytosis

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Esterified Cholesterolin Bruch's Membrane

Filipin fluorescence, digital microscopy, normal eyesCurcio et al., IOVS 42:265, 2001

0

4

8

12

16

0 20 40 60 80 100

Me

an

flu

ore

sce

nce

inte

nsi

ty

Age, years

0 20 40 60 80 100

Age, years

MACULAp = 0.0002

PERIPHERYp = 0.004

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Bruch’s Membrane Cholesterol

* x 1000 nmol/g dry wt

Region Tissue N EC* %EC/TC

Macula Bruch's 10 39.1 59.6

Retina 7 2.0 3.6

Choroid 7 7.4 15.0

Periphery Bruch's 3 8.3 26.9

Enzymatic fluorimetric assay; chloroform/ methanol extracts; mechanically isolated Bruch’s

membrane; preserved normal eyesCurcio et al., IOVS 42:265, 2001

Vessels included % of ECRecovered

Enrichment(vs Plasma)

Choriocapillaris 2.5% 40-fold

Choriocapillaris+veins 6.1% 17-fold

AV

c

* x 1000 nmol/ gm

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Esterified Cholesterol-rich ParticlesConventional TEM Lipid-preserving (OTAP)

• Solid 100 nm particles• Extractable by chloro/ meth• Increase with age

– Occupy >30% of Bruch’s in eyes > 60 years

• Dense band external to RPE basal lamina in eyes > 60 yr

OTAP = osmium tannic acid p-phenylenediamine post-fixationCurcio et al., IOVS 42:265, 2001

RPERPEBLBL

RPERPE

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Lipid Wall in Inner Bruch’sTEM QFDE TEM QFDE

41 year old donor 76 year old donorRuberti, Curcio, Millican, Menco, Huang, Johnson, IOVS 44:1753 (2003)

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Particle Structure

Ruberti, Curcio, Millican, Menco, Huang, Johnson IOVS 44:1753 (2003)78 year old donor

SurfaceSurface

CoreCore

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Solid Particles in BlamD

Curcio, Presley, Millican, Medeiros, Exp. Eye Res., 80:761-775

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Lipid Particles in Lesions

2% osmium and osmium tannic acid p-phenylenediamine post-fixation methods. Curcio, Presley, Millican, Medeiros, Exp. Eye Res., 80:761-775

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Drusen Contain Cholesterol

Curcio, Presley, Medeiros,

Malek, Avery, Kruth

Exp Eye Res 2005, in press

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Drusen Contain ApoB

Malek et al, Am J Pathol 162:413 (2003)

Apo B immunoreactivity ControlAutofluorescence

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Retina & RPE: Apo B/ MTP

Pathway

Li, Presley, Zhang, Dashti, Chung, Medeiros,Guidry, Curcio; 2005,J. Lipid Res, 46:628 - 640

• mRNA detected by RT-PCR

• mRNA sequence: apo B-100

• ApoB and MTP proteins detected by western blot

• Native retina & RPE, ARPE-19, and HepG2 cell lines

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De novo Neutral Lipid Secretion

Li, Presley, Zhang, Dashti, Chung, Medeiros,Guidry, Curcio; J. Lipid Res, 2005, 46:628 - 640

20051028, Curcio-2 24

Plasma vs Eye Lipoproteins

Li, Chung, Presley, Malek, Zhang, Dashti, Li, Chen, Bradley, Kruth, Curcio; IOVS, 2005, 46:2576-2586

20051028, Curcio-2 25

ApoB and ApoA-I Expression

Li, Chung, Presley, Malek, Zhang, Dashti, Li, Chen, Bradley, Kruth, Curcio; 2005, IOVS, 46:2576-2586

Pk 1 2 Pk 1 2

Pk 1 2 Pk 1 2

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Abetalipoproteinemia• MIM2001001, Bassen-Kornzweig disease (1950)

– Autosomal recessive inheritance– No apo B containing lipoproteins in plasma

• Fat malabsorption/ steatorrhea

– Acanthocytosis of red blood cells– Ataxic neuropathy & pigmentary retinopathy

• Caused by lack of functional MTP– Absence of protein in ABL intestinal biopsy (1992)– Null mutation of MTP gene (1993)

• Variable clinical response to vitamin E (1982-1986)– Retinopathy persists, progresses despite long-term supplementation

(2001).– Retinas can be normal despite absence of plasma vitamin E or

abnormal despite normal plasma vitamin E• Re-interpretion: ABL is a naturally occurring loss-of-function mutation of

MTP at the level of the RPE signifying that lipoprotein assembly is constitutive and important for outer retinal health

58-year-old maleHomozygote forS590I missense mutationAl-Shali et alClin Gen 63:135 (2003)

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Model/ Hypothesis

20051028, Curcio-2 28

Hypothesis• Age-related maculopathy, like coronary artery disease, involves the

Response-to-Retention of apolipoprotein B-containing lipoprotein particles in a vascular intima, with the twist that the apo B-containing lipoprotein comes largely from the RPE rather than plasma.

BiologicalProcess

ApoB-Lipoprotein

Source

ApoB-Lipoprotein

Particle

Retention inIntima/ BrM

LipoproteinModification

CellularResponse

ThreateningComplications

Age-related Maculopathy

Coronary Artery Disease

Fuel transport Liver,Intestine

VLDL/ LDL,Chylomicrons/

remnants

Oxidative,Non-oxidative

Macrophages,Smooth muscle

Endothelium

Neovascularization,Rupture, thrombosis

RPE?MacrophagesEndothelium Neovascularization

Bindingof apo B

OS lipiddisposal? RPE? RPE lipoprotein?

Binding,Molecular sieve?

Oxidative,Non-oxidative

20051028, Curcio-2 29

AcknowledgmentsCurcio LaboratoryDina AveryKelley Bradley Tammy BaileyMelissa ChimentoMark ClarkChuan-Ming Li, MD, Ph.DGoldis MalekLeigh MillicanBrett PresleyLan Wang, MDXueming ZhangOphthalmologyClyde Guidry, PhDMedicineB.H. Chung, PhDNassrin Dashti, PhDLing Li, DVM, PhD

Retina Specialists of North Alabama Nancy Medeiros, MDNorthwestern UniversityMark Johnson, PhDJiahn-Dar HuangJeff Ruberti, PhDWake Forest UniversityDawn Schwenke, PhDNHLBIHoward Kruth, MD

ALABAMAEYE BANK

20051028, Curcio-2 30

Support• National Eye Institute• Research to Prevent Blindness, Inc.• International Retinal Research Foundation• EyeSight Foundation of Alabama• Roger Johnson Prize in Macular

Degeneration Research• Macula Vision Research Foundation