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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
20051028, Curcio-2 3
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
20051028, Curcio-2 6
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
20051028, Curcio-2 7
Lipoprotein Classes
Vance et al, Biochemistry of Lipids, Lipoproteins and Membranes 2002;
nm nmnmnmnm
20051028, Curcio-2 8
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
20051028, Curcio-2 9
Lipoprotein Metabolism
Lusis et al. Circulation.
2004;110:1868
20051028, Curcio-2 10
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
20051028, Curcio-2 12
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
20051028, Curcio-2 13
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
20051028, Curcio-2 14
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
20051028, Curcio-2 15
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
20051028, Curcio-2 16
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)
20051028, Curcio-2 17
Particle Structure
Ruberti, Curcio, Millican, Menco, Huang, Johnson IOVS 44:1753 (2003)78 year old donor
SurfaceSurface
CoreCore
20051028, Curcio-2 18
Solid Particles in BlamD
Curcio, Presley, Millican, Medeiros, Exp. Eye Res., 80:761-775
20051028, Curcio-2 19
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
20051028, Curcio-2 20
Drusen Contain Cholesterol
Curcio, Presley, Medeiros,
Malek, Avery, Kruth
Exp Eye Res 2005, in press
20051028, Curcio-2 21
Drusen Contain ApoB
Malek et al, Am J Pathol 162:413 (2003)
Apo B immunoreactivity ControlAutofluorescence
20051028, Curcio-2 22
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
20051028, Curcio-2 23
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
20051028, Curcio-2 26
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)
20051028, Curcio-2 27
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