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Mechanism of Cholesterol Deposition in Bruch’s Membrane

Mechanism of Cholesterol Deposition in Bruch’s Membrane. Christine A. Curcio, Ph.D. Department of Ophthalmology School of Medicine University of Alabama. Outline. Conclusion Intro to apoB/MTP system; role in atherosclerosis Evidence for an intra-ocular apoB lipoprotein Conclusion & models.

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Mechanism of Cholesterol Deposition in Bruch’s Membrane

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  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. References Curcio 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. Age-related Maculopathy RPE? Macrophages Endothelium OS lipid disposal? Binding, Molecular sieve? Oxidative, Non-oxidative Neovascularization RPE? RPE lipoprotein? Biological Process ApoB- Lipoprotein Source ApoB- Lipoprotein Particle Retention in Intima/ BrM Lipoprotein Modification Cellular Response Threatening Complications Macrophages, Smooth muscle Endothelium Liver, Intestine VLDL/ LDL, Chylomicrons/ remnants Binding of apo B Oxidative, Non-oxidative Neovascularization, Rupture, thrombosis Fuel transport Coronary Artery Disease 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. 20051028, Curcio-2

  5. Oil red O stain 35 years 74 years Lipids in Bruch’s Membrane Pauleikhoff, Harper, Marshall, & Bird (1990) Ophthalmology 97:171 Sheraidah, Steinmetz, Maguire, Pauleikhoff, Marshall, & Bird (1993). Ophthalmology 100: 47. Holz, Sheraidah, Pauleikhoff, & Bird (1994) Arch. Ophthalmol. 112: 402. 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 nm nm nm nm nm 20051028, Curcio-2 Vance et al, Biochemistry of Lipids, Lipoproteins and Membranes 2002;

  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-b OH group (esterified, EC) • Intracellular storage • Transport 20051028, Curcio-2

  9. Lipoprotein Metabolism Lusis et al. Circulation. 2004;110:1868 20051028, Curcio-2

  10. 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 a-amphipathic, b-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 ApoB Segrest, Jones, De Loof, Dashti J Lipid Res 2001, 42:1346 20051028, Curcio-2

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

  12. Accumulated cholesterol Foam cell formation Binding & retention Uptake of modified lipoproteins Smooth muscle cells PG Chemotaxis, stimulation Extravasation Transcytosis Remnants LDL Adhesion molecule expression Neovascularization Rupture Hemorrhage Monocyte attachment Chylomicrons VLDL Apo B-48 Apo B-100 Response to Retention Hypothesis Williams & Tabas. ATVB 1995, 15:551 Figure after Proctor et al. Curr Opin Lipidol 2002, 13:461. 20051028, Curcio-2

  13. Esterified Cholesterolin Bruch's Membrane Filipin fluorescence, digital microscopy, normal eyes Curcio et al., IOVS 42:265, 2001 20051028, Curcio-2

  14. c V A Bruch’s Membrane Cholesterol * x 1000 nmol/ gm Enzymatic fluorimetric assay; chloroform/ methanol extracts; mechanically isolated Bruch’s membrane; preserved normal eyes Curcio et al., IOVS 42:265, 2001 * x 1000 nmol/g dry wt 20051028, Curcio-2

  15. RPE RPE BL Esterified Cholesterol-rich Particles Conventional 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-fixation Curcio et al., IOVS 42:265, 2001 20051028, Curcio-2

  16. TEM QFDE TEM QFDE 41 year old donor 76 year old donor Lipid Wall in Inner Bruch’s Ruberti, Curcio, Millican, Menco, Huang, Johnson, IOVS 44:1753 (2003) 20051028, Curcio-2

  17. Surface Core 78 year old donor Particle Structure Ruberti, Curcio, Millican, Menco, Huang, Johnson IOVS 44:1753 (2003) 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 Apo B immunoreactivity Autofluorescence Control 20051028, Curcio-2 Malek et al, Am J Pathol 162:413 (2003)

  22. Retina & RPE: Apo B/ MTP Pathway • 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 Li, Presley, Zhang, Dashti, Chung, Medeiros, Guidry, Curcio; 2005, J. Lipid Res, 46:628 - 640 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. Pk 1 2 Pk 1 2 Pk 1 2 Pk 1 2 ApoB and ApoA-I Expression Li, Chung, Presley, Malek, Zhang, Dashti, Li, Chen, Bradley, Kruth, Curcio; 2005, IOVS, 46:2576-2586 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 male Homozygote for S590I missense mutation Al-Shali et al Clin Gen63:135 (2003) 20051028, Curcio-2

  27. Model/ Hypothesis 20051028, Curcio-2

  28. Age-related Maculopathy RPE? Macrophages Endothelium OS lipid disposal? Binding, Molecular sieve? Oxidative, Non-oxidative Neovascularization RPE? RPE lipoprotein? Biological Process ApoB- Lipoprotein Source ApoB- Lipoprotein Particle Retention in Intima/ BrM Lipoprotein Modification Cellular Response Threatening Complications Macrophages, Smooth muscle Endothelium Liver, Intestine VLDL/ LDL, Chylomicrons/ remnants Binding of apo B Oxidative, Non-oxidative Neovascularization, Rupture, thrombosis Fuel transport Coronary Artery Disease 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. 20051028, Curcio-2

  29. ALABAMA EYE BANK Acknowledgments Curcio Laboratory Dina Avery Kelley Bradley Tammy Bailey Melissa Chimento Mark Clark Chuan-Ming Li, MD, Ph.D Goldis Malek Leigh Millican Brett Presley Lan Wang, MD Xueming Zhang Ophthalmology Clyde Guidry, PhD Medicine B.H. Chung, PhD Nassrin Dashti, PhD Ling Li, DVM, PhD Retina Specialists of North Alabama Nancy Medeiros, MD Northwestern University Mark Johnson, PhD Jiahn-Dar Huang Jeff Ruberti, PhD Wake Forest University Dawn Schwenke, PhD NHLBI Howard Kruth, MD 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 20051028, Curcio-2

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