1. 20051028, Curcio-21 Mechanism of Cholesterol Deposition in Bruch’s Membrane Christine A. Curcio, Ph.D. Department of Ophthalmology School of Medicine University of Alabama

2. 20051028, Curcio-22 Outline Conclusion Intro to apoB/MTP system; role in atherosclerosis Evidence for an intra-ocular apoB lipoprotein Conclusion & models

3. 20051028, Curcio-23 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:

4. 20051028, Curcio-24 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. Biological Process ApoB- Lipoprotein Source ApoB- Lipoprotein Particle Retention in Intima/ BrM Lipoprotein Modification Cellular Response Threatening Complications 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? Macrophages Endothelium Neovascularization Binding of apo B OS lipid disposal? RPE?RPE lipoprotein? Binding, Molecular sieve? Oxidative, Non-oxidative

5. 20051028, Curcio-25 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 years74 years Pauleikhoff, Harper, Marshall, & Bird (1990) Ophthalmology 97:171

6. 20051028, Curcio-26 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

7. 20051028, Curcio-27 Lipoprotein Classes Vance et al, Biochemistry of Lipids, Lipoproteins and Membranes 2002; nm

8. 20051028, Curcio-28 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

9. 20051028, Curcio-29 Lipoprotein Metabolism Lusis et al. Circulation. 2004;110:1868

10. 20051028, Curcio-210 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

11. 20051028, Curcio-211 Segrest 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

12. 20051028, Curcio-212 Response to Retention Hypothesis Accumulated cholesterol Chylomicrons VLDL Apo B-48 Apo B-100 Remnants LDL Binding & retention PG Chemotaxis, stimulation Adhesion molecule expression Monocyte attachment Foam cell formation Uptake of modified lipoproteins Smooth muscle cells Extravasation Neovascularization Rupture Hemorrhage Williams & Tabas. ATVB 1995, 15:551 Figure after Proctor et al. Curr Opin Lipidol 2002, 13:461. Transcytosis

13. 20051028, Curcio-213 Esterified Cholesterol in Bruch's Membrane Filipin fluorescence, digital microscopy, normal eyes Curcio et al., IOVS 42:265, 2001

14. 20051028, Curcio-214 Bruch’s Membrane Cholesterol * x 1000 nmol/g dry wt Enzymatic fluorimetric assay; chloroform/ methanol extracts; mechanically isolated Bruch’s membrane; preserved normal eyes Curcio et al., IOVS 42:265, 2001 A V c * x 1000 nmol/ gm

15. 20051028, Curcio-215 Esterified Cholesterol-rich Particles Conventional TEMLipid-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, 2001RPEBLRPE

16. 20051028, Curcio-216 Lipid Wall in Inner Bruch’s TEMQFDETEMQFDE 41 year old donor76 year old donor Ruberti, Curcio, Millican, Menco, Huang, Johnson, IOVS 44:1753 (2003)

17. 20051028, Curcio-217 Particle Structure Ruberti, Curcio, Millican, Menco, Huang, Johnson IOVS 44:1753 (2003) 78 year old donor SurfaceCore

18. 20051028, Curcio-218 Solid Particles in BlamD Curcio, Presley, Millican, Medeiros, Exp. Eye Res., 80:761-775

19. 20051028, Curcio-219 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

20. 20051028, Curcio-220 Drusen Contain Cholesterol Curcio, Presley, Medeiros, Malek, Avery, Kruth Exp Eye Res 2005, in press

21. 20051028, Curcio-221 Drusen Contain ApoB Malek et al, Am J Pathol 162:413 (2003) Apo B immunoreactivityControlAutofluorescence

22. 20051028, Curcio-222 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

23. 20051028, Curcio-223 De novo Neutral Lipid Secretion Li, Presley, Zhang, Dashti, Chung, Medeiros, Guidry, Curcio; J. Lipid Res, 2005, 46:628 - 640

24. 20051028, Curcio-224 Plasma vs Eye Lipoproteins Li, Chung, Presley, Malek, Zhang, Dashti, Li, Chen, Bradley, Kruth, Curcio; IOVS, 2005, 46:2576-2586

25. 20051028, Curcio-225 ApoB and ApoA-I Expression Li, Chung, Presley, Malek, Zhang, Dashti, Li, Chen, Bradley, Kruth, Curcio; 2005, IOVS, 46:2576-2586 Pk 1 2

26. 20051028, Curcio-226 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 Gen 63:135 (2003)

27. 20051028, Curcio-227 Model/ Hypothesis

28. 20051028, Curcio-228 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. Biological Process ApoB- Lipoprotein Source ApoB- Lipoprotein Particle Retention in Intima/ BrM Lipoprotein Modification Cellular Response Threatening Complications 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? Macrophages Endothelium Neovascularization Binding of apo B OS lipid disposal? RPE?RPE lipoprotein? Binding, Molecular sieve? Oxidative, Non-oxidative

29. 20051028, Curcio-229 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 ALABAMA EYE BANK

30. 20051028, Curcio-230 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