Dislipidemia 2009: Is there something new?

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Dislipidemia 2009: Is there something new?

  1. 1. Dyslipidemia 2009: Is there something new? Scavenger Receptor Class B Type I (SR-BI) in Humans Annabelle Rodriguez, M.D. Associate Professor of Medicine Division of Endocrinology and Metabolism Johns Hopkins University School of Medicine Baltimore, Maryland
  2. 2. HDL Cholesterol Transport
  3. 3. Scavenger Receptor Class B Type I Connelly M et al. Trends in Endo Metab 2003;14:467
  4. 4. Scavenger Receptor Class B Type I • SR-BI expressed in liver, steroidogenic tissues, endothelium, macrophages • Elevated HDL in KO mice • KO have accelerated atherosclerosis in the background of apoE or LDLR deficiency • Female KO mice are infertile, but can be reversed with the cholesterol medication, probucol • Much less is known regarding the role of SR-BI in humans • We have shown that a subset of infertile women (approximately 24%) with low SR-BI RNA levels had significantly lower plasma estradiol levels M Velasco… A Rodriguez. Fertil & Steril 2006;85:1391
  5. 5. Hyperalphalipoproteinemic (HALP) Study • Adults with HDL-C levels > 60 mg/dl • Isolated monocyte-derived macrophages as source of SR-BI protein • Isolated genomic DNA, RNA, cell lysates, serum and plasma for lipoprotein subfraction analysis (apoproteins, NMR, and 2D gel) • Differential diagnosis: hepatic lipase, CETP activity, LCAT activity • Carotid IMT
  6. 6. Characteristics of the HALP Study Population Age (years) 57.2  10.9 Gender Men (%) 14 (22) Women (%) 51 (78) Race White (%) 45 (69) Black (%) 17 (26) Asian (%) 2 (3) Hispanic (%) 1 (2) Body mass index (kg/m2) 25.6  5.7 Total cholesterol (mg/dl) 220  35 Triglycerides (mg/dl) 78  3 HDL cholesterol (mg/dl) 89  19 LDL cholesterol (mg/dl) 116  31
  7. 7. HDL-C levels are inversely correlated with macrophage SR-BI protein levels 140 130 HDL-C (mg/dl) 120 110 100 90 80 70 60 50 0 .1 .2 .3 .4 .5 .6 Macrophage SR-BI protein normalized to -actin p=0.02, r=0.32
  8. 8. HDL particle size is inversely correlated with macrophage SR-BI protein levels 11 R=0.29 10.5 HDL size (nm) 10 9.5 9 0 .1 .2 .3 .4 .5 .6 SR-BI protein (OD, normalized to -actin) p=0.03
  9. 9. SR-BI function is correlated with macrophage SR-BI protein levels 70 R=0.38 60 CE uptake (%) 50 40 30 20 10 0 0 .1 .2 .3 .4 .5 .6 SR-BI protein (OD, normalized to -actin) p=0.01
  10. 10. Multiple regression of independent predictors for HDL-C levels • Variable(s) p-values • Gender p=0.001 • A1c p=0.04 • TG p=0.007 • Fish oil use p=0.008 • SR-BI p=0.01 • Entire model (age, gender, BMI, p=0.0001, r=0.70 exercise, alcohol use, HT use, TG, hemoglobin A1c, fish oil use, smoking, and SR-BI protein by western blotting)
  11. 11. Multiple regression of independent predictors for SR-BI protein levels • Variable(s) p-values • BMI p=0.008 • Rs4238001 p=0.02 • Rs2278986 p=0.02 • Entire model p=0.01, R2=0.35 (gender, BMI, HT use, ethnicity, aspirin use, CETP and LCAT activities, rs4238001 [exon 1 G2S], rs2278986 [intron 3], and rs5888 [exon 8]).
  12. 12. B. WT G2S SR-BI (OD, normalized to actin) A. -actin SR-BI protein levels 0.7 0.6 0.5 0.4 p=0.007, r=0.36, 0.3 0.2 r2=0.13 0.1 0 AA/AG GG rs4238001 C. SR-BI protein levels 0.7 0.6 0.5 0.4 0.3 p=0.02, r=0.33, 0.2 r2=0.11 0.1 0 CC/TC TT rs2278986
  13. 13. Exon 1 G2S variant increases degradation of SR-BI protein A. WT G2S WT G2S B. 1 2 3 4 5 6 7 8 9 1-3: pCDNA3.1 empty 4-6: pCDNA3.1-SR-BI WT 7-9: pCDNA3.1-SR-BI G2S C. WT G2S Hours: 0 2 4 7 0 2 4 7 SR-BI -actin
  14. 14. Exon 1 G2S variant decreases SR-BI function A. 120000 cpm/mg cell protein 100000 Wild-type 80000 * G2S 60000 40000 20000 0 Degradation B. 9000 C. 600000 cpm/mg cell protein dpm/mg cell protein 8000 500000 7000 6000 400000 5000 4000 300000 * 3000 2000 * 200000 1000 100000 0 0 125 I Specific Cell Association [3H]CE Specific Cell Association * p<0.0004 compared to wild-type
  15. 15. VARIANTS IN SCAVENGER RECEPTOR CLASS B TYPE I GENE ARE ASSOCIATED WITH HDL CHOLESTEROL LEVELS IN YOUNGER WOMEN Roberts CGP, Shen H, Damcott CM, Shuldiner A, Mitchell BD, Rodriguez A. Scavenger receptor class B type I variants associated with glycemic and lipid traits: The Amish Family Diabetes Study. Hum Hered 2007;64:107-113.
  16. 16. VARIANTS IN SCAVENGER RECEPTOR CLASS B TYPE I GENE ARE ASSOCIATED WITH HDL CHOLESTEROL LEVELS IN YOUNGER WOMEN Rs5891 80 75 P= NS P <0.001 70 HDL-C (mg/dL) 65 60 55 50 45 40 35 30 GG GA AA GG GA AA Rs5888 65 60 P= NS P= 0.04 HDL-C (mg/dL) 55 50 45 40 35 30 CC CT TT CC CT TT Men Women Roberts et al. Hum Hered 2007
  17. 17. SCARB1 exon 8 synonymous SNP associated with lower SR-BI protein expression in transiently transfected COS cells 0.18 Relative intensity SR-BI/-actin 0.16 Wild type C Mutant T 0.14 0.12 0.1 (*p<0.04) 0.08 0.06 0.04 0.02 0 wild-type exon 8 SNP M-FOLD 20 RNA Structure prediction SR-BI protein Jason Constantineau, Erin Greason, Caroline Roberts, Lane Christensen, JLR, under revision
  18. 18. RS5888 RNA exhibits enrichment in monosomal fractions A 25 * p<0.04 compared to WT 20 15 10 5 0 10 12 12 13 11 13 2 9 6 7 11 3 5 4 10 1 1 2 8 6 8 4 7 3 5 9 SNP WT RS5888 B relative SR-BI levels (normalized to 18S) 2.5 Total SR-BI 2.0 1.5 1.0 0.5 0.0 WT SNP
  19. 19. Multi-Ethnic Study of Atherosclerosis (MESA) A prospective study of subclinical atherosclerosis and cardiovascular outcomes in ~6500 participants in 6 communities throughout the United States. Ascertainments: coronary artery calcium score, carotid IMT, lipid measurements, anthropometric measurements, genomic DNA, among other measurements.
  20. 20. Multi-Ethnic Study of Atherosclerosis (MESA) • Forty-three SCARB1 SNPS were genotyped • Significant associations were found between SNPs and lipids, with most being associated with TG levels • Significant associations were found between SNPs and common carotid IMT in three racial groups • Conclusion: SCARB1 SNPs associated with increased subclinical atherosclerosis
  21. 21. The Role of Scavenger Receptor Class B type I in Human Infertility
  22. 22. SR-BI knockdown in immortalized human granulosa cells (HGL5) SR-BI GAPDH
  23. 23. Pilot study: Media progesterone levels significantly lower in SR-BI knockdown HGL5 cells DMSO Progesterone levels (ng/ml) 70 Forskolin (10 uM) 60 Forksolin + 10% serum Forksolin + HDL (50 g protein/ml) 50 40 30 p=0.001 20 p=0.04 10 p=0.04 0 Scrambled SR-BI knockdown
  24. 24. Summary • SR-BI protein levels are independent predictors of HDL-C levels in HALP subjects • We have identified two SNPs that are independent predictors of SR-BI protein levels • Certain SCARB1 SNPs are associated with subclinical atherosclerosis (MESA) • Low SR-BI levels are associated with lower progesterone levels in vitro.
  25. 25. Acknowledgments • Funding (Rodriguez): NHLBI RO1, Burroughs Wellcome Clinical Translational Scientist Award • JHU: Rodriguez lab: Erin Greason BS, Angela Green BA, Tonya Kolmakova PhD, Michael West MD PhD, Adam Naj, PhD Reproductive Endocrinology: Jairo Garcia, MD, Yulian Zhao PhD, Melissa Yates MD Cardiology: Wendy Post M.D • University of Colorado: Jeffrey Kieft PhD • University of Kansas: Lane Christenson PhD • University of Maryland: Charles Chaffin PhD

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