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
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. The Role of Scavenger Receptor
Class B type I in Human
Infertility
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. 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
