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Accelerated atherosclerosis development in C57Bl6 mice by overexpressing AAV-
mediated PCSK9 and partial carotid ligation
Sandeep Kumar, Dong-Won Kang, Amir Rezvan and Hanjoong Jo
SUPPLEMENTARY MATERIAL
Supplementary Table 1. Plasma lipid profile in C57 animals after AAV-PCSK9 injection
and high-fat diet feeding
Total
Cholesterol
(mg/dL)
Triglyceride
s
(mg/dL)
HDL
(mg/dL)
LDL
(mg/dL)
C57
(1 week)
Control 139.0±16.0 55.0±11.6 71.1±16.4 87.0±56.7
AAV-PCSK9 689.2±12.1 264.5±62.6 23.52±12.4 241.0±49.3
C57
(3 weeks)
Control 229±62.2 65±16.2 62.4±14.5 87±56.7
AAV-PCSK9 704.2±3.1 295±120.1 13.5±6.6 328±66.3
C57
(12 weeks)
Control 126.4±13.4 51±17.4 32.1±26.0 35.7±19.4
AAV-PCSK9 759.7±4.2 298.7±9.1 18.5±3.3 535±49.4
LDL, low-density lipoprotein; HDL, high-density lipoprotein; AAV-PCSK9, recombinant adeno-
associated virus serotype 9-gain-of-function mutant of murine PCSK9 gene.
Supplementary Figure 1. Quantification of plaque lesion in the lesser curvature vs.
greater curvature in C57BL6 mice injected with PCSK9 and fed a high-fat diet for 3
months
Greater Curvature
Lesser Curvature
Thoracic Aorta
0.0000.0250.050
0.5
1.0
1.5
2.0
2.5
Oil
Red-
O p
ositi
ve a
rea
(mm
2 )
ControlAAV9-PCSK9
Plaque Coverage*
C57Bl6 mice were injected with AAV-PCSK9 or AAV-Luc (Control) (1 X 1011 VG/mouse) and fed
a high-fat diet for 3 month. Mice were sacrificed and the aortic tree was dissected out. The
aortae were opened longitudinally, pinned on black wax and en-face Oil-Red-O staining was
performed. Plaque lesion area in the lesser curvature, greater curvature and thoracic aorta was
quantified by Image J. Data represented as means ± S.E.M from 3 animals; * P<0.05.
Supplementary Figure 2. Plasma levels of mouse PCSK9 in animals treated with AAV-
PCSK9 and high-fat diet feeding
Graphs show quantification of plasma PCSK9 (ng/mL) obtained from mice treated with AAV9-
PCSK9 or Control at the end of study duration from (A) C57 mice subjected to partial carotid
ligation and fed a high-fat diet for 3 weeks, (B) eNOS-/- mice on C57 background subjected to
partial carotid ligation and fed a high-fat diet for 3 weeks and (C) NOX1-/y mice on C57
background subjected to partial carotid ligation and fed a high-fat diet for 3 weeks, respectively.
Data shown as means ± S.E.M from 5 different animals; * P<0.05.
Supplementary Figure 3. Plasma lipid profile of mice treated with AAV-PCSK9 and high-
fat diet feeding for 3 weeks
Graphs show quantification of plasma triglycerides, cholesterol, HDL and LDL (mg/dL) obtained
from mice treated with AAV9-PCSK9 or Control at the end of study duration from (A) C57 mice
subjected to partial carotid ligation and fed a high-fat diet for 3 weeks, (C) eNOS-/- mice on C57
background subjected to partial carotid ligation and fed a high-fat diet for 3 weeks and (D)
NOX1-/y mice on C57 background subjected to partial carotid ligation and fed a high-fat diet for 3
weeks, respectively. Data shown as means ± S.E.M from 5 different animals; * P<0.05.
Supplementary Figure 4. Atherosclerosis development in additional C57 mice treated
with AAV9-PCSK9 and subjected to partial carotid ligation
Mice were injected with AAV-PCSK9 or AAV-Luc (Control) (1 X 1011 VG/mouse) and fed a high-
fat diet for the entire duration of the study. After 1 week, partial carotid ligation was performed
and animals were sacrificed at 3 weeks post surgery. RCAs and LCAs were dissected out and
atherosclerotic plaques were imaged using dissection microscopy.
Supplementary Figure 5. Atherosclerosis development in additional eNOS-/- mice treated
with AAV9-PCSK9 and subjected to partial carotid ligation
Mice were injected with AAV-PCSK9 or AAV-Luc (Control) (1 X 1011 VG/mouse) and fed a high-
fat diet for the entire duration of the study. After 1 week, partial carotid ligation was performed
and animals were sacrificed at 3 weeks post surgery. RCAs and LCAs were dissected out and
atherosclerotic plaques were imaged using dissection microscopy.
Supplementary Figure 6. Atherosclerosis development in additional NOX1-/y mice treated
with AAV9-PCSK9 and subjected to partial carotid ligation
Mice were injected with AAV-PCSK9 or AAV-Luc (Control) (1 X 1011 VG/mouse) and fed a high-
fat diet for the entire duration of the study. After 1 week, partial carotid ligation was performed
and animals were sacrificed at 3 weeks post surgery. RCAs and LCAs were dissected out and
atherosclerotic plaques were imaged using dissection microscopy.