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.