Page 104 - GTM-2-1

Global Translational Medicine GalNAc AGT ASO reduce atherosclerosis

adventitial tissues were removed, and the intimal surface 2.8. Statistical analysis
was exposed by a longitudinal cut and pinned on a black There were two types of data in this study: non-repeated
rubber surface. Images of en face aortas were taken using measures after termination, and repeated measures
a digital camera (Nikon Digital Sight DS-Ri1), with a ruler during the study. Prism v9 (GraphPad Software Inc.,
for calibration. La Jolla) was used for non-repeated measures, while R
An en face method was used to measure atherosclerotic version 4.2.1 was used for repeated measures. Before
lesions on the intimal surface of the aorta in accord with analyzing non-repeated measures, normality and
the American Heart Association (AHA) statement and homogeneous variance assumptions were tested with
as detailed in our standard protocol [16,17] . Atherosclerotic Shapiro–Wilk and Brown-Forsythe tests, respectively.
lesions were traced manually from the ascending aorta to Since these assumptions were satisfied, all non-repeated
the proximal part of the descending thoracic aorta (1 mm data were analyzed using one-way analysis of variance
distal from the orifice of the left subclavian artery) using (ANOVA) to compare means among three or more
Nikon NIS-Elements software (NIS-Elements AR 5.11.00.) groups, followed by the Sidak post hoc test. For repeated
under a dissecting microscope. measures, mixed-effect models with inverse-variance
weights were used with random intercept and slope for
2.5. Histology time. A piecewise model was fitted to estimate separate
At termination, a piece of each liver sample was fixed slopes for distinct time points (-2 to 0 and 0 to 11 weeks)
in paraformaldehyde (4% wt/vol) overnight and then in plasma AGT concentrations. The mixed-effect models
embedded in paraffin. Five-micron sections were used were run using the lme function in the nlme R package.
for hematoxylin and eosin (H&E) staining. In addition, Data of non-repeated measures were represented as
a piece of liver (fresh frozen) was embedded in optimal individual data points and mean ± standard error of the
cutting temperature compound (OCT; Cat # 14-373-65, mean (SEM). P < 0.05 or Bonferroni-corrected P < 0.05
Fisher Scientific), sectioned using a cryostat (Leica CM was considered statistically significant.
1850, Leica) at 10 µm/section, and stained with Oil Red O 3. Results
to visualize neutral lipid accumulation.
3.1. N-acetylgalactosamine-conjugated antisense
2.6. Quantification of liver steatosis oligonucleotides targeting angiotensinogen
Liver weights were recorded at termination. A small reduced blood pressure, atherosclerosis, and
piece of liver was snap-frozen in liquid nitrogen. Liver Western diet-induced liver steatosis
lipids were extracted, solubilized with Triton X-100, and In the vehicle group, plasma AGT remained unaltered
quantified using enzymatic assays kits for total cholesterol (Figure 1B) in mice fed either normal laboratory diet
(Cat # 23-66-201, Pointe Scientific Cholesterol reagent), (from week -2 to week 0) or Western diet (from week
and triglycerides (Regents 1 and 2: Cat # 994-02891 and 0 through week 12). GalNAc AGT ASO significantly
Cat # 990-0299, Fujifilm Healthcare) . reduced plasma AGT concentrations in mice when
[18]
they were fed normal laboratory diet; Western diet
2.7. RNA isolation and quantitative polymerase did not alter this outcome. All three doses of GalNAc
chain reaction (PCR) AGT ASO yielded the same degree of AGT lowering,

Total RNA was extracted from liver and kidney samples using maximally reducing plasma AGT by ~90%. GalNAc
a commercial kit (Cat # AS1280, Promega) and the automated AGT ASO reduced systolic BP in a dose-dependent
Maxwell RSC 48 Instrument (Promega). To quantify manner versus vehicle (Figure 1C), with the effect
®
mRNA abundance, total RNA was reversely transcribed with at 5 mg/kg being significantly larger than that at
iScript cDNA Synthesis kit (Cat # 170-8891, Bio-Rad), and 1 mg/kg (P < 0.001). Similarly, a dose-dependent
quantitative PCR (qPCR) was performed using the TaqMan reduction in atherosclerotic lesion size was observed
Fast Advances Master Mixes kit (Cat # A44359, Thermo Fisher (Figure 1D and E).
Scientific) on a Bio-Rad CFX96 cycler. TaqMan assay primers: LDL receptor mice fed a Western diet developed liver
-/-
Agt (ID: Mm00599662_m1), Ren1 (ID: Mm02342889_g1), steatosis with increased liver weight and liver cholesterol
Gapdh (ID: Mm99999915_g1), Actb (ID: Mm01205647_g1), and triglyceride content [8,19-22] . The results obtained in this
and Ppia (ID: Mm02342429_g1). Data were analyzed using study (Figure 2A–C) confirmed this outcome. GalNAc
the ∆∆Ct method and normalized with the geometric mean AGT ASO administration reduced liver weight and liver
of the three reference genes: Gapdh, Actb, and Ppia. total cholesterol and triglyceride content (Figures 2A–C)

Volume 2 Issue 1 (2023) 4 https://doi.org/10.36922/gtm.288