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Global Translational Medicine GalNAc AGT ASO reduce atherosclerosis

1. Introduction feeding (diet # TD.88137, Envigo). Week -2 represents the
start of subcutaneous injection of either PBS or GalNAc
Angiotensinogen (AGT) is cleaved by renin to generate AGT ASO, while week 0 represents the 1 week of Western
st
angiotensin (Ang) I and des(AngI)AGT [1,2] . Subsequently, diet feeding (Figure 1A). On week -2, the mice were
AngI is cleaved by angiotensin-converting enzyme injected with vehicle or any of the 3 doses of GalNAc AGT
(ACE) to release AngII. AngII, through binding to AngII ASO on days 1, 3, and 5, and then the 1 day of week -1.
st
type 1 (AT1) receptor, contributes to blood pressure (BP) Two weeks after the initiation of subcutaneous injections
regulation and atherosclerosis [3-5] . In previous studies, the (week 0), the mice were fed Western diet for 12 weeks,
pharmacological inhibition of renin, ACE, or AT1 receptor while vehicle or GalNAc AGT ASO was injected once
reduced BP and atherosclerosis in low-density lipoprotein every week. GalNAc AGT ASO was provided by Ionis
(LDL) receptor mice fed a Western diet [3-7] . Whole-body Pharmaceuticals Inc. (Carlsbad, CA, USA).
-/-
reduction of AGT, hepatocyte-specific AGT deficiency
(hepAGT ), and global AGT antisense oligonucleotides We then tested the rapidity of GalNAc AGT ASO
-/-
(ASO) exerted similar effects on BP and atherosclerosis in reduction in plasma AGT concentrations. Male C57BL/6J
LDL receptor mice . mice (~8 weeks old) were injected with GalNAc AGT ASO
[8]
-/-
10 mg/kg once (Table S3), and their plasma samples were
While the AngII-dependent functions of AGT have
been well-documented [1,9] , the function of the des(AngI) collected sequentially for 10 days. Subsequently, GalNAc
AGT ASO and losartan were compared. Eight-week-old
AGT portion of AGT has not been extensively studied. male LDL receptor mice were randomly assigned to three
-/-
Recently, the biological function of des(AngI)AGT has groups (Table S4). The mice in Group 1 (vehicle) were
been investigated by infecting hepAGT mice with an subcutaneously injected with PBS (on days 1 and 3, then
-/-
adeno-associated virus (AAV) encoding des(AngI)AGT. once every week) and implanted a pump to deliver water
The expression of des(AngI)AGT in hepAGT mice
-/-
increased Western diet-induced body weight gain and liver subcutaneously; mice in Group 2 (GalNAc AGT ASO) were
subcutaneously injected with GalNAc AGT ASO (5 mg/kg on
steatosis but had no effects on BP and atherosclerosis . days 1 and 3, then once every week) and implanted a pump
[8]
These data support the notion that the effects of AGT to deliver water subcutaneously; mice in Group 3 (losartan)
deletion on BP and atherosclerosis are AngII-dependent, were subcutaneously injected with PBS (on days 1 and 3, then
whereas des(AngI)AGT has effects on metabolic disorders
in an AngII-independent manner. once every week) and implanted a pump to deliver losartan
(15 mg/kg/day; Cat # 61188-100 mg, Millipore Sigma). The
Although hepatocytes are the major source of plasma mini osmotic pump ALZET Model 2006 (Durect Corp.)
AGT, its synthesis has also been reported at non-hepatic was used to deliver water or losartan subcutaneously. The
sites [10-13] . The degree to which non-hepatic sources of first mini osmotic pumps were replaced by second ones
AGT contribute to the above effects remains unknown. after 6 weeks of infusion. Week -1 represents the start of
Studies have shown that adipocyte- or macrophage- subcutaneous injection, while week 0 represents the first week
derived AGT deficiency has marginal or no effect on of Western diet feeding. On week -1, the mice were injected
BP and atherosclerosis [8,10,11] . To verify the importance with PBS or GalNAc AGT ASO on days 1 and 3, and then
of hepatic AGT from a pharmacological approach, once each week. Mini osmotic pumps were implanted on
we targeted hepatocyte AGT mRNA, making use of day 5 of week -1 to deliver either water or losartan. One week
N-acetylgalactosamine (GalNAc)-conjugated AGT after the initiation of subcutaneous injections (week 0), the
ASO , in LDL receptor mice fed a Western diet for mice were fed Western diet for 12 weeks.
-/-
[14]
12 weeks. A comparison was made versus losartan, a classic
AT1 receptor blocker, to distinguish AngII-dependent and All animal experiments (Table S5 and S6) reported in this
independent effects. article were performed with the approval of the University
of Kentucky Institutional Animal Care and Use Committee
2. Materials and methods (IACUC protocol number 2018-2968 or 2015-2050).
2.1. Animals 2.2. Systolic blood pressure

Male LDL receptor deficient mice (LDL receptor , strain Systolic BP was measured in conscious mice using a non-
-/-
# 002207) were purchased from The Jackson Laboratory invasive tail-cuff system (BP-2000, Visitech Systems)
(Table S1). Eight-week-old male LDL receptor mice following our standard protocol and was calculated
[15]
-/-
(Table S2) were subcutaneously injected with phosphate- based on 20 measurements per mouse per day for 3
buffered saline (PBS; vehicle) or GalNAc AGT ASO (1, consecutive days. The mean systolic BP of each mouse
2.5, or 5 mg/kg) beginning 2 weeks before Western diet from the 3-day measurements was used for data analysis.

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