Glucagon-like peptide-1 inhibits the progression of abdominal aortic aneurysm in mice:The earlier the better

doi:10.21203/rs.3.rs-2502990/v1

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Glucagon-like peptide-1 inhibits the progression of abdominal aortic aneurysm in mice:The earlier the better

https://doi.org/10.21203/rs.3.rs-2502990/v1

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published 05 May, 2023

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ObjectivesGlucagon‑like peptide‑1 (GLP-1) has a cardiovascular-protective effect in preventing Abdominal Aortic Aneurysm (AAA) formation. However, the administration time to maximize the optimal effect has not been determined. We conducted this study to determine whether the GLP-1 receptor agonist, liraglutide, inhibited AAA progression of mice more efficiently during the earlier stages.

Methods Mice were treated with 300ug/kg/day liraglutide 7, 14 and 28 days after aneurysm preparation. The morphology of abdominal aorta were followed up with 7.0 T magnetic resonance imaging (MRI) during the liraglutide administration. 28 days after administration, the AAA dilatation ratio calculation and histopathological examination were then conducted. Oxidative stress levels were evaluated by the expression of malondialdehyde (MDA). The inflammatory responses were also determined.

Results Treatment with liraglutide decreased AAA formation. Specifically, expansion of the abdominal aorta, elastin lamina degradation and vascular inflammation by leukocyte infiltration decreased. Additionally, MDA expression and matrix metalloproteinase (MMP) activity reduced. Furthermore, the dilatation rate of aortic, MDA expression, leukocyte infiltration, and MMPs activity in the vascular wall decreased significantly in early stage.

Conclusions GLP-1 treatment inhibited AAA progression in mice through its anti-inflammatory and anti-oxidant effects, especially during the earlier stages of AAA formation. Thus, GLP-1 may be a potential pharmacological target for AAA treatment.

Abdominal aortic aneurysm

Glucagon like peptide 1

Pharmacological treatment

Anti-inflammatory and anti-oxidant effects

Abdominal Aortic Aneurysm (AAA) is a potentially fatal condition because of the risk of rupture; a ruptured AAA is the cause of 1%−2% of all deaths worldwide¹. Although still unclear concerning the etiology of AAAs, the current literature suggests that elastin, extracellular matrix (ECM), and vascular smooth muscle cells (VSMCs) degradation play key roles in the pathogenesis of this condition^2,3. Changes in their content and/or quality are likely to play a key role in the progression of AAA⁴. Although reactive oxygen species (ROS) are important in the physiological regulation of vascular signal transduction, animal and human studies have found that excessive ROS expression in aneurysm walls was associated with localized inflammatory responses⁵. AAA formation is an inflammatory process involving infiltration of macrophages and lymphocytes, the release of pro-inflammatory cytokines, and the eventual activation of matrix metalloproteinases (MMPs), which degrade the ECM^6–9. There is currently no effective prophylaxis for AAA except invasive surgical or endovascular therapies, and intervention is costly and associated with morbidity and mortality. Two large randomized controlled trials have indicated that survival was not improved by elective surgery for AAAs < 5.5 cm in diameter, and small AAAs were at risk of rupture^10,11. This highlights the need for further research to facilitate the development of AAAs medical therapies.

Glucagon-like peptide 1 (GLP-1), a peptide mainly produced in the entero-endocrine L-intestinal cells, is a major incretin hormone that stimulates insulin secretion after nutrient ingestion. It plays an important role in lowering levels of blood glucose and lipids. Epidemiological studies have reported that patients treated with anti-diabetic drugs targeting GLP-1 pathways had significantly reduced risks of major cardiovascular complications^12,13. Liraglutide is a type of GLP-1 receptor agonist approved for the management of hyperglycemia in type 2 diabetes; it is also approved at a higher dose for the treatment of obesity¹⁴. Studies have reported mechanisms underlying the anti-atherosclerotic effects of GLP-1 receptor agonist through reduction in MMP-2 levels or inhibition of vascular smooth muscle cell proliferation, involving their anti-inflammatory effects, and their effects on endothelial function resulting in nitric oxide-induced vasodilation and reduced oxidative stress^15,16.

The GLP-1 receptor agonist, liraglutide, a novel anti-diabetic drug, has been reported to exert a cardiovascular-protective effect on preventing AAA formation^17–19. However, the administration time to maximize the optimal protective effect for AAA has not been determined. In the present study, we tested the hypothesis that the GLP-1 receptor agonist, liraglutide, prevents aneurysm progression more efficiently during the earlier stages because of its anti-inflammatory and anti-oxidant effects, in an experimental mouse AAA model.

AAA animal model preparation

Establishment of an experimental AAA model in mice

Fifty CS7BL/6 wild-type male mice, 8 − 10 weeks of age, were used in the present study. All animal procedures were approved by the Animal Experimentation Ethics Committee of Beijing Anzhen Hospital and conformed to the NIH Guide for the Care and Use of Laboratory Animals. All mice were kept in the same room in a light-controlled environment with a 12:12 h light-dark cycle and with free access to standard mouse chow and water. The mice were divided into the sham-operated group (Group Sham), control group (Group C), and three treated groups, including the early group (Group E), middle group (Group M), and late group (Group L). Local elastin damage in the infrarenal abdominal aortic region in mice of the experimental groups was induced by perivascular application of calcium chloride (CaCl₂) and elastase incubation²⁰. Briefly, after mice were anesthetized, the infrarenal abdominal aorta was exposed and treated periadventitially by placing a piece of CaCl₂ (0.50 mol/L) and elastase (1 U/µL)-soaked sterile cotton gauze (0.5 cm × 0.5 cm) on the aorta for 20 min. The treated area was flushed with warm saline, and the abdominal incision was closed with sutures. The abdominal incision was sterilized by iodine cotton balls. In all cases for imaging and therapeutic studies, mice in the sham-operated group were surgically treated with saline instead of CaCl₂ and elastase, following similar procedures described in a previous report²⁰.

GLP1 RA (liraglutide) administration

Mice were infused subcutaneously with liraglutide (300 µg/kg/day; Sigma-Aldrich, St. Louis, MO, USA) or normal saline (NS) using an insulin needle. Liraglutide-treated groups were administered liraglutide at a dose of 300 µg/kg/day subcutaneously on days 7, 14, and 28 after AAA preparation, and the Group Sham and Group C were administered an equal dose (300 µg/kg/day) of NS as vehicle, 7 days after the sham operation or AAA preparation (Fig. 1). The administration of liraglutide or NS was 28 days in all groups. At the end of the study, all mice were euthanized by inhaling 3%−5% isoflurane.

Magnetic resonance imaging (MRI)

MRI examinations were conducted using a NOVA 7.0 T preclinical horizontal MRI system (Time Medical, Burlingame, CA, USA). Pretreatment MRI acquisition was performed 1 day before the AAA preparation. Post-treatment MRI acquisition was performed again at 28 days after administration. Animals underwent induction using 3% isoflurane, inserted in a 30 mm diameter radio frequency volume resonator. During MRI acquisitions, 1 − 1.5% isoflurane in a 300 mL/min air flow was administered through an adapted nose cone, assuring stable sleep under physiological conditions. An angio FLASH sequence (TE = 2 ms, TR = 120 ms, flip angle 90°, FOV 30 mm, matrix 256 × 256, two averages) was used to visualize the aorta in two longitudinal planes for perpendicular slice positioning of the vessel wall imaging sequence. Multi-slice FLASF imaging was then used to acquire positive-blood-contrast images from the same slices (TR = 60 ms, TE-2.6 = ms, flip angle = 30º, FOV = 20 mm, matrix size = 256 × 256 µm², slice thickness = 0.5 mm, interslice distance = 1 mm), with the total duration of the protocol being 30 min.

Macroscopic assessments

The diameter of the aneurysm at the infrarenal proximal neck was defined as the maximal aneurysm diameter, and was measured in post-treatment MR images. The diameter of the referenced aorta at the same location of the aneurysm, defined as the normal aortic diameter, was measured in pretreatment MR images. All MR images were analyzed using RadiAnt DICOM Viewer software. Diameter measurements were recorded at short-axis views, and the overall morphology of the abdominal aorta was obtained by multiplanar reconstruction. The dilation ratio (%) was calculated according to the following formula: dilation ratio (%) = (maximal aneurysm diameter/normal aortic diameter) × 100%.

Biochemical measurements

All mice fasted overnight, and the blood glucose levels were measured with a blood sugar measuring instrument (YUWELL, Jiangsu, China).

ELISA assay

Blood samples were collected from the ophthalmic vein and stored at 4℃ overnight before centrifugation for 15 min at 1,000 × g. Levels of IL-1β, TNF-α, TGF-β, and Arg-1 in plasma were measured with mouse ELISA kits (all from Animalunion, China) according to the manufacturer’s instructions, respectively.

Histological analysis

At 28 days after administration, the middle segments of abdominal aortas were excised from each mouse, and were fixed through trans-cardiac perfusion with saline, followed by 4% paraformaldehyde (PFA). Then, the abdominal aorta samples were immersed into 4% PFA and fixed for 24 h. Later, the aortic samples were embedded into paraffin blocks and cut into 5 µm thickness sections. Hematoxylin & eosin (H&E) staining and EVG staining (Vector Laboratories, Newark, NJ, USA) were performed following standard protocols²¹. Images were acquired using a IX73 imaging microscope (Olympus, Tokyo, Japan) and assessed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Areas of elastin in a cross-sectional aortic wall were measured using ImageJ software. Elastin content was calculated by dividing the elastin positive area by the cross-sectional media area of the aortic wall, which was measured using the EVG-stained sections to highlight internal elastic lamina and external elastic lamina, and expressed as a percentage. Microscopic digital images of five random fields on each section were scanned in a 680 × 512 µm frame.

Quantitative RT-PCR analysis

Total RNA was isolated from aneurysm samples using a RNeasy fibrous tissue mini-kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The RNA was reverse transcribed to complimentary DNA with Prime Script™ RT Master Mix (Takara, Shiga, Japan). Real-time PCR was performed using an Applied Biosystems 7500 (Life Technologies, Carlsbad, CA, USA). Relative gene expressions of IL-6, TNF-α, Arg-1, and Ym-1 were normalized to GAPDH using the ΔΔCt method. The primer sequences are listed in Supporting Information Table S1.

Western blot analysis

Abdominal aortas were homogenized and lysed. The concentration of soluble proteins in cell lysates was determined using the BCA assay. Total protein (30 µg) of each sample was separated by SDS-PAGE, transferred to 0.2 µm PVDF membranes (Millipore, Burlington, MA, USA) and probed with primary antibodies against α-SMA (YM3365; Immunoway Biotechnology, Plano, TX, USA), MMP2 (SC-13595; Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (sc-13520; Santa Cruz Biotechnology), and GAPDH ( ET1601-4; HUABIO, Woburn, MA, USA). The ECL system was used for detection. GAPDH was used as an internal control, and the results are expressed as the mean ± standard deviation.

Statistical analysis

Continuous variables are expressed as the mean ± standard deviation. Comparisons between two groups were made using Student’s t-test, while comparisons among multiple groups were performed using one-way analysis of variance. If one-way analysis of variance used post hoc analysis, the Games-Howell test was used to calculate significance among the groups. A probability value of P < 0.05 was considered significant. Data were analyzed using SPSS 23 software (SPSS, Chicago, IL, USA).

The success of surgical procedures was 98% (49/50); one case of technical failure occurred in Group M during the surgical procedures. The remaining mice in each group were used for biochemical determinations of blood glucose, histological examination (H&E and EVG staining), oxidative stress response expression, and measurement of inflammation levels using RT-PCR analysis.

Blood glucose assessment

Because liraglutide is one of the drugs used to treat diabetes, we examined blood glucose levels of mice in each group before and after treatment. As shown in Table 1, treatment with liraglutide did not cause hypoglycemia in mice, and there was no significant difference in blood glucose levels between each group.

Table 1

Effects of liraglutide on blood glucose and inflammation cytokines levels in plasma
	Sham	Group C	Group E	Group M	Group L
Glu (mmol/L)	12.1 ± 1.4	11.3 ± 1.0	12.9 ± 2.5	12.2 ± 0.7	11.7 ± 1.4
IL-1(pg/ml)	2225.8 ± 683.7	1691.7 ± 302.8	1951.1 ± 199.5	1654.7 ± 190.1	1932.1 ± 347.3
TNF-α (pg/ml)	7089.5 ± 3198.6	5758.7 ± 857.6	835.1 ± 373.5	723.9 ± 361.9	897.2 ± 448.6
TGF-β (pg/ml)	2204.5 ± 758.3	1119.9 ± 373.1*	1906.5 ± 292.1**	1768.3 ± 188.1	1318.3 ± 594.9
ARG-1 (pg/ml)	11992 ± 9516.6	6945.3 ± 2050.9	862.7 ± 385.8	1348.7 ± 674.4	2768.8 ± 1384.4

Glu: glucose; IL-1: interleukin-1; TNF-α: tumor necrosis factor-α; TGF-β: transforming growth factor-β; Arg-1: arginase-1. Data are expressed by mean ± standard deviation.

^*P < 0.05 Group C vs. Sham; ^**P < 0.05 Group E vs. C.

Macroscopic assessments

Macroscopic findings on day 28 after administration are shown in Fig. 2a,b. In our study, the incidences of AAAs (excluding AAA-associated deaths) in each group were the following: 0% (0/10) in Group Sham, 50% (5/10) in Group C, 0% (0/10) in Group E, 22% (2/9) in Group M, and 30% (3/10) in Group L (Fig. 2c). AAA dilatation ratios were the following: 103.6 ± 2.8% in Group Sham, 141.3 ± 14.3% in Group C, 121.4 ± 8.4% in Group E, 132.2 ± 13.0% in Group M, and 137.6 ± 14.4% in Group L. Dilatation ratios of abdominal aortas in Group C were significantly higher than in Group Sham (P < 0.05). Aortic dilatation ratios in Group E were lower than that in Group C (P < 0.05). Aortic dilatation ratios among liraglutide-treated groups were the following: dilatation ratios in Group E and M were lower than that in Group L, and there was a significant difference between Group E and L (P < 0.01 for Group E vs. Group L) (Fig. 2d).

Microscopic assessments

To investigate the effect of liraglutide on alterations of elastin and collagen, H&E and EVG staining were performed. The H&E-stained sections showed that inflammation in the aortic wall increased significantly in Group C, and the inflammation decreased with liraglutide treatment, especially in Groups E and M (Fig. 3a). EVG-stained sections showed disruption of elastin fibers in the aortic wall in Group C, but these disruptions were much less following liraglutide treatment (Fig. 3b). Elastin content was 16.3 ± 0.1% in Group Sham, 5.3 ± 1.1% in Group C, 11.4 ± 0.9% in Group E, 7.9 ± 2.5% in Group M, and 6.7 ± 0.4% in Group L. Elastin content decreased significantly in Group C compared with Group Sham (P<0.05). Elastin content increased gradually from Group L to Group E, and the content in Group C was significantly higher than that in Group C (P < 0.05) (Fig. 3c).

Effect of GLP1 RA on inflammation

Inflammatory factors in aortic walls

The mRNA expression of Ym-1 was higher in the liraglutide-treated groups, but there was no significant difference of Arg-1 among the groups (Fig. 4a,b). The expression of Ym-1 was 2.9 ± 0.1 in Group E, 1.7 ± 0.7 in Group M, 1.9 ± 0.1 in Group L, and 1.9 ± 0.1 in Group C (P < 0.05 for Group E vs. Group C) (Fig. 4a). The mRNA expressions of TNF-α and IL-6 were lower in the liraglutide-treated groups (Fig. 4c,d). The expression of TNF-α was 1.5 ± 0.3 in Group E, 1.6 ± 0.3 in Group M, 2.5 ± 0.4 in Group L, and 2.6 ± 0.5 in Group C (P < 0.05 for Group E and M vs. Group C). Among the liraglutide-treated groups, expressions of TNF-α in Groups E and M were significantly lower than that in Group L (P < 0.01) (Fig. 4c). The expression of IL-6 was 1.4 ± 0.1 in Group E, 1.5 ± 0.4 in Group M, 1.7 ± 0.3 in Group L, and 2.2 ± 0.4 in Group C (P < 0.05 for Group E vs. Group C), and there was no significant difference of IL-6 among the liraglutide-treated groups (Fig. 4d).

Inflammatory factors in the plasma

ELISAs were performed to monitor the plasma levels of inflammation promoters (IL-1 and TNF-α) and protectors (Arg-1 and TGF-β). Only the TGF-β concentration showed significant differences among groups. The plasma TGF-β concentration decreased in Group C, when compared to Group Sham, and treatment with liraglutide increased the plasma TGF-β concentration in Group E (TGF-β concentration: Group Sham: 2,204.5 ± 758.3 pg/mL, Group C: 1,119.9 ± 373.1 pg/mL, Group E: 1906.5 ± 292.1 pg/mL, Group M: 1,768.3 ± 188.1 pg/mL, Group L: 1,318.3 ± 594.9 pg/mL; P < 0.05 for Group C vs. Sham, P < 0.05 for Group E vs. C), but there was no significant difference among the liraglutide-treated groups (Table 1).

Effect of activation of α-SMA and MMPs

We examined the effects of liraglutide on MMPs in homogenates of aneurysmal tissues. The expressions of MMP-2 and MMP-9 were significantly lower in the liraglutide-treated groups than in Group C (Fig. 5a). The relative expressions of MMP-2 and MMP-9 in Groups E and M were significantly lower than that in Group C (MMP-2: 1.6 ± 0.2 in Group E, 1.5 ± 0.3 in Group M, 1.5 ± 0.3 in Group L, 2.6 ± 0.1 in Group C, P < 0.01 for Group E vs. Group C, P < 0.01 for Group M vs. Group C; MMP-9: 1.1 ± 0.2 in Group E, 1.6 ± 0.1 in Group M, 1.8 ± 0.2 in Group L, 2.1 ± 0.2 in Group C, P < 0.01 for Group E vs. Group C, P < 0.05 for Group M vs. Group C) (Fig. 5b,c). Additionally, expression of MMP-2 in Group E was significantly lower than that of Group L (P < 0.01), and expressions of MMP-9 in Group E was significantly lower than in other liraglutide-treated groups (P < 0.01). Considering the role of α-SMA in formation and stability of VSMCs, we examined the effect of liraglutide on α-SMA activation in homogenates of aneurysmal tissues²². The relative expression of α-SMA in Group E was significantly lower than that in Group C (1.1 ± 0 in Group E, 2.3 ± 0.5 in Group M, 1.7 ± 0.5 in Group L, 2.2 ± 0.4 in Group C; Group E vs. Group C: P < 0.05), and there was no significant difference of α-SMA among liraglutide-treated groups (Fig. 5d,e).

MDA expression in AAA walls

MDA is a lipid peroxide of ROS, so it may affect the mitochondrial respiratory chain and the activity of key enzymes in mitochondria associated with H₂O₂-induced ROS generation²³. To evaluate the oxidative stress response expression in aneurysm walls on day 28 after administration, ELISAs were performed to determine the MDA content of the aorta from each group. As shown in Fig. 6, MDA content was significantly elevated in Group C, when compared with the Group Sham on day 28 after NS injection (P < 0.05), the MDA content was significantly lower in Group E than that in Group C (P < 0.05), and the content in Group E was significantly lower than those in other liraglutide-treated groups (P < 0.05 for Group E vs. Group M, P < 0.01 for Group E vs. Group M) (Group Sham: 0.43 ± 0.18 nmol/mL, Group C: 5.3 ± 1.1 nmol/mL, Group E: 2.1 ± 0.7 nmol/mL, Group M: 3.7 ± 0.7 nmol/mL, and Group L: 4.9 ± 0.9 nmol/mL).

The results showed that treatment with liraglutide exerted important protective effects against AAA formation in a mice model. Treatment with liraglutide reduced the aortic dilatation ratio, and the earlier liraglutide-treated group showed a better effect. The reduction in aortic dilatation ratio associated from liraglutide treatment was, at least in part, due to a decrease in the extent of leukocyte infiltration, inflammatory reaction, and oxidative stress response expression. The results therefore suggested that liraglutide is an effective pharmacological agent for AAA treatment in clinical practice, especially during the earlier stages of AAA formation.

Previous studies reported that GLP-1 elevation inhibited AAA development in mice through its anti-oxidant and anti-inflammatory effects^17–19. Oxidative stress can cause the excessive generation of ROS and has various adverse effects on muscular mitochondria^24,25, which plays a key role in the pathogenesis of AAA formation and development²⁶. MDA, one of the toxic substances produced by increased ROS, is closely related to leukemia, cardiovascular and cerebrovascular diseases, and tumors³¹. The maintenance-integrity structure of elastin lamina in tissues, including arteries, were disrupted by infiltrated macrophages, which secreted ECM-degradation factors, and the inflammatory responses and expressions of MMP-2 and MMP-9 were also increased in aneurysm walls^32–34. In this study, liraglutide suppressed MDA expression, reflecting the decrease of oxidative stress, but also the recruitment of leukocytes and the secretion of inflammatory cytokines and MMP-2, as well as MMP-9 protein expression, which prevented destruction of elastin and the ECM when using the current AAA model. The changes of VSMC phenotype were characterized by the expressions of α-SMA and other contractile proteins. The expression of α-SMA decreased when VSMCs shifted from a contractile to a proliferative state³⁵. In the present study, α-SMA in the aortic wall was effectively decreased by liraglutide treatment, which prevented degradation of VSMCs. These findings confirmed that the GLP-1 receptor agonist, liraglutide, ameliorated aortic wall degeneration by reducing oxidative stress and inflammation, resulting in the prevention of aortic dilatation.

The Society for Vascular Surgery practice suggests that surveillance imaging at 10, 3, 1, or 0.5-year intervals for patients with AAAs of 2.5 − 2.9 cm, 3.0 − 3.9 cm, 4.0 − 4.9 cm, and 5.0 − 5.4 cm in diameter, and recommends elective repair for patients with an AAA diameter ≥ 5.5 cm³⁶. There is no mention of drug therapy for small AAAs, especially for the timing of drug treatments. The classification of observation groups in previous studies^17–19 was based on doses of GLP-1 receptor agonists, with protective effects positively correlated with dose. Therefore, the appropriate timing of drug administration needs further study, and is very relevant to clinical practice. In the present study, early administration inhibited AAA expansion more effectively, when compared with late administration. MMP-2 expression in the early liraglutide-treated group was significantly reduced, when compared with the late liraglutide-treated group, and MMP-9 and MDA expressions in early liraglutide-treated groups were significantly reduced, when compared with the middle and late liraglutide-treated groups. Pro-inflammatory cytokine (TNF-α) expression in the early liraglutide-treated group significantly increased, when compared with the late liraglutide-treated group, and anti-inflammatory cytokine (Ym-1) expression in the early liraglutide-treated group significantly decreased, when compared with middle and late liraglutide-treated groups. These differences were not significant between the middle and late liraglutide-treated groups. Therefore, we speculate that liraglutide used during the early stages of abdominal aortic aneurysm formation is likely to play a better protective effect. This is the first time that the administration timing of liraglutide has been investigated, and compensates for the lack of clear medical treatment strategies for early small AAA. We also plan to adjust the dose of liraglutide, based on the size of AAA in subsequent studies, to obtain the optimal drug strategy for AAA.

In previous studies^17–19, acquisition of the aortic diameter was from autopsy of mice after euthanasia, and maximal aneurysm and normal aortic diameters were obtained from different parts of the aorta after AAA formed. In the present study, the introduction of MRI enabled us to determine diameter changes noninvasively, before and after the formation of AAA in the same location. This is the first time MRI has been used to monitor the progression of AAA, which makes our data more comparable and convincing.

Except for the walls of vessels, the plasma inflammatory cytokines were measured in the present study, which suggested that liraglutide may increase the content of TGF-β in plasma. TGF-β is a guardian of vascular integrity and immune homeostasis; TGF-β blockade enhances leukocyte infiltration, both in the aortic wall and the intraluminal thrombus, and aggravates extracellular matrix degradation³⁷. Other inflammatory cytokine levels in plasma were also measured between groups, but there was no significant difference. The potential of using circulating cytokine levels as biomarkers of coronary artery disease has been discussed³⁸. It will be interesting to examine whether plasma inflammatory cytokine activity may be useful in promoting features of human-like AAA in other experimental models.

This study had some limitations. The experiments were performed using a mice AAA model. Because this AAA model in mice was prepared by treatment with elastase and calcium chloride, it does not completely mimic human AAA because it lacks several prominent features of human lesions, such as atherosclerosis and intraluminal thrombosis. The GLP-1 receptor agonist and DPP-4 inhibitor have certain side effects, such as headache and dizziness³⁹, but no side effects were noted using 300 µg/kg/day in this study with mice, or in a previous report¹⁹.

In conclusion, the present study showed that the GLP-1 receptor agonist, liraglutide, attenuated aneurysm progression in a mouse AAA model through its anti-inflammatory and anti-oxidant effects, especially during the earlier stages of AAA formation. These findings suggested that GLP-1 could be an effective pharmacological target for the treatment of AAA.

AAA

Abdominal Aortic Aneurysm

α-SMA

alpha-smooth muscle actin

blood glucose

DDP-4

dipeptidyl peptidase-4

ECM

extracellular matrix

GLP-1

Glucagonlike peptide1

Group E

early group

Group M

middle group

Group L

late group

H&E

Hematoxylin Eosin

MRI

magnetic resonance imaging

MDA

malondialdehyde

MMPs

matrix metalloproteinase

MPR

multiplanar reconstruction

normal saline

PFA

paraformaldehyde

ROS

reactive oxygen species

VSMCs

vascular smooth muscle cells.

Ethics approval and consent to participate

All animal procedure were approved by the Animal Experimentation Ethics Committee of Beijing Anzhen Hospital and conformed to the NIH Guide for the Care and Use of Laboratory Animals.

Consent for publication

All authors have read this manuscript and would like to have it considered a research exclusively for publication in Cardiovascular Diabetology.

Availability of data and materials

All data generated or analysed during this study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by National Natural Science Foundation of China (82071875), Beijing Natural Science Foundation (7212025) and Natural Science Foundation of Sichuan Privince (2022 NSFSC1364).

Authors’ contributions

X.H Zhao helped conceive of the study, and participated in its design, analysis, interpretation. Q.H Wu and Pengfei Ye participated in the design and coordination of the study. X.H Ma and Y.K Guo participated in the design and coordination of the study and helped carry out the cell biology and histological studies. Z Cheng helped conceive of the study, L Zhao, C Zhang, and H.B Zhang participated in its design, analysis, interpretation and helped draft the manuscript.. All authors read and approved the final manuscript.

Acknowledgements

We thank Y Han for financial and psychological assistance, Y Ding for for providing inspiration for the title, W.H WU and J.Z Pu for providing medical writing assistance for this manuscript.

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Glucagon-like peptide-1 inhibits the progression of abdominal aortic aneurysm in mice:The earlier the better

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Methods

AAA animal model preparation

Establishment of an experimental AAA model in mice

GLP1 RA (liraglutide) administration

Magnetic resonance imaging (MRI)

Macroscopic assessments

Biochemical measurements

ELISA assay

Histological analysis

Quantitative RT-PCR analysis

Western blot analysis

Statistical analysis

Results

Blood glucose assessment

Macroscopic assessments

Microscopic assessments

Effect of GLP1 RA on inflammation

Inflammatory factors in aortic walls

Inflammatory factors in the plasma

Effect of activation of α-SMA and MMPs

MDA expression in AAA walls

Discussion

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1