GLP-1 receptor agonists alleviate inflammatory responses and endothelial dysfunction in atherosclerosis by activating the Sema3A/NRP1 pathway

Objective This study aimed to investigate the mechanism by which GLP-1 RAs activate the Sema3A/NRP1 signaling pathway to alleviate inflammation and endothelial dysfunction in atherosclerosis (AS). Methods Ten 8-week-old male C57BL/6J mice served as controls (CON) and received intraperitoneal injections of saline every 2 days. Forty ApoE −/− mice were fed a 60 kcal% fat high-fat diet for 12 weeks. From weeks 13 to 24, the ApoE −/− mice were randomly divided into 4 groups ( n = 10): AS model group (AS, saline injections), low/high-dose GLP-1 RAs group (L/H-GLP-1 RAs, 30/60 μg/kg semaglutide injections) and atorvastatin group (ATO, 1.3 mg/kg atorvastatin injections as a positive control). For rescue experiments, endothelial-specific NRP1 knockout ( NRP1 EC-KO ) mice were generated. Thirty NRP1 WT mice were divided into 3 groups ( n = 10): NRP1 WT control group ( NRP1 WT CON), the NRP1 WT AS model group ( NRP1 WT AS) and the NRP1 WT AS model + high dose GLP-1 RAs group ( NRP1 WT + H-GLP-1 RAs). While twenty NRP1 EC-KO mice were divided into NRP1 EC-KO AS and NRP1 EC-KO + H-GLP-1 RAs groups ( n = 10). Mouse body weight was monitored weekly during interventions, and mice were euthanized with 120 mg/kg pentobarbital sodium at the end of experiments. HUVECs were divided into five groups ( n = 3): CON, ox-LDL, L-GLP-1 RAs, H-GLP-1 RAs, ATO. Except for controls, cells were treated with 100 μg/mL ox-LDL for 24 h to establish the model. CCK-8 assays determined low/high semaglutide doses (1/2 μM), while atorvastatin was applied at 10 μM. Rescue experiments included CON, ox-LDL, H-GLP-1 RAs, and NRP1 inhibition + H-GLP-1 RAs groups, with the latter pre-treated with 0.5 μM NRP1 inhibitor EG01377 for 2 h before interventions. Results Compared to controls, AS group mice exhibited significant weight gain from week 15 ( P < 0.001), while H-GLP-1 RAs and ATO groups showed reduced weight from week 21 ( P < 0.05). The AS group had elevated serum TC, LDL-C, IL-6, HDL-C, TNF-α, ET-1, TG levels, and percentage of plaque collagen-positive area ( P < 0.001), alongside decreased NO levels ( P < 0.001), all of which were improved by GLP-1 RAs ( P < 0.05). H&E staining revealed reduced inflammatory cell infiltration in aortic roots of semaglutide- and atorvastatin-treated mice. Aortic tissues from AS group mice showed decreased Sema3A/NRP1 expression and binding ( P < 0.05), increased p-ERK1/2/ERK1/2 and p-NF-κB p65/NF-κB p65 ratios ( P < 0.05), which were reversed by semaglutide, with higher doses showing greater effects. Immunofluorescence confirmed that Sema3A and NRP1 mainly localized in vascular endothelial cells. In ox-LDL-induced HUVECs, GLP-1 RAs improved cell viability, migration capacity, and tubule numbers ( P < 0.05), reduced IL-6 and TNF-α levels ( P < 0.05), upregulated eNOS ( P < 0.05), and downregulated VCAM-1 and ICAM-1 ( P < 0.05). Western blot and co-immunoprecipitation results aligned with in vivo trends. In vitro , EG01377 reversed GLP-1 RAs' protective effects ( P < 0.05). In vivo , GLP-1 RAs' therapeutic efficacy was significantly weakened in NRP1 EC-KO mice ( P < 0.05). Conclusion GLP-1 RAs alleviate inflammation and endothelial dysfunction to reduce the atherosclerosis progression by activating the Sema3A/NRP1 pathway and inhibiting downstream ERK1/2-NF-κB signaling.