Liraglutide Attenuates Endothelial Dysfunction by Inhibiting Hyperglycemia-Induced Endothelial Cell Senescence Through the LARP7/SIRT1 Pathway

HUVECs, procured from Shanghai FuHeng (FH1122), were cultured in an ECM-specific medium (FH-HUVEC, FuHeng, Shanghai, China). The cells were passaged in a controlled environment at 37°C with 5% CO₂, ensuring that HUVECs used for experiments were within 25 passages. Cells were divided into three groups and cultured for 72 hours (the NG group (exposed to 5.5 mM normal glucose), the HG group (exposed to 30 mM high glucose), and the HG+LIR group (30 mM high glucose + 500 μM LIR. LARP7 knockdown and overexpression vectors acquired from GenePharma (Shanghai, China) with their RNA sequences are detailed in Table S1 in Supplementary Materials. These vectors were transfected using Lipofectamine™ 3000 (L3000150, Thermo, Waltham, USA) at 50 nM for 4 h at 37°C. Cells were harvested 72 h post-transfection for subsequent RNA and protein analysis.

HUVECs, at a density of 2 × 10³ cells per well, were seeded in 96-well plates. Cell viability was determined 3 h post CCK-8 solution treatment (CA1210, Solarbio, Beijing, China), adhering to the manufacturer's guidelines. Absorbance readings at 450 nm were obtained using a microplate reader, and the optical density (OD) values of the normal glucose group (NG group) served as the reference for percentage calculations.

Following the manufacturer's protocol, cell senescence was ascertained using an SA-β-gal staining kit (K146501, Thermo). Briefly, 5 × 10⁴ cells were incubated per well in a 6-well plate for 72 h. Post-culture medium removal, cells were washed once with 1× phosphate buffered saline (PBS) and fixed with 1 mL of β-Gal Fixative Solution A for 15 min at ambient temperature. The fixed cells were washed thrice with 1 × PBS, each lasting 3 min. The staining solution was formulated in a 5:1:1:93 ratio using components B, C, D, and E. After removing the β-Gal wash solution, 1 mL of the prepared staining solution was added to each well and incubated overnight at 37°C. Senescent cells were identified under an optical microscope (CKX41, Olympus, Tokyo, Japan) by blue cytoplasmic staining.

Total protein from HUVECs was extracted utilizing the Whole Protein Extraction Kit (90079, Thermo). Proteins were separated on a Bis-Tris gel system (NW00107BOX, Thermo) and transferred to a polyvinylidene fluoride (PVDF) membrane (PB0117, Thermo). The membranes were blocked with 5% non-fat milk for 2 h at 37°C and incubated sequentially with primary antibodies overnight at 4°C and secondary antibodies for 2 h at 37°C. Immunoblot images were captured using the iBright™ CL750 Imaging System (A44116, Thermo, USA) and analyzed quantitatively with ImageJ 6.0 (Bethesda, USA). Employed antibodies included: Anti-LARP7 (17067-1-AP, Proteintech, Rosemont, USA), 1:1000), Anti-p53 (60283-2-Ig, Proteintech, 1:5000), Anti-p21 (10355-1-AP, Proteintech, 1:1000), Anti-p16 (10883-1-AP, Proteintech, 1:1000), Anti-SIRT1 (13161-1-AP, Proteintech, 1:1000), Anti-EP300 (20695-1-AP, Proteintech, 1:500), Anti-p-AMPK (Phosphorylated AMPK) (Thr172) (D4D6D, CST, Danvers, USA, 1:1000), Anti-AMPK (10929-2-AP, Proteintech, 1:2000), Anti-lgG (PR30012, Proteintech).

Total RNA was extracted from HUVEC cells using the RNA Extraction Kit (R1200, Solarbio), and its concentration was quantified with a BioPhotometer Plus spectrophotometer (Eppendorf Ltd., Hamburg, Germany). Complementary DNA (cDNA) synthesis was performed using the Reverse Transcription Kit (T2240, Solarbio). The messenger ribonucleic acid (mRNA) expression levels were assessed utilizing the One-Step SYBR Green RT-qPCR Kit (11736051, Thermo). Primer sequences are detailed in Table S2 in the Supplementary Materials. All results were normalized against the glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Intracellular ROS levels were detected using DCFH-DA fluorescence staining per the manufacturer's protocol (CA1410, Solarbio). Cells were cultured in dulbecco's modified eagle's medium (DMEM) supplemented with 1% fetal bovine serum (FBS) for 24 h, incubated with DCFH-DA at 37°C for 60 min, and visualized under a fluorescence microscope (SZ51, Olympus).

HUVEC cells were lysed and processed for malondialdehyde (MDA) content analysis as described in the MDA Assay Kit (EY-01H360, Yansheng, China). The supernatant (1 mL) was combined with MDA detection reagents (Dilution Work Solution A, Storage Work Solution B, and Antioxidant Work Solution C at a 10:3:1 ratio), thoroughly dissolved at 70°C, heated in a boiling water bath for 20 min, and then cooled to room temperature. After centrifugation (1000 rpm, 10 min), the absorbance of the supernatant was measured at 532 nm.

As instructed by the manufacturer (BL902A, Bioshrap, Hefei, China), 1 × 10⁶ cells were cultured in a 6-well plate for 72 h, followed by a cold bath (4°C, 10 min). After adding 200 μL of superoxide dismutase (SOD) sample preparation solution and centrifuging (4°C, 12,000 rpm), the supernatant was collected for protein quantification using the BCA Protein Assay Kit (P0011, Beyotime, Shanghai, China). A mixture of 160 μL WST-1/enzyme working solution (ratio of 150:8:1 of SOD Assay Buffer, WST, and enzyme solution) and 40 μL reaction initiation solution (ratio of 39:1 of SOD Buffer and reaction initiation solution) was prepared. The assay involved the sequential addition of the test samples, SOD Assay Buffer, WST-1/enzyme working solution, and reaction initiation solution, incubated at 37°C for 30 min. Absorbance at 450 nm was measured using a spectrophotometer, and SOD activity was calculated according to the formula provided in the kit.

Nitric oxide (NO) levels were determined following the protocol of the NO Assay Kit (AAT-B16350, Qiyuan, Shanghai, China). As described earlier, HUVEC cell supernatants were incubated with Work Solutions A–B (37°C, 60 min), followed by Work Solutions C–D (37°C, 40 min) and centrifugation (1000 rpm, 10 min). After adding 1 mL of color-developing reagent and incubating (37°C, 10 min), absorbance at 550 nm was measured using a spectrophotometer. The NO content was calculated based on the established standard curve.

Matrigel (BD Biosciences, Franklin Lakes, USA) was dissolved at 4°C for 24 h, diluted, and mixed with pre-cooled serum-free medium in a 1:3 ratio. After allowing Matrigel to solidify for 2 h at 37°C, HUVECs were seeded to overlay the Matrigel. Post 6-h incubation, tube formation by the cells was observed and quantified in terms of number and length using a CKX41 microscope (Olympus).

The migratory capacity of HUVECs was assessed using a Transwell assay, as described in Abdel Ghafar et al. (2020). Briefly, cells were placed in the upper chamber of the Transwell (in DMEM without FBS), while the lower chamber contained 10% FBS. After 72 h of incubation, non-migrating cells in the upper chamber were carefully removed. Cells that migrated were stained with crystal violet and counted under a microscope.

Statistical analyses and graph plotting were performed using GraphPad, San Diego, USA, Prism 9 software (GraphPad Software Inc.). All experiments were replicated at least three times, with results expressed as mean ± standard deviation. Two-sample comparisons were conducted using t-tests, while analysis of variance (ANOVA) was employed for analyses involving three or more groups. A p-value of <0.05 was considered statistically significant (^*p < 0.05, ^**p < 0.01, ^#p < 0.05, ^##p < 0.01).

To ascertain the induction of cellular senescence by HG and the counteracting effects of LIR, HUVECs were treated with a high concentration of glucose (30 mM) and LIR. The CCK-8 assay results demonstrated a marked decline in cell viability under HG conditions, which was effectively reversed by LIR (Figure 1a). Following 25 generations of culture under high-glucose conditions and subsequent SA-β-gal staining, pronounced senescence features were evident in HUVECs, whereas LIR treatment slowed cellular aging (Figure 1b). To elucidate the role of LARP7 in HG-induced HUVEC senescence, we examined the protein and mRNA expression levels of LARP7 and aging-related genes, including cellular tumor antigen p53 (p53), cyclin-dependent kinase inhibitor 1A (p21), and cyclin-dependent kinase inhibitor 2A (p16), using Western blot and qPCR. LARP7 expression decreased in senescent HUVECs, showing a negative correlation with age-related gene expression (Figure 1c). Furthermore, quantitative polymerase chain reaction (qPCR) analysis of senescence-associated secretory phenotype (SASP) genes, including interleukin (IL)-1α, IL-1β, C-X-C motif chemokine ligand 8 (IL-8), C-C motif chemokine ligand 2 (MCP-1), and intercellular cell adhesion molecule 1 (ICAM-1), indicated elevated secretion of SASP proteins in HUVECs under HG conditions. LIR treatment effectively reduced SASP secretion (Figure 1d).

Fig 1.

LIR augments LARP7 expression and inhibits HG-induced HUVEC senescence. Cells were categorized into four groups: NG group (5.5 mM glucose), HG group (30 mM glucose), and HG + LIR group (30 mM glucose, 500 μM LIR). (A) CCK-8 assay to assess cell viability. (B) SA-β-gal staining to evaluate cellular senescence. (C) Western blot and qPCR analysis for changes in LARP7, p53, and p21 expression levels. (D) qPCR to quantify SASP gene expression levels of IL-1α, IL-1β, IL-8, MCP-1, and ICAM-1. n = 3 for each group. Scale bar: 50 μM. ^**p < 0.01 vs. NG, ^#p < 0.05, ^##p < 0.01 vs. HG. HG, high glucose; HUVECs, human umbilical vein endothelial cells; LIR, Liraglutide; MCP-1, motif chemokine ligand 2; SASP, senescence-associated secretory phenotype.

To elucidate LARP7's role in HG-induced HUVEC senescence, we developed a LARP7 knockdown HUVEC model. LARP7 mRNA and protein levels in this model were confirmed to be substantially reduced through qPCR (Figure 2a) and Western blot (Figure 2b) analyses. Oxidative stress is commonly associated with cellular aging. Studies have highlighted oxidative stress and DNA damage accumulation as critical contributors to cellular senescence (Lee et al. 2024). Therefore, HG-induced HUVECs were divided into four treatment groups: HG induction alone, LIR treatment, and LIR treatment with concurrent LARP7 knockdown or overexpression. ROS levels in these groups were measured using the DCFH-DA fluorescent probe. Results indicated that HG treatment significantly increased ROS levels in cells, effectively mitigated by LIR. Intriguingly, the therapeutic effect of LIR was almost nullified following LARP7 knockdown, while the overexpression of LARP7 had the opposite effect (Figure 2c). This suggests that LIR's inhibition of HG-induced oxidative stress in HUVECs depends on LARP7. Similarly, assay kit measurements of oxidative stress-related factors, including MDA, SOD, catalase, and NO, revealed that LIR effectively controlled the onset of oxidative stress in HUVECs under HG conditions. However, this effect was significantly compromised after LARP7 knockdown, but LARP7 overexpression amplified the therapeutic effects of LIR (Figure 2d). This suggests that LARP7's presence is essential for LIR to reduce oxidative stress in HG-induced HUVECs effectively, and its absence significantly impairs LIR's therapeutic efficacy, with enhanced LARP7 expression boosting LIR's therapeutic effects.

Fig 2.

LIR alleviates oxidative stress in senescent ECs via LARP7. Two distinct LARP7 knockdown vectors were constructed to mitigate off-target responses, with knockdown efficiency subsequently validated. (A,B) qPCR and Western blot for assessing LARP7 mRNA and protein expression post-transfection. The impact of LARP7 knockdown on HUVECs was examined in four groups: HG, HG + LIR, HG + LIR + siLARP7, and HG + LIR + oeLARP7. (C) DCFH-DA fluorescent probe for measuring cellular ROS content. (D) Assay kits to determine oxidative stress-related factors, specifically MDA/NO content and SOD and CAT activities. n = 3 for each group. Scale bar: 200 μM. ^*p < 0.05, ^**p < 0.01 vs. Control or HG, ^#p < 0.05, ^##p < 0.01 vs. HG + LIR. CAT, catalase; ECs, endothelial cells; HG, high glucose; HUVECs, human umbilical vein endothelial cells; LIR, Liraglutide; MDA, malondialdehyde; NO, nitric oxide; ROS, reactive oxygen species; SOD, superoxide dismutase.

To delve deeper into the specific mechanisms by which LIR functions via LARP7, we employed Western blot analysis to assess the downstream genes of LARP7, namely, SIRT1 and age-related signaling molecule EP300 (1A Binding Protein P300) and phosphorylated AMP-activated protein kinase (p-AMPK; Figure 3a) (Kalinin et al. 2023). Our findings revealed that in HUVECs treated with HG, there was a notable decrease in SIRT1 expression, a significant upregulation of EP300, and inhibition of AMPK phosphorylation. After we knocked down or overexpressed LARP7, we examined the above protein expression levels again. The results showed that SIRT1 expression was restored after LIR treatment, EP300 expression was significantly inhibited, and AMPK phosphorylation was reactivated. However, alterations induced by LIR treatment were almost completely nullified following LARP7 knockdown, and LARP7 overexpression enhanced the effects of LIR (Figure 3b). To ascertain if LIR's impact on cellular senescence is associated with LARP7, we conducted CCK-8 assays on cells from these varied treatment groups. The results indicated that LIR's protective effect on cell viability was partially diminished due to LARP7 knockdown and enhanced by LARP7 overexpression (Figure 3c). Similarly, SA-β-gal staining demonstrated that LIR effectively mitigated the cellular senescence phenotype; however, it could not be effectively controlled following LARP7 knockdown. Critically, LARP7 overexpression further promoted the mitigation of cellular senescence by LIR (Figure 3d). Through qPCR analysis of SASP genes, including IL-1α, IL-1β, IL-8, MCP-1, and ICAM-1 mRNA expression, it was observed that SASP mRNA levels in HUVECs under HG conditions were significantly reduced by LIR, an effect also hindered by LARP7 knockdown, and conversely, it was enhanced by LARP7 overexpression (Figure 3e). These findings suggest that the efficacy of LIR in enhancing HUVEC cell vitality and inhibiting cellular senescence necessitates the involvement of LARP7. SIRT1 and AMPK phosphorylation are activated in this context, while EP300 is inhibited. The significant attenuation of LIR's effect following LARP7 knockdown, coupled with the re-inhibition of SIRT1 and AMPK phosphorylation and partial reactivation of EP300, and the opposite effect following LARP7 overexpression. It is further elucidated that LIR may regulate the senescence process of HUVEC cells through LARP7. The involvement of LARP7 in this process was significantly correlated with the SIRT1/EP300/AMPK signaling axis.

Fig 3.

LIR regulates EC senescence through the LARP7/SIRT1 pathway. Cells were initially segregated into NG, HG, and HG + LIR groups. (A) Western blot analysis for alterations in the known senescence signaling axes, focusing on SIRT1, EP300, p-AMPK (Thr172), and AMPK. Subsequent assessments in the HG, HG + LIR, HG + LIR + siLARP7, and HG + LIR + oeLARP7 groups were conducted to determine the effects on the senescence signaling axis and cellular phenotype. ^**p < 0.01 vs. NG, ^##p < 0.01 vs. HG. (B) Western blot analysis for SIRT1, EP300, p-AMPK (Thr172), and AMPK. (C) CCK-8 assay for evaluating cell viability. (D) SA-β-gal staining to determine cellular senescence. (E) qPCR to measure SASP gene expression levels of IL-1α, IL-1β, IL-8, MCP-1, and ICAM-1. n = 3 for each group. Scale bar: 50 μM. ^*p < 0.05, ^**p < 0.01 vs. HG, ^#p < 0.05, ^##p < 0.01 vs. HG + LIR. EC, endothelial cell; HG, high glucose; LIR, Liraglutide; MCP-1, motif chemokine ligand 2; SASP, senescence-associated secretory phenotype.

Having established LIR's positive impact on HUVEC cell vitality and senescence, we extended our investigation to explore its role in facilitating endothelial dysfunction. We evaluated EC functional characteristics, including tube formation and migration capabilities. The tube formation assay results demonstrated that LIR treatment significantly enhanced the tube-forming ability of HUVEC cells. However, the extent of vascular formation was negated mainly by LARP7 knockdown, and the upregulation of LARP7 similarly improved the effects of LIR in promoting vascular formation (Figure 4a). We then assessed the mRNA expression levels of vascular endothelial growth factor A (VEGFA) and kinase insert domain receptor (KDR), both critical in tube formation processes. The results indicated that LIR effectively boosted the mRNA expression levels of VEGFA and KDR, which, however, declined again following LARP7 knockdown, but the overexpression of LARP7 had the opposite effect (Figure 4b). Furthermore, the results from the transwell assay, which assessed HUVEC cell migration capabilities, revealed that LIR significantly promoted HUVEC cell migration, a capability that was suppressed upon LARP7 knockdown, and the upregulation of LARP7 similarly enhanced the ability of LIR to improve cell migration (Figure 4c). The above results demonstrated that LIR could effectively promote the functional properties of ECs and that the upregulation of LARP7 effectively enhanced the efficacy of LIR.

Fig 4.

LIR restores endothelial function impaired by LARP7 deficiency. Further investigation into the impact of LIR and LARP7 on HUVEC function was conducted in the HG, HG + LIR, HG + LIR + siLARP7, and HG + LIR + oeLARP7 groups. (A) Tube formation assay to measure angiogenic capacity. (B) qPCR is used to evaluate mRNA changes in VEGFA and KDR. (C) Transwell assay for assessing HUVEC migration capabilities. n = 3 for each group. Scale bar: 200 μM. ^**p < 0.01 vs. HG, ^#p < 0.05, ^##p < 0.01 vs. HG + LIR. HG, high glucose; HUVECs, human umbilical vein endothelial cells; KDR, kinase insert domain receptor; LIR, Liraglutide; VEGFA, vascular endothelial growth factor A.