Liraglutide reduces high sugar–caused abnormal movement, growth, and death of blood vessel muscle cells by activating GLP-1 receptors and blocking ERK1/2 and PI3K/Akt signals

Male Sprague Dawley rats (n = 4; 5–8 wks) were provided by the Laboratory Animal Center of Harbin Medical University, China. All procedures were performed in accordance with guidelines set by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Harbin Medical University, which is in compliance with the ARRIVE guidelines on animal research [49].

LIRA was purchased from Novo Nordisk (Bagsvaerd, Denmark). The ERK1/2 inhibitor (PD98059), PI3K inhibitor (LY294002), and FITC-conjugated anti-α-smooth muscle actin (α-SMA) monoclonal antibody were obtained from Sigma-Aldrich (St Louis, MO, USA). The GLP-1R antagonist, Exendin (9–39) (Exe(9–39)), was purchased from AnaSpec (San Jose, CA, USA). Trypsin, fetal bovine serum (FBS), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco (of Thermo Fisher Scientific, Waltham, MA, USA). Rabbit polyclonal antibodies against ERK1/2, Akt and phosphorylated (p)-Akt, and the mouse monoclonal antibody against p-ERK1/2 were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Rabbit polyclonal antibodies against β-actin, Bax, Bcl-2, and caspase-3 were purchased from Abcam (Cambridge, UK). Secondary antibodies were purchased from Vector Labs (Burlingame, CA, USA). Western blot bands were detected using the ECL Advance Western blotting detection kit from GE Healthcare (Chalfont St Giles, UK). Cell Counting Kit-8 was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Transwell plates were purchased from Millipore (Bedford, MA, USA). Annexin V-FITC Kit was purchased from BD Biosciences (San Jose, CA, USA).

VSMCs were prepared from thoracic aorta of Sprague Dawley rats as previously described, with minor modifications [50]. Each rat was sacrificed and the whole thoracic aorta was isolated and washed several times in phosphate buffered saline (PBS). After the adventitia and intima were carefully removed, the aortic tissue was cut into small pieces (1 mm²). Theses explants were plated into a tissue culture flask and cultured in DMEM supplemented with 15% FBS and maintained in a humidified incubator at 37°C and 5% CO₂. The cells were passaged by trypsinization and reseeded into new flasks approximately 4–8 times before use in subsequent experiments. VSMCs were identified by α-SMA staining.

All cultures were incubated with serum-free media for 24 h prior to treatment. Cultures of VSMCs were divided into the following groups: control (5 mM glucose); HG (25 mM glucose); LIRA (100 nM; previously described dose [51]); HG + LIRA (25 mM glucose and 100 nM LIRA); osmotic (MG; 25 mM mannitol); HG + LY294002 (25 mM glucose and 50 μM LY294002 [52]); HG + LIRA + LY294002 (25 mM glucose, 100 nM LIRA, and 50 μM LY294002); HG + PD98059 (25 mM glucose and 50 μM PD98059 [53]); HG + LIRA + PD98059 (25 mM glucose, 100 nM LIRA, and 50 μM PD98059); and HG + LIRA + Exe(9–39) (25 mM glucose, 100 nM LIRA, and 200 nM Exe(9–39) [54,55]). All VSMCs cultures were pretreated with the indicated drugs 1 h prior to HG treatment with exception of Exe(9–39) treatment, which was added 30 min prior to HG treatment.

Cells were plated in 96-well culture plates and incubated until reaching 80% confluency; the culture media was then replaced with serum-free DMEM and incubated for an additional 24 h. Cells were then treated with the media containing the indicated concentration of various purposely designed chemical(s) for an additional 48 h. Cell proliferation was then assessed using a Cell Counting Kit-8, following the manufacturer’s instructions. Briefly, the colorimetric reagent, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt, was added to each sample and incubated for 1 h at 37°C. Proliferation was then assessed by measuring absorbance of each sample at the wavelength of 450 nm.

Cell migration was determined using the Transwell migration assay as previous described, with minor modification [56]. Briefly, VSMCs were treated with the indicated chemical(s) for 12 h at 37°C. The cells were washed three times with PBS after the culture media was removed and then trypsinized with 0.25% (v/v) trypsin and resuspended in serum-free DMEM at 37°C. These cells were then counted and the upper chamber of each Transwell was seeded with 1 × 10⁵ cells per chamber in 0.2 mL serum-free DMEM. As a chemoattractant, 0.8 mL of DMEM supplemented with 20% FBS was added to the lower chamber of each Transwell. Chambers were incubated for 12 h at 37°C with 5% CO₂. Cells that migrated to the underside of the Transwell filter were fixed with 4% formaldehyde (w/v) for 20 min at room temperature and then immersed into a hematoxylin staining solution for 15 min. After washing with distilled water, membranes were mounted on glass slides and examined by microscopy at 200× magnification.

The migration capacity of VSMCs was also characterized using a well-established in vitro scratch wound model, with minor modifications [57]. VSMCs were grown to confluence and then subjected to scratching using a 200 μL sterile pipette tip. The scratch wound was allowed to heal for 24 h in the presence of the indicated chemical(s). Micrographs were captured for each sample at 0 and 24 h, and the capacity of VSMC migration was evaluated by measuring the width of the scratch wound at both time points using ImageJ [58].

Cell apoptosis was measured using the Annexin V-FITC kit, following the manufacturer’s instructions. Briefly, cells treated with the indicated chemical(s) for 48 h and then harvested by trypsinization. Cells were washed twice by centrifugation and re-suspended in PBS. Cells were then collected and re-suspended in 500 μL of the binding buffer. These cells were then stained with 5 μL of Annexin V-FITC and 5 μL of the propidium iodide staining solution for 15 min at room temperature in the dark. The percentage of Annexin V-FITC- and propidium iodide-positive cells was measured by flow cytometry (FACSAria, BD Biosciences, San Jose, USA).

All cells were collected and lysed in 200 μL radioimmunoprecipitation assay buffer with the protease inhibitor phenylmethylsulfonyl fluoride (100 mM) for 1 h on ice. Subsequently, the lysate was centrifuged at 12,000 × g for 5 min at 4°C. The supernatant was collected and the total protein concentration was determined using a bicinchoninic acid kit (Thermo Fisher Scientific). Ten μg of total protein from each sample was separated by electrophoresis using 12% SDS-PAGE gels and transferred onto nitrocellulose membranes. The target proteins were measured using the primary antibodies (1:10,000) and their corresponding secondary antibodies (1:50,000), followed by development with an ECL Advance Western blotting detection kit. β-actin was detected as a loading control. Signals were developed on X-ray films following exposure to ECL advance luminescence. The intensity of each band was quantified using Quantity One 4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Levels of phosphorylated proteins were determined as a ratio of total protein: p-Akt relative to Akt and p-ERK1/2 relative to ERK1/2.

All results were from three independent experiments and are expressed as mean ± standard deviation. Data were analyzed by one-way analysis of variance using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). A p < 0.05 was regarded as statistically significant.

The migration capacity of VSMCs was measured via Transwell migration and in vitro scratch assays.

The scratch wound assay showed that treatment with HG resulted in significantly more wound healing than the control group (85.61 ± 6.36 vs. 37.25 ± 4.78%; p < 0.01). LIRA pretreatment was able to attenuate the effects of HG on VSMC migration (67.03 ± 5.61%; p < 0.05 vs. HG) (Figure 3C,D). Treatment with LIRA or mannitol alone had no effect on scratch width, as compared to the control group.

To further investigate the mechanism of the HG-induced reduction in apoptosis, the expression of relevant apoptotic proteins was investigated by Western blotting (Figure 4C,D). The anti-apoptotic protein Bcl-2 was significantly upregulated (p < 0.01 vs. control) and the pro-apoptotic proteins cleaved caspase-3 and BAX were significantly downregulated after HG treatment (p < 0.01 vs. control). LIRA pretreatment reduced those effects by HG treatment, resulting in increased cleaved caspase-3 and Bax levels, and decreased Bcl-2 levels (p < 0.01 vs. HG) (Figure 4C,D). PD98059, LY294002 or Exe(9–39) alone had no effect on cleaved caspase-3 expression (all p > 0.05 vs. control).

Application of the GLP-1R antagonist Exe(9–39) ameliorated the effect of LIRA on VSMCs under HG treatment, including a increased cell proliferation (1.94 ± 0.17 vs. 1.62 ± 0.15; p < 0.05) (Figure 7A) and migration detected by the Transwell (86.2 ± 4.6 vs. 72.4 ± 2.07 cells; p < 0.01) (Figure 7B) and wound healing (80.79 ± 7.99 vs. 63.07 ± 5.61%; p < 0.05) (Figure 7C) assays. Moreover, Exe(9–39) reduced apoptosis (1.54 ± 0.29 vs. 3.44 ± 0.89%; p < 0.05) (Figure 7D) and cleaved caspase-3 (p < 0.01) (Figure 4C) in HG + LIRA-treated cells. Finally, inhibition of GLP-1R increased phosphorylation of ERK1/2 and Akt in HG + LIRA-treated cells (both p < 0.05) (Figure 5).

Previous studies have shown that the ERK1/2 signaling pathway plays an important role in VSMC proliferation [65], and insulin [66,67] or HG [13,14] could facilitate proliferation via activation of this pathway. HG can also induce dynamic changes in VSMCs through activation of the PI3K/Akt pathway [16,20-23]. Therefore, these pathways likely play a crucial role in the tissue damage induced by hypertension and atherosclerosis.

To explore whether these signaling pathways are involved in the HG-induced altered physiology of VSMCs, we first investigated whether HG concentrations altered ERK1/2 and PI3K/Akt activation. We then used specific inhibitors to block these signaling pathways. We show, in agreement with previous studies [13,15,17,23], that HG treatment of VSMCs significantly increases the phosphorylation of ERK1/2 and Akt. Furthermore, inhibition of these enzymes reduces HG-induced cell proliferation and migration. Pretreatment with ERK1/2 or PI3K inhibitors also effectively prevents the ability of HG to reduce VSMC apoptosis.

Thus, our data demonstrate that HG treatment of VSMCs alters cell migration, proliferation, and apoptosis via ERK1/2 and PI3K/Akt signaling pathways. These findings open a new avenue to further explore the underlying mechanism by which HG regulates VSMC cell migration in diabetic patients. Furthermore, our study identifies new research areas that may prove critical in deciphering the pathogenesis of, and provides a novel means to prevent, atherosclerosis in DM.

GLP-1 analogues and receptor agonists have been used in the treatment of DM, increasing pancreatic protein content and mass via boosting S6 kinase phosphorylation and acinar cell mass [68]. LIRA, a GLP-1 analogue, effectively lowers the body weight of diabetic patients, which was associated with enhancing plasma cardiac natriuretic peptides levels [69]. Long-acting GLP-1 mimetics, such as domain antibodies to serum albumin, can sustain the activation of GLP-1R and reduce myocardial infarct size and injury in acute coronary syndrome [70]. Another GLP-1 agonist, exenatide, increases hydrogen sulfide, carbon monoxide, and nitric oxide production, and then reduces central arterial pressure in an animal model [71]. In carotid endarterectomy of diabetic patients, preoperational use of GLP-1R agonists significantly increases the expression of sirtuin-6, a protein involved in the inflammatory pathway of diabetic atherosclerotic lesions and plaque stabilization [72]. A prospective pilot clinical trial has demonstrated that LIRA decreases carotid intima-media thickness in patients with type 2 diabetes [73].

In this study, we show that HG-induced VSMC alterations are prevented by treatment with LIRA. It is possible that in DM patients, LIRA protects the vascular system against atherosclerotic changes by similar mechanisms. Our results also demonstrate that LIRA by itself does not induce measurable physiologic changes in VSMCs, but only blocks HG-induced effects.

Previous studies have shown that GLP-1 protects cardiomyocytes and endothelial cells against cell apoptosis by activating ERK1/2 and/or PI3K/Akt [31,44,74]. However, our data demonstrate that LIRA inhibits this activation, observed as reduced phosphorylation levels in VSMCs. Moreover, synergistic effects are observed when LIRA pretreatment is combined with ERK1/2 or PI3K inhibitors. Although GLP-1 shows protections on cardiomyocytes and endothelial cells by activating ERK1/2 and/or PI3K/Akt [31,44,74], our data suggest that LIRA exerts the beneficial effects on VSMCs in part via inhibiting ERK1/2 and PI3K/Akt pathways. As VSMCs play a distinct role in the development of diabetic atherosclerosis, the involvement of these signaling pathways may be cell-type specific, which requires further investigation.

Cardiomyocytes, endothelial cells, macrophages, and VSMCs all express the GLP-1R, which mediates the anti-inflammatory and anti-proliferation effects of GLP-1 [31,40,48]. To confirm that the effects of LIRA in HG-treated cells occur via GLP-1R activation, VSMCs were pretreated with Exe(9–39). Indeed, Exe(9–39) treatment abolished the beneficial effects of LIRA treatment, and reversed the suppression of Akt and ERK1/2 activation.