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. 2024 Feb 23;7(3):733–742. doi: 10.1021/acsptsci.3c00288

Calcitonin Inhibits Phenotypic Switching of Aortic Smooth Muscle Cells and Neointimal Hyperplasia through the AMP-Activated Protein Kinase/Mechanistic Target of Rapamycin Pathway

Mingyu Liu †,‡, Fei Xiang †, Tao Yang †, Yujia Sun †, Jiemei Yang †, Tengyu Wang †, Sixuan Chen †, Yingjie Xu †, Gaojun Shan †, Yuanqi Shi †,‡,*, Zengxiang Dong †,‡,∥,*, Yuanyuan Guo †,§,*
PMCID: PMC10928884  PMID: 38481691

Abstract

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Calcitonin (CT) is a peptide hormone secreted by the parafollicular C cells of the thyroid gland, salmon calcitonin was originally extracted from the hind cheek of salmon. Neointimal hyperplasia refers to the excessive proliferation and migration of vascular smooth muscle cells (VSMCs). In this study, a rat model of restenosis was employed to explore the impact of calcitonin on neointima proliferation. Calcitonin was administered via continuous injections for a duration of 14 days postsurgery, and the expression of proteins associated with proliferation, migration, and phenotypic switching was assessed using the vascular smooth muscle cells. Additionally, metabolomic analyses were conducted to shed light on the mechanisms that underlie the role of calcitonin in the development of cardiovascular disease. In our study, we found that calcitonin possesses the capability to dispute the proliferation, migration, and phenotypic transformation of VSMCs induced by platelet-derived growth factor-BB (PDGF-BB) and 15% fetal bovine serum in vitro. Calcitonin has demonstrated a favorable impact on smooth muscle cells, both in vitro and in vivo. More specifically, it has been observed to mitigate phenotypic switching, proliferation, and migration of these cells. Moreover, calcitonin has been identified as a protective factor against phenotypic switching and the formation of neointima, operating through the AMP-activated protein kinase/mechanistic target of rapamycin (mTOR) pathway.

Keywords: calcitonin, vascular smooth muscle cells, phenotypic switching, neointimal hyperplasia, AMPK/mTOR pathway

Coronary artery disease, particularly neointimal hyperplasia (NIH), stands as the predominant cause of global mortality and morbidity.1,2 NIH, characterized by the thickening of arterial intima leading to subsequent arterial lumen narrowing, constitutes a major pathological hallmark of restenosis.3 Various therapeutic interventions are available for NIH patients, including balloon angioplasty, stent implantation, and surgical bypass grafting. However, the efficacy of these interventions in preventing NIH and restenosis remains suboptimal, and effective postoperative adjuvant therapies have yet to be established.4 VSMCs are the primary cell type present in all stages of NIH. The phenotypic switching of VSMCs during restenosis plays a pivotal role in NIH.5 VSMCs transition from a contractile phenotype to a synthetic phenotype characterized by high migratory and proliferative activity.6 Recognizing the contribution of VSMCs phenotype switching to NIH progression, blocking this process may effectively mitigate the disease.7 Our investigations have identified a short peptide and its binding site on VSMCs, laying the foundation for the rational design of a new generation of anti-NIH agents.

PDGF-BB serves as a pivotal mediator in VSMCs proliferation, migration, and phenotype switching. PDGF receptors (PDGFR) are divided into platelet-derived growth factor receptor-alpha (PDGFR-α) and platelet-derived growth factor receptor-beta (PDGFR-β) with PDGFR-β being predominantly implicated in vascular pathology. Following vascular injury and angioplasty, medial vessel VSMCs migrate to the intima, assuming a synthetic phenotype in a PDGFR-β-dependent manner. Multiple studies have revealed an association between vascular diseases and the overexpression of PDGF-BB and its receptors. Consequently, the inhibition of PDGF-BB and PDGF-BB receptors has been demonstrated to attenuate neointimal thickening.8,9 Additionally, it has been established that elevated serum levels trigger the differentiation, proliferation, and migration of VSMCs.10 Consequently, we utilized PDGF-BB and a 15% FBS concentration for the described experimental functions.

AMPK plays an important role in regulating cellular energy metabolism, primarily activated by low levels of Adenosine triphosphate (ATP). Mitochondria constitute the principal source of ATP production.11 Recent studies conducted over the past several years have unveiled the involvement of AMPK in NIH pathogenesis. Both AMPK and mTOR have been demonstrated to modulate processes such as proliferation, migration, and phenotype switching.12,13 These two proteins, AMPK and mTOR, represent key components in the VSMCs phenotype switching during NIH progression. Nevertheless, the precise connection between calcitonin and AMPK–mTOR signaling remains incompletely elucidated.

The peptide hormone CT is comprised of 32 amino acids and is secreted by the parafollicular or C cells within the human thyroid gland.14 There are four kinds of calcitonin, but salmon calcitonin extracted from salmon is the most used and the best effect. Recent evidence has highlighted the involvement of CT in interactions with its receptor, the calcitonin receptor (CTR), and subsequent signal transduction.15 Previous research has revealed that CT inhibits osteoclast activity and bone resorption in osteogenesis. It also reduces the reabsorption of calcium, sodium, potassium, chloride, and phosphate in cases of renal disease. Additionally, CT induces analgesia, gastric acid secretion, and anorexia via the central nervous system.16−18 Recent studies have demonstrated that CT is produced by the atrial myocardium, playing a role in the regulation of fibrotic remodeling within the atrium.19 However, whether CT participates in the phenotypic switching of VSMCs, and arterial remodeling remains unknown. Furthermore, the mechanism underlying CT’s effects on NIH remains unclear.

In this investigation, we have found that CT effectively reduces in vitro migration, proliferation, and phenotypic switching of VSMCs induced by PDGF-BB and 15% FBS. Moreover, CT has shown in vivo protective effects against NIH. By activating mTOR and increasing AMPK Thr-172 phosphorylation, CT effectively suppresses the phenotypic transition of aortic smooth muscle cells and subsequently mitigates NIH. The reduction in NIH by CT can be attributed to its binding with CTR and PDGFR, acting through the AMPK/mTOR axis. Consequently, CT emerges as a potential polypeptide hormone for the management of restenotic artery diseases.

Materials and Methods

Animals

The Animal Center of the Second Affiliated Hospital of Harbin Medical University donated 30 male SD rats, each weighing between 350 and 400 g. The rats were reared in the Harbin Medical University’s First Affiliated Hospital’s Animal Center. All animal testing was done in compliance with the guidelines established by the committee of use and the research institution. Three groups of ten mice each were randomly assigned: the CT group (n = 10), the surgical group (n = 10), and the sham group (n = 10). For the mice in the CT group, the carotid artery was damaged by a balloon, and then CT was injected intraperitoneally for 14 days at a dose of 20 IU/kg/day.

Rat Carotid Artery Injury Model

The common carotid artery, external carotid artery, and internal carotid artery were all exposed after the rats were given general anesthesia, shaved, cleaned, and had their skin sliced along the front midline of the neck. A nylon rope was used to ligate the external carotid artery’s distal end. Micromaterials were used to temporarily constrict the external and common carotid arteries. At the distal end of the external carotid artery, a V-shaped incision was made at the distal end of the external carotid artery to allow the insertion of a 3–4 cm long 1.25F Fogarty balloon catheter into the blood vessel. The balloon was inflated to a pressure of 4–5 atm using a pressure pump, dragged backward three times to induce injury, and then the incision was ligated. After releasing the microclamps to restore normal blood flow in the neck blood vessels, the neck incision was sutured. The neck incision was then stitched up after the micro material clamp was loosened and the normal blood flow of the neck blood vessels was restored. The animals were brought back to the animal breeding facility when they had awoken, where they were given a regular food for 2 weeks. The same procedure was carried out without ligating the cervical vessels in the sham operation group.

Rat Tail Blood Pressure Measurement

Systemic blood pressure was measured in conscious rats using the noninvasive tail-cuff method (BP-98A, Tokyo, Japan). The rats were placed in a dark cloth bag, and a measuring instrument was attached to the tail for blood pressure measurement.

Doppler Ultrasound Imaging

Using the Vevo 2100 ultrasound imaging equipment (Philips L15-7io 15–7 MHz), the aortic diameter and pulse wave velocity were measured at 0 and 14 days. Rats were put under anesthesia and positioned supinely on the operating table. After shaving the neck area, applying a gel, and fixing the acquisition probe, the neck vascular image was captured.

H&E Staining

For paraffin section preparation, a sample of the left external carotid artery was obtained. The samples were embedded in paraffin, cut into slices, and baked after being fixed with 4% paraformaldehyde, dehydrated with graded alcohol, cleaned with xylene, and embedded in paraffin. After 2 h of baking at 60 °C, the slices underwent xylene dewaxing and gradient alcohol dehydration. The slices were then stained with hematoxylin after being washed with distilled water for a minute. After 4 min of staining, the area was washed for 15 min under running water. The tissue slices were stained for 4 min with eosin dye, washed for 3 min with tap water, dehydrated for 4 min with serial alcohol concentration, rinsed for 2 min with xylene, and then sealed with neutral resin.

Immunohistochemistry Assay

The carotid arteries were imbedded in optimal cutting temperature (OCT). After being recovered in 3% H2O2 for 15 min, they underwent a 15 min antigen repair incubation in citrate buffer at 95 °C. After sealing the artery tissues with 5% goat serum at room temperature for 30 min, the primary antibody was incubated with the tissues overnight at 4 °C. Then tissues were treated with fluorescent secondary antibodies for an hour at room temperature. The nuclei were stained for 10 min with 4′,6-diamidino-2-phenylindole (DAPI) and then cleaned three times with PBS.

Cell Culture

Primary rat VSMCs were derived from the aortas of adult SD wild-type rats aged 8 to 10 weeks. Following the excision, the aortic tissue was finely minced, and the tissue fragments were uniformly distributed at the base of sterile culture flasks. Subsequently, dulbecco’s modified Eagle medium (DMEM) supplemented with 20% FBS was added to initiate cell growth. The culture flasks were gently inverted after a 2 h incubation period, and over the course of 5–8 days, cells proliferated in close proximity to the tissue fragments. A10, human arterial smooth muscle cells (HASMCs), and human umbilical vein endothelial cells (HUVECs) were purchased from Otwo Biotech Inc. (Shenzhen, China) and Qingqi Biotechnology Development Co. Ltd. (Shanghai, China). The cells were cultured in DMEM supplemented with 10% FBS and 1% mixture of penicillin–streptomycin in a humidified incubator at 37 °C and 5% carbon dioxide (CO2). The cells were stimulated with calcitonin (Beyotime, China) for 48 h after reaching confluence in a 3.5 cm Petri dish.

Cell Viability Measurement

Cell viability was assessed using the Cell Couting Kit-8 (CCK-8) assay. Briefly, cells were seeded in 96-well flat-bottomed plates at 5 × 104 cells per well, treated with or without calcitonin for 48 h, and then incubated with CCK-8 at 37 °C for 2 h. Then use a plate reader to measure the light absorption value of each hole at 450 nm wavelength and analyze the results.

Transfection of siRNA (6-Well Plate as an Example)

For transfection, 105 cells were inoculated in a 6-well plate and cultured for 14–18 h to a cell density of 80–90%. Before transfection, the culture media was changed to DMEM medium with serum-free and double antibody-free. Following this, 200u of buffer was immediately mixed with 4u of reagent and 4u of siRNA, which was then added dropwise to the cell culture plate for a subsequent 6 h culture. Western Blot analysis was used to measure the knockdown efficiency 48 h after transfection. (si-AMPK:5′-GUUUAGAUGUUGUUGGAAATTUUUCCAACAACAUCUAAACTT-3′).

Live and Dead Cell Staining

The live and dead cells were detected using the LIVE/DEAD Viability/Cytotoxicity Assay Kit (ab287858, Abcam). Briefly, the cells were seeded into the six-well plate at 5 × 104 cells per well and incubated with Calcein acetoxymethyl (AM)/propidium iodide (PI) at 37 °C for 15 min. The labeled cells were visualized using a fluorescence microscope (Zeiss, German) at ×20 magnification and counted using ImagePro Plus image analysis software.

Wound-Induced Migration Assay

Cells were seeded into a six-well plate at 5 × 104 cells per well and cultured in DMEM supplemented with 15% FBS and 1% penicillin–streptomycin. 90% confluence was reached before a vertical scratch with a 200 μL pipet tip was used to make a cell-free zone that spanned the horizontal lines drew on the back of the plate prior to cell seeding to determine an exact location. The cells were gently washed twice with PBS to get rid of the debris. The horizontal lines served as a baseline and were used to obtain the wound healing images under a light microscope (German manufacturer Zeiss) at ×5 magnification. At 0, 24, and 48 h after wounding, microscope photographs of the wound healing were collected. Cell migration was quantified by measuring the area of the cell-free zone using ImagePro Plus image analysis software.

EdU Assay

5-Ethynyl-2′-deoxyuridine (EdU) labeling was used to identify proliferating cells. A labeling medium containing EdU was briefly incubated with cells for 4 h at 37 °C with 5% CO2. Following the manufacturer’s technique, cells were then fixed and prepared for EdU detection.EdU-positive cells were detected by immunofluorescence staining using a peroxidase-coupled antibody against EdU. The cells were counted and examined using ImagePro Plus image analysis software after being randomly observed under a fluorescent microscope at ×20 magnification.

ATP Assay

The ATP Assay Kit (Beyotime, cat. S0026) was used to measure the amount of cellular ATP in accordance with the manufacturer’s guidelines. The lysed VSMCs were centrifuged at 12,000g for 5 min at 4 °C after being treated. After then, the supernatant was gathered. The reagents were added to the 96-well plate after the supernatant, in the order recommended by the manufacturer, and then the mixture was incubated for 15 min. At room temperature, the absorbance was calculated using a fluorescence microplate reader.

Western Blot Analysis

After being collected, lysed in RIPA buffer, and centrifuged, the cells’ protein content was measured using a BCA assay. Equal amounts of protein were electrophoretic ally separated on 7.5–12.5% SDS-PAGE gels and then transferred to nitrocellulose membranes. The membrane was then sealed with 5% skimmed milk, incubated with the primary antibody for an overnight period at 4 °C, rinsed with PBST, and then incubated with the secondary antibody for an hour and a half. Enhanced chemiluminescence (ECL)substrate solution was then added for color development at room temperature.

ELISA

Using the Human CT ELISA kit, the levels of CT in the plasma of the two rat groups were determined. The level of plasma CT was adjusted to ng/L.

Molecular Docking

The Protein Data Bank PDB database (http://www.rcsb.org) was used to download the protein structural domain in PDB format. GRAMMX was used to analyze the molecular docking between the calcitonin model, CTR, PDGFRB, and their complexes in order to uncover the protein interaction. Structures were rendered using PyMOL (PyMOL Molecular Graphics System, version 2.3.0).

Metabolites Analysis

Based on the AB Sciex QTRAP 6500 LC-MS/MS platform, MetWare (http://www.metware.cn/) was able to identify every metabolite.

Statistical Analysis

All statistical analyses were performed using the GraphPad Prism 9 (GraphPad Software Inc., USA) software. All data were expressed as the mean ± SD. Differences between the two groups were determined using Student’s t-test. One-way ANOVA followed by Holm–Sidak test or Tukey’s test was used for multiple group comparisons. P value < 0.05 was considered statistically significant.

Results

Expression of CT and CTR

LM19 reported the abundant secretion of calcitonin (CT) in cardiomyocytes of the atrium, acting through the CT-CTR axis. Calcitonin receptor (CTR), characterized by seven transmembrane domains and a G protein-coupled nature, initiates intracellular signaling via cyclic adenosine monophosphate (cAMP) and calcium-mediated second messenger pathways. CTR exhibits a wide distribution in various tissues, including osteoclasts, the brain, ovary, kidney, stomach, and skeletal muscle, suggesting its broad influence on diverse biological functions.16−18,20 Abnormal blood calcium concentrations have been linked to cardiovascular disease, although investigations into the relationship between CT and cardiovascular disease remain limited. In our study, we observed elevated calcitonin levels in both arterial and venous blood samples from patients with defective vascular walls, despite the typical serum calcitonin value being less than 100 ng/L (Figure 1a). This suggests a potential role for CT in the development of vascular diseases. CTR was consistently detected in human carotid artery tissues, regardless of whether they were bifurcated or nonbifurcated (Figure 1b). However, CT was only expressed in two bifurcated samples but was absent in nonbifurcated areas, which may be due to different blood flow velocities or differences affected by shear forces (Figure 1b). The blood vessels are mainly composed of smooth muscle cells and endothelial cells. We discovered that CTR and CT were expressed in human umbilical vein endothelial cells and human aortic smooth muscle cells. Remarkably, CT was found to be overexpressed in human umbilical vein endothelial cells (Figure 1b), leading us to hypothesize that CT may be released by HUVECs and affect HAVSMCs. In rat carotid arteries, CTR was consistently detected in normal, PDGF-BB-stimulated, and balloon-injured conditions (Figure 1c,e). However, CT remained undetected in carotid atherosclerosis in rats.

Figure 1.

Figure 1

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Expression of CT and CTR. (a) Expression of calcitonin in human arterial and venous blood (ELISA). (b) Protein representation of human aorta, HUVECs and HVSMC. (c) Protein representation of rat aorta, PDGF-BB stimulated aorta and balloon injured aorta. (d) Protein representation of CT and CTR in VSMCs. (e) Immunofluorescence images of rat aorta of CTR, Scale bar = 50 μm. (f) Immunofluorescence images of VSMCs of CTR and statistical chart of C. *P < 0.05 vs Con group, ##P < 0.01 vs PDGF-BB group. (n = 5) Scale bar = 1000 μm.

Lastly, VSMCs were found to express CTR but not CT (Figure 1d). Intriguingly, CT treatment resulted in a gradual increase in CTR expression by VSMCs stimulated with PDGF-BB (Figure 1f).

Effects of Calcitonin on Cell Viability

Although atrial cardiomyocytes and parafollicular thyroid cells both produce CT, it is unknown what level of calcitonin is present in individual cells. Hence, we started by looking for a therapeutic CT concentration that is not harmful to cells. The CCK-8 test was used to investigate the cytotoxic effects of calcitonin on VSMCs and HUVECs at doses of 10 nmol/L to 100 μmol/L. HUVECs were incubated for 48 h in 10% FBS and VSMCs were incubated for 48 h in a full medium comprising 0.2 and 15% at 37 °C. With a CT concentration of less than 1 μmol/L, there was no discernible impact on cellular viability. However, CT concentrations greater than 10 mol/L caused a 50% increase in cell death. For smooth muscle and endothelial cells, the median fatal dose of calcitonin was around 10 μmol/L (Figure 2a–c). These results suggest that VSMCs are affected within a specific dose range of calcitonin. However, endothelial cells were not affected by higher CT concentrations.

Figure 2.

Figure 2

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Effects of calcitonin on cell viability. (a,b) Effects of calcitonin on VSMCs viability evaluated by CCK-8. The IC50 value was measured under 0.2 (a) and 15% (b) FBS conditions (n = 6). (c) Effects of calcitonin on cell viability of HUVECs evaluated by CCK-8. The IC50 value was assessed under 10% FBS conditions (n = 6). (d–f) The cytotoxicity of calcitonin on VSMCs was evaluated by using the LIVE/DEAD Viability/Cytotoxicity Assay. The live cells were stained green with calcein AM, and the dead cells were stained red with EthD-1. The cells were cultured under 0.2 (d), 15% (e) and 10% FBS (f) conditions. Scale bar = 1000 μm.

We further assessed the cytotoxicity of calcitonin using the LIVE/DEAD Viability/Cytotoxicity assay (Figure 2d–f), and the results were similar. Therefore, we chose a relatively safe concentration of 600 nmol/L of CT for VSMCs and HUVECs.

CT Inhibited Proliferation, Migration, and Phenotypic Transformation of PDGF-BB-Induced VSMCs

First, the proliferation of VSMCs was assessed using EdU. As shown in Figure 3a, PDGF-BB significantly increased DNA synthesis after 48 h of treatment. The proliferation of VSMCs was dramatically slowed down by CT therapy. Proliferation-related proteins’ expression was likewise downregulated by CT treatment (Figure 3c,d). Further analyses revealed that CT reduced PDGF-BB-induced migration of VSMCs (Figure 3b). The expression cell migration-related proteins matrix metallopeptidase 2 (MMP2) and matrix metallopeptidase 9 (MMP9) also showed a similar trend (Figure 3e,f). The above results suggest that CT treatment inhibits the migration of VSMCs by modulating the expression of MMP2 and MMP9. Finally, we investigated the effect of CT on the phenotypic transformation of VSMCs. The expression of smooth muscle protein 22alpha (SM22α) and alpha-smooth muscle actin (α-SMA) was downregulated by PDGF-BB, but this effect was corrected by CT therapy. Additionally, CT inhibited VSMCs phenotypic alteration (Figure 3g,h). Similarly, CT can also inhibit the proliferation and migration of VSMCs under the stimulation of 15% serum (Figure S1).

Figure 3.

Figure 3

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CT inhibited proliferation, migration and phenotypic transformation of PDGF-BB-induced VSMCs. VSMCs were incubated with PDGF-BB (30 ng/mL) in the absence or presence of CT (600 nM) for 48 h. (a) The representative image of EdU (n = 5). (b) The representative image of Wound-induced migration assay (n = 5). (c,d) The representative image of Cyclin D1, PCNA, Cyclin D1 level was normalized to that of Actin protein level in (c), PCNA level was normalized to that of Actin protein level in (d) (n = 5). ***P < 0.001 vs PDGF-BB group. (e,f) The representative image of MMP2, MMP9. MMP2 level was normalized to that of GAPDH protein level in (e), MMP9 level was normalized to that of GAPDH protein level in (f) (n = 5). (g,h) The representative image of α-SMA, SM22α. α-SMA level was normalized to that of GAPDH protein level in (g), SM22α level was normalized to that of GAPDH protein level in (h) (n = 5). *P < 0.05 vs Con group, **P < 0.01 vs Con group. #P < 0.05 vs PDGF-BB group, ##P < 0.01 vs PDGF-BB group. Scale bar = 1000 μm.

Metabolomic Changes in VSMCs after Treatment with CT

It was also determined how PDGF-BB and PDGF-BB + CT affected the expression of a number of metabolites (Figure 4a). A heat map (Figure 4d) was used to show the metabolites that were significantly different between the PDGF-BB and the PDGF-BB + CT groups. One of the more prevalent metabolites in group CT was ATP (Figure 4b). In particular, CT changed how ATP was expressed (Figure 4c). Low ATP had an immediate impact on AMPK alterations. Therefore, CT influences the alterations in AMPK, which in turn affects the proliferation, migration, and phenotype switching of VSMCs. Energy homeostasis is controlled by a crucial molecule called AMPK.21 The AMPK signaling pathway plays a key role in the proliferation and migration of vascular smooth muscle cells.22 Changes in ATP dysregulate AMPK expression. Further analyses revealed that CT (600 nmol/L) activated the AMPK pathway (Figure 4e). The mTOR pathway, one of the mitotic signaling pathways linked to VSMCs proliferation, is crucial for controlling cell cycle and growth.8 We found that CT inhibited mTOR activation (Figure 4f). However, inhibiting AMPK expression using si-AMPK inhibited the CT-induced mTOR activation inhibition (Figure 4g,h). According to the findings, CT therapy prevented VSMCs proliferation, migration, and phenotypic change by encouraging phosphorylation of AMPK changes upon treatment and blocking the mTOR pathway.

Figure 4.

Figure 4

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Metabolomic changes in VSMCs after treatment with CT. (a) Representative heatmap from a hierarchical clustering analysis shows significant differences in metabolism between groups (n = 4). (b) Box diagram of ATP changes of Con, PDGF-BB and PDGF-BB + CT. (c) The ATP expression levels of Con, PDGF-BB and PDGF-BB + CT (n = 5). (d) Functional enrichment analysis of differentially expressed metabolites. (e) The representative image of p-AMPK/AMPK, statistical chart of p-AMPK/AMPK (n = 5). (f) The representative image of p-mTOR/mTOR, statistical chart of p-mTOR/mTOR (n = 5). (g) The representative image of p-AMPK/AMPK, statistical chart of p-AMPK/AMPK (n = 5). (h) The representative image of p-mTOR/mTOR, statistical chart of p-mTOR/mTOR (n = 5). ****P < 0.0001 vs Con group, ####P < 0.0001 vs PDGF-BB group. Add si-AMPK in (g,h).

Relationship between CTR and PDGFR-β

Contrary to what we anticipated, we discovered that CT therapy boosted the expression of its receptor, CTR (Figure 5d). We chose PDGF-BB as a stimulator, and PDGF-BB and PDGFR-β function when combined, to create a cell model. In this work, we discovered that the expression of PDGFR- β decreased after CT administration, and we hypothesized that CTR and PDGFR-β interaction. Therefore, to confirm the interaction between CTR and PDGFR-β, we used the molecular docking function. Based on the X-ray crystal structure from the protein database, a computational three-dimensional complex structure model was created (Figure 5a). The docking simulation data revealed 13 sites for the interaction between CTR and PDGFR-β. As shown in Figure 5b, CTR and PDGFR-β formed a stable protein docking model. Then we docked the CT with the CTR-PDGFR-β complex and found that the combination of them was very strong (Figure 5c). In summary, CT and CTR/PDGFR-β form a trimeric complex. Therefore, the overexpression of CTR was due to a combined effect of CT and PDGFR-β. Western Blot results (Figure 5d,f) showed that a combination of CT and PDGFR-β strongly inhibited CTR expression. The CO-IP experiment provides evidence of a robust interaction between them (Figure 5l,m). Subsequently, inhibiting AMPK—using AMPK inhibitor reversed the above phenomenon and activated CTR (Figure 5h–k).

Figure 5.

Figure 5

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The relationship between CTR and PDGFR-β (a) Three-dimensional structure diagram of CTR and PDGFR-β. (b) Schematic diagram and enlarged detail of Interaction between CTR and PDGFR-β. (c) Schematic diagram of the combination of CTR and PDGF-BB with CT. (d) The representative image of CTR in VSMCs (n = 5). (e) The statistical chart of CTR. (f) The representative image of PDGFR-β in VSMCs. (g) The statistical chart of PDGFR-β. (h) The representative image of CTR (n = 5). (i) Statistical chart of CTR (n = 5). (j) The representative image of PDGFR-β in VSMCs (n = 5). (k) The statistical chart of PDGFR-β. (l) Co-IP assays were performed using CTR antibody and then Western blotting was performed for CTR and P PDGFR-β. (m) Co-IP assays were performed using PDGFR-β antibody and then Western Blotting was performed for CTR and PDGFR-β. *P < 0.05 vs Con group, **P < 0.01 vs Con group. #P < 0.05 vs PDGF-BB group, ##P < 0.01 vs PDGF-BB group, &&&P < 0.001 vs PDGF-BB + CT group. Add AMPK inhibitor, compound C (40 μM, 90 min) in (h,j).

CT Attenuates NIH of the Carotid Artery

NIH or restenosis is mainly attributed to excessive proliferation and migration of VSMCs.1,23 Herein, we investigated the effect of CT on the neointima of rat carotid artery after balloon injury and 14 days of CT treatment. Following effective balloon damage modeling, CT (20 IU/kg) was subcutaneously given for 14 days straight (Figure 6a,c). After receiving CT treatment for 14 days, the carotid artery neointima proliferation in balloon-injured rats was dramatically reduced (Figure 6c). Neointimal thickness significantly decreased (Figure 6d,e), suggesting that CT may be a possible remodeling agent. The proliferation and migration of smooth muscle were greatly slowed down by CT therapy, according to immunofluorescence results for MMP9 and proliferating cell nuclear antigen (PCNA) (Figure 6f,g). CT affects blood pressure because it regulates the concentration of calcium ions. However, in our study, there was no difference in calcium ions and blood pressure between the treatment group and the model group (Figure S2a–c).

Figure 6.

Figure 6

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CT attenuates NIH of carotid artery in balloon-injured rats (a) M-mode ultrasound was performed on the carotid arteries of the rats in each group at 0 and 14 days, respectively. (b) Statistical chart of (a). (c) Rat carotid artery H&E staining. (d) Quantitative analysis for the value of NIA. (e) The ratio of NIA to MIA. (f) Immunofluorescence images of PCNA of Rat carotid artery. (g) Immunofluorescence images of MMP9 of Rat carotid artery. ***P < 0.001 vs Sham group, ****P < 0.0001 vs Sham group, ###P < 0.001 vs Model group, ####P < 0.0001 vs Model group. Scale bar = 50 μm.

Mechanism of CT

In this study, we found that CT and CTR synergistically affect NIH. The CT-CTR/PDGFR-β axis affects cell metabolism, which is involved in metabolic activities such as ATP and adenosine diphosphate (ADP), which in turn affects the metabolism-related signaling pathway AMPK-mTOR. The CT-CTR/PDGFR-β axis activates AMPK and inhibits the mTOR pathway. Inhibiting AMPK activity using si-AMPK blocked the expression of activated mTOR. In conclusion, by activating AMPK and blocking the mTOR pathway, the CT-CTR/PDGFR-β axis prevents VSMCs proliferation, migration, and phenotypic switching.

Conclusions

In this study, we investigated CT’s impact on VSMCs phenotypic switching, proliferation, and migration, as well as its role in NIH. We discovered that CT, targeting the CTR and PDGFR-β both in vivo and in vitro, effectively inhibited the phenotypic switching of arterial VSMCs.

According to a recent study by Lucia M19 reported that CT reduced the proliferation and migration of human atrial cardiac fibroblasts. These findings are consistent with our observations in VSMCs, suggesting a similar role for CT across different cell types or tissues. Lucia M19 also found that human atrial cardiomyocytes secrete myocardial CT, which acts on atrial cardiac fibroblasts in a paracrine manner by binding to CTR. It remains a subject of interest whether endothelial cells are a source of vascular CT in vascular remodeling diseases. In our study, we detected the presence of CTR in both arteries, but CT was not found in either uninjured or balloon-damaged rat carotid arteries (Figure 1). Moreover, CTR expression was diminished in balloon-injured rat carotid arteries (Figure 1). Recent research has shown that CTR expression is associated with the stabilization of atherosclerotic plaques.24 The binding of CT to CTR/PDGFR-β exerting a protective effect in vascular diseases. In line with these findings, Western blot and immunofluorescent analyses demonstrated that CTR was expressed in VSMCs and was lower in PDGF-BB-induced VSMCs remodeling (Figure 1). CT’s binding to CTR/PDGFR-β effectively inhibited PDGF-BB-induced phenotypic switching, proliferation, migration of VSMCs (Figure 3), and mitigated NIH (Figure 6).

In light of our findings, CT emerges as a protective factor in vascular diseases, shedding new light on the mechanisms underlying CT-mediated suppression of vascular remodeling. PDGF-BB and 15% FBS trigger mitochondrial dysfunction in VSMCs, leading to excessive ATP production and suppression of p-AMPK, ultimately activating mTOR. Our study demonstrated that CT counteracted mTOR activation, reduced ATP levels, and promoted p-AMPK activation. We performed extensive experiments to explore this relationship further. Initially, we used Western blot to confirm that CT treatment reduced the expression of proliferation and migration markers in PDGF-BB and 15% FBS-induced VSMCs remodeling (Figures 3 and S1). Targeted metabolic analysis revealed that PDGF-BB reduced ATP production, which was restored by CT administration (Figure 4). CT appears to mediate VSMCs growth and migration through an ATP-related mechanism. Studies have identified signaling pathways involving Akt, Stat3, Erk1/2, and AMPK in vascular remodeling. CT relaxes arteries by opening ATP-sensitive potassium channels in vascular smooth muscle cells.25,26 As such, CT attenuates VSMC phenotypic switching, proliferation, and migration, possibly through AMPK activation induced by ATP. Our study demonstrated that CT specifically increased p-AMPK protein at Thr-172 but had no impact on AMPK levels induced by PDGF-BB. Moreover, inhibiting AMPK intensified p-AMPK expression and reduced mTOR expression in PDGF-BB-induced VSMCs remodeling, effectively mitigating proliferation, migration, and phenotypic switching (Figure 4). These findings confirm our initial hypothesis.

On the other hand, although the mechanism underlying PDGF-BB and 15% FBS-induced phenotypic switching, proliferation, and migration of VSMCs is different, CT blocks all these processes. Furthermore, CT-induced repression was associated with ATP reduction and mitochondrial dysfunction through the CT and CTR/PDGFR-β dependent mechanism instead of modeling agent-related pathways. AMPK induces p-AMPK phosphorylation at Thr-172, inhibits the mTOR signaling pathway, and attenuates NIH.27 Studies have revealed that CT binding CTR activation, NO and camp pathway in endothelial cells activation, ATP -sensitive potassium channel in VSMCs, overexpression of sodium channel in nerves tissues, and activation of wnt-β-catenin signaling in osteoclast inhibit bone formation.28−30 There had never been any research on the connection between CT and AMPK/mTOR in VSMCs before to our work. Thus, our findings revealed a new CT target, which could be targeted to prevent vascular remodeling.

It is interesting to note that contrary to our findings, a number of research have found that CT lowers blood calcium levels via raising intracellular calcium. We found that CT (600 nmol/L) does not significantly affect the intracellular calcium levels of VSMCs. CT (20 IU/kg) through intraperitoneal administration had no significant effects on rat blood pressure (Figure S2). This indicates that the effects of a low CT concentration (600 nmol/L) could be calcium independent. As a result, CT-induced CTR/PDGFR-β binding may not be connected to intracellular calcium. The weighing of calcium in vivo and in vitro models is similar. Our subsequent studies will focus on the CT-induced through the protein kinase B (Akt), signal transducer and activator of transcription 3 (STAT3), extracellular signal-related kinases 1 and 2 (Erk1/2), and wnt-β-catenin signaling pathways.

CT is used clinically to treat most hypercalcemia, such as bony metastases, vitamin-D intoxication, Paget’s disease, and high-bone turnover osteoporosis. Recent studies have revealed that CT is an emerging agent for treating cardiovascular diseases because of its antiproliferative properties in cardiovascular tissues. However, its pro-proliferative properties in osteoporosis limit its potential effects in vivo. Therefore, it is necessary to assess the suitable concentrations of CT for treating NIH. Herein, the IC50 of CT is similar in serum-free and under 10% FBS and PDGF-BB conditions. We further examined the cytotoxic effect of CT using the LIVE/DEAD Cell Viability assay. CT-induced cell death only at the higher concentration, at low concentration showed no significant cytotoxic effect (Figure 2). It is challenging to administer CT orally since it is a peptide. Thus, the balloon-injured rat carotid arteries induced NIH were treated through intraperitoneal injection, which revealed that CT has good therapeutic effects and advantages for NIH treatment (Figure 6). Therefore, we initially assessed for a suitable therapeutic concentration for treating NIH.

In summary, we demonstrated that binding of CT to CTR/PDGFR-β attenuates phenotypic switching, proliferation, and migration of VSMCs and NIH. The mechanism of action CT might be regulated via the AMPK/mTOR pathway. Overall, CT is a therapy option for NIH.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (nos. 81900366, 82000397), Foundation of the First Affiliated Hospital for Distinguished Young Scholars (HYD2020YQ0005), The First Affiliated Hospital of Harbin Medical University Funding program for training outstanding young medical talents (2021J01, 2021J03), Natural Science Foundation of Heilongjiang Province (YQ2021H015), Research Project of the First Affiliated Hospital of Harbin Medical University (2021Y09), China Postdoctoral Science Foundation Project (2023MD744208).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00288.

  • The impact of calcitonin on VSMCs proliferation was assessed using EdU incorporation assay, while changes in proliferative proteins Cyclin D1 and PCNA were evaluated via Western Blot analysis. Migration ability of VSMCs was examined using a scratch test, with expression levels of migration-associated proteins MMP2 and MMP9, as well as contraction-specific proteins α-SMA and SM22α, determined through Western Blot analysis. Blood pressure in rats was measured using a noninvasive blood pressure monitor, and the effect of calcitonin on intracellular calcium ion concentration in VSMCs was determined using the Fluo-4AM kit (PDF)

Author Contributions

# M.-Y.L and F.X. contributed equally. Ming-Yu Liu and Fei Xiang: Conception, design, data acquisition, analysis, and interpretation, Tao Yang: Animal model construction. Yujia Sun: Molecular biology experiment. Jiemei Yang: Animal ultrasonic image acquisition. Tengyu Wang: Collection of human blood samples and tissues. Sixuan Chen: Molecular biology experiment. Yingjie Xu: Data acquisition, analysis. Gaojun Shan: Collection of human blood samples and tissues. Yuan-Qi Shi, Zeng-Xiang Dong, Yuan-Yuan Guo: drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of this work.

The authors declare no competing financial interest.

Supplementary Material

pt3c00288_si_001.pdf (296.2KB, pdf)

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Supplementary Materials

pt3c00288_si_001.pdf (296.2KB, pdf)

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