Vagus nerve stimulation‐induced stromal cell‐derived factor‐l alpha participates in angiogenesis and repair of infarcted hearts

Abstract

Aims

We aim to explore the role and mechanism of vagus nerve stimulation (VNS) in coronary endothelial cells and angiogenesis in infarcted hearts.

Methods and results

Seven days after rat myocardial infarction (MI) was prepared by ligation of the left anterior descending coronary artery, the left cervical vagus nerve was treated with electrical stimulation 1 h after intraperitoneal administration of the α7‐nicotinic acetylcholine inhibitor mecamylamine or the mAChR inhibitor atropine or 3 days after local injection of Ad‐shSDF‐1α into the infarcted heart. Cardiac tissue acetylcholine (ACh) and serum ACh, tumour necrosis factor α (TNF‐α), interleukin 1β (IL‐1β) and interleukin 6 (IL‐6) levels were detected by ELISA to determine whether VNS was successful. An inflammatory injury model in human coronary artery endothelial cells (HCAECs) was established by lipopolysaccharide and identified by evaluating TNF‐α, IL‐1β and IL‐6 levels and tube formation. Immunohistochemistry staining was performed to evaluate CD31‐positive vessel density and stromal cell‐derived factor‐l alpha (SDF‐1α) expression in the MI heart in vivo and the expression and distribution of SDF‐1α, C‐X‐C motif chemokine receptor 4 and CXCR7 in HCAECs in vitro. Western blotting was used to detect the levels of SDF‐1α, V‐akt murine thymoma viral oncogene homolog (AKT), phosphorylated AKT (pAKT), specificity protein 1 (Sp1) and phosphorylation of Sp1 in HCAECs. Left ventricular performance, including left ventricular systolic pressure, left ventricular end‐diastolic pressure and rate of the rise and fall of ventricular pressure, should be evaluated 28 days after VNS treatment. VNS was successfully established for MI therapy with decreases in serum TNF‐α, IL‐1β and IL‐6 levels and increases in cardiac tissue and serum ACh levels, leading to increased SDF‐1α expression in coronary endothelial cells of MI hearts, triggering angiogenesis of MI hearts with increased CD31‐positive vessel density, which was abolished by the m/nAChR inhibitors mecamylamine and atropine or knockdown of SDF‐1α by shRNA. ACh promoted SDF‐1α expression and its distribution along with the branch of the formed tube in HCAECs, resulting in an increase in the number of tubes formed in HCAECs. ACh increased the levels of pAKT and phosphorylation of Sp1 in HCAECs, resulting in inducing SDF‐1α expression, and the specific effects could be abolished by mecamylamine, atropine, the PI3K/AKT blocker wortmannin or the Sp1 blocker mithramycin. Functionally, VNS improved left ventricular performance, which could be abolished by Ad‐shSDF‐1α.

Conclusions

VNS promoted angiogenesis to repair the infarcted heart by inducing SDF‐1α expression and redistribution along new branches during angiogenesis, which was associated with the m/nAChR‐AKT‐Sp1 signalling pathway.

Introduction

Myocardial infarction (MI) shows the characteristics of autonomic nerve imbalance with an increased ratio of sympathetic nerve to vagus nerve activity, accompanied by an inflammatory response and recruitment of leukocytes, leading to myocardial injury.¹ Basic and clinical studies have shown that vagal nerve stimulation (VNS) balances autonomic nerve activity in injured hearts, providing a novel therapeutic strategy for autonomic dysfunction by activating cholinergic anti-inflammatory pathways.^1–4 However, whether VNS-mediated anti-inflammatory roles are beneficial to angiogenesis of the infarcted heart is still unclear.

Angiogenesis is essential to the surviving myocardium and the recovery of ventricular function after MI by reshaping the blood supply to the heart.⁵ The reduced levels of α7-nicotinic acetylcholine (ACh) receptor (α7-nAChR) expression hamper angiogenesis in ischaemic or infarcted hearts.^6,7 Indeed, coinciding with vagal innervations in the conduction system and coronary vessels of the heart,⁸ VNS increased the concentration of ACh in the ventricle.^9–11 Meanwhile, VNS restored and even enhanced the levels of α7-nAChR and m3-AChR expression in the injured heart,¹² indicating that VNS could have beneficial involvement in angiogenesis of infarcted heart through released ACh and its activated α7-nAChR and/or m3-AChR. Unfortunately, its potential mechanism has not been fully explored.

Our and other scholars' results have shown that vascular endothelial growth factor (VEGF) is involved in the angiogenesis of ischaemic or infarcted hearts.^12–14 Of interest, VEGF-A gene therapy alone does not effectively result in sufficient angiogenesis to match the improvement in cardiac function,¹⁵ implying that other underlying factors may also be required for angiogenesis in injured hearts. Indeed, in addition to typically regulating leukocyte trafficking, recent evidence has shown that chemokines are cardioprotective and participate in angiogenesis.^16-18 As a classical chemokine molecule, stromal cell-derived factor-l alpha (SDF-1α) can activate and/or induce the migration of haematopoietic/endothelial progenitor cells and endothelial cells, in addition to most leukocytes.^19–22 However, whether SDF-1α is involved in VNS-mediated anti-inflammatory effects and angiogenesis has yet to be determined.

In this study, rats with MI were treated with VNS, and increased SDF-1α expression and CD31-positive vessel density in the infarcted heart were found, which could be abolished by the m/nAChR inhibitors mecamylamine and atropine or knockdown of SDF-1α by shRNA. In vitro, ACh induced SDF-1α expression and its redistribution along the branch of the formed tube in human coronary artery endothelial cells (HCAECs), which could be related to the m/nAChR-AKT-Sp1 signalling pathway. Taken together, these results indicate that vagus nerve stimulation (VNS)-induced SDF-1α promotes angiogenesis and repair in infarcted hearts through the m/nAChR-AKT-Sp1 signalling pathway.

Methods

Animals

In accordance with the Guide for the Care and Use of Laboratory Animals published by China and US National Institutes of Health, adult male Sprague–Dawley (SD) rats (250–300 g) were supplied by the Experimental Animal Centre of the Hubei University of Medicine. All animal protocols were approved by the Institutional Animal Care and Use Committee of Hubei University of Medicine (HBUM2018-142).

Myocardial infarction model establishment

The left anterior descending coronary artery in rats was ligated to prepare the MI model as previously described.²³ In brief, ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) were used to anaesthetize SD rats (250–300 g). Descending coronary artery ligation was promptly performed after thoracotomy on the left fourth intercostal space after tracheal ventilation remained stable using a Colombus ventilator (HX-300, Taimeng Instruments, Chengdu, China). When confirming the occurrence of MI with blanching of the myocardium as well as electrocardiography, the open thoracic cavity was sutured layer by layer immediately.

Method of vagus nerve stimulation

Surviving rats were randomized into groups with sham or stimulation on the seventh day of MI. After the left vagal nerve in the neck was gently exposed and separated, the vagal nerve was looped with Teflon-coated stainless-steel wires (UL1330; Triumph Cable Co, Ltd, China) and ligated into the stimulator output part (BL-420S; Chengdu Tme Technology Co, Ltd, China) for electrical stimulation (20 Hz for 10 s every minute for 4 h) as previously described.¹³ Regular pulse stimulation in the vagal nerve was executed in the VNS group.¹³ Similar operations were performed without initiating stimulation in the sham group (MI). A 10% reduction in heart rate was used as a criterion for VNS. A mixture of white petrolatum (Vaseline) and paraffin was used to immerse the vagus nerve and connected electrodes to prevent drying.

Acetylcholine receptor inhibitor in vivo treatment before vagus nerve stimulation

One hour before VNS, mecamylamine (10 mg/kg, ip) or atropine (10 mg/kg, ip) was used to assess whether the role of VNS in SDF-1α expression in the infarcted heart was linked to mACh-R and α7-nAChR, as described previously.^12,13

Knockdown of stromal cell-derived factor-l alpha by shRNA in vivo treatment in myocardial infarction hearts before vagus nerve stimulation

SDF-1α shRNA (shSDF-1) was designed using a dedicated program provided by our published data.¹⁵ To determine the effect of SDF-1α on angiogenesis in the infarcted heart, Ad-shCtrl and Ad-shSDF-1α (1 × 10⁹ pfu in 200 μL) were injected into four sites of the infracted hearts (50 μL per site, 12 rats per group) with a 30-gauge tuberculin syringe 3 days before VNS. Two injections were in the myocardium bordering the ischaemic area, and two were within the ischaemic area. Penicillin (150 000 U/mL, i.v.) was given before each procedure. Buprenorphine hydrochloride (0.05 mg/kg, s.c.) was administered twice a day for the first 48 h after the procedure.

Measurement of coronary flow and haemodynamic parameters

Six SD rats (250–300 g)/group were anaesthetized with ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). A thoracotomy was performed, and hearts were rapidly excised into ice-cold perfusion fluid. The aorta was cannulated on a shortened and blunted 14-gauge needle, and perfusion initiated at a constant pressure of 80 mmHg on the Langendorff system. A fluid-filled balloon constructed from polyvinyl chloride film was introduced into the left ventricle through an incision in the left atrial appendage. Two tubes were introduced into the right atrial appendage through the superior and inferior caval veins. Hearts were immersed in warmed perfusate in a jacketed bath maintained at 37°C, and perfusate delivered to the coronary circulation was maintained at the same temperature using a super constant temperature water bath. The organ bath and perfusate temperatures were continuously monitored using a digital thermometer. Langendorff-perfused hearts were allowed to equilibrate until heart rate and contractility reached steady state for 30 min. Total coronary flow within 30 min was collected in the cylinder through tubes linked to the right atrial appendage for evaluating coronary flow percentage/min. Krebs–Henseleit perfusion fluid (containing NaCl, 119 mM; glucose, 11 mM; NaHCO₃, 22 mM; KCl, 4.7 mM; MgCl₂, 1.2 mM; KH₂PO₄, 1.2 mM; CaCl₂, 2.5 mM; EDTA, 0.5 mM; and pyruvate, 2 mM) was used in all experiments. The fluid with a pH of 7.4 was bubbled with a mixture of 95% O₂ and 5% CO₂ at 37°C and was filtered through a 0.45 μm filter before delivery to the heart.¹⁴

Twenty-eight days after VNS treatment, heart functions, including left ventricular systolic pressure, left ventricular end-diastolic pressure, and rate of the rise and fall of ventricular pressure (+dP/dt_max and –dP/dt_max), were evaluated as described previously.²³ Following anaesthesia for rats with the application of ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.), one end of the catheter was connected to a pressure transducer, and the other end of the catheter filled with heparinized (10 U/mL) saline solution was advanced into the left ventricle to record ventricular pressure while it was inserted into the isolated left carotid artery. After evaluating cardiac function, the heart was immediately collected for subsequent detection and analysis.

Heart tissue immunostaining

Serial transverse sections of heart tissue (5 μm) were prepared as previously described.¹⁵ Before adding primary antibodies, a blocking buffer (PBS containing 5% goat serum and 0.1% Triton X-100) was used to treat these sections at room temperature for 1 h. The primary antibodies (diluted in blocking buffer), including goat anti-rat SDF-1α (sc-6193, 1:150; Santa Cruz), mouse anti-rat CD31 (ab24590, 1:250; Abcam), rabbit anti-rat CD68 (1:250, GB11067, ServiceBio), and rabbit anti-rat peNOS (#9575, 1:250, CST), were incubated at 4°C overnight; then, the secondary antibodies, including horseradish peroxidase (HRP)-labelled goat anti-mouse IgG, goat-anti-rabbit IgG, FITC-conjugated anti-rabbit IgG, or TRITC-conjugated anti-mouse IgG (Jackson ImmunoResearch), were incubated at room temperature for 2 h.¹⁵ Eventually, these indicated results were quantified by densitometry analysis (Image Pro, USA) after taking pictures under a microscope (MF43-N, Olympus, Japan).

Establishment of an inflammatory injury model in human coronary artery endothelial cells induced by lipopolysaccharide

HCAECs (Jennio Biotech Co., Ltd., Guangzhou, China) were cultured in complete medium for 24 h as previously mentioned.¹⁴ Then, lipopolysaccharide (LPS, 1 mg/μL) was added to the medium for 24 h. Successfully embellishment of the LPS-induced HCAEC inflammatory injury model in vitro was confirmed by evaluation of HCAEC-mediated tube formation and inflammatory responses. Evaluation of tube formation was shown in the HCAEC tube formation method. Inflammatory responses were assessed by detecting NF-κB p65 by using immunofluorescence staining and tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) by using ELISA.^24,25

Human coronary artery endothelial cells tube formation

HCAEC tube formation was carried out as described,²⁶ and HCAECs (4 × 10⁵/mL) were cultured in 24-well plates coated with Matrigel™ for 12 h. To confirm the relationship between ACh and LPS during angiogenesis, ACh was added to the tube formation medium with or without LPS. To further confirm whether the role of ACh in HCAEC tube formation is associated with mACh-R and α7-nAChR, atropine (1 μM) or mecamylamine (7 μM) was used for 30 min before changing the tube formation medium. To ensure the reliability of the results, three independent experiments were performed. Three duplicated wells/groups were executed every time. The total number of tube branches (in pixels) per well was quantified using the Image-Pro Plus software package (Media Cybernetics, Carlsbad, CA) and averaged. The cells were finally fixed in formalin, washed with PBS, and imaged using microscopy.

Processing method of distribution characteristics of stromal cell derived factor 1 alpha/C-X-C motif chemokine receptor 4/7 in human coronary artery endothelial cells

The processing method of the distribution characteristics of SDF-1α/C-X-C motif chemokine receptor 4/7 (CXCR4/7) in HCAECs precultured in Matrigel was as follows: After HCAECs were cultured with complete medium in 6-well plates coated with Matrigel™ for 24 h, the cells were digested, collected and inoculated into 24-well plates with tube formation medium for 12 h. To confirm the relationship between ACh and LPS during angiogenesis, ACh was added to the tube formation medium with or without LPS. To further confirm whether the role of ACh in the expression and distribution characteristics of SDF-1α and CXCR4/7 is associated with mACh-R and α7-nAChR, atropine (1 μM) or mecamylamine (7 μM) was used for 30 min before changing the tube formation medium. The cells were finally fixed in formalin, washed with PBS, and subjected to immunofluorescence staining of SDF-1α/CXCR4/7. The cells were imaged to observe the expression and distribution characteristics of SDF-1α and CXCR4/7. Three independent experiments were performed.

The distribution characteristics of SDF-1α/CXCR4/7 in HCAECs were not precultured in Matrigel as follows: After HCAECs were cultured with complete medium in 6-well plates for 24 h, the cells were digested, collected and inoculated into 24-well plates with complete medium for 24 h. To confirm the relationship between ACh and LPS during angiogenesis, ACh was added to the tube formation medium with or without LPS. To further confirm whether the role of ACh in the expression and distribution characteristics of SDF-1α and CXCR4/7 is associated with mACh-R and α7-nAChR, atropine (1 μM) or mecamylamine (7 μM) was used for 30 min before changing the complete medium. The cells were finally fixed in formalin, washed with PBS, and subjected to immunofluorescence staining of SDF-1α/CXCR4/7. The cells were imaged to observe the distribution characteristics of SDF-1α and CXCR4/7. Three independent experiments were performed.

Immunofluorescence staining

HCAECs were incubated in blocking buffer (PBS containing 5% goat serum and 0.1% Triton X-100) at room temperature for 1 h. Incubation with antibodies (diluted 1:250 in blocking buffer) was performed at 4°C overnight for primary antibodies and at room temperature for 2 h for secondary antibodies. The primary antibodies used were mouse/rabbit anti-human SDF-1α (ab18919, 1:250; Abcam; sc-74271, 1:150, Santa Cruz), rabbit anti-human CXCR4 (sc-74271, NB100-74396, 1:300, NOVUS), rabbit anti-human CXCR7 (GB11256, 1:250; Servicebio), and WGA (L4895, 1:500, Sigma). The secondary antibodies were FITC-conjugated anti-rabbit IgG or TRITC-conjugated anti-mouse IgG (Jackson ImmunoResearch) (Tang et al., 2011; van Gils et al., 2013). Double staining of SDF-1α and CXCR4/7 was used to confirm the relationship of distribution between SDF-1α and CXCR4/7 during tube formation. Two rounds of wheat germ agglutinin (WGA-FITC) and SDF-1α/CXCR4/7 were used to confirm the indicated protein internalization and trafficking.²⁷

Administration of inhibitors or adenovirus overexpression in human coronary artery endothelial cells

HCAECs (Jennio Biotech Co., Ltd., Guangzhou, China) were cultured in complete medium for 48 h. To further assess the mechanisms underlying ACh-induced SDF-1α expression in HCAECs, the mACh-R blocker atropine (1 μM) or the nACh-R blocker mecamylamine (7 μM), the PI3K/AKT blocker wortmannin (50 nM), the MEK/ERK1/2 inhibitor PD98059 (50 μM), the p38MAPK inhibitor SB203580 (30 μM) and the Sp1 blocker mithramycin (1 μM, Sp1-I) were inoculated for 1 h before treatment with 10⁻⁵ M ACh for 24 h. To further assess the role of Sp1 in ACh-induced SDF-1α expression in HCAECs, HCAECs were transfected with Ad-Sp1 at 100 MOI (Vigenebio, Shandong, China) for 24 h before treatment with 10⁻⁵ M ACh for 24 h. For subsequent target molecular detection, harvested cells were lysed in RIPA buffer containing protease and phosphatase inhibitors.

ELISA

ELISA for ACh was performed in cardiac tissue and serum treated with the cholinesterase inhibitor eserine (100 μM) after VNS using a commercial kit by following the manufacturer's protocol (ab65345, Abcam). ELISA for TNF-α (900-M73, PeproTech), IL-6 (900-M86, PeproTech) and IL-1β (900-M91, PeproTech) was performed in cardiac tissue and serum after VNS using a commercial kit by following the manufacturer's protocol. TNF-α (PT518, Beyotime, CN), IL-6 (PI330, Beyotime, CN) and IL-1β (PI305, Beyotime, CN) in the supernatant of HCAECs were detected by ELISA following the manufacturer's protocol.

Measurement of vascular density

The numbers of CD31-positive vessels in each section were analysed in 6 ~ 8 equally distributed areas of 0.1 mm² in the peri-infarction or infarction area. The values were then expressed as the number of vessels per square millimetre. Two independent examiners analysed these results using the Image-Pro Plus software package (Media Cybernetics, Carlsbad, CA).²⁸

Western blot

Western blotting was performed as previously described.²⁹ Fifty micrograms of protein was resolved in a 10% SDS–PAGE gel and transferred onto a polyvinylidene fluoride membrane (Millipore). After blocking with 5% nonfat milk, the membrane was incubated overnight with primary antibodies, including rabbit anti-human SDF-1α (ab18919, 1:1000; Abcam), mouse anti-human SDF-1α (sc-74271, 1:500; Santa Cruz), Sp1 (sc-420, 1:500; Santa Cruz), rabbit anti-human p-Sp1 (ab37707, 1:1000; Abcam), rabbit anti-human V-akt murine thymoma viral oncogene homolog (AKT) (9272S, 1:500; Cell Signaling), rabbit anti-human phosphorylated AKT (pAKT) (9271S, 1:500; Cell Signaling), and mouse anti-human α-tubulin (YM3035, 1:500, Immunoway). Then, incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG, anti-mouse IgG, 1:10 000, Jackson ImmunoResearch) was carried out for 90 min. Eventually, the expression of the indicated proteins was quantified by densitometry analysis (Image Pro, USA) after visualization with an enhanced chemiluminescence reaction (Amersham Pharmacia Biotech).²⁹

Transmission electron microscopy

Cardiac tissues were collected for transmission electron microscopic observation of endothelial cell morphology and its linkage and myocardial cell mitochondrial structure according to the manufacturer's protocol (Servicebio, CN).

Statistical analyses

Data shown are the mean ± SD. Statistical significance between two groups was determined by paired or unpaired Student's t test. The results for more than two experimental groups were evaluated by one-way ANOVA to specify differences between groups. P < 0.05 was considered significant.

Results

Vagus nerve stimulation promotes angiogenesis in the infarcted heart through m/n-ACh-R

To first identify whether VNS was successful in the rat MI model, ACh levels in heart tissues and serum were detected by ELISA, showing that ACh levels were increased after VNS (Figure 1A,B), indicating that VNS caused the cardiac vagus nerve to release more ACh. To further confirm whether VNS was successful in the rat MI model, TNF-α, IL-1β and IL-6 levels, as typical markers for the anti-inflammatory role of VNS in MI hearts, were analysed by ELISA, showing that TNF-α, IL-1β and IL-6 levels were obviously decreased in MI hearts treated with VNS (Figure 1C–E). These results demonstrated that VNS model establishment was successful for MI therapy.

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Subsequently, to explore the role of VNS in angiogenesis in the infarcted heart, we used immunohistochemical staining for the endothelial cell marker CD31. As shown in Figure 1F,G, VNS increased the numbers of CD31-positive vessels in the infarction area and peri-infarction area of infarcted hearts, indicating that VNS induced angiogenesis in MI hearts. Meanwhile, VNS improved CD31-positive cell function characterized by increased eNOS levels (Figure 2A–C). Furthermore, transmission electron microscopy showed that VNS improved EC function with a relatively complete cell membrane and normal intercellular space (Figure 2D), resulting in the recovery of the morphology and structure of myocardial mitochondria adjacent to ECs (Figure 2E). These results demonstrated that successful VNS treatment promoted angiogenesis in the infarcted heart.

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Finally, to further confirm whether VNS-induced angiogenesis involved cholinergic receptors, the m-AChR inhibitor atropine (0.5 mg/kg, ip) and the α7-nAChR mecamylamine (1.0 mg/kg, ip) were used 1 h before VNS. As shown in Figure 3A–C, atropine and mecamylamine obviously abolished the specific effects of VNS on angiogenesis (Figure 3A–D). These results demonstrated that VNS promoted angiogenesis in the infarcted heart through m/n-ACh-R.

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Vagus nerve stimulation-induced angiogenesis involves stromal cell derived factor 1 alpha in the infarcted heart through m/n-ACh-R

Previous reports have shown that SDF-1α could play a crucial role in angiogenesis in the infarcted heart.³⁰ We first found that VNS induced SDF-1α expression in CD31-positive cells, especially in epicardial endothelial cells (Figure 4A–C), leading to increased numbers of CD31-positive vessels in the infarcted hearts (Figure 3). Then, to confirm whether VNS-induced SDF-1α expression involved cholinergic receptors, the m-AChR inhibitor atropine (0.5 mg/kg, ip) and the α7-nAChR mecamylamine (1.0 mg/kg, ip) were used 1 h before VNS. As shown in Figure 4A–C, atropine and mecamylamine obviously abolished the specific effects of VNS on SDF-1α, indicating that m/n-AChR was involved in VNS-induced SDF-1α expression in coronary vessels. These results suggested that VNS-induced angiogenesis involves SDF-1α in the infarcted heart through m/n-ACh-R.

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Stromal cell derived factor 1 alpha is involved in vagus nerve stimulation-induced angiogenesis in the infarcted heart

To further determine whether SDF-1α is involved in VNS-induced angiogenesis in the infarcted heart, local injection of Ad-shSDF-1α into the infarcted hearts was performed 3 days before MI. As shown in Figure 5A,B, application of Ad-shSDF-1α effectively knocked down SDF-1α expression in the infarcted hearts. Furthermore, with the knockdown of SDF-1α, we found that Ad-shSDF-1α significantly reduced the CD31-positive vascular density (Figure 5C,D). Functionally, the beneficial effects of VNS on coronary blood flow (Figure 5E) could be eliminated by the application of Ad-shSDF-1α. These data indicated that VNS-induced SDF-1α expression promoted angiogenesis through m/n-ACh-R.

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Acetylcholine promoted human coronary artery endothelial cells tube formation while inhibiting the inflammatory response in vitro through m/n-AChR

To simulate the inflammatory reaction of MI, LPS (1 mg/μL) was added to the culture medium of HCAECs for 24 h. Nuclear translocation of NF-κB p65 and the levels of TNF-α, IL-1β and IL-6 were evaluated to identify whether the HCAEC inflammatory injury model was successfully established. As shown in Figure 6A,B, LPS promoted NF-κB p65 nuclear translocation in HCAECs, leading to an increase in TNF-α, IL-1β and IL-6 levels (Figure 6C–E). Meanwhile, LPS inhibited HCAEC tube formation in vitro (Figure 6F,G). These data indicated that the LPS-mediated HCAEC inflammatory injury model was successfully established.

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Subsequently, to confirm whether VNS and the neurotransmitter ACh promoted angiogenesis, ACh was added to the HCAEC tube formation model in vitro to mimic the role of VNS in angiogenesis in vivo. As shown in Figure 6E,F, ACh in vitro completely reversed the inhibitory effect of LPS on tube formation, and the specific effect could be abolished by atropine or mecamylamine, indicating that ACh in vitro, similar to VNS in vivo, promoted angiogenesis in the inflammatory damage environment of MI through m/n-ACh-R.

Acetylcholine induced stromal cell derived factor 1 alpha distribution along new branches during tube formation

In addition to SDF-1α expression related to angiogenesis, immunofluorescence staining showed that SDF-1α was distributed along new branches of tube formation. Furthermore, ACh enhanced the specific distribution of SDF-1α during tube formation (Figure 7A,B). Under LPS stimulation, the typical characteristics of an SDF-1α distribution were altered, showing the distribution around the nucleus within round, conical, rod-shaped HCAECs. More importantly, ACh recovered the LPS-induced SDF-1α redistribution along new branches during the formation of the vascular ring (Figure 7A,B). These effects could be markedly cancelled by atropine or mecamylamine. These results suggested that ACh promoted angiogenesis by restoring SDF-1α distribution along new branches.

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Acetylcholine induced C-X-C motif chemokine receptor 4 distribution along a parallel direction with stromal cell derived factor 1 alpha through m/n-AChR

To confirm the relationship between ACh and CXCR4 during HCAEC tube formation mediated by SDF-1α, double staining of SDF-1/CXCR4 by immunofluorescence showed CXCR4 distribution within normal HCAECs along a parallel direction with SDF-1α. LPS treatment altered CXCR4 distribution along the branch direction of tube formation; ACh could substantially eliminate the inhibitory role of LPS in CXCR4 distribution (Figure 8A,B). Furthermore, the ACh-mediated effect on CXCR4 distribution could be memorably abolished by atropine or mecamylamine. These results suggested that ACh recovers CXCR4 distribution in parallel with SDF-1α during tube formation through m/n-AChR.

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CXCR7, another receptor for SDF-1α, showed changes similar to those of CXCR4 when facing LPS treatment with or without ACh, with the addition of atropine or mecamylamine (Figure S1).

Acetylcholine promoted stromal cell derived factor 1 alpha expression in human coronary artery endothelial cells through the AKT-Sp1 signalling pathway

To explore the possible molecular mechanism of VNS in SDF-1α expression, gene chip analysis was previously performed, showing that the PI3K-AKT signalling pathway was obviously altered in infarcted hearts following treatment with VNS and ACh.¹³ We first found that ACh dose-dependently increased the protein level of SDF-1α (Figure S2). Further findings showed that ACh did not alter the total AKT level but significantly increased the phosphorylated AKT level in HCAECs (Figure 9A,B). Moreover, the typical PI3K/AKT inhibitor wortmannin decreased ACh-mediated SDF-1α expression (Figure 7A,B). Meanwhile, ACh obviously enhanced the levels of p-Sp1 in HCAECs along with ACh-induced AKT phosphorylation (Figure 9C,D), and the specific role could almost be entirely blocked by atropine or mecamylamine (Figure 9C,D). Importantly, ACh-induced SDF-1α expression could almost entirely be abolished by the Sp1-specific inhibitor mithramycin A, while Sp1 overexpression enhanced ACh-induced SDF-1α expression (Figure 9E–H), indicating that ACh increased Sp1 phosphorylation and activity through pAKT. These results suggested that ACh-induced SDF-1α expression is involved in the m/nAChR/AKT/Sp1 signalling pathway.

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Vagus nerve stimulation and acetylcholine improved cardiac function

Functional analysis was performed to explore whether VNS-induced angiogenesis improved infarcted heart function. The results showed that coronary blood flows and LV function, including left ventricular systolic pressure, left ventricular end-diastolic pressure, +dP/dt_max, and –dP/dt_max, was significantly improved in VNS-treated hearts compared to MI-treated hearts. SDF-1α shRNA, however, abolished VNS-improved LV function (Figure 10A–D, Figure S3).

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Discussion

Our studies made three novel observations. First, we found that VNS-induced SDF-1α expression regulated angiogenesis in infarcted hearts. Second, ACh recovered SDF-1α/CXCR4 distribution along new branches during the formation of the vessel. Finally, SDF-1α expression was regulated by the ACh/m/nAChR/AKT/Sp1 signalling cascade.

Accumulated data have shown that vigorous inflammatory responses generally cause endothelial dysfunction in various human diseases, including MI. As a typical phenotype of endothelial dysfunction, the disruption of endothelial cell-branching morphogenesis and angiogenesis are evidently triggered by inflammatory cytokines such as IL-1β, IL-6, IFN-γ, and/or TNF-α.^31,32 Indeed, IL-1β and IL-6 promote SDF-1α expression, while IFN-γ and TNF-α reduce its expression in various cells,^31,32 indicating that inflammatory cytokines should positively and negatively control SDF-1α levels. Herein, accompanied by a decrease in these inflammatory cytokines, we further found that VNS induced SDF-1α expression in endothelial cells of infarcted hearts. This is in accordance with a recent report that SDF-1α acted as an anti-inflammatory chemokine during autoimmune inflammatory responses,³³ suggesting that VNS and the vagal neurotransmitter ACh, which exert an anti-inflammatory effect, could have an independent role in SDF-1α expression. Initially, we provide novel evidence that ACh induces SDF-1α expression in CECs under noninflammatory conditions. More importantly, VNS-induced SDF-1α in infarcted hearts participated in the enhanced process of angiogenesis similar to SDF-1α-mediated microvascular network remodelling.³⁴ Significantly, VNS induced the differential expression distribution characterized by SDF-1α in the epicardium and VEGF in the endocardium. SDF-1α has unique effects on promoting vascular reassembly in the infarcted heart,³⁵ and the combined effect of epicardial SDF-1α and endocardial VEGF on cardiomyocyte replenishment initially occurs through the guidance of coronary sprouting.³⁶ In summary, VNS could play a unique role in the angiogenesis of the infarcted heart by inducing SDF-1α and VEGF expression.

Indeed, ECs in the heart contain both m1 and m3-AChR apart from α7-nACh-R, the selective m1AChR inhibitor pirenzepine, and the selective m3AChR inhibitor 4-DAMP, which could better prove the role of VNS and ACh in SDF-1α expression and angiogenesis. Herein, we used the nonselective mAChR inhibitor atropine and found that atropine effectively eliminated VNS- and ACh-induced SDF-1α expression and angiogenesis, indirectly indicating that atropine could block m1 and m3AChRs. More accurate experiments in the future need to be designed to prove the relationship between ACh and m1/m3AChR in ECs. Meanwhile, using a selective α7-nACh-R inhibitor, memamylamine, we found that atropine and mecamylamine abolished the effect of VNS and ACh on SDF-1α expression and angiogenesis. In any case, we draw a preliminary conclusion that ACh induced SDF-1α expression and angiogenesis through m/n-AChR.

Published data have shown that NF-κB, PI3K/AKT, p38 MAPK, ERK1/2, and eNOS signalling can regulate SDF-1α expression in ECs, endothelial progenitor cells (EPCs), and mesenchymal stem cells.^37–42 In fact, in infarcted hearts, apart from nutrient deficiency, hypoxia is an important characteristic that induces SDF-1α expression and cell migration through hypoxia inducible factor 1 alpha (HIF1α) and NF-κB p65.^43–46 ACh acted as a cardioprotective agent by increasing HIF1a levels in the infarcted heart, suggesting that HIF1a-mediated SDF-1α expression may likely participate in the process of repairing the infarcted heart. Herein, under normoxic conditions, P13K/AKT signalling activated by ACh is involved in SDF-1α expression. Similar to a previous report that pAKT could phosphorylate transcription factor Sp1 and increase its activity,⁴⁷ increased pAKT levels in ACh-treated HCAECs induced phosphorylation of Sp1, indicating that ACh promoted SDF-1α expression through the P13K/AKT-Sp1 signalling pathway.

In addition, increased LPS in the infarcted heart significantly inhibited angiogenesis and damaged the structure and function of the heart.^{24,25,48–50} After VNS, gene chip analysis showed a pronounced decrease in LPS-induced inflammatory responses in infarcted hearts in vivo, accompanied by an increase in angiogenesis.¹³ Herein, we found that ACh induced SDF-1α expression and tube formation in LPS-injured HCAECs.

CXCR4, as a receptor for SDF-1α, is the only chemokine receptor for which knockout mice die perinatally.⁵¹ Mice lacking CXCR4 show lethally defective cardiac ventricular septa and embryonic haematopoiesis and neurogenesis, a phenotype similar to that of SDF-1α knockout mice.⁵² Of interest, unlike previous results showing that LPS upregulated SDF-1α and CXCR4 expression in macrophages and fibroblast cells,^53,54 LPS decreased SDF-1α and CXCR4 expression in HCAECs. ACh abolished the inhibitory effect of LPS on SDF-1α and CXCR4 expression, and a m/n-AChR blocker abolished these specific effects (Figures S4, S5 and S6). More importantly, ACh did not alter SDF-1α and CXCR4/7 distribution within HCAECs in proliferation medium (Figures S3, S4, and S5). However, under the vascular tube formation system, we found for the first time that ACh altered the distribution pattern of SDF-1α and its receptor CXCR4 in HCAECs. LPS reconstituted the distribution of SDF-1α around the nucleus accompanied by CXCR4, not CXCR7, leading to disorganization of endothelial cell-branching morphogenesis. ACh partially reversed these effects. Meanwhile, ACh-mediated SDF-1α redistribution and angiogenesis could be abolished by a m/n-AChR blocker. These results suggested that VNS and the vagal neurotransmitter ACh promoted angiogenesis and repaired the infarcted heart by altering the expression and distribution of SDF-1α/CXCR4.

Limitations

Clinically, VNS has attracted much attention due to its cholinergic anti-inflammatory and anti-adrenergic effects on MI and HF. Herein, we provide novel evidence that the effect of VNS on MI and HF is attributed to the unique synergy of epicardial SDF-1α and endocardial VEGF,^13,14 especially in the coronary artery. In the past, we used the combined treatment of VEGF and SDF-1α genes to better repair MI,⁵⁵ but how VNS cooperates with the expression pattern of VEGF and SDF-1α in the endocardium and epicardium to facilitate the repair of MI still needs to be further clarified in the future. In addition, using wired and radio-controlled pulse generators to stimulate the vagal nerve, the progression of MI and HF has been obviously delayed, with a better prognosis of HF.^1–4 Thus, whether VNS in different ways is equivalent to driving synergetic expression patterns of epicardial SDF-1α and endocardial VEGF to facilitate the repair of MI still needs further clarification in the future. The solution of these problems will be more conducive to promoting the clinical research and application of VNS in the treatment of MI and HF.

Taken together, our studies demonstrated that VNS improves heart function by promoting angiogenesis in the infarcted heart via activation of SDF-1α signalling, which was regulated by the P13K/AKT-Sp1 signalling cascade (Figure 11).

[IMAGE OMITTED. SEE PDF]

Author contributions

Y.W., X.Y.L. and Y.L. carried out the main animal and cell experiments and drafted the manuscript. L.L.S. and Y.X.L. carried out the animal experiments. X.B. and L.Y. carried out protein detection; X.B. and Y.W. carried out the HCAEC cultures. L.Y.Y. and L.C. participated in the immunoassaying. J.X.Z. and S.J.C. carried out data evaluation. S.Y.C. and M.N.M. helped draft and revise the manuscript. J.M.T., J.L. and L.L.S. conceived of the study, participated in the experimental design and coordination of the study, and helped draft the manuscript. All authors read and approved the final manuscript.

Conflict of interest

The authors declare that they have no competing interests.

Funding

This study was supported by grants from the Hubei Provincial Technology Innovation Project (2018ACA162 and 2021DFE026 to J.M.T.), the Foundation of Hubei University of Medicine (HBMUPI201807 and FDFR201601 to J.M.T.), Hubei Province's Outstanding Medical Academic Leader Program, the National Natural Science Foundation of China (81670272 and 82270299 to J.M.T. and 82000285 to J.X.Z.) and the Natural Science Foundation of Hubei Province (2022CFB934 to L.L.S.). [Correction added on 28 September 2023, after first online publication: The funding number of ‘Natural Science Foundation of Hubei Province’ has been corrected from ‘2020CFB624 to Y.W’ to ‘2022CFB934 to L.L.S.’ in this version.]

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