Elabela gene therapy promotes angiogenesis after myocardial infarction

Abstract This study was aimed at investigating whether Elabela (ELA) gene therapy can promote angiogenesis in the treatment of myocardial infarction (MI). The fusion expression plasmid pAAV‐3 × Flag/ELA‐32 was successfully constructed using molecular cloning technique. The model of acute MI was established by ligating the left anterior descending coronary artery in mice. Adeno‐associated virus serotype 9 (AAV9) was injected into the surrounding myocardium and tail vein immediately after the model was established. AAV was injected again from the tail vein one week later. Compared with the MI+PBS (control) group, the serum N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) concentration, and the values of left ventricular end‐diastolic diameter (LVDd) and left ventricular end‐systolic diameter (LVDs) of the MI+AAV‐ELA (gene therapy) group were significantly decreased, while the value of left ventricular ejection fraction was significantly increased at 2 and 4 weeks after operation. Compared with the control group, the expression of CD105 and vWF and the percentage of CD31‐ and Ki67–co‐positive cells were significantly increased in the gene therapy group. Moreover, the expressions of apelin peptide jejunum (APJ) receptor, vascular endothelial growth factor (VEGF), VEGFR2, Jagged1 and Notch3 in the heart tissue around the infarction were up‐regulated in mice with gene therapy. The results suggest that ELA activates VEFG/VEGFR2 and Jagged1/Notch3 pathways through APJ to promote angiogenesis after myocardial infarction. ELA gene therapy may be used in the treatment of ischaemic cardiomyopathy in future.

therapeutic strategy for myocardial infarction. The basis for therapeutic angiogenesis is to improve myocardial perfusion and cardiac function by promoting the formation of neovascularization in ischaemic areas of the heart. 2 Elabela (apela or toddler) is a newly discovered non-coding RNA transcription gene. This gene can transcribe and translate to produce a 55-amino acid secretory hormone. After enzyme cleavage, it forms a 32-amino acid mature polypeptide . Through the study of zebrafish, a model animal, it was found that the hormone acts on the apelin peptide jejunum (APJ) receptor as apelin and plays an important role in the development of the embryonic cardiovascular system. ELA appears earlier than apelin in the process of embryonic development, and it is the earliest ligand acting with the APJ receptor. The embryos with ELA gene or APJ gene knockout had a similar phenotype of cardiac developmental malformation, suggesting that ELA plays an important role in embryonic cardiovascular development. 3,4 A basic research showed that ELA-32 could not only promote the formation of vascular tubular structures in human umbilical vein endothelial cells in vitro but also improve the cardiac function of mice with acute myocardial infarction (MI). 5,6 This study further explored whether ELA-32 gene therapy can promote angiogenesis in the treatment of myocardial infarction.

| Establishment of myocardial infarction model in mice
Eight-week-old male SPF-grade C57BL/6 mice were purchased from Model Animal Research Center of Nanjing University. Mice were raised in 22℃ ± 2℃ environment, with food and water available ad libitum.
After anaesthesia, the skin and subcutaneous tissue were cut along the anterior midline of the neck of the mice, the tracheal intubation was inserted, and the ventilator was connected to control the breathing (90-100 breaths/min, tidal volume 0.4-0.5 ml). The chest was cut longitudinally to expose the heart. The left anterior descending coronary artery was ligated with a 7-0 suture about 1.5 mm below its junction, and the skin was sutured gradually. After waking up, the mice were extubated and observed for 15 min. Animal experiment was approved by the Committee on Ethics in the Care and Use of Laboratory Animals of Nanjing Medical University.

| Grouping of experimental animals
The experiment was divided into the following groups: (1) the sham group, wherein the heart of mouse was exposed without ligation; (2) the MI+PBS group, wherein 100µl PBS was injected into the tail vein and the borders of the infarction immediately after successful modelling and another 100 μl PBS was injected into the tail vein one week later in the same MI mouse; and (3) the MI+AAV-ELA group, wherein 100 μl 6 × 10 11 genome copies/animal AAV-ELA was injected into the tail vein and the borders of the infarction immediately after successful modelling and another 100 μl 6 × 10 11 genome copies/animal AAV-ELA was injected into the tail vein one week later in the same MI mouse. In our present study, the injected AAV dose was calculated according to the animal bodyweight (3 × 10 13 genome copies/kilogram/animal). 7 The average weight of the 8-week-old mice was 20 g; thus, the viral copy number used was determined as 6 × 10 11 genome copies/animal.

| Echocardiography examination
Echocardiography was performed at 2 and 4 weeks after model preparation, as previously described. The mice were anaesthetized with 1.5%-2% isoflurane and fixed in supine position to maintain sponta-

| Detection of N-terminal pro-brain natriuretic peptide (NT-proBNP)
Blood samples of mice were collected before modelling and 2 weeks and 4 weeks after intervention. Serum endogenous NT-proBNP (Cloud-Clone Corp) was measured by ELISA according to the manufacturer's instructions.

| Immunohistochemical and immunofluorescence test
The dried paraffin sections were dewaxed with xylene and hydrated Real Time PCR System (Applied Biosystems). Analysis of relative gene expression was performed using the comparative Ct method.

| Western blot
The cultured cells or heart tissue in each group was lysed in RIPA buffer. The protein concentration was detected using a BCA protein quantitative detection kit. The protein sample (30 μg) was loaded on a 10% SDS-PAGE gel. Electrophoresis was performed, followed by electrophoretic transfer of the proteins to a PVDF membrane.
After blocking with 5% skim milk, the membrane was incubated of each band was quantified using Quantity One software.

| Statistical analyses
Data were analysed using GraphPad Prism 8 statistical software. Data of normal distribution were presented as mean ± SEM. A non-paired t test was used in the comparison of fewer than three groups. Statistical differences among more than three groups were evaluated by one-way ANOVA and Tukey's test. p < 0.05 was considered statistically significant.

| The expression of 3×Flag/ELA-32 after AAV intervention in vivo
At the fourth week after the establishment of the MI model and AAV intervention, total RNA and protein were extracted from heart tissue of experimental mice for RT-PCR ( Figure 2A) and Western blot ( Figure 2B), respectively. The results showed that 3 × Flag/ELA was expressed only in the hearts of MI mice intervened by AAV-3 × Flag/ ELA injection. Further, 3 × Flag/ELA was not detected in the hearts of mice in the MI +PBS or sham group. In addition, total RNA was extracted from the heart, kidney, liver, brain, lung and testis tissue of MI mice 4 weeks after AAV9 injection. RT-PCR analysis showed that the expression of 3 × Flag/ELA mRNA was strongest in the heart with mild expression found in the liver and there was no expression in lung, kidney, brain and testis ( Figure 2C).

| ELA gene therapy improves cardiac function in mice with myocardial infarction
As shown in Figure 3, there was no significant difference in values of LVDd ( Figure 3C), LVDs ( Figure 3B) and LVEF ( Figure 3D) and serum levels of NT-proBNP ( Figure 3A) before operation among the groups. At

| ELA gene therapy promotes angiogenesis in mice with myocardial infarction
At the fourth week after intervention, the hearts of the three groups of mice were taken out for immunostaining examination.

| ELA gene therapy increases VEGF/ VEFGR2 and Jagged1/Notch3 expression in mice with myocardial infarction
At the fourth week after the intervention, the hearts of the three groups of mice were taken out to extract the total protein for Western blotting analysis ( Figure 5A). In MI+PBS and MI+AAV-ELA groups, the myocardial tissues around the infarction were extracted.
The expression levels of the ELA receptor APJ ( Figure 5B), VEGF ( Figure 5C) and its receptor VEGFR2 ( Figure 5D) and Jagged1 and its receptor Notch3 were detected. The expression of APJ, VEGF, VEGFR2, Jagged1 ( Figure 5E) and Notch 3 ( Figure 5F) was higher in the MI+PBS group than in the sham group. After ELA gene therapy,

| DISCUSS ION
Ischaemic cardiomyopathy is among the main causes of heart failure. In addition to drug therapy, percutaneous coronary revascularization and surgical coronary artery bypass grafting are also the main treatment methods. However, drug treatment has side effects, and minimally invasive intervention and surgery also have complications, such as in-stent restenosis and coronary bridge occlusion. 8 Therefore, cardiologists have been trying to treat Stem cell transplantation has seen new progress in the treatment of ischaemic cardiomyopathy in recent years, and good re-

sults have been achieved in animal experiments. Researchers in
France also reported the application of embryonic stem cells in the treatment of patients with end-stage heart failure. 9 However, issues such as ethical and oncogenic controversy limit the clinical application. 10 Gene therapy is a hot spot in the treatment of ischaemic cardiomyopathy. Plasmids carrying genes encoding antioxidant, endothelial nitric oxide synthase, mitogen protein kinase, hepatocyte growth factor and vascular endothelial growth factor were transfected into animals via an adenovirus, and biological effects, such as heart protection and angiogenesis, were The apelin/APJ system is involved in the regulation of the vascular diameter during angiogenesis in apelin-knockout mice. 12 The animal model of myocardial ischaemia also showed that apelin could up-regulate the expression of phosphorylated endothelial nitric oxide synthase and vascular endothelial factor (VEGF) in ischaemic myocardium. 13 The biological effects of apelin on mitosis and angiogenesis of endothelial cells suggest that apelin has a potential role in therapeutic angiogenesis. 14  In this study, the AAV-ELA plasmid was successfully constructed by gene recombination technology. After injected into mice via the tail vein and local intra-myocardial injection, the cardiac function of mice with myocardial infarction was significantly improved, and the angiogenesis around the infarction was promoted. In our previous study, we continuously injected exogenous recombinant ELA in mice with MI by osmotic pump. We found that ELA inhibited myocardial fibrosis and apoptosis of myocardial and F I G U R E 5 ELA gene therapy increases VEGF/VEFGR2 and Jagged1/Notch3 expression in mice with myocardial infarction. AAV was injected into the surrounding myocardium and tail vein immediately after the model was established. Then, AAV was injected again from the tail vein one week later. The expression of apelin peptide jejunum (APJ) receptor (A and B), vascular endothelial growth factor (VEGF, A and C), VEGFR2 (A and D), Jagged1 (A and E) and Notch3 (A and F) from the indicated group (each group, n = 5) was detected by Western blot assay at 4 weeks after operation.
Values are mean ± SEM. *p < 0.05, compared with the sham group;**p < 0.01, compared with the MI+PBS group kidney cells and improved the heart and kidney function of mice with MI. 6 Considering the multiple effects of ELA on the cardiovascular system, we speculate that cardiac function improvement is related to promoting angiogenesis, inhibiting myocardial fibrosis and apoptosis of myocardial cells.
We further observed that the expression of VEGF and VEGFR2 in the myocardial infarction control group was slightly up-regulated, while that in the Ela gene therapy group was significantly up-regulated. VEGF is a highly specific vascular endothelial growth factor, which can promote vascular permeability and endothelial cell migration, proliferation and angiogenesis.
However, it also mediates angiogenesis in tumour growth, metastasis, diabetic retinopathy, myocardial ischaemia and other pathological states. 23 VEGFR belongs to the receptor tyrosine protein kinase family and has five isomers. VEGFR2 is mainly expressed by endothelial cells and endothelial progenitor cells.
The biological function of VEGF is mainly exercised by its interaction with VEGFR2. VEGF was expressed at low levels in the physiological state. In acute myocardial ischaemia, VEGF secretion could be enhanced by the stimulation of mechanical tension and local inflammatory factors. 24,25 Previous studies have shown that exogenous apelin can induce endothelial progenitor cells homing to ischaemic myocardium and promote angiogenesis. 13 Combined with the results of this study, we speculate that ELA gene therapy up-regulates VEGF/VEGFR2 in infarcted myocardium, which may be associated with promoting endothelial progenitor cells homing to ischaemic myocardium and promoting angiogenesis.
In this study, the expression of CD105 and vWF was significantly increased after ELA gene therapy. Immunofluorescence ting results also showed that the expression of Jagged1/Notch3 in infarcted myocardium was increased after ELA gene therapy.
Notch signalling pathway is a highly conserved signalling pathway widely existing in vertebrates and invertebrates. It regulates the differentiation and development of cells, tissues and organs through the interaction between adjacent cells. 26,27 In 1995, the ligand that can activate the Notch signalling pathway was cloned and named Jagged1. Jagged1/Notch system is related to heart development, and Jagged1 gene mutation can cause congenital heart disease. 28 Notch3 gene mutation can reduce the diameter of the cerebral artery and microvessel density. 29 In recent years, more and more attention has been paid to the role of Jagged1/ Notch3 system in angiogenesis, particularly in tumour angiogenesis. 30  we also preliminarily observed that ELA-32, a novel APJ ligand, can also up-regulate Jagged1/Notch3 expression. It is speculated that there is a crosstalk between ELA/APJ pathway and Jagged1/ Notch3 pathway, and the underlying mechanism needs to be further studied.
Several limitations of our study warrant discussion. First, we currently lack imaging technology to evaluate myocardial angiogenesis in vivo. Second, because of the significant effect of angiogenesis and the improvement of cardiac function at 4 weeks after AAV9 injection, we concluded the animal experiment at 4 weeks. Whether or not AAV9 gene therapy has an off-target effect in the long term needs to be studied in the future.

| CON CLUS IONS
In summary, our results suggest that ELA activates VEFG/VEGFR2 and Jagged1/Notch3 pathways through APJ to promote angiogenesis after myocardial infarction. ELA gene therapy may be used in the treatment of ischaemic cardiomyopathy in future.

ACK N OWLED G EM ENTS
The work was supported from the National Natural Science

CO N FLI C T S O F I NTE R E S T
The authors declare no any conflicts of interest in this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.