Gene Therapy with Hepatocyte Growth Factor Improves Left Ventricular Systolic Function After Myocardial Infarction: A Systematic Review and Meta-analysis of Pig Models

Background: Hepatocyte growth factor (HGF) is an angiogenic cytokine which can promote angiogenesis and inhibit brosis. Previous studies have shown that HGF may have signicant therapeutic effects on ischaemic diseases, such as ischaemic heart disease and peripheral arterial occlusive disease. Due to insucient clinical study evidence, we conducted a quantitative meta-analysis of HGF therapy in pigs with myocardial infarction (MI) to provide more reliable evidence for the feasibility and effectiveness of HGF therapy for MI patients. We also analysed the ecacy and characteristics of different gene therapy vectors and drug delivery routes. Methods: PubMed, EMBASE, and the China National Knowledge Infrastructure were searched for randomised studies that corresponded to our subject. The search terms included (hepatocyte growth factor OR HGF) AND (heart failure OR HF OR myocardial infarction OR MI OR AMI OR coronary heart disease OR CHD). The retrieved articles were screened strictly according to the inclusion criteria. The endpoints were the left ventricular ejection fraction (LVEF) and capillary density in the ischaemic regions in the model pigs. Results: A total of nine studies were eventually included in this meta-analysis. Our analysis showed that LVEF (with mean difference [MD]:9.73, 95%CI :8.70, 10.76, P < 0.00001) and capillary density (MD:79.98, 95%CI :24.58,135.39, P=0.005) in the HGF group were signicantly higher than those in the control group several weeks after HGF treatment. Further analysis showed that there was no statistically signicant difference in the improvement of LVEF caused by intracoronary adenovirus 5 -mediated HGF (Ad5-HGF) gene transfer and intramyocardial plasmid HGF injection (12.63±3.2 vs 9.4±1.09, P=0.06), while intramyocardial injection of plasmid HGF had a stronger angiogenic capacity than intramyocardial administration of Ad5-HGF and HGF in hydrogel (117.34±27.82 vs 26.45±22.11 vs 11.50±5.28, P (cid:0) 0.00001). Conclusions: HGF therapy can effectively promote angiogenesis and recovery of cardiac function and is a promising cardiac repair method. Intracoronary Ad5 vector transfer and intramyocardial injection of plasmid vectors can be used as effective means of gene therapy, and hydrogel as a vector also has potential applications. HGF on angiogenesis in of MI involving 64 pigs, of which with HGF and were used as controls. Angiogenesis was measured by capillary density (capillaries/mm 2 ). signicant heterogeneity between studies (I 2 =98% (cid:0) P<0.00001), used a random-effect model to analyse the difference in capillary density between the HGF group and the control group. The results showed the capillary density in the ischaemic area of the HGF group signicantly than of control P=0.005; an intramyocardial injection factor 1α, in hydrogels and delivered them to the peri-infarct regions, and found that the release time of the cytokines was signicantly prolonged and the therapeutic effects were enhanced when the cytokines were encapsulated in hydrogels. These unique advantages of hydrogels potentially make them a more attractive method of exogenous administration.


Background
Myocardial infarction (MI), the most severe form of ischaemic heart disease (IHD), causes irreversible apoptosis and necrosis of myocardial cells [1]. The infarcted area is then replaced by brous scar tissue, leading to ventricular remodelling and heart failure (HF) [2,3]. In the process of ventricular remodelling, the oxygen consumption of the hypertrophic ventricular wall increases, the number of capillaries decreases, and the myocardial ischaemia is further aggravated [4,5]. Although early revascularisation can improve blood supply in ischaemic area to a certain extent, cardiac remodelling is still ongoing, resulting in HF approximately 50% of the time [6]. The only way to reverse this process is heart transplantation, which is limited by donor organ shortage, high cost, and the need for immunosuppressive medications [7]. Therefore, new therapeutic strategies should be explored for inhibiting cardiac remodelling and improving cardiac repair, and promoting angiogenesis is one promising approach. Among numerous angiogenic strategies, cytokine-based gene therapy has been extensively studied.
At present, a variety of vascular growth factors have been attempted for use in gene therapy after MI, among which vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) have attracted wide attention [8]. It has been reported that HGF could regulate the expression of cardiomyocyte-speci c transcription factors and structural genes through its unique tyrosine kinase receptor, c-Met [9], and then perform functions such as promoting angiogenesis, regulating in ammation, inhibiting brosis, and even activating tissue regeneration [10]. HGF, and the activation of c-Met, could alleviate chronic myocardial injury in myocarditis, cardiomyopathy and other disease models [11]. In animal experiments, local HGF administration has been mainly conducted through intracoronary gene transfer using a catheter and intramyocardial injection via thoracotomy [12,13]. At present, there have been relatively few clinical studies on the local application of HGF for MI treatment which, to some extent, hinders the prospects for application of HGF therapy. In contrast, HGF has been extensively studied in animal models. However, the e cacy of HGF has varied between studies, and researchers have used different carriers and routes of administration. Considering that pigs and humans are relatively similar species, we pooled the preclinical experimental data to provide more reliable evidence for the feasibility of clinical transformation of HGF therapy. In addition, the prospects of different treatment methods were analysed.
This article aims to analyse and summarise the applicability of HGF therapy after MI, as well as to compare and systematically review different approaches, including delivery routes and vehicles, and introduce new ones. Finally, more effective treatment options and future research directions will be discussed.

Search strategy
Two investigators retrieved literature through the PubMed database, the Excerpta Medica Database (Embase), and the China National Knowledge Information database. The search terms used were as follows: (hepatocyte growth factor OR HGF) AND (heart failure OR HF OR myocardial infarction OR MI OR AMI OR coronary heart disease OR CHD). We did not set a starting date for our search, but the end date was set at 28 March 2020. The publication language was not limited. The retrieved studies were carefully examined to exclude similar articles, and the studies related to the topic in the references of each article were manually retrieved to prevent omissions.

Study selection
Two investigators independently reviewed all of the retrieved studies, read the full text of each study, including titles and abstracts, and then extracted relevant data. Only randomised studies examining the effect of HGF therapy on cardiac function and angiogenesis in swine models with MI were included. The included studies had to be designed with experimental controls, and article types such as reviews and reports were excluded. Studies were included as long as either LVEF or capillary density was quantitatively assessed.
Quality assessment and data extraction Two authors independently assessed the quality of the included studies according to ve aspects: randomisation and control, adequate allocation, adequate method of randomisation, blinding of the operator, and blinding of the functional analysis. The following information was extracted from the full text: pig breed, gender, weight, number of pigs, intervention form, follow-up time, and any other relevant information. Any ambiguities were resolved by a more experienced third individual.

Data analysis and statistical methods
Our primary endpoints were the differences in mean LVEF (%) and capillary density (capillaries/mm 2 ) at follow-up between the HGF group and the control group. We also measured the effects of different delivery methods and vehicles on cardiac function and angiogenesis. The mean difference (MD) and 95% con dence interval (CI) were used to calculate and assess evaluation data of the continuous results between HGF treatment and control groups. Statistical analysis was performed using Review Manager (version 5.3). Furthermore, I 2 values were used to assess the heterogeneity among the included articles. If I 2 50%, the random-effect model would be used; if I 2 50%, the xed-effect model would be adopted. To test the robustness of the results, sensitivity analysis was performed by excluding the studies one by one. In addition, we performed subgroup analysis according to different conditions and used funnel plots to describe whether there was potential publication bias.

Search results
Using the above query method, 1,224 articles were retrieved. We excluded studies that did not address our research topic or did not focus on pigs.
Non-controlled studies were also dismissed. We nally included nine studies after excluding similar and identical articles by analysing the full text. The detailed selection process for included studies is represented in Figure 1.

Risk of bias assessment and study characteristics
All nine studies included in the meta-analysis met our selection criteria. The methodological quality of each study was evaluated, and the speci c content of the quality assessment is shown in Table 1. In all included studies, MI pig models were established, and were randomly divided into an HGF treatment group and a control group, which conformed to the randomisation and control method. However, not all studies indicated whether blinded analysis of cardiac function and angiogenesis was used. After completing the study quality assessment, we extracted basic characteristics of each study, including pig breed, gender, weight, number of pigs, intervention form, follow-up time, and other relevant information (Table 2). Finally, 115 pigs with MI were analysed, 57 of which received HGF treatment and 58 that received control treatment.

Meta-analysis
Six studies [14][15][16][17][18][19] provided data on LVEF (%) and included 81 MI pigs, of which 40 were treated with HGF and 41 were used as controls. Since there was no signi cant heterogeneity (I 2 =2%, P=0.40) among studies, the data analysis was based on the xed-effect model. The results of the analysis suggested that, compared with the control group, local application of HGF signi cantly increased LVEF (MD:9.73, 95% CI:8.70, 10.76, P 0.00001; Figure 2). In addition, of the six included studies, three were conducted with intracoronary adenovirus 5 -mediated HGF (Ad5-HGF) transferred into the myocardium via the right coronary artery, and the other three were conducted with plasmid HGF intramyocardial injection. Therefore, we performed a subgroup analysis based on these two methods, and the results showed that there was no statistically signi cant difference in the improvement of LVEF between intracoronary injection of Ad5-HGF and intramuscular injection of plasmid HGF (MD: 12.63, 95% CI:9.43, 15.83 vs MD:9.40, 95% CI:8.31, 10.48, P=0.06; Figure 3).
The effects of HGF on angiogenesis in ischaemic areas of MI pigs were studied in ve articles [12,17,18,20,21] involving 64 pigs, of which 32 were treated with HGF and 32 were used as controls. Angiogenesis was measured by capillary density (capillaries/mm 2 ). Considering the signi cant heterogeneity between studies (I 2 =98% P<0.00001), we used a random-effect model to analyse the difference in capillary density between the HGF group and the control group. The results showed that the capillary density in the ischaemic area of the HGF group was signi cantly higher than that of the control group (MD:79.98, 95% CI:24.58,135.39, P=0.005; Figure 4). In addition, an intramyocardial injection delivery method was used in these ve studies, but the HGF vehicles used were different. Among them, plasmid vectors were used in three studies, an Ad5 vector in one study, and a hydrogel vector in one study. Therefore, we performed a subgroup analysis to compare the therapeutic effects of different vectors under the same route of administration. The results showed that plasmid-mediated HGF therapy had a stronger angiogenic effect than Ad5 and hydrogel when intramyocardial administration was applied, with a statistically signi cant difference (117.34±27.82 vs 26.45±22.11 vs 11.50±5.28, P 0.00001; Figure 5).
In addition, sensitivity analysis, performed by excluding the studies one by one, demonstrated the same results, which indicated the meta-analysis was robust. However, the funnel plot indicated that there might be some publication bias, as the values were not completely and symmetrically distributed around the overall estimate ( Figure 6).

Discussion
Chronic myocardial ischaemia and ventricular remodelling after MI are important causes of HF. In recent years, gene therapy based on angiogenic factors has become a potential method to promote angiogenesis, inhibit ventricular remodelling, and improve cardiac function [22,23]. The cytokines that have been studied in preclinical and clinical research include VEGF, HGF, broblast growth factor, and insulin-like growth factor, and some studies have achieved encouraging results [24,25]. Recently, Wang et al. [26] injected adenovirus-mediated VEGF165 into the peri-infarct myocardium in an MI rat heart, and their results suggested that VEGF165 gene therapy could improve cardiac function by inducing angiogenesis and inhibiting cardiomyocyte apoptosis.
HGF and its potential angiogenic, anti-apoptotic, and anti-brotic effects [27,28] have been extensively studied in various models of ischaemic disease, such as MI [29] and peripheral artery occlusion disease [30]. A Phase III clinical trial of intramuscular injection of plasmid HGF for the treatment of severe limb ischaemia has been successful [31], suggesting that HGF may also be used for the treatment of IHD. The biological function of HGF is mediated by its unique tyrosine kinase receptor c-Met [32]. Activation of the c-Met receptor further activates many intracellular signalling pathways, including RAS-mitogen activated protein kinase, signal transducer and activator of transcription, phosphatidylinositol-3 kinase, protein kinase B, mammalian target of rapamycin, and β-catenin pathway [33,34]. Our group has previously shown that HGF therapy could promote cardiac repair and improve cardiac function in MI rats through the above mechanisms [35].
Although HGF has been widely studied in some large animal MI models, the clinical trials related to HGF therapy have just started [36], which prompted us to conduct a meta-analysis on the preclinical data. Our study showed that the cardiac pump function of the HGF group was signi cantly better than that of the control group within 1-2 months after MI, with an increase in LVEF of about 9.73%. In addition to the improvement of LVEF, local application of HGF promoted angiogenesis and increased blood supply to the ischaemic area. Compared with the control group, the capillary density in the HGF treatment group was signi cantly increased (about 97.33 capillaries/mm 2 difference). Our study fully demonstrates that HGF treatment after MI can promote angiogenesis and improve the cardiac pump function. Neovascularization can improve the blood supply in ischaemic areas, provide preconditions for the tissue repair of the heart and, to some extent, reverse the brosis induced by hypoxia [12].
In terms of vectors and routes of delivery, plasmids, adenoviruses and injectable hydrogels have been widely accepted for gene therapy [37]. As mature and traditional vectors, plasmid-and adenovirus-mediated HGF gene transfer can signi cantly improve HGF expression in myocardial tissue. Wang et al. [15] transferred Ad5-HGF (4×10 9 pfu) into the myocardium via the right coronary artery four weeks after left anterior descending coronary artery (LAD) ligation in pigs, and detected the expression level of HGF by ELISA three weeks later. The results showed that the expression level of HGF in the experimental group increased to nearly 18 times that of the control group (109.3±7.8 vs 6.2±2.6), and the LVEF value increased by nearly 45% (43.9±4.3 vs 30.4±2.8). Similarly, Funatsu et al. [38] injected 125 microg of plasmid encoding human HGF (hHGF) into the peri-infarct regions of canines four weeks after LAD ligation, and the expression of hHGF was speci cally detected by ELISA after four weeks (endogenous HGF could not be detected). The results showed that hHGF expression in the HGF group was 4.7±1.7 ng/g, while no hHGF protein expression was found in the control group, and the number of capillaries in the ischaemic area of the HGF group also increased to 140% of that in the normal area. Therefore, both plasmids and adenoviruses as vectors can effectively improve the expression of HGF. In addition, studies have shown that local application of plasmid HGF or Ad5-HGF is reliable in terms of safety [39]. As illustrated by the above points, plasmids and adenoviruses are ideal vectors for gene therapy. Our analysis showed that intracoronary Ad5-HGF gene transfer and intramyocardial plasmid HGF injection had similar effects on cardiac pump function at conventional doses, and both treatments could bring signi cant improvement in LVEF (12.63±3.2 and 9.4±1.09; P=0.06). Injectable hydrogel is a promising synthetic biomaterial, with advantages such as mild gelation and cardiac-compatible properties [40,41]. Recent studies have shown that the injection of hydrogel in ischaemic areas after MI could promote cardiac repair and improve cardiac function [42]. Hydrogels can also encapsulate therapeutic drugs and deliver them to target areas to achieve sustained therapeutic effects [43]. Steele et al. [44] encapsulated two cytokines, dimeric fragment of HGF and engineered stromal cell-derived factor 1α, in hydrogels and delivered them to the peri-infarct regions, and found that the release time of the cytokines was signi cantly prolonged and the therapeutic effects were enhanced when the cytokines were encapsulated in hydrogels. These unique advantages of hydrogels potentially make them a more attractive method of exogenous administration.
All of the studies included in this meta-analysis adopted local administration. The traditional route of local administration is either intracoronary delivery of therapeutic drugs through a catheter or intramyocardial injection through thoracotomy, which can be used locally in coronary artery bypass grafting [45]. Our analysis showed that intramyocardial injection of plasmid HGF had a stronger angiogenic capacity than intramyocardial injection of Ad5-HGF and HGF in hydrogel (P 0.00001). Recently, Shi et al. [46] designed phase-transition microneedles coated with adenoassociated virus (AAV), which achieved uniform distribution of AAV delivery and were superior to direct muscular injection. This new delivery system may further optimise the e cacy of intramuscular injection. The advantage of local administration is that it effectively avoids the accumulation of drugs in other tissues and makes the concentration of drugs in the heart reach its peak. The disadvantage is that it limits the convenience and repeatability of its application. In contrast, systemic administration is more convenient, but it is necessary to nd and apply heartspeci c vectors to reduce the distribution of drugs in other tissues and organs.
This meta-analysis had certain strengths and limitations. First of all, this is the rst meta-analysis pooling data from preclinical large animal studies, which provides strong evidence for the feasibility of HGF therapy in the treatment of MI. A second strength was that we analysed the therapeutic effects and characteristics of different vectors and delivery routes, and introduced a new intramuscular delivery system, providing a methodological basis for HGF gene therapy and other drug gene therapy. In terms of limitations, some studies did not provide relevant statistical data (three without LVEF data, four without capillary density data), which may partially affect the accuracy of the results. Secondly, most studies only used western blot to qualitatively analyse the expression difference of HGF between the experimental group and the control group without quantifying HGF expression. Therefore, we were unable to compare the differences in the expression of HGF between studies. In addition, there was some methodological heterogeneity in the included studies; however, they all observed the effects of HGF on cardiac function and angiogenesis in the chronic phase of MI, so we relaxed this aspect of our inclusion criteria and conducted some valid subgroup analyses to address the issue.

Conclusion
The safety of local HGF application has been preliminarily con rmed by previous Phase I clinical studies, which should be further veri ed by multicentre studies with more rigorous experimental design and longer follow-up times. Based on the current evidence, we demonstrated that HGF therapy after MI could promote angiogenesis and improve cardiac function, and that intramyocardial injection of plasmid vector and intracoronary Ad5 vector transfer are effective means to achieve these effects. Although differences exist between humans and pigs, our study provides reliable evidence for clinical transformation of HGF therapy. In addition, HGF can combine with other therapeutic factors to have a synergistic effect. For example, a stronger therapeutic effect may be achieved through combined application of HGF with bone marrow mesenchymal stem cells, VEGF, and other therapeutic factors, although this needs to be con rmed by further preclinical and clinical trials.

Declaration
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.