Hepatocyte Growth Factor Gene Therapy for Ischemic Diseases

Abstract

Stem cells and gene therapy have become promising strategies for treating ischemic diseases and regenerating tissue. Hepatocyte growth factor (HGF) is an angiogenic growth factor with multiple functions, including promoting angiogenesis, regulating inflammation, inhibiting fibrosis, and activating tissue regeneration. Numerous preclinical experiments and clinical trials have demonstrated the feasibility and efficacy of HGF gene therapy in the treatment of ischemic diseases and tissue regeneration. This review summarizes the current advances of therapeutic angiogenesis using HGF gene transfer and modified stem cells. The physiological roles of HGF in angiogenesis and tissue regeneration are revisited. The current advances of clinical trials of plasmid and adenovirus HGF in the treatment of critical limb ischemia and coronary heart disease in China are introduced. Furthermore, valuable insight is provided into the prospective future of novel regenerative strategies using HGF-modified mesenchymal stem cells. HGF gene therapy is presented as a promising therapeutic approach in the treatment of ischemic diseases and regenerative medicine.

Introduction

Therapeutic angiogenesis using angiogenic growth factor gene transfer and stem cells to augment the collateral circulation and enhance blood flow to ischemic tissues is becoming a novel approach to treat ischemic diseases.^1
–3 Numerous angiogenic growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) have been extensively studied in gene therapy for ischemic diseases such as peripheral arterial occlusive disease (PAOD) and coronary heart disease (CHD).^3

–6 Among these angiogenic growth factors, HGF is a multifunctional cytokine with important roles in various cellular processes such as proliferation, survival, motility, and morphogenesis.⁷ The clinical studies using plasmid HGF gene therapy to treat PAOD have achieved promising results and have entered Phase III clinical trials.⁸ Compared to other known angiogenic growth factors such as VEGF and angiopoietin, HGF has noted advantages in promoting angiogenesis, reducing inflammation, fibrosis, and cell senescence under pathological conditions.⁹ These features present the clinical benefits of HGF gene therapy in PAOD patients.¹⁰

Mesenchymal stem cells (MSCs) derived from a variety of sources are promising tools in regenerative cell therapy. It is known that MSCs perform regenerative effects possibly through the secretion of exosomes and soluble growth factors, including HGF, FGF-B, and VEGF.¹¹ Overexpressing these growth factors enhances the regenerative activity of MSCs. Thus, gene modification of MSCs with growth factors is a novel way to improve their therapeutic effectiveness. This review summarizes the recent advances of plasmid HGF and adenovirus HGF in the treatment of ischemic diseases, and introduces an innovative strategy using HGF-modified MSCs in therapeutic angiogenesis and tissue regeneration.

Physiological Roles of Hgf in Angiogenesis and Tissue Regeneration

HGF, also known as scatter factor (SF), is an angiogenic growth factor with multiple functions, including angiogenesis, mitogenesis, morphogenesis, and organ regeneration activities.⁷ The biological functions of HGF are mediated through its unique tyrosine kinase receptor, c-Met. The activation of c-Met by autophosphorylation recruits adaptor molecules and activates several intracellular signaling pathways, including the RAS-mitogen activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K)-protein kinase B (PKB or AKT), mammalian target of rapamycin, signal transducer and activator of transcription (STAT), and beta-catenin pathways.^12,13 The activation of HGF/c-Met signaling cascade results in specific cellular programs such as promoting angiogenesis, inhibiting fibrosis and apoptosis, regulating inflammation, and stimulating tissue regeneration.¹⁴

Promoting angiogenesis

Angiogenesis is a physiological process through which new blood vessels form from pre-existing vessels. It is highly regulated by signal networks of various growth factors, proteolytic enzymes, and interactions between cellular components, vascular factors, and extracellular matrix.^15,16 Both physiological and pathological angiogenesis are involved in the proliferation, migration, and survival of endothelial cells. HGF functions as a potent mitogen and mediates angiogenesis by directly acting on vascular endothelial cells, including promoting proliferation, migration, and cell survival. The HGF receptor c-Met is reported to be expressed in the vascular endothelial cells of various origins.¹⁷ HGF/c-Met signaling induces vascular endothelial cell DNA synthesis and proliferation through MAPK/ERK and STAT3 pathways.¹⁸ Tyrosine phosphorylation of a line of signal molecules such as Gab1, SHP-2, and PI3K is required for HGF-induced MAPK/ERK activation, which mediates cell migration, proliferation, and stabilization of endothelial cells.¹⁹ Shp-2 mediated sphingosine kinase-S1P lipid signal activation is involved in HGF/c-Met-induced endothelial cell migration.^20,21 In addition to the direct effect on stimulating endothelial cells to form tubular structures, HGF can recruit bone marrow (BM) endothelial progenitor cells to participate in angiogenesis in the ischemic area.^22,23 Furthermore, HGF also promotes vascular structure remodeling by regulating smooth-muscle cells.²⁴ The molecular mechanisms of HGF-induced angiogenesis are strongly associated with HIF-1α and the secretion of angiogenic growth factors such as VEGF and interleukin (IL)-8.²⁵ HGF increases the stability and activity of HIF-1 protein and enhances VEGF-A expression.²⁶ The E-twenty-six (ETS) family of transcription factors is known to regulate gene expression in response to multiple developmental and mitogenic signals.²⁷ Several reports reveal that ETS mediates the HGF-induced expression of proangiogenic molecules IL-8 and VEGF.^25,27 HGF and other angiogenic growth factors play synergistic roles in promoting the proliferation and survival of vascular endothelial cells and the degradation and remodeling of matrix. HGF-induced angiogenesis is the physiological basis for the treatment of ischemic diseases.

Anti-fibrosis

Fibrosis is a key pathophysiology of chronic inflammatory diseases such as cirrhosis, chronic renal failure, and radioactive pulmonary fibrosis. It is regulated through the proliferation and differentiation of fibroblasts.²⁸ HGF plays an important role in the inhibition of tissue fibrosis. It inhibits the proliferation of fibroblasts, reduces the secretion of fibrosis-related molecules, and enhances the expression of matrix metalloproteinases. The molecular mechanisms of the anti-fibrosis activity of HGF are strongly associated with the inhibition of transforming growth factor (TGF)-β pathway, which plays a significant role in regulating gene expression involved in fibroblast differentiation and inflammatory disorders. HGF functions as a negative regulator of TGF-β1-induced fibroblast transformation and inhibits fibrosis in many organs, such as pulmonary, renal, and liver.²⁹ In fibrosis of a variety of organs and dysfunctional animal models, HGF gene therapy can improve tissue blood supplement and reduce tissue fibrosis.^30,31

HGF ameliorates experimental tissue fibrosis through many mechanisms, including degradation of accumulated collagen and decreased expression of fibrotic genes.^32,33 It functions as a key ligand to elicit myofibroblast apoptosis and extracellular matrix degradation. HGF suppresses profibrogenic signal transduction via nuclear export of Smad3 with galectin-7.³⁴ Thus, the anti-fibrosis function of HGF plays important roles in regulating tissue regeneration. Because a low level of HGF is one of the causes of tissue fibrosis, HGF alternative therapy has become an important measure of fibrosis treatment.

Anti-inflammatory effect

It was recently recognized that HGF plays an important regulatory role in inflammatory response and autoimmune diseases and in the protection of many types of inflammatory and autoimmune diseases.³⁵ HGF affects the pathophysiological processes of inflammatory responses, such as immune cell migration, maturation, cytokine secretion, antigen presenting to T cells, and other functions. HGF functions as an antagonist of TGF-β1 signal and is usually considered to have an immunosuppressive effect. HGF therapy may have potential in the treatment of autoimmune dysfunctions because it regulates the immune response induced by potent immunomodulatory factors such as IL-10, TGF-β1, and glucocorticoids.³⁶ A line of experimental evidence suggests that HGF has protective autoimmune activity, improves the process of autoimmune diseases, reduces IFN-γ production, and increases Th2 cytokine responses in experimental models. Many reports have revealed that HGF exhibits anti-inflammatory potential in oral traumatic ulcer through the reduction of epithelial apoptosis, connective tissue TNF-α expression, and the induction of c-Met expression.³⁷ By using tissue-specific Met knockout mice models, HGF has further been proved to be characterized as a modulator of immune cell functions and also plays an inhibitory role in the progression of chronic inflammation and fibrosis.³⁸

Anti-apoptosis

Another important role of HGF on angiogenesis is the inhibition of endothelial cell apoptosis. HGF/C-Met has anti-apoptotic and nutritional effects on various types of cells, including endothelial cells. In addition to the direct effect on vascular endothelial cells, HGF also induces the phosphorylation of cell survival factors Akt and Erk1/2, which are linked to its anti-apoptotic and angiogenic properties.^39,40 HGF also upregulates the expression of anti-apoptotic protein Bcl-2 or Bcl-xL.⁴⁰ HGF could prevent cardiomyocytes from apoptosis after hypoxia via upregulating cell autophagy.⁴¹ Furthermore, it protects cardiac myocytes against oxidative stress partly through activating the MEK/MAPK pathway.⁴²

Tissue remodeling and regeneration

HGF is a pleotropic factor required for normal organ development during embryogenesis. In adults, basal expression of HGF maintains tissue homeostasis and is upregulated in response to tissue injury.⁴³ Three-dimensional (3D) structure formation is important for tissue regeneration and structural restoration. It is known that HGF is a 3D-forming protein that stimulates cell proliferation, migration, and 3D structure formation. The use of conditional knockout c-Met mice reveals that HGF deletion significantly affects tissue remodeling during liver regeneration. This is associated with a decrease in matrix metalloproteinase 9 and a lack of stromal cell–derived cells.

Clinical Trial of Plasmid Hgf for Treatment of Paod Patients

Critical limb ischemia (CLI) is the most severe form of PAOD, including arteriosclerosis obliterans, thromboangitis obliterans, diabetic arteriosclerosis obliterans, and foot ulcers, and causes disability and impaired quality of life due to atherosclerotic occlusion of the lower extremities. CLI is an ideal candidate for plasmid HGF gene therapy because the plasmid can be directly injected into the ischemic area. In 2004, Morishita et al. completed the first human clinical trial of plasmid HGF gene therapy for PAOD in Japan.⁴⁴ Although the trial population was small, the initial clinical outcome indicated that intramuscular injection of naked HGF plasmid is safe and feasible, and improvement of pain, ankle-brachial index (ABI), and ischemic ulcers was achieved. A 2-year follow-up study demonstrated that HGF gene therapy achieved long-term efficacy in continuous improvement in clinical symptoms, such as the healing of ulcers and reduction in rest pain, and might decrease the amputation rate and mortality up to 2 years.⁴⁵ All the published clinical data suggest that HGF plasmid is safe and beneficial for improvement of rest pain and ulcer size.^{46

–53}

Different plasmid constructs were tested in these clinical trials. Some trials used the plasmid DNA expressing single HGF,^45

–49 while others used the plasmid DNA co-expressing two isomers of HGF: 728 amino acids (known as HGF or HGF728) and 723 amino acids (known as deleted HGF or HGF723).^50
–52 In these clinical trials, different injection strategies were tested. For example, Powell et al. injected the plasmid DNA into the muscle surrounding occluded tibial vessels guided by ultrasound. The volume and dose per injection site increased with the increase in total dosage. The plasmid DNA delivery utilized ultrasound-guided injection into the muscle surrounding the occluded tibial vessels. Other studies were designed to increase the number of injection sites as the total dosage increased.

Two plasmids (pCK-HGF-X7 and pUDK-HGF) expressing HGF were entered into a clinical trial in China. pCK-HGF-X7 expressing two isoforms of HGF (HGF728 and HGF723) has completed a Phase I clinical trial. Gu et al. reported the Phase I clinical trial results of pCK-HGF-X7 in critical CLI patients. The safety and preliminary efficacy, including reduction of pain, decreasing visual analog scale (VAS), increasing ABI or TcPO2 value, support the performance of the Phase II randomized controlled trial.⁵¹

The other plasmid enrolled in a clinical trial for the treatment of CLI is pUDK-HGF encoding HGF only consisting of 728 amino acids. Based on the preclinical findings, clinical trials were conducted to determine the safety and therapeutic effects of pUDK-HGF in patients with PAOD. The study involved 21 patients with PAOD who were divided into four groups and treated with direct intramuscular injection of 4, 8, 12, or 16 mg of pUDK-HGF into the sites of limb ischemia twice. All doses of pUDK-HGF (4, 8, 12, and 16 mg) were well tolerated by the patients. No serious adverse events caused by injection of pUDK-HGF were found over 3 months follow-up. Safety, VAS scores, and resting pain were evaluated before and 3 months after treatment. At the end points of observation, mean VAS in all cases reduced from 4.52 to 0.30. Rest pain disappeared completely in 14/17 cases. Of the five cases that were complicated with ischemic ulcer on the affected side, three healed and two improved.⁵² These encouraging clinical trial data are consistent with other studies that have demonstrated the safety and efficacy of pUDK-HGF in the treatment of ischemic diseases.^46,53

In pUDK-HGF Phase II clinical trials, 120 patients with ischemic rest pain and 120 patients with ischemic ulcers were enrolled and randomly divided into four groups to receive placebo or HGF plasmid (4 mg low dose, 6 mg middle dose, and 8 mg high dose) intramuscular injection on days 0, 14, and 28. Patients were evaluated for safety, changes of pain severity score, wound healing, changes in TcPO2, and ankle/toe pressure and amputation throughout the 6-month follow-up period. The results showed that intramuscular injection of all dose of pUDK-HGF was safe and well tolerated. pUDK-HGF injection significantly relieved the pain and obviously improved ulcer healing. Currently, both Phase III clinical trials of pCK-HGF-X7 and pUDK-HGF are approved by the China Food and Drug Administration (CFDA).

Adenovirus-Hgf Gene Therapy for Chd

Currently, both adenovirus and plasmid HGF are being developed toward gene therapy drugs for the treatment of ischemic diseases in China. Adenovirus is an ideal vector for myocardial gene transfer and has proven its potential in gene therapy of CHD, which is among the leading causes of mortality in the industrial world. Adenovirus-HGF (Ad-HGF) is a replication defective type 5 adenovirus carrying the human HGF gene. Transfer of Ad-HGF to primary myocardial cells results in overexpression of HGF protein, which stimulates the growth and migration of endothelial cells. Preclinical studies demonstrated the therapeutic effects of Ad-HGF by using rat, canine, and swine myocardial ischemia models. In rat and canine acute myocardial ischemia models, Ad-HGF-induced therapeutic angiogenesis was observed on day 14 after injection. The myocardial ischemia area was significantly reduced on day 21 after Ad-HGF therapy. The therapeutic effects of Ad-HGF were further validated in a minipig model of chronically myocardium ischemia in which an ameroid constrictor was placed around the left circumflex branch of the coronary artery. The data of 18 minipigs showed that Ad-HGF significantly improves the heart function and blood supply in chronic myocardial ischemia models.⁵⁴ Furthermore, catheter-based intramyocardial delivery (NavX) of Ad-HGF was proved safe and accurate for Ad-HGF delivery in pigs. Intramyocardial injection guided by the NavX system provides a method of feasible and safe percutaneous gene transfer to myocardial infarct regions.⁵⁵

Based on the conclusions of preclinical studies using animal models that Ad-HGF administered by direct intramuscular injection is safe and effective, Ad-HGF was approved by the CFDA for Phase I safety studies in CHD patients. Two strategies of adenovirus injection have been tried for CHD therapy: intramuscular myocardium direct injection while undergoing coronary artery bypass surgery, and intracoronary administration. An open-label safety and tolerance trial of Ad-HGF by direct multipoint injection into the myocardium in 18 patients who suffer from coronary heart disease was conducted. Three groups, each with six patients, received 5 × 10⁸ (low dosage), 1.5 × 10⁹ (medium dosage), or 5 × 10⁹ (high dosage) pfu of Ad-HGF by direct multipoint injection into the myocardium while undergoing coronary artery bypass surgery. The safety data showed that no subjective or objective adverse reactions were demonstrated in any of the 18 patients. There was evidence of revascularization in ischemic regions confirmed by instrumental examination and physical symptoms.⁵⁴ Clinical trials of Ad-HGF demonstrated that intramyocardial injection of Ad-HGF is feasible and safe. Based on these safety data, a Phase II clinical trial was subsequently started, and currently 27 patients with CHD have been recruited. A large randomized, placebo-controlled, double-blind study is currently ongoing in China.

Another clinical study using a catheter-based intramyocardial injection guided by the NavX system is ongoing. Patients with ischemic heart disease were enrolled. Under the guidance of the NavX system, Ad-HGF was injected into the endocardium in the lesion vascular supply area through three to eight injection sites using a myocardial injector. Patients were divided into low-, medium-, and high-dose groups. EF, left ventricular end diastolic diameter, myocardial perfusion integral (SRS), and New York Heart Association (NYHA) classification were recorded before treatment and at 1, 3, and 6 months post treatment. Changes in cardiac function and safety indexes were observed at 6 months after surgery, and the results were compared. Fifteen patients were included in the trial group, and all of them had complete follow-up data. Among them, 12 cases received the low or medium dose of Ad-HGF, and three cases received the high dose of Ad-HGF. The clinical results showed that the EF value of the high-dose group was 6.1% (echocardiography) and 4% (ECT) higher than that before the operation, while the average increase in the low-dose group was only 3.45% (echocardiography) and 2.67% (ECT). These encouraging data demonstrate that Ad-HGF injection intramyocardially can improve the cardiac function–related indexes such as the NHYA classification, EF value, left ventricular end diastolic diameter, and myocardial perfusion score in patients with CHD (unpublished data). To date, more than 10 clinical trials (Phase I–III) using HGF gene therapy for PAOD and CHD have been reported (Table 1).^{8,23,44

–54,56

–59} The clinical data show the advantages of HGF gene therapy compared to other angiogenic growth factors.

Table 1.

Clinical trials for HGF gene therapy

No.	Vector type	Disease	Phase	Open label?	Administration route	Publication year	Country	Details	Refs.
1	Adenovirus vector	Coronary heart disease	I	Yes	Intramyocardial and intracoronary injection	2008, 2009	China	An open-label safety and tolerance trial of Ad-HGF in patients suffering from coronary heart disease was performed. The conclusion indicates that direct intramyocardial or intracoronary administration of Ad-HGF was well tolerated and could improve myocardial perfusion with a dose–effect relationship, encouraging larger and randomized efficacy trials.	54,56
2	Adenovirus vector	Coronary heart disease	I	Yes	Intracoronary injection	2009	China	Twenty-one patients with severe coronary artery disease were recruited to the study. In conclusion, HGF gene therapy may play an important role in the regulation of cytokines and the induction of endothelial progenitor cell mobilization in patients with coronary heart disease.	23
3	Plasmid	Critical limb ischemia	II	No	Intramuscular injection	2004, 2010, 2011, 2011, 2012	Japan	Intramuscular injection of naked HGF plasmid DNA was performed in ischemic limbs of critical limb ischemia patients. It is safe and feasible, and successful improvement in ischemic limbs can be achieved.	44–46,48,57
4	Plasmid	Critical limb ischemia	II	No	Intramuscular injection	2008, 2010, 2011, 2016	United States/Korea	A double-blind, placebo-controlled study was conducted to assess the safety of intramuscular injection of hepatocyte growth factor plasmid to improve limb perfusion in patients with critical limb ischemia. VM202 is safe and may provide therapeutic bioactivity in critical limb ischemia patients.	8,49,47,53
5	Plasmid	Critical limb ischemia	I	No	Intramuscular injection	2011	Romania/Germany	The clinical trial analyzed the safety of gene therapy in 43 patients using plasmid constructs expressing vascular endothelial and hepatocyte growth factors in patients with critical limb ischemia. Gene therapy with vascular endothelial and hepatocyte growth factors is therapeutically safe and reduces the rate of major amputations and relieves pain at rest in patients with critical limb ischemia.	58
6	Plasmid	Critical limb ischemia	I	Yes	Intramuscular injection	2011, 2015	China	Twenty-one patients with critical limb ischemia were consecutively assigned to receive increasing doses of pCK-HGF-X7, which was administered into the ischemic calf and/or thigh muscle. No serious adverse events were observed in any of the 21 patients during the 3-month follow-up period. A significant reduction in pain was observed in the treated patients.	52,53
7	Plasmid	Ischemic heart disease	I	Yes	Intramyocardial injection	2013	Korea	Nine patients were assigned to receive increasing doses (0.5–2.0 mg) of VM202 (naked DNA expressing two isoforms of hepatocyte growth factor) injected into the right coronary artery territory following completion of a coronary artery bypass graft for the left coronary artery territory. Intramyocardial injection of VM202 can be safely used in patients with ischemic heart disease and might appear to have improved regional myocardial perfusion and wall thickness in the injected region.	59
8	Plasmid	Painful diabetic peripheral neuropathy	I/II	Yes	Intramuscular injection	2013	United States	Twelve patients with painful diabetic peripheral neuropathy in three cohorts received two sets of VM202 injections separated by 2 weeks. VM202 treatment appeared to be safe, well tolerated, and sufficient to provide long-term symptomatic relief and improvement in the quality of life in patients with painful diabetic peripheral neuropathy.	50

HGF, hepatocyte growth factor.

Hgf-Modified Stem Cells for Treatment of Ischemic Diseases

MSCs have been identified as a therapeutic option to induce angiogenesis and tissue regeneration.^60,61 Their therapeutic effect is likely correlated to the number of MSCs homing and their abilities secreting soluble growth factors and releasing exosomes.^11,62,63 The use of stem cells alone remains limited therapeutic effects because of insufficient expression of angiogenic factors and low cell viability after transplantation. HGF is an essential factor in tissue repair and regeneration, which were clarified by using tissue-specific knockout mouse models.⁶⁴ HGF gene modification could enhance therapeutic effects of transplanted MSCs by increasing autocrine HGF and promoting their proliferation, migration, engraftment, and differentiation ability. Scientists have explored applying this novel strategy of combining HGF and MSCs in the treatment of various diseases caused by tissue or organ injury, including ischemic disease, radiation damage, and lung injury. The application and effects of HGF-MSCs on ischemic diseases are listed in Table 2.^{65

–70,76,77,79}

Table 2.

Applications and effects of HGF-modified MSCs on ischemic diseases

Disease	Cell type	Vector type	Treatment route	Effects	Ref.
Myocardial ischemia/myocardial infarction	BM-MSCs (rat)	Adenovirus vector	Intramyocardial injection	A ligation model of proximal left anterior descending coronary artery of rats was used. Implantation of HGF-modified BM-MSCs into left anterior descending risk areas improved the functions of impaired myocardium, including diminishing the area of ischemia, increasing the number of capillaries, and reducing collagen content.	65
	BM-MSCs (human)	Lentivector	Intramyocardial injection	Myocardial infarction was induced by coronary ligation with subsequent injection of BM-MSCs/HGF, BM-MSCs, HGF, or saline into the border zone under echocardiography guidance. Four weeks after transplantation therapy, better ejection fractions, smaller scar sizes, less collagen deposition, increased microvessel densities, and more regenerative cardiomyocytes were observed in the BM-MSC/HGF-treated animals than in those receiving HGF or BM-MSCs alone.	66
	BM-MSCs (swine)	Adenoviral vector	Coronary vein injection	A porcine myocardial infarction model was created by balloon occlusion of the distal left anterior descending artery for 90 min followed by reperfusion. MSCs (alone or transfected with HGF) delivered into the infarcted porcine heart via the coronary vein had better cardiac function, accompanied by smaller infarct sizes, increased cell survival, less collagen deposition, greater blood vessel densities in the damaged area, and cardiac perfusion than in the saline control group. MSCs + HGF was superior to MSCs alone.	67
	BM-MSCs	Lentiviral vectors	Intramyocardial injection	Murine acute myocardial infarction was induced by coronary ligation, and cells were injected into the border zone. MSC-HGF-treated animals showed smaller scar sizes, increased peri-infarct vessel densities, and better preserved left ventricular function compared to MSCs transfected with empty vector.	79
	BM-MSCs (swine)	Adenoviral vector	Noninfarct-relative artery injection	A host myocardial infarction swine model was created by ligating the distal left anterior descending artery. BM-MSC transplantation via a noninfarct-relative artery stimulates cardiomyocyte regeneration and angiogenesis and improves cardiac function, but it does not stimulate collateral artery growth. BM-MSC transplantation combined with HGF therapy is not superior to the transplantation of BM-MSCs alone.	77
	AD-MSCs (swine)	Lentiviral vectors	Intramyocardial injection	Intramyocardial transplant in a porcine infarct model demonstrated the safety of paMSC in short-term treatments. Treatment with paMSC-IGF-1/HGF (1:1) compared to the other groups showed a clear reduction in inflammation in some sections analyzed and promoted angiogenic processes in ischemic tissue. Cardiac function parameters were not significantly improved.	76
	UC-MSCs (human)	Adenoviral vector	Intramyocardial injection	The left descending anterior artery was balloon occluded to establish a myocardial infarction model. Four weeks later, the randomly grouped swine were administered MSCs, phosphate-buffered saline, or HGF-MSCs via thoracotomy. The HGF-MSC group displayed the highest vessel density and Cx43 expression levels, and the lowest levels of apoptosis, tyrosine hydroxylase, and growth associated protein 43 (GAP43) expression. Moreover, the HGF-MSC group exhibited a decrease in the number of sympathetic nerve fibers, substantial decreases in the low frequency and the low-/high-frequency ratio, and increases in the root mean square of successive differences (rMSSD) and the percentage of successive normal sinus R-R intervals >50 ms (pNN50) compared to the other two groups. Finally, the HGF-MSC group displayed the lowest susceptibility to developing ventricular arrhythmia.	68
Limb ischemia	BM-MSCs (rat)	Lentiviral vector	Ischemic thigh muscle injection	The left femoral artery of each rat was resected, and its side branches were dissected and excised. Cells were injected into the ischemic thigh muscles 7 days post operation. Three weeks after injection, angiogenesis was significantly induced, and capillary density, the number of transplanted cell-derived endothelial cells, and the expression of angiogenic cytokines such as HGF and VEGF in local ischemic muscles was higher in the HGF-MSC group than in the MSC group.	69
Stroke	BM-MSCs (rat)	Herpes simplex virus type 1 vector	Intracerebral injection	A rat transient middle cerebral artery occlusion model was established, and gene-transferred MSCs were intracerebrally transplanted into the rats' ischemic brains. The results showed a significant improvement in neurological deficits and decrease in infarction areas. The percentage of apoptosis-positive cells in the ischemic boundary zone (IBZ) was significantly decreased, while that of remaining neurons in the cortex of the IBZ was significantly increased in the MSC-HGF group compared to others.	70

MSCs, mesenchymal stem cells; BM-MSCs, bone marrow–derived mesenchymal stem cells; UC-MSCs, umbilical cord–derived mesenchymal stem cells; AD-MSCs, adipose-derived mesenchymal stem cells.

Besides angiogenic growth factor gene therapy, MSCs have been identified as another potential new option to induce therapeutic angiogenesis.^61,71 Currently, more than 30 registered clinical studies have been conducted using MSCs derived from various resources such as BM, adipose tissue, umbilical cord, and even cardiac stem/progenitor cells to treat myocardial ischemia. However, clinical outcome data suggest improvement in the therapeutic effect of MSCs is needed. The biological basis for the clinical benefit of therapeutic angiogenesis is vascular remodeling, maturation, and stability. Both stem cells and HGF play important roles in these processes. Thus, the combination of HGF gene transfer and stem-cell transplantation would be the optimal approach to induce therapeutic angiogenesis.

HGF-modified stem cells for CHD

The HGF/Met pathway has a prominent role in cardiovascular remodeling after tissue injury.⁷² HGF induces cytoskeletal rearrangement and cell migration, and expresses cardiac-specific markers in MSCs.⁷³ It synergizes with IGF-1 to stimulate the differentiation of MSCs and myoblasts for 3D skeletal muscle tissue engineering.⁷⁴ HGF presents as a soluble factor during muscle regeneration and contributes to the proliferation and migration of myoblasts. Based on these functions of HGF on cardiac myocytes and MSCs, a growing body of preclinical evidence suggests that HGF-modified MSCs are effective approaches for CHD. MSCs infected by Ad-HGF release soluble HGF protein at an elevated level, which can be maintained at least for 2 weeks. In 2003, Duan et al.⁶⁵ proved the synergistic effect of HGF-modified MSCs on restoring local blood flow, regenerating lost cardiomyocytes, and reducing myocardial fibrosis in an acute rat myocardial ischemia model. Recently, Chang et al. evaluated the anti-apoptotic responses and therapeutic angiogenesis activities of genome-edited MSCs with inducible secreting HGF.⁷⁵ Furthermore, using different myocardial ischemia models such as murine AMI, rat post myocardial infarction, and porcine myocardial infarction, many reports support that HGF-modified MSCs could improve cardiac function, stimulate angiogenesis, and reduce myocardial fibrosis.^66,67,76 Yang et al. also elucidated that HGF-modified BM-MSCs stimulate cardiomyocyte regeneration and angiogenesis and improve cardiac function via a noninfarct-relative artery in a swine myocardial infarction model.⁷⁷ Furthermore, HGF modification enhances the anti-arrhythmic properties of human BM-MSCs.⁶⁸ Combining stem-cell therapy with HGF gene therapy is an innovative strategy in angiogenesis and tissue regeneration for CHD.

HGF-modified stem cells for ischemic stroke and tissue repair

Occlusive cerebrovascular disease often causes global ischemia of the brain and results in neuropathological changes. Several strategies have been proposed to augment brain reorganization, including the stimulation of endogenous processes through gene therapy and cell therapy. MSCs have the ability of homing in on the central nervous system in vivo. Many reports have demonstrated that MSCs improve the functional recovery of the brain after a stroke. The rationale for the use of MSCs in stroke is based on their capacity to reduce inflammation and increase neurogenesis and angiogenesis by secreting a large variety of bioactive molecules such as growth factors and chemokines.⁷⁸ HGF administration also inhibits destruction of the blood–brain barrier, decreases brain edema, and provides a neuroprotective effect after brain ischemia.¹³ MSCs-HGF are valuable sources for increasing cytokine production and maximizing the beneficial effect of MSCs-based regenerative strategies.⁷⁹

HGF-MSCs therapy is also suitable for treating various diseases caused by tissue injury and degeneration. HGF-modified MSCs improve ischemia/reperfusion-induced acute lung injury in rats,⁸⁰ promote blood vessel regeneration,⁸¹ ameliorate radiation-induced liver damage, prevent bone loss in the early phase of ovariectomy-induced osteoporosis,⁸² and protect radiation-induced lung injury, intestinal injury,⁸³ cardiac injury,⁸⁴ and hematopoietic damage.⁸⁵

Current Challenges and Future Prospects

Even though plasmid HGF gene therapy is therapeutically effective on ischemic diseases in clinical trials, a large group of randomized, placebo-controlled, double-blind Phase III studies is needed and is currently ongoing. The preclinical and Phase I clinical data highlight the safety and significant therapeutic effects of Ad-HGF in the treatment of CHD. Its clinical benefits need to be evaluated in Phase II and III clinical studies. Furthermore, a more efficient and convenient system for delivering the gene therapy drug to the myocardium needs to be developed. The combination of stem cells and HGF gene therapy shows a synergistic effect on repairing tissue and represents a promising therapeutic tool for regeneration medicine.

Footnotes

Acknowledgments

This work was supported in part by a grant from the National High Technology Research and Development Program of China (863 Program; no. 2012AA020807) and The National Key Research and Development Program of China (no. 2017YFA0105303).

Author Disclosure

No competing financial interests exist.

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