Fig 2 - uploaded by Genta Narazaki
Content may be subject to copyright.
Time course of HGF (A), VEGF (B), and FGF-2 (C) production in ischemic muscle after treatment with saline, aMNCs, or Ad-HGF. NI, nonischemic. *P 0.05 vs. NI; †P 0.05 and † †P 0.01 vs. the respective ischemic saline-treated groups.
Source publication
Autologous cell implantation and angiogenic gene therapy have been evaluated in critical limb ischemic patients. Here, we compared the features of these strategies individually and in combination. C57BL/6J mice with ischemic hindlimbs were injected with adherent mononuclear cells (aMNCs) from bone marrow or adenovirus encoding the hepatocyte growth...
Contexts in source publication
Context 1
... factor production. In vitro studies have shown that aMNCs produce VEGF and HGF; however, FGF-2 was below detectable levels (VEGF: 63.8 7.1 pg/ml and HGF: 1.3 0.4 ng/ml, n 3). HGF production in ischemic muscle was elevated after 12 h and lasted for 5 days ( Fig. 2A). HGF production was significantly stimulated by aMNC treatment compared with controls, and immunohistochemical analysis showed that HGF was detected in endothelial cells (Fig. 3, A-C). Ad-HGF stimulated HGF production in the ischemic limb for 14 days, and the maximal amount was 10 times greater than the aMNC-treated group on day 5. ...
Context 2
... with controls, and immunohistochemical analysis showed that HGF was detected in endothelial cells (Fig. 3, A-C). Ad-HGF stimulated HGF production in the ischemic limb for 14 days, and the maximal amount was 10 times greater than the aMNC-treated group on day 5. VEGF production in ischemic muscle increased 12 h after the induction of ischemia (Fig. 2B). VEGF production was stimulated by aMNC treat- ment but not by Ad-HGF transfection, and the increase induced by aMNC treatment lasted for 28 days. FGF-2 production in ischemic muscle increased 5 days after the induction of ische- mia (Fig. 2C). FGF-2 production was stimulated by aMNC treatment 14 days after implantation, and the ...
Context 3
... group on day 5. VEGF production in ischemic muscle increased 12 h after the induction of ischemia (Fig. 2B). VEGF production was stimulated by aMNC treat- ment but not by Ad-HGF transfection, and the increase induced by aMNC treatment lasted for 28 days. FGF-2 production in ischemic muscle increased 5 days after the induction of ische- mia (Fig. 2C). FGF-2 production was stimulated by aMNC treatment 14 days after implantation, and the effects lasted for 28 days. The expression of FGF-2 was observed in capillaries and skeletal muscle (Fig. 3, G-I). Ad-HGF transfection stim- ulated FGF-2 production 14 days after treatment. There were no differences in HGF, VEGF, and FGF-2 production ...
Context 4
... most injected aMNCs disappeared from the in- jected site 7 days after the injection (Fig. 4), aMNC therapy influenced the production of angiogenic factors in ischemic tissue in the chronic phase (Fig. 2, A-C). Several investigations have suggested that the angiogenic effects of cell therapy are due to the secretion of angiogenic factors by the injected cells (6, 18). However, Tateno et al. (28) suggested that ischemic skeletal muscle (at the site of injection) is responsible for the production of angiogenic factors and cytokines. Our ...
Context 5
... Tateno et al. (28) suggested that ischemic skeletal muscle (at the site of injection) is responsible for the production of angiogenic factors and cytokines. Our results are partially compatible with the latter model, and we believe that both the transplanted cells and ischemic tissue (skeletal muscle and endothelial cells) produce angiogenic factors (Figs. 2 and 3). Note that we confirmed the stability of the PKH fluorescent marker after 28 days of culture (Supplemental Fig. III). ...
Citations
... There are multiple physiological and immunological barriers against the grafting of donor tissue [2]. Even syngeneic transplantation, donor cells, or cell sheets have been reported to disappear rapidly from the host, for example, after cell injection in a mouse model of hindlimb ischemia [24] and cell sheet transplantation in the heart of rats with myocardial infarction [25]. First, because of the absence of existing blood vessels in cellular grafts, the cell graft results in hypoxic and low-nutrient conditions a few hours post-transplantation. ...
Background
We previously established a human mesenchymal stem cell (MSC) line that was modified to express trophic factors. Transplanting a cell sheet produced from this line in an amyotrophic lateral sclerosis mouse model showed a beneficial trend for mouse life spans. However, the sheet survived for less than 14 days, and numerous microglia and macrophages were observed within and adjacent to the sheet. Here, we examined the roles of microglia and macrophages as well as acquired antibodies in cell sheet transplantation.
Methods
We observed the effects of several MSC lines on macrophages in vitro, that is, phenotype polarization (M1 or M2) and migration. We then investigated how phenotypic polarization affected MSC survival using antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). We also confirmed the role of complement on cytotoxicity. Lastly, we selectively eliminated microglia and macrophages in vivo to determine whether these cells were cytoprotective to the donor sheet.
Results
In vitro co-culture with MSCs induced M2 polarization in macrophages and facilitated their migration toward MSCs in vitro. There was no difference between M1 and M2 phenotypes on ADCC and ADCP. Cytotoxicity was observed even in the absence of complement. Eliminating microglia/macrophage populations in vivo resulted in increased survival of donor cells after transplantation.
Conclusions
Acquired antibodies played a role in ADCC and ADCP. MSCs induced M2 polarization in macrophages and facilitated their migration toward MSCs in vitro. Despite these favorable characteristics of microglia and macrophages, deletion of these cells was advantageous for the survival of donor cells in vivo.
... In contrast, HGF/MSC therapy was more effective than either alone. 26,44,47,50,[57][58][59] Table S1. The main mechanisms of HGF/MSC therapy are summarized in Figure 1, and they are further demonstrated below. ...
... 66 It was reported that HGF/MSCs promoted vascular endothelial cell proliferation and blood vessel regeneration more efficiently than MSCs and HGF protein. 46,48,51,58,59,67,68 The endothelial ...
Mesenchymal stem cell (MSC) therapy is considered a new treatment for a wide range of diseases and injuries, but challenges remain, such as poor survival, homing and engraftment rates, thus limiting the therapeutic efficacy of the transplanted MSCs. Many strategies have been developed to enhance the therapeutic efficacy of MSCs, such as preconditioning, co‐transplantation with graft materials and gene modification. Hepatocyte growth factor (HGF) is secreted by MSCs, which plays an important role in MSC therapy. It has been reported that the modification of the HGF gene is beneficial to the therapeutic efficacy of MSCs, including diseases of the heart, lung, liver, urinary system, bone and skin, lower limb ischaemia and immune‐related diseases. This review focused on studies involving HGF/MSCs both in vitro and in vivo. The characteristics of HGF/MSCs were summarized, and the mechanisms of their improved therapeutic efficacy were analysed. Furthermore, some insights are provided for HGF/MSCs' clinical application based on our understanding of the HGF gene and MSC therapy.
... In the present study, paracrine effects of myo-critical factors that promote angiogenesis and inhibit cell apoptosis (11). HGF has been reported to enhance angiogenesis by stimulating migration and proliferation of endothelial cells and inhibiting their apoptosis (21). Cultured ASCs secreted both HGF and VEGF as reported previously (12). ...
... Interestingly, ASC sheets expressed significantly higher mRNA and protein levels of HGF, VEGF, and bFGF than myoblast cell sheets under hypoxic condition, associated with the less prevalence of cell apoptosis in ASC sheets than myoblast cell sheets. bFGF also augments neovascularization and inhibits cellular apoptosis (3,21). Since preventing apoptosis of transplanted cells leads to improvement of Fig. 4 Comparison of the parameters of cardiac systolic and diastolic functions of MI hearts transplanted with ASC sheets or myoblast cell sheets to untreated hearts estimated by Langendorff techniques in the absence and presence of β-adrenergic stimulants. ...
Adipose stem cells (ASCs) are a source of regenerative cells available for autologous transplantation to hearts. We compared protective actions of ASC sheets on rat myocardial infarction (MI) in comparison with those of skeletal myoblast cell sheets. Their effects on infarcted hearts were evaluated by biological, histochemical as well as physiological analyses. ASC sheets secreted higher concentrations of angiogenic factors (HGF, VEGF, and bFGF; P < 0.05) under normoxic and hypoxic conditions than those of myoblast cell sheets, associated with reduction of cell apoptosis (P < 0.05). Like myoblast cell sheets, ASC sheets improved cardiac function (P < 0.05) and decreased the plasma level of ANP (P < 0.05) in MI hearts. ASC sheets restored cardiac remodeling characterized by fibrosis, cardiac hypertrophy and impaired angiogenesis (P < 0.05), which was associated with increases in angiogenic factors (P < 0.05). In isolated perfused rat hearts, ASC sheets improved both systolic and diastolic functions, which was comparable to cardiac functions of myoblast cell sheets, while both cell sheets failed to restore cardiac contractile response to either isoproterenol, pimobendan or dibutyryl cAMP. These results indicated that ASC sheets improved cardiac function and remodeling of MI hearts mediated by their paracrine action and this improvement was comparable to those by myoblast cell sheets.
... The merits of such a pro-angiogenic approach could be directly applicable to not only NVAMD but also non-NVAMD when one considers the postulated association between vascular dropout within the choriocapillaris and the pathogenesis of AMD [43,44]. Growth factors such as platelet-derived growth factor (PDGF), the angiopoietins (Ang), and hepatocyte growth factor (HGF) promote these different components of vascular maturation and therefore may have a role to play in promoting tissue repair in NVAMD [102][103][104]. Ultimately, from a wound healing perspective, combination therapy may provide a realistic and sustained benefit in the future. ...
Recently, anti-vascular endothelial growth factor therapies for neovascular age-related macular degeneration have been developed. These agents, originally developed for their anti-angiogenic mechanism of action, probably also work through an anti-permeability effect in preventing or reducing the amount of leakage from submacular neovascular tissue. Other treatment modalities include laser photocoagulation, photodynamic therapy with verteporfin, and submacular surgery. In reality, these latter treatments can be similarly categorized as anti-angiogenic because their sole aim is destroying or removing choroidal neovascularization (CNV). At the cellular level, CNV resembles stereotypical tissue repair that consists of several matricellular components in addition to neovascularization. In the retina, the clinical term CNV is a misnomer since the term may more appropriately be referred to as aberrant submacular repair. Furthermore, CNV raises a therapeutic conundrum: To complete or correct any reparative process in the body, angiogenesis becomes an essential component. Anti-angiogenic therapy, in all its guises, arrests repair and causes the hypoxic environment to persist, thus fueling pro-angiogenesis and further development of CNV as a component of aberrant repair. However, we realize that anti-vascular endothelial growth factor therapy preserves vision in patients with age-related macular degeneration, albeit temporarily and therefore, repeated treatment is needed. More importantly, however, anti-angiogenic therapy demonstrates that we can at the very least tolerate neovascular tissue beneath the macula and preserve vision in contrast to our historical approach of total vascular destruction. In this clinical scenario, it may be possible to look beyond anti-angiogenesis if our goal is facilitating submacular repair without destroying the neurosensory retina. Thus, in this situation of neovascular tolerance, it may be timely to consider treatments that facilitate vascular maturation, rather than its arrest or destruction. This would neutralize hypoxia, thus removing the stimulus that drives neovascularization and in turn the need for repeated lifelong intravitreal therapy. A pro-angiogenic approach would eliminate neovascular leakage and ultimately complete repair and preserve the neurosensory retina.
... Several authors have described the synergistic effects of these growth factors to induce the development of mature blood vessels either in vitro or in vivo [2,12]. The local administration of VEGF and bFGF in a rabbit ischemic hind limb model resulted in a higher capillary density and capillary vs muscle fiber ratio than either VEGF or bFGF alone [12]. ...
The development of a scaffold able to mimic the mechanical properties of elastic tissues and to induce local angiogenesis by controlled release of angiogenic growth factors could be applied in the treatment of several ischemic diseases. For this purpose a composite scaffold made of a poly(ether)urethane-polydimethylsiloxane (PEtU-PDMS) semi-interpenetrating polymeric network (semi-IPN) and fibrin loaded growth factors (GFs), such as VEGF and bFGF, was manufactured using spray, phase-inversion technique. To evaluate the contribution of each scaffold component with respect to tissue response and in particular to blood vessel formation, three different scaffold formulations were developed as follows: 1) bare PEtU-PDMS; 2) PEtU-PDMS/Fibrin; and 3) PEtU-PDMS/Fibrin + GFs. Scaffolds were characterized in vitro respect to their morphology, VEGF and bFGF release kinetics and bioactivity. The induction of in vivo angiogenesis after subcutaneous and ischemic hind limb scaffold implantation in adult Wistar rats was evaluated at 7 and 14 days by immunohistological analysis (IHA), while Laser Doppler Perfusion Imaging (LDPI) was performed in the hind limbs at 0, 3, 7, 10 and 14 days. IHA of subcutaneously implanted samples showed that at 7 and 14 days the PEtU-PDMS/Fibrin + GFs scaffold induced a statistically significant increase in number of capillaries compared to bare PEtU-PDMS scaffold. IHA of ischemic hind limb showed that at 14 days the capillary number induced by PEtU-PDMS/Fibrin + GFs scaffolds was higher than that of PEtU-PDMS/Fibrin scaffolds. Moreover, at both time-points PEtU-PDMS/Fibrin scaffolds induced a significant increase in number of capillaries compared to bare PEtU-PDMS scaffolds. LDPI showed that at 10 and 14 days the ischemic/non-ischemic blood perfusion ratio was significantly greater in the PEtU-PDMS/Fibrin + GFs than in the other scaffolds. In conclusion, this study showed that the semi-IPN composite scaffold acting as a pro-angiogenic GFs delivery system has therapeutic potential for the local treatment of ischemic tissue and wound healing.
Critical limb ischemia (CLI) is a highly morbid disease with many patients considered poor surgical candidates. The lack of treatment options for CLI has driven interest in developing molecular therapies within recent years. Through these translational medicine studies in CLI, much has been learned about the pathophysiology of the disease. Here, we present an overview of the macrovascular and microvascular changes that lead to the development of CLI, including impairment of angiogenesis, vasculogenesis, and arteriogenesis. We summarize the randomized clinical controlled trials that have used molecular therapies in CLI, and discuss the novel imaging modalities being developed to assess the efficacy of these therapies.
Progress in liver tissue engineering depends on the ability to reliably culture hepatocytes in vitro. In this study, we designed an environment that can help support liver-specific functions of primary hepatocytes for long enough periods of time to use them in therapeutic devices and in vitro modeling. This was accomplished by encapsulating hepatocytes with endothelial cells (ECs) and pericytes in a cell-adhesive and enzymatically degradable poly(ethylene glycol) (PEG) hydrogel scaffold. We used these hydrogels to investigate the long-term effects of 3D co-culture of hepatocytes with non-parenchymal ECs and pericytes in the context of vascular networks. We found that 3D co-culture of hepatocytes with tubule-forming ECs and pericytes leads to the development of robust microvascular tubules with close approximation to hepatocytes, similar to structures found in vivo. Furthermore, hepatocytes help support vasculogenesis and long-term tubule stability. We show that 3D co-culture of hepatocytes with cells in these networks enhances and retains hepatocyte function and phenotype. Hepatocytes in our constructs were able to synthesize 6.3 ± 1.1 pg albumin/hepatocyte/day at day 7 and increased over 3-fold to 22.6 ± 1.5 pg albumin/hepatocyte/day by day 28. Other essential liver functions, like urea secretion and cytochrome P450 activity, were also retained in our constructs for at least 4 weeks. Finally, we found that some of the hepatocytes in these organoids expressed the proliferation marker, Ki-67. These results are promising for the development of new liver disease treatments, organ engineering, and drug-testing models. Open image in new window
Lay Summary
Hepatocytes are the parenchymal cells of the liver, and they are responsible for performing many essential liver functions. When they are isolated from the body and cultured without the correct biochemical cues, they tend to lose their liver-specific functions and dedifferentiate into fibroblast-like cells. Advancement in tissue engineering liver disease therapies and in vitro drug-testing models depends on the development of hepatocyte culture methods that can maintain hepatocyte phenotype and function. Here, we tailored a polymeric hydrogel in which we can 3D co-culture hepatocytes with stabilizing non-parenchymal cells and achieve organ-level albumin synthesis as well maintenance of urea secretion and cytochrome P450 enzymatic activity for at least a month.
Hepatocyte growth factor (HGF) was originally identified as a potent mitogen of hepatocytes in 1980s. HGF induces mitogenic, motogenic and morphogenic activities in epithelial cells through tyrosine phosphorylation of its receptor, c-Met. HGF-c-Met axis is necessary for embryogenesis, organogenesis and tissue repair of almost epithelial organs. Indeed, a loss in HGF-c-Met signaling pathways leads to organ damage and dysfunction during acute and chronic diseases. In the early 1990s’, HGF was shown to be an angiogenic regulator via direct effects on endothelial cells (ECs). HGF plays an important part for vascular branching tubular formation via mitogenic, motogenic and morphogenic activities. HGF stabilizes endothelial barrier function via Rac1-dependent cascades. HGF is an anti-inflammatory ligand through inhibiting NF-κB activation in ECs. HGF protects ECs from injurious stresses. However, local HGF production is impaired due to cylic AMP depletion under an ischemic condition. In contrast, c-Met expression is up-regulated, in response to a local hypoxia. When HGF is exogenously injected to hypoxic regions, angiogenic regeneration is induced, associated with an increase in blood flow. This is a rationale why HGF supplemental therapy improves ischemic organ diseases, such as peripheral arterial disease (PAD) and cardial arterial disease (CAD) at least in animal models. Now, clinical trials are ongoing worldwide to determine an optimal condition of HGF supplemental therapy for the treatments of PAD and CAD. In this review, a therapeutic potential of HGF will be discussed, with a focus on biological mechanisms and preclinical or clinical outcomes during ischemic organ diseases.
Introduction:
Gene therapy has emerged as a novel therapy to promote angiogenesis in patients with critical limb ischemia (CLI) caused by peripheral artery disease. Researchers working in this area have focused on pro-angiogenic factors, such as VEGF, fibroblast growth factor (FGF) and hepatocyte growth factor (HGF). Based on the elaborate studies and favorable results of basic research using naked plasmid DNA (pDNA) encoding these growth factors, some clinical Phase I and Phase II trials have been performed. The results of these studies demonstrate the safety of these approaches and their potential for symptomatic improvement in CLI patients. However, the Phase III clinical trials have so far been limited to HGF gene therapy. Because one pitfall of the Phase III trials has been the limited transgene expression achieved using naked pDNA alone, the development of more efficient gene transfer systems, such as ultrasound microbubbles and the needleless injector, as well as the addition of other genes will make these novel therapies more effective and ease the symptoms of CLI.
Areas covered:
This study reviews the previously published basic research and clinical trials that have studied VEGF, FGF and HGF gene therapies for the treatment of CLI. Adjunctive therapies, such as the addition of prostacyclin synthase genes and the development of more efficient gene transfer techniques for pDNA, are also reviewed.
Expert opinion:
To date, clinical studies have demonstrated the safety of gene therapy in limb ischemia but the effectiveness of this treatment has not been determined. Larger clinical studies, as well as the development of more effective gene therapy, are needed to achieve and confirm beneficial effects.
Since their initial discovery, endothelial progenitor cells (EPCs) have held tremendous promise for cell therapy for a variety of cardiovascular diseases including pulmonary hypertension. The clinical experience to date suggests that circulating or bone marrow mononuclear cells and EPCs can induce neovascularization, and enhance cardiac repair after myocardial function, as well as improvements in the hemodynamic and functional status of patients with idiopathic pulmonary arterial hypertension. Although these results are promising, the overall magnitude of the clinical benefits seen in these trials appear to be rather modest. Indeed, strong experimental evidence points towards a reduction in mobilization and impairment in function of EPCs in preclinical models and patients with cardiac disease or with cardiovascular risk factors such as advanced age, type I and II diabetes, hypercholesterolemia, coronary artery disease, as well as other conditions such as pulmonary hypertension. Genetic engineering of EPCs ex vivo, prior to transplantation, is a promising cell-enhancement strategy for restoring the angiogenic potential of autologous, patient-derived cells. This review provides an update of the experimental studies that have used gene-modified EPC therapy to treat ischemic cardiovascular disease and pulmonary hypertension.
