Therapeutic myocardial angiogenesis with vascular endothelial growth factors

Emerging evidence has shown that administration of angiogenic growth factors, either as recombinant protein or by gene transfer, can augment tissue perfusion through neovascularization in animal models of myocardial and hindlimb ischemia. Many cytokines have angiogenic activity; one of those that have been best studied in animal models and clinical trials is vascular endothelial growth factor (VEGF). VEGF has been known to be a key regulator of physiologic and pathologic angiogenesis associated with tumor. Recently the effect of VEGF is not restricted to the direct angiogenic effect in vivo but includes mobilization of bone-marrow-derived endothelial progenitor cells and augmentation of postnatal vasculogenesis in situ. Clinical trials of therapeutic angiogenesis with VEGF in patients with end-stage coronary artery disease have shown increases in exercise time and reductions in anginal symptoms and have provided objective evidence of improved perfusion and left ventricular function. Larger scale placebo-controlled trials with recombinant protein (rhVEGF₁₆₅) have been limited to intracoronary and intravenous administration and have shown favorable trends in exercise time and angina frequency. Small-scale, placebo-controlled, randomized clinical trials of gene transfer (phVEGF-2) via thoracotomy or percutaneous intramyocardial delivery demonstrated significant improvement of both subjective symptoms and objective measures of myocardial ischemia. Both therapeutic modalities appear to be safe and well tolerated. Further studies are required to determine the optimal dose, formulation, route of administration, and combinations of growth factors and the utility of adjunctive endothelial progenitor cell or other stem cell supplementation, to provide safe and effective therapeutic myocardial neovascularization. (Mol Cell Biochem 264: 63–74, 2004)     

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.     

Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

Background aimsTransplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media.MethodsThe effects of prolonged hypoxic culture on growth and pro-angiogenic properties were investigated using human ASC cultured at 1%, 5% and 21% oxygen. The effect of trypsinization on the expression of pro-angiogenic genes was also determined.ResultsTrypsinization induced up-regulation of the vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) genes independent of oxygen concentration. The expression of VEGF and IGF-1 was up-regulated in ASC cultured at 1% oxygen for 13 days compared with 4 days. The VEGF concentration in ASC-conditioned media was higher after prolonged hypoxic culture compared with short-term culture, while the IGF-1 and chemokine (CXC motif) ligand 12 (CXCL12) concentrations were unchanged. The VEGF receptor blocker SU5416 abolished angiogenesis in a cultured rat aortic ring model. Media from cells exposed to hypoxia increased angiogenesis, an effect that was dependent on factors other than just the VEGF concentration in the added media.ConclusionsOptimization of the angiogenic potential of stem cell-based therapy in the treatment of vascular disease is important. We have demonstrated that prolonged hypoxic culture and trypsinization augment the therapeutic angiogenic potential of ASC.     

Changing the logic of therapeutic angiogenesis for ischemic disease

The worldwide epidemic of ischemic disease urgently requires innovative treatments. Recently, therapeutic angiogenesis has emerged as a noninvasive supply-side approach, aimed at promoting neovascularization in underperfused tissues through the local delivery of angiogenic growth factors. Successful preclinical studies paved the way for the first clinical trials, with single growth factors given as recombinant proteins or genes. However, clinical results have not matched the initial promises. Our opinion is that the logic of therapeutic angiogenesis needs profound revision. Here, we introduce the concept that pleiotropic agents can stimulate the healing of all the components of ischemic tissue. We also propose prophylactic interventions to delay vascular senescence. The optimization of therapeutic angiogenesis will open unprecedented opportunities for the care of life-threatening ischemic disease.     

Therapeutic myocardial angiogenesis with vascular endothelial growth factors

Emerging evidence has shown that administration of angiogenic growth factors, either as recombinant protein or by gene transfer, can augment tissue perfusion through neovascularization in animal models of myocardial and hindlimb ischemia. Many cytokines have angiogenic activity; one of those that have been best studied in animal models and clinical trials is vascular endothelial growth factor (VEGF). VEGF has been known to be a key regulator of physiologic and pathologic angiogenesis associated with tumor. Recently the effect of VEGF is not restricted to the direct angiogenic effect in vivo but includes mobilization of bone-marrow-derived endothelial progenitor cells and augmentation of postnatal vasculogenesis in situ. Clinical trials of therapeutic angiogenesis with VEGF in patients with end-stage coronary artery disease have shown increases in exercise time and reductions in anginal symptoms and have provided objective evidence of improved perfusion and left ventricular function. Larger scale placebo-controlled trials with recombinant protein (rhVEGF165) have been limited to intracoronary and intravenous administration and have shown favorable trends in exercise time and angina frequency. Small-scale, placebo-controlled, randomized clinical trials of gene transfer (phVEGF-2) via thoracotomy or percutaneous intramyocardial delivery demonstrated significant improvement of both subjective symptoms and objective measures of myocardial ischemia. Both therapeutic modalities appear to be safe and well tolerated. Further studies are required to determine the optimal dose, formulation, route of administration, and combinations of growth factors and the utility of adjunctive endothelial progenitor cell or other stem cell supplementation, to provide safe and effective therapeutic myocardial neovascularization.     

Hypoxia Augments Outgrowth Endothelial Cell (OEC) Sprouting and Directed Migration in Response to Sphingosine-1-Phosphate (S1P)

Therapeutic angiogenesis provides a promising approach to treat ischemic cardiovascular diseases through the delivery of proangiogenic cells and/or molecules. Outgrowth endothelial cells (OECs) are vascular progenitor cells that are especially suited for therapeutic strategies given their ease of noninvasive isolation from umbilical cord or adult peripheral blood and their potent ability to enhance tissue neovascularization. These cells are recruited to sites of vascular injury or tissue ischemia and directly incorporate within native vascular endothelium to participate in neovessel formation. A better understanding of how OEC activity may be boosted under hypoxia with external stimulation by proangiogenic molecules remains a challenge to improving their therapeutic potential. While vascular endothelial growth factor (VEGF) is widely established as a critical factor for initiating angiogenesis, sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, has recently gained great enthusiasm as a potential mediator in neovascularization strategies. This study tests the hypothesis that hypoxia and the presence of VEGF impact the angiogenic response of OECs to S1P stimulation in vitro. We found that hypoxia altered the dynamically regulated S1P receptor 1 (S1PR1) expression on OECs in the presence of S1P (1.0 μM) and/or VEGF (1.3 nM). The combined stimuli of S1P and VEGF together promoted OEC angiogenic activity as assessed by proliferation, wound healing, 3D sprouting, and directed migration under both normoxia and hypoxia. Hypoxia substantially augmented the response to S1P alone, resulting in ~6.5-fold and ~25-fold increases in sprouting and directed migration, respectively. Overall, this report highlights the importance of establishing hypoxic conditions in vitro when studying ischemia-related angiogenic strategies employing vascular progenitor cells.     

Mechanisms and targets for angiogenic therapy after stroke

Stroke remains a major health problem worldwide, and is the leading cause of serious long-term disability. Recent findings now suggest that strategies to enhance angiogenesis after focal cerebral ischemia may provide unique opportunities to improve clinical outcomes during stroke recovery. In this mini-review, we survey emerging mechanisms and potential targets for angiogenic therapies in brain after stroke. Multiple elements may be involved, including growth factors, adhesion molecules and progenitor cells. Furthermore, cross talk between angiogenesis and neurogenesis may also provide additional substrates for plasticity and remodeling in the recovering brain. A better understanding of the molecular interplay between all these complex pathways may lead to novel therapeutic avenues for tackling this difficult disease.Key words: angiogenic therapy, stroke, neuroprotection, neurogenesis, angiogenesis, neurovascular unit, cerebral ischemia, stroke recovery     

Hypoxic preconditioning reinforces cellular functions of autologous peripheral blood-derived cells in rabbit hindlimb ischemia model

Peripheral blood mononuclear cell (PBMNC) is one of powerful tools for therapeutic angiogenesis in hindlimb ischemia. However, traditional approaches with transplanted PBMNCs show poor therapeutic effects in severe ischemia patients. In this study, we used autograft models to determine whether hypoxic pretreatment effectively enhances the cellular functions of PBMNCs and improves hindlimb ischemia. Rabbit PBMNCs were cultured in the hypoxic condition. After pretreatment, cell adhesion, stress resistance, and expression of angiogenic factor were evaluated in vitro. To examine in vivo effects, we autografted preconditioned PBMNCs into a rabbit hindlimb ischemia model on postoperative day (POD) 7. Preconditioned PBMNCs displayed significantly enhanced functional capacities in resistance to oxidative stress, cell viability, and production of vascular endothelial growth factor. In addition, autologous transplantation of preconditioned PBMNCs significantly induced new vessels and improved limb blood flow. Importantly, preconditioned PBMNCs can accelerate vessel formation despite transplantation on POD 7, whereas untreated PBMNCs showed poor vascularization. Our study demonstrated that hypoxic preconditioning of PBMNCs is a feasible approach for increasing the retention of transplanted cells and enhancing therapeutic angiogenesis in ischemic tissue.     

Therapeutic angiogenesis for cardiovascular disease

The identification of angiogenic growth factors, such as vascular endothelial growth factor and fibroblast growth factor, has fueled interest in using such factors to induce therapeutic angiogenesis. The results of numerous animal studies and clinical trials have offered promise for new treatment strategies for various ischemic diseases. Increased understanding of the cellular and molecular biology of vessel growth has, however, prompted investigators and clinicians alike to reconsider the complexity of therapeutic angiogenesis. The realization that formation of a stable vessel is a complex, multistep process may provide useful insights into the design of the next generation of angiogenesis therapy.     

Dormant Tumors Awaken by a Short-Term Angiogenic Burst: The Spike Hypothesis

Tumor dormancy, a complex and still poorly understood phenomenon observed both in experimental models and in patients, has been associated with insufficient angiogenic capacity. A defined event, termed "angiogenic switch" and characterized by an imbalance between pro- and anti-angiogenic factors, often marks interruption of the dormant state, thus triggering invasive tumor growth. In our current view, sustained angiogenesis is considered essential in promoting this transition. Recently, we demonstrated that co-administration of proliferation-arrested Kaposi's sarcoma cells or recombinant angiogenic factors interrupts dormancy of poorly angiogenic leukemia cells by providing a brief angiogenic burst. These findings indicate that even a transient angiogenic switch can prime progressive tumor growth and suggest that tumor angiogenesis is a process requiring a higher amount of angiogenic factors for its induction than maintenance. Here we discuss the implications of these observations on our view of tumor angiogenesis and on the therapeutic potential of angiogenesis inhibitors.     

Enhancement of Ischemic Wound Healing by Spheroid Grafting of Human Adipose-Derived Stem Cells Treated with Low-Level Light Irradiation

We investigated whether low-level light irradiation prior to transplantation of adipose-derived stromal cell (ASC) spheroids in an animal skin wound model stimulated angiogenesis and tissue regeneration to improve functional recovery of skin tissue. The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31⁺, KDR⁺, and CD34⁺. On the other hand, monolayer-cultured hASCs were negative for these markers. PBS, human adipose tissue-derived stromal cells, and the ASC spheroid were transplanted into a wound bed in athymic mice to evaluate the therapeutic effects of the ASC spheroid in vivo. The ASC spheroid transplanted into the wound bed differentiated into endothelial cells and remained differentiated. The density of vascular formations increased as a result of the angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site. These results indicate that the transplantation of the ASC spheroid significantly improved functional recovery relative to both ASC transplantation and PBS treatment. These findings suggest that transplantation of an ASC spheroid treated with low-level light may be an effective form of stem cell therapy for treatment of a wound bed.     

Therapeutic neovascularization for peripheral arterial diseases: advances and perspectives

Recently, with the better understanding of the mechanisms of neovascularization, many new therapeutic approaches to enhance neovascularization have emerged. Of these diverse emerging methods, use of growth factors and cells are the two major ones. This review will provide an update on the present understanding of the basic mechanisms of angiogenesis, vasculogenesis, and arteriogenesis, as a basis for designing future pro-neovascularization treatments. Several angiogenic factors including vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) have been implicated in augmenting the neovascularization process. However, single growth factors are not sufficient to generate functional vessels. In synergistic or complementary manner, these factors may be used in harmony to form long-term functional vessels. Cell therapy has the potential to supply stem/progenitor cells and multiple angiogenic factors to the region of ischemia. However, the efficacy of stem cells transplantation may be impaired by low survival rate, insufficient cell number and impaired function in aging and diseases. Combination of cells or cells primed with growth factor(s) or genetic modification may augment the therapeutic efficacy. This paper reviews critical literature in depth to elucidate the mechanism of therapeutic neovascularization, angiogenic factor therapy and cell transplantation. Based on past experience and actual knowledge, we propose future strategies for clinical application and discuss the problems and controversies that need to be addressed in order to fully exploit the potential of growth factors and/or cell transplantation with clinical relevance.     

Enrichment of vascular endothelial growth factor secreting mesenchymal stromal cells enhances therapeutic angiogenesis in a mouse model of hind limb ischemia

Critical limb ischemia, a severe manifestation of peripheral artery disease, is emerging as a major concern in aging societies worldwide. Notably, cell-based gene therapy to induce angiogenesis in ischemic tissue has been investigated as treatment. Despite many studies demonstrating the efficacy of this approach, better therapies are required to prevent serious sequelae such as claudication, amputation and other cardiovascular events. We have now established a simplified method to enhance the effects of therapeutic transgenes by selecting for and transplanting only transduced cells. Herein, mesenchymal stromal cells were transfected to co-express vascular endothelial growth factor as angiogenic factor and enhanced green fluorescent protein as marker. Transfected cells were then collected using flow cytometry based on green fluorescence and transplanted into ischemic hind limbs in mice. Compared with unsorted or untransfected cells, purified cells significantly improved blood perfusion within 21days, suggesting that transplanting only cells that overexpress vascular endothelial growth factor enhances therapeutic angiogenesis. Importantly, this approach may prove to be useful in cell-based gene therapy against a wide spectrum of diseases, simply by replacing the gene to be delivered or the cell to be transplanted.     

Gene modification with integrin-linked kinase improves function of endothelial progenitor cells in pre-eclampsia in vitro

Integrin-linked kinase (ILK), a multifunctional serine-threonine protein kinase, has been shown to have implications for the treatment of ischemia vascular diseases by promoting angiogenesis in various tissues. However, whether this kinase has therapeutic potential in pre-eclampsia is not well studied. In this report, we determined the changes in the production and action of ILK on endothelial progenitor cells (EPCs) isolated from patients with pre-eclampsia. The effects of ILK transfection on proliferation, migration, and angiogenesis of EPCs were investigated. We showed that EPCs transfected with the ILK gene expressed high levels of ILK protein and mRNA. Transfection with ILK also enhanced the proliferative, migratory, and angiogenic capabilities of EPCs, and promoted the production of VEGF. These results suggest that ILK gene transfection is an effective approach to augment angiogenic properties of EPCs in vitro and providing basis for clinical cell-based gene therapy in patients with pre-eclampsia.     

Therapeutic angiogenesis using tumor cell‐conditioned medium

Stem cell‐conditioned medium (CM), which contains angiogenic factors that are secreted by stem cells, represents a potential therapy for ischemic diseases. Along with stem cells, tumor cells also secrete various angiogenic factors. Here, tumor cells as a cell source of CM for therapeutic angiogenesis was evaluated and the therapeutic efficacy of tumor cell CM in mouse hindlimb ischemia models was demonstrated. CM obtained from a human fibrosarcoma HT1080 cell line culture was compared with CM obtained from a human bone marrow‐derived mesenchymal stem cell (MSC) culture. HT1080 CM contained higher concentrations of angiogenic factors compared with MSC CM, which was attributable to the higher cell density that resulted from a much faster growth rate of HT1080 cells compared with MSCs. For use in in vitro and in vivo angiogenesis studies, HT1080 CM was diluted such that HT1080 CM and MSC CM would have the same cell number basis. The two types of CMs induced the same extent of human umbilical vein endothelial cell (HUVEC) proliferation in vitro. The injection of HT1080 CM into mouse ischemic limbs significantly improved capillary density and blood perfusion compared with the injection of fresh medium. Although the therapeutic outcome of HT1080 CM was similar to that of MSC CM, the preparation of CM by tumor cell line culture would be much more efficient due to the faster growth and unlimited life‐time of the tumor cell line. These data suggest the potential application of tumor cell CM as a therapeutic modality for angiogenesis and ischemic diseases. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:456–464, 2016     

Adipose stromal cells--plastic type of cells with high therapeutic potential

Much effort has been made in searching for multipotent cell types with high therapeutic potentials for repair of damaged tissue. Through enzymatic digestion of fat tissue, it is possible to obtain a large number of stromal cells. Isolated cells show a high proliferate capacity in culture. All this makes adipose stromal cells (ASC) promising candidates for their use in cell therapy. This review is focused on analyzing the surface antigen profile of isolated population of ASC, expression of angiogenic factors by these cells, as well as on their differentiation potential. A high percentage of ASC population initially express the progenitor cell marker CD34, but during culturing, cells exhibit a mesenchymal cell phenotype and express CD29, CD105, CD106, CD166. Culturing ASC in specific differentiation media induces expression of early markers of differentiated mesenchymal cells, such as adipocytes, chondrocytes and osteoblasts, as well as myoblasts, cardiomyocytes and neural cells. It has been also shown that ASC have a strong pro-angiogenic potential, they are able to secret growth factors, such as VEGF, HGF, bFGF and others, which stimulate survival and proliferation of endothelial cells. In addition, systemic or local delivery of ASC to mice with hindlimb ischemia stimulates recovery of injured tissue and blood flow. Potential clinical uses of ASCs are discussed in the review.     

Non-peptidic Thrombospondin-1 Mimics as Fibroblast Growth Factor-2 Inhibitors: AN INTEGRATED STRATEGY FOR THE DEVELOPMENT OF NEW ANTIANGIOGENIC COMPOUNDS*

Endogenous inhibitors of angiogenesis, such as thrombospondin-1 (TSP-1), are promising sources of therapeutic agents to treat angiogenesis-driven diseases, including cancer. TSP-1 regulates angiogenesis through different mechanisms, including binding and sequestration of the angiogenic factor fibroblast growth factor-2 (FGF-2), through a site located in the calcium binding type III repeats. We hypothesized that the FGF-2 binding sequence of TSP-1 might serve as a template for the development of inhibitors of angiogenesis. Using a peptide array approach followed by binding assays with synthetic peptides and recombinant proteins, we identified a FGF-2 binding sequence of TSP-1 in the 15-mer sequence DDDDDNDKIPDDRDN. Molecular dynamics simulations, taking the full flexibility of the ligand and receptor into account, and nuclear magnetic resonance identified the relevant residues and conformational determinants for the peptide-FGF interaction. This information was translated into a pharmacophore model used to screen the NCI2003 small molecule databases, leading to the identification of three small molecules that bound FGF-2 with affinity in the submicromolar range. The lead compounds inhibited FGF-2-induced endothelial cell proliferation in vitro and affected angiogenesis induced by FGF-2 in the chicken chorioallantoic membrane assay. These small molecules, therefore, represent promising leads for the development of antiangiogenic agents. Altogether, this study demonstrates that new biological insights obtained by integrated multidisciplinary approaches can be used to develop small molecule mimics of endogenous proteins as therapeutic agents.     

No influence of tumor growth by intramuscular injection of hepatocyte growth factor plasmid DNA: safety evaluation of therapeutic angiogenesis gene therapy in mice

Recently, a novel therapeutic treatment for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As intramuscular injection of naked human hepatocyte growth factor (HGF) plasmid DNA induced therapeutic angiogenesis in several animal test subjects, we have started a clinical trial to treat peripheral arterial disease. However, one might assume that over-expression of angiogenic growth factors could enhance tumor growth. To resolve this issue, we examined the over-expression of HGF in tumor bearing mice. Tumors on their backs were prepared with an intradermal inoculation of A431, human epidermoid cancer cells expressing c-Met. These mice were intramuscularly injected with human HGF plasmid or control plasmid into the femoral muscle. Human HGF concentration was increased only in the femoral muscle, but not in blood. Although recombinant HGF stimulated the growth of A431 cells in vitro, temporally and locally HGF elevation in hindlimb had no effect on tumor growth in mice.     

Mechanisms and targets for angiogenic therapy after stroke

Stroke remains a major health problem worldwide, and is the leading cause of serious long-term disability. Recent findings now suggest that strategies to enhance angiogenesis after focal cerebral ischemia may provide unique opportunities to improve clinical outcomes during stroke recovery. In this mini-review, we survey emerging mechanisms and potential targets for angiogenic therapies in brain after stroke. Multiple elements may be involved, including growth factors, adhesion molecules and progenitor cells. Furthermore, cross talk between angiogenesis and neurogenesis may also provide additional substrates for plasticity and remodeling in the recovering brain. A better understanding of the molecular interplay between all these complex pathways may lead to novel therapeutic avenues for tackling this difficult disease.     

Erythropoietin augments the efficacy of therapeutic angiogenesis induced by allogenic bone marrow stromal cells in a rat model of limb ischemia

Transplantation of adult marrow stromal cells (MSCs) has been developed as a new method of treating severe ischemia diseases by therapeutic angiogenesis. Erythropoietin (EPO) is capable of inducing angiogenesis and inhibiting MSCs apoptosis. The effect of EPO on the therapeutic potency of MSCs transplantation in a rat model of limb ischemia was investigated in the current study. The results indicate that the combined treatment with MSC transplantation and EPO infusion is superior to MSC transplantation alone in the treatment of limb ischemia. MSCs transplantation and EPO infusion could enhance the angiogenic effect of each other to achieve a better therapeutic effect. This combination therapy may become a more effective approach for ischemia diseases of the limbs.