The latest review of therapeutic angiogenesis using angiogenic growth factors to peripheral arterial diseases

Therapeutic angiogenesis using angiogenic growth factors is expected to be a new treatment of patients with severe ischemic diseases. Indeed, human gene therapy for peripheral arterial disease(PAD) using VEGF gene demonstrated the beneficial effects. In contrast, we have reported the potent angiogenic activity of hepatocyte growth factor (HGF) in animal study and we planned gene therapy for ASO and Buerger disease using HGF gene (TREAT-HGF). In a prospective, open-labeled clinical trial, we investigated the safety and biological efficiency of this gene therapy in patients with peripheral arterial disease(PAD) who had failed conventional therapy.     

Angiogenic and antiangiogenic gene therapy

Gene therapy is thought to be a promising method for the treatment of various diseases. One gene therapy strategy involves the manipulations on a process of formation of new vessels, commonly defined as angiogenesis. Angiogenic and antiangiogenic gene therapy is a new therapeutic approach to the treatment of cardiovascular and cancer patients, respectively. So far, preclinical and clinical studies are successfully focused mainly on the treatment of coronary artery and peripheral artery diseases. Plasmid vectors are often used in preparations in angiogenic gene therapy trials. The naked plasmid DNA effectively transfects the skeletal muscles or heart and successfully expresses angiogenic genes that are the result of new vessel formation and the improvement of the clinical state of patients. The clinical preliminary data, although very encouraging, need to be well discussed and further study surely continued. It is really possible that further development of molecular biology methods and advances in gene delivery systems will cause therapeutic angiogenesis as well as antiangiogenic methods to become a supplemental or alternative option to the conventional methods of treatment of angiogenic diseases.     

Therapeutic angiogenesis for critical limb ischemia: invited commentary.

Lower extremity arterial occlusive disease results in tissue ischemia of the legs and is relatively common in the elderly. Clinically, it may be asymptomatic, cause muscle pain during exercise, or progress to a severe degree of ischemia that may result in limb loss. Although bypass surgery and angioplasty have increased the rate of limb salvage in these patients, amputation of the affected limb remains a common outcome for many patients. Therapeutic angiogenesis is the administration of angiogenic factors, or genes encoding these factors, to promote neovascularization and thereby increase blood flow to the ischemic leg. We have developed an animal model of hindlimb ischemia in which to study therapeutic angiogenesis. We chose nitric oxide as the angiogenic factor for our experiments because of its ability to induce angiogenesis, vasodilation, and inhibit inflammation. In this review, we will discuss our experience with our model of hindlimb ischemia, as well as discuss our results of gene therapy for therapeutic angiogenesis using nitric oxide.     

Therapeutic angiogenesis for ischemic diseases

The clinical consequences of peripheral arterial disease include pain on walking, pain at rest and loss of tissue integrity in the distal ischemic limbs. Although development of beneficial drugs and intervention devices do contribute to the treatment of this disease, critical limb ischemia is estimated to develop in 500 to 1,000 individuals per million per year. As angiogenic growth factors can stimulate the development of collateral arteries, a concept called "therapeutic angiogenesis" is now evaluated in the clinical fields. Recent progress in molecular biology has led to the development of gene therapy as a new strategy to treat a variety of cardiovascular diseases using angiogenic growth factors such as vascular endothelial growth factor (VEGF). Therapeutic angiogenesis using angiogenic growth factors is expected to be a new treatment for patients with severe ischemic heart or peripheral arterial disease.     

An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis

A mouse model involving the human papillomavirus type-16 oncogenes develops cervical cancers by lesional stages analogous to those in humans. In this study the angiogenic phenotype was characterized, revealing intense angiogenesis in high-grade cervical intraepithelial neoplasias (CIN-3) and carcinomas. MMP-9, a proangiogenic protease implicated in mobilization of VEGF, appeared in the stroma concomitant with the angiogenic switch, expressed by infiltrating macrophages, similar to what has been observed in humans. Preclinical trials sought to target MMP-9 and angiogenesis with a prototypical MMP inhibitor and with a bisphosphonate, zoledronic acid (ZA), revealing both to be antiangiogenic, producing effects comparable to a Mmp9 gene KO in impairing angiogenic switching, progression of premalignant lesions, and tumor growth. ZA therapy increased neoplastic epithelial and endothelial cell apoptosis without affecting hyperproliferation, indicating that ZA was not antimitotic. The analyses implicated cellular and molecular targets of ZA's actions: ZA suppressed MMP-9 expression by infiltrating macrophages and inhibited metalloprotease activity, reducing association of VEGF with its receptor on angiogenic endothelial cells. Given its track record in clinical use with limited toxicity, ZA holds promise as an "unconventional" MMP-9 inhibitor for antiangiogenic therapy of cervical cancer and potentially for additional cancers and other diseases where MMP-9 expression by infiltrating macrophages is evident.     

Anti-angiogenesis in cancer therapy

OBJECTIVE: To review tumor angiogenesis, identify potential targets for anti-angiogenic cancer therapy, and highlight certain anti-angiogenic agents in clinical trials. DATA SOURCE: Research articles, abstracts, review articles, and book chapters. CONCLUSION: Tumor angiogenesis is a complex, multistep process that provides a target for antineoplastic therapy whereby tumor neovasculature is interrupted at various steps in the angiogenic process. Clinical trials are investigating the application and efficacy of anti-angiogenic agents. IMPLICATIONS FOR NURSING PRACTICE: Oncology nurses must continually increase their knowledge with the onset of newer, targeted agents. This will provide a background for educating and caring for the patient who is receiving anti-angiogenic therapy.     

Gene-therapy for peripheral vascular diseases

The prognosis for patients with chronic critical leg ischemic is often poor. The treatment of peripheral vascular disease, although greatly improved over recent decades by drug medication, surgical and minimally-invasive techniques, remains limited by vascular proliferative lesions and by our inability to modulate the progression of native disease. The therapeutic angiogenesis is now the most expected therapy for peripheral vascular diseases. This review explores some of concepts and methods of therapeutic angiogenesis including gene therapy using angiogenic growth factor such as VEGF and basic FGF and an implantation of bone marrow derived endothelial progenitor cells.     

The CXC chemokine, monokine induced by interferon-gamma, inhibits non-small cell lung carcinoma tumor growth and metastasis

Angiogenesis is an absolute requirement for tumor growth beyond 2 mm3 in size. The balance in expression between opposing angiogenic and angiostatic factors controls the angiogenic process. The CXC chemokines are a group of chemotactic cytokines that possess disparate activity in the regulation of angiogenesis. Non-small cell lung carcinoma (NSCLC) has an imbalance in expression of ELR+ (angiogenic) compared with ELR- (angiostatic) CXC chemokines that favors angiogenesis and progressive tumor growth. We found that the level of the ELR- CXC chemokine MIG (monokine induced by interferon gamma) in human specimens of NSCLC was not significantly different from that found in normal lung tissue. These results suggested that the increased expression of ELR+ CXC chemokines found in these tumor samples is not counterregulated by a concomitant increase in the expression of the angiostatic ELR-CXC chemokine MIG. This would result in an even more profound imbalance in the expression of regulatory factors of angiogenesis that would favor neovascularization. We hypothesized that MIG might be an endogenous inhibitor of NSCLC tumor growth in vivo and that reconstituion of MIG in the tumor microenvironment would result in the inhibition of tumor growth and metastasis. In support of this hypothesis, we demonstrate here that overexpression of the ELR-CXC chemokine MIG, by three different strategies including gene transfer, results in the inhibition of NSCLC tumor growth and metastasis via a decrease in tumor-derived vessel density. These findings support the importance of the ELR- CXC chemokine MIG in inhibiting NSCLC tumor growth by attenuation of tumor-derived angiogenesis. Furthermore, these findings demonstrate the potential of gene therapy as an alternative means to deliver and overexpress a potent angiostatic CXC chemokine.     

Combined angiopoietin-1 and vascular endothelial growth factor gene transfer restores cavernous angiogenesis and erectile function in a rat model of hypercholesterolemia.

Hypercholesterolemia-related endothelial cell dysfunction and decreased endothelium-derived nitric oxide formation may account for impaired angiogenesis and subsequent erectile dysfunction. Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular maturation and enhances vascular endothelial growth factor (VEGF)-induced angiogenesis in a complementary manner. We hypothesized that combined adenovirus-delivered human Ang1 (ad-Ang1) and VEGF165 (ad-VEGF165) gene transfer might promote angiogenesis cooperatively in a rat model of hypercholesterolemic erectile dysfunction and result in a recovery of erectile function. Ad-Ang1 and ad-VEGF165 were injected either alone or in combination into the corpus cavernosum of the penis. Combined gene transfer of both ad-Ang1 and ad-VEGF165 significantly increased cavernous angiogenesis, eNOS phosphorylation, and cGMP expression compared with that in the groups treated with either therapy alone. Erectile function, as evaluated by electrical stimulation of the cavernous nerve 2 and 8 weeks after treatment, was completely restored in the combined treatment group, whereas intracavernous injection of either ad-Ang1 or ad-VEGF165 alone elicited partial improvement. The results indicate that combined application of angiogenic factors may enhance cavernous angiogenesis cooperatively by reinforcing the endothelium both structurally and functionally, which results in an additive effect on erectile function in hypercholesterolemic rats.     

Anti-angiogenic inhibition of tumor growth by systemic delivery of PEI-g-PEG-RGD/pCMV-sFlt-1 complexes in tumor-bearing mice.

Vascular endothelial growth factor (VEGF) is an endogenous mediator of tumor angiogenesis. Blocking associations of the VEGF with its corresponding receptors (Flt-1, KDR/flk-1) have become critical for anti-tumor angiogenesis therapy. Previously, we synthesized PEI-g-PEG-RGD conjugate and evaluated as an angiogenic endothelial polymeric gene carrier. In this study, PEI-g-PEG-RGD/pCMV-sFlt-1 complexes are evaluated in terms of tumor growth inhibition in vivo. Complexes were repeatedly injected systemically via tail vein into subcutaneous tumor-bearing mice. As a result, tumor growth was inhibited in the PEI-g-PEG-RGD/pCMV-sFlt-1 injected group. However, this effect was not identified in PEI-g-PEG/pCMV-sFlt-1 or PEI-g-PEG-RGD/pCMV-GFP control groups. Moreover, the survival rate increased in the PEI-g-PEG-RGD/pCMV-sFlt-1 group compared with the controls group. These results suggest that delivery of pCMV-sFlt-1 using PEG-g-PEG-RGD may be effective for anti-angiogenic gene therapy.     

IRES-based Vector Coexpressing FGF2 and Cyr61 Provides Synergistic and Safe Therapeutics of Lower Limb Ischemia

Due to the lack of an adequate conventional therapy against lower limb ischemia, gene transfer for therapeutic angiogenesis is seen as an attractive alternative. However, the possibility of side effects, due to the expression of large amounts of angiogenic factors, justifies the design of devices that express synergistic molecules in low controlled doses. We have developed an internal ribosome entry site (IRES)–based bicistronic vector expressing two angiogenic molecules, fibroblast growth factor 2 (FGF2), and Cyr61. Through electrotransfer into the ApoE^−/− mice hindlimb ischemic muscle model, we show that the IRES-based vector gives more stable expression than either monocistronic plasmid. Furthermore, laser Doppler analysis, arteriography, and immunochemistry clearly show that the bicistronic vector promotes a more abundant and functional revascularization than the monocistronic vectors, despite the fact that the bicistronic system produces 5–10 times less of each angiogenic molecule. Furthermore, although the monocistronic Cyr61 vector accelerates B16 melanoma growth in mice, the bicistronic vector is devoid of such side effects. Our results show an active cooperation of FGF2 and Cyr61 in therapeutic angiogenesis of hindlimb ischemia, and validate the use of IRES-based bicistronic vectors for the coexpression of controlled low doses of therapeutic molecules, providing perspectives for a safer gene therapy of lower limb ischemia.     

Prevention of angiogenesis by naked DNA IL-12 gene transfer: angioprevention by immunogene therapy

IL-12 is thought to induce a cytokine cascade with antiangiogenic effects mediated by IFN-gamma and angiostatic CXCR3 chemokine ligands. Naked DNA intramuscular injection of an expression vector plasmid producing IL-12 resulted in significant, well-tolerated elevation of serum IL-12 levels. Injection of the IL-12 plasmid at least 2 days, and up to 20 days, before subcutaneous injection of matrigel with angiogenic factors resulted in strong prevention of angiogenesis in both C57/bl and nude mice. A single injection of the IL-12 plasmid contemporarily with the matrigel or 2 days after resulted in partial, statistically not significant, inhibition. Control plasmid injection did not affect either angiogenesis or angiogenesis inhibition by IL-12 protein in vivo. Angiogenesis inhibition was observed in NK cell-depleted C57/bl and nude mice as well as in IFN-gamma(-/-) and CXCR3(-/-) knockout mice, indicating that NK- and/or T-cell-initiated IFN-gamma-chemokine cascades were not involved in the angiogenesis inhibition observed in vivo. Finally, IL-12 plasmid DNA gene transfer significantly prevented the growth and vascularization of highly angiogenic KS-Imm Kaposi's sarcoma and TS/A murine mammary carcinoma tumors in nude and/or syngeneic mice. These data suggest that a preventive gene therapy approach using antiangiogenic cytokines can effectively inhibit tumor angiogenesis and KS, representing an example of angioimmunoprevention.     

Gene therapy for therapeutic angiogenesis in critically ischaemic lower limb - on the way to the clinic

Currently, no effective pharmacological treatment is available for vascularisation defects in lower limbs. Many patients presenting with persistent pain and ischaemic ulcers are not suitable candidates for surgical or endovascular approaches. Further refinement of the available methods will undoubtedly lead to a more active approach towards treatment of peripheral arterial occlusive disease (PAOD). Recently, therapeutic angiogenesis, in the form of recombinant growth factor administration or gene therapy, has emerged as a novel tool to treat these patients. However, improved gene transfer methods and better understanding of blood vessel formation are required to bring therapeutic angiogenesis to clinical practice. Here we review the clinical problem (PAOD), mechanisms of blood vessel formation (angiogenesis, vasculogenesis and arteriogenesis), experimental evidence and clinical trials for therapeutic angiogenesis in critically ischaemic lower limbs. Also, angiogenic growth factors, including vascular endothelial growth factors (VEGFs) and fibroblast growth factors (FGFs), delivery methods, and vectors for gene transfer in skeletal muscle, are discussed. In addition to vascular growth, gene transfer of growth factors may enhance regeneration, survival, and innervation of ischaemic skeletal muscle. Nitric oxide (NO) appears to be a key mediator in vascular homeostasis and growth, and a reduction in its production by age, hypercholesterolemia or diabetes leads to the impairment of ischaemic disorders.     

Targeted delivery of small interfering RNA to angiogenic endothelial cells with liposome-polycation-DNA particles

Angiogenesis is an attractive target for cancer therapy, due to its central position in tumor growth and development. Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFRs) play a key role in the angiogenic process. A promising strategy for targeting VEGF-mediated angiogenesis is RNA interference (RNAi) using short interfering RNA (siRNA). However, for efficacious RNAi a well-designed siRNA delivery system is crucial. Liposome-Polycation-DNA (LPD) particles form a promising system for siRNA delivery to tumors. In order to target angiogenic endothelial cells, LPD particles may be modified with a targeting ligand, such as a cyclic Arg-Gly-Asp (RGD) peptide that specifically binds to integrins expressed on tumor-associated endothelial cells. In the current study, RGD-targeted PEGylated LPD particles containing VEGFR-2 siRNA were prepared and optimized with respect to their size and charge by varying protamine content, carrier DNA content for stronger complexation, and PEGylation density. The size of the optimized particles was around 200 nm and the ζ-potential was approximately +20 mV. The uptake and silencing efficacy of the RGD-targeted PEGylated LPD particles were evaluated in H5V cells (murine endothelial cells) and Human Umbilical Vein Endothelial cells (HUVECs). When compared to non-targeted LPD particles, enhanced uptake and silencing of VEGFR-2 expression was observed for RGD-targeted PEGylated LPD particles. In conclusion, the RGD-targeted PEGylated LPD particles containing VEGFR-2 siRNA presented here may be a promising approach for targeting VEGF-mediated angiogenesis in cancer therapy.     

Combinatorial therapy with three‐dimensionally cultured adipose‐derived stromal cells and self‐assembling peptides to enhance angiogenesis and preserve cardiac function in infarcted hearts

Even though stem cell therapy is a promising angiogenic strategy to treat ischaemic diseases, including myocardial infarction (MI), therapeutic efficacy is limited by low survival and retention of transplanted cells in ischaemic tissues. In addition, therapeutic angiogenesis depends on stimulating host angiogenesis with paracrine factors released by transplanted cells rather than on direct blood vessel formation by transplanted cells. In the present study, to overcome these limitations and to enhance the therapeutic efficacy of MI treatment, combinatorial therapy with three‐dimensional cell masses (3DCMs) and self‐assembling peptides (SAPs) was tested in a rat MI model. Spheroid‐type 3DCMs, which are vascular differentiation‐induced cells, were prepared by culturing human adipose‐derived stromal cells on a fibroblast growth factor‐immobilized surface. The SAPs were used as the carrier material to increase engraftment of transplanted cells. After coronary artery ligation, 3DCMs were combined with SAPs and were transplanted into ischaemic lesions. The therapeutic potential was evaluated 4 weeks after treatment. By combining 3DCMs and SAPs, survival and retention of transplanted cells increased threefold when compared with treatment with 3DCMs alone and transplanted cells established vascular networks in infarcted hearts. In addition, the size of the infarct in the 3DCM + SAP group was reduced to 6.09 ± 2.83% by the promotion of host angiogenesis and cardiac function was preserved, as demonstrated by a 54.25 ± 4.42% increase in the ejection fraction. This study indicates that combinatorial therapy with 3DCM and SAPs could be a promising strategy for therapeutic angiogenesis to treat MI. Copyright © 2016 John Wiley & Sons, Ltd.     

Long-term safety of intramuscular gene transfer of non-viral FGF1 for peripheral artery disease

Peripheral artery disease is a progressive disease. Primary ischemic leg symptoms are muscle fatigue, discomfort or pain during ambulation, known as intermittent claudication. The most severe manifestation of peripheral artery disease is critical limb ischemia (CLI). The long-term safety of gene therapy in peripheral artery disease remains unclear. This four center peripheral artery disease registry was designed to evaluate the long-term safety of the intramuscular non-viral fibroblast growth factor-1 (NV1FGF), a plasmid-based angiogenic gene for local expression of fibroblast growth factor-1 versus placebo in patients with peripheral artery disease who had been included in five different phase I and II trials. Here we report a 3-year follow-up in patients suffering from CLI or intermittent claudication. There were 93 evaluable patients, 72 of them in Fontaine stage IV (47 NV1FGF versus 25 placebo) and 21 patients in Fontaine stage IIb peripheral artery disease (15 NV1FGF versus 6 placebo). Safety parameters included rates of non-fatal myocardial infarction (MI), stroke, death, cancer, retinopathy and renal dysfunction. At 3 years, in 93 patients included this registry, there was no increase in retinopathy or renal dysfunction associated with delivery of this angiogenic factor. There was also no difference in the number of strokes, MI or deaths, respectively, for NV1FGF versus placebo. In the CLI group, new cancer occurred in two patients in the NV1FGF group. Conclusions that can be drawn from this relatively small patient group are limited because of the number of patients followed and can only be restricted to safety. Yet, data presented may be valuable concerning rates in cancer, retinopathy, MI or strokes following angiogenesis gene therapy in the absence of any long-term data in angiogenesis gene therapy. It may take several years until data from larger patient populations will become available.