Challenges in Translating from Bench to Bed-Side: Pro-Angiogenic Peptides for Ischemia Treatment.

We describe progress and obstacles in the development of novel peptide-hydrogel therapeutics for unmet medical needs in ischemia treatment, focusing on the development and translation of therapies specifically in peripheral artery disease (PAD). Ischemia is a potentially life-threatening complication in PAD, which affects a significant percentage of the elderly population. While studies on inducing angiogenesis to treat PAD were started two decades ago, early results from animal models as well as clinical trials have not yet been translated into clinical practice. We examine some of the challenges encountered during such translation. We further note the need for sustained angiogenic effect involving whole growth factor, gene therapy and synthetic growth factor strategies. Finally, we discuss the need for tissue depots for de novo formation of microvasculature. These scaffolds can act as templates for neovasculature development to improve circulation and healing at the preferred anatomical location.

Aggf1 attenuates neuroinflammation and BBB disruption via PI3K/Akt/NF-κB pathway after subarachnoid hemorrhage in rats.

BACKGROUND: Neuroinflammation and blood-brain barrier (BBB) disruption are two critical mechanisms of subarachnoid hemorrhage (SAH)-induced brain injury, which are closely related to patient prognosis. Recently, angiogenic factor with G-patch and FHA domain 1 (Aggf1) was shown to inhibit inflammatory effect and preserve vascular integrity in non-nervous system diseases. This study aimed to determine whether Aggf1 could attenuate neuroinflammation and preserve BBB integrity after experimental SAH, as well as the underlying mechanisms of its protective roles. METHODS: Two hundred forty-nine male Sprague-Dawley rats were subjected to the endovascular perforation model of SAH. Recombinant human Aggf1 (rh-Aggf1) was administered intravenously via tail vein injection at 1 h after SAH induction. To investigate the underlying neuroprotection mechanism, Aggf1 small interfering RNA (Aggf1 siRNA) and PI3K-specific inhibitor LY294002 were administered through intracerebroventricular (i.c.v.) before SAH induction. SAH grade, neurological score, brain water content, BBB permeability, Western blot, and immunohistochemistry were performed. RESULTS: Expression of endogenous Aggf1 was markedly increased after SAH. Aggf1 was primarily expressed in endothelial cells and astrocytes, as well as microglia after SAH. Administration of rh-Aggf1 significantly reduced brain water content and BBB permeability, decreased the numbers of infiltrating neutrophils, and activated microglia in the ipsilateral cerebral cortex following SAH. Furthermore, rh-Aggf1 treatment improved both short- and long-term neurological functions after SAH. Meanwhile, exogenous rh-Aggf1 significantly increased the expression of PI3K, p-Akt, VE-cadherin, Occludin, and Claudin-5, as well as decreased the expression of p-NF-κB p65, albumin, myeloperoxidase (MPO), TNF-α, and IL-1ß. Conversely, knockdown of endogenous Aggf1 aggravated BBB breakdown, inflammatory response and neurological impairments at 24 h after SAH. Additionally, the protective roles of rh-Aggf1 were abolished by LY294002. CONCLUSIONS: Taken together, exogenous Aggf1 treatment attenuated neuroinflammation and BBB disruption, improved neurological deficits after SAH in rats, at least in part through the PI3K/Akt/NF-κB pathway.

Pro-angiogenic peptides in biomedicine.

Pro-angiogenic therapy provides a promising new perspective in tackling of many common and severe pathological conditions, such as central and peripheral vascular diseases. Pro-angiogenic therapy also finds interesting applications in the regenerative medicine for the treatment of chronic wounds and in tissue engineering. However, clinical studies on therapeutic angiogenesis, mainly performed by administrating growth factors, have not led to convincing results until now, mainly due to the unfavorable pharmacokinetic and to safety concerns. Thus, the research of new pro-angiogenic molecules endowed of improved pharmacological profile is strongly encouraged. This review focuses on Vascular Endothelial Growth Factor (VEGF) mimetic peptides exerting a pro-angiogenic activity, which are considered among the most promising alternatives to the VEGF based therapy. Peptides show a great potential in drug discovery, as they feature straightforward development approaches, robust and cheap synthetic methodologies for their preparation and functionalization, improved safety and efficacy profiles. Thus, pro-angiogenic peptides represent a valuable alternative to traditional drugs for biomedical applications in cardiovascular diseases and regenerative medicine.

Supramolecular nanostructures that mimic VEGF as a strategy for ischemic tissue repair.

There is great demand for the development of novel therapies for ischemic cardiovascular disease, a leading cause of morbidity and mortality worldwide. We report here on the development of a completely synthetic cell-free therapy based on peptide amphiphile nanostructures designed to mimic the activity of VEGF, one of the most potent angiogenic signaling proteins. Following self-assembly of peptide amphiphiles, nanoscale filaments form that display on their surfaces a VEGF-mimetic peptide at high density. The VEGF-mimetic filaments were found to induce phosphorylation of VEGF receptors and promote proangiogenic behavior in endothelial cells, indicated by an enhancement in proliferation, survival, and migration in vitro. In a chicken embryo assay, these nanostructures elicited an angiogenic response in the host vasculature. When evaluated in a mouse hind-limb ischemia model, the nanofibers increased tissue perfusion, functional recovery, limb salvage, and treadmill endurance compared to controls, which included the VEGF-mimetic peptide alone. Immunohistological evidence also demonstrated an increase in the density of microcirculation in the ischemic hind limb, suggesting the mechanism of efficacy of this promising potential therapy is linked to the enhanced microcirculatory angiogenesis that results from treatment with these polyvalent VEGF-mimetic nanofibers.

Computational protein design to reengineer stromal cell-derived factor-1α generates an effective and translatable angiogenic polypeptide analog.

BACKGROUND: Experimentally, exogenous administration of recombinant stromal cell-derived factor-1α (SDF) enhances neovasculogenesis and cardiac function after myocardial infarction. Smaller analogs of SDF may provide translational advantages including enhanced stability and function, ease of synthesis, lower cost, and potential modulated delivery via engineered biomaterials. In this study, computational protein design was used to create a more efficient evolution of the native SDF protein. METHODS AND RESULTS: Protein structure modeling was used to engineer an SDF polypeptide analog (engineered SDF analog [ESA]) that splices the N-terminus (activation and binding) and C-terminus (extracellular stabilization) with a diproline segment designed to limit the conformational flexibility of the peptide backbone and retain the relative orientation of these segments observed in the native structure of SDF. Endothelial progenitor cells (EPCs) in ESA gradient, assayed by Boyden chamber, showed significantly increased migration compared with both SDF and control gradients. EPC receptor activation was evaluated by quantification of phosphorylated AKT, and cells treated with ESA yielded significantly greater phosphorylated AKT levels than SDF and control cells. Angiogenic growth factor assays revealed a distinct increase in angiopoietin-1 expression in the ESA- and SDF-treated hearts. In addition, CD-1 mice (n=30) underwent ligation of the left anterior descending coronary artery and peri-infarct intramyocardial injection of ESA, SDF-1α, or saline. At 2 weeks, echocardiography demonstrated a significant gain in ejection fraction, cardiac output, stroke volume, and fractional area change in mice treated with ESA compared with controls. CONCLUSIONS: Compared with native SDF, a novel engineered SDF polypeptide analog (ESA) more efficiently induces EPC migration and improves post-myocardial infarction cardiac function and thus offers a more clinically translatable neovasculogenic therapy.

Tetrahedral framework nucleic acid carrying angiogenic peptide prevents bisphosphonate-related osteonecrosis of the jaw by promoting angiogenesis.

The significant clinical feature of bisphosphonate-related osteonecrosis of the jaw (BRONJ) is the exposure of the necrotic jaw. Other clinical manifestations include jaw pain, swelling, abscess, and skin fistula, which seriously affect the patients' life, and there is no radical cure. Thus, new methods need to be found to prevent the occurrence of BRONJ. Here, a novel nanoparticle, tFNA-KLT, was successfully synthesized by us, in which the nanoparticle tetrahedral framework nucleic acid (tFNA) was used for carrying angiogenic peptide, KLT, and then further enhanced angiogenesis. TFNA-KLT possessed the same characteristics as tFNA, such as simple synthesis, stable structure, and good biocompatibility. Meanwhile, tFNA enhanced the stability of KLT and carried more KLT to interact with endothelial cells. First, it was confirmed that tFNA-KLT had the superior angiogenic ability to tFNA and KLT both in vitro and in vivo. Then we apply tFNA-KLT to the prevention of BRONJ. The results showed that tFNA-KLT can effectively prevent the occurrence of BRONJ by accelerating angiogenesis. In summary, the prepared novel nanoparticle, tFNA-KLT, was firstly synthesized by us. It was also firstly confirmed by us that tFNA-KLT significantly enhanced angiogenesis and can effectively prevent the occurrence of BRONJ by accelerating angiogenesis, thus providing a new avenue for the prevention of BRONJ and a new choice for therapeutic angiogenesis.

Specific angiogenic peptide binding with injectable cardiac ECM collagen gel promotes the recovery of myocardial infarction in rat.

Restoring blood supply is an effective way for the therapy of myocardial infarction (MI). It was reported a specific angiogenic peptide (VMP) derived from vascular endothelial growth factor (VEGF) could activate its receptor to mimic the biological activity of VEGF. In this study, in order to improve the local concentration in infarction region, a collagen-binding domain was synthesized with VMP to construct collagen binding domain (CBD)-VMP peptides. The fused CBD-VMP could bind specifically to collagen which was rich in cardiac extracellular matrix (c-ECM), without impacting the biological activity of VMP peptides. When the CBD-VMP peptides loaded on collagen scaffold and implanted into the rats subcutaneously, significant vascularization was observed. Then, CBD-VMP peptides binding with injectable c-ECM injected into the MI rat by intramuscular administration, significant blood vessels regeneration, and decrease of cell apoptosis were observed, that corelated with the recovery of cardiac function. It might be an alternative promising strategy for the clinical application of MI.

The wound healing potential of a pro-angiogenic peptide purified from Indian Russell's viper (Daboia russelii) venom.

The cutaneous wound healing property of a pro-angiogenic venom peptide (RVVAP) in a cream-based formulation was evaluated using the excision wound healing model on Wistar strain rats. The wound healing potency and modest antibacterial activity of RVVAP was enhanced significantly (p < 0.05) when combined with Aloe vera extract. RVVAP was also found to be non-toxic at the tested dose of 1.0 mg/kg. Nevertheless, the release of inflammatory cytokines such as IL-1, IL-6, IL-10, and TNF-α in RVVAP-treated mice was suppressed, compared to the untreated controls. This is the first report assessing the wound healing potential of a low-molecular mass, non-enzymatic, pro-angiogenic peptide purified from snake venom.

Angiogenic Growth Factors for Coronary Artery Disease: Current Status and Prospects.

Although there have been advances in coronary artery bypass grafting and percutaneous coronary intervention, some patients who have ischemic coronary artery disease (CAD) are ineligible for revascularization due to suboptimal anatomy. Cardiac angiogenesis is not only a physiological response to ischemia or hypoxia but also a potential target of therapeutic strategies. Preclinical studies have shown a great enthusiasm on therapeutic angiogenesis for ischemic CAD. However, the latest trials provided the limited evidence on its efficacy. This article aims to discuss the physiological process of angiogenesis, the characteristic of angiogenic growth factors, delivery system, and clinical and preclinical studies, which can provide a novel insight into the therapeutic angiogenesis for CAD.

Granulocyte-macrophage colony-stimulating factor-induced vessel growth restores cerebral blood supply after bilateral carotid artery occlusion.

BACKGROUND AND PURPOSE: Hemodynamic compromise due to occlusive cerebrovascular disease is associated with an increased stroke risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been suggested to stimulate collateral blood vessel growth in various models of hemodynamic compromise. The purpose of this study was to investigate the effects of GM-CSF on cerebral hemodynamics and vessel growth in a rat model of chronically impaired cerebral blood flow (CBF). METHODS: Male Sprague-Dawley rats underwent sequential bilateral carotid artery occlusion (BCO) and were treated with GM-CSF or saline for 6 weeks. Sham-occluded animals served as a control group. Baseline CBF was measured by iodo[(14)C]antipyrine autoradiography, and cerebrovascular reserve capacity was assessed by laser-Doppler flowmetry after application of 20 mg/kg body weight acetazolamide. The capillary density and arterioles immunopositive for alpha-smooth muscle actin were counted on brain sections. The cerebral angioarchitecture was visualized with a latex perfusion technique. RESULTS: Baseline CBF as measured by iodo[(14)C]antipyrine autoradiography was not affected by BCO. The cerebrovascular reserve capacity, however, was significantly impaired 1 week after BCO. CBF and cerebrovascular reserve capacity recovered completely in GM-CSF-treated animals but not in solvent-treated animals. Histologic analysis of the hippocampus revealed integrity of the hypoxia-vulnerable neurons in all animals. The capillary density showed a very mild increase in GM-CSF-treated animals. However, the number of intraparenchymal and leptomeningeal arterioles was significantly higher in GM-CSF-treated animals than in both other groups. CONCLUSIONS: Long-term GM-CSF treatment in a BCO model in rats leads to restoration of impaired cerebral hemodynamics and accompanies structural changes in the resistance-vessel network.

Angiogenic growth factors in the treatment of peripheral arterial disease.

Peripheral arterial disease (PAD) remains a major cause of morbidity. Despite advances in revascularisation procedures and medical treatment, limb salvage and relief of pain are still not satisfactory in patients with severe disease. This has prompted the exploration of alternative modes of treatment including enhancement of new vessel formation (angiogenesis). Angiogenic Growth Factors (AGF), mainly Vascular Endothelial Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF) and Hepatocyte Growth Factor (HGF) have emerged as exciting therapeutic modalities. Both experimental and clinical studies have demonstrated that topical (mainly intramuscular) AGF gene therapy results in improved peripheral vasculature and alleviation of symptoms. However, most clinical work is limited to small patient series and the long-term safety and efficacy are still unclear. Clinical benefit must be balanced against potential untoward effects, such as tumour growth and atherosclerotic plaque angiogenesis leading to plaque instability. VEGF is important in the pathogenesis of diabetic microvascular disease. Further studies are required before implementation of AGF therapy in clinical practice.

Treatment of osteonecrosis of the femoral head: combination of operation and multiple cellular mediators.

The goal in the treatment of osteonecrosis of the femoral head is to preserve, not replace, the femoral head. Although many methods have been proposed, none has proved completely satisfactory. Cellular mediators as a supplement to bone grafting and decompression is an attractive approach to this problem because it combines the desirable features of other procedures, each of which has shown a certain degree of effectiveness in stimulating bone growth and repair. Basic and clinical researches have shown the efficacy of various cellular mediators (bone morphogenetic proteins, interleukins, angiogenic growth factors, etc.) in healing bone defects. The potential application of these cellular mediators to other musculoskeletal conditions, including osteonecrosis of the femoral head, only recently has been explored. The surgical alternatives may include core decompression, osteotomy, nonvascularized, and vascularized bone grafting, which might be enhanced with the use of cellular mediators. At least three of these factors are potential candidates as therapeutic modalities: cytokines, bone morphogenetic proteins, and angiogenic factors. Therefore, we hypothesized that the combination of operation and multiple cellular mediators is an attractive method to preserve the femoral head for the therapy of osteonecrosis of the femoral head.

Potential therapeutic implications of intracrine angiogenesis.

Angiogenesis, in most cases, is a requirement for tumor growth beyond a diameter of a few millimeters and is, therefore, a major target for cancer therapy. The intracellular actions of certain extracellular signaling proteins (intracrines) have been reported, and it is clear that intracrines such as vascular endothelial growth factor, basic fibroblast growth factor, angiogenin, angiotensin, and endothelin, among others, are involved in angiogenesis. We have proposed that intracrine networks play an important role in angiogenesis, and have suggested that very similar intracrine networks exist in some tumor cells. These notions have implications for the development of anti-angiogenesis therapies because they suggest that the inhibition of intracellular intracrine trafficking pathways may be an effective therapeutic target. Here the participation and regulation of intracrines in angiogenesis is explored, as are the actions of various anti-angiogenic factors.

Physician-initiated first-in-human clinical study using a novel angiogenic peptide, AG30/5C, for patients with severe limb ulcers.

AIM: In patients with diabetes or ischemia, angiogenesis and infection control are required for chronic leg ulcers, which substantially impair patients' quality of life. We developed a novel functional peptide, named AG30/5C, with angiogenic and anti-microbial properties. Treatment with AG30/5C significantly accelerated the wound healing of full-thickness defects in mice. To evaluate the safety of AG30/5C in the treatment of leg ulcers, a physician-initiated clinical study was carried out. METHODS: The first-in-human trial was designed as an open-label treatment with AG30/5C (0.1 mg/mL) given twice per day for 11 days, and with a follow-up period of 17 days. The inclusion criteria for severe skin ulcers were: (i) diabetes or critical limb ischemia; (ii) resistance to standard therapy for 1 month; and (iii) detection of methicillin-resistant Staphylococcus aureus in the skin ulcer. RESULTS: Four patients were enrolled in this study, and two patients met these criteria. For the evaluation of safety, three adverse effects were reported as possibly related to AG30/5C treatment; however, these adverse effects were not severe and resolved during or after treatment. Thus, there were no safety concerns. In both patients, the size of the ulcer decreased after treatment (44.62% and 10.23% decrease), and further decreased after the follow-up period (73.85% and 10.23% decrease). The former patient was diagnosed as Werner syndrome and the skin ulcer was resistant to standard therapy; however, it was sensitive to AG30/5C treatment. CONCLUSIONS: Topical treatment with AG30/5C for severe leg ulcers was safe, well tolerated and effective. Geriatr Gerontol Int 2017; 17: 2150-2156.

Cerebral ischemia and angiogenesis.

Angiogenesis occurs in a wide range of conditions. As ischemic tissue usually depends on collateral blood flow from newly produced vessels, acceleration of angiogenesis should be of therapeutic value to ischemic disorders. Indeed, therapeutic angiogenesis reduced tissue injury in myocardial or limb ischemia. In ischemic stroke, on the other hand, angiogenic factors often increase vascular permeability and thus may deteriorate tissue damage. In order to apply safely the therapeutic angiogenesis for ischemic stroke treatment, elucidating precise mechanism of brain angiogenesis is mandatory. In the present article, we review previous reports which investigated molecular mechanisms of angiogenesis. Endothelial cell mitogens, enzymes that degrade surrounding extracellular matrix, and molecules implicated in endothelial cells migration are induced rapidly in the ischemic brain. Their possible neuroprotective or injury exacerbating effects are discussed. Because therapeutic potential of angiogenic factors application had gained much attention, we here extensively reviewed relevant previous reports. In the future however, there is a need to consider angiogenesis in relation with regenerative medicine, as angiogenic factors sometimes possess neuron producing property.

Angiogenesis--a new target for future therapy.

Development of blood vessels from in situ differentiating endothelial cells (EC) is called vasculogenesis, whereas sprouting of new blood vessels from the pre-existing ones is termed angiogenesis or neovascularisation. Angiogenesis, the growth of new blood vessels, is essential during tissue repair, foetal development, and female reproductive cycle. In contrast, uncontrolled angiogenesis promotes tumor and retinopathies, while inadequate angiogenesis can lead to coronary artery disease. A balance between pro-angiogenic and anti-angiogenic growth factors and cytokines tightly controls angiogenesis. With the identification of several proangiogenic molecules such as the vascular endothelial cell growth factor (VEGF), the fibroblast growth factors (FGFs), and the angiopoietins, and the recent description of specific inhibitors of angiogenesis such as platelet factor-4, angiostatin, endostatin, and vasostatin, it is recognized that therapeutic interference with vasculature formation offers a tool for clinical applications in various pathologies. Inhibition of angiogenesis can prevent diseases such as cancer, diabetic nephropathy, arthritis, psoriasis, whereas stimulation of angiogenesis is beneficial in the treatment of coronary artery disease (CAD), cardiac failure, tissue injury, etc. One of the most specific and critical regulators of angiogenesis is vascular endothelial growth factor (VEGF), which regulates endothelial proliferation, permeability, and survival. Substantial evidence also implicates VEGF as an angiogenic mediator in tumors and intraocular neovascular syndromes, and numerous clinical trials are presently testing the hypothesis that inhibition of VEGF may have therapeutic value.

Principles and therapeutic implications of angiogenesis, vasculogenesis and arteriogenesis.

The vasculature is the first organ to arise during development. Blood vessels run through virtually every organ in the body (except the avascular cornea and the cartilage), assuring metabolic homeostasis by supplying oxygen and nutrients and removing waste products. Not surprisingly therefore, vessels are critical for organ growth in the embryo and for repair of wounded tissue in the adult. Notably, however, an imbalance in angiogenesis (the growth of blood vessels) contributes to the pathogenesis of numerous malignant, inflammatory, ischaemic, infectious and immune disorders. During the last two decades, an explosive interest in angiogenesis research has generated the necessary insights to develop the first clinically approved anti-angiogenic agents for cancer and blindness. This novel treatment is likely to change the face of medicine in the next decade, as over 500 million people worldwide are estimated to benefit from pro- or anti-angiogenesis treatment. In this following chapter, we discuss general key angiogenic mechanisms in health and disease, and highlight recent developments and perspectives of anti-angiogenic therapeutic strategies.

Angiogenesis for the treatment of inoperable coronary disease: the future.

Improved treatment options and better management of cardiovascular risk factors have resulted in improved outcomes for patients suffering from severe coronary artery disease. However, coronary artery disease may be of such a diffuse and severe manner that repeated attempts at catheter-based interventions and coronary artery bypass grafting may be unsuccessful at restoring normal myocardial blood flow. It is the goal of therapeutic angiogenesis to restore perfusion to chronically ischemic myocardium using protein growth factors, gene therapy, or, more recently, cell-based therapy, without intervening on the epicardial coronary arteries. However, angiogenesis has not yet provided significant clinical benefit and is still reserved as an experimental treatment for patients who have failed conventional therapies. Once potential endogenous inhibitors of vascular development can be modified, angiogenesis may become more useful for therapeutic purposes. It is hoped that angiogenesis for therapeutic purposes will one day effectively re-create the potent natural processes of vascularization that every human being undergoes during growth and development and become a major modality for the treatment of coronary artery disease.

PEGylated nanoliposomes encapsulating angiogenic peptides improve perfusion defects: Radionuclide imaging-based study.

INTRODUCTION: Although liposomes hold promise for cancer therapy, the effectiveness of treating myocardial ischemia by promoting angiogenesis has yet to be proved. Nanoliposomes loaded with therapeutic agents can effectively target ischemic myocardium via enhanced permeability and retention. Surface polyethylene glycol (PEG) modification can further facilitate effective targeting by prolonging liposomal circulation. This study aimed to determine whether PEGylated nanoliposomes are effective in facilitating targeted drug delivery and treating myocardial ischemia. METHODS: Rats subjected to 30min of myocardial ischemia were given (99m)Tc-hexamethylpropyleneamine oxime- or (99m)Tc-diethylenetriamine pentaacetate-labeled liposomes with mean diameters of ~100nm or ~600nm with or without PEG modifications to determine the extent of myocardial uptake in the different conditions. Therapeutic effectiveness was assessed by studying changes in myocardial perfusion defects with (99m)Tc-tetrofosmin autoradiography and vascular density with immunohistochemistry at 7days post-treatment. RESULTS: The liver and spleen showed the largest capacity for liposome uptake. Uptake by the liver and spleen was more pronounced when the liposomes were larger. Conversely, myocardial liposome uptake was significantly greater when the liposomes were ~100nm rather than ~600nm in diameter. Surface modification with PEG significantly augmented myocardial uptake of ~100nm liposomes. PEG modification did not affect the size dependence. To investigate therapeutic efficacy, hearts subjected to ischemia received PEGylated nanoliposomes encapsulated with angiogenic peptides. Our data demonstrated that PEGylated nanoliposomes loaded with angiogenic peptides improved myocardial perfusion defects and increased vascular density. A 10-fold increase in liposomal concentration did not further benefit myocardial ischemia. CONCLUSIONS: Liposomal angiogenic formulation with size control and PEG modification may be effective treatment strategy for myocardial ischemia. Increasing the concentration of liposomes does not necessarily benefit myocardial ischemia.