Avner Adini

An orally delivered small-molecule formulation with antiangiogenic and anticancer activity

Targeting angiogenesis, the formation of blood vessels, is an important modality for cancer therapy. TNP-470, a fumagillin analog, is among the most potent and broad-spectrum angiogenesis inhibitors. However, a major clinical limitation is its poor oral availability and short half-life, necessitating frequent, continuous parenteral administration. We have addressed these issues and report an oral formulation of TNP-470, named Lodamin. TNP-470 was conjugated to monomethoxy-polyethylene glycol–polylactic acid to form nanopolymeric micelles. This conjugate can be absorbed by the intestine and selectively accumulates in tumors. Lodamin significantly inhibits tumor growth, without causing neurological impairment in tumor-bearing mice. Using the oral route of administration, it first reaches the liver, making it especially efficient in preventing the development of liver metastasis in mice. We show that Lodamin is an oral nontoxic antiangiogenic drug that can be chronically administered for cancer therapy or metastasis prevention.

Angiogenesis in Wounds Treated by Microdeformational Wound Therapy

BACKGROUND Mechanical forces play an important role in tissue neovascularization and are a constituent part of modern wound therapies. The mechanisms by which vacuum assisted closure (VAC) modulates wound angiogenesis are still largely unknown. OBJECTIVE To investigate how VAC treatment affects wound hypoxia and related profiles of angiogenic factors as well as to identify the anatomical characteristics of the resultant, newly formed vessels. METHODS Wound neovascularization was evaluated by morphometric analysis of CD31-stained wound cross-sections as well as by corrosion casting analysis. Wound hypoxia and mRNA expression of HIF-1α and associated angiogenic factors were evaluated by pimonidazole hydrochloride staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Vascular endothelial growth factor (VEGF) protein levels were determined by western blot analysis. RESULTS VAC-treated wounds were characterized by the formation of elongated vessels aligned in parallel and consistent with physiological function, compared to occlusive dressing control wounds that showed formation of tortuous, disoriented vessels. Moreover, VAC-treated wounds displayed a well-oxygenated wound bed, with hypoxia limited to the direct proximity of the VAC-foam interface, where higher VEGF levels were found. By contrast, occlusive dressing control wounds showed generalized hypoxia, with associated accumulation of HIF-1α and related angiogenic factors. CONCLUSIONS The combination of established gradients of hypoxia and VEGF expression along with mechanical forces exerted by VAC therapy was associated with the formation of more physiological blood vessels compared to occlusive dressing control wounds. These morphological changes are likely a necessary condition for better wound healing.

Matrigel cytometry: A novel method for quantifying angiogenesis in vivo

Many of the current in vivo methods to evaluate angiogenesis are poorly quantifiable. Recently, the Matrigel plug assay has become the method of choice in many studies involving in vivo testing for angiogenesis. When known angiogenic factors are mixed with Matrigel and injected subcutaneously into mice, endothelial cells migrate into the gel plug. These endothelial cells form vessel-like structures, a process that mimics the formation of capillary networks. Here, we present a modification of the traditional Matrigel assay with improved method to quantify the amount of endothelial cells that incorporate into the plug. The removed plugs were subjected to a mild protease treatment, yielding intact cells. The liberated cells were then stained using an endothelial cell-specific markers, and counted by flow cytometry. This novel combination of FACS analysis with the traditional Matrigel assay improves the ability to quantify in vivo angiogenesis, and for the first time enables to determine the number of migrating and proliferating endothelial cells which reflects the angiogenesis rate.

Melanocyte-secreted fibromodulin promotes an angiogenic microenvironment

Studies have established that pigmentation can provide strong, protective effects against certain human diseases. For example, angiogenesis-dependent diseases such as wet age-related macular degeneration and infantile hemangioma are more common in light-skinned individuals of mixed European descent than in African-Americans. Here we found that melanocytes from light-skinned humans and albino mice secrete high levels of fibromodulin (FMOD), which we determined to be a potent angiogenic factor. FMOD treatment stimulated angiogenesis in numerous in vivo systems, including laser-induced choroidal neovascularization, growth factor-induced corneal neovascularization, wound healing, and Matrigel plug assays. Additionally, FMOD enhanced vascular sprouting during normal retinal development. Deletion of Fmod in albino mice resulted in a marked reduction in the amount of neovascularization induced by retinal vein occlusion, corneal growth factor pellets, and Matrigel plugs. Our data implicate the melanocyte-secreted factor FMOD as a key regulator of angiogenesis and suggest an underlying mechanism for epidemiological differences between light-skinned individuals of mixed European descent and African-Americans. Furthermore, inhibition of FMOD in humans has potential as a therapeutic strategy for treating angiogenesis-dependent diseases.

The stem cell marker prominin-1/CD133 interacts with vascular endothelial growth factor and potentiates its action.

Prominin-1, a pentaspan transmembrane protein, is a unique cell surface marker commonly used to identify stem cells, including endothelial progenitor cells and cancer stem cells. However, recent studies have shown that prominin-1 expression is not restricted to stem cells but also occurs in modified forms in many mature adult human cells. Although prominin-1 has been studied extensively as a stem cell marker, its physiological function of the protein has not been elucidated. We investigated prominin-1 function in two cell lines, primary human endothelial cells and B16-F10 melanoma cells, both of which express high levels of prominin-1. We found that prominin-1 directly interacts with the angiogenic and tumor survival factor vascular endothelial growth factor (VEGF) in both the primary endothelial cells and the melanoma cells. Knocking down prominin-1 in the endothelial cells disrupted capillary formation in vitro and decreased angiogenesis in vivo. Similarly, tumors derived from prominin-1 knockdown melanoma cells had a reduced growth rate in vivo. Further, melanoma cells with knocked down prominin-1 had diminished ability to interact with VEGF, which was associated with decreased bcl-2 protein levels and increased apoptosis. In vitro studies with soluble prominin-1 showed that it stabilized dimer formation of VEGF164, but not VEGF121. Taken together, our findings support the notion that prominin-1 plays an active role in cell growth through its ability to interact and potentiate the anti-apoptotic and pro-angiogenic activities of VEGF. Additionally, prominin-1 promotes tumor growth by supporting angiogenesis and inhibiting tumor cell apoptosis.

A morphometric study of mechanotransductively induced dermal neovascularization.

Background: Mechanical stretch has been shown to induce vascular remodeling and increase vessel density, but the pathophysiologic mechanisms and the morphologic changes induced by tensile forces to dermal vessels are poorly understood. Methods: A custom computer-controlled stretch device was designed and applied to the backs of C57BL/6 mice (n = 38). Dermal and vascular remodeling was studied over a 7-day period. Corrosion casting and three-dimensional scanning electron microscopy and CD31 staining were performed to analyze microvessel morphology. Hypoxia was assessed by immunohistochemistry. Western blot analysis of vascular endothelial growth factor (VEGF) and mRNA expression of VEGF receptors was performed. Results: Skin stretching was associated with increased angiogenesis as demonstrated by CD31 staining and vessel corrosion casting where intervascular distance and vessel diameter were decreased (p < 0.01). Immediately after stretching, VEGF dimers were increased. Messenger RNA expression of VEGF receptor 1, VEGF receptor 2, neuropilin 1, and neuropilin 2 was increased starting as early as 2 hours after stretching. Highly proliferating epidermal cells induced epidermal hypoxia starting at day 3 (p < 0.01). Conclusions: Identification of significant hypoxic cells occurred after identification of neovessels, suggesting an alternative mechanism. Increased expression of angiogenic receptors and stabilization of VEGF dimers may be involved in a mechanotransductive, prehypoxic induction of neovascularization.

Poly-N-acetyl glucosamine fibers are synergistic with vacuum-assisted closure in augmenting the healing response of diabetic mice.

BACKGROUND Vacuum-assisted closure (VAC) has become the preferred modality to treat many complex wounds but could be further improved by methods that minimize bleeding and facilitate wound epithelialization. Short fiber poly-N-acetyl glucosamine nanofibers (sNAG) are effective hemostatic agents that activate platelets and facilitate wound epithelialization. We hypothesized that sNAG used in combination with the VAC device could be synergistic in promoting wound healing while minimizing the risk of bleeding. METHODS Membranes consisting entirely of sNAG nanofibers were applied immediately to dorsal excisional wounds of db/db mice followed by application of the VAC device. Wound healing kinetics, angiogenesis, and wound-related growth factor expression were measured. RESULTS The application of sNAG membranes to wounds 24 hours before application of the VAC device was associated with a significant activation of wounds (expression of PDGF, TGFβ, EGF), superior granulation tissue formation rich in Collagen I as well as superior wound epithelialization (8.6% ± 0.3% vs. 1.8% ± 1.1% of initial wound size) and wound contraction. CONCLUSIONS The application of sNAG fiber-containing membranes before the application of the polyurethane foam interface of VAC devices leads to superior healing in db/db mice and represents a promising wound healing adjunct that can also reduce the risk of bleeding complications.

A novel strategy to enhance angiogenesis in vivo using the small VEGF-binding peptide PR1P

Therapeutic angiogenesis is an experimental frontier in vascular biology that seeks to deliver angiogenic growth factors to ischemic or injured tissues to promote targeted formation of new blood vessels as an alternative approach to surgical revascularization procedures. Vascular endothelial growth factor (VEGF) is a potent angiogenic signal protein that is locally upregulated at sites of tissue injury. However, therapies aimed at increasing VEGF levels experimentally by injecting VEGF gene or protein failed to improve outcomes in human trials in part due to its short half-life and systemic toxicity. We recently designed a novel 12-amino acid peptide (PR1P) whose sequence was derived from an extracellular VEGF-binding domain of the pro-angiogenic glycoprotein prominin-1. In this study, we characterized the molecular binding properties of this novel potential therapeutic for targeted angiogenesis and provided the foundation for its use as an angiogenic molecule that can potentiate endogenous VEGF. We showed that PR1P bound VEGF directly and enhanced VEGF binding to endothelial cells and to VEGF receptors VEGFR2 and neuropilin-1. PR1P increased angiogenesis in the murine corneal micropocket assay when combined with VEGF, but had no activity without added VEGF. In addition, PR1P also enhanced angiogenesis in murine choroidal neovascularization and wound-healing models and augmented reperfusion in a murine hind-limb ischemia model. Together our data suggest that PR1P enhanced angiogenesis by potentiating the activity of endogenous VEGF. In so doing, this novel therapy takes advantage of endogenous VEGF gradients generated in injured tissues and may improve the efficacy of and avoid systemic toxicity seen with previous VEGF therapies.

Quantitative Study of In Vivo Angiogenesis and Vasculogenesis Using Matrigel-Based Assays

Neovascularization, the formation of new blood vessels, is critical for various physiological and pathological events such as wound repair and tumor growth as well as for functional tissue engineering as it ensures proper nutrient and oxygen supply to newly forming tissues. Neovascularization can occur via angiogenesis, where new capillaries sprout from pre-existing vessels, and/or vasculogenesis, where endothelial cells spontaneously self-assemble into vascular structures. Because neovascularization is the rate-limiting step in new tissue formation, there is great interest in developing robust quantitative approaches that facilitate greater understanding of this complex process. Matrigel-based assays are particularly useful for studying neovascularization as it provides a more natural environment for endothelial cell recruitment and capillary formation. In addition, Matrigel-based assays are reproducible and easy to perform when compared to other available in vivo angiogenesis assays. We have recently developed a novel matrigel assay that permits quantification of the endogenous angiogenic response in vivo by determining the number of recruited endothelial cells using fluorescence-activated cell sorting (FACS). We have also developed a technique to quantitatively evaluate vasculogenesis where endothelial cells and mesenchymal stem cells are suspended together in Matrigel prior to subcutaneous injection and vascular network formation is quantitatively analyzed from whole-mount confocal images of lectin-perfused Matrigel implants. Here, we outline the utility of these Matrigel assays for quantitative analyses of both angiogenic response and vasculogenesis and provide a detailed description of the methodology involved.

160A: MECHANOTRANSDUCTIVE INDUCED RECEPTOR MODULATION AS A PREHYPOXIC MECHANISM OF NEOVASCULARIZATION

Methods: A custom computer controlled stretch device was designed and applied to the backs of C57BL/6 mice (n=38). Mice were stretched continuously with a 50 g force. Seven days after stretching, corrosion casting and three-dimensional (3D) scanning electron microscopy (SEM) were performed to analyze microvessel morphology. Hypoxia was assessed by immunhistochemistry using pimonidazole hydrochloride as an in vivo marker. Western blot analysis of VEGF and mRNA expression of vascular endothelial growth factor receptor 1 (VEGFR1) and 2 (VEGFR2), neuropilin receptor 1 (NP1) and 2 (NP2) was performed.