PR1P, a VEGF-stabilizing peptide, reduces injury and inflammation in acute lung injury and ulcerative colitis animal models.

Acute Respiratory Distress Syndrome (ARDS) and Ulcerative Colitis (UC) are each characterized by tissue damage and uncontrolled inflammation. Neutrophils and other inflammatory cells play a primary role in disease progression by acutely responding to direct and indirect insults to tissue injury and by promoting inflammation through secretion of inflammatory cytokines and proteases. Vascular Endothelial Growth Factor (VEGF) is a ubiquitous signaling molecule that plays a key role in maintaining and promoting cell and tissue health, and is dysregulated in both ARDS and UC. Recent evidence suggests a role for VEGF in mediating inflammation, however, the molecular mechanism by which this occurs is not well understood. We recently showed that PR1P, a 12-amino acid peptide that binds to and upregulates VEGF, stabilizes VEGF from degradation by inflammatory proteases such as elastase and plasmin thereby limiting the production of VEGF degradation products (fragmented VEGF (fVEGF)). Here we show that fVEGF is a neutrophil chemoattractant in vitro and that PR1P can be used to reduce neutrophil migration in vitro by preventing the production of fVEGF during VEGF proteolysis. In addition, inhaled PR1P reduced neutrophil migration into airways following injury in three separate murine acute lung injury models including from lipopolysaccharide (LPS), bleomycin and acid. Reduced presence of neutrophils in the airways was associated with decreased pro-inflammatory cytokines (including TNF-α, IL-1ß, IL-6) and Myeloperoxidase (MPO) in broncho-alveolar lavage fluid (BALF). Finally, PR1P prevented weight loss and tissue injury and reduced plasma levels of key inflammatory cytokines IL-1ß and IL-6 in a rat TNBS-induced colitis model. Taken together, our data demonstrate that VEGF and fVEGF may each play separate and pivotal roles in mediating inflammation in ARDS and UC, and that PR1P, by preventing proteolytic degradation of VEGF and the production of fVEGF may represent a novel therapeutic approach to preserve VEGF signaling and inhibit inflammation in acute and chronic inflammatory diseases.

Fibromodulin Ablation Exacerbates the Severity of Acute Colitis.

Introduction: Epidemiological studies have associated pigment production with protection against certain human diseases. In contrast to African Americans, European descendants are more likely to suffer from angiogenesis-dependent and inflammatory diseases, such as wet age-related macular degeneration (ARMD) and ulcerative colitis (UC), respectively. Methods: In a mouse model of dextran sulfate sodium (DSS)-induced acute colitis, the effect of fibromodulin (FMOD) depletion was examined on colitis severity. Results: In this study, albino mice that produce high levels of FMOD developed less severe acute colitis compared with mice lacking in FMOD as assessed by clinical symptoms and histopathological changes. FMOD depletion affected the expression of tight junction proteins, contributing to the destruction of the epithelial barrier. Furthermore, this study revealed a stronger inflammatory response after DSS treatment in the absence of FMOD, where FMOD depletion led to an increase in activated T cells, plasmacytoid dendritic cells (pDCs), and type I interferon (IFN) production. Discussion: These findings point to FMOD as a potential biomarker of disease severity in UC among light-skinned individuals of European descent.

RIP1/RIP3/MLKL Mediates Myocardial Function Through Necroptosis in Experimental Autoimmune Myocarditis.

Cardiomyopathy often leads to dilated cardiomyopathy (DCM) when caused by viral myocarditis. Apoptosis is long considered as the principal process of cell death in cardiomyocytes, but programmed necrosis or necroptosis is recently believed to play an important role in cardiomyocyte cell death. We investigated the role of necroptosis and its interdependency with other processes of cell death, autophagy, and apoptosis in a rat system of experimental autoimmune myocarditis (EAM). We successfully created a rat model system of EAM by injecting porcine cardiac myosin (PCM) and showed that in EAM, all three forms of cell death increase considerably, resulting in the deterioration of cardiac conditions with an increase in inflammatory infiltration in cardiomyocytes. To explore whether necroptosis occurs in EAM rats independent of autophagy, we treated EAM rats with a RIP1/RIP3/MLKL kinase-mediated necroptosis inhibitor, Necrostatin-1 (Nec-1). In Nec-1 treated rats, cell death proceeds through apoptosis but has no significant effect on autophagy. In contrast, autophagy inhibitor 3-Methyl Adenine (3-MA) increases necroptosis, implying that blockage of autophagy must be compensated through necroptosis. Caspase 8 inhibitor zVAD-fmk blocks apoptosis but increases both necroptosis and autophagy. However, all necroptosis, apoptosis, and autophagy inhibitors independently reduce inflammatory infiltration in cardiomyocytes and improve cardiac conditions. Since apoptosis or autophagy is involved in many important cellular aspects, instead of suppressing these two major cell death processes, Nec1 can be developed as a potential therapeutic target for inflammatory myocarditis.

The Prominin-1-Derived Peptide Improves Cardiac Function Following Ischemia.

Myocardial infarction (MI) remains the leading cause of death in the western world. Despite advancements in interventional revascularization technologies, many patients are not candidates for them due to comorbidities or lack of local resources. Non-invasive approaches to accelerate revascularization within ischemic tissues through angiogenesis by providing Vascular Endothelial Growth Factor (VEGF) in protein or gene form has been effective in animal models but not in humans likely due to its short half-life and systemic toxicity. Here, we tested the hypothesis that PR1P, a small VEGF binding peptide that we developed, which stabilizes and upregulates endogenous VEGF, could be used to improve outcome from MI in rodents. To test this hypothesis, we induced MI in mice and rats via left coronary artery ligation and then treated animals with every other day intraperitoneal PR1P or scrambled peptide for 14 days. Hemodynamic monitoring and echocardiography in mice and echocardiography in rats at 14 days showed PR1P significantly improved multiple functional markers of heart function, including stroke volume and cardiac output. Furthermore, molecular biology and histological analyses of tissue samples showed that systemic PR1P targeted, stabilized and upregulated endogenous VEGF within ischemic myocardium. We conclude that PR1P is a potential non-invasive candidate therapeutic for MI.

PR1P Stabilizes VEGF and Upregulates Its Signaling to Reduce Elastase-induced Murine Emphysema.

Emphysema is a progressive and fatal lung disease with no cure that is characterized by thinning, enlargement, and destruction of alveoli, leading to impaired gas exchange. Disease progression is due in part to dysregulation of VEGF (vascular endothelial growth factor) signaling in the lungs and increased lung-cell apoptosis. Here we asked whether PR1P (Prominin-1-derived peptide), a novel short peptide we designed that increases VEGF binding to endothelial cells, could be used to improve outcome in in vitro and in vivo models of emphysema. We used computer simulation and in vitro and in vivo studies to show that PR1P upregulated endogenous VEGF receptor-2 signaling by binding VEGF and preventing its proteolytic degradation. In so doing, PR1P mitigated toxin-induced lung-cell apoptosis, including from cigarette-smoke extract in vitro and from LPS in vivo in mice. Remarkably, inhaled PR1P led to significantly increased VEGF concentrations in murine lungs within 30 minutes that remained greater than twofold above that of control animals 24 hours later. Finally, inhaled PR1P reduced acute lung injury in 4- and 21-day elastase-induced murine emphysema models. Taken together, these results highlight the potential of PR1P as a novel therapeutic agent for the treatment of emphysema or other lung diseases characterized by VEGF signaling dysregulation.

PR1P ameliorates neurodegeneration through activation of VEGF signaling pathway and remodeling of the extracellular environment.

Neurodegenerative diseases affect millions of people worldwide. Optic neuropathies are the most commonly occurring neurodegenerative diseases, characterized by progressive retinal ganglion cell (RGC) degeneration. We recently reported that Prominin-1, a protein found on the surface of stem cells, interacts with VEGF and enhances its activity. VEGF is known to have various protective roles in the nervous system. Subsequently, we have developed a 12-mer peptide derived from Prominin-1, named PR1P, and investigated its effects on neuronal survival of damaged RGCs in a rat model of optic nerve crush (ONC). PR1P prevented RGC apoptosis resulting in improvement of retinal function in the rat ONC model. PR1P treatment significantly increased phosphorylation of ERK and AKT and expression its downstream proteins c-fos and Egr-1 in the retina. Additionally, PR1P beneficially increased the MMP-9/TIMP-1 ratio and promoted glial activation in the retina of ONC rats. Thus, PR1P displayed neuroprotective effects through enhanced VEGF-driven neuronal survival and reconstruction of the extracellular environment in ONC model. Our data indicate that PR1P may be a promising new clinical candidate for the treatment of neurodegenerative diseases.

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.

Melanocyte pigmentation inversely correlates with MCP-1 production and angiogenesis-inducing potential.

The incidence of certain angiogenesis-dependent diseases is higher in Caucasians than in African Americans. Angiogenesis is amplified in wound healing and cornea models in albino C57 mice compared with black C57 mice. Moreover, mouse and human melanocytes with low pigmentation stimulate endothelial cell (EC) proliferation and migration in vitro more than melanocytes with high pigmentation. This effect is due, in part, to the secretion of an angiogenic protein called fibromodulin (FMOD) from lowly pigmented melanocytes. Herein, we expand upon the mechanism contributing to increased angiogenesis in lighter skin and report that monocyte chemotactic protein-1 (MCP-1) is secreted by nonpigmented mouse melanocytes by 5- to 10-fold more than pigmented melanocytes. MCP-1 protein stimulates EC proliferation and migration in vitro and angiogenesis in vivo. Mechanistic studies determine that FMOD is upstream of MCP-1 and promotes its secretion from both melanocytes and activated ECs via stimulation of NF-κB activity. Mice injected with FMOD-neutralizing antibodies show 2.3-fold decreased levels of circulating MCP-1. Human studies confirmed that, on average, Caucasians have 2-fold higher serum levels of MCP-1 than African Americans. Taken together, this study implicates the FMOD/MCP-1 pathway in the regulation of angiogenesis by local melanocytes and suggests that melanogenic activity may protect against aberrant angiogenic diseases.

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, TGFß, 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.

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.

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.

Dendritic cells support angiogenesis and promote lesion growth in a murine model of endometriosis.

Endometriosis affects 10-15% of women and is associated with pelvic pain and infertility. Angiogenesis plays an essential role in its pathogenesis. Dendritic cells (DCs) were recently implicated in supporting tumor angiogenesis. As both tumors and endometriosis lesions depend on angiogenesis, we investigated the possibility that DCs may also play a role in endometriosis. We induced endometriosis in 8-wk-old female C57BL/6 mice by implantation of autologous endometrium into the peritoneal cavity. We observed an abundance of CD11c(+) DCs infiltrating sites of angiogenesis in endometriosis lesions. We noticed a similar pattern of infiltrating DCs at sites of angiogenesis in the peritoneal Lewis lung carcinoma tumor model. These DCs were immature (major histocompatability complex class II(low)) and expressed vascular endothelial growth factor receptor 2. Peritoneal implanted bone marrow-derived DCs (BMDCs) incorporated into both endometriosis lesions and into B16 melanoma tumors and enhanced their growth at 8 days compared with controls (5.1+/-2.5 vs. 1.5+/-0.5 mm(2), n=4 and 4, P<0.0001 for endometriosis; 67.6+/-15.1 vs. 22.7+/-14.6 mm(2), n=5 and 7, P=0.0004 for mouse melanoma). Finally, immature BMDCs but not mature BMDCs enhanced microvascular endothelial cell migration in vitro (219+/-51 vs. 93+/-32 cells, P=0.02). Based on these findings, we suggest a novel role for DCs in supporting angiogenesis and promoting lesion growth both in endometriosis and in tumors.

Placental growth factor is a survival factor for tumor endothelial cells and macrophages.

The vascular endothelial growth factor (VEGF)-related factor, placental growth factor (PlGF),has been shown recently to play an important role in pathological VEGF-driven angiogenesis. In this study, we examine the effects of mPlGF/PlGF-2 overexpression in tumors grown from glioma cells containing a tetracycline-regulated mPlGF cDNA. Overexpression of mPlGF leads to increased tumor growth and vascular survival. When tetracycline is used to abruptly withdraw mPlGF overexpression, we see increased apoptosis in both vascular cells and macrophages. In addition, PlGF-2 induces survival gene expression and inhibits apoptosis in vitro. Thus, we propose that PlGF-2 contributes to tumor angiogenesis by providing increased survival function to endothelial cells and macrophages.