Summary Background Data Mechanical forces play an important role in tissue neovascularisation 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 RT-PCR, respectively. 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 physiologically 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.

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.

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 AfricanAmericans. 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. Relative to what is found in light-skinned individuals of mixed European descent, exudative AMD is 55% less frequent in African-Americans and 46% less frequent in Asian-Americans. These findings support the hypothesis that lower pigmentation is a risk factor for neovascular AMD (1-3). Studies examining the correlation between race, ethnicity, and incidence of uveal melanoma indicate an influence of iris color on the disease. The frequency of uveal melanoma is highest in light-skinned individuals of mixed European descent, followed by Hispanics, Asians, and African-Americans, with an 18:1 ratio of incidence between light-skinned individuals of mixed European descent and African-Americans (4-6). Similar to the trend of occurrence seen with wet AMD, cutaneous melanoma and infantile hemangioma are much more prevalent in light-skinned individuals of mixed European descent than AfricanAmericans. These facts suggest that low levels of melanin correlate with the development of angiogenic ocular and skin diseases. Since intracellular pigment levels of retinal pigment epithelium (RPE) cells do not differ greatly among races, our studies focused on melanocytes, the main source of ocular pigment.

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.

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.

No abstract

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.

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.