Genetic alteration of endothelial heparan sulfate selectively inhibits tumor angiogenesis.

To examine the role of endothelial heparan sulfate during angiogenesis, we generated mice bearing an endothelial-targeted deletion in the biosynthetic enzyme N-acetylglucosamine N-deacetylase/N-sulfotransferase 1 (Ndst1). Physiological angiogenesis during cutaneous wound repair was unaffected, as was growth and reproductive capacity of the mice. In contrast, pathological angiogenesis in experimental tumors was altered, resulting in smaller tumors and reduced microvascular density and branching. To simulate the angiogenic environment of the tumor, endothelial cells were isolated and propagated in vitro with proangiogenic growth factors. Binding of FGF-2 and VEGF(164) to cells and to purified heparan sulfate was dramatically reduced. Mutant endothelial cells also exhibited altered sprouting responses to FGF-2 and VEGF(164), reduced Erk phosphorylation, and an increase in apoptosis in branching assays. Corresponding changes in growth factor binding to tumor endothelium and apoptosis were also observed in vivo. These findings demonstrate a cell-autonomous effect of heparan sulfate on endothelial cell growth in the context of tumor angiogenesis.

Anti-fungal activity of cathelicidins and their potential role in Candida albicans skin infection.

Cathelicidins have broad anti-microbial capacity and are important for host defense against skin infections by some bacterial and viral pathogens. This study investigated the activity of cathelicidins against Candida albicans. The human cathelicidin LL-37, and mouse cathelicidin mCRAMP, killed C. albicans, but this fungicidal activity was dependent on culture conditions. Evaluation of the fungal membrane by fluorescent dye penetration after incubation with cathelicidins correlated membrane permeabilization and inhibition of fungal growth. Anti-fungal assays carried out in an ionic environment that mimicked human sweat and with the processed forms of cathelicidin such as are present in sweat found that the cleavage of LL-37 to forms such as RK-31 conferred additional activity against C. albicans. C. albicans also induced an increase in the expression of cathelicidin in mouse skin, but this induction did not confer systemic or subcutaneous resistance as mCRAMP-deficient mice were not more susceptible to C. albicans in blood-killing assays or in an intradermal infection model. Therefore, cathelicidins appear active against C. albicans, but may be most effective as a superficial barrier to infection.

Acquired progressive lymphangioma in an HIV-positive patient.

Acquired progressive lymphangioma (APL) is a rare condition characterized by benign proliferation of thin-walled vessels lined by flattened endothelial cells.(1-4) Although benign, the acquired nature of this tumor may lead to misdiagnosis as a malignant vascular tumor. This is especially true if the patient has risk factors, such as immunodeficiency. In this article, the authors present a case of APL in an HIV-positive man.

Expression and potential function of cathelicidin antimicrobial peptides in dermatophytosis and tinea versicolor.

OBJECTIVES: This study was designed to characterize the role of the human cathelicidin LL-37 in fungal skin infections such as dermatophytosis and tinea versicolor. METHODS: The in vitro antimicrobial activity of synthetic antimicrobial peptides including the human cathelicidin LL-37 against Malassezia furfur and several dermatophytes was determined. Immunostaining was performed to determine expression of cathelicidin in skin biopsies from patients with tinea pedis, tinea corporis and tinea versicolor. Cathelicidin peptide expression was evaluated by western blotting and mRNA expression was studied in keratinocytes exposed to M. furfur or Trichophyton rubrum. RESULTS: LL-37 inhibits the growth of fungi with an MIC of 20-30 microM for M. furfur and 12.5 microM for Trichophyton mentagrophytes and T. rubrum. LL-37 also shows fungicidal activity with a minimum fungicidal concentration (MFC) of 12.5 and 25 microM for T. mentagrophytes and T. rubrum, respectively. An increase in cathelicidin expression was observed in human skin tissue infected with fungi compared with healthy skin. Western blotting of skin scrapings demonstrated that human cathelicidin is processed from its precursor into an active peptide in both healthy and infected plantar skin. CONCLUSIONS: These findings support a hypothesis that antimicrobial peptides such as cathelicidins can play a role in skin defence against dermatophytes and M. furfur.

Expression of an additional cathelicidin antimicrobial peptide protects against bacterial skin infection.

Cathelicidin antimicrobial peptides are effectors of innate immune defense in mammals. Humans and mice have only one cathelicidin gene, whereas domesticated mammals such as the pig, cow, and horse have multiple cathelicidin genes. We hypothesized that the evolution of multiple cathelicidin genes provides these animals with enhanced resistance to infection. To test this, we investigated the effects of the addition of cathelicidins by combining synthetic cathelicidin peptides in vitro, by producing human keratinocytes that overexpress cathelicidins in culture, or by producing transgenic mice that constitutively overexpress cathelicidins in vivo. The porcine cathelicidin peptide PR-39 acted additively with human cathelicidin LL-37 to kill group A Streptococcus (GAS). Lentiviral delivery of PR-39 enhanced killing of GAS by human keratinocytes. Finally, transgenic mice expressing PR-39 under the influence of a K14 promoter showed increased resistance to GAS skin infection (50% smaller necrotic ulcers and 60% fewer surviving bacteria). Similarly constructed transgenic mice designed to overexpress their native cathelicidin did not show increased resistance. These findings demonstrate that targeted gene transfer of a xenobiotic cathelicidin confers resistance against infection and suggests the benefit of duplication and divergence in the evolution of antimicrobial peptides.

Dermatan sulfate proteoglycan and glycosaminoglycan synthesis is induced in fibroblasts by transfer to a three-dimensional extracellular environment.

Composition and architecture of the extracellular matrix dictate cell behavior. Proteoglycans bind multiple components of the extracellular matrix by serving as important regulators of cell behavior. Given the influence of culture architecture on cell function, we investigated whether switching NIH3T3 fibroblasts from growth on type 1 collagen in monolayer to a collagen gel might influence dermatan sulfate expression. Immunofluorescent staining, immunoblot, and Western blot demonstrated an induction in decorin expression in cells switched to collagen gels. This induction was associated with a 40-fold increase in decorin transcript expression determined by quantitative real time PCR. Disaccharide analysis of extracted glycosaminoglycans from collagen gels showed an increase in total glycosaminoglycan and in the ratio of chondroitin sulfate to heparan sulfate compared with monolayer culture. The ratio of chondroitin sulfate to heparan sulfate likewise increased on syndecan-1 from gel culture. Digestion with chondroitinase B showed that this induced chondroitin sulfate was dermatan sulfate. Syndecan-1 extracted from wounded mouse skin also displayed an increase in dermatan sulfate synthesis compared with unwounded skin. Furthermore, glycosaminoglycans from collagen gel culture activated keratinocyte growth factor, whereas glycosaminoglycans from monolayer culture lacked this ability. These findings suggest that regulation of dermatan sulfate and dermatan sulfate proteoglycan is dependent on extracellular matrix architecture. The ability of collagen gel culture to mimic better the in vivo dermal environment may be due in part to this influence on dermatan sulfate and dermatan sulfate proteoglycan synthesis.

HB-107, a nonbacteriostatic fragment of the antimicrobial peptide cecropin B, accelerates murine wound repair.

Antimicrobial peptides are essential to innate host defense as effectors of pathogen clearance and can modify host cell behaviors to promote wound repair. While these two functions appear interrelated, it is unclear whether the ability to aid in wound repair requires inherent antimicrobial function. We hypothesized that the influence of antimicrobial peptides on wound repair is not dependent on antimicrobial function. To explore this, we analyzed the microbial killing activity of peptide fragments and correlated this with the ability to influence wound repair in mice. HB-107, a peptide lacking antimicrobial activity and originally derived from the antimicrobial cecropin B, showed up to 64 percent improvement in wound repair compared to scrambled peptide and vehicle controls, an effect comparable to treatment with recombinant human platelet-derived growth factor-BB (formulated as Regranex). Wounds treated with HB-107 showed keratinocyte hyperplasia and increased leukocyte infiltration. Furthermore, HB-107 stimulated interleukin-8 secretion from cultured endothelial cells, an effect that may explain the increase in leukocyte migration. These findings confirm that antimicrobial peptides can function as effectors of cutaneous wound repair. Moreover, this study furthers our understanding of antimicrobial peptides by showing that their wound repair properties can be independent of antimicrobial function.