Human Platelets Take up Anti-VEGF Agents.

PURPOSE: Growing evidence suggests different systemic exposure of anti-vascular endothelial growth factor (anti-VEGF) agents with repeated intravitreal application. Since the penetration of anti-VEGF agents through vascular barrier was reported, the interaction of anti-VEGF with nonresident platelets has become a topic of interest. The purpose of this study was to evaluate, with the help of visualization techniques, whether platelets take up the anti-VEGF agents ranibizumab, aflibercept, and bevacizumab. METHODS: The uptake of anti-VEGF agents with or without VEGF treatment was investigated using immunofluorescence and immunogold staining in human platelets. The role of actin filaments and clathrin-coated vesicles in the transport of ranibizumab, aflibercept, and bevacizumab was evaluated by two pharmacologic inhibitors: staurosporine (protein kinase C inhibitor) and cytochalasin D. RESULTS: All three anti-VEGF agents were taken up by platelets and colocalized with VEGF. Ranibizumab and aflibercept were mainly detected in alpha-granules; however, bevacizumab was equally localized in alpha-granules and in platelet vesicles. Both staurosporine and cytochalasin D completely inhibited the uptake of aflibercept into platelets. Both pharmacological inhibitors also decreased the transport of ranibizumab and bevacizumab into platelets. Bevacizumab was significantly more frequently colocalized within clathrin-coated vesicles than ranibizumab and aflibercept. CONCLUSION: All three anti-VEGF agents are taken up by platelets and internalized in alpha-granules, which may result in a higher local exposure of anti-VEGF after the activation of platelets, potentially contributing to arterial thromboembolic events. Clathrin-coated vesicles seem to be more prominent in the transport of bevacizumab than ranibizumab and aflibercept. Nevertheless, whether the different localization and transport of bevacizumab are truly related to specific differences of receptor-mediated endocytosis has to be revealed by further research.

Dermasence refining gel modulates pathogenetic factors of rosacea in vitro.

BACKGROUND: Over the counter cosmetics sold for local treatment of slight to moderate rosacea often state the claim of actively modulating rosacea pathogenesis. Factors involved in the pathogenesis of this common yet complex skin disorder include kallikrein-related peptidase 5 (KLK5), LL-37, as well as protease-activated receptor 2 (PAR2) and vascular endothelial growth factor (VEGF). OBJECTIVE: The objective was to prove the modulating effect of the cosmetic skin care agent Dermasence Refining Gel (DRG) on factors involved in rosacea pathogenesis. METHODS: We analyzed the effect of DRG on the expression of KLK5, LL-37, PAR2, and VEGF in an in vitro skin model of human reconstituted epidermis. RESULTS: The expression of CAMP (LL-37 gene, fold change -4.19 [±0.11]), VEGFA (fold change -2.55 [±0.12]) and PAR2 (-1.33 [±0.12]) was reduced, KLK5 expression increased (fold change 2.06 (±0.08)) after 18 h of treatment with DRG in comparison to treatment with the matrix gel only. The reduction in CAMP expression was significant (P<.01). The protein expression of all four inflammatory markers was markedly reduced after 18 hours of DRG treatment in comparison to baseline (0 hour), by measure of fluorescence intensity. CONCLUSION: We show evidence explaining the anti-inflammatory effect of Dermasence Refining Gel in rosacea pathogenesis in vitro. The adjunctive use of DRG in mild to moderate rosacea as a topical cosmetic seems medically reasonable.

The nuclear aryl hydocarbon receptor is involved in regulation of DNA repair and cell survival following treatment with ionizing radiation.

In the present study, we explored the role of the aryl hydrocarbon receptor (AhR) for γ-H2AX associated DNA repair in response to treatment with ionizing radiation. Ionizing radiation was able to stabilize AhR protein and to induce a nuclear translocation in a similar way as described for exposure to aromatic hydrocarbons. A comparable AhR protein stabilization was obtained by treatment with hydroxyl-nonenal-generated by radiation-induced lipid peroxidation. AhR knockdown resulted in significant radio-sensitization of both A549- and HaCaT cells. Under these conditions an increased amount of residual γ-H2AX foci and a delayed decline of γ-H2AX foci was observed. Knockdown of the co-activator ARNT, which is essential for transcriptional activation of AhR target genes, reduced AhR-dependent CYP1A expression in response to irradiation, but was without effect on the amount of residual γ-H2AX foci. Nuclear AhR was found in complex with γ-H2AX, DNA-PK, ATM and Lamin A. AhR and γ-H2AX form together nuclear foci, which disappear during DNA repair. Presence of nuclear AhR protein is associated with ATM activation and chromatin relaxation indicated by acetylation of histone H3. Taken together, we could show, that beyond the function as a transcription factor the nuclear AhR is involved in the regulation of DNA repair. Reduction of nuclear AhR inhibits DNA-double stand repair and radiosensitizes cells. First hints for its molecular mechanism suggest a role during ATM activation and chromatin relaxation, both essential for DNA repair.

Ultrastructural localization of alpha-L-fucose, factor VIII related antigen and vimentin in endothelial cells of human skin blood vessels using the low temperature embedding technique with Lowicryl K4M.

The present study was performed to investigate the ultrastructural localization of the binding sites of the lectin Ulex europaeus agglutinin, Type I (UEA) and the antibody against factor VIII related antigen (F VIII RAG), both widely used as vascular markers in human skin capillaries at light microscopy level. In addition the ultrastructural localization of vimentin, the major protein of the endothelial intermediate filaments, was demonstrated in human vascular endothelial cells (EC) in situ. The low temperature postembedding technique with Lowicryl K4M with subsequent application of gold labelled antibodies provided both well preserved antigenicity and morphology. The antigenic site of UEA, alpha-L-fucose containing glycohydrate residues, was primarily found in or near the luminal plasma membrane of EC. Gold particles representing the sites of F VII RAG were located in the Weibel-Palade bodies (WPB) of EC. The ultrastructural staining pattern of the anti-vimentin antibody showed an exclusive labelling of the intermediate filaments of EC.

Ultrastructural localization of actin in normal human skin.

Normal human skin was embedded in Lowicryl K4M. Actin microfilaments were localized by applying a postembedding immunogold technique using the monoclonal anti-actin antibody HHF35. Actin microfilaments are part of the cytoskeleton in muscle and nonmuscle cells. Together with myosin they produce contraction. The antibody labelled myofilaments in smooth muscle arrector pili cells, myoepithelial cells and pericytes. In sweat gland cells the microvilli system, a zone beneath the cytoplasma membrane corresponding to the adhesion belt region, and apocrine decapitation formations showed labelling.

Techniques in immuno-electron microscopy. I. Cryosubstitution.

Normal skin was cryoprotected by submerging it in a mixture of 30% dimethylformamide (DMF) in PBS or RPMI. Subsequently it was frozen in liquid propane gas. Cryosubstitution was carried out at -90 degrees C by using methanol to which uranyl acetate or osmium tetroxide were added. The tissue was embedded in either Lowicryl K4M at -40 degrees C or in Epon at +60 degrees C. The tissue was evaluated by its overall preservation of ultrastructural details and by its labeling intensity after incubation with either anti-desmoglein or anti-type VII collagen monoclonal antibodies. The mixture of DMF and PBS caused an electron-dense precipitate within the cell. The overall morphology was better in Epon-embedded material than in K4M-embedded material. However, the labeling was best in K4M material. Regardless of whether the tissue was embedded in Epon or K4M, the addition of osmium tetroxide markedly reduced the degree of labeling.

Congenital self-healing reticulohistiocytosis--a benign Langerhans cell disease.

An-11-day old girl was seen with brownish nodular lesions scattered over the body with emphasis on the face and scalp. Several lesions had started to involute. Tissue was studied by histopathology, immunohistopathology, routine electron microscopy, and immuno-electron microscopy using cryosubstitution and embedding in K4M. Immunohistopathology revealed that the cells of the dermal infiltrate were Langerhans cells. They expressed Leu 6 and HLA-DR. On routine electron microscopy no Birbeck granules were found in the dermal cells. Birbeck granules in epidermal Langerhans cells were deformed and often situated next to laminated dense bodies. The latter expressed Leu 6 and lysozyme on immuno-electron microscopy. It was concluded that congenital self-healing reticulohistiocytosis is a benign Langerhans cell disease in which Birbeck granules are transformed to laminated dense bodies and possibly degraded by lysosomal enzymes.

Melanocytes in nevi and melanomas synthesize basement membrane and basement membrane-like material. An immunohistochemical and electron microscopic study including immunoelectron microscopy.

Light microscopic studies have shown that nevus cell nests and melanoma nests are surrounded by basement membrane (BM) material containing type IV collagen and laminin. This study confirms this by electron microscopy and relates it to proteins which interact with the basement membrane. Nevi except for dysplastic and Spitz nevi, malignant melanomas, and melanoma metastases were studied by immunohistopathology, routine electron microscopy (EM), and immunoelectron microscopy. The lesions were incubated with monoclonal antibody (moAb) against type IV collagen, laminin, and the integrin alpha6 and studied by light microscopy. In addition, melanomas were studied by immuno-EM after incubation with a moAb against matrix metalloproteinase-2 (MMP-2). Nevus cell nests and melanoma nests are surrounded by BM material containing type IV collagen and laminin by immuno-EM. The BM material various in thickness and is amorphous. Type IV collagen, laminin, and MMP-2 are synthesized by melanoma cells as well as adjacent fibroblasts. Destruction or loss of the BM is not mandatory for melanoma invasion or even metastasis. Possibly the BM material is a protective wall for melanoma cells. Interactions between melanocytes and the extracellular matrix of which the BM is a part, can be traced back to the migration of melanocytes from the neural crest.