Comparison of cytokine expression and ultrastructural alterations in fresh-frozen and dried electron beam-irradiated human amniotic membrane and chorion.

The purpose of the current study was to compare the effects of drying and fresh-freezing on human amniotic membrane (HAM) and amnion/chorion membrane (HACM) in terms of histological and structural characteristics and cytokine levels. HAM and HACM samples, obtained from six placentae, were investigated. HAM and HACM were dried, electron beam-irradiated (dehydration group; d-HAM/d-HACM), or fresh-frozen (freezing group; f-HAM/f-HACM). Luminex assay was used to assay the levels of 15 cytokines. The ultrastructural characteristics of HAM and HACM were evaluated using light and transmission electron microscopies. Total cytokine contents did not show the statistical difference between dehydration and fresh-freezing process. Significantly higher levels of total cytokines were observed in HACM than in HAM. Epidermal growth factor (EGF) level was significantly higher in d-HAM than in the other samples. The levels of most of the other growth factors were higher in HACM than in HAM, but there was no statistical difference between the dehydration process and the fresh-freezing process. The levels of the cytokines, other than the growth factors, were higher in HACM than in HAM, and higher concentrations of cytokines were observed in the freezing group than in the dehydration group. Histological examination revealed that the dehydration group had thinner tissues than the freezing group, but the structural stability, including the basement membrane, did not differ between the two groups. Microscopic structures such as microvilli and nuclei were well-preserved in the freezing group, based on the results of the transmission electron microscopy. Our dehydration process maintained the histological structure of HAM/HACM and a variety of growth factors and cytokines were identified. Especially, the HAM, processed with the dehydration method, had a higher EGF level than that processed with the fresh-freezing method. Therefore, dehydration method can be used to effectively promote wound repair.

Irradiated C6 glioma cells induce angiogenesis in vivo and activate endothelial cells in vitro.

Malignant gliomas are angiogenesis dependent and present a remarkable degree of resistance to radiotherapy. In the present work, we studied the effect of irradiation of C6 glioma cells on their proliferation and activation in vitro and on glioma cell-induced angiogenesis in vivo and in vitro. Irradiation of C6 glioma cells decreased cell proliferation in a dose-dependent manner. Interestingly, metalloproteinase-2 and -9 expression and secretion, as well as integrin alpha(v) expression, increased with elevated doses of X rays 48 hr after irradiation and was mostly evident at the higher doses used. When pre-irradiated C6 cells were implanted on nonirradiated chicken embryo chorioallantoic membranes (CAMs), there was a significant dose-dependent increase in tumor induced angiogenesis, compared to angiogenesis induced by nonirradiated cells. Similar results were obtained when C6 cells were irradiated 48 hr after their inoculation onto nonirradiated CAMs. In the same line, conditioned medium from irradiated C6 cells significantly increased endothelial cell proliferation and migration in vitro, in a manner dependent on the dose of X rays. These results explain at least in part the low effectiveness of radiation therapy of malignant gliomas and support the notion that inhibition of angiogenesis in parallel with radiotherapy may represent a new therapeutic approach.

0.2 T magnetic field inhibits angiogenesis in chick embryo chorioallantoic membrane.

Inhibition of angiogenesis is a major target in the fight against cancer and other diseases. Although the effects of static magnetic fields on cancer development and cell growth have been investigated, effects on angiogenesis have received no attention so far. In this study we report the effects on angiogenesis of exposure to 0.2 T static magnetic field. Angiogenesis was analyzed using the chick embryo chorioallantoic membrane assay. Exposure to 0.2 T static magnetic field was achieved by placing the eggs for 3 hr in the isocentre of the magnet of a sectorial magnetic resonance tomograph used in clinical practice. In sham exposed specimens treated with phosphate buffered saline (negative control), no significant vascular reaction was detectable; 3 hr exposure to 0.2 T static magnetic field did not affect the basal pattern of vascularization or chick embryo viability. Prostaglandin E1 and fetal calf serum elicited a strong angiogenic response in sham exposed eggs. This angiogenic response was significantly inhibited by 3 hr exposure to 0.2 T static magnetic field. These findings point to possible use of static magnetic field in inhibiting angiogenesis; this effect could be exploited for treatment of cancer and other diseases where excessive angiogenesis is involved.

Effects of paclitaxel in combination with ionizing radiation on angiogenesis in the chick embryo chorioallantoic membrane. A radiobiological study.

PURPOSE: To investigate the combined effects of paclitaxel and single or fractionated doses of ionizing radiation on the angiogenesis process, using as a model the chick embryo choriallantoic membrane (CAM). MATERIAL AND METHODS: Experiments were performed on 9-day CAM, when membranes were irradiated with various single or fractionated doses of X-rays, either alone or in combination with paclitaxel (6.4 micro g/disk). RESULTS: Single doses of irradiation (5, 10, or 15 Gy) produced a significant antiangiogenic effect, which was not dose-dependent. Fractionated doses of X-rays (two doses of 2.5, 5, or 7.5 Gy, each 12 h apart) exerted a dose-dependent reduction of the vascular density index. Paclitaxel was not shown to provoke radiosensitization in this model, i. e., to inhibit angiogenesis of the 9-day CAM. CONCLUSION: These data confirm that the CAM system can be conveniently and properly used for radiobiological studies and indicate that paclitaxel in combination with ionizing radiation does not inhibit further angiogenesis in the system used.

Application of an ex ovo chicken chorioallantoic membrane model for two-photon excitation photodynamic therapy of age-related macular degeneration.

Two-photon excitation photodynamic therapy (TPE-PDT) is being investigated as a clinical treatment for age-related macular degeneration (AMD). TPE-PDT has the potential to provide a more specific and therefore advantageous therapy regime than traditional one-photon excitation PDT. The highly vascularized 8 to 9-day-old chicken chorioallantoic membrane (CAM) is used to model the rapid growth of blood vessels in the wet form of AMD. Using an ex ovo model system for the CAM, ablation studies were successful in mimicking the leaky vessels found in AMD. In addition, the distribution and localization of liposomal Verteporfin were investigated in order to characterize the photosensitizing drug in vivo. Localization of the photosensitizer appears to be greatest on the upper vessel wall, which indicates a potentially strong treatment locale for TPE-PDT.

Ionizing radiation modulates the exposure of the HUIV26 cryptic epitope within collagen type IV during angiogenesis.

PURPOSE: The majority of the research on the biologic effects of ionizing radiation has focused on the impact of radiation on cells in terms of gene expression, DNA damage, and cytotoxicity. In comparison, little information is available concerning the direct effects of radiation on the extracellular microenvironment, specifically the extracellular matrix and its main component, collagen. We have developed a series of monoclonal antibodies that bind to cryptic epitopes of collagen Type IV that are differentially exposed during matrix remodeling and are key mediators of angiogenesis. We have hypothesized that ionizing radiation might affect the process of angiogenesis through a direct effect on the extracellular matrix and specifically on collagen Type IV. METHODS AND MATERIALS: Angiogenesis was induced in a chick chorioallantoic membrane (CAM) model; 24 h later, a single-dose treatment with ionizing radiation (0.5, 5, and 20 cGy) was administered. Angiogenesis was assessed, and the exposure of two cryptic regulatory epitopes within collagen Type IV (HUI77 and HUIV26) was studied in vitro by solid-phase ELISA and in vivo by immunofluorescence staining. RESULTS: A dose-dependent reduction of angiogenesis with maximum inhibition (85%-90%) occurring at 20 cGy was demonstrated in the CAM model. Exposure of the cryptic HUIV26 site, an angiogenesis control element, was inhibited both in vitro and in vivo by the same radiation dose, whereas little if any change was observed for the HUI77 cryptic epitope. CONCLUSIONS: A dose-dependent alteration of the functional exposure of the HUIV26 cryptic epitope is induced by radiation in vitro and in the CAM model in vivo. This radiation-induced change in protein structure and function may contribute to the inhibitory effects of ionizing radiation on new blood vessel growth and warrants further studies in other models.

Nitration of cytoskeletal proteins in the chicken embryo chorioallantoic membrane.

Protein tyrosine nitration is one of the post-translational modifications that alter the biological function of proteins. Two important mechanisms are involved: peroxynitrite formation and myeloperoxidase or eosinophil peroxidase (EPO) activity. In the present work we studied the nitration of proteins in the in vivo system of chicken embryo chorioallantoic membrane (CAM). 3-Nitrotyrosine was detected only in the insoluble fraction of the CAM homogenate. By immunoprecipitation, Western blot analysis, and double immunofluorescence, we identified two major polypeptides that were nitrated: actin and alpha-tubulin. Quantification of actin and alpha-tubulin nitration revealed that they are differentially nitrated during normal development of the chicken embryo CAM. After irradiation, although they were both increased, they required different time periods to return to the physiological levels of nitration. It seems that both peroxynitrite formation and EPO activity are involved in the in vivo tyrosine nitration of cytoskeletal proteins. These data suggest that tyrosine nitration of cytoskeletal proteins has a physiological role in vivo, which depends on the protein involved and is differentially regulated.

Photodynamic parameters in the chick chorioallantoic membrane (CAM) bioassay for topically applied photosensitizers.

The relative efficacy of Photofrin-based photodynamic therapy (PDT) has been compared with that of the second-generation photosensitizers 5-aminolevulinic acid (ALA), sulfonated chloro-aluminum phthalocyanine (AlPcSn), benzoporphyrin derivative monoacid ring A (BPD-MA), and lutetium texaphyrin (Lutex). PDT-induced vascular damage in the chick chorioallantoic membrane (CAM) is measured following topical application of the photosensitizers. In order to make meaningful comparisons, care is taken to keep treatment variables the same. These include light dose (5 and 10 J/cm2), power density (33 and 100 mW/cm2), and drug uptake time (30 and 90 min). The drug dose ranges from 0.1 microgram/cm2 for BPD to 5000 micrograms/cm2 for ALA. Results are also analyzed statistically according to CAM vessel type (arterioles versus venules), vessel diameter, and vessel development (embryonic age). For each photosensitizer, the order of importance for the various PDT parameters is found to be unique. The differences between the sensitizers are most likely due to variation in biophysical and biochemical characteristics, biodistribution, and uptake kinetics.

In vivo experimental evidence that the nitric oxide pathway is involved in the X-ray-induced antiangiogenicity.

We have investigated both the effects of X-rays on angiogenesis and the possible role of nitric oxide (NO) on the observed antiangiogenic effect of X-rays, using as an in vivo model the chick embryo chorioallantoic membrane (CAM). These effects were assessed both morphologically and biochemically, by measuring vascular density and collagenous protein biosynthesis, respectively, on days 9 and 14 of the chick embryo development. Vascular density and cytotoxicity of the CAM were also evaluated histologically. We have shown that X-rays have an antiangiogenic effect on the system used and that the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) promoted angiogenesis of the non-irradiated CAM and reversed the antiangiogenic effect of irradiation. D-NAME, which is an inactive enantiomer of L-NAME, showed no such effects. L-Arginine, which is the substrate for NO synthase, had a modest antiangiogenic effect on the non-irradiated CAM, no effect on the irradiated CAM and abolished the angiogenic effect of L-NAME on these CAM preparations. These results suggest that NO is involved in the antiangiogenic mechanism of X-rays and that pharmacological manipulation of NO firstly, may offer a better understanding of these mechanisms and, secondly, may also prove to be an alternative therapeutic approach for treating pathological conditions involving angiogenesis.

Prenatal diagnosis of xeroderma pigmentosum and Cockayne syndrome.

In a study of fetal cells from a series of 12 pregnancies in ten families at risk for the ultraviolet light-sensitive, DNA repair-deficient diseases xeroderma pigmentosum (XP) and Cockayne syndrome (CS), we detected one XP and two CS homozygote fetuses. The diagnoses were confirmed by analysis of fetal skin fibroblasts or second amniotic samples after termination of the pregnancies. The measurement of ultraviolet light sensitivity and DNA repair depended on properties common to the seven excision repair-deficient XP complementation groups (A-G) and the two CS complementation groups (A, B). No XP variant families were included in the study, because the variant requires different testing techniques. Reliable and rapid diagnosis proved possible in all but one of the 12 pregnancies, supporting the use of these methods until the spectrum of mutations in the various XP and CS genes of the U.S. population is fully characterized and a DNA sequence-based diagnostic procedure becomes available.

Demonstration of synergistic effects of hyperthermia and photodynamic therapy using the chick chorioallantoic membrane model.

The chick chorioallantoic membrane (CAM) model was used to study vascular effects of photodynamic therapy (PDT) and hyperthermia (HPT) and the synergism of these modalities. The CAM is a convenient medium for monitoring the modifications of the vasculature. It is possible to view the CAM and to examine structural changes of individual blood vessels in real time. Moreover, the CAM is a closed system which lends itself to mathematical modeling of the temporal and spatial temperature profile and in which HPT can be performed quantitatively and to a selected depth, using different lasers. A porphyrin-type photosensitizer solution was applied to areas of the CAM, defined by teflon O-rings placed on the surface. Uptake dynamics of the sensitizer into the CAM was determined by analyzing its fluorescence in vivo. The CAM area was irradiated with a dual-wavelength laser system composed of a dye laser at 644 nm (to induce PDT) and a CO2 laser at 10.6 microns (to bring about HPT). Damage to the CAM vasculature, due to combined PDT+HPT, was compared to the outcome of the separate modalities, and a synergistic effect of about 40% was observed.

Effect of beta-irradiation by a 106 ruthenium plaque on the rabbit eye choroid.

Fourteen Chinchilla gray rabbit eyes were treated with a ruthenium plaque, receiving 200, 400, or 800 Gy, to assess the effect of beta-irradiation on the normal rabbit choroid. Radiation effects were evaluated using fundus photography, fluorescein angiography, fluorophotometry, and histology. Fluorophotometry showed a fluorescein leakage into the vitreous 1 day after irradiation. Leakage values returned to normal within 1 month after irradiation. Fluorescein angiography showed nonperfusion of the choroid after beta-irradiation; the time between irradiation and the onset of nonperfusion was found to be dose dependent. Five months after 200 Gy irradiation, choroidal atrophy had developed but some vessels still stained with fluorescein; 400 Gy irradiation induced subtotal choroidal nonperfusion within 3 months and 800 Gy within 1 week.