ABSTRACT
Cis-stilbene combretastatin A-4 (CA-4) and a large group of its derivant compounds have been shown significant anti-angiogenesis activity. However the side effects even the toxicities of these chemicals were not evaluated adequately. The zebrafish model has become an important vertebrate model for evaluating drug effects. The testing of CA-4 on zebrafish is so far lacking and assessment of CA-4 on this model will provide with new insights of understanding the function of CA-4 on angiogenesis, the toxicities and side effects of CA-4. We discovered that 7-9 ng/ml CA-4 treatments resulted in developmental retardation and morphological malformation, and led to potent angiogenic defects in zebrafish embryos. Next, we demonstrated that intraperitoneal injection of 5, 10 and 20 mg/kg CA-4 obviously inhibited vessel plexus formation in regenerated pectoral fins of adult zebrafish. Interestingly, we proved that CA-4 treatment induced significant cell apoptosis in central nervous system of zebrafish embryos and adults. Furthermore, it was demonstrated that the neuronal apoptosis induced by CA-4 treatment was alleviated in p53 mutants. In addition, notch1a was up-regulated in CA-4 treated embryos, and inhibition of Notch signaling by DAPT partially rescued the apoptosis in zebrafish central nervous system caused by CA-4.
Subject(s)
Apoptosis/drug effects , Neovascularization, Pathologic/drug therapy , Stilbenes/adverse effects , Animals , Central Nervous System/drug effects , Central Nervous System/metabolism , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental/drug effects , Models, Animal , Morphogenesis/drug effects , Neovascularization, Pathologic/metabolism , Receptors, Notch/metabolism , Signal Transduction/drug effects , Tumor Suppressor Protein p53/metabolism , Up-Regulation/drug effects , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
OBJECTIVE: To investigate modulatory role of Rac1 protein in epidermal stem cell (ESC) migration during wound healing, in order to provide a reference for enriching basic theory of wound healing and guiding clinical application. METHODS: Constitutively active mutant of Rac1 protein (Rac1Q61L) or dominant negative isoform of Rac1 protein (Rac1T17N) was transfected into ESC using a retroviral vector FUGW, and retroviral vector FUGW transfected into ESC in singles was used as blank control. The cells were divided into 3 parts according to the random number table and treated as follows. First, equal numbers of cells were inoculated into 24-well plates coated with collagen I (20 µg/mL), collagen IV (20 µg/mL) or fibronectin (10 µg/mL). Cells adhered to above matrices were quantitated using CytoTox 96 colorimetric kit. Second, 1000 cells adhered to collagen IV, after being stained with tetramethyl rhodamine isothiocyanate-phalloidin, were collected for observation of cell morphology and comparison of spreading area under confocal laser scanning microscope. Third, ESC with density of 2 × 10(5) cells per well were placed in upper compartment of Transwell chamber, DK-SFM culture medium alone or that containing stromal cell derived factor 1 (SDF-1) was added into lower compartment of Transwell chamber. Migration of ESC was observed using inverted phase contrast microscope, and the result was denoted as migration rate. Lastly, ESC with density of 7.5 × 10(5) cells per well was inoculated into 6-well plates for 12 hours, and treated with 4 µg/mL mitomycin C for 2 hours. The remaining scratch width of monolayer was respectively measured 6 hours or 12 hours after scratching to calculate the percentage of remaining scratch width. Data were processed with t test. RESULTS: Compared with that of blank control, the number of Rac1Q61L-transfected cells adhered to collagen I was significantly increased (t = 5.302,P < 0.05), while the number of Rac1T17N-transfected cells adhered to collagen I, IV, and fibronectin were all obviously decreased (with t value respectively 13.741, 15.676, 8.256, P values all below 0.05). Confocal laser scanning microscope showed that spreading area of Rac1Q61L-transfected ESC (with laminate pseudopodia on edge) and Rac1T17N-transfected ESC was respectively larger and smaller as compared with that of blank control. With SDF-1 effect, the migration rate of Rac1T17N-transfected ESC was decreased by 78.0% and Rac1Q61L-transfected ESC was increased by 43.4% as compared with that of blank control. Without SDF-1 effect, the migration rate of Rac1T17N-transfected ESC was decreased by 55.2%, while the migration rate of Rac1Q61L-transfected ESC was close to that of blank control. Six or 12 hours after scratching, the percentage of remaining scratch width in Rac1Q61L-transfected ESC was lower as compared with that in blank control [(39 ± 9)% vs. (43 ± 5)%, (6 ± 5)% vs. (18 ± 7)%, with t value respectively 1.027, 4.389, with P value respectively above and below 0.05], while that in Rac1T17N-transfected ESC [(81 ± 9)%, (71 ± 11)%, respectively] was obviously higher as compared with that in blank control (with t value respectively 11.386, 11.726, P values all below 0.05). CONCLUSIONS: Rac1 protein may control the migration of ESC by regulating its adhesion, spreading, and chemotaxis, and it plays an active role in wound healing accelerated by ESC.
Subject(s)
Cell Movement , Epidermal Cells , Stem Cells/cytology , Wound Healing , rac1 GTP-Binding Protein/metabolism , Cell Proliferation , Epithelial Cells , Humans , Mutation , Transfection , rac1 GTP-Binding Protein/geneticsABSTRACT
OBJECTIVE: To study the promotion effect of stromal cell-derived factor 1 (SDF-1) on the migration of epidermal stem cells (ESC) in the healing process of frostbite-wound model ex vivo. METHODS: A three-dimensional model of full-thickness frostbite of skin was constructed (with slot-like wound) out of skin equivalent. The expression of SDF-1 in wound stroma was observed with immunohistochemistry staining on post injury days (PID) 3 and 7. The model frostbite wounds were divided into control group (treated with PBS 50 microL per wound), SDF-1 group (treated with 100 ng/mL SDF-1, 50 microL per wound), and AMD3100 group [treated with 100 ng/mL AMD3100 (50 microL per wound) for 30 minutes, and then SDF-1 50 microL was added per wound]. The redistribution of ESC around wound was observed. RESULTS: The expression of SDF-1 in wound stroma increased gradually on PID 3 and 7. Compared with those in control and AMD3100 groups, there were more ESC and epithelial cell layers, and more integrin beta(1)-positive cells appeared at the basal layer of wound in SDF-1 group, and some of the positive cells migrated upward to epidermis. CONCLUSIONS: SDF-1 contributes to wound repair through promoting ESC to migrate toward and gather around wound edge. This may be one of the mechanisms of ESC participating in wound repair.
