Effect of palmitoylethanolamide on degeneration of a human-derived retinal pigment epithelial cell induced by all-trans retinal.

AIM: To study the effect of palmitoylethanolamide (PEA) on apoptosis of retinal pigment epithelial (RPE) cells induced by all-trans retinal (atRAL) and to explore the possible molecular mechanism. METHODS: CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) was used to detect the effect of PEA on human-derived retinal epithelial cells (ARPE-19) viability induced by atRAL. A Leica DMi8 inverted microscope was used to observe cell morphology. Reactive oxygen species (ROS) production was evaluated with 2',7'-dichlorodihydrof-luorescein diacetate (H2DCFDA) staining and fluorescence microscopy. Expression of c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), c-Jun, phosphorylated c-Jun (p-c-Jun), Bak, cleaved caspase-3, C/EBP homologous protein (CHOP), and binding (Bip) protein levels were tested by Western blot. Abca4 -/- Rdh8 -/- mice, mouse models of atRAL clearance defects which displays some symbolic characteristics of dry age-related macular degeneration (AMD) and Stargardt disease (STGD1). In the animal models, PEA was injected intraperitoneally. The full-field electroretinogram was used to detect visual function under scotopic conditions traced from mice. Optical coherence tomography showed reconstitution or thickening of the retinal pigment epithelium layer. Effect of PEA on fundus injury induced by light in Abca4-/-Rdh8-/- mice was observed by fundus photography. RESULTS: PEA ameliorated ARPE-19 cells apoptosis and inhibited ROS (including mitochondrial ROS) production induced by atRAL. PEA improved the retinal functional, prohibited both RPE and photoreceptor from death, ameliorates light-induced fundus impairment in Abca4 -/- Rdh8 -/- mice. In vitro and in vivo, PEA inhibited JNK, p-JNK, c-Jun, p-c-Jun, Bak, cleaved caspase-3, CHOP, and Bip protein levels induced by all-trans retinal in ARPE-19 cells. CONCLUSION: PEA has effect on treating RPE cells apoptosis in retinopathy caused by atRAL accumulation. PEA is a potential treatment strategy for dry AMD and STGD1. The molecular mechanism is affecting the ROS-JNK-CHOP signaling pathway partly.

[Effects of bone morphogenetic protein-2 gene therapy on the bone-implant interface: an experimental study with dogs].

OBJECTIVE: To investigate the effects of bone morphogenetic protein-2 (BMP-2) gene therapy on the bone-implant interface in the reconstruction of periprosthetic bone defect. METHODS: Transverse defects were caused in the external condylae of both femurs of 14 adult Beagle dogs. Titanium alloy implants were inserted and a bone defect 3 mm wide around the titanium alloy implant was preserved. Then the total 28 defects were divided into 4 groups: 8 bone defects remained untreated (blank control group); 8 bone defects were implanted with heterogeneous freeze-dried bone by impaction grafting technique (non-cell group); 8 bone defects were implanted with heterogeneous freeze-dried bone loaded with autogenous bone marrow stromal cells (BMSCs) from the greater trochanter of the same dog (cell group); and 10 bone defects were implanted with freeze-dried allograft loaded with autogenous BMSCs from the greater trochanter of the same dog which were transfected by Adv-BMP-2 gene (gene group). Three, 6, and 12 weeks after implantation X-ray examination was carried out to observe the place of the implant and the absorption of the implants. Six and 12 weeks after the dogs were killed and their bone defects were taken out to undergo histological, histomorphometric and biomechanical examination to observe the healing and oseeointegration of the bone-implant interface. RESULTS: Histological examination showed that 6 weeks after implantation new bone formation was found on the implant surface and there was point contact between the bone and implant in the gene group with the bone-to-impact contact (BIC) of about 10%; and continuous soft tissue was found at bone-implant interface in all other groups. Twelve weeks after, there was thick soft tissue membrane between the new bone and implant in the blank control group; most of the interface was connective fibrous tissue in the non-cell group and cell group with point contact between the bone and implant and a BIC lower than 10%; and in the gene group the interface consisted mainly of bone tissue and continuous bone-implant contact was found with the BIC of 50%, significantly higher than those of the other 2 groups (both P < 0.01). The mechanical strength of interface increased time-dependently in all groups, that of the gene group being significantly higher than those of the other 2 groups at any time-points (both P < 0.01). CONCLUSION: BMP-2 gene therapy can improve the osseointegration of bone-implant interface.

Ectopic bone formation of human bone morphogenetic protein-2 gene transfected goat bone marrow-derived mesenchymal stem cells in nude mice.

OBJECTIVE: To evaluate the osteogenic potential of bone morphogenetic protein (BMP)-2 gene transfected goat bone marrow-derived mesenchymal stem cells (MSCs). METHODS: Goat bone marrow-derived MSCs were transfected by Adv-human bone morphogenetic protein (hBMP)-2 gene (Group 1), Adv-beta gal transfected MSCs (Group 2) and uninfected MSCs (Group 3). Western blot analysis, alkaline phosphatase staining, Von Kossa staining and transmission electron microscopy were adopted to determine the phenotype of MSCs. Then the cells were injected into thigh muscles of the nude mice. Radiographical and histological evaluations were performed at different intervals. RESULTS: Only Adv-hBMP-2 transfected MSCs produced hBMP-2. These cells were positive for alkaline phosphatase staining at the 12th day and were positive for Von Kossa staining at the 16th day after gene transfer. Electron microscopic observation showed that there were more rough endoplasmic reticulum, mitochondria and lysosomes in Adv-hBMP-2 transfected MSCs compared to MSCs of other two groups. At the 3rd and 6th weeks after cell injection, ectopic bones were observed in muscles of nude mice of Group 1. Only fibrous tissue or a little bone was found in other two groups. CONCLUSIONS: BMP-2 gene transfected MSCs can differentiate into osteoblasts in vitro and induce bone formation in vivo.

[BMP-2 gene modified tissue-engineered bone repairing segmental tibial bone defects in goats].

OBJECTIVE: To evaluate the effectiveness of the tissue-engineered bone substitute loaded with adenovirus mediated human bone morphogenetic protein-2 gene (Adv-hBMP-2) transfected bone marrow derived mesenchymal stem cells (BMSC) in the repair of diaphyseal segmental bone defect of large animal. METHODS: The right tibial bone defects (2.6 cm) model of 26 goats were established and divided into 5 groups: I. Adv-hBMP-2 transfected BMSC/calcined bone (CB) group (n = 9); II. adenovirus-beta-galactosidase (Adv-betagal) gene transfected BMSC/CB group (n = 6); III. untransfected BMSC/CB group (n = 6); IV. single CB group (n = 3); VI. untreated group (n = 2). The above tissue-engineered bone substitutes were implanted in the bone defects respectively except group VI. Roentgenography, histomorphometrical analysis and biomechanical measurement were studied at various times. RESULTS: X-ray: at 4 - 8th weeks after implantation, more bony callus was found in the bone defects of group I. The complete healing rates of group I, II, III, IV, and V were 5/8, 1/5, 0/5, 0/2, 0/1 respectively at 26th week after implantation. Histomorphometrical analysis showed much more new bony callus including cortical bone formed in group I than those of other groups. The compression strength of the implanted bone substitute of group I is significantly higher than those of group II and III. CONCLUSION: The tissue-engineered bone substitute loaded with human BMP-2 gene transfected BMSC can repair diaphyseal segmental bone defect of large animal (goat).