Axon guidance molecules, slit glycoprotein (Slit) and Roundabout receptors (Robo) were firstly identified in the central neural system of Drosophila melanogaster. The Slit-Robo signal plays a crucial role in axon guidance, inflammation, tumor metastasis and angiogenesis, of which the role of Slit2-Robo pathway in angiogenesis has drawn a greater attention and still remains conflicting. Herein, we provide a review on the role of Slit2-Robo pathway in ocular angiogenesis and vascularization of other organs and systems. We hope this review will be the basis of further study on the mechanism of Slit2-Robo signaling on angiogenesis and provide new target for the therapy on ocular vascular disease
With the rapid development of fundus imaging technology, it is of great significance to establish a new naming system for neovascular age-related macular degeneration (nAMD) based on the multi-mode imaging. In 2020, an international panel of retina specialists, imaging and image reading center experts, and ocular pathologists reached a consensus after repeated discussions, a new name for nAMD subtype and related lesions was established based on the previous knowledge of fundus fluorescein angiography and pathology, combining indocyanine green angiography, optical coherence tomography and optical coherence tomography angiography with current pathological knowledge, in order to help ophthalmologists to study nAMD. The consensus proposed the term "macular neovascularization" and classified it into type 1, type 2 and type 3. Many lesions related to macular neovascularization, such as pigment epithelial detachment, hemorrhage, fibrosis, rip of retinal pigment epithelium and so on, were named. The new designation will help improve clinical communication between different studies, establish standard definitions and terms between reading centers and researchers, and further promote the understanding and communication of nAMD among ophthalmologists.
ObjectiveTo observe the effects of NDRG1 on proliferation, migration and lumen formation of retinal vascular endothelial cells (RF/6A cells) in monkeys under high glucose condition. MethodsRF/6A cells were divided into normal group, mannitol group, high glucose group, small interfering RNA (siRNA) negative control group without target gene (siRNA group), 30 nmol/L siRNA down-regulated NDRG1 genome (siNDRG1 group) and 50 nmol/L siNDRG1 group. Normal group cells were cultured conventionally. The mannitol group was added with 25 mmol/L mannitol, and the high-glucose group was added with 25 mmol/L glucose. In the siRNA group, 25 mmol/L glucose was added, and then blank siRNA was added for induction. The 30 and 50 nmol/L siNDRG1 groups were added with 25 mmol/L glucose and induced with 30 and 50 nmol/L siRNDRG1, respectively. All cells were incubated for 24 h for follow-up experiments. Cell proliferation was observed by 4', 6-diaminidine 2-phenylindole staining. Cell counting kit-8 staining was used to detect cell activity. The expression level of NDRG1 mRNA and protein was detected by Western blot and real-time quantitative polymerase chain reaction. Cell migration was observed by cell scratch assay. Cell lumen formation assay was used to detect lumen formation. The two-tailed Student t test was used to compare the two groups. One-way analysis of variance was used to compare groups. ResultsThere were significant differences in cell proliferation rate (t=36.659, 57.645) mobility rate (t=24.745, 33.638) and lumen formation number (t=41.276, 22.867) between high glucose group and normal group and mannitol group (P＜0.01). Compared with normal group and mannitol group, the relative expression levels of NDRG1gene mRNA and protein in high glucose group were significantly decreased, with statistical significance (t=46.145, 21.541, 36.738, 32.976; P＜0.001). Compared with the siRNA negative group, the relative expression levels of NDRG1gene mRNA and protein in 30 nmol/L siNDRG1 group and 50 nmol/L siNDRG1 group were significantly decreased, and the differences were statistically significant (t=44.275, 40.7577, 57.167, 25.877; P＜0.01). Compared with normal group and siRNA group, cell mobility in 30 nmol/LsiNDRG1 group was increased, and the difference was statistically significant (t=57.562, 49.522; P＜0.01). Compared with normal group and siRNA group, the number of cell lumen formation in 30 nmol/LsiNDRG1 group was significantly increased in the same field of vision, and the difference was statistically significant (t=63.446, 42.742; P＜0.01). ConclusionDown-regulation of NDRG1 gene can improve the activity, migration and lumen formation of RF/6A cells under hyperglycemia.
Objective To observe the effect of Twist gene interference on the migration and pAkt protein expression of Rhesus retinal vascular endothelial cell line. Methods The Rhesus retinal vascular endothelial cells (RF/6A) were divided into Twist interference plasmid group, negative control group, and phosphate buffered solution (PBS) group; plasmid vectors were transfected via liposome gene transfection method. Migrated endothelial cells was detected and counted by Transwell chamber assay. Matrigel was used in endothelialcell tube formation; the inhibitory effect of Twist gene interference on endothelial cell tube formation was observed.The effect of Twist gene interference on the expression of pAkt protein in RF/6Acells was measured by Western blot. Results The number of migrated endothelial cells in Twist interference plasmid group was lower than that in the negative control and PBS group (F=23.786，P=0.000).The number of endothelial cell tubes in Twist interference plasmid group was apparently less than that in the negative control and PBS gorup (F=7.159，P=0.014). The expression of pAkt protein in Twist interference plasmid group decreased markedly.Conclusion Twist gene interference can suppress the migration of retinal endothelial cells via inhibiting the expression of pAkt protein.
ObjectiveTo review the osteoclasts (OC) function beyond bone resorption. MethodsThe related literature on OC function beyond bone resorption was reviewed, analyzed, and summarized. ResultsOC control the bone formation through releasing of matrix-derived growth factors, bidirectional cell-to-cell signals, and secreting OC-coupling factors, and play an important role in the niche formation, hematopoietic stem cells mobilization, and maintenance of its quantity and function;besides, OCs also regulate angiogenesis. ConclusionThese discoveries greatly enrich the current knowledge of OC function and open up an all-new research domain. However, the exact regulatory mechanism of OC affecting the hematopoiesis is still lack in-depth understood. Additionally, it remains to be elucidated how OC-coupling factors act on osteoblast lineage differentiation and how OC-induced angiogenesis participates in physiological and pathological processes. Unclosing the underlying mechanisms will facilitate providing scientific therapeutic strategies for treatment of many OC-related diseases.
Purpose To investigate the status of proliferation and activation of vascular endothelial cells in preretinal neovascular membranes from patients with insulin dependent diabetetes mellitus(IDDM)by means of immunohistochemical techniques. Methods Status of vascular endothelial cells in 18 preretinal neovascular membranes from 18 patients with IDDM was studied by double-immunofluorescence technique and the alkaline phosphataes-anti-alkaline phosphatase(APAAP)technique and compared the findings with the main clinical features of the patients. Results Of 18 vascularized membranes,16(88.9%)contained proliferating endothelial cells (positive for proliferating vascular endothelial cell marker EN 7/44) and 14 (77.8%) were positive for endothelial cell activation marker anti-VCAM-1;furthermore,by using a double staining technique,we found that in 14 of the 16 cases(87.5%) the proliferating vascular endothelial cells were activated (expressing VCAM-1). Conclusion The proliferation and activation of the vascular endothelial cells of the newly formed vessels in preretinal neovascular membranes suggests the significance of the vascular endothelial cells in the pathophysiology and the progress of proliferative diabetic retinopathy. (Chin J Ocul Fundus Dis,1998,14:141-143)
Purpose To estabalish a quantifying model of retinal neovascularization suitable for the study of pathogenesis and therapeutic intervention for the retinal neovascularization． Methods Sixteen one-week-old C57BL/6 mice were exposed to 75% oxygen for 5 days and then to room air and 16 mice of the same age kept in room air as controls.Ink-perfused retinal flatmount was examined to assess the oxygen-induced changes of retinal vessels.The proliferated neovascular response was quantitated by counting the nuclei of endothelial cells of new vessels extending from the retina into the vitreous in 6 mu;m sagittal cross sections． VEGF and bFGF were determined on the cross-sections after immunohistochemcal stain. Results Constriction and closure of the blood vessels were found under the hyperoxia condition,and dilation and proliferation were found under the relatively hypoxia status.There was a mean of 24 neovascular nuclei per cross-section in the oxygen-treated retina and less than 1 nucleus in the control group (P＜0.001).VEGF stain was found ber in the inner retinal layer of oxygen-treated mouse than in that of the controls. Conclusion The quantifying model of retinal neovascularization may fascilitate the further researches of medical intervention and pathogenesis of retinal neovacularization. (Chin J Ocul Fundus Dis，2000，16：213-284)
Objective To compare two kinds of myofascial flap encapsulating adi pose-derived stromal cells (ADSCs) in adi pogenic efficacy in vivo, and to provide experimental basis for the efficient transplantation of free adi pose tissue. Methods ADSCs were isolated from the subcutaneous adipose tissue in the neck of 10 New Zealand rabbits (aged 3-4 months old, male and female, weighing 2.0-2.5 kg), and primary culture and subculture of ADSCs were conducted. When the cells at passage 3 covered 70%-80% of the bottom of the culture flask, BrdU (10 μg/mL) was appl ied to label the cells for 48 hours before performing immunofluorescence staining. Oil red O staining observation was conducted to thosecells 2 weeks after being induced towards adi pocyte, al izarin red staining observation was performed 3 weeks after being induced towards osteoblast, and alcian blue staining was conducted 2 weeks after being induced towards chondrocyte. Besides, after being induced towards adipocyte for 2 weeks, 1 × 107 ADSCs/piece at passage 3 labeled by BrdU was seeded into Col I (10 mm × 10 mm × 5 mm/piece) to prepare cell carrier complex. The experiment was divided into two groups: group A in which vascular pedicled dextral latissimus dorsi fascial flap was adopted to encapsulate the complex; group B in which dextral gluteus maximus fascial flap with no specific vessel pedicle was appl ied to encapsulate the complex. Rabbits in each group went through autogenous ADSCs transplant and self control. The implants were dislodged 8 weeks after operation, HE staining and immunohistochemistry staining were performed to testify cambium, the wet weight and micro vessel count of the cambium in each group were tested, immunofluorescence staining was performed to determine the origin of cambium and microvascular endothel ium. Results The nucleus of ADSCs positive for BrdU label ing showed green fluorescence under fluorescence microscope, with the positive label ing ratio of ADSCs above 90%. For ADSCs at passage 3, the formation of red l ipid droplets within cells was observed 2 weeks after being induced towards adipocyte, red calcium nodules were evident 3 weeks after being induced towards osteoblast, and highly congregated cell mass positive for alcian blue staining appeared 2 weeks after being induced towards chondrocyte. Eight weeks after operation, neogenetic blood vessel grew into scaffolds and no obvious fibreencapsulation was observed in group A, while few blood vessel grew into scaffolds in group B. The wet weight of cambium in group A and B was (0.149 5 ± 0.017 3) g and (0.095 3 ± 0.012 7) g, respectively, indicating there was a significant difference between two groups (P lt; 0.01). HE staining showed the formation of neogenetic adipose tissue and the growth of micrangium in the implant, and the degradation and absorption of scaffold. The micro vessel count of group A and B was 31.2 ± 4.5 and 19.3 ± 2.6, respectively, indicating there was a significant difference between two groups (P lt; 0.01). Eight weeks after operation, the immunofluorescence staining of cambium showed that the cell nucleus of regenerated adi pocytes and partial capillary endothel ium in groups A and B presented green fluorescence. Conclusion ADSCs encapsulated by vascular pedicled latissimus dorsi fascial flap and collagen protein scaffold complex has a higher adi pogenic efficacy in vivo than the gluteus maximus fascial flap with no specific vessel pedicle.
Purpose To evaluate the efficacy of vitreous surgery and endolaser in a series of patients with retinal vein occlusion(RVO)with vitreous hemorrhage,neovascular membranes(NVM) and/or traction retinal detachment(TRD). Methods Clinical records were reviewed on 37 consecutive patients(38 eyes)who underwent vitreous surgery and endolaser for RVO with persistent vitreous hemorrhage,NVM and/or TRD.There were 19 patients(20 eyes)with retinal branch vein occlusion (BRVO)and 18 patients(18 eyes)with central retinal vein occlusion(CRVO). Results NVM and TRD were confirmed during operation in 27 and 23 eyes,respectively.Visual acuity improved postoperatively in 34 eyes(89.5%)including 22 eyes with 0.1 or better vision,and 4 eyes remained unchanged.CRVO group had longer history and less visual improvement after surgery. Conclusions Vitreous surgery and endolaser photocoagulation can improve the outcome in the majority of patients with RVO with vitreous hemorrage,NVM and/or TRD. (Chin J Ocul Fundus Dis,1998,14:3-6)
ObjectiveTo explore an optimized protocol of decellularization to fabricate an ideal scaffold derived from porcine skeletal muscle acellular matrix. MethodsSerial-step protocol of homogenating-milling-detergent method was used to fabricate decellularized porcine muscle tissue (DPMT) derived from native porcine skeletal muscle tissue from adult pig waist. Histological method was used to assess the effects of decellularization and degreasing. Sirius red staining was used to analyze collagen components. Scanning electron microscopy, BCA assay, and PicoGreen assay were used to evaluate the ultrastructure, total protein content, and DNA content in DPMT. The adipose derived stem cells (ADSCs), NIH3T3 cells, and human umbilical vein endothelial cells (HUVECs) were cultured in extraction liquor of DPMT in different concentrations for 1, 3, and 5 days, then the relative growth rate was calculated with cell counting kit 8 to assess the toxicity in vitro. Live/dead cell staining was used to evaluate the cytocompatibility by seeding HUVECs on the surface of DPMT and co-cultured in vitro for 3 days. For in vivo test, DPMT was subcutaneously implanted at dorsal site of male specific-pathogen free Sprague Dawley rats and harvested after 3, 7, 14, and 28 days. Gross obersvation was done and transverse diameter of remained DPMT in vivo was determined. HE staining and immunohistochemical staining of CD31 were used to assess inflammatory response and new capillary rings formation. ResultsDecellularization of the porcine skeletal muscle tissue by homogenating-milling-detergent serial steps protocol was effective, time-saving, and simple, which could be finished within only 1 day. The decellularizarion and degreasing effect of DPMT was complete. The main component of DPMT was collagen type I and type IV. The DNA content in DPMT was (15.902±1.392) ng/mg dry weight, the total protein content was 68.94% of DPMT dry weight, which was significantly less than those of fresh skeletal muscle tissue[(140.727±10.422) ng/mg and 93.14%] (P<0.05). The microstructure of DPMT was homogeneous and porous. The result of cytocompatibility revealed that the cytotoxicity of DPMT was 0-1 grade, and HUVECs could stably grow on DPMT. In vivo study revealed DPMT could almost maintain its structural integrity at 14 days and it degraded completely at 28 days after implantation. The inflammatory response peaked at 3 days after implantation, and reduced obviously at 7 days. Difference was significant in the number of inflammatory cells between 2 time points (P<0.05). Neovascularization was observed at 7 days after implantation and the number of new vessels increased at 14 days, showing significant difference between at 7 and 14 days (P<0.05). ConclusionThe homogenating-milling-detergent serial-steps protocol is effective, time-saving, and reproducible. The DPMT reveals to be cell and lipid free, with highly preserved protein component. DPMT has good biocompatibility both in vitro and in vivo and may also have potential in promoting neovascularization.