| PurposeThe retinal pigment epithelial (RPE) cell is a main element of some inflammatory and non-inflammatory ocular diseases, including uveitis, rhegmatogenous retinal detachment (RRD), proliferative vitreoretinopathy (PVR) and age-related macular degeneration (AMD)[1, 2]. Adhesion and migration of RPE cells play key roles in those above diseases. It is known that cells can migrate, proliferate and play roles only when they are attached to other cells and to extracellular matrix (ECM). In the process of ocular diseases mentioned above, RPE cells were exposed to pathological microenvironment which was composed of some cytokines, such as tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ)[3], monocytes/macrophages, vitreous and shear stress, and these stimulants are crucial in the behavior and function alterations of RPE cells.Adhesion is of importance in many physical processes, such as morphology modulation, immunological response and wound repair[2]. Adhesion connection is based on the formation of transmembrane adhesion proteins of the cytoskeleton[3]. Our previous study showed that syndecan-1, a transmembrane adhesion molecule, participates in the pathophysical process of RPE cells[5], and may play roles in the occurrence and development of PVR[6].The early gene changes are crucial in the subsequent structure and function alterations after extracellular environments were changed. Early growth response gene-1 (Egr-1) is a member of the family of immediate-early genes, which regulates a wide range of cellular processes, including cell growth, differentiation and apoptosis, chemotaxis of inflammatory cells and immune irritation, etc.This study aimed :①to investigate the levels of soluble syndecan-1 in the subretinal fluid (SRF) and the vitreous from eyes with RRD, and the correlation between its levels with clinical parameters of patients with RRD.②to study the effects of stimulants, e.g., TNF-α, lipopolysaccharide (LPS), IFN-γ, supernatant of monocytes/macrophages (THP-1 cells), the vitreous and shear stress, on the expression of syndecan-1 and Egr-1 in cultured human RPE cells. The results may provide us with the roles of syndecan-1 and Egr-1 in RPE cells and their related ocular diseases, and new ideas for their prevention and treatment.Methods1. With ELISA, soluble syndecan-1 levels were detected in the SRF and the vitreous fluid from patients with RRD, and potential correlations between the syndecan-1 levels with clinical parameters [age, gender, left or right eye, status of vision refraction, extent of the detachment (quadrants), duration of retinal detachment, whether or not the RRD was complicated with PVR] were tested by multiple linear regression model.2.(1) Cytokine-activated RPE cells were obtained by incubation of the cells with D-F12 dissolved with final concentrations of 40 ng/ml TNF-α, 20μg/ml LPS, 10 U/ml IFN-γ, 30% supernatant of THP-1 cells or 50% vitreous for 0, 10 h, 24 h and 48 h, respectively. Immunofluorescence staining and western blot were used to test the protein expression of syndecan-1, RT-PCR was used to detect the syndecan-1 mRNA, and ELISA was to detect the level of soluble syndecan-1 in the supernatant of RPE cells.(2) RPE cells were exposed to shear stress of 2 dyns/cm2 for 0, 15 min, 30 min, 45 min, 1 h and 3 h. With immunofluorescence staining, the changes of cytoskeleton actin were observed. Immunofluorescence staining and western blot were used to detect protein expression of syndecan-1, and RT-PCR was used to detect the syndecan-1 mRNA.3.(1) RPE cells were incubated with 40 ng/ml TNF-α, 20μg/ml LPS, 10 U/ml IFN-γ, 30% supernatant of THP-1 cells or 50% vitreous for 0, 10 min, 20 min, 30 min, 40 min and 60 min, respectively. Protein expression of Egr-1 were detected with immunofluorescence staining and western blot. By RT-PCR, Egr-1 mRNA was detected.(2) After RPE cells were exposed to shear stress for 0, 15 min, 30 min, 45 min, 1 h and 3 h, western blot and RT-PCR were used to observe the protein and mRNA expression of Egr-1, respectively. Results1. The levels of soluble syndecan-1 in the controls, the SRF and the vitreous fluid were 0.2236±0.0951 ng/ml,1.4989±0.1836 ng/ml and 2.5770±0.5777 ng/ml, respectively. Compared with the controls, RRD was associated with a 6-fold increase in the SRF and a 11-fold enhancement in the vitreous fluid of soluble syndecan-1 concentration. Compared with the controls, differences of syndecan-1 levels in the SRF and the vitreous fluid were significant (P=0.006, P=0.000). Difference of soluble syndecan-1 levels between the SRF and the vitreous was also significant (P=0.015). An increase in the soluble syndecan-1 concentrations in SRF samples correlated with a longer duration of retinal detachment (P<0.0001, r = 0.737).2.(1) In unstimulated RPE cells, strong syndecan-1 positive green fluorescence was localized in the cell membrane and cytoplasm. With exposure to TNF-α, LPS, IFN-γ, supernatant of THP-1 cells and vitreous, weaker staining with slight green fluorescence was detected. By western blot and RT-PCR, it showed that TNF-α, LPS, IFN-γ, supernatant of THP-1 cells and the vitreous down-regulated syndecan-1 protein and mRNA on RPE cells, and increased the shedding of soluble syndecan-1 ectodomain to the supernatant.(2) After treated with shear stress, morphology of RPE cells changed, such as paracellular spaces widened, cells crippled and detached, cytoskeleton actin increased, thickened, and accorded with the force direction. And shear stress down-regulated syndecan-1 expression.3.(1) RPE cells at 0-time point revealed faint labelling of Egr-1 in the cytoplasm. With exposure to stimulants, Egr-1 protein level in the cytoplasm increased obviously above the 0-time point within 1 h, and it parallelled with Egr-1 located in some nuclei of RPE cells. The results of western blot and RT-PCR showed that TNF-α, LPS, IFN-γ, supernatant of THP-1 cells and the vitreous enhanced Egr-1 protein and mRNA on RPE cells quickly within 1 h.(2) When treated with shear stress of 2 dyns/cm2, Egr-1 mRNA of RPE cells increased fast within a short period and declined to the baseline at 3 h.Conclusion1. Compared with the low level of soluble syndecan-1 in the vitreous fluid from normal eyes, RRD was associated with a significant increase in the SRF and the vitreous. Moreover, in SRF, an enhanced soluble syndecan-1 level correlated positively with the duration of RD. These suggest that the high levels of soluble syndecan-1 in the SRF and the vitreous fluid is a reflection of the duration of wounding and inflammation in RRD.2. Common stimulants in the microenvironment, TNF-α, LPS, IFN-γ, supernatant of monocytes/macrophages (THP-1 cells), vitreous and shear stress, could down-regulate syndecan-1 on cultured human RPE cells, and increase the shedding of soluble syndecan-1 ectodomain to the supernatant. This result was in accordance with the results that syndecan-1 decreased or absence in proliferative membranes of PVR[6] and soluble syndecan-1 enhanced in SRF and the vitreous from patients suffered from RRD. These indicates that syndecan-1 decrease or absence can decline cell adhesion and increase paracellular permeability, and thus alter cell structure and function. After treated with common stimulants, the immediate-early gene of Egr-1 was activated shortly and maintained for a short time. Activation of Egr-1 may participate in regulation of cell differention, proliferation and apoptosis.3. After exposed to shear stress, morphology of RPE cells changed, syndecan-1 was down-regulated, and Egr-1 increased shortly and declined to the baseline in a short time. These changes will alter adhesion, morphology and function of RPE cells, and enhance paracellular permeability. Regulations of Egr-1 in the cell transcription will affect cell organization and function, and play roles in the pathophysical process.
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