| Background and purpose Proliferative vitreoretinopathy (PVR) is a common sequel to rhegmatogenous retinal detachment (RRD) and is the leading cause of failed surgery repair. It is well known that retinal pigment epithelial (RPE) cells are predominant proliferative cells in PVR which is an excessively wound healing response occurring after RPE cell wounding. The activated RPE cells migrate from their normal, sessile state to the vitreous and onto both surfaces of the retina, in which they begin to dedifferentiate, migrate, proliferate, change phenotype and secrete extracellular matrice (ECM). Multiple proteins and growth factors are involved in this process. But it is not completely clear that the mechanism of modulation on RPE cells growth in the pathogenesis of PVR, which delays the development of prevention and treatment of PVR.Endothelin(ET), 2.5D in molecular weight, was originally identified in conditioned medium from umbilical vein endothelial cells, the mature form is 21 amino acids in length, there is a constractive effect on blood vessels. In succedent study, not only was it a constractive agent, but also a multifunctional regulatory peptide, which has extensive biological effects. It mediates inflammation, stimulates the secretion of other hormones, facilitates caryomitosis, stimulates cell proliferation, cell migration and cell adhesion by autocrine andparacrine through its recepters. The cytokines including TGF- β , IL-1, TNF- α , IFN- γ and thrombin can induce both transcription of the prepro forms of ET as well as release of mature ET-1. In contrast, natriuretic factors including atrial natriuretic peptide (ANP) and brain derived natriuretic peptide (BNP) along with nitric oxide donors have been shown to inhibit secretion of ET-1 from endothelial cells. Released endothelin acts on the ET receptors that exist mainly as ETA and ETB receptor subtypes. The second messenger systems associated with ocular ET-1 includ intracellular calcium, prostaglandins, cyclic AMP, nitric oxide, and cyclic GMP.The biological behaviors of ET-1 are of many consistency with RPE cells changes in PVR pathological process, the proliferation, adhesion and migration of RPE cells are three important steps in PVR, while ET-1 facilitates vascular smoothe muscle cells proliferation and migration, mononuclear cell and platelet adhesion, and in ocular tissues, ET-1 can stimulate optic nerve head astrocytes, corneal epithelium, corneal endothelium and retinal pericytes proliferation, however, it has not been reported in the studies related to PVR.The purposes of our studies are to investigate the expression of ET-1 in clinical proliferative retinal membrane and the content of vitreous body fluid of RRD with PVR, and detect the time of expression of ET-1, the relationship with outer blood retinal barrier and the effect of DEX on the expression of ET-1 through the animal model of the retinal detachment, and research its effect on the RPE cells adhesion, proliferation and migration, and probe its expression in the RPE cells after traction, its auto-regulation, and its effect on [Ca2+]i through RPE cells culture in vitro in order to investigate its effect on PVR and probe a new way for prevention and treatment of PVR. Methods (1) Twenty vitreous samples and 24 periretinal membranes(PCM) collected from RRD patients with PVR were determined the expression and content of ET-1 by radioimmunoassay, immunohistochemistry and situ hybridization, 4 normal retinae obtained from donor eyes were detected the expression of ET-1 by immunohistochemistry, immunofluorescence and in situ hybridization. (2) To establish an animal model of retinal detachment ( RD ) and identify the proliferation of RPE cells and the tissue origin of the proliferative cells by immunohisto- chemistry of proliferating cell nuclear antigen(PCNA) and cytokeratin (CK) and detect the expression and content of ET-1 in the detached retina and the vitreous body fluid by radioimmunoassay, immunohistochemistry and in situ hybridization at intervals ranging froml/2hr to 14 days, and observe the effect of DEX on the expression of ET-1 by immunohistochemistry, in situ hybridization and radioimmunoassay at intervals of l/2hr to 6hr. (3) To investigate the adherent effects of ET-1 on the hRPE cells by attachment test of cells, and quantify the content of 6-Keto-PGFal, the metabolic product of PGI2, by radioimmunoassay in the hRPE cell conditioned medium; and estimate the hRPE cell proliferation after treated by ET-1 by MTT and immunohistochemistry of PCNA; and study the hRPE cell migration after treated by ET-1 by the migratory test and immunohistochemistry of a-SMA. (4) The hRPE cells attached to collagen-coated magnetic beads were tractioned under a fixed magnetic field and detected ET-1 expression in RPE cells and ET-1 content in the cell-conditioned medium by radioimmunoassay, in situ hybridization and immunohistochemistry. The hRPE cells stimulated by ET-1 were detected the expression of ET-1 and nitric oxide synthase 1 (NOS1) by immunocytochemistry, in situ hybridizition, and the secreted ET-1 and 6-Keto-PGFal estimated by radioimmunoassay in the cell-conditioned medium. And the fluo-3/- AM loaded RPE cellstreated by ET-1, DEX and genistein were observed the cytoplasmic free Ca2+ concentration ([Ca2+]i) response using a laser scanning confocal microscope(LSCM).Results (1) CD The ET-1 was not detectable from the vitreous samples in all of the normal controls, and 3 of 10 samples of PVR grade C and D . The level of ET-1 in the vitreous body of PVR had significant differences compared to normal control (F=345.98. p<0.01). ?Twenty four PRMs obtained from PVR patients by vitrectomy, including 10 cases of PVR/ C3 and 14 cases of PVR/D, all expressed ET-1 except for one PRM of PVR/D. In the various grades of the PRMs, the cells in the center of PRM showed weaker expression of ET-1 than those at the rim of PRM, the center of PRM was distributed more cells less matrix than the rim of PRM in which the cells shaped fusiform and slimmer and showed its parallel with surface of PRM.? In the human normal retina, ET-1 mainly expressed in retinal and choroidal vascular endothelial cells, retinal ganglial cells, external granular layer and glial cell, and weakly expressed in hRPE cells. (2) (D The retinal detachment showed in all 32eyes of 16 experimental rabbits, including 2 eyes with vitreous heamorrhage, one eye with injury of lens posterior capsule. The detached area was over a half of the retina. The detached retina remained for 3 to 14days. (2) there were some gathered cells in the RPE cell layer in 3 days group, some hypertrophy like macrophages in 7 days group and and poly-layer RPE cells in 14 days group of spontaneous reattachment occurring. The macrophage-like cells were originated from RPE in microscope and stained cytokeratin positive, and RPE cells began to stain PCNA positive at 3 days in the model. (3) The level of ET-1 in vitreous body were detectable from 3hr and gradually increased at 6hr ,24hr and 3d. ET-1-like immunoreactivity appeared distinctlylocated at RPE cells at 24hour-old retinal detachment. The ET-1 mRNA expression were mainly situated in the nucleous of RPE cells at 3hr, and apparently in cytoplasma at 24hr. (4) The ERK was phosphorylated and entered into the cell nucleus of RPE at l/2hr-old retinal detachment in the simple model groups but not in the model and DEX groups by intravitreal injection of lmg DEX. The concentration of ET-1 were detectable at 3hr and 6hr in the simple model groups and not in the model and DEX groups in vitreous body. The ET-1 mRNA expression were mainly situated in the nucleous of RPE cells and other cells of neural retina at 3hr and 6hr in the simple model groups and did not in the model and DEX groups. (3) (D The RPE cell adhesion and the contents of 6-Keto-PGFal in RPE cell conditioned medium increased in a dose-dependent manner after stimulated by five ET-1 doses from 10(-ll) to 10(-7)M (F=9.547, p<0.01, F=:33.929, p<0.01 respectively ), there were a positive linear correlation between the RPE cell adhesion and the content of 6-Keto-PGFal (r=0.770,p<0.01) (2)10 (-11) -10 (-9) MET-1 stimulated the RPE cell proliferation in a dose-dependent manner (F=4.400, p<0.05), the stimulated rates were 6.53%,13.54%,35.93%, respectively. The PCNA expression showed a aparently increase in the RPE cells treated by the doses of ET-1 of 10 (-10) and10 (-8) M. (3)10 (-11) -10 (-9) MET-1 stimulated RPE cell migration in a dose-dependent manner (F-4.153, p<0.05), the stimulated rates were 18.45%, 30.55%, 59.25%, respectively. DXM notably inhibited the 10(-9)M ET-1-induced migration of RPE cells in a dose-dependent manner (F=7.838, p<0.01), the inhibitory rate of 2, 20, 200ug/mL three doses of DEX were 11.70%,14.42%, 33.01%, respectively. The expression of a-SMA in hRPE cells showed obviously enhanced withthe increase of concentration of ET-1. (4) (1) The shape of RPE cells tractioned by magnetic field changed with the prolonging time of traction, RPE cells obviously expressed ET-1 at 24hr after traction.The content of ET-1 in the cell-conditioned medium also showed rising notably at 24hr and 48hr after 10min,30min and 2hr megnitic traction (p<0.05 ) .(2) The expression of NOS1 and ET-1 gradually accentuated in hRPE cells with treated by 10(-11), 10(-9), 10(-7) M ET-1. There was a dose-dependent increase of the contents of ET-1 and 6-Keto-PGFal in the cell-conditioned medium in response to 10(-l 1), 10(-9) , 10(-7) M ET-1 (F=23.67, p<0.01; F=44.431, p<0.01, respectively), and a positive linear correlation between the contents of ET-land 6-Keto-PGFal (r=0.85, p<0.01). (3) The RPE cells induced a significant increase in [Ca2+]i under exposure to 10(-12)~10(-7)M ET-1 solved by DMEM (F=8.228, p<0.01), did not in D-Hank solved ET-1(F=1.843, p>0.05). DEX and genistein suppressed the 10(-9)M ET-1-induced [Ca2+]i elevation in RPE cells ( F=9.945, p<0.01, F=l 7.864, p<0.01, respectively).Conclusions (1 )ET-1 exists in vitreous body fluid and the cells of the PCM of RRD with PVR, it hints that ET-1 is involved in the formation PVR. (2)The animal model of RRD is convenient and available. In this model, the RPE cells showed proliferation and the macrophage-like cells were originated from the RPE cells. The expression of ET-1 in the detached retina and assaying of ET-1 in vitreous body after RD indicate that origin of ET-1 contains two pathways by which one is blood due to the destruction of the blood retinal barrier (BRB), the other is its own release. The study of intravitreal injection of DEX after retina being detached indicats that lmg DEX not only inhibits the ET-1 entrance into sub- retinal fluid from blood, but its expression in...
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