| BackgroundDiabetic retinopathy is one of the most important microvascular complications in diabetes and is a leading cause of blindness among the people of occupational age. The early diabetic retinopathy is characterized by loss of pericytes and increased vascular permeability, followed by progress microvascular occlusion in the retina. However, the precise mechanism leading to retinal vasculature and tissue damage in diabetic retinopathy has not been fully elucidated.Inner blood retinal barrier (iBRB) is composed of three elementary structures: endothelial cells, basement membrane tubes, and intramural pericytes, which are located within the basement membrane. The integrity of iBRB is determined by a water-tight apical junctional complex that is composed of tight junctions and adherent junctions. Another important determinant for the maintenance of barrier function is the actin cytoskeleton.The adhesion junction of cell-cell and cell-ECM (extracellular matrix) is mainly mediated by cadherins and integrins which undergo a conformational change upon activation to recruit structural and signaling molecules. The cadherin which mediates homophilic calcium-dependent cell–cell-adhesion is mainly VE-cadherin and N-cadherin in the retina, forming focal adhesion. However, N-cadherin is also a critical mediator of cell-cell adhesion between EC and pericyte, forming special adherent junction with heterologous peg-and-socket contacts. N-cadherin is connected to actin cystoskeleton byβ-catenin. Dynamic cell (including EC and PC)-ECM interactions is medicated by integrins.Integrin-mediated cell adhesion regulates a wide variety of biological processes, including the formation of focal adhesions, cell migration, differentiation, survival, proliferation and transmembrane signal transduction via signaling molecules recruited to focal adhesions. One protein that plays a central role in integrin activation and signal transduction is integrin-linked kinase (ILK). ILK is an ankyrin-repeat containing serine/threonine protein kinase that interacts with the cytoplasmic domain ofβ1 andβ3 integrins. It regulates integrin-dependent functions as a crucial regulator of cell responding to signaling mediated by integrins and growth factor. ILK is involved in the formation of cystoskeleton, the regulation of cell adhesion, growth, gene expression, differentiation, cell shape change, and ECM assembly. It links integrins to actin cystoskeleton, transduce signal from integrin to ECM and various subcellular components, and from extracellular environment to intracellular signal. ILK also regulates activity of some signaling intermediate including Akt/FOXO, GSK-3β, ERK,β-catenin and cadherin.However, so far no biological function of ILK and its related signaling molecule in diabetic retinopathy has been demonstrated. The purpose of this study was to investigate the expression and activity of ILK, N-cadherin,β-catenin and Akt/FOXO1A in the retina of streptozotocin (STZ)-induced diabetic rats and in the cultured bovine retina pericytes under the condition of high glucose and to gain insight into their roles in the progression of diabetic retinopathy, and to elucidate the possible regulation of ILK in the diabetic retinopathy associated with diabetes mellitus, and to further explore the clues for prevention of diabetic retinopathy and new pathways for treatment of diabetic retinopathy.Objective1. To investigate whether ILK is involved in the pathogenesis of diabetic retinopathy by analyzing the expression and the activity of ILK in the retina and retinal microvasculature from a STZ-induced rat model of diabetes.2. To investigate the possible roles of N-cadherin,β-catenin and FOXO1A in the progression of diabetic retinopathy, by examining the expression of N-cadherin,β-catenin and FOXO1A in the retina and retinal microvasculature from a STZ-induced diabetic rat.3. To investigate whether ILK and FOXO1A are involved in apoptosis of pericytes induced by high glucose, by analyzing the expression of ILK and FOXO1A in the pericytes of bovine retina cultured under the condition of high glucose.MethodsThe model of STZ-induced diabetic rat was set up and the microvascular pericytes of bovine retina was cultured. The vascular permeability in the retina from both the diabetic rats at 4, 8 and 12 weeks after induction of diabetes and control rats was quantified by measuring albumin leakage from blood vessels into the retina using the Evans blue method. The levels of ILK mRNA in the retina at 4, 8 and 12 weeks after induction of diabetes were examined by reverse transcription polymerase chain reaction (RT-PCR). The immunoreactivities of ILK, N-cadherin,β-catenin and FOXO1A in the retina and retinal vasculature at 12 weeks after induction of diabetes were examined by immunohistochemistry (IHC). The expression of ILK, N-cadherin,β-catenin and FOXO1A protein levels in the retina at 4, 8 and 12 weeks after induction of diabetes was detected by Western blot. The expressions of ILK and FOXO1A protein levels in the pericytes cultured in 30 mmol/L glucose for 24 h, 48 h and 72 h and in 5 mmol/L glucose for 72 h were examined by using Western blot, respectively. The immunoreactivities of ILK and FOXO1A in the pericytes cultured in 30 mmol/L glucose and in 5 mmol/L glucose for 72 h were also examined by immunocytochemistry. The apoptosis of pericytes cultured in 30 mmol/L glucose for 24 h, 48 h and 72 h was detected by the Annexin V-FITC Apoptosis Detection Kit.Results1. In the STZ-diabetic rats, body weight was significantly lighter than that of the age-matched controls. After 12 weeks, the body weight of the diabetic rats (236±14.1 g) was significantly different (P=0.000) from the controls (body weight 496±10.2 g). Meanwhile, there was significant increase in blood glucose levels in the diabetic rats compared to the controls. After 12 weeks, the blood glucose level of the diabetic rats was up to 30.2±4.8 mmol/L, which was significantly different (P=0.000) from the controls (blood glucose levels 4.9±0.12 mmol/L).2. The vascular permeability was increased by 68%, 91% and 125%, respectively (each P <0.05), in the retinas from 4-week, 8-week and 12-week diabetic rats after induction of diabetes compared with that from the controls.3. The results of semiquantitative RT-PCR analysis showed that the levels of ILK mRNA expression was increased by 12.2%, 35.8% and 45.6%, respectively (each P <0.05), in the retinas from 4-week, 8-week and 12-week diabetic rats after induction of diabetes compared with that from the controls.4. Immunohistochemical analysis showed that the immunostaining of ILK occurred in the retinal vasculature including the endothelial cells and pericytes and in the outer plexiform layer (OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL), and the ganglion cell layer (GCL) of retinas from both the 12-week diabetic rats and the controls. However, ILK immunoreactivity was significantly increased in the retinas and retinal vasculature at 12 weeks after induction of diabetes compared with that in the controls. Meanwhile the results also showed that the FOXO1A immunostaining occurred in the retinal vasculature and OPL, INL, IPL and GCL from both the 12-week diabetic rats and the controls, but its immunoreactivity was significantly increased in the retinas at 12 weeks after induction of diabetes.Theβ-catenin immunostaining occurred in the retinal vasculature and the PL, OPL, IPL, GCL and ILM from both the 12-week diabetic and control rats, but its immunoreactivity was significantly increased in the retinas at 12 weeks after induction of diabetes. However, the N-cadherin immunostaining occurred in the retinal vasculature and OPL, INL, IPL, GCL and ILM of retinas from both the 12-week diabetic and control rats, but its immunoreactivity was significantly decreased in the retinas at 12 weeks after induction of diabetes..5. Immunobloting analyses showed that the ILK protein levels were increased by 35.6%, 77.6% and 89.8%, andβ-catenin protein levels by 13.2%, 15.8% and 35.1%, respectively (each P <0.05), in the retinas at 4, 8 and 12 weeks after induction of diabetes compared with that in the controls. However, N-cadherin protein levels were decreased by 15.4%, 28.4% and 37.2%, respectively (each P <0.05), in the retinas of diabetes compared with that from the controls at the same observation times. The Akt protein levels were reduced by 15.2% and 22.1%, (both P <0.05) in the retinas at 8 and 12 weeks after induction of diabetes compared with that in the controls. However, the Akt protein levels were reduced by 15.2% and 22.1%, respectively (both P<0.05), in the retinas at 8 and 12 weeks after induction of diabetes compared with that in the controls, with a molecular weight of approximately 65 kDa. The Akt protein levels were increased by 20.6% (P<0.05) in the retinas at 4 weeks after induction of diabetes compared with that in the controls. The Akt activity was significantly reduced in diabetic retinas. Two phosphorylation Akt (serine-473 and threonine-308) protein levels were reduced by 38.5% and 65.4%, 8.8% and 63.4%, respectively (each P<0.05), in the retinas at 8 and 12 weeks, respectively (both P<0.05), and increased by 36.6% and 64.8%, respectively (both P<0.05), in the retinas at 4 weeks after induction of diabetes compared with that in the controls. Moreover, the FOXO1A protein levels were increased by 25.6% and 44.8%, respectively (both P<0.05), in the retinas at 8 and 12 weeks after induction of diabetes compared with that from the controls. Meanwhile the phospho-FOXO1A protein levels were decreased by 5.8% and 25.8%, respectively (both P<0.05), in the retinas of diabetes compared with that from the controls at the same observation times. Interestingly, like the Akt activity, the FOXO1A protein levels were decreased by 26.3% (P<0.05) while the phospho-FOXO1A were increased by 55.8% (P<0.05) in the retina from 4-week diabetic rats compared with that in the controls.6. Immuocytochemisry analysis showed that ILK immunoreactivity was not detected in the retinal microvascular pericytes cultured in low glucose for 72 h, but was significantly increased in the retinal microvascular pericytes cultured in high glucose and H2O2 for 72 h and mainly distributed in the cytoplasm of pericytes. Similarly, there was weak immunoreactivity of the FOXO1A in the retinal microvascular pericytes cultured in low glucose for 72 h, but it was significantly increased in the retinal microvascular pericytes cultured in high glucose and H2O2 for 72 h and was mainly distributed in the cytoplasm and nucleus of pericytes.7. Flow cytometry showed that the apoptosis of pericyte cultured in 30 mmol/L glucose was increased by 47.6%,58.4% and 68.7%, respectively (each P <0.05), in time-dependent manner comparing with that in 5 mmol/L glucose for 24 h, 48 h and 72 h.8. Western blot analysis showed that the ILK protein levels were increased by 27.2%,48.6% and 54.5%, respectively (each P <0.05), in the pericytes cultured in 30 mmol/L glucose for 24 h, 48 h and 72 h compared with that in 5 mmol/L glucose. Moreover, the expression levels of FOXO1A protein were increased by 45% and 51%, respectively (both P <0.05), but the expression levels of phospho-FOXO1A protein were decreased by 44% and 63%, respectively (both P <0.05), in the pericytes cultured in 30 mmol/L glucose for 48 h and 72 h. Meanwhile, the expression levels of FOXO1A and phospho-FOXO1A protein were reduced by 17% and increased by 40%, respectively (P <0.05), in 30 mmol/L glucose for 24 h compared with that in 5 mmol/L glucose glucose.Conclusions1. The present results demonstrated for the first time that the up-regulation of ILK mRNA and protein expression occurred in the retinas and retinal microvasculatures from the STZ-induced diabetic rats. This may be involved in the progression and the damage of neural and microvascular structures at the earlier stage of diabetic retinopathy.2. The results firstly demonstrated that the up-regulation ofβ-catenin and FOXO1A expressions and down-regulation of N-cadherin occurred in the retinas and retinal microvasculatures from STZ-induced diabetic rats. It suggests that the alterations of N-cadherin,β-catenin and FOXO1A expression may be affected by hyperglycaemia in the retinas and retinal microvasculatures from the STZ-induced diabetic rats, and may contribute to the early diabetic retinopathy.3. The results demonstrated for the first time that the expressions of the ILK and FOXO1A were increased in the pericytes cultured in 30 mmol/L glucose. It indicates that the up-regulation of ILK and FOXO1A expressions may be associated with the apoptosis of pericytes. |
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