TWEAK Associated With Proliferative Diabetic Retinopathy And Promotes Proliferation And Collagen Synthesis In Retinal Cells

Investigate goalThe goal of the current study was to determine the expression of the TWEAK/Fn14 pathway in PDR,and to confirm the correlation between TWEAK expression and clinicopathological characteristics of PDR.To investigate the regulatory roles of the TWEAK/Fn14 pathway in cell proliferation and collagen synthesis in retinal ARPE-19 cells.INTRODUCTIONDiabetic retinopathy(DR)is ranked as the most severe ocular complication of diabetes mellitus(DM)and remains a major cause of vision loss in many countries.Chronic inflammation develops in the retinal microvasculature under sustained hyperglycemiaand is implicated in the pathogenesis of diabetic retinopathy.Proliferative DR(PDR)is followed by recurrent vitreous hemorrhages,traction retinal detachment,retinal and vitreous fibrosis,and optic nerve atrophy,which finally lead to severe visual impairments.Retinal neovascularization is the most striking characteristic of PDR and involves various angiogenic factorssuch as cytokines,inflammatory cells,and growth factors.Previous studies have also emphasized the role of vascular endothelial growth factor and other factors with angiogenic properties in the pathogenesis of PDR.It is known that disturbances or functional imbalance of the immune system as well as activation of vascular proliferation contribute to the pathogenesis of PDR.However,the interactionsbetween the pro/anti-inflammatory cytokines and angiogenic factors in PDR remain to be explored.Several members of the tumor necrosis factor(TNF)superfamily have recently been found to regulate inflammation and neovascularization.In particular,TNF-like weak inducer of apoptosis(TWEAK)activates the nuclear factor-?B signal transduction pathway by binding to fibroblast growth factor-inducible molecule 14(Fn14),a member of the TNF receptor superfamily,and exerts critical control of the inflammatory response and proangiogenic reactions.In addition,TWEAK has been reported to promote the levels of pro-inflammatory molecules such as matrix metalloproteinase-9,intercellular adhesion molecule-1,and interleukin,all of which are involved in the pathogenesis of PDR.Furthermore,considerable evidence suggested a proangiogenic role for TWEAK.Therefore,we speculate that the TWEAK/Fn14 pathway may modulate PDR.The goal of the current study was to determine the expression of the TWEAK/Fn14 pathway in PDR,and to confirm the correlation between TWEAK expression and clinicopathological characteristics of PDR.We also wanted to investigate the regulatory roles of the TWEAK/Fn14 pathway in cell proliferation and collagen synthesis in retinal ARPE-19 cells.MATERIAL and METHODSVitreous samplesVitreous fluid samples were obtainedfrom 16 PDR patients and 21 type 2 diabetic(T2D)patients who had undergone vitrectomy.ELISA analysis of TWEAK and Fn14 in vitreous samplesELISA analysis of TWEAK and Fn14 in vitreous samples of two groups.Western blot analysis of TWEAK and Fn14 in vitreous samplesThe levels of TWEAK and Fn14 in vitreous samples of two groups have been presented as a percent gray value normalized to GAPDH.Acquisition of target genesObtained total DNA from 293 T cells,TWEAK coding sequence was amplified by PCR.ARPE-19 cell culture,and TWEAK overexpressionARPE-19 cells were purchased from the American Type Culture Collection and were cultured.Cells at greater than 90%confluence were sub-cultured at a ratio of 1:3.To overexpress TWEAK in ARPE-19 cells,the human TWEAK coding sequence was amplified and cloned into a pc DNA3.1(+)vector.Enhanced green fluorescence protein(EGFP)coding sequence was also cloned into the vector as a control pc DNA3.1(+).TWEAK-pc DNA3.1(+)or control pc DNA3.1(+)plasmids were transfected into ARPE-19 cells.Cells with successful integration of the target genes TWEAK or EGFP were selected by G418,and contained.RNA extraction and reverse transcription polymerase chain reactionReverse transcription PCR(RT-PCR)was performed following the manufacturer protocol,with GAPDH as an internal control.Western blot analysis of TWEAK in overexpression cells.Protein bands were visualized via electrochemiluminescence.The levels of TWEAK in different stages of cells passage have been presented as a percent gray value normalized to GAPDH.Cell count assay,MTT assay,and colony formation assayGrowth curves of ARPE-19(TWEAK+)and ARPE-19(control)cells were determined via cell count assays.Cellular viability was assayed using the MTT method.Colony formation assay was used for cells proliferation.Western blot analysis of ?-SMA,collagen I and collagen IV in overexpression cells.The levels of ?-SMA,collagen I and collagen IV in different stages of cells passage have been analysised by Westernblot.Statistical analysisStatistical analyses were performed using Graph Pad Prism.Quantitative results were presented as mean ± standard error of the mean(SEM).Comparisons between the two groups were carried out using the Student t-test.Comparisons of several groups were carried out using ANOVA.Statistical significance was reached when P <0.05.RESULTSLevel of TWEAK and Fn14 in vitreous samplesIn the present study,21 patients with T2DM(without PDR)and 16 patients with PDR were recruited.In order to determine a possible role of TWEAK in PDR,we examined the vitreous level of TWEAK in each group of patients by using ELISA.The TWEAK level in control T2 DM patients was 1.64 ± 0.56 ng/m L(N=21),whereas in the PDR patients,it was 2.96± 1.14 ng/m L(N=16),which wassignificant higher(P < 0.001).The level of the receptor for TWEAK,Fn14,was also significantly higher in the PDR group as compared to the control group(5.37 ± 1.64 vs 3.85 ± 0.78 ng/m L,P < 0.01).We further examined protein expression of these two molecules via western blot assays.There was also a significant difference in the levels of TWEAK and Fn14 of the PDR and control T2 DM groups(P < 0.001).Therefore,it is possible that TWEAK/Fn14 signaling is upregulated in vitreous fluids of PDR patients.Construction of a TWEAK-overexpressing ARPE-19 cell lineWe obtained total DNA from 293 T cells,and TWEAK coding sequence was amplified by PCR.We overexpressed TWEAK in retinal ARPE-19 cellsin order to determine the role of TWEAK in ARPE-19 cell proliferation.The TWEAK-coding sequence was cloned into the pc DNA3.1(+)vector,and ARPE-19 cells were transfected with the recombinant TWEAK-pc DNA3.1(+)plasmid.Successful transfections were selected under the using the antibiotic G418.Expression of TWEAK was significantly elevated in ARPE-19(TWEAK+)cells as compared to that in ARPE-19(control)cells(P < 0.001).This effect persisted despite further serial cell passages.TWEAK overexpression promotes cell proliferation and collagen synthesis of retinal cellsTo investigate the regulatory role of TWEAK on the proliferation of ARPE-19 cells,we curved the growth of ARPE-19(TWEAK+)and ARPE-19(control)cells.ARPE-19(TWEAK+)experienced faster growth as compared to ARPE-19(control)cells at 3 and 5 days post inoculation(P <0.01).We also examined the viability of ARPE-19(TWEAK+)and ARPE-19(control)cells in media supplemented with 2% FBS.ARPE-19(TWEAK+)cells were more viable than ARPE-19(control)cells at both 24 h and 48 h post inoculation(P <0.05).To further evaluate the growth difference between the two types of cells,we performed colony-forming assays on these cells.We found that regardless of the number of initially inoculated cells,significantly more colonies were formed by ARPE-19(TWEAK+)cells as compared to ARPE-19(control)cells(either P <0.01).Furthermore,colonies formed by ARPE-19(TWEAK+)cells were larger than those formed by ARPE-19(control)cells.Thus,we confirmed that overexpression of TWEAK leads to increased cell proliferation in retinal ARPE-19 cells.CONCLUSIONWe have found increased expression of the TWEAK/Fn14 pathway in PDR.Our findings imply that TWEAK/Fn14 mediates persistent inflammation and modulation of pathological neovascularization associated with PDR.