Glucosamine inhibits epithelial‐to‐mesenchymal transition and migration of retinal pigment epithelium cells in culture and morphologic changes in a mouse model of proliferative vitreoretinopathy

Purpose: To explore the effect of glucosamine (GlcN) on transforming growth factor (TGF)‐β signalling and several processes involved in proliferative vitreoretinopathy (PVR). Methods: We evaluated the surface levels of TGF‐β receptor and its binding of TGF‐β in ARPE‐19 cells. Release of cytokines and collagen, and expression of signalling intermediates were quantified. Migration was qualitatively and quantitatively examined. The morphology of cells undergoing PVR in vitro and in a mouse PVR model was observed. Results: Glucosamine reduced the surface levels of TGF‐β receptor and the ability of ARPE‐19 cells to bind TGF‐β. In ARPE‐19 cells, TGF‐β1 plus epidermal growth factor (EGF) or TGF‐β2 increased the expression of alpha‐smooth muscle actin (α‐SMA) and decreased the expression of zona occludens protein (ZO‐1). Transforming growth factor‐(β2) also caused the release of platelet‐derived growth factor (PDGF), connective tissue growth factor (CTGF) and type 1 collagen and increased the phosphorylation of SMAD2 and SMAD3. Platelet‐derived growth factor and CTGF stimulated cell migration, and TGF‐β2 stimulated wound closure, contraction of collagen and changes in cell morphology. Conclusions: Treatment with GlcN counteracted all of these effects, and its administration in the mouse model reduced the morphologic appearance of PVR. Glucosamine could inhibit the TGF‐β signalling pathway in retinal pigment epithelium cells and several of the downstream events associated with epithelial–mesenchymal transition and PVR.     

High glucose‐induced apoptosis in bovine retinal pericytes is associated with transforming growth factor β and βIG‐H3: βIG‐H3 induces apoptosis in retinal pericytes by releasing Arg‐Gly‐Asp peptides

Background: Transforming growth factor β (TGF‐β) plays an important role in diabetic retinopathy. βIG‐H3 is a downstream target molecule of TGF‐β that may participate in the pathogenesis of diabetic retinopathy and in particular in the loss of pericytes during early pathological changes. Methods: We observed bovine retinal pericytes apoptosis and the increased expression of TGF‐β and βIG‐H3 induced by high concentrations of glucose in the cell culture media. An anti‐TGF‐β antibody was used to block glucose‐induced retinal pericytes apoptosis. Retinal pericytes were also transfected with cDNA encodings either wild‐type or mutant βIG‐H3 lacking Arg‐Gly‐Asp (RGD) sequences in order to validate the effects of βIG‐H3 and RGD signalling on retinal pericytes apoptosis. Results: A cell death‐detecting enzyme‐linked immunosorbent assay revealed that 25 mM glucose significantly increased cell death compared with 5.5 mM glucose after 5 or 7 days of exposure (P < 0.01). High glucose significantly increased the TGF‐β levels as compared with 5.5 mM glucose after 5 days, and βIG‐H3 levels after 3, 5 and 7 days of exposure (P < 0.01). TGF‐β increased cell death and βIG‐H3 levels in a dose‐dependent manner, with a maximal effect observed at 1 ng/mL. An anti‐TGF‐β antibody nearly completely blocked high glucose‐induced cell death. Wild‐type βIG‐H3‐transfected cells showed a significant increase in cell death as compared with mutant βIG‐H3‐transfected (Mycb‐c) cells, untransfected or mock‐transfected cells. Conclusion: These results suggest that hyperglycaemia‐induced expression of TGF‐β and βIG‐H3 contributes to accelerated retinal pericytes apoptosis. βIG‐H3 induces pericytes apoptosis through its RGD motif, which may constitute an important pathogenic mechanism leading to pericytes loss in diabetic retinopathy.     

Circulating bone‐marrow‐derived endothelial precursor cells contribute to neovascularization in diabetic epiretinal membranes

Purpose: The role of vasculogenesis, recruitment and differentiation of circulating bone‐marrow‐derived endothelial precursor cells into mature endothelium in proliferative diabetic retinopathy (PDR) remains undefined. We investigated the presence of bone‐marrow‐derived endothelial precursor cells and the expression of the chemotactic pathway SDF‐1/CXCL12?CXCR4 in PDR epiretinal membranes. Methods: Membranes from eight patients with active PDR and nine patients with inactive PDR were studied by immunohistochemistry using antibodies against CD133, vascular endothelial growth factor receptor‐2 (VEGFR‐2), CD14, SDF‐1 and CXCR4. Results: Blood vessels expressed CD133, VEGFR‐2, CD14, SDF‐1 and CXCR4 in 10, 10, 10, seven and seven out of 17 membranes, respectively. There were significant correlations between number of blood vessels expressing CD34 and number of blood vessels expressing CD133 (r_s = 0.646; p = 0.005), VEGFR‐2 (r_s = 0.704; p = 0.002), CD14 (r_s = 0.564; p = 0.018) and SDF‐1 (r_s = 0.577; p = 0.015). Stromal cells in close association with blood vessels expressed CD133, VEGFR‐2, CD14 and CXCR4 in 10, 12, 13 and 14 membranes, respectively. The number of blood vessels expressing CD133 (p = 0.013), VEGFR‐2 (p = 0.005), CD14 (p = 0.008) and SDF‐1 (p = 0.005), and stromal cells expressing CD133 (p = 0.003), VEGFR‐2 (p = 0.013) and CD14 (p = 0.002) was significantly higher in active membranes than in inactive membranes. Conclusion: Bone‐marrow‐derived CD133⁺ endothelial progenitor cells and CD14⁺ monocytes may contribute to vasculogenesis in PDR.     

Transforming growth factor-beta1 induces alpha-smooth muscle actin expression and fibronectin synthesis in cultured human retinal pigment epithelial cells

Background : Proliferative vitreoretinopathy is a serious complication of retinal detachment, yet its pathogenesis is not fully understood. Retinal pigment epithelial cells and glial cells are found in the fibrous membranes in proliferative vitreoretinopathy. Many cytokines are involved in the pathology. Transforming growth factor (TGF)‐β₁, a cytokine found in serum, has been shown to be an important factor regulating the synthesis of fibrous extracellular matrix in proliferative vitreoretinopathy. Methods : Cultured human retinal pigment epithelial cells were used in the experiments. The effects of TGF‐β₁ on phenotype and function in retinal pigment epithelial cells were recorded as changes in the expression of α‐smooth muscle actin and fibronectin synthesis using immunohistochemistry and enzyme‐linked immunosorbent assay, respectively. Results : TGF‐β₁ induced the expression of α‐smooth muscle actin (P < 0.0001, n = 3), and significantly increased the synthesis of fibronectin by cultured human retinal pigment epithelial cells (P < 0.01, n = 4). Conclusions : Elevated levels of TGF‐β₁ in proliferative vitreoretinopathy may contribute to phenotype changes in retinal pigment epithelial cells leading to matrix deposition and contraction. Since the elevated levels of TGF‐β₁ may emanate from a number of diverse sources in proliferative vitreoretinopathy, developing an antagonist to TGF‐β₁ may offer an approach to the treatment of proliferative vitreoretinopathy.     

NGF and TGF‐β mRNA expression during pregnancy in a rat corneal wound healing model

Background: Growth factors seem to play a major role in corneal wound healing and TGF‐β seems to be associated with abnormal healing after corneal surgical procedures. Few studies have analysed the role of NGF and TGF‐β on corneal wound healing during pregnancy. The aim of the present study was to create an animal model to evaluate the expression of NGF and TGF‐βs during corneal wound healing in two groups: control and pregnant rats. Methods: Corneal mRNA for NGF and the three isoforms of TGF‐β were analysed by RT‐PCR, in a time‐course experiment on different days after epithelial wounding (2, 7, 14 days) in pregnant and control groups Results: The results show high corneal mRNA expression for NGF and TGF‐β1 without any variation throughout the healing process or pregnancy evolution. However, we detected a different expression of corneal mRNAs for TGF‐β2 and TGF‐β3 in the control group. This data was not detected in the pregnant group. Discussion: Our results suggested that pregnancy could have a relevant role on TGF‐β2 and TGF‐β3 mRNA expression during the corneal wound healing process. Additional research should be performed to corroborate these findings.     

Expression of angiogenic and fibrogenic factors in proliferative vitreoretinal disorders

Purpose To investigate the expression of connective tissue growth factor (CTGF) in the retina of human subjects with diabetes mellitus, and CTGF, CD105, and gelatinase B in proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR) epiretinal membranes. Methods Twelve donor eyes from six subjects with diabetes mellitus, 10 eyes from five nondiabetic subjects, 14 PDR membranes, and five PVR membranes were studied by immunohistochemical techniques. In situ zymography was used to examine gelatinolytic activity in four PDR membranes. Results In nondiabetic retinas, there was no immunoreactivity for CTGF. Diabetic retinas showed immunoreactivity for CTGF in ganglion cells and microglia. Vascular endothelial cells in PDR membranes expressed CTGF, CD105, and gelatinase B in 10 (71.4%), 11 (78.6%), and 5 (35.7%) membranes, respectively. Myofibroblasts in PDR membranes expressed CTGF, and gelatinase B in 14 (100%), and 6 (42.9%) membranes, respectively. There was a significant correlation between the number of blood vessels expressing the panendothelial marker CD34 and the number of blood vessels expressing CTGF (r = 0.7884; P = 0.0008), and CD105 (r = 0.6901; P = 0.0063), and the number of myofibroblasts expressing CTGF (r = 0.5922; P = 0.0257). There was a significant correlation between the number of myofibroblasts expressing α-smooth muscle actin and the number of myofibroblaasts expressing CTGF (r = 0.8393; P = 0.0002). In situ zymography showed the presence of gelatinolytic activity in vascular endothelial cells in PDR membranes. Myofibroblasts in PVR membranes expressed CTGF, and gelatinase B. Conclusions These results suggest a possible role of CTGF, CD105, and gelatinase B in the pathogenesis of proliferative vitreoretinal disorders.     

Profile of intraocular tumour necrosis factor‐α and interleukin‐6 in diabetic subjects with different degrees of diabetic retinopathy

Purpose: To assess and correlate the levels of inflammatory mediators in the eyes from non‐diabetic and diabetic subjects without retinopathy (NDR), with non‐proliferative diabetic retinopathy (NPDR) or with proliferative diabetic retinopathy (PDR) to corresponding erum levels. Methods: The levels of interleukin 1β, interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α) were analysed by an ELISA‐mimicking technique in the vitreous from 26 diabetic subjects with active PDR and 27 non‐diabetic subjects, or by a multiplex bead assay in the aqueous humour from 35 diabetic subjects with NDR/NPDR and 40 non‐diabetic subjects. Intraocular protein production was estimated in vitreous specimens by calculating a vitreous/serum ratio. Results: In the vitreous, IL‐6 was higher in diabetic [157.5 (25.0–1401.0) pg/ml; median (min–max)] than in non‐diabetic subjects [44.0 (5.0–4425) pg/ml; p = 0.021]. The vitreous/serum ratio was high (55.5:1 and 16:1, respectively), suggesting intraocular production. TNF‐α was lower in diabetic [18.0 (8.0–46.0) pg/ml] than in non‐diabetic subjects [22.0 (13.0–47.0) pg/ml; p = 0.034], but the vitreous/serum ratio was elevated in both groups (2:1 and 3.4:1, respectively). TNF‐α levels were higher in serum from diabetic subjects [9.0 (5.0–53.0) pg/ml versus 6.7 (3.0–11.0) pg/ml; p < 0.001]. Aqueous levels of inflammatory mediators did not differ between diabetic subjects with NDR/NPDR and non‐diabetic subjects despite elevated TNF‐α in serum [27.8 (6.8–153.7) pg/ml versus 16.4 (4.1–42.4) pg/ml; p = 0.021]. Conclusion: Intraocular inflammation seems to be involved in PDR but does not seem to be prominent in early retinopathy stages, i.e. NDR or NPDR. Diabetic subjects have an overall increased inflammatory activity compared to non‐diabetic subjects, as demonstrated by increased serum levels of TNF‐α.     

Expression of myofibroblast activation molecules in proliferative vitreoretinopathy epiretinal membranes

Purpose: Fibrotic disorders are associated with activation of fibroblasts into extracellular matrix‐secreting myofibroblasts expressing α‐smooth muscle actin (α‐SMA). Myofibroblasts are the predominant cellular component of proliferative vitreoretinopathy (PVR) epiretinal membranes. We investigated the expression of molecules involved in myofibroblast activation, migration and proliferation in PVR epiretinal membranes. Methods: Fifteen membranes were studied by immunohistochemical techniques using monoclonal and polyclonal antibodies directed against snail, fibroblast activation protein (FAP), CD44, hydrogen peroxide‐inducible clone‐5 (Hic‐5), galectin‐3, interleukin‐13 receptor α2 (IL‐13Rα2) and receptor for advanced glycation end products (RAGE). Results: Myofibroblasts expressing α‐SMA were present in all membranes. Myofibroblasts expressed nuclear immunoreactivity for Snail and Hic‐5, cytoplasmic immunoreactivity for FAP, IL‐13Rα2 and RAGE and membranous immunoreactivity for CD44. There was no immunoreactivity for galectin‐3. The number of cells expressing α‐SMA correlated significantly with the number of cells expressing Snail (r = 0.56; p = 0.03), Hic‐5 (r = 0.526; p = 0.044), IL‐13Rα2 (r = 0.773; p = 0.001) and RAGE (r = 0.734; p = 0.002). Conclusions: Snail, FAP, CD44, Hic‐5, IL13Rα2 and RAGE may be involved in proliferative events occurring in PVR.     

Silibinin inhibits myofibroblast transdifferentiation in human tenon fibroblasts and reduces fibrosis in a rabbit trabeculectomy model

Purpose: To investigate the effect of silibinin in myofibroblast transdifferentiation and in animal trabeculectomy models. Methods: The effect of silibinin on the expression of α‐smooth muscle actin (α‐SMA) and vimentin in response to transforming growth factor‐β1 (TGF‐β1) was determined in human tenon fibroblasts (HTFs). Cell migration and collagen contraction arrays were used to demonstrate the functionality of silibinin‐modulated HTFs. ELISA analysis was used to determine the effect of silibinin on the release of type 1 collagen and connective tissue growth factor (CTGF). The effect of silibinin on the activation of the TGF‐β receptor–related pathway was evaluated by Western blotting. A rabbit model of trabeculectomy was established to assess the effect of silibinin in vivo. Results: TGF‐β1 elevated the expression of α‐SMA and vimentin in HTFs; this elevation was inhibited by silibinin. TGF‐β1 increased cell migration and collagen contraction of HTFs, which were also suppressed by silibinin. The production of both CTGF and type 1 collagen in TGF‐β1‐treated HTFs was inhibited by silibinin. The effects of silibinin on TGF‐β1‐stimulated HTFs were mediated via the down‐regulation of TGF‐β receptor–related SMAD signalling pathways. In the rabbit model of trabeculectomy, silibinin increased the period of decreasing intraocular pressure after trabeculectomy and reduced the production of collagen and α‐SMA at the site of blebs in vivo. Conclusion: Silibinin inhibited the TGF‐β receptor–related signalling pathway in TGF‐β‐treated HTFs and several of the downstream events associated with myofibroblast transdifferentiation. Silibinin also improved the outcome of trabeculectomies by reducing the fibrotic response in the bleb tissue in vivo.     

Differential effects of TGF‐β and FGF‐2 on in vitro proliferation and migration of primate retinal endothelial and Müller cells

Purpose: During retinal development, the pattern of blood vessel formation depends upon the combined effects of proliferation and migration of endothelial cells, astrocytes and Müller cells. In this study, we investigated the potential for transforming growth factor‐β (TGF‐β) and fibroblast growth factor (FGF‐2) to influence this process by regulating proliferation and migration of retinal endothelial and macroglial cells. Methods: We assessed the effects of exogenous TGF‐β and FGF‐2 on the proliferation and migration of cultured endothelial (RF/6A) and Müller cell (MIO‐M1) lines. Cell proliferation was measured using a MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) colorimetric assay over 72 hr. Cell migration was measured using a scratch‐wound assay over 72 hr. Results: Transforming growth factor‐β inhibited the proliferation of endothelial and Müller cells and inhibited the migration of Müller cells, but not endothelial cells, compared to untreated controls. Conversely, FGF‐2 increased endothelial cell proliferation but inhibited endothelial cell migration. Fibroblast growth factor‐2 increased migration of Müller cells but had little effect on proliferation except at higher concentrations (20 ng/ml). Conclusion: Taken together, these observations indicate that TGF‐β and FGF could work in concert to inhibit endothelial cell proliferation and migration, respectively; this may have implications for establishing and maintaining the avascular zone of primate fovea.     

Effect of TGFβ and PDGF-B blockade on corneal myofibroblast development in mice

The purpose of this study was to investigate the role of transforming growth factor beta (TGFβ) and/or platelet-derived growth factor-B (PDGF-B) blockade on the differentiation of vimentin and alpha-smooth muscle actin (αSMA)-expressing myofibroblasts associated with haze in mice. Mouse corneas had haze-generating irregular PTK (phototherapeutic keratectomy) and topical treatment with the vectors. Six study groups of PTK treated corneas, with four corneas per group in each experiment, were Group 1) treated with TGFβ-KDEL vector interfering with TGFβ signaling through anomalous sorting of cytokine bound to the expressed altered receptor; Group 2) treated with PDGF-B-KDEL vector interfering with PDGF signaling through anomalous sorting of cytokine bound to the expressed altered receptor; Group 3) treated with both TGFβ-KDEL vector and PDGF-B-KDEL vector to interfere with signaling of both cytokines; Group 4) empty pGFPC1 vector; Group 5) empty pCMV vector; and Group 6) no vector treatment control. At one month after surgery, the corneas were analyzed by immunocytochemistry (IHC) for central stromal cells expressing myofibroblast markers vimentin and αSMA. The stroma of corneas treated with the TGFβ-KDEL vector alone (p < 0.05) or both the TGFβ-KDEL and PDGF-B-KDEL vectors (P < 0.05) had significantly lower density of vimentin-positive cells compared to the corresponding control group. The central stroma of corneas treated with the TGFβ-KDEL vector (p < 0.05) or the PDGF-B-KDEL vector (p < 0.05) had lower density of αSMA-positive cells compared to the corresponding control group. The density of αSMA-positive stromal cells was also significantly lower (p < 0.05) when both the TGFβ-KDEL and PDGF-B-KDEL and vectors were applied together compared to the corresponding control groups. This study provides in situ evidence that TGFβ and PDGF-B have important roles in modulating myofibroblast generation in the mouse cornea after haze-associated injury.     

Regional Immunity of the Eye

This article reviews molecular mechanism of intraocular inflammation in animal models and in humans, and the immunological defence system of the eye with particular attention to ocular pigment epithelium. In experimental autoimmune uveitis (EAU), T lymphocytes, particularly CD4⁺ T lymphocytes, play a central role in its immunopathogenic mechanisms. In humans, activated CD4⁺ T cells also play a central role in the immunopathogenic mechanisms. This notion is demonstrated in two human diseases: one is Vogt–Koyanagi–Harada disease, and the other is human T‐cell leukemia virus type 1 (HTLV‐1) uveitis. Activated CD4⁺ T cells infiltrating the eye are harmful to vision‐related cells and tissues in the eye and cause sight‐threatening conditions. However, the eye has regional defence systems to protect itself from these harmful activated T cells. We focus on ocular pigment epithelium (PE) and demonstrate immunoregulatory activity of iris PE and retinal PE. Iris PE suppresses activated CD4⁺ T cells by cell‐to‐cell contact with a crucial role played by B7‐2 molecule on iris PE and CTLA4 on T cells. The actual immunosuppressive factor being membrane bound TGF‐β. In contrast, retinal PE suppresses activated CD4⁺ T cells by soluble factors, such as soluble TGF‐β and thrombospondin 1. In addition to the direct T‐cell suppression by ocular PE, ocular PE has the capacity to promote activated T cells to regulatory T cells and use them as a tool to amplify the immune down regulation in the eye. The molecular mechanisms of generation of T regulatory cells by iris PE and retinal PE is also discussed.     

各种不同因素对视网膜脱离术后视力恢复的影响

目的:探讨各种不同因素对视网膜脱离(retinal detachment,RD)术后视力恢复的影响。方法:回顾分析我院以RD为第一诊断并经手术治疗视网膜复位成功的病例资料,共119例119眼。其中采用巩膜环扎手术38例、玻璃体联合视网膜复位手术81例。观察RD患者的发病年龄、RD时间、RD范围、有无裂孔、黄斑状态、增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)程度、手术前后视力、术后视网膜复位情况,用χ2检验对其进行分析,根据结果选择差异具有统计学意义的因素进行Spearman等级相关检验。结果:视网膜复位术后患者视力提高65例(54.6%),视力不变34例(28.6%),视力下降20例(16.8%)。RD患者发病的不同年龄、RD时间、RD范围、黄斑状态、PVR分级及有无裂孔对术后视力恢复的影响差异具有统计学意义(P<0.05)。对有统计学差异的因素进行Spearman等级相关检验,发现上述术前因素与术后视力关联程度从大到小分别为:PVR分级(r s=-0.493,P=0.000)、RD范围(r s=-0.476,P=0.000)、有无裂孔(r s=-0.411,P=0.000)、黄斑是否脱离(r s=-0.360,P=0.000)、RD时间(r s=-0.334,P=0.000)、患者年龄(r s=-0.241,P=0.008)。结论:RD患者术前PVR分级、RD范围、有无裂孔、黄斑状态、RD时间和年龄是影响术后视力恢复的重要因素,其中术前PVR分级、RD范围、有无裂孔对术后视力的恢复影响最为显著。     

Expression of autotaxin and acylglycerol kinase in proliferative vitreoretinal epiretinal membranes

Purpose: Lysophosphatidic acid (LPA)/LPA₁ receptor pathway is involved in inflammation, angiogenesis and fibrosis. This study was conducted to analyse the expression of LPA‐producing enzymes, autotaxin (ATX) and acylglycerol kinase (AGK) and LPA₁ receptor, in proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR) epiretinal membranes. Methods: Nine active and 13 inactive membranes from patients with PDR and 21 membranes from patients with PVR were studied by immunohistochemistry. Results: In PDR membranes, vascular endothelial cells expressed ATX and AGK in 16 and 19 membranes, respectively. Stromal cells expressed ATX and AGK in 19 and 22 membranes, respectively. Immunoreactivity for LPA₁ receptor was noted in vascular endothelial cells and stromal cells in the five membranes stained for LPA₁ receptor. Numbers of blood vessels and stromal cells expressing CD34, ATX and AGK were significantly higher in active membranes than in inactive membranes. Significant correlations were detected between number of blood vessels expressing the panendothelial cell marker CD34 and number of blood vessels and stromal cells expressing ATX and AGK. In PVR membranes, spindle‐shaped myofibroblasts expressing α‐smooth muscle actin co‐expressed ATX, AGK and LPA₁ receptor. Conclusions: The LPA/LPA₁ receptor pathway may be involved in inflammatory, angiogenic and fibrotic responses in proliferative vitreoretinal disorders.     

Intraocular expression of thymosin β4 in proliferative diabetic retinopathy

Purpose: To examine an association between thymosin β4 as potentially angioproliferative factor and proliferative diabetic retinopathy. Methods: The clinical study part included 62 patients with proliferative diabetic retinopathy (PDR) (study group) and 24 patients with non‐diabetic pre‐retinal membranes (control group). All patients underwent pars plana vitrectomy. We examined the thymosin β4 concentration in vitreous and plasma; and the expression of thymosin β4, glial fibrillary acidic protein (GFAP) and CD31 (PECAM‐1 or Platelet Endothelial Cell Adhesion Molecule) and the levels of thymosin β4 mRNA and vascular endothelial growth factor (VEGF) mRNA in the excised membranes. The experimental study part consisted of 24 Sprague–Dawley rats with streptozotocin‐induced diabetes mellitus and 24 age‐matched control animals without diabetes. We determined the mRNA concentrations of thymosin β4, VEGF and GFAP in the rat retinas. Results: In the clinical study part, the vitreal and plasma thymosin β4 concentrations were significantly higher in the study group than control group (p = 0.04 and p = 0.01, respectively), and were significantly (p = 0.028) correlated with each other. Co‐expression of thymosin β4 and CD31 was observed in the diabetic fibrovascular membranes. Thymosin β4 mRNA and VEGF mRNA levels were significantly (p < 0.01) higher in diabetic membranes than in non‐diabetic membranes. In the experimental study part, the diabetic retinas showed co‐localization of thymosin β4 and GFAP. The mRNA levels of thymosin β4, VEGF and GFAP were significantly (p < 0.01) higher in diabetic rats than in control animals. Conclusions: Thymosin β4 was produced in intraocular fibrovascular membranes of patients with PDR and in rats with experimental diabetes mellitus. Thymosin β4 may play a role in diabetic retinal neovascularization.     

Ang‐2 upregulation correlates with increased levels of MMP‐9, VEGF,EPO and TGFβ1 in diabetic eyes undergoing vitrectomy

Purpose: Angiogenesis in diabetic retinopathy (DR) is a multifactorial process regulated by hypoxia‐induced growth factors and inflammatory cytokines. In addition to the angiogenic switch, the proteolytic processing and altered synthesis of the extracellular matrix are critical steps in this disease. This study was performed to evaluate the levels of matrix metalloproteinase‐2 and matrix metalloproteinase‐9 (MMP‐2 and MMP‐9), angiopoietin‐1 and angiopoietin‐2 (Ang‐1 and Ang‐2), vascular endothelial growth factor (VEGF), erythropoietin (EPO) and transforming growth factor‐β1 (totalTGFβ1) in the vitreous of diabetic eyes undergoing vitrectomy compared with control eyes operated because of macular hole or pucker. Methods: Prospective consecutive controlled observational study performed in the unit of vitreoretinal surgery in Finland during the years 2006–2008. Vitreous samples were collected before the start of the conventional 3‐ppp vitrectomy. Vitreous MMP‐2 and MMP‐9, Ang‐1 and Ang‐2, VEGF, EPO and TGFβ1 concentrations were measured from 69 patients with Type 1 or 2 diabetes and 40 controls. Results: Comparison of eyes with DR with controls revealed that the mean vitreous concentrations of proMMP‐2 (p = 0.0015), totalMMP‐2 (p = 0.0011), proMMP‐9 (p = 0.00001), totalMMP‐9 (p < 0.00001), Ang‐2 (p < 0.00001), VEGF (p < 0.00001), EPO (p < 0.00001) and totalTGFβ1 (p = 0.000026) were significantly higher in the former group. A multivariate logistic regression analysis suggested intravitreal Ang‐2 concentration being the key marker of PDR (p = 0.00025) (OR = 1507.9). Conclusion: The main new finding is that the intravitreal concentrations of Ang‐2 correlated significantly with MMP‐9, VEGF, EPO and TGFβ1 levels in diabetic eyes undergoing vitrectomy. Thus, these factors could promote retinal angiogenesis synergistically.     

Association of tumour necrosis factor-alpha -308 G/A polymorphism with primary open-angle glaucoma

Background: Tumour necrosis factor‐alpha (TNF‐α) is an important proinflammatory cytokine driving axonal degeneration and retinal ganglion cell apoptosis in glaucoma. The aim of the study was to evaluate the association of TNF‐α ‐308 G/A and ‐238 G/A polymorphisms with primary open‐angle glaucoma (POAG). Design: A prospective, case–control study, university hospital setting. Participants: Eighty‐six POAG patients and 193 healthy unrelated controls. Methods: TNF‐α polymorphisms were screened by using direct gene sequencing. Main Outcome Measures: Frequency of TNF‐α ‐308 G/A and TNF‐α ‐238 G/A promoter polymorphisms in glaucoma and healthy subjects. Results: The frequencies of TNF‐α ‐308 GA genotype and ‘A’ allele were higher in patients with POAG (22.1% and 12.2%, respectively) in comparison with the control group (10.9% and 6%, respectively) (P = 0.046 and 0.02, respectively), with odds ratios of 2.45 (P = 0.01, 95% CI = 1.23–4.87) and 2.19 (P = 0.013, 95% CI = 1.18–4.08), respectively. Genotype distribution of the TNF‐α ‐238 variants did not yield a statistically significant difference between the two groups (P = 0.87). Conclusion: TNF‐α ‐308 G/A polymorphism seems to be associated with POAG in Turkish population. However, population‐based studies with large number of subjects and long‐term follow‐up are needed to verify the association of TNF‐α ‐308 G/A polymorphism with glaucoma susceptibility.     

基于深度学习的睑板腺功能障碍图像分析模型研究和评价

目的：构建一个基于卷积神经网络(CNN)的人工智能(AI)系统,能够全自动地评价睑板腺功能障碍(MGD)患者的睑板腺形态变化。

方法：选取2021-01/11在温州医科大学附属眼视光医院杭州院区就诊的145名受试者右眼纳入研究。随机选择其中60名受试者的睑板腺照相用于AI训练。收集睑板腺图像后首先标注出睑板区域和每一根睑板腺腺体。使用残差神经网络(ResNet)结合U-Net模型进行数据训练,获得成熟的AI系统; 85名受试者包括阻塞性MGD患者53名和睑板腺正常的志愿者32名,使用AI系统自动分析其各项睑板腺形态参数。同时观察临床指标包括眼表疾病指数(OSDI)、泪河高度(TMH)、泪膜破裂时间(TBUT)、角膜荧光素染色(CFS)、睑缘评分、睑板腺评分和睑板腺分泌能力评分。分析睑板腺参数与临床指标的相关性。

结果：通过多次版本迭代,最终获得了交并比达92.0%的AI系统。使用该AI系统,发现上眼睑的睑板腺密度与OSDI(r_s=-0.320)、TBUT(r_s=0.484)、睑缘评分(r_s=-0.350)、睑板腺评分(r_s=-0.749)和睑板腺分泌能力评分(r_s=0.425)存在显著相关性(均P<0.05); 下眼睑的睑板腺密度与OSDI(r_s=-0.420)、TBUT(r_s=0.598)、睑缘评分(r_s=-0.396)、睑板腺评分(r_s=-0.720)和睑板腺分泌能力评分(r_s=0.438)存在显著相关性(均P<0.05); 总眼睑的睑板腺密度与OSDI(r_s=-0.404)、TBUT(r_s=0.601)、睑缘评分(r_s=-0.416)、睑板腺评分(r_s=-0.805)和睑板腺分泌能力评分(r_s=0.480)存在显著相关性(均P<0.05)。

结论：基于CNN的AI系统是一个准确、高效的睑板腺形态学评价系统,能够方便地采用我们建立的睑板腺密度这一指标对MGD患者的睑板腺形态进行快速准确地评价。睑板腺密度这一指标比目前通用的睑板腺评分更精确,是评价睑板腺萎缩程度的全新定量指标。     

角膜生物力学与光密度相关性研究

目的:探究角膜生物力学与角膜光密度的相关性。方法:前瞻性研究。选取2019-03/06在云南省第二人民医院拟行角膜屈光手术术前检查的患者为研究对象。采用Pentacam HR眼前节分析系统进行角膜光密度测量,以角膜顶点为中心,分为0~2mm、> 2~6mm、> 6~10mm直径范围3个区域,以角膜厚度分为前、中、后3层。选取Pentacam HR中角膜最薄点厚度值纳入研究。采用Corvis ST角膜生物力学分析仪测量,相关参数包括第一次压平的长度(AP1L)和速率(AP1V)、第2次压平的长度(AP2L)和速率(AP2V)、最大凹陷时顶点距离(PD)、曲率半径(HCR)和形变幅度(DA)。运用Pentacam&Corvis ST生物力学联合诊断平台软件综合分析检查结果,得出综合角膜生物力学参数(CBI)以及其它独立参数包括硬度参数(SP)、综合半径(IR)、Ambrosio相关厚度-水平方向(ARTh)、形变幅度比(DAR)。各区域光密度间差异采用方差分析,角膜生物力学各项参数与各区域光密度的相关性采用Pearson或Spearman分析。结果:不同直径范围、不同层面间光密度有差异(F=35.101,P<0.01;F=1002.897,P<0.01),CBI与独立生物力学参数中AP2L、AP2V、PD、DA、SP、IR、ARTh、DAR具有相关性(rs=-0.502,P<0.01;rs=-0.457,P=0.001;rs=0.428,P=0.002;rs=0.539,P<0.01;rs=-0.687,P<0.01;rs=0.716,P<0.01;rs=-0.728,P<0.01;rs=0.750,P<0.01)。CBI与角膜0~2mm范围内光密度呈正相关(r=0.343,P=0.015)。0~2mm范围内光密度与独立生物力学参数中AP2L、IR、ARTh、DAR有相关性(rs=-0.298,P=0.035;rs=0.368,P=0.009;rs=-0.419,P=0.002;rs=0.493,P<0.01)。结论:角膜中央区域光密度与角膜生物力学具有显著的关联,临床中可以通过光密度和生物力学对角膜健康状况进行综合评价。     

孔源性视网膜脱离并发脉络膜脱离患者的角膜内皮形态学参数

目的：探讨孔源性视网膜脱离并发脉络膜脱离(RRD-CHD)患者的角膜内皮细胞形态学参数变化。

方法：连续收集RRD-CHD患者70例70眼,分为无高度近视组(A组)38眼38眼、高度近视组(B组)32例32眼,另收集正常组(C组)36例36眼。使用角膜内皮镜检测各组角膜内皮细胞最小面积(S_min)、最大面积(S_max)、平均面积、平均密度(CD)、细胞面积标准差(SD)、变异系数(CV)及六角形细胞比例(HG)。

结果：RRD-CHD患者和正常组的角膜内皮细胞CD、HG均有差异(P<0.001)。A组的CD与B组、C组均有差异(P<0.05),B组与C组有差异(P<0.001)。在A组中,SD、CV与眼轴(r_s=-0.426、0.494, 均P<0.01),CD与眼压、眼轴(r_s=-0.025、0.368, 均P<0.05),HG与病程(r_s=0.552, P<0.05)均相关。在RRD-CHD患者中,SD、CV与眼轴(r_s=0.236、0.159, 均P<0.05),HG与病程(r_s=0.142, P<0.05),S_max与眼压(r_s=-0.314, P<0.01)均相关。

结论：RRD-CHD患者持续低眼压状态下角膜内皮细胞的HG和CD均明显降低,眼轴、病程和眼压可影响角膜内皮的形态学参数。