Macrophage-Myofibroblast Transition Contributes to Myofibroblast Formation in Proliferative Vitreoretinal Disorders.

Inflammation and fibrosis are key features of proliferative vitreoretinal disorders. We aimed to define the macrophage phenotype and investigate the role of macrophage-myofibroblast transition (MMT) in the contribution to myofibroblast populations present in epiretinal membranes. Vitreous samples from proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR) and nondiabetic control patients, epiretinal fibrovascular membranes from PDR patients and fibrocellular membranes from PVR patients, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by ELISA, immunohistochemistry and flow cytometry analysis. Myofibroblasts expressing α-SMA, fibroblast activation protein-α (FAP-α) and fibroblast-specific protein-1 (FSP-1) were present in all membranes. The majority of CD68+ monocytes/macrophages co-expressed the M2 macrophage marker CD206. In epiretinal membranes, cells undergoing MMT were identified by co-expression of the macrophage marker CD68 and myofibroblast markers α-SMA and FSP-1. Further analysis revealed that CD206+ M2 macrophages co-expressed α-SMA, FSP-1, FAP-α and ß-catenin. Soluble (s) CD206 and sFAP-α levels were significantly higher in vitreous samples from PDR and PVR patients than in nondiabetic control patients. The proinflammatory cytokine TNF-α and the hypoxia mimetic agent cobalt chloride induced upregulation of sFAP-α in culture media of Müller cells but not of HRMECs. The NF-Ä¸ß inhibitor BAY11-7085 significantly attenuated TNF-α-induced upregulation of sFAP-α in Müller cells. Our findings suggest that the process of MMT might contribute to myofibroblast formation in epiretinal membranes, and this transition involved macrophages with a predominant M2 phenotype. In addition, sFAP-α as a vitreous biomarker may be derived from M2 macrophages transitioned to myofibroblasts and from Müller cells.

CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

We aimed to investigate the role of the CD40-CD40 ligand (CD40L) pathway in inflammation-mediated angiogenesis in proliferative diabetic retinopathy (PDR). We analyzed vitreous fluids and epiretinal fibrovascular membranes from PDR and nondiabetic patients, cultures of human retinal microvascular endothelial cells (HRMECs) and Müller glial cells and rat retinas with ELISA, immunohistochemistry, flow cytometry and Western blot analysis. Functional tests included measurement of blood-retinal barrier breakdown, in vitro angiogenesis and assessment of monocyte-HRMEC adherence. CD40L and CD40 levels were significantly increased in PDR vitreous samples. We demonstrated CD40L and CD40 expression in vascular endothelial cells, leukocytes and myofibroblasts in epiretinal membranes. Intravitreal administration of soluble (s)CD40L in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, VEGF, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). sCD40L induced upregulation of VEGF, MMP-9, MCP-1 and HMGB1 in cultured Müller cells and phospo-ERK1/2, p65 subunit of NF-ĸB, VCAM-1 and VEGF in cultured HRMECS. TNF-α induced significant upregulation of CD40 in HRMECs and Müller cells and VEGF induced significant upregulation of CD40 in HRMECs. sCD40L induced proliferation and migration of HRMECs. We provide experimental evidence supporting the involvement of the CD40L-CD40 pathway and how it regulates inflammatory angiogenesis in PDR.

Proprotein convertase furin is a driver and potential therapeutic target in proliferative diabetic retinopathy.

BACKGROUND: Furin converts inactive proproteins into bioactive forms. By activating proinflammatory and proangiogenic factors, furin might play a role in pathophysiology of proliferative diabetic retinopathy (PDR). METHODS: We studied vitreous samples from PDR and nondiabetic patients, epiretinal membranes from PDR patients, retinal microvascular endothelial cells (HRMECs), retinal Müller cells and rat retinas by ELISA, Western blot analysis, immunohistochemistry and immunofluorescence microscopy. We performed in vitro angiogenesis assays and assessed adherence of monocytes to HRMECs. RESULTS: Furin levels were significantly increased in PDR vitreous samples. In epiretinal membranes, immunohistochemistry analysis revealed furin expression in monocytes/macrophages, vascular endothelial cells and myofibroblasts. Furin was significantly upregulated in diabetic rat retinas. Hypoxia and TNF-α induced significant upregulation of furin in Müller cells and HRMECs. Furin induced upregulation of phospho-ERK1/2, p65 subunit of NF-κB, ADAM17 and MCP-1 in cultured Müller cells and phospho-ERK1/2 in cultured HRMECs and induced HRMECs migration. Treatment of monocytes with furin significantly increased their adhesion to HRMECs. Intravitreal administration of furin in normal rats induced significant upregulation of p65 subunit of NF-κB, phospho-ERK1/2 and ICAM-1 in the retina. Inhibition of furin with dec-CMK significantly decreased levels of MCP-1 in culture medium of Müller cells and HRMECs and significantly attenuated TNF-α-induced upregulation of p65 subunit of NF-κB, ICAM-1 and VCAM-1 in HRMECs. Dec-CMK significantly decreased adherence of monocytes to HRMECs and TNF-α-induced upregulation of adherence of monocytes to HRMECs. Treatment of HRMECs with dec-CMK significantly attenuated migration of HRMECs. CONCLUSIONS: Furin is a potential driver molecule of PDR-associated inflammation and angiogenesis.

Matrix metalloproteinase-14 is a biomarker of angiogenic activity in proliferative diabetic retinopathy.

Purpose: Matrix metalloproteinase-14 (MMP-14) is a transmembrane MMP that plays a critical role in promoting angiogenesis. We investigated the expression levels of MMP-14 and correlated the levels with clinical disease activity and with the levels of the angiogenic factors vascular endothelial growth factor (VEGF) and MMP-9 in proliferative diabetic retinopathy (PDR). To reinforce the findings at the functional level, we examined the expression of MMP-14 in the retinas of diabetic rats. Methods: Vitreous samples from 34 patients with PDR and 18 nondiabetic patients and epiretinal membranes from 13 patients with PDR and the retinas of rats were studied with enzyme-linked immunosorbent assay, immunohistochemistry, western blotting, and real-time reverse transcription PCR (RT-PCR). Results: The MMP-14, VEGF, and MMP-9 levels were statistically significantly higher in the vitreous samples from patients with PDR than in the samples from the nondiabetic controls (p<0.001 for all comparisons). The MMP-14 levels in patients with PDR with active neovascularization were statistically significantly higher than those in patients with inactive PDR (p<0.001). There were statistically significant positive correlations between levels of MMP-14 and levels of VEGF (r = 0.3; p = 0.032) and MMP-9 (r = 0.54; p<0.001). In the epiretinal membranes, MMP-14 was expressed in vascular endothelial cells, leukocytes, and myofibroblasts. Statistically significant positive correlations were detected between the numbers of blood vessels expressing CD31 and the numbers of blood vessels (r = 0.74; p = 0.004) and stromal cells (r = 0.72; p = 0.005) expressing MMP-14. Statistically significant increases of MMP-14 mRNA and protein were detected in rat retinas after induction of diabetes. Conclusions: These results suggest that MMP-14 is involved in PDR angiogenesis.

Pretreatment ocular blood flow and retinal oxygen metabolism in the acute uveitic phase is associated with final outcome in Vogt-Koyanagi-Harada disease.

PURPOSE: To investigate the association between pretreatment blood flow velocity in the choroid and optic nerve head (ONH) and retinal oxygen metabolism in the acute uveitic phase and the development of 'sunset glow fundus' in Vogt-Koyanagi-Harada (VKH) disease. METHODS: Retrospective analysis of 41 patients (82 eyes). Laser speckle flowgraphy and retinal oximetry measurements were performed at the presentation. The main outcome measure was the development of 'sunset glow fundus'. RESULTS: Twenty patients (40 eyes) presented in the phase preceding anterior segment inflammation (early presentation), and 21 patients (42 eyes) presented with anterior segment inflammation (late presentation). In ONH, mean blur rate (MBR)-vessel, representing blood flow velocity in retinal vessels, was significantly lower in the late presentation group, while choroidal MBR was not significantly different. The late presentation group had significantly lower oxygen saturation in retinal venules, a higher arteriovenous oxygen saturation difference and a smaller calibre of retinal arterioles compared with the early presentation group. Eyes that subsequently developed 'sunset glow fundus' had significantly lower ONH MBR-vessels, lower oxygen saturation in retinal venules, a higher arteriovenous oxygen saturation difference and a smaller calibre of retinal arterioles compared with eyes without 'sunset glow fundus'. ONH MBR-vessel had a significant negative correlation with arteriovenous oxygen saturation difference and a significant positive correlation with calibre of retinal arterioles. CONCLUSIONS: In the acute uveitic phase of VKH disease, the development of 'sunset glow fundus' is associated with pretreatment reduced retinal blood flow velocity, calibre of retinal arterioles and oxygen saturation in retinal venules, as well as an increased arteriovenous oxygen saturation difference.

Comparisons of choroidal blood flow velocity between initial-onset acute uveitis associated with Vogt-Koyanagi-Harada disease and acute central serous chorioretinopathy.

OBJECTIVES: Acute central serous chorioretinopathy (CSC) and Vogt-Koyanagi-Harada (VKH) disease in the acute uveitic phase are characterized by serous retinal detachment caused by dysfunction of the choroid. The aim of this study is to compare blood flow velocity and pulse waveform parameters in the choroid between these two diseases. METHODS: In this study, 25 patients (50 eyes) with VKH disease, 21 patients (27 eyes) with CSC and 15 healthy controls (30 eyes) were studied. Laser speckle flowgraphy (LSFG) was performed at presentation. RESULTS: Choroidal mean blur rate (MBR), representing blood flow velocity in choroidal vessels, was significantly lower in the eyes affected by VKH disease compared with the healthy control and CSC eyes. CSC eyes had a significantly higher MBR compared with healthy controls. Among the analyzed pulse waveform parameters, blow-out time (BOT), falling rate (FR) and flow acceleration index (FAI) changed significantly. BOT value was significantly lower in CSC eyes than in healthy control and VKH eyes. FR and FAI values were significantly lower in VKH eyes than in healthy control and CSC eyes. There was a strong positive correlation between MBR and FAI. CONCLUSIONS: Our findings confirm different pathophysiology of these two diseases. Assessment of choroidal blood flow velocity and haemodynamics with LSFG provides useful information to differentiate acute CSC and initial-onset acute uveitis associated with VKH disease.

Acute uveitic phase of Vogt-Koyanagi-Harada disease: optic nerve head swelling, ocular blood flow and retinal oxygen metabolism.

OBJECTIVES: To investigate the association of optic nerve head (ONH) swelling in the acute uveitic phase of Vogt-Koyanagi-Harada (VKH) disease with blood flow velocity in the choroid and ONH and oxygen saturation and diameter of retinal vessels. METHODS: In this prospective study, 25 patients (50 eyes) were studied. Thirteen patients (26 eyes) had ONH swelling and 12 patients (24 eyes) had no ONH swelling. Laser speckle flowgraphy (LSFG) and retinal oximetry measurements were performed at presentation. RESULTS: In the ONH, mean blur rate (MBR)-vessel, representing blood flow velocity in retinal vessels, was significantly lower in the eyes affected by ONH swelling, while choroidal MBR was not significantly different. Eyes with ONH swelling had a significantly lower oxygen saturation in retinal venules, a significantly higher arteriovenous oxygen saturation difference and a significantly smaller calibre of retinal arterioles compared with eyes without ONH swelling. There were significant positive correlations between the MBR-vessel of the ONH and venular oxygen saturation and calibre of retinal arterioles. In addition, MBR-vessel of the ONH had a significant negative correlation with arteriovenous oxygen saturation difference. CONCLUSIONS: The occurrence of ONH swelling in the acute uveitic phase of VKH disease is associated with lower retinal blood flow velocity and smaller calibre of retinal arterioles as well as lower oxygen saturation in retinal venules and higher arteriovenous difference in oxygen saturation.

Therapeutic window of opportunity in the acute uveitic phase of Vogt-Koyanagi-Harada disease: Prevention of late autoimmune complications by early intervention.

PURPOSE: To determine relationship between timing of treatment initiation and disease outcomes and whether a therapeutic window of opportunity exists in initial-onset acute uveitis associated with Vogt-Koyanagi-Harada disease. METHODS: Retrospective analysis of 112 patients (224 eyes). Main outcome measures were final visual acuity, progression to chronic recurrent evolution, development of complications, particularly 'sunset glow fundus', and drug-free remission cure of uveitis. RESULTS: Forty-six patients (92 eyes) presented in the phase preceding anterior segment (AS) inflammation (early presentation) and 66 patients (132 eyes) had AS inflammation at presentation (late presentation). In significantly more eyes in the early presentation group (85.9%), final visual acuity of 20/20 was achieved compared with those in the late presentation group (66.7%) (p = 0.001). None of the eyes in the early presentation group progressed to chronic recurrent evolution and none developed 'sunset glow fundus', whereas in the late presentation group, 28.8% of the eyes progressed to chronic recurrent evolution (p < 0.001) and 56.1% developed 'sunset glow fundus' (p < 0.001). Patients in the early presentation group were able to discontinue treatment without relapse of inflammation at significantly shorter time intervals compared to patients in the delayed presentation group (p < 0.001). In the late presentation group, logistic regression analysis demonstrated that presenting clinical features predicting unfavourable outcomes were posterior synechiae (odds ratio = 4.03; 95% confidence interval [CI] = 1.29-12.23), bullous exudative retinal detachment extending to the periphery (odds ratio = 3.35; 95% CI = 1.53-7.32) and female gender (odds ratio = 2.05; CI = 1.08-3.90). CONCLUSIONS: Our findings suggest that the window of opportunity lies in the phase preceding AS inflammation and initiation of effective treatment during this phase results in cure of uveitis and prevents blinding complications.

Clinical Findings Predicting Posttraumatic Endophthalmitis after Repair of Open Globe Injuries.

BACKGROUND: To evaluate risk factors for developing endophthalmitis after repair of open globe injuries. METHODS: Retrospective chart analysis of 1303 patients from May 1996 till December 2019. RESULTS: All patients received prophylactic intravenous broad-spectrum antibiotics for 5-7 days. Endophthalmitis was clinically suspected in 37 (2.8%) eyes and was culture proven in 14 of these eyes (1.1%). Univariate analysis identified poor initial visual acuity at presentation, rural setting of injury, contaminated wound and lens injury as significant predictors for the development of clinically suspected endophthalmitis. Intravitreal antibiotics at the time of primary repair in eyes with high-risk characteristics decreased risk of developing endophthalmitis (OR: 2.28;95% CI,1.07-4.86; p = .033). CONCLUSIONS: Poor initial visual acuity, rural setting of injury, contaminated wound, and lens injury increased risk of suspected posttraumatic endophthalmitis. Prophylactic intravitreal antibiotics at the time of primary repair in eyes with high-risk characteristics reduced the risk of posttraumatic endophthalmitis.

Long-term Outcomes of Uveitis Associated with Vogt-Koyanagi-Harada Disease in the Pediatric Age Group.

PURPOSE: To investigate the outcomes of uveitis associated with Vogt-Koyanagi-Harada (VKH) disease in pediatric age group (aged 16 years and under). METHODS: A retrospective review of patients with VKH disease. RESULTS: Among the 244 patients identified, 38 (76 eyes) were children. Among them, five had insulin-dependent diabetes mellitus. 21 presented with initial-onset acute disease and 17 with chronic recurrent disease. The mean follow-up period was 59.1 months. At presentation, chronic recurrent disease was associated with more severe inflammation as indicated by the presence of mutton-fat keratic precipitates (p < .001), iris nodules (p = .005) and posterior synechiae (p < .001). During follow-up, the rate of complications was higher in children with chronic recurrent disease compared with initial-onset acute disease (p < .001). 92.4% of the eyes with initial-onset acute disease achieved a final visual acuity of ≥20/40 compared with 70.6% of the eyes with chronic recurrent disease (p = .013). CONCLUSIONS: Chronic recurrent VKH disease in children is associated with worse outcomes.

Effect of immunosuppressive therapy on ocular blood flow in initial-onset acute uveitis associated with Vogt-Koyanagi-Harada disease.

PURPOSE: To investigate the effect of immunosuppressive therapy on blood flow and waveform parameters in the choroid and optic nerve head (ONH) in patients with initial-onset acute uveitis associated with Vogt-Koyanagi-Harada (VKH) disease. METHODS: In this prospective study, 18 patients (36 eyes) were studied. Laser speckle flowgraphy was performed at baseline and at 4 weeks, 8 weeks and 12 weeks after treatment. We analysed longitudinal changes in mean blur rate (MBR), blow-out time, blow-out score (BOS), acceleration time index (ATI), flow acceleration index (FAI), resistivity index (RI) and blood flow fluctuation. RESULTS: After immunosuppressive therapy, MBR, representing blood flow velocity, in the choroid and ONH significantly increased at each post-treatment time point compared to baseline values. Among the analysed pulse waveform parameters, BOS significantly increased, while RI and fluctuation significantly decreased. Increased BOS and decreased RI indicate decreased vascular resistance following treatment. There was a strong negative correlation between BOS and RI. Additionally, FAI increased in the choroid and ATI increased in ONH. CONCLUSIONS: Immunosuppressive therapy in the acute uveitic phase of VKH disease improved inflammation-related impairment in choroidal and ONH blood flow.

Effect of immunosuppressive therapy on oxygen saturation and diameter of retinal vessels in initial onset acute uveitis associated with Vogt-Koyanagi-Harada disease.

PURPOSE: To demonstrate changes in oxygen saturation and calibre of retinal vessels in initial-onset acute uveitis associated with Vogt-Koyanagi-Harada (VKH) disease at baseline and during immunosuppressive therapy. METHODS: In this prospective study, 22 patients (44 eyes) were studied. Retinal oximetry measurements were performed using the noninvasive spectrophotometric retinal oximeter (Oxymap T1) at baseline and at 1-3 months, 4-6 months, 5-7 months and more than 9 months after treatment. RESULTS: At baseline, mean logarithm of the minimum angle of resolution (logMAR) best-corrected visual acuity (BCVA) was 1.12 ± 0.78 (Snellen equivalent, 20/265). Arteriolar and venular oxygen saturations were 108 ± 7% and 70 ± 9%, respectively and calibres of arterioles and venules were 12.1 ± 1.1 pixels and 16.9 ± 1.4 pixels, respectively. At 4-6 months of follow-up, logMAR BCVA was almost maximum (0.08 ± 0.1, Snellen equivalent 20/24; p < 0.001) and thereafter remained almost unchanged. After immunosuppressive therapy, arteriolar and venular oxygen saturation values continued to decrease up to >9 months of follow-up (92 ± 7% and 56 ± 10%, respectively; p < 0.001 for both arterioles and venules). Similarly, arteriolar and venular calibres continued to decrease up to >9 months of follow-up to 11.4 ± 0.9 pixels (p = 0.006) and 15.6 ± 1.3 pixels (p = 0.001), respectively. CONCLUSIONS: Eyes with initial-onset acute uveitis associated with VKH disease have increased oxygen saturation and calibres of retinal vessels at baseline. Immunosuppressive therapy normalizes these changes and in a similar pattern improves BCVA.

Incidence, Risk Factors and Surgical Outcomes of Cataract among Patients with Vogt-Koyanagi-Harada Disease.

Purpose: To investigate risk factors and surgical outcomes of cataract in Vogt-Koyanagi-Harada (VKH) disease.Methods: Review of 187 patients (374 eyes).Results: At presentation, cataract was diagnosed in 56 (14.9%) eyes all had chronic recurrent VKH. During follow-up, cataract developed in additional 51 (13.6%) eyes. Fifteen (13.6%) of these had initial-onset acute VKH with anterior segment (AS) inflammation and 36 (19.4%) had chronic recurrent VKH. No patient with initial-onset acute VKH without AS inflammation developed cataract. Risk factors for cataract development during follow-up included female gender, keratic precipitates, anterior chamber reaction ≥2+, chronic recurrent VKH, posterior synechiae, iris nodules, glaucoma, glaucoma surgery, choroidal neovascular membrane, "sunset glow fundus" and chorioretinal atrophy. Thirty-two eyes underwent cataract extraction. Fourteen (43.8%) eyes achieved ≥20/40. Posterior segment complications of chronic recurrent VKH accounted for <20/40 outcome.Conclusions: Poor outcome after surgery is secondary to posterior segment complications of chronic recurrent VKH.

CD146/Soluble CD146 Pathway Is a Novel Biomarker of Angiogenesis and Inflammation in Proliferative Diabetic Retinopathy.

Purpose: Inflammation, angiogenesis and fibrosis are pathological hallmarks of proliferative diabetic retinopathy (PDR). The CD146/sCD146 pathway displays proinflammatory and proangiogenic properties. We investigated the role of this pathway in the pathophysiology of PDR. Methods: Vitreous samples from 41 PDR and 27 nondiabetic patients, epiretinal fibrovascular membranes from 18 PDR patients, rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by ELISA, Western blot analysis, immunohistochemistry and immunofluorescence microscopy analysis. Blood-retinal barrier breakdown was assessed with fluorescein isothiocyanate-conjugated dextran. Results: sCD146 and VEGF levels were significantly higher in vitreous samples from PDR patients than in nondiabetic patients. In epiretinal membranes, immunohistochemical analysis revealed CD146 expression in leukocytes, vascular endothelial cells and myofibroblasts. Significant positive correlations were detected between numbers of blood vessels expressing CD31, reflecting angiogenic activity of PDR, and numbers of blood vessels and stromal cells expressing CD146. Western blot analysis showed significant increase of CD146 in diabetic rat retinas. sCD146 induced upregulation of phospho-ERK1/2, NF-κB , VEGF and MMP-9 in Müller cells. The hypoxia mimetic agent cobalt chloride, VEGF and TNF-α induced upregulation of sCD146 in HRMECs. The MMP inhibitor ONO-4817 attenuated TNF-α-induced upregulation of sCD146 in HRMECs. Intravitreal administration of sCD146 in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, intercellular adhesion molecule-1 and VEGF in the retina. sCD146 induced migration of HRMECs. Conclusions: These results suggest that the CD146/sCD146 pathway is involved in the initiation and progression of PDR.

Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation.

Purpose: Endogenous tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) has powerful regulatory effects on inflammation and angiogenesis. In this study, we investigated the role of TIMP-3 in regulating inflammation in the diabetic retina. Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic patients were subjected to Western blot analysis. Streptozotocin-treated rats were used as a preclinical diabetic retinopathy (DR) model. Blood-retinal barrier (BRB) breakdown was assessed with fluorescein isothiocyanate (FITC)-conjugated dextran. Rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by Western blot analysis and ELISA. Adherence of human monocytes to HRMECs was assessed and in vitro angiogenesis assays were performed. Results: Tissue inhibitor of matrix metalloproteinase-3 in vitreous samples was largely glycosylated. Intravitreal injection of TIMP-3 attenuated diabetes-induced BRB breakdown. This effect was associated with downregulation of diabetes-induced upregulation of the p65 subunit of NF-κB, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF), whereas phospho-ERK1/2 levels were not altered. In Müller cell cultures, TIMP-3 significantly attenuated VEGF upregulation induced by high-glucose (HG), the hypoxia mimetic agent cobalt chloride (CoCl2) and TNF-α and attenuated MCP-1 upregulation induced by CoCl2 and TNF-α, but not by HG. TIMP-3 attenuated HG-induced upregulation of phospho-ERK1/2, caspase-3 and the mature form of ADAM17, but not the levels of the p65 subunit of NF-κB and the proform of ADAM17 in Müller cells. TIMP-3 significantly downregulated TNF-α-induced upregulation of ICAM-1 and VCAM-1 in HRMECs. Accordingly, TIMP-3 significantly decreased spontaneous and TNF-α- and VEGF-induced adherence of monocytes to HRMECs. Finally, TIMP-3 significantly attenuated VEGF-induced migration, chemotaxis and proliferation of HRMECs. Conclusion: In vitro and in vivo data point to anti-inflammatory and anti-angiogenic effects of TIMP-3 and support further studies for its applications in the treatment of DR.

Pediatric open-globe injury in a university-based tertiary hospital.

BACKGROUND: To investigate epidemiology, etiology, and outcomes after repair of pediatric open-globe injury. METHODS: We retrospectively reviewed medical records of patients ⩽18 years who underwent primary open-globe repair. RESULTS: A total of 213 patients were identified. Male-female ratio was 1.44:1. Type of injury was penetration in 157 (74.4%) cases, rupture in 52 (24.4%) cases, and perforation in 2 (0.9%) cases. Knife injuries were the most common cause, affecting 38/196 (19.4%), followed by metallic object in 37/196 (18.9%) patients, glass in 26/196 (13.3%) patients, and pen or pencil in 24/196 (12.8%). Predictors of good visual outcome defined as (⩾20/40) were good initial visual acuity (⩾20/40; p < 0.0001), time from injury to arrival at the emergency room >24 h (p = 0.038), size of wound less than 10 mm (p < 0.0001), absence of iris prolapse (p < 0.0001), deep anterior chamber at presentation (p < 0.0001), absence of hyphema (p = 0.043), intact lens (p < 0.0001), and no retinal detachment during follow-up (p < 0.0001). A total of 27 (12.7%) cases were documented to have retinal detachment at any time during follow-up period. Predictors of retinal detachment were perforation and rupture (p < 0.0001), whereas penetration was not associated with development of retinal detachment, size of the wound ⩾10 mm (p < 0.0001), initial visual acuity ⩽20/200 (p < 0.0001), lens injury (p < 0.0001), and development of endophthalmitis (p < 0.027). Eight (3.7%) eyes had the clinical diagnosis of posttraumatic endophthalmitis. CONCLUSIONS: The most common type of injury was penetration and the most common tool was knife. Visual outcome was affected by the initial presentation. Retinal detachment was a significant predictor of a worse final visual outcome.

Local Cytokine Expression Profiling in Patients with Specific Autoimmune Uveitic Entities.

Purpose: To evaluate expression of cytokines GM-CSF, IL-11, IL-12p40, IL-12p70, IL-27p28, IL-35, APRIL, BAFF, TWEAK, and LIGHT in uveitis.Methods: Aqueous humor samples from patients with active uveitis associated with Behçet's disease (BD), sarcoidosis, HLA-B27-related inflammation, and Vogt-Koyanagi-Harada (VKH) disease and control patients were assayed with a multiplex assay.Results: Comparing all patients to controls, GM-CSF, IL-11, IL-12p40, APRIL, and BAFF were significantly increased, whereas LIGHT was significantly decreased. IL-11 and BAFF were the most strongly upregulated, being elevated 19.7-fold and 14.1-fold, respectively, compared with controls. IL-11 was significantly highest in HLA-B27 uveitis. GM-CSF, IL-11, and IL-12p40 were significantly higher in nongranulomatous uveitis (BD and HLA-B27) than in granulomatous uveitis (sarcoidosis and VKH), whereas APRIL and TWEAK were significantly higher in granulomatous uveitis.Conclusions: IL-11-driven immune responses might be more potent in nongranulomatous uveitis, particularly in HLA-B27 uveitis. BAFF and APRIL might contribute to B cell-driven autoimmune response in uveitis.

Interleukin-11 Overexpression and M2 Macrophage Density are Associated with Angiogenic Activity in Proliferative Diabetic Retinopathy.

PURPOSE: To investigate the expression of IL-11 and its receptor IL-11Rα and to quantify density of CD163+ M2 macrophages in proliferative diabetic retinopathy (PDR). METHODS: Vitreous samples from 29 PDR and 19 nondiabetic patients, epiretinal fibrovascular membranes from 15 patients with PDR and Müller cells were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. RESULTS: We showed a significant increase in expression of IL-11, soluble(s) IL-11Rα, sCD163 and VEGF in vitreous samples from PDR patients compared to nondiabetic controls. Significant positive correlations were found between levels of VEGF and levels of IL-11 and sCD163. Significant positive correlations were found between microvessel density and number of blood vessels and stromal cells expressing IL-11, IL-11Rα and CD163 in PDR epiretinal membranes. The hypoxia mimetic agent cobalt chloride induced upregulation of IL-11 and IL-11Ra in cultured Müller cells. CONCLUSIONS: IL-11/IL-11Rα signaling and CD163+ M2 macrophages might be involved in PDR angiogenesis.

Soluble cytokine receptor levels in aqueous humour of patients with specific autoimmune uveitic entities: sCD30 is a biomarker of granulomatous uveitis.

PURPOSE: Soluble cytokine receptors are potential biomarkers for immune activation and have a promising potential as immunotherapeutic agents. We investigated the levels of soluble cytokine receptors in aqueous humour (AH) samples from patients with specific autoimmune uveitic entities. METHODS: Patients with active uveitis associated with Behçet's disease (BD) (n = 13), sarcoidosis (n = 8), HLA-B27-related inflammation (n = 12), Vogt-Koyanagi-Harada (VKH) disease (n = 12) and control subjects (n = 9) were included. AH samples were analyzed with the use of multiplex assays for the proinflammatory cytokine tumour necrosis factor (TNF)-α and the soluble cytokine receptors sCD30, sCD163, sgp130, sIL-6 receptor-α (sIL-6R), sTNFRI and sTNFRII. RESULTS: TNF-α and soluble cytokine receptor AH levels were significantly higher in uveitis patients (n = 45) compared with controls (n = 9). When nongranulomatous uveitis (BD and HLA-B27-associated uveitis) was compared with granulomatous uveitis (sarcoidosis and VKH disease), the levels of sCD30 and sTNFRI/TNF-α and sTNFRII/TNF-α ratios were significantly enhanced in granulomatous uveitis. Finally, when comparing the profile in the specific uveitis entities, sCD30 levels were highest in patients with VKH disease. sgp130, sCD163, sIL-6R, sTNFRI and sTNFRII did not differ significantly between the four different clinical uveitic subgroups. CONCLUSIONS: Soluble cytokine receptors are significantly upregulated in autoimmune uveitis. CD30+ T cells might contribute to the inflammatory process in granulomatous uveitis, particularly in VKH disease. Granulomatous uveitis is also characterized by significantly higher sTNFRs/TNF-α ratios than nongranulomatous uveitis.

Evaluation of Proteoforms of the Transmembrane Chemokines CXCL16 and CX3CL1, Their Receptors, and Their Processing Metalloproteinases ADAM10 and ADAM17 in Proliferative Diabetic Retinopathy.

The transmembrane chemokine pathways CXCL16/CXCR6 and CX3CL1/CX3CR1 are strongly implicated in inflammation and angiogenesis. We investigated the involvement of these chemokine pathways and their processing metalloproteinases ADAM10 and ADAM17 in the pathophysiology of proliferative diabetic retinopathy (PDR). Vitreous samples from 32 PDR and 24 non-diabetic patients, epiretinal membranes from 18 patients with PDR, rat retinas, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. In vitro angiogenesis assays were performed and the adherence of leukocytes to CXCL16-stimulated HRMECs was assessed. CXCL16, CX3CL1, ADAM10, ADAM17 and vascular endothelial growth factor (VEGF) levels were significantly increased in vitreous samples from PDR patients. The levels of CXCL16 were 417-fold higher than those of CX3CL1 in PDR vitreous samples. Significant positive correlations were found between the levels of VEGF and the levels of CXCL16, CX3CL1, ADAM10 and ADAM17. Significant positive correlations were detected between the numbers of blood vessels expressing CD31, reflecting the angiogenic activity of PDR epiretinal membranes, and the numbers of blood vessels and stromal cells expressing CXCL16, CXCR6, ADAM10 and ADAM17. CXCL16 induced upregulation of phospho-ERK1/2, p65 subunit of NF-κB and VEGF in cultured Müller cells and tumor necrosis factor-α induced upregulation of soluble CXCL16 and ADAM17 in Müller cells. Treatment of HRMECs with CXCL16 resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and increased leukocyte adhesion to HRMECs. CXCL16 induced HRMEC proliferation, formation of sprouts from HRMEC spheroids and phosphorylation of ERK1/2. Intravitreal administration of CXCL16 in normal rats induced significant upregulation of the p65 subunit of NF-κB, VEGF and ICAM-1 in the retina. Our findings suggest that the chemokine axis CXCL16/CXCR6 and the processing metalloproteinases ADAM10 and ADAM17 might serve a role in the initiation and progression of PDR.