Fibrovascular Epiretinal Membranes Research Articles

LL37/FPR2 regulates neutrophil mPTP promoting the development of neutrophil extracellular traps in diabetic retinopathy.

Diabetic retinopathy (DR) is characterized by chronic, low-grade inflammation. This state may be related to the heightened production of neutrophil extracellular traps (NETs) induced by high glucose (HG). Human cathelicidin antimicrobial peptide (LL37) is an endogenous ligand of G protein-coupled chemoattractant receptor formyl peptide receptor 2 (FPR2), expressed on neutrophils and facilitating the formation and stabilization of the structure of NETs. In this study, we detected neutrophils cultured under different conditions, the retinal tissue of diabetic mice, and fibrovascular epiretinal membranes (FVM) samples of patients with proliferative diabetic retinopathy (PDR) to explore the regulating effect of LL37/FPR2 on neutrophil in the development of NETs during the process of DR. Specifically, HG or NG with LL37 upregulates the expression of FPR2 in neutrophils, induces the opening of mitochondrial permeability transition pore (mPTP), promotes the increase of reactive oxygen species and mitochondrial ROS, and then leads to the rise of NET production, which is mainly manifested by the release of DNA reticular structure and the increased expression of NETs-related markers. The PI3K/AKT signaling pathway was activated in neutrophils, and the phosphorylation level was enhanced by FPR2 agonists invitro. Invivo, increased expression of NETs markers was detected in the retina of diabetic mice and in FVM, vitreous fluid, and serum of PDR patients. Transgenic FPR2 deletion led to decreased NETs in the retina of diabetic mice. Furthermore, invitro, inhibition of the LL37/FPR2/mPTP axis and PI3K/AKT signaling pathway decreased NET production induced by high glucose. These results suggested that FPR2 plays an essential role in regulating the production of NETs induced by HG, thus may be considered as one of the potential therapeutic targets.

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.

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-ĸß 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.

The G-Protein-Coupled Formyl Peptide Receptor 2 Promotes Endothelial-Mesenchymal Transition in Diabetic Retinopathy

Introduction: In proliferative diabetic retinopathy (PDR), retinal neovascularization is the essential pathogenic process that is linked to endothelial-to-mesenchymal transition (EndoMT) induced by high glucose (HG). This pathophysiological process may be regulated by a G-protein-coupled chemoattractant receptor FPR2 (mouse Fpr2), involved in inflammatory cell migration and proliferation. In the current study, we investigated the role of Fpr2 in regulating EndoMT and the underlying mechanisms during diabetic retinopathy progression. Methods: FPR2 agonist or inhibitor was added to human microvascular endothelial cells (HMECs) exposed to normal glucose or HG. Morphologic, phenotypic, and functional changes of HMECs as well as the formation of microvasculature related to EndoMT were assessed. EndoMT biomarkers were detected in the retinal tissues of diabetic mice and fibrovascular epiretinal membranes (FVMs) from patients with PDR. Results: HG upregulated FPR2 in HMECs, which triggered morphological changes, and the cells acquired mesenchymal phenotype, with enhanced cell migration, viability, and angiogenic process shown by tube formation and aortic ring sprouting. Inhibition of FPR2 attenuated HG-induced EndoMT and endothelial cell migration to form vessel-like tube structures. RNA sequence and protein analysis further revealed that inhibition of FPR2 decreased the expression of genes associated with EndoMT. ERK1/2 and P38 signaling pathway was activated in HMECs, promoting neovascularization in HG-induced EndoMT of HMECs. In vivo, increased expression of mesenchymal markers was detected in the retina of diabetic mice and FVMs from patients with PDR. FPR2 deficiency was associated with diminished EndoMT-related phenotypic changes in the retina of diabetic mice. Conclusions: FPR2 is actively involved in the progression of EndoMT that may contribute to the pathogenesis of PDR. Thus, FPR2 may be a potential therapeutic target for PDR.

Differential Expression and Localization of ADAMTS Proteinases in Proliferative Diabetic Retinopathy.

We analyzed the expression of ADAMTS proteinases ADAMTS-1, -2, -4, -5 and -13; their activating enzyme MMP-15; and the degradation products of proteoglycan substrates versican and biglycan in an ocular microenvironment of proliferative diabetic retinopathy (PDR) patients. Vitreous samples from PDR and nondiabetic patients, epiretinal fibrovascular membranes from PDR patients, rat retinas, retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied. The levels of ADAMTS proteinases and MMP-15 were increased in the vitreous from PDR patients. Both full-length and cleaved activation/degradation fragments of ADAMTS proteinases were identified. The amounts of versican and biglycan cleavage products were increased in vitreous from PDR patients. ADAMTS proteinases and MMP-15 were localized in endothelial cells, monocytes/macrophages and myofibroblasts in PDR membranes, and ADAMTS-4 was expressed in the highest number of stromal cells. The angiogenic activity of PDR membranes correlated significantly with levels of ADAMTS-1 and -4 cellular expression. ADAMTS proteinases and MMP-15 were expressed in rat retinas. ADAMTS-1 and -5 and MMP-15 levels were increased in diabetic rat retinas. HRMECs and Müller cells constitutively expressed ADAMTS proteinases but not MMP-15. The inhibition of NF-κB significantly attenuated the TNF-α-and-VEGF-induced upregulation of ADAMTS-1 and -4 in a culture medium of HRMECs and Müller cells. In conclusion, ADAMTS proteinases, MMP-15 and versican and biglycan cleavage products were increased in the ocular microenvironment of patients with PDR.

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

ABSTRACT 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.

Galectin-1 studies in proliferative diabetic retinopathy.

Galectin-1 regulates endothelial cell function and promotes angiogenesis. We investigated the hypothesis that galectin-1 may be involved in the pathogenesis of proliferative diabetic retinopathy (PDR). Vitreous samples from 36 PDR and 20 nondiabetic patients, epiretinal fibrovascular membranes from 13 patients with PDR, rat retinas and human retinal Müller glial cells were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and Western blot analysis. In vitro angiogenesis assays were performed and the adherence of leukocytes to galectin-1-stimulated human retinal microvascular endothelial cells (HRMECs) was assessed. The ELISA analysis revealed that galectin-1 and vascular endothelial growth factor (VEGF) levels were significantly higher in vitreous samples from PDR patients than in those from nondiabetics (p<0.001 for both comparisons). A significant positive correlation was found between the levels of galectin-1 and VEGF (r=0.354; p=0.022). In epiretinal membranes, immunohistochemical analysis showed that galectin-1 was expressed in vascular endothelial cells expressing CD31, myofibroblasts expressing α-smooth muscle actin and leukocytes expressing CD45. The galectin-1 receptor neuropilin-1 was expressed on vascular endothelial cells. CD31 staining was used as a marker to assess microvessel density (MVD). Significant positive correlation was detected between MVD in epiretinal membranes and the number of blood vessels expressing galectin-1 (r=0.848; p<0.001). Western blot analysis demonstrated significant increase of galectin-1 protein in rat retinas after induction of diabetes. ELISA analysis revealed that hydrogen peroxide and cobalt chloride (CoCl2 ) induced upregulation of galectin-1 in Müller cells. Treatment with galectin-1 induced upregulation of VEGF in Müller cells and increased leukocyte adhesion to HRMECs. The galectin-1 inhibitor OTX008 attenuated VEGF-induced HRMECs migration and CoCl2 -induced upregulation of NF-κB, galectin-1 and VEGF in Müller cells. These results suggest that galectin-1is involved in the pathogenesis of PDR.

APOPTOSIS AND ANGIOFIBROSIS IN DIABETIC TRACTIONAL MEMBRANES AFTER VASCULAR ENDOTHELIAL GROWTH FACTOR INHIBITION: Results of a Prospective Trial. Report No. 2.

We sought to characterize the angiofibrotic and apoptotic effects of vascular endothelial growth factor (VEGF)-inhibition on fibrovascular epiretinal membranes in eyes with traction retinal detachment because of proliferative diabetic retinopathy. Membranes were excised from 20 eyes of 19 patients (10 randomized to intravitreal bevacizumab, 10 controls) at vitrectomy. Membranes were stained with antibodies targeting connective tissue growth factor (CTGF) or VEGF and colabeled with antibodies directed against endothelial cells (CD31), myofibroblasts, or retinal pigment epithelium markers. Quantitative and colocalization analyses of antibody labeling were obtained through immunofluorescence confocal microscopy. Masson trichrome staining, cell counting of hematoxylin and eosin sections, and terminal dUTP nick-end labeling staining were performed. High levels of fibrosis were observed in both groups. Cell apoptosis was higher (P = 0.05) in bevacizumab-treated membranes compared with controls. The bevacizumab group had a nonsignificant reduction in colocalization in CD31-CTGF and cytokeratin-VEGF studies compared with controls. Vascular endothelial growth factor in extracted membranes was positively correlated with vitreous levels of VEGF; CTGF in extracted membranes was negatively correlated with vitreous levels of CTGF. Bevacizumab suppresses vitreous VEGF levels, but does not significantly alter VEGF or CTGF in diabetic membranes that may be explained by high baseline levels of fibrosis. Bevacizumab may cause apoptosis within fibrovascular membranes.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) in diabetic retinopathy: latest evidence and clinical considerations.

Diabetic retinopathy (DR) is considered as a diabetes-related complication that can render severe visual impairments and is also a risk factor for acquired blindness in both developed as well as developing countries. Through fibrovascular epiretinal membranes (ERMs), this condition can similarly lead to tractional retinal detachment. Laboratory efforts evaluating the DR pathogenesis can be provided by ocular vitreous fluid and ERMs resulting from vitrectomy. The clinical stages of DR are significantly associated with expression levels of certain chemokines, including monocyte chemotactic protein-1 (MCP-1) in the intraocular fluid. The MCP-1 is also a known potent chemotactic factor for monocytes and macrophages that can stimulate them to produce superoxide and other mediators. Following hyperglycemia, retinal pigmented epithelial (RPE) cells, endothelial cells, and Müller's glial cells are of utmost importance for MCP-1 production, and vitreous MCP-1 levels rise in patients with DR. Increased expression of the MCP-1 in the eyes can also play a significant role in the pathogenesis of DR. In this review, current clinical and laboratory progress achieved on the MCP-1 and the DR concerning neovascularization and inflammatory responses in vitreous and/or aqueous humor of DR patients was summarized. It was suggested that further exploration of the MCP-1/CCR2 axis association between clinical stages of DR and expression levels of inflammatory and angiogenic cytokines and chemokines, principally the MCP-1 might lead to potential therapies aiming at neutralizing antibodies and viral vectors.

Feline Neovascular Vitreoretinopathy and Anterior Segment Dysgenesis With Concurrent Glaucoma in Domestic Cats.

Feline neovascular vitreoretinopathy (FNV) is a newly recognized rare condition affecting kittens and young domestic cats. This study investigated the clinical and pathologic findings in 22 cats with FNV. In affected cats, ophthalmoscopy of the fundus (when visible) revealed avascular peripheral retinae and epiretinal vascular membranes. Frequent nonspecific clinical findings were buphthalmos ( n = 21), medically uncontrollable glaucoma ( n = 22), and lenticular abnormalities ( n = 13). Anterior segment dysgenesis (ASD) was detected clinically in affected cats ( n = 6). The fellow eye was affected in 11 of 18 cats to a variable degree or appeared clinically normal in 7 of 18 cats. The globes were examined histologically and using immunohistochemistry for vimentin, glial fibrillary acidic protein (GFAP), synaptophysin, neurofilament, laminin, factor VIII-related antigen (FVIII-RA), and smooth muscle actin (SMA). Histologically, diagnostic features included laminin-positive epiretinal vascular membranes affecting the central retina, with an avascular peripheral retina and gliosis. Enucleated globes exhibited multiple additional abnormalities, including corneal disease ( n = 15), anterior segment dysgenesis ( n = 21), lymphoplasmacytic anterior uveitis ( n = 19), peripheral anterior synechiae ( n = 20), retinal degeneration ( n = 22), and retinal detachment ( n = 19). Gliotic retinae labeled strongly for GFAP and vimentin with reduced expression of synaptophysin and neurofilament, consistent with degeneration or lack of differentiation. While an avascular peripheral retina and epiretinal fibrovascular membranes are also salient features of retinopathy of prematurity, there is no evidence to support hyperoxic damage in cats with FNV. The cause remains unknown.

Identification of proteins associated with clinical and pathological features of proliferative diabetic retinopathy in vitreous and fibrovascular membranes.

PurposeTo identify the protein profiles in vitreous associated with retinal fibrosis, angiogenesis, and neurite formation in epiretinal fibrovascular membranes (FVMs) in patients with proliferative diabetic retinopathy (PDR).MethodsVitreous samples of 5 non-diabetic control patients with vitreous debris and 7 patients with PDR membranes were screened for 507 preselected proteins using the semi-quantitative RayBio® L-series 507 antibody array. From this array, 60 proteins were selected for a custom quantitative antibody array (Raybiotech, Human Quantibody® array), analyzing 7 control patients, 8 PDR patients with FVMs, and 5 PDR patients without FVMs. Additionally, mRNA levels of proteins of interest were measured in 10 PDR membranes and 11 idiopathic membranes and in retinal tissues and cells to identify possible sources of protein production.ResultsOf the 507 proteins screened, 21 were found to be significantly elevated in PDR patients, including neurogenic and angiogenic factors such as neuregulin 1 (NRG1), nerve growth factor receptor (NGFR), placental growth factor (PlGF) and platelet derived growth factor (PDGF). Angiopoietin-2 (Ang2) concentrations were strongly correlated to the degree of fibrosis and the presence of FVMs in patients with PDR. Protein correlation analysis showed PDGF to be extensively co-regulated with other proteins, including thrombospondin-1 and Ang2. mRNA levels of glial-derived and brain/derived neurotrophic factor (GDNF and BDNF) were elevated in PDR membranes. These results were validated in a second study of 52 vitreous samples of 32 PDR patients and 20 control patients.ConclusionsThis exploratory study reveals protein networks that potentially contribute to neurite outgrowth, angiogenesis and fibrosis in the formation of fibrovascular membranes in PDR. We identified a possible role of Ang2 in fibrosis and the formation of FVMs, and of the neurotrophic factors NRG1, PDGF and GDNF in neurite growth that occurs in all FVMs in PDR.

Protective effect of miR-200b/c by inhibiting vasohibin-2 in human retinal microvascular endothelial cells

AimsProliferative diabetic retinopathy (PDR), characterized by angiogenesis, can cause serve vision loss and even blindness. Recent studies have suggested a pivotal role of vasohibin-2 (VASH2) in the promotion of angiogenesis in tumor tissues. Here we further investigated the role of VASH2 in the proliferation and migration of retinal endothelial cells. Main methodsThe expression of VASH2 in vascular endothelial cells of epiretinal fibrovascular membranes (FVMs) from PDR patients were detected by immunofluorescence. VASH2 gene interfering lentiviral vectors (VASH2-shRNA) and miR-200b/c were constructed for the evaluation of the VASH2 effect on high glucose induced human retinal microvascular endothelial cell line (HRMECs). Cell proliferation, cell cycle and cell migration were carried out subsequently. The relationship between VASH2 and miR-200b/c was determined by luciferase reporter gene assays. Key findingsA positive expression of VASH2 was identified in vascular endothelial cells of FVMs from PDR patients. In HRMECs, cells transfected with shRNA or miR-200b/c mimics showed a significantly reduced VASH2 expression compared with negative control group by real time-polymerase chain reaction and western-blot analysis. Inhibition of VASH2 was demonstrated to suppress cell proliferation and migration from Day 2 to Day 4. The luciferase reporter gene assays confirmed the post-transcriptional regulation of VASH2 by miR-200b/c in HRMECs. SignificanceThe present study suggests a protective effect of miR-200b/c on high glucose induced HRMECs dysfunction by inhibiting VASH2. It could be a potential therapeutic strategy to inhibit angiogenesis for the treatment of retinal vascular disease.

Role of High‐mobility group box‐1 in diabetic retinal vasculopathy and neuropathy

SummaryHigh‐mobility mobility group box‐1 (HMGB1) is a non‐histone DNA‐binding protein that plays an important role in chromatin organization and transcriptional regulation. Extracellular HMGB1 functions as a proinflammatory cytokine and triggers the inflammatory response through the activation of multiple receptors such as the receptor for advanced glycation end products (RAGE), toll‐like receptor‐2 (TLR2), TLR4 and TLR9. We demonstrated that HMGB1 and RAGE were expressed in fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy (PDR). In addition, we demonstrated increased levels of HMGB1 in the vitreous samples from patients with PDR and that there were significant positive correlations between the vitreous levels and HMGB1 and the levels of the biomarkers of inflammation and oxidative injury. Furthermore, we demonstrated that diabetes induced significant upregulation of the expression of HMGB1 and RAGE in the retinas of rats and mice and that HMGB1 mediates diabetes‐induced oxidative stress, activation of inflammatory signaling pathways, breakdown of the blood‐retinal barrier and neuropathy in the retina. Therefore, compounds inhibiting HMGB1 may be novel therapeutic agents for diabetic retinopathy.

Myofibroblasts in proliferative diabetic retinopathy can originate from infiltrating fibrocytes and through endothelial‐to‐mesenchymal transition (EndoMT)

PurposeThe fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) are characterized by the accumulation of a large number of myofibroblasts. We explored the hypothesis that proliferating endothelial cells via endothelial‐to‐mesenchymal transition (EndoMT) and/or bone marrow‐derived circulating fibrocytes contribute to the myofibroblast population present in PDR membranes.MethodsEpiretinal membranes from 14 patients with PDR were studied by immunohistochemistry. In addition, we investigated the phenotypic changes that take place in human retinal microvascular endothelial cells following exposure to transforming growth factor‐β1 (TGF‐ β1), connective tissue growth factor (CTGF) and the proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐a (TNF‐a).ResultsAll membranes contained neovessels expressing the endothelial cell marker CD31. CD31+ endothelial cells co‐expressed the fibroblast/myofibroblast markers fibroblast‐specific protein‐1 (FSP‐1) and a‐SMA, indicative for the occurrence of endoMT. In the stroma, cells expressing FSP‐1, a‐SMA, the leukocyte common antigen CD45, and the myelomonocytic marker CD11b were detected. Double labeling showed co‐localization of CD45 with FSP‐1 and a‐SMA and co‐localization of CD11b with a‐SMA and matrix metalloproteinase‐9, demonstrating the presence of infiltrating fibrocytes. Retinal microvascular endothelial cells changed morphology upon cytokine exposure, lost the expression of endothelial cell markers (endothelial nitric oxide synthase and vascular endothelial‐cadherin) and started to express mesenchymal markers (calponin, snail, transgelin and FSP‐1).ConclusionsThese results suggest that endothelial cells as well as circulating fibrocytes may differentiate into myofibroblasts in the diabetic eye and contribute to pathologic fibrosis in PDR.

Myofibroblasts in proliferative diabetic retinopathy can originate from infiltrating fibrocytes and through endothelial‐to‐mesenchymal transition (EndoMT)

PurposeThe fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) are characterized by the accumulation of a large number of myofibroblasts. We explored the hypothesis that proliferating endothelial cells via endothelial‐to‐mesenchymal transition (EndoMT) and/or bone marrow‐derived circulating fibrocytes contribute to the myofibroblast population present in PDR membranes.MethodsEpiretinal membranes from 14 patients with PDR were studied by immunohistochemistry. In addition, we investigated the phenotypic changes that take place in human retinal microvascular endothelial cells following exposure to transforming growth factor‐β (TGF‐β1), connective tissue growth factor (CTGF) and the proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐a (TNF‐a).ResultsAll membranes contained neovessels expressing the endothelial cell marker CD31. CD31+ endothelial cells co‐expressed the fibroblast/myofibroblast markers fibroblast‐specific protein‐1 (FSP‐1) and a‐SMA, indicative for the occurrence of endoMT. In the stroma, cells expressing FSP‐1, a‐SMA, the leukocyte common antigen CD45, and the myelomonocytic marker CD11b were detected. Double labeling showed co‐localization of CD45 with FSP‐1 and a‐SMA and co‐localization of CD11b with a‐SMA and matrix metalloproteinase‐9, demonstrating the presence of infiltrating fibrocytes. Retinal microvascular endothelial cells changed morphology upon cytokine exposure, lost the expression of endothelial cell markers (endothelial nitric oxide synthase and vascular endothelial‐cadherin) and started to express mesenchymal markers (calponin, snail, transgelin and FSP‐1).ConclusionsThese results suggest that endothelial cells as well as circulating fibrocytes may differentiate into myofibroblasts in the diabetic eye and contribute to pathologic fibrosis in PDR.

Myofibroblasts in proliferative diabetic retinopathy can originate from infiltrating fibrocytes and through endothelial-to-mesenchymal transition (EndoMT)

Myofibroblasts expressing α-smooth muscle actin (α-SMA) are the key cellular mediator of fibrosis. Fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) are characterized by the accumulation of a large number of myofibroblasts. We explored the hypothesis that proliferating endothelial cells via endothelial-to-mesenchymal transition (EndoMT) and/or bone marrow-derived circulating fibrocytes contribute to the myofibroblast population present in PDR epiretinal membranes. Epiretinal membranes from 14 patients with PDR were studied by immunohistochemistry. All membranes contained neovessels expressing the endothelial cell marker CD31. CD31+ endothelial cells co-expressed the fibroblast/myofibroblast markers fibroblast-specific protein-1 (FSP-1) and α-SMA, indicative for the occurrence of endoMT. In the stroma, cells expressing FSP-1, α-SMA, the leukocyte common antigen CD45, and the myelomonocytic marker CD11b were detected. Double labeling showed co-localization of CD45 with FSP-1 and α-SMA and co-localization of CD11b with α-SMA and matrix metalloproteinase-9, demonstrating the presence of infiltrating fibrocytes. In addition, we investigated the phenotypic changes that take place in human retinal microvascular endothelial cells following exposure to transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF) and the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Retinal microvascular endothelial cells changed morphology upon cytokine exposure, lost the expression of endothelial cell markers (endothelial nitric oxide synthase and vascular endothelial-cadherin) and started to express mesenchymal markers (calponin, snail, transgelin and FSP-1). These results suggest that endothelial cells as well as circulating fibrocytes may differentiate into myofibroblasts in the diabetic eye and contribute to pathologic fibrosis in PDR.

A simple method for establishing adherent ex vivo explant cultures from human eye pathologies for use in subsequent calcium imaging and inflammatory studies.

A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes (fvERMs) from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh) stimulation and indicate presence of ACh receptors in these cells. Furthermore, an ex vivo study model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment.

Functional and Molecular Characterization ofEx VivoCultured Epiretinal Membrane Cells from Human Proliferative Diabetic Retinopathy

Characterization of the cell surface marker phenotype of ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherently ex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNFα activation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when cultured ex vivo, which were negative for endothelial cell markers (CD31, VEGFR2), partially positive for hematopoietic- (CD34, CD47) and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFRβ), and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1) were detected upon TNFα activation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDR ex vivo.

Expression of stem cell factor/c‐kit signaling pathway components in diabetic fibrovascular epiretinal membranes

Abstract Purpose Stem cell factor (SCF)/c‐kit signaling promotes recruitment of endothelial progenitor cells and contributes to ischemia‐induced new vessel formation. We investigated the expression of the components of this pathway including c‐kit, SCF, granulocyte colony‐stimulating factor (G‐CSF), endothelial nitric oxide synthase (eNOS), and the chemokine receptor CXCR4 in proliferative diabetic retinopathy (PDR) epiretinal membranes. Methods Membranes from 8 patients with active PDR and 12 patients with inactive PDR were studied by immunohistochemistry. Results Blood vessels expressed c‐kit, SCF, G‐CSF, eNOS, and CXCR4 in 18, 15, 19, 20 and 20 out of 20 membranes, respectively. Significant correlations were detected between number of blood vessels expressing CD34 and number of blood vessels expressing SCF (r=0.463; p=0.04), G‐CSF (r=0.87; p&lt;0.001), eNOS (r=0.864; p&lt;0.001), and CXCR4 (r=0.864; p&lt;0.001). Stromal cells expressed c‐kit, SCF, eNOS, and CXCR4 in 19, 15, 20, and 20 membranes, respectively. The numbers of blood vessels expressing CD34 (p=0.005), c‐kit (p=0.03), G‐CSF (p=0.007), eNOS (p=0.001), and CXCR4 (p=0.018), and stromal cells expressing c‐kit (p=0.013), SCF (p&lt;0.001), eNOS (p=0.048), and CXCR4 (p=0.003) were significantly higher in active membranes than in inactive membranes. Conclusion SCF/c‐kit signaling might contribute to neovascularization in PDR.

Expression of advanced glycation end products and related molecules in diabetic fibrovascular epiretinal membranes

To investigate associations between expressions of advanced glycation end products (AGEs), transforming growth factor-beta (TGF-beta), tumour necrosis factor-alpha (TNF-alpha) and integrins and correlations between their expression and level of vascularization and proliferative activity in diabetic fibrovascular epiretinal membranes. Membranes from eight patients with active proliferative diabetic retinopathy and nine patients with inactive proliferative diabetic retinopathy were studied by immunohistochemistry. Blood vessels expressed AGEs, TGF-beta, TNF-alpha and alpha(v)beta(3) integrin in 5, 13, 8 and 8 membranes, respectively. Stromal cells expressed AGEs, TNF-alpha and alpha(v)beta(3) integrin in 15, 13 and 3 membranes, respectively. There was no immunoreactivity for alpha(v)beta(5), alpha(5)beta(1) and alpha(2)beta(1) integrins. There were significant correlations between number of blood vessels expressing CD34 and number of blood vessels expressing AGEs (r(s) = 0.496; P = 0.043), TGF-beta (r(s) = 0.777; P < 0.001) and TNF-alpha (r(s) = 0.699; P = 0.002). There were significant correlations between number of blood vessels expressing AGEs and number of blood vessels expressing TGF-beta (r(s) = 0.532; P = 0.028) and TNF-alpha (r(s) = 0.626; P = 0.007). The correlation between number of blood vessels expressing TNF-alpha and alpha(v)beta(3) integrin was significant (r(s) = 0.617; P = 0.008). Number of blood vessels expressing CD34 (P = 0.001), TGF-beta (P = 0.006) and TNF-alpha (P = 0.002) and stromal cells expressing AGEs (P = 0.001) and TNF-alpha (P = 0.004) were significantly higher in active membranes than in inactive membranes. Interactions of AGEs, TGF-beta, TNF-alpha and alpha(v)beta(3) integrin might be involved in pathogenesis of proliferative diabetic retinopathy fibrovascular proliferation.