Idelalisib inhibits vitreous-induced Akt activation and proliferation of retinal pigment epithelial cells from epiretinal membranes.

Proliferative vitreoretinopathy (PVR) is a blinding fibrotic eye disease that develops in 8-10% of patients who undergo primary retinal detachment-reparative surgery and in 40-60% of patients with open-globe injury. At present, there is no pharmacological treatment for this devastating disease. Vitreal growth factors activate their respective receptors of cells in the vitreous, trigger their downstream signaling transduction (e.g. phosphoinositide 3 kinases (PI3Ks)/Akt), and drive cellular responses intrinsic to the pathogenesis of PVR. PI3Ks play a central role in experimental PVR. However, which isoform(s) are involved in PVR pathogenesis remain unknown. Herein, we show that p110δ, a catalytic subunit of receptor-regulated PI3K isoform δ, is highly expressed in epiretinal membranes from patients with PVR, and that idelalisib, a specific inhibitor of PI3Kδ, effectively inhibits vitreous-induced Akt activation, proliferation, migration and contraction of retinal pigment epithelial cells derived from an epiretinal membrane of a PVR patient. Small molecules of kinase inhibitors have shown great promise as a class of therapeutics for a variety of human diseases. The data herein suggest that idelalisib is a promising PVR prophylactic.

Involvement of premacular mast cells in the pathogenesis of macular diseases.

We previously reported on the elevated intravitreal activities of tryptase and chymase in association with idiopathic epiretinal membrane (ERM) and idiopathic macular hole (MH). In this present study, we investigated the potential intraocular production of these serine proteases, and measured and compared tryptase and chymase activities in the vitreous body and serum in ERM, MH, proliferative diabetic retinopathy (PDR), and rhegmatogenous retinal detachment (RRD) patients. In addition, nuclear staining with hematoxylin and eosin (H&E) and mast-cell staining with toluidine blue were performed on samples of the vitreous core and bursa premacularis (BPM) of MH. We also performed immunostaining on the above two regions of vitreous samples for MH with anti-tryptase antibody, anti-chymase antibody, anti-podoplanin antibody, anti-lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) antibody, and anti-fibroblast antibody. Moreover, we performed immunostaining with anti-tryptase antibody and anti-chymase antibody on ERMs collected intraoperatively. Tryptase activity in the vitreous body was significantly higher in ERM and MH than in PDR. However, no significant differences were observed in the tryptase activity in the serum among these four diseases. Chymase activity in the vitreous body was significantly higher in MH than in the other three diseases, yet chymase activity in the serum was below detection limit in any of the diseases. Nuclear staining with H&E revealed an abundance of nuclei in the BPM region, but few in the surrounding area. Mast-cell staining with toluidine blue revealed that the BPM showed metachromatic staining. In immunostaining with anti-fibroblasts antibody, anti-tryptase antibody, anti-chymase antibody, anti-podoplanin antibody, and anti-LYVE-1 antibody, the BPM stained more strongly than the vitreous core. Tryptase and chymase-positive cells were also observed in ERM. These findings revealed that the presence of mast cells in the BPM potentially represent the source of these serine proteases. Moreover, the BPM, as a lymphatic tissue, may play an important role in the pathogenesis of macular disease.

Expression of autotaxin and acylglycerol kinase in proliferative vitreoretinal epiretinal membranes.

PURPOSE: Lysophosphatidic acid (LPA)/LPA(1) 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 LPA(1) 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 LPA(1) receptor was noted in vascular endothelial cells and stromal cells in the five membranes stained for LPA(1) 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 LPA(1) receptor. CONCLUSIONS: The LPA/LPA(1) receptor pathway may be involved in inflammatory, angiogenic and fibrotic responses in proliferative vitreoretinal disorders.

Cyclo-oxygenase-2 expression in human idiopathic epiretinal membrane.

PURPOSE: The purpose of this study was to examine the expression of cyclo-oxygenase (COX)-2 in the idiopathic epiretinal membrane (IERM), inner limiting membrane (ILM), and proliferative diabetic retinopathy membrane. METHODS: Twenty membranes, consisting of eight IERMs, four ILMs, and eight proliferative diabetic retinopathy membranes, were surgically removed. Formalin-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry using anti-COX-2 antibody. The nuclear density showing the density of cells situated in IERM and ILM specimens was calculated under high-power fields using a light microscope. RESULTS: The IERM comprised flattened cells with oval nuclei constituting a monolayer. The ILM contained a few cells with abundant collagenous tissues. Neither endothelial nor inflammatory cells were observed in the IERM and ILM. COX-2 immunoreactivity was markedly detected in cells located in the IERM. In contrast, COX-2 immunoreactivity was faintly detected in the ILM. The COX-2-positive rate was 65.4 +/- 15.5% and 34.3 +/- 20.3% in the IERM and ILM, respectively, being significantly higher in the former (P = 0.046). The nuclear density was 39.3 +/- 10.3 and 8.6 +/- 7.2 in the IERM and ILM, respectively, being significantly higher in the former (P = 0.0003). The proliferative diabetic retinopathy membranes consisted of many vascular endothelial and stromal cells. Cytoplasmic immunoreactivity for COX-2 was detected in endothelial and stromal cells in the proliferative diabetic retinopathy membranes. CONCLUSION: These results suggest that COX-2 plays a potential role in the formation of avascular and vascularized epiretinal membranes if an epiphenomenon of COX-2 expression within these epiretinal membranes has been ruled out in future studies.

Expression of cyclo-oxygenase-2 and downstream enzymes in diabetic fibrovascular epiretinal membranes.

BACKGROUND/AIMS: The inducible enzyme cyclo-oxygense-2 (COX-2) and its metabolic products are important mediators for angiogenesis. We investigated the expression of COX-2 and its downstream enzymes microsomal prostaglandin-E synthase (mPGES)-1, cytosolic PGES (cPGES) and thromboxane synthase (TXS), and correlated it with vascular endothelial growth factor (VEGF) expression and level of vascularisation in proliferative diabetic retinopathy (PDR) epiretinal membranes. METHODS: Membranes from five patients with active PDR and nine patients with inactive PDR were studied by immunohistochemistry. RESULTS: Vascular endothelial cells expressed COX-2, mPGES-1 and VEGF in 11, nine and six membranes, respectively. TXS was expressed in stromal cells in 12 membranes. There was no immunoreactivity for cPGES. There were significant correlations between number of blood vessels expressing CD34 and number of blood vessels expressing COX-2 (r(s) = 0.858; p<0.001), mPGES-1 (r(s) = 0.743; p = 0.002) and VEGF (r(s) = 0.845; p = 0.001) and number of cells expressing TXS (r(s) = 0.74; p = 0.002). The number of blood vessels expressing CD34 (p = 0.007), COX-2 (p = 0.027) and VEGF (p = 0.008) and stromal cells expressing mPGES-1 (p = 0.003), TXS (p = 0.04) and VEGF (p = 0.017) were significantly higher in active membranes than in inactive membranes. CONCLUSION: COX-2 and its metabolic products might contribute to PDR angiogenesis.

Expression of glutamine synthetase and cell proliferation in human idiopathic epiretinal membrane.

BACKGROUND/AIM: The mechanisms of the cellular origin and cell proliferation in the idiopathic epiretinal membrane (ERM) are unsolved. The aim of this study was to examine the expression of cell cycle related molecules and glutamine synthetase (GS), which is expressed in Müller cells and their processes, in ERM tissues. METHODS: The ERMs were surgically removed using pars plana vitrectomy. Formalin fixed, paraffin embedded ERM tissues were analysed by immunohistochemistry with anti-cyclin D1, p27 (KIP1), proliferating cell nuclear antigen (PCNA), and GS antibodies. RESULTS: The histopathological findings showed that all the ERMs consisted of oval or spindle mononuclear cells with thin collagen-like tissues. Immunoreactivity for GS was detected in collagen-like tissues of ERM, presenting a continuous, isodense pattern. GS immunopositive cells in all cases expressed PCNA in their nuclei. Nuclear immunoreactivity for cyclin D1 was noted in the ERM constituent cells, whereas p27 (KIP1) positive nuclei were not detected. CONCLUSION: Cyclin D1 and PCNA were expressed in the idiopathic ERM, which was mainly derived from Müller cells and extensions of their processes.

Measurement of activities in two different angiotensin II generating systems, chymase and angiotensin-converting enzyme, in the vitreous fluid of vitreoretinal diseases: a possible involvement of chymase in the pathogenesis of macular hole patients.

PURPOSE: To investigate possible involvement of chymase and angiotensin-converting enzyme (ACE) in the pathogenesis of vitreoretinal diseases, both of which are related to the production of angiotensin II. METHODS: We measured chymase and ACE activities in the vitreous in the 54 affected eyes of 54 patients who had undergone vitreous surgery for idiopathic macular holes (MH, n = 14), proliferative diabetic retinopathy (PDR, n = 14), idiopathic epiretinal membranes (ERM, n = 13), and rhegmatogenous retinal detachment (RRD, n = 13). RESULTS: Chymase activities in the vitreous from patients with MH, PDR, ERM, and RRD were 1.87 +/- 0.53, 0.06 +/- 0.04, 0.40 +/- 0.12, and 0.08 +/- 0.03 (mean +/- SE) mU/mg protein, respectively, and ACE activities in the vitreous humor were 0.18 +/- 0.09, 0.30 +/- 0.07, 0.01 +/- 0.01, and 0.03 +/- 0.02 (mean +/- SE) mU/mg protein, respectively. Chymase activity was significantly elevated in MH among these diseases (p < 0.01, Scheffe), and ACE was significantly activated in PDR compared to ERM and RRD (p < 0.05, Scheffe). CONCLUSIONS: Our results suggest that two different angiotensin II generating systems are activated in human vitreous humor; an increased activity of chymase may play a possible role in the formation of macular holes.

Matrix metalloproteinases in human diabetic and nondiabetic vitreous.

PURPOSE: To compare matrix metalloproteinase (MMP) activities in human vitreous samples from patients with diabetic retinopathy (DR) and other vitreoretinal diseases, and to investigate the factors influencing the MMP activities in human DR vitreous samples. METHODS: Thirty-one diabetic and 17 nondiabetic vitreous samples (from nine patients with macular holes and eight patients with epiretinal membranes) were examined. Samples collected at the time of pars plana vitrectomy were subjected to substrate zymography to conduct a quantitative analysis of MMP activity. Immunoblotting against antihuman MMP-1, 2, and 9 was performed to identify MMP in vitreous samples. The effects of posterior vitreous detachment (PVD), vitreous hemorrhage, proliferative membrane, traction detachment, and cystoid macular edema on MMP activities were investigated. RESULTS: All vitreous samples from both DR and non-DR patients showed a single band at the position of 72 kD, corresponding to MMP-2. Another band at 99 kD, corresponding to MMP-9, was detected significantly more often in DR samples than in non-DR samples: 45.2% and 0%, respectively (P = 0.0007). The number of samples showing a band from MMP-9 was significantly higher in partial PVD samples than in complete PVD samples: 66.7% and 15.4%, respectively (P = 0.001). CONCLUSION: The results indicated that MMP-9 may be involved in DR and that partial PVD may be related to the MMP-9 activity in DR.

Matrix metalloproteinases and their natural inhibitors in fibrovascular membranes of proliferative diabetic retinopathy.

AIM: To examine epiretinal membranes of proliferative diabetic retinopathy (PDR) for the presence of selective matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), in order to determine whether neovascularisation and fibrosis, characteristic of this complication of diabetes mellitus, are associated with specific anomalies of MMP or TIMP expression. METHODS: The presence of selected MMPs and TIMPs was investigated in 24 fibrovascular epiretinal membranes of PDR, and the findings compared with that observed in 21 avascular epiretinal membranes of proliferative vitreoretinopathy (PVR) and five normal retinas. Specimens were examined for deposition of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), and three tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, and TIMP-3). RESULTS: The results showed that unlike normal retina, which constitutively expresses MMP-1 and TIMP-2, a large proportion of PDR membranes (> 62%) stained for MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, and TIMP-3. There were no differences in the expression of these molecules when compared with PVR membranes. A characteristic staining for MMP-9 was observed within the perivascular matrix of PDR membranes, and there was a significant increase in TIMP-2 expression by PDR membranes (p= 0.036) when compared with PVR membranes. CONCLUSIONS: The findings that MMPs involved in degradation of fibrovascular tissue matrix, as well as TIMP-1 and TIMP-2, are found in a large proportion of PDR membranes, and that their expression does not differ from that of PVR membranes, suggest the existence of common pathways of extracellular matrix degradation in pathological processes leading to retinal neovascularisation and fibrosis.

Human diabetic neovascular membranes contain high levels of urokinase and metalloproteinase enzymes.

PURPOSE: Retinal neovascularization is one of the leading causes of blindness. A crucial event in this process is the remodeling and penetration of the capillary basement membrane by migrating endothelial cells. This process requires proteolysis of basement membrane components by a variety of proteinases. The objective of the present study was to determine the expression of proteinases in human retinal tissues showing active neovascularization. METHODS: Epiretinal neovascular membranes surgically removed from patients with proliferative diabetic retinopathy were analyzed by zymography, and the types and amounts of proteinases present in the tissues were determined. Retinas from nondiabetic donor eyes served as control specimens. RESULTS: Both the high- (54 kDa) and low- (33 kDa) molecular-weight forms of urokinase were present at significantly higher levels in neovascular membranes than in normal retinas. The pro forms of the matrix metalloproteinases (MMP) MMP-2 and MMP-9 were significantly elevated in the neovascular membranes in comparison with levels in normal retinas. In addition, the active forms of these enzymes were present in the membranes, whereas there was no detectable level of the active forms in normal retinas. CONCLUSIONS: Human diabetic neovascular membranes contain high levels of urokinase and MMP. The increased activity of proteinases in the final common pathway of retinal neovascularization indicates that inhibition of these enzymes may be a useful therapeutic target as an alternative approach in the management of proliferative retinopathies.