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Purpose: The COMRADE studies are the first randomized controlled head‐to‐head trials comparing the efficacy and safety of intravitreal ranibizumab versus dexamethasone (DEX) in patients with macular oedema secondary to retinal vein occlusion (RVO). The COMRADE extension trial was designed to provide additional 6‐month data of patients who completed the core studies. Methods: In this open‐label, phase IV study patients who completed the COMRADE core studies were prospectively enrolled. Overall, 92 branch RVO (BRVO) patients (ranibizumab 52, DEX 40) and 83 central RVO (CRVO) patients (ranibizumab 61, DEX 22) were treated, and 94.6% of BRVO patients and 97.6% of CRVO patients completed the extension study. Patients were assigned to the same treatment group as in the core studies. Patients were monitored monthly and received either 0.5 mg ranibizumab or a 0.7 mg DEX implant as needed. Results: Over the course of the extension, treatment‐emergent adverse events (TEAEs) of the study eye occurred in 55.8% of BRVO patients on ranibizumab and in 62.5% of those on DEX. Among CRVO patients, 65.5% in the ranibizumab group and 59.1% in the DEX group developed TEAEs. Overall, elevated intraocular pressure (IOP) was more frequent with DEX than ranibizumab treatment. Mean average change in best‐corrected visual acuity (BCVA) in BRVO patients was significantly better for ranibizumab than DEX (p = 0.0249). The CRVO results were consistent with BRVO's, although not significant (p = 0.1119). Conclusion: When used according to the European labels, ranibizumab revealed a better ocular safety profile and produced greater average BCVA gains than DEX. By the end of the additional 6‐month study period, this difference in BCVA was more pronounced in BRVO as in CRVO patients. The main limitation of the COMRADE studies was that DEX patients received only a single intravitreal treatment during the first 6 months, which is presumably not adequate. However, frequent DEX implants could lead to more steroid‐related side effects, especially to an increased intraocular pressure. [ABSTRACT FROM AUTHOR]

Background/Aims: Hallmark of diabetic macular edema is the enhanced permeability of retinal endothelial cells (REC) induced by vascular endothelial growth factor (VEGF-A165), which acts through activating specific receptors. To improve the predictability of inhibitors' potentials to block harmful effects of VEGF-A165, we investigated if its signaling pathways triggered in REC are redundant. Methods: Immortalized bovine REC monolayers were treated with inhibitors specific for various protein kinases in combination with VEGF-A165. Permeability was monitored continuously by measurements of the cell index (Cl) to reveal even subtle and transient changes. Expression of tight junction (TJ) proteins was determined as additional indicator of barrier stability. Results: After a sharp but transient Cl drop caused by VEGF-A155 early after its addition, further exposure resulted in a continuous Cl decline over several days associated with loss of TJ protein claudin-1. Both phases were blocked by inhibition of VEGF receptor 2. Tested inhibitors of intracellular kinases had a limited or no effect, or were efficient only in certain phases of exposure to VEGF-A165, e.g. inhibiting protein kinase C only prevented the early response. High concentrations of some inhibitors even resulted in VEGF-independent barrier destabilization. Conclusions: Specific kinase inhibitors differently affect VEGF-A165-triggered processes in distinct phases of its action. VEGF-A165-initiated signaling is redundant and blocking of key proteins of single pathways is not sufficient to suppress REC barrier breakdown. [ABSTRACT FROM AUTHOR]

Background/Aims: Hallmark of diabetic macular edema is the enhanced permeability of retinal endothelial cells (REC) induced by vascular endothelial growth factor (VEGF-A165), which acts through activating specific receptors. To improve the predictability of inhibitors' potentials to block harmful effects of VEGF-A165, we investigated if its signaling pathways triggered in REC are redundant. Methods: Immortalized bovine REC monolayers were treated with inhibitors specific for various protein kinases in combination with VEGF-A165. Permeability was monitored continuously by measurements of the cell index (Cl) to reveal even subtle and transient changes. Expression of tight junction (TJ) proteins was determined as additional indicator of barrier stability. Results: After a sharp but transient Cl drop caused by VEGF-A155 early after its addition, further exposure resulted in a continuous Cl decline over several days associated with loss of TJ protein claudin-1. Both phases were blocked by inhibition of VEGF receptor 2. Tested inhibitors of intracellular kinases had a limited or no effect, or were efficient only in certain phases of exposure to VEGF-A165, e.g. inhibiting protein kinase C only prevented the early response. High concentrations of some inhibitors even resulted in VEGF-independent barrier destabilization. Conclusions: Specific kinase inhibitors differently affect VEGF-A165-triggered processes in distinct phases of its action. VEGF-A165-initiated signaling is redundant and blocking of key proteins of single pathways is not sufficient to suppress REC barrier breakdown. [ABSTRACT FROM AUTHOR]

Purpose: The bradykinin 1 receptor may be important in inflammatory retinal vascular leakage in diabetic macular edema. BI 1026706 is an antagonist of bradykinin 1 receptor that has demonstrated efficacy in preclinical studies. Boehringer Ingelheim trial 1320.22 (NCT02732951) was a randomized, double-blind, placebo-controlled study. The pharmacodynamics, safety, and tolerability of oral BI 1026706 for 12 weeks were evaluated in patients with type 1 or type 2 diabetes mellitus and mild visual impairment owing to center-involved diabetic macular edema., Methods: Patients ( n = 105) were randomized to receive either oral BI 1026706 100 mg twice daily (morning and evening) or placebo for 12 weeks. The primary end point of the study was week 12 change from baseline in central subfield foveal thickness (CSFT) by spectral domain optical coherence tomography. Additional end points included absolute CSFT values, safety, and pharmacokinetics., Results: After 12 weeks of treatment, there was no meaningful change from baseline in the adjusted mean CSFT in either treatment group (BI 1026706, 10.3 µm; placebo, -6.2 µm; adjusted mean treatment difference, 16.5 µm [95% confidence interval, -16.2 to 49.1]). There were also no differences in best-corrected visual acuity outcomes between treatment groups. Most reported adverse events were of mild or moderate intensity, and were balanced between treatment groups., Conclusions: BI 1026706 was not superior to placebo in CSFT week-12 change from baseline. Therefore, BI 1026706 does not reduce CSFT, a morphologic sign of diabetic macular edema., Translational Relevance: Kinin-kallikrein inhibition effects may not be apparent over 12 weeks for bradykinin 1 receptor inhibition alone., Competing Interests: Disclosure: G.E. Lang, Novartis Pharma GmbH (F, C, S), Allergan (C, S), Alcon Pharma GmbH (C, S), Bayer AG (F, S), Boehringer Ingelheim (C, S), Carl Zeiss Meditec (F, S), Alimera Sciences (S); R. Tadayoni, Alcon Laboratories Inc. (C), Alimera Sciences, Inc. (C), Allergan (C), Bausch & Lomb (C), Bayer HealthCare Pharmaceuticals (C), Carl Zeiss Meditec (C), Chibret International (C), FCI Ophthalmics (C), Genentech (C), Moria (C), Novartis (C), Roche (C), ThromboGenics, Inc. (C); W. Tang, Boehringer Ingelheim (E); C. Barth, Boehringer Ingelheim (E); C. Weiss-Haljiti, Boehringer Ingelheim (E); V. Chong, Boehringer Ingelheim (E), (Copyright 2020 The Authors.)

Purpose: Pathogenesis of diabetic macular edema is driven by deregulated expression of VEGF. A study of long-term exposure of immortalized bovine retinal endothelial cells (iBRECs) to VEGF(165) clearly confirmed the role of the tight junction protein claudin-1, which almost completely disappeared within 24 hours, an effect that was completely reversed by addition of the VEGF-binding Fab fragment ranibizumab. This study was conducted to investigate whether the VEGF(165)-induced loss of claudin-1 is regulated by protein kinase C (PKC) and indeed affects the barrier function of iBRECs., Methods: The effects of various PKC inhibitors on claudin-1 expression and cellular localization in iBRECs treated with VEGF(165) for up to 2 days were studied by Western blot analyses and immunofluorescence microscopy. The permeability of the cell layers was determined by transendothelial electrical resistance measurements., Results: Activation of PKC led to decreased expression of claudin-1, which was blocked by inhibitors of PKCdelta. However, none of the PKC inhibitors significantly affected VEGF(165)-induced effects on cellular localization or expression of claudin-1. Also VEGF(165)-induced higher permeability of iBREC layers could be reversed or prevented by ranibizumab but not by PKC inhibitors. In addition, low claudin-1 expression and its delocalization from the plasma membrane were significantly associated with elevated permeability., Conclusions: In iBRECs, PKC isoforms are not crucially involved in the VEGF(165)-initiated signal transduction that affects permeability and expression of claudin-1. This finding is in contrast to published results concerning only short-term effects of VEGF(165). The results also confirmed that claudin-1 is a highly relevant component of functional tight junctions in retinal endothelial cells.

Background: Diabetic retinopathy and microalbuminuria are often thought of as distinct disease entities despite their common pathophysiology. Many studies have addressed the prognostic significance of these conditions and their treatment., Methods: Medline was selectively searched for articles published from 1948 to 2008 containing the terms "angiotensin," "microalbuminuria," and "retinopathy." The results were further amplified by screening the reference sections of the retrieved articles., Results: Diabetic retinopathy and microalbuminuria are expressions of microvascular damage. They are promoted by hypertension, hyperglycemia, dyslipidemia, and elevated levels of angiotensin II. They are treated by adjusting these risk factors to the near-normal range. In the IDNT study, angiotensin II blockade with irbesartan was found to lead to an absolute reduction of renal events by 7.4% as compared to standard treatment, and by 9.5% as compared to amlodipine. In the DIRECT study, candesartan reduced the progression of retinopathy by 13% and effected a regression by 34%. In the Steno-2 study, an intensive program of multifactorial risk reduction significantly lowered the rate of microvascular complications over a mean follow-up interval of 3.8 years (hazard ratios for different complications varying from 0.27 to 0.45). Over the longer term (13.3 years), this approach also led to a reduction of macrovascular events (HR 0.54 for mortality of all causes, 0.43 for cardiovascular mortality, and 0.41 for cardiovascular events)., Conclusions: Diabetic retinopathy and microalbuminuria are expressions of microvascular damage. They often appear together and point toward possible future macrovascular events. Multifactorial intervention can lessen the consequences of these pathological conditions.

Members of the tetraspanin protein family are modulators of several fundamental cellular processes in various cell types. However, expression and function of these proteins have not been studied in microvascular endothelial cells despite their (patho-)physiological importance. Western blotting, FACS or RT-PCR analyses confirmed that CD9 and other tetraspanins are expressed in immortalized microvascular endothelial cells of the bovine retina (iBREC). In subconfluent cultures, most of the detected CD9 was located intracellularly as well as in the plasma membrane at cell-cell contact sites and in long spike-like extensions, whereas cells in confluent cultures predominantly showed plasma membrane staining. In wound healing assays, CD9 delocalized from the plasma membrane to its intracellular compartment in cells located at the gap border, and the gap closure was retarded by the addition of an anti-CD9 antibody. Migration of iBREC towards fibronectin and their adhesion to fibronectin were also strongly inhibited in the presence of an anti-CD9 antibody whereas other anti-tetraspanin antibodies had no effect. In summary, iBREC express members of the tetraspanin family of which CD9 was demonstrated to have a function in migration and adhesion of these cells.

Transforming growth factor beta (TGFbeta) both inhibits proliferation of macrovascular endothelial cells and promotes their transdifferentiation to alpha-smooth-muscle-actin (alphaSMA)-expressing mesenchymal cells in vitro. Recently, we have confirmed that proliferation of immortalized bovine retinal microvascular endothelial cells (iBREC) is strongly inhibited by TGFbeta2. We now demonstrate a complete transition of both parental iBREC and single cell-derived subclones from cobblestone morphology to a ragged appearance as a consequence of incubation for a few days with 10 ng/ml TGFbeta1 or TGFbeta2. Depending on the type of culture medium, 5-40% of these cells strongly expressed alphaSMA after approximately 6 days whereas expression of the endothelial cell-specific marker proteins von Willebrand factor and VE Cadherin (CD144) declined. Expression of alphaSMA, associated with formation of stress fibers, was first detected in single cells and then spread to adjacent cells, and declined slowly after prolonged cultivation in medium without TGFbeta2. However, re-constitution of vWF expression was not observed. TGFbeta2-induced phenotypic alterations were specific, as they were not caused by treatment of iBREC with VEGF, IGF-1 or bFGF. Induction of alphaSMA expression but not effects on morphology was strongly inhibited by bFGF, whereas IGF-1 enhanced TGFbeta2-induced alphaSMA expression. These findings may have an important impact on the understanding of development of microvascular complications of diabetes such as diabetic retinopathy.

Primary retina-derived endothelial cells isolated from bovine and other animal tissues are extremely useful for studying the molecular mechanisms involved in the pathogenesis of diabetic retinopathy. Preparations of primary bovine retinal endothelial cells (BREC), however, usually contain a number of contaminating non-endothelial cells, e.g. pericytes which have been shown to inhibit proliferation of endothelial cells. To overcome this major drawback, we immortalized BREC by reconstitution of telomerase activity as a consequence of CMV promoter-driven expression of the catalytic domain of human telomerase. Immortal BREC (iBREC) have the capacity to proliferate for >90 passages over 10 months and constantly express endothelial marker proteins. In addition, iBREC behave like BREC with respect to stimulation of proliferation with VEGF, IGF-1 and bFGF, indicating that we have generated a stable endothelial cell line closely related to the corresponding primary cells.