The microenvironment of proliferative diabetic retinopathy supports lymphatic neovascularization.

Proliferative diabetic retinopathy (PDR) is a major diabetic microvascular complication characterized by pathological angiogenesis. Several retinopathy animal models have been developed to study the disease mechanisms and putative targets. However, knowledge on the human proliferative disease remains incomplete, relying on steady-state results from thin histological neovascular tissue sections and vitreous samples. New translational models are thus required to comprehensively understand the disease pathophysiology and develop improved therapeutic interventions. We describe here a clinically relevant model, whereby the native multicellular PDR landscape and neo(fibro)vascular processes can be analysed ex vivo and related to clinical data. As characterized by three-dimensional whole-mount immunofluorescence and electron microscopy, heterogeneity in patient-derived PDR neovascular tissues included discontinuous capillaries coupled with aberrantly differentiated, lymphatic-like and tortuous endothelia. Spatially confined apoptosis and proliferation coexisted with inflammatory cell infiltration and unique vascular islet formation. Ex vivo-cultured explants retained multicellularity, islet patterning and capillary or fibrotic outgrowth in response to vitreoretinal factors. Strikingly, PDR neovascular tissues, whose matched vitreous samples enhanced lymphatic endothelial cell sprouting, contained lymphatic-like capillaries in vivo and developed Prox1+ capillaries and sprouts with lymphatic endothelial ultrastructures ex vivo. Among multiple vitreal components, vascular endothelial growth factor C was one factor found at lymphatic endothelium-activating concentrations. These results indicate that the ischaemia-induced and inflammation-induced human PDR microenvironment supports pathological neolymphovascularization, providing a new concept regarding PDR mechanisms and targeting options. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Development of diabetic macular oedema shows associations with systemic medication - An epidemiological study.

PURPOSE: To identify associations between systemic drugs and the incidence of diabetic macular oedema (DME). Of interest was to find beneficial and/or deleterious associations of used drugs. METHODS: A historic cohort design based on administrative data. Study population consisted of 150 353 individuals with diabetes. Endpoint event was the development of DME (ICD-10 H36.01), censoring events were death or study end December 2017. The follow-up started between 1997 and 2010. The systemic medication consisted of 95 substances. We constructed a nested case-control study design comparing 2630 cases with DME to 13 144 age- and sex-matched controls without DME. Results are reported as odds ratios (ORs) with 95% confidence intervals (CIs) based on conditional logistic regression models. RESULTS: Incidence rate for DME was 1.80 per 1000 person-years (95% CI 1.73-1.87). In all, we observed a lower incidence rate of DME in females (IRR 0.57; 95% CI 0.52-0.62) compared to males. Exposure to hormone replacement therapy estradiol (OR 0.42; 0.25-0.68), temazepam (0.23; 0.08-0.62) and allopurinol (0.61; 0.43-0.86) were associated with lower risk of DME, while use of insulin or insulin analogue (3.30; 2.99-3.64), sulfonylureas (1.21; 1.05-1.40), diuretic furosemide (1.90; 1.61-2.24), calcium channel blocker amlodipine (1.53; 1.34-1.75), ACE inhibitors ramipril (1.66; 1.46-1.89) and enalapril (1.38; 1.16-1.64) were associated with an increased risk of DME. CONCLUSIONS: Large-scale studies examining the incidence of DME are lacking. Our findings suggest that associations of systemic medications with the incidence of DME may shed light on the pathogenesis of complex DME, encouraging further studies.

Risk of glaucoma after vitreoretinal surgery - Findings from a population-based cohort study.

PURPOSE: To investigate the association between different types of vitrectomy and risk of different types of glaucoma and to determine the effect of systemic medication and diabetes status on this risk. METHODS: A population-based nested case-control study included individuals of age ≥ 18 years who had undergone single vitrectomy, vitrectomy with retinal procedure, or combined phaco-vitrectomy between 2001 and 2010. End of follow-up was 2017. Odds ratio (OR) for the development of glaucoma after different types of vitrectomy and 95% confidence interval (CI) were based on conditional logistic regression models. For every glaucoma case, five controls were matched by age, sex, start of follow-up year, and hospital district. RESULTS: The cohort (n = 37 687), of which 52.8% was female, consisted of 6552 individuals diagnosed with glaucoma and 31 135 controls matched by age, sex, and hospital district. Vitrectomy was performed on 103 eyes in the glaucoma group and 158 eyes in the control group. As regards the risk of any glaucoma, the risk was lowest in eyes that underwent combined phaco-vitrectomy (OR: 2.7, 95% CI: 1.8-4.1), followed by single vitrectomy (OR: 3.15, 95% CI: 2.1-4.8), and highest in eyes that underwent vitrectomy with retinal procedure (OR: 4.5, 95% CI: 2.7-7.4). Diabetes had no effect (OR: 0.96, 95% CI: 0.92-1.01), but 5-year systemic statin use slightly decreased glaucoma risk (OR: 0.86, 95% CI: 0.77-0.97). CONCLUSIONS: Vitreoretinal surgery was associated with an increased glaucoma risk; the risk being related to the complexity of vitrectomy. Long-term systemic statin therapy may decrease glaucoma risk, while diabetes had no association.

Proliferative diabetic retinopathy transcriptomes reveal angiogenesis, anti-angiogenic therapy escape mechanisms, fibrosis and lymphatic involvement.

Proliferative diabetic retinopathy (PDR) is a sight-threatening diabetic complication in urgent need of new therapies. In this study we identify potential molecular mechanisms and target candidates in the pathogenesis of PDR fibrovascular tissue formation. We performed mRNA sequencing of RNA isolated from eleven excised fibrovascular membranes of type 1 diabetic PDR patients and two non-diabetic patients with rhegmatogenous retinal detachment with proliferative vitreoretinopathy. We determined differentially expressed genes between these groups and performed pathway and gene ontology term enrichment analyses to identify potential underlying mechanisms, pathways, and regulators. Multiple pro-angiogenic processes, including VEGFA-dependent and -independent pathways, as well as processes related to lymphatic development, epithelial to mesenchymal transition (EMT), wound healing, inflammation, fibrosis, and extracellular matrix (ECM) composition, were overrepresented in PDR. Overrepresentation of different angiogenic processes may help to explain the transient nature of the benefits that many patients receive from current intravitreal anti-angiogenic therapies, highlighting the importance of combinatorial treatments. Enrichment of genes and pathways related to lymphatic development indicates that targeting lymphatic involvement in PDR progression could have therapeutic relevance. Together with overrepresentation of EMT and fibrosis as well as differential ECM composition, these findings demonstrate the complexity of PDR fibrovascular tissue formation and provide avenues for the development of novel treatments.

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Soluble and membrane-bound adenylate kinase and nucleotidases augment ATP-mediated inflammation in diabetic retinopathy eyes with vitreous hemorrhage.

ATP and adenosine are important signaling molecules involved in vascular remodeling, retinal function, and neurovascular coupling in the eye. Current knowledge on enzymatic pathways governing the duration and magnitude of ocular purinergic signaling is incompletely understood. By employing sensitive analytical assays, this study dissected ocular purine homeostasis as a complex and coordinated network. Along with previously characterized ecto-5'-nucleotidase/CD73 and adenylate kinase activities, other enzymes have been identified in vitreous fluids, including nucleoside triphosphate diphosphohydrolase (NTPDase), adenosine deaminase, and alkaline phosphatase. Strikingly, activities of soluble adenylate kinase, adenosine deaminase, ecto-5'-nucleotidase/CD73, and alkaline phosphatase, as well as intravitreal concentrations of ATP and ADP, were concurrently upregulated in patients suffering from diabetic retinopathy (DR) with non-clearing vitreous hemorrhage (VH), when compared to DR eyes without VH and control eyes operated due to macular hole or pucker. Additional histochemical analysis revealed selective distribution of key ecto-nucleotidases (NTPDase1/CD39, NTPDase2, ecto-5'-nucleotidase/CD73, and alkaline phosphatase) in the human sensory neuroretina and optic nerve head, and also in pathological neofibrovascular tissues surgically excised from patients with advanced proliferative DR. Collectively, these data provide evidence for specific hemorrhage-related shifts in purine homeostasis in DR eyes from the generation of anti-inflammatory adenosine towards a pro-inflammatory and pro-angiogenic ATP-regenerating phenotype. In the future, identifying the exact mechanisms by which a broad spectrum of soluble and membrane-bound enzymes coordinately regulates ocular purine levels and the further translation of purine-converting enzymes as potential therapeutic targets in the treatment of proliferative DR and other vitreoretinal diseases will be an area of intense interest. KEY MESSAGES: NTPDase, alkaline phosphatase, and adenosine deaminase circulate in human vitreous. Purinergic enzymes are up-regulated in diabetic eyes with vitreous hemorrhage. Soluble adenylate kinase maintains high ATP levels in diabetic retinopathy eyes. Ecto-nucleotidases are co-expressed in the human retina and optic nerve head. Alkaline phosphatase is expressed on neovascular tissues excised from diabetic eyes.