Possible retinotoxicity of long-term vardenafil treatment.

Phosphodiesterase (PDE) inhibitors - such as vardenafil - are used primarily for treating erectile dysfunction via increasing cyclic guanosine monophosphate (cGMP) levels. Recent studies have also demonstrated their significant cardioprotective effects in several diseases, including diabetes, upon long-term, continuous application. However, PDE inhibitors are not specific for PDE5 and also inhibit the retinal isoform. A sustained rise in cGMP in photoreceptors is known to be toxic; therefore, we hypothesized that long-term vardenafil treatment might result in retinotoxicity. The hypothesis was tested in a clinically relevant animal model of type 2 diabetes mellitus. Histological experiments were performed on lean and diabetic Zucker Diabetic Fatty rats. Half of the animals were treated with vardenafil for six months, and the retinal effects were evaluated. Vardenafil treatment alleviated rod outer segment degeneration but decreased rod numbers in some positions and induced changes in the interphotoreceptor matrix, even in control animals. Vardenafil treatment decreased total retinal thickness in the control and diabetic groups and reduced the number of nuclei in the outer nuclear layer. Müller cell activation was detectable even in the vardenafil-treated control animals, and vardenafil did not improve gliosis in the diabetic group. Vardenafil-treated animals showed complex retinal alterations with improvements in some parameters while deterioration in others. Our results point towards the retinotoxicity of vardenafil, even without diabetes, which raises doubts about the retinal safety of long-term continuous vardenafil administration. This effect needs to be considered when approving PDE inhibitors for alternative indications.

Imaging mass cytometry for high-dimensional tissue profiling in the eye.

BACKGROUND: Imaging mass cytometry (IMC) combines the principles of flow cytometry and mass spectrometry (MS) with laser scanning spatial resolution and offers unique advantages for the analysis of tissue samples in unprecedented detail. In contrast to conventional immunohistochemistry, which is limited in its application by the number of possible fluorochrome combinations, IMC uses isoptope-coupled antibodies that allow multiplex analysis of up to 40 markers in the same tissue section simultaneously. METHODS: In this report we use IMC to analyze formalin-fixed, paraffin-embedded conjunctival tissue. We performed a 18-biomarkers IMC analysis of conjunctival tissue to determine and summarize the possibilities, relevance and limitations of IMC for deciphering the biology and pathology of ocular diseases. RESULTS: Without modifying the manufacturer's protocol, we observed positive and plausible staining for 12 of 18 biomarkers. Subsequent bioinformatical single-cell analysis and phenograph clustering identified 24 different cellular clusters with distinct expression profiles with respect to the markers used. CONCLUSIONS: IMC enables highly multiplexed imaging of ocular samples at subcellular resolution. IMC is an innovative and feasible method, providing new insights into ocular disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics.

Transcriptional Profiling Identifies Prognostic Gene Signatures for Conjunctival Extranodal Marginal Zone Lymphoma.

This study characterizes the transcriptional profile and the cellular tumor microenvironment of conjunctival extranodal marginal zone lymphoma (EMZL) and identifies prognostically relevant biomarkers. Ten formalin-fixed and paraffin-embedded conjunctival EMZL and eight healthy conjunctival specimens were analyzed by Massive Analysis of cDNA Ends (MACE) RNA sequencing. The 3417 upregulated genes in conjunctival EMZL were involved in processes such as B cell proliferation and Rac protein signaling, whereas the 1188 downregulated genes contributed most significantly to oxidative phosphorylation and UV protection. The tumor microenvironment, as determined by deconvolution analysis, was mainly composed of multiple B cell subtypes which reflects the tumor's B cell lineage. However, several T cell types, including T helper 2 cells and regulatory T cells, as well as innate immune cell types, such as anti-inflammatory macrophages and plasmacytoid dendritic cells, were also strongly enriched in conjunctival EMZL. A 13-biomarker prognostic panel, including S100A8 and S100A9, classified ocular and extraocular tumor recurrence, exceeded prognostic accuracy of Ann Arbor and American Joint Committee on Cancer (AJCC) staging, and demonstrated prognostic value for patient survival in 21 different cancer types in a database of 12,332 tumor patients. These findings may lead to new options of targeted therapy and may improve prognostic prediction for conjunctival EMZL.

Single-Cell Protein and Transcriptional Characterization of Epiretinal Membranes From Patients With Proliferative Vitreoretinopathy.

Purpose: Proliferative vitreoretinopathy (PVR) remains an unresolved clinical challenge and can lead to frequent revision surgery and blindness vision loss. The aim of this study was to characterize the microenvironment of epiretinal PVR tissue, in order to shed more light on the complex pathophysiology and to unravel new treatment options. Methods: A total of 44 tissue samples were analyzed in this study, including 19 epiretinal PVRs, 13 epiretinal membranes (ERMs) from patients with macular pucker, as well as 12 internal limiting membranes (ILMs). The cellular and molecular microenvironment was assessed by cell type deconvolution analysis (xCell), RNA sequencing data and single-cell imaging mass cytometry. Candidate drugs for PVR treatment were identified in silico via a transcriptome-based drug-repurposing approach. Results: RNA sequencing of tissue samples demonstrated distinct transcriptional profiles of PVR, ERM, and ILM samples. Differential gene expression analysis revealed 3194 upregulated genes in PVR compared with ILM, including FN1 and SPARC, which contribute to biological processes, such as extracellular matrix (ECM) organization. The xCell and IMC analyses showed that PVR membranes were composed of macrophages, retinal pigment epithelium, and α-SMA-positive myofibroblasts, the latter predominantly characterized by the co-expression of immune cell signature markers. Finally, 13 drugs were identified as potential therapeutics for PVR, including aminocaproic acid and various topoisomerase-2A inhibitors. Conclusions: Epiretinal PVR membranes exhibit a unique and complex transcriptional and cellular profile dominated by immune cells and myofibroblasts, as well as a variety of ECM components. Our findings provide new insights into the pathophysiology of PVR and suggest potential targeted therapeutic options.

Characterization of the Cellular Microenvironment and Novel Specific Biomarkers in Pterygia Using RNA Sequencing.

With a worldwide prevalence of ~12%, pterygium is a common degenerative and environmentally triggered ocular surface disorder characterized by wing-shaped growth of conjunctival tissue onto the cornea that can lead to blindness if left untreated. This study characterizes the transcriptional profile and the cellular microenvironment of conjunctival pterygia and identifies novel pterygia-specific biomarkers. Formalin-fixed and paraffin-embedded pterygia as well as healthy conjunctival specimens were analyzed using MACE RNA sequencing (n = 8 each) and immunohistochemistry (pterygia n = 7, control n = 3). According to the bioinformatic cell type enrichment analysis using xCell, the cellular microenvironment of pterygia was characterized by an enrichment of myofibroblasts, T-lymphocytes and various antigen-presenting cells, including dendritic cells and macrophages. Differentially expressed genes that were increased in pterygia compared to control tissue were mainly involved in autophagy (including DCN, TMBIM6), cellular response to stress (including TPT1, DDX5) as well as fibroblast proliferation and epithelial to mesenchymal transition (including CTNNB1, TGFBR1, and FN1). Immunohistochemical analysis confirmed a significantly increased FN1 stromal immunoreactivity in pterygia when compared to control tissue. In addition, a variety of factors involved in apoptosis were significantly downregulated in pterygia, including LCN2, CTSD, and NISCH. Furthermore, 450 pterygia-specific biomarkers were identified by including transcriptional data of different ocular surface pathologies serving as controls (training group), which were then validated using transcriptional data of cultured human pterygium cells. Among the most pterygia-specific factors were transcripts such as AHNAK, RTN4, TPT1, FSTL1, and SPARC. Immunohistochemical validation of SPARC revealed a significantly increased stromal immunoreactivity in pterygia when compared to controls, most notably in vessels and intravascular vessel wall-adherent mononuclear cells. Taken together, the present study provides new insights into the cellular microenvironment and the transcriptional profile of pterygia, identifies new and specific biomarkers and in addition to fibrosis-related genes, uncovers autophagy, stress response and apoptosis modulation as pterygium-associated processes. These findings expand our understanding of the pathophysiology of pterygia, provide new diagnostic tools, and may enable new targeted therapeutic options for this common and sight-threatening ocular surface disease.

In-Depth Molecular Characterization of Neovascular Membranes Suggests a Role for Hyalocyte-to-Myofibroblast Transdifferentiation in Proliferative Diabetic Retinopathy.

Background: Retinal neovascularization (RNV) membranes can lead to a tractional retinal detachment, the primary reason for severe vision loss in end-stage disease proliferative diabetic retinopathy (PDR). The aim of this study was to characterize the molecular, cellular and immunological features of RNV in order to unravel potential novel drug treatments for PDR. Methods: A total of 43 patients undergoing vitrectomy for PDR, macular pucker or macular hole (control patients) were included in this study. The surgically removed RNV and epiretinal membranes were analyzed by RNA sequencing, single-cell based Imaging Mass Cytometry and conventional immunohistochemistry. Immune cells of the vitreous body, also known as hyalocytes, were isolated from patients with PDR by flow cytometry, cultivated and characterized by immunohistochemistry. A bioinformatical drug repurposing approach was applied in order to identify novel potential drug options for end-stage diabetic retinopathy disease. Results: The in-depth transcriptional and single-cell protein analysis of diabetic RNV tissue samples revealed an accumulation of endothelial cells, macrophages and myofibroblasts as well as an abundance of secreted ECM proteins such as SPARC, FN1 and several types of collagen in RNV tissue. The immunohistochemical staining of cultivated vitreal hyalocytes from patients with PDR showed that hyalocytes express α-SMA (alpha-smooth muscle actin), a classic myofibroblast marker. According to our drug repurposing analysis, imatinib emerged as a potential immunomodulatory drug option for future treatment of PDR. Conclusion: This study delivers the first in-depth transcriptional and single-cell proteomic characterization of RNV tissue samples. Our data suggest an important role of hyalocyte-to-myofibroblast transdifferentiation in the pathogenesis of diabetic vitreoretinal disease and their modulation as a novel possible clinical approach.