Elucidating the molecular basis of PPCD: Effects of decreased ZEB1 expression on corneal endothelial cell function.

PURPOSE: To investigate the functional role that the zinc e-box binding homeobox 1 (ZEB1) gene, which underlies the genetic basis of posterior polymorphous corneal dystrophy 3 (PPCD3), plays in corneal endothelial cell proliferation, apoptosis, migration, and barrier function. METHODS: A human corneal endothelial cell line (HCEnC-21T) was transfected with siRNA targeting ZEB1 mRNA. Cell proliferation, apoptosis, migration, and barrier assays were performed: Cell proliferation was assessed with cell counting using a hemocytometer; cell apoptosis, induced by either ultraviolet C (UVC) radiation or doxorubicin treatment, was quantified by measuring cleaved caspase 3 (cCASP3) protein levels; and cell migration and barrier function were monitored with electric cell-substrate impedance sensing (ECIS). RESULTS: ZEB1 knockdown in HCEnC-21T cells transfected with siRNA targeting ZEB1 did not result in a significant difference in cell proliferation when compared with control. Although knockdown of ZEB1 in HCEnC-21T cells sensitized the cells to UV-induced apoptosis, ZEB1 knockdown did not alter the cells' susceptibility to doxorubicin-induced apoptosis, as measured with cCASP3 protein levels, compared with controls. Similarly, no difference was observed in cell migration following ZEB1 knockdown. However, cell barrier function increased significantly following ZEB1 knockdown. CONCLUSIONS: The corneal endothelium in PPCD3 is characterized by morphologic, anatomic, and molecular features that are more consistent with an epithelial-like rather than an endothelial-like phenotype. Although these characteristics have been well documented, we demonstrate for the first time that susceptibility to UV-induced apoptosis and cell barrier function are significantly altered in the setting of reduced ZEB1. The significance of an altered cellular response to apoptotic stimuli and increased cell barrier function in the pathobiology of PPCD remains to be fully elucidated.

Antibiotic and antitussive prescribing among urgent care and emergency department visits for respiratory diagnoses in a large health system.

Objective: Urgent care centers (UCs) commonly evaluate patients with respiratory infections, and patients increasingly prefer UCs to emergency departments (EDs) because of their customer-centric approach. The aim of this study is to describe antibiotic and opioid prescribing among UC and ED visits with respiratory diagnoses. Methods: This is a cross-sectional study of visits to 7 EDs and 6 UCs in the greater Chicago area. We included visits from July 1, 2017, to June 30, 2019, with a primary diagnosis of upper or lower respiratory infection. We describe the proportion of visits resulting in an antibiotic or antitussive prescription as well as the most frequently prescribed medications in these categories. We also describe the demographic and clinical characteristics of visits. Results: Of 9134 ED visits, 32.9% were prescribed an antibiotic and 14.4% an antitussive (6.6% opioid). Of 41,380 UC visits for respiratory diagnoses, 57.9% were prescribed an antibiotic and 25.0% an antitussive (9.3% opioid). The most frequently prescribed antibiotics among ED and UC visits were penicillins (36.6% and 44.5%, respectively) and macrolides (44.1% and 35.3%, respectively). The most commonly prescribed opioid was codeine (55.6% and 91.0%, respectively). Median waiting room time was 16 and 5 minutes for ED and UC visits, respectively; median length of stay was 178 and 41 minutes, respectively. Conclusions: Antibiotics and antitussives, including opioids, are frequently prescribed for ED and UC visits with non-bacterial respiratory diagnoses. These findings suggest greater attention to the appropriateness of antibiotic prescribing in both settings and the incorporation of specific guidance on codeine products in opioid-prescribing guidelines.

ZEB1 insufficiency causes corneal endothelial cell state transition and altered cellular processing.

The zinc finger e-box binding homeobox 1 (ZEB1) transcription factor is a master regulator of the epithelial to mesenchymal transition (EMT), and of the reverse mesenchymal to epithelial transition (MET) processes. ZEB1 plays an integral role in mediating cell state transitions during cell lineage specification, wound healing and disease. EMT/MET are characterized by distinct changes in molecular and cellular phenotype that are generally context-independent. Posterior polymorphous corneal dystrophy (PPCD), associated with ZEB1 insufficiency, provides a new biological context in which to understand and evaluate the classic EMT/MET paradigm. PPCD is characterized by a cadherin-switch and transition to an epithelial-like transcriptomic and cellular phenotype, which we study in a cell-based model of PPCD generated using CRISPR-Cas9-mediated ZEB1 knockout in corneal endothelial cells (CEnCs). Transcriptomic and functional studies support the hypothesis that CEnC undergo a MET-like transition in PPCD, termed endothelial to epithelial transition (EnET), and lead to the conclusion that EnET may be considered a corollary to the classic EMT/MET paradigm.

Confirmation of the OVOL2 Promoter Mutation c.-307T>C in Posterior Polymorphous Corneal Dystrophy 1.

PURPOSE: To identify the genetic basis of posterior polymorphous corneal dystrophy (PPCD) in families mapped to the PPCD1 locus and in affected individuals without ZEB1 coding region mutations. METHODS: The promoter, 5' UTR, and coding regions of OVOL2 was screened in the PPCD family in which linkage analysis established the PPCD1 locus and in 26 PPCD probands who did not harbor a ZEB1 mutation. Copy number variation (CNV) analysis in the PPCD1 and PPCD3 intervals was performed on DNA samples from eight probands using aCGH. Luciferase reporter assays were performed in human corneal endothelial cells to determine the impact of the identified potentially pathogenic variants on OVOL2 promoter activity. RESULTS: OVOL2 mutation analysis in the first PPCD1-linked family demonstrated segregation of the c.-307T>C variant with the affected phenotype. In the other 26 probands screened, one heterozygous coding region variant and five promoter region heterozygous variants were identified, though none are likely pathogenic based on allele frequency. Array CGH in the PPCD1 and PPCD3 loci excluded the presence of CNV involving either OVOL2 or ZEB1, respectively. The c.-307T>C variant demonstrated increased promoter activity in corneal endothelial cells when compared to the wild-type sequence as has been demonstrated previously in another cell type. CONCLUSIONS: Previously identified as the cause of PPCD1, the OVOL2 promoter variant c.-307T>C was herein identified in the original family that established the PPCD1 locus. However, the failure to identify presumed pathogenic coding or non-coding OVOL2 or ZEB1 variants, or CNV involving the PPCD1 and PPCD3 loci in 26 other PPCD probands suggests that other genetic loci may be involved in the pathogenesis of PPCD.