Evaluation of trabecular meshwork-specific promoters in vitro and in vivo using scAAV2 vectors expressing C3 transferase.

AIM: To evaluate the potential of two trabecular meshwork (TM)-specific promoters, Chitinase 3-like 1 (Ch3L1) and matrix gla protein (MGP), for improving specificity and safety in glaucoma gene therapy based on self-complementary AAV2 (scAAV2) vector technologies. METHODS: An scAAV2 vector with C3 transferase (C3) as the reporter gene (scAAV2-C3) was selected. The scAAV2-C3 vectors were driven by Ch3L1 (scAAV2-Ch3L1-C3), MGP (scAAV2-MGP-C3), enhanced MGP (scAAV2-eMGP-C3) and cytomegalovirus (scAAV2-CMV-C3), respectively. The cultured primary human TM cells were treated with each vector at different multiplicities of infections. Changes in cell morphology were observed by phase contrast microscopy. Actin stress fibers and Rho GTPases/Rho-associated protein kinase pathway-related molecules were assessed by immunofluorescence staining, real-time quantitative polymerase chain reaction and Western blot. Each vector was injected intracamerally into the one eye of each rat at low and high doses respectively. In vivo green fluorescence was visualized by a Micron III Retinal Imaging Microscope. Intraocular pressure (IOP) was monitored using a rebound tonometer. Ocular responses were evaluated by slit-lamp microscopy. Ocular histopathology analysis was examined by hematoxylin and eosin staining. RESULTS: In TM cell culture studies, the vector-mediated C3 expression induced morphologic changes, disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rho-associated protein kinase signaling pathway. At the same dose, these changes were significant in TM cells treated with scAAV2-CMV-C3 or scAAV2-Ch3L1-C3, but not in cells treated with scAAV2-eMGP-C3 or scAAV2-MGP-C3. At low-injected dose, the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGP-C3-injected and scAAV2-eMGP-C3-injected eyes. At high-injected dose, significant IOP reduction was observed in the scAAV2-eMGP-C3-injected eyes but not in scAAV2-MGP-C3-injected eyes. Similar to scAAV2-CMV-C3, scAAV2-Ch3L1-C3 vector showed efficient transduction both in the TM and corneal endothelium. In anterior segment tissues of scAAV2-eMGP-C3-injected eyes, no obvious morphological changes were found except for the TM. Inflammation was absent. CONCLUSION: In scAAV2-transduced TM cells, the promoter-driven efficiency of Ch3L1 is close to that of cytomegalovirus, but obviously higher than that of MGP. In the anterior chamber of rat eye, the transgene expression pattern of scAAV2 vector is presumably affected by MGP promoter, but not by Ch3L1 promoter. These findings would provide a useful reference for improvement of specificity and safety in glaucoma gene therapy using scAAV2 vector.

CircZNF532 knockdown protects retinal pigment epithelial cells against high glucose-induced apoptosis and pyroptosis by regulating the miR-20b-5p/STAT3 axis.

INTRODUCTION: The loss of retinal pigment epithelial (RPE) cells is associated with the etiology of diabetic retinopathy (DR). This study investigated the effects of circular RNA ZNF532 (circZNF532) on apoptosis and pyroptosis of RPE cells. MATERIALS AND METHODS: Blood samples were collected from patients with DR and healthy volunteers. A human RPE cell line ARPE-19 was induced by high glucose (HG) and assayed for cell viability, apoptosis, and pyroptosis. The binding of miR-20b-5p with circZNF532 and STAT3 was confirmed by a luciferase activity assay. A mouse model of diabetic retinopathy was established. RESULTS: CircZNF532 and STAT3 were upregulated but miR-20b-5p was downregulated in the serum samples of patients with DR and HG-induced ARPE-19 cells. Elevated miR-20b-5p or CircZNF532 knockdown enhanced proliferation but reduced apoptosis and pyroptosis of ARPE-19 cells. CircZNF532 sponged miR-20b-5p and inhibited its expression. STAT3 was verified as a target of miR-20b-5p. MiR-20b-5p modulated ARPE-19 cell viability, apoptosis, and pyroptosis by targeting STAT3. Mice with STZ-induced diabetes showed elevated expressions of circZNF532 and STAT3 but decreased the level of miR-20b-5p compared with the controls. Knockdown of circZNF532 inhibited apoptosis and pyroptosis in mouse retinal tissues. CONCLUSION: CircZNF532 knockdown rescued human RPE cells from HG-induced apoptosis and pyroptosis by regulating STAT3 via miR-20b-5p.

Esophageal/gastric cancer screening in high-risk populations in Henan Province, China.

OBJECTIVE: To summarize the endoscopic screening findings in high-risk population of esophageal and gastric carcinoma and analyze influential factors related to screening. METHODS: In seven selected cities and counties with high incidences of esophageal carcinoma, people at age of 40-69 were set as the target population. Those with gastroscopy contradictions were excluded, and all who were voluntary and willing to comply with the medical requirements were subjected to endoscopic screening and histological examination for esophageal, gastric cardia and gastric carcinoma in accordance with national technical manual for early detection and treatment of cancer. RESULTS: In three years, 36,154 people were screened, and 16,847 (46.60%) cases were found to have precancerous lesions. A total of 875 cases were found to have cancers (2.42%), and among them 739 cases had early stage with an early diagnosis rate is 84.5%. Some 715 patients underwent prompt treatment and the success rate was 81.8%. CONCLUSIONS: In a high-risk population of esophageal and gastric carcinoma, it is feasible to implement early detection and treatment by endoscopic screening. Screening can identify potential invasive carcinoma, early stage carcinoma and precancerous lesions, improving efficacy through early detection and treatment. The exploratory analysis of related influential factors will help broad implementation of early detection and treatment for esophageal and gastric carcinoma.