Optimal Humanized Scg3-Neutralizing Antibodies for Anti-Angiogenic Therapy of Diabetic Retinopathy.

Secretogranin III (Scg3) is a diabetic retinopathy (DR)-restricted angiogenic factor identified in preclinical studies as a target for DR therapy. Previously, our group generated and characterized ML49.3, an anti-Scg3 monoclonal antibody (mAb) which we then converted into an EBP2 humanized antibody Fab fragment (hFab) with potential for clinical application. We also generated anti-Scg3 mT4 mAb and related EBP3 hFab. In this study, to identify the preferred hFab for DR therapy, we compared all four antibodies for binding, neutralizing and therapeutic activities in vitro and in vivo. Octet binding kinetics analyses revealed that ML49.3 mAb, EBP2 hFab, mT4 mAb and EBP3 hFab have Scg3-binding affinities of 35, 8.7, 0.859 and 0.116 nM, respectively. Both anti-Scg3 EBP2 and EBP3 hFabs significantly inhibited Scg3-induced proliferation and migration of human umbilical vein endothelial cells in vitro, and alleviated DR vascular leakage and choroidal neovascularization with high efficacy. Paired assays in DR mice revealed that intravitreally injected EBP3 hFab is 26.4% and 10.3% more effective than EBP2 hFab and aflibercept, respectively, for ameliorating DR leakage. In conclusion, this study confirms the markedly improved binding affinities of hFabs compared to mAbs and further identifies EBP3 hFab as the preferred antibody to develop for anti-Scg3 therapy.

Anti-Scg3 Gene Therapy to Treat Choroidal Neovascularization in Mice.

Neovascular age-related macular degeneration (nAMD) with choroidal neovascularization (CNV) is a leading cause of blindness in the elderly in developed countries. The disease is currently treated with anti-angiogenic biologics, including aflibercept, against vascular endothelial growth factor (VEGF) but with limited efficacy, treatment resistance and requirement for frequent intravitreal injections. Although anti-VEGF gene therapy may provide sustained therapy that obviates multiple injections, the efficacy and side effects related to VEGF pathway targeting remain, and alternative strategies to block angiogenesis independently of VEGF are needed. We recently reported that secretogranin III (Scg3) induces only pathological angiogenesis through VEGF-independent pathways, and Scg3-neutralizing antibodies selectively inhibit pathological but not physiological angiogenesis in mouse proliferative retinopathy models. Anti-Scg3 antibodies synergize dose-dependently with VEGF inhibitors in a CNV model. Here, we report that an adeno-associated virus-8 (AAV8) vector expressing anti-Scg3 Fab ameliorated CNV with an efficacy similar to that of AAV-aflibercept in a mouse model. This study is the first to test an anti-angiogenic gene therapy protocol that selectively targets pathological angiogenesis via a VEGF-independent mechanism. The findings support further safety/efficacy studies of anti-Scg3 gene therapy as monotherapy or combined with anti-VEGF to treat nAMD.

CircFAT1 regulates retinal pigment epithelial cell pyroptosis and autophagy via mediating m6A reader protein YTHDF2 expression in diabetic retinopathy.

Diabetic retinopathy (DR) is a serious blinding complication of diabetes. At present, the therapeutic intervention effect is limited. We aimed to investigate the circRNA expression profiles in retinal proliferative fibrovascular membranes of patients with DR and explore the effect of circFAT1 on pyroptosis and autophagy of high glucose (HG)-induced retinal pigment epithelial (RPE) cells and its molecualr mechanism. In this study, circRNA sequencing was performed to determine the expression profiles of circRNAs in DR patients. The expression of circFAT1 was measured by qRT-PCR. Cell counting kit-8, transmission electron microscope, western blot, immunofluorescence and enzyme-linked immunosorbent assay were conducted to explore the roles of HG and circFAT1 in RPE cell pyroptosis and autophagy. RNA pull down was used to determine the binding protein of circFAT1. Our data showed that HG significantly reduced the viability of RPE cells, inhibited cell autophagy and contributed to cell pyroptosis. In addition, a total of 189 differentially expressed circRNAs (DEcircRNAs) were identified between DR patients and non-DR patients, including 93 upregulated and 96 downregulated DEcircRNAs in the retinal proliferative fibrovascular membranes of DR patients. Pathway analysis showed that DEcircRNAs were mainly involved in MAPK signaling pathway, TGF-beta signaling pathway and adherens junction. Moreover, circFAT1 was significantly downregulated in retinal proliferative fibrovascular membranes of DR patients and HG-induced RPE cells. CircFAT1 overexpression remarkably enhanced the expression of LC3B, while reduced the expression of GSDMD in HG-induced RPE cells. RNA pull down combined with western blot analysis indicated that circFAT1 bound to m6A reader YTHDF2. YTHDF2 overexpression significantly increased the protein expression of LC3B in HG-induced RPE cells. In summary, circFAT1 promoted autophagy and inhibited pyroptosis of RPE cells induced by HG, and could combine with YTHDF2. This study provides new ideas for DR prevention and treatment.

Secretogranin III stringently regulates pathological but not physiological angiogenesis in oxygen-induced retinopathy.

Conventional angiogenic factors, such as vascular endothelial growth factor (VEGF), regulate both pathological and physiological angiogenesis indiscriminately, and their inhibitors may elicit adverse side effects. Secretogranin III (Scg3) was recently reported to be a diabetes-restricted VEGF-independent angiogenic factor, but the disease selectivity of Scg3 in retinopathy of prematurity (ROP), a retinal disease in preterm infants with concurrent pathological and physiological angiogenesis, was not defined. Here, using oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP, we quantified an exclusive binding of Scg3 to diseased versus healthy developing neovessels that contrasted sharply with the ubiquitous binding of VEGF. Functional immunohistochemistry visualized Scg3 binding exclusively to disease-related disorganized retinal neovessels and neovascular tufts, whereas VEGF bound to both disorganized and well-organized neovessels. Homozygous deletion of the Scg3 gene showed undetectable effects on physiological retinal neovascularization but markedly reduced the severity of OIR-induced pathological angiogenesis. Furthermore, anti-Scg3 humanized antibody Fab (hFab) inhibited pathological angiogenesis with similar efficacy to anti-VEGF aflibercept. Aflibercept dose-dependently blocked physiological angiogenesis in neonatal retinas, whereas anti-Scg3 hFab was without adverse effects at any dose and supported a therapeutic window at least 10X wider than that of aflibercept. Therefore, Scg3 stringently regulates pathological but not physiological angiogenesis, and anti-Scg3 hFab satisfies essential criteria for development as a safe and effective disease-targeted anti-angiogenic therapy for ROP.

The application of non-invasive and invasive mechanical ventilation in the first episode of acute respiratory failure.

Acute respiratory failure (RF) is a life-threatening syndrome. This study investigated the application of two major clinical strategies, non-invasive mechanical ventilation (NIV) and invasive mechanical ventilation (IMV), in the first episode of acute RF. Data from the longitudinal health insurance database, which included 1,000,000 insured citizens, were used. The NIV group consisted of 1201 patients and the IMV group consisted of 16,072 patients. Chi-square test and t test were applied to determine the differences in categorical and continuous variables. Further analysis was performed by using univariate and multivariable logistic regression and Poisson regression. There was a significant increase of 733% in the number of NIV users from 2000 to 2012. NIV use was frequently observed in old-age persons (aOR 3.99, 95% CI 3.06-5.21 for those aged ≥ 80 years), women (aOR 1.33, 95% CI 1.18-1.50), patients admitted to a high-level hospital (aOR 1.95, 95% CI 1.63-2.34 for those admitted to a medical center), and patients with a higher Charlson comorbidity index (CCI, aOR 1.38-1.66 for those CCI ≥ 2). In addition, patients with chronic pulmonary disease, cancer, and congestive heart failure were predominant in NIV users and were significantly associated with NIV use. Overall, the use of NIV has markedly increased over the past few years. Persons of advanced age, women, patients admitted to a high-level hospital, and patients with multiple comorbidities were associated with more frequent NIV use. Chronic pulmonary disease, cancer, and congestive heart failure were most important comorbidities for NIV use.

Protective roles of autophagy in retinal pigment epithelium under high glucose condition via regulating PINK1/Parkin pathway and BNIP3L.

BACKGROUND: Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells). METHODS: The morphological changes and reactive oxygen species (ROS) generation in ARPE-19 cells under high glucose treatment were respectively detected using the transmission electron microscopy and flow cytometry. The expression levels of Parkin, PINK1, BNIP3L, LC3-I and LC3-II in ARPE-19 cells received high glucose treatment were measured by western blot after pretreatment of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 3-methyladenine (3-MA), N-acetyl cysteine (NAC) or cyclosporin A (CsA) followed by high glucose treatment. RESULTS: ARPE-19 cells subjected to high glucose stress showed an obvious reduction in the LC3-I expression and significant increase in the number of autophagosomes, in the intracellular ROS level, and in the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). Pretreatment with CCCP significantly reduced the LC3-I expression and increased the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). ARPE-19 cells pretreated with CsA under high glucose stress showed markedly down-regulated expressions of Parkin, PINK1 and BNIP3L compared with the cells treated with high glucose (p < 0.05). Pretreatment of ARPE-19 cells with NAC or 3-MA under high glucose stress resulted in a marked reduction in the expression levels of PINK1, BNIP3L and LC3-II (p < 0.05). Meanwhile, the expression level of Parkin in the ARPE-19 cells pretreated with NAC under high glucose stress was comparable with that in the control cells. CONCLUSION: Autophagy might have protective roles against high glucose induced injury in ARPE19 cells via regulating PINK1/Parkin pathway and BNIP3L.

Protective roles of autophagy in retinal pigment epithelium under high glucose condition via regulating PINK1/Parkin pathway and BNIP3L

BACKGROUND: Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells). METHODS: The morphological changes and reactive oxygen species (ROS) generation in ARPE-19 cells under high glucose treatment were respectively detected using the transmission electron microscopy and flow cytometry. The expression levels of Parkin, PINK1, BNIP3L, LC3-I and LC3-II in ARPE-19 cells received high glucose treatment were measured by western blot after pretreatment of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 3-methyladenine (3-MA), N-acetyl cysteine (NAC) or cyclosporin A (CsA) followed by high glucose treatment. RESULTS: ARPE-19 cells subjected to high glucose stress showed an obvious reduction in the LC3-I expression and significant increase in the number of autophagosomes, in the intracellular ROS level, and in the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). Pretreatment with CCCP significantly reduced the LC3-I expression and increased the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). ARPE-19 cells pretreated with CsA under high glucose stress showed markedly down-regulated expressions of Parkin, PINK1 and BNIP3L compared with the cells treated with high glucose (p < 0.05). Pretreatment of ARPE-19 cells with NAC or 3-MA under high glucose stress resulted in a marked reduction in the expression levels of PINK1, BNIP3L and LC3-II (p < 0.05). Meanwhile, the expression level of Parkin in the ARPE-19 cells pretreated with NAC under high glucose stress was comparable with that in the control cells. CONCLUSION: Autophagy might have protective roles against high glucose induced injury in ARPE19 cells via regulating PINK1/Parkin pathway and BNIP3L.