Yonghao Li

Both Internalization and AIP1 Association Are Required for Tumor Necrosis Factor Receptor 2-Mediated JNK Signaling

Objective—The proinflammtory cytokine tumor necrosis factor (TNF), primarily via TNF receptor 1 (TNFR1), induces nuclear factor-&kgr;B (NF-&kgr;B)–dependent cell survival, and c-Jun N-terminal kinase (JNK) and caspase-dependent cell death, regulating vascular endothelial cell (EC) activation and apoptosis. However, signaling by the second receptor, TNFR2, is poorly understood. The goal of this study was to dissect how TNFR2 mediates NF-&kgr;B and JNK signaling in vascular EC, and its relevance to in vivo EC function. Methods and Results—We show that TNFR2 contributes to TNF-induced NF-&kgr;B and JNK signaling in EC as TNFR2 deletion or knockdown reduces the TNF responses. To dissect the critical domains of TNFR2 that mediate the TNF responses, we examine the activity of TNFR2 mutant with a specific deletion of the TNFR2 intracellular region, which contains conserved domain I, domain II, domain III, and 2 TNFR-associated factor-2-binding sites. Deletion analyses indicate that different sequences on TNFR2 have distinct roles in NF-&kgr;B and JNK activation. Specifically, deletion of the TNFR-associated factor-2-binding sites (TNFR2–59) diminishes the TNFR2-mediated NF-&kgr;B, but not JNK activation; whereas, deletion of domain II or domain III blunts TNFR2-mediated JNK but not NF-&kgr;B activation. Interestingly, we find that the TNFR-associated factor-2-binding sites ensure TNFR2 on the plasma membrane, but the di-leucine LL motif within the domain II and aa338–355 within the domain III are required for TNFR2 internalization as well as TNFR2-dependent JNK signaling. Moreover, domain III of TNFR2 is responsible for association with ASK1-interacting protein-1, a signaling adaptor critical for TNF-induced JNK signaling. While TNFR2 containing the TNFR-associated factor-2-binding sites prevents EC cell death, a specific activation of JNK without NF-&kgr;B activation by TNFR2–59 strongly induces caspase activation and EC apoptosis. Conclusion—Our data reveal that both internalization and ASK1-interacting protein-1 association are required for TNFR2-dependent JNK and apoptotic signaling. Controlling TNFR2-mediated JNK and apoptotic signaling in EC may provide a novel strategy for the treatment of vascular diseases.

Facing the challenges in ophthalmology clerkship teaching: Is flipped classroom the answer?

Recent reform of medical education highlights the growing concerns about the capability of the current educational model to equip medical school students with essential skills for future career development. In the field of ophthalmology, although many attempts have been made to address the problem of the decreasing teaching time and the increasing load of course content, a growing body of literature indicates the need to reform the current ophthalmology teaching strategies. Flipped classroom is a new pedagogical model in which students develop a basic understanding of the course materials before class, and use in-class time for learner-centered activities, such as group discussion and presentation. However, few studies have evaluated the effectiveness of the flipped classroom in ophthalmology education. This study, for the first time, assesses the use of flipped classroom in ophthalmology, specifically glaucoma and ocular trauma clerkship teaching. A total number of 44 international medical school students from diverse background were enrolled in this study, and randomly divided into two groups. One group took the flipped glaucoma classroom and lecture-based ocular trauma classroom, while the other group took the flipped ocular trauma classroom and lecture-based glaucoma classroom. In the traditional lecture-based classroom, students attended the didactic lecture and did the homework after class. In the flipped classroom, students were asked to watch the prerecorded lectures before the class, and use the class time for homework discussion. Both the teachers and students were asked to complete feedback questionnaires after the classroom. We found that the two groups did not show differences in the final exam scores. However, the flipped classroom helped students to develop skills in problem solving, creative thinking and team working. Also, compared to the lecture-based classroom, both teachers and students were more satisfied with the flipped classroom. Interestingly, students had a more positive attitude towards the flipped ocular trauma classroom than the flipped glaucoma classroom regarding the teaching process, the course materials, and the value of the classroom. Therefore, the flipped classroom model in ophthalmology teaching showed promise as an effective approach to promote active learning.

Carbamoylating Activity Associated with the Activation of the Antitumor Agent Laromustine Inhibits Angiogenesis by Inducing ASK1-Dependent Endothelial Cell Death

The anticancer agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine), upon decomposition in situ, yields methyl isocyanate and the chloroethylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE). 90CE has been shown to kill tumor cells via a proposed mechanism that involves interstrand DNA cross-linking. However, the role of methyl isocyanate in the antineoplastic function of laromustine has not been delineated. Herein, we show that 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), an analog of laromustine that generates only methyl isocyanate, activates ASK1-JNK/p38 signaling in endothelial cells (EC). We have previously shown that ASK1 forms a complex with reduced thioredoxin (Trx1) in resting EC, and that the Cys residues in ASK1 and Trx1 are critical for their interaction. 101MDCE dissociated ASK1 from Trx1, but not from the phosphoserine-binding inhibitor 14-3-3, in whole cells and in cell lysates, consistent with the known ability of methyl isocyanate to carbamoylate free thiol groups of proteins. 101MDCE had no effect on the kinase activity of purified ASK1, JNK, or the catalytic activity of Trx1. However, 101MDCE, but not 90CE, significantly decreased the activity of Trx reductase-1 (TrxR1). We conclude that methyl isocyanate induces dissociation of ASK1 from Trx1 either directly by carbamoylating the critical Cys groups in the ASK1-Trx1 complex or indirectly by inhibiting TrxR1. Furthermore, 101MDCE (but not 90CE) induced EC death through a non-apoptotic (necroptotic) pathway leading to inhibition of angiogenesis in vitro. Our study has identified methyl isocyanates may contribute to the anticancer activity in part by interfering with tumor angiogenesis.

Functional analyses of TNFR2 in physiological and pathological retina angiogenesis.

PURPOSE To determine the function of tumor necrosis factor receptor-2 (TNFR2) in retinal development and ischemia-induced revascularization in an oxygen-induced retinopathy (OIR) model. METHODS Mice with a global deletion of TNFR2 (TNFR2-KO) or with a vascular endothelial cell (EC)-specific TNFR2 transgene (TNFR2-TG) were compared to wild-type C57BL/6 mice (WT). Retinal vasculature development was visualized by whole-mount and cross-sectional isolectin staining. In the OIR model, neonatal mice were subjected to 75% oxygen from postnatal day (P)7 to P12 and then returned to normoxia from P12 to P17. Immunostaining and biochemical analyses were performed to assess the effects of TNFR2 deletion and TNFR2 transgenesis on retinal vascular repair. RESULTS TNFR2 deletion slightly delayed, while TNFR2 transgenesis weakly promoted, intraretinal vascular development and intraretinal vessel growth. TNFR2 deletion enhanced, while TNFR2 transgene reduced, hyperoxia-induced vaso-obliteration. However, hypoxia-induced revascularization and development of deep intraretinal vessels at P17 were reduced in TNFR2-KO but increased in TNFR2-TG mice without significant increase in preretinal neovascularization (NV). Moreover, TNFR2-TG/KO mice in which only vascular EC express TNFR2 sufficiently rescued the vascular defects of TNFR2-KO in the OIR model. Biochemical analyses of retina tissues showed that the phenotypic changes in retina correlated with TNFR2-dependent activation of Nuclear factor-κB (NF-κB) survival and bone marrow kinase (Bmx)-VEGFR2 angiogenic pathways. CONCLUSIONS TNFR2 plays a marginal role during retinal vascular development. TNFR2 in vascular EC strongly prevents hyperoxia-induced vaso-obliteration by inhibiting cell apoptosis, and promotes retinal repair by enhancing hypoxia-induced revascularization without increasing pathological neovascular tufts. Therefore, activation of TNFR2 signaling may be an ideal strategy for the treatment of OIR.

Quantitative analysis of rhegmatogenous retinal detachment associated with choroidal detachment in Chinese using UBM.

Purpose: To investigate the characteristics of rhegmatogenous retinal detachment associated with choroidal detachment (RRDCD) in a Chinese population by using ultrasound biomicroscope (UBM). Methods: A random prospective study was used to assess 1,389 patients (1,408 eyes) with rhegmatogenous retinal detachment. Comprehensive standardized ophthalmic examinations included intraocular pressure (IOP) by Goldmann applanation tonometer and the entire ocular fundus by Goldmann three-mirror lens, B-type ultrasound, and UBM. Results: A total of 1,389 patients with rhegmatogenous retinal detachment participated in the study, including 906 men and 483 women. Two hundred and sixty-three eyes (18.79%) had RRDCD and the age of patients ranged from 9 to 82 years (median, 50.8 ± 17.7 years). Patients with high myopia (−6.00 diopters or more) represented 31.94% and more frequently experienced low intraocular pressure and multiple retinal breaks. In 263 RRDCD eyes, the results of UBM showed that ranges of choroidal detachment (CD) were <1 quadrant in 9 (3.42%) eyes, from 1 to 2 quadrants in 37 (14.07%) eyes, from 2 to 3 quadrants in 13 (4.94%) eyes, and from 3 to 4 quadrants in 204 (77.57%) eyes. ultrasound biomicroscope also showed that the angular ranges of CD were between 3.31° and 45.65° (mean, 15.98 ± 9.29°). In addition, UBM showed the mean values of CD angle for the superior (15.42 ± 9.40°), nasal (14.08 ± 8.66°), inferior (15.03 ± 9.05°), and temporal (15.96 ± 9.22°) sides. No statistically significant differences were found among any of these comparisons (F value = 2.011, P > 0.05). Conclusion: Retinal detachment associated with CD occurs more frequently in older people or in patients with high myopia, who usually present with low intraocular pressure and multiple retinal breaks. UBM examination can reduce false-negative rate of RRDCD, determine CD degree quantitatively, and guide classification and treatment. UBM combined with B ultrasound testing can provide a precise diagnosis of rhegmatogenous retinal detachment degree and may have important clinical significance for preoperative diagnosis of anterior proliferative vitreoretinopathy and determining operative strategy and postoperative prognosis.

MACULAR BUCKLING USING A THREE-ARMED SILICONE CAPSULE FOR FOVEOSCHISIS ASSOCIATED WITH HIGH MYOPIA.

Purpose: To investigate the safety and efficacy of a novel macular buckling technique on foveoschisis in highly myopic eyes. Methods: Highly myopic eyes with foveoschisis, posterior staphyloma, and axial length greater than 26.5 mm, but without a full-thickness macular hole, were included. Macular buckling was performed in the included eyes using a three-armed adjustable silicon capsule. Results: Eight eyes from eight patients (five women) were enrolled in this study. The mean follow-up period was 11.6 (range 9–14) months. After surgery, the best-corrected visual acuity was improved in 7/8 (87.5%) eyes, optical coherence tomography imaging showed gradual anatomic improvement of macula over time. The final best-corrected visual acuity gained 21.5 early treatment diabetes retinopathy study letters from baseline on average (P = 0.014). Postoperatively, the most common complications were transiently elevated intraocular pressure (62.5%) and asymptomatic abduction limitation (100%), and the most serious complication was hemorrhagic choroidal detachment (25%). Conclusion: Macular buckling with a three-armed adjustable silicone capsule resulted in anatomic and visual improvement in eyes with myopic foveoschisis.

Protection of Retina by Mini-αA in NaIO3-Induced Retinal Pigment Epithelium Degeneration Mice

Background: Studies have shown that mini-αA can protect retinal pigment epithelium (RPE) cells from apoptosis. However, no in vivo study concerning the anti-apoptotic function of mini-αA has been conducted yet. Methods: MTT assay, HE staining and TUNEL assay were used to assess levels of cells, and an animal model was established to examine the protective effects of mini-αA against NaIO3-induced RPE cell apoptosis. Western blot analysis and RT-qPCR were performed to explore the possible mechanism of mini-αA’s protective function against NaIO3-induced RPE cell apoptosis. Results: Results from in vivo and animal experiments showed that mini-αA antagonized NaIO3-induced RPE cell apoptosis. Further investigation into how mini-αA provided protection against NaIO3-induced RPE cell apoptosis showed that mini-αA reduced NaIO3-induced RPE cell apoptosis and autophagy. In addition, unfolded protein response was also involved in the protective effects of mini-αA against NaIO3-induced RPE cell apoptosis. Conclusions: mini-αA can antagonize RPE cell apoptosis induced by NaIO3. A possible mechanism is by inhibition of apoptosis by repressing autophagy and endoplasmic reticulum stress.

Comparison of Clinical Features After 20-Gauge Vitrectomy Versus 23-Gauge Vitrectomy.

PurposeTo compare the clinical features after 20-gauge (20G) versus 23-gauge (23G) pars plana vitrectomy (PPV). DesignThis was a prospective observational study. MethodsPatients who underwent 20G or 23G PPV participated in the study. Sutures were used in all patients who had 20G and as necessary in patients who had 23G. All patients were examined with ultrasound biomicroscopy and tonometry for intraocular pressure (IOP) preoperatively and postoperatively at 1 day, 1 week, 1 month, and 6 months. ResultsForty-nine eyes underwent 20G PPV and 97 eyes underwent 23G PPV. Hypotony appeared more frequently in the 23G group (9 patients of 97, 9%) than in the 20G group (1 patient of 49, 2%) 1 day after surgery. Mean IOP was statistically significantly lower in the 23G group 1 day after surgery (P = 0.000). Postoperatively, choroidal detachment (CD) was found in both the 23G group (22/97, 23%) and the 20G group (1/49, 2%). In the 23G group, the mean IOP of eyes with CD was significantly lower than those without CD. There was no statistically significant difference in vitreous incarceration between the 2 groups (P = 0.317). ConclusionsChoroidal detachment and hypotony were common complications in the early stages after 23G PPV. The incidence of postoperative vitreous incarceration was similar in both groups.

AIP1 Expression in Tumor Niche Suppresses Tumor Progression and Metastasis

Studies from tumor cells suggest that tumor-suppressor AIP1 inhibits epithelial-mesenchymal transition (EMT). However, the role of AIP1 in the tumor microenvironment has not been examined. We show that a global or vascular endothelial cell (EC)-specific deletion of the AIP1 gene in mice augments tumor growth and metastasis in melanoma and breast cancer models. AIP1-deficient vascular environment not only enhances tumor neovascularization and increases premetastatic niche formation, but also secretes tumor EMT-promoting factors. These effects from AIP1 loss are associated with increased VEGFR2 signaling in the vascular EC and could be abrogated by systemic administration of VEGFR2 kinase inhibitors. Mechanistically, AIP1 blocks VEGFR2-dependent signaling by directly binding to the phosphotyrosine residues within the activation loop of VEGFR2. Our data reveal that AIP1, by inhibiting VEGFR2-dependent signaling in tumor niche, suppresses tumor EMT switch, tumor angiogenesis, and tumor premetastatic niche formation to limit tumor growth and metastasis.

ASK1-dependent endothelial cell activation is critical in ovarian cancer growth and metastasis

We have recently reported that tumor-associated macrophages (TAMs) promote early transcoelomic metastasis of ovarian cancer by facilitating TAM-ovarian cancer cell spheroid formation. ASK1 is known to be important for macrophage activation and inflammation-mediated tumorigenesis. In the present study, we show that ASK1 deficiency attenuates TAM-spheroid formation and ovarian cancer progression in an orthotopic ovarian cancer model. Interestingly, ASK1 in stroma, but not in TAMs, is critical for peritoneal tumor growth of ovarian cancer. Moreover, overexpression of an ASK1 inhibitory protein (suppressor of cytokine signaling-1; SOCS1) in vascular endothelium attenuates vascular permeability, TAM infiltration, and ovarian cancer growth. Mechanistically, we show that ASK1 mediates degradation of endothelial junction protein VE-cadherin via a lysosomal pathway to promote macrophage transmigration. Importantly, a pharmacological ASK1 inhibitor prevents tumor-induced vascular leakage, macrophage infiltration, and tumor growth in two mouse models. Since transcoelomic metastasis is also associated with many other cancers, such as pancreatic and colon cancers, our study provides ASK1 as a therapeutic target for the treatment of ovarian cancer and other transcoelomic metastasis cancers.