Long-term Risks of Cirrhosis and Hepatocellular Carcinoma Across Steatotic Liver Disease Subtypes.

INTRODUCTION: The prospective study aimed to investigate the long-term associated risks of cirrhosis and hepatocellular carcinoma (HCC) across various subtypes of steatotic liver disease (SLD). METHODS: We enrolled 332,175 adults who participated in a health screening program between 1997 and 2013. Participants were categorized into various subtypes, including metabolic dysfunction-associated SLD (MASLD), MASLD with excessive alcohol consumption (MetALD), and alcohol-related liver disease (ALD), based on ultrasonography findings, alcohol consumption patterns, and cardiometabolic risk factors. We used computerized data linkage with nationwide registries from 1997 to 2019 to ascertain the incidence of cirrhosis and HCC. RESULTS: After a median follow-up of 16 years, 4,458 cases of cirrhosis and 1,392 cases of HCC occurred in the entire cohort, resulting in an incidence rate of 86.1 and 26.8 per 100,000 person-years, respectively. The ALD group exhibited the highest incidence rate for cirrhosis and HCC, followed by MetALD, MASLD, and non-SLD groups. The multivariate adjusted hazard ratios for HCC were 1.92 (95% confidence interval [CI] 1.51-2.44), 2.91 (95% CI 2.11-4.03), and 2.59 (95% CI 1.93-3.48) for MASLD, MetALD, and ALD, respectively, when compared with non-SLD without cardiometabolic risk factors. The pattern of the associated risk of cirrhosis was similar to that of HCC (all P value <0.001). The associated risk of cirrhosis for ALD increased to 4.74 (95% CI 4.08-5.52) when using non-SLD without cardiometabolic risk factors as a reference. DISCUSSION: This study highlights elevated risks of cirrhosis and HCC across various subtypes of SLD compared with non-SLD, emphasizing the importance of behavioral modifications for early prevention.

Critical Role of VEGF as a Direct Regulator of Photoreceptor Function.

Vascular endothelial growth factor (VEGF or VEGF-A), a major pathogenic factor for diabetic and hypoxic blood-retina barrier (BRB) diseases, has been shown to act as a direct functional regulator for neurons in the peripheral and central nerve systems. To determine if VEGF plays a direct role in regulating retinal neuronal function, we established specific experimental procedures and examined the effect of recombinant VEGF (rVEGF) on photoreceptor function with electroretinography (ERG) in mice. In our case, rVEGF caused a significant reduction of scotopic ERG a-wave and b-wave amplitudes and photopic ERG b-wave amplitudes in a dose-dependent manner in dark-adapted wild-type (WT) mice, shortly after the intravitreal delivery of rVEGF in dark. However, the effect of rVEGF on photoreceptor function was nullified in adult Akita diabetic mice. Our data strongly suggest that VEGF is a direct regulator of photoreceptor function and VEGF upregulation contributes significantly to the diabetes-induced reduction of photoreceptor function. In this chapter, we will discuss the relevant background, key experimental procedures and results, and clinical significance of our work.

The efficacy and feasibility of neoadjuvant immunotherapy plus chemotherapy followed by McKeown minimally invasive oesophagectomy for locally advanced oesophageal squamous cell carcinoma.

Introduction: In immunotherapy, antibodies are activated to block immune checkpoints, resist tumour immunosuppression, shrink tumours and prevent a recurrence. As the science behind tumour immunotherapy continuously develops and improves, neoadjuvant immunotherapy bears more prominent advantages: antigen exposure not only enhances the degree of tumour-specific T-cell response but also prolongs the duration of actions. In this study, we evaluated the efficacy and safety of McKeown minimally invasive oesophagectomy (McKeown MIO) following neoadjuvant immunotherapy combined with chemotherapy (NICT) in patients with locally advanced oesophageal cancer (OC). Patients and Methods: In this retrospective study, 94 patients underwent either NICT or neoadjuvant chemotherapy (NCT) followed by MIO at our institution from January 2020 to October 2022. We assessed the therapy-related adverse events and perioperative outcomes and compared them between the two groups. Results: After completing at least two cycles of neoadjuvant therapy, all patients underwent McKeown MIO with negative margins within 4-7 weeks. Demographic data of the two cohorts were similar. Regarding perioperative characteristics, the median intraoperative blood loss was 50 ml in the NICT group, lower than that of the NCT group (100 ml, P < 0.05). In addition, the NICT group had significantly more harvested lymph nodes than the NCT group (P < 0.05). No significant differences were found in post-operative complications. The rate of objective response rate in the NICT group was higher than that in the NCT group (88.3% vs. 58.8%). Regarding tumour regression, the number of patients with TRG Grades 1-3 in the NICT group was more than that in the NCT. Adverse events experienced by the two groups included anaemia and elevated transaminase. We found no difference in the adverse events between the two groups. Conclusions: This study showed the efficacy and feasibility of NICT followed by McKeown MIO in treating locally advanced OC.

Differences in susceptibility to chlorantraniliprole between Chilo suppressalis (Lepidoptera: Crambidae) and two dominant parasitic wasps collected from Sichuan Province, China.

Chilo suppressalis Walker (Lepidoptera: Crambidae) is one of the most destructive pests occurring in the rice-growing regions of Asia. Parasitoids, mainly egg parasitoids, have been of interest for several years even with practical used cases. Therefore, the potential impact of insecticides on natural enemies needs great attention. In this study, chlorantraniliprole was evaluated for its impact on C. suppressalis and two dominant parasitic wasps. Bioassays showed that chlorantraniliprole had negligible toxicity to Eriborus terebrans but was significantly toxic to Chelonus munakatae; the mortality exceeded 50% when the concentration reached 46.83 ng/cm2. Enzyme assays suggested that the significantly different carboxylesterase activity may be involved in the high-level detoxification metabolism of E. terebrans. According to the results of enzyme gene correlation analysis, P450s may be the dominant factor in the detoxification metabolism of C. munakatae. In addition, the ryanodine receptor C-terminus of C. suppressalis (CsRyR), C. munakatae (CmRyR) and E. terebrans (EtRyR) were successfully cloned. Different amino acids at resistance mutation I4758 M between susceptible C. suppressalis (I) and parasitic wasps (M) may be related to susceptibility differences. Simulated docking showed that CsRyR and CmRyR can interact with chlorantraniliprole but not EtRyR. More interaction forces were formed between CsRyR and chlorantraniliprole than CmRyR. Furthermore, a Pi-Pi T-shape formed between 73PHE in CsRyR and the benzene ring in chlorantraniliprole. These results indicated that both detoxification metabolism and the target site could mediate the susceptibility difference between C. suppressalis and its parasitic wasps.

Loss of X-box binding protein 1 in Müller cells augments retinal inflammation in a mouse model of diabetes.

AIMS/HYPOTHESIS: Müller glia (MG) are major sources of retinal cytokines, and their activation is closely linked to retinal inflammation and vascular leakage in diabetic retinopathy. Previously, we demonstrated that X-box binding protein 1 (XBP1), a transcription factor activated by endoplasmic reticulum (ER) stress in diabetic retinopathy, is involved in regulation of inflammation in retinal endothelial cells. Now, we have explored the role of XBP1 and ER stress in the regulation of MG-derived proinflammatory factors, and their influence on vascular permeability in diabetic retinopathy. METHODS: MG-specific conditional Xbp1 knockout (Xbp1Müller-/-) mice were generated by crossing Xbp1 flox/flox mice with Müller-Cre transgenic mice. Diabetes was modelled by induction with streptozotocin, and retinal vascular permeability was measured with FITC-conjugated dextran 2 months after induction. Primary Müller cells were isolated from Xbp1Müller-/- and Xbp1Müller+/+ mice and exposed to hypoxia and high levels of glucose. Levels of ER-stress and inflammatory factors were examined by real-time PCR, western blotting or immunohistochemistry. RESULTS: Xbp1Müller-/- mice exhibited normal retinal development and retinal function and expressed similar levels of ER-stress and inflammatory genes to Xbp1Müller+/+ littermates. In diabetes-inducing conditions, compared with Xbp1Müller+/+ mice, Xbp1Müller-/- mice had higher mRNA levels of retinal Vegf (also known as Vegfa) and Tnf-α (also known as Tnf) and ER-stress marker genes Grp78 (also known as Hspa5), Atf4, Chop (also known as Ddit3) and Atf6 and higher protein levels of vascular endothelial growth factor (VEGF), TNF-α, phospho-c-Jun N-terminal kinase (JNK), 78 kDa glucose-regulated protein (GRP78), phospho-eukaryotic translation initiation factor (eIF)2α and activating transcription factor (ATF)6. Retinal vascular permeability was significantly higher in diabetic Xbp1Müller-/- mice than in diabetic Xbp1Müller+/+ mice (p < 0.01). Results obtained in vitro with primary Müller cells isolated from Xbp1Müller-/- mice confirmed higher expression levels of inflammatory and ER-stress markers (but not GRP78) than in cells from Xbp1Müller+/+ mice. Moreover, XBP1-deficient Müller cells were more susceptible to high-glucose- or hypoxia-induced ER stress and inflammation than cells from Xbp1Müller+/+ mice. Inhibition of ER stress with chemical chaperones suppressed hypoxia-induced VEGF and TNF-α production in XBP1-deficient Müller cells. CONCLUSIONS/INTERPRETATION: Our results have revealed an important role of XBP1 and ER stress in MG-driven retinal inflammation, and suggest that targeting ER stress may represent a promising approach for the prevention and treatment of diabetic retinopathy.

Identification of novel cell glycolysis related gene signature predicting survival in patients with endometrial cancer.

BACKGROUND: Endometrial cancer (EC) is one of the three major gynecological malignancies. Numerous biomarkers that may be associated with survival and prognosis have been identified through database mining in previous studies. However, the predictive ability of single-gene biomarkers is not sufficiently specific. Genetic signatures may be an improved option for prediction. This study aimed to explore data from The Cancer Genome Atlas (TCGA) to identify a new genetic signature for predicting the prognosis of EC. METHODS: mRNA expression profiling was performed in a group of patients with EC (n = 548) from TCGA. Gene set enrichment analysis was performed to identify gene sets that were significantly different between EC tissues and normal tissues. Cox proportional hazards regression models were used to identify genes significantly associated with overall survival. Quantitative real-time-PCR was used to verify the reliability of the expression of selected mRNAs. Subsequent multivariate Cox regression analysis was used to establish a prognostic risk parameter formula. Kaplan-Meier survival estimates and the log-rank test were used to validate the significance of risk parameters for prognosis prediction. RESULT: Nine genes associated with glycolysis (CLDN9, B4GALT1, GMPPB, B4GALT4, AK4, CHST6, PC, GPC1, and SRD5A3) were found to be significantly related to overall survival. The results of mRNA expression analysis by PCR were consistent with those of bioinformatics analysis. Based on the nine-gene signature, the 548 patients with EC were divided into high/low-risk subgroups. The prognostic ability of the nine-gene signature was not affected by other factors. CONCLUSION: A nine-gene signature associated with cellular glycolysis for predicting the survival of patients with EC was developed. The findings provide insight into the mechanisms of cellular glycolysis and identification of patients with poor prognosis in EC.

Noninvasive Diagnosis of Regional Alteration of Retinal Morphology and Structure with Optical Coherence Tomography in Rodents.

Spectral-domain optical coherence tomography (SD-OCT) produces high-resolution images of retinal cross sections and is becoming a method of choice for in vivo analyses of retinal morphology in rodents. We have adopted this technology to identify and analyze alterations of retinal structure, particularly those with regional and subtle changes. In this technical brief, we will demonstrate the use of SD-OCT in identifying subtle changes in retinal structure and morphology due to the effect of mosaic gene deletion in conditional knockout mice and of uneven distribution of intravitreally delivered compounds, review the application of SD-OCT in measuring pathological lesion volumes, and discuss the major benefits of SD-OCT technology over the traditional histological methods.

Critical Role of Trophic Factors in Protecting Müller Glia: Implications to Neuroprotection in Age-Related Macular Degeneration, Diabetic Retinopathy, and Anti-VEGF Therapies.

The concept that Müller glia (MG) are major retinal supporting cells for neuroprotection under various stresses is well established. However, the detailed molecular and cellular mechanisms of MG-mediated neuroprotection remain elusive. Particularly, the role and mechanism of MG in neuroprotection under diabetic and hypoxic stresses are largely unknown. In this article, we will discuss the role and mechanisms of a major growth factor, vascular endothelial growth factor (VEGF), in mediating MG viability and its potential impact on neuronal integrity in diabetes and hypoxia, demonstrate results on alternative mechanisms to VEGF signaling for MG and neural protection, and highlight the relevance of our work to the treatment of neovascular age-related macular degeneration, diabetic retinopathy, wet age-related macular degeneration, and other hypoxic retinal vascular diseases.

Endoplasmic reticulum (ER) Ca2+-channel activity contributes to ER stress and cone death in cyclic nucleotide-gated channel deficiency.

Endoplasmic reticulum (ER) stress and mislocalization of improperly folded proteins have been shown to contribute to photoreceptor death in models of inherited retinal degenerative diseases. In particular, mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model for achromatopsia, display both early-onset ER stress and opsin mistrafficking. By 2 weeks of age, these mice show elevated signaling from all three arms of the ER-stress pathway, and by 1 month, cone opsin is improperly distributed away from its normal outer segment location to other retinal layers. This work investigated the role of Ca2+-release channels in ER stress, protein mislocalization, and cone death in a mouse model of CNG-channel deficiency. We examined whether preservation of luminal Ca2+ stores through pharmacological and genetic suppression of ER Ca2+ efflux protects cones by attenuating ER stress. We demonstrated that the inhibition of ER Ca2+-efflux channels reduced all three arms of ER-stress signaling while improving opsin trafficking to cone outer segments and decreasing cone death by 20-35%. Cone-specific gene deletion of the inositol-1,4,5-trisphosphate receptor type I (IP3R1) also significantly increased cone density in the CNG-channel-deficient mice, suggesting that IP3R1 signaling contributes to Ca2+ homeostasis and cone survival. Consistent with the important contribution of organellar Ca2+ signaling in this achromatopsia mouse model, significant differences in dynamic intraorganellar Ca2+ levels were detected in CNG-channel-deficient cones. These results thus identify a novel molecular link between Ca2+ homeostasis and cone degeneration, thereby revealing novel therapeutic targets to preserve cones in inherited retinal degenerative diseases.

VEGF as a Trophic Factor for Müller Glia in Hypoxic Retinal Diseases.

Age-related macular degeneration (AMD) and diabetic retinopathy (DR), leading causes of blindness, share a common retinal environment: hypoxia which is a major stimulator for the upregulation of vascular endothelial growth factor (VEGF), a cardinal pathogenic factor for the breakdown of blood-retina barrier (BRB). As a result of intensive studies on VEGF pathobiology, anti-VEGF strategy has become a major therapeutics for wet AMD and DR. To investigate the potential impact of anti-VEGF strategy on major retinal supporting cells, Müller glia (MG), we disrupted VEGF receptor-2 (VEGFR2) in MG with conditional knockout (CKO) and examined the effect of VEGFR2-null on MG viability and neuronal integrity in mice. VEGFR2 CKO mice demonstrated a significant loss of MG density in diabetes/hypoxia, which in turn resulted in accelerated retinal degeneration. These defects appear similar to the clinical characteristics in a significant portion of wet-AMD patients with long-term anti-VEGF therapies. In this article, we will discuss the potential relevance of these clinical characteristics to the critical role of VEGF signaling in MG viability and neuronal integrity in hypoxia.

Early AMD-like defects in the RPE and retinal degeneration in aged mice with RPE-specific deletion of Atg5 or Atg7.

PURPOSE: To examine the effects of autophagy deficiency induced by RPE-specific deletion of Atg5 or Atg7 in mice as a function of age. METHODS: Conditional knockout mice with a floxed allele of Atg5 or Atg7 were crossed with inducible VMD2-rtTA/Cre transgenic mice. VMD2-directed RPE-specific Cre recombinase expression was induced with doxycycline feeding in the resulting mice. Cre-mediated deletion of floxed Atg5 or Atg7 resulted in RPE-specific inactivation of the Atg5 or Atg7 gene. Plastic and thin retinal sections were analyzed with light and electron microscopy for histological changes. Photoreceptor outer segment (POS) thickness in plastic sections was measured using the Adobe Photoshop CS4 extended ruler tool. Autophagic adaptor p62/SQSTM1 and markers for oxidatively damaged lipids, proteins, and DNA were examined with immunofluorescence staining of cryosections. Fluorescence signals were quantified using Image J software. RESULTS: Accumulation of p62/SQSTM1 reflecting autophagy deficiency was observed in the RPE of the Atg5ΔRPE and Atg7ΔRPE mice. 3-nitrotyrosine, advanced glycation end products (AGEs), and 8-hydroxy-2'-deoxyguanosine (8-OHdG), markers for oxidatively damaged proteins and DNA, were also found to accumulate in the RPE of these mice. We observed retinal degeneration in 35% of the Atg5ΔRPE mice and 45% of the Atg7ΔRPE mice at 8 to 24 months old. Degeneration severity and the number of mice with degeneration increased with age. The mean POS thickness of these mice was 25 µm at 8-12 months, 15 µm at 13-18 months, and 3 µm at 19-24 months, compared to 35 µm, 30 µm, and 24 µm in the wild-type mice, respectively. Early age-related macular degeneration (AMD)-like RPE defects were found in all the Atg5ΔRPE and Atg7ΔRPE mice 13 months old or older, including vacuoles, uneven RPE thickness, diminished basal infoldings, RPE hypertrophy/hypotrophy, pigmentary irregularities, and necrosis. The severity of the RPE defects increased with age and in the mice with retinal degeneration. RPE atrophy and choroidal neovascularization (CNV) were occasionally observed in the Atg5ΔRPE and Atg7ΔRPE mice with advanced age. CONCLUSIONS: Autophagy deficiency induced by RPE-specific deletion of Atg5 or Atg7 predisposes but does not necessarily drive the development of AMD-like phenotypes or retinal degeneration.

Conditional Ablation of Retinol Dehydrogenase 10 in the Retinal Pigmented Epithelium Causes Delayed Dark Adaption in Mice.

Regeneration of the visual chromophore, 11-cis-retinal, is a crucial step in the visual cycle required to sustain vision. This cycle consists of sequential biochemical reactions that occur in photoreceptor cells and the retinal pigmented epithelium (RPE). Oxidation of 11-cis-retinol to 11-cis-retinal is accomplished by a family of enzymes termed 11-cis-retinol dehydrogenases, including RDH5 and RDH11. Double deletion of Rdh5 and Rdh11 does not limit the production of 11-cis-retinal in mice. Here we describe a third retinol dehydrogenase in the RPE, RDH10, which can produce 11-cis-retinal. Mice with a conditional knock-out of Rdh10 in RPE cells (Rdh10 cKO) displayed delayed 11-cis-retinal regeneration and dark adaption after bright light illumination. Retinal function measured by electroretinogram after light exposure was also delayed in Rdh10 cKO mice as compared with controls. Double deletion of Rdh5 and Rdh10 (cDKO) in mice caused elevated 11/13-cis-retinyl ester content also seen in Rdh5(-/-)Rdh11(-/-) mice as compared with Rdh5(-/-) mice. Normal retinal morphology was observed in 6-month-old Rdh10 cKO and cDKO mice, suggesting that loss of Rdh10 in the RPE does not negatively affect the health of the retina. Compensatory expression of other retinol dehydrogenases was observed in both Rdh5(-/-) and Rdh10 cKO mice. These results indicate that RDH10 acts in cooperation with other RDH isoforms to produce the 11-cis-retinal chromophore needed for vision.

Di-retinoid-pyridinium-ethanolamine (A2E) Accumulation and the Maintenance of the Visual Cycle Are Independent of Atg7-mediated Autophagy in the Retinal Pigmented Epithelium.

Autophagy is an evolutionarily conserved catabolic mechanism that relieves cellular stress by removing/recycling damaged organelles and debris through the action of lysosomes. Compromised autophagy has been implicated in many neurodegenerative diseases, including retinal degeneration. Here we examined retinal phenotypes resulting from RPE-specific deletion of the autophagy regulatory gene Atg7 by generating Atg7(flox/flox);VMD2-rtTA-cre+ mice to determine whether autophagy is essential for RPE functions including retinoid recycling. Atg7-deficient RPE displayed abnormal morphology with increased RPE thickness, cellular debris and vacuole formation indicating that autophagy is important in maintaining RPE homeostasis. In contrast, 11-cis-retinal content, ERGs and retinal histology were normal in mice with Atg7-deficient RPE in both fasted and fed states. Because A2E accumulation in the RPE is associated with pathogenesis of both Stargardt disease and age-related macular degeneration (AMD) in humans, deletion of Abca4 was introduced into Atg7(flox/flox);VMD2-rtTA-cre+ mice to investigate the role of autophagy during A2E accumulation. Comparable A2E concentrations were detected in the eyes of 6-month-old mice with and without Atg7 from both Abca4(-/-) and Abca4(+/+) backgrounds. To identify other autophagy-related molecules involved in A2E accumulation, we performed gene expression array analysis on A2E-treated human RPE cells and found up-regulation of four autophagy related genes; DRAM1, NPC1, CASP3, and EIF2AK3/PERK. These observations indicate that Atg7-mediated autophagy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in coping with stress caused by A2E accumulation.

Cre Recombinase: You Can't Live with It, and You Can't Live Without It.

The development of conditional gene targeting has greatly advanced our knowledge of human retinal diseases, but issues have arisen related to the use of some Cre-expressing mouse lines. In this article, we discuss potential problems associated with transgenic Cre expression-induced degeneration and alteration of rod photoreceptors and retinal pigment epithelium (RPE). Our strategy for circumventing RPE degeneration by induced transient Cre expression uses a single intravitreal doxycycline injection in a tetracycline-inducible RPE-specific Cre mouse line, which results in productive Cre-mediated recombination efficiently in the RPE. As constitutive expression of Cre in the RPE alters RPE biology, this inducible Cre/lox system provides an opportunity for conditional gene targeting in the RPE, a tissue that is closely related to photoreceptor degeneration, age-related macular degeneration, and diabetic retinopathy.

Numb regulates the polarized delivery of cyclic nucleotide-gated ion channels in rod photoreceptor cilia.

The development and maintenance of protein compartmentalization is essential for neuronal function. A striking example is observed in light-sensing photoreceptors, in which the apical sensory cilium is subdivided into an inner and outer segment, each containing specific proteins essential for vision. It remains unclear, however, how such polarized protein localization is regulated. We report here that the endocytic adaptor protein Numb localizes to the inner, but not the outer segment of mouse photoreceptor cilia. Rod photoreceptor-specific inactivation of numb in vivo leads to progressive photoreceptor degeneration, indicating an essential role for Numb in photoreceptor cell biology. Interestingly, we report that loss of Numb in photoreceptors does not affect the localization of outer segment disk membrane proteins, such as rhodopsin, Peripherin-rds, Rom-1, and Abca4, but significantly disrupts the localization of the rod cyclic nucleotide-gated (Cng) channels, which accumulates on the inner segment plasma membrane in addition to its normal localization to the outer segments. Mechanistically, we show that Numb interacts with both subunits of the Cng channel and promotes the trafficking of Cnga1 to the recycling endosome. These results suggest a model in which Numb prevents targeting of Cng channels to the inner segment, by promoting their trafficking through the recycling endosome, where they can be sorted for specific delivery to the outer segment. This study uncovers a novel mechanism regulating polarized protein delivery in light-sensing cilia, raising the possibility that Numb plays a part in the regulation of protein trafficking in other types of cilia.

Autophagy protects the retina from light-induced degeneration.

Autophagy is a conserved feature of lysosome-mediated intracellular degradation. Dysregulated autophagy is implicated as a contributor in neurodegenerative diseases; however, the role of autophagy in retinal degeneration remains largely unknown. Here, we report that the photo-activated visual chromophore, all-trans-retinal, modulated autophagosome formation in ARPE19 retinal cells. Increased formation of autophagosomes in these cells was observed when incubated with 2.5 µM all-trans-retinal, a condition that did not cause cell death after 24 h in culture. However, autophagosome formation was decreased at concentrations, which caused cell death. Increased expression of activating transcription factor 4 (Atf4), which indicates the activation of oxidative stress, was recorded in response to light illumination in retinas of Abca4(-/-)Rdh8(-/-) mice, which showed delayed clearance of all-trans-retinal after light exposure. Expression of autophagosome marker LC3B-II and mitochondria-specific autophagy, mitophagy, regulator Park2, were significantly increased in the retinas of Abca4(-/-)Rdh8(-/-) mice after light exposure, suggesting involvement of autophagy and mitophagy in the pathogenesis of light-induced retinal degeneration. Deletion of essential genes required for autophagy, including Beclin1 systemically or Atg7 in only rod photoreceptors resulted in increased susceptibility to light-induced retinal damage. Increased photoreceptor cell death was observed when retinas lacking the rod photoreceptor-specific Atg7 gene were coincubated with 20 µM all-trans-retinal. Park2(-/-) mice also displayed light-induced retinal degeneration. Ultra-structural analyses showed mitochondrial and endoplasmic reticulum impairment in retinas of these model animals after light exposure. Taken together, these observations provide novel evidence implicating an important role of autophagy and mitophagy in protecting the retina from all-trans-retinal- and light-induced degeneration.

7-Ketocholesterol induces autophagy in vascular smooth muscle cells through Nox4 and Atg4B.

Oxidized lipoproteins stimulate autophagy in advanced atherosclerotic plaques. However, the mechanisms underlying autophagy induction and the role of autophagy in atherogenesis remain to be determined. This study was designed to investigate the mechanisms by which 7-ketocholesterol (7-KC), a major component of oxidized lipoproteins, induces autophagy. This study was also designed to determine the effect of autophagy induction on apoptosis, a central event in the development of atherosclerosis. Exposure of human aortic smooth muscle cells to 7-KC increased autophagic flux. Autophagy induction was suppressed by treating the cells with either a reactive oxygen species scavenger or an antioxidant. Administration of 7-KC concomitantly up-regulated Nox4 expression, increased intracellular hydrogen peroxide levels, and inhibited autophagy-related gene 4B activity. Catalase overexpression to remove hydrogen peroxide or Nox4 knockdown with siRNA reduced intracellular hydrogen peroxide levels, restored autophagy-related gene 4B activity, and consequently attenuated 7-KC-induced autophagy. Moreover, inhibition of autophagy aggravated both endoplasmic reticulum (ER) stress and cell death in response to 7-KC. In contrast, up-regulation of autophagic activity by rapamycin had opposite effects. Finally, activation of autophagy by chronic rapamycin treatment attenuated ER stress, apoptosis, and atherosclerosis in apolipoprotein E knockout (ApoE(-/-)) mouse aortas. In conclusion, we demonstrate that up-regulation of autophagy is a cellular protective response that attenuates 7-KC-induced cell death in human aortic smooth muscle cells.

Efficient induction of productive Cre-mediated recombination in retinal pigment epithelium.

PURPOSE: To dissect gene functions in the retinal pigment epithelium (RPE), we previously generated a tetracycline-inducible RPE-specific Cre mouse line. Although this Cre mouse line was useful for several conditional gene targeting studies that were conducted by different laboratories, its potential has not been fully exploited, presumably due to a lack of knowledge or procedure for inducing Cre expression appropriately in this mouse line. The goal of the current study is to establish a procedure that will improve the reproducibility of Cre-mediated recombination in this mouse line. METHODS: Analysis of Cre expression and function was performed in double transgenic mice derived from inducible RPE-specific Cre and Cre-activatable ROSA26 lacZ reporter mice. A tetracycline derivative, doxycycline, was supplied to mice intravitreally to induce Cre expression. Cre expression and function were examined with reverse transcription-PCR, immunoblotting, immunostaining, and in situ enzymatic assay for ß-galactosidase. Retinal integrity was examined with electroretinography and morphometry. RESULTS: Intravitreal Dox injection elevated Cre expression significantly and resulted in productive Cre-mediated recombination in approximately 60% of the RPE cells in this mouse line with no apparent change in retinal integrity. CONCLUSIONS: Our results suggest that productive Cre-mediated recombination in this mouse line can be induced efficiently with intravitreal Dox delivery, with no apparent Dox or Cre toxicity. Therefore, our inducible RPE-specific Cre mice are suitable for Cre/lox-based gene activation and inactivation in adult RPE, which is critical to the effectiveness and suitability of this Cre mouse line in long-term studies requiring conditional gene targeting.

Simplified system to investigate alteration of retinal neurons in diabetes.

Diabetic retinopathy (DR) is traditionally considered as a microvascular complication in diabetic retinas. Emerging evidences suggest that the alteration of neuronal function and the death of retinal neurons are part of DR pathology. However, surprisingly little is known about how retinal neurons behave in DR. As diabetic animals are chronicle models that are difficult and expensive to maintain, we used a chemical hypoxia model that mimics the later stage of diabetes and investigated its potential in predicting retinal cell behaviors in diabetes in an efficient manner. In this chapter, we discuss the similarities and differences between diabetic and hypoxic models and the usefulness and limitation of the cobalt-chloride-generated hypoxia system in mice for studying retinal neurobiology in diabetes.

Investigating the role of retinal Müller cells with approaches in genetics and cell biology.

Müller cells are major macroglia and play many essential roles as a supporting cell in the retina. As Müller cells only constitute a small portion of retinal cells, investigating the role of Müller glia in retinal biology and diseases is particularly challenging. To overcome this problem, we first generated a Cre/lox-based conditional gene targeting system that permits the genetic manipulation and functional dissection of gene of interests in Müller cells. To investigate diabetes-induced alteration of Müller cells, we recently adopted methods to analyze Müller cells survival/death in vitro and in vivo. We also used normal and genetically altered primary cell cultures to reveal the mechanistic insights for Müller cells in biological and disease processes. In this article, we will discuss the applications and limitations of these methodologies, which may be useful for research in retinal Müller cell biology and pathophysiology.