Proteomic and Cytokine Profiling in Plasma from Patients with Normal-Tension Glaucoma and Ocular Hypertension.

Primary open-angle glaucoma (POAG) is subdivided depending on eye pressure. Patients with normal-tension glaucoma (NTG) have never had high intraocular pressure (IOP) measured while patients with ocular hypertension (OHT) have high eye pressure but no signs of glaucoma. Although IOP is considered to be a risk factor for all glaucoma patients, it is reasonable to assume that other risk factors such as inflammation play a role. We aimed to characterize the proteome and cytokine profile during hypoxia in plasma from patients with NTG (n = 10), OHT (n = 10), and controls (n = 10). Participants were exposed to hypoxia for two hours, followed by 30 min of normoxia. Samples were taken before ("baseline"), during ("hypoxia"), and after hypoxia ("recovery"). Proteomics based on liquid chromatography coupled with mass spectrometry (LC-MS) was performed. Cytokines were measured by Luminex assays. Bioinformatic analyses indicated the involvement of complement and coagulation cascades in NTG and OHT. Regulation of high-density lipoprotein 3 (HDL3) apolipoproteins suggested that changes in cholesterol metabolism are related to OHT. Hypoxia decreased the level of tumor necrosis factor-α (TNF-α) in OHT patients compared to controls. Circulating levels of interleukin-1ß (IL-1ß) and C-reactive protein (CRP) were decreased in NTG patients compared to controls during hypoxia. After recovery, plasma interleukin-6 (IL-6) was upregulated in patients with NTG and OHT. Current results indicate an enhanced systemic immune response in patients with NTG and OHT, which correlates with pathogenic events in glaucoma. Apolipoproteins may have anti-inflammatory effects, enabling OHT patients to withstand inflammation and development of glaucoma despite high IOP.

Comparison of recombinant protein Z with natural protein Z derived from malt: From structure to functional properties.

Protein Z (PZ) is a prominent albumin found in the endosperm of barley seeds with a molecular weight of approximately 40 kDa. Its multifaceted functional attributes, including trypsin- and thrombin-inhibiting bioactivities and superior foaming properties, have garnered significant attention in research. Considering the post-translational modifications of PZ natural in barley malt, we tried to express recombinant protein Z (rPZ) in E. coli. The present study aims to undertake a comparative analysis between natural PZ and rPZ in order to elucidate their respective characteristics. After spectral analysis, there are significant differences in their secondary and tertiary structures. In addition, rPZ showed superior foamability and foam stability. As for the serpin-like activity, the inhibition rate of rPZ is much higher than that of PZ. In contrast with the inhibition activity, the digestability of rPZ is much lower than that of PZ. As for the cargo carrier properties, rPZ showed an excellent ability to stabilize astaxanthin at 37 °C. These results suggest that rPZ is more suitable as protein carrier, due to the high foamability, serpin-like activity and low digestive stability, which not only give a brief view of recombinant protein, but also give a direction for PZ in cargo delivery.

ROS-responsive biomimetic nanosystem camouflaged by hybrid membranes of platelet-exosomes engineered with neuronal targeting peptide for TBI therapy.

Recovery and survival following traumatic brain injury (TBI) depends on optimal amelioration of secondary injuries at lesion site. Delivering mitochondria-protecting drugs to neurons may revive damaged neurons at sites secondarily traumatized by TBI. Pioglitazone (PGZ) is a promising candidate for TBI treatment, limited by its low brain accumulation and poor targetability to neurons. Herein, we report a ROS-responsive nanosystem, camouflaged by hybrid membranes of platelets and engineered extracellular vesicles (EVs) (C3-EPm-|TKNPs|), that can be used for targeted delivery of PGZ for TBI therapy. Inspired by intrinsic ability of macrophages for inflammatory chemotaxis, engineered M2-like macrophage-derived EVs were constructed by fusing C3 peptide to EVs membrane integrator protein, Lamp2b, to confer them with ability to target neurons in inflamed lesions. Platelets provided hybridized EPm with capabilities to target hemorrhagic area caused by trauma via surface proteins. Consequently, C3-EPm-|PGZ-TKNPs| were orientedly delivered to neurons located in the traumatized hemisphere after intravenous administration, and triggered the release of PGZ from TKNPs via oxidative stress. The current work demonstrate that C3-EPm-|TKNPs| can effectively deliver PGZ to alleviate mitochondrial damage via mitoNEET for neuroprotection, further reversing behavioral deficits in TBI mice. Our findings provide proof-of-concept evidence of C3-EPm-|TKNPs|-derived nanodrugs as potential clinical approaches against neuroinflammation-related intracranial diseases.

Pathological high intraocular pressure induces glial cell reactive proliferation contributing to neuroinflammation of the blood-retinal barrier via the NOX2/ET-1 axis-controlled ERK1/2 pathway.

BACKGROUND: NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat. METHODS: Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2 -/- mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study. RESULTS: We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst. CONCLUSIONS: Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.

The Impact of Aging on the Function of Retinal Ganglion Cells.

Aging is a major risk factor for retinal neurodegenerative diseases. Aged mammalian retinal ganglion cells (RGCs) lack the ability to regenerate axons after injury. Rodent models suggest that older age increases the vulnerability of RGCs to injury and impairs RGC function as well as their functional recovery. Molecular changes - including decreased circulating levels of brain-derived neurotrophic factor (BDNF) - might contribute to impaired RGC dendritic extension during aging. Moreover, age-related mitochondrial dysfunction plays a major role in aging processes, as it leads to reduced adenosine triphosphate and increased generation of reactive oxygen species. Autophagy activity is necessary for the maintenance of cellular homeostasis and decreases with aging in the central nervous system. During aging, vascular insufficiency may lead to impaired oxygen and nutrient supply to RGCs. Microglial cells undergo morphological changes and functional impairment with aging, which might compromise retinal homeostasis and promote an inflammatory environment. Addressing these age-related changes by means of a low-energy diet, exercise, and neurotrophic factors might prevent age-related functional impairment of RGCs. This review focuses on the current understanding of aging RGCs and key players modulating those underlying mechanisms.

Suppressing Endoplasmic Reticulum Stress Alleviates LPS-Induced Acute Lung Injury via Inhibiting Inflammation and Ferroptosis.

Acute lung injury (ALI) is a life-threatening clinical disorder with high mortality rate. Ferroptosis is a new type of programmed cell death with lipid peroxidation and iron ion overloading as the main characteristics. Endoplasmic reticulum (ER) stress and ferroptosis play pivotal roles in the pathogenesis of ALI. The study aimed to investigate the underlying relationship between ER stress and ferroptosis in ALI. The ER stress inhibitor 4-phenylbutyric acid (4-PBA) alleviated LPS-induced inflammation, and decreased IL-1ß, IL-6, and TNF-α levels in BALF and lungs. The increased MDA and decreased GSH induced by LPS were partially reversed by 4-PBA, which also inhibited the expressions of ferroptosis-related protein ACSL4, COX-2, and FTH1. TEM further confirmed the ferroptosis within airway epithelia cells was ameliorated by 4-PBA. Moreover, 4-PBA reduced the production of ROS and lipid ROS in LPS-exposed BEAS-2B cells in a concentration-dependent way. Meanwhile, 4-PBA mitigated LPS-induced cell apoptosis in vivo and in vitro. Mechanistically, the MAPK signaling pathway activated by LPS was downregulated by 4-PBA. Collectively, these findings suggested that 4-PBA protected against ALI by inhibiting inflammation and ferroptosis through downregulating ER stress, thus providing a potential intervention for ALI and revealing the possible interaction between ER stress and ferroptosis in ALI.

Jagged 2 inhibition attenuates hypoxia-induced mitochondrial damage and pulmonary hypertension through Sirtuin 1 signaling.

Notch pathway has played a significant role in the pathophysiology of pulmonary hypertension (PH). However, the role of Jagged 2 (Jag2), one ligand of Notch, remains to be elucidated.Therefore, determining the contribution of Jag2 to PH and its impact on pulmonary artery smooth muscle cells (PASMCs) was the aim of this investigation. Adeno-associated virus-mediated Jag2 inhibition was used to explore the role of Jag2 in peripheral pulmonary vascular remodeling assessed in a rat model of chronic hypoxia (10% O2, 4 weeks) induced pulmonary hypertension. In vitro, the effect of Jag2 silencing on hypoxia (1% O2, 24h) induced rat PASMCs was determined. Group differences were assessed using a 2-sided unpaired Student's t-test for two groups and one-way ANOVA for multiple groups. Jag2 upregulation was first confirmed in rats with sustained hypoxia-induced PH using publicly available gene expression data, experimental PH rat models and hypoxia induced rat PASMCs. Jag2 deficiency decreased oxidative stress injury, peripheral pulmonary vascular remodeling (0.276±0.020 vs. 0.451±0.033 µm, P<0.001, <50µm), and right ventricular systolic pressure (36.8±3.033 vs. 51.8±4.245 mmHg, P<0.001) in the chronic hypoxia-induced rat model of PH. Moreover, Jag2 knockdown decreased proliferation (1.227±0.051 vs. 1.45±0.07, P = 0.012), increased apoptosis (16.733%±0.724% vs. 6.56%±0.668%, P<0.001), and suppressed mitochondrial injury in hypoxia-treated rat PASMCs. Jag2 inhibition restored the activity of the Nrf2/HO-1 pathway, which was abolished by Sirtuin 1 deficiency. These findings show that Jag2 is essential for modulating pulmonary vascular dysfunction and accelerating PH, and that inhibition of Jag2 expression suppresses the progression and development of PH.

Cytokine Profiling in Aqueous Humor of Glaucoma Patients and in Retinas from an Ex Vivo Glaucoma Animal Model.

BACKGROUND: Although the current role of cytokines and neuroinflammation in glaucoma remains obscure, it represents an expanding field in research. The purpose of this study was to analyze cytokines in the aqueous humor (AH) of glaucoma patients and in retinas from an ex vivo glaucoma animal model, to aid in determining the role of neuroinflammation in glaucoma. METHODS: AH samples were collected from 20 patients during cataract surgeries (controls: n = 10, age = 70.3 ± 9.742; glaucoma: n = 10, age: 66.5 ± 8.073) in Shanghai East Hospital, an affiliate of Tongji University, between September 2018 and March 2019 and analyzed in duplicate by Luminex cytokine polystyrene color bead-based multiplex assay. Retinas from female Sprague-Dawley rats (n = 6) were harvested ex vivo and cultured with or without 60 mmHg of hydrostatic pressure for 24 hours. Retinal ganglion cells (RGCs) were quantified using Brn3a staining. Cytokines in the retina and culture medium were analyzed by rat cytokine array (Abcam). RESULTS: At baseline, patients with primary angle closure glaucoma (PACG) have significantly lower levels of IL-6 and IP-10 and a higher level of PDGF-BB in their AH, compared to the controls. Postoperatively, patients with PACG have significantly higher levels of IL-1ra, IL-13, and MIP-1α and a lower level of IL-6. Elevated hydrostatic pressure led to significant RGC loss in the retina, ex vivo, as well as the upregulation of ciliary neurotrophic factor (CNTF), IL-6, IL-10, IL-4, and TIMP-1 alongside the downregulation of PDGF-AA, MMP-8, TNF-α, and IFN-γ. Furthermore, eight cytokines were detected as being downregulated in the culture medium, including PDGF-AA, MMP-8, and IL-4. CONCLUSIONS: Proinflammatory cytokines showed changes in both AH and ex vivo. Further studies are needed on the role of these cytokines and their corresponding signaling pathways in both neurodegeneration and glaucoma.

cGAS-STING Pathway Activation and Systemic Anti-Tumor Immunity Induction via Photodynamic Nanoparticles with Potent Toxic Platinum DNA Intercalator Against Uveal Melanoma.

The cGAS-STING pathway, as a vital innate immune signaling pathway, has attracted considerable attention in tumor immunotherapy research. However, STING agonists are generally incapable of targeting tumors, thus limiting their clinical applications. Here, a photodynamic polymer (P1) is designed to electrostatically couple with 56MESS-a cationic platinum (II) agent-to form NPPDT -56MESS. The accumulation of NPPDT -56MESS in the tumors increases the efficacy and decreases the systemic toxicity of the drugs. Moreover, NPPDT -56MESS generates reactive oxygen species (ROS) under the excitation with an 808 nm laser, which then results in the disintegration of NPPDT -56MESS. Indeed, the ROS and 56MESS act synergistically to damage DNA and mitochondria, leading to a surge of cytoplasmic double-stranded DNA (dsDNA). This way, the cGAS-STING pathway is activated to induce anti-tumor immune responses and ultimately enhance anti-cancer activity. Additionally, the administration of NPPDT -56MESS to mice induces an immune memory effect, thus improving the survival rate of mice. Collectively, these findings indicate that NPPDT -56MESS functions as a chemotherapeutic agent and cGAS-STING pathway agonist, representing a combination chemotherapy and immunotherapy strategy that provides novel modalities for the treatment of uveal melanoma.

The Association between Vascular Abnormalities and Glaucoma-What Comes First?

Glaucoma is a leading cause of irreversible blindness worldwide. While intraocular pressure (IOP) presents a major risk factor, the underlying pathophysiology still remains largely unclear. The correlation between vascular abnormalities and glaucoma has been deliberated for decades. Evidence for a role played by vascular factors in the pathogenesis of glaucomatous neurodegeneration has already been postulated. In addition, the fact that glaucoma causes both structural and functional changes to retinal blood vessels has been described. This review aims to investigate the published evidence concerning the relationship between vascular abnormalities and glaucoma, and to provide an overview of the "chicken or egg" dilemma in glaucoma. In this study, several biomarkers of glaucoma progression from a vascular perspective, including endothelin-1 (ET-1), nitric oxide, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), were identified and subsequently assessed for their potential as pharmacological intervention targets.

Nintedanib inhibits normal human vitreous-induced epithelial-mesenchymal transition in human retinal pigment epithelial cells.

PURPOSE: In this study, we aim to investigate the potential of nintedanib as a therapeutic approach to proliferative vitreoretinopathy (PVR), which is the leading cause of failure in retinal detachment repair. PVR is characterized by the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, and understanding the effects of nintedanib on EMT in the normal human vitreous (HV)-induced RPE cells is crucial. METHODS: Our research focuses on assessing the impact of nintedanib on HV-induced EMT in human retinal pigment epithelial (ARPE-19) cells in vitro. We employed various techniques, including quantitative real-time PCR (qPCR), western blot analysis, and immunofluorescence staining, to evaluate the mRNA and protein expression of EMT biomarkers in HV-induced ARPE-19 cells. Additionally, we measured the proliferation of RPE cells using cell counting, CCK-8, and Ki-67 assays. Migration was assessed through wound healing and transwell migration assays, while contraction was determined using a collagen gel contraction assay. Morphological changes were examined using phase-contrast microscopy. RESULTS: Our results demonstrate that nintedanib selectively attenuates the upregulation of mesenchymal markers in HV-induced ARPE-19 cells, at both the mRNA and protein levels. Furthermore, nintedanib effectively suppresses the HV-induced proliferation, migration, and contraction of ARPE-19 cells, while maintaining the cells' basal activity. These findings strongly suggest that nintedanib exhibits protective effects against EMT in ARPE-19 cells and could be a promising therapeutic option for PVR. CONCLUSIONS: By elucidating the anti-EMT effects of nintedanib in HV-induced RPE cells, our study highlights the potential of this oral triple tyrosine kinase inhibitor in the treatment of PVR. These findings contribute to the growing body of research aimed at developing novel strategies to prevent and manage PVR, ultimately improving the success rates of retinal detachment repair.

Efficacy of citicoline as a supplement in glaucoma patients: A systematic review.

PURPOSE: Glaucoma is a leading cause of irreversible blindness worldwide. Retinal ganglion cells (RGC), the neurons that connect the eyes to the brain, specifically die in glaucoma, leading to blindness. Elevated intraocular pressure (IOP) is the only modifiable risk factor, however, many patients progress despite excellent IOP control. Thus, alternative treatment strategies to prevent glaucoma progression are an unmet need. Citicoline has demonstrated neuroprotective properties in central neurodegenerative diseases. However, conclusive evidence of the effect of citicoline on glaucoma progression is missing. This systematic review investigates first-time the therapeutic potential of citicoline in glaucoma patients. METHODS: The present study was conducted according to the PRISMA 2020 statement. PubMed, Web of Science, Google Scholar, and Embase were accessed in July 2023 to identify all clinical studies investigating the efficacy of citicoline on IOP, the mean deviation of the 24-2 visual field testing (MD 24-2), retinal nerve fibre layer (RNFL), and the pattern electroretinogram (PERG) P50-N95 amplitude in glaucoma patients. The risk of bias was assessed using the Review Manager 5.3 software (The Nordic Cochrane Collaboration, Copenhagen) and the Risk of Bias in Non-randomised Studies of Interventions (ROBINS-I) tool. RESULTS: Ten studies were eligible for this systematic review, including 424 patients. The mean length of the follow-up was 12.1 ± 11.6 months. The overall risk of bias was low to moderate. The mean age of the patients was 56.7 years. There were no significant differences in the IOP, MD 24-2, RNFL, or PERG P50-N95 amplitude between patients receiving citicoline and the control group. There was no improvement from baseline to the last follow-up in IOP, MD 24-2, RNFL, or PERG P50-N95 amplitude. CONCLUSION: There is a lack of sufficient evidence to support that citicoline slows the progression of glaucoma.

Insight into the lncRNA-mRNA Co-Expression Profile and ceRNA Network in Lipopolysaccharide-Induced Acute Lung Injury.

Long non-coding RNAs (lncRNAs) participate in acute lung injury (ALI). However, their latent biological function and molecular mechanism have not been fully understood. In the present study, the global expression profiles of lncRNAs and mRNAs between the control and lipopolysaccharide (LPS)-stimulated groups of human normal lung epithelial cells (BEAS-2B) were determined using high-throughput sequencing. Overall, a total of 433 lncRNAs and 183 mRNAs were differentially expressed. A lncRNA-mRNA co-expression network was established, and then the top 10 lncRNAs were screened using topological methods. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis results showed that the key lncRNAs targeting mRNAs were mostly enriched in the inflammatory-related biological processes. Gene set variation analysis and Pearson's correlation coefficients confirmed the close correlation for the top 10 lncRNAs with inflammatory responses. A protein-protein interaction network analysis was conducted based on the key lncRNAs targeting mRNAs, where IL-1ß, IL-6, and CXCL8 were regarded as the hub genes. A competing endogenous RNA (ceRNA) modulatory network was created with five lncRNAs, thirteen microRNAs, and twelve mRNAs. Finally, real-time quantitative reverse transcription-polymerase chain reaction was employed to verify the expression levels of several key lncRNAs in BEAS-2B cells and human serum samples.

Identification and validation of ferroptosis-related genes in lipopolysaccharide-induced acute lung injury.

BACKGROUND: Emerging evidence reveals the important role of ferroptosis in the pathophysiological process of acute lung injury (ALI). We aimed to identify and validate the potential ferroptosis-related genes of ALI through bioinformatics analysis and experimental validation. METHODS: Murine ALI model was established via intratracheal instillation with LPS and confirmed by H&E staining and transmission electronic microscopy (TEM). RNA sequencing (RNA-seq) was used to screen differentially expressed genes (DEGs) between control and ALI model mice. The potential differentially expressed ferroptosis-related genes of ALI were identified using the limma R package. Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, gene set enrichment analysis (GSEA), and protein-protein interactions (PPI) were applied for the differentially expressed ferroptosis-related genes. CIBERSORT tool was used to conduct immune cell infiltration analysis. Finally, protein expressions and RNA expression of ferroptosis DEGs were validated in vivo and in vitro by western blots and RT-qPCR. RESULTS: Among 5009 DEGs, a total of 86 differentially expressed ferroptosis-related genes (45 up-regulated genes and 41 down-regulated genes) were identified in the lungs between control and ALI. GSEA analysis showed that the genes enriched were mainly involved in response to molecule of bacterial origin and fatty acid metabolic process. The GO and KEGG enrichment analysis indicated that the top 40 ferroptosis DEGs were mainly enriched in reactive oxygen species metabolic process, HIF-1signaling pathway, lipid and atherosclerosis, and ferroptosis. The PPI results and Spearman correlation analysis suggested that these ferroptosis-related genes interacted with each other. Immune infiltration analysis confirmed that ferroptosis DEGs were closely related to immune response. Consistent with the RNA-seq data, the western blot and RT-qPCR unveiled increased mRNA expressions of Cxcl2, Il-6, Il-1ß, and Tnfα, and protein expressions of FTH1, TLR4 as well as decreased ACSL3 in LPS-induced ALI. In vitro, the upregulated mRNA levels of CXCL2, IL-6, SLC2A1, FTH1, TNFAIP3, and downregulated NQO1 and CAV1 in LPS-stimulated BEAS-2B and A549 cells were verified. CONCLUSION: We identified 86 potential ferroptosis-related genes of LPS-induced ALI through RNA-seq. Several pivotal ferroptosis-related genes involved in lipid metabolism and iron metabolism were implicated in ALI. This study may be helpful to expand our understanding of ALI and provide some potential targets to counteract ferroptosis in ALI.

SIRT6 ameliorates LPS-induced apoptosis and tight junction injury in ARDS through the ERK1/2 pathway and autophagy.

Sirtuin6 (SIRT6) has been demonstrated to be involved in a range of physiological processes and diseases, while its role in acute respiratory distress syndrome (ARDS) remains unclear. Therefore, this study focused on the role and underlying mechanism of SIRT6 in ARDS with the aim of identifying potential therapeutic targets. In this study, we found that SIRT6 was significantly decreased in lipopolysaccharide (LPS)-induced A549 cells and a murine model. In vitro overexpression of SIRT6 restored the expression of tight junction proteins (ZO-1 and occludin) and alleviated cell apoptosis and inflammation, while knockdown of SIRT6 aggravated the loss of tight junction proteins (ZO-1 and occludin) and promoted cell apoptosis and inflammation in LPS-induced A549 cells. Furthermore, the overexpression of SIRT6 enhanced autophagy and inhibited the ERK1/2 pathway, while the knockdown of SIRT6 inhibited autophagy and activated the ERK1/2 pathway. The autophagy activator rapamycin and the ERK1/2 inhibitor PD98059 rescued the effects of SIRT6 knockdown on tight junction proteins, apoptosis, and inflammation. Mechanistically, SIRT6 deacetylated histone 3 at Lys9 to negatively regulate the ERK1/2 pathway. In vivo, the SIRT6-specific inhibitor OSS_128167 also significantly accelerated LPS-induced loss of tight junction proteins, lung inflammation, and apoptosis. Meanwhile, the SIRT6-specific inhibitor OSS_128167 also activated the ERK1/2 pathway and inhibited lung autophagy. These results suggested that SIRT6 could ameliorate the loss of tight junction proteins, inflammation, and apoptosis in LPS-induced ARDS by inhibiting the ERK1/ 2 pathway and enhancing autophagy, indicating that SIRT6 plays a beneficial role in ARDS and might be a potential therapeutic target for ARDS.

Nintedanib prevents TGF-ß2-induced epithelial-mesenchymal transition in retinal pigment epithelial cells.

Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a key fibrosis pathogenesis in proliferative vitreoretinopathy (PVR). However, few medicines can prevent proliferative membranes and cell proliferation in the clinic. Nintedanib, a tyrosine kinase inhibitor, has been shown to prevent fibrosis and be anti-inflammatory in multiple organ fibrosis. In our study, 0.1, 1, 10 µM nintedanib was added to 20 ng/mL transforming growth factor beta 2 (TGF-ß2)-induced EMT in ARPE-19 cells. Western blot and immunofluorescence assay showed that 1 µM nintedanib suppressed TGF-ß2-induced E-cadherin expression decreased and Fibronectin, N-cadherin, Vimentin, and α-SMA expression increased. Quantitative real-time PCR results showed that 1 µM nintedanib decreased TGF-ß2-induced increase in SNAI1, Vimentin, and Fibronectin expression and increased TGF-ß2-induced decrease in E-cadherin expression. In addition, the CCK-8 assay, wound healing assay, and collagen gel contraction assay also showed that 1 µM nintedanib ameliorated TGF-ß2-induced cell proliferation, migration, and contraction, respectively. These results suggested that nintedanib inhibits TGF-ß2-induced EMT in ARPE-19 cells, which may be a potential pharmacological treatment for PVR.

Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy.

BACKGROUND: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. METHODS: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. RESULTS: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. CONCLUSIONS: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.

The novel role of lymphatic vessels in the pathogenesis of ocular diseases.

Historically, the eye has been considered as an organ free of lymphatic vessels. In recent years, however, it became evident, that lymphatic vessels or lymphatic-like vessels contribute to several ocular pathologies at various peri- and intraocular locations. The aim of this review is to outline the pathogenetic role of ocular lymphatics, the respective molecular mechanisms and to discuss current and future therapeutic options based thereon. We will give an overview on the vascular anatomy of the healthy ocular surface and the molecular mechanisms contributing to corneal (lymph)angiogenic privilege. In addition, we present (i) current insights into the cellular and molecular mechanisms occurring during pathological neovascularization of the cornea triggered e.g. by inflammation or trauma, (ii) the role of lymphatic vessels in different ocular surface pathologies such as dry eye disease, corneal graft rejection, ocular graft versus host disease, allergy, and pterygium, (iii) the involvement of lymphatic vessels in ocular tumors and metastasis, and (iv) the novel role of the lymphatic-like structure of Schlemm's canal in glaucoma. Identification of the underlying molecular mechanisms and of novel modulators of lymphangiogenesis will contribute to the development of new therapeutic targets for the treatment of ocular diseases associated with pathological lymphangiogenesis in the future. The preclinical data presented here outline novel therapeutic concepts for promoting transplant survival, inhibiting metastasis of ocular tumors, reducing inflammation of the ocular surface, and treating glaucoma. Initial data from clinical trials suggest first success of novel treatment strategies to promote transplant survival based on pretransplant corneal lymphangioregression.

Inhibition of Iron Release from Donkey Spleen Ferritin through Malt-Derived Protein Z-Ferulic Acid Interactions.

Protein-small molecule interactions naturally occur in foodstuffs, which could improve the properties of protein and small molecules. Meanwhile, they might affect the bioavailability and nutritional value of proteins. Ferritin, as an iron-storage protein, has been a focus of research. However, the complexity of foodstuffs enables the interaction between ferritin and food components, especially polyphenols, which can induce iron release from ferritin. Thus, the application of ferritin in food is limited. Inspired by the natural-occurring, strong protein-polyphenol interactions in beer, to inhibit the iron release of ferritin, the malt-derived protein Z (PZ) was chosen to interact with ferulic acid (FA), an abundant reductant in malt, beer, and other foodstuffs. The analysis of the interaction between PZ and FA was carried out using fluorescence spectroscopy, the results of which suggest that one PZ molecule can bind with 22.11 ± 2.13 of FA, and the binding constant is (4.99 ± 2.13) × 105 M-1. In a molecular dynamics (MD) simulation, FA was found to be embedded in the internal hydrophobic pocket of PZ, where it formed hydrogen bonds with Val-389 and Tyr-234. As expected, compared to iron release induced by FA, the iron release from donkey spleen ferritin (DSF) induced by FA decreased by 86.20% in the presence of PZ. Meanwhile, based on the PZ-FA interaction, adding PZ in beer reduced iron release from DSF by 40.5% when DSF:PZ was 1:40 (molar ratio). This work will provide a novel method of inhibiting iron release from ferritin.

Microarray profiling of circular RNAs in LPS-induced Beas-2B cells and identification of circ_2979 as a potential biomarker for ARDS.

Acute respiratory distress syndrome (ARDS) is a major cause of high mortality and morbidity in critically ill patients. Circular RNAs (CircRNAs) are widely expressed in numerous tissues and are associated with various diseases. However, the role of circRNAs in ARDS remains unclear. In this study, we found that cell viability and proliferation were reduced in lipopolysaccharide (LPS)-induced Beas-2B cells. Microarray analysis identified 1131 differentially expressed circRNAs in LPS-treated Beas-2B cells, with 623 circRNAs significantly upregulated and 508 circRNAs strongly downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed significant enrichment and indicated potential functions and pathways of differentially expressed circRNAs. Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that expression of circ_2979, circ_5438, circ_4557 and circ_2066 in LPS-induced Beas-2B cells was consistent with the results obtained by RNA sequencing (RNA-seq). Additionally, we recruited 17 patients with ARDS and 13 healthy volunteers and found that expression of circ_2979 in serum was significantly increased in the patients with ARDS compared with healthy volunteers. Spearman's analyses indicated that circ_2979 was correlated with partial pressure of carbon dioxide in arterial blood (PaCO2), the ratio of partial pressure of arterial oxygen to the fraction of inspired oxygen (PaO2/FiO2), interleukin 2 receptor (IL-2R) and fibrinogen (FIB). The results suggested that circRNAs may play an important role in the progression of ARDS, and that circ_2979 may serve as a diagnosis and prognosis biomarker for ARDS.