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Investigative Ophthalmology & Visual... Apr 2024Fuchs endothelial corneal dystrophy (FECD) is a progressive blinding disorder, characterized by increased corneal endothelial excrescences (guttae), corneal endothelial...
PURPOSE
Fuchs endothelial corneal dystrophy (FECD) is a progressive blinding disorder, characterized by increased corneal endothelial excrescences (guttae), corneal endothelial cell loss, and edema. These symptoms are hypothesized to be caused by changes in the extracellular matrix (ECM) and mitochondrial dysfunction in the corneal endothelium. Despite this clinical and biological relevance, a comprehensive animal model that recapitulates all the major disease characteristics is currently unavailable. In this study, we develop such a model to improve our understanding of the signaling pathways involved in the FECD progression and develop strategies for early intervention.
METHOD
To generate a comprehensive FECD model, we generated a double mutant mouse bearing tamoxifen-inducible knockdown of Slc4a11 and the Col8a2 (Q455K) mutation. We performed optical coherence tomography (OCT) and in vivo confocal microscopy using the Heidelberg Retinal Tomography 3 - Rostock Cornea module (HRT3-RCM) on the mice at 5 weeks of age before tamoxifen feeding to establish baseline values for corneal thickness, endothelial cell density, and test for the presence of guttae. We measured these parameters again post-tamoxifen treatment at 16 weeks of age. We collected corneas at 16 weeks to perform histopathology, immunofluorescence staining for tight junctions, adherens junctions, and oxidative stress. We evaluated endothelial pump function using a lactate assay.
RESULTS
The double mutant tamoxifen-fed animals showed the presence of guttae, and displayed increased corneal thickness and decreased endothelial cell density. Endothelial cells showed altered morphology with disrupted adherens junctions and elevated reactive oxygen species (ROS). Finally, we found that stromal lactate concentrations were elevated in the double mutant mice, indicative of compromised endothelial pump function.
CONCLUSIONS
Overall, this mouse model recapitulates all the important phenotypic features associated with FECD.
Topics: Animals; Mice; Fuchs' Endothelial Dystrophy; Endothelial Cells; Disease Models, Animal; Lactic Acid; Tamoxifen; Anion Transport Proteins; Symporters
PubMed: 38587441
DOI: 10.1167/iovs.65.4.18 -
BioRxiv : the Preprint Server For... Mar 2024Intracellular pH (pHi) dynamics are linked to cell processes including proliferation, migration, and differentiation. The adherens junction (AJ) and signaling protein...
Intracellular pH (pHi) dynamics are linked to cell processes including proliferation, migration, and differentiation. The adherens junction (AJ) and signaling protein β-catenin has decreased abundance at high pHi due to increased proteasomal-mediated degradation. However, the effects of low pHi on β-catenin abundance and functions have not been characterized. Here, we show that low pHi stabilizes β-catenin in epithelial cells using population-level and single-cell assays. β-catenin abundance is increased at low pHi and decreased at high pHi. We also assay single-cell protein degradation rates to show that β-catenin half-life is longer at low compared to high pHi. Importantly, we show that AJs are not disrupted by β-catenin loss at high pHi due to rescue by plakoglobin. Finally, we show that low pHi increases β-catenin transcriptional activity in single cells and is indistinguishable from a Wnt-on state. This work characterizes pHi as a rheostat regulating β-catenin abundance, stability, and function and implicates β-catenin as a molecular mediator of pHi-dependent cell processes.
PubMed: 38585883
DOI: 10.1101/2024.03.22.586349 -
BioRxiv : the Preprint Server For... Mar 2024SGEF, a RhoG specific GEF, can form a ternary complex with the Scribble polarity complex proteins Scribble and Dlg1, which regulates the formation and maintenance of...
SGEF, a RhoG specific GEF, can form a ternary complex with the Scribble polarity complex proteins Scribble and Dlg1, which regulates the formation and maintenance of adherens junctions and barrier function of epithelial cells. Notably, silencing SGEF results in a dramatic downregulation of the expression of both E-cadherin and ZO-1. However, the molecular mechanisms involved in the regulation of this pathway are not known. Here, we describe a novel signaling pathway governed by the Scribble/SGEF/Dlg1 complex. Our results show that an intact ternary complex is required to maintain the stability of the apical junctions, the expression of ZO-1, and TJ permeability. In contrast, only SGEF is necessary to regulate E-cadherin expression. The absence of SGEF destabilizes the E-cadherin/catenin complex at the membrane, triggering a positive feedback loop that exacerbates the phenotype through the repression of E-cadherin transcription in a process that involves the internalization of E-cadherin by endocytosis, β-catenin signaling and the transcriptional repressor Slug.
PubMed: 38585765
DOI: 10.1101/2024.03.26.586884 -
FASEB Journal : Official Publication of... Apr 2024Neurotensin (NTS) is a 13-amino acid peptide which is highly expressed in the mammalian ovary in response to the luteinizing hormone surge. Antibody neutralization of...
Neurotensin (NTS) is a 13-amino acid peptide which is highly expressed in the mammalian ovary in response to the luteinizing hormone surge. Antibody neutralization of NTS in the ovulatory follicle of the cynomolgus macaque impairs ovulation and induces follicular vascular dysregulation, with excessive pooling of red blood cells in the follicle antrum. We hypothesize that NTS is an essential intrafollicular regulator of vascular permeability. In the present study, follicle injection of the NTS receptor antagonist SR142948 also resulted in vascular dysregulation. To measure vascular permeability changes in vitro, primary macaque ovarian microvascular endothelial cells (mOMECs) were enriched from follicle aspirates and studied in vitro. When treated with NTS, permeability of mOMECs decreased. RNA sequencing (RNA-Seq) of mOMECs revealed high mRNA expression of the permeability-regulating adherens junction proteins N-cadherin (CDH2) and K-cadherin (CDH6). Immunofluorescent detection of CDH2 and CDH6 confirmed expression and localized these cadherins to the cell-cell boundaries, consistent with function as components of adherens junctions. mOMECs did not express detectable levels of the typical vascular endothelial cadherin, VE-cadherin (CDH5) as determined by RNA-Seq, qPCR, western blot, and immunofluorescence. Knockdown of CDH2 or CDH6 via siRNA abrogated the NTS effect on mOMEC permeability. Collectively, these data suggest that NTS plays an ovulation-critical role in vascular permeability maintenance, and that CDH2 and CDH6 are involved in the permeability modulating effect of NTS on the ovarian microvasculature. NTS can be added to a growing number of angiogenic regulators which are critical for successful ovulation.
Topics: Female; Animals; Ovary; Endothelial Cells; Neurotensin; Adherens Junctions; Capillary Permeability; Cadherins; Macaca; Permeability; Endothelium, Vascular; Mammals
PubMed: 38581236
DOI: 10.1096/fj.202302652RR -
International Immunopharmacology May 2024Secondary brain injury exacerbates neurological dysfunction and neural cell death following intracerebral hemorrhage (ICH), targeting the pathophysiological mechanism of...
Secondary brain injury exacerbates neurological dysfunction and neural cell death following intracerebral hemorrhage (ICH), targeting the pathophysiological mechanism of the secondary brain injury holds promise for improving ICH outcomes. Adjudin, a potential male contraceptive, exhibits neuroprotective effects in brain injury disease models, yet its impact in the ICH model remains unknown. In this study, we investigated the effects of adjudin on brain injury in a mouse ICH model and explored its underlying mechanisms. ICH was induced in male C57BL/6 mice by injecting collagenase into the right striatum. Mice received adjudin treatment (50 mg/kg/day) for 3 days before euthanization and the perihematomal tissues were collected for further analysis. Adjudin significantly reduced hematoma volume and improved neurological function compared with the vehicle group. Western blot showed that Adjudin markedly decreased the expression of MMP-9 and increased the expression of tight junctions (TJs) proteins, Occludin and ZO-1, and adherens junctions (AJs) protein VE-cadherin. Adjudin also decreased the blood-brain barrier (BBB) permeability, as indicated by the reduced albumin and Evans Blue leakage, along with a decrease in brain water content. Immunofluorescence staining revealed that adjudin noticeably reduced the infiltration of neutrophil, activation of microglia/macrophages, and reactive astrogliosis, accompanied by an increase in CD206 positive microglia/macrophages which exhibit phagocytic characteristics. Adjudin concurrently decreased the generation of proinflammatory cytokines, such as TNF-α and IL-1β. Additionally, adjudin increased the expression of aquaporin 4 (AQP4). Furthermore, adjudin reduced brain cell apoptosis, as evidenced by increased expression of anti-apoptotic protein Bcl-2, and decreased expression of apoptosis related proteins Bax, cleaved caspase-3 and fewer TUNEL positive cells. Our data suggest that adjudin protects against ICH-induced secondary brain injury and may serve as a potential neuroprotective agent for ICH treatment.
Topics: Animals; Male; Cerebral Hemorrhage; Blood-Brain Barrier; Mice, Inbred C57BL; Neuroprotective Agents; Mice; Neuroinflammatory Diseases; Disease Models, Animal; Matrix Metalloproteinase 9; Cytokines; Microglia; Brain; Hydrazines; Indazoles
PubMed: 38565042
DOI: 10.1016/j.intimp.2024.111962 -
Lab on a Chip Apr 2024Three-dimensional (3D) models, superior in simulating physiological conditions compared to 2D models, offer intricate cell-cell and cell-ECM interactions with diverse...
Three-dimensional (3D) models, superior in simulating physiological conditions compared to 2D models, offer intricate cell-cell and cell-ECM interactions with diverse signaling cues like fluid shear stress and growth factor gradients. Yet, developing 3D tissue barrier models, specifically perfusable luminal structures with dense, multicellular constructs maintained for extended durations with oxygen and nutrients, remains a technical challenge. Here, we describe a molding-based approach for the fabrication of free-standing, perfusable, high cellular density tissue constructs using a self-assembly and migration process to form functional barriers. This technique utilizes a polytetrafluoroethylene (PTFE)-coated stainless-steel wire, held by stainless steel needles, as a template for a perfusable channel within an elongated PDMS well. Upon adding a bio-ink mix of cells and collagen, it self-assembles into a high cell density layer conformally around the wire. Removing the wire reveals a hollow construct, connectable to an inlet and outlet for perfusion. This scalable method allows creating varied dimensions and multicellular configurations. Notably, post-assembly, cells such as human umbilical vein endothelial cells (HUVECs) migrate to the surface and form functional barriers with adherens junctions. Permeability tests and fluorescence imaging confirm that these constructs closely mimic endothelial barrier permeability, exhibiting the lowest permeability among all models in the literature. Unlike traditional methods involving uneven post-seeding of endothelial cells leading to subpar barriers, our approach is a straightforward alternative for fabricating complex perfusable 3D tissue constructs and effective tissue barriers for use in various applications, including tissue engineering, drug screening, and disease modeling.
Topics: Humans; Cell Movement; Human Umbilical Vein Endothelial Cells; Polytetrafluoroethylene; Membranes, Artificial; Dimethylpolysiloxanes; Equipment Design
PubMed: 38563430
DOI: 10.1039/d4lc00006d -
BioRxiv : the Preprint Server For... Mar 2024Transporting epithelial cells in the gut and kidney rely on protocadherin-based apical adhesion complexes to organize microvilli that extend into the luminal space. In...
Transporting epithelial cells in the gut and kidney rely on protocadherin-based apical adhesion complexes to organize microvilli that extend into the luminal space. In these systems, CDHR2 and CDHR5 localize to the distal ends of microvilli, where they form an intermicrovillar adhesion complex (IMAC) that links the tips of these structures, promotes the formation of a well-ordered array of protrusions, and in turn maximizes apical membrane surface area. Recently, we discovered that IMACs can also form between microvilli that extend from neighboring cells, across cell-cell junctions. As an additional point of physical contact between cells, transjunctional IMACs are well positioned to impact the integrity of canonical tight and adherens junctions that form more basolaterally. Here, we sought to test this idea using cell culture and mouse models that lacked CDHR2 expression and were unable to form IMACs. CDHR2 knockout perturbed cell and junction morphology, led to loss of key components from tight and adherens junctions, and impaired barrier function and wound healing. These results indicate that, in addition to organizing apical microvilli, IMACs provide a layer of cell-cell contact that functions in parallel with canonical tight and adherens junctions to support the physiological functions of transporting epithelia.
PubMed: 38562895
DOI: 10.1101/2024.03.19.585733 -
BMC Genomics Apr 2024Cow milk fat is an essential indicator for evaluating and measuring milk quality and cow performance. Growing research has identified the molecular functions of circular...
BACKGROUND
Cow milk fat is an essential indicator for evaluating and measuring milk quality and cow performance. Growing research has identified the molecular functions of circular RNAs (circRNAs) necessary for mammary gland development and lactation in mammals.
METHOD
The present study analyzed circRNA expression profiling data in mammary epithelial cells (MECs) from cows with highly variable milk fat percentage (MFP) using differential expression analysis and weighted gene co-expression network analysis (WGCNA).
RESULTS
A total of 309 differentially expressed circRNAs (DE-circRNAs) were identified in the high and low MFP groups. WGCNA analysis revealed that the pink module was significantly associated with MFP (r = - 0.85, P = 0.007). Parental genes of circRNAs in this module were enriched mainly in lipid metabolism-related signaling pathways, such as focal adhesion, ECM-receptor interaction, adherens junction and AMPK. Finally, six DE-circRNAs were screened from the pink module: circ_0010571, circ_0007797, circ_0002746, circ_0003052, circ_0004319, and circ_0012840. Among them, circ_0002746, circ_0003052, circ_0004319, and circ_0012840 had circular structures and were highly expressed in mammary tissues. Subcellular localization revealed that these four DE-circRNAs may play a regulatory role in the mammary glands of dairy cows, mainly as competitive endogenous RNAs (ceRNAs). Seven hub target genes (GNB1, GNG2, PLCB1, PLCG1, ATP6V0C, NDUFS4, and PIGH) were obtained by constructing the regulatory network of their ceRNAs and then analyzed by CytoHubba and MCODE plugins in Cytoscape. Functional enrichment analysis revealed that these genes are crucial and most probable ceRNA regulators in milk fat metabolism.
CONCLUSIONS
Our study identified several vital circRNAs and ceRNAs affecting milk fat synthesis, providing new research ideas and a theoretical basis for cow lactation, milk quality, and breed improvement.
Topics: Female; Cattle; Animals; RNA, Circular; Milk; RNA, Competitive Endogenous; Lactation; Lipid Metabolism; Gene Regulatory Networks; MicroRNAs; Mammals
PubMed: 38561663
DOI: 10.1186/s12864-024-10252-y -
Journal of Hazardous Materials May 2024Cadmium (Cd) is a well-known testis toxicant. The blood-testis barrier (BTB) is a crucial component of the testis. Cd can disrupt the integrity of the BTB and...
Cadmium (Cd) is a well-known testis toxicant. The blood-testis barrier (BTB) is a crucial component of the testis. Cd can disrupt the integrity of the BTB and reproductive function. However, the mechanism of Cd-induced disruption of BTB and testicular damage has not been fully elucidated. Here, our study investigates the effects of Cd on BTB integrity and testicular dysfunction. 80 (aged 1 day) Hy-Line white variety chickens were randomly designed into 4 groups and treated for 90 days, as follows: control group (essential diet), 35 Cd, 70 Cd and 140 Cd groups (35, 70 and 140 mg/kg Cd). The results found that Cd exposure diminished volume of the testes and induced histopathological lesions in the testes. Exposure to Cd induced an inflammatory response, disrupted the structure and function of the FAK/occludin/ZO-1 protein complex and disrupted the tight junction and adherens junction in the BTB. In addition, Cd exposure reduced the expression of steroid-related proteins and inhibited testosterone synthesis. Taken together, these data elucidate that Cd disrupts the integrity of the BTB and further inhibits spermatogenesis by dissociating the FAK/occludin/ZO-1 complex, which provides a basis for further investigation into the mechanisms of Cd-induced impairment of male reproductive function and pharmacological protection.
Topics: Animals; Male; Blood-Testis Barrier; Cadmium; Chickens; Focal Adhesion Kinase 1; Occludin; Spermatogenesis; Testis; Testosterone; Zonula Occludens-1 Protein
PubMed: 38554509
DOI: 10.1016/j.jhazmat.2024.134126 -
Biochemical and Biophysical Research... May 2024The microRNA-200 (miR-200) family is a potent suppressor of epithelial-to-mesenchymal transition (EMT). While its role as a tumor suppressor has been well documented,...
The microRNA-200 (miR-200) family is a potent suppressor of epithelial-to-mesenchymal transition (EMT). While its role as a tumor suppressor has been well documented, recent studies suggested that it can promote cancer progression in several stages. In this study, we investigated whether the miR-200 family members play a role in the acquisition of a hybrid epithelial/mesenchymal (E/M) state, which is reported to be associated with cancer malignancy, in mesenchymal MDA-MB-231 cells. Our results demonstrated that the induction of miR-200c-141, a cluster of the miR-200 family member, can induce the expression of epithelial gene and cell-cell junction while mesenchymal markers are retained. Moreover, induction of miR-200c-141 promoted collective migration accompanied by the formation of F-actin cables anchored by adherens junction. These results suggest that the miR-200 family can induce a hybrid E/M state and endows with the ability of collective cell migration in mesenchymal cancer cells.
Topics: Cell Line, Tumor; MDA-MB-231 Cells; MicroRNAs; Genes, Tumor Suppressor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic
PubMed: 38552553
DOI: 10.1016/j.bbrc.2024.149829