scholarly journals Primary cilia and the reciprocal activation of AKT and SMAD2/3 regulate stretch-induced autophagy in trabecular meshwork cells

2021 ◽  
Vol 118 (13) ◽  
pp. e2021942118
Author(s):  
Myoung Sup Shim ◽  
April Nettesheim ◽  
Angela Dixon ◽  
Paloma B. Liton
Keyword(s):  
Trabecular Meshwork ◽  
Primary Cilia ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Mechanical Stretch ◽  
Cell Types ◽  
Elevated Pressure ◽  
Compensatory Response ◽  
Protein Levels ◽  
Trabecular Meshwork Cells

Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.

Mechanical stretch upregulates connexin43 in human trabecular meshwork cells

2019 ◽  
Author(s):  
Nikoleta Tellios ◽  
Mary Feng ◽  
Nancy Chen ◽  
Hong Liu ◽  
Vasiliki Tellios ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Mechanical Stretch ◽  
Trabecular Meshwork Cells

scholarly journals Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma

2020 ◽  
Vol 10 (1) ◽  
pp. 78
Author(s):  
April Nettesheim ◽  
Myoung Sup Shim ◽  
Angela Dixon ◽  
Urmimala Raychaudhuri ◽  
Haiyan Gong ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Cathepsin B ◽  
Molecular Mechanisms ◽  
Culture Media ◽  
Ecm Remodeling ◽  
Trabecular Meshwork Cells ◽  
Proteolytic Cascade

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.

Na-K-Cl cotransport regulates intracellular volume and monolayer permeability of trabecular meshwork cells

1995 ◽  
Vol 268 (4) ◽  
pp. C1067-C1074 ◽  
Author(s):  
M. E. O'Donnell ◽  
J. D. Brandt ◽  
F. R. Curry
Keyword(s):  
Trabecular Meshwork ◽  
Ethacrynic Acid ◽  
Vascular Endothelial Cells ◽  
Critical Role ◽  
Cell Types ◽  
Cell Monolayers ◽  
Aqueous Humor Outflow ◽  
Trabecular Meshwork Cells ◽  
Intracellular Volume ◽  
Underlying Mechanisms

The trabecular meshwork (TM) of the eye plays a critical role in modulating intraocular pressure (IOP) through regulation of aqueous humor outflow, although the underlying mechanisms remain unknown. Ethacrynic acid, an agent known to inhibit Na-K-Cl cotransport of a number of cell types, recently has been reported to increase aqueous outflow and lower IOP through an unknown effect on the TM. In vascular endothelial cells and a variety of other cell types, the Na-K-Cl cotransporter functions to regulate intracellular volume. The present study was conducted to evaluate TM cells for the presence of Na-K-Cl cotransport activity and to test the hypothesis that modulation of cotransport activity alters intracellular volume and, consequently, permeability of the TM. We demonstrate here that bovine and human TM cells exhibit robust Na-K-Cl cotransport activity that is inhibited by bumetanide and by ethacrynic acid. Our studies also show that TM cell Na-K-Cl cotransport is modulated by a variety of hormones and neurotransmitters. Inhibition of the cotransporter either by bumetanide, ethacrynic acid, or inhibitory hormones reduces TM intracellular volume, whereas stimulatory hormones increase cell volume. In addition, shrinkage of the cells by hypertonic media stimulates cotransport activity and initiates a subsequent regulatory volume increase. Permeability of TM cell monolayers, assessed as transmonolayer flux of [14C]sucrose, is increased by hypertonicity-induced cell shrinkage and by bumetanide. These findings suggest that Na-K-Cl cotransport of TM cells is of central importance to regulation of intracellular volume and TM permeability. Defects of Na-K-Cl cotransport may underlie the pathophysiology of glaucoma.

scholarly journals Expression of mRNAs, miRNAs, and lncRNAs in Human Trabecular Meshwork Cells Upon Mechanical Stretch

2020 ◽  
Vol 61 (5) ◽  
pp. 2
Author(s):  
Hannah Youngblood ◽  
Jingwen Cai ◽  
Michelle D. Drewry ◽  
Inas Helwa ◽  
Eric Hu ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Mechanical Stretch ◽  
Trabecular Meshwork Cells

scholarly journals Genome-wide Expression Profiling and Pathway Analysis in Cyclic Stretched Human Trabecular Meshwork Cells

2018 ◽  
Author(s):  
Michelle D. Drewry ◽  
Jingwen Cai ◽  
Inas Helwa ◽  
Eric Hu ◽  
Sabrina Liu ◽  
...  
Keyword(s):  
Mechanical Stress ◽  
Pathway Analysis ◽  
Trabecular Meshwork ◽  
Target Genes ◽  
Mechanical Stretch ◽  
Differentially Expressed ◽  
Receptor Protein ◽  
Trabecular Meshwork Cells ◽  
Differentially Expressed Mirnas ◽  
Significant Enrichment

AbstractPurposeRegulation of intraocular pressure is dependent upon homeostatic responses of trabecular meshwork (TM) cells to mechanical stretch. Despite the important roles of miRNAs in regulating TM function and aqueous outflow, it remains unclear how miRNA and their target genes interact in response to physiological cyclic mechanical stretch. We aimed to identify differentially expressed miRNAs and their potential targets in human TM cells in response to cyclic mechanical stress.MethodsMonolayers of TM cells from non-glaucomatous donors (n=3-6) were cultured in the presence or absence of 15% mechanical stretch, 1 cycle/s, for 6 or 24-hours using computer-controlled Flexcell Unit. We profiled the expression of 800 miRNAs using NanoString Human miRNA assays and identified differentially expressed miRNAs using the Bioconductor Limma package. We identified differentially expressed genes using Operon Human Oligo Arrays with GeneSpring software. Pathway analysis with WebGestalt identified stretch-related pathways. We used Integrative miRNA Target Finder from Ingenuity Pathway Analysis to identify potential miRNA-mRNA regulations.ResultsWe identified 540 unique genes and 74 miRNAs with differential expression in TM cells upon cyclic mechanical stretch. Pathway analysis indicated the significant enrichment of genes involved in Wnt-signaling, receptor protein serine/threonine kinase signaling, TGF-β pathway, and response to unfolded protein. We also identified several miRNA master regulators, including miR-19b-3p and miR-93-5p, which may act as switches to control several mechano-responsive genes.ConclusionsThis study suggests that cyclic mechanical stress of TM cells triggers alterations in the expression of both mRNAs and miRNAs implicated in glaucoma-associated pathways.

scholarly journals Characterization of primary cilia features reveal cell-type specific variability in in vitro models of osteogenic and chondrogenic differentiation

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9799
Author(s):  
Priyanka Upadhyai ◽  
Vishal Singh Guleria ◽  
Prajna Udupa
Keyword(s):  
Cell Lines ◽  
Chondrogenic Differentiation ◽  
Primary Cilia ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Cell Types ◽  
Treatment Modalities ◽  
Cell Type ◽  
Cell Type Specific

Primary cilia are non-motile sensory antennae present on most vertebrate cell surfaces. They serve to transduce and integrate diverse external stimuli into functional cellular responses vital for development, differentiation and homeostasis. Ciliary characteristics, such as length, structure and frequency are often tailored to distinct differentiated cell states. Primary cilia are present on a variety of skeletal cell-types and facilitate the assimilation of sensory cues to direct skeletal development and repair. However, there is limited knowledge of ciliary variation in response to the activation of distinct differentiation cascades in different skeletal cell-types. C3H10T1/2, MC3T3-E1 and ATDC5 cells are mesenchymal stem cells, preosteoblast and prechondrocyte cell-lines, respectively. They are commonly employed in numerous in vitro studies, investigating the molecular mechanisms underlying osteoblast and chondrocyte differentiation, skeletal disease and repair. Here we sought to evaluate the primary cilia length and frequencies during osteogenic differentiation in C3H10T1/2 and MC3T3-E1 and chondrogenic differentiation in ATDC5 cells, over a period of 21 days. Our data inform on the presence of stable cilia to orchestrate signaling and dynamic alterations in their features during extended periods of differentiation. Taken together with existing literature these findings reflect the occurrence of not only lineage but cell-type specific variation in ciliary attributes during differentiation. These results extend our current knowledge, shining light on the variabilities in primary cilia features correlated with distinct differentiated cell phenotypes. It may have broader implications in studies using these cell-lines to explore cilia dependent cellular processes and treatment modalities for skeletal disorders centered on cilia modulation.

scholarly journals Effects of sub-chronic amylin receptor activation on alcohol-induced locomotor stimulation and monoamine levels in mice

2020 ◽  
Vol 237 (11) ◽  
pp. 3249-3257 ◽  
Author(s):  
Aimilia Lydia Kalafateli ◽  
Cajsa Aranäs ◽  
Elisabet Jerlhag
Keyword(s):  
Locomotor Activity ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Receptor Activation ◽  
Dopamine Turnover ◽  
Protein Levels ◽  
Locomotor Stimulation ◽  
Term Administration ◽  
Alcohol Response ◽  
Receptor Activity Modifying Proteins

Abstract Rationale Amylin receptors consist of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs). The identification of amylin receptors in areas processing reward, namely laterodorsal tegmental area (LDTg), ventral tegmental area (VTA), and nucleus accumbens (NAc), has attributed them a role as reward regulators. Indeed, acute activation of amylin receptors by the amylin receptor agonist salmon calcitonin (sCT) attenuates alcohol-induced behaviours in rodents. Objectives The effects of long-term administration of sCT on alcohol-related behaviours and the molecular mechanisms underlying these processes are not yet elucidated. To fill this knowledge gap, we investigated the effects of sub-chronic sCT treatment on the locomotor stimulatory responses to alcohol in mice and the molecular pathways involved. Methods We assessed the behavioural effects of sub-chronic sCT treatment by means of locomotor activity experiments in mice. We used western blot to identify changes of the CTR levels and ex vivo biochemical analysis to detect changes in monoamines and their metabolites. Results After discontinuation for 5 days of sCT treatment, alcohol did not induce locomotor stimulation in mice pre-treated with sCT when compared with vehicle, without altering secondary behavioural parameters of the locomotor activity experiment or the protein levels of the CTR in reward-related areas in the same set of animals. Moreover, repeated sCT treatment altered monoaminergic neurotransmission in various brain areas, including increased serotonin and decreased dopamine turnover in the VTA. Lastly, we identified a differential effect of repeated sCT and acute alcohol administration on alcohol-induced locomotion in mice, where sCT initially attenuated and later increased this alcohol response. It was further found that this treatment combination did not affect secondary behavioural parameters measured in this locomotor activity experiments. Conclusions These data suggest that sub-chronic sCT treatment differentially alters the ability of alcohol to cause locomotor stimulation, possibly through molecular mechanisms involving various neurotransmitter systems and not the CTR levels per se.

scholarly journals Telomere associated gene expression as well as TERT protein level and telomerase activity are altered in the ovarian follicles of aged mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Esra Gozde Kosebent ◽  
Saffet Ozturk
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Structural Integrity ◽  
Cell Types ◽  
Telomerase Activity ◽  
Ovarian Follicles ◽  
Biological Aging ◽  
Aged Mice ◽  
Protein Levels ◽  
Ovarian Aging

AbstractTelomeres cap the ends of eukaryotic chromosomes to maintain genomic stability and integrity during an organism’s lifespan. The length of telomeres inevitably shortens due to DNA replication, genotoxic agents, and biological aging. A limited number of cell types, e.g., stem cells, germline cells, and early embryos can elongate shortened telomeres via the enzymatic action of telomerase, which is composed of telomerase reverse transcriptase (TERT) and telomerase RNA component (Terc). Additionally, telomere-associated proteins including telomeric repeat binding factor 1 (TRF1) and 2 (TRF2), as well as protection of telomeres 1a (POT1a), bind to telomeres to maintain their structural integrity and length. During ovarian aging in mammals, telomeres progressively shorten, accompanied by fertility loss; however, the molecular mechanism underlying this attrition during follicle development remains unclear. In this study, the primary, secondary, preantral, and antral follicles were obtained either from 6-week-old adult (n = 19) or 52-week-old aged (n = 12) mice. We revealed that the Tert, Terc, Trf1, Trf2, and Pot1a gene expression (P < 0.001) and TERT protein (P < 0.01) levels significantly decreased in certain ovarian follicles of the aged group when compared to those of the adult group. Also, telomerase activity exhibited remarkable changes in the follicles of both groups. Consequently, altered telomere-associated gene expression and reduced TERT protein levels in the follicles of aged mice may be a determinant of telomere shortening during ovarian aging, and infertility appearing in the later decades of reproductive lifespan. Further investigations are required to determine the molecular mechanisms underlying these alterations in the follicles during ovarian aging.

scholarly journals Mechanical stretch induces Ca2+ influx and extracellular release of PGE2 through Piezo1 activation in trabecular meshwork cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takatoshi Uchida ◽  
Shota Shimizu ◽  
Reiko Yamagishi ◽  
Suzumi M. Tokuoka ◽  
Yoshihiro Kita ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Intraocular Pressure ◽  
Trabecular Meshwork ◽  
Pressure Fluctuations ◽  
Mechanical Stretch ◽  
Cell Contraction ◽  
Trabecular Meshwork Cells ◽  
Extracellular Release ◽  
Main Pathway ◽  
Outflow Pathway

AbstractThe trabecular meshwork (TM) constitutes the main pathway for aqueous humor drainage and is exposed to complex intraocular pressure fluctuations. The mechanism of homeostasis in which TM senses changes in intraocular pressure and leads to normal levels of outflow resistance is not yet well understood. Previous reports have shown that Piezo1, a mechanically-activated cation channel, is expressed in TM and isolated TM cells. Therefore, we tested hypothesis that Piezo1 may function in response to membrane tension and stretch in TM. In human trabecular meshwork (hTM) cells, PIEZO1 was showed to be abundantly expressed, and Piezo1 agonist Yoda1 and mechanical stretch caused a Piezo1-dependent Ca2+ influx and release of arachidonic acid and PGE2. Treatment with Yoda1 or PGE2 significantly inhibited hTM cell contraction. These results suggest that mechanical stretch stimuli in TM activates Piezo1 and subsequently regulates TM cell contraction by triggering Ca2+ influx and release of arachidonic acid and PGE2. Thus, Piezo1 could acts as a regulator of intraocular pressure (IOP) within the conventional outflow pathway and could be a novel therapeutic strategy to modulate IOP in glaucoma patients.

scholarly journals Mesenchymal stem cell-derived exosomes protect trabecular meshwork from oxidative stress

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ying-chao Li ◽  
Juan Zheng ◽  
Xi-zi Wang ◽  
Xin Wang ◽  
Wen-jing Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Survival Rate ◽  
Candidate Genes ◽  
Inflammatory Cytokines ◽  
Trabecular Meshwork ◽  
Control Groups ◽  
Beneficial Effects ◽  
Protein Levels ◽  
Trabecular Meshwork Cells ◽  
And Control

AbstractThis study aims to investigate the beneficial effects of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) on trabecular meshwork cells under oxidative stress and predict candidate genes associated with this process. Trabecular meshwork cells were pretreated with BMSC-derived exosomes for 24 h, and exposed to 0.1 mM H2O2 for 6 h. Survival rate of trabecular meshwork cells was measured with CCK-8 assay. Production of intracellular reactive oxygen species (iROS) was measured using a flow cytometer. RT-PCR and ELISA were used to detect mRNA and protein levels of inflammatory cytokines and matrix metalloproteinases (MMPs). Sequencing of RNA and miRNA for trabecular meshwork cells from Exo and control groups was performed on BGISEQ500 platform. Phenotypically, pretreatment of BMSC-derived exosomes improves survival rate of trabecular meshwork cells exposed to H2O2, reduces production of iROS, and inhibits expression of inflammatory cytokines, whereas increases expression of MMPs. There were 23 miRNAs, 307 lncRNAs, and 367 mRNAs differentially expressed between Exo and control groups. Exosomes derived from BMSCs may protect trabecular meshwork cells from oxidative stress. Candidate genes responsible for beneficial effects, such as DIO2 and HMOX1, were predicted.

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