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.

scholarly journals A 3D Model of Human Trabecular Meshwork for the Research Study of Glaucoma

2020 ◽  
Vol 11 ◽  
Author(s):  
Sara Tirendi ◽  
Sergio Claudio Saccà ◽  
Stefania Vernazza ◽  
Carlo Traverso ◽  
Anna Maria Bassi ◽  
...  
Keyword(s):  
Animal Models ◽  
Trabecular Meshwork ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Cellular Level ◽  
Research Progress ◽  
New Therapeutic Approaches

Glaucoma is a multifactorial syndrome in which the development of pro-apoptotic signals are the causes for retinal ganglion cell (RGC) loss. Most of the research progress in the glaucoma field have been based on experimentally inducible glaucoma animal models, which provided results about RGC loss after either the crash of the optic nerve or IOP elevation. In addition, there are genetically modified mouse models (DBA/2J), which make the study of hereditary forms of glaucoma possible. However, these approaches have not been able to identify all the molecular mechanisms characterizing glaucoma, possibly due to the disadvantages and limits related to the use of animals. In fact, the results obtained with small animals (i.e., rodents), which are the most commonly used, are often not aligned with human conditions due to their low degree of similarity with the human eye anatomy. Although the results obtained from non-human primates are in line with human conditions, they are little used for the study of glaucoma and its outcomes at cellular level due to their costs and their poor ease of handling. In this regard, according to at least two of the 3Rs principles, there is a need for reliable human-based in vitro models to better clarify the mechanisms involved in disease progression, and possibly to broaden the scope of the results so far obtained with animal models. The proper selection of an in vitro model with a “closer to in vivo” microenvironment and structure, for instance, allows for the identification of the biomarkers involved in the early stages of glaucoma and contributes to the development of new therapeutic approaches. This review summarizes the most recent findings in the glaucoma field through the use of human two- and three-dimensional cultures. In particular, it focuses on the role of the scaffold and the use of bioreactors in preserving the physiological relevance of in vivo conditions of the human trabecular meshwork cells in three-dimensional cultures. Moreover, data from these studies also highlight the pivotal role of oxidative stress in promoting the production of trabecular meshwork-derived pro-apoptotic signals, which are one of the first marks of trabecular meshwork damage. The resulting loss of barrier function, increase of intraocular pressure, as well the promotion of neuroinflammation and neurodegeneration are listed as the main features of glaucoma. Therefore, a better understanding of the first molecular events, which trigger the glaucoma cascade, allows the identification of new targets for an early neuroprotective therapeutic approach.

scholarly journals Low Intensity Shockwave Treatment Modulates Macrophage Functions Beneficial to Healing Chronic Wounds

2021 ◽  
Vol 22 (15) ◽  
pp. 7844
Author(s):  
Jason S. Holsapple ◽  
Ben Cooper ◽  
Susan H. Berry ◽  
Aleksandra Staniszewska ◽  
Bruce M. Dickson ◽  
...  
Keyword(s):  
Macrophage Activation ◽  
Molecular Mechanisms ◽  
Chronic Wounds ◽  
Immunohistochemical Analysis ◽  
Therapeutic Effects ◽  
Gene Expressions ◽  
Extracorporeal Shock Wave

Extracorporeal Shock Wave Therapy (ESWT) is used clinically in various disorders including chronic wounds for its pro-angiogenic, proliferative, and anti-inflammatory effects. However, the underlying cellular and molecular mechanisms driving therapeutic effects are not well characterized. Macrophages play a key role in all aspects of healing and their dysfunction results in failure to resolve chronic wounds. We investigated the role of ESWT on macrophage activity in chronic wound punch biopsies from patients with non-healing venous ulcers prior to, and two weeks post-ESWT, and in macrophage cultures treated with clinical shockwave intensities (150–500 impulses, 5 Hz, 0.1 mJ/mm2). Using wound area measurements and histological/immunohistochemical analysis of wound biopsies, we show ESWT enhanced healing of chronic ulcers associated with improved wound angiogenesis (CD31 staining), significantly decreased CD68-positive macrophages per biopsy area and generally increased macrophage activation. Shockwave treatment of macrophages in culture significantly boosted uptake of apoptotic cells, healing-associated cytokine and growth factor gene expressions and modulated macrophage morphology suggestive of macrophage activation, all of which contribute to wound resolution. Macrophage ERK activity was enhanced, suggesting one mechanotransduction pathway driving events. Collectively, these in vitro and in vivo findings reveal shockwaves as important regulators of macrophage functions linked with wound healing. This immunomodulation represents an underappreciated role of clinically applied shockwaves, which could be exploited for other macrophage-mediated disorders.

Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Perike Srikanth ◽  
Andrielle E Capote ◽  
Alsina Katherina M ◽  
Benjamin Levin ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

Abstract P458: Myosin Light Chain Dephosphorylation By Ppp1r12c Promotes Atrial Hypocontractility In Atrial Fibrillation

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Srikanth Perike ◽  
Frederick Damen ◽  
Andrielle Capote ◽  
Katherina M Alsina ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Introduction: Atrial fibrillation (AF), is the most common sustained arrhythmia, with an estimated prevalence in the U.S. of 2.7 million to 6.1 million and is predictive to increase to 12.1 million in 2030. AF increases the chances of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. Objective: The overexpression of PPP1R12C, causes hypophosphorylation of atrial myosin light chain 2 (MLC2a), decreasing atrial contractility. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluated the role of PP1c-PPP1R12C interaction in MLC2a de-phosphorylation we used Western blots, coimmunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A) PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The Overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, that cause a reduction in atrial contractility and increases AF inducibility. All these discoveries advocate that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

scholarly journals Linc00426 accelerates lung adenocarcinoma progression by regulating miR-455-5p as a molecular sponge

2020 ◽  
Vol 11 (12) ◽  
Author(s):  
Hongli Li ◽  
Qingjie Mu ◽  
Guoxin Zhang ◽  
Zhixin Shen ◽  
Yuanyuan Zhang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Molecular Mechanisms ◽  
Epithelial Mesenchymal Transition ◽  
Ectopic Expression ◽  
Tumor Development ◽  
Biological Significance ◽  
Mesenchymal Transition

AbstractIncreasing lines of evidence indicate the role of long non-coding RNAs (LncRNAs) in gene regulation and tumor development. Hence, it is important to elucidate the mechanisms of LncRNAs underlying the proliferation, metastasis, and invasion of lung adenocarcinoma (LUAD). We employed microarrays to screen LncRNAs in LUAD tissues with and without lymph node metastasis and revealed their effects on LUAD. Among them, Linc00426 was selected for further exploration in its expression, the biological significance, and the underlying molecular mechanisms. Linc00426 exhibits ectopic expression in LUAD tissues and cells. The ectopic expression has been clinically linked to tumor size, lymphatic metastasis, and tumor differentiation of patients with LUAD. The deregulation of Linc00426 contributes to a notable impairment in proliferation, invasion, metastasis, and epithelial–mesenchymal transition (EMT) in vitro and in vivo. Mechanistically, the deregulation of Linc00426 could reduce cytoskeleton rearrangement and matrix metalloproteinase expression. Meanwhile, decreasing the level of Linc00426 or increasing miR-455-5p could down-regulate the level of UBE2V1. Thus, Linc00426 may act as a competing endogenous RNA (ceRNA) to abate miR-455-5p-dependent UBE2V1 reduction. We conclude that Linc00426 accelerates LUAD progression by acting as a molecular sponge to regulate miR-455-5p, and may be a potential novel tumor marker for LUAD.

scholarly journals METTL3 promotes the initiation and metastasis of ovarian cancer by inhibiting CCNG2 expression via promoting the maturation of pri-microRNA-1246

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Xuehan Bi ◽  
Xiao Lv ◽  
Dajiang Liu ◽  
Hongtao Guo ◽  
Guang Yao ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Molecular Mechanisms ◽  
High Expression ◽  
Ovarian Cancer Cells ◽  
Inadequate Knowledge ◽  
And Migration ◽  
Cancer Tissues

AbstractOvarian cancer is a common gynecological malignant tumor with a high mortality rate and poor prognosis. There is inadequate knowledge of the molecular mechanisms underlying ovarian cancer. We examined the expression of methyltransferase-like 3 (METTL3) in tumor specimens using RT-qPCR, immunohistochemistry, and Western blot analysis, and tested the methylation of METTL3 by MSP. Levels of METTL3, miR-1246, pri-miR-1246 and CCNG2 were then analyzed and their effects on cell biological processes were also investigated, using in vivo assay to validate the in vitro findings. METTL3 showed hypomethylation and high expression in ovarian cancer tissues and cells. Hypomethylation of METTL3 was pronounced in ovarian cancer samples, which was negatively associated with patient survival. Decreased METTL3 inhibited the proliferation and migration of ovarian cancer cells and promoted apoptosis, while METTL3 overexpression exerted opposite effects. Mechanistically, METTL3 aggravated ovarian cancer by targeting miR-1246, while miR-1246 targeted and inhibited CCNG2 expression. High expression of METTL3 downregulated CCNG2, promoted the metabolism and growth of transplanted tumors in nude mice, and inhibited apoptosis. The current study highlights the promoting role of METTL3 in the development of ovarian cancer, and presents new targets for its treatment.

scholarly journals Downregulation of CRABP2 Inhibit the Tumorigenesis of Hepatocellular Carcinoma In Vivo and In Vitro

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Qingmin Chen ◽  
Ludong Tan ◽  
Zhe Jin ◽  
Yahui Liu ◽  
Ze Zhang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Retinoic Acid ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Tumor Stage ◽  
Migration And Invasion ◽  
Hcc Cells

Cellular retinoic acid-binding protein 2 (CRABP2) binds retinoic acid (RA) in the cytoplasm and transports it into the nucleus, allowing for the regulation of specific downstream signal pathway. Abnormal expression of CRABP2 has been detected in the development of several tumors. However, the role of CRABP2 in hepatocellular carcinoma (HCC) has never been revealed. The current study aimed to investigate the role of CRABP2 in HCC and illuminate the potential molecular mechanisms. The expression of CRABP2 in HCC tissues and cell lines was detected by western blotting and immunohistochemistry assays. Our results demonstrated that the expression levels of CRABP2 in HCC tissues were elevated with the tumor stage development, and it was also elevated in HCC cell lines. To evaluate the function of CRABP2, shRNA-knockdown strategy was used in HCC cells. Cell proliferation, metastasis, and apoptosis were analyzed by CCK-8, EdU staining, transwell, and flow cytometry assays, respectively. Based on our results, knockdown of CRABP2 by shRNA resulted in the inhibition of tumor proliferation, migration, and invasion in vitro, followed by increased tumor apoptosis-related protein expression and decreased ERK/VEGF pathway-related proteins expression. CRABP2 silencing in HCC cells also resulted in the failure to develop tumors in vivo. These results provide important insights into the role of CRABP2 in the development and development of HCC. Based on our findings, CRABP2 may be used as a novel diagnostic biomarker, and regulation of CRABP2 in HCC may provide a potential molecular target for the therapy of HCC.

Role of macrophage CD44 in the disposal of inflammatory cell corpses*

2002 ◽  
Vol 103 (5) ◽  
pp. 441-449 ◽  
Author(s):  
Sharon VIVERS ◽  
Ian DRANSFIELD ◽  
Simon P. HART
Keyword(s):  
Molecular Mechanisms ◽  
Inflammatory Diseases ◽  
Surrounding Tissue ◽  
Human Macrophage ◽  
Neutrophil Granulocytes ◽  
Apoptotic Cells ◽  
Tissue Destruction

Understanding the cellular and molecular mechanisms that determine whether inflammation resolves or progresses to scarring and tissue destruction should lead to the development of effective therapeutic strategies for inflammatory diseases. Apoptosis of neutrophil granulocytes is an important determinant of the resolution of inflammation, providing a mechanism for down-regulation of function and triggering clearance by macrophages without inducing a pro-inflammatory response. However, if the rate of cell death by apoptosis is such that the macrophage clearance capacity is exceeded, apoptotic cells may progress to secondary necrosis, resulting in the release of harmful cellular contents and in damage to the surrounding tissue. There are many possible ways in which the rate and capacity of the macrophage-mediated clearance of apoptotic cells may be enhanced or suppressed. Ligation of human macrophage surface CD44 by bivalent monoclonal antibodies rapidly and profoundly augments the capacity of macrophages to phagocytose apoptotic neutrophils in vitro. The molecular mechanism behind this effect and its potential significance in vivo is a current focus of research.

scholarly journals Oxidative stress and ageing of the post-ovulatory oocyte

Reproduction ◽  
2013 ◽  
Vol 146 (6) ◽  
pp. R217-R227 ◽  
Author(s):  
Tessa Lord ◽  
R John Aitken
Keyword(s):  
Oxidative Stress ◽  
Molecular Mechanisms ◽  
Embryo Quality ◽  
Fertilization Rates ◽  
Molecular Events ◽  
Post Implantation ◽  
Potential Use

With extended periods of time following ovulation, the metaphase II stage oocyte experiences deterioration in quality referred to as post-ovulatory oocyte ageing. Post-ovulatory ageing occurs both in vivo and in vitro and has been associated with reduced fertilization rates, poor embryo quality, post-implantation errors and abnormalities in the offspring. Although the physiological consequences of post-ovulatory oocyte ageing have largely been established, the molecular mechanisms controlling this process are not well defined. This review analyses the relationships between biochemical changes exhibited by the ageing oocyte and the symptoms associated with the ageing phenotype. We also discuss molecular events that are potentially involved in orchestrating post-ovulatory ageing with a particular focus on the role of oxidative stress. We propose that oxidative stress may act as the initiator for a cascade of events that create the aged oocyte phenotype. Specifically, oxidative stress has the capacity to cause a decline in levels of critical cell cycle factors such as maturation-promoting factor, impair calcium homoeostasis, induce mitochondrial dysfunction and directly damage multiple intracellular components of the oocyte such as lipids, proteins and DNA. Finally, this review addresses current strategies for delaying post-ovulatory oocyte ageing with a particular focus on the potential use of compounds such as caffeine or selected antioxidants in the development of more refined media for the preservation of oocyte integrity during IVF procedures.

scholarly journals A comprehensive protein–protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1

2014 ◽  
Vol 25 (14) ◽  
pp. 2199-2215 ◽  
Author(s):  
Desiree DeMille ◽  
Benjamin T. Bikman ◽  
Andrew D. Mathis ◽  
John T. Prince ◽  
Jordan T. Mackay ◽  
...  
Keyword(s):  
Glucose Homeostasis ◽  
Protein Modification ◽  
Molecular Mechanisms ◽  
Binding Partners ◽  
Pas Kinase ◽  
Protein Interactome ◽  
Deficient Mice

Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein–protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase–deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis.

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