scholarly journals Microfluidic organ-on-a-chip model of the outer blood–retinal barrier with clinically relevant read-outs for tissue permeability and vascular structure

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Yusuf B. Arık ◽  
Wesley Buijsman ◽  
Joshua Loessberg-Zahl ◽  
Carlos Cuartas-Vélez ◽  
Colin Veenstra ◽  
...  
Keyword(s):  
Complex Disease ◽  
Vascular Structure ◽  
Blood Retinal Barrier ◽  
Future Studies ◽  
Disease Mechanisms ◽  
Visual Disorders ◽  
Organ On A Chip ◽  
Tissue Permeability

This organ-on-a-chip device of the outer blood retinal barrier will allow future studies of complex disease mechanisms and treatments of visual disorders using clinically relevant endpoints in vitro.

Usefulness of collaboration between mathematical models and cell engineering for elucidating complex disease mechanisms and discover effective drugs

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
H Kohjitani ◽  
A Kashiwa ◽  
T Makiyama ◽  
F Toyoda ◽  
Y Yamamoto ◽  
...  
Keyword(s):  
Mathematical Model ◽  
In Silico ◽  
Complex Disease ◽  
Ion Selectivity ◽  
Public Institution ◽  
Cell Engineering ◽  
Funding Source ◽  
Patch Clamp Technique ◽  
In Silico Model

Abstract Background A missense mutation, CACNA1C-E1115K, located in the cardiac L-type calcium channel (LTCC), was recently reported to be associated with diverse arrhythmias. Several studies reported in-vivo and in-vitro modeling of this mutation, but actual mechanism and target drug of this disease has not been clarified due to its complex ion-mechanisms. Objective To reveal the mechanism of this diverse arrhythmogenic phenotype using combination of in-vitro and in-silico model. Methods and results Cell-Engineering Phase: We generated human induced pluripotent stem cell (hiPSC) from a patient carrying heterozygous CACNA1C-E1115K and differentiated into cardiomyocytes. Spontaneous APs were recorded from spontaneously beating single cardiomyocytes by using the perforated patch-clamp technique. Mathematical-Modeling Phase: We newly developed ICaL-mutation mathematical model, fitted into experimental data, including its impaired ion selectivity. Furthermore, we installed this mathematical model into hiPSC-CM simulation model. Collaboration Phase: Mutant in-silico model showed APD prolongation and frequent early afterdepolarization (EAD), which are same as in-vitro model. In-silico model revealed this EAD was mostly related to robust late-mode of sodium current occurred by Na+ overload and suggested that mexiletine is capable of reducing arrhythmia. Afterward, we applicated mexiletine onto hiPSC-CMs mutant model and found mexiletine suppress EADs. Conclusions Precise in-silico disease model can elucidate complicated ion currents and contribute predicting result of drug-testing. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists

scholarly journals Urine-Derived Epithelial Cells as Models for Genetic Kidney Diseases

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1413
Author(s):  
Tjessa Bondue ◽  
Fanny O. Arcolino ◽  
Koenraad R. P. Veys ◽  
Oyindamola C. Adebayo ◽  
Elena Levtchenko ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Kidney Diseases ◽  
Cell Types ◽  
Cell Models ◽  
Tubular Epithelial Cells ◽  
Disease Mechanisms ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular ◽  
Disease Cell

Epithelial cells exfoliated in human urine can include cells anywhere from the urinary tract and kidneys; however, podocytes and proximal tubular epithelial cells (PTECs) are by far the most relevant cell types for the study of genetic kidney diseases. When maintained in vitro, they have been proven extremely valuable for discovering disease mechanisms and for the development of new therapies. Furthermore, cultured patient cells can individually represent their human sources and their specific variants for personalized medicine studies, which are recently gaining much interest. In this review, we summarize the methodology for establishing human podocyte and PTEC cell lines from urine and highlight their importance as kidney disease cell models. We explore the well-established and recent techniques of cell isolation, quantification, immortalization and characterization, and we describe their current and future applications.

scholarly journals Influences of advanced glycosylation end products on the inner blood–retinal barrier in a co-culture cell model in vitro

2020 ◽  
Vol 15 (1) ◽  
pp. 619-628
Author(s):  
Chen Yuan ◽  
Ya Mo ◽  
Jie Yang ◽  
Mei Zhang ◽  
Xuejun Xie
Keyword(s):  
Electrical Resistance ◽  
Cell Model ◽  
Polyclonal Antibodies ◽  
Time Dependent ◽  
Dependent Manner ◽  
Blood Retinal Barrier ◽  
Advanced Glycosylation End Products ◽  
End Products ◽  
Advanced Glycosylation

AbstractAdvanced glycosylation end products (AGEs) are harmful factors that can damage the inner blood–retinal barrier (iBRB). Rat retinal microvascular endothelial cells (RMECs) were isolated and cultured, and identified by anti-CD31 and von Willebrand factor polyclonal antibodies. Similarly, rat retinal Müller glial cells (RMGCs) were identified by H&E staining and with antibodies of glial fibrillary acidic protein and glutamine synthetase. The transepithelial electrical resistance (TEER) value was measured with a Millicell electrical resistance system to observe the leakage of the barrier. Transwell cell plates for co-culturing RMECs with RMGCs were used to construct an iBRB model, which was then tested with the addition of AGEs at final concentrations of 50 and 100 mg/L for 24, 48, and 72 h. AGEs in the in vitro iBRB model constructed by RMEC and RMGC co-culture led to the imbalance of the vascular endothelial growth factor (VEGF) and pigment epithelial derivative factor (PEDF), and the permeability of the RMEC layer increased because the TEER decreased in a dose- and time-dependent manner. AGEs increased VEGF but lowered PEDF in a dose- and time-dependent manner. The intervention with AGEs led to the change of the transendothelial resistance of the RMEC layer likely caused by the increased ratio of VEGF/PEDF.

scholarly journals Janus-faced EPHB4-associated disorders: novel pathogenic variants and unreported intrafamilial overlapping phenotypes

2021 ◽  
Author(s):  
Silvia Martin-Almedina ◽  
Kazim Ogmen ◽  
Ege Sackey ◽  
Dionysios Grigoriadis ◽  
Christina Karapouliou ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Fetal Hydrops ◽  
Clinical Phenotypes ◽  
Functional Studies ◽  
Pathogenic Variants ◽  
Disease Mechanisms ◽  
Novel Variants ◽  
Uncertain Significance ◽  
Clinical Phenotyping

Abstract Purpose Several clinical phenotypes including fetal hydrops, central conducting lymphatic anomaly or capillary malformations with arteriovenous malformations 2 (CM-AVM2) have been associated with EPHB4 (Ephrin type B receptor 4) variants, demanding new approaches for deciphering pathogenesis of novel variants of uncertain significance (VUS) identified in EPHB4, and for the identification of differentiated disease mechanisms at the molecular level. Methods Ten index cases with various phenotypes, either fetal hydrops, CM-AVM2, or peripheral lower limb lymphedema, whose distinct clinical phenotypes are described in detail in this study, presented with a variant in EPHB4. In vitro functional studies were performed to confirm pathogenicity. Results Pathogenicity was demonstrated for six of the seven novel EPHB4 VUS investigated. A heterogeneity of molecular disease mechanisms was identified, from loss of protein production or aberrant subcellular localization to total reduction of the phosphorylation capability of the receptor. There was some phenotype–genotype correlation; however, previously unreported intrafamilial overlapping phenotypes such as lymphatic-related fetal hydrops (LRFH) and CM-AVM2 in the same family were observed. Conclusion This study highlights the usefulness of protein expression and subcellular localization studies to predict EPHB4 variant pathogenesis. Our accurate clinical phenotyping expands our interpretation of the Janus-faced spectrum of EPHB4-related disorders, introducing the discovery of cases with overlapping phenotypes.

scholarly journals Mechanical Strain-Enabled Reconstitution of Dynamic Environment in Organ-on-a-Chip Platforms: A Review

Micromachines ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 765
Author(s):  
Qianbin Zhao ◽  
Tim Cole ◽  
Yuxin Zhang ◽  
Shi-Yang Tang
Keyword(s):  
Cell Culture ◽  
Shear Stress ◽  
Cultured Cells ◽  
Mechanical Strain ◽  
3D Cell Culture ◽  
Small Scale ◽  
Mechanical Stimuli ◽  
Human Organ ◽  
Organ On A Chip

Organ-on-a-chip (OOC) uses the microfluidic 3D cell culture principle to reproduce organ- or tissue-level functionality at a small scale instead of replicating the entire human organ. This provides an alternative to animal models for drug development and environmental toxicology screening. In addition to the biomimetic 3D microarchitecture and cell–cell interactions, it has been demonstrated that mechanical stimuli such as shear stress and mechanical strain significantly influence cell behavior and their response to pharmaceuticals. Microfluidics is capable of precisely manipulating the fluid of a microenvironment within a 3D cell culture platform. As a result, many OOC prototypes leverage microfluidic technology to reproduce the mechanically dynamic microenvironment on-chip and achieve enhanced in vitro functional organ models. Unlike shear stress that can be readily generated and precisely controlled using commercial pumping systems, dynamic systems for generating proper levels of mechanical strains are more complicated, and often require miniaturization and specialized designs. As such, this review proposes to summarize innovative microfluidic OOC platforms utilizing mechanical actuators that induce deflection of cultured cells/tissues for replicating the dynamic microenvironment of human organs.

scholarly journals Plant Extracts and Reactive Oxygen Species as Two Counteracting Agents with Anti- and Pro-Obesity Properties

2019 ◽  
Vol 20 (18) ◽  
pp. 4556 ◽  
Author(s):  
Hanna Zielinska-Blizniewska ◽  
Przemyslaw Sitarek ◽  
Anna Merecz-Sadowska ◽  
Katarzyna Malinowska ◽  
Karolina Zajdel ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Plant Extracts ◽  
Complex Disease ◽  
Reactive Oxygen ◽  
Complementary Therapy ◽  
Biologically Active ◽  
Mitochondrial Activity ◽  
Oxygen Species

Obesity is a complex disease of great public health significance worldwide: It entails several complications including diabetes mellitus type 2, cardiovascular dysfunction and hypertension, and its prevalence is increasing around the world. The pathogenesis of obesity is closely related to reactive oxygen species. The role of reactive oxygen species as regulatory factors in mitochondrial activity in obese subjects, molecules taking part in inflammation processes linked to excessive size and number of adipocytes, and as agents governing the energy balance in hypothalamus neurons has been examined. Phytotherapy is the traditional form of treating health problems using plant-derived medications. Some plant extracts are known to act as anti-obesity agents and have been screened in in vitro models based on the inhibition of lipid accumulation in 3T3-L1 cells and activity of pancreatic lipase methods and in in vivo high-fat diet-induced obesity rat/mouse models and human models. Plant products may be a good natural alternative for weight management and a source of numerous biologically-active chemicals, including antioxidant polyphenols that can counteract the oxidative stress associated with obesity. This review presents polyphenols as natural complementary therapy, and a good nutritional strategy, for treating obesity without serious side effects.

scholarly journals Effects of Hyperglycemia on Vascular Smooth Muscle Ca2+Signaling

2017 ◽  
Vol 2017 ◽  
pp. 1-16 ◽  
Author(s):  
Nahed El-Najjar ◽  
Rashmi P. Kulkarni ◽  
Nancy Nader ◽  
Rawad Hodeify ◽  
Khaled Machaca
Keyword(s):  
Insulin Resistance ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Vascular Smooth Muscle ◽  
Complex Disease ◽  
Prolonged Exposure ◽  
In Vitro System ◽  
A7r5 Cells

Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca2+signaling in an isolated in vitro system using the A7r5 rat aortic cell line as a model. We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca2+signaling, including most prominently an inhibition of the passive ER Ca2+leak and the sarcoplasmic reticulum Ca2+-ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca2+leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca2+signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca2+signaling machinery are different.

Abstract 70: Protein Phosphatase 1 Contributes to Atrial Stunning in Atrial Fibrillation

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Srikanth Perike ◽  
Xander Wehrens ◽  
Dawood Darbar ◽  
Mark McCauley
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain ◽  
Stroke Risk ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Future Studies ◽  
Hek Cells

Background: Atrial fibrillation (AF) is the most common cardiac arrhythmia, and increases a patient’s stroke risk five-fold. Reduced atrial contractility (stunning) is observed in AF and contributes to stroke risk; however, the mechanisms responsible for atrial stunning in AF are unknown. Recent data from our laboratory indicate that protein phosphatase 1 (PP1) dephosphorylation of myosin light chain 2a (MLC2a) may contribute to atrial stunning in AF. Objective: To determine how the PP1 regulatory subunit 12C (PPP1R12C) and catalytic (PPP1c) subunits modify atrial sarcomere phosphorylation in AF. Methods: We evaluated the protein expression, binding and phosphorylation among PPP1R12C, PPP1c, and MLC2a in transfected HL-1 cells, murine atrial tissue (Pitx2null +/– mice, with a genetic predisposition AF), and in HEK cells. An inhibitor of PPP1R12C phosphorylation, BDP5290, was used to enhance the PPP1R12C-PPP1C interaction. Results: In Pitx2 null +/– mice, PPP1R12C was increased by 2-fold ( P <0.01) and associated with a 40% reduction in S-19-MLC2a phosphorylation versus WT mice ( P <0.058). BDP5290 increased PPP1R12C-PPP1C binding by >3-fold in HL-1 cells ( P <0.01). BDP5290 reduced MLC2a phosphorylation by 40% through an enhanced interaction with PPP1R12C by >3-fold in HEK cells ( P <0.01). Conclusion: In Pitx2 null+/- mice, increased expression of PPP1R12C is associated with PP1 holoenzyme targeting to sarcomeric MLC2a, and is associated with reduced S19-MLC2a phosphorylation. Additionally, BDP5290 enhances the PPP1R12C-PPP1C interaction and models PP1 activity in AF. Future studies will examine the effects of both AF and BDP5290 upon atrial contractility in vitro.

Linear plasmids in Klebsiella and other Enterobacteriaceae

2021 ◽  
Author(s):  
Jane Hawkey ◽  
Hugh Cottingham ◽  
Alex Tokolyi ◽  
Ryan R Wick ◽  
Louise M Judd ◽  
...  
Keyword(s):  
Plasmid Stability ◽  
Bacterial Species ◽  
Salmonella Typhi ◽  
Multidrug Resistant ◽  
Linear Plasmid ◽  
Extrachromosomal Dna ◽  
Linear Plasmids ◽  
Future Studies ◽  
And Function

Linear plasmids are extrachromosomal DNA that have been found in a small number of bacterial species. To date, the only linear plasmids described in the Enterobacteriaceae family belong to Salmonella, first found in Salmonella Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. We used this collection to search public sequence databases and discovered an additional 74 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function, however each phylogroup carried its own unique toxin-antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.

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