scholarly journals Alternative Splicing of FN (Fibronectin) Regulates the Composition of the Arterial Wall Under Low Flow

2020 ◽  
Author(s):  
Patrick A. Murphy ◽  
Noor Jailkhani ◽  
Sarah-Anne Nicholas ◽  
Amanda M. Del Rosario ◽  
Jeremy L. Balsbaugh ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Alternative Splicing ◽  
Low Flow ◽  
Matrix Composition ◽  
Splice Isoforms ◽  
Vitro Assay ◽  
Disturbed Flow ◽  
The Impact

Objective: Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage. However, the impact of alternative splicing of FN on extracellular matrix composition remains unknown. Approach and Results: Here, we perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. We also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. We found flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in our in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments. Conclusions: Our results reveal the extent of the dynamic alterations in the matrisome in the acute response to low and disturbed flow and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, can affect matrix composition.

scholarly journals Scutellarin ameliorates pulmonary fibrosis through inhibiting NF-κB/NLRP3-mediated epithelial–mesenchymal transition and inflammation

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Ling Peng ◽  
Li Wen ◽  
Qing-Feng Shi ◽  
Feng Gao ◽  
Bin Huang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Pulmonary Fibrosis ◽  
Epithelial Mesenchymal Transition ◽  
Matrix Metalloproteinase 2 ◽  
Mesenchymal Transition ◽  
The Impact ◽  
Anti Inflammation ◽  
Iκbα Degradation

AbstractIdiopathic pulmonary fibrosis (IPF) is featured with inflammation and extensive lung remodeling caused by overloaded deposition of extracellular matrix. Scutellarin is the major effective ingredient of breviscapine and its anti-inflammation efficacy has been reported before. Nevertheless, the impact of scutellarin on IPF and the downstream molecular mechanism remain unclear. In this study, scutellarin suppressed BLM-induced inflammation via NF-κB/NLRP3 pathway both in vivo and in vitro. BLM significantly elevated p-p65/p65 ratio, IκBα degradation, and levels of NLRP3, caspase-1, caspase-11, ASC, GSDMDNterm, IL-1β, and IL-18, while scutellarin reversed the above alterations except for that of caspase-11. Scutellarin inhibited BLM-induced epithelial–mesenchymal transition (EMT) process in vivo and in vitro. The expression levels of EMT-related markers, including fibronectin, vimentin, N-cadherin, matrix metalloproteinase 2 (MMP-2) and MMP-9, were increased in BLM group, and suppressed by scutellarin. The expression level of E-cadherin showed the opposite changes. However, overexpression of NLRP3 eliminated the anti-inflammation and anti-EMT functions of scutellarin in vitro. In conclusion, scutellarin suppressed inflammation and EMT in BLM-induced pulmonary fibrosis through NF-κB/NLRP3 signaling.

scholarly journals Mechanobiology of Epithelia From the Perspective of Extracellular Matrix Heterogeneity

2020 ◽  
Vol 8 ◽  
Author(s):  
Aleksandra N. Kozyrina ◽  
Teodora Piskova ◽  
Jacopo Di Russo
Keyword(s):  
Extracellular Matrix ◽  
Three Dimensional ◽  
Cell Sheets ◽  
Ecm Remodeling ◽  
Cellular Behavior ◽  
New Concepts ◽  
Matrix Heterogeneity ◽  
The Impact

Understanding the complexity of the extracellular matrix (ECM) and its variability is a necessary step on the way to engineering functional (bio)materials that serve their respective purposes while relying on cell adhesion. Upon adhesion, cells receive messages which contain both biochemical and mechanical information. The main focus of mechanobiology lies in investigating the role of this mechanical coordination in regulating cellular behavior. In recent years, this focus has been additionally shifted toward cell collectives and the understanding of their behavior as a whole mechanical continuum. Collective cell phenomena very much apply to epithelia which are either simple cell-sheets or more complex three-dimensional structures. Researchers have been mostly using the organization of monolayers to observe their collective behavior in well-defined experimental setups in vitro. Nevertheless, recent studies have also reported the impact of ECM remodeling on epithelial morphogenesis in vivo. These new concepts, combined with the knowledge of ECM biochemical complexity are of key importance for engineering new interactive materials to support both epithelial remodeling and homeostasis. In this review, we summarize the structure and heterogeneity of the ECM before discussing its impact on the epithelial mechanobiology.

scholarly journals The Impact of the Extracellular Matrix Environment on Sost Expression by the MLO-Y4 Osteocyte Cell Line

2022 ◽  
Vol 9 (1) ◽  
pp. 35
Author(s):  
Robert T. Brady ◽  
Fergal J. O’Brien ◽  
David A. Hoey
Keyword(s):  
Extracellular Matrix ◽  
Cell Line ◽  
Three Dimensional ◽  
3D Culture ◽  
Specific Gene ◽  
Collagen Scaffolds ◽  
Sost Gene ◽  
The Impact

Bone is a dynamic organ that can adapt its structure to meet the demands of its biochemical and biophysical environment. Osteocytes form a sensory network throughout the tissue and orchestrate tissue adaptation via the release of soluble factors such as a sclerostin. Osteocyte physiology has traditionally been challenging to investigate due to the uniquely mineralized extracellular matrix (ECM) of bone leading to the development of osteocyte cell lines. Importantly, the most widely researched and utilized osteocyte cell line: the MLO-Y4, is limited by its inability to express sclerostin (Sost gene) in typical in-vitro culture. We theorised that culture in an environment closer to the in vivo osteocyte environment could impact on Sost expression. Therefore, this study investigated the role of composition and dimensionality in directing Sost expression in MLO-Y4 cells using collagen-based ECM analogues. A significant outcome of this study is that MLO-Y4 cells, when cultured on a hydroxyapatite (HA)-containing two-dimensional (2D) film analogue, expressed Sost. Moreover, three-dimensional (3D) culture within HA-containing collagen scaffolds significantly enhanced Sost expression, demonstrating the impact of ECM composition and dimensionality on MLO-Y4 behaviour. Importantly, in this bone mimetic ECM environment, Sost expression was found to be comparable to physiological levels. Lastly, MLO-Y4 cells cultured in these novel conditions responded accordingly to fluid flow stimulation with a decrease in expression. This study therefore presents a novel culture system for the MLO-Y4 osteocyte cell line, ensuring the expression of an important osteocyte specific gene, Sost, overcoming a major limitation of this model.

Further Considerations Towards an Effective and Efficient Oncology Drug Discovery DMPK Strategy

2020 ◽  
Vol 21 (2) ◽  
pp. 145-162 ◽  
Author(s):  
Beth Williamson ◽  
Nicola Colclough ◽  
Adrian John Fretland ◽  
Barry Christopher Jones ◽  
Rhys Dafydd Owen Jones ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Drug Exposure ◽  
Phase Iii ◽  
Human Pharmacokinetics ◽  
Vitro Assay ◽  
Pharmacodynamic Modelling ◽  
Oncology Drug ◽  
The Impact

Background: DMPK data and knowledge are critical in maximising the probability of developing successful drugs via the application of in silico, in vitro and in vivo approaches in drug discovery. Methods: The evaluation, optimisation and prediction of human pharmacokinetics is now a mainstay within drug discovery. These elements are at the heart of the ‘right tissue’ component of AstraZeneca’s ‘5Rs framework’ which, since its adoption, has resulted in increased success of Phase III clinical trials. With the plethora of DMPK related assays and models available, there is a need to continually refine and improve the effectiveness and efficiency of approaches best to facilitate the progression of quality compounds for human clinical testing. Results: This article builds on previously published strategies from our laboratories, highlighting recent discoveries and successes, that brings our AstraZeneca Oncology DMPK strategy up to date. We review the core aspects of DMPK in Oncology drug discovery and highlight data recently generated in our laboratories that have influenced our screening cascade and experimental design. We present data and our experiences of employing cassette animal PK, as well as re-evaluating in vitro assay design for metabolic stability assessments and expanding our use of freshly excised animal and human tissue to best inform first time in human dosing and dose escalation studies. Conclusion: Application of our updated drug-drug interaction and central nervous system drug exposure strategies are exemplified, as is the impact of physiologically based pharmacokinetic and pharmacokinetic-pharmacodynamic modelling for human predictions.

Physiologically-Based Pharmacokinetic (PBPK) Modelling of Transporter Mediated Drug Absorption, Clearance and Drug-Drug Interactions

2021 ◽  
Vol 21 ◽  
Author(s):  
Kunal S. Taskar ◽  
Isobel Harada ◽  
Ravindra V. Alluri
Keyword(s):  
Drug Interactions ◽  
Intestinal Absorption ◽  
Drug Disposition ◽  
Pbpk Modelling ◽  
Vitro Assay ◽  
Pbpk Models ◽  
In Vitro Systems ◽  
The Impact

Abstarct: Membrane transporters play an important role in intestinal absorption, distribution and clearance of drugs. Additionally transporters along with enzymes regulate tissue exposures (e.g. liver, kidney and brain), which are important for safety and efficacy considerations. Early identification of transporters involved guides generation of in vitro and in vivo data needed to gain mechanistic understanding on the role of transporters in organ clearance, tissue exposures and enables development of physiological-based pharmacokinetic (PBPK) models. A lot of progress has been made in developing several in vitro assay systems and mechanistic in silico models to determine kinetic parameters for transporters, which are incorporated into PBPK models. Although, intrinsic clearance and inhibition data from in vitro systems generally tend to underpredict in vivo clearance and magnitude of drug-drug interactions (DDIs), empirical scaling factors derived from a sizable dataset are often used to offset underpredictions. PBPK models are increasing used to predict the impact of transporters on intestinal absorption, clearance, victim and perpetrator DDIs prior to first in human clinical trials. The models are often refined when clinical data is available and are used to predict pharmacokinetics in untested scenarios such as the impact of polymorphisms, ontogeny, ethnicity, disease states and DDIs with other perpetrator drugs. The aim of this review is to provide an overview of (i) regulatory requirements around transporters, (ii) in vitro systems and their limitations in predicting transporter mediated drug disposition and DDIs, (iii) PBPK modelling tactics and case studies used for internal decision making and/or for regulatory submissions.

scholarly journals Perlecan Knockdown Significantly Alters Extracellular Matrix Composition and Organization During Cartilage Development

2020 ◽  
Vol 19 (7) ◽  
pp. 1220-1235 ◽  
Author(s):  
Alexander R. Ocken ◽  
Madeline M. Ku ◽  
Tamara L. Kinzer-Ursem ◽  
Sarah Calve
Keyword(s):  
Extracellular Matrix ◽  
Matrix Composition ◽  
Pericellular Matrix ◽  
Post Translational Modifications ◽  
Healthy Cartilage ◽  
Liquid Chromatography Tandem Mass ◽  
Cartilage Biomechanics ◽  
Insight Into

Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo. This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y−Neo. Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y−Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS.

An in Vitro Assay Using Cultured Kupffer Cells Can Predict the Impact of Drug Conjugation on in Vivo Antibody Pharmacokinetics

2020 ◽  
Vol 17 (3) ◽  
pp. 802-809
Author(s):  
David W Meyer ◽  
Lauren B Bou ◽  
Sara Shum ◽  
Mechthild Jonas ◽  
Martha E Anderson ◽  
...  
Keyword(s):  
Kupffer Cells ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Drug Conjugation ◽  
Antibody Pharmacokinetics ◽  
The Impact

scholarly journals Impact of Nitrogen Fertilization on Phytophthora cinnamomi Root-related Damage in Juglans regia Saplings

HortScience ◽  
2019 ◽  
Vol 54 (12) ◽  
pp. 2188-2194
Author(s):  
Javiera Morales ◽  
Ximena Besoain ◽  
Italo F. Cuneo ◽  
Alejandra Larach ◽  
Laureano Alvarado ◽  
...  
Keyword(s):  
Phytophthora Cinnamomi ◽  
Juglans Regia ◽  
N Fertilization ◽  
Vitro Assay ◽  
Positive Effects ◽  
In Vivo Assay ◽  
The Impact ◽  
Canopy Damage

Excessive nitrogen (N) use in agriculture has been associated with increased severity of the damage caused by Phytophthora species. In this study, we investigated the impact in vitro and in vivo of N about Phytophthora cinnamomi. The preliminary in vitro assay showed the effect of different N sources on the mycelial growth of P. cinnamomi. This assay indicated that ammonium nitrate (NH4NO3) and ammonium sulfate [(NH4)2SO4] allowed for greater control of P. cinnamomi mycelia in comparison with calcium nitrate [Ca(NO3)2] and potassium nitrate (KNO3) when used with 1000 ppm N. The in vivo assay showed the severity of P. cinnamomi in 5-month-old Juglans regia saplings grown under greenhouse conditions. We selected NH4NH3 as the source for N for the greenhouse assay, considering the inhibitory effect on the ingrowth of P. cinnamomi and the intensive use of this fertilizer in agriculture. Walnut saplings were fertilized with 0, 35, 70, 140, 210, and 1050 ppm N and were inoculated with zoospores of P. cinnamomi 45 d after the application of nitrogen treatment (DAA). They were harvested at 90 DAA. We found that a 70-ppm N fertilization reduced the development of P. cinnamomi, resulting in lower root and canopy damage indices (DIs) than the unfertilized inoculated treatments and fertilized treatments greater than 140 ppm. The results of the in vitro and in vivo assay agree that increased N concentrations were associated with reduced mycelium growth of P. cinnamomi, providing further evidence that N fertilization can mitigate this disease. Greater root and canopy damage was observed in saplings fertilized with 1050 ppm N, regardless of whether they were inoculated with P. cinnamomi, as a result of N phytotoxicity (verified through foliar analysis). In contrast, inoculated and unfertilized saplings (N0) also showed high root and canopy DIs associated either with the inoculation with P. cinnamomi or the no fertilization treatment. We postulate that 70 ppm N is the best fertilization rate for J. regia saplings because the positive effects of N on growth are maximized and the damage caused by P. cinnamomi is mitigated.

scholarly journals Bacillus anthracis Exosporium Protein BclA Affects Spore Germination, Interaction with Extracellular Matrix Proteins, and Hydrophobicity

2007 ◽  
Vol 75 (11) ◽  
pp. 5233-5239 ◽  
Author(s):  
Trupti N. Brahmbhatt ◽  
Brian K. Janes ◽  
E. Scott Stibitz ◽  
Stephen C. Darnell ◽  
Patrick Sanz ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Bacillus Anthracis ◽  
Spore Germination ◽  
Extracellular Matrix Proteins ◽  
Matrix Proteins ◽  
Wild Type ◽  
Host Matrix ◽  
The Impact

ABSTRACT Bacillus collagen-like protein of anthracis (BclA) is the immunodominant glycoprotein on the exosporium of Bacillus anthracis spores. Here, we sought to assess the impact of BclA on spore germination in vitro and in vivo, surface charge, and interaction with host matrix proteins. For that purpose, we constructed a markerless bclA null mutant in B. anthracis Sterne strain 34F2. The growth and sporulation rates of the ΔbclA and parent strains were nearly indistinguishable, but germination of mutant spores occurred more rapidly than that of wild-type spores in vitro and was more complete by 60 min. Additionally, the mean time to death of A/J mice inoculated subcutaneously or intranasally with mutant spores was lower than that for the wild-type spores even though the 50% lethal doses of the two strains were similar. We speculated that these in vitro and in vivo differences between mutant and wild-type spores might reflect the ease of access of germinants to their receptors in the absence of BclA. We also compared the hydrophobic and adhesive properties of ΔbclA and wild-type spores. The ΔbclA spores were markedly less water repellent than wild-type spores, and, probably as a consequence, the extracellular matrix proteins laminin and fibronectin bound significantly better to mutant than to wild-type spores. These studies suggest that BclA acts as a shield to not only reduce the ease with which spores germinate but also change the surface properties of the spore, which, in turn, may impede the interaction of the spore with host matrix substances.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

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