scholarly journals Primary Human Trabecular Meshwork Model for Pseudoexfoliation

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3448
Author(s):  
Munmun Chakraborty ◽  
Prity Sahay ◽  
Aparna Rao
Keyword(s):  
Trabecular Meshwork ◽  
Human Disease ◽  
In Vitro Model ◽  
Transforming Growth Factor ◽  
Aggregate Formation ◽  
Mesenchymal Transition ◽  
Time Points ◽  
Vitro Model ◽  
Tgf Β1

The lack of an animal model or an in vitro model limits experimental options for studying temporal molecular events in pseudoexfoliation syndrome (PXF), an age related fibrillopathy causing trabecular meshwork damage and glaucoma. Our goal was to create a workable in vitro model of PXF using primary human TM (HTM) cell lines simulating human disease. Primary HTM cells harvested from healthy donors (n = 3), were exposed to various concentrations (5 ng/mL, 10 ng/mL, 15 ng/mL) of transforming growth factor-beta1 (TGF-β1) for different time points. Morphological change of epithelial–mesenchymal transition (EMT) was analyzed by direct microscopic visualization and immunoblotting for EMT markers. Expression of pro-fibrotic markers were analyzed by quantitative RT-PCR and immunoblotting. Cell viability and death in treated cells was analyzed using FACS and MTT assay. Protein complex and amyloid aggregate formation was analyzed by Immunofluorescence of oligomer11 and amyloid beta fibrils. Effect of these changes with pharmacological inhibitors of canonical and non-canonical TGF pathway was done to analyze the pathway involved. The expression of pro-fibrotic markers was markedly upregulated at 10 ng/mL of TGF-β1 exposure at 48–72 h of exposure with associated EMT changes at the same time point. Protein aggregates were seen maximally at these time points that were found to be localized around the nucleus and in the extracellular matrix (ECM). EMT and pro-fibrotic expression was differentially regulated by different canonical and non-canonical pathways suggesting complex regulatory mechanisms. This in vitro model using HTM cells simulated the main characteristics of human disease in PXF like pro-fibrotic gene expression, EMT, and aggregate formation.

scholarly journals 2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study

PLoS ONE ◽  
2019 ◽  
Vol 14 (9) ◽  
pp. e0221942 ◽  
Author(s):  
Stefania Vernazza ◽  
Sara Tirendi ◽  
Sonia Scarfì ◽  
Mario Passalacqua ◽  
Francesco Oddone ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
In Vitro Model ◽  
Preliminary Assessment ◽  
3D Cultures ◽  
Trabecular Meshwork Cells ◽  
Vitro Model ◽  
2D And 3D

scholarly journals Obesity attenuates the effect of sleep apnea on active TGF-ß1 levels and tumor aggressiveness in patients with melanoma

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Carolina Cubillos-Zapata ◽  
Miguel Ángel Martínez-García ◽  
Elena Díaz-García ◽  
Ana Jaureguizar ◽  
Francisco Campos-Rodríguez ◽  
...  
Keyword(s):  
Sleep Apnea ◽  
Transforming Growth Factor ◽  
Serum Levels ◽  
Transforming Growth Factor Β1 ◽  
Multicenter Observational Study ◽  
Obstructive Sleep ◽  
Obese Subjects ◽  
Vitro Model ◽  
Tgf Β1

Abstract Active transforming growth factor-β1 (TGF-β1), a cytokine partially regulated by hypoxia and obesity, has been related with poor prognosis in several tumors. We determine whether obstructive sleep apnea (OSA) increases serum levels of active TGF-β1 in patients with cutaneous melanoma (CM), assess their relationship with melanoma aggressiveness and analyze the factors related to TGF-β1 levels in obese and non-obese OSA patients. In a multicenter observational study, 290 patients with CM were underwent sleep studies. TGF-β1 was increased in moderate-severe OSA patients vs. non-OSA or mild OSA patients with CM. In OSA patients, TGF-β1 levels correlated with mitotic index, Breslow index and melanoma growth rate, and were increased in presence of ulceration or higher Clark levels. In CM patients, OSA was associated with higher TGF-β1 levels and greater melanoma aggressiveness only in non-obese subjects. An in vitro model showed that IH-induced increases of TGF-β1 expression in melanoma cells is attenuated in the presence of high leptin levels. In conclusion, TGF-β1 levels are associated with melanoma aggressiveness in CM patients and increased in moderate-severe OSA. Moreover, in non-obese patients with OSA, TGF-β1 levels correlate with OSA severity and leptin levels, whereas only associate with leptin levels in obese OSA patients.

scholarly journals Microcarriers Based on Glycosaminoglycan-Like Marine Exopolysaccharide for TGF-β1 Long-Term Protection

Marine Drugs ◽  
2019 ◽  
Vol 17 (1) ◽  
pp. 65 ◽  
Author(s):  
Agata Zykwinska ◽  
Mélanie Marquis ◽  
Mathilde Godin ◽  
Laëtitia Marchand ◽  
Corinne Sinquin ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Target Location ◽  
Cartilage Regeneration ◽  
Transforming Growth Factor Β1 ◽  
Hydrogel Matrix ◽  
Vitro Model ◽  
Tgf Β1

Articular cartilage is an avascular, non-innervated connective tissue with limited ability to regenerate. Articular degenerative processes arising from trauma, inflammation or due to aging are thus irreversible and may induce the loss of the joint function. To repair cartilaginous defects, tissue engineering approaches are under intense development. Association of cells and signalling proteins, such as growth factors, with biocompatible hydrogel matrix may lead to the regeneration of the healthy tissue. One current strategy to enhance both growth factor bioactivity and bioavailability is based on the delivery of these signalling proteins in microcarriers. In this context, the aim of the present study was to develop microcarriers by encapsulating Transforming Growth Factor-β1 (TGF-β1) into microparticles based on marine exopolysaccharide (EPS), namely GY785 EPS, for further applications in cartilage engineering. Using a capillary microfluidic approach, two microcarriers were prepared. The growth factor was either encapsulated directly within the microparticles based on slightly sulphated derivative or complexed firstly with the highly sulphated derivative before being incorporated within the microparticles. TGF-β1 release, studied under in vitro model conditions, revealed that the majority of the growth factor was retained inside the microparticles. Bioactivity of released TGF-β1 was particularly enhanced in the presence of highly sulphated derivative. It comes out from this study that GY785 EPS based microcarriers may constitute TGF-β1 reservoirs spatially retaining the growth factor for a variety of tissue engineering applications and in particular cartilage regeneration, where the growth factor needs to remain in the target location long enough to induce robust regenerative responses.

scholarly journals Antifibrotic effect of Ocimum basilicum L. and linalool on arecoline-induced fibrosis in human buccal fibroblasts

2018 ◽  
Vol 3 ◽  
pp. 2057178X1876447 ◽  
Author(s):  
Pooja Adtani ◽  
Narasimhan Malathi ◽  
Kannan Ranganathan ◽  
Sivaswamy Lokeswari ◽  
Alan Mathew Punnoose
Keyword(s):  
Transforming Growth Factor Beta ◽  
In Vitro Model ◽  
Transforming Growth Factor ◽  
Oral Submucous Fibrosis ◽  
Ocimum Basilicum ◽  
Morphological Alterations ◽  
Antifibrotic Effect ◽  
Submucous Fibrosis ◽  
Vitro Model

Aim: To explore Ocimum basilicum L. (sweet basil) and linalool for their antifibrotic activity in an arecoline-induced in vitro fibrotic model. Methods: Leaf extract of O. basilicum L. (LEOB) and linalool were used as experimental agents to test their antifibrogenic activity in vitro. Half-maximal inhibitory concentration (IC50) for arecoline, ethanolic LEOB, and linalool was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To evaluate the antifibrotic effect of ethanolic LEOB and linalool on pretreatment, that is, both the testing agents were added to the human buccal fibroblasts (HBFs) prior to induction with arecoline, and reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to study the response of transforming growth factor beta (TGFβ), collagen 1 subtype A2 (COL1A2), and collagen 3 subtype A1 (COL3A1). To appreciate the morphological alterations in HBFs on treatment with arecoline, ethanolic LEOB, and linalool, Masson’s trichrome staining was performed. Results: Arecoline enhanced fibrotic activity by upregulating TGFβ1, COL1A2, and COL3A1 levels, whereas ethanolic LEOB and linalool on pretreatment significantly downregulated the increased levels of TGFβ1, COL1A2, and COL3A1 in primary HBF cell cultures. Conclusion and implication to clinic: Both ethanolic LEOB and linalool exhibited significant antifibrotic activity in an in vitro model. Further studies in an in vitro model can help attain a foundation for an herbal formulation in gel form that can be prescribed to patients diagnosed with oral submucous fibrosis for topical application. It can also be used synergistically with Western medicine.

scholarly journals Metformin activity in an in vitro model of posterior capsule opacification

2018 ◽  
Vol 17 (4) ◽  
pp. 105-112
Author(s):  
Jade Marie Lasiste ◽  
Pablo Zoroquiain ◽  
Denise Miyamoto ◽  
Miguel Burnier
Keyword(s):  
Growth Factor ◽  
Western Blot ◽  
In Vitro Model ◽  
Transforming Growth Factor ◽  
Posterior Capsule Opacification ◽  
Posterior Capsule ◽  
Vitro Model ◽  
And Migration ◽  
E Cadherin

Purpose: To determine the activity of metformin in an in vitro model of posterior capsule opacification (PCO).Design: Experimental laboratory research. Methods: The HLE-B3 lens epithelial cell line was treated with PCO induction media (PCOM) supplemented with transforming growth factor-beta (TGF-β) and fibroblast growth factor (FGF). Different metformin concentrations (0-100 mM) were used. The following cellular parameters were assessed: (1) survival, using a viability assay; (2) morphology, via microscopy and image analysis; (3) migration, using the wound assay; (4) and expression of epithelial (Pax6, E-cadherin) and mesenchymal (α-smooth muscle actin or α-SMA, fibronectin) markers via Western blot. Expression of the uptake receptor SLC22A1 was evaluated in HLE-B3 and in human donor eyes with Western blot and immunohistochemistry, respectively. Statistical analysis of variance (ANOVA) with Tukey post-hoc test was done for analysis of cytotoxicity, morphology and migration data. Results: Metformin was lethal to half (LC50) of the cells at 30 mM, and a decrease in viability (P<0.05) was noted at 5 mM. LECs in PCOM treated with 1 mM metformin showed increased Pax6 and E-cadherin and decreased α-SMA and fibronectin expression. LECs in PCOM treated with metformin also maintained epithelial morphology. Migration was inhibited with 0.5 mM metformin (P<0.05). Both HLE-B3 and the lens epithelium in donor eyes were found to express SLC22A1.Conclusion: Metformin decreased survival and migration in LECs, maintaining epithelial phenotype and reducing mesenchymal marker expression. Metformin therefore has potential as an adjunct in PCO prevention.Financial Disclosures: This work was partially funded by Mitacs Canada.

scholarly journals The effect of TGF-β1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells

2007 ◽  
Vol 12 (3) ◽  
Author(s):  
Guo-Ping Xu ◽  
Qing-Quan Li ◽  
Xi-Xi Cao ◽  
Qi Chen ◽  
Zhong-Hua Zhao ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Epithelial Cells ◽  
Transforming Growth Factor ◽  
Alveolar Epithelial Cells ◽  
Ultrastructural Changes ◽  
Type Ii ◽  
Mesenchymal Transition ◽  
Alveolar Epithelial ◽  
Tgf Β1

AbstractThe aim of this study was to investigate whether transforming growth factor-β1 (TGF-β1) could induce alveolar epithelial-mesenchymal transition (EMT) in vitro, and whether Smad7 gene transfer could block this transition. We also aimed to elucidate the possible mechanisms of these processes. The Smad7 gene was transfected to the rat type II alveolar epithelial cell line (RLE-6TN). Expression of the EMT-associated markers was assayed by Western Blot and Real-time PCR. Morphological alterations were examined via phase-contrast microscope and fluorescence microscope, while ultrastructural changes were examined via electron microscope. TGF-β1 treatment induced a fibrotic phenotype of RLE-6TN with increased expression of fibronectin (FN), α-smooth muscle actin (α-SMA) and vimentin, and decreased expression of E-cadherin (E-cad) and cytokeratin19 (CK19). After transfecting the RLE-6TN with the Smad7 gene, the expression of the mesenchymal markers was downregulated while that of the epithelial markers was upregulated. TGF-β1 treatment for 48 h resulted in the separation of RLE-6TN from one another and a change into elongated, myofibroblast-like cells. After the RLE-6TN had been transfected with the Smad7 gene, TGF-β1 treatment had no effect on the morphology of the RLE-6TN. TGF-β1 treatment for 48 h resulted in an abundant expression of α-SMA in the RLE-6TN. If the RLE-6TN were transfected with the Smad7 gene, TGF-β1 treatment for 48 h could only induce a low level of α-SMA expression. Furthermore, TGF-β1 treatment for 12 h resulted in the degeneration and swelling of the osmiophilic multilamellar bodies, which were the markers of type II alveolar epithelial cells. TGF-β1 can induce alveolar epithelialmesenchymal transition in vitro, which is dependent on the Smads signaling pathway to a certain extent. Overexpression of the Smad7 gene can partially block this process

scholarly journals An In Vitro Model That Recapitulates the Epithelial to Mesenchymal Transition (EMT) in Human Breast Cancer

PLoS ONE ◽  
2011 ◽  
Vol 6 (2) ◽  
pp. e17083 ◽  
Author(s):  
Elad Katz ◽  
Sylvie Dubois-Marshall ◽  
Andrew H. Sims ◽  
Philippe Gautier ◽  
Helen Caldwell ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Human Breast Cancer ◽  
In Vitro Model ◽  
Epithelial To Mesenchymal Transition ◽  
Human Breast ◽  
Mesenchymal Transition ◽  
Vitro Model
Sign in / Sign up

Export Citation Format

Share Document