Icariin Promotes Extracellular Matrix Synthesis and Gene Expression of Chondrocytes In Vitro

[Objective] Runx2, a key transcription factor in osteoblast differentiation, is expressed in calcified atherosclerotic plaques. We have recently shown that Runx2 represses vascular smooth muscle cells (VSMCs) differentiation and promotes their osteogenic differentiation. Connective tissue growth factor (CTGF) has been implicated in the progression to vulnerable plaque by inducing mononuclear cell chemotaxis and VSMCs apoptosis despite of its potent stimulatory effect on connective tissue cell the proliferation and extracellular matrix synthesis. To assess the role of Runx2 in the process of plaque development, we investigated the molecular mechanism of the CTGF gene expression by Runx2 in VSMCs. [Methods and Results] RT-PCR analyses showed that adenovirally overexpressed Runx2 significantly repressed the basal expression of the CTGF gene in human aortic SMCs (HASMCs). Consistent with this, knockdown of the Runx2 expression in HASMCs by small interfering RNA (siRNA) increased CTGF mRNA levels. Luciferase assays showed that Runx2 reduced the transcriptional activity of the CTGF promoter. Transfection of a series of 5′-deletion constructs revealed that Runx2 inhibited CTGF expression through the sequence element located at 5′ untranslated region of CTGF mRNA. We next examined the effects of Runx2 on the TGFβ-induced CTGF expression. Runx2 overexpression significantly repressed CTGF expression in HASMCs stimulated with TGFβ, and knockdown of Runx2 by siRNA enhanced the induction of CTGF expression in response to TGFβ. Runx2 repressed TGFβ-induced CTGF promoter activity through the sequence including Smad binding element (SBE). Overexpression of Runx2 significantly reduced TGFβ- and Smad3-mediated luciferase activity of Smad-dependent promoter which contains four copies of SBE. Biotinylated DNA pulldown assay using SBE of CTGF promoter showed that Runx2 formed a complex with Smad3 and Smad4. [Conclusion] Runx2 repressed basal and TGFβ-induced CTGF gene expression in VSMCs. Thus, in addition to the potential for inducing vascular calcification, Runx2 may affect plaque stability by modulating extracellular matrix synthesis through inhibiting CTGF gene expression and TGFβ signaling.

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Blocking Sp1 Transcription Factor Broadly Inhibits Extracellular Matrix Gene Expression In Vitro and In Vivo: Implications for the Treatment of Tissue Fibrosis

Journal of Investigative Dermatology ◽

10.1046/j.1523-1747.2001.01326.x ◽

2001 ◽

Vol 116 (5) ◽

pp. 755-763 ◽

Cited By ~ 92

Author(s):

Franck Verrecchia ◽

Alain Mauviel ◽

Jérôme Rossert

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Transcription Factor ◽

Tissue Fibrosis ◽

Sp1 Transcription Factor ◽

Matrix Gene

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P0658MATRIX AND FLOW MODULATE GENE EXPRESSION IN A 3D VASCULARISED TUBULE MODEL

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0658 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Julie Williams ◽

Sanlin Robinson ◽

Babak Alaei ◽

Kimberly Homan ◽

Maryam Clausen ◽

...

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Impact Analysis ◽

Cell Types ◽

Drug Uptake ◽

Shear Stresses ◽

The Matrix ◽

And Function

Abstract Background and Aims Questions abound regarding the translation of in vitro 2D cell culture systems to the human setting. This is especially true of the kidney in which there is a complex hierarchical structure and a multitude of cell types. While it is well accepted that extracellular matrix plays a large part in directing cellular physiology emerging research has highlighted the importance of shear stresses and flow rates too. To fully recapitulate the normal gene expression and function of a particular renal cell type how important is it to completely reconstitute their in vivo surroundings? Method To answer this question, we have cultured proximal tubular (PT) epithelial cells in a 3-dimensional channel embedded within an engineered extracellular matrix (ECM) under physiological flow that is colocalised with an adjacent channel lined with renal microvascular endothelial cells that mimic a peritubular capillary. Modifications to the system were made to allow up to 12 chips to be run in parallel in an easily handleable form. After a period of maturation under continuous flow, both cell types were harvested for RNAseq analyses. RNA expression data was compared with cells cultured under static 2-dimensional conditions on plastic or the engineered ECM. Additionally, the perfusion of glucose through this 3D vascularised PT model has been investigated in the presence and absence of known diabetes modulating agents. Results PCA of RNAseq data showed that a) static non-coated, b) static matrix-coated and c) flow matrix-coated conditions separated into 3 distinct groups, while cell co-culture had less impact. Analysis of transcriptomic signatures showed that many genes were modulated by the matrix with additional genes influenced under flow conditions. Several of these genes, classified as transporters, are of particular importance when using this model to assess drug uptake and safety implications. Co-culture regulated some interesting genes, but fewer than anticipated. Preliminary experiments are underway to monitor glucose uptake and transport between tubules under different conditions. Conclusion We have developed a medium throughput system in which matrix and flow modulate gene expression. This system can be used to study the physiology of molecular cross-talk between cells. Ongoing analysis will further consider relevance to human physiology.

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Combining Innovative Bioink and Low Cell Density for the Production of 3D-Bioprinted Cartilage Substitutes: A Pilot Study

Stem Cells International ◽

10.1155/2020/2487072 ◽

2020 ◽

Vol 2020 ◽

pp. 1-16 ◽

Cited By ~ 3

Author(s):

Christel Henrionnet ◽

Léa Pourchet ◽

Paul Neybecker ◽

Océane Messaoudi ◽

Pierre Gillet ◽

...

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Cell Encapsulation ◽

Type Ii Collagen ◽

Mitochondrial Activity ◽

Type Ii ◽

3D Bioprinting ◽

Matrix Synthesis ◽

M Cell ◽

Low Concentration

3D bioprinting offers interesting opportunities for 3D tissue printing by providing living cells with appropriate scaffolds with a dedicated structure. Biological advances in bioinks are currently promising for cell encapsulation, particularly that of mesenchymal stem cells (MSCs). We present herein the development of cartilage implants by 3D bioprinting that deliver MSCs encapsulated in an original bioink at low concentration. 3D-bioprinted constructs (10×10×4 mm) were printed using alginate/gelatin/fibrinogen bioink mixed with human bone marrow MSCs. The influence of the bioprinting process and chondrogenic differentiation on MSC metabolism, gene profiles, and extracellular matrix (ECM) production at two different MSC concentrations (1 million or 2 million cells/mL) was assessed on day 28 (D28) by using MTT tests, real-time RT-PCR, and histology and immunohistochemistry, respectively. Then, the effect of the environment (growth factors such as TGF-β1/3 and/or BMP2 and oxygen tension) on chondrogenicity was evaluated at a 1 M cell/mL concentration on D28 and D56 by measuring mitochondrial activity, chondrogenic gene expression, and the quality of cartilaginous matrix synthesis. We confirmed the safety of bioextrusion and gelation at concentrations of 1 million and 2 million MSC/mL in terms of cellular metabolism. The chondrogenic effect of TGF-β1 was verified within the substitute on D28 by measuring chondrogenic gene expression and ECM synthesis (glycosaminoglycans and type II collagen) on D28. The 1 M concentration represented the best compromise. We then evaluated the influence of various environmental factors on the substitutes on D28 (differentiation) and D56 (synthesis). Chondrogenic gene expression was maximal on D28 under the influence of TGF-β1 or TGF-β3 either alone or in combination with BMP-2. Hypoxia suppressed the expression of hypertrophic and osteogenic genes. ECM synthesis was maximal on D56 for both glycosaminoglycans and type II collagen, particularly in the presence of a combination of TGF-β1 and BMP-2. Continuous hypoxia did not influence matrix synthesis but significantly reduced the appearance of microcalcifications within the extracellular matrix. The described strategy is very promising for 3D bioprinting by the bioextrusion of an original bioink containing a low concentration of MSCs followed by the culture of the substitutes in hypoxic conditions under the combined influence of TGF-β1 and BMP-2.

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LncRNA GAS5 as miR-26a-5p Sponge Regulates the PTEN/PI3K/Akt Axis and Affects Extracellular Matrix Synthesis in Degenerative Nucleus Pulposus Cells in vitro

Frontiers in Neurology ◽

10.3389/fneur.2021.653341 ◽

2021 ◽

Vol 12 ◽

Author(s):

Liang Tan ◽

Yifang Xie ◽

Ye Yuan ◽

Kai Hu

Keyword(s):

Extracellular Matrix ◽

Nucleus Pulposus ◽

Intervertebral Disc Degeneration ◽

Nucleus Pulposus Cell ◽

Nucleus Pulposus Cells ◽

Akt Inhibitor ◽

Matrix Synthesis ◽

Lncrna Gas5

The role of lncRNA growth arrest specific 5 (GAS5) in degenerative nucleus pulposus cell (NPC) apoptosis has been reported, but the mechanism of GAS5 in extracellular matrix (ECM) synthesis in intervertebral disc degeneration (IDD) remains unknown. We aimed to investigate the mechanism of GAS5 in ECM synthesis in degenerative NPCs. GAS5 expression was measured in degenerative NPCs (CP-H170) and normal NPCs (CP-H097). siRNA-mediated GAS5 knockdown was transfected to NPCs to detect cell viability and the expression of ECM-related genes (Collagen II, aggrecan, Collagen I, and MMP-3). Subcellular localization of GAS5 was analyzed. The downstream gene and pathway of GAS5 in degenerative NPCs were explored. As our results indicated, lncRNA GAS5 was upregulated in degenerative NPCs. Silencing GAS5 improved the viability of degenerative NPCs and increased ECM synthesis. GAS5 was mainly located in the cytoplasm of NPCs. LncRNA GAS5 sponged miR-26a-5p to regulate PTEN. Overexpression of miR-26a-5p promoted ECM synthesis in degenerative NPCs. Akt inhibitor LY294002 reversed the promotion of silencing GAS5 on ECM synthesis of degenerative NPCs. In conclusion, lncRNA GAS5 sponged miR-26a-5p to upregulate PTEN and inhibit the PI3K/Akt pathway, thus inhibiting ECM synthesis of degenerative NPCs.

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Glucose Availability Affects Extracellular Matrix Synthesis During Chondrogenesis In Vitro

Tissue Engineering Part A ◽

10.1089/ten.tea.2020.0144 ◽

2021 ◽

Author(s):

Yi Zhong ◽

Arnold I Caplan ◽

Jean F. Welter ◽

Harihara Baskaran

Keyword(s):

Extracellular Matrix ◽

Matrix Synthesis ◽

Glucose Availability

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Human endothelial cell morphology and autacoid expression

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1995.268.4.h1613 ◽

1995 ◽

Vol 268 (4) ◽

pp. H1613-H1620

Author(s):

C. J. de Groot ◽

V. A. Chao ◽

J. M. Roberts ◽

R. N. Taylor

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Morphological Changes ◽

Umbilical Vein ◽

Three Dimensional ◽

Endothelin 1 ◽

And Function ◽

Cellular Fibronectin ◽

Cells Cultured

Human umbilical vein endothelial (HUVE) cells plated on plastic or gelatin-coated dishes grow as a “cobblestone” monolayer. By contrast, endothelial cells cultured on a complex matrix (e.g., Matrigel) form three-dimensional, capillary-like structures. In the current study, we verified the capillary phenotype of the latter structures and asked whether the morphological changes induced by extracellular matrix also affect human endothelial gene expression and function in vitro. Concentrations of cellular fibronectin, prostacyclin, and endothelin-1 were measured in the conditioned media by enzyme-linked immunosorbent and radioimmunoassays. Steady-state concentrations of HUVE mRNA were estimated by reverse transcription-polymerase chain reaction and quantified by Northern analyses to assess fibronectin and endothelin-1 gene expression. We found that the subjacent extracellular matrix affects the morphology, proliferation, and differentiation of HUVE cells in vitro. Cells cultured on gelatin were more mitotically active, expressed significantly less cellular fibronectin, made similar amounts of prostacyclin, and secreted significantly more endothelin-1 compared with the same cells grown on a Matrigel substrate.

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