3D matrix‐embedding inhibits cycloheximide‐mediated sensitization to TNF‐alpha‐induced apoptosis of human endothelial cells

Saemisch, Michael; Nickmann, Markus; Riesinger, Lisa; Edelman, Elazer R.; Methe, Heiko

doi:10.1002/term.2609

Cited by 3 publications

(4 citation statements)

References 57 publications

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“…Genes that were upregulated in 3D ECs relative to those of ECs cultured in 2D TCPs were enriched within the GO BP categories vasculature development, cardiovascular system development, and regulation of cell proliferation. In addition, the GO BP terms 'the integrin-mediated signalling pathway' and 'cell surface receptor signalling pathway' , and the GO MF terms 'integrin binding' , 'collagen binding involved in cell-matrix adhesion' and 'ECM binding' were enriched, which was proven in a recent study showing that 3D matrix-embedded ECs exhibit altered integrin expression and express significantly reduced phosphorylated focal adhesion kinase compared with those of ECs cultured on 2D TCPs [27]. Zhang et al also reported that ECs cultured on TCPs adopt a proliferative phenotype compared with ECs cultured on top of or encapsulated in PEG hydrogels and gelled Matrigel, which was related to the BP term 'regulation of cell proliferation' that was enriched in our results [8].…”

Section: Discussionmentioning

confidence: 81%

A comparative study unraveling the effects of TNF-α stimulation on endothelial cells between 2D and 3D culture

Wang

Chen²,

Gao

et al. 2020

Biomed. Mater.

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Endothelial cell (EC) dysfunction is an important predictor of and contributor to the pathobiology of cardiovascular diseases. However, most in vitro studies are performed using monolayer cultures of ECs on 2D tissue polystyrene plates (TCPs), which cannot reflect the physiological characteristics of cells in vivo. Here, we used 2D TCPs and a 3D culture model to investigate the effects of dimensionality and cardiovascular risk factors in regulating endothelial dysfunction. Cell morphology, oxidative stress, inflammatory cytokines and endothelial function were investigated in human umbilical vein endothelial cells (HUVECs) cultured in 2D/3D. The differentially expressed genes in 2D/3D-cultured HUVECs were analysed using Enrichr, Cytoscape and STRING services. Finally, we validated the proteins of interest and conﬁrmed their relevance to TNF-α and the culture microenvironment. Compared with 2D TCPs, 3D culture increased TNF-α-stimulated oxidative stress and the inflammatory response and changed the mediators secreted by ECs. In addition, the functional characteristics, important pathways and key proteins were determined by bioinformatics analysis. Furthermore, we found that some key proteins, notably ACE, CD40, Sirt1 and Sirt6, represent a critical link between endothelial dysfunction and dimensionality, and these proteins were screened by bioinformatics analysis and verified by western blotting. Our observations provide insight into the interdependence between endothelial dysfunction and the complex microenvironment, which enhances our understanding of endothelial biology or provides a therapeutic strategy for cardiovascular-related diseases.

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Section: Discussionmentioning

confidence: 81%

A comparative study unraveling the effects of TNF-α stimulation on endothelial cells between 2D and 3D culture

Wang

Chen²,

Gao

et al. 2020

Biomed. Mater.

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“…An immunoprecipitation kit (Invitrogen) was used to quantify association of receptorinteracting protein (RIP) with focal adhesion kinase (FAK). Immunoprecipitation and Western blot analysis were performed as previously described (52). Antibody specifications are outlined in Table 1.…”

Section: Methodsmentioning

confidence: 99%

“…Composition of the subendothelial extracellular matrix undergoes several important changes during evolution of atherosclerosis (31,48). In addition we recently demonstrated that the spatial formation of the tissue environment impacts proneness of ECs to undergo apoptosis with a three-dimensional environment exerting protection over a two-dimensional environment (52).…”

Section: Introductionmentioning

confidence: 96%

Subendothelial matrix components influence endothelial cell apoptosis in vitro

Saemisch

Balcells

Riesinger

et al. 2019

American Journal of Physiology-Cell Physiology

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The programmed form of cell death (apoptosis) is essential for normal development of multicellular organisms. Dysregulation of apoptosis has been linked with embryonal death and is involved in the pathophysiology of various diseases. Specifically, endothelial apoptosis plays pivotal roles in atherosclerosis whereas prevention of endothelial apoptosis is a prerequisite for neovascularization in tumors and metastasis. Endothelial biology is intertwined with the composition of subendothelial basement membrane proteins. Apoptosis was induced by addition of tumor necrosis factor-α to cycloheximide-sensitized endothelial cells. Cells were either grown on polystyrene culture plates or on plates precoated with healthy basement membrane proteins (collagen IV, fibronectin, or laminin) or collagen I. Our results reveal that proteins of healthy basement membrane alleviate cytokine-induced apoptosis whereas precoating with collagen type I had no significant effect on apoptosis by addition of tumor necrosis factor-α to cycloheximide-sensitized endothelial cells compared with cells cultured on uncoated plates. Yet, treatment with transforming growth factor-β1 significantly reduced the rate of apoptosis endothelial cells grown on collagen I. Detailed analysis reveals differences in intracellular signaling pathways for each of the basement membrane proteins studied. We provide additional insights into the importance of basement membrane proteins and the respective cytokine milieu on endothelial biology. Exploring outside-in signaling by basement membrane proteins may constitute an interesting target to restore vascular function and prevent complications in the atherosclerotic cascade.

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“…Endothelial dysfunction is an important contributor to atherosclerosis [1]. Zanotelli et al found that ECs can maintain good activity and form a capillary-like network when embedded in a polyethylene glycol hydrogel for 3D culture, which allow for better evaluation of the morphogenesis of vascular ECs [11] In recent years, Saemisch et al used microarray chips to detect significant differences in the expression of integrins α1, α2, β3, and other subunits in human aortic ECs cultured in three dimensions, which are also related to cytoskeleton, migration, adhesion, and matrix remodelling [12]. The above studies indicate that the biological characteristics and functions of ECs may change owing to the 3D culture environment, and the changes in EC functions in 3D culture may be related to the differential expression of these genes.…”

Section: Introductionmentioning

confidence: 99%

Effects of hydrogen peroxide on endothelial function in three-dimensional hydrogel vascular model and regulation mechanism of polar protein Par3

Gao

Wang

Chen³

et al. 2022

Biomed. Mater.

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Three-dimensional (3D) cell cultures better reflect the function of endothelial cells (ECs) than two-dimensional (2D) cultures. In recent years, studies have found that ECs cultured in a 3D luminal structure can mimic the biological characteristics and phenotypes of vascular ECs, thus making it more suitable for endothelial dysfunction research. In this study, we used a 3D model and 2D tissue culture polystyrene (TCP) to investigate the effects of cell polarity on hydrogen peroxide (H2O2)-induced endothelial dysfunction and its related mechanisms. We observed the cell morphology, oxidative stress, and barrier and endothelial function of human umbilical vein endothelial cells (HUVECs) in 3D and 2D cultures. We then used Illumina to detect the differentially expressed genes in the 3D-cultured HUVEC with and without H2O2 stimulation, using ClusterProfiler for Gene Ontology (GO) function enrichment analysis and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes. Finally, we explored the role and mechanism of polar protein partitioning defective protein 3 (Par3) in the regulation of ECs. ECs were inoculated into the 3D hydrogel channel; after stimulation with H2O2, the morphology of HUVECs changed, the boundary was blurred, the expression of intercellular junction proteins decreased, and the barrier function of the EC layer was damaged. 3D culture increased the oxidative stress response of cells stimulated by H2O2 compared to 2D TCPs. The polarity-related protein Par3 and cell division control protein 42 (CDC42) were screened using bioinformatics analysis, and western blotting was used to verify the results. Par3 knockdown significantly suppressed claudin1 (CLDN1) and vascular endothelial cadherin (VE-cadherin). These results suggest that the polar protein Par3 can protect H2O2-induced vascular ECs from damage by regulating CLDN1 and VE-cadherin.

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3D matrix‐embedding inhibits cycloheximide‐mediated sensitization to TNF‐alpha‐induced apoptosis of human endothelial cells

Cited by 3 publications

References 57 publications

A comparative study unraveling the effects of TNF-α stimulation on endothelial cells between 2D and 3D culture

A comparative study unraveling the effects of TNF-α stimulation on endothelial cells between 2D and 3D culture

Subendothelial matrix components influence endothelial cell apoptosis in vitro

Effects of hydrogen peroxide on endothelial function in three-dimensional hydrogel vascular model and regulation mechanism of polar protein Par3

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