Gene Expression: How their environment influences endothelial cells
Cell culture is a widely used technique in biology (Jensen and Teng, 2020), and cells from the endothelium – the single layer of cells that lines the inside of blood vessels – are often employed in cardiovascular research. Endothelial cells are unique in that they are in direct contact with the blood, and the flow of blood exerts a mechanical force (known as shear stress) that can activate signaling pathways and modify gene expression in the cells. Moreover, endothelial cells are also close to smooth muscle cells (Figure 1), and these two types of cells communicate with each other to maintain healthy blood vessels (Li et al., 2018).
Most of the endothelial cells used in cell culture experiments are taken from the umbilical cord of newborn babies, but we do not fully understand how removing these cells from their native environment impacts their behavior and function (Aird, 2007). In particular, it is not clear how the absence of two factors – shear stress and communication between different cell types – changes the behavior of endothelial cells in cell culture experiments. Furthermore, is it possible to change the conditions in cell culture experiments to make them more similar to the conditions in vivo?
Now, in eLife, Luisa Iruela-Arispe (Northwestern University) and colleagues – including Yalda Afshar (University of California) as first author – report that gene expression in endothelial cells is altered when the cells are cultured in vitro, and can be partially restored if the cells are exposed to shear stress and smooth muscle cells (Afshar et al., 2023).
Using endothelial cells taken from the umbilical cords of seven human donors, Afshar et al. compared the gene expression profiles of cells cultured under in vitro conditions with the profiles of cells freshly isolated from the same donor (hereafter called cord cells). RNA sequencing showed that nearly half of the genes expressed in the culture condition were different from those expressed in the freshly isolated cells: in particular, genes sensitive to blood flow were expressed less, and genes related to cell proliferation were expressed more (Figure 1).
Afshar et al. then modified the culture conditions to see if the gene expression profile could be made more similar to the profile of the cord cells. First, the cultured cells were placed under flow conditions for 48 hours to simulate the shear stress caused by blood flow (Chiu and Chien, 2011). This led to the expression of a number of genes that were not expressed in culture without flow, including genes which belong to two signaling pathways – BMP and NOTCH – that are known to be sensitive to flow (Souilhol et al., 2020).
Next, Afshar et al. co-cultured endothelial cells alongside smooth muscle cells from the same donor. Single-cell RNA sequencing showed that this restored the expression of various genes (including genes for cytoskeleton proteins) that were not expressed when endothelial cells were cultured on their own. Moreover, the expression of genes related to cell proliferation was reduced. Furthermore, the team also built “Flow Profiler”, an open-source website to display the analyzed datasets and allow further exploration of the behavior of genes under flow conditions.
The results of Afshar et al. demonstrate the impact of culture conditions on gene expression in a systematic and quantitative fashion, and highlight the importance of contextual information when interpreting experimental results. Going forward it would be interesting to explore if introducing flow and smooth muscle cells at the same time would restore even more of the gene expression profile. Furthermore, endothelial cells are found in a variety of vessels in the body, and the flow pattern (and hence the shear stress) will be different in different vessels: future work could explore the impact of different locations and flow patterns on gene expression profiles. It would also be interesting to investigate the influence of DNA methylation and histone modification on gene expression in endothelial cells. The knowledge and insights gained from such studies will help researchers to develop more representative cell culture models for the study of cells and organs.
Researchers are also exploring a range of other techniques that mimic the in vivo environment, such as 3D bioprinting (Neufeld et al., 2022), organoids (LeSavage et al., 2022) and organ-on-a-chip technologies (Tolabi et al., 2023). Combining all these techniques, including improved cell culture models, should lead to a better understanding of cell behavior and disease mechanisms, and thus help researchers working to improve medical outcomes in scenarios where endothelial function is impaired.
Is it time to start transitioning from 2D to 3D cell culture?Frontiers in Molecular Biosciences 7:33.https://doi.org/10.3389/fmolb.2020.00033
Next-generation cancer organoidsNature Materials 21:143–159.https://doi.org/10.1038/s41563-021-01057-5
Endothelial-vascular smooth muscle cells interactions in atherosclerosisFrontiers in Cardiovascular Medicine 5:151.https://doi.org/10.3389/fcvm.2018.00151
3D bioprinted cancer models: from basic biology to drug developmentNature Reviews Cancer 22:679–692.https://doi.org/10.1038/s41568-022-00514-w
Endothelial responses to shear stress in atherosclerosis: a novel role for developmental genesNature Reviews Cardiology 17:52–63.https://doi.org/10.1038/s41569-019-0239-5
Article and author information
- Version of Record published: May 9, 2023 (version 1)
© 2023, Liu and Bouman Chen
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
- Page views
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
- Cell Biology
- Chromosomes and Gene Expression
Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA, and R-loops, even in the absence of cohesin. Our results place SA1 on chromatin upstream of the cohesin ring and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease.
- Cell Biology
Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF. PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-b/PGE2 balance in vitro in control and IPF human lung fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 expression in a PDGFR dependent manner in control ones. PRRX1 inhibition decreased human lung fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-β driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-β response global decrease. Finally, targeted inhibition of Prrx1 attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using human and mouse precision-cut lung slices. Our results identified PRRX1 as a key mesenchymal transcription factor during lung fibrogenesis.