1. Cell Biology
  2. Developmental Biology

Transcriptional drifts associated with environmental changes in endothelial cells

  1. Yalda Afshar  Is a corresponding author
  2. Feyiang Ma
  3. Austin Quach
  4. Anhyo Jeong
  5. Hannah L Sunshine
  6. Vanessa Freitas
  7. Yasaman Jami-Alahmadi
  8. Raphael Helaers
  9. Xinmin Li
  10. Matteo Pellegrini
  11. James A Wohlschlegel
  12. Casey E Romanoski
  13. Miikka Vikkula
  14. Luisa Iruela-Arispe
  1. University of California, Los Angeles, United States
  2. Northwestern University, United States
  3. University of São Paulo, Brazil
  4. University of Louvain, Belgium
  5. University of Arizona, United States
https://doi.org/10.7554/eLife.81370
Share this article
Cite this article
  1. Yalda Afshar
  2. Feyiang Ma
  3. Austin Quach
  4. Anhyo Jeong
  5. Hannah L Sunshine
  6. Vanessa Freitas
  7. Yasaman Jami-Alahmadi
  8. Raphael Helaers
  9. Xinmin Li
  10. Matteo Pellegrini
  11. James A Wohlschlegel
  12. Casey E Romanoski
  13. Miikka Vikkula
  14. Luisa Iruela-Arispe
(2023)
Transcriptional drifts associated with environmental changes in endothelial cells
eLife 12:e81370.
https://doi.org/10.7554/eLife.81370
  2. Download BibTeX
  3. Download .RIS

Abstract

Environmental cues, such as physical forces and heterotypic cell interactions play a critical role in cell function, yet their collective contributions to transcriptional changes are unclear. Focusing on human endothelial cells, we performed broad individual sample analysis to identify transcriptional drifts associated with environmental changes that were independent of genetic background. Global gene expression profiling by RNAseq and protein expression by LC-MS directed proteomics distinguished endothelial cells in vivo from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes. Inclusion of heterotypic interactions by co-culture of endothelial cells with smooth muscle cells normalized approximately 9% of the original in vivo signature. We also identified novel flow dependent genes, as well as genes that necessitate heterotypic cell interactions to mimic the in vivo transcriptome. Our findings highlight specific genes and pathways that rely on contextual information for adequate expression from those that are agnostic of such environmental cues.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file, Source Data files have been provided

The following previously published data sets were used

Article and author information

Author details

  1. Yalda Afshar

    Department of Obstetrics and Gynecology, University of California, Los Angeles, Los Angeles, United States
    For correspondence
    YAfshar@mednet.ucla.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3807-7022

  2. Feyiang Ma

    Molecular Biology Institute, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Austin Quach

    Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Anhyo Jeong

    Department of Obstetrics and Gynecology, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Hannah L Sunshine

    Department of Cell and Developmental Biology, Northwestern University, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Vanessa Freitas

    Departament of Cell and Developmental Biology, University of São Paulo, São Paulo, Brazil
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9613-8626

  7. Yasaman Jami-Alahmadi

    Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8289-2222

  8. Raphael Helaers

    Human Molecular Genetics, University of Louvain, Brussels, Belgium
    Competing interests
    The authors declare that no competing interests exist.

  9. Xinmin Li

    Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.

  10. Matteo Pellegrini

    Molecular Biology Institute, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.

  11. James A Wohlschlegel

    Departament of Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.

  12. Casey E Romanoski

    Department of Cellular and Molecular Medicine, University of Arizona, Tucson, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0149-225X

  13. Miikka Vikkula

    Human Molecular Genetics, University of Louvain, Brussels, Belgium
    Competing interests
    The authors declare that no competing interests exist.

  14. Luisa Iruela-Arispe

    Department of Cell and Developmental Biology, Northwestern University, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3050-4168

Funding

National Institutes of Health (R35HL140014)

  • Luisa Iruela-Arispe

National Institutes of Health (R01HL147187)

  • Casey E Romanoski

Foundation for the National Institutes of Health (FAPESP 2016/19968-3)

  • Vanessa Freitas

National Institutes of Health (K12 HD000849)

  • Yalda Afshar

National Institutes of Health (T32HL069766)

  • Yalda Afshar

Fondation Leducq (21CVD03)

  • Miikka Vikkula
  • Luisa Iruela-Arispe

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Ilse S Daehn, Icahn School of Medicine at Mount Sinai, United States

Ethics

Human subjects: Human umbilical cords were collected under Institutional Review Board (UCLA IRB#16-001694) at time of the delivery and processed 2-4 hours from time of birth. All samples were collected from patients who provided signed informed consent.

Version history

  1. Received: June 24, 2022
  2. Preprint posted: July 9, 2022 (view preprint)
  3. Accepted: March 26, 2023
  4. Accepted Manuscript published: March 27, 2023 (version 1)
  5. Accepted Manuscript updated: March 29, 2023 (version 2)
  6. Version of Record published: May 9, 2023 (version 3)

Copyright

© 2023, Afshar et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,966
    views
  • 300
    downloads
  • 13
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

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)

  1. Yalda Afshar
  2. Feyiang Ma
  3. Austin Quach
  4. Anhyo Jeong
  5. Hannah L Sunshine
  6. Vanessa Freitas
  7. Yasaman Jami-Alahmadi
  8. Raphael Helaers
  9. Xinmin Li
  10. Matteo Pellegrini
  11. James A Wohlschlegel
  12. Casey E Romanoski
  13. Miikka Vikkula
  14. Luisa Iruela-Arispe
(2023)
Transcriptional drifts associated with environmental changes in endothelial cells
eLife 12:e81370.
https://doi.org/10.7554/eLife.81370

Share this article

https://doi.org/10.7554/eLife.81370

Categories and tags

Research organism

Further reading

    1. Cell Biology

    Gene Expression: How their environment influences endothelial cells

    Xuejing Liu, Zhen Bouman Chen
    Insight

    Changes in gene expression in cultured endothelial cells can be partially reversed by simulating in vivo conditions.

    1. Cancer Biology
    2. Cell Biology

    Actin networks modulate heterogenous NF-κB dynamics in response to TNFα

    Francesca Butera, Julia E Sero ... Chris Bakal
    Research Article

    The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in PDAC tumours. In this study, we characterised previously unexplored endogenous RELA-GFP dynamics in PDAC cell lines through live single cell imaging. Our observations revealed that TNFα stimulation induces rapid, sustained, and non-oscillatory nuclear translocation of RELA. Through Bayesian analysis of single cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. RNA-seq analysis identified distinct clusters of RELA-regulated gene expression in PDAC cells, including TNFα-induced RELA upregulation of the actin regulators NUAK2 and ARHGAP31. Further, siRNA-mediated depletion of ARHGAP31 and NUAK2 altered TNFα-stimulated nuclear RELA dynamics in PDAC cells, establishing a novel negative feedback loop that regulates RELA activation by TNFα. Additionally, we characterised the NF-κB pathway in PDAC cells, identifying how NF-κB/IκB proteins genetically and physically interact with RELA in the absence or presence of TNFα. Taken together, we provide computational and experimental support for interdependence between the F-actin network and the NF-κB pathway with RELA translocation dynamics in PDAC.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    The ORP9-ORP11 dimer promotes sphingomyelin synthesis

    Birol Cabukusta, Shalom Borst Pauwels ... Jacques Neefjes
    Research Article

    Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.