Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis
Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified Kallikrein-Related Peptidase 10 (Klk10, a secreted serine protease) as a flow-sensitive gene in mouse arterial endothelial cells, but its role in endothelial biology and atherosclerosis was unknown. Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo& mouse models and in vitro studies with cultured endothelial cells (ECs). Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated Klk10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced permeability dysfunction and inflammation in human artery ECs (HAECs), as determined by NFkB activation, expression of vascular cell adhesion molecule 1 (VCAM1) and intracellular adhesion molecule 1 (ICAM1), and monocyte adhesion. Further, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibited atherosclerosis in Apoe-/- mice. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. KLK10 is a flow-sensitive endothelial protein that serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.
All data generated or analyzed during this study are included in the manuscript and supporting file; Source Data files for all western blots and gels have been provided for all applicable figures.Previously Published Datasets: Endothelial reprogramming by disturbed flow revealed by single-cell RNAseq and chromatin accessibility study: Andueza A, Kumar S, Kim J, Kang DW, Mumme HL, Perez JI, Villa-Roel N, Jo H, 2020, https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA646233, NCBI Bioproject, PRJNA646233
Article and author information
National Heart, Lung, and Blood Institute (HL119798)
- Hanjoong Jo
National Heart, Lung, and Blood Institute (HL145974)
- Darian Williams
National Heart, Lung, and Blood Institute (HL139757)
- Hanjoong Jo
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All animal studies were performed with male C57BL/6 or ApoE-/- mice (Jackson Laboratory), were approved by Institutional Animal Care and Use Committee by Emory University (PROTO201700428), and were performed in accordance with the established guidelines and regulations consistent with federal assurance.
Human subjects: Human coronary arteries were obtained from de-identified human hearts not suitable for cardiac transplantation donated to LifeLink of Georgia. Therefore, Emory University determined that this study was an IRB-exempt study.
- Edward A Fisher, New York University Grossman School of Medicine, United States
- Received: July 28, 2021
- Preprint posted: August 10, 2021 (view preprint)
- Accepted: January 8, 2022
- Accepted Manuscript published: January 11, 2022 (version 1)
- Version of Record published: February 1, 2022 (version 2)
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
- 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
Full-length mRNAs transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the 'transferome') is unknown. Here, we analyzed the transferome in an in vitro human-mouse cell co-culture model using RNA-sequencing. We found that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells. Typically, <1% of endogenous mRNAs undergo transfer. Non-selective, expression-dependent RNA transfer was further validated using synthetic reporters. RNA transfer appears contact-dependent via TNTs, as exemplified for several mRNAs. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.
- Cell Biology
Sorting nexins (SNX) are a family of proteins containing the Phox homology domain, which shows a preferential endo-membrane association and regulates cargo sorting processes. Here, we established that SNX32, an SNX-BAR (Bin/Amphiphysin/Rvs) sub-family member associates with SNX4 via its BAR domain and the residues A226, Q259, E256, R366 of SNX32, and Y258, S448 of SNX4 that lie at the interface of these two SNX proteins mediate this association. SNX32, via its PX domain, interacts with the transferrin receptor (TfR) and Cation-Independent Mannose-6-Phosphate Receptor (CIMPR), and the conserved F131 in its PX domain is important in stabilizing these interactions. Silencing of SNX32 leads to a defect in intracellular trafficking of TfR and CIMPR. Further, using SILAC-based differential proteomics of the wild-type and the mutant SNX32, impaired in cargo binding, we identified Basigin (BSG), an immunoglobulin superfamily member, as a potential interactor of SNX32 in SHSY5Y cells. We then demonstrated that SNX32 binds to BSG through its PX domain and facilitates its trafficking to the cell surface. In neuroglial cell lines, silencing of SNX32 leads to defects in neuronal differentiation. Moreover, abrogation in lactate transport in the SNX32-depleted cells led us to propose that SNX32 may contribute to maintaining the neuroglial coordination via its role in BSG trafficking and the associated monocarboxylate transporter activity. Taken together, our study showed that SNX32 mediates the trafficking of specific cargo molecules along distinct pathways.