Related Experiment Video
Updated: May 21, 2026
Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications
Published on: June 10, 2022
Identification of unannotated microproteins involved in endothelial cell homeostasis, dysfunction, and vascular
Mauro Siragusa1,2, Johannes Graumann3,4,5, Carsten Kuenne6
1Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany.
Aims:
Microproteins (miPs) translated from small open reading frames (smORFs) are crucial regulators of cell function. However, the expression and function of miPs in endothelial cells and alterations in miP expression linked with inflammation and cardiovascular disease, remain largely unexplored.
Methods And Results:
An optimized proteogenomic approach combining RiboTag RNA-sequencing and mass spectrometry of the small molecular mass proteome was utilized to identify endothelial cell-specific miPs. Heart, lung, and blood vessels from endothelial cell-specific RiboTag mice and human endothelial cells were studied under homeostatic and inflammatory conditions. We identified 2739 murine as well as 1365 intracellular and 607 extracellular human endothelial cell miPs encoded from previously non-canonical (unannotated) smORFs. Vascular inflammation induced in vitro by interleukin-1β (IL-1β) and in vivo through PCSK9 overexpression, high-fat diet, and partial carotid artery ligation significantly altered smORF expression. An additional 347 miPs were detected in human serum, 23 decreasing and 31 increasing, after cardiac damage. The expression of an inflammation-induced miP encoded by an internal smORF within the proline-serine-threonine phosphatase interacting protein 2 (PSTPIP2) transcript, that is, miP-PSTPIP2, was assessed using a custom antibody. miP-PSTPIP2 expression was upregulated in IL-1β-treated human endothelial cells, in pre-atherosclerotic murine carotid arteries and detected in carotid arteries from patients with atherosclerosis. The relevance of 250 miPs for endothelial cell growth and viability was demonstrated using a high-throughput clustered regularly interspaced Short palindromic Repeats (CRISPR)/Cas9 screen.
Conclusion:
Taken together, we document the existence of a large number of human and murine miPs encoded by non-canonical smORFs and their altered expression in inflammatory conditions. The identification of secreted miPs suggests that they may also exert autocrine or paracrine functions. These novel small peptides modulate cell proliferation and survival in endothelial cells and may play a significant role in human cardiovascular disease.
Insights
Researchers discovered numerous microproteins (miPs) from small open reading frames (smORFs) in endothelial cells. Their expression changes with inflammation, suggesting a role in cardiovascular disease.
Area of Science:
- Molecular Biology
- Cardiovascular Research
- Proteomics
Background:
- Microproteins (miPs) from small open reading frames (smORFs) are vital for cellular functions.
- Their role in endothelial cells and connection to inflammation/cardiovascular disease are understudied.
Purpose of the Study:
- To identify endothelial cell-specific miPs.
- To investigate miP expression changes under inflammatory conditions.
- To explore the functional significance of miPs in endothelial cells.
Main Methods:
- Utilized a proteo-genomic approach (RiboTag RNA-sequencing, mass spectrometry).
- Studied endothelial cells from mice and humans under homeostatic and inflammatory states.
- Employed CRISPR/Cas9 screening to assess miP function.
Main Results:
- Identified 2,739 murine and 1,962 human endothelial cell miPs from novel smORFs.
- Observed significant alterations in smORF expression during vascular inflammation (in vitro and in vivo).
- Found miP-PSTPIP2 upregulated in inflamed conditions and in atherosclerotic patients' arteries; 250 miPs impacted endothelial cell growth/viability.
Conclusions:
- A substantial number of miPs encoded by non-canonical smORFs exist in humans and mice.
- Altered miP expression is linked to inflammatory conditions, with potential autocrine/paracrine roles for secreted miPs.
- These miPs influence endothelial cell proliferation and survival, implicating them in cardiovascular disease pathogenesis.
