Zilberberg, L. | bioRxiv Science

bioRxiv2025

Single Cell Proteomics Reveals Novel Cell Phenotypes in Marfan Mouse Aneurysm

BackgroundSingle-cell omics technology is a powerful tool in biomedical research. However, single cell proteomics has lagged due to an inability to amplify peptides in a similar fashion to nucleotide strings. Single cell proteomics is important because proteins are the main functional unit in cells, and they often poorly correlate with mRNA quantities. In this paper we describe the first single cell proteomic analysis of complex tissue, comparing aneurysmal and normal mouse aorta from males and females. We also compare and integrate our single cell proteomic profiles with a matching single cell transcriptomics dataset. MethodsWe compared single cell proteomes between male and female, wild-type and Fbn1C1041G/+ Marfan mice (N=3 per group). Individual cells from mouse aortic root single cell suspensions were deposited in 384 well plates and subjected to ultra-sensitive nanoflow liquid chromatography-ion mobility-time of flight-mass spectrometry. The data were analyzed with leiden clustering to identify cell types. Statistical analyses were performed to detect differential proteins within cell types and multi-omics analysis integrated single cell proteomics with published single cell RNA-seq. ResultsWe identified all major aortic cell types including 7 distinct smooth muscle cell subtypes. The proportion of these cells varied based on sex and the Fbn1C1041G/+ genotype. Differentially expressed proteins between male and female in addition to wild-type and Marfan samples uncovered enhanced endothelial to mesenchymal transition patterns in endothelial cells from male Marfan mice. Comparisons between single cell RNA and single cell proteomic profiles showed similarities in major subtypes but not smooth muscle cell subtypes. Multi-omics analysis of these two single cell platforms demonstrated a potential novel role for smooth muscle cell derived angiotensin signaling in the Marfan phenotype. ConclusionsSingle cell proteomics identified new subpopulations of vascular smooth muscles cells and novel cell type specific protein signatures related to sex differences and aneurysm formation. AbbreviationsNext generation sequencing (NGS), Mass spectrometer (MS), Single cell proteomics by Mass Spectrometry (ScOPE-MS), Marfans syndrome (MFS), Fibrillin 1 (FBN1), Transforming growth factor {beta} (TGF{beta}), Smooth muscle cell (SMC), Single cell proteomic (scProteomic), Differentially expressed proteins (DEPs), Wild-type (WT), Hanks balanced salt solution (HBSS), Fetal bovine serum (FBS), Dulbeccos Modified Eagle Medium (DMEM), Data-independent acquisition parallel accumulation-serial fragmentation (DIA-PASEF), Magnetic assisted cell sorted (MACS), Single Cell Analysis in Python (Scanpy), Kyoto Encyclopedia of Genes and Genomes (KEGG), Principal component analysis (PCA), Uniform manifold projection (UMAP), Single cell transcriptomic (scTranscriptomic), Smoothelin (Smtn), Transgelin (Tagln), Myosin heavy chain 11 (Myh11), Platelet endothelial cell adhesion molecule 1 (Pecam1), Dipeptidase 1 (Dpep1), Uncoupling protein 1 (Ucp1), Low-density lipoprotein receptor-related protein (Lrp1), DNA ligase 3 (Lig3), Capsaicin channel transient receptor potential vanilloid 1 (Trpv1), Endothelial to mesenchymal transition (endMT), Intercellular adhesion molecule 1 (Icam1), Intercellular adhesion molecule 2 (Icam2), Endothelial cell-selective adhesion molecule (Esam), Calponin 1 (Cnn1), Vimentin (Vim), Zinc finger E-box-binding homeobox 1 (Zeb1), Snail family transcriptional repressor 1 (Snai1), Tropomyosin alpha-4 chain (Tpm4), Angiotensin converting enzyme (Ace)

systems biology↗

bioRxiv2023

17 β-estradiol impedes aortic root dilation and rupture in male Marfan mice

Marfan syndrome causes a hereditary form of thoracic aortic aneurysms with dilation of the aortic root. Human and animal models suggest a worse phenotype for males compared to females with respect to aneurysm size and risk of dissection. In this study we examine the effects of 17 {beta}-estradiol on aortic dilation and rupture in a Marfan mouse model. Marfan male mice were administered 17 {beta}-estradiol and the growth in aortic root size along with the risk of aortic rupture or dissection with the addition of angiotensin II was measured. Transcriptomic profiling was used to identify enriched pathways from 17 {beta}-estradiol treatment. Aortic smooth muscle cells were then treated with cytokines in order to validate the mechanism of 17 {beta}-estradiol protection. We show that 17 {beta}-estradiol decreased the size and rate of aortic root dilation and improved survival from rupture and dissection after treatment with angiotensin II. The Marfan transcriptome was enriched in inflammatory genes and the addition of 17 {beta}-estradiol modulated a set of genes that function through TNF mediated NF-{kappa}B signaling. These included many proteins known to play a role in the phenotypic shift of aortic smooth muscle cells from a contractile to a more inflammatory-like state such as Vcam-1, Mcp-1, Lgals3, Il-6, Il-1b, and C3. In addition, 17 {beta}-estradiol suppressed the induction of these TNF induced genes in aortic smooth muscle cells in vitro and this effect appears to be NF-{kappa}B dependent. In conclusion, 17 {beta}-estradiol protects against the dilation and rupture of aortic roots in Marfan male mice through the inhibition of TNF -NF-{kappa}B signaling and thus prevents the phenotypic switch of aortic smooth muscle cells from a contractile to an inflammatory state.

genetics↗