preparation-single-cell-suspension-from-mouse-carotid-arteries-for

Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are ...

Soochow University

Accumulating evidence suggests a role of semaphorins in vascular homeostasis. Here, we investigate the role of Sema7A (semaphorin 7A) in atherosclerosis and its underlying mechanism.

Vascular Semaphorin 7A Upregulation by Disturbed Flow Promotes Atherosclerosis Through Endothelial β1 Integrin.

Endothelial to mesenchymal transition (EndMT) is an important pathological change in many diseases. Semaphorin7A (Sema7A) has been reported to regulate nerve and vessel homeostasis, but its role in EndMT remains unclear. Here we investigate the effect of Sema7A on EndMT and the underlying mechanism. Sema7A-overexpressed human umbilical vein endothelial cells (Sema7A-HUVECs) were generated and showed lower levels of endothelial cell markers and higher levels of mesenchymal cell markers indicating the occurrence of EndMT. RNA-sequencing analysis showed a total of 1168 upregulated genes and 886 downregulated genes. Among them, most of the molecules associated with EndMT were upregulated in Sema7A-HUVECs. Mechanistically, Sema7A-HUVECs showed a higher TGF-β2 expression and activated TGF-β/Smad Signaling. Importantly, Sema7A overexpression upregulated activating transcription factor 3 (ATF3) that was found to selectively bind the promotor region of TGF-β2, but not TGF-β1, promoting TGF-β2 transcription, which was further confirmed by ATF3-siRNA knockdown approach. Blocking β1 integrin, a known Sema7A receptor, alleviated the expression of ATF3, TGF-β2, and EndMT in Sema7A-overexpressed HUVECs, implying a role of β1 integrin/ATF3/TGF-β2 axis in mediating Sema7A-induced EndMT. Using Sema7A-deficient mice and the partial carotid artery ligation (PCL) model, we showed that Sema7A deletion attenuated EndMT induced by blood flow disturbance in vivo. In conclusion, Sema7A promotes TGF-β2 secretion by upregulating transcription factor ATF3 in a β1 integrin-dependent manner, and thus facilitates EndMT through TGF/Smad signaling, implying Sema7A as a potential therapeutic target for EndMT-related vascular diseases.

Semaphorin 7A promotes endothelial to mesenchymal transition through ATF3 mediated TGF-β2/Smad signaling.

Disturbed blood flow (d-flow) has been known to induce changes of the cells in the arterial wall, increasing the risk of atherosclerosis. However, the heterogeneity of the vascular cell populations under d-flow remains less understood. To generate d-flow in vivo, partial carotid artery ligation (PCL) was performed. Seven days after ligation, single-cell RNA sequencing of nine left carotid arteries (LCA) from the PCL group (10,262 cells) or control group (14,580 cells) was applied and a single-cell atlas of gene expression was constructed. The integrated analysis identified 15 distinct carotid cell clusters, including 10 d-flow-relevant subpopulations. Among endothelial cells, at least four subpopulations were identified, including Klk8 ECs, Lrp1 ECs, Dkk2 ECs, and Cd36 ECs. Analysis of GSVA and single-cell trajectories indicated that the previously undescribed Dkk2 ECs subpopulation was mechanosensitive and potentially transformed from Klk8 ECs under d-flow. D-flow-induced Spp1 VSMCs subpopulation that appeared to be endowed with osteoblast differentiation, suggesting a role in arterial stiffness. Among the infiltrating cell subpopulations, Trem2 Mφ, Birc5 Mφ, DCs, CD4 T cells, CXCR6 T cells, NK cells, and granulocytes were identified under d-flow. Of note, the novel Birc5 Mφ was identified as a potential contributor to the accumulation of macrophages in atherosclerosis. Finally, Dkk2 ECs, and Cd36 ECs were also found in the proatherosclerotic area of the aorta where the d-flow occurs. In conclusion, we presented a comprehensive single-cell atlas of all cells in the carotid artery under d-flow, identified previously unrecognized cell subpopulations and their gene expression signatures, and suggested their specialized functions.

Single-cell RNA-seq reveals cellular heterogeneity of mouse carotid artery under disturbed flow.

The differences in plaque histology between symptomatic and asymptomatic patients have been widely accepted. Whether there is a heterogeneity of cells between symptomatic and asymptomatic plaques remains largely unclear. To reveal the potential heterogeneity within different plaques, which may contribute to different stroke incidences, we obtained the scRNA-seq data from symptomatic and asymptomatic patients and identified eight cell types present in plaques. Further analysis of endothelial cells (ECs) revealed three distinct EC subpopulations appeared to be endowed with specific biological functions such as antigen processing and presentation, cell adhesion, and smooth muscle cell proliferation. Of note, the differentially expressed genes of the EC 2 subpopulation showed that the genes involved in cell adhesion were up-regulated in asymptomatic plaques compared to symptomatic plaques. Integrating the data of intraplaque haemorrhage and plaque stability, the 5th top-enriched biological process was cell adhesion in the stable or non-haemorrhaged plaques compared to unstable plaques or haemorrhaged plaques. Among these cell adhesion-related genes, the intersection gene AOC3 may play a vital role in plaque haemorrhage and plaque stability. Targeting cell adhesion and the specialized genes may provide potential new therapeutic directions to prevent asymptomatic patients from stroke.

Single-cell transcriptional profiling of human carotid plaques reveals a subpopulation of endothelial cells associated with stroke incidences.

Obesity and related diseases are becoming a growing risk for public health around the world due to the westernized lifestyle. Sema7A, an axonal guidance molecule, has been known to play a role in neurite growth, bone formation, and immune regulation. Whether Sema7A participates in obesity and metabolic diseases is unknown. As several SNPs in SEMA7A and its receptors were found to correlate with BMI and metabolic parameters in the human population, we investigated the potential role of Sema7A in obesity and hepatic steatosis.

Sema7A protects against high-fat diet-induced obesity and hepatic steatosis by regulating adipo/lipogenesis.

Corrigendum to "Sema7A protects against high-fat diet-induced obesity and hepatic steatosis by regulating adipo/lipogenesis" [Mol Metab. 70 (2023) 101698]∗.

Article	Year
Vascular Semaphorin 7A Upregulation by Disturbed Flow Promotes Atherosclerosis Through Endothelial β1 Integrin. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 29269512	2018
Semaphorin 7A promotes endothelial to mesenchymal transition through ATF3 mediated TGF-β2/Smad signaling. Cell death & disease\| PubMed ID: 32826874	2020
Single-cell RNA-seq reveals cellular heterogeneity of mouse carotid artery under disturbed flow. Cell death discovery\| PubMed ID: 34282126	2021
Single-cell transcriptional profiling of human carotid plaques reveals a subpopulation of endothelial cells associated with stroke incidences. Journal of cellular and molecular medicine\| PubMed ID: 35527426	2022
Sema7A protects against high-fat diet-induced obesity and hepatic steatosis by regulating adipo/lipogenesis. Molecular metabolism\| PubMed ID: 36842496	2023
Corrigendum to "Sema7A protects against high-fat diet-induced obesity and hepatic steatosis by regulating adipo/lipogenesis" [Mol Metab. 70 (2023) 101698]∗. Molecular metabolism\| PubMed ID: 37871636	2023

Fengchan Li

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