A cell atlas of thoracic aortic perivascular adipose tissue: a focus on mechanotransducers

Abstract

Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. However, major gaps remain in our understanding of the cells present in PVAT, as well as how different cells contribute to mechanotransduction. We hypothesized that snRNA-seq would reveal the expression of mechanotransducers, and test one (PIEZO1) to illustrate the expression and functional agreement between single-nuclei RNA sequencing (snRNA-seq) and physiological measurements. To contrast two brown tissues, subscapular brown adipose tissue (BAT) was also examined. We used snRNA-seq of the thoracic aorta PVAT (taPVAT) and BAT from male Dahl salt-sensitive (Dahl SS) rats to investigate cell-specific expression mechanotransducers. Localization and function of the mechanostransducer PIEZO1 were further examined using immunohistochemistry (IHC) and RNAscope, as well as pharmacological antagonism. Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNA-seq, identifying eight major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. The presence of PIEZO1 in the PVAT but not the adventitia was confirmed by RNAscope and IHC in male and female rats. Importantly, antagonism of PIEZO1 by GsMTX4 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.NEW & NOTEWORTHY This study describes the atlas of cells in the thoracic aorta perivascular adipose tissue (taPVAT) of the Dahl-SS rat, an important hypertension model. We show that mechanotransducers are widely expressed in these cells. Moreover, PIEZO1 expression is shown to be restricted to the taPVAT and is functionally implicated in stress relaxation. These data will serve as the foundation for future studies investigating the role of taPVAT in this model of hypertensive disease.

Keywords: Dahl-SS rat; Piezo1; brown adipose tissue; mechanotransduction; perivascular adipose tissue.

Figure 1.

Procedure overview used to derive transcript data to inform cell-based clusters. A: nuclear isolation from thoracic aorta perivascular adipose tissue (taPVAT) and brown adipose tissue (BAT) from male Dahl salt-sensitive (Dahl SS) rat. B: use of Chromium Next GEM Single Cell 3′ kit to produce amplified cDNA. C: construction of a 3′ gene expression library. D: sequencing and analyses of cell-based clusters (see materials and methods for more details). All panels of the workflow overview figure were created using a licensed version of BioRender.com, and some parts of the diagram were modified from the 10× Chromium Nuclei Isolation Kit and Chromium Next GEM Single Cell 3′ Kits user guide.

Figure 2.

Characterization of the male Dahl salt-sensitive (Dahl SS) rat thoracic aorta perivascular adipose tissue (taPVAT) and brown adipose tissue (BAT) adipose tissue depots. A: uniform manifold approximation and projection (UMAP) visualization of nuclei from the taPVAT and BAT following integration using single-cell variational inference (scVI) and manual annotation of cell types. B: UMAP visualization for nuclei from each individual adipose tissue depot. C: relative proportion of each cell type for each adipose tissue depot. Proportions are shown on a log scale for comparison of high-abundance and low-abundance cell types. Bars represent mean values, whereas individual points represent proportions in individual samples. D: top 5 marker genes for each cell type (listed at top) identified common to both taPVAT and BAT.

Figure 3.

Comparison of thoracic aorta perivascular adipose tissue (taPVAT) and brown adipose tissue (BAT) adipose tissue depot gene expression. A: UpSet plot of differentially expressed genes (|fold-change| ≥ 2, adjusted P value ≤ 0.05) for individual cell types between taPVAT and BAT. Total number of differentially expressed genes for each cell type is shown as horizontal bars on the left and the intersecting list of differentially expressed genes for the cell types markers by a black dot is shown as vertical bar. B: heatmap of the top 5 taPVAT and BAT-enriched genes. C: top 10 enriched functional groups determined using GSEApy (see materials and methods) where a positive normalized enrichment score (NES; top) represents taPVAT-enriched functions and negative NES (bottom) represents BAT-enriched functions.

Figure 4.

Analysis of mechanotransduction-related gene expression. A: dot plot of the most highly expressed mechanotransduction genes in thoracic aorta perivascular adipose tissue (taPVAT) and brown adipose tissue (BAT). Dot size represents percentage of nuclei expressing the gene, and color intensity represents expression level. The mesothelium of BAT was removed for visualization as it only included 1 nucleus. B: cell-specific enrichment of mechanotransduction-related Gene Ontology (GO) terms (see materials and methods). C: uniform manifold approximation and projection (UMAP) visualization of expression for Ddr2, Slc12a2, Piezo1, and Piezo2. D: heatmap of fold-change for differentially expressed mechanotransduction-related genes (|fold-change| ≥ 2, adjusted P value ≤ 0.05).

Figure 5.

The mechanotransducer Piezo1 is expressed in and functionally serves thoracic aorta perivascular adipose tissue (taPVAT). A: Brightfield of Piezo1 mRNA ZZ-probe detection (RNAscope) in the thoracic aorta of the Dahl salt-sensitive (Dahl SS) male rat. Arrows indicate positive dot detection. P, PVAT (anterior); A, adventitia; M, media. Horizontal bar indicates 50 µm. Photomicrograph represents a ×20 magnification (1st full image) overview of the aorta and taPVAT. Box drawn around a region of interest shows ×40 magnification (2nd full image). To the right are tissue-specific positive (+) controls for housekeeping gene peptidylprolyl isomerase B (PPIB) and negative (−) controls for bacterial gene diaminopimelate (DapB) are shown. Images represent 3 male rats. B: fluorescent immunohistochemical detection of PVAT. Images of sections incubated with (1st full image) and without primary (2nd full image) PIEZO1 antibody. P, PVAT (anterior); M, media; L, lumen; E, endothelium. Kidney nephron tubule epithelium is shown on the far right stained for PIEZO1 in the presence (top) and absence (bottom) of PIEZO1 primary antibody. Representative of 3 male rats. C, left: representative tracing of the response of the aorta (top) or its surrounding ring of PVAT (bottom) to a 4-g passive tension addition in the absence (black) or presence of PIEZO1 inhibitor GsMTx4 (5 µM). C, right: quantifies tension relaxed to after application of a baseline tension of 2 g (no inhibitors present but legend indicates which group tissues will be in); after addition of 2 g tension to achieve 4 g total (after 1-h incubation with vehicle/inhibitor); and after addition of 4 g tension to achieve 8 g total (after reincubation with vehicle/inhibitor). Measurements were repeated on the same tissue without the inhibitor and after addition 4 and 8 g of tension. Bars are means ± SE with individual scattered values. *P < 0.05, statistically significant differences within group members as marked by lines.

Figure 6.

Adipocyte stem and progenitor cells subtypes in perivascular adipose tissue (PVAT) from Dahl salt-sensitive (Dahl SS). A: expression dot plot of ASPC marker genes Dcn, Fbn1, Cd34, and Pdgfra in individual cell types for thoracic aorta perivascular adipose tissue (taPVAT) and brown adipose tissue (BAT) combined. Dot size represents percentage of genes expressing the gene, whereas color represents mean expression. B: uniform manifold approximation and projection (UMAP) visualization of reintegrated adipocyte stem and progenitor cells (ASPCs) as described in materials and methods. Nuclei were reclustered using Leiden clustering at a resolution of 0.1, identifying a total of 3 subpopulations. C: top 5 markers genes for each ASPC Leiden cluster with a minimum |fold-change| of 2. Only 2 genes met the threshold criteria for cluster 0. D: median expression and distribution of expression for previously identified markers Bmper, Pi16, and Gdf10 are shown as violin plots for each Leiden cluster identified in B. Piezo1 expression is also shown for each Leiden cluster.