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

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.

Perivascular Adipose Tissue Remodels Only after Elevation of Blood Pressure in the Dahl SS Rat Fed a High-Fat Diet.

INTRODUCTION: Tunica media extracellular matrix (ECM) remodeling is well understood to occur in response to elevated blood pressure, unlike the remodeling of other tunicas. We hypothesize that perivascular adipose tissue (PVAT) is responsive to hypertension and remodels as a protective measure. METHODS: The adventitia and PVAT of the thoracic aorta were used in measuring ECM genes from 5 pairs of Dahl SS male rats on 8 or 24 weeks of feeding from weaning on a control (10% Kcal fat) or high-fat (HF; 60%) diet. A PCR array of ECM genes was performed with cDNA from adventitia and PVAT after 8 and 24 weeks. A gene regulatory network of the differentially expressed genes (DEGs) (HF 2-fold > con) was created using Cytoscape. RESULTS: After 8 weeks, 29 adventitia but 0 PVAT DEGs were found. By contrast, at 24 weeks, PVAT possessed 47 DEGs while adventitia had 3. Top DEGs at 8 weeks in adventitia were thrombospondin 1 and collagen 8a1. At 24 weeks, thrombospondin 1 was also a top DEG in PVAT. The transcription factor Adarb1 was identified as a regulator of DEGs in 8-week adventitia and 24-week PVAT. CONCLUSION: These data support that PVAT responds biologically once blood pressure is elevated.

Integrins play a role in stress relaxation of perivascular adipose tissue.

Perivascular adipose tissue (PVAT) is known for being anti-contractile in healthy tissues. We discovered a new function of PVAT, the ability to stress relax and maintain a tone in response to a stretch. This is of note because stress relaxation has been attributed to smooth muscle, of which PVAT has none that is organized in a functional layer. We test the hypothesis the interactions of integrins with collagen play a role in stress relaxation. Our model is the thoracic aorta of the male Dahl SS rat. The PVAT and aorta were physically separated for most assays. Results from single nuclei RNA sequencing (snRNAseq) experiments, histochemistry and isometric contractility were also used. Masson Trichrome staining made evident the expression of collagen in PVAT. From snRNA seq experiments of the PVAT, mRNA for multiple collagen and integrin isoforms were detected: the α1 and ß1 integrin were most highly expressed. Pharmacological inhibition of integrin/collagen interaction was effected by the specific α1ß1 distintegrin obtustatin or general integrin inhibitor RGD peptide. RGD peptide but not obtustatin increased the stress relaxation. Cell-cell communication inference identified integrins αv and α5, two major RGD motif containing isoforms, as potential signaling partners of collagens. Collectively, these findings validate that stress relaxation can occur in a non-smooth muscle tissue, doing so in part through integrin-collagen interactions that may not include α1ß1 heterodimers. The importance of this lies in considering PVAT as a vascular layer that possesses mechanical functions.

A Cell Atlas of Thoracic Aortic Perivascular Adipose Tissue: a focus on mechanotransducers.

Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. To examine the cell-specificity of recognized mechanotransducers we used single nuclei RNA sequencing (snRNAseq) of the thoracic aorta PVAT (taPVAT) from male Dahl SS rats compared to subscapular brown adipose tissue (BAT). Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNAseq, identifying 8 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. Presence of PIEZO1 in the PVAT was confirmed by RNAscope® and IHC; antagonism of PIEZO1 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.