Poster T12 - T cell- Dependent Regional Variations in Perivascular Adipose Tissue and Vascular Remodeling in Response to Angiotensin II-Induced Hypertension

Introduction: Hypertension is a leading cause of cardiovascular disease and a significant contributor to mortality in the United States. The increased hemodynamic load from high blood pressure can prompt remodeling of the vascular extracellular matrix, leading to arterial stiffening due to impaired vascular elasticity and increased resistance. Notably, regional stiffening along the aorta varies under angiotensin II (AngII)-induced hypertensive conditions. Additionally, regional immunological responses to hypertension, including perivascular recruitment and infiltration of CD45+ leukocytes and CD3+ T cells, differ along the aorta with increased infiltration reported in the thoracic aorta compared to the abdominal aorta in AngII-induced hypertensive mice.
Perivascular adipose tissue (PVAT) is a specialized tissue surrounding the aorta that acts as an endocrine organ to regulate vascular properties including vasoconstriction, endothelial function, and vessel remodeling through the secretion of cytokines and adipokines. PVAT also serves as a reservoir for immune cells that can influence vascular biology based on environmental stimuli. Interestingly, PVAT exhibits distinct characteristics and functions in different regions along the aorta due to varied distributions and proportions of adipose phenotypes (e.g., white, brown, or beige) based on structural and functional tissue attributes.
Given these observations, we hypothesize that regional differences in PVAT and its capacity to differentially recruit immune cells may modulate the regional variations in immune-mediated vascular remodeling associated with hypertension. This hypothesis underscores the significance of PVAT in regulating aortic stiffness and immune cell dynamics, thus highlighting its potential contribution to hypertension-induced vascular remodeling.

Materials and

Methods: Male wild-type (WT) and Rag1-/- mice on a C57BL/6J background at 8-12 weeks of age were maintained on a standard diet with access to water ad libitum. Control mice received saline (DPBS) delivered via subcutaneous osmotic minipump, while the experimental group was administered AngII at 490 ng/kg/min to induce hypertension. After 14 days of treatment, the descending thoracic aorta (DTA) and infrarenal abdominal aorta (IAA), were harvested with or without their respective thoracic (t-PVAT) and abdominal (a-PVAT) PVAT depots.
Bulk RNA sequencing and gene ontology analysis of DTA and IAA samples from WT and Rag1-/- mice identified transcriptional changes resulting from hypertension. To evaluate hypertensive vascular remodeling, biaxial mechanical testing was performed on isolated DTA and IAA segments. Oil Red O staining was employed to examine regional in PVAT area due to hypertension. Additionally, immunofluorescence staining for UCP-1 (brown adipocytes) and Perilipin-1 (total adipocytes) was performed on vascular sections with surrounding t-PVAT and a-PVAT to identify hypertension-induced adipocyte phenotypic shifts. To discern gene expression differences between t-PVAT and a-PVAT, real-time quantitative PCR for select genes was conducted. Furthermore, differences protein secretion between t-PVAT and a-PVAT from WT and Rag1-/- mice was assessed using an adipokine array.
Data are presented as mean ± SEM. For two group comparisons (effect of AngII), a student’s t-test for normally distributed variables was employed. For four group comparisons (effect of genotype and AngII), two-way ANOVA was used with post-hoc Tukey HSD testing for multiple comparisons was performed with a significance threshold of p < 0.05.

Results, Conclusions, and Discussions: Differential gene expression analysis highlighted several immunological genes, specifically related to immune regulation and T cell activation and proliferation, that were upregulated in the WT-DTA comparison group (e.g., Cd44, Cd33, Ptpn6, etc.) Ptpn6 was significantly altered in Rag-DTA under AngII treatment, despite Rag1-/- mice lacking mature B and T cells. The gene encoding the hematopoietic cell adhesion protein CD45 (gene: Ptprc) was also significantly up-regulated in the AngII-treated Rag-DTA but not in Rag-IAA, underscoring its role in immune cell activation and signaling (Fig.1, A). Genes related to brown adipocyte differentiation were up-regulated in AngII-treated Rag-DTA and Rag-IAA. At the transcriptional level, hypertensive WT t-PVAT is more inflamed, whereas WT a-PVAT exhibits more expression of matrix turnover genes (Fig.1, B). Additionally, hypertensive WT PVAT showed a slight decrease in Ucp1 gene expression, suggesting a shift towards a white adipose-like state. Gene expression patterns are less pronounced in Rag1-/- groups under hypertension (Fig.1, C).
Histological analysis revealed a significant increase in PVAT area around the hypertensive WT-DTA, but not WT-IAA. In contrast, PVAT area was unchanged in Rag-DTA mice, and significantly decreased in Rag-IAA mice under hypertension (Fig.2, top). Despite differences in PVAT area, aortic wall thickness was significantly increased in hypertensive WT-DTA and Rag-DTA mice, with no change observed in the IAA groups (Fig.2, bottom). To confirm the role of T cells in PVAT morphological changes observed in WT mice, we adoptively transferred splenic CD3+ T cells from WT donors into Rag1-/- recipients and treated them with AngII for 14 days. Consistent with WT mice, there was a significant increase in PVAT area and a trend toward significant increase wall thickness in the DTA, but not IAA, of T cell recipient Rag1-/- mice (Fig.2, bottom). These results suggest AngII-induced hypertension leads to T cell-mediated expansion of PVAT and aortic wall thickness in WT-DTA and reconstituted Rag1-/- mice.
Together, these findings underscore the distinct molecular changes in regional PVAT composition and immune cell recruitment associated with regional aortic remodeling in response to hypertension and highlights the potential for PVAT to influence vascular remodeling and contribute to the pathophysiology of hypertension.

Acknowledgements (Optional): We thank the Genome Technology Access Center at the McDonnell Genome Institute at Washington University School of Medicine for help with genomic analysis. The Center is partially supported by NCI Cancer Center Support Grant #P30 CA91842 to the Siteman Cancer Center and by ICTS/CTSA Grant# UL1TR002345 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research. This publication is solely the responsibility of the authors and does not necessarily represent the official view of NCRR or NIH.
This work was supported by the American Heart Association (AHA) Career Development Award 941138 to MRB.