S22 - CL316,243 Induced Perivascular Adipose Tissue Browning Affects Murine Aortic Biomechanics

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

JE

Jack Esche

Undergraduate Student
Texas A&M University, United States

Co-Author(s)

JE

Jack Esche

Undergraduate Student
Texas A&M University, United States
Matthew R. Bersi, PhD

Assistant Professor, Mechanical Engineering & Materials Science
Washington University in St. Louis
St. Louis, Missouri, United States

Last Author(s)

Matthew R. Bersi, PhD

Assistant Professor, Mechanical Engineering & Materials Science
Washington University in St. Louis
St. Louis, Missouri, United States

Primary Investigator(s)

Matthew R. Bersi, PhD

Assistant Professor, Mechanical Engineering & Materials Science
Washington University in St. Louis
St. Louis, Missouri, United States

Co-Author(s)

Yujun Xu, PhD

PhD Candidate
Washington University in St. Louis
St. Louis, Missouri, United States

Introduction: : Cardiovascular disease (CVD) is a leading cause of mortality in humans that is strongly associated with obesity and insulin resistance which can drive pathologies like atherosclerosis and hypertension [1]. Murine models are valuable for mechanistic CVD research due to their rapid disease progression, genetic tractability, and compatibility with biaxial biomechanical testing to quantify clinically relevant functional metrics, such as vascular stiffness [2].

Perivascular adipose tissue (PVAT) is a metabolically active layer surrounding blood vessels that acts as a paracrine organ to locally regulate vascular tone, inflammation, and remodeling [3]. PVAT exhibits beige adipose tissue (BeAT) plasticity by dynamically shifting between energy-storing white adipose tissue (WAT) and thermogenic brown adipose tissue (BAT) phenotypes [4]. Critically, the BeAT phenotype varies anatomically with thoracic PVAT resembling BAT (multilocular, UCP1-rich) and abdominal PVAT resembling WAT (unilocular, less thermogenic) [5, 6]. The activation of BAT can enhance systemic metabolism via uncoupled respiration, improving glucose uptake and insulin sensitivity, which are key protective mechanisms against cardiometabolic disease [7].

However, aging and metabolic stress drives a PVAT "whitening," characterized by adipocyte hypertrophy, mitochondrial dysfunction, and pro-inflammatory cytokine secretion [6]. This dysfunction impairs the beneficial effects of PVAT. Stimulating PVAT through cold exposure or activation of the β3 adrenergic receptor (β3AR) can induce “browning” and restoration of BAT-like characteristics [4, 6].

This study investigates how targeted pharmacological activation of β3AR using the compound CL316,243 induces PVAT browning and subsequently alters aortic biomechanics. We hypothesize CL316,243 mediated browning will improve aortic properties and potentially mitigate CVD risk factors.

Materials and

Methods: : Male wild type C57BL/6J mice received daily subcutaneous injections of 0.03 mg/kg CL316,243 or saline (vehicle control) for 8 days. Following euthanasia, infrarenal abdominal aorta (IAA) and descending thoracic aorta (DTA) samples with surrounding intact PVAT were then collected. Aortic passive biomechanical properties and remodeling were assessed using biaxial testing, optical coherence tomography (OCT), and histology/immunofluorescence.

Arterial stiffness and in vivo stretch were evaluated using a custom biaxial testing system. Unloaded geometry (outer diameter, axial length) was recorded at 2 mmHg. During testing, circumferential pressure and axial force were monitored while measuring outer diameter and length changes, respectively. Pressure-diameter relationships (0–140 mmHg at constant axial stretch) and force-length relationships (axial stretch ratio λ = 95%, 100%, and 105% of the estimated in vivo axial stretch at constant pressure) were recorded. Passive mechanical behavior was characterized using a four-fiber-family strain energy function [2]. Unloaded aorta segments were imaged with OCT to generate 3D volumetric reconstructions (~0.8 mm length). Wall thickness was measured from representative 2D transverse cross-sections, providing baseline vessel structural dimensions.

Immunofluorescent staining of aortic cryosections quantified medial composition: elastic fiber and α-smooth muscle actin (αSMA) content via mean fluorescence intensity. PVAT browning was assessed by adipocyte density (nuclei count per tissue area).

A custom machine learning model was developed in Cellpose for semi-automatic PVAT adipocyte detection and quantification. Pretrained on 435 manually annotated adipocytes from 10 BAT and WAT images, the custom model enabled consistent segmentation across PVAT samples with minimal post-processing correction.

Results, Conclusions, and Discussions:: Biaxial testing results (Fig. 1) revealed a significant overall increase in axial and circumferential stiffness in both DTA and IAA groups treated with CL316,243 compared to saline (e.g., p(treatment) < 0.05). Region-specific responses were observed with DTA samples having a significant elevation in axial and circumferential stiffness at higher pressures (100 and 120 mmHg) but not at lower pressures (80 and 93 mmHg). Additionally, IAA samples only exhibited significant differences in circumferential stiffness due to treatment at pressures of 93, 100, and 120 mmHg with no difference in axial stiffness at any pressure. Overall axial and circumferential Cauchy stresses were also significantly increased in DTA and IAA samples from CL316,243 treated mice (e.g., p(treatment) < 0.01). Notably, the DTA exhibited significantly higher circumferential and axial Cauchy stresses at all pressures post-treatment, an effect not observed in IAA samples.

Additionally, DTA samples from treated mice had a higher preferred in vivo axial stretch (at 120 mmHg) than untreated mice (p < 0.001), whereas IAA samples had no significant difference between treated and control groups. A significant decrease in wall thickness was observed in CL316,243 treated DTA samples (p < 0.01), but not IAA samples, at 120 mmHg.

Histological analysis of immunofluorescent stain intensity (Fig. 2) revealed a significant increase in medial αSMA expression in CL316,243 treated IAA samples, indicating potential smooth muscle cell activation or phenotypic modulation. No significant difference was observed in elastin between treated and control IAA samples. Similarly, no significant difference was observed in αSMA or elastin between treated and control DTA samples.

The custom machine learning model successfully quantified increasing adipocyte size (perimeter) across adipose depots (Fig. 3), confirming the expected morphological spectrum from BAT (smallest) to WAT (largest) in test datasets (BAT, thoracic/abdominal PVAT, and WAT).

Overall, CL316,243 induced PVAT browning significantly increased arterial stress and stiffness, particularly in the DTA. These findings suggest CL316,243 treatment may not uniformly improve aortic biomechanics, emphasizing the need to further investigate the implications of PVAT browning on vascular function and CVD risk. Additionally, the machine learning approach to adipocyte quantification demonstrates feasibility for semi-automated adipocyte morphometry for PVAT browning assessment in the future.

Acknowledgements and/or References (Optional): : [1] T. M. Powell-Wiley et al., "Obesity and Cardiovascular Disease: A Scientific Statement From the American Heart Association," Circulation, vol. 143, no. 21, pp. e984-e1010, Mar 2021, doi: 10.1161/CIR.0000000000000973.
[2] J. Ferruzzi, M. R. Bersi, and J. D. Humphrey, "Biomechanical phenotyping of central arteries in health and disease: advantages of and methods for murine models," Ann Biomed Eng, vol. 41, no. 7, pp. 1311-30, Jul 2013, doi: 10.1007/s10439-013-0799-1.
[3] E. K. Oikonomou and C. Antoniades, "The role of adipose tissue in cardiovascular health and disease," Nat Rev Cardiol, vol. 16, no. 2, pp. 83-99, Feb 2019, doi: 10.1038/s41569-018-0097-6.
[4] Y. Adachi et al., "Beiging of perivascular adipose tissue regulates its inflammation and vascular remodeling," Nature Communications, vol. 13, no. 1, p. 5117, Sept 2022, doi: 10.1038/s41467-022-32658-6.
[5] N. K. Brown et al., "Perivascular adipose tissue in vascular function and disease: a review of current research and animal models," Arterioscler Thromb Vasc Biol, vol. 34, no. 8, pp. 1621-30, Aug 2014, doi: 10.1161/atvbaha.114.303029.
[6] P. Aldiss, G. Davies, R. Woods, H. Budge, H. S. Sacks, and M. E. Symonds, "'Browning' the cardiac and peri-vascular adipose tissues to modulate cardiovascular risk," Int J Cardiol, vol. 228, pp. 265-274, Feb 2017, doi: 10.1016/j.ijcard.2016.11.074.
[7] K. I. Stanford et al., "Brown adipose tissue regulates glucose homeostasis and insulin sensitivity," J Clin Invest, vol. 123, no. 1, pp. 215-23, Jan 2013, doi: 10.1172/jci62308.