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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster I1 - Paracrine Signaling by Distinct Adipose Tissue Depots Regulate Fibroblast-to-Myofibroblast Transition

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Ryan W. Catlett (he/him/his)

Undergraduate Researcher
University of Missouri-Columbia
Saint Louis, Missouri, United States

Co-Author(s)

Yujun Xu, PhD

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

Primary Investigator(s)

Matthew R. Bersi, PhD (he/him/his)

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

Introduction: Hypertension is the leading risk factor for mortality worldwide. Adipose tissue, or fat, has been linked to increasing hypertension rates. Adipose is an endocrine gland that regulates tissue functions and exists on a spectrum from white adipose (energy storage) to brown adipose (thermoregulatory). Perivascular adipose tissue (PVAT) is a specialized adipose depot that surrounds blood vessels outside of the brain. PVAT is a non-uniform mixture of brown and white adipocytes and has different phenotypes based on anatomical location. Here, we are interested in the regulatory effects on hypertension exerted by different adipose depots through examination of the impact of chemical, structural, and secretory differences on fibroblast activation. PVAT first signals to the adventitial layer composed of collagen, nerve endings, and cells. Thus, we are focused on crosstalk between adipose tissue and fibroblast mechanobiology since the activation and transition of fibroblasts into myofibroblasts has been associated with hypertension and vascular fibrosis. Myofibroblasts gain expression of alpha smooth muscle actin (αSMA), while fibroblasts do not. In the current study, we performed co-cultures between fibroblasts and distinct adipose tissue depots (brown adipose tissue, BAT; thoracic PVAT, TF; abdominal PVAT, AF; and white adipose tissue, WAT) and measured αSMA expression as an indicator of adipose-mediated phenotypic changes in cultured fibroblasts. Our goal is to bridge the knowledge gap regarding adipose phenotypes and fibroblast mechanobiology to enhance our understanding of how PVAT influences vascular health. This knowledge could contribute to developing new therapeutic strategies for hypertension or other cardiovascular diseases.

Materials and

Methods: In this experiment we used wild type (WT) and Rag1-deficient (Rag) male mice (n=4) at 8-12 weeks old. Rag mice were utilized since they lack mature B and T lymphocytes and previous reports have found minimal hypertensive complications in Rag mice, compared to WT. To perform adipose-fibroblast co-cultures, we first dissected the interscapular, epididymal, thoracic PVAT, and abdominal PVAT adipose depots from WT and Rag mice to obtain BAT, WAT, TF, and AF adipose tissues, respectively. To observe the crosstalk between adipose tissue and fibroblasts, at least 3 mg of each adipose depot were added to each well of a 24 well plate and floated in 2 mL of media in contact with NIH 3T3 fibroblasts. Before plating for co-culture, 3T3 fibroblasts were seeded on cell cover slips to allow for immunofluorescence staining. Once plated, co-cultures were maintained under standard culture conditions in Dulbecco’s Modification of Eagle’s Medium (DMEM) with 10% FBS and 1% P/S and incubated at 37°C and 5% CO2 for 48 hours. After 48 hours the co-culture media was extracted and stored, the adipose tissue was removed, and co-cultured 3T3 cells underwent immunofluorescence (IF) staining and imaging. During IF staining, cells were first fixed and permeabilized using 4% paraformaldehyde and 0.1% Triton X-100, followed by blocking with 1% bovine serum albumin and 3% goat serum, and staining with a Cy3-conjugated αSMA antibody (1:300). Finally, after incubation overnight at 4°C, cover slips were mounted to stained cells using ProLongTM Gold antifade reagent containing DAPI.

Results, Conclusions, and Discussions: Figure 1 compares αSMA staining intensity across each genotype (WT and Rag) and treatment group (untreated 3T3, BAT, TF, AF, and WAT) included in the study. The increase in normalized αSMA intensity, relative to untreated 3T3 control, was more pronounced in WT adipose-treated cells than in Rag adipose-treated cells. There was a trend toward increased intensity in WT BAT, TF, and AF-treated cells, with a significant increase observed in TF-treated cells (p=0.0380) as determined by a two-way Analysis of Variance (ANOVA). Figure 2 shows comparisons across adipose depots within each genotype. While significant increases in αSMA staining intensity were identified for both WT TF (p=0.0363) and WT AF (p=0.0349) treatment relative to control, no differences from control were observed for adipose depots from Rag mice. Representative images for αSMA expression from each genotype and adipose depot treatment are shown in Figure 3.

Our results yielded minimal changes in αSMA expression in fibroblasts cultured with Rag adipose depots. This is consistent with prior reports of minimal hypertensive remodeling and fibrosis in Rag mice lacking mature lymphocytes. Minimal αSMA changes in Rag mice indicates less fibroblast-to-myofibroblast transition and suggests an important role for lymphocytes in this process. Fibroblasts treated with WT adipose had significantly higher αSMA than Rag-treated cells indicating a higher myofibroblast expression. Again, this is consistent with observations of excess hypertensive remodeling in WT mice. It is interesting that PVAT depots (TF and AF) induced significantly higher αSMA expression since we expect them to have the greatest regulatory effect on hypertension. PVAT composition is similar to WAT tissue that behaves like BAT; which is consistent with our αSMA expression data. In the future, we will perform analysis of the adipose conditioned media using an adipokine array to explore what signals are transmitted to the 3T3 cells and better understand why PVAT produces a more significant upregulation αSMA expression in fibroblasts than BAT and WAT.

Deepening our understanding of the relationship between adipose tissue and myofibroblast activation in the context of cardiovascular disease has the potential to produce advancing therapeutics on key issues like hypertensive remodeling and increase life expectancies.

Acknowledgements (Optional): Research reported in this poster was supported by the American Heart Association Career Development Award (941138 to MRB) and the WashU Cardiovascular Summer Research (CardS) program. I would like to especially thank Dr. Matthew Bersi and Yujun Xu for their immense guidance and support this summer.