Poster E5 - Characterizing the Influence of Adipose Tissue on Fibroblast Biophysical Properties Using Micropipette Aspiration

Thursday, October 24, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

TT

Tata A. Toure

Undergraduate Researcher
Washington University in St. Louis
St. Louis, Missouri, United States

Co-Author(s)

Ryan Catlett

Undergraduate Researcher
Washington University in St. Louis, United States
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: Vascular fibrosis is a pathological condition characterized by reduced lumen diameter and arterial wall thickening attributed to excessive deposition of extracellular matrix (ECM) (Lan, 2013). Vascular fibrosis can lead to hemodynamic damage of peripheral tissue, may impair endothelial function, and may promote vascular rarefaction (Harvey, 2016). The specific factors that drive vascular fibrosis are not completely known, but we suspect that perivascular adipose tissue (PVAT) plays a role. PVAT is a specialized adipose tissue depot comprised of immune cells, stromal cells, and both brown and white adipocytes that surrounds the arterial wall and provides both external support and biochemical signaling that regulates vascular biology. Thus, we hypothesize that understanding the influence of adipose tissue on promoting cellular biophysical changes associated vascular fibrosis, such as changes in cell stiffness, may give way to treatments for fibrotic vascular remodeling. The primary objective of this project was to study the effect of adipose tissue on the potential for vascular fibrosis through investigating the influence of nonphysical adipose interactions (i.e., paracrine signaling) on fibroblast phenotypes. Although PVAT is our primary adipose tissue depot of interest, here we have elected to begin our analysis with a focus on traditional brown adipose tissue (BAT) and white adipose tissue (WAT) depots. Using micropipette aspiration, we measured the elastic and viscoelastic properties of individual NIH 3T3 fibroblasts. We took baseline (untreated) measurements, as well as measurements following co-culture in the presence of either BAT or WAT from wildtype or immunocompromised (Rag1-deficient) mice.

Materials and

Methods: For our experiments, NIH 3T3 fibroblast cells were cultured for use in a 10 cm dish using DMEM Media with 10% FBS and 1% P/S. Elastic and viscoelastic tests were performed on a micropipette aspiration system. Briefly, micropipette tips were pulled from 1 mm glass capillary tubes using a micropipette puller, and the tips were broken using a Microforge to be approximately half the diameter of the cells (~10 microns for 3T3s). For the purposes of our experiment, we looked at the Young’s Modulus (E), critical pressure (Pc), and cortical tension (T) derived from elastic tests, as well as the steady state Young’s Modulus (E_ss), instantaneous Young’s Modulus (E_inst), relaxation time constant (τ), and the viscosity (μ) derived from viscoelastic tests. Note that elastic tests were defined by a linear ramp in pressure while viscoelastic tests involved a step increase in pressure. Calculations for each of these biophysical quantities were performed using either Equation 1 (Hochmuch, 2000) or Equation 2 (Guilak, 2000), respectively (Figure 1).

Adipose tissues were collected from two genotypes of male mice between 8-12 weeks of age: wild-type C57BL/6J and 〖Rag1〗^(-/-). Interscapular/brown adipose tissue (BAT) and epidydimal/white adipose tissue (WAT) were isolated and divided into small pieces (~5 mg). The adipose tissues were placed in the upper portion of trans-well inserts in a 24-well plate in order to be co-cultured with the 3T3 cells in the absence of physical interactions. Micropipette aspiration was performed after the cells had been in co-culture for 48 hours.

Results, Conclusions, and Discussions: Figure 2 shows statistically significant comparisons determined by one-way Analysis of Variance (ANOVA) between baseline and co-culture measurements. Wildtype BAT-treated cells showed a significant decrease in stiffness and increase in time constant, while the Rag1 BAT-treated cells showed a significant decrease in cortical tension compared to baseline. Figure 3 shows statistically significant comparisons determined by two-way ANOVA between co-culture measurements from different genotypes. Again, wildtype BAT-treated cells were most different from other groups. They were less stiff than both wildtype WAT-treated and Rag1 BAT-treated cells, had lower critical pressure and cortical tension than Rag1 BAT-treated cells, had higher time constant and viscosity than wildtype WAT-treated cells, and had higher time constant than Rag1 BAT-treated cells. From both figures, we can conclude that BAT treatment leads to changes in elastic and viscoelastic properties of fibroblasts, particularly in wildtype mice. The reduced effect of BAT in Rag1-deficient mice suggests, there may be an influence of lymphocytes on BAT biology. The next step for this project would be to compare these results with findings from PVAT co-cultures and potentially treat 3T3s with specific signaling proteins found in adipose tissues. This would provide more targeted treatments for the 3T3s, revealing insights into specific signaling pathways that may play a role in modulating fibroblast biophysical properties and fibroblast-to-myofibroblast conversion.

Arterial stiffening as a result of vascular fibrosis has long been associated with hypertension. Increasing our understanding of the underlying mechanobiological causes of this stiffening could lead to better treatments to minimize vascular remodeling. Since arterial stiffening has been associated with changes in vascular fibroblast phenotype to activated myofibroblasts, variations in the biophysical properties measured in the current study may be an early indicator of increased fibroblast-to-myofibroblast conversion following adipose tissue co-culture. Results from this study indicate that there may be decreased conversion of fibroblasts to myofibroblasts in the presence of BAT, however this remains to be confirmed using other identification or functional measurements. Additionally, we observed differences in fibroblast properties between wildtype and Rag1-deficient mice, suggesting lymphocytes may be an important mediator of adipose-mediated changes in fibroblast phenotypes.

Acknowledgements (Optional): This work was supported, in part, by the American Heart Association Career Development Award (941138 to MRB). I would like to thank Dr. Matthew Bersi for giving me the opportunity to work and learn in his lab. Special thanks to Yujun Xu, who has let me assist her in her research on the mechanobiology of the interactions between adipose tissue and fibroblasts. In addition, she has been a great research mentor, I couldn’t have done this project without her help.