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Biomechanics

Biomechanics - Poster Session A

Poster E6 - Cluster model for fibroblast activation in heart

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Fibrotic diseases are responsible for over 800,000 deaths worldwide each year, with lung and cardiac fibrosis being the leading contributors.1 Many organs in the human body can develop fibrosis, a condition characterized by the excessive deposition and remodeling of extracellular matrix (ECM) material. This process increases tissue stiffness and can ultimately impair organ function.2 A crucial aspect of fibrosis is the transformation of normal fibroblast cells into myofibroblasts. Fibroblasts are resident cells that maintain ECM homeostasis. The activation of fibroblasts into myofibroblasts requires mechanical signaling and soluble growth factors.3 Fibroblasts adapt to the mechanical properties of their surrounding environment.4,5 They detect the mechanical force or tension, which can lead to deviations from their normal, baseline homeostatic states, resulting in the transformation into myofibroblasts. Myofibroblasts exhibit properties including ECM secretion and enhanced contractility.6,7 While myofibroblasts play a beneficial role in wound healing, their dysregulation can lead to fibrosis. In the heart, myofibroblasts can stiffen the heart muscle by producing excessive ECM and increasing contractility. This increased stiffness can impair heart function,8,9 and disrupt electrical transmission among cardiomyocytes, potentially causing fatal re-entrant arrhythmias.10,11
One of the phenomena of cell clustering drives fibroblast activation, increasing ECM stiffness. Cells adhere, influenced by environmental signals, exerting stronger contractile forces that transform fibroblasts into myofibroblasts, further stiffening the ECM. In this study, we aim to develop an integrated experimental and theoretical approach to study the factors influencing cell clustering.

Materials and

Methods:  Our investigation included examining the impact of cell density and drug treatment. In the first set of experiments, we cultured tissue samples containing collagen type I and fibroblast cells at densities of 0.3 and 0.5 million cells per milliliter. For the second set of experiments, tissues were cultured at a density of 0.5 million cells per milliliter in three groups: one treated with the microtubule inhibitor nocodazole before culturing (pre-treatment), another treated with nocodazole 24 hours after culturing (post-treatment), and a control group left untreated. All samples had a collagen concentration of 1 mg/ml and were analyzed after 24 hours of tissue contraction. Tissue stacks were imaged using a Zeiss microscope (Carl Zeiss Microscopy), and clustering levels were quantified using Zeiss Zen microscopy software.
To understand the cell clustering mechanism, we developed a model (Fig 1) and validated it with experiments. Our model is based on the fact that myofibroblasts stiffen the ECM locally by exerting contractile force. This stiffening increases the likelihood of nearby normal fibroblasts transforming into myofibroblasts. As more myofibroblasts form, they intensify ECM stiffening. The initial transformation is assumed to occur randomly and with low probability across the tissue volume.

Results, Conclusions, and Discussions: In the first set of experiments, we examined how cell density affects cell clustering by measuring the number of clusters of varying sizes in tissues with two different cell densities: 0.3 million cells per milliliter (13 samples) and 0.5 million cells per milliliter (6 samples). As shown in Fig 2, most cells did not form large clusters due to the relatively low densities in both groups. However, the group with the higher cell density exhibited a greater degree of clustering (Fig 2B). Specifically, 5% of cells in the higher density group formed three-cell clusters, none seen in the low-density group (Fig 2A). Isolated cells comprised 83% of the higher density group, versus 93% in the lower. These results match our model, showing that higher cell densities promote larger and more frequent cell clusters due to increased cell-cell interactions.
In the second set of experiments, we investigated how microtubule polymerization affects cell clustering. We used nocodazole, a known inhibitor of microtubule polymerization, to treat cells in tissues with a density of 0.5 million cells per milliliter at two different times: before culturing and after culturing. This approach is based on our observations: pre-treating fibroblasts with nocodazole inhibits protrusion growth and ECM alignment, while applying it 12-24 hours post-culturing enhances collagen remodeling and alignment. As shown in Fig 3, the control group (no nocodazole treatment) (5 samples) displayed similar clustering behavior to the previous experiment with the same cell density (Fig 3B, Fig 2B). However, tissues pre-treated with nocodazole (5 samples) showed lower clustering levels compared to the control group (Fig 3A). Post-treated tissues (5 samples) exhibited a significant increase in clustering level, with cells forming a notable number of large clusters and only 26% of isolated cells (Fig 3C). These findings highlight how nocodazole treatment timing and microtubule dynamics crucially influence cell clustering and interaction within tissues.
Our study demonstrates that cell density and microtubule dynamics significantly influence cell clustering and ECM stiffening. These findings help us understand the mechanism behind cardiac fibrosis development and other related conditions.

Acknowledgements (Optional):  Aliza Mujahidab, Yuxuan Huangac, Mohammad Jafariab, Elliot L. Elsonae, Guy M. Geninacd, Farid Alisafaeiab
a NSF Science and Technology Center for Engineering Mechanobiology (CEMB)
b Department of Mechanical and Industrial Engineering, New Jersey Institute of Technology
c Department of Mechanical Engineering and Materials Science, Washington University in St. Louis
d Department of Biomedical Engineering, Washington University in St. Louis
e Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis