T14 - TMAO-Induced Collagen Production in VICs via Sirius Red and Colorimetric Quantification

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

aniyah D. dickson

Undergraduate
The University of Georgia
Stone Mountain, Georgia, United States

Co-Author(s)

SS

Samanvitha Sudi

PHd Student
The University of Florida
Gainesville, Florida, United States
Ana M. Porras

Assistant Professor
University of Florida
GAINESVILLE, Florida, United States

Primary Investigator(s)

Ana M. Porras

Assistant Professor
University of Florida
GAINESVILLE, Florida, United States

Introduction: : Calcific aortic valve disease (CAVD) is a progressive disease that currently lacks any working clinical treatments; valve replacement surgery is the only option for TMAO treatment. However, recent studies have identified the gut microbiome as one of the contributing factors to cardiovascular disease through the production of metabolic byproducts like trimethylamine-N-oxide (TMAO). TMAO has emerged as a possible risk factor for late-stage valve disease. Despite this, there is limited information on how TMAO affects early disease progression at the cellular level. Valvular interstitial cells ( VICs), the primary cells found in aortic valves, are usually found in a quiescent state, but can become activated under diseased conditions and contribute to fibrotic remodeling through increased collagen production. Our study investigates whether TMAO influences VIC behavior by promoting collagen deposition, thereby contributing to the development of early stage CAVD, or sclerotic valve disease.

Materials and

Methods: : Primary valvular interstitial cells were isolated from healthy porcine aortic valves and cultured in vitro. Cells were treated with varying concentrations of TMAO and we compared these to untreated VICs and a positive control, transforming growth factor (TGF-β) positive control. We chose this specific control to focus on due to it being known to activate VICs and induce collagen production. To effectively assess collagen levels in both the cell matrix and the secreted media, my mentor and I used Sirius Red staining and a colorimetric assay ( Chondrex kit). Sirius Red specifically binds to collagen types I and III, which are commonly found in connective tissues. Stained cell plates were then imaged and analyzed for intensity of red coloration, which directly correlates with the amount of collagen present. For secreted collagen, absorbance was measured at a specific wavelength using a spectrophotometer, which allowed us to gage a more detailed quantitative comparison between treatment groups. Experiments were repeated to ensure consistency, and the resulting data were analyzed using a one-way ANOVA to assess whether TMAO induced a dose-dependent increase in collagen synthesis.

Results, Conclusions, and Discussions:: We observed an increase in collagen production after TMAO treatment in VICs. Visual analysis of the Sirius Red- stained samples disclosed darker red staining in TMAO-treated groups compared to controls, indicating increased collagen deposition (Figure 1). Similarly, the TGF-β group also exhibited strong collagen staining, consistent with its role as a positive control for fibrosis. Additionally, to support these visual findings, we used a colorimetric assay to measure collagen secretion from the media. Absorbance readings increased with higher TMAO concentrations and were also elevated in the TGF-B group. This implied that both treatments enhance collagen secretion that is dependent on the dose provided ( Figure 2). These findings support our hypothesis that TMAO activates VICs and promotes fibrotic ECM remodeling by increasing collagen content.

In conclusion, our study demonstrates that TMAO can stimulate collagen production in porcine VICs, potentially contributing to the early stages of CAVD. The use of both Sirius Red staining and a colorimetric assay provided a vigorous and reproducible method to evaluate collagen dynamics in vitro. These results highlight the importance of microbiome-derived metabolites in cardiovascular health and disease and emphasized the need for further investigation into non-surgical therapies for CAVD.

Acknowledgements and/or References (Optional): :