W2 - Inflammatory serum factors from patients with mosaic loss of Y chromosome modulate valvular myofibroblast activation

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Nicole E. Felix-Velez

Graduate Student Researcher
University of California, San Diego
La Jolla, California, United States

Last Author(s)

Brian A. Aguado

Assistant Professor
University of California San Diego, United States

Primary Investigator(s)

Brian A. Aguado

Assistant Professor
University of California San Diego, United States

Introduction: : Mosaic loss of Y chromosome (mLOY) is an age-related phenomenon defined as the loss of the Y chromosome in a subset of leukocytes. mLOY is the most common acquired somatic mutation in human blood cells and increasing evidence links higher incidence of mLOY with increased risk of developing age-related diseases such as prostate cancer, neurodegenerative diseases, macular degeneration, and cardiovascular disease. Emerging clinical observations reveal mLOY is associated with increased mortality in patients with aortic valve stenosis (AVS) and has been linked to increased cardiac fibrosis. Despite its link to increased cardiovascular disease risk, the male-dependent signaling pathways by which mLOY promotes fibrosis remain unclear. Recognizing the central role of inflammation in valvular fibrosis, we investigated the effect of inflammatory factors in serum from mLOY+ AVS patients modulate senescence in valvular interstitial cells (VICs), a hallmark of aortic valve fibrosis. We hypothesized mLOY+ serum contains elevated profibrotic factors that pathologically induce senescence and myofibroblast activation in VICs. Here, we combine omics and biomaterials-based in vitro modeling to understand how mLOY-specific factors can drive valvular myofibroblast activation.

Materials and

Methods: : Primary blood mononuclear cells (PBMCs) and serum were isolated from whole blood of 24 participants (21 AVS patients, 3 healthy donors). Genomic DNA was extracted from PBMCs and submitted to droplet digital PCR (ddPCR) where a TaqMan-based method was performed to quantify the relative number of X and Y chromosomes in a sample. The percentage for loss of Y chromosome (% LOY) was calculated for each sample using (Avg_X -Avg_Y)/(Avg_X)×100. Sera proteome for each sample was profiled using a DNA-based aptamer array targeting 1,500 proteins. For in vitro studies, poly(ethylene glycol) norbornene (PEG-Nb) hydrogels functionalized with RGD were synthesized and crosslinked with a PEG-dithiol crosslinker via click chemistry reaction. Male porcine VICs were seeded on top of soft (3 kPa) and stiff (14 kPa) PEG-Nb hydrogels that recapitulate healthy and fibrotic valve stiffness, respectively, and treated with 1% patient serum or fetal bovine serum (FBS) control for 3 days. Gels were fixed and stained for senescence-associated -galactosidase, a marker of cell senescence.

Results, Conclusions, and Discussions:: We identified 5 mLOY+ patients within our cohort using ddPCR. Correlative analysis between proteomic data and clinical markers of AVS severity revealed distinct protein clusters between mLOY+ and mLOY- samples, suggesting divergent molecular signatures between the groups. Pathway enrichment analysis identified elevated proteins involved in wound healing and tissue remodeling in mLOY+ serum, indicating that mLOY+ AVS serum contains profibrotic cues. To assess whether profibrotic cues in mLOY+ sera modulate valvular fibroblast senescence in vitro, VICs were cultured on soft and stiff hydrogels and treated with patient serum for 3 days. We observed increased senescence-associated -galactosidase on VICs cultured on soft gels with mLOY+ serum relative to both FBS and mLOY- treatment. Taken together, these findings suggest that mLOY+ sera contains profibrotic cues that can induce senescence in quiescent valvular fibroblasts. Future work will integrate RNA sequencing of patient samples and serum-treated cells to identify differentially regulated genes driving fibroblast activation and fibrosis, providing insight into how mLOY contributes to AVS progression.

Acknowledgements and/or References (Optional): :