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BMES High School Student Chapter Member

HS Poster

Poster 87 - Identification and Validation of Novel Secreted Biomarkers in Atherosclerosis Using scRNA-seq and Disturbed Flow Models

Saturday, October 26, 2024
10:00 AM – 12:00 PM EST
Location: Exhibit Hall E, F & G

Introduction: Atherosclerosis is a leading cause of cardiovascular morbidity and mortality worldwide, characterized by the progressive accumulation of lipids, inflammatory cells, and fibrous elements within the arterial wall. Despite advancements in understanding its pathophysiology, the early detection, risk stratification, and effective management of atherosclerosis remain challenging. Although traditional biomarkers like C-reactive protein and low-density lipoprotein cholesterol provide some insight into the disease, there is a critical need for more specific and sensitive markers to better capture the early stages and progression of atherosclerosis.

The intricate pathogenesis of atherosclerosis involves a complex interplay of lipid metabolism, inflammation, endothelial dysfunction, and immune responses. These processes are reflected in the secretion of various biomolecules that could serve as potential markers for the disease. However, many of these secreted markers have yet to be fully explored or validated in the context of atherosclerosis.

This study aims to investigate a comprehensive panel of secreted markers associated with atherosclerosis, with the goal of identifying novel biomarkers that could improve the early detection and risk stratification of this condition. By leveraging advanced techniques in single-RNA sequencing (scRNA-seq), VerSeDa (a specialized tool for secretome data analysis), and quantitative polymerase chain reaction (qPCR), we seek to expand the current biomarker repertoire and uncover new pathways involved in atherosclerotic plaque development and progression. The outcomes of this research have the potential to inform the development of targeted therapies and personalized medicine approaches, ultimately reducing the global burden of cardiovascular disease.

Materials and

Methods: We analyzed scRNA-seq data collected by Dr. Jo lab at Emory from C57BL/6 mice subjected to partial carotid ligation surgery to induce atherosclerosis. Sequencing data were processed and analyzed to identify differentially expressed genes associated with atherosclerosis. For the secretome analysis, we utilized VerSeDa to analyze scRNA-seq data, focusing on the identification of secreted proteins significantly elevated in the atherosclerotic carotid artery compared to the control. We selected secretomes based on the differential expression of the identified secreted proteins in three data groups: (1) disturbed flow group from mice subjected to partial carotid ligation surgery, (2) cholesterol group from mice subjected to a high-fat diet, (3) disturbed flow + cholesterol group from mice subjected to partial carotid ligation surgery with a high-fat diet. Secretomes were selected based on their differential expression in atherosclerosis.

Moreover, we studied an in vitro model to confirm the effect of the disturbed flow on the secretion of atherosclerosis-related secretomes. Immortalized human artery endothelial cells (iHAECs) were cultured in endothelial growth medium. A cone-and-plate system was used to apply disturbed flow conditions to iHAECs for 24 hours, and cells exposed to static conditions served as controls. Total RNA was extracted from iHAECs post-treatment. qPCR was performed to quantify the expression of VCAM1 and KLF4 as markers of atherosclerosis induction and the secretomes identified from the scRNA-seq analysis and VerSeDa to assess whether the secretome genes were upregulated or downregulated in alignment with the in vivo findings.

Results, Conclusions, and Discussions: Results and Discussions:
In our study, we utilized single-cell RNA sequencing (scRNA-seq) data from a mouse model subjected to partial carotid ligation surgery to identify secretomes associated with atherosclerosis. The analysis, performed using VerSeDa, highlighted secreted factors that were significantly elevated in the atherosclerotic environment compared to control tissues: showing 97 secretomes in group 1, 216 in group 2, 87 group 3 (Fig. 1). These secretomes were primarily involved in inflammatory pathways, extracellular matrix remodeling, and endothelial cell dysfunction—key processes implicated in the development and progression of atherosclerosis. GRN (Granulin, which modulate inflammation), ENO1 (Enolase 1, showing a proatherogenic role in endothelial cells), and PSAP (Prosaposin, showing inflammatory activity in macrophages) were upregulated in three groups, serving as new potential biomarkers for atherosclerosis.

To validate these findings, we employed an in vitro model using a cone-and-plate system to simulate disturbed flow conditions, a well-known inducer of atherosclerosis, in iHAECs (Fig. 2). The qPCR analysis revealed that the cells under disturbed flow (oscillatory shear stress, OSS group) significantly upregulated VCAM1 expression but downregulated KLF4 expression compared to the unidirectional laminar shear stress (ULS) group, confirming the induction of a pathogenic atherosclerotic response in vitro. Importantly, the qPCR data also showed that the three key secretomes, GRN, ENO1, and PSAP, identified in the scRNA-seq analysis and VerSeDa were consistently upregulated in the iHAECs under disturbed flow, supporting their role in atherogenesis. TINAGL1 was also consistently upregulated in OSS group compared to ULS group, needing further systematic analysis.

Conclusion:
The results of this study underscore the importance of secreted markers in the pathophysiology of atherosclerosis. The secretomes identified through scRNA-seq and VerSeDa and validated in our in vitro model point to key players in the inflammatory and remodeling processes that drive the disease. These findings align with recent research highlighting the role of inflammation and endothelial dysfunction in atherosclerosis.

In conclusion, this study highlights the utility of combining advanced genomic techniques with functional validation to uncover novel biomarkers for atherosclerosis. The secreted markers identified here could pave the way for more precise risk assessment , ultimately improving outcomes for patients with atherosclerosis.

Acknowledgements (Optional): We would like to express our sincere gratitude to Christian Park and Dr. Hanjoong Jo for providing the scRNA-seq data, and to Dr. Young Hoon Son and Dr. Jihee Won for their invaluable support, including cell-culture and qPCR training, as well as guidance throughout the course of this study.

References (Optional):