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    Cellular and Molecular Bioengineering

    Lipid Comparison Between Lab-Made and Murine In Vivo Clots and Correlation with Immune Response

    (I-342) Lipid Comparison Between Lab-Made and Murine In Vivo Clots and Correlation with Immune Response

    Saturday, October 14, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row I - Poster # 342
    Introduction:: Venous thromboembolism (VTE) impacts over 900,000 Americans each year with one in three cases resulting in fatality [1]. An understanding of the molecular composition of the thrombi, with a focus on the immune system response, is necessary to develop better treatment options. Mass spectrometry imaging (MSI) is a powerful tool that allows for the identification of molecules while maintaining spatial distribution of the molecules in a sample [2]. This work will focus on utilizing MSI and Liquid Chromatography-Mass Spectrometry (LC-MS) to identify lipids. Additionally, ImageJ will be used to determine the spatial distribution of the lipids and compare clot types. The goal of this work is to determine lipids that correspond with the immune system response by comparing lab-made clots to murine model clots. We hypothesize that lab-made clots will not present an immune response, and changes in lipid metabolism in murine clots will correspond to immune response.

    Materials and Methods:: Lab-made clots were fabricated by first centrifuging whole bovine blood (Lampire Biologic) to separate platelet-rich plasma (PRP) and red blood cells (RBCs). The blood was reconstituted to a 40% hematocrit and calcium chloride was introduced for coagulation. Clots were formed in either stationary well plates or in a dynamic Chandler loop system at 37°C. Murine clots were surgically induced using a ligation technique [3]. Clots were removed after euthanasia at 24 hours. Both lab-made and murine clots were flash frozen for sectioning at 10 microns onto glass slides at -25°C. Samples were then sprayed with either a positive or negative matrix for matrix-assisted laser desorption/ionization (MALDI) MSI. Slides were run with a pixel-size of 100 µm and a m/z range of 50-2000. The samples were then analyzed with HD Imaging to look at their spatial heterogeneity and ImageJ to quantify and compare their lipid distributions (Figure 1A). Additionally, LC-MS was performed on serial sectioned clot samples (lab-made and murine). This data was utilized for statistical analysis and to identify lipidome differences between lab-made and murine clots.

    Results, Conclusions, and Discussions:: LC-MS identified 193 lipid features. Partial least-squares discriminant analysis (PLS-DA) resulted in significant differences between lab-made and murine clots with 95% confidence (Figure 1B). Volcano plot analysis further supported the differences found with PLS-DA. When comparing the lipids found between clot types, lipids with two or less degrees of unsaturation had a lower abundance in murine clots. Lipids with greater than 2 degrees of unsaturation were increased within murine models [4]. It is hypothesized that these levels of abundance are due to the immune response being present for murine clots. With ImageJ analysis, murine clots were generally found to have higher percentages of lipids within a given sample. When analyzing lipids found only in murine samples, the lipids were located centrally. This was determined with concentric circles starting in the center of the sample and moving outward in the sample and confirmed with visual analysis. It is likely that these lipids are located in the center of the clot’s structure due to how a clot forms over time. This centralization is indicative of lipids being relevant to the immune response as macrophages and T-lymphocytes accumulate in the center of clots [5].
    Exploration of how lipids are related to the immune response will be beneficial to the continued research of VTE. Current findings strongly suggest that lipids within clots have a correlation with the immune response. By observing two different clot types, it is possible to determine what may or may not be a part of the immune response due to the way they are formed (in vitro versus in vivo). Additionally, LC-MS provided further insight as to how these two clot types are different, and which lipids need further investigation. Overall, lipid relation to the immune system in the context of VTE is a necessary step in determining potential new treatment methods.


    Acknowledgements (Optional): :

    Thank you to the people at Crouch Imaging Lab: Thomas Paterson, Ziven Noorani, Ryan Thompson, Kylee Karpowich, Amin Jarrahi, and Allison Jones.



    References (Optional): : [1] Impact of Blood Clots on the United States. Centers for Disease Control and Prevention, https://www.cdc.gov/ncbddd/dvt/infographic-impact.html. [2] Caldwell RL, Mol. Cell. Proteomics. 2005. p. 394–401. [3] Sasano, T., J Thromb Haemost. 2022; 20:104-114. [4] McDonald, Riley G, et al. “A MALDI Mass Spectrometry Imaging Sample Preparation Method for Venous Thrombosis with Initial Lipid Characterization of Lab-Made and Murine Clots.” ACS Publications, 13 July 2023, pubs.acs.org/doi/10.1021/jasms.3c00079. [5] J. Matthew Nicklas, International Journal of Molecular Sciences. 2020.