VCOM Louisiana Research Day Program Book 2024

Biomedical Research: Section 1

13 EXPRESSION OF PULMONARY ENDOTHELIAL CELL COMPONENTS IN PSEUDOMONAS INFECTION

Elizabeth Kibodeaux, BS; Sarah Voth, PhD; Adam Morrow, PhD; Rebekah Morrow, PhD; VCOM-Louisiana

Background: Pulmonary endothelial cells are crucial for regular maintenance of both the gas exchanger barrier and pulmonary vascular resistance. Therefore, describing the molecular components of the cells and how they might be altered in both acute and chronic pathological processes could assist in the development of new treatment strategies for several pulmonary diseases. This preliminary study aims to quantify the levels of several cellular components—legumain, a mammalian asparaginyl endopeptidase; ICAM, a cellular adhesion molecule; and NF-KB, a DNA transcription factor involved in immunity; and SirT1, a nicotinamide adenosine dinucleotide-dependent deacetylase—across several lines of rat pulmonary endothelial cells to better understand their role in pulmonary function. Toll-like receptors (TLR) are pattern recognition receptors that act as part of the innate immune response in endothelial cells. We treated rat pulmonary microvascular endothelial cells with either a strain of Pseudomonas (∆PcrV) or TLR agonists to initiate immune responses and collected whole cell lysates. Protein levels were measured via Western Blot of whole cell lysates. Objective: This preliminary study aims to quantify the levels of several cellular components—legumain, a mammalian asparaginyl endopeptidase; ICAM, a cellular adhesion molecule; and NF-KB, a DNA transcription factor involved in immunity; and SirT1, a nicotinamide adenosine dinucleotide-dependent deacetylase— across several lines of rat pulmonary endothelial cells to better understand their role in pulmonary function. Because these molecules are found extensively in the cells of mammals, we anticipate being able to isolate them in each strain and infection group; however,

Western Blots will assist in determining if the molecule levels are higher or lower in the different experimental groups. This study will therefore highlight proteins of particular interest for future studies. Methods: Rat pulmonary microvascular endothelial cells were treated with either a strain of Pseudomonas (∆PcrV) or TLR agonists (FSL, PAM) to initiate desired immune responses. Whole cell lysates were collected for Western Blot to measure protein levels. Densitometry was performed to quantify the level of each protein. Fold change was calculated using GAPDH as a standard comparison. The densitometry and fold change calculations were both performed in Excel. Results were graphed and analyzed in Excel, as well. The proteins investigated include legumain, an asparaginyl endopeptidase that is involved in tissue homeostasis and is associated with several disease states; ICAM, an adhesion molecule with the vital function of cellular recruitment at sites of inflammation; NF-KB, a DNA transcription factor critical to immunity due to its impacts on cell signaling; and SirT1, a nicotinamide adenosine dinucleotide-dependent deacetylase that is associated with many physiological functions. The control groups are labeled as GRNA #3 and HBSS. Results: The investigated proteins demonstrated expression in each cell line or treatment group, with some differences in quantity between groups. The results demonstrate preliminary data. Even though each target protein was expressed in each treatment group, densitometry was used to further delineate which treatment groups had higher expression of a given protein. No one treatment group had higher

expression of more than one protein, i.e., each protein was expressed the highest in a different treatment group. For ICAM, the APP KO #3 treatment group had the highest level; however, MVR1D had the highest level of legumain. For NK-KB, the MVR1D group treated with Pseudomonas had the highest level while the MVR1D group treated with FSL 100 had the highest level of SirT1. Of particular interest is the high level of NF-KB in the MVR1D group treated with Pseudomonas. This finding is relevant for the development of therapies against Pseudomonas that target the NF-KB signaling pathway. Conclusions: ICAM, legumain, NF-KB, and SirT1 were present across all strains with differences in protein levels in different cell lines or treatment groups. These results represent preliminary findings to help guide future studies of pulmonary endothelial cell components, especially which components are appearing more frequently in cell types associated with certain inflammatory disease states. The next steps include GAPDH reprobes of the blots to calculate fold change; examination of other proteins involved in pulmonary disease states, particularly Pseudomonas infection, by performing more Western Blots; PCR studies to evaluate specific DNA fragments of the MVR1D cell line; and further examination of the treatment groups with particularly high levels of a particular protein. Repeat Western Blots of the experiments featured in this poster would also be helpful for increasing the sample size on further statistical analysis. Results from this study can also be used for investigations of Pseudomonas infection and its relationship to amyloid proteins.

25 2024 Via Research Recognition Day