VCOM Louisiana Research Day Program Book 2024

Biomedical Research: Section 1

Melissa Ruiz, MS, OMS-III 1 *; Chung-Sik Choi, PhD 2 ; Mike Lin, PhD 3,4 ; Troy Stevens, PhD 3,4 ; Rebekah Morrow, PhD 1 ; K. Adam Morrow, PhD 1 ; Sarah Voth, PhD 1 1 Department of Cell Biology and Physiology, VCOM-Louisiana; 2 Department of Physiology and Cell Biology, University of South Alabama College of Medicine; 3 Center for Lung Biology, University of South Alabama College of Medicine; 4 Department of Internal Medicine, University of South Alabama College of Medicine 8 AMYLOID PRECURSOR PROTEIN CONTRIBUTES TO LUNG ENDOTHELIAL IMMUNE RESILIENCE

Background: Innate immune resilience refers to a tissue’s ability to maintain functional integrity during a microbial insult and to repair itself following resolution of the active infection. The innate resilience of the air-blood barrier is fundamental to preventing alveolar flooding and hypoxemia during nosocomial pneumonia. Bacterial breach of the endothelial barrier promotes permeability, bacteremia, and acute respiratory distress syndrome (ARDS). The inflammatory milieu characteristic of ARDS is partially comprised of endothelial-derived cytotoxic amyloids. Pathogenic amyloids elicited from infected lung endothelium contribute to the long-term cognitive and physical impairments that describe post-intensive care syndrome. Cytotoxic amyloids arise from the corruption of their innate physiologic function by specific bacterial toxins. Physiologically functional amyloids – including amyloid-beta (A β ) – are innately bacteriostatic and bactericidal. Non-pathogenic A β is generated from the amyloidogenic processing of amyloid precursor protein (APP), which has been reported to contribute to vascular resilience in the cerebrovascular compartment. However, there has been little elucidation on the role of APPs in the resilience aspect of lung endothelial host defense. Objective: We hypothesize that APP promotes lung endothelial barrier resilience by regulating the endothelial immune response.

Methods: Rat PMVECs utilized in these studies included a wildtype (WT), CRISPR/Cas9 control (APP-expressing subclone from CRISPR/Cas9), and two subclones in which APP was deleted (APP-/-). Subconfluent PMVECs were lifted, and 100,000 cells were seeded into 6-well plates for growth curves to assess whether APP contributes to PMVEC proliferation. Three wells from each clone were lifted, counted, and counts averaged at the same time each day. Averages were plotted over time and the doubling time calculated. Wound closure assays were used to determine APP’s contribution to cell migration and endothelial barrier repair. For wound closure assays, PMVECs were grown to confluence in 6-well plates and a 100 µl pipette tip was used to scratch the monolayer to induce injury. Images were taken over time and analyzed with ImageJ to quantify wound area. RT-qPCR measured the basal level of pro-inflammatory mediators IL-6, MCP-1, TNF α , and CCL5. Angiogenesis assays were utilized to assess the potential role of APP in vascular network formation. Subconfluent PMVECs were identically seeded in Matrigel-containing 48-well plates in full media and imaged over time. ImageJ was used to quantify the number of closed loops, intersections, and distance between intersections of vascular networks. Results: Although there was no difference between the doubling time of WT and control PMVECs (ns;

p < 0.3500), the rate of APP-/- cell doubling was significantly impaired as compared to the control (p < 0.0027) and WT cells (n ≥ 3; 3 replicates, p < 0.0001; one-way ANOVA with Tukey’s post hoc). WT PMVECs demonstrated a decreased ability to repair a disrupted endothelial barrier as compared to APP-/- cells. No difference in wound area between WT, control or APP-/- PMVECs immediately following injury (ns; p < 0.8743, 0.5007, and 0.6677) was noted. At 18 h post-injury, however, WT PMVECs exhibited a decreased ability to repair the injury (p < 0.0166) as compared to both control PMVECs (p < 0.0106) and APP-/- cells (p < 0.8117). Moreover, no notable differences in angiogenic capacity (ns) or wound closure was noted between control and APP-/- PMVECs (p < 0.8117; n ≥ 3; 3 replicates, two-way ANOVA with Tukey’s post-hoc). Interestingly, APP null cells significantly overexpress cytokine CCL5 as compared to control PMVECs (p < 0.001) or WT (n ≥ 2, two-way ANOVA with Tukey’s post-hoc analysis) PMVECs. The dysregulation of CCL5 has been heavily implicated as integral to the development of neurodegenerative disease. Conclusions: APP is fundamental to lung endothelial barrier resilience by promoting cell proliferation, stability, and regulating the production of CCL5.

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