768.10 - Amyloid-Beta Precursor Protein: Essential to Lung Capillary Barrier Defense During Acute Infection
Sunday, April 3, 2022
9:15 AM – 9:30 AM
Room: 203 B - Pennsylvania Convention Center
Introduction: Mortality in critically ill patients has decreased due to improvements in hemodynamic support, mechanical ventilation strategies and bundled care. However, patients surviving to discharge have staggering rates of mortality, ~50%, during the next year of life. Furthermore, survivors have significant morbidity due to end-organ dysfunction. An NHBLI report suggests two erroneous paradigms that have limited our understanding of the role of pneumonia on health: that pneumonia is a 1) lt;emgt;localizedlt;/emgt; and 2) lt;emgt;acutelt;/emgt; disease. In fact, pneumonia causes end-organ injury by poorly understood mechanisms and the clinical manifestations persist long after resolution of the primary infection. Biological changes occurring during pneumonia that induce long-term morbidity are poorly understood. In summary, we propose to discuss exciting new mechanisms and hypotheses elucidating mechanisms of long-term morbidity and end-organ dysfunction in survivors of pneumonia that suffer from chronic critical illness.
Sarah Voth (Edward Via College of Osteopathic Medicine - Louisiana), Chung-Sik Choi (University of South Alabama College of Medicine), K. Morrow (Edward Via College of Osteopathic Medicine - Louisiana), Meredith Gwin (University of South Alabama College of Medicine), Jennifer Knighten (University of South Alabama College of Medicine), C. Francis (University of South Alabama College of Medicine), Mike Lin (University of South Alabama College of Medicine), Samir Gautam (Yale University School of Medicine), Dara Frank (Medical College of Wisconsin), Jonathan Audia (University of South Alabama College of Medicine), Troy Stevens (University of South Alabama College of Medicine), Lokesh Sharma (Yale University School of Medicine), Charles Cruz (Yale University School of Medicine)
Presenting Author Edward Via College of Osteopathic Medicine - Louisiana
Rationale: Pneumonia is a leading cause of acute respiratory distress syndrome (ARDS) and sepsis. Despite resolution of active infection, pro-inflammatory mediators and cytotoxins elicited during the acute phase promote secondary tissue injury, impede recovery, and contribute to poor patient prognoses. Lung infection with Pseudomonas aeruginosa, Klebsiella pneumoniae, and influenza elicit pathogenic amyloids that propagate injury and likely contribute to this inflammatory milieu. However, clinical trials targeting amyloid-beta (Aβ) have consistently failed. Lung endothelial-derived amyloids have been implicated as innate antimicrobial effectors yet the contribution of Aβ to host defense of the lung capillary barrier remains poorly resolved. We hypothesized that lung endothelial Aβ contributes to 1) the attenuation of active infection, and 2) endothelial barrier resilience to infection.
Methods: To test our hypotheses, CRISPR/Cas9 was targeted to Aβ precursor protein (APP) in rat pulmonary microvascular endothelial cells (PMVECs), and the deletion was confirmed via PCR, sequencing, and immunoblotting. The contribution of APP expression to cell proliferation, self-replication, wound closure, angiogenesis, and barrier integrity (transwell permeability, bacterial dissemination, ECIS) both at baseline and during virulent infection was assessed in both WT and APP-/- PMVECs. Amyloid content was monitored with immunoblotting and Thioflavin T staining. Antimicrobicity was measured with kinetic bactericidal assays, agglutination, plating, and SYTO 9/propidium iodide.
Results: APP-/- cells were ~49% less effective in reducing the bacterial load of an active infection as compared to APP expressing PMVECs. Immunodepletion of Aβ from WT endothelial cell supernatant reduced bactericidal efficacy in a comparable manner. APP-/- cells also exhibited impaired proliferation (WT: 0.59 days, APP-/- 0.91 days), a ~2.2-fold reduction in replication capacity, poor wound closure and barrier formation, and little resilience to infection with loss of barrier integrity in roughly half the time of infected WT PMVECs. Moreover, conditioning of naïve cells with supernatant derived from WT but not APP-/- PMVECs significantly increased proliferation and angiogenic capacity.
Conclusions: Lung endothelial APP is an important contributor to host defense. Both APP and its Aβ variant(s) appear to constrain infective bacterial burden whereas APP products appear to function as mitogens/morphogens within the microvascular milieu, likely contributing to tissue repair and recovery post-insult.
