Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors of the immunoglobin superfamily prominently expressed by lung epithelium. Previous experiments demonstrated that over-expression of RAGE by murine alveolar epithelium throughout embryonic development causes neonatal lethality coincident with significant lung hypoplasia. In the current study, we evaluated the expression of NKX2.1 (also referred to as TTF-1), a homeodomain-containing transcription factor critical for branching morphogenesis, in mice that differentially expressed RAGE. We also contextualized NKX2.1 expression with the abundance of FoxA2, a winged double helix DNA binding protein that influences respiratory epithelial cell differentiation and surfactant protein expression. Conditional RAGE over-expression was induced in mouse lung throughout gestation (embryonic day E0–18.5), as well as during the critical saccular period of development (E15.5–18.5), and analyses were conducted at E18.5. Histology revealed markedly less lung parenchyma beginning in the canalicular stage of lung development and continuing throughout the saccular period. We discovered consistently decreased expression of both NKX2.1 and FoxA2 in lungs from transgenic (TG) mice compared to littermate controls. We also observed diminished surfactant protein C in TG mice, suggesting possible hindered differentiation and/or proliferation of alveolar epithelial cells under the genetic control of these two critical transcription factors. These results demonstrate that RAGE must be specifically regulated during lung formation. Perturbation of epithelial cell differentiation culminating in respiratory distress and perinatal lethality may coincide with elevated RAGE expression in the lung parenchyma.

The receptor for advanced glycation end products (RAGE) is a key contributor to the immune and inflammatory response in a myriad of diseases. RAGE is a transmembrane pattern recognition receptor with special interest in pulmonary anomalies due to its naturally abundant expression in the lungs. Our previous studies demonstrated a role for RAGE in inflammation following acute exposure to secondhand smoke (SHS). However, chronic inflammatory mechanisms associated with RAGE have yet to be fully elucidated. In this study, we address the impact of long-term SHS exposure on RAGE signaling. RAGE knockout (RKO) and wild type (WT) mice were exposed to SHS five times weekly via a nose-only delivery system (Scireq Scientific, Montreal, Canada) for six months. SHS animals were compared to mice exposed to room air only. Immunoblot and colorimetric high throughput FACE assays (Active Motif) were used to assess phospho-AKT and NF-κB, respectively. A mouse cytokine antibody array (Abcam) was used to screen secreted cytokines in bronchoalveolar lavage fluid (BALF). Phospho-AKT was decreased and NF-κB was elevated in both groups of SHS exposed mice, with RKO+SHS mice demonstrating tempered outcomes for both intermediates compared to WT+SHS exposed mice. BALF contained increased levels of pro-inflammatory cytokines including IFNγ, IL-13, MIP-1γ and Eotaxin1 in exposed groups and diminished secretion was observed in exposed RKO mice. These results validate a role for RAGE in the mediation of chronic pulmonary inflammatory responses and suggest AKT signaling as a viable pathway of RAGE dependent inflammatory responses. Additional characterization of RAGE-mediated pulmonary responses to prolonged exposure will provide valuable insight into cellular mechanisms of lung diseases such as chronic obstructive pulmonary disease. This work was supported by funding from the National Institutes of Health (NIH 1R15-HL152257, PRR and JAA). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exposure to cigarette smoke is known to induce disease during pregnancy. Recent evidence showed that exposure to secondhand smoke (SHS) negatively impacts fetal and placental weights leading to the development of intrauterine growth restriction (IUGR) in mice. Hypertension and proteinuria are two important hallmarks of obstetric pathology leading to the development of preeclampsia (PE). In the present study, we wanted to determine the effects SHS exposure at two different important gestational points during mouse pregnancy. C57/Bl6 mice were exposed to SHS via a nose-only delivery system (Scireq) for 4 days (from 14.5 gestational day (dGA) to 17.5 dGA) or for 6 days (from 12.5 dGA to 17.5 dGA). At the time of necropsy (18.5 dGA) placental and fetal weights were recorded. Maternal blood pressure was determined with a tail occlusion cuff (Kent Scientific) and dip stick test for proteinuria was obtained. Treatment with SHS showed: 1) a significant decrease in placental weight (p<0.0001) and fetal weight (p<0.0002) following 4 days of exposure, 2) higher systolic (p<0.02) and diastolic (p<0.02) blood pressure following 6 days of exposure, and 3) increased proteinuria after 6 days of exposure. We conclude that detrimental effects of SHS coincides with the length of maternal exposure. We confirmed that 4 days of exposure resulted in metrics common to IUGR while 6 days of exposure more closely resembled PE pathology. These results could be beneficial in understanding the long-term effects of SHS exposure and the development of placental diseases. NIH 1R15HD108743 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors of the immunoglobulin superfamily predominantly expressed by lung epithelium. Previous experiments showed that RAGE upregulation throughout pregnancy caused significant fetal lung hypoplasia via elevated apoptosis and misregulation of the transcription factors thyroid transcription factor 1 (TTF1) and forkhead box protein A2 (Fox-A2). Our current research focused on characterizing the contributors to these abnormalities. We confirmed poor development of murine lungs through a time course of RAGE upregulation from conception to sacrifice on embryonic day (E)15.5, E16.5, E17.5, E18.5, and specific upregulation from E15.5 to E18.5 only. H&E staining confirmed significant simplification. We hypothesized that this malformation was in part due to aberrant levels of inflammation and apoptosis. Blotting revealed significantly increased levels of phosphorylated AKT and ERK1/2 in RAGE TG pups which cause NF-κB mediated transcription of inflammatory genes. Due to potential roles for these signaling intermediates, we sought to assess RAGE-mediated impact on mitochondrial efficiency. Oxidative phosphorylation was assessed in lungs from RAGE TG pups and controls by measuring oxygen consumption per unit mass when lungs were exposed to the substrates malate, glutamate, ADP, succinate, and FCCP which activate sequential components of the electron transport chain. We discovered significantly increased levels of oxidative phosphorylation in transgenic pups compared to controls. Levels of ATP and reactive oxidative species (ROS) in relation to altered oxidative phosphorylation and inflammation provided additional mechanistic clarity. These results support the importance of proper RAGE regulation in the development of lung tissue and elucidate mechanisms possibly causing such damage. This work was supported by funding from the National Institutes of Health (NIH 1R15-HL152257, PRR and JAA). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Preeclampsia (PE) is an obstetric complication associated with significant health implications for the fetus and mother. Studies have shown a correlation between lung disease development and PE. Gas6 protein is expressed in the lung and placenta, and binds to the AXL Tyrosine kinase receptor. Recently, our laboratory utilized Gas6 to induce preeclamptic-like conditions in rats. Our objective was to determine the role of Gas6/AXL signaling in the maternal lung during PE development. Briefly, pregnant rats were divided into control, Gas6, or Gas6 + R428 (an AXL inhibitor). Immunofluorescence was performed to determine AXL expression. Bronchoalveolar lavage fluid (BALF) was procured for the assessment of inflammatory cell secretion. Western blot was performed to detect signaling molecules and ELISA determined inflammatory cytokines. We observed increased proteinuria and increased blood pressure in Gas6-treated animals. AXL was increased in the lungs of the treated animals and BALF fluid revealed elevated total protein abundance in Gas6 animals. Extracellular-signal regulated kinase (ERK) and protein kinase B (AKT) signaling in the lung appeared to be mediated by Gas6 as well as the secretion of inflammatory cytokines. We conclude that Gas6 signaling is capable of inducing PE and that this is associated with increased lung inflammation.