Intrauterine growth restriction (IUGR) is a disease affecting 10% of all pregnancies. IUGR is associated with maternal, fetal, or placental abnormalities. Studies investigating the effects of secondhand smoke (SHS) exposure and IUGR are limited. The receptor for advanced glycation end-products (RAGE) is a pro-inflammatory transmembrane receptor increased by SHS in the placenta. We tested the hypothesis that inhibition of RAGE during SHS exposure protects from smoke-induced IUGR. C57BL/6 mice were exposed to SHS or SHS + semi-synthetic glycosaminoglycan ethers (SAGEs) known to inhibit RAGE signaling. Trophoblast cells were treated with cigarette smoke extract (CSE) with or without SAGEs in order to address the effects of RAGE inhibition during trophoblast invasion in vitro. SHS-treated mice demonstrated a significant reduction in fetal weight (7.35-fold, P ≤ 0.0001) and placental weight (1.13-fold, P ≤ 0.0001) compared with controls. Mice co-treated with SHS and SAGEs were protected from SHS-induced fetal weights decreases. SHS treatment of C57BL/6 mice activated placental extracellular signal-regulated kinase (ERK) (3.0-fold, P ≤ 0.05), JNK (2.4-fold, P ≤ 0.05) and p38 (2.1-fold, P ≤ 0.05) and the expression of inflammatory mediators including TNF-α (1.34-fold, P ≤ 0.05) and IL-1β (1.03-fold, P ≤ 0.05). SHS-mediated activation of these molecules was reduced to basal levels when SAGE was co-administered. Invasion of trophoblast cells decreased 92% (P < 0.002) when treated with CSE and CSE-mediated invasion was completely reversed by SAGEs. We conclude that RAGE inhibition protects against fetal weight loss during SHS-induced IUGR. These studies provide insight into tobacco-mediated IUGR development and clarify avenues that may be helpful in the alleviation of placental complications.
These data reveal that tight junctional proteins such as Cldn6 are differentially regulated by tobacco-smoke exposure and that Cldns are potentially targeted when epithelial cells respond to tobacco smoke. Further research may show that Cldns expressed in tight junctions between parenchymal cells contribute to impaired structural integrity of the lung coincident with smoking.
It has long been understood that increased epithelial permeability contributes to inflammation observed in many respiratory diseases. Recently, evidence has revealed that environmental exposure to noxious material such as cigarette smoke reduces tight junction barrier integrity, thus enhancing inflammatory conditions. Claudin-6 (Cldn6) is a tetraspanin transmembrane protein found within the tight junctional complex and is implicated in maintaining lung epithelial barriers. To test the hypothesis that increased Cldn6 ameliorates inflammation at the respiratory barrier, we utilized the Tet-On inducible transgenic system to conditionally over-express Clnd6 in the distal lung. Cldn6 transgenic (TG) and control mice were continuously provided doxycycline from postnatal day (PN) 30 until euthanasia date at PN90. A subset of Cldn6 TG and control mice were also subjected to daily secondhand tobacco smoke (SHS) via a nose only inhalation system from PN30-90 and compared to room air (RA) controls. Animals were euthanized on PN90 and lungs were harvested for histological and molecular characterization. Bronchoalveolar lavage fluid (BALF) was procured for the assessment of inflammatory cells and molecules. Quantitative RT-PCR and immunoblotting revealed increased Cldn6 expression in TG vs. control animals and SHS decreased Cldn6 expression regardless of genetic up-regulation. Histological evaluations revealed no adverse pulmonary remodeling via Hematoxylin and Eosin (H&E) staining or any qualitative alterations in the abundance of type II pneumocytes or proximal non-ciliated epithelial cells via staining for cell specific propeptide of Surfactant Protein-C (proSP-C) or Club Cell Secretory Protein (CCSP), respectively. Immunoblotting and qRT-PCR confirmed the differential expression of Cldn6 and the pro-inflammatory cytokines TNF-α and IL-1β. As a general theme, inflammation induced by SHS exposure was influenced by the availability of Cldn6. These data reveal captivating information suggesting a role for Cldn6 in lungs exposed to tobacco smoke. Further research is critically necessary in order to fully explain roles for tight junctional components such as Cldn6 and other related molecules in lungs coping with exposure.
Intrauterine growth restriction (IUGR) is a disease affecting 10% of all pregnancies and is often characterized by hypoxia, asphyxia, and fetal demise. Primary and secondhand cigarette smoke (SHS) during pregnancy is known to induce IUGR. Receptors for Advanced Glycation End‐products (RAGE) are transmembrane receptors increased by SHS in the placenta and RAGE signaling is related to the induction of inflammation. We tested the hypothesis that inhibition of RAGE during SHS exposure protects from smoke‐induced decreases in fetal growth. C57/Bl6 mice (n=8) were exposed to SHS alone or SHS + semi‐synthetic glycosaminoglycan ethers (SAGEs; an inhibitor of RAGE) for 4 days from day 14 to day 17 of gestation (dGA). RAGE −/− mice were also exposed to SHS for 4 days from day 14 to day 17 dGA. At the time of necropsy (18 dGA) placental and fetal weights were recorded and tissues were immediately frozen for histological and protein analysis. To address the effects of the RAGE inhibition and cigarette smoke in trophoblast cell invasion, first trimester trophoblast cells were treated with cigarette smoke extract (CSE) alone or CSE in the presence of SAGEs for 24 hours. Mice treated with SHS alone demonstrated a 4.6‐fold reduction (p<0.001) in fetal weight and 1.4‐fold reduction in (p<0.002) in placental weight when compared with controls. When mice were co‐treated with SHS and SAGEs, a reduction of SHS‐induced fetal weights (1.7‐fold, p<0.001) and placental weights (1.3‐fold, p<0.03) were determined when compared to controls. When RAGE −/− mice were exposed to SHS, no differences in fetal weights and placental weights were determined. Invasion of trophoblast cells decreased 92% (p<0.002) when treated with CSE for 24 hours. This inhibition of invasion was completely reversed when SAGEs were added to the CSE media added to trophoblasts. We concluded that inhibition of RAGE protects against fetal and placental weight loss during SHS‐induced IUGR. This conclusion was also supported by the discovery that hindered trophoblast invasion was reversed by SAGE treatment. Our results further suggested that there is a direct correlation between RAGE activation and the development of IUGR during SHS. These studies provide insight into tobacco‐mediated IUGR progression and clarify possible avenues for alleviating placental complications during SHS exposure.
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