Cigarette smoke exposure is associated with an increased risk of cardiovascular complications. The role of advanced glycation end products (AGEs) is already well established in numerous comorbidities, including cardiomyopathy. Given the role of AGEs and their receptor, RAGE, in activating inflammatory pathways, we sought to determine whether ceramides could be a mediator of RAGE-induced altered heart mitochondrial function. Using an in vitro model, we treated H9C2 cardiomyocytes with the AGE carboxy-methyllysine before mitochondrial respiration assessment. We discovered that mitochondrial respiration was significantly impaired in AGE-treated cells, but not when cotreated with myriocin, an inhibitor of de novo ceramide biosynthesis. Moreover, we exposed wild-type and RAGE knockout mice to secondhand cigarette smoke and found reduced mitochondrial respiration in the left ventricular myocardium from wild-type mice, but RAGE knockout mice were protected from this effect. Finally, conditional overexpression of RAGE in the lungs of transgenic mice elicited a robust increase in left ventricular ceramides in the absence of smoke exposure. Taken together, these findings suggest a RAGE-ceramide axis as an important contributor to AGE-mediated disrupted cardiomyocyte mitochondrial function.
Background Acquired lymphangioma circumscriptum of the vulva is rare and can occur subsequent to malignancies of the anogenital and pelvic region. We sought to investigate the clinicopathologic characteristics of malignancy‐associated acquired vulvar lymphangioma circumscriptum (AVLC). Methods We identified all cases of AVLC within our institution with history of prior malignancy between 2005 and 2021. A similar search was performed in the PubMed database to identify published cases to date. The clinical and histopathologic information was recorded. Results A total of 71 cases were identified. The most common preceding malignancy was cervical carcinoma (71.8%, 51/71). Radiation therapy was given to 91.4% (64/70) of the patients and lymph node dissection was made on 70.2% (40/57). Median interval between the diagnosis of malignancy and the AVLC was 10 years (range 0‐32 years). AVLC frequently presented as vesicular (31.6%, 18/57) or verrucous (28.1%, 16/57) lesions clinically. Common treatments for AVLC included excision (53.1%, 26/49) and laser therapy (16.3%, 8/49), with an overall recurrence rate of 42.9% (24/56) at a median follow‐up interval of 1.8 years (range 0.04‐32.3 years). Conclusion AVLC is a rare, late complication of anogenital and pelvic malignancies causing debilitating physical symptoms and psychological stress. Further studies are warranted to determine the most effective treatment modalities to mitigate recurrence.
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.
BackgroundReceptors for advanced glycation end-products (RAGE) are immunoglobulin-like pattern recognition receptors abundantly localized to lung epithelium. Our research demonstrated that primary tobacco smoke exposure increases RAGE expression and that RAGE partly mediates pro-inflammatory signaling during exposure. However, the degree to which RAGE influences developing lungs when gestating mice are exposed to secondhand smoke (SHS) has not been determined to date.MethodsTimed pregnant RAGE null and wild type control mice were exposed to 4 consecutive days of SHS from embryonic day (E) 14.5 through E18.5 using a state of the art nose-only smoke exposure system (Scireq, Montreal, Canada). RAGE expression was assessed using immunofluorescence, immunoblotting, and quantitative RT-PCR. TUNEL immunostaining and blotting for caspase-3 were performed to evaluate effects on cell turnover. Matrix abnormalities were discerned by quantifying collagen IV and MMP-9, a matrix metalloprotease capable of degrading basement membranes. Lastly, TNF-α and IL-1β levels were assessed in order to determine inflammatory status in the developing lung.ResultsPulmonary RAGE expression was elevated in both dams exposed to SHS and in fetuses gestating within mothers exposed to SHS. Fetal weight, a measure of organismal health, was decreased in SHS-exposed pups, but unchanged in SHS-exposed RAGE null mice. TUNEL assessments suggested a shift toward pulmonary cell apoptosis and matrix in SHS-exposed pups was diminished as revealed by decreased collagen IV and increased MMP-9 expression. Furthermore, SHS-exposed RAGE null mice expressed less TNF-α and IL-1β when compared to SHS-exposed controls.ConclusionsRAGE augmentation in developing pups exposed to maternal SHS weakens matrix deposition and influences lung inflammation.
Claudin-6 (Cldn6) is a tetraspanin transmembrane protein that contributes to tight junctional complexes and has been implicated in the maintenance of lung epithelial barriers. In the present study, we tested the hypothesis that genetic up-regulation of Cldn-6 influences inflammation in mice exposed to short-term environmental diesel particulate matter (DPM). Mice were subjected to ten exposures of nebulized DPM (PM2.5) over a period of 20 days via a nose-only inhalation system (Scireq, Montreal, Canada). Using real-time RT-PCR, we discovered that the Cldn6 gene was up-regulated in control mice exposed to DPM and in lung-specific transgenic mice that up-regulate Cldn-6 (Cldn-6 TG). Interestingly, DPM did not further enhance Cldn-6 expression in Cldn-6 TG mice. DPM caused increased cell diapedesis into bronchoalveolar lavage fluid (BALF) from control mice; however, Cldn-6 TG mice had less total cells and PMNs in BALF following DPM exposure. Because Cldn-6 TG mice had diminished cell diapedesis, other inflammatory intermediates were screened to characterize the impact of increased Cldn-6 on inflammatory signaling. Cytokines that mediate inflammatory responses including TNF-α and IL-1β were differentially regulated in Cldn6 TG mice and controls following DPM exposure. These results demonstrate that epithelial barriers organized by Cldn-6 mediate, at least in part, diesel-induced inflammation. Further work may show that Cldn-6 is a key target in understanding pulmonary epithelial gateways exacerbated by environmental pollution.
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.
Claudins are tight junctional proteins that are implicated in cell polarity and in establishing and maintaining epithelial barrier function. Our published research has also revealed that claudin misregulation adversely impacts cell differentiation and proliferation. Impairment of such critical functions related to various claudins has been linked to anomalous barriers in abnormal lung development and diseases such as acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and cancer. The current research evaluated expression of claudins 1‐8 in order to elucidate expression patterns within the developing murine lung. Claudins 1‐8 were selected based on their phylogenetic similarity and published reports of potentially diverse pulmonary expression patterns. Wild type mice from embryonic days 12.5, 14.5, 16.5, 18.5 and post‐natal day 2 were sacrificed and assessed to demonstrate cell‐specific expression via immunohistochemical analysis. Protein and RNA expression was quantitatively confirmed using immunoblotting and qPCR techniques. The results suggest increasing and decreasing patterns of expression that included redundancies and temporal transition between family members. Furthermore, cell‐specific expression of individual claudins implicated a subset as orchestrators of proximal vs. distal lung barrier establishment. These data support the need for further studies using claudin‐specific transgenic mice that knock‐in/out specific claudins so that precise functions in the normal and diseased lung can be determined. This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).
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