Experimental congenital diaphragmatic hernia features an alteration of DNA sensing targets cGAS and STING.

BACKGROUND: The pathogenesis of congenital diaphragmatic hernia (CDH) depends on multiple factors. Activation of the DNA-sensing cyclic-GMP-AMP-synthase (cGAS) and Stimulator-of-Interferon-Genes (STING) pathway by double-stranded DNA (dsDNA) links environmental stimuli and inflammation. We hypothesized that nitrofen exposure alters cGAS and STING in human bronchial epithelial cells and fetal rat lungs. METHODS: We used the Quant-IT™-PicoGreen™ assay to assess dsDNA concentration in BEAS-2B cells after 24 h of nitrofen-exposure and performed immunofluorescence of cGAS/STING. We used nitrofen to induce CDH and harvested control and CDH lungs at embryonic day E15, E18 and E21 for cGAS/STING immunofluorescence, RT-qPCR and RNA-Scope™ in-situ-hybridization (E18, E21). RESULTS: We found a higher concentration of dsDNA following nitrofen treatment. Nitrofen-exposure to BEAS-2B cells increased cGAS and STING protein abundance. cGAS abundance was higher in nitrofen lungs at E15, E18 and E21. RNA-Scope in-situ-hybridization showed higher cGAS and STING expression in E18 and E21 lungs. RT-qPCR revealed higher mRNA expression levels of STING in E21 nitrofen-induced lungs. CONCLUSION: Our data suggest that nitrofen-exposure increases dsDNA content which leads to stimulation of the cGAS/STING pathway in human BEAS-2B cells and the nitrofen rat model of CDH. Consequently, DNA sensing and the cGAS-STING-pathway potentially contribute to abnormal lung development in CDH. IMPACT STATEMENT: We found an alteration of DNA sensing targets cGAS and STING in human BEAS-2B cells and experimental congenital diaphragmatic hernia with higher protein abundance and mRNA expression in cells and lung sections of nitrofen-treated rat pups. This is the first study to investigate DNA sensing, a potential link between environmental stimuli and inflammation, in experimental CDH. Our study extends the knowledge on the pathogenesis of experimental CDH.

The RNA-binding protein quaking is upregulated in nitrofen-induced congenital diaphragmatic hernia lungs at the end of gestation.

BACKGROUND: The RNA-binding protein Quaking (QKI) increases during epithelial-to-mesenchymal transition and its expression is controlled by microRNA-200 family members. Here, we aimed to describe the expression of QKI in the developing lungs of control and nitrofen-induced congenital diaphragmatic hernia lungs (CDH). METHODS: To investigate the expression of QKI, we dissected lungs from control and nitrofen-induced CDH rats on embryonic day 15, 18, 21 (E15, E18, E21). We performed immunofluorescence (IF) and quantitative reverse transcription PCR (RT-qPCR) for QKI expression. Additionally, we assessed Interleukin-6 (IL-6) abundance using IF. RESULTS: On E21, IF showed that the abundance of all three QKI isoforms and IL-6 protein was higher in CDH lungs compared to control lungs (QKI5: p = 0.023, QKI6: p = 0.006, QKI7: p = 0.014, IL-6: p = 0.045, respectively). Furthermore, RT-qPCR data showed increased expression of QKI5, QKI6, and QKI7 mRNA in E21 nitrofen lungs by 1.63 fold (p = 0.001), 1.63 fold (p = 0.010), and 1.48 fold (p = 0.018), respectively. CONCLUSIONS: Our data show an increase in the abundance and expression of QKI at the end of gestation in nitrofen-induced CDH lungs. Therefore, a disruption in the regulation of QKI during the late stage of pregnancy could be associated with the pathogenesis of abnormal lung development in CDH.

Dysregulation of CITED2 in abnormal lung development in the nitrofen rat model.

PURPOSE: CITED2 both modulates lung, heart and diaphragm development. The role of CITED2 in the pathogenesis of congenital diaphragmatic hernia (CDH) is unknown. We aimed to study CITED2 during abnormal lung development in the nitrofen model. METHODS: Timed-pregnant rats were given nitrofen on embryonic day (E) 9 to induce CDH. Fetal lungs were harvested on E15, 18 and 21. We performed RT-qPCR, RNAscope™ in situ hybridization and immunofluorescence staining for CITED2. RESULTS: We observed no difference in RT-qPCR (control: 1.09 ± 0.22 and nitrofen: 0.95 ± 0.18, p = 0.64) and in situ hybridization (1.03 ± 0.03; 1.04 ± 0.03, p = 0.97) for CITED2 expression in E15 nitrofen and control pups. At E18, CITED2 expression was reduced in in situ hybridization of nitrofen lungs (1.47 ± 0.05; 1.14 ± 0.07, p = 0.0006), but not altered in RT-qPCR (1.04 ± 0.16; 0.81 ± 0.13, p = 0.33). In E21 nitrofen lungs, CITED2 RNA expression was increased in RT-qPCR (1.04 ± 0.11; 1.52 ± 0.17, p = 0.03) and in situ hybridization (1.08 ± 0.07, 1.29 ± 0.04, p = 0.02). CITED2 protein abundance was higher in immunofluorescence staining of E21 nitrofen lungs (2.96 × 109 ± 0.13 × 109; 4.82 × 109 ± 0.25 × 109, p < 0.0001). CONCLUSION: Our data suggest that dysregulation of CITED2 contributes to abnormal lung development of CDH, as demonstrated by the distinct spatial-temporal distribution in nitrofen-induced lungs.

MicroRNA-200b deficiency is not sufficient to increase susceptibility to allergen-induced airway inflammation and dysfunction in mice.

MicroRNA-200b (miR-200b) has emerged as a therapeutic option for reducing inflammation and airway dysfunction in asthma. miR-200b belongs to a family of miRNAs that regulate epithelial-to-mesenchymal (EMT) transition and IL-33 abundance. In asthma, miR-200b abundance is reduced in the airways and is correlated with disease severity. In addition, prophylactic treatment with a miR-200b mimetic reduces airway inflammation and airway dysfunction in a mouse model. However, it is unclear whether miR-200b deficiency is sufficient to drive airway dysfunction and airway inflammation in asthma. Here, we show that male and female mice deficient in miR-200b do not display heightened airway inflammation or alterations in lung function that are characteristic of asthma. Following sensitization with house dust mite (HDM), female miR-200b knockout (KO) mice have elevated total lung resistance and male miR-200b KO have increased airway resistance. However, neither male nor female miR-200b mice display any changes in methacholine sensitivity or responsiveness and do not have enhanced HDM-induced airway inflammation. Collectively, these findings suggest that loss of miR-200b does not drive airway inflammation and airway dysfunction in mice. Thus, although treatment with exogenous miR-200b may ameliorate inflammation in asthma, deficiency of miR-200b is not likely driving pathobiology in asthma.NEW & NOTEWORTHY MicroRNA-200b regulates the abundance of key asthma-related genes. However, loss of miR-200b does not potentiate allergic asthma in a mouse model, suggesting that miR-200b deficiency may not be sufficient to drive of asthma pathogenesis.

Proteomic Profiling of Hypoplastic Lungs Suggests an Underlying Inflammatory Response in the Pathogenesis of Abnormal Lung Development in Congenital Diaphragmatic Hernia.

The pathogenesis of lung hypoplasia in congenital diaphragmatic hernia (CDH), a common birth defect, is poorly understood. The diaphragmatic defect can be repaired surgically, but the abnormal lung development contributes to a high mortality in these patients. To understand the underlying pathobiology, we compared the proteomic profiles of fetal rat lungs at the alveolar stage (E21) that were either exposed to nitrofen in utero (CDH lungs, n=5) or exposed to vehicle only (non-CDH control lungs, n=5). Pathway analysis of proteomic datasets showed significant enrichment in inflammatory response proteins associated with cytokine signaling and Epstein Barr Virus in nitrofen CDH lungs. Among the 218 significantly altered proteins between CDH and non-CDH control lungs were Tenascin C, CREBBP, LYN, and STAT3. We showed that Tenascin C was decreased around the distal airway branches in nitrofen rat lungs and human CDH lungs, obtained from stillborn fetuses that did not receive pre- or postnatal treatment. In contrast, STAT3 was significantly increased in the airway epithelium of nitrofen lungs at E21. STAT3 inhibition after direct nitrofen exposure to fetal rat lung explants (E14.5) partially rescued the hypoplastic lung phenotype ex vivo by increasing peripheral lung budding. Moreover, we demonstrated that several STAT3-associated cytokines (IL-15, IL-9, andIL-2) are increased in fetal tracheal aspirates of CDH survivors compared with nonsurvivors after fetoscopic endoluminal tracheal occlusion. With our unbiased proteomics approach, we showed for the first time that downstream inflammatory processes are likely involved in the pathogenesis of abnormal lung development in CDH.

Yes-associated protein is dysregulated during nitrofen-induced hypoplastic lung development due to congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a birth defect associated with abnormal lung development. Yes-associated protein (YAP) is a core kinase of the Hippo pathway, which controls organ size during development. The absence of YAP protein during lung development results in hypoplastic lungs comparable to the lung phenotype in CDH (Mahoney, Dev Cell 30(2):137-150, 2014). We aimed to describe the expression of YAP during normal and nitrofen-induced abnormal lung development. METHODS: Intra-gastric administration of dams with 100 mg of nitrofen was used to induce CDH and abnormal lung development in the embryos. Immunofluorescence was performed to visualize the localization of YAP and p-YAP during lung development (E15, E18, E21). Western Blotting was used to determine the abundance of YAP and p-YAP in E21 control and nitrofen-induced hypoplastic CDH lungs. RESULTS: Immunofluorescence demonstrated cytoplasmic localization of YAP protein in airway epithelial and mesenchymal cells of nitrofen-induced hypoplastic lungs compared to nuclear localization in control lungs. Western Blotting showed a decrease (p = 0.0188) in abundance of YAP (active form) and increase in p-YAP (inactive form) in hypoplastic lungs compared to control lungs. CONCLUSION: Our results demonstrate that YAP protein is mostly phosphorylated, inactive, and expressed in the cytoplasm at the later stages of nitrofen-induced hypoplastic lung development indicating that the alteration in regulation of YAP can be associated with the pathogenesis of abnormal lung development in experimental CDH.

Tenascin C is dysregulated in hypoplastic lungs of miR-200b-/- mice.

PURPOSE: We previously demonstrated that absence of miR-200b results in abnormal lung development in congenital diaphragmatic hernia due to imbalance between epithelial and mesenchymal cells. Tenascin C is a highly conserved extracellular matrix protein involved in epithelial to mesenchymal transition, tissue regeneration and lung development. Considering the involvement of Tenascin C and miR-200b and their potential interaction, we aimed to study Tenascin C during lung development in the absence of miR-200b. METHODS: We collected lungs of miR-200b-/- mice (male, 8 weeks). We performed Western blot (WB) analysis (N = 6) and immunofluorescence (N = 5) for Tenascin C and alpha smooth muscle actin and RT-qPCR for Tenascin C gene expression (N = 4). RESULTS: Using WB analysis, we observed a decreased total protein abundance of Tenascin C in miR-200b-/- lungs (miR-200b+/+: 3.8 × 107 ± 1 × 107; miR-200b-/-: 1.9 × 107 ± 5 × 106; p = 0.002). Immunofluorescence confirmed decreased total Tenascin C in miR-200b-/- lungs. Tenascin C was significantly decreased in the mesenchyme but relatively increased in the airways of mutant lungs. Total lung RNA expression of Tenascin C was higher in miR-200b-/- lungs. CONCLUSION: We report dysregulation of Tenascin C in lungs of miR-200b-/- mice. This suggests that absence of miR-200b results in abnormal Tenascin C abundance contributing to the lung hypoplasia observed in miR-200b-/- mice.

The effect of tracheal occlusion in congenital diaphragmatic hernia in the nitrofen rat lung explant model.

PURPOSE: Here, we establish a tracheal occlusion (TO) model with rat lung explants in nitrofen-induced pulmonary hypoplasia in the congenital diaphragmatic hernia (CDH). METHODS: We extracted lungs from rats on an embryonic day 18. We mimicked TO in the lung explants by tying the trachea. We assessed lung weight, morphometry, and abundance of Ki-67, Active caspase-3, and Prosurfactant Protein C (proSP-C) with immunofluorescence. RESULTS: Lung weight was higher in TO + than TO - on day 1. Abundance of Ki-67 was higher in TO + than TO - (0.15 vs. 0.32, p = 0.009 for day 1, 0.07 vs. 0.17, p = 0.004 for day 2, 0.07 vs. 0.12, p = 0.044 for day 3), and Active caspase-3 was higher in TO + than TO - on day 2 and day 3 (0.04 vs. 0.03 p = 0.669 for day 1, 0.03 vs. 0.13 p < 0.001 for day 2, 0.04 vs. 0.17 p = 0.008 for day3). However, proSP-C protein abundance was lower in TO + than TO - (67.9 vs. 59.1 p = 0.033 for day 1, 73.5 vs. 51.6 p = 0.038 for day 2, 83.1 vs. 56.4 p = 0.009 for day 3). CONCLUSIONS: The TO model in lung explants mimics the outcomes of current surgical models of TO and further studies can reveal the cellular and molecular effects of TO in CDH lungs.

The RNA-binding protein Quaking regulates multiciliated and basal cell abundance in the developing lung.

RNA-binding proteins (RBPs) form complexes with RNA, changing how the RNA is processed and thereby regulating gene expression. RBPs are important sources of gene regulation during organogenesis, including the development of lungs. The RBP called Quaking (QK) is critical for embryogenesis, yet it has not been studied in the developing lung. Here, we show that QK is widely expressed during rat lung development and into adulthood. The QK isoforms QK5 and QK7 colocalize to the nuclei of nearly all lung cells. QK6 is present in the nuclei and cytoplasm of mesenchymal cells and is only present in the epithelium during branching morphogenesis. QK knockdown in embryonic lung explants caused a greater number of multiciliated cells to appear in the airways, at the expense of basal cells. The mRNA of multiciliated cell genes and the abundance of FOXJ1/SOX2+ cells increased after knockdown, whereas P63/SOX2+ cells decreased. The cytokine IL-6, a known regulator of multiciliated cell differentiation, had increased mRNA levels after QK knockdown, although protein levels remained unchanged. Further studies are necessary to confirm whether QK acts as a blocker for the IL-6-induced differentiation of basal cells into multiciliated cells, and a conditional QK knockout would likely lead to additional discoveries on QK's role during lung development.

Epithelial cell-adhesion protein cadherin 26 is dysregulated in congenital diaphragmatic hernia and congenital pulmonary airway malformation.

BACKGROUND: Congenital diaphragmatic hernia (CDH) and congenital pulmonary airway malformation (CPAM) are two inborn pathologies of the lung of unknown origin. Alterations of gene expression in airway epithelial cells are involved in the pathobiology of both diseases. We previously found decreased expression of the epithelial cell adhesion protein cadherin 26 (CDH26) in hypoplastic mice lungs. Here, our objective was to describe the expression and localization of CDH26 in hypoplastic CDH lungs and hyperproliferative CPAM tissues. METHODS: After ethical approval, we used human lung tissues from CDH and CPAM cases and age-matched control samples from a previously established biobank. Furthermore, lungs from the nitrofen rat model of CDH were included in the study. We performed immunohistochemistry and western blot analysis with antibodies against CDH26 to examine protein localization and abundance. Statistical analysis was performed using Mann-Whitney U test with significance set at p < 0.05. RESULTS: We observed an overexpression of CDH26 within the epithelium of cystic CPAM lesions compared to normal airways within the same lung and compared to control lungs. Western blot demonstrated a downregulation of CDH26 in the nitrofen rat model of CDH compared to healthy controls. Immunohistochemistry could not show consistent differences between CDH and control in human and rat lungs. In the studied human lung samples, CDH26 was localized to the apical part of the airway epithelial cells. CONCLUSION: CDH26 is differentially expressed in human CPAM lung tissues and may be downregulated in nitrofen-induced hypoplastic rat lungs compared to control lungs. Disruption of CDH26 associated pathways in lung development may be involved in the pathogenesis of lung hypoplasia or cystic lung disease.

A QSAR study that compares the ability of bisdioxopiperazine analogs of the doxorubicin cardioprotective agent dexrazoxane (ICRF-187) to protect myocytes with DNA topoisomerase II inhibition.

The cardiotoxicity caused by doxorubicin and extravasation injury caused by anthracyclines is reduced by the clinically approved bisdioxopiperazine drug dexrazoxane. Dexrazoxane is a rings-closed analog of EDTA and is hydrolyzed in vivo to a form that strongly binds iron. Its protective effects were originally thought to be due to the ability of its metabolite to remove iron from the iron-doxorubicin complex, thereby preventing oxygen radical damage to cellular components. More recently it has been suggested that dexrazoxane may exert its protective effects by inhibiting topoisomerase IIß in the heart and inducing a reduction in its protein levels through induction of proteasomal degradation. The ability of dexrazoxane, other bisdioxopiperazines, and mitindomide to protect against doxorubicin-induced damage was determined in primary neonatal rat myocytes. This QSAR study showed that the protection that a series of bisdioxopiperazine analogs of dexrazoxane and the bisimide mitindomide offered against doxorubicin-induced myocyte damage was highly correlated with the ability of these compounds to catalytically inhibit the decatenation activity of topoisomerase II. The structural features of the dexrazoxane analogs that contribute to the binding and inhibition of topoisomerase II have been identified. These results suggest that the inhibition of topoisomerase II in myocytes by dexrazoxane is central to its role in its activity as an anthracycline cardioprotective agent. Additionally, sequence identity analysis of the amino acids surrounding the dexrazoxane binding site showed extremely high identity, not only between both invertebrate topoisomerase II isoforms, but also with yeast topoisomerase II as well.

First steps in the development of a liquid biopsy in situ hybridization protocol to determine circular RNA biomarkers in rat biofluids.

PURPOSE: Epigenetic factors are involved in the pathogenesis of congenital diaphragmatic hernia (CDH). Circular RNAs (circRNAs) are epigenetic regulators amenable to biomarker profiling. Here, we aimed to develop a liquid biopsy protocol to detect pathognomonic circRNA changes in biofluids. METHODS: Our protocol is adapted from the existing BaseScope™ in situ hybridization technique. Rat biofluids were fixed in a gelatin-coated 96-well plate with formalin. Probes were designed to target circRNAs with significant fold change in nitrofen-induced CDH. FastRED fluorescence was assessed using a plate reader and confirmed with confocal microscopy. We tested maternal serum and amniotic fluid samples from control and nitrofen-treated rats. RESULTS: We detected circRNAs in rat serum and amniotic fluid from control and CDH (nitrofen-treated) rats using fluorescent readout. CircRNA signal was observed in fixed biofluids as fluorescent punctate foci under confocal laser scanning microscopy. This was confirmed by comparison to BaseScope™ lung tissue sections. Signal was concentration dependent and DNase resistant. CONCLUSION: We successfully adapted BaseScope™ to detect circRNAs in rat biofluids: serum and amniotic fluid. We detected signal from probes targeted to circRNAs that are dysregulated in rat CDH. This work establishes the preliminary feasibility of circRNA detection in prenatal diagnostics.

Disulfiram is a slow-binding partial noncompetitive inhibitor of 20S proteasome activity.

The alcohol abuse drug disulfiram has also been shown to exhibit potent cell growth inhibitory and anticancer activity. While a number of cellular and animal studies have suggested that disulfiram exhibits its anticancer activity through interaction with the proteasome, direct evidence for inhibition of proteasome activity is lacking. In this study we show that disulfiram potently inhibits the chymotrypsin-like activity of purified human 20S proteasome at low micromolar pharmacological concentrations. The enzyme progress curves displayed characteristics of a slow-binding reaction, similar to that observed for the FDA-approved proteasomal-targeted anticancer drugs bortezomib and carfilzomib. The apparent second order rate constant for reaction with 20s proteasome that was derived from an analysis of the progress curves was about 250-fold smaller than for bortezomib and carfilzomib. The concentration dependence of the enzyme kinetics was consistent with partial noncompetitive inhibition, whereby the putative disulfiram-proteasome adduct retains, partial but decreased enzyme activity. Disulfiram, which is known to have a high affinity for protein thiols, likely reacted with a non-critical cysteine residue, and not at the proteasome substrate binding site.

Mechanisms of Action and Reduced Cardiotoxicity of Pixantrone; a Topoisomerase II Targeting Agent with Cellular Selectivity for the Topoisomerase IIα Isoform.

Pixantrone is a new noncardiotoxic aza-anthracenedione anticancer drug structurally related to anthracyclines and anthracenediones, such as doxorubicin and mitoxantrone. Pixantrone is approved in the European Union for the treatment of relapsed or refractory aggressive B cell non-Hodgkin lymphoma. This study was undertaken to investigate both the mechanism(s) of its anticancer activity and its relative lack of cardiotoxicity. Pixantrone targeted DNA topoisomerase IIα as evidenced by its ability to inhibit kinetoplast DNA decatenation; to produce linear double-strand DNA in a pBR322 DNA cleavage assay; to produce DNA double-strand breaks in a cellular phospho-histone γH2AX assay; to form covalent topoisomerase II-DNA complexes in a cellular immunodetection of complex of enzyme-to-DNA assay; and to display cross-resistance in etoposide-resistant K562 cells. Pixantrone produced semiquinone free radicals in an enzymatic reducing system, although not in a cellular system, most likely due to low cellular uptake. Pixantrone was 10- to 12-fold less damaging to neonatal rat myocytes than doxorubicin or mitoxantrone, as measured by lactate dehydrogenase release. Three factors potentially contribute to the reduced cardiotoxicity of pixantrone. First, its lack of binding to iron(III) makes it unable to induce iron-based oxidative stress. Second, its low cellular uptake may limit its ability to produce semiquinone free radicals and redox cycle. Finally, because the ß isoform of topoisomerase II predominates in postmitotic cardiomyocytes, and pixantrone is demonstrated in this study to be selective for topoisomerase IIα in stabilizing enzyme-DNA covalent complexes, the attenuated cardiotoxicity of this agent may also be due to its selectivity for targeting topoisomerase IIα over topoisomerase IIß.

Structure-based design, synthesis and biological testing of piperazine-linked bis-epipodophyllotoxin etoposide analogs.

Drugs that target DNA topoisomerase II, such as the epipodophyllotoxin etoposide, are a clinically important class of anticancer agents. A recently published X-ray structure of a ternary complex of etoposide, cleaved DNA and topoisomerase IIß showed that the two intercalated etoposide molecules in the complex were separated by four DNA base pairs. Thus, using a structure-based design approach, a series of bis-epipodophyllotoxin etoposide analogs with piperazine-containing linkers was designed to simultaneously bind to these two sites. It was hypothesized that two-site binding would produce a more stable cleavage complex, and a more potent anticancer drug. The most potent bis-epipodophyllotoxin, which was 10-fold more growth inhibitory toward human erythroleukemic K562 cells than etoposide, contained a linker with eight methylene groups. All of the mono- and bis-epipodophyllotoxins, in a variety of assays, showed strong evidence that they targeted topoisomerase II. COMPARE analysis of NCI 60-cell GI50 endpoint data was also consistent with these compounds targeting topoisomerase II.

Structure-based design, synthesis and biological testing of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA.

Drugs that target DNA topoisomerase II isoforms and alkylate DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Guided by molecular modeling and docking a series of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds were designed, synthesized and biologically characterized. These hybrids were designed to alkylate nucleophilic protein residues on topoisomerase II and thus produce inactive covalent adducts and to also alkylate DNA. The most potent hybrid had a mean GI(50) in the NCI-60 cell screen 17-fold lower than etoposide. Using a variety of in vitro and cell-based assays all of the hybrids tested were shown to target topoisomerase II. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles matching both etoposide and the N-mustard compounds from which they were derived. These results supported the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA.

Microinjection With Nanoparticles to Deliver Drugs in Prenatal Lung Explants - A Pilot Study for Prenatal Therapy in Congenital Diaphragmatic Hernia.

BACKGROUND: Fetoscopic endoluminal tracheal occlusion (FETO) improves the survival rate in fetuses with severe congenital diaphragmatic hernia (CDH). We hypothesize that prenatal therapies into the trachea during FETO can further improve outcomes. Here, we present an ex vivo microinjection technique with rat lung explants to study prenatal therapy with nanoparticles. METHODS: We used microsurgery to isolate lungs from rats on embryonic day 18. We injected chitosan nanoparticles loaded with fluorescein (FITC) into the trachea of the lung explants. We compared the difference in biodistribution of two types of nanoparticles, functionalized IgG-conjugated nanoparticles (IgG-nanoparticles) and bare nanoparticles after 24 h culture with immunofluorescence (IF). We used IF to mark lung epithelial cells with E-cadherin and to investigate an apoptosis (Active-caspase 3) and inflammatory marker (Interleukin, IL-6) and compared its abundance between the two experimental groups and control lung explants. RESULTS: We detected the presence of nanoparticles in the lung explants, and the relative number of nanoparticles to cells was 2.49 fold higher in IgG-nanoparticles than bare nanoparticles (p < 0.001). Active caspase-3 protein abundance was similar in the control, bare nanoparticles (1.20 fold higher), and IgG-nanoparticles (1.34 fold higher) groups (p = 0.34). Similarly, IL-6 protein abundance was not different in the control, bare nanoparticles (1.13 fold higher), and IgG-nanoparticles (1.12 fold higher) groups (p = 0.33). CONCLUSIONS: Functionalized nanoparticles had a higher presence in lung cells and this did not result in more apoptosis or inflammation. Our proof-of-principle study will guide future research with therapies to improve lung development prenatally. LEVELS OF EVIDENCE: N/A TYPE OF STUDY: Animal and laboratory study.

The maternal-fetal transfer of passive immunity as a mechanism of transplacental nanoparticle drug delivery for prenatal therapies.

Nanoparticles administered into the maternal circulation and across the placenta are a potential clinical therapy to treat congenital diseases. The mechanism by which nanoparticles can safely cross the placenta for targeted drug delivery to the fetus remains poorly understood. We demonstrate that the maternal-fetal transfer of passive immunity through the neonatal Fc Receptor (FcRn) can induce the transplacental transfer of chitosan nanoparticles modifed with IgG antibodies (414 ± 27 nm). The transfer of FITC-tagged IgG-modified chitosan nanoparticles was 2.8 times higher (p = 0.0264) compared to similarly-sized unmodified chitosan nanoparticles (375 ± 17 nm). Co-administration of free IgG competitively diminished the transplacental transfer of IgG-modified nanoparticles, yet unmodified nanoparticles remained unaffected. Colocalization of the FcRn and the IgG-modified chitosan nanoparticles were observed with confocal microscopy. Barrier function before and after nanoparticle administration remained intact as determined by TEER (75-79 Ω cm2) and immmunofluorescence of ZO-1 tight junction proteins. The results provide insight into the clinical applications of nanoparticles for prenatal therapies using the mechanism of the maternal-fetal transfer of passive immunity.

The lack of target specificity of small molecule anticancer kinase inhibitors is correlated with their ability to damage myocytes in vitro.

Many new targeted small molecule anticancer kinase inhibitors are actively being developed. However, the clinical use of some kinase inhibitors has been shown to result in cardiotoxicity. In most cases the mechanisms by which they exert their cardiotoxicity are not well understood. We have used large scale profiling data on 8 FDA-approved tyrosine kinase inhibitors and 10 other kinase inhibitors to a panel of 317 kinases in order to correlate binding constants and kinase inhibitor binding selectivity scores with kinase inhibitor-induced damage to neonatal rat cardiac myocytes. The 18 kinase inhibitors that were the subject of this study were: canertinib, dasatinib, dovitinib, erlotinib, flavopiridol, gefitinib, imatinib, lapatinib, midostaurin, motesanib, pazopanib, sorafenib, staurosporine, sunitinib, tandutinib, tozasertib, vandetanib and vatalanib. The combined tyrosine kinase and serine-threonine kinase selectivity scores were highly correlated with the myocyte-damaging effects of the kinase inhibitors. This result suggests that myocyte damage was due to a lack of target selectivity to binding of both tyrosine kinases and serine-threonine kinases, and was not due to binding to either group specifically. Finally, the strength of kinase inhibitor binding for 290 kinases was examined for correlations with myocyte damage. Kinase inhibitor binding was significantly correlated with myocyte damage for 12 kinases. Thus, myocyte damage may be multifactorial in nature with the inhibition of a number of kinases involved in producing kinase inhibitor-induced myocyte damage.