Kathryn M. Heyob

Deficits in lung alveolarization and function after systemic maternal inflammation and neonatal hyperoxia exposure

Systemic maternal inflammation contributes to preterm birth and is associated with development of bronchopulmonary dysplasia (BPD). Infants with BPD exhibit decreased alveolarization, diffuse interstitial fibrosis with thickened alveolar septa, and impaired pulmonary function. We tested the hypothesis that systemic prenatal LPS administration to pregnant mice followed by postnatal hyperoxia exposure is associated with prolonged alterations in pulmonary structure and function after return to room air (RA) that are more severe than hyperoxia exposure alone. Timed-pregnant C3H/HeN mice were dosed with LPS (80 microg/kg) or saline on gestation day 16. Newborn pups were exposed to RA or 85% O2 for 14 days and then to RA for an additional 14 days. Data were collected and analyzed on postnatal days 14 and 28. The combination of prenatal LPS and postnatal hyperoxia exposure generated a phenotype with more inflammation (measured as no. of macrophages per high-power field) than either insult alone at day 28. The combined exposures were associated with a diffuse fibrotic response [measured as hydroxyproline content (microg)] but did not induce a more severe developmental arrest than hyperoxia alone. Pulmonary function tests indicated that hyperoxia, independent of maternal exposure, induced compliance decreases on day 14 that did not persist after RA recovery. Either treatment alone or combined induced an increase in resistance on day 14, but the increase persisted on day 28 only in pups receiving the combined treatment. In conclusion, the combination of systemic maternal inflammation and neonatal hyperoxia induced a prolonged phenotype of arrested alveolarization, diffuse fibrosis, and impaired lung mechanics that mimics human BPD. This new model should be useful in designing studies of specific mechanisms and interventions that could ultimately be utilized to define therapies to prevent BPD in premature infants.

Prenatal inflammation exacerbates hyperoxia-induced functional and structural changes in adult mice

Maternally derived inflammatory mediators, such as IL-6 and IL-8, contribute to preterm delivery, low birth weight, and respiratory insufficiency, which are routinely treated with oxygen. Premature infants are at risk for developing adult-onset cardiac, metabolic, and pulmonary diseases. Long-term pulmonary consequences of perinatal inflammation are unclear. We tested the hypothesis that a hostile perinatal environment induces profibrotic pathways resulting in pulmonary fibrosis, including persistently altered lung structure and function. Pregnant C3H/HeN mice injected with LPS or saline on embryonic day 16. Offspring were placed in room air (RA) or 85% O(2) for 14 days and then returned to RA. Pulmonary function tests, microCTs, molecular and histological analyses were performed between embryonic day 18 and 8 wk. Alveolarization was most compromised in LPS/O(2)-exposed offspring. Collagen staining and protein levels were increased, and static compliance was decreased only in LPS/O(2)-exposed mice. Three-dimensional microCT reconstruction and quantification revealed increased tissue densities only in LPS/O(2) mice. Diffuse interstitial fibrosis was associated with decreased micro-RNA-29, increased transforming growth factor-β expression, and phosphorylation of Smad2 during embryonic or early fetal lung development. Systemic maternal LPS administration in combination with neonatal hyperoxic exposure induces activation of profibrotic pathways, impaired alveolarization, and diminished lung function that are associated with prenatal and postnatal suppression of miR-29 expression.

DHA Suppresses Primary Macrophage Inflammatory Responses via Notch 1/ Jagged 1 Signaling

Persistent macrophages were observed in the lungs of murine offspring exposed to maternal LPS and neonatal hyperoxia. Maternal docosahexaenoic acid (DHA) supplementation prevented the accumulation of macrophages and improved lung development. We hypothesized that these macrophages are responsible for pathologies observed in this model and the effects of DHA supplementation. Primary macrophages were isolated from adult mice fed standard chow, control diets, or DHA supplemented diets. Macrophages were exposed to hyperoxia (O2) for 24 h and LPS for 6 h or 24 h. Our data demonstrate significant attenuation of Notch 1 and Jagged 1 protein levels in response to DHA supplementation in vivo but similar results were not evident in macrophages isolated from mice fed standard chow and supplemented with DHA in vitro. Co-culture of activated macrophages with MLE12 epithelial cells resulted in the release of high mobility group box 1 and leukotriene B4 from the epithelial cells and this release was attenuated by DHA supplementation. Collectively, our data indicate that long term supplementation with DHA as observed in vivo, resulted in deceased Notch 1/Jagged 1 protein expression however, DHA supplementation in vitro was sufficient to suppress release LTB4 and to protect epithelial cells in co-culture.

DHA suppresses chronic apoptosis in the lung caused by perinatal inflammation

We have previously shown that an adverse perinatal environment significantly alters lung growth and development and results in persistently altered cardiopulmonary physiology in adulthood. Our model of maternal LPS treatment followed by 14 days of neonatal hyperoxia exposure causes severe pulmonary disease characterized by permanent decreases in alveolarization and diffuse interstitial fibrosis. The current investigations tested the hypothesis that dysregulation of Notch signaling pathways contributes to the permanently altered lung phenotype in our model and that the improvements we have observed previously with maternal docosahexaenoic acid (DHA) supplementation are mediated through normalization of Notch-related protein expression. Results indicated that inflammation (IL-6 levels) and oxidation (F2a-isoprostanes) persisted through 8 wk of life in mice exposed to LPS/O2 perinatally. These changes were attenuated by maternal DHA supplementation. Modest but inconsistent differences were observed in Notch-pathway proteins Jagged 1, DLL 1, PEN2, and presenilin-2. We detected substantial increases in markers of apoptosis including PARP-1, APAF-1, caspase-9, BCL2, and HMGB1, and these increases were attenuated in mice that were nursed by DHA-supplemented dams during the perinatal period. Although Notch signaling is not significantly altered at 8 wk of age in mice with perinatal exposure to LPS/O2, our findings indicate that persistent apoptosis continues to occur at 8 wk of age. We speculate that ongoing apoptosis may contribute to persistently altered lung development and may further enhance susceptibility to additional pulmonary disease. Finally, we found that maternal DHA supplementation prevented sustained inflammation, oxidation, and apoptosis in our model.

Glutathione reductase targeted to type II cells does not protect mice from hyperoxic lung injury.

Exposure of the lung epithelium to reactive oxygen species without adequate antioxidant defenses leads to airway inflammation, and may contribute to lung injury. Glutathione peroxidase catalyzes the reduction of peroxides by oxidation of glutathione (GSH) to glutathione disulfide (GSSG), which can in turn be reduced by glutathione reductase (GR). Increased levels of GSSG have been shown to correlate negatively with outcome after oxidant exposure, and increased GR activity has been protective against hyperoxia in lung epithelial cells in vitro. We tested the hypothesis that increased GR expression targeted to type II alveolar epithelial cells would improve outcome in hyperoxia-induced lung injury. Human GR with a mitochondrial targeting sequence was targeted to mouse type II cells using the SPC promoter. Two transgenic lines were identified, with Line 2 having higher lung GR activities than Line 1. Both transgenic lines had lower lung GSSG levels and higher GSH/GSSG ratios than wild-type. Six-week-old wild-type and transgenic mice were exposed to greater than 95% O2 or room air (RA) for 84 hours. After exposure, Line 2 mice had higher right lung/body weight ratios and lavage protein concentrations than wild-type mice, and both lines 1 and 2 had lower GSSG levels than wild-type mice. These findings suggest that GSSG accumulation in the lung may not play a significant role in the development of hyperoxic lung injury, or that compensatory responses to unregulated GR expression render animals more susceptible to hyperoxic lung injury.

Mir-29b Supplementation Decreases Expression of Matrix Proteins and Improves Alveolarization in Mice Exposed to Maternal Inflammation and Neonatal Hyperoxia

Even with advances in the care of preterm infants, chronic lung disease or bronchopulmonary dysplasia (BPD) continues to be a significant pulmonary complication. Among those diagnosed with BPD, a subset of infants develop severe BPD with disproportionate pulmonary morbidities. In addition to decreased alveolarization, these infants develop obstructive and/or restrictive lung function due to increases in or dysregulation of extracellular matrix proteins. Analyses of plasma obtained from preterm infants during the first week of life indicate that circulating miR-29b is suppressed in infants that subsequently develop BPD and that decreased circulating miR-29b is inversely correlated with BPD severity. Our mouse model mimics the pathophysiology observed in infants with severe BPD, and we have previously reported decreased pulmonary miR-29b expression in this model. The current studies tested the hypothesis that adeno-associated 9 (AAV9)-mediated restoration of miR-29b in the developing lung will improve lung alveolarization and minimize the deleterious changes in matrix deposition. Pregnant C3H/HeN mice received an intraperitoneal LPS injection on embryonic day 16 and newborn pups were exposed to 85% oxygen from birth to 14 days of life. On postnatal day 3, AAV9-miR-29b or AAV9-control was administered intranasally. Mouse lung tissues were then analyzed for changes in miR-29 expression, alveolarization, and matrix protein levels and localization. Although only modest improvements in alveolarization were detected in the AAV9-miR29b-treated mice at postnatal day 28, treatment completely attenuated defects in matrix protein expression and localization. Our data suggest that miR-29b restoration may be one component of a novel therapeutic strategy to treat or prevent severe BPD in prematurely born infants.

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and Is Modified by DHA Supplementation

Profilin 1, cofilin 1, and vasodialator-stimulated phosphoprotein (VASP) are actin-binding proteins (ABP) that regulate actin remodeling and facilitate cancer cell metastases. miR-17-92 is highly expressed in metastatic tumors and profilin1 and cofilin1 are predicted targets. Docosahexaenoic acid (DHA) inhibits cancer cell proliferation and adhesion. These studies tested the hypothesis that the metastatic phenotype is driven by changes in ABPs including alternative phosphorylation and/or changes in subcellular localization. In addition, we tested the efficacy of DHA supplementation to attenuate or inhibit these changes. Human lung cancer tissue sections were analyzed for F-actin content and expression and cellular localization of profilin1, cofilin1, and VASP (S157 or S239 phosphorylation). The metastatic phenotype was investigated in A549 and MLE12 cells lines using 8 Br-cAMP as a metastasis inducer and DHA as a therapeutic agent. Migration was assessed by wound assay and expression measured by Western blot and confocal analysis. miR-17-92 expression was measured by qRT-PCR. Results indicated increased expression and altered cellular distribution of profilin1/VASPpS157, but no changes in cofilin1/VASPpS239 in the human malignant tissues compared with normal tissues. In A549 and MLE12 cells, the expression patterns of profilin1/VASPpS157 or cofilin1/VASPpS239 suggested an interaction in regulation of actin dynamics. Furthermore, DHA inhibited cancer cell migration and viability, ABP expression and cellular localization, and modulated expression of miR-17-92 in A549 cells with minimal effects in MLE12 cells. Further investigations are warranted to understand ABP interactions, changes in cellular localization, regulation by miR-17-92, and DHA as a novel therapeutic. Mol Cancer Ther; 15(9); 2220–31. ©2016 AACR.

Perinatal Inflammation Induces Sex-related Differences in Cardiovascular Morbidities in Mice

Sex-related differences in cardiovascular health and disease have been identified, with males having a higher incidence of cardiovascular events but females more likely to develop arrhythmias. Adverse fetal environments are now accepted as a cause for the development of cardiovascular diseases in adulthood, but sex-related differences in response to adverse fetal environments have not been extensively explored. The combination of both in utero and postnatal exposure to inflammation is highly relevant for the infant that is born preterm or has clinical complications at birth or in early postnatal life. We have previously observed cardiac contractile deficiencies and dysregulation of Ca2+-handling proteins in our model of maternal lipopolysaccharide (LPS) and neonatal hyperoxia exposures (LPS/O2). This investigation tested the hypothesis that there are sex-related differences in the adult pathologies after exposure to perinatal inflammation. Using pressure-volume assessments, males exposed to LPS/O2 had more pronounced contractile deficiencies than similarly exposed females, but females tended to have long PR intervals. While both sexes demonstrated decreases in α-myosin heavy chain and connexin 43 after LPS/O2 exposure compared with saline/room air controls, females indicated aberrant increases in microRNA 208a, microRNA 208b, and desmin expression. Our study supports our hypothesis that early life exposure to inflammation results in sex-dependent deficits in cardiovascular function. NEW & NOTEWORTHY Sex-specific differences in cardiovascular disease are recognized, but the mechanisms and origins are not well understood. Adverse maternal environments can influence cardiac development and later cardiovascular disease. This study identifies sex-dependent differences in cardiac disease associated with perinatal inflammation.

DHA-mediated regulation of lung cancer cell migration is not directly associated with Gelsolin or Vimentin expression

AIMS Deaths associated with cancer metastasis have steadily increased making the need for newer, anti-metastatic therapeutics imparative. Gelsolin and vimentin, actin binding proteins expressed in metastatic tumors, participate in actin remodelling and regulate cell migration. Docosahexaenoic acid (DHA) limits cancer cell proliferation and adhesion but the mechanisms involved in reducing metastatic phenotypes are unknown. We aimed to investigate the effects of DHA on gelsolin and vimentin expression, and ultimately cell migration and proliferation, in this context. MAIN METHODS Non-invasive lung epithelial cells (MLE12) and invasive lung cancer cells (A549) were treated with DHA (30μmol/ml) or/and 8 bromo-cyclic adenosine monophosphate (8 Br-cAMP) (300μmol/ml) for 6 or 24h either before (pre-treatment) or after (post-treatment) plating in transwells. Migration was assessed by the number of cells that progressed through the transwell. Gelsolin and vimentin expression were measured by Western blot and confocal microscopy in cells, and by immunohistochemistry in human lung cancer biopsy samples. KEY FINDINGS A significant decrease in cell migration was detected for A549 cells treated with DHA verses control but this same decrease was not seen in MLE12 cells. DHA and 8 Br-cAMP altered gelsolin and vimentin expression but no clear pattern of change was observed. Immunofluorescence staining indicated slightly higher vimentin expression in human lung tissue that was malignant compared to control. SIGNIFICANCE Collectively, our data indicate that DHA inhibits cancer cell migration and further suggests that vimentin and gelsolin may play secondary roles in cancer cell migration and proliferation, but are not the primary regulators.

Changes in Vasodilator-Stimulated Phosphoprotein Phosphorylation, Profilin-1, and Cofilin-1 in Accreta and Protection by DHA

Accreta and gestational trophoblastic disease (ie, choriocarcinoma) are placental pathologies characterized by hyperproliferative and invasive trophoblasts. Cellular proliferation, migration, and invasion are heavily controlled by actin-binding protein (ABP)-mediated actin dynamics. The ABP vasodilator-stimulated phosphoprotein (VASP) carries key regulatory role. Profilin-1, cofilin-1, and VASP phosphorylated at Ser157 (pVASP-S157) and Ser239 (pVASP-S239) are ABPs that regulate actin polymerization and stabilization and facilitate cell metastases. Docosahexaenoic acid (DHA) inhibits cancer cell migration and proliferation. We hypothesized that analogous to malignant cells, ABPs regulate these processes in extravillous trophoblasts (EVTs), which exhibit aberrant expression in placenta accreta. Placental–myometrial junction biopsies of histologically confirmed placenta accreta had significantly increased immunostaining levels of cofilin-1, VASP, pVASP-S239, and F-actin. Treatment of choriocarcinoma-derived trophoblast (BeWo) cells with DHA (30 µM) for 24 hours significantly suppressed proliferation, migration, and pVASP-S239 levels and altered protein profiles consistent with increased apoptosis. We concluded that in accreta changes in the ABP expression profile were a response to restore homeostasis by counteracting the hyperproliferative and invasive phenotype of the EVT. The observed association between VASP phosphorylation, apoptosis, and trophoblast proliferation and migration suggest that DHA may offer a therapeutic solution for conditions where EVT is hyperinvasive.