Siow Teng Chan

Human amnion epithelial cells mediate lung repair by directly modulating macrophage recruitment and polarization

Human amnion epithelial cells (hAECs) have been shown to modulate inflammation and restore normal lung structure and respiratory function following bleomycin challenge in immune-competent mice. These effects are exerted despite a lack of significant engraftment of hAECs, suggesting that immunomodulatory effect mechanisms are at play. In this study, using the bleomycin model of injury, we explored the interactions between hAECs and macrophages. We administered 4 million hAECs intraperitoneally to C57Bl6 mice 24 h following a bleomycin challenge. Using FACS analysis and qPCR, we showed that hAEC administration significantly reduced macrophage infiltration into the lungs and that the majority of the pulmonary macrophages were of the M2 phenotype. Using bone marrow-derived macrophages, we then showed that hAEC-conditioned media could alter macrophage polarization, migration, and phagocytosis, without affecting macrophage survival or proliferation in vitro. This study provides the first evidence that hAECs directly influence macrophage behavior in a proreparative manner and suggests that hAECs are able to mediate these effects independently of other immune cell types.

Human amnion epithelial cells do not abrogate pulmonary fibrosis in mice with impaired macrophage function.

Since current treatments for both acute and chronic lung diseases are less than ideal, there has been recent interest in the use of cell-based therapies for inflammatory lung disease. Specifically, human amnion epithelial cells (hAECs) have been shown to reduce bleomycin-induced lung injury and prevent subsequent loss of respiratory function, primarily through modulation of the host immune response. The precise mechanisms of this effect remain unclear. We aimed to investigate the potential of hAECs to mitigate bleomycin-induced lung injury in surfactant protein C deficient (Sftpc-/-) mice, which are highly susceptible to pulmonary injury as a result of impairment of macrophage function. Primary hAECs were administered to wild-type (Sftpc+/+) and Sftpc-/- mice 24 h after exposure to bleomycin. Compared to Sftpc+/+ mice receiving bleomycin alone, Sftpc+/+ mice administered hAECs 24 h after bleomycin exposure had decreased expression of proinflammatory genes, decreased macrophage and neutrophil infiltration, fibrosis, collagen content, and α-smooth muscle actin as well as a significant improvement in lung function. Compared to Sftpc-/- mice given bleomycin alone, Sftpc-/- mice administered hAECs 24 h after bleomycin did not have a decrease in inflammatory gene expression or a reduction in macrophage pulmonary infiltration. Subsequently, Sftpc-/- mice did not show any decrease in pulmonary fibrosis or improvement of lung function after hAEC administration. The ability of hAECs to mitigate bleomycin-induced lung injury is abolished in Sftpc-/- mice, suggesting that hAECs require normal host macrophage function to exert their reparative effects.

Human amnion epithelial cells repair established lung injury.

With a view to developing a cell therapy for chronic lung disease, human amnion epithelial cells (hAECs) have been shown to prevent acute lung injury. Whether they can repair established lung disease is unknown. We aimed to assess whether hAECs can repair existing lung damage induced in mice by bleomycin and whether the timing of cell administration influences reparative efficacy. In addition, we aimed to characterize the effect of hAECs on fibroblast proliferation and activation, investigating possible mechanisms of reparative action. hAECs were administered intraperitoneally (IP) either 7 or 14 days after bleomycin exposure. Lungs were assessed 7 days after hAEC administration. Bleomycin significantly reduced body weight and induced pulmonary inflammation and fibrosis at 14 and 21 days. Delivery of hAECs 7 days after bleomycin had no effect on lung injury, whereas delivery of hAECs 14 days after bleomycin normalized lung tissue density, collagen content, and α-SMA production, in association with a reduction in pulmonary leucocytes and lung expression of TGF-β, PDGF-α, and PDGF-β. In vitro, hAECs reduced proliferation and activation of primary mouse lung fibroblasts. Our findings suggest that the timing of hAEC administration in the course of lung disease may impact on the ability of hAECs to repair lung injury.

Preterm human amnion epithelial cells have limited reparative potential

The collection and use of stem cells from the fetal membranes as cell therapy for a variety of lung diseases, including preterm lung disease, have been previously proposed. To date, only cells from term amnion have been assessed. In the setting of a future therapy for the preterm neonate, it would be ideal if autologous cells could be given. However, the reparative and anti-inflammatory actions of stem cells isolated from preterm amnions have not been evaluated. In this study, with a view to developing an autologous cell therapy for preterm lung injury, we compared the differentiation potential and efficacy of term versus preterm human amnion epithelial cells (hAECs) to protect against inflammation and fibrosis in a bleomycin mouse model of lung injury. We found that, unlike term hAECs, preterm hAECs did not differentiate into a lung lineage following culture in small airway growth media. Preterm hAECs also exerted significantly less protective effects than term hAEC following acute lung injury. Specifically, preterm hAEC did not improve Ashcroft scoring or collagen deposition in the lung despite a reduction in activated myofibroblasts. Term hAECs expressed double the levels of HLA-G compared to preterm hAECs. These findings indicate that while hAECs can be isolated from term and preterm amnions in similar numbers, they bear distinctive characteristics, which may impact upon their clinical use.

Human Mesenchymal Stem Cells Reduce Lung Injury in Immunocompromised Mice but Not in Immunocompetent Mice

Background: The immunomodulatory and immunosuppressive capacity of human mesenchymal stem cells (hMSC) is well recognized, but efficacies of hMSC in immunocompetent and immunocompromised animals have never been directly compared. Objectives: We aimed to compare the efficacy of hMSC in preventing bleomycin-induced lung injury in immunocompromised SCID and immunocompetent C57Bl/6 mice. Methods: SCID and C57Bl/6 mice were subjected to a single bolus intranasal instillation of bleomycin to induce lung injury. One million hMSC were administered intravenously 24 h following the induction of bleomycin lung injury. Results: hMSC xenotransplantation into SCID mice resulted in transient improvements in lung weight and tidal volume and to persistent improvement in inspiratory duty cycle, inspiratory flow rate and inspiration/expiration ratio. We did not observed protective effects in C57Bl/6 mice. This correlated with histological changes, where hMSC administration reduced Ashcroft scores, collagen deposition and inflammatory influx in the lungs of SCID mice, but not in those of C57Bl/6 mice. Conclusion: The application of hMSC for the treatment of acute and chronic lung injury is significantly affected by the immune status of the recipient. Lack of hMSC-mediated repair observed in C57Bl/6 mice was likely to be due to limitations of their immune privilege and differential priming of hMSC in immunocompetent versus immunocompromised hosts.

Evaluating the Impact of Human Amnion Epithelial Cells on Angiogenesis

The effects of human amnion epithelial cells (hAECs) on angiogenesis remain controversial. It is yet unknown if the presence of inflammation and/or gestational age of hAEC donors have an impact on angiogenesis. In this study, we examined the differences between term and preterm hAECs on angiogenesis in vitro and in vivo. Conditioned media from term hAECs induced the formation of longer huVEC tubules on Matrigel. Both term and preterm hAECs expressed VEGFA, PDGFB, ANGPT1, and FOXC1, which significantly increased after TNFα and IFNγ stimulation. In the presence of TNFα and IFNγ, coculture with term hAECs reduced gene transcription of Tie-2 and Foxc1 in huVECs, while coculture with preterm hAECs increased gene transcription of PDGFRα and PDGFRβ and reduced gene transcription of FOXC1 in huVECs. In vivo assessment of angiogenesis using vWF immunostaining revealed that hAEC treatment decreased angiogenesis in a bleomycin model of lung fibrosis but increased angiogenesis in a neonatal model of hyperoxia-induced lung injury. In summary, our findings suggested that the impact of hAECs on angiogenesis may be influenced by the presence of inflammation and underlying pathology.

Activin A contributes to the development of hyperoxia-induced lung injury in neonatal mice

Background:Bronchopulmonary dysplasia (BPD) is one of the leading causes of morbidity and mortality in babies born prematurely, yet there is no curative treatment. In recent years, a number of inhibitors against TGFβ signaling have been tested for their potential to prevent neonatal injury associated with hyperoxia, which is a contributing factor of BPD. In this study, we assessed the contribution of activin A—a member of the TGFβ superfamily—to the development of hyperoxia-induced lung injury in neonatal mice.Methods:We placed newborn C57Bl6 mouse pups in continuous hyperoxia (85% O2) to mimic many aspects of BPD including alveolar simplification and pulmonary inflammation. The pups were administered activin A receptor type IIB-Fc antagonist (ActRIIB-Fc) at 5 mg/kg or follistatin at 0.1 mg/kg on postnatal days 4, 7, 10, and 13.Results:Treatment with ActRIIB-Fc and follistatin protected against hyperoxia-induced growth retardation. ActRIIB-Fc also reduced pulmonary leukocyte infiltration, normalized tissue: airspace ratio and increased septal crest density. These findings were associated with reduced phosphorylation of Smad3 and decreased matrix metalloproteinase (MMP)-9 activity.Conclusion:This study suggests that activin A signaling may contribute to the pathology of bronchopulmonary dysplasia.

Measuring respiratory function in mice using unrestrained whole-body plethysmography.

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

Role of activin A in the pathogenesis of endothelial cell dysfunction in preeclampsia

Circulating markers for endothelial activation such as endothelin-1 (ET-1), ICAM-1 and VCAM-1 are elevated in women with preeclampsia. Using human umbilical vein endothelial cells (HUVECs) as an in vitro model of the maternal vasculature, we show that activin A and preeclamptic serum upregulate ET-1, ICAM-1, and VCAM-1 in HUVECs. Further, we show that follistatin, a specific binding protein for activin, mitigates the upregulation of ET-1, ICAM-1 and VCAM-1 in HUVECs exposed to either activin A or preeclamptic serum. These data are consistent with activin A contributing to the pathophysiology of preeclampsia and suggest that therapies targeting activin signalling are worth exploring.

Exosomal miRNA in pre-eclampsia: good things come in small packages

Background: Studies have shown circulating miRNA dysregulation in pre-eclampsia, however difficulties in reproducibility have limited their use as biomarkers. Exosomal miRNA are a promising avenue to identify reproducibly dysregulated miRNA biomarkers for pre-eclampsia. Aims: To optimise exosome isolation from maternal serum in pregnancy and examine serum-derived exosomal miR-1 and miR-210 dysregulation in pre-eclampsia. Methods: Exosome yields from maternal serum isolated by ultracentrifugation and ExoQuickTM isolation methods were compared using electron microscopy and BCA assay-measured protein content. Exosomal RNA was extracted using a modified and manufacturers mirVanaTM PARISTM protocol and TRIzol® and SeraMirTM protocols. qRT-PCR was performed on SeraMirTM-extracted RNA for U6 small nuclear RNA, miR-1 and miR-210. Results: ExoQuickTM isolated the highest exosome yield as indicated by electron microscopy visualisation and protein content; 11215 &mgr;g vs ultracentrifugation ⩽ 12 &mgr;g. SeraMirTM extracted the highest and only exosomal RNA concentration above the limit of detection (15.7 ng/&mgr;L). No amplification products were detected by qRT-PCR. Discussion: This is the first study to isolate serum-derived exosomes and exosomal RNA in pregnancy and provides a workflow to discover exosomal miRNA biomarkers for pre-eclampsia and other pregnancy complications. Findings suggest more sensitive PCR techniques; pre-amplification or digital PCR, are required to demonstrate exosomal miRNA dysregulation and discover these biomarkers.