Experimental Model Of Bronchopulmonary Dysplasia Research Articles

Comparative Effects of Bone Marrow-derived Versus Umbilical Cord Tissue Mesenchymal Stem Cells in an Experimental Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a life-threatening condition in preterm infants with few effective therapies. Mesenchymal stem or stromal cells (MSCs) are a promising therapeutic strategy for BPD. The ideal MSC source for BPD prevention is however unknown. The objective of this study was to compare the regenerative effects of MSC obtained from bone marrow (BM) and umbilical cord tissue (UCT) in an experimental BPD model. In vitro, UCT-MSC demonstrated greater proliferation and expression of anti-inflammatory cytokines as compared to BM-MSC. Lung epithelial cells incubated with UCT-MSC conditioned media (CM) had better-wound healing following scratch injury. UCT-MSC CM and BM-MSC CM had similar pro-angiogenic effects on hyperoxia-exposed pulmonary microvascular endothelial cells. In vivo, newborn rats exposed to normoxia or hyperoxia (85% O2) from postnatal day (P) 1 to 21 were given intra-tracheal (IT) BM or UCT-MSC (1 × 106 cells/50 μL), or placebo (PL) on P3. Hyperoxia PL-treated rats had marked alveolar simplification, reduced lung vascular density, pulmonary vascular remodeling, and lung inflammation. In contrast, administration of both BM-MSC and UCT-MSC significantly improved alveolar structure, lung angiogenesis, pulmonary vascular remodeling, and lung inflammation. UCT-MSC hyperoxia-exposed rats however had greater improvement in some morphometric measures of alveolarization and less lung macrophage infiltration as compared to the BM-MSC-treated group. Together, these findings suggest that BM-MSC and UCT-MSC have significant lung regenerative effects in experimental BPD but UCT-MSC suppresses lung macrophage infiltration and promotes lung epithelial cell healing to a greater degree.

Amniotic fluid-derived extracellular vesicles: characterization and therapeutic efficacy in an experimental model of bronchopulmonary dysplasia

Background aimsExtracellular vesicles (EVs) are being tested for their use as novel therapeutics. However, the optimal source of EVs is currently under investigation. Amniotic fluid (AF) is a natural source of EVs that can be easily obtained for use in regenerative medicine, yet AF-EV characterization has not been fully explored. MethodsHere the authors demonstrate AF as a rich source of EVs and identify the microRNA and proteomic cargo. Bioinformatics analysis of this cargo revealed multiple pathway targets, including immunomodulatory, anti-inflammatory and free radical scavenging networks. The authors further demonstrated the therapeutic potential of this EV product as a novel preventative agent for bronchopulmonary dysplasia (BPD). ResultsIntra-tracheal administration of AF-EVs preserved alveolar development, attenuated vascular remodeling and pulmonary hypertension, decreased lung pro-inflammatory cytokine expression and reduced macrophage infiltration in an experimental BPD model. ConclusionsThe authors’ results suggest that AF is a viable biological fluid for EV harvest and that AF-EVs have strong therapeutic potential for pulmonary diseases, such as BPD, warranting further development to transition this novel EV product into the clinic.

Abstract 16158: Comparative Effects of Bone Marrow Derived versus Umbilical Cord Tissue Mesenchymal Stem Cells in an Experimental Model of Bronchopulmonary Dysplasia

Introduction: Bronchopulmonary dysplasia (BPD) or chronic lung injury of newborn is a life-threatening condition in preterm infants with few effective therapies. Mesenchymal stem cells (MSC) are a promising therapeutic strategy for BPD. The ideal MSC source for BPD prevention is however unknown. Umbilical cord tissue (UCT) MSC may potentially have more anti-inflammatory and pro-angiogenic properties. Hypothesis: We hypothesized that the UCT-MSC will have superior lung regenerative effect in BPD owing to superior anti-inflammatory and pro-angiogenic properties as compared to BM-MSC. Methods: Newborn rats (n=10-12/group) were randomly assigned to normoxia or hyperoxia (85% O 2 ) from postnatal day (P) 1 to 21 were given intra-tracheal (IT) BM or UCT-MSC (1 x 10 6 cells/50 μl), or placebo (PL) on P3. Results: Hyperoxia PL-treated pups had marked alveolar simplification, vascular rarefaction, remodeling and lung inflammation. Administration of both BM-MSC and UCT-MSC significantly improved alveolar structure, vascular density, pulmonary hypertension, vascular remodeling and lung inflammation. However, the improvement in alveolar structure and lung inflammation was more marked in hyperoxic pups who received UCT-MSC. In vitro studies demonstrated greater TSG-6 and IL-10 expression in UCT-MSC as compared to BM-MSC. Moreover, lung epithelial cells incubated with UCT-MSC conditioned media had greater wound healing following scratch injury. Conclusion: These findings demonstrate that while both BM and UCT-MSC have lung regenerative effects, UCT-MSC have greater anti-inflammatory and alveolar protective effects in experimental BPD. These findings have significant implications for cellular therapy in BPD, as it suggests that UCT-MSC are a superior cellular source for preterm lung protection.

Impaired Autophagic Activity Contributes to the Pathogenesis of Bronchopulmonary Dysplasia. Evidence from Murine and Baboon Models.

Bronchopulmonary dysplasia (BPD) is a common and serious complication associated with preterm birth. The pathogenesis of BPD is incompletely understood, and there is an unmet clinical need for effective treatments. The role of autophagy as a potential cytoprotective mechanism in BPD remains to be fully elucidated. In the present study, we investigated the role and regulation of autophagy in experimental models of BPD. Regulation and cellular distribution of autophagic activity during postnatal lung development and in neonatal hyperoxia-induced lung injury (nHILI) were assessed in the autophagy reporter transgenic GFP-LC3 (GFP-microtubule-associated protein 1A/1B-light chain 3) mouse model. Autophagic activity and its regulation were also examined in a baboon model of BPD. The role of autophagy in nHILI was determined by assessing lung morphometry, injury, and inflammation in autophagy-deficient Beclin 1 heterozygous knockout mice (Becn1+/-). Autophagic activity was induced during alveolarization in control murine lungs and localized primarily to alveolar type II cells and macrophages. Hyperoxia exposure of neonatal murine lungs and BPD in baboon lungs resulted in impaired autophagic activity in association with insufficient AMPK (5'-AMP-activated protein kinase) and increased mTORC1 (mTOR complex 1) activation. Becn1+/- lungs displayed impaired alveolarization, increased alveolar septal thickness, greater neutrophil accumulation, and increased IL-1β concentrations when exposed to nHILI. Becn1+/- alveolar macrophages isolated from nHILI-exposed mice displayed increased expression of proinflammatory genes. In conclusion, basal autophagy is induced during alveolarization and disrupted during progression of nHILI in mice and BPD in baboons. Becn1+/- mice are more susceptible to nHILI, suggesting that preservation of autophagic activity may be an effective protective strategy in BPD.

Mesenchymal Stem Cell (MSC) Derived Exosomes alleviate Pulmonary Hypertension in an Experimental Model of Bronchopulmonary Dysplasia (BPD) Through the Vascular Endothelial Growth Factor Signaling Pathway.

Title: Mesenchymal Stem Cell (MSC) Derived Exosomes Attenuate Pulmonary Hypertension (PH) in an Experimental Model of Bronchopulmonary Dysplasia (BPD) through the Vascular Endothelial Growth Factor Signaling Pathway. Background: Pulmonary Hypertension (PH) is a significant cause of morbidity and mortality in preterm infants with severe bronchopulmonary dysplasia (BPD). Exosomes are nano-sized vesicles that play a key role in intercellular and inter-organ communications. Here, we test the hypothesis that intra-tracheal administration of exosomes derived from Wharton’s jelly (WJ) or bone marrow (BM) MSCs would attenuate PH and vascular remodeling in an experimental model of severe …

Mesenchymal Stem Cell (MSC) Derived Exosomes alleviate Pulmonary Hypertension in an Experimental Model of Bronchopulmonary Dysplasia (BPD) Through the Vascular Endothelial Growth Factor Signaling Pathway.

Mesenchymal Stem Cell (MSC) Derived Exosomes alleviate Pulmonary Hypertension in an Experimental Model of Bronchopulmonary Dysplasia (BPD) Through the Vascular Endothelial Growth Factor Signaling Pathway.

Modulation of thymic architecture and T cell function by Mesenchymal stromal cell extracellular vesicles in a model of neonatal hyperoxia

Abstract Therapies based on mesenchymal stem/stromal cell extracellular vesicles (MEx) have shown promise in experimental models of bronchopulmonary dysplasia (BPD), a disease of disrupted lung development that can result from neonatal exposure to hyperoxia. The effects of oxygen exposure on other developing organs in the neonatal period remain poorly understood and whether MEx treatment may modify these responses is not known. Our research aimed at understanding the effects of neonatal exposure to hyperoxia and the immunomodulatory properties of MEx on thymic architecture and T cell development. Using a mouse model of neonatal hyperoxia, we showed that, in addition to lung injury resulting in BPD, there was involution of the thymic medulla with altered T cell function. Administration of a single dose of MEx resulted in preservation of thymic architecture, and restoration of normal thymic function through modulation of the thymic medullary antigen presentation network and T cell phenotypes. Consequently, MEx reinstated the mechanisms of thymic-driven central tolerance that are perturbed upon hyperoxia exposure, as demonstrated by increased FoxP3+ regulatory T cell generation and decreased T cell autoreactivity. Overall, our study demonstrates that MEx represent a promising cell-free therapeutic option to restore thymic architecture and ensure the development of central tolerance by preventing thymic injury associated with neonatal hyperoxia exposure.

1174: IL-6 Knockout decreases severity of lung injury in an experimental model of bronchopulmonary dysplasia (BPD)

1174: IL-6 Knockout decreases severity of lung injury in an experimental model of bronchopulmonary dysplasia (BPD)

Advance on stem cells therapy for bronchopulmonary dysplasia

The postnatal lung contains populations of stem cells with the capacity to reconstitute the lung after injury.Prematurity and its antecedent factors may alter lung stem cell programming leading to dysfunctional repair.Preterm infants who develop bronchopulmonary dysplasia exhibit decreased and/or dysfunctional stem cells.Preclinical studies provide robust evidence that stem cells reduce lung injury and improve survival in experimental models of bronchopulmonary dysplasia via paracrine-mediated mechanisms.Mesenchymal stem cells are an attractive population for lung repair as they are immunoprivileged, easily isolated, and have pleiotropic therapeutic effects.Early clinical evidence demonstrates the safety and feasibility of mesenchymal stem cell therapy for bronchopulmonary dysplasia.However, barriers for implementation, including optimal dosing, patient, timing, and culture techniques, need to be overcome for successful translation. Key words: Bronchopulmonary dysplasia; Stem cells; Mesenchymal stem cells; Therapy

Immune System Regulation Affected by a Murine Experimental Model of Bronchopulmonary Dysplasia: Genomic and Epigenetic Findings

Background: Bronchopulmonary dysplasia (BPD) is a common cause of abrupted lung development after preterm birth. BPD may lead to increased rehospitalization, more severe and frequent respiratory infections, and life-long reduced lung function. The gene regulation in lungs with BPD is complex, with various genetic and epigenetic factors involved. Objectives: The aim of this study was to examine the regulatory relation between gene expression and the epigenome (DNA methylation) relevant for the immune system after hyperoxia followed by a recovery period in air using a mouse model of BPD. Methods: Newborn mice pups were subjected to an immediate hyperoxic condition from birth and kept at 85% O₂ levels for 14 days followed by a 14-day period in room air. Next, mice lung tissue was used for RNA and DNA extraction with subsequent microarray-based assessment of lung transcriptome and supplementary methylome analysis. Results:The immune system-related transcriptomeregulation was affected in mouse lungs after hyperoxia. A high proportion of genes relevant in the immune system exhibited significant expression alterations, e.g., B cell-specific genes central to the cytokine-cytokine receptor interaction, the PI3K-AKT, and the B cell receptor signaling pathways. The findings were accompanied by significant DNA hypermethylation observed in the PI3K-AKT pathway and immune system-relevant genes. Conclusions: Oxygen damage could be partly responsible for the increased susceptibility and abnormal response to respiratory viruses and infections seen in premature babies with BPD through dysregulated genes.

Human amnion cell-derived extracellular vesicles in the treatment of bronchopulmonary dysplasia and associated pulmonary hypertension

Background & Aim We previously showed that human amnion epithelial cells (hAECs) are a viable source of cell therapy for established bronchopulmonary dysplasia (BPD). This work has culminated in a first-in-human clinical trial in babies with established BPD. In this current study, we sought to assess the therapeutic potential of hAEC-derived EVs (hAEC-EVs) in an experimental model of BPD. Methods, Results & Conclusion Methods Lipopolysaccharide (LPS) was introduced to C57bl6 mouse fetuses intra-amniotically at E16 prior to exposure to 65% oxygen (hyperoxia) at birth. hAEC-EVs were injected intravenously on postnatal day (PND) 4. One cohort of mice were culled on either postnatal day PND7 or PND14, while another cohort were kept in hyperoxia until PND 28, following which they were recovered under normoxia conditions for a further 2 weeks or 6 weeks for cardiovascular assessment. Results The isolated hAEC-EVs had distinct cup shaped morphology with average size of 80-140nm. ALIX, Grp94 and HLA-G were expressed in EVs, and Pathway enrichment analysis showed that endothelin signaling pathway, Wnt signaling pathway, and inflammation mediated by chemokine and cytokine signaling pathway were enriched in hAEC-EVs. In the mouse model of experimental BPD, hAEC-EV administration improved tissue-to-airspace ratio and septal crest density in a dose-dependent manner. hAEC-EVs reduced the levels of inflammatory cytokines such as interleukin (IL)-1β and tumour necrosis factor-alpha (TNF-α) on PND7. The improvement of lung injury was associated with the increase of the percentage of type II alveolar cells (AT2s) at both PND7 and 14. Surprisingly, neonatal hAEC-EV delivery reduced airway hyper-responsiveness, mitigated pulmonary hypertension and prevented right ventricle hypertrophy that associated with BPD-like lung injury at week 6. These improvements persisted till week 10. All these observations showed that HAEC-EVs mitigate the BPD lung injury through decreasing inflammation, activating local AT2 niche and reversing pulmonary artery remodeling. Conclusions hAEC-derived EVs had similar therapeutic effects to hAECs in this mouse model of experimental BPD. hAEC-derived EVs improved lung structure and reduced lung inflammation in a manner similar to that achieved using hAECs. EVs repaired lung injury partly through the activation of local stem/progenitor cells. Furthermore, hAEC-EV administration had long term benefit in improving lung function, preventing pulmonary hypertension and right ventricle hypertrophy.

TNFα-stimulated protein 6 (TSG-6) reduces lung inflammation in an experimental model of bronchopulmonary dysplasia.

Inflammation is a key factor in the pathogenesis of bronchopulmonary dysplasia (BPD). Tumor necrosis factor-stimulated protein 6 (TSG-6) is a glycoprotein that modulates inflammation. Here we tested the hypothesis that intra-tracheal (IT) administration of an adenovirus overexpressing TSG-6 (AdTSG-6) would decrease inflammation and restore lung structure in experimental BPD. Newborn Sprague-Dawley rats exposed to normoxia (RA) or hyperoxia (85% O2) from postnatal day (P) 1-P14 were randomly assigned to receive IT AdTSG-6 or placebo (PL) on P3. The effect of IT AdTSG-6 on lung inflammation, alveolarization, angiogenesis, apoptosis, pulmonary vascular remodeling, and pulmonary hypertension were evaluated on P14. Data were analyzed by two-way ANOVA. TSG-6 mRNA was significantly increased in pups who received IT AdTSG-6. Compared to RA, hyperoxia PL-treated pups had increased NF-kβ activation and lung inflammation. In contrast, IT AdTSG-6 hyperoxia-treated pups had decreased lung phosphorylated NF-kβ expression and markers of inflammation. This was accompanied by an improvement in alveolarization, angiogenesis, pulmonary vascular remodeling, and pulmonary hypertension. IT AdTSG-6 decreases lung inflammation and improves lung structure in neonatal rats with experimental BPD. These findings suggest that therapies that increase lung TSG-6 expression may have beneficial effects in preterm infants with BPD.

Stromal derived factor-1 mediates the lung regenerative effects of mesenchymal stem cells in a rodent model of bronchopulmonary dysplasia

BackgroundMesenchymal stem cells (MSCs) attenuate lung injury in experimental models of bronchopulmonary dysplasia (BPD). Stromal derived factor-1 (SDF-1), a chemokine secreted by MSCs, modulates angiogenesis and stem cell recruitment. Here we tested the hypothesis that SDF-1 mediates MSC protective effects in experimental BPD by modulating angiogenesis.MethodsSDF-1 was knocked down in MSCs using lentiviral vectors carrying anti-SDF-1 short hairpin RNA (MSC-SDF KD). Non-silencing short hairpin RNA was used as control (MSC-NS control). Newborn rats exposed to normoxia or hyperoxia (FiO2 = 0.85) for 3 weeks, were randomly assigned to receive a single intra-tracheal injection (IT) of MSC-NS control or MSC-SDF KD (1 × 106 cells/50 μl) or placebo on postnatal day 7. The degree of alveolarization, lung angiogenesis, inflammation, and pulmonary hypertension (PH) were assessed at postnatal day 21.ResultsAdministration of IT MSC-NS control improved lung alveolarization, angiogenesis and inflammation, and attenuated PH in newborn rats with hyperoxia-induced lung injury (HILI). In contrast, knockdown of SDF-1 in MSCs significantly reduced their beneficial effects on alveolarization, angiogenesis, inflammation and PH.ConclusionsThe therapeutic benefits of MSCs in neonatal HILI are in part mediated by SDF-1, through anti-inflammatory and angiogenesis promoting mechanisms. Therapies directly targeting this chemokine may provide a novel strategy for the treatment of BPD.

The Effect of Gender on Mesenchymal Stem Cell (MSC) Efficacy in Neonatal Hyperoxia-Induced Lung Injury

BackgroundMesenchymal stem cells (MSC) improve alveolar and vascular structures in experimental models of bronchopulmonary dysplasia (BPD). Female MSC secrete more anti-inflammatory and pro-angiogenic factors as compared to male MSC. Whether the therapeutic efficacy of MSC in attenuating lung injury in an experimental model of BPD is influenced by the sex of the donor MSC or recipient is unknown. Here we tested the hypothesis that female MSC would have greater lung regenerative properties than male MSC in experimental BPD and this benefit would be more evident in males.ObjectiveTo determine whether intra-tracheal (IT) administration of female MSC to neonatal rats with experimental BPD has more beneficial reparative effects as compared to IT male MSC.MethodsNewborn Sprague-Dawley rats exposed to normoxia (RA) or hyperoxia (85% O2) from postnatal day (P) 2- P21 were randomly assigned to receive male or female IT bone marrow (BM)-derived green fluorescent protein (GFP+) MSC (1 x 106 cells/50 μl), or Placebo on P7. Pulmonary hypertension (PH), vascular remodeling, alveolarization, and angiogenesis were assessed at P21. PH was determined by measuring right ventricular systolic pressure (RVSP) and pulmonary vascular remodeling was evaluated by quantifying the percentage of muscularized peripheral pulmonary vessels. Alveolarization was evaluated by measuring mean linear intercept (MLI) and radial alveolar count (RAC). Angiogenesis was determined by measuring vascular density. Data are expressed as mean ± SD, and analyzed by ANOVA.ResultsThere were no significant differences in the RA groups. Exposure to hyperoxia resulted in a decrease in vascular density and RAC, with a significant increase in MLI, RVSP, and the percentage of partially and fully muscularized pulmonary arterioles. Administration of both male and female MSC significantly improved vascular density, alveolarization, RVSP, percent of muscularized vessels and alveolarization. Interestingly, the improvement in PH and vascular remodeling was more robust in the hyperoxic rodents who received MSC from female donors. In keeping with our hypothesis, male animals receiving female MSC, had a greater improvement in vascular remodeling. This was accompanied by a more significant decrease in lung pro-inflammatory markers and a larger increase in anti-inflammatory and pro-angiogenic markers in male rodents that received female MSC. There were no significant differences in MSC engraftment among groups.ConclusionsFemale BM-derived MSC have greater therapeutic efficacy than male MSC in reducing neonatal hyperoxia-induced lung inflammation and vascular remodeling. Furthermore, the beneficial effects of female MSC were more pronounced in male animals. Together, these findings suggest that female MSC maybe the most potent BM-derived MSC population for lung repair in severe BPD complicated by PH.

Antenatal endotoxin disrupts lung vitamin D receptor and 25-hydroxyvitamin D 1α-hydroxylase expression in the developing rat.

Vitamin D [vit D; 1,25-(OH)2D] treatment improves survival and lung alveolar and vascular growth in an experimental model of bronchopulmonary dysplasia (BPD) after antenatal exposure to endotoxin (ETX). However, little is known about lung-specific 1,25-(OH)2D3 regulation during development, especially regarding maturational changes in lung-specific expression of the vitamin D receptor (VDR), 1α-hydroxylase (1α-OHase), and CYP24A1 during late gestation and the effects of antenatal ETX exposure on 1,25-(OH)2D3 metabolism in the lung. We hypothesized that vit D regulatory proteins undergo maturation regulation in the late fetal and early neonatal lung and that prenatal exposure to ETX impairs lung growth partly through abnormal endogenous vit D metabolism. Normal fetal rat lungs were harvested between embryonic day 15 and postnatal day 14. Lung homogenates were assayed for VDR, 1α-OHase, and CYP24A1 protein contents by Western blot analysis. Fetal rats were injected on embryonic day 20 with intra-amniotic ETX, ETX + 1,25-(OH)2D3, or saline and delivered 2 days later. Pulmonary artery endothelial cells (PAECs) from fetal sheep were assessed for VDR, 1α-OHase, and CYP24A1 expression after treatment with 25-(OH)D3, 1,25-(OH)2D3, ETX, ETX + 25-(OH)D3, or ETX + 1,25-(OH)2D3. We found that lung VDR, 1α-OHase, and CYP2741 protein expression dramatically increase immediately before birth (P < 0.01 vs. early fetal values). Antenatal ETX increases CYP24A1 expression (P < 0.05) and decreases VDR and 1α-OHase expression at birth (P < 0.001), but these changes are prevented with concurrent vit D treatment (P < 0.001). ETX-induced reduction of fetal PAEC growth and tube formation and lung 1α-OHase expression are prevented by vit D treatment (P < 0.001). We conclude that lung VDR, 1α-OHase, and CYP24A1 protein content markedly increase before birth and that antenatal ETX disrupts lung vit D metabolism through downregulation of VDR and increased vit D catabolic enzyme expression, including changes in developing endothelium. We speculate that endogenous vitamin D metabolism modulates normal fetal lung development and that prenatal disruption of vit D signaling may contribute to impaired postnatal lung growth at least partly through altered angiogenic signaling.

Stem Cells and Their Mediators - Next Generation Therapy for Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) remains a major complication of premature birth. Despite great achievements in perinatal medicine over the past decades, there is no treatment for BPD. Recent insights into the biology of stem/progenitor cells have ignited the hope of regenerating damaged organs. Animal experiments revealed promising lung protection/regeneration with stem/progenitor cells in experimental models of BPD and led to first clinical studies in infants. However, these therapies are still experimental and knowledge on the exact mechanisms of action of these cells is limited. Furthermore, heterogeneity of the therapeutic cell populations and missing potency assays currently limit our ability to predict a cell product’s efficacy. Here, we review the therapeutic potential of mesenchymal stromal, endothelial progenitor, and amniotic epithelial cells for BPD. Current knowledge on the mechanisms behind the beneficial effects of stem cells is briefly summarized. Finally, we discuss the obstacles constraining their transition from bench-to-bedside and present potential approaches to overcome them.

Fractal analysis of alveolarization in hyperoxia-induced rat models of bronchopulmonary dysplasia

Many morphometric approaches have been proposed for quantitative evaluation of alveolarization in experimental models of bronchopulmonary dysplasia (BPD), such as alveolar number, alveolar size and variability, mean linear intercept (Lm), surface-to-volume ratio, or radial alveolar count. Conversely, no papers are available about potentiality of fractal analysis in this matter. Thus, in the present study, we performed a comparative analysis between Lm and fractal parameters (fractal dimension (D) and lacunarity (λ)), in experimental hyperoxia-induced models of BPD. At birth the newborn rats were randomly distributed between the following experimental groups: a) rats raised in ambient air for 2 weeks; b) exposed to 60% oxygen for 2 weeks; c) raised in normoxia for 6 weeks; d) exposed to 60% hyperoxia for 2 weeks and then to room air for further 4 weeks. Normoxic 6-weeks rats showed a significant decrease of Lm and a significant increase of D and λ with respect to normoxic 2-weeks rats, indicative of alveolar development. Hyperoxia-exposed rats of 2 weeks did not show significant changes in Lm and D with respect to normoxic rats equal in age, although λ was significantly lower in hyperoxic group. Conversely, in the comparison between 6-week rats, the hyperoxia-exposed group showed higher value of Lm and lower values of D and λ, when compared to the normoxic group, confirming a lower alveolar gas exchange surface-to-volume ratio and an airspace morphology of lower complexity. The analysis of the ROC curves showed a comparable discriminatory power of Lm and D as binary classifiers of the airspace morphology (areas under the curves of 0.815 and 0.753, respectively). Moreover, the values exhibited by the parameters when different segmentation thresholds were applied suggested that fractal parameters are less sensitive (D and λ showed a coefficient of variation of 0.005 and 0.009, respectively) than Lm (exhibiting a coefficient of variation of 0.05) to the bias eventually introduced in the analysis by approximations or errors in tissue segmentation. In conclusion, the present study demonstrated the ability of fractal analysis (D and λ) to identify developmental and hyperoxia-induced changes in alveolarization with discriminatory power similar to Lm. The fractal approach, however, appears more fit to automatic image analysis, being less influenced by approximations in thresholding and image editing.

MSC Microvesicles for the Treatment of Lung Disease: A New Paradigm for Cell-Free Therapy

Bronchopulmonary dysplasia (BPD), also known as chronic lung disease of infancy, is a major complication of preterm birth that, despite improvements in neonatal respiratory support and perinatal care, remains an important cause of morbidity and mortality, often with severe adverse neurodevelopmental sequelae. Even with major advances in our understanding of the pathogenesis of this disease, BPD remains essentially without adequate treatment. Cell-based therapies arose as a promising treatment for acute and chronic lung injury in many experimental models of disease. Currently, more than 3000 human clinical trials employing cell therapy for the treatment of diverse diseases, including cardiac, neurologic, immune, and respiratory conditions, are ongoing or completed. Among the treatments, mesenchymal stem cells (MSCs) are the most studied and have been extensively tested in experimental models of BPD, pulmonary hypertension, pulmonary fibrosis, and acute lung injury. Despite the promising potential, MSC therapy for human lung disease still remains at an experimental stage and optimal transplantation conditions need to be determined. Although the mechanism of MSC action can be manifold, accumulating evidence suggests a predominant paracrine, immunomodulatory, and cytoprotective effect. The current review summarizes the effect of MSC treatment in models of lung injury, including BPD, and focuses on the MSC secretome and, specifically, MSC-derived microvesicles as potential key mediators of therapeutic action that can be the focus of future therapies.

Vitamin D treatment improves survival and infant lung structure after intra-amniotic endotoxin exposure in rats: potential role for the prevention of bronchopulmonary dysplasia

Vitamin D (vit D) has anti-inflammatory properties and modulates lung growth, but whether vit D can prevent lung injury after exposure to antenatal inflammation is unknown. We hypothesized that early and sustained vit D treatment could improve survival and preserve lung growth in an experimental model of bronchopulmonary dysplasia induced by antenatal exposure to endotoxin (ETX). Fetal rats (E20) were exposed to ETX (10 μg), ETX + Vit D (1 ng/ml), or saline (control) via intra-amniotic (IA) injections and delivered 2 days later. Newborn pups exposed to IA ETX received daily intraperitoneal injections of vit D (1 ng/g) or saline for 14 days. Vit D treatment improved oxygen saturations (78 vs. 87%; P < 0.001) and postnatal survival (84% vs. 57%; P < 0.001) after exposure to IA ETX compared with IA ETX alone. Postnatal vit D treatment improved alveolar and vascular growth at 14 days by 45% and 25%, respectively (P < 0.05). Vit D increased fetal sheep pulmonary artery endothelial cell (PAEC) growth and tube formation by 64% and 44%, respectively (P < 0.001), and prevented ETX-induced reductions of PAEC growth and tube formation. Vit D directly increased fetal alveolar type II cell (ATIIC) growth by 26% (P < 0.001) and enhanced ATIIC growth in the presence of ETX-induced growth suppression by 73% (P < 0.001). We conclude that antenatal vit D therapy improved oxygenation and survival in newborn rat pups and enhanced late lung structure after exposure to IA ETX in vivo, which may partly be due to direct effects on vascular and alveolar growth.

Endothelial colony-forming cell conditioned media promote angiogenesis in vitro and prevent pulmonary hypertension in experimental bronchopulmonary dysplasia

Late-outgrowth endothelial colony-forming cells (ECFCs), a type of circulating endothelial progenitor cell (EPC), may contribute to pulmonary angiogenesis during development. Cord blood ECFCs from preterm newborns proliferate more rapidly than term ECFCs but are more susceptible to the adverse effects of hyperoxia. Recent studies suggest that bone marrow-derived EPCs protect against experimental lung injury via paracrine mechanisms independent of vascular engraftment. To determine whether human umbilical cord blood ECFCs from preterm and term newborns have therapeutic benefit in experimental neonatal lung injury, we isolated cord blood ECFCs from full-term and preterm newborns and prepared ECFC-conditioned medium (CM) to test its therapeutic benefit on fetal pulmonary artery endothelial cell (PAEC) proliferation and function as well as alveolar type 2 (AT2) cell growth. PAECs and AT2 cells were isolated from late-gestation fetal sheep. Additionally, we administered both ECFCs and ECFC-CM to bleomycin-exposed newborn rats, an experimental model of bronchopulmonary dysplasia (BPD). Both term ECFC-CM and preterm ECFC-CM promoted cell growth and angiogenesis in vitro. However, when ECFC-CM was collected during exposure to mild hyperoxia, the benefit of preterm ECFC-CM was no longer observed. In the bleomycin model of BPD, treatment with ECFC-CM (or CM from mature EC) effectively decreased right ventricular hypertrophy but had no effect on alveolar septation. We conclude that term ECFC-CM is beneficial both in vitro and in experimental BPD. During oxidative stress, preterm ECFC-CM, but not term ECFC-CM, loses its benefit. The inability of term ECFC-CM to promote alveolarization may limit its therapeutic potential.