Antenatal Endotoxin Induces Dysanapsis in Experimental Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, is characterized by impaired lung development with sustained functional abnormalities due to alterations of airways and the distal lung. Although clinical studies have shown striking associations between antenatal stress and BPD, little is known about the underlying pathogenetic mechanisms. Whether dysanapsis, the concept of discordant growth of the airways and parenchyma, contributes to late respiratory disease as a result of antenatal stress is unknown. We hypothesized that antenatal endotoxin (ETX) impairs juvenile lung function as a result of altered central airway and distal lung structure, suggesting the presence of dysanapsis in this preclinical BPD model. Fetal rats were exposed to intraamniotic ETX (10 µg) or saline solution (control) 2 days before term. We performed extensive structural and functional evaluation of the proximal airways and distal lung in 2-week-old rats. Distal lung structure was quantified by stereology. Conducting airway diameters were measured using micro-computed tomography. Lung function was assessed during invasive ventilation to quantify baseline mechanics, response to methacholine challenge, and spirometry. ETX-exposed pups exhibited distal lung simplification, decreased alveolar surface area, and decreased parenchyma-airway attachments. ETX-exposed pups exhibited decreased tracheal and second- and third-generation airway diameters. ETX increased respiratory system resistance and decreased lung compliance at baseline. Only Newtonian resistance, specific to large airways, exhibited increased methacholine reactivity in ETX-exposed pups compared with controls. ETX-exposed pups had a decreased ratio of FEV in 0.1 second to FVC and a normal FEV in 0.1 second, paralleling the clinical definition of dysanapsis. Antenatal ETX causes abnormalities of the central airways and distal lung growth, suggesting that dysanapsis contributes to abnormal lung function in juvenile rats.

Acute kidney injury decreases pulmonary vascular growth and alveolarization in neonatal rat pups.

BACKGROUND: Acute kidney injury (AKI) is common in sick neonates and associated with poor pulmonary outcomes, however, the mechanisms responsible remain unknown. We present two novel neonatal rodent models of AKI to investigate the pulmonary effects of AKI. METHODS: In rat pups, AKI was induced surgically via bilateral ischemia-reperfusion injury (bIRI) or pharmacologically using aristolochic acid (AA). AKI was confirmed with plasma blood urea nitrogen and creatinine measurements and kidney injury molecule-1 staining on renal immunohistochemistry. Lung morphometrics were quantified with radial alveolar count and mean linear intercept, and angiogenesis investigated by pulmonary vessel density (PVD) and vascular endothelial growth factor (VEGF) protein expression. For the surgical model, bIRI, sham, and non-surgical pups were compared. For the pharmacologic model, AA pups were compared to vehicle controls. RESULTS: AKI occurred in bIRI and AA pups, and they demonstrated decreased alveolarization, PVD, and VEGF protein expression compared controls. Sham pups did not experience AKI, however, demonstrated decreased alveolarization, PVD, and VEGF protein expression compared to controls. CONCLUSION: Pharmacologic AKI and surgery in neonatal rat pups, with or without AKI, decreased alveolarization and angiogenesis, producing a bronchopulmonary dysplasia phenotype. These models provide a framework for elucidating the relationship between AKI and adverse pulmonary outcomes. IMPACT: There are no published neonatal rodent models investigating the pulmonary effects after neonatal acute kidney injury, despite known clinical associations. We present two novel neonatal rodent models of acute kidney injury to study the impact of acute kidney injury on the developing lung. We demonstrate the pulmonary effects of both ischemia-reperfusion injury and nephrotoxin-induced AKI on the developing lung, with decreased alveolarization and angiogenesis, mimicking the lung phenotype of bronchopulmonary dysplasia. Neonatal rodent models of acute kidney injury provide opportunities to study mechanisms of kidney-lung crosstalk and novel therapeutics in the context of acute kidney injury in a premature infant.

Understanding the role of placental pathophysiology in the development of bronchopulmonary dysplasia.

The associations between bronchopulmonary dysplasia (BPD) and the gestational pathologies of chorioamnionitis (CA) and hypertensive disorders of pregnancy (HDP) have become increasingly well recognized. However, the mechanisms through which these antenatal conditions cause increased risk of BPD remain less well characterized. The objective of this review is to discuss the role of the placenta in BPD predisposition as a primary driver of intrauterine alterations adversely impacting fetal lung development. We hypothesize that due to similarities in structure and function, placental disorders during pregnancy can uniquely impact the developing fetal lung, creating a unique placental-pulmonary connection. In the current review, we explore this hypothesis through analysis of clinical literature and preclinical model systems evaluating BPD predisposition, discussion of BPD phenotypes, and an overview on strategies to incorporate placental investigation into research on fetal lung development. We also discuss important concepts learned from research on antenatal steroids as a modulator fetal lung development. Finally, we propose that the appropriate selection of animal models and establishment of in vitro lung developmental model systems incorporating primary human placental components are key in continuing to understand and address antenatal predisposition to BPD.

Antenatal mesenchymal stromal cell extracellular vesicle treatment preserves lung development in a model of bronchopulmonary dysplasia due to chorioamnionitis.

Antenatal stressors such as chorioamnionitis (CA) increase the risk for bronchopulmonary dysplasia (BPD). Studies have shown that experimental BPD can be ameliorated by postnatal treatment with mesenchymal stromal cell-derived extracellular vesicles (MEx). However, the antenatal efficacy of MEx to prevent BPD is unknown. To determine whether antenatal MEx therapy attenuates intrauterine inflammation and preserves lung growth in a rat model of CA-induced BPD. At embryonic day (E)20, rat litters were treated with intra-amniotic injections of saline, endotoxin (ETX) to model chorioamnionitis, MEx, or ETX plus MEx followed by cesarean section delivery with placental harvest at E22. Placental and lung evaluations were conducted at day 0 and day 14, respectively. To assess the effects of ETX and MEx on lung growth in vitro, E15 lung explants were imaged for distal branching. Placental tissues from ETX-exposed pregnancies showed increased expression of inflammatory markers NLRP-3 and IL-1ß and altered spiral artery morphology. In addition, infant rats exposed to intrauterine ETX had reduced alveolarization and pulmonary vessel density (PVD), increased right ventricular hypertrophy (RVH), and decreased lung mechanics. Intrauterine MEx therapy of ETX-exposed pups reduced inflammatory cytokines, normalized spiral artery architecture, and preserved distal lung growth and mechanics. In vitro studies showed that MEx treatment enhanced distal lung branching and increased VEGF and SPC gene expression. Antenatal MEx treatment preserved distal lung growth and reduced intrauterine inflammation in a model of CA-induced BPD. We speculate that MEx may provide a novel therapeutic strategy to prevent BPD due to antenatal inflammation.

Multicolor structured illumination microscopy and quantitative control of polychromatic light with a digital micromirror device.

Linear structured illumination microscopy (SIM) is a super-resolution microscopy technique that does not impose photophysics requirements on fluorescent samples. Multicolor SIM implementations typically rely on liquid crystal on silicon (LCoS) spatial light modulators (SLM's) for patterning the excitation light, but digital micromirror devices (DMD's) are a promising alternative, owing to their lower cost and higher speed. However, existing coherent DMD SIM implementations use only a single wavelength of light, limited by the lack of efficient approaches for solving the blazed grating effect for polychromatic light. We develop the requisite quantitative tools, including a closed form solution of the blaze and diffraction conditions, forward models of DMD diffraction and pattern projection, and a model of DMD aberrations. Based on these advances, we constructed a three-color DMD microscope, quantified the effect of aberrations from the DMD, developed a high-resolution optical transfer function measurement technique, and demonstrated SIM on fixed and live cells. This opens the door to applying DMD's in polychromatic applications previously restricted to LCoS SLM's.

Perinatal Hypoxia-Inducible Factor Stabilization Preserves Lung Alveolar and Vascular Growth in Experimental Bronchopulmonary Dysplasia.

Rationale: Antenatal inflammation with placental dysfunction is strongly associated with high bronchopulmonary dysplasia (BPD) risk in preterm infants. Whether antenatal or postnatal HIF (hypoxia-inducible factor) augmentation can preserve lung structure and function and prevent pulmonary hypertension after intrauterine inflammation is controversial.Objectives: To determine whether antenatal or postnatal prolyl-hydroxylase inhibitor (PHi) therapy increases lung HIF expression, preserves lung growth and function, and prevents pulmonary hypertension in a rat model of chorioamnionitis-induced BPD caused by antenatal inflammation.Methods: Endotoxin (ETX) was administered to pregnant rats by intraamniotic injection at Embryonic Day 20, and pups were delivered by cesarean section at Embryonic Day 22. Selective PHi drugs, dimethyloxalylglycine or GSK360A, were administered into the amniotic space at Embryonic Day 20 or after birth by intraperitoneal injection for 2 weeks. Placentas and lung tissue were collected at birth for morphometric and Western blot measurements of HIF-1a, HIF-2a, VEGF (vascular endothelial growth factor), and eNOS (endothelial nitric oxide synthase) protein contents. At Day 14, lung function was assessed, and tissues were harvested to determine alveolarization by radial alveolar counts, pulmonary vessel density, and right ventricle hypertrophy (RVH).Measurements and Main Results: Antenatal PHi therapy preserves lung alveolar and vascular growth and lung function and prevents RVH after intrauterine ETX exposure. Antenatal administration of PHi markedly upregulates lung HIF-1a, HIF-2a, VEGF, and eNOS expression after ETX exposure.Conclusions: HIF augmentation improves lung structure and function, prevents RVH, and improves placental structure following antenatal ETX exposure. We speculate that antenatal or postnatal PHi therapy may provide novel strategies to prevent BPD due to antenatal inflammation.

Maternal Vitamin D Deficiency Causes Sustained Impairment of Lung Structure and Function and Increases Susceptibility to Hyperoxia-induced Lung Injury in Infant Rats.

Vitamin D deficiency (VDD) during pregnancy is associated with increased respiratory morbidities and risk for chronic lung disease after preterm birth. However, the direct effects of maternal VDD on perinatal lung structure and function and whether maternal VDD increases the susceptibility of lung injury due to hyperoxia are uncertain. In the present study, we sought to determine whether maternal VDD is sufficient to impair lung structure and function and whether VDD increases the impact of hyperoxia on the developing rat lung. Four-week-old rats were fed VDD chow and housed in a room shielded from ultraviolet A/B light to achieve 25-hydroxyvitamin D concentrations <10 ng/ml at mating and throughout lactation. Lung structure was assessed at 2 weeks for radial alveolar count, mean linear intercept, pulmonary vessel density, and lung function (lung compliance and resistance). The effects of hyperoxia for 2 weeks after birth were assessed after exposure to fraction of inspired oxygen of 0.95. At 2 weeks, VDD offspring had decreased alveolar and vascular growth and abnormal airway reactivity and lung function. Impaired lung structure and function in VDD offspring were similar to those observed in control rats exposed to postnatal hyperoxia alone. Maternal VDD causes sustained abnormalities of distal lung growth, increases in airway hyperreactivity, and abnormal lung mechanics during infancy. These changes in VDD pups were as severe as those measured after exposure to postnatal hyperoxia alone. We speculate that antenatal disruption of vitamin D signaling increases the risk for late-childhood respiratory disease.

Maternal vitamin D deficiency induces transcriptomic changes in newborn rat lungs.

Vitamin D deficiency (VDD) during pregnancy is common and related to several maternal and fetal morbidities. Vitamin D (VD) plays a role in normal lung development and VDD causes abnormal airway, alveolar, and vascular growth in newborn rats. Here we use an unbiased transcriptomic approach to identify pathways altered in the lungs of offspring from VDD dams. The lungs of newborn offspring from VD replete and VDD dams were removed and RNA from these samples were analyzed using Affymetrix microarrays. Data were RMA normalized, differential gene expression was determined using Significance Analysis of Microarrays (5 % FDR) and pathway enrichment analysis was assessed. There were 2233 differentially expressed transcripts between the VDD and control lungs (1889 up, 344 down). Consistent with the suppression of lung growth in the VDD group, there were significant suppression of signal transduction pathways related to vascular biology and anabolic signaling pathways, e.g. the insulin-like growth factor-1 receptor (IGF-1R), fibroblast growth factor (FGF), cell cycle control. A major, enriched functional category was upregulation of pathways related to the innate immune system, including pathways for granulocyte and macrophage development, chemotaxis, and activation of cytokine signaling through Jak/Stat (e.g. resulting in higher IL1 α and ß). We conclude that VDD during fetal development alters multiple pathways beyond the predicted angiogeneic alterations. These changes either contribute to, or reflect, the abnormal airway, alveolar, and vascular growth seen in the neonatal lung resulting from maternal VDD. The pattern also suggests abnormal lung development caused by maternal VDD creates a proinflammatory milieu that could contribute to the suppression of lung growth and development.

rhIGF-1/BP3 Preserves Lung Growth and Prevents Pulmonary Hypertension in Experimental Bronchopulmonary Dysplasia.

Rationale: Antenatal factors, such as chorioamnionitis, preeclampsia, and postnatal injury, are associated with an increased risk for bronchopulmonary dysplasia (BPD) and pulmonary hypertension (PH) after preterm birth. IGF-1 (insulin-like growth factor-1) is markedly decreased in normal preterm infants, but whether IGF-1 treatment can prevent BPD or PH is unknown.Objectives: To evaluate whether postnatal treatment with rhIGF-1 (recombinant human IGF-1)/BP3 (binding peptide 3) improves lung growth and prevents PH in two antenatal models of BPD induced by intraamniotic exposure to endotoxin (ETX) or sFlt-1 (soluble fms-like tyrosine kinase 1), and in a postnatal model due to prolonged hyperoxia.Methods: ETX or sFlt-1 were administered into the amniotic sac of pregnant rats at Embryonic Day 20 to simulate antenatal models of chorioamnionitis and preeclampsia, respectively. Pups were delivered by cesarean section at Embryonic Day 22 and treated with rhIGF-1/BP3 (0.02-20 mg/kg/d intraperitoneal) or buffer for 2 weeks. Study endpoints included radial alveolar counts (RACs), vessel density, and right ventricular hypertrophy (RVH). Direct effects of rhIGF-1/BP3 (250 ng/ml) on fetal lung endothelial cell proliferation and tube formation and alveolar type 2 cell proliferation were studied by standard methods in vitro.Measurements and Main Results: Antenatal ETX and antenatal sFlt-1 reduced RAC and decreased RVH in infant rats. In both models, postnatal rhIGF-1/BP3 treatment restored RAC and RVH to normal values when compared with placebo injections. rhIGF-1/BP3 treatment also preserved lung structure and prevented RVH after postnatal hyperoxia. In vitro studies showed that rhIGF-1/BP3 treatment increased lung endothelial cell and alveolar type 2 cell proliferation.Conclusions: Postnatal rhIGF-1/BP3 treatment preserved lung structure and prevented RVH in antenatal and postnatal BPD models. rhIGF-1/BP3 treatment may provide a novel strategy for the prevention of BPD in preterm infants.

Antenatal Endotoxin Impairs Lung Mechanics and Increases Sensitivity to Ventilator-Induced Lung Injury in Newborn Rat Pups.

Perinatal inflammation due to chorioamnionitis and ventilator-induced lung injury (VILI) at birth is independent risk factors for the development of bronchopulmonary dysplasia (BPD). We have previously shown that antenatal endotoxin (ETX) causes abnormal lung structure and function in 2-week-old rats, but whether ETX impairs lung mechanics at birth and increases risk for VILI is unknown. Fetal rats were exposed to 10 µg endotoxin or saline via intra-amniotic injection. At birth (D0) or 7 days (D7), rats received 90 min of lung protective ventilation [PROTECT group; tidal volume (Vt) = 6 ml/kg with positive end expiratory pressure (PEEP) = 2 cmH2O]; P20 ventilation [plateau pressure (Pplat) = 20 cmH2O, PEEP = 0]; or P24 ventilation (Pplat = 24 cmH2O, PEEP = 0, only applied to D7). Prior to prolonged ventilation at D0, endotoxin-exposed rats had decreased compliance and inspiratory capacity (IC) compared to controls. At D7, endotoxin was associated with reduced compliance. High-pressure ventilation (P20 and P24) tended to increase IC and compliance in all saline-treated groups. Ventilation at D0 with P20 increased IC and compliance when applied to saline-treated but not endotoxin-exposed pups. At D7, P24 ventilation of endotoxin-exposed pups increased elastance, bronchoalveolar lavage protein content, and IL-1b and TEN-C mRNA expression in comparison to the saline group. In summary, antenatal endotoxin exposure alters lung mechanics at birth and 1 week of life and increases susceptibility to VILI as observed in lung mechanics, alveolocapillary barrier injury, and inflammatory mRNA expression. We speculate that antenatal inflammation primes the lung for a more marked VILI response, suggesting an adverse synergistic effect of antenatal and postnatal exposures.

Heterogeneous response of endothelial cells to insulin-like growth factor 1 treatment is explained by spatially clustered sub-populations.

A common strategy to measure the efficacy of drug treatment is the in vitro comparison of ensemble readouts with and without treatment, such as proliferation and cell death. A fundamental assumption underlying this approach is that there exists minimal cell-to-cell variability in the response to a drug. Here, we demonstrate that ensemble and non-spatial single-cell readouts applied to primary cells may lead to incomplete conclusions due to cell-to-cell variability. We exposed primary fetal pulmonary artery endothelial cells (PAEC) isolated from healthy newborn sheep and persistent pulmonary hypertension of the newborn (PPHN) sheep to the growth hormone, insulin-like growth factor 1 (IGF-1). We found that IGF-1 increased proliferation and branch points in tube formation assays but not angiogenic signaling proteins at the population level for both cell types. We hypothesized that this molecular ambiguity was due to the presence of cellular sub-populations with variable responses to IGF-1. Using high throughput single-cell imaging, we discovered a spatially localized response to IGF-1. This suggests localized signaling or heritable cell response to external stimuli may ultimately be responsible for our observations. Discovering and further exploring these rare cells is critical to finding new molecular targets to restore cellular function.

Intrauterine growth restriction decreases NF-κB signaling in fetal pulmonary artery endothelial cells of fetal sheep.

Intrauterine growth restriction (IUGR) in premature newborns increases the risk for bronchopulmonary dysplasia, a chronic lung disease characterized by disrupted pulmonary angiogenesis and alveolarization. We previously showed that experimental IUGR impairs angiogenesis; however, mechanisms that impair pulmonary artery endothelial cell (PAEC) function are uncertain. The NF-κB pathway promotes vascular growth in the developing mouse lung, and we hypothesized that IUGR disrupts NF-κB-regulated proangiogenic targets in fetal PAEC. PAECs were isolated from the lungs of control fetal sheep and sheep with experimental IUGR from an established model of chronic placental insufficiency. Microarray analysis identified suppression of NF-κB signaling and significant alterations in extracellular matrix (ECM) pathways in IUGR PAEC, including decreases in collagen 4α1 and laminin α4, components of the basement membrane and putative NF-κB targets. In comparison with controls, immunostaining of active NF-κB complexes, NF-κB-DNA binding, baseline expression of NF-κB subunits p65 and p50, and LPS-mediated inducible activation of NF-κB signaling were decreased in IUGR PAEC. Although pharmacological NF-κB inhibition did not affect angiogenic function in IUGR PAEC, angiogenic function of control PAEC was reduced to a similar degree as that observed in IUGR PAEC. These data identify reductions in endothelial NF-κB signaling as central to the disrupted angiogenesis observed in IUGR, likely by impairing both intrinsic PAEC angiogenic function and NF-κB-mediated regulation of ECM components necessary for vascular development. These data further suggest that strategies that preserve endothelial NF-κB activation may be useful in lung diseases marked by disrupted angiogenesis such as IUGR.

Antenatal Vitamin D Preserves Placental Vascular and Fetal Growth in Experimental Chorioamnionitis Due to Intra-amniotic Endotoxin Exposure.

BACKGROUND: Chorioamnionitis (CA) is associated with a high risk for the development of bronchopulmonary dysplasia (BPD) after preterm birth, but mechanisms that increase susceptibility for BPD and strategies to prevent BPD are uncertain. As a model of CA, antenatal intra-amniotic (IA) endotoxin (ETX) exposure alters placental structure, causes fetal growth restriction, increases perinatal mortality, and causes sustained cardiorespiratory abnormalities throughout infancy. Vitamin D (Vit D) has been shown to have both anti-inflammatory and proangiogenic properties. Antenatal IA treatment with Vit D (1,25-(OH)2D3) during IA ETX exposure improves survival and increases vascular and alveolar growth in infant rats. Whether IA ETX causes decreased placental vascular development and if the protective effects of prenatal Vit D treatment are due to direct effects on the fetus or to improved placental vascular development remain unknown. OBJECTIVE: The objective of this study was to determine if IA ETX impairs placental vascular development and Vit D metabolism, and whether 1,25-(OH)2D3 treatment improves placental vascularity after IA ETX exposure during late gestation in pregnant rats. DESIGN/METHODS: Fetal rats were exposed to ETX (10 mg), ETX + 1,25-(OH)2D3 (1 ng/mL), 1,25-(OH)2D3 (1 ng/mL), or saline (control) via IA injection at E20 and delivered 2 days later. To assess placental vascular development, histologic sections from the placenta were stained for CD31 and vessel density per high power field (HPF) was determined and analyzed using Matlab software. To determine the effects of ETX on placental Vit D metabolism, Vit D receptor (VDR) and activity of the Vit D conversion enzyme, CYP27B1, were assayed from placental homogenates. Angiogenic mediators were measured by reverse transcription polymerase chain reaction by RNA extracted from placental tissue. RESULTS: IA ETX reduced placenta and newborn birth weights by 22 and 20%, respectively, when compared with controls (placental weight: 0.60 vs. 0.47 g; p < 0.0001; birth weight: 4.68 vs. 5.88 g; p < 0.0001). IA 1,25-(OH)2D3 treatment increased birth weight by 12% in ETX-exposed pups (5.25 vs. 4.68 g; p < 0.001). IA ETX decreased placental vessel density by 24% in comparison with controls (1,114 vs. 848 vessels per HPF; p < 0.05). Treatment with IA 1,25-(OH)2D3 increased placenta vessel density twofold after ETX exposure (1,739 vs. 848); p < 0.0001), and increased vessel density compared with saline controls by 56% (1,739 vs. 1,114; p < 0.0001). IA ETX decreased both VDR and CYP27B1 expression by 83 and 35%, respectively (p < 0.01). CONCLUSION: IA ETX decreases placental growth and vessel density and decreases placental VDR and CYP27B1 protein expression, and that antenatal 1,25-(OH)2D3 restores placental weight and vessel density, as well as birth weight. We speculate that 1,25-(OH)2D3 treatment preserves placental function in experimental CA and that these effects may be mediated by increased vascular growth.

Anti-sFlt-1 Therapy Preserves Lung Alveolar and Vascular Growth in Antenatal Models of Bronchopulmonary Dysplasia.

RATIONALE: Pregnancies complicated by antenatal stress, including preeclampsia (PE) and chorioamnionitis (CA), increase the risk for bronchopulmonary dysplasia (BPD) in preterm infants, but biologic mechanisms linking prenatal factors with BPD are uncertain. Levels of sFlt-1 (soluble fms-like tyrosine kinase 1), an endogenous antagonist to VEGF (vascular endothelial growth factor), are increased in amniotic fluid and maternal blood in PE and associated with CA. OBJECTIVES: Because impaired VEGF signaling has been implicated in the pathogenesis of BPD, we hypothesized that fetal exposure to sFlt-1 decreases lung growth and causes abnormal lung structure and pulmonary hypertension during infancy. METHODS: To test this hypothesis, we studied the effects of anti-sFlt-1 monoclonal antibody (mAb) treatment on lung growth in two established antenatal models of BPD that mimic PE and CA induced by intraamniotic (i.a.) injections of sFlt-1 or endotoxin, respectively. In experimental PE, mAb was administered by three different approaches, including antenatal treatment by either i.a. instillation or maternal uterine artery infusion, or by postnatal intraperitoneal injections. RESULTS: With each strategy, mAb therapy improved infant lung structure as assessed by radial alveolar count, vessel density, right ventricular hypertrophy, and lung function. As found in the PE model, the adverse lung effects of i.a. endotoxin were also reduced by antenatal or postnatal mAb therapy. CONCLUSIONS: We conclude that treatment with anti-sFlt-1 mAb preserves lung structure and function and prevents right ventricular hypertrophy in two rat models of BPD of antenatal stress and speculate that early mAb therapy may provide a novel strategy for the prevention of BPD.

Automatic and adaptive heterogeneous refractive index compensation for light-sheet microscopy.

Optical tissue clearing has revolutionized researchers' ability to perform fluorescent measurements of molecules, cells, and structures within intact tissue. One common complication to all optically cleared tissue is a spatially heterogeneous refractive index, leading to light scattering and first-order defocus. We designed C-DSLM (cleared tissue digital scanned light-sheet microscopy) as a low-cost method intended to automatically generate in-focus images of cleared tissue. We demonstrate the flexibility and power of C-DSLM by quantifying fluorescent features in tissue from multiple animal models using refractive index matched and mismatched microscope objectives. This includes a unique measurement of myelin tracks within intact tissue using an endogenous fluorescent reporter where typical clearing approaches render such structures difficult to image. For all measurements, we provide independent verification using standard serial tissue sectioning and quantification methods. Paired with advancements in volumetric image processing, C-DSLM provides a robust methodology to quantify sub-micron features within large tissue sections.Optical clearing of tissue has enabled optical imaging deeper into tissue due to significantly reduced light scattering. Here, Ryan et al. tackle first-order defocus, an artefact of a non-uniform refractive index, extending light-sheet microscopy to partially cleared samples.

Quantifying three-dimensional rodent retina vascular development using optical tissue clearing and light-sheet microscopy.

Retinal vasculature develops in a highly orchestrated three-dimensional (3-D) sequence. The stages of retinal vascularization are highly susceptible to oxygen perturbations. We demonstrate that optical tissue clearing of intact rat retinas and light-sheet microscopy provides rapid 3-D characterization of vascular complexity during retinal development. Compared with flat mount preparations that dissect the retina and primarily image the outermost vascular layers, intact cleared retinas imaged using light-sheet fluorescence microscopy display changes in the 3-D retinal vasculature rapidly without the need for point scanning techniques. Using a severe model of retinal vascular disruption, we demonstrate that a simple metric based on Sholl analysis captures the vascular changes observed during retinal development in 3-D. Taken together, these results provide a methodology for rapidly quantifying the 3-D development of the entire rodent retinal vasculature.

Endothelin-1-Rho kinase interactions impair lung structure and cause pulmonary hypertension after bleomycin exposure in neonatal rat pups.

Bronchopulmonary dysplasia (BPD) is the chronic lung disease associated with premature birth, characterized by impaired vascular and alveolar growth. In neonatal rats bleomycin decreases lung growth and causes pulmonary hypertension (PH), which is poorly responsive to nitric oxide. In the developing lung, through Rho kinase (ROCK) activation, ET-1 impairs endothelial cell function; however, whether ET-1-ROCK interactions contribute to impaired vascular and alveolar growth in experimental BPD is unknown. Neonatal rats were treated daily with intraperitoneal bleomycin with and without selective ETA (BQ123/BQ610) and ETB (BQ788) receptor blockers, nonselective ET receptor blocker (ETRB) (bosentan), or fasudil (ROCK inhibitor). At day 14, lungs were harvested for morphometrics, and measurements of Fulton's index (RV/LV+S), medial wall thickness (MWT), and vessel density. Lung ET-1 protein and ROCK activity (phospho-MYPT-1:total MYPT-1 ratio) were also measured by Western blot analysis. Bleomycin increased lung ET-1 protein expression by 65%, RV/LV+S by 60%, mean linear intercept (MLI) by 212%, and MWT by 140% and decreased radial alveolar count (RAC) and vessel density by 40 and 44%, respectively (P < 0.01 for each comparison). After bleomycin treatment, fasudil and bosentan partially restored RAC and vessel density and decreased MLI, RV/LV+S, and MWT to normal values. Bleomycin increased ROCK activity by 120%, which was restored to normal values by bosentan but not selective ETRB. We conclude that ET-1-ROCK interactions contribute to decreased alveolar and vascular growth and PH in experimental BPD. We speculate that nonselective ETRB and ROCK inhibitors may be effective in the treatment of infants with BPD and PH.

Increased Lung Expression of Anti-Angiogenic Factors in Down Syndrome: Potential Role in Abnormal Lung Vascular Growth and the Risk for Pulmonary Hypertension.

BACKGROUND AND AIMS: Infants with Down syndrome (DS) or Trisomy 21, are at high risk for developing pulmonary arterial hypertension (PAH), but mechanisms that increase susceptibility are poorly understood. Laboratory studies have shown that early disruption of angiogenesis during development impairs vascular and alveolar growth and causes PAH. Human chromosome 21 encodes known anti-angiogenic factors, including collagen18a1 (endostatin, ES), ß-amyloid peptide (BAP) and Down Syndrome Critical Region 1 (DSCR-1). Therefore, we hypothesized that fetal lungs from subjects with DS are characterized by early over-expression of anti-angiogenic factors and have abnormal lung vascular growth in utero. METHODS: Human fetal lung tissue from DS and non-DS subjects were obtained from a biorepository. Quantitative reverse transcriptase PCR (qRT-PCR) was performed to assay 84 angiogenesis-associated genes and individual qRT-PCR was performed for ES, amyloid protein precursor (APP) and DSCR1. Western blot analysis (WBA) was used to assay lung ES, APP and DSCR-1 protein contents. Lung vessel density and wall thickness were determined by morphometric analysis. RESULTS: The angiogenesis array identified up-regulation of three anti-angiogenic genes: COL18A1 (ES), COL4A3 (tumstatin) and TIMP3 (tissue inhibitor of metallopeptidase 3) in DS lungs. Single qRT-PCR and WBA showed striking elevations of ES and APP mRNA (p = 0.022 and p = 0.001) and protein (p = 0.040 and p = 0.002; respectively). Vessel density was reduced (p = 0.041) and vessel wall thickness was increased in DS lung tissue (p = 0.033) when compared to non-DS subjects. CONCLUSIONS: We conclude that lung anti-angiogenic factors, including COL18A1 (ES), COL4A3, TIMP3 and APP are over-expressed and fetal lung vessel growth is decreased in subjects with DS. We speculate that increased fetal lung anti-angiogenic factor expression due to trisomy 21 impairs lung vascular growth and signaling, which impairs alveolarization and contributes to high risk for PAH during infancy.

Hepatocyte growth factor as a downstream mediator of vascular endothelial growth factor-dependent preservation of growth in the developing lung.

Impaired vascular endothelial growth factor (VEGF) signaling contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). We hypothesized that the effects of VEGF on lung structure during development may be mediated through its downstream effects on both endothelial nitric oxide synthase (eNOS) and hepatocyte growth factor (HGF) activity, and that, in the absence of eNOS, trophic effects of VEGF would be mediated through HGF signaling. To test this hypothesis, we performed an integrative series of in vitro (fetal rat lung explants and isolated fetal alveolar and endothelial cells) and in vivo studies with normal rat pups and eNOS(-/-) mice. Compared with controls, fetal lung explants from eNOS(-/-) mice had decreased terminal lung bud formation, which was restored with recombinant human VEGF (rhVEGF) treatment. Neonatal eNOS(-/-) mice were more susceptible to hyperoxia-induced inhibition of lung growth than controls, which was prevented with rhVEGF treatment. Fetal alveolar type II (AT2) cell proliferation was increased with rhVEGF treatment only with mesenchymal cell (MC) coculture, and these effects were attenuated with anti-HGF antibody treatment. Unlike VEGF, HGF directly stimulated isolated AT2 cells even without MC coculture. HGF directly stimulates fetal pulmonary artery endothelial cell growth and tube formation, which is attenuated by treatment with JNJ-38877605, a c-Met inhibitor. rHGF treatment preserves alveolar and vascular growth after postnatal exposure to SU-5416, a VEGF receptor inhibitor. We conclude that the effects of VEGF on AT2 and endothelial cells during lung development are partly mediated through HGF-c-Met signaling and speculate that reciprocal VEGF-HGF signaling between epithelia and endothelia is disrupted in infants who develop BPD.

VEGF and endothelium-derived retinoic acid regulate lung vascular and alveolar development.

Prevention or treatment of lung diseases caused by the failure to form, or destruction of, existing alveoli, as observed in infants with bronchopulmonary dysplasia and adults with emphysema, requires understanding of the molecular mechanisms of alveolar development. In addition to its critical role in gas exchange, the pulmonary circulation also contributes to alveolar morphogenesis and maintenance by the production of paracrine factors, termed "angiocrines," that impact the development of surrounding tissue. To identify lung angiocrines that contribute to alveolar formation, we disrupted pulmonary vascular development by conditional inactivation of the Vegf-A gene during alveologenesis. This resulted in decreased pulmonary capillary and alveolar development and altered lung elastin and retinoic acid (RA) expression. We determined that RA is produced by pulmonary endothelial cells and regulates pulmonary angiogenesis and elastin synthesis by induction of VEGF-A and fibroblast growth factor (FGF)-18, respectively. Inhibition of RA synthesis in newborn mice decreased FGF-18 and elastin expression and impaired alveolarization. Treatment with RA and vitamin A partially reversed the impaired vascular and alveolar development induced by VEGF inhibition. Thus we identified RA as a lung angiocrine that regulates alveolarization through autocrine regulation of endothelial development and paracrine regulation of elastin synthesis via induction of FGF-18 in mesenchymal cells.