Nanoparticle Delivery of Proangiogenic Transcription Factors into the Neonatal Circulation Inhibits Alveolar Simplification Caused by Hyperoxia.
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ABSTRACT: Rationale: Advances in neonatal critical care have greatly improved the survival of preterm infants, but the long-term complications of prematurity, including bronchopulmonary dysplasia (BPD), cause mortality and morbidity later in life. Although VEGF (vascular endothelial growth factor) improves lung structure and function in rodent BPD models, severe side effects of VEGF therapy prevent its use in patients with BPD.Objectives: To test whether nanoparticle delivery of proangiogenic transcription factor FOXM1 (forkhead box M1) or FOXF1 (forkhead box F1), both downstream targets of VEGF, can improve lung structure and function after neonatal hyperoxic injury.Methods: Newborn mice were exposed to 75% O2 for the first 7 days of life before being returned to a room air environment. On Postnatal Day 2, polyethylenimine-(5) myristic acid/polyethylene glycol-oleic acid/cholesterol nanoparticles containing nonintegrating expression plasmids with Foxm1 or Foxf1 cDNAs were injected intravenously. The effects of the nanoparticles on lung structure and function were evaluated using confocal microscopy, flow cytometry, and the flexiVent small-animal ventilator.Measurements and Main Results: The nanoparticles efficiently targeted endothelial cells and myofibroblasts in the alveolar region. Nanoparticle delivery of either FOXM1 or FOXF1 did not protect endothelial cells from apoptosis caused by hyperoxia but increased endothelial proliferation and lung angiogenesis after the injury. FOXM1 and FOXF1 improved elastin fiber organization, decreased alveolar simplification, and preserved lung function in mice reaching adulthood.Conclusions: Nanoparticle delivery of FOXM1 or FOXF1 stimulates lung angiogenesis and alveolarization during recovery from neonatal hyperoxic injury. Delivery of proangiogenic transcription factors has promise as a therapy for BPD in preterm infants.
Project description:Dysmorphic pulmonary vascular growth and abnormal endothelial cell (EC) proliferation are paradoxically observed in premature infants with bronchopulmonary dysplasia (BPD), despite vascular pruning. The pentose phosphate pathway (PPP), a metabolic pathway parallel to glycolysis, generates NADPH as a reducing equivalent and ribose 5-phosphate for nucleotide synthesis. It is unknown whether hyperoxia, a known mediator of BPD in rodent models, alters glycolysis and the PPP in lung ECs. We hypothesized that hyperoxia increases glycolysis and the PPP, resulting in abnormal EC proliferation and dysmorphic angiogenesis in neonatal mice. To test this hypothesis, lung ECs and newborn mice were exposed to hyperoxia and allowed to recover in air. Hyperoxia increased glycolysis and the PPP. Increased PPP, but not glycolysis, caused hyperoxia-induced abnormal EC proliferation. Blocking the PPP reduced hyperoxia-induced glucose-derived deoxynucleotide synthesis in cultured ECs. In neonatal mice, hyperoxia-induced abnormal EC proliferation, dysmorphic angiogenesis, and alveolar simplification were augmented by nanoparticle-mediated endothelial overexpression of phosphogluconate dehydrogenase, the second enzyme in the PPP. These effects were attenuated by inhibitors of the PPP. Neonatal hyperoxia augments the PPP, causing abnormal lung EC proliferation, dysmorphic vascular development, and alveolar simplification. These observations provide mechanisms and potential metabolic targets to prevent BPD-associated vascular dysgenesis.
Project description:BackgroundProlonged exposure to high oxygen concentrations in premature infants, although lifesaving, can induce lung oxidative stress and increase the risk of developing BPD, a form of chronic lung disease. The lung alveolar epithelium is damaged by sustained hyperoxia, causing oxidative stress and alveolar simplification; however, it is unclear what duration of exposure to hyperoxia negatively impacts cellular function.MethodsHere we investigated the role of a very short exposure to hyperoxia (95% O2, 5% CO2) on mitochondrial function in cultured mouse lung epithelial cells and neonatal mice.ResultsIn epithelial cells, 4 h of hyperoxia reduced oxidative phosphorylation, respiratory complex I and IV activity, utilization of mitochondrial metabolites, and caused mitochondria to form elongated tubular networks. Cells allowed to recover in air for 24 h exhibited a persistent global reduction in fuel utilization. In addition, neonatal mice exposed to hyperoxia for only 12 h demonstrated alveolar simplification at postnatal day 14.ConclusionA short exposure to hyperoxia leads to changes in lung cell mitochondrial metabolism and dynamics and has a long-term impact on alveolarization. These findings may help inform our understanding and treatment of chronic lung disease.ImpactMany studies use long exposures (up to 14 days) to hyperoxia to mimic neonatal chronic lung disease. We show that even a very short exposure to hyperoxia leads to long-term cellular injury in type II-like epithelial cells. This study demonstrates that a short (4 h) period of hyperoxia has long-term residual effects on cellular metabolism. We show that neonatal mice exposed to hyperoxia for a short time (12 h) demonstrate later alveolar simplification. This work suggests that any exposure to clinical hyperoxia leads to persistent lung dysfunction.
Project description:Supplemental oxygen used to treat infants born prematurely disrupts angiogenesis and is a risk factor for persistent pulmonary disease later in life. Although it is unclear how neonatal oxygen affects development of the respiratory epithelium, alveolar simplification and depletion of type II cells has been observed in adult mice exposed to hyperoxia between postnatal Days 0 and 4. Because hyperoxia inhibits cell proliferation, we hypothesized that it depleted the adult lung of type II cells by inhibiting their proliferation at birth. Newborn mice were exposed to room air (RA) or hyperoxia, and the oxygen-exposed mice were recovered in RA. Hyperoxia stimulated mRNA expressed by type II (Sftpc, Abca3) and type I (T1α, Aquaporin 5) cells and inhibited Pecam expressed by endothelial cells. 5-Bromo-2'-deoxyuridine labeling and fate mapping with enhanced green fluorescence protein controlled statically by the Sftpc promoter or conditionally by the Scgb1a1 promoter revealed increased Sftpc and Abca3 mRNA seen on Day 4 reflected an increase in expansion of type II cells shortly after birth. When mice were returned to RA, this expanded population of type II cells was slowly depleted until few were detected by 8 weeks. These findings reveal that hyperoxia stimulates alveolar epithelial cell expansion when it disrupts angiogenesis. The loss of type II cells during recovery in RA may contribute to persistent pulmonary diseases such as those reported in children born preterm who were exposed to supplemental oxygen.
Project description:Transplantation of endothelial cells (ECs) for therapeutic vascularization or tissue engineering is a promising method for increasing tissue perfusion. Here, we report on a new approach for enhanced EC transplantation using targeted nanoparticle transfection to deliver proangiogenic microRNA-132 (miR-132) to cultured ECs before their transplantation, thereby sensitizing cells to the effects of endogenous growth factors. We synthesized biodegradable PLGA polymer nanoparticles (NPs) that were loaded with miR-132 and coated with cyclic RGD (cRGD) peptides that target integrin ?v?3 expressed on cultured human umbilical vein ECs (HUVECs), increasing NP uptake through clathrin-coated pits. Unlike previously reported NPs for miR delivery, these NPs slowly release RNA for several weeks. The endocytosed NPs remain in clathrin-coated vesicles from which they mediate intracellular delivery of siRNA or miRNA. Transfection of HUVECs with miR-132 enhances growth factor-induced proliferation and migration in 2D culture, producing a 1.8- and 5-fold increase, respectively. However, while the effects of conventional transfection were short-lived, NP transfection produced protein knockdown and biological effects that were significantly longer in duration (? 6 d). Transfection of HUVECs with miR-132 NP resulted in a 2-fold increase in the number of microvessels per square millimeter compared to lipid after transplantation into immunodeficient mice and led to a higher number of mural cell-invested vessels than control transfection. These data suggest that sustained delivery of miR-132 encapsulated in a targeted biodegradable polymer NP is a safe and efficient strategy to improve EC transplantation and vascularization.
Project description:Neonatal hyperoxia impairs vascular and alveolar growth in mice and decreases endothelial progenitor cells. To determine the role of bone marrow-derived cells in restoration of neonatal lung structure after injury, we studied a novel bone marrow myeloid progenitor cell population from Tie2-green fluorescent protein (GFP) transgenic mice (bone marrow-derived angiogenic cells; BMDAC). We hypothesized that treatment with BMDAC would restore normal lung structure in infant mice during recovery from neonatal hyperoxia. Neonatal mice (1-day-old) were exposed to 80% oxygen for 10 days. BMDACs (1 x 10(5)), embryonic endothelial progenitor cells, mouse embryonic fibroblasts (control), or saline were then injected into the pulmonary circulation. At 21 days of age, saline-treated mice had enlarged alveoli, reduced septation, and a reduction in vascular density. In contrast, mice treated with BMDAC had complete restoration of lung structure that was indistinguishable from room air controls. BMDAC comprised 12% of distal lung cells localized to pulmonary vessels or alveolar type II (AT2) cells and persist (8.8%) for 8 wk postinjection. Coculture of AT2 cells or lung endothelial cells (luEC) with BMDAC augmented AT2 and luEC cell growth in vitro. We conclude that treatment with BMDAC after neonatal hyperoxia restores lung structure in this model of bronchopulmonary dysplasia.
Project description:Preterm birth increases the risk for pulmonary hypertension and heart failure in adulthood. Oxygen therapy can damage the immature cardiopulmonary system and may be partially responsible for the cardiovascular disease in adults born preterm. We previously showed that exposing newborn mice to hyperoxia causes pulmonary hypertension by 1 year of age that is preceded by a poorly understood loss of pulmonary vein cardiomyocyte proliferation. We now show that hyperoxia also reduces cardiomyocyte proliferation and survival in the left atrium and causes diastolic heart failure by disrupting its filling of the left ventricle. Transcriptomic profiling showed that neonatal hyperoxia permanently suppressed fatty acid synthase (Fasn), stearoyl-CoA desaturase 1 (Scd1), and other fatty acid synthesis genes in the atria of mice, the HL-1 line of mouse atrial cardiomyocytes, and left atrial tissue explanted from human infants. Suppressing Fasn or Scd1 reduced HL-1 cell proliferation and increased cell death, while overexpressing these genes maintained their expansion in hyperoxia, suggesting that oxygen directly inhibits atrial cardiomyocyte proliferation and survival by repressing Fasn and Scd1. Pharmacologic interventions that restore Fasn, Scd1, and other fatty acid synthesis genes in atrial cardiomyocytes may, thus, provide a way of ameliorating the adverse effects of supplemental oxygen on preterm infants.
Project description:Liver metastasis is a leading cause of cancer morbidity and mortality. Thus, there has been strong interest in the development of therapeutics that can effectively prevent liver metastasis. One potential strategy is to utilize molecules that have broad effects on the liver microenvironment, such as miR-122, a liver-specific miRNA that is a key regulator of diverse hepatic functions. Here we report the development of a nanoformulation miR-122 as a therapeutic agent for preventing liver metastasis. We engineered a galactose-targeted lipid calcium phosphate (Gal-LCP) nanoformulation of miR-122. This nanotherapeutic elicited no significant toxicity and delivered miR-122 into hepatocytes with specificity and high efficiency. Across multiple colorectal cancer liver metastasis models, treatment with Gal-LCP miR-122 treatment effectively prevented colorectal cancer liver metastasis and prolonged survival. Mechanistic studies revealed that delivery of miR-122 was associated with downregulation of key genes involved in metastatic and cancer inflammation pathways, including several proinflammatory factors, matrix metalloproteinases, and other extracellular matrix degradation enzymes. Moreover, Gal-LCP miR-122 treatment was associated with an increased CD8+/CD4+ T-cell ratio and decreased immunosuppressive cell infiltration, which makes the liver more conducive to antitumor immune response. Collectively, this work presents a strategy to improve cancer prevention and treatment with nanomedicine-based delivery of miRNA. SIGNIFICANCE: Highly specific and efficient delivery of miRNA to hepatocytes using nanomedicine has therapeutic potential for the prevention and treatment of colorectal cancer liver metastasis.
Project description:We performed miRNA array analysis from 2 groups (neonatal lung control, neonatal lung after hyperoxia). We used pools of every 100ng of total RNA of three samples for each groups.
Project description:BackgroundPulmonary hypertension (PH) is a common complication in patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with mutations in the FOXF1 gene. Although the loss of alveolar microvasculature causes PH in patients with ACDMPV, it is unknown whether increasing neonatal lung angiogenesis could prevent PH and right ventricular (RV) hypertrophy.MethodsWe used echocardiography, RV catheterization, immunostaining, and biochemical methods to examine lung and heart remodeling and RV output in Foxf1WT/S52F mice carrying the S52F Foxf1 mutation (identified in patients with ACDMPV). The ability of Foxf1WT/S52F mutant embryonic stem cells to differentiate into respiratory cell lineages in vivo was examined using blastocyst complementation. Intravascular delivery of nanoparticles with a nonintegrating Stat3 expression vector was used to improve neonatal pulmonary angiogenesis in Foxf1WT/S52F mice and determine its effects on PH and RV hypertrophy.ResultsFoxf1WT/S52F mice developed PH and RV hypertrophy after birth. The severity of PH in Foxf1WT/S52F mice directly correlated with mortality, low body weight, pulmonary artery muscularization, and increased collagen deposition in the lung tissue. Increased fibrotic remodeling was found in human ACDMPV lungs. Mouse embryonic stem cells carrying the S52F Foxf1 mutation were used to produce chimeras through blastocyst complementation and to demonstrate that Foxf1WT/S52F embryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying Stat3 cDNA protected Foxf1WT/S52F mice from RV hypertrophy and PH, improved survival, and decreased fibrotic lung remodeling.ConclusionsNanoparticle therapies increasing neonatal pulmonary angiogenesis may be considered to prevent PH in ACDMPV.
Project description:Bronchopulmonary dysplasia (BPD), a long-term respiratory morbidity of prematurity, is characterized by attenuated alveolar and vascular development. Supplemental oxygen and immature antioxidant defenses contribute to BPD development. Our group identified thioredoxin reductase-1 (TXNRD1) as a therapeutic target to prevent BPD. The present studies evaluated the impact of the TXNRD1 inhibitor aurothioglucose (ATG) on pulmonary responses and gene expression in newborn C57BL/6 pups treated with saline or ATG (25 mg/kg ip) within 12 h of birth and exposed to room air (21% O2) or hyperoxia (>95% O2) for 72 h. Purified RNA from lung tissues was sequenced, and differential expression was evaluated. Hyperoxic exposure altered ~2,000 genes, including pathways involved in glutathione metabolism, intrinsic apoptosis signaling, and cell cycle regulation. The isolated effect of ATG treatment was limited primarily to genes that regulate angiogenesis and vascularization. In separate studies, pups were treated as described above and returned to room air until 14 days. Vascular density analyses were performed, and ANOVA indicated an independent effect of hyperoxia on vascular density and alveolar architecture at 14 days. Consistent with RNA-seq analyses, ATG significantly increased vascular density in room air, but not in hyperoxia-exposed pups. These findings provide insights into the mechanisms by which TXNRD1 inhibitors may enhance lung development.