TGF-ß controls alveolar type 1 epithelial cell plasticity and alveolar matrisome gene transcription in mice.

Premature birth disrupts normal lung development and places infants at risk for bronchopulmonary dysplasia (BPD), a disease disrupting lung health throughout the life of an individual and that is increasing in incidence. The TGF-ß superfamily has been implicated in BPD pathogenesis, however, what cell lineage it impacts remains unclear. We show that TGFbr2 is critical for alveolar epithelial (AT1) cell fate maintenance and function. Loss of TGFbr2 in AT1 cells during late lung development leads to AT1-AT2 cell reprogramming and altered pulmonary architecture, which persists into adulthood. Restriction of fetal lung stretch and associated AT1 cell spreading through a model of oligohydramnios enhances AT1-AT2 reprogramming. Transcriptomic and proteomic analyses reveal the necessity of TGFbr2 expression in AT1 cells for extracellular matrix production. Moreover, TGF-ß signaling regulates integrin transcription to alter AT1 cell morphology, which further impacts ECM expression through changes in mechanotransduction. These data reveal the cell intrinsic necessity of TGF-ß signaling in maintaining AT1 cell fate and reveal this cell lineage as a major orchestrator of the alveolar matrisome.

Lung biopsy in infants with severe bronchopulmonary dysplasia.

INTRODUCTION: Lung biopsy is infrequently performed in the population of infants with severe bronchopulmonary dysplasia (BPD). Yet, its presentation may overlap with other infant diffuse lung diseases, including those within the spectrum of childhood interstitial lung diseases (chILD). Lung biopsy might differentiate between these entities or identify those with an extremely poor prognosis. Both might alter the clinical management of some infants diagnosed with BPD. METHODS: In this tertiary referral center, we drew on a retrospective cohort of 308 preterm infants with severe BPD. Of these, nine underwent lung biopsy between 2012 and 2017. We aimed to assess the indication for lung biopsy, the prior clinical history, safety of the procedure, and describe the biopsy findings. Finally, we considered management decisions in relation to the biopsy results in these patients. RESULTS: All nine infants undergoing biopsy survived the procedure. The mean gestational age and birth weight of the nine patients were 30 ± 3 (range 27-34) weeks and 1421 ± 571 (range 611-2140) grams. All infants received serial echocardiograms to assess pulmonary hypertension, genetic testing, and computed tomography angiography (CTA) before biopsy. In all nine patients moderate to severe alveolar simplification was present and eight had some degree of pulmonary interstitial glycogenosis (PIG) ranging from focal to diffuse. Following biopsy, two infants with PIG received high dose systemic steroids and two separate infants had care redirected. CONCLUSION: In our cohort, lung biopsy was safe and well tolerated. Findings from lung biopsy may aid decision making in selected patients as a part of a step-wise diagnostic algorithm.

TGFß controls alveolar type 1 epithelial cell plasticity and alveolar matrisome gene transcription.

Premature birth disrupts normal lung development and places infants at risk for bronchopulmonary dysplasia (BPD), a disease increasing in incidence which disrupts lung health throughout the lifespan. The TGFß superfamily has been implicated in BPD pathogenesis, however, what cell lineage it impacts remains unclear. We show that Tgfbr2 is critical for AT1 cell fate maintenance and function. Loss of Tgfbr2 in AT1 cells during late lung development leads to AT1-AT2 cell reprogramming and altered pulmonary architecture, which persists into adulthood. Restriction of fetal lung stretch and associated AT1 cell spreading through a model of oligohydramnios enhances AT1-AT2 reprogramming. Transcriptomic and proteomic analysis reveal the necessity of Tgfbr2 expression in AT1 cells for extracellular matrix production. Moreover, TGFß signaling regulates integrin transcription to alter AT1 cell morphology, which further impacts ECM expression through changes in mechanotransduction. These data reveal the cell intrinsic necessity of TGFß signaling in maintaining AT1 cell fate and reveal this cell lineage as a major orchestrator of the alveolar matrisome.

Temporal and spatial staging of lung alveolar regeneration is determined by the grainyhead transcription factor Tfcp2l1.

Alveolar epithelial type 2 (AT2) cells harbor the facultative progenitor capacity in the lung alveolus to drive regeneration after lung injury. Using single-cell transcriptomics, software-guided segmentation of tissue damage, and in vivo mouse lineage tracing, we identified the grainyhead transcription factor cellular promoter 2-like 1 (Tfcp2l1) as a regulator of this regenerative process. Tfcp2l1 loss in adult AT2 cells inhibits self-renewal and enhances AT2-AT1 differentiation during tissue regeneration. Conversely, Tfcp2l1 blunts the proliferative response to inflammatory signaling during the early acute injury phase. Tfcp2l1 temporally regulates AT2 self-renewal and differentiation in alveolar regions undergoing active regeneration. Single-cell transcriptomics and lineage tracing reveal that Tfcp2l1 regulates cell fate dynamics across the AT2-AT1 differentiation and restricts the inflammatory program in murine AT2 cells. Organoid modeling shows that Tfcp2l1 regulation of interleukin-1 (IL-1) receptor expression controlled these cell fate dynamics. These findings highlight the critical role Tfcp2l1 plays in balancing epithelial cell self-renewal and differentiation during alveolar regeneration.

Oxygen-mediated lung injury in mice lacking the gene for NRF2: Rescue with the cytochrome P4501A-inducer, beta-naphthoflavone (BNF), and differential sex-specific effects.

BACKGROUND: Acute respiratory distress syndrome (ARDS) leads to progressive lung injury, which significantly impacts patient morbidity and mortality but may differ clinically between the sexes. Cytochrome P450 (CYP) 1A enzymes are protective against hyperoxic lung injury and may contribute to sex-dependent pathology. NRF2 is a critical transcriptional regulator of antioxidants and loss of NRF2 leads to severe hyperoxic lung injury and mortality in mice. NRF2 deficiencies and polymorphisms have been observed in patients with pulmonary diseases such as chronic obstructive pulmonary disease and severe asthma. No prior studies have evaluated whether there are sex-specific differences in oxygen-mediated lung injury in Nrf2-/- mice and there are few rescue studies. OBJECTIVE: To test the hypothesis that hyperoxia induces greater lung injury and inflammation in Nrf2-/- mice compared to wild type (WT) that differs between sexes, and that this phenotype will be rescued by the administration of the cytochrome P450 (CYP) 1A inducer beta-naphthoflavone (BNF). DESIGN/METHODS: Male and female 8-10-week-old WT or Nrf2-/- C57BL/6 mice were pre-treated with BNF (40 mg/kg) or corn oil control and exposed to hyperoxia (95% O2) for 68 h. Survival, pulmonary edema, neutrophil recruitment, and lung injury scores were evaluated. Gene expression of phase II detoxification enzymes, pulmonary cytokines, and Cyp1a1/2 was quantified. CYP1A1/2 protein expression and catalytic activities were also measured. RESULTS: Hyperoxia exposure greatly reduced survival in Nrf2-/- mice, particularly in females. BNF treatment improved survival by 182.8% in Nrf2-/- females and by 41.4% in Nrf2-/- males as well as in WT females by 85.7%. Females had greater pulmonary edema as measured by lung weight to body weight ratios but was attenuated in all groups except Nrf2-/- females by BNF. Neutrophils doubled in Nrf2-/- lungs compared to WT in hyperoxia but were decreased in BNF-treated females of both genotypes. Pulmonary cytokine gene expression including Il-6 and Tnf-α increased in hyperoxia especially in Nrf2-/- mice and was unaffected by BNF. Pulmonary and hepatic Nqo1 gene expression w-as decreased in Nrf2-/- mice and was largely unaffected by BNF; however pulmonary Ho-1 did not vary significantly between the genotypes and was decreased in WT animals treated with BNF. Activities and protein expression of pulmonary and hepatic CYP1A1/2 were induced via BNF across all groups. Although hepatic Cyp1a2 gene expression was higher in Nrf2-/- males, the catalytic activity was higher in Nrf2-/- females. CONCLUSIONS: Hyperoxia augmented lung injury in Nrf2-/- mice, and pre-treatment with BNF was protective against mortality and injury, eliminating the sex-dependent survival difference in both genotypes. Our results support the hypothesis that NRF2 protects mice against lung injury, and the fact that BNF rescues the lung injury phenotype in Nrf2-/- mice suggests that augmented CYP1A expression by BNF may contribute to the beneficial effects. Further studies could lead to the development of BNF and other flavonoids for the prevention/treatment of hyperoxic lung injury, particularly in vulnerable patients with relative NRF2 deficiency, regardless of sex.

Prioritization of Candidate Genes for Congenital Diaphragmatic Hernia in a Critical Region on Chromosome 4p16 using a Machine-Learning Algorithm.

Wolf-Hirschhorn syndrome (WHS) is caused by partial deletion of the short arm of chromosome 4 and is characterized by dysmorphic facies, congenital heart defects, intellectual/developmental disability, and increased risk for congenital diaphragmatic hernia (CDH). In this report, we describe a stillborn girl with WHS and a large CDH. A literature review revealed 15 cases of WHS with CDH, which overlap a 2.3-Mb CDH critical region. We applied a machine-learning algorithm that integrates large-scale genomic knowledge to genes within the 4p16.3 CDH critical region and identified FGFRL1 , CTBP1 , NSD2 , FGFR3 , CPLX1 , MAEA , CTBP1-AS2 , and ZNF141 as genes whose haploinsufficiency may contribute to the development of CDH.

Prematurity disrupts glomeruli development, whereas prematurity and hyperglycemia lead to altered nephron maturation and increased oxidative stress in newborn baboons.

BackgroundPremature birth occurs when nephrogenesis is incomplete and has been linked to increased renal pathologies in the adult. Metabolic factors complicating preterm birth may have additional consequences for kidney development. Here, we evaluated the effects of prematurity and hyperglycemia on nephrogenesis in premature baboons when compared with those in term animals.MethodsBaboons were delivered prematurely (67% gestation; n=9) or at term (n=7) and survived for 2-4 weeks. Preterm animals were classified by glucose control during the first 5 days of life: normoglycemic (PtN; serum glucose 50-100 mg/dl, n=6) and hyperglycemic (PtH; serum glucose 150-250 mg/dl, n=3). Kidneys were assessed histologically for glomeruli relative area, maturity, size, and overall morphology. Kidney lysates were evaluated for oxidative damage with 4-hydroxynonenal (4-HNE) antibody.ResultsHistological examination revealed decreased glomeruli relative area (P<0.05), fewer glomerular generations (P<0.01), and increased renal corpuscle area (P<0.001) in preterm compared with those in term animals. Numbers of apoptotic glomeruli were similar between groups. PtH kidneys exhibited reduced nephrogenic zone width (P<0.0001), increased numbers of mature glomeruli (P<0.05), and increased 4-HNE staining compared with those in PtN kidneys.ConclusionPrematurity interrupts normal kidney development, independent of glomerular cell apoptosis. When prematurity is complicated by hyperglycemia; kidney development shifts toward accelerated maturation and increased oxidative stress.

Reactive oxygen species and oxidative stress in osteoclastogenesis, skeletal aging and bone diseases.

Osteoclasts are cells derived from bone marrow macrophages and are important in regulating bone resorption during bone homeostasis. Understanding what drives osteoclast differentiation and activity is important when studying diseases characterized by heightened bone resorption relative to formation, such as osteoporosis. In the last decade, studies have indicated that reactive oxygen species (ROS), including superoxide and hydrogen peroxide, are crucial components that regulate the differentiation process of osteoclasts. However, there are still many unanswered questions that remain. This review will examine the mechanisms by which ROS can be produced in osteoclasts as well as how it may affect osteoclast differentiation and activity through its actions on osteoclastogenesis signaling pathways. In addition, the contribution of ROS to the aging-associated disease of osteoporosis will be addressed and how targeting ROS may lead to the development of novel therapeutic treatment options.

Caspase-2 modulates osteoclastogenesis through down-regulating oxidative stress.

The loss of caspase-2 (Casp-2) in mice results in an osteopenic phenotype associated with increased numbers of osteoclasts in vivo. In this study, we show that Casp-2 is involved in osteoclastogenesis. Protein levels of Casp-2 decrease during the differentiation of macrophages to osteoclasts. Furthermore, siRNA-mediated Casp-2 knockdown in osteoclast precursors or differentiation of bone marrow macrophage (BMM) precursors from Casp2(-/-) mice results in increased osteoclast numbers and tartrate-resistant acid phosphatase (TRAP) activity. Casp2(-/-) osteoclasts are larger in size compared to wild-type osteoclasts and exhibited increased numbers of nuclei, perhaps due to increased precursor fusion. The loss of Casp-2 did not alter earlier stages of differentiation, but had a greater consequence on later stages involving NFATc1 auto-amplification and pre-osteoclast fusion. We have previously shown that the loss of Casp-2 results in increased oxidative stress in the bone. Reactive oxygen species (ROS) is known to play a critical role in late osteoclast differentiation and we show that total ROS and specifically, mitochondrial ROS, significantly increased in Casp2(-/-) BMM precursors after RANKL administration, with a concomitant reduction in FoxO3a and its target antioxidant enzymes, catalase and superoxide 2 (SOD2). Because mitochondrial ROS has been identified as a putative regulator of the later stages of differentiation, the heightened ROS levels in Casp2(-/-) cells likely promote precursor fusion and increased osteoclast numbers. In conclusion, our results indicate a novel role of Casp-2 in the osteoclast as a modulator of total and mitochondrial ROS and osteoclast differentiation.

A nonapoptotic role for CASP2/caspase 2: modulation of autophagy.

CASP2/caspase 2 plays a role in aging, neurodegeneration, and cancer. The contributions of CASP2 have been attributed to its regulatory role in apoptotic and nonapoptotic processes including the cell cycle, DNA repair, lipid biosynthesis, and regulation of oxidant levels in the cells. Previously, our lab demonstrated CASP2-mediated modulation of autophagy during oxidative stress. Here we report the novel finding that CASP2 is an endogenous repressor of autophagy. Knockout or knockdown of CASP2 resulted in upregulation of autophagy in a variety of cell types and tissues. Reinsertion of Caspase-2 gene (Casp2) in mouse embryonic fibroblast (MEFs) lacking Casp2 (casp2(-/-)) suppresses autophagy, suggesting its role as a negative regulator of autophagy. Loss of CASP2-mediated autophagy involved AMP-activated protein kinase, mechanistic target of rapamycin, mitogen-activated protein kinase, and autophagy-related proteins, indicating the involvement of the canonical pathway of autophagy. The present study also demonstrates an important role for loss of CASP2-induced enhanced reactive oxygen species production as an upstream event in autophagy induction. Additionally, in response to a variety of stressors that induce CASP2-mediated apoptosis, casp2(-/-) cells demonstrate a further upregulation of autophagy compared with wild-type MEFs, and upregulated autophagy provides a survival advantage. In conclusion, we document a novel role for CASP2 as a negative regulator of autophagy, which may provide important insight into the role of CASP2 in various processes including aging, neurodegeneration, and cancer.