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.

Women who pre-game: A study of stress and alcohol use in female collegiate predrinkers.

Introduction: This study examined the relationship between stress and pre-gaming (i.e., drinking prior to going out to an event) in female college students. Methods: Thirty-four female college students were grouped as pre-gamers or non-pre-gamers based on self-reported drinking patterns. They completed surveys about alcohol use and mental health and provided a set of salivary cortisol samples upon waking, 30 min later, and at 10am on the same day. Results: Pre-gamers and non-pre-gamers did not differ on demographics or psychosocial variables. Pre-gamers reported riskier drinking overall and had greater endorsement of social, coping, and enhancement drinking motives. Pre-gamers also had lower cortisol levels 30 min after waking and exhibited attenuated CAR. Conclusions: Female collegiate pre-gamers may differ from their peers not only in terms of alcohol consumption and drinking motives, but also on attenuated CAR, a physiological biomarker associated with stress dysregulation and vulnerability to addictive behaviors.

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.

Extreme thrombocytosis is associated with critical illness and young age, but not increased thrombotic risk, in hospitalized pediatric patients.

BACKGROUND: Extreme thrombocytosis (EXT, platelet count > 1000 × 103 /µL) is an uncommon but potentially clinically significant finding. Primary EXT in the setting of myeloproliferative disorders is linked to thrombotic and/or bleeding complications more frequently than secondary EXT, which typically occurs in reaction to infection, inflammation, or iron deficiency. However, comorbidities have been reported in adults with secondary EXT. Clinical implications of EXT in children are not well defined, as prior studies targeted small and/or specialized pediatric populations. OBJECTIVES: Our objectives were to determine etiologies and sequelae of EXT in a hospitalized general pediatric patient population. PATIENTS AND METHODS: We retrospectively analyzed EXT cases from a single-center pediatric cohort of ~80 000 patients over 8 years. RESULTS: Virtually all cases (99.8%) were secondary in nature, and most were multifactorial. Many cases of EXT occurred in children under 2 years old (47%) and/or during critical illness (55%). No thrombotic or bleeding events directly resulted from EXT, confirming a paucity of clinical complications associated with EXT in pediatric patients. There were indications that neonatal hematopoiesis and individual genetic variation influenced some cases, in addition to certain diagnoses (eg, sickle cell anemia) and clinical contexts (eg, asplenia). CONCLUSION: Our findings confirm that thrombotic events related to EXT are rare in pediatric patients, which can inform the use of empiric anti-platelet therapy.

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.

Prevention of carcinogen and inflammation-induced dermal cancer by oral rapamycin includes reducing genetic damage.

Cancer prevention is a cost-effective alternative to treatment. In mice, the mTOR inhibitor rapamycin prevents distinct spontaneous, noninflammatory cancers, making it a candidate broad-spectrum cancer prevention agent. We now show that oral microencapsulated rapamycin (eRapa) prevents skin cancer in dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) carcinogen-induced, inflammation-driven carcinogenesis. eRapa given before DMBA/TPA exposure significantly increased tumor latency, reduced papilloma prevalence and numbers, and completely inhibited malignant degeneration into squamous cell carcinoma. Rapamycin is primarily an mTORC1-specific inhibitor, but eRapa did not reduce mTORC1 signaling in skin or papillomas, and did not reduce important proinflammatory factors in this model, including p-Stat3, IL17A, IL23, IL12, IL1ß, IL6, or TNFα. In support of lack of mTORC1 inhibition, eRapa did not reduce numbers or proliferation of CD45(-)CD34(+)CD49f(mid) skin cancer initiating stem cells in vivo and marginally reduced epidermal hyperplasia. Interestingly, eRapa reduced DMBA/TPA-induced skin DNA damage and the hras codon 61 mutation that specifically drives carcinogenesis in this model, suggesting reduction of DNA damage as a cancer prevention mechanism. In support, cancer prevention and DNA damage reduction effects were lost when eRapa was given after DMBA-induced DNA damage in vivo. eRapa afforded picomolar concentrations of rapamycin in skin of DMBA/TPA-exposed mice, concentrations that also reduced DMBA-induced DNA damage in mouse and human fibroblasts in vitro. Thus, we have identified DNA damage reduction as a novel mechanism by which rapamycin can prevent cancer, which could lay the foundation for its use as a cancer prevention agent in selected human populations.

Connexin 43 channels are essential for normal bone structure and osteocyte viability.

Connexin (Cx) 43 serves important roles in bone function and development. Targeted deletion of Cx43 in osteoblasts or osteocytes leads to increased osteocyte apoptosis, osteoclast recruitment, and reduced biomechanical properties. Cx43 forms both gap junction channels and hemichannels, which mediate the communication between adjacent cells or between cell and extracellular environments, respectively. Two transgenic mouse models driven by a DMP1 promoter with the overexpression of dominant negative Cx43 mutants were generated to dissect the functional contribution of Cx43 gap junction channels and hemichannels in osteocytes. The R76W mutant blocks the gap junction channel, but not the hemichannel function, and the Δ130-136 mutant inhibits activity of both types of channels. Δ130-136 mice showed a significant increase in bone mineral density compared to wild-type (WT) and R76W mice. Micro-computed tomography (µCT) analyses revealed a significant increase in total tissue and bone area in midshaft cortical bone of Δ130-136 mice. The bone marrow cavity was expanded, whereas the cortical thickness was increased and associated with increased bone formation along the periosteal area. However, there is no significant alteration in the structure of trabecular bone. Histologic sections of the midshaft showed increased apoptotic osteocytes in Δ130-136, but not in WT and R76W, mice which correlated with altered biomechanical and estimated bone material properties. Osteoclasts were increased along the endocortical surface in both transgenic mice with a greater effect in Δ130-136 mice that likely contributed to the increased marrow cavity. Interestingly, the overall expression of serum bone formation and resorption markers were higher in R76W mice. These findings suggest that osteocytic Cx43 channels play distinctive roles in the bone; hemichannels play a dominant role in regulating osteocyte survival, endocortical bone resorption, and periosteal apposition, and gap junction communication is involved in the process of bone remodeling.

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.

Caspase-2 maintains bone homeostasis by inducing apoptosis of oxidatively-damaged osteoclasts.

Osteoporosis is a silent disease, characterized by a porous bone micro-structure that enhances risk for fractures and associated disabilities. Senile, or age-related osteoporosis (SO), affects both men and women, resulting in increased morbidity and mortality. However, cellular and molecular mechanisms underlying senile osteoporosis are not fully known. Recent studies implicate the accumulation of reactive oxygen species (ROS) and increased oxidative stress as key factors in SO. Herein, we show that loss of caspase-2, a cysteine aspartate protease involved in oxidative stress-induced apoptosis, results in total body and femoral bone loss in aged mice (20% decrease in bone mineral density), and an increase in bone fragility (30% decrease in fracture strength). Importantly, we demonstrate that genetic ablation or selective inhibition of caspase-2 using zVDVAD-fmk results in increased numbers of bone-resorbing osteoclasts and enhanced tartrate-resistant acid phosphatase (TRAP) activity. Conversely, transfection of osteoclast precursors with wild type caspase-2 but not an enzymatic mutant, results in a decrease in TRAP activity. We demonstrate that caspase-2 expression is induced in osteoclasts treated with oxidants such as hydrogen peroxide and that loss of caspase-2 enhances resistance to oxidants, as measured by TRAP activity, and decreases oxidative stress-induced apoptosis of osteoclasts. Moreover, oxidative stress, quantified by assessment of the lipid peroxidation marker, 4-HNE, is increased in Casp2-/- bone, perhaps due to a decrease in antioxidant enzymes such as SOD2. Taken together, our data point to a critical and novel role for caspase-2 in maintaining bone homeostasis by modulating ROS levels and osteoclast apoptosis during conditions of enhanced oxidative stress that occur during aging.