Background & Aims-Premature neonates are predisposed to necrotizing enterocolitis (NEC), an idiopathic, inflammatory bowel necrosis. We investigated the hypothesis that NEC occurs in the preterm intestine due to incomplete 'non-inflammatory' differentiation of intestinal macrophages, which increases the risk of a severe mucosal inflammatory response to bacterial products.

Hypoxia causes abnormal neonatal pulmonary artery remodeling (PAR) and inhibition of alveolar development (IAD). Transforming growth factor (TGF)-beta is an important regulator of lung development and repair from injury. We tested the hypothesis that inhibition of TGF-beta signaling attenuates hypoxia-induced PAR and IAD. Mice with an inducible dominant-negative mutation of the TGF-beta type II receptor (DNTGFbetaRII) and nontransgenic wild-type (WT) mice were exposed to hypoxia (12% O(2)) or air from birth to 14 days of age. Expression of DNTGFbetaRII was induced by 20 microg/g ZnSO(4) given intraperitoneally daily from birth. PAR, IAD, cell proliferation, and expression of extracellular matrix (ECM) proteins were assessed. In WT mice, hypoxia led to thicker, more muscularized resistance pulmonary arteries and impaired alveolarization, accompanied by increases in active TGF-beta and phosphorylated Smad2. Hypoxia-induced PAR and IAD were greatly attenuated in DNTGFbetaRII mice given ZnSO(4) compared with WT control mice and DNTGFbetaRII mice not given ZnSO(4). The stimulatory effects of hypoxic exposure on pulmonary arterial cell proliferation and lung ECM proteins were abrogated in DNTGFbetaRII mice given ZnSO(4). These data support the conclusion that TGF-beta plays an important role in hypoxia-induced pulmonary vascular adaptation and IAD in the newborn animal model.

Transforming growth factor (TGF)-beta mediates hypoxia-induced inhibition of alveolar development in the newborn lung. TGF-beta is regulated primarily at the level of activation of latent TGF-beta. Fibroblasts expressing Thy-1 (CD90) inhibit TGF-beta activation. We hypothesized that loss of Thy-1 due to hypoxia may be a mechanism by which hypoxia increases TGF-beta activation and that animals deficient in Thy-1 will simulate the effects of hypoxia on lung development. To determine if loss of Thy-1 occurred during hypoxia, non-transgenic (C57BL/6) wild-type (WT) mice exposed to hypoxia were evaluated for Thy-1 mRNA and protein. To determine if Thy-1 deficiency simulated hypoxia, WT and Thy-1 null (Thy-1(-/-)) mice were exposed to air or hypoxia from birth to 2 wk, the critical period of lung development, and lung histology, function, parameters related to TGF-beta signaling, and extracellular matrix protein content were measured. To test if the phenotype in Thy-1(-/-) mice was due to excessive TGF-beta signaling, measurements were also performed in Thy-1(-/-) mice administered TGF-beta neutralizing antibody (1D11). We observed that hypoxia reduced Thy-1 mRNA and Thy-1 staining in WT mice. Thy-1(-/-) mice had impaired alveolarization, increased TGF-beta signaling, reduced lung epithelial and endothelial cell proliferation but increased fibroblast proliferation, and increased collagen and elastin. Lung compliance was lower, and tissue but not airway resistance was higher in Thy-1(-/-) mice at 2 wk. Thy-1(-/-) mice given 1D11 had improved alveolar development and lung function. These data support the hypothesis that hypoxia, by reducing Thy-1, increases TGF-beta activation, and thereby inhibits normal alveolar development.

Objective To determine the optimal dose of vitamin D supplementation to achieve biochemical vitamin D sufficiency in extremely low gestational age newborns in a masked randomized controlled trial. Study design 100 infants 23 0/7 to 27 6/7 weeks gestation were randomized to vitamin D intakes of placebo (n=36), 200 IU (n=34), and 800 IU/day (n=30) (approximating 200, 400, or 1000 IU/day, respectively, when vitamin D routinely included in parenteral or enteral nutrition is included). The primary outcomes were serum 25 (OH) vitamin D concentrations on postnatal day 28 and the number of days alive and off respiratory support in the first 28 days. Results At birth, 67% of infants had 25(OH) vitamin D < 20 ng/mL suggesting biochemical vitamin D deficiency. Vitamin D concentrations on day 28 were (Median [25th–75th centiles], ng/mL): Placebo: 22 [13–47], 200 IU: 39 [26–57], 800 IU: 84.5 [52–99], p < 0.001. There were no differences in days alive and off respiratory support (Median (25th–75th centiles), days: Placebo: 1 (0–11), 200 IU: 0 (0–8), 800 IU: 0.5 (0–22), p=0.63), or other respiratory outcomes among groups. Conclusion At birth, most extremely preterm infants have biochemical vitamin D deficiency. This biochemical deficiency is reduced on day 28 by supplementation with 200 IU/day and prevented by 800 IU/day. Larger trials are required to determine if resolution of biochemical vitamin D deficiency improves clinical outcomes. ClinicalTrials.gov: NCT01600430 Trial registration ClinicalTrials.gov: NCT01600430

Laser capture microdissection (LCM)-enabled region-specific tissue analyses are critical to better understand complex multicellular processes. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, impacting measurement robustness, quantification and throughput. Here, we coupled LCM with a proteomics workflow that provides fully automated analysis of proteomes from microdissected tissues. Benchmarking against the current state-of-the-art in ultrasensitive global proteomics (FASP workflow), our approach demonstrated significant improvements in quantification (~2-fold lower variance) and throughput (>5 times faster). Using our approach we for the first time characterized, to a depth of >3,400 proteins, the ontogeny of protein changes during normal lung development in microdissected alveolar tissue containing only 4,000 cells. Our analysis revealed seven defined modules of coordinated transcription factor-signaling molecule expression patterns, suggesting a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes.