Recent investigations have suggested an active role for endothelial cells in organ development, including the lung. Herein, we investigated some of the molecular mechanisms underlying normal pulmonary vascular development and their influence on epithelial branching morphogenesis. Because the lung in utero develops in a relative hypoxic environment, we first investigated the influence of low oxygen on epithelial and vascular branching morphogenesis. Two transgenic mouse models, the C101-LacZ (epithelial-LacZ marker) and the Tie2-LacZ (endothelial-LacZ marker), were used. At embryonic day 11.5, primitive lung buds were dissected and cultured at either 20 or 3% oxygen. At 24-h intervals, epithelial and endothelial LacZ gene expression was visualized by X-galactosidase staining. The rate of branching of both tissue elements was increased in explants cultured at 3% oxygen compared with 20% oxygen. Low oxygen increased expression of VEGF, but not that of the VEGF receptor (Flk-1). Expression of two crucial epithelial branching factors, fibroblast growth factor-10 and bone morphogenetic protein-4, were not affected by low oxygen. Epithelial differentiation was maintained at low oxygen as shown by surfactant protein C in situ hybridization. To explore epithelial-vascular interactions, we inhibited vascular development with antisense oligonucleotides targeted against either hypoxia inducible factor-1α or VEGF. Epithelial branching morphogenesis in vitro was dramatically abrogated when pulmonary vascular development was inhibited. Collectively, the in vitro data show that a low-oxygen environment enhances branching of both distal lung epithelium and vascular tissue and that pulmonary vascular development appears to be rate limiting for epithelial branching morphogenesis.

The transcriptional inhibitor Snail is a critical regulator for epithelial–mesenchymal transition (EMT). Although low oxygen induces Snail transcription, thereby stimulating EMT, a direct role of hypoxia-inducible factor (HIF) in this process remains to be demonstrated. Here we show that hypoxia induces the expression of Snail via HIF. In silico analysis identified a potential hypoxia-response element (HRE) close to the minimal promoter of the human and mouse genome of the snail gene. Gel shift assays demonstrated that a specific hypoxia-inducible complex is formed with the putative HRE and that the complex contains HIF proteins. ChIP assays confirmed the interaction of HIF proteins with the putative HRE in vivo. Reporter gene analyses showed that the putative HRE responds to hypoxia in its natural position as well as in front of a heterologous promoter and that the HRE is directly activated by HIF-1α or HIF-2α. HIF knockdown with siRNA at 2% oxygen and overexpression of an oxygen-insensitive HIF (HIF-ΔODD) mutant at 21% oxygen showed that HIF regulates Snail activation and subsequent cell migration. Our findings identify snail as a HIF target gene and provide novel insights into the regulation of snail and hypoxia-induced EMT. Mol Cancer Res; 9(2); 234–45. ©2011 AACR.

Efficient differentiation of pluripotent cells to proximal and distal lung epithelial cell populations remains a challenging task. The 3D extracellular matrix (ECM) scaffold is a key component that regulates the interaction of secreted factors with cells during development by often binding to and limiting their diffusion within local gradients. Here we examined the role of the lung ECM in differentiation of pluripotent cells in vitro and demonstrate the robust inductive capacity of the native lung matrix alone. Extended culture of stem cell-derived definitive endoderm on decellularized lung scaffolds in defined, serum-free medium resulted in differentiation into mature airway epithelia, complete with ciliated cells, club cells, and basal cells with morphological and functional similarities to native airways. Heparitinase I, but not chondroitinase ABC, treatment of scaffolds revealed that the differentiation achieved is dependent on heparan sulfate proteoglycans and its bound factors remaining on decellularized scaffolds.

BACKGROUND: Iron is an essential micronutrient for cell survival and growth; however, excess of this metal drives ferroptosis. Although maternal iron imbalance and placental hypoxia are independent contributors to the pathogenesis of preeclampsia, a hypertensive disorder of pregnancy, the mechanisms by which their interaction impinge on maternal and placental health remain elusive. METHODS: We used placentae from normotensive and preeclampsia pregnancy cohorts, human H9 embryonic stem cells differentiated into cytotrophoblast-like cells, and placenta-specific Phd2 ^−/− preeclamptic mice. Lipid peroxidation and iron cargo of placenta-derived small extracellular vesicles (sEVs) isolated from the maternal circulation of control and preeclampsia individuals were examined by mass spectrometry, flow cytometry, and colorimetry. Human microvascular endothelial cells’ angiogenic capacity and function were examined after exposure to control and pathological sEVs. RESULTS: Placentae from preeclampsia pregnancies contain increased ferrous iron and lipid peroxidation byproduct, malondialdehyde. Antioxidant capacity is significantly lower in preeclampsia placentae, with decreased glutathione content, and GPx4 (glutathione peroxidase 4) expression and activity. Hypoxia triggers the occurrence of ferroptosis in human trophoblast cells and mouse Phd2 ^{− /−} placentae. Disrupted placental iron homeostasis in preeclampsia is accompanied by improper extrusion of iron through sEVs mediated by the pentaspan protein prominin-2. Heightened lipid peroxidation content was found in villous explants and maternal circulating sEVs of preeclampsia individuals. Exposure of human microvascular endothelial cells to preeclampsia-derived placental sEVs results in endothelial activation and impaired angiogenesis, which is rescued by treatment with hinokitiol, a compound known to restore tissue iron balance. CONCLUSIONS: In pregnancy, iron and oxygen work synergistically to conserve an operative antioxidant system to maintain iron homeostasis and protect the placenta from ferroptotic death. Hindrance to this system due to hypoxia results in heightened ferroptosis rates and sEV-mediated extrusion of harmful lipid peroxides from trophoblast cells into the circulation thereby contributing to maternal endothelial dysfunction characterizing preeclampsia.

High‐tidal volume ventilation has been shown to increase the expression of several inflammation‐associated genes prior to overt physiologic lung injury. Herein, using an in vitro stretch system, we investigated the mechanotransduction pathways involved in ventilation‐induced expression of these early response genes (i.e., early growth response gene (Egr)1, heat‐shock protein (HSP)70, and the pro‐inflammatory cytokines interleukin (IL)‐1β, IL‐6, and MIP‐2). Mechanical stretch of fetal lung epithelial cells activated various signaling pathways, resulting in transient or progressive increases in gene expression of the early response genes. The transient increase in Egr1 and IL‐6 expression was mediated via p44/42 mitogen‐activated protein kinase (p44/42 MAPK), while nuclear factor‐kappaB (NF‐κB) was responsible for the sustained and progressive increase in expression of HSP70 and MIP‐2. Blockage of Egr‐1 expression did not affect the upregulation of IL‐6, HSP70, MIP‐2, and itself by stretch. Inhibition of calcium mobilization abolished stretch‐induced p44/42 MAPK activation and NF‐κB nuclear translocation as well as increased expression of all early response genes. Similar results were obtained with an inhibitor of Ras. These results suggest that mechanical stretch of fetal lung epithelial cells evokes a complex network of signaling molecules, which diverge downstream to regulate the temporal expression of a unique set of early response genes, but upstream converge at calcium. Thus, calcium mobilization may be a point of hierarchical integration of mechanotransduction in lung epithelial cells. J. Cell. Physiol. 210: 133–143, 2007.