Quanfu Mao

Endoglin (CD105) Up-regulation in Pulmonary Microvasculature of Ventilated Preterm Infants

RATIONALE Preterm infants exposed to mechanical ventilation and oxygen are at risk for bronchopulmonary dysplasia (BPD), a multifactorial chronic lung disorder characterized by arrested alveolar development. Studies have described disruption of microvascular development in BPD, characterized by primitive angioarchitectural patterns reminiscent of the canalicular/saccular stages of lung development. The molecular regulation of this BPD-associated dysangiogenesis remains undetermined. OBJECTIVES Endoglin (CD105), a hypoxia-inducible transforming growth factor-beta coreceptor, has been implicated as an important regulator of angiogenesis in various neoplastic and nonneoplastic conditions. The aim of this study was to investigate the expression of endoglin and other angiogenesis-related factors in ventilated preterm human lungs. METHODS We have studied endoglin protein and mRNA expression in postmortem lungs of short-term and long-term ventilated preterm infants. Control subjects were age-matched infants who had lived for less than 1 hour. MEASUREMENTS AND MAIN RESULTS Lungs of short-term ventilated preterm infants showed significant upregulation of endoglin mRNA and protein levels, immunolocalized to the microvasculature. Similar but more variable endoglin upregulation was noted in lungs of long-term ventilated infants with BPD. The mRNA levels of vascular endothelial growth factor, angiopoietin-1, and their respective receptors were significantly lower in ventilated lungs than in age-matched nonventilated control lungs. CONCLUSIONS BPD is associated with a shift from traditional angiogenic growth factors (vascular endothelial growth factor, angiopoietin-1) to alternative regulators such as endoglin, which may contribute to BPD-associated microvascular dysangiogenesis.

Angiogenesis-related gene expression profiling in ventilated preterm human lungs

ABSTRACT Preterm infants exposed to oxygen and mechanical ventilation are at risk for bronchopulmonary dysplasia (BPD), a multifactorial chronic lung disorder characterized by arrested alveolar development and nonsprouting, dysmorphic microvascular angiogenesis. The molecular regulation of this BPD-associated pathological angiogenesis remains incompletely understood. In this study, the authors used focused microarray technology to characterize the angiogenic gene expression profile in postmortem lung samples from short-term ventilated preterm infants (born at 24 to 27 weeks’ gestation) and age-matched control infants. Microarray analysis identified differential expression of 13 of 112 angiogenesis-related genes. Genes significantly up-regulated in ventilated lungs included the antiangiogenic genes thrombospondin-1, collagen XVIII alpha-1, and tissue inhibitor of metalloproteinase-1 (TIMP1), as well as endoglin, transforming growth factor-α, and monocyte chemoattractant protein-1 (CCL2). Increased expression of thrombospondin-1 in ventilated lungs was verified by real-time polymerase chain reaction (PCR) and immunolocalized primarily to intravascular platelets and fibrin aggregates. Down-regulated genes included proangiogenic angiogenin and midkine, as well as vascular endothelial growth factor (VEGF)-B, VEGF receptor-2, and the angiopoietin receptor TEK/Tie-2. In conclusion, short-term ventilated lungs show a shift from traditional angiogenic growth factors to alternative, often antisprouting regulators. This angiogenic shift may be implicated in the regulation of dysmorphic angiogenesis and, consequently, deficient alveolarization characteristic of infants with BPD.

Thyrotropin releasing hormone (TRH) in the hippocampus of Alzheimer patients

Thyrotropin-releasing hormone (TRH) is best known for its hypothalamic neuroendocrine role in regulating thyroid function. In extra-hypothalamic regions in vitro, we have shown TRH to have a protective effect against synaptic loss and neuronal apoptosis. A role for TRH in Alzheimers disease (AD) has not been established previously. In this study, we examined the content of the TRH peptide in the hippocampus of elderly controls (n=5) and AD patients (n=7) by radioimmunoassay (RIA). The TRH concentration was decreased in the AD hippocampus compared to normal elderly controls (p < 0.01). In a separate series of experiments utilizing primary cell cultures made from rat hippocampus, TRH peptide concentration was depleted by the addition of TRH antiserum. TRH withdrawal was found to enhance the activity of glycogen synthetase kinase-3 (GSK-3beta), a critical enzyme necessary for the phosphorylation of tau, as well as the phosphorylation of the tau protein itself. This TRH depletion induced upregulation in phosphorylation that was observed to initiate axonal retraction in cultured neurons. These data suggest that TRH within the hippocampus can regulate the activity of various proteins by phosphorylation/dephosphorylation that may be involved in the pathogenesis of AD.

Fas-Ligand-Induced Apoptosis of Respiratory Epithelial Cells Causes Disruption of Postcanalicular Alveolar Development

Premature infants are at risk for bronchopulmonary dysplasia, a complex condition characterized by impaired alveolar development and increased alveolar epithelial apoptosis. The functional involvement of pulmonary apoptosis in bronchopulmonary dysplasia- associated alveolar disruption remains undetermined. The aims of this study were to generate conditional lung-specific Fas-ligand (FasL) transgenic mice and to determine the effects of FasL-induced respiratory epithelial apoptosis on alveolar remodeling in postcanalicular lungs. Transgenic (TetOp)(7)-FasL responder mice, generated by pronuclear microinjection, were bred with Clara cell secretory protein (CCSP)-rtTA activator mice. Doxycycline (Dox) was administered from embryonal day 14 to postnatal day 7, and lungs were studied between embryonal day 19 and postnatal day 21. Dox administration induced marked respiratory epithelium-specific FasL mRNA and protein up-regulation in double-transgenic CCSP-rtTA(+)/(TetOp)(7)-FasL(+) mice compared with single-transgenic CCSP-rtTA(+) littermates. The Dox-induced FasL up-regulation was associated with dramatically increased apoptosis of alveolar type II cells and Clara cells, disrupted alveolar development, decreased vascular density, and increased postnatal lethality. These data demonstrate that FasL-induced alveolar epithelial apoptosis during postcanalicular lung remodeling is sufficient to disrupt alveolar development after birth. The availability of inducible lung-specific FasL transgenic mice will facilitate studies of the role of apoptosis in normal and disrupted alveologenesis and may lead to novel therapeutic approaches for perinatal and adult pulmonary diseases characterized by dysregulated apoptosis.

Postmortem RNA and protein stability in perinatal human lungs.

The availability of fetal and neonatal lung tissue is an invaluable resource to elucidate the molecular regulation of human lung development. In this study, we have investigated the mRNA and protein stability of perinatal lung tissues treated with RNA later (Ambion Inc., Austin, TX) or snap frozen in liquid nitrogen (LN2). Lung samples were obtained from 25 consecutive perinatal autopsies of live-born and stillborn infants (median gestational age, 23 weeks) with various clinical presentations. Treatment of lung tissue with RNA later yielded more total RNA and protein than LN2 freezing. The integrity of RNA, assessed by spectrophotometry and gel electrophoresis, was equivalent between both tissue preservation methods, and both methods produced RNA suitable for reverse transcriptase–polymerase chain reaction analysis of representative genes (&bgr;-actin and surfactant protein-B [SP-B]). Similarly, the protein integrity of RNA later-treated tissues was equivalent to that of LN2-frozen tissues, as judged by Western blot analysis of SP-B/actin protein expression. Although the total yield was similar in live-born, nonmacerated stillborn and macerated stillborn infants, only RNA and protein from live-born or nonmacerated stillborn infants was suitable for subsequent molecular analyses. Within the 41-hour range studied, the duration of the postmortem interval did not affect the yield or integrity of RNA and protein with either tissue preservation method. In summary, high-quality RNA and protein, suitable for routine molecular analyses, can be obtained from postmortem lung tissue from live-born and nonmacerated stillborn infants, even with prolonged postmortem intervals. RNA later is equivalent, if not superior, to LN2 for preservation of postmortem RNA and protein in developing human lungs.

Alveolar Epithelial Cell Therapy with Human Cord Blood―Derived Hematopoietic Progenitor Cells

The role of umbilical cord blood (CB)-derived stem cell therapy in neonatal lung injury remains undetermined. We investigated the capacity of human CB-derived CD34(+) hematopoietic progenitor cells to regenerate injured alveolar epithelium in newborn mice. Double-transgenic mice with doxycycline (Dox)-dependent lung-specific Fas ligand (FasL) overexpression, treated with Dox between embryonal day 15 and postnatal day 3, served as a model of neonatal lung injury. Single-transgenic non-Dox-responsive littermates were controls. CD34(+) cells (1 × 10(5) to 5 × 10(5)) were administered at postnatal day 5 by intranasal inoculation. Engraftment, respiratory epithelial differentiation, proliferation, and cell fusion were studied at 8 weeks after inoculation. Engrafted cells were readily detected in all recipients and showed a higher incidence of surfactant immunoreactivity and proliferative activity in FasL-overexpressing animals compared with non-FasL-injured littermates. Cord blood-derived cells surrounding surfactant-immunoreactive type II-like cells frequently showed a transitional phenotype between type II and type I cells and/or type I cell-specific podoplanin immunoreactivity. Lack of nuclear colocalization of human and murine genomic material suggested the absence of fusion. In conclusion, human CB-derived CD34(+) cells are capable of long-term pulmonary engraftment, replication, clonal expansion, and reconstitution of injured respiratory epithelium by fusion-independent mechanisms. Cord blood-derived surfactant-positive epithelial cells appear to act as progenitors of the distal respiratory unit, analogous to resident type II cells. Graft proliferation and alveolar epithelial differentiation are promoted by lung injury.

Fate and Effects of Adult Bone Marrow Cells in Lungs of Normoxic and Hyperoxic Newborn Mice

Cell-based therapy in adult lung injury models is associated with highly variable donor cell engraftment and epithelial reconstitution. The role of marrow-derived cell therapy in neonatal lung injury is largely unknown. In this study, we determined the fate and effects of adult bone marrow cells in a model of neonatal lung injury. Wild-type mice placed in a normoxic or hyperoxic (95% O(2)) environment received bone marrow cells from animals expressing green fluorescent protein (GFP) at Postnatal Day (P)5. Controls received vehicle buffer. Lungs were analyzed between Post-Transplantation (TPX) Day 2 and Week 8. The volume of GFP-immunoreactive donor cells, monitored by stereologic volumetry, remained constant between Post-TPX Weeks 1 and 8 and was similar in normoxic and hyperoxia-exposed recipients. Virtually all marrow-derived cells showed colocalization of GFP and the pan-macrophage marker, F4/80, by double immunofluorescence studies. Epithelial transdifferentiation was not seen. Marrow cell administration had adverse effects on somatic growth and alveolarization in normoxic mice, while no effects were discerned in hyperoxia-exposed recipients. Reexposure of marrow-treated animals to hyperoxia at P66 resulted in significant expansion of the donor-derived macrophage population. In conclusion, intranasal administration of unfractionated bone marrow cells to newborn mice does not achieve epithelial reconstitution, but establishes persistent alveolar macrophage chimerism. The predominantly adverse effects of marrow treatment in newborn lungs are likely due to macrophage-associated paracrine effects. While this model and route of cell therapy may not achieve epithelial reconstitution, the role of selected stem cell populations and/or alternate routes of administration for cell-based therapy in injured newborn lungs deserve further investigation.

The Fas System Confers Protection against Alveolar Disruption in Hyperoxia-Exposed Newborn Mice

The functional significance of the Fas/Fas-ligand (FasL) system in hyperoxia-induced lung injury and alveolar disruption in newborn lungs in vivo remains undetermined. To assess the role of the Fas/FasL system, we compared the effects of hyperoxia (95% O2 from birth to Postnatal Day [P]7) in Fas-deficient lpr mice and wild-type mice. Alveolar disruption was more severe in hyperoxic lpr mice than in wild-type mice. In addition, a transient alveolarization defect was noted in normoxic lpr mice. Hyperoxia induced marked up-regulation of pulmonary Fas expression in wild-type mice, as well as elevated mRNA levels of pro-apoptotic Bax, Bad, and Bak. Pulmonary apoptotic activity was similar in hyperoxic wild-type and lpr mice. In contrast, lung growth and proliferation, assessed by stereologic volumetry and Ki67 proliferation studies, were significantly higher in hyperoxic wild-type mice compared with lpr mice, suggesting the Fas/FasL system has a pro-proliferative role in hyperoxic conditions. Levels of the prosurvival MAPkinase, pERK1/2, were significantly higher in hyperoxic wild-type mice compared with lpr mice, while pAkt levels were similar. These data suggest that the primary role of the Fas/FasL system in hyperoxic newborn lungs is pro-proliferative, rather than pro-apoptotic, and likely mediated through a Fas-ERK1/2 pathway. Fas-induced proliferation and lung growth in hyperoxic newborn lungs may counteract, in part, the detrimental effects of apoptosis mediated by non-Fas pathways, such as pro-apoptotic Bax/Bcl-2 family members. The capacity of the Fas/FasL signaling pathway to mediate protective rather than destructive functions in hyperoxic newborn lungs highlights the versatility of this complex pathway.

Efficacy of a Non-Hypercalcemic Vitamin-D2 Derived Anti-Cancer Agent (MT19c) and Inhibition of Fatty Acid Synthesis in an Ovarian Cancer Xenograft Model

Background Numerous vitamin-D analogs exhibited poor response rates, high systemic toxicities and hypercalcemia in human trials to treat cancer. We identified the first non-hypercalcemic anti-cancer vitamin D analog MT19c by altering the A-ring of ergocalciferol. This study describes the therapeutic efficacy and mechanism of action of MT19c in both in vitro and in vivo models. Methodology/Principal Finding Antitumor efficacy of MT19c was evaluated in ovarian cancer cell (SKOV-3) xenografts in nude mice and a syngenic rat ovarian cancer model. Serum calcium levels of MT19c or calcitriol treated animals were measured. In-silico molecular docking simulation and a cell based VDR reporter assay revealed MT19c–VDR interaction. Genomewide mRNA analysis of MT19c treated tumors identified drug targets which were verified by immunoblotting and microscopy. Quantification of cellular malonyl CoA was carried out by HPLC-MS. A binding study with PPAR-Y receptor was performed. MT19c reduced ovarian cancer growth in xenograft and syngeneic animal models without causing hypercalcemia or acute toxicity. MT19c is a weak vitamin-D receptor (VDR) antagonist that disrupted the interaction between VDR and coactivator SRC2-3. Genome-wide mRNA analysis and western blot and microscopy of MT19c treated xenograft tumors showed inhibition of fatty acid synthase (FASN) activity. MT19c reduced cellular levels of malonyl CoA in SKOV-3 cells and inhibited EGFR/phosphoinositol-3kinase (PI-3K) activity independently of PPAR-gamma protein. Significance Antitumor effects of non-hypercalcemic agent MT19c provide a new approach to the design of vitamin-D based anticancer molecules and a rationale for developing MT19c as a therapeutic agent for malignant ovarian tumors by targeting oncogenic de novo lipogenesis.

The human fetal lung xenograft: validation as model of microvascular remodeling in the postglandular lung.

Coordinated remodeling of epithelium and vasculature is essential for normal postglandular lung development. The value of the human‐to‐rodent lung xenograft as model of fetal microvascular development remains poorly defined.