Sharon L. Ryan

Bone marrow-derived angiogenic cells restore lung alveolar and vascular structure after neonatal hyperoxia in infant mice

Neonatal hyperoxia impairs vascular and alveolar growth in mice and decreases endothelial progenitor cells. To determine the role of bone marrow-derived cells in restoration of neonatal lung structure after injury, we studied a novel bone marrow myeloid progenitor cell population from Tie2-green fluorescent protein (GFP) transgenic mice (bone marrow-derived angiogenic cells; BMDAC). We hypothesized that treatment with BMDAC would restore normal lung structure in infant mice during recovery from neonatal hyperoxia. Neonatal mice (1-day-old) were exposed to 80% oxygen for 10 days. BMDACs (1 x 10(5)), embryonic endothelial progenitor cells, mouse embryonic fibroblasts (control), or saline were then injected into the pulmonary circulation. At 21 days of age, saline-treated mice had enlarged alveoli, reduced septation, and a reduction in vascular density. In contrast, mice treated with BMDAC had complete restoration of lung structure that was indistinguishable from room air controls. BMDAC comprised 12% of distal lung cells localized to pulmonary vessels or alveolar type II (AT2) cells and persist (8.8%) for 8 wk postinjection. Coculture of AT2 cells or lung endothelial cells (luEC) with BMDAC augmented AT2 and luEC cell growth in vitro. We conclude that treatment with BMDAC after neonatal hyperoxia restores lung structure in this model of bronchopulmonary dysplasia.

Endothelial Colony-forming Cells from Preterm Infants Are Increased and More Susceptible to Hyperoxia

RATIONALE Preterm birth and hyperoxic exposure increase the risk for bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by impaired vascular and alveolar growth. Endothelial progenitor cells, such as self-renewing highly proliferative endothelial colony-forming cells (ECFCs), may participate in vascular repair. The effect of hyperoxia on ECFC growth is unknown. OBJECTIVES We hypothesize that umbilical cord blood (CB) from premature infants contains more ECFCs with greater growth potential than term CB. However, preterm ECFCs may be more susceptible to hyperoxia. METHODS ECFC colonies were quantified by established methods and characterized by immunohistochemistry and flow cytometry. Growth kinetics were assessed in room air and hyperoxia (FI(O(2)) = 0.4). MEASUREMENTS AND MAIN RESULTS Preterm CB (28-35 wk gestation) yielded significantly more ECFC colonies than term CB. Importantly, we found that CD45(-)/CD34(+)/CD133(+)/VEGFR-2(+) cell number did not correlate with ECFC colony count. Preterm ECFCs demonstrated increased growth compared with term ECFCs. Hyperoxia impaired growth of preterm but not term ECFCs. Treatment with superoxide dismutase and catalase enhanced preterm ECFC growth during hyperoxia. CONCLUSIONS Preterm ECFCs appear in increased numbers and proliferate more rapidly but have an increased susceptibility to hyperoxia compared with term ECFCs. Antioxidants protect preterm ECFCs from hyperoxia.

Cord blood angiogenic progenitor cells are decreased in bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, is associated with impaired vascular and alveolar growth. Antenatal factors contribute to the risk for developing BPD by unclear mechanisms. Endothelial progenitor cells, such as angiogenic circulating progenitor cells (CPCs) and late-outgrowth endothelial colony-forming cells (ECFCs), may contribute to angiogenesis in the developing lung. We hypothesise that cord blood angiogenic CPCs and ECFCs are decreased in preterm infants with moderate and severe BPD. We quantified ECFCs and the CPC/nonangiogenic-CPC ratio (CPC/non-CPC) in cord blood samples from 62 preterm infants and assessed their relationships to maternal and perinatal risk factors as well as BPD severity. The CPC/non-CPC ratio and ECFC number were compared between preterm infants with mild or no BPD and those with moderate or severe BPD. ECFC number (p<0.001) and CPC/non-CPC ratio (p<0.05) were significantly decreased in cord blood samples of preterm infants who subsequently developed moderate or severe BPD. Gestational age and birth weight were not associated with either angiogenic marker. Circulating vascular progenitor cells are decreased in the cord blood of preterm infants who develop moderate and severe BPD. These findings suggest that prenatal factors contribute to late respiratory outcomes in preterm infants.

Endothelial colony-forming cell conditioned media promote angiogenesis in vitro and prevent pulmonary hypertension in experimental bronchopulmonary dysplasia

Late-outgrowth endothelial colony-forming cells (ECFCs), a type of circulating endothelial progenitor cell (EPC), may contribute to pulmonary angiogenesis during development. Cord blood ECFCs from preterm newborns proliferate more rapidly than term ECFCs but are more susceptible to the adverse effects of hyperoxia. Recent studies suggest that bone marrow-derived EPCs protect against experimental lung injury via paracrine mechanisms independent of vascular engraftment. To determine whether human umbilical cord blood ECFCs from preterm and term newborns have therapeutic benefit in experimental neonatal lung injury, we isolated cord blood ECFCs from full-term and preterm newborns and prepared ECFC-conditioned medium (CM) to test its therapeutic benefit on fetal pulmonary artery endothelial cell (PAEC) proliferation and function as well as alveolar type 2 (AT2) cell growth. PAECs and AT2 cells were isolated from late-gestation fetal sheep. Additionally, we administered both ECFCs and ECFC-CM to bleomycin-exposed newborn rats, an experimental model of bronchopulmonary dysplasia (BPD). Both term ECFC-CM and preterm ECFC-CM promoted cell growth and angiogenesis in vitro. However, when ECFC-CM was collected during exposure to mild hyperoxia, the benefit of preterm ECFC-CM was no longer observed. In the bleomycin model of BPD, treatment with ECFC-CM (or CM from mature EC) effectively decreased right ventricular hypertrophy but had no effect on alveolar septation. We conclude that term ECFC-CM is beneficial both in vitro and in experimental BPD. During oxidative stress, preterm ECFC-CM, but not term ECFC-CM, loses its benefit. The inability of term ECFC-CM to promote alveolarization may limit its therapeutic potential.

Adenovirus serotype 5 E1A sensitizes tumor cells to NKG2D-dependent NK cell lysis and tumor rejection

The expression of the Adenovirus serotype 5 (Ad5) E1A oncogene sensitizes tumor cells to natural killer (NK) cell–mediated killing and tumor rejection in vivo. These effects are dependent on the ability of E1A to bind the transcriptional coadaptor protein p300. To test the hypothesis that E1A up-regulates ligands recognized by the NKG2D-activating receptor, we stably transfected the highly tumorigenic mouse fibrosarcoma cell line MCA-205 with Ad5-E1A or a mutant form of E1A that does not interact with p300 (E1A-Δp300). Ad5-E1A, but not E1A-Δp300, up-regulated the expression of the NKG2D ligand retinoic acid early inducible (RAE)-1, but not murine ULBP-like transcript 1, another NKG2D ligand, in four independently derived MCA-205 transfectants. The up-regulation of RAE-1 by E1A targeted MCA-205 tumor cells to lysis by NK cells, resulting in NKG2D-dependent tumor rejection in vivo. Moreover, the up-regulation of NKG2D ligands by E1A was not limited to mouse tumor cells, as E1A also increased the expression of NKG2D ligands on primary baby mouse kidney cells, human MB435S breast cancer cells, and human H4 fibrosarcoma cells.

Adenovirus E1A Oncogene Expression in Tumor Cells Enhances Killing by TNF-Related Apoptosis-Inducing Ligand (TRAIL)

Expression of the adenovirus serotype 5 (Ad5) E1A oncogene sensitizes cells to apoptosis by TNF-α and Fas-ligand. Because TNF-related apoptosis-inducing ligand (TRAIL) kills cells in a similar manner as TNF-α and Fas ligand, we asked whether E1A expression might sensitize cells to lysis by TRAIL. To test this hypothesis, we examined TRAIL-induced killing of human melanoma (A2058) or fibrosarcoma (H4) cells that expressed E1A following either infection with Ad5 or stable transfection with Ad5-E1A. E1A-transfected A2058 (A2058-E1A) or H4 (H4-E1A) cells were highly sensitive to TRAIL-induced killing, but Ad5-infected cells expressing equally high levels of E1A protein remained resistant to TRAIL. Infection of A2058-E1A cells with Ad5 reduced their sensitivity to TRAIL-dependent killing. Therefore, viral gene products expressed following infection with Ad5 inhibited the sensitivity to TRAIL-induced killing conferred by transfection with E1A. E1B and E3 gene products have been shown to inhibit TNF-α- and Fas-dependent killing. The effect of these gene products on TRAIL-dependent killing was examined by using Ad5-mutants that did not express either the E3 (H5dl327) or E1B-19K (H5dl250) coding regions. A2058 cells infected with H5dl327 were susceptible to TRAIL-dependent killing. Furthermore, TRAIL-dependent killing of A2058-E1A cells was not inhibited by infection with H5dl327. Infection with H5dl250 sensitized A2058 cells to TRAIL-induced killing, but considerably less than H5dl327-infection. In summary, expression of Ad5-E1A gene products sensitizes cells to TRAIL-dependent killing, whereas E3 gene products, and to a lesser extent E1B-19K, inhibit this effect.

Hyperoxia disrupts vascular endothelial growth factor-nitric oxide signaling and decreases growth of endothelial colony-forming cells from preterm infants

Exposure of preterm infants to hyperoxia impairs vascular growth, contributing to the development of bronchopulmonary dysplasia and retinopathy of prematurity. Disruption of vascular endothelial growth factor (VEGF)-nitric oxide (NO) signaling impairs vascular growth. Endothelial progenitor cells (EPCs) may play an important role in vascular growth. Endothelial colony-forming cells (ECFCs), a type of EPC, from human preterm cord blood are more susceptible to hyperoxia-induced growth impairment than term ECFCs. Therefore, we hypothesized that hyperoxia disrupts VEGF-NO signaling and impairs growth in preterm ECFCs and that exogenous VEGF or NO preserves growth in hyperoxia. Growth kinetics of preterm cord blood-derived ECFCs (gestational ages, 27-34 wk) were assessed in room air (RA) and hyperoxia (40-50% oxygen) with or without VEGF, NO, or N(omega)-nitro-l-arginine. VEGF, VEGF receptor-2 (VEGFR-2), and endothelial NO synthase (eNOS) protein expression and NO production were compared. Compared with RA controls, hyperoxia significantly decreased growth, VEGFR-2 and eNOS expression, and NO production. VEGF treatment restored growth in hyperoxia to values measured in RA controls and significantly increased eNOS expression in hyperoxia. NO treatment also increased growth in hyperoxia. N(omega)-nitro-l-arginine treatment inhibited VEGF-augmented growth in RA and hyperoxia. We conclude that hyperoxia decreases growth and disrupts VEGF-NO signaling in human preterm ECFCs. VEGF treatment restores growth in hyperoxia by increasing NO production. NO treatment also increases growth during hyperoxia. Exogenous VEGF or NO may protect preterm ECFCs from the adverse effects of hyperoxia and preservation of ECFC function may improve outcomes of preterm infants.

Macrophages Kill Human Papillomavirus Type 16 E6-Expressing Tumor Cells by Tumor Necrosis Factor Alpha- and Nitric Oxide-Dependent Mechanisms

ABSTRACT The expression of adenovirus serotype 2 or 5 (Ad2/5) E1A sensitizes cells to killing by NK cells and activated macrophages, a property that correlates with the ability of E1A to bind the transcriptional coadaptor proteins p300-CBP. The E6 oncoproteins derived from the high-risk human papillomaviruses (HPV) interact with p300 and can complement mutant forms of E1A that cannot interact with p300 to induce cellular immortalization. Therefore, we determined if HPV type 16 (HPV16) E6 could sensitize cells to killing by macrophages and NK cells. HPV16 E6 expression sensitized human (H4 and C33A) and murine (MCA-102) cell lines to lysis by macrophages but not by NK cells. The lysis of cells that expressed E6 by macrophages was p53 independent but dependent on the production of tumor necrosis factor alpha (TNF-α) or nitric oxide (NO) by macrophages. Unlike cytolysis assays with macrophages, E6 expression did not significantly sensitize cells to lysis by the direct addition of NO or TNF-α. Like E1A, E6 has been reported to sensitize cells to lysis by TNF-α by inhibiting the TNF-α-induced activation of NF-κB. We found that E1A, but not E6, blocked the TNF-α-induced activation of NF-κB, an activity that correlated with E1A-p300 binding. In summary, Ad5 E1A and HPV16 E6 sensitized cells to lysis by macrophages. Unlike E1A, E6 did not block the ability of TNF-α to activate NF-κB or sensitize cells to lysis by NK cells, TNF-α, or NO. Thus, there appears to be a spectrum of common and unique biological activities that result as a consequence of the interaction of E6 or E1A with p300-CBP.

Adenovirus E1A, Not Human Papillomavirus E7, Sensitizes Tumor Cells to Lysis by Macrophages Through Nitric Oxide- and TNF-α-Dependent Mechanisms Despite Up-Regulation of 70-kDa Heat Shock Protein

Expression of adenovirus (Ad) serotype 2 or 5 (Ad2/5) E1A or human papillomavirus (HPV)16 E7 reportedly sensitizes cells to lysis by macrophages. Macrophages possess several mechanisms to kill tumor cells including TNF-α, NO, reactive oxygen intermediates (ROI), and Fas ligand (FasL). E1A sensitizes cells to apoptosis by TNF-α, and macrophages kill E1A-expressing cells, in part through the elaboration of TNF-α. However, E1A also up-regulates the expression of 70-kDa heat shock protein, a protein that inhibits killing by TNF-α and NO, thereby protecting cells from lysis by macrophages. Unlike E1A, E7 does not sensitize cells to killing by TNF-α, and the effector mechanism(s) used by macrophages to kill E7-expressing cells remain undefined. The purpose of this study was to further define the capacity of and the effector mechanisms used by macrophages to kill tumor cells that express Ad5 E1A or HPV16 E7. We found that Ad5 E1A, but not HPV16 E7, sensitized tumor cells to lysis by macrophages. Using macrophages derived from mice unable to make TNF-α, NO, ROI, or FasL, we determined that macrophages used NO, and to a lesser extent TNF-α, but not FasL or ROI, to kill E1A-expressing cells. Through the use of S-nitroso-N-acetylpenicillamine, which releases NO upon exposure to an aqueous environment, E1A was shown to directly sensitize tumor cells to NO-induced death. E1A sensitized tumor cells to lysis by macrophages despite up-regulating the expression of 70-kDa heat shock protein. In summary, E1A, but not E7, sensitized tumor cells to lysis by macrophages. Macrophages killed E1A-expressing cells through NO- and TNF-α-dependent mechanisms.

Hepatocyte Growth Factor as a Downstream Mediator of Vascular Endothelial Growth Factor-Dependent Preservation of Growth in the Developing Lung

Impaired vascular endothelial growth factor (VEGF) signaling contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). We hypothesized that the effects of VEGF on lung structure during development may be mediated through its downstream effects on both endothelial nitric oxide synthase (eNOS) and hepatocyte growth factor (HGF) activity, and that, in the absence of eNOS, trophic effects of VEGF would be mediated through HGF signaling. To test this hypothesis, we performed an integrative series of in vitro (fetal rat lung explants and isolated fetal alveolar and endothelial cells) and in vivo studies with normal rat pups and eNOS(-/-) mice. Compared with controls, fetal lung explants from eNOS(-/-) mice had decreased terminal lung bud formation, which was restored with recombinant human VEGF (rhVEGF) treatment. Neonatal eNOS(-/-) mice were more susceptible to hyperoxia-induced inhibition of lung growth than controls, which was prevented with rhVEGF treatment. Fetal alveolar type II (AT2) cell proliferation was increased with rhVEGF treatment only with mesenchymal cell (MC) coculture, and these effects were attenuated with anti-HGF antibody treatment. Unlike VEGF, HGF directly stimulated isolated AT2 cells even without MC coculture. HGF directly stimulates fetal pulmonary artery endothelial cell growth and tube formation, which is attenuated by treatment with JNJ-38877605, a c-Met inhibitor. rHGF treatment preserves alveolar and vascular growth after postnatal exposure to SU-5416, a VEGF receptor inhibitor. We conclude that the effects of VEGF on AT2 and endothelial cells during lung development are partly mediated through HGF-c-Met signaling and speculate that reciprocal VEGF-HGF signaling between epithelia and endothelia is disrupted in infants who develop BPD.