R. N. N. Han

Defective lung vascular development and fatal respiratory distress in endothelial NO synthase-deficient mice: A model of alveolar capillary dysplasia?

Abstract— Endothelium-derived NO plays a critical role in the regulation of cardiovascular function and structure, as well as acting as a downstream mediator of the angiogenic response to numerous vascular growth factors. Although endothelial NO synthase (eNOS)–deficient mice are viable, minor congenital cardiac abnormalities have been reported and homozygous offspring exhibit high neonatal mortality out of proportion to the severity of these defects. The aim of the present report was to determine whether abnormalities of the pulmonary vascular development could contribute to high neonatal loss in eNOS-deficient animals. We now report that eNOS-deficient mice display major defects in lung morphogenesis, resulting in respiratory distress and death within the first hours of life in the majority of animals. Histological and molecular examination of preterm and newborn mutant lungs demonstrated marked thickening of saccular septae, with evidence of reduced surfactant material. Lungs of eNOS-deficient mice also exhibited a striking paucity of distal arteriolar branches and extensive regions of capillary hypoperfusion, together with misalignment of pulmonary veins, which represent the characteristic features of alveolar capillary dysplasia. We conclude that eNOS plays a previously unrecognized role in lung development, which may have relevance for clinical syndromes of neonatal respiratory distress.

Changes in structure, mechanics, and insulin-like growth factor-related gene expression in the lungs of newborn rats exposed to air or 60% oxygen.

Exposure of neonatal rats to ≥95% O2 for 2 wk, a widely used model of oxidant/antioxidant interactions in neonatal lung injury, results in arrested lung growth without the dysplastic lesions observed in chronic human neonatal lung injury. To determine whether dysplastic lung cell growth would be seen at lesser O2 concentrations, we exposed newborn rats to either 95% O2 for 1 wk followed by 60% O2 for 1 wk, or to 60% O2 for 2 wk. Exposure to 95% O2 for 1 wk profoundly inhibited lung DNA synthesis. Recovery of synthesis did not occur during the 2nd wk in 60% O2, nor were areas of dysplastic growth evident in lung tissue. In contrast, a continuous 2-wk exposure to 60% O2 resulted in a slight increase in lung weight with a significant reduction in lung volume over a range of inflation pressures. Also seen was an overall, but inhomogeneous, reduction in lung cell DNA synthesis. A preliminary analysis of affected cell types suggested that inhibition of DNA synthesis affected endothelial cells more than interstitial cells, whereas DNA synthesis increased in type II pneumocytes. Areas of reduced DNA synthesis were interspersed with patchy areas of parenchymal thickening and active DNA synthesis. These areas of parenchymal thickening, but not other areas, had increased immunoreactive IGF-I and the type I IGF receptor. These data are consistent with a direct effect of O2 on growth factor and growth factor receptor expression in causing dysplastic lung cell growth in chronic neonatal lung injury.

Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects sperm from DNA damage and apoptosis caused by benzo(a)pyrene☆

Benzo(a)pyrene (BaP), an aryl hydrocarbon receptor (AhR) ligand present in cigarette smoke and car exhaust, is thought to have negative effects on male reproduction. We hypothesized that BaP damages sperm through AhR activation, phase I enzyme induction, DNA adduct formation, and increased germ cell apoptosis in the testis, and that resveratrol, a natural competitive inhibitor of the AhR found in some red wines, could prevent the adverse effects of BaP on sperm. Male Balb C mice were injected subcutaneously (s.c.) for 5 weeks with a range of BaP doses (0.5 mg/kg to 50 mg/kg). Live sperm were obtained from the vas deferens, counted, and stained to measure annexin-V positive (apoptotic) cells. In a subsequent study, mice were injected for 5 weeks with corn oil (control), BaP (5 mg/kg/week), or BaP plus resveratrol (50 mg/kg/week) (n = 3 per group). Immunohistochemistry (IHC) was performed on testis sections for the determination of CYP1A1, BaP diol epoxide (BPDE) DNA adducts, and apoptosis and the results quantified by using the HSCORE, a semiquantitative scoring system. Our results demonstrated that sperm counts after 5 weeks were inversely correlated to BaP dosage. BaP (0.5 to 5 mg/week) positively correlated with sperm apoptosis while higher doses increased sperm necrosis. CYP1A1 protein was observed mainly in interstitial cells of some testis sections, but there was no significant induction by BaP. BPDE DNA adducts were induced in all components of the seminiferous tubules by BaP and suppressed by resveratrol: median HSCORE (interquartile range) control 61 (52-71.5); BaP 213 (192-248), P = 0.01 compared to control; BaP plus resveratrol 83 (70-90). BaP significantly increased apoptosis, mainly in spermatogonia: medain HSCORE (interquartile range) BaP 189 (161-223) versus control 83 (57-93), P < 0.01; and this effect was abrogated by resveratrol. Median HSCORE for BaP plus resveratrol was 112 (range 99-121). In summary, BaP caused increased sperm cell BPDE DNA adduct formation and apoptosis in the mouse. The natural AhR antagonist, resveratrol diminished BaP-induced DNA adducts and apoptosis in seminiferous tubules.

Expression of Basic Fibroblast Growth Factor and Receptor: Immunolocalization Studies in Developing Rat Fetal Lung

ABSTRACT: To study the role of basic fibroblast growth factor (bFGF) in fetal lung development, the distribution of bFGF peptide and FGF receptor (FGF-R) was examined by immunohistochemistry in embryonic and fetal rat lung [d 12 to term (term = 22 d)). Throughout development bFGF was localized to airway epithelial cells, their basement membranes, and their extracellular matrix. FGF-R was also detected in airway epithelial cells, especially in the branching areas, and in interstitial cells as early as d 13. The number of FGF-R immunoreactive cells increased during the embryonic and pseudoglandular stages of lung development, followed by fluctuations in reactivity during the canalicular stage. No FGF-R was detected in tissue from the saccular stage of lung development. The presence of bFGF and FGF-R in developing airway epithelium and mesenchyme is compatible with a role for this growth factor during fetal lung development. In the developing lung, bFGF seems to be sequestered and stored in the extracellular matrix, and may be released at times of need. Furthermore, FGF-R up- and down-regulation offers another mechanism by which the growth of specific cell populations may be controlled during fetal lung development.

Platelet-Derived Growth Factors and Growth-Related Genes in Rat Lung. III. Immunolocalization during Fetal Development

ABSTRACT: To further study the role of platelet-derived growth factor (PDGF) in fetal lung development, the distribution of the PDGF homodimers PDGF-AA and PDGF-BB was examined by immunohistochemistry in embryonic and fetal rat lung from d 12 to 22 of gestation (term = 22 d). PDGF-AA and PDGF-BB were localized to airway epithelial cells as early as d 12 of gestation, 2 d before their appearance in mesenchymal cells. Both PDGF homodimer immunoreactivities increased until the late pseudoglandular stage of lung development, followed by fluctuations in reactivity during the canalicular stage. Only weak immunoreactivity to either PDGF homodimer was evident during the saccular stage of lung development. Immunodetection by Western blotting revealed that PDGF-AA and PDGF-BB homodimer protein concentrations were high during the embryonic and pseudoglandular stage of lung development and decreased with advancing gestation. We conclude that the presence of PDGF in both developing airway epithelial cells and mesenchymal cells, as well as gestation-dependent changes of PDGF homodimers, is compatible with a role for this growth factor during fetal lung development.

Focal adhesion kinase is a key mediator of human trophoblast development.

Trophoblast differentiation during the first trimester of pregnancy involves cell proliferation and invasion and extracellular matrix (ECM) remodeling. Reports have indicated that, in a variety of cell types, processes such as proliferation, invasion, and ECM remodeling require the turnover of focal adhesions mediated by a cytoplasmic tyrosine kinase named focal adhesion kinase (FAK). Therefore, in the present study we examined the expression and spatial localization of FAK during early human placental development. Immunocytochemical and immunoblot analysis showed that FAK and a focal adhesion-associated protein named paxillin were highly expressed between the 5th and 8th weeks of gestation, specifically in villous cytotrophoblast and extravillous trophoblast (EVT) cells. Activated FAK, phosphorylated on Tyr-397, colocalized with α5 integrin and matrix metalloproteinase-2 (MMP2) expression in EVT cells within a previously characterized intermediate, invasive-restrained region. FAK and paxillin expression dramatically decreased after 10 to 12 weeks of gestation coincident with increasing pO2 levels. Exposure of human villous explants of 5 to 8 weeks to a 3% O2 environment resulted in increased trophoblast outgrowth, cell proliferation, and detection of α5 integrin and MMP2, as well as increased activation of FAK in EVT cells compared with explants grown in a 20% O2 environment. To determine whether FAK was a key requisite for trophoblast differentiation, villous explants of 5 weeks gestation were grown in Matrigel in a 3% O2 environment and incubated with 20-mer antisense FAK oligonucleotides. A dramatic reduction of trophoblast outgrowth was observed in antisense-treated explants compared with missense and control cultures, and, in addition, cell proliferation and MMP2 activity in antisense-treated explants were dramatically reduced. These data suggest that FAK is a key kinase involved in early trophoblast cell differentiation and plays a role in regulating cell proliferation and motility during early placental development.

Alpha 1-antitrypsin protects neonatal rats from pulmonary vascular and parenchymal effects of oxygen toxicity.

ABSTRACT: We investigated whether α1-antitrypsin (α1-AT) might protect neonatal rats from the pulmonary parenchymal and vascular effects resulting from hyperoxic exposure. Neonatal rats born into and maintained in hyperoxia (60% fraction of inspired oxygen) or room air were injected with a loading dose of α1AT (72 mg/kg) followed by 36 mg/kg every 72 h or with vehicle during the first 14 d of life. At the end of the experimental period, we measured body weight, lung compliance, lung volume, alveoli per mm2, and total number of alveoli and assessed right ventricular hypertrophy and vascular changes consisting of medial hypertrophy, muscular extension into peripheral, normally nonmuscular arteries, and number of peripheral arteries relative to alveoli. Our data show that α1-AT treatment prevented the reduced lung compliance observed in the untreated hyperoxia- exposed neonatal rats, as well as the right ventricular hypertrophy and the associated vascular changes of medial hypertrophy of muscular arteries and muscularization of distal arteries. Reduced lung compliance in the hyperoxic but α1-AT-untreated rats was associated with a reduction in lung elastin compared with room-air or α1-AT-treated rats. In room-air rats, α1-AT treatment increased lung compliance but also reduced the number of arteries relative to the number of alveoli, a feature that was not, however, associated with right ventricular hypertrophy. Our data suggest that supplemental α1-AT might restore the imbalance in elastolytic activity induced by hyperoxia and thereby alleviate the toxic effects on lung parenchymal and vascular development.

Changes in expression of platelet-derived growth factor and its receptors in the lungs of newborn rats exposed to air or 60 % O2

PDGF-related gene expression has been well characterized during fetal rat lung development and adult rat lung injury, but not during normal postnatal lung growth or injury. Lung expression of the mRNA for PDGF-A, -B, -αR, and -βR and immunoreactive PDGF-AA, -BB, -αR, and -βR were assessed in rat pups raised in air or 60% O2 for up to 14 d after birth. Expression of mRNA and immunoreactive ligand did not correlate for pups raised in air. Immunoreactive PDGF-αR and -βR, but not PDGF-AA and -BB, were evident throughout the lung at birth. Both PDGF-AA and -BB were evident in airway epithelium, PDGF-BB in alveolar epithelial cells and PDGF-AA was widely distributed in parenchymal tissue at 4 d. PDGF-αR was localized to airway epithelium, and PDGF-βR to subendothelial perivascular regions and to airway and alveolar epithelium at 4 d. Immunoreactive PDGF ligands all declined after 4 d. Intraperitoneal injection of neutralizing antibodies or truncated soluble receptors to PDGF-BB reduced lung DNA synthesis in air. Exposure to 60% O2 significantly increased mRNA for PDGF-B, -βR, and -αR, but not PDGF-A, relative to air-exposed lung at various time points after birth. PDGF-A, -B, and -αR immunoreactivities in these lungs were reduced and delayed, consistent with a global inhibition of lung growth. Pups exposed to 60% O2 had a similar distribution of PDGF-βR to that seen in air, except that at 14 d PDGF-βR was distributed throughout the lung parenchyma. We conclude that PDGF ligands and receptors are important for normal postnatal lung growth and that their expression is delayed by O2 exposure.

Growth of distal fetal rat lung epithelial cells in a defined serum-free medium.

SummaryFetal rat distal lung epithelial cells, in contrast to adult type II pneumocytes, will divide readily in culture in the presence of 10% (vol:vol) fetal bovine serum. The presence of serum makes purification of uncontaminated cell-derived growth factors difficult and modifies cellular responses to oxidant injury. We report the development of a defined serum-free medium that will support growth of fetal distal lung epithelial cells in primary culture. Initial studies used a low-serum (2%; vol:vol) to determine the effect of basal media, substrata, and various additives. Subsequent studies demonstrated growth on a poly-d-lysine substratum under serum-free culture conditions in Dulbecco’s modified minimal essential medium with insulin (50 µg/ml), endothelial cell growth supplement (20 µg/ml), bovine pituitary extract (100 µg/ml), bovine serum albumin (50 µg/ml), selenous acid (4 ng/ml), reduced glutathione (500 ng/ml), soybean trypsin inhibitor (100 µg/ml), transferrin (5 µg/ml), HEPES buffer (2.6 mg/ml), and cholera toxin (5 µg/ml). Growth was enhanced by reducing the gas phase oxygen concentration from 21 to 3%. The undefined components of this medium, bovine pituitary extract and endothelial cell growth supplement, could be replaced by platelet-derived growth factor (20 ng/ml) with prostaglandin E1 (25 nM). The response of fetal distal lung epithelial cells to known growth factors differs substantially from that observed with type II pneumocytes from adult lung and is similar in many, though not all, respects to the responses reported for proximal airway cells from adult lung.

Posttranscriptional Regulation of Human Leukocyte Antigen G During Human Extravillous Cytotrophoblast Differentiation

Abstract Human maternal tolerance to a semiallogenic fetus may be maintained, in part, by the unusual expression pattern of antigen-presenting molecules in placental trophoblast cells. Extravillous cytotrophoblast (EVC) cells, which invade the maternal decidua, express high levels of human leukocyte antigen G (HLA-G), a nonclassical, major histocompatibility complex (MHC) class I molecule. HLA-G transcripts have been detected in tumors and other tissues, yet protein accumulation is rare. We show that, within EVC cells themselves, the mRNA is more broadly expressed than the protein. Specifically, accumulation of HLA-G protein was markedly delayed during EVC cell differentiation. To elucidate this mechanism, we performed a comprehensive analysis comparing the expression of HLA-G and proteins essential for MHC class I expression at the cell surface. The transporter for antigen processing proteins TAP1 and TAP2, as well as tapasin and β2-microglobulin, appeared to be coordinately expressed throughout EVC cell columns. Strikingly, they all accumulated well in advance of the HLA-G protein but concurrently with its mRNA. A similar delay in the accumulation of the HLA-G protein was observed in vitro, using cultures of chorionic villi. We conclude that posttranscriptional regulation of HLA-G is fundamental to EVC cell development and is achieved independently of the peptide loading system. This represents a novel mechanism of MHC class I regulation.