Sonic hedgehog is required for cardiac outflow tract and neural crest cell development

The Hedgehog signaling pathway is critical for a significant number of developmental patterning events. In this study, we focus on the defects in pharyngeal arch and cardiovascular patterning present in Sonic hedgehog (Shh) null mouse embryos. Our data indicate that, in the absence of Shh, there is general failure of the pharyngeal arch development leading to cardiac and craniofacial defects. The cardiac phenotype results from arch artery and outflow tract patterning defects, as well as abnormal development of migratory neural crest cells (NCCs). The constellation of cardiovascular defects resembles a severe form of the human birth defect syndrome tetralogy of Fallot with complete pulmonary artery atresia. Previous studies have demonstrated a role for Shh in NCC survival and proliferation at later stages of development. Our data suggest that SHH signaling does not act directly on NCCs as a survival factor, but rather acts to restrict the domains that NCCs can populate during early stages (e8.5-10.5) of cardiovascular and craniofacial development.     

Highly specific expression of luciferase gene in lungs of naïve nude mice directed by prostate-specific antigen promoter

PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10(9)PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.     

AdTGF-β1223/225气管内注射制备大鼠肺间质纤维化动物模型

目的观察TGF-β1基因过表达诱发大鼠肺间质纤维化动物模型的组织病理学变化.方法成年健康SD大鼠30只,随机分为空白对照组10只,模型组10只和空载体对照组10只,其中对模型组大鼠气管内一次性注射携带TGF-β1基因腺病毒载体(AdTGF-β1 223/225,1×109 pfu,0.4 mL),空白对照组大鼠气管内注射等量的生理盐水,空载体对照组大鼠气管内注射等量的腺病毒空载体.结果一次性肺气管内注射 AdTGF-β1 223/225可造成肺急性炎症和肺间质纤维化,而对照(腺病毒空载体)并不引起相应的变化.结论肺组织局部分泌的细胞因子TGF-β1可以单独诱发肺间质纤维化.     

成人不典型肺结核的CT 表现及鉴别诊断分析( 附30 例报告)

目的：回顾性分析成人不典型肺结核的CT表现,探讨与其他同影性疾病的鉴别方法,做到早诊断早治疗,减低病人的治疗成本和思想负担。方法：选择性收集病变不典型。CT影像检壹曾误诊或诊断困难的病例30例做回顾性分析。所有病例均有最后治疗结果或术后病理结果证实。结果：节段性阴影12例,局灶性斑片及斑点状影8例,散在少数小结节样影4例,多发结节并空洞影3例,肺内弥漫分布的粟粒结节影3例,肺内阴影伴有肺门淋巴结及纵隔淋巴结肿大者9例以青壮年病人吸收较快,病程较短,以粟粒影和大片影表现者吸收最快。讨论：对于不典型的成人肺结核,不能片面的强调或突出某一方面征象,年龄因素、临床症状、实验室检查如结核茵素试验（PPD）、痰结核茵培养、抗结核抗体阳性在鉴别诊断中有着重要意义。进一步正确认识及探究病变的影像特点,对于正确的诊断提供了强有力的理论根据。     

Activation of Glucose-6-phosphate Dehydrogenase Promotes Acute Hypoxic Pulmonary Artery Contraction

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to a decrease in airway O₂ tension, but the underlying mechanism is incompletely understood. We studied the contribution of glucose-6-phosphate dehydrogenase (Glc-6-PD), an important regulator of NADPH redox and production of reactive oxygen species, to the development of HPV. We found that hypoxia (95% N₂, 5% CO₂) increased contraction of bovine pulmonary artery (PA) precontracted with KCl or serotonin. Depletion of extracellular glucose reduced NADPH, NADH, and HPV, substantiating the idea that glucose metabolism and Glc-6-PD play roles in the response of PA to hypoxia. Our data also show that inhibition of glycolysis and mitochondrial respiration (indicated by an increase in NAD⁺ and decrease in the ATP-to-ADP ratio) by hypoxia, or by inhibitors of pyruvate dehydrogenase or electron transport chain complexes I or III, increased generation of reactive oxygen species, which in turn activated Glc-6-PD. Inhibition of Glc-6-PD decreased Ca²⁺ sensitivity to the myofilaments and diminished Ca²⁺-independent and -dependent myosin light chain phosphorylation otherwise increased by hypoxia. Silencing Glc-6-PD expression in PA using a targeted small interfering RNA abolished HPV and decreased extracellular Ca²⁺-dependent PA contraction increased by hypoxia. Similarly, Glc-6-PD expression and activity were significantly reduced in lungs from Glc-6-PD^mut(−/−) mice, and there was a corresponding reduction in HPV. Finally, regression analysis relating Glc-6-PD activity and the NADPH-to-NADP⁺ ratio to the HPV response clearly indicated a positive linear relationship between Glc-6-PD activity and HPV. Based on these findings, we propose that Glc-6-PD and NADPH redox are crucially involved in the mechanism of HPV and, in turn, may play a key role in increasing pulmonary arterial pressure, which is involved in the development of pulmonary hypertension.     

Loss of βarrestin1 and βarrestin2 contributes to pulmonary hypoplasia and neonatal lethality in mice

Less is known about the connection between the malfunction of βarrestins and developmental defects as the mice with either of two βarrestin isoforms knockout appear normal. In order to address the biological function of βarrestins during developmental process, we generate βarrestin1/2 double knockout mice. We found that βarrestin1/2 dual-null mice developed respiratory distress and atelectasis that subsequently caused neonatal death. Morphological examination revealed type II pneumocyte immaturity. Our results indicate that not only βarrestin1/2 double knockout lung tissue show disturbances in cell proliferation but βarrestin1 and βarrestin2 contribute to pulmonary surfactant complex generation during pulmonary maturation. Intra-amniotic delivery of recombinant adenovirus expressing βarrestin1 or βarrestin2 enhances surfactant-associated proteins synthesis in vivo. Our mRNA microarray data further reveal that βarrestin1/2 double knockout results in downregulation of a significant proportion of genes involved in several lung morphogenesis processes. Together, our study demonstrates that βarrestin1 and βarrestin2 collaborate in embryonic development processes for epithelial pneumocyte differentiation and lung maturation.     

Pulmonary Collectins Protect Macrophages against Pore-forming Activity of Legionella pneumophila and Suppress Its Intracellular Growth

Pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), play important roles in innate immunity of the lung. Legionella pneumophila is a bacterial respiratory pathogen that can replicate within macrophages and causes opportunistic infections. L. pneumophila possesses cytolytic activity, resulting from insertion of pores in the macrophage membrane upon contact. We examined whether pulmonary collectins play protective roles against L. pneumophila infection. SP-A and SP-D bound to L. pneumophila and its lipopolysaccharide (LPS) and inhibited the bacterial growth in a Ca²⁺-dependent manner. The addition of LPS in the culture blocked the inhibitory effects on L. pneumophila growth by the collectins, indicating the importance of LPS-collectin interaction. When differentiated THP-1 cells were infected with L. pneumophila in the presence of SP-A and SP-D, the number of permeable cells was significantly decreased, indicating that pulmonary collectins inhibit pore-forming activity of L. pneumophila. The number of live bacteria within the macrophages on days 1–4 after infection was significantly decreased when infection was performed in the presence of pulmonary collectins. The phagocytosis experiments with the pH-sensitive dye-labeled bacteria revealed that pulmonary collectins promoted bacterial localization to an acidic compartment. In addition, SP-A and SP-D significantly increased the number of L. pneumophila co-localized with LAMP-1. These results indicate that pulmonary collectins protect macrophages against contact-dependent cytolytic activity of L. pneumophila and suppress intracellular growth of the phagocytosed bacteria. The promotion of lysosomal fusion with Legionella-containing phagosomes constitutes a likely mechanism of L. pneumophila growth suppression by the collectins.     

Increase in the growth inhibition of bovine pulmonary artery smooth muscle cells by an O-hexanoyl low-molecular-weight heparin derivative

Proliferation of pulmonary artery smooth muscle cells (PASMCs) appears to play a significant role in chronic pulmonary hypertension. The proliferation of PASMCs is strongly inhibited by some commercial heparin preparations. Heparin fragments were prepared by periodate treatment, followed by sodium borohydride reduction, to enhance potency. The tributylammonium salt of this fragmented heparin was O-acylated with hexanoic anhydride. Gradient polyacrylamide gel electrophoresis showed that the major heparin fragment contained eight disaccharide units. NMR analysis showed that approximately one hexanoyl group per disaccharide residue was present. The O-hexanoyl heparin fragments were assayed for growth inhibitory effect on bovine PASMCs in culture. This derivative was found to be more effective in growth inhibition of bovine PASMCs in culture than the heparin from which it was derived. In the future, it is envisioned that this or similar derivatives may be an effective treatment for pulmonary hypertension.     

Intratracheal gene transfer of tissue factor pathway inhibitor attenuates pulmonary fibrosis

Activation of the coagulation system and increased expression of tissue factor (TF) in pulmonary fibrosis associated with acute and chronic lung injury have been previously documented. In the present study, we evaluated the effect of TF inhibition with intratracheal gene transfer of tissue factor pathway inhibitor (TFPI), a potent and highly specific endogenous inhibitor of TF-dependent coagulation activation, in a rat model of bleomycin-induced lung fibrosis. Significant lung fibrotic changes as assessed by histologic findings and hydroxyproline content, and increased procoagulant activity and thrombin generation in bronchoalveolar lavage fluid were detected in rats after intratracheal injection of bleomycin. Intratracheal administration of an adenovirus vector expressing TFPI significantly decreased bleomycin-induced procoagulant and thrombin generation resulting in a strong inhibition of pulmonary fibrosis. TFPI-overexpression in the lung was associated with a significant reduction in gene expression of the connective tissue growth factor, a potent profibrotic growth factor. This is the first report showing that direct inhibition of TF-mediated coagulation activation abrogates bleomycin-induced pulmonary fibrosis.     

An N-glycan structure correlates with pulmonary metastatic ability of cancer cells

N-Glycan structures on the surface of cancer cells have diverse structures and play significant roles in metastatic process. However, little is known about their roles in organ-selective metastasis. Our study revealed that an alpha1,6-fucosylated biantennary N-glycan structure designated A2G2F is characteristic of lungs, with far more abundant expression in normal human and murine lungs than in other organs. In this study, we further examined the role of A2G2F in pulmonary metastasis. We stained metastatic cancers by alpha1,6-fucose-specific Lens culinaris agglutinin lectin and revealed that pulmonary metastatic nodules more abundantly expressed alpha1,6-fucosylated N-glycans than hepatic metastatic nodules from common primary cancers. The most specific alpha1,6-fucosylated N-glycan structure in pulmonary metastatic cancer was identified to be A2G2F. Using a B16 melanoma cell metastasis model, we showed that A2G2F-rich B16 cells formed more pulmonary metastatic nodules than A2G2F-poor cells. Our results suggest that A2G2F plays a critical role in pulmonary metastasis.