Disrupted pulmonary vascular development and pulmonary hypertension in transgenic mice overexpressing transforming growth factor-alpha

Pulmonary vascular disease plays a major role in morbidity and mortality in infant and adult lung diseases in which increased levels of transforming growth factor (TGF)-alpha and its receptor EGFR have been associated. The aim of this study was to determine whether overexpression of TGF-alpha disrupts pulmonary vascular development and causes pulmonary hypertension. Lung-specific expression of TGF-alpha in transgenic mice was driven with the human surfactant protein (SP)-C promoter. Pulmonary arteriograms and arterial counts show that pulmonary vascular development was severely disrupted in TGF-alpha mice. TGF-alpha mice developed severe pulmonary hypertension and vascular remodeling characterized by abnormally extensive muscularization of small pulmonary arteries. Pulmonary vascular development was significantly improved and pulmonary hypertension and vascular remodeling were prevented in bi-transgenic mice expressing both TGF-alpha and a dominant-negative mutant EGF receptor under the control of the SP-C promoter. Vascular endothelial growth factor (VEGF-A), an important angiogenic factor produced by the distal epithelium, was decreased in the lungs of TGF-alpha adults and in the lungs of infant TGF-alpha mice before detectable abnormalities in pulmonary vascular development. Hence, overexpression of TGF-alpha caused severe pulmonary vascular disease, which was mediated through EGFR signaling in distal epithelial cells. Reductions in VEGF may contribute to the pathogenesis of pulmonary vascular disease in TGF-alpha mice.     

Increased pulmonary vascular resistance and defective pulmonary artery filling in caveolin-1-/- mice

Caveolin-1, the structural and signaling protein of caveolae, is an important negative regulator of endothelial nitric oxide synthase (eNOS). We observed that mice lacking caveolin-1 (Cav1(-/-)) had twofold increased plasma NO levels but developed pulmonary hypertension. We measured pulmonary vascular resistance (PVR) and assessed alterations in small pulmonary arteries to determine the basis of the hypertension. PVR was 46% greater in Cav1(-/-) mice than wild-type (WT), and increased PVR in Cav1(-/-) mice was attributed to precapillary sites. Treatment with NG-nitro-l-arginine methyl ester (l-NAME) to inhibit NOS activity raised PVR by 42% in WT but 82% in Cav1(-/-) mice, indicating greater NO-mediated pulmonary vasodilation in Cav1(-/-) mice compared with WT. Pulmonary vasculature of Cav1(-/-) mice was also less reactive to the vasoconstrictor thromboxane A2 mimetic (U-46619) compared with WT. We observed redistribution of type I collagen and expression of smooth muscle alpha-actin in lung parenchyma of Cav1(-/-) mice compared with WT suggestive of vascular remodeling. Fluorescent agarose casting also showed markedly decreased density of pulmonary arteries and artery filling defects in Cav1(-/-) mice. Scanning electron microscopy showed severely distorted and tortuous pulmonary precapillary vessels. Thus caveolin-1 null mice have elevated PVR that is attributed to remodeling of pulmonary precapillary vessels. The elevated basal plasma NO level in Cav1(-/-) mice compensates partly for the vascular structural abnormalities by promoting pulmonary vasodilation.     

Acute effects on the lung and the liver of oral administration of cerium chloride on adult, neonatal and fetal mice

We evaluated tissue changes associated with cerium chloride administration via gavage to adult mice, via milk to neonatal mice and transplacentally to fetal mice. Change in adults consisted of extensive pulmonary hemorrhage, pulmonary venous congestion, thickened alveolar septae, hepatic necrosis and neutrophil infiltrations. Those in fetal mice consisted of pulmonary and hepatic congestion. These results indicate that gavage cerium administration elicited subtle tissue changes, though oral toxicity is rather low. These changes were less severe in neonatal and fetal mice. When cerium was injected into adult mice through the tail vein, cerium was distributed mainly to the liver, spleen and lung dose-dependently with the cerium concentration gradually decreasing after 3 days. A study of cerium anticoagulation in mouse plasma showed that clotting time was significantly prolonged when cerium was added to plasma. These results suggest that cerium may disturb blood coagulation and cause pulmonary and hepatic vascular congestion.     

Inbred and outbred mice have equivalent variability in a cockroach allergen-induced model of asthma

Outbred mice traditionally are considered to display high variability, thereby limiting their use in some studies. Researchers frequently are encouraged to use inbred strains of mice because of the greater homogeneity of these experimental animals. We compared the pulmonary inflammatory response of inbred BALB/cJ mice to that of outbred HSD-ICR mice by measuring multiple variables, including cytokines, chemokines, number of pulmonary inflammatory cells, and respiratory parameters. Cockroach allergens induced significant pulmonary inflammation in both BALB and ICR mice. Our comparisons of the coefficients of variance for 148 discrete data sets for each strain or stock indicated that BALB and ICR mice have roughly equivalent intrastrain or -stock variability in our model of asthma-like pulmonary inflammation. The average coefficient of variance, calculated as the ratio of the SD to the mean of a data set, was 0.35 ± 0.34 for BALB mice compared with 0.31 ± 0.32 for ICR mice. In conclusion, inbred BALB and outbred ICR mice have roughly equivalent intrastrain or -stock variability in a murine model of asthma-like pulmonary inflammation.     

Role of IFN-gamma in regulating T2 immunity and the development of alternatively activated macrophages during allergic bronchopulmonary mycosis

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of a chronic pulmonary fungal infection accompanied by an "allergic" response (T2) to the infection, i.e., a model of an allergic bronchopulmonary mycosis. Our objective was to determine whether IFN-gamma plays a role in regulating the pulmonary T2 immune response in C. neoformans-infected C57BL/6 mice. Long-term pulmonary fungistasis was lost in IFN-gamma knockout (KO) mice, resulting in an increased pulmonary burden of fungi at wk 3. IFN-gamma was required for the early influx of leukocytes into the lungs but was not required later in the infection. By wk 3, eosinophil and macrophage numbers were elevated in the absence of IFN-gamma. The inducible NO synthase to arginase ratio was lower in the lungs of IFN-gamma KO mice and the macrophages had increased numbers of intracellular cryptococci and YM1 crystals, indicative of alternatively activated macrophages in these mice. There was evidence of pulmonary fibrosis in both wild-type and IFN-gamma KO mice by 5 wk postinfection. IFN-gamma production was not required for the development of T2 cytokine (IL-4, IL-5, IL-13) producing cells in the lungs and lung-associated lymph nodes or induction of an IgE response. At a number of time points, T2 cytokine production was enhanced in IFN-gamma KO mice. Thus, in the absence of IFN-gamma, C57BL/6 mice develop an augmented allergic response to C. neoformans, including enhanced generation of alternatively activated macrophages, which is accompanied by a switch from a chronic to a progressive pulmonary cryptococcal infection.     

Expression of 150-kDa oxygen-regulated protein (ORP150) stimulates bleomycin-induced pulmonary fibrosis and dysfunction in mice

Idiopathic pulmonary fibrosis (IPF) involves pulmonary injury associated with inflammatory responses, fibrosis and dysfunction. Myofibroblasts and transforming growth factor (TGF)-β1 play major roles in the pathogenesis of this disease. Endoplasmic reticulum (ER) stress response is induced in the lungs of IPF patients. One of ER chaperones, the 150-kDa oxygen-regulated protein (ORP150), is essential for the maintenance of cellular viability under stress conditions. In this study, we used heterozygous ORP150-deficient mice (ORP150(+/-) mice) to examine the role of ORP150 in bleomycin-induced pulmonary fibrosis. Treatment of mice with bleomycin induced the expression of ORP150 in the lung. Bleomycin-induced inflammatory responses were slightly exacerbated in ORP150(+/-) mice compared to wild-type mice. On the other hand, bleomycin-induced pulmonary fibrosis, alteration of lung mechanics and respiratory dysfunction was clearly ameliorated in the ORP150(+/-) mice. Bleomycin-induced increases in pulmonary levels of both active TGF-β1 and myofibroblasts were suppressed in ORP150(+/-) mice. These results suggest that although ORP150 is protective against bleomycin-induced lung injury, this protein could stimulate bleomycin-induced pulmonary fibrosis by increasing pulmonary levels of TGF-β1 and myofibroblasts.     

BMPR-II heterozygous mice have mild pulmonary hypertension and an impaired pulmonary vascular remodeling response to prolonged hypoxia

Heterozygous mutations of the bone morphogenetic protein type II receptor (BMPR-II) gene have been identified in patients with primary pulmonary hypertension. The mechanisms by which these mutations contribute to the pathogenesis of primary pulmonary hypertension are not fully elucidated. To assess the impact of a heterozygous mutation of the BMPR-II gene on the pulmonary vasculature, we studied mice carrying a mutant BMPR-II allele lacking exons 4 and 5 (BMPR-II(+/-) mice). BMPR-II(+/-) mice had increased mean pulmonary arterial pressure and pulmonary vascular resistance compared with their wild-type littermates. Histological analyses revealed that the wall thickness of muscularized pulmonary arteries (<100 mum in diameter) and the number of alveolar-capillary units were greater in BMPR-II(+/-) than in wild-type mice. Breathing 11% oxygen for 3 wk increased mean pulmonary arterial pressure, pulmonary vascular resistance, and hemoglobin concentration to similar levels in BMPR-II(+/-) and wild-type mice, but the degree of muscularization of small pulmonary arteries and formation of alveolar-capillary units were reduced in BMPR-II(+/-) mice. Our results suggest that, in mice, mutation of one copy of the BMPR-II gene causes pulmonary hypertension but impairs the ability of the pulmonary vasculature to remodel in response to prolonged hypoxic breathing.     

Defense mechanisms against primary influenza virus infection in mice. I. The roles of interferon and neutralizing antibodies and thymus dependence of interferon and antibody production.

To investigate the defensive roles and production of interferon and antibodies, C3H/He mice were subjected to various immunosuppressive treatments and infected with influenza virus. In infected normal control mice the pattern of pulmonary viral growth can be divided into three phases. The first phase is characterized by an exponential increase of virus titer, the second by a rapid decrease, and the third by a moderate decrease. At the time of transition from the first phase to the second in pulmonary virus growth, interferon could be detected in the tracheobronchial washings of infected mice, but neutralizing antibodies could not. In infected B cell-deprived mice and infected anti-mu-treated mice, the transition from the first phase to the second occurred without any detectable antibody production, and interferon could be induced in the early stage of infection. However, the pulmonary virus in these mice increased again exponentially until the death of the mice. In infected T cell-deprived mice which could not induce interferon, but produced IgM-neutralizing antibodies, the second phase was not observed after the first phase, but a transient plateau phase could be demonstrated, and then the pulmonary virus increased again exponentially until the death of the mice. In anti-gamma-treated infected mice, pulmonary virus growth and production of interferon and neutralizing antibody were almost similar to those of infected normal control mice except for the absence of IgG neutralizing antibody production. Although anti-alpha-treated infected mice produced interferon and no IgA antibody, the transition from the first exponential increase of pulmonary virus to the second rapid decrease was seen, but then the virus increased exponentially again until the death of the mice. These results suggest that interferon plays an important role in the transition from the first phase to the second, and that T cells are required for interferon induction in mice infected with influenza virus. These data also suggest that IgA antibodies play an important role in the inhibition of virus propagation in the lungs after the disappearance of interferon. Moreover, infected T cell-deprived mice could produce only IgM neutralizing antibodies, but not IgG and IgA antibodies. Therefore, T cells are required for the production of IgG and IgA antibodies and even     

Basal lung mechanics and airway and pulmonary vascular responsiveness in different inbred mouse strains.

Little is known about interstrain variations in baseline lung functions or smooth muscle contractility in murine lungs. We therefore examined basal lung mechanics and airway, as well as vascular reactivity to methacholine, thromboxane (using U-46619), and endothelin-1 (ET-1), A/J, AKR, BALB/c, C3H/HeN, C57BL/6, and SCID mice. All experiments were performed with isolated perfused mouse lungs. Except AKR mice (which were excluded from further analysis), all other strains showed stable pulmonary compliance, pulmonary resistance, and pulmonary arterial pressure within a control period of 45 min. Among these strains, C3H/HeN mice exhibited higher dynamic pulmonary compliance and lower pulmonary resistance, whereas SCID mice had higher baseline pulmonary resistance than the other strains. Concentration-response experiments with methacholine showed a lower airway reactivity for C57BL/6 mice compared with the other strains. Perfusion with 1 microM U-46619 or 100 nM ET-1 revealed a similar pattern: the agonist-inducible broncho- and vasoconstriction was lower in C57BL/6 mice than in all other strains, whereas it tended to be higher in SCID mice. The present study demonstrates a correlation between airway and vascular responsiveness in all tested strains. SCID mice are hyperreactive, whereas C57BL/6 mice are hyporeactive, to smooth muscle constrictors. Lung mechanics, as well as airway and vascular responsiveness, appear to be genetically controlled.     

Reversibility of pulmonary abnormalities by conditional replacement of surfactant protein D (SP-D) in vivo

Surfactant protein D (SP-D) gene-targeted mice develop severe pulmonary disease associated with emphysema, pulmonary lipidosis, and foamy macrophage infiltrations. To determine the potential reversibility of these abnormalities, transgenic mice were developed in which SP-D was conditionally replaced in the respiratory epithelium of SP-D(-/-) mice. SP-D was not detected in the absence of doxycycline. Treatment with doxycycline after birth restored pulmonary SP-D concentrations and corrected pulmonary pathology at adulthood. When SP-D was replaced in adult SP-D(-/-) mice, alveolar SP-D was restored within 3 days, pulmonary lipid abnormalities were corrected, but emphysema persisted. In corrected adult SP-D(-/-) mice, loss of SP-D caused focal emphysema and pulmonary inflammation but did not cause phospholipid abnormalities characteristic of SP-D(-/-) mice. Thus, abnormalities in surfactant phospholipid homeostasis and alveolar macrophage abnormalities were readily corrected by restoration of SP-D. However, once established, emphysema was not reversed by SP-D. SP-D-dependent processes regulating surfactant lipid homeostasis were disassociated from those mediating emphysema.     

Overexpression of tumor necrosis factor-alpha produces an increase in lung volumes and pulmonary hypertension

Tumor necrosis factor (TNF)-alpha is a key proinflammatory cytokine that is thought to be important in the development of pulmonary fibrosis, whereas its role in pulmonary emphysema has not been as thoroughly documented. In the present study, TNF-alpha was overexpressed in alveolar type II cells under the control of the human surfactant protein C promoter. In this report, we further characterized the pulmonary abnormalities and provided a physiological assessment of these mice. Histopathology of the lungs revealed chronic inflammation, severe alveolar air space enlargement and septal destruction, and bronchiolitis. However, pulmonary fibrosis was very limited and only seen in the subpleural, peribronchiolar, and perivascular regions. Physiological assessment showed an increase in lung volumes and a decrease in elastic recoil characteristic of emphysema; there was no evidence of restrictive lung disease characteristic of pulmonary fibrosis. In addition, the mice raised in ambient conditions in Denver developed pulmonary hypertension. Gelatinase activity was increased in the lavage fluid from these lungs. These results suggest that in these mice TNF-alpha contributed to the development of pulmonary emphysema through chronic lung inflammation and activation of the elastolytic enzymes but by itself was unable to produce significant pulmonary fibrosis.     

Pulmonary hypertension in TNF-alpha-overexpressing mice is associated with decreased VEGF gene expression.

Tumor necrosis factor-alpha (TNF-alpha) transgenic mice have previously been found to have characteristics consistent with emphysema and severe pulmonary hypertension. Lungs demonstrated alveolar enlargement as well as interstitial thickening due to chronic inflammation and perivascular fibrosis. In the present report, we sought to determine potential mechanisms leading to development of pulmonary hypertension in TNF-alpha transgenic mice. To determine whether sustained vasoconstriction was an important component of this pulmonary hypertension, nitric oxide was administered and hemodynamics were measured. Nitric oxide (25 ppm) failed to normalize right ventricular pressure in transgene-positive mice, suggesting that the pulmonary hypertension was not due to sustained vasoconstriction. Structural analysis of the pulmonary arteries found adventitial thickening and a trend toward medial hypertrophy in pulmonary arteries of transgene-positive mice, suggesting that vascular remodeling had occurred. Echocardiographic measurement of the percent fractional shortening of the left ventricle as a measurement of ventricular function in vivo revealed that left ventricular dysfunction was not contributing to pulmonary hypertension. We examined expression of genes known to be important in regulation of vascular tone and structure. Messenger RNA expression of vascular endothelial growth factor and its receptor flk-1 was reduced compared with transgene-negative littermates at all ages. Endothelial and inducible nitric oxide synthase mRNA levels were similar in both groups. Endothelin-1 mRNA was also decreased in TNF-alpha transgenic mice. Interestingly, female transgenic mice had decreased survival rate compared with male transgenic mice. We conclude that chronic overexpression of TNF-alpha is associated with decreased vascular endothelial growth factor and flk-1 gene expression, pulmonary vascular remodeling, and severe pulmonary hypertension, although the precise mechanism is unknown.     

Enalapril protects mice from pulmonary hypertension by inhibiting TNF-mediated activation of NF-kappaB and AP-1

The present study was undertaken to investigate the effects of treatment with the angiotensin-converting enzyme (ACE) inhibitor enalapril in a mouse model of pulmonary hypertension induced by bleomycin. Bleomycin-induced lung injury in mice is mediated by enhanced tumor necrosis factor-alpha (TNF) expression in the lung, which determines the murine strain sensitivity to bleomycin, and murine strains are sensitive (C57BL/6) or resistant (BALB/c). Bleomycin induced significant pulmonary hypertension in C57BL/6, but not in BALB/c, mice; average pulmonary arterial pressure (PAP) was 26.4 +/- 2.5 mmHg (P < 0.05) vs. 15.2 +/- 3 mmHg, respectively. Bleomycin treatment induced activation of nuclear factor (NF)-kappaB and activator protein (AP)-1 and enhanced collagen and TNF mRNA expression in the lung of C57BL/6 but not in BALB/c mice. Double TNF receptor-deficient mice (in a C57BL/6 background) that do not activate NF-kappaB or AP-1 in response to bleomycin did not develop bleomycin-induced pulmonary hypertension (PAP 14 +/- 3 mmHg). Treatment of C57BL/6 mice with enalapril significantly (P < 0.05) inhibited the development of pulmonary hypertension after bleomycin exposure. Enalapril treatment inhibited NF-kappaB and AP-1 activation, the enhanced TNF and collagen mRNA expression, and the deposition of collagen in bleomycin-exposed C57BL/6 mice. These results suggest that ACE inhibitor treatment decreases lung injury and the development of pulmonary hypertension in bleomycin-treated mice.     

Effect of Z-100, an immunomodulator extracted from human type tubercle bacilli,on the pulmonary metastases of lewis lung carcinoma in attempt to regulate suppressor T cells and suppressor factor,IL-4

In the present study, anti-metastatic effect of Z-100 on the spontaneous pulmonary metastases of Lewis lung carcinoma (3LL) was examined in an attempt to regulate suppressor T cells. When Z-100 (10 mg/kg) was daily injected i.p. after 3LL inoculation, survival rate of these mice was increased significantly (p<0.05). In addition, the number of pulmonary metastatic colonies of 3LL in Z-100-treated mice were significantly decreased by 38% at 21 days, as compared with that of control mice (p<0.05). Along with the decrease of pulmonary metastases, suppressor cell activity was also gradually reduced in these mice, as compared with that of control mice. When splenic suppressor cells (5×10⁷ cells) from 3LL-bearing mice were adoptively transferred into normal mice (recipients) just before inoculation of 3LL, the development of pulmonary metastases in recipients was significantly accelerated. However, splenocytes from 3LL-bearing mice treated with Z-100 did not affect the development of pulmonary metastasis. The potential to accelerate the metastasis of splenic mononuclear cells from 3LL-bearing mice was decreased significantly by the treatment with anti-Thy 1.2 monoclonal antibody (mAb), anti-Lyt 2.2 mAb or anti-CD11b mAb followed by complement. IL-4 activity in the sera of 3LL-bearing mice was detected 15 days after tumor inoculation (13 pg/ml) and gradually increased (18 pg/ml) 20 days after tumor inoculation. However, when Z-100 (10 mg/kg) was daily injected i.p., IL-4 activity in sera was decreased significantly, and the IL-4 activity was not detected in these mice on day 20. These results suggest that Z-100 could inhibit the pulmonary metastases in 3LL-bearing mice through the inhibition of suppressor T cell activity and a possible candidate of its effector molecule, IL-4.     

清热消炎复方制剂抗流感病毒作用的研究

为评价清热消炎复方制剂(简称AI)的抗流感病毒活性,我们以病毒唑为对照,通过在体外观察病毒致细胞病变效应(CPE)、MTT细胞染色检查病毒抑制率和检测病毒血凝滴度;在体内观察其对染毒小鼠的死亡保护作用,对小鼠流感病毒性肺炎的抑制作用,以及对小鼠肺内病毒增殖的影响,从而判定其抗流感病毒作用。结果发现AI在160ug/mL时能完全抑制流感病毒在MDCK细胞内的增殖复制作用。体内实验中0.1g/kg,0.5g/kg,1.2g/kg3个剂量均能明显降低染毒小鼠的致死率,延长平均存活时间:降低肺炎小鼠的肺指数和血凝滴度(P<0.01)。其作用与病毒唑相当。结论认为清热消炎复方制剂是一种有效的体内、体外抗流感病毒中药复方制剂。     

p53 Gene deficiency promotes hypoxia-induced pulmonary hypertension and vascular remodeling in mice

Chronic hypoxia induces pulmonary arterial remodeling, resulting in pulmonary hypertension and right ventricular hypertrophy. Hypoxia has been implicated as a physiological stimulus for p53 induction and hypoxia-inducible factor-1α (HIF-1α). However, the subcellular interactions between hypoxic exposure and expression of p53 and HIF-1α remain unclear. To examine the role of p53 and HIF-1α expression on hypoxia-induced pulmonary arterial remodeling, wild-type (WT) and p53 knockout (p53KO) mice were exposed to either normoxia or hypoxia for 8 wk. Following chronic hypoxia, both genotypes demonstrated elevated right ventricular pressures, right ventricular hypertrophy as measured by the ratio of the right ventricle to the left ventricle plus septum weights, and vascular remodeling. However, the right ventricular systolic pressures, the ratio of the right ventricle to the left ventricle plus septum weights, and the medial wall thickness of small vessels were significantly greater in the p53KO mice than in the WT mice. The p53KO mice had lower levels of p21 and miR34a expression, and higher levels of HIF-1α, VEGF, and PDGF expression than WT mice following chronic hypoxic exposure. This was associated with a higher proliferating cell nuclear antigen expression of pulmonary artery in p53KO mice. We conclude that p53 plays a critical role in the mitigation of hypoxia-induced small pulmonary arterial remodeling. By interacting with p21 and HIF-1α, p53 may suppress hypoxic pulmonary arterial remodeling and pulmonary arterial smooth muscle cell proliferation under hypoxia.     

Interaction between granulocytes and antibodies in the enhancement of lung defenses against Staphylococcus aureus after intranasal immunization of mice with live-attenuated bacteria

Abstract Immunization with live-attenuated Staphylococcus aureus induced measurable levels of specific IgG and IgA in the lungs, but the pulmonary clearance of S. aureus in immunized mice did not differ from that of control mice. Aerosol exposure of mice to Pseudomonas aeruginosa induced a significant recruitment of polymorphonuclear leukocytes (PMNL) to the lungs in both immunized and control mice, whereas S. aureus challenge did not. However, challenge with a mixture of P. aeruginosa-S. aureus or exposure to an aerosol of Escherichia coli lipopolysaccharide (LPS) before S. aureus challenge induced PMNL migration and a significant enhancement of pulmonary clearance of S. aureus in immunized mice. The presence of both antibodies and PMNL was required for enhancement of S. aureus pulmonary clearance.