Tsc1 deficiency-mediated mTOR hyperactivation in vascular endothelial cells causes angiogenesis defects and embryonic lethality.

This is a study on the role of tuberous sclerosis complex1 (TSC1) mutation and mTOR activation in endothelial cells during angiogenic and embryonic development. Past studies had shown that Tsc1/Tsc2 mutant genes lead to overactivation of mTOR in the regulating pathways in developing fetus. We used conditional Cre-loxp gene knockout approach to delete Tsc1 in mice's endothelial cells in our experimental models. Similarly, activation of mTOR signaling in endothelial cells of these embryos (Tie2-Cre/Tsc1(-/-)) was found. Majority of Tie2-Cre/Tsc1(-/-) embryos died at embryonic day 14.5 in utero. Cardiovascular defects, subcutaneous edema and hemorrhage were present among them. Whole-mount immunostaining in these embryos revealed a disorganized vascular network, defective sprouting of vessels in yolk sac and thickening of the labyrinth layer in the placenta. A thinner ventricular wall with disorganized trabeculae was present in the hearts of Tie2-Cre/Tsc1(-/-) embryos. Endothelial cells in Tsc1-deficient mice showed defective mitochondrial and endoplasmic reticular morphology, but no significant change was observed in cell junctions. The mutant embryos displayed significantly reduced cell proliferation, increased apoptosis and disturbed expression of angiogenic factors. A cohort of mice was treated prenatally with mTOR inhibitor rapamycin. The offspring of these mutant mice survived up to 22 days after birth. It was concluded that physiological TSC1-mTOR signaling in endothelial cells is crucial for vascular development and embryogenesis. We postulated that disruption of normal angiogenic pathways through hyperactive mTOR signaling maybe the mechanism that lead to deranged vascular pathogenesis in the tuberous sclerosis complex.

Characteristics of serum lipid metabolism in patients with autoimmune pulmonary alveolar proteinosis.

OBJECTIVE: To study the serum lipid panels in consecutive autoimmune pulmonary alveolar proteinosis(APAP)patients and analyze their relationship with anti-granulocyte macrophage-colony stimulating factor(GM-CSF)antibody and other markers. METHODS: Thirty-two non-diabetic APAP patients were enrolled in the study. Serum lipids of these patients and 100 healthy volunteers were tested after an overnight fasting. Anti-GM-CSF antibody levels were measured with enzyme-linked immunosorbent assay. The correlation of serum lipids with lactate dehydrogenase,carcinoembryonic antigen,pulmonary function,and artery blood gas parameters were analyzed. RESULTS: Total cholesterol and low-density lipoprotein cholesterol levels [(5.54±0.99)and(3.73±0.83)mmol/L respectively] were significantly higher in APAP patients than in healthy volunteers [(5.05±0.97)and(3.17±0.89)mmol/L respectively](all P<0.05). High-density lipoprotein cholesterol(HDL-C)level of the APAP group [(1.10±0.18)mmol/L ]was significantly lower than that of the healthy group(P<0.05). Low-density lipoprotein/HDL and total cholesterol/HDL ratios in the APAP group(3.47±0.90 and 5.14±1.12 respectively)were significantly higher than those in the healthy group[(2.63±0.87)and(4.18±1.12)](all P<0.05). There was no significant difference in triglyceride level between the two groups(P>0.05). HDL-C level was negatively correlated with alveolar-arterial oxygen pressure difference(r=-0.436,P<0.05)and positively correlated with arterial oxygen saturation(r=0.459,P<0.05). None of the lipid markers correlated with serum anti-GM-CSF antibody levels(all P>0.05). CONCLUSIONS: APAP patients were likely to suffer from disturbed lipid metabolism,which was correlated with disease severity to some degree. Lipid markers deserved more attention in the management of APAP patients.

[Clinical analysis of 123 cases of chylous effusion].

OBJECTIVE: To analyze the clinical characteristics of chylous effusion and boost its diagnostic and therapeutic level. METHODS: A retrospective analysis was conducted for 123 cases of chylous effusion at our hospital between January 1990 and December 2009. RESULTS: The main clinical manifestations of chylous effusion included dyspnea (55.3%), edema (26.8%), abdominal distention (22.8%) and loss of weight (17.1%). Hypoalbuminemia was common (45.5%) and it was even more so in patients with idiopathic lymphopathies or of unknown causes (95.2%). The positive rates of identifying chylous effusion by Sudan III test, high triglyceride levels (> 1.25 mmol/L), lymphangiography or lymphangioscintigraphy were 89.1%, 80.6%, 70.6% and 89.5% respectively. Its common etiologies included injury (14.6%), malignancy (9.0%), infections (7.3%), lymphatic disorders or idiopathic lymphopathies (40%), drug-associated (2.4%) or associated with underlying disorders (16.3%). Efficacy was achieved in 63.6% of the patients and 21.8% died. CONCLUSIONS: Chylous effusion is a special type of serous effusion with multiple causes. Its clinical manifestations are often nonspecific. But malnutrition is common. Its causes are identified after a systematic evaluation. Treatment modalities include dietary modification, management of underlying causes and surgical approaches.

The St. George's Respiratory Questionnaire in lymphangioleiomyomatosis.

OBJECTIVE: To examine the correlation between the health-related quality of life measured by the St. George's Respiratory Questionnaire (SGRQ) and the commonly used physiological measures in lymphangioleiomyomatosis (LAM). METHODS: This study retrospectively analyzed the SGRQ scores and other measures (the Borg scale of breathlessness at rest, 6-minute walking distance, blood oxygen levels, and pulmonary function) of patients diagnosed and confirmed with LAM. Altogether 38 patients between June 2007 and November 2009 were included. RESULTS: The mean values of the SGRQ three components (symptoms, activity, and impacts) and total scores in the LAM patients were 46.95 +/- 28.90, 58.47 +/- 25.41, 47.89 +/- 29.66, and 51.11 +/- 26.35, respectively. The SGRQ total or component scores were correlated well with the Borg scale of breathlessness, 6-minute walking distance, partial pressure of oxygen in arterial blood, spirometry and diffusion capacity of lung. There were poor correlations between SGRQ score and residual volume or total lung capacity. In our preliminary observation, sirolimus improved the SGRQ total and three component scores and the Borg scale of breathlessness significantly after 101-200 days of treatment (n = 6). CONCLUSIONS: The SGRQ score in LAM is correlated well with physiological measures (Borg scale of breathlessness, 6-minute walking distance, blood oxygen levels, and pulmonary function tests). The SGRQ could therefore be recommended in baseline and follow-up evaluation of patients with LAM. Treatment with sirolimus, an inhibitor of mammalian target of rapamycin, may improve the quality of life and patient's perception of breathlessness in LAM.

mTOR Overactivation and Compromised Autophagy in the Pathogenesis of Pulmonary Fibrosis.

The mammalian target of rapamycin (mTOR) signaling pathway in pulmonary fibrosis was investigated in cell and animal models. mTOR overactivation in alveolar epithelial cells (AECs) was achieved in the conditional and inducible Tsc1 knock-down mice SPC-rtTA/TetO-Cre/Tsc1(fx/+) (STT). Doxycycline caused Tsc1 knock-down and consequently mTOR activation in AECs for the STT mice. Mice treated with bleomycin exhibited increased mortality and pulmonary fibrosis compared with control mice. In wild-type C57BL/6J mice, pretreatment with rapamycin attenuated the bleomycin-mediated mortality and fibrosis. Rapamycin-mediated mouse survival benefit was inhibited by chloroquine, an autophagy inhibitor. Autophagosomes were decreased in the lungs after bleomycin exposure. Rapamycin induced the production of autophagosomes and diminished p62. We concluded that mTOR overactivation in AECs and compromised autophagy in the lungs are involved in the pathogenesis of pulmonary fibrosis. The suppression of mTOR and enhancement of autophagy may be used for treatment of pulmonary fibrosis.

The role of vascular endothelial growth factor-D in diagnosis of lymphangioleiomyomatosis (LAM).

BACKGROUND: Definite diagnosis of lymphangioleiomyomatosis (LAM) depends on either transbronchial lung biopsy or video-assisted thoracic surgery, unless there is a history of chylothorax, kidney angiomyolipoma (AML), or tuberous sclerosis complex (TSC). Vascular endothelial growth factor-D (VEGF-D) was recently considered as a novel diagnostic marker for LAM. Herein, we evaluated diagnostic value of serum VEGF-D in LAM patients. METHODS: Serum samples were obtained from 78 cases of LAM (50 definite and 28 probable LAM based on European Respiratory Society guidelines), and 40 healthy female volunteers. VEGF-D was measured using enzyme-linked immunosorbant assay according to product instruction (R&D). RESULTS: Serum VEGF-D was significantly increased in definite LAM group, compared with that of health control (median: 3841.9 pg/mL vs 405.5 pg/mL respectively, p < 0.001). The optimal cut-off point for definite LAM diagnosis was 850.7 pg/mL. In probable LAM group, the majority of patients (92.9%) had serum VEGF-D level over 850.7 pg/mL. The serum levels of VEGF-D in LAM patients with pulmonary cystic lesions only were lower than that in patients with any of evidences of AML, chylous effusions, adenopathy, lymphangioleiomyomas, or TSC, but higher than that in the health control. In addition, VEGF-D levels were correlated with disease severity measured as LAM CT grade, and presentations of chylous effusions and/or lymphatic involvement (p < 0.05). CONCLUSION: Serum VEGF-D should be added to the current diagnosis algorithm to enhance definitive diagnosis for LAM.

SPC-Cre-ERT2 transgenic mouse for temporal gene deletion in alveolar epithelial cells.

Although several Cre-loxP-based gene knockout mouse models have been generated for the study of gene function in alveolar epithelia in the lung, their applications are still limited. In this study, we developed a SPC-Cre-ER(T2) mouse model, in which a tamoxifen-inducible Cre recombinase (Cre-ER(T2)) is under the control of the human surfactant protein C (SPC) promoter. The specificity and efficiency of Cre-ER(T2) activity was first evaluated by crossing SPC-Cre-ER(T2) mouse with ROSA26R mouse, a ß-galactosidase reporter strain. We found that Cre-ER(T2) was expressed in 30.7% type II alveolar epithelial cells of SPC-Cre-ER(T2)/ROSA26R mouse lung tissues in the presence of tamoxifen. We then tested the tamoxifen-inducible recombinase activity of Cre-ER(T2) in a mouse strain bearing TSC1 conditional knockout alleles (TSC1(fx/fx)). TSC1 deletion was detected in the lungs of tamoxifen treated SPC-Cre-ER(T2)/TSC1(fx/fx) mice. Therefore this SPC-Cre-ER(T2) mouse model may be a valuable tool to investigate functions of genes in lung development, physiology and disease.

Inactivation of mammalian target of rapamycin (mTOR) by rapamycin in a murine model of lipopolysaccharide-induced acute lung injury.

BACKGROUND: The mammalian target of rapamycin (mTOR) pathway, a key cellular signaling pathway associated with various cellular functions, has distinct roles in the inflammatory process. In this study, the mTOR inhibitor rapamycin (Rapa) was used to test whether inhibition of mTOR activation attenuates lipopolysaccharide (LPS)-induced acute lung injury (ALI) in a murine model. METHODS: Mice pretreated with Rapa or vehicle were given LPS intratracheally. Local cell numbers and inflammatory cytokines present in the bronchoalveolar lavage fluid (BAL), wet-to-dry weight ratio, histopathology of the lungs, and survival were evaluated. RESULTS: The phosphorylation of S6, a major downstream target of mTOR, had a 3-fold increase in lung tissue after LPS stimulation, but the increase was blocked by Rapa. Rapa reduced the levels of TNF-α (LPS vs. LPS + Rapa, (1672.74 ± 193.73) vs. (539.17 ± 140.48) pg/ml, respectively; P < 0.01) and IL-6 (LPS vs. LPS + Rapa: (7790.88 ± 1170.54) vs. (1968.57 ± 474.62) pg/ml, respectively; P < 0.01) in the BAL fluid. However, Rapa had limited effects on the overall severity of ALI, as determined by the wet-to-dry weight ratio of the lungs, number of neutrophils in the BAL fluid, and changes in histopathology. In addition, Rapa failed to reduce mortality in the LPS-induced ALI model. CONCLUSIONS: We confirmed that mTOR was activated during LPS-induced ALI and strongly inhibited by Rapa. Although Rapa reduced the levels of the mediators of inflammation, the overall severity and survival of the ALI murine model were unchanged.