Kai-Feng Xu

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.

mTORC1 enhancement of STIM1-mediated store-operated Ca2+ entry constrains tuberous sclerosis complex-related tumor development

The protein complex of tuberous sclerosis complex (TSC)1 and TSC2 tumor suppressors is a key negative regulator of mammalian target of rapamycin (mTOR). Hyperactive mTOR signaling due to the loss-of-function of mutations in either TSC1 or TSC2 gene causes TSC, an autosomal dominant disorder featured with benign tumors in multiple organs. As the ubiquitous second messenger calcium (Ca2+) regulates various cellular processes involved in tumorigenesis, we explored the potential role of mTOR in modulation of cellular Ca2+ homeostasis, and in turn the effect of Ca2+ signaling in TSC-related tumor development. We found that loss of Tsc2 potentiated store-operated Ca2+ entry (SOCE) in an mTOR complex 1 (mTORC1)-dependent way. The endoplasmic reticulum Ca2+ sensor, stromal interaction molecule 1 (STIM1), was upregulated in Tsc2-deficient cells, and was suppressed by mTORC1 inhibitor rapamycin. In addition, SOCE repressed AKT1 phosphorylation. Blocking SOCE either by depleting STIM1 or ectopically expressing dominant-negative Orai1 accelerated TSC-related tumor development, likely because of restored AKT1 activity and enhanced tumor angiogenesis. Our data, therefore, suggest that mTORC1 enhancement of store-operated Ca2+ signaling hinders TSC-related tumor growth through suppression of AKT1 signaling. The augmented SOCE by hyperactive mTORC1-STIM1 cascade may contribute to the benign nature of TSC-related tumors. Application of SOCE agonists could thus be a contraindication for TSC patients. In contrast, SOCE agonists should attenuate mTOR inhibitors-mediated AKT reactivation and consequently potentiate their efficacy in the treatment of the patients with TSC.

mTORC1 Is Involved in Hypoxia-Induced Pulmonary Hypertension Through the Activation of Notch3

Hypoxia‐induced pulmonary hypertension (HPH) is a clinical syndrome associated with high morbidity and mortality. However, the underlying mechanisms remain unclear. Both the mammalian target of rapamycin (mTOR) and the Notch3 signaling pathways have been reported to be involved in HPH; however, it is unknown whether there is a connection between these two signaling pathways in HPH. This study was designed to investigate the relationship between mTOR and Notch3 in HPH. After treatment with 10% O2 for 4 weeks, male C57BL/6 mice developed HPH with gradually increased right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary arteriolar remodeling accompanied by the activation of mTOR complex 1 (mTORC1) and Notch3 in the lung tissue and pulmonary arterioles. Pretreatment with the mTORC1 inhibitor rapamycin not only alleviated pulmonary arterial pressure and pulmonary arteriolar remodeling but also suppressed hypoxia‐induced mTORC1 and Notch3 activation. Prophylactic N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT) administration, a Notch signaling inhibitor, protected against the effects of hypoxia. These in vivo data were confirmed by in vitro experiments on human pulmonary arterial smooth muscle cell (PASMC) exposed to 3% O2. Furthermore, overexpression of Notch3 intracellular domain partially abrogated the inhibitory effects of rapamycin on human PASMC proliferation. These data indicate that both mTORC1 and Notch3 signaling are involved in HPH and the downstream effects of mTORC1 activation in HPH are partially dependent on the activation of Notch3 signaling. J. Cell. Physiol. 229: 2117–2125, 2014.

Characterization of gene mutations and phenotypes of cystic fibrosis in Chinese patients.

Cystic fibrosis (CF) is a relatively common autosomal recessive disorder in Caucasians. CF is considered a very rare disease in Asians, and fewer than 30 Chinese CF patients are reported in the literature. We enrolled seven patients of Chinese Han origin diagnosed with CF at the Peking Union Medical College Hospital, to characterize gene mutations and phenotypes of CF in Chinese patients.

Lymphangioleiomyomatosis: differential diagnosis and optimal management

Lymphangioleiomyomatosis (LAM) is an uncommon disease presented as diffuse thin-walled cystic changes in the lung. The main differential diagnoses include pulmonary Langerhans’ histiocytosis (PLCH), Birt-Hogg-Dubé syndrome (BHD), lymphoid interstitial pneumonia (LIP), and amyloidosis. A combination of clinical, radiological, and pathological approaches as well as genetic testing will clarify the diagnosis in most cases. LAM is a disease almost exclusively in women. Dyspnea, pneumothorax, and hemoptysis are common presentations in LAM patients. LAM is also a lymphatic disorder affecting lymphatic vessels and lymph nodes. Chylothorax, chylous ascites, and lymphangiomyomas are frequently seen. LAM can present sporadically as a single entity or as part of tuberous sclerosis complex (TSC). Angiomyolipoma (AML) is a characteristic extra-pulmonary lesion, either found in association with sporadic or TSC-related LAM. High-risk populations should be screened for LAM, including adult women with TSC and female patients with spontaneous pneumothorax, AMLs in the kidney, and diffuse cystic lung diseases. Definitive diagnosis of LAM is based on a high level of clinical suspicion on presentation supported by pathological findings or by a distinct feature, such as a history of TSC, AMLs in the kidney, chylothorax, or chylous ascites. Vascular endothelial growth factor-D (VEGF-D) in serum is a noninvasive and reliable diagnostic biomarker. In experienced centers, trans-bronchial lung biopsy (TBLB) provides a convenient and safe way to obtain lung specimens for diagnostic purposes. An effective treatment for LAM is now available, namely using a mechanistic target of rapamycin (mTOR) inhibitor such as sirolimus. Efficacy of sirolimus has been confirmed in clinical trials. Research in other molecular-targeted therapies is under investigation. A previously little-known rare disease with no cure is now better understood with regards to its pathogenesis, diagnosis, and management. In this review, current knowledge in diagnosis and differential diagnosis of LAM will be discussed, followed by the discussion of therapy with mTOR inhibitors.

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 mices 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.

Pneumocystis Pneumonia in Patients with Autoimmune Diseases: A Retrospective Study Focused on Clinical Characteristics and Prognostic Factors Related to Death

Background With the increasing use of immunosuppressive agents, the number of opportunistic infections has risen in patients with autoimmune diseases. Pneumocystis pneumonia (PCP) is one of these opportunistic infections that have a high mortality rate. However, only a few studies have described PCP in these patients, and these studies are limited in scope. We conducted this retrospective study to describe the clinical characteristics and factors associated with outcomes of PCP in patients with autoimmune diseases. Methods A retrospective study was performed in laboratory diagnosed PCP patients with autoimmune diseases in an academic hospital over a 10-year period. Patients with human immunodeficiency virus (HIV) infection were not included. Clinical characteristics were collected and the factors related to death were analysed. Results A total of 69 patients with PCP during the study period were included. Common clinical features included fever (81%), cough (56%), and dyspnea (35%). Ground glass opacity (81%) and reticulation (52%) were the most common radiological findings. Concurrent pulmonary infections including bacterium, aspergillus and cytomegalovirus were found in 34% of the patients. The overall in-hospital mortality rate was 32%. High mortality was associated with lower PaO2/FiO2 ratios and albumin levels. The lymphocyte count, CD4+ T cell count, previous usage of immunosuppressive agents, the duration and dose of glucocorticoids did not affect the outcome. Conclusions The mortality rate in PCP patients with autoimmune diseases is high. Low PaO2/FiO2 ratios and albumin levels are independent prognostic factors of mortality.

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-ERT2 mouse model, in which a tamoxifen-inducible Cre recombinase (Cre-ERT2) is under the control of the human surfactant protein C (SPC) promoter. The specificity and efficiency of Cre-ERT2 activity was first evaluated by crossing SPC-Cre-ERT2 mouse with ROSA26R mouse, a β-galactosidase reporter strain. We found that Cre-ERT2 was expressed in 30.7% type II alveolar epithelial cells of SPC-Cre-ERT2/ROSA26R mouse lung tissues in the presence of tamoxifen. We then tested the tamoxifen-inducible recombinase activity of Cre-ERT2 in a mouse strain bearing TSC1 conditional knockout alleles (TSC1fx/fx). TSC1 deletion was detected in the lungs of tamoxifen treated SPC-Cre-ERT2/TSC1fx/fx mice. Therefore this SPC-Cre-ERT2 mouse model may be a valuable tool to investigate functions of genes in lung development, physiology and disease.

A case report: The efficacy of pirfenidone in a Chinese patient with progressive systemic sclerosis-associated interstitial lung disease: A CARE-compliant article.

AbstractSystemic sclerosis (SSc)-associated interstitial lung disease (SSc-ILD) has become the leading SSc-related cause of death. Although various types of immunosuppressive therapy have been attempted for patients with SSc-ILD, no curative or effective treatment strategies for SSc-ILD have been developed. Therefore, management of patients with SSc-ILD remains a challenge. Here, we report a Chinese, female, SSc-ILD patient who was negative for Scl-70 and showed an excellent response to pirfenidone without obvious adverse effects. She had been suffered from dry cough and exertional dyspnea for 2 months. The chest computed tomography manifestation was consistent with a pattern of fibrotic nonspecific interstitial pneumonia. The pulmonary function test showed isolated impaired diffusion. After 11 weeks of administration of pirfenidone, the dry cough and dyspnea had disappeared. Both of the lung shadows and the pulmonary diffusion function were improved. Pirfenidone might be an effective option for early SSc-ILD treatment. A well-controlled clinical trial is expected in the future.

The St. George's Respiratory Questionnaire in lymphangioleiomyomatosis.

OBJECTIVE To examine the correlation between the health-related quality of life measured by the St. Georges 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 patients perception of breathlessness in LAM.