Publications by authors named "Ning Lai"

30 Publications

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Upregulation of Calcium Homeostasis Modulators in Contractile-To-Proliferative Phenotypical Transition of Pulmonary Arterial Smooth Muscle Cells.

Front Physiol 2021 2;12:714785. Epub 2021 Aug 2.

Section of Physiology, Division of Pulmonary, Critical Care and Sleep Medicine, La Jolla, CA, United States.

Excessive pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and migration are implicated in the development of pathogenic pulmonary vascular remodeling characterized by concentric arterial wall thickening and arteriole muscularization in patients with pulmonary arterial hypertension (PAH). Pulmonary artery smooth muscle cell contractile-to-proliferative phenotypical transition is a process that promotes pulmonary vascular remodeling. A rise in cytosolic Ca concentration [(Ca) ] in PASMCs is a trigger for pulmonary vasoconstriction and a stimulus for pulmonary vascular remodeling. Here, we report that the calcium homeostasis modulator (CALHM), a Ca (and ATP) channel that is allosterically regulated by voltage and extracellular Ca, is upregulated during the PASMC contractile-to-proliferative phenotypical transition. Protein expression of CALHM1/2 in primary cultured PASMCs in media containing serum and growth factors (proliferative PASMC) was significantly greater than in freshly isolated PA (contractile PASMC) from the same rat. Upregulated CALHM1/2 in proliferative PASMCs were associated with an increased ratio of pAKT/AKT and pmTOR/mTOR and an increased expression of the cell proliferation marker PCNA, whereas serum starvation and rapamycin significantly downregulated CALHM1/2. Furthermore, CALHM1/2 were upregulated in freshly isolated PA from rats with monocrotaline (MCT)-induced PH and in primary cultured PASMC from patients with PAH in comparison to normal controls. Intraperitoneal injection of CGP 37157 (0.6 mg/kg, q8H), a non-selective blocker of CALHM channels, partially reversed established experimental PH. These data suggest that CALHM upregulation is involved in PASMC contractile-to-proliferative phenotypical transition. Ca influx through upregulated CALHM1/2 may play an important role in the transition of sustained vasoconstriction to excessive vascular remodeling in PAH or precapillary PH. Calcium homeostasis modulator could potentially be a target to develop novel therapies for PAH.
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http://dx.doi.org/10.3389/fphys.2021.714785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8364962PMC
August 2021

Halofuginone, a promising drug for treatment of pulmonary hypertension.

Br J Pharmacol 2021 Sep 31;178(17):3373-3394. Epub 2021 Jul 31.

Section of Physiology, Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of California, San Diego, La Jolla, California, USA.

Background And Purpose: Halofuginone is a febrifugine derivative originally isolated from Chinese traditional herb Chang Shan that exhibits anti-hypertrophic, anti-fibrotic and anti-proliferative effects. We sought to investigate whether halofuginone induced pulmonary vasodilation and attenuates chronic hypoxia-induced pulmonary hypertension (HPH).

Experimental Approach: Patch-clamp experiments were conducted to examine the activity of voltage-dependent Ca channels (VDCCs) in pulmonary artery smooth muscle cells (PASMCs). Digital fluorescence microscopy was used to measure intracellular Ca concentration in PASMCs. Isolated perfused and ventilated mouse lungs were used to measure pulmonary artery pressure (PAP). Mice exposed to hypoxia (10% O ) for 4 weeks were used as model of HPH for in vivo experiments.

Key Results: Halofuginone increased voltage-gated K (K ) currents in PASMCs and K currents through KCNA5 channels in HEK cells transfected with KCNA5 gene. HF (0.03-1 μM) inhibited receptor-operated Ca entry in HEK cells transfected with calcium-sensing receptor gene and attenuated store-operated Ca entry in PASMCs. Acute (3-5 min) intrapulmonary application of halofuginone significantly and reversibly inhibited alveolar hypoxia-induced pulmonary vasoconstriction dose-dependently (0.1-10 μM). Intraperitoneal administration of halofuginone (0.3 mg·kg , for 2 weeks) partly reversed established PH in mice.

Conclusion And Implications: Halofuginone is a potent pulmonary vasodilator by activating K channels and blocking VDCC and receptor-operated and store-operated Ca channels in PASMCs. The therapeutic effect of halofuginone on experimental PH is probably due to combination of its vasodilator effects, via inhibition of excitation-contraction coupling and anti-proliferative effects, via inhibition of the PI3K/Akt/mTOR signalling pathway.
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http://dx.doi.org/10.1111/bph.15442DOI Listing
September 2021

Revisiting the mechanism of hypoxic pulmonary vasoconstriction using isolated perfused/ventilated mouse lung.

Pulm Circ 2020 Oct-Dec;10(4):2045894020956592. Epub 2020 Nov 25.

Section of Physiology, Division of Pulmonary, Critical Care and Sleep Medicine, University of California, San Diego, CA, USA.

Hypoxic Pulmonary Vasoconstriction (HPV) is an important physiological mechanism of the lungs that matches perfusion to ventilation thus maximizing O saturation of the venous blood within the lungs. This study emphasizes on principal pathways in the initiation and modulation of hypoxic pulmonary vasoconstriction with a primary focus on the role of Ca signaling and Ca influx pathways in hypoxic pulmonary vasoconstriction. We used an ex vivo model, isolated perfused/ventilated mouse lung to evaluate hypoxic pulmonary vasoconstriction. Alveolar hypoxia (utilizing a mini ventilator) rapidly and reversibly increased pulmonary arterial pressure due to hypoxic pulmonary vasoconstriction in the isolated perfused/ventilated lung. By applying specific inhibitors for different membrane receptors and ion channels through intrapulmonary perfusion solution in isolated lung, we were able to define the targeted receptors and channels that regulate hypoxic pulmonary vasoconstriction. We show that extracellular Ca or Ca influx through various Ca-permeable channels in the plasma membrane is required for hypoxic pulmonary vasoconstriction. Removal of extracellular Ca abolished hypoxic pulmonary vasoconstriction, while blockade of L-type voltage-dependent Ca channels (with nifedipine), non-selective cation channels (with 30 µM SKF-96365), and TRPC6/TRPV1 channels (with 1 µM SAR-7334 and 30 µM capsazepine, respectively) significantly and reversibly inhibited hypoxic pulmonary vasoconstriction. Furthermore, blockers of Ca-sensing receptors (by 30 µM NPS2143, an allosteric Ca-sensing receptors inhibitor) and Notch (by 30 µM DAPT, a γ-secretase inhibitor) also attenuated hypoxic pulmonary vasoconstriction. These data indicate that Ca influx in pulmonary arterial smooth muscle cells through voltage-dependent, receptor-operated, and store-operated Ca entry pathways all contribute to initiation of hypoxic pulmonary vasoconstriction. The extracellular Ca-mediated activation of Ca-sensing receptors and the cell-cell interaction via Notch ligands and receptors contribute to the regulation of hypoxic pulmonary vasoconstriction.
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http://dx.doi.org/10.1177/2045894020956592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691930PMC
November 2020

Overexpression of p53 due to excess protein O-GlcNAcylation is associated with coronary microvascular disease in type 2 diabetes.

Cardiovasc Res 2020 05;116(6):1186-1198

Department of Physiology, The University of Arizona, 1501 N. Campbell Ave., Tucson, AZ 85724, USA.

Aims: We previously reported that increased protein O-GlcNAcylation in diabetic mice led to vascular rarefaction in the heart. In this study, we aimed to investigate whether and how coronary endothelial cell (EC) apoptosis is enhanced by protein O-GlcNAcylation and thus induces coronary microvascular disease (CMD) and subsequent cardiac dysfunction in diabetes. We hypothesize that excessive protein O-GlcNAcylation increases p53 that leads to CMD and reduced cardiac contractility.

Methods And Results: We conducted in vivo functional experiments in control mice, TALLYHO/Jng (TH) mice, a polygenic type 2 diabetic (T2D) model, and EC-specific O-GlcNAcase (OGA, an enzyme that catalyzes the removal of O-GlcNAc from proteins)-overexpressing TH mice, as well as in vitro experiments in isolated ECs from these mice. TH mice exhibited a significant increase in coronary EC apoptosis and reduction of coronary flow velocity reserve (CFVR), an assessment of coronary microvascular function, in comparison to wild-type mice. The decreased CFVR, due at least partially to EC apoptosis, was associated with decreased cardiac contractility in TH mice. Western blot experiments showed that p53 protein level was significantly higher in coronary ECs from TH mice and T2D patients than in control ECs. High glucose treatment also increased p53 protein level in control ECs. Furthermore, overexpression of OGA decreased protein O-GlcNAcylation and down-regulated p53 in coronary ECs, and conferred a protective effect on cardiac function in TH mice. Inhibition of p53 with pifithrin-α attenuated coronary EC apoptosis and restored CFVR and cardiac contractility in TH mice.

Conclusions: The data from this study indicate that inhibition of p53 or down-regulation of p53 by OGA overexpression attenuates coronary EC apoptosis and improves CFVR and cardiac function in diabetes. Lowering coronary endothelial p53 levels via OGA overexpression could be a potential therapeutic approach for CMD in diabetes.
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http://dx.doi.org/10.1093/cvr/cvz216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177511PMC
May 2020

Chloroquine differentially modulates coronary vasodilation in control and diabetic mice.

Br J Pharmacol 2020 01 8;177(2):314-327. Epub 2020 Jan 8.

Department of Medicine, University of California, San Diego, La Jolla, California.

Background And Purpose: Chloroquine is a traditional medicine to treat malaria. There is increasing evidence that chloroquine not only induces phagocytosis but regulates vascular tone. Few reports investigating the effect of chloroquine on vascular responsiveness of coronary arteries have been made. In this study, we examined how chloroquine affected endothelium-dependent relaxation in coronary arteries under normal and diabetic conditions.

Experimental Approach: We isolated coronary arteries from mice and examined endothelium-dependent relaxation (EDR). Human coronary endothelial cells and mouse coronary endothelial cells isolated from control and diabetic mouse (TALLYHO/Jng [TH] mice, a spontaneous type 2 diabetic mouse model) were used for the molecular biological or cytosolic NO and Ca measurements.

Key Results: Chloroquine inhibited endothelium-derived NO-dependent relaxation but had negligible effect on endothelium-derived hyperpolarization (EDH)-dependent relaxation in coronary arteries of control mice. Chloroquine significantly decreased NO production in control human coronary endothelial cells partly by phosphorylating eNOS (an inhibitory phosphorylation site of eNOS) and attenuating the rise of cytosolic Ca concentration after stimulation. EDR was significantly inhibited in diabetic mice in comparison to control mice. Interestingly, chloroquine enhanced EDR in diabetic coronary arteries by, specifically, increasing EDH-dependent relaxation due partly to its augmenting effect on gap junction activity in diabetic mouse coronary endothelial cells.

Conclusions And Implications: These data indicate that chloroquine affects vascular relaxation differently under normal and diabetic conditions. Therefore, the patients' health condition such as coronary macrovascular or microvascular disease, with or without diabetes, must be taken account into the consideration when selecting chloroquine for the treatment of malaria.
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http://dx.doi.org/10.1111/bph.14864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989957PMC
January 2020

Rho kinase and Na /H exchanger mediate endothelin-1-induced pulmonary arterial smooth muscle cell proliferation and migration.

Physiol Rep 2018 05;6(9):e13698

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD.

Excessive production of endothelin-1 (ET-1) has been observed in almost all forms of pulmonary hypertension. ET-1, a highly potent vasoconstrictor, can also potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. Increasing evidence indicates that alkalinization of intracellular pH (pH ), typically due to activation of Na /H exchange (NHE), is associated with enhanced PASMC proliferation and migration. We recently demonstrated that application of exogenous ET-1 increased NHE activity in murine PASMCs via a mechanism requiring Rho kinase (ROCK). However, whether ROCK and/or increased NHE activity mediate ET-1-induced migration and proliferation in PASMCs remains unknown. In this study, we used fluorescent microscopy in transiently cultured PASMCs from distal pulmonary arteries of the rat and the pH-sensitive dye, BCECF-AM, to measure changes in resting pH and NHE activity induced by exposure to exogenous ET-1 (10  mol/L) for 24 h. Cell migration and proliferation in response to ET-1 were also measured using Transwell assays and BrdU incorporation, respectively. We found that application of exogenous ET-1 had no effect on NHE1 expression, but increased pH , NHE activity, migration, and proliferation in rat PASMCs. Pharmacologic inhibition of NHE or ROCK prevented the ET-1-induced changes in cell function (proliferation and migration). Our results indicate that ET-1 modulates PASMC migration and proliferation via changes in pH homeostasis through a pathway involving ROCK.
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http://dx.doi.org/10.14814/phy2.13698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5949284PMC
May 2018

Orai1, 2, 3 and STIM1 promote store-operated calcium entry in pulmonary arterial smooth muscle cells.

Cell Death Discov 2017 27;3:17074. Epub 2017 Nov 27.

State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510180, China.

Previous studies have demonstrated that besides the classic canonical transient receptor potential channel family, Orai family and stromal interaction molecule 1 (STIM1) might also be involved in the regulation of store-operated calcium channels (SOCCs). An increase in cytosolic free Ca concentration promoted by store-operated Ca entry (SOCE) in pulmonary arterial smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction and proliferation and migration of PASMCs. In this study, our data revealed the following: (1) in both rat distal pulmonary arteries and PASMCs, chronic hypoxia exposure upregulated the expression of Orai1 and Orai2, without affecting Orai3 and STIM1; (2) either heterozygous knockout of HIF-1 in mice or knockdown of HIF-1 in PASMCs abolished the hypoxic upregulation of Orai2, but not Orai1, suggesting the hypoxic upregulation of Orai2 depends on HIF-1; and (3) using small interference RNA knockdown strategies, Orai1, 2, 3 and STIM1 were all shown to mediate SOCE in hypoxic PASMCs. Together, these results suggested that the components of SOCCs, including Orai1, 2, 3 and STIM1, may lead to novel therapeutic targets for the treatment of chronic hypoxia-induced pulmonary hypertension.
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http://dx.doi.org/10.1038/cddiscovery.2017.74DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702854PMC
November 2017

Genetic Variants in the Hedgehog Interacting Protein Gene Are Associated with the FEV1/FVC Ratio in Southern Han Chinese Subjects with Chronic Obstructive Pulmonary Disease.

Biomed Res Int 2017 27;2017:2756726. Epub 2017 Aug 27.

State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China.

Background: Convincing evidences have demonstrated the associations between and polymorphisms and COPD in non-Asian populations. Here genetic variants in and were investigated in Southern Han Chinese COPD.

Methods: A case-control study was conducted, including 989 cases and 999 controls. The associations between SNPs genotypes and COPD were performed by a logistic regression model; for SNPs and COPD-related phenotypes such as lung function, COPD severity, pack-year of smoking, and smoking status, a linear regression model was employed. Effects of risk alleles, genotypes, and haplotypes of the 3 significant SNPs in the gene on FEV/FVC were also assessed in a linear regression model in COPD.

Results: The mean FEV/FVC% value was 46.8 in combined COPD population. None of the 8 selected SNPs apparently related to COPD susceptibility. However, three SNPs (rs12509311, rs13118928, and rs182859) in were associated significantly with the FEV/FVC% ( = 4.1 × 10) in COPD adjusting for gender, age, and smoking pack-years. Moreover, statistical significance between risk alleles and the FEV/FVC% ( = 2.3 × 10), risk genotypes, and the FEV/FVC% ( = 3.5 × 10) was also observed in COPD.

Conclusions: Genetic variants in were related with FEV/FVC in COPD. Significant relationships between risk alleles and risk genotypes and FEV/FVC in COPD were also identified.
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http://dx.doi.org/10.1155/2017/2756726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591965PMC
March 2018

Aquaporin 1-mediated changes in pulmonary arterial smooth muscle cell migration and proliferation involve β-catenin.

Am J Physiol Lung Cell Mol Physiol 2017 Nov 10;313(5):L889-L898. Epub 2017 Aug 10.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland

Exposure to hypoxia induces migration and proliferation of pulmonary arterial smooth muscle cells (PASMCs), leading to vascular remodeling and contributing to the development of hypoxic pulmonary hypertension. The mechanisms controlling PASMC growth and motility are incompletely understood, although aquaporin 1 (AQP1) plays an important role. In tumor, kidney, and stem cells, AQP1 has been shown to interact with β-catenin, a dual function protein that activates the transcription of crucial target genes (i.e., c-Myc and cyclin D1) related to cell migration and proliferation. Thus the goal of this study was to examine mechanisms by which AQP1 mediates PASMC migration and proliferation, with a focus on β-catenin. Using primary rat PASMCs from resistance level pulmonary arteries infected with adenoviral constructs containing green fluorescent protein (control; AdGFP), wild-type AQP1 (AdAQP1), or AQP1 with the COOH-terminal tail deleted (AdAQP1M), we demonstrated that increasing AQP1 expression upregulated β-catenin protein levels and the expression (mRNA and protein) of the known β-catenin targets c-Myc and cyclin D1. In contrast, infection with AdAQP1M had no effect on any of these variables. Using silencing approaches to reduce β-catenin levels prevented both hypoxia- and AQP1-induced migration and proliferation of PASMCs, as well as induction of c-Myc and cyclin D1 by AQP1. Thus our results indicate that elevated AQP1 levels upregulate β-catenin protein levels, via a mechanism requiring the AQP1 COOH-terminal tail, enhancing expression of β-catenin targets and promoting PASMC proliferation and migration.
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http://dx.doi.org/10.1152/ajplung.00247.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792177PMC
November 2017

A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry.

Cytotechnology 2016 Oct 3;68(5):2015-25. Epub 2016 Aug 3.

Department of Technology R&D, Nexcelom Bioscience LLC, 360 Merrimack St. Building 9, Lawrence, MA, 01843, USA.

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.
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http://dx.doi.org/10.1007/s10616-016-0015-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023569PMC
October 2016

Clinical characteristics and risk factors of pulmonary hypertension associated with chronic respiratory diseases: a retrospective study.

J Thorac Dis 2016 Mar;8(3):350-8

1 State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The Affiliated Hospital of Guangzhou Medical University, Guangdong 510120, China ; 2 Geisinger Medical Center, 100 North Academy Avenue, Danville, USA ; 3 Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, USA.

Background: Chronic respiratory disease-associated pulmonary hypertension (PH) is an important subtype of PH, which lacks clinical epidemiological data in China.

Methods: Six hundred and ninety three patients hospitalized from 2010 to 2013 were classified by echocardiography according to pulmonary arterial systolic pressure (PASP): mild (36≤ PASP <50 mmHg); moderate (50≤ PASP <70 mmHg) and severe (PASP ≥70 mmHg).

Results: Dyspnea (93.51%) was the most common symptom. Hemoptysis observed in the severe group (6.42%) was significantly higher than the other two groups (P<0.05). COPD (78.35%), lung bullae (44.16%), tuberculosis (including obsolete pulmonary tuberculosis) (38.82%), and bronchiectasis (30.45%) were frequently present. Mild group occupied the highest proportion (84.7%) in COPD, while severe group occupied the highest proportion (19.3%) in pulmonary embolism (P<0.01). Age, partial pressure of oxygen (PaO2), hematocrit (HCT), partial pressure of carbon dioxide (PaCO2), increase of N-terminal pro brain natriuretic peptide (NT-proBNP) and right ventricular (RV) diameter (>20 mm) were associated with moderate-to-severe PH, while RV [odds ratio (OR) =3.53, 95% CI, 2.17-5.74], NT-proBNP (OR=2.44, 95% CI, 1.51-3.95), HCT (OR=1.03, 95% CI, 1.00-1.07) and PaCO2 (OR=1.01, 95% CI, 1.00-1.03) were independent risk factors.

Conclusions: PH related to respiratory diseases is mostly mild to moderate, and the severity is associated with the category of respiratory disease. Increased HCT can be an independent risk factor for PH related to chronic respiratory diseases.
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http://dx.doi.org/10.21037/jtd.2016.02.58DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4805801PMC
March 2016

Leydig cell tumor with lung metastasis diagnosed by lung biopsy.

Int J Clin Exp Pathol 2015 1;8(10):12972-6. Epub 2015 Oct 1.

State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Diseases, The 1st Affiliated Hospital of Guangzhou Medical University Guangzhou 510120, Guangdong, People's Republic of China.

Leydig cell tumors are very rare and account for only 3% of testicular tumors and are generally benign. Only less than 0.2% of all testicular cancers were evidenced by metastatic spread. We report a 34-year-old man visited hospital because of coughing sputum mixed with blood. His chest CT showed bilateral patch clouding opacity. He was suspected with allergic alveolitis and treated with methylprednisolone. However, his symptoms and general condition deteriorated, and he visited our hospital. He had no abnormal findings on physical examination. A chest radiograph showed pneumonia in whole lung and CT showed multiple nodules and diffused ground glass opacities in both lung fields. Lung biopsy confirmed a diagnosis of Leydig cell tumor with lung metastasis. The diagnosis is based on the histopathology and immunohistochemistry.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4680438PMC
October 2016

Study Design and Interim Outcomes of Guangzhou Institute of Respiratory Disease COPD Biobank.

COPD 2016 1;13(2):203-13. Epub 2015 Dec 1.

a State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital, Guangzhou Medical University , Guangzhou , Guangdong , China.

Background: GIRD COPD Biobank is a multicenter observational study blood-based database with local characteristics, in order to investigate the causes, risk factors, pathogenesis, prevalence patterns and trends of COPD and promote new pathogenic insights in China.

Methods: We enrolled 855 clinically COPD patients and 660 controls with normal lung function. Extensive data collection has been undertaken with questionnaires, clinical measurements, and collection and storage of blood specimens, following Standard Operating Procedures (SOP). All surveys had similar quality controls, supervisions, and training of the investigator team.

Results: Since September 2010, a total of 1515 subjects (1116 [73.7%] males; 855 [56.4%] diagnosed with COPD) were enrolled. Analyses of the design and interim results of the GIRD COPD Biobank Study identified patients with COPD were older, lower educational level, a longer history of pack-year smoking, less in kitchen fan usage, X-ray exposure, and history of disease (P < 0.01 for all); Most of the COPD subjects belonged to moderately severe or worse, stratified according to Global Lung Function Initiative (GLI); COPD patients had relatively more co-morbidities than controls; Environmental hazard exposures might be the main contributors to the reported respiratory symptoms; Cold air, haze, and influenza acted the top three factors to induce respiratory symptoms in both COPD cases and controls.

Conclusion: The GIRD COPD Biobank Study has the potential to provide substantial novel insights into the genetics, biomarkers, environmental and lifestyle aspects of COPD. It is expected to provide new insights for pathogenesis and the long-term progression of COPD.
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http://dx.doi.org/10.3109/15412555.2015.1069807DOI Listing
January 2017

Ca(2+) and ion channels in hypoxia-mediated pulmonary hypertension.

Int J Clin Exp Pathol 2015 1;8(2):1081-92. Epub 2015 Feb 1.

State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University China.

Alveolar hypoxia, a consequence of many lung diseases, can have adverse effects on the pulmonary vasculature. The changes that occur in the pulmonary circulation with exposure to chronic hypoxia include reductions in the diameter of the pulmonary arteries due to structural remodeling of the vasculature. Although the structural and functional changes that occur in the development of pulmonary hypertension have been well investigated, less is known about the cellular and molecular mechanisms of this process. This review will discuss the role of several potassium and calcium channels in hypoxic pulmonary vasoconstriction, both in elevating calcium influx into pulmonary artery smooth muscle cells (PASMCs). In addition to other signal transduction pathways, Ca(2+) signaling in PASMCs plays an important role in the development and progression of pulmonary hypertension due to its central roles in vasoconstriction and vascular remodeling. This review will focus on the effect of chronic hypoxia on ion channels and the potential pathogenic role of Ca(2+) signaling and regulation in the progression of pulmonary hypertension.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396234PMC
March 2016

Intravascular large B-cell lymphoma confirmed by lung biopsy.

Int J Clin Exp Pathol 2014 15;7(9):6301-6. Epub 2014 Aug 15.

State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University China.

Intravascular lymphoma is a very rare form of large B-cell non-Hodgkin's lymphoma which is characterized by selective growth of lymphoma cells within the lumina of small blood vessels. We report a 64-year-old woman visited hospital because of persistent cough, intermittent high fever as high as 38.7 °C and occasional shortness of breath. Her chest CT showed left upper lobe pneumonia and tuberculosis skin test (PPD test) was positive. She was suspected with tuberculosis and treated with anti-tuberculosis drugs. However, her symptoms and general condition deteriorated, and she visited our hospital. She had no abnormal findings on physical examination, but had abnormal laboratory findings, including decreased hemoglobin, elevated LDH and C-reactive protein. Arterial blood gas analysis showed moderate hypoxaemia. A chest radiograph showed pneumonia in whole lung and CT showed diffused ground glass opacities in both lung fields. Lung biopsy confirmed a diagnosis of intravascular large B-cell lymphoma. Primary pulmonary manifestation is very rare. The diagnosis is based on the histopathology and immunohistochemistry.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203254PMC
July 2015

Effects of chronic exposure to cigarette smoke on canonical transient receptor potential expression in rat pulmonary arterial smooth muscle.

Am J Physiol Cell Physiol 2014 Feb 11;306(4):C364-73. Epub 2013 Dec 11.

State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, People's Republic of China;

To clarify the possible mechanism of cigarette smoke (CS)-induced pulmonary hypertension and furthermore provide effective targets for prevention and treatment, the effects of chronic CS on rat pulmonary arterial smooth muscle in vivo and nicotine treatment on rat pulmonary arterial smooth muscle cells (PASMCs) in vitro were investigated. In this study, we demonstrated that chronic CS exposure led to rat weight loss, right ventricular hypertrophy, and pulmonary arterial remodeling. A fluorescence microscope was used to measure intracellular calcium concentration ([Ca(2+)]i) in rat distal PASMCs. Results showed that basal [Ca(2+)]i and store-operated calcium entry (SOCE) levels in PASMCs from 3- and 6-mo CS-exposed rats were markedly higher than those in cells from the unexposed control animals (the increases in 6-mo CS group were more significant than that in 3-mo group), accompanied with increased canonical transient receptor potential 1 (TRPC1) and TRPC6 expression at both mRNA and protein levels in isolated distal PA. Simultaneously, in vitro study showed that nicotine treatment (10 nM) significantly increased basal [Ca(2+)]i and SOCE and upregulated TRPC1 and TRPC6 expression in cultured rat distal PASMCs. TRPC siRNA knockdown strategies revealed that the elevations of basal [Ca(2+)]i and SOCE induced by nicotine in PASMCs were TRPC1 and TRPC6 dependent. These results suggested that chronic CS-induced changes in vascular tone and structure in PA and the development of pulmonary hypertension might be largely due to upregulation of TRPC1 and TRPC6 expression in PASMCs, in which nicotine played an important role.
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http://dx.doi.org/10.1152/ajpcell.00048.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919980PMC
February 2014

The aquaporin 1 C-terminal tail is required for migration and growth of pulmonary arterial myocytes.

Am J Respir Cell Mol Biol 2014 Jun;50(6):1010-20

1 Guangzhou Institute of Respiratory Diseases, State Key Laboratory of Respiratory Diseases, The First Affiliated Hospital, Guangzhou Medical University, Guangzhou, People's Republic of China; and.

Pulmonary arterial smooth muscle cell (PASMC) proliferation and migration are important contributors to the vascular remodeling that occurs during development of pulmonary hypertension. We previously demonstrated that aquaporin (AQP)1, a member of the water channel family of proteins, was expressed in PASMCs and was necessary for hypoxia-induced migration; however, the mechanism by which AQP1 controls this response is unclear. The C-terminal tail of AQP1 contains putative calcium (EF-hand) and protein binding sites. Thus, we wanted to explore whether the C-terminal tail or the EF-hand motif of AQP1 was required for migration and proliferation. Rat PASMCs were isolated from distal pulmonary arteries, and proliferation and migration were measured using BrdU incorporation and transwell filters, respectively. To deplete AQP1, PASMCs were transfected with AQP1 small interference RNA (siRNA) or nontargeting siRNA. Knockdown of AQP1 reduced basal proliferation and hypoxia-induced migration and proliferation in PASMCs. In subsequent experiments, wild-type AQP1, AQP1 lacking the entire cytoplasmic C-terminal tail, or AQP1 with a mutation in the EF-hand motif were expressed in PASMCs using adenoviral constructs. For all AQP1 constructs, infection increased AQP1 protein levels, water permeability, and the change in cell volume induced by hypotonic challenge. Infection with wild-type and EF-hand mutated AQP1, but not C-terminal-deleted AQP1, increased PASMC migration and proliferation. Our results suggest that AQP1 controls proliferation and migration in PASMCs and that the mechanism requires the C-terminal tail of the protein but is independent of water transport or the EF-hand motif.
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http://dx.doi.org/10.1165/rcmb.2013-0374OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946682PMC
June 2014

Sildenafil inhibits hypoxia-induced transient receptor potential canonical protein expression in pulmonary arterial smooth muscle via cGMP-PKG-PPARγ axis.

Am J Respir Cell Mol Biol 2013 Aug;49(2):231-40

State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China.

Transient receptor potential canonical (TRPC) proteins play important roles in chronically hypoxic pulmonary hypertension (CHPH). Previous results indicated that sildenafil inhibited TRPC1 and TRPC6 expression in rat distal pulmonary arteries (PAs). However, the underlying mechanisms remain unknown. We undertook this study to investigate the downstream signaling of sildenafil's regulation on TRPC1 and TRPC6 expression in pulmonary arterial smooth muscle cells (PASMCs). Hypoxia-exposed rats (10% O2 for 21 d) and rat distal PASMCs (4% O2 for 60 h) were taken as models to mimic CHPH. Real-time PCR, Western blotting, and Fura-2-based fluorescent microscopy were performed for mRNA, protein, and Ca(2+) measurements, respectively. The cellular cyclic guanosine monophosphate (cGMP) analogue 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate sodium salt (CPT-cGMP) (100 μM) inhibited TRPC1 and TRPC6 expression, store-operated Ca(2+) entry (SOCE), and the proliferation and migration of PASMCs exposed to prolonged hypoxia. The inhibition of CPT-cGMP on TRPC1 and TRPC6 expression in PASMCs was relieved by either the inhibition or knockdown of cGMP-dependent protein kinase (PKG) and peroxisome proliferator-activated receptor γ (PPARγ) expression. Under hypoxic conditions, CPT-cGMP increased PPARγ expression. This increase was abolished by the PKG antagonists Rp8 or KT5823. PPARγ agonist GW1929 significantly decreased TRPC1 and TRPC6 expression in PASMCs. Moreover, hypoxia exposure decreased, whereas sildenafil treatment increased, PKG and PPARγ expression in PASMCs ex vivo, and in rat distal PAs in vivo. The suppressive effects of sildenafil on TRPC1 and TRPC6 in rat distal PAs and on the hemodynamic parameters of CHPH were inhibited by treatment with the PPARγ antagonist T0070907. We conclude that sildenafil inhibits TRPC1 and TRPC6 expression in PASMCs via cGMP-PKG-PPARγ-dependent signaling during CHPH.
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http://dx.doi.org/10.1165/rcmb.2012-0185OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3824028PMC
August 2013

Bone morphogenetic protein 2 decreases TRPC expression, store-operated Ca(2+) entry, and basal [Ca(2+)]i in rat distal pulmonary arterial smooth muscle cells.

Am J Physiol Cell Physiol 2013 May 27;304(9):C833-43. Epub 2013 Feb 27.

State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, Guangzhou, China.

Recent studies indicate that multiple bone morphogenetic protein (BMP) family ligands and receptors are involved in the development of pulmonary arterial hypertension, yet the underlying mechanisms are incompletely understood. Although BMP2 and BMP4 share high homology in amino acid sequence, they appear to exert divergent effects on chronic hypoxic pulmonary hypertension (CHPH). While BMP4 promotes vascular remodeling, BMP2 prevents CHPH. We previously demonstrated that BMP4 upregulates the expression of canonical transient receptor potential channel (TRPC) proteins and, thereby, enhances store-operated Ca(2+) entry (SOCE) and elevates intracellular Ca(2+) concentration ([Ca(2+)]i) in pulmonary arterial smooth muscle cells (PASMCs). In this study, we investigated the effects of BMP2 on these variables in rat distal PASMCs. We found that treatment with BMP2 (50 ng/ml, 60 h) inhibited TRPC1, TRPC4, and TRPC6 mRNA and protein expression. Moreover, BMP2 treatment led to reduced SOCE and decreased basal [Ca(2+)]i in PASMCs. These alterations were associated with decreased PASMC proliferation and migration. Conversely, knockdown of BMP2 with specific small interference RNA resulted in increased cellular levels of TRPC1, TRPC4, and TRPC6 mRNA and protein, enhanced SOCE, elevated basal [Ca(2+)]i, and increased proliferation and migration of PASMCs. Together, these results indicate that BMP2 participates in regulating Ca(2+) signaling in PASMCs by inhibiting TRPC1, TRPC4, and TRPC6 expression, thus leading to reduced SOCE and basal [Ca(2+)]i and inhibition of cell proliferation and migration.
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http://dx.doi.org/10.1152/ajpcell.00036.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651608PMC
May 2013

Sodium tanshinone IIA sulfonate inhibits canonical transient receptor potential expression in pulmonary arterial smooth muscle from pulmonary hypertensive rats.

Am J Respir Cell Mol Biol 2013 Jan 11;48(1):125-34. Epub 2012 Oct 11.

Guangzhou Institute of Respiratory Diseases, State Key Laboratory of Respiratory Diseases, The First Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, People's Republic of China.

Danshen, the dried root of Salvia miltiorrhiza, is widely used in clinics in China for treating various diseases, including cardiovascular diseases. Sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of tanshinone IIA isolated as the major active component from Danshen, was recently reported to be effective in attenuating the characteristic pulmonary vascular changes associated with chronically hypoxic pulmonary hypertension (CHPH); however, the underlying detailed mechanisms are poorly understood. In this study, we investigated the effects of STS on basal intracellular Ca(2+) concentration ([Ca(2+)](i)) and store-operated Ca(2+) entry (SOCE) in distal pulmonary arterial smooth muscle cells (PASMCs) exposed to prolonged hypoxia or isolated from CHPH rats. SOCE measured by Mn(2+) quenching of Fura-2 fluorescence in PASMCs from rats exposed to chronic hypoxia (10% O(2), 21 d) was increased by 59%, and basal [Ca(2+)](i) was increased by 119%; this effect was inhibited by intraperitoneal injection of STS. These inhibitory effects of STS on hypoxic increases of SOCE and basal [Ca(2+)](i) were associated with reduced expression of canonical transient receptor potential (TRPC)1 and TRPC6 in distal pulmonary arterial smooth muscle and decreases on right ventricular pressure, right ventricular hypertrophy, and peripheral pulmonary vessel thickening. In ex vivo cultured distal PASMCs from normoxic rats, STS (0-25 μM) dose-dependently inhibited hypoxia-induced cell proliferation and migration, paralleled with attenuation in increases of basal [Ca(2+)](i), SOCE, mRNA, and protein expression of TRPC1 and TRPC6. STS also relieved right ventricular systolic pressure, right ventricular hypertrophy, and TRPC1 and TRPC6 protein expression in distal pulmonary arteries in a monocrotaline-induced rat model of pulmonary arterial hypertension. These results indicate that STS prevents pulmonary arterial hypertension development likely by inhibiting TRPC1 and TRPC6 expression, resulting in normalized basal [Ca(2+)](i) and attenuated proliferation and migration of PASMCs.
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http://dx.doi.org/10.1165/rcmb.2012-0071OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3547081PMC
January 2013

A novel image-based cytometry method for autophagy detection in living cells.

Autophagy 2012 Sep 16;8(9):1371-82. Epub 2012 Aug 16.

Department of Technology R&D, Nexcelom Bioscience, LLC, Lawrence, MA, USA.

Autophagy is an important cellular catabolic process that plays a variety of important roles, including maintenance of the amino acid pool during starvation, recycling of damaged proteins and organelles, and clearance of intracellular microbes. Currently employed autophagy detection methods include fluorescence microscopy, biochemical measurement, SDS-PAGE and western blotting, but they are time consuming, labor intensive, and require much experience for accurate interpretation. More recently, development of novel fluorescent probes have allowed the investigation of autophagy via standard flow cytometry. However, flow cytometers remain relatively expensive and require a considerable amount of maintenance. Previously, image-based cytometry has been shown to perform automated fluorescence-based cellular analysis comparable to flow cytometry. In this study, we developed a novel method using the Cellometer image-based cytometer in combination with Cyto-ID(®) Green dye for autophagy detection in live cells. The method is compared with flow cytometry by measuring macroautophagy in nutrient-starved Jurkat cells. Results demonstrate similar trends of autophagic response, but different magnitude of fluorescence signal increases, which may arise from different analysis approaches characteristic of the two instrument platforms. The possibility of using this method for drug discovery applications is also demonstrated through the measurement of dose-response kinetics upon induction of autophagy with rapamycin and tamoxifen. The described image-based cytometry/fluorescent dye method should serve as a useful addition to the current arsenal of techniques available in support of autophagy-based drug discovery relating to various pathological disorders.
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http://dx.doi.org/10.4161/auto.21028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3442883PMC
September 2012

Rapid image-based cytometry for comparison of fluorescent viability staining methods.

J Fluoresc 2012 Sep 21;22(5):1301-11. Epub 2012 Jun 21.

Department of Technology R&D, Nexcelom Bioscience LLC, 360 Merrimack St. Building 9, Lawrence, MA 01843, USA,

The ability to accurately measure cell viability is important for any cell-based research. Traditionally, viability measurements have been performed using trypan blue exclusion method on hemacytometer, which allowed researchers to visually distinguish viable from nonviable cells. However, the trypan blue method is often limited to only cell lines or primary cells that have been rigorously purified. In the recent years, small desktop image-based cell counters have been developed for rapid cell concentration and viability measurement due to advances in imaging and optics technologies as well as novel fluorescent stains. In this work, we employed the Cellometer image-based cytometer to demonstrate the ability to simplify viability detection compared to the current methods. We compared various fluorescence viability detection methods using single- or dual-staining technique. Single-staining method using nucleic acid stains including ethidium bromide, propidium iodide, 7AAD, DAPI, Sytox Green and Sytox Red, and enzymatic stains including CFDA and Calcein AM were performed. All stains produced comparable results to trypan blue exclusion method for cell line samples. Dual-staining method using AO/PI, CFDA/PI, Calcein AM/PI and Hoechst 33342/PI that enumerates viable and non-viable cells was tested on primary cell samples with high debris contents. This method allowed exclusion of cellular debris and non-nucleated cells from analysis, which can eliminate the need to perform purification step during sample preparation, and improves the efficiency of viability detection method. Overall, these image-based fluorescent cell counters can simplify assay procedures as well as capture images for visual confirmation.
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http://dx.doi.org/10.1007/s10895-012-1072-yDOI Listing
September 2012

Hypoxia-induced migration in pulmonary arterial smooth muscle cells requires calcium-dependent upregulation of aquaporin 1.

Am J Physiol Lung Cell Mol Physiol 2012 Aug 8;303(4):L343-53. Epub 2012 Jun 8.

Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, 5501 Hopkins Bayview Circle, JHAAC 4A.52, Baltimore, MD 21224, USA.

Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration ([Ca(2+)](i)) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca(2+) dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, [Ca(2+)](i), and migration in PASMCs. Blockade of Ca(2+) entry through voltage-dependent Ca(2+) or nonselective cation channels prevented the hypoxia-induced increase in PASMC [Ca(2+)](i), AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in [Ca(2+)](i) that results in increased AQP1 protein levels and cell migration.
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http://dx.doi.org/10.1152/ajplung.00130.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423828PMC
August 2012

Adipocyte induction of preadipocyte differentiation in a gradient chamber.

Tissue Eng Part C Methods 2012 Dec 20;18(12):958-67. Epub 2012 Sep 20.

Department of Chemical and Biological Engineering, Tufts University, Medford, MA 02155, USA.

Adipose tissue expansion involves enlargement of mature adipocytes and the formation of new adipocytes through the differentiation of locally resident preadipocytes. Factors released by the enlarged adipocytes are potential cues that induce the differentiation of the preadipocytes. Currently, there are limited options to investigate these cues in isolation from confounding systemic influences. A gradient generating microfluidic channel-based cell culture system was designed to enable solution patterning, while supporting long-term culture and differentiation of preadipocytes. Solution patterning was confirmed by selectively staining a fraction of uniformly seeded preadipocytes. An adipogenic cocktail gradient was used to induce the differentiation of a fraction of uniformly seeded preadipocytes and establish a spatially defined coculture of adipocytes and preadipocytes. Varying the adipogenic cocktail gradient generated cocultures of preadipocytes and adipocytes with different compositions. Transient application of the cocktail gradient, followed by basal medium treatment showed a biphasic induction of differentiation. The two phases of differentiation correlated with a spatial gradient in adipocyte size. Our results provide in vitro data supporting the size-dependent release of preadipocyte differentiation factors by enlarged adipocytes. Prospectively, the coculture system developed in this study could facilitate controlled, yet physiologically meaningful studies on paracrine interactions between adipocytes and preadipocytes during adipose tissue development.
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http://dx.doi.org/10.1089/ten.TEC.2012.0168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3491637PMC
December 2012

A rapid detection method for apoptosis and necrosis measurement using the Cellometer imaging cytometry.

Apoptosis 2011 Dec;16(12):1295-303

Department of Technology R&D, Nexcelom Bioscience LLC, Lawrence, MA 01843, USA.

Apoptosis and necrosis play an important role in various aspects of preclinical pharmaceutical drug discovery and validation. The ability to quickly determine the cytotoxic effect of chemical compounds on cancer cells allows researchers to efficiently identify potential drug candidates for further development in the pharmaceutical discovery pipeline. Recently, a new imaging cytometry system has been developed by Nexcelom Bioscience LLC (Lawrence, MA, USA) for fluorescence-based cell population analysis. Currently, fluorescence-based cell death assays have not been demonstrated by the Cellometer system, which can potentially provide a quick, simple, and inexpensive alternative method for smaller biomedical research laboratories. In this study, we demonstrate for the first time the use of Cellometer imaging cytometry for necrosis/apoptosis detection by studying the dose-response effect of heat and drug-induced cell death in Jurkat cells labeled with annexin V-FITC (apoptotic) and propidium iodide (necrotic). The experimental results were evaluated to validate the imaging cytometric capabilities of the Cellometer system as compared to the conventional flow cytometry. Similar cell population results were obtained from the two methods. The ability of Cellometer to rapidly and cost-effectively perform fluorescent cell-based assays has the potential of improving research efficiency, especially where a flow or laser scanning cytometer is not available or in situations where a rapid analysis of data is desired.
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http://dx.doi.org/10.1007/s10495-011-0651-8DOI Listing
December 2011

Bone morphogenetic protein 4 enhances canonical transient receptor potential expression, store-operated Ca2+ entry, and basal [Ca2+]i in rat distal pulmonary arterial smooth muscle cells.

Am J Physiol Cell Physiol 2010 Dec 15;299(6):C1370-8. Epub 2010 Sep 15.

Guangzhou Institute of Respiratory Diseases, State Key Laboratory of Respiratory Diseases, The First Affiliated Hospital, Guangzhou Medical University, Guangzhou, People's Republic of China.

Recent advances have identified an important role of bone morphogenetic protein 4 (BMP4) in pulmonary vascular remodeling, yet the underlying mechanisms remain largely unexplored. We have previously found that Ca(2+) influx through store-operated calcium channels (SOCC), which are mainly thought to be composed of canonical transient receptor potential (TRPC) proteins, likely contribute to the pathogenic development of chronic hypoxic pulmonary hypertension. In this study, we investigated the effect of BMP4 on expression of TRPC and store-operated Ca(2+) entry (SOCE) in pulmonary arterial smooth muscle cells (PASMCs). Real-time quantitative PCR and Western blotting revealed that treatment with BMP4 (50 ng/ml, 60 h) increased TRPC1, TRPC4, and TRPC6 mRNA and protein expression in growth-arrested rat distal PASMCs. Moreover, in comparison to vehicle control, cells treated with BMP4 also exhibited enhanced SOCE, and elevated basal intracellular calcium concentration ([Ca(2+)](i)) as determined by fluorescent microscopy using the Ca(2+) indicator Fura-2 AM. Perfusing cells with Ca(2+)-free Krebs-Ringer bicarbonate solution (KRBS) or KRBS containing SOCC antagonists SKF-96365 or NiCl(2) attenuated the increases in basal [Ca(2+)](i) caused by BMP4. Specific knockdown of BMP4 by small interference RNA significantly decreased the mRNA and protein expression of TRPC1, TRPC4, and TRPC6 and reduced SOCE and basal [Ca(2+)](i) in serum-stimulated PASMCs. We conclude that BMP4 regulates calcium signaling in PASMCs likely via upregulation of TRPC expression, leading to enhanced SOCE and basal [Ca(2+)](i) in PASMCs, and by this mechanism contributes to pulmonary vascular remodeling during pulmonary arterial hypertension.
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http://dx.doi.org/10.1152/ajpcell.00040.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3006336PMC
December 2010

Enhanced proliferation of human umbilical vein endothelial cells and differentiation of 3T3-L1 adipocytes in coculture.

Tissue Eng Part A 2009 May;15(5):1053-61

Department of Chemical and Biological Engineering, Tufts University, Medford, Massachusetts 02155, USA.

The interactions between adipocytes and endothelial cells in adipose tissue development are poorly understood. In this study, we characterized the growth and differentiation of 3T3-L1 preadipocytes and human umbilical vein endothelial cells (HUVECs) in planar and collagen gel cocultures. In planar coculture, preadipocyte proliferation was up to three times as great as in the control culture with only preadipocytes, where the increase was proportional to the HUVEC fraction in the seeding mixture. In the collagen gel coculture, triglyceride (TG) content (per adipocyte) was up to 3.4 times as much as in the control with only adipocytes. This effect depended on the total density and composition of the seeding mixture, with the largest increase observed at the highest density (2 x 10(6) cells/mL collagen) and preadipocyte:HUVEC ratio (90:10) tested in this study. Immunostaining showed that the collagen gel coculture also supported the elongation of endothelial cells. Blockade of vascular endothelial growth factor receptor 2 (VEGFR2) abolished the adipogenesis- and neovascularization-related effects of the coculture. Taken together, our results indicate that endothelial cell-mediated enhancement of adipocyte differentiation requires the activation of VEGFR2.
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http://dx.doi.org/10.1089/ten.tea.2008.0101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2767334PMC
May 2009

[A single factor analysis of the prognosis of 301 hepatitis failure cases and a study of a scoring system on their prognostic assessment].

Zhonghua Gan Zang Bing Za Zhi 2005 Aug;13(8):586-9

Department of Infectious Diseases, Second Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400010, China.

Objectives: To analyze the factors related to prognosis of hepatitis failure and to determine the factors which significantly affect it, and to build a scoring system for assessment of the prognosis of hepatitis failure and also to examine its efficacy for clinical use.

Methods: Clinical data from 301 patients were analyzed retrospectively. The correlated degree between those single factors and prognosis of hepatitis failure was explored by logistic regression analysis. Independent risk factors of prognosis and those correlated coefficients, which were from logistic regression analysis, were used to build a scoring system. This system was used in analyzing the clinical data of 275 patients to examine its efficacy of the prognostic assessment.

Results: The factors that significantly affected the prognosis of hepatitis failure included age, clinical typing, hepatic coma, total bilirubin, and others (P < 0.01). Some factors, including PTA, blood urea, sodium and hepatic coma, were independent risk factors of prognosis. The scoring system built gave different scores between the effective treatment group and ineffective treatment group with statistical significance (P < 0.01). When the score was less than 40, the probability of a recovery was 76.9%; when the score was 40 to 80, the probability of a recovery was only 12.5%. When the score was more than 80, the probability of a recovery dropped to 0%.

Conclusions: The factors, including PTA, blood urea and sodium and hepatic coma, are important in building a scoring system to assess the prognosis of hepatitis failure. The scoring system we built is very effective in evaluating the prognosis of hepatitis failure.
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August 2005

[Diagnosis and therapy of 186 spontaneous bacterial peritonitis patients with end-stage liver disease].

Zhonghua Gan Zang Bing Za Zhi 2004 Jun;12(6):350-2

Department of Infectious Diseases, the Second Affiliated Hospital of Chongqing University of Medical Sciences, Chongqing 400010, China.

Objective: To improve the diagnosis and treatment level of spontaneous bacterial peritonitis (SBP) in the patients with advanced liver disease, get better curative effect and prognosis.

Methods: Registered the body temperature, symptoms and signs in the abdomen, and blood routine test, the polymorphonuclear (PMN) cell count, and ascites culture in the patients with cirrhosis and fulminant hepatitis. These patients were given supporting therapies including use plasma and albumin as well as antibiotics treatment according to drug sensitivity or empiric. Changes of the body temperature, symptoms and signs were used to evaluate the effect of therapy.

Results: 186 of 275 inward patients with end-stage liver disease during this period were considered as SBP by ascites culture or clinical experience with various degree symptoms and signs such as pain, distention, higher tension and touch pain in the abdomen. Infective rate was 67.6%. Among them 138 patients had abnormal body temperature more than 37.4 degrees C. 106 patients with leukocyte count in the peripheral blood more than 10 x 10(9)/L; 137 patients with PMN more than 80% in differential cell count; 103 patients with PMN more than 250/mm(3) in ascites. Only 29 patients were culture positive. 82 patients were cured, 17 patients with improvement, 18 patients with inefficacy or deterioration. 42 patients died of hepatic-renal failure and 27 patients died because of upper alimentary tract bleeding, respectively.

Conclusion: Signs and symptoms of SBP were atypical in the patients with end-stage liver disease. Ascites culture positive rate was not high. Early diagnosis and proper use antibiotics according to culture and empirics were important to increase effect and improve prognosis
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June 2004

[Etiology and clinical characters of hepatitis caused by non-hepatotropic virus].

Zhonghua Gan Zang Bing Za Zhi 2004 Mar;12(3):129-30

Department of Infectious Diseases, the Second Affiliated Hospital of Chongqing University Medical Sciences, Chongqing 400010, China.

Objective: To explore the etiology and clinical characters of hepatitis caused by non-hepatotropic virus.

Methods: 68 non-hepatotrophic viral hepatitis patients with negative anti-HAV-anti-HEV were diagnosed by detecting antibodies of anti-HSV IgM, anti-EBV IgM, anti-CMV IgM, anti-CSV IgM and anti-ANA, anti-mitochondrion antibody. Their clinical symptoms and signs were compared with that of acute viral hepatitis patients at the same time.

Results: Among the 68 patients, 9 were infected by HSV, 12 by EBV, 8 by CMV, 14 by CSV, and the other 13 patients and 12 patients were positive for anti-ANA and anti-mitochondrion antibody, respectively. 35 of 43 non-hepatotrophic viral hepatitis patients were infected in winter and spring season. Their clinical symptoms and signs were milder than that of acute viral hepatitis patients.

Conclusion: Liver damage and dysfunction may be the prominent phenomenon during HSV, EBV, CMV and CSV infection, just like that of acute viral hepatitis but with milder clinical symptom and signs.
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March 2004
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