Yanxia Chu

Cartilage Oligomeric Matrix Protein in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5–3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes (Fold change 13, p-value <0.05) in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-β1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-β1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-β1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-β1 signaling should be persuaded.

E2F1 effects on osteoblast differentiation and mineralization are mediated through up-regulation of frizzled-1☆

Frizzled homolog 1 (FZD1) is a transmembrane receptor that mediates Wnt signaling. The transcriptional regulation of FZD1 and the role of FZD1 in osteoblast biology are not well understood. We examined the role of E2F1 in FZD1 promoter activation and osteoblast differentiation and mineralization. A putative E2F1 binding site in the FZD1 promoter region was initially identified in silico and characterized further in Saos2 cells in vitro by chromatin immunoprecipitation (ChIP), electrophoretic mobility shift (EMSA) and promoter reporter assays. Over-expression of E2F1 transactivated the FZD1 promoter and increased endogenous FZD1 mRNA and protein levels in Saos2 cells. Over-expression of E2F1 in Saos2 cells up-regulated osteoblast differentiation markers alkaline phosphatase (ALP), type I collagen α (COL1A), and osteocalcin (OCN). Furthermore, E2F1 over-expression enhanced mineralization of differentiated Saos2 cells, whereas siRNA knockdown of FZD1 diminished the effects of E2F1 on osteoblast mineralization. The effects of E2F1 on FZD1 expression and osteoblast mineralization were further confirmed in normal human FOB osteoblasts. Taken together, our experiments demonstrate a role of E2F1 in osteoblast differentiation and mineralization and suggest that FZD1 is required, in part, for E2F1 regulation of osteoblast mineralization.

Elevated NT-Pro-Brain Natriuretic Peptide Level Is Independently Associated with All-Cause Mortality in HIV-Infected Women in the Early and Recent HAART Eras in the Women’s Interagency HIV Study Cohort

Background HIV-infected individuals are at increased risk of right and left heart dysfunction. N-terminal-pro-brain natriuretic peptide (NT-proBNP), a marker of cardiac ventricular strain and systolic dysfunction, may be associated with all-cause mortality in HIV-infected women. The aim of this study was to determine if elevated levels of NT-proBNP is associated with increased mortality in HIV-infected women. Design Prospective cohort study. Methods and Results We measured NT-proBNP in 936 HIV-infected and 387 age-matched HIV-uninfected women early (10/11/94 to 7/17/97) and 1082 HIV-infected and 448 HIV-uninfected women late (4/1/08 to 10/7/08) in the highly active antiretroviral therapy (HAART) periods in the Women’s Interagency HIV Study. An NT-proBNP >75th percentile was more likely in HIV-infected persons, but only statistically significant in the late period (27% vs. 21%, unadjusted p = 0.03). In HIV-infected participants, NT-proBNP>75th percentile was independently associated with worse 5-year survival in the early HAART period (HR 1.8, 95% CI 1.3–2.4, p<0.001) and remained a predictor of mortality in the late HAART period (HR 2.8, 95% CI 1.4–5.5, p = 0.002) independent of other established risk covariates (age, race/ethnicity, body mass index, smoking, hepatitis C serostatus, hypertension, renal function, and hemoglobin). NT-proBNP level was not associated with mortality in HIV-uninfected women. Conclusion NT-proBNP is a novel independent marker of mortality in HIV-infected women both when HAART was first introduced and currently. As NT-proBNP is often associated with both pulmonary hypertension and left ventricular dysfunction, these findings suggest that these conditions may contribute significantly to adverse outcomes in this population, requiring further definition of causes and treatments of elevated NT-proBNP in HIV-infected women.

Loss of Twist1 in the Mesenchymal Compartment Promotes Increased Fibrosis in Experimental Lung Injury by Enhanced Expression of CXCL12

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by the accumulation of apoptosis-resistant fibroblasts in the lung. We have previously shown that high expression of the transcription factor Twist1 may explain this prosurvival phenotype in vitro. However, this observation has never been tested in vivo. We found that loss of Twist1 in COL1A2+ cells led to increased fibrosis characterized by very significant accumulation of T cells and bone marrow–derived matrix-producing cells. We found that Twist1-null cells expressed high levels of the T cell chemoattractant CXCL12. In vitro, we found that the loss of Twist1 in IPF lung fibroblasts increased expression of CXCL12 downstream of increased expression of the noncanonical NF-κB transcription factor RelB. Finally, blockade of CXCL12 with AMD3100 attenuated the exaggerated fibrosis observed in Twist1-null mice. Transcriptomic analysis of 134 IPF patients revealed that low expression of Twist1 was characterized by enrichment of T cell pathways. In conclusion, loss of Twist1 in collagen-producing cells led to increased bleomycin-induced pulmonary fibrosis, which is mediated by increased expression of CXCL12. Twist1 expression is associated with dysregulation of T cells in IPF patients. Twist1 may shape the IPF phenotype and regulate inflammation in fibrotic lung injury.

Thrombospondin-1 gene polymorphism is associated with estimated pulmonary artery pressure in patients with sickle cell anemia

tion linearity (Table 1). At a 1003 dilution for ERFE levels, we did not observe a correlation between ERFE and EPO, or ERFE and hepcidin (Supporting Information Figure 1A,B). Furthermore, in blood donors there was no correlation between delta-(day 0-4) hepcidin and delta(day 0-4) ERFE levels (hepcidin (nM)58 3 10 ERFE (pg/mL)11.93, Pearson R50.06), neither between delta (day 0-4) EPO and delta (day 0-4) ERFE levels (EPO (mU/mL)53 3 10 ERFE (pg/mL)26.29, Pearson R50.005). We cannot fully exclude that the absence of correlations between ERFE and EPO, and ERFE and hepcidin in these samples may be attributed to ERFE instability, since our samples of b-thalassemia intermedia patients were stored at 2808C for 6-7 years, and thawed 2-5 times before ERFE measurement, and not much is known on the effects of prolonged storage and freeze-thawing on ERFE integrity. However, ERFE measurements in blood donors were performed in aliquots stored at 2808C for only 1-2 years, and are therefore less likely to be affected by ERFE degradation. The absence of a correlation between ERFE and hepcidin plasma levels in our study differs from a previous report on associations between both analytes that were observed using the same FAM132B ELISA in a study among hemodialysed patients. However, our observations on lack of correlations of ERFE plasma levels and plasma hepcidin and EPO levels are in agreement with those obtained in a study among patients with chronic mountain sickness (as defined by excessive erythrocytosis, hemoglobin 21 g/dL, and hypoxemia with no other medical explanation), who underwent isovolemic venesection of 500 mL on four consecutive days (days 1-4). Using two other plasma FAM132B ELISA kits, the authors found no significant rise in plasma FAM132B at three different time points up till day 20 after venesection, whereas, comparable to our observations in blood donors after blood donation, hematocrit and plasma hepcidin decreased, and EPO levels increased. Taken together, our data obtained in whole blood donors and patients diagnosed with b-thalassemia intermedia with the commercially available kit for human ERFE measurements of MyBiosource does not corroborate the concept of the increased EPO—increased ERFE—lower hepcidin axis as observed in mouse models. The full comprehension of the role of this axis in men therefore awaits the development of an analytically and biologically validated assay for human plasma ERFE levels.

Transcriptional Regulation of Frizzled-1 in Human Osteoblasts by Sp1

The wingless pathway has a powerful influence on bone metabolism and is a therapeutic target in skeletal disorders. Wingless signaling is mediated in part through the Frizzled (FZD) receptor family. FZD transcriptional regulation is poorly understood. Herein we tested the hypothesis that Sp1 plays an important role in the transcriptional regulation of FZD1 expression in osteoblasts and osteoblast mineralization. To test this hypothesis, we conducted FZD1 promoter assays in Saos2 cells with and without Sp1 overexpression. We found that Sp1 significantly up-regulates FZD1 promoter activity in Saos2 cells. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift (EMSA) assays identified a novel and functional Sp1 binding site at -44 to -40 from the translation start site in the FZD1 promoter. The Sp1-dependent activation of the FZD1 promoter was abolished by mithramycin A (MMA), an antibiotic affecting both Sp1 binding and Sp1 protein levels in Saos2 cells. Similarly, down-regulation of Sp1 in hFOB cells resulted in less FZD1 expression and lower alkaline phosphatase activity. Moreover, over-expression of Sp1 increased FZD1 expression and Saos2 cell mineralization while MMA decreased Sp1 and FZD1 expression and Saos2 cell mineralization. Knockdown of FZD1 prior to Sp1 overexpression partially abolished Sp1 stimulation of osteoblast differentiation markers. Taken together, our results suggest that Sp1 plays a role in human osteoblast differentiation and mineralization, which is at least partially mediated by Sp1-dependent transactivation of FZD1.

Association of genetic variation in COMT gene with pain related to sickle cell disease in patients from the walk-PHaSST study

Background Vaso-occlusive pain episodes (VOEs) are the hallmark of sickle cell disease (SCD), and our current understanding of disease biology, treatment, and psychological covariates does not adequately explain the variability of pain in SCD. Functional variants in catechol-O-methyltransferase (COMT) gene contribute to variability in pain perception, but their impact on pain perception in African American SCD patients is not well known. Methods We studied COMT single-nucleotide polymorphisms (SNPs) rs6269, rs4633, rs4818, rs4680, and rs165599 to determine their relationship to patient self-reported pain, the number of acute VOEs, and their impact on daily life and health care utilization in 438 hemoglobin SS patients who participated in the walk-PHaSST study. Results In women, two risk SNPs (rs4633 and rs165599) and the corresponding haplotype (ATCAA) were associated with increased frequency of pain-related emergency room visit. Conclusion COMT functional variants may predispose SCD patients to worse acute pain in women. The association of COMT variants with the intensity of self-reported acute pain warrants further genetic study of pain perception in SCD.