[Show abstract][Hide abstract] ABSTRACT: Objective
Versican is a versatile and highly interactive chondroitin sulfate proteoglycan that is found in the extracellular matrix (ECM) of many tissues and is a major component of developing and developed lesions in atherosclerotic vascular disease. In this paper, we present data to indicate that versican may have important intracellular functions in addition to its better known roles in the ECM.
Methods and Results
Rat aortic smooth muscle cells were fixed and immunostained for versican and images of fluorescently labeled cells were obtained by confocal microscopy. Intracellular versican was detected in the nucleus and cytosol of vascular smooth muscle cells. The use of a synthetic neutralizing peptide eliminated versican immunostaining, demonstrating the specificity of the antibody used in this study. Western blot of pure nuclear extracts confirmed the presence of versican in the nucleus, and multi-fluorescent immunostaining showed strong colocalization of versican and nucleolin, suggesting a nucleolar localization of versican in non-dividing cells. In dividing valve interstitial cells, a strong signal for versican was observed in and around the condensed chromosomes during the various stages of mitosis. Multi-fluorescent immunostaining for versican and tubulin revealed versican aggregated at opposing poles of the mitotic spindle during metaphase. Knockdown of versican expression using siRNA disrupted the organization of the mitotic spindle and led to the formation of multipolar spindles during metaphase.
Collectively, these data suggest an intracellular function for versican in vascular cells where it appears to play a role in mitotic spindle organization during cell division. These observations open a new avenue for studies of versican, suggesting even more diverse roles in vascular health and disease.
[Show abstract][Hide abstract] ABSTRACT: Background
Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts.
Methods and Results
The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle α-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-β signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2.
Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.
PLoS ONE 07/2015; 10(7). DOI:10.1371/journal.pone.0133056 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic obstructive pulmonary disease (COPD) is one of the major causes of morbidity and mortality in the world. Regrettably, there are no biomarkers to objectively diagnose COPD exacerbations, which are the major drivers of hospitalization and deaths from COPD. Moreover, there are no biomarkers to guide therapeutic choices or to risk-stratify patients for imminent exacerbations and no objective biomarkers of disease activity or disease progression. While there has been a tremendous investment in COPD biomarker discovery over the past two decades, clinical translation and implementation has not matched these efforts. In this article, we outline the challenges of biomarker development in COPD and provide an overview of a developmental pipeline that may be able to surmount these challenges and bring novel biomarker solutions to accelerate therapeutic discoveries and to improve the care and outcomes of the millions of individuals worldwide with COPD.
American Journal of Respiratory and Critical Care Medicine 07/2015; DOI:10.1164/rccm.201505-0871PP · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidemiological and interventional studies have suggested a protective role for vitamin D in cardiovascular disease, and basic research has implicated vitamin D as a potential inhibitor of fibrosis in a number of organ systems; yet little is known regarding direct effects of vitamin D on human cardiac cells. Given the critical role of fibrotic responses in end stage cardiac disease, we examined the effect of active vitamin D treatment on fibrotic responses in primary human adult ventricular cardiac fibroblasts (HCF-av), and investigated the relationship between circulating vitamin D (25(OH)D3) and cardiac fibrosis in human myocardial samples.
Interstitial cardiac fibrosis in end stage HF was evaluated by image analysis of picrosirius red stained myocardial sections. Serum 25(OH)D3 levels were assayed using mass spectrometry. Commercially available HCF-av were treated with transforming growth factor (TGF)β1 to induce activation, in the presence or absence of active vitamin D (1,25(OH)2D3). Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH)2D3 treatment significantly inhibited TGFβ1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH)2D3. Treatment with 1,25(OH)2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation.
Our results demonstrate that active vitamin D can prevent TGFβ1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts. An inverse relationship between vitamin D status and cardiac fibrosis in end stage heart failure was observed. Collectively, our data support an inhibitory role for vitamin D in cardiac fibrosis.
PLoS ONE 06/2015; 10(6):e0128655. DOI:10.1371/journal.pone.0128655 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Up-to-date estimates of burden of diseases are required for evidence-based policy making. The objectives of this study were to determine the excess costs of chronic obstructive pulmonary disease (COPD) and its trend from 2001 to 2010 in British Columbia, Canada.
We used British Columbia's administrative health data to construct a cohort of patients with COPD and a matched comparison cohort of non-COPD subjects. We followed each individual from the time of first COPD-related health care event (or equivalent time for the comparison cohort). Direct medical costs (in 2010 Canadian dollars [$]) were calculated based on billing records pertaining to hospital admissions, outpatient services use, medication dispensations, and community care services. We determined the excess medical costs of COPD by calculating the difference in overall medical costs between the COPD and the comparison cohort.
153,570 and 246,801 individuals constituted the COPD and comparison cohorts, respectively (for both cohorts: mean age at entry 66.9, 47.2% female). The excess costs of COPD during the study period were $5,452 per patient-year. Inpatient, outpatient, medication, and community care costs were responsible for 57%, 16%, 22%, and 5% of the excess costs, respectively. Excess costs increased by $296/person-year (P<0.01), with hospital costs demonstrating the largest increase over time ($258/person-year, P<0.01).
The direct economic burden of COPD is high and has increased significantly between 2001 and 2010 over and above the increase in the health care costs of the general population. Further investigation is required to elucidate the underlying reasons for the temporal increase in COPD direct costs.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT Viruses such as coxsackievirus B3 (CVB3) are entirely host cell-dependent parasites. Indeed, they must cleverly exploit various compartments of host cells to complete their life cycle, and consequently launch disease. Evolution has equipped this pico-rna-virus, CVB3, to use different strategies, including CVB3-induced direct damage to host cells followed by a host inflammatory response to CVB3 infection, and cell death to super-additively promote target organ tissue injury, and dysfunction. In this update, the patho-stratagems of CVB3 are explored from molecular, and systems-level approaches. In summarizing recent developments in this field, we focus particularly on mechanisms by which CVB3 can harness different host cell processes including kinases, host cell-killing and cell-eating machineries, matrix metalloproteinases and miRNAs to promote disease.
[Show abstract][Hide abstract] ABSTRACT: AimsAnderson–Fabry disease (AFD) is an important X-linked metabolic disease resulting in progressive end-organ involvement, with cardiac disease being the dominant determinant of survival in a gender-dependent manner. Recent epidemiological screening for AFD suggests the prevalence is much higher than previously recognized, with estimates of 1:3000. Our aim was to discover novel plasma biomarker signatures in adult patients with AFD.Methods and resultsWe used an unbiased proteomic screening approach to discover novel plasma biomarker signatures. In the discovery cohort of 46 subjects, 14 healthy controls and 32 patients with AFD, we used a mass spectrometry iTRAQ proteomic approach followed by multiple reaction monitoring (MRM) assays to identify biomarkers. Of the 38 protein groups discovered by iTRAQ, 18 already had existing MRM assays. Based on MRM, we identified an eight-protein biomarker panel (22 kDa protein, afamin, α1 antichymotrypsin, apolipoprotein E, β-Ala His dipeptidase, haemoglobin α-2, isoform 1 of sex hormone-binding globulin, and peroxiredoxin 2) that was very specific and sensitive for male AFD patients. In female AFD patients, we identified a nine-marker panel of proteins with only three proteins, apolipoprotein E, haemoglobin α-2, and peroxiredoxin 2, common to both genders, suggesting a gender-specific alteration in plasma biomarkers in patients with AFD. The biomarkers were validated in plasma samples from 48 subjects using MRM, and they performed inferiorly in patients with heart failure.Conclusions
We have identified gender-specific plasma protein biomarker panels that are specific and sensitive for the AFD phenotype. The gender-specific panels offer important insight into potential differences in pathophysiology and prognosis between males and females with AFD.
European Journal of Heart Failure 01/2015; 17(3). DOI:10.1002/ejhf.230 · 6.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Multi-omics research is a key ingredient of data-intensive life sciences research, permitting measurement of biological molecules at different functional levels in the same individual. For a complete picture at the biological systems level, appropriate statistical techniques must however be developed to integrate different 'omics' data sets (e.g., genomics and proteomics). We report here multivariate projection-based analyses approaches to genomics and proteomics data sets, using the case study of and applications to observations in kidney transplant patients who experienced an acute rejection event (n=20) versus non-rejecting controls (n=20). In this data sets, we show how these novel methodologies might serve as promising tools for dimension reduction and selection of relevant features for different analytical frameworks. Unsupervised analyses highlighted the importance of post transplant time-of-rejection, while supervised analyses identified gene and protein signatures that together predicted rejection status with little time effect. The selected genes are part of biological pathways that are representative of immune responses. Gene enrichment profiles revealed increases in innate immune responses and neutrophil activities and a depletion of T lymphocyte related processes in rejection samples as compared to controls. In all, this article offers candidate biomarkers for future detection and monitoring of acute kidney transplant rejection, as well as ways forward for methodological advances to better harness multi-omics data sets.
Omics A Journal of Integrative Biology 11/2014; 18(11):682-95. DOI:10.1089/omi.2014.0062 · 2.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Leiomyosarcoma (LMS) is a mesenchymal cancer that occurs throughout the body. Although LMS is easily recognized histopathologically, the cause of the disease remains unknown. Versican, an extracellular matrix (ECM) proteoglycan, increases in LMS. Microarray analyses of 80 LMS and 24 leiomyomas show a significant elevated expression of versican in human LMS vs. benign leiomyomas. To explore the importance of versican in this smooth muscle cell tumor, we used versican-directed siRNA to knockdown versican expression in a LMS human cell line, SK-LMS-1. Decreased versican expression was accompanied by slower rates of LMS cell proliferation and migration, increased adhesion, and decreased accumulation of the ECM macromolecule hyaluronan. Addition of purified versican to cells expressing versican siRNA restored cell proliferation to the level of LMS controls, increased the pericellular coat and the retention of hyaluronan, and decreased cell adhesion in a dose-dependent manner. The presence of versican was not only synergistic with hyaluronan in increasing cell proliferation, but the depletion of versican decreased hyaluronan synthase expression and decreased the retention of hyaluronan. When LMS cells stably expressing versican siRNA were injected into nude mice, the resulting tumors displayed significantly less versican and hyaluronan staining, had lower volumes, and reduced levels of mitosis as compared to controls. Collectively, these results suggest a role for using versican as a point of control in the management and treatment of LMS.
[Show abstract][Hide abstract] ABSTRACT: Background
Measurement-unit conflicts are a perennial problem in integrative research domains such as clinical meta-analysis. As multi-national collaborations grow, as new measurement instruments appear, and as Linked Open Data infrastructures become increasingly pervasive, the number of such conflicts will similarly increase.
We propose a generic approach to the problem of (a) encoding measurement units in datasets in a machine-readable manner, (b) detecting when a dataset contained mixtures of measurement units, and (c) automatically converting any conflicting units into a desired unit, as defined for a given study.
We utilized existing ontologies and standards for scientific data representation, measurement unit definition, and data manipulation to build a simple and flexible Semantic Web Service-based approach to measurement-unit harmonization. A cardiovascular patient cohort in which clinical measurements were recorded in a number of different units (e.g., mmHg and cmHg for blood pressure) was automatically classified into a number of clinical phenotypes, semantically defined using different measurement units.
We demonstrate that through a combination of semantic standards and frameworks, unit integration problems can be automatically detected and resolved.
[Show abstract][Hide abstract] ABSTRACT: Chronic heart failure is a costly epidemic that affects up to 2% of people in developed countries. The purpose of this study was to discover novel blood proteomic biomarker signatures of recovered heart function that could lead to more effective heart failure patient management by both primary care and specialty physicians.
The discovery cohort included 41 heart transplant patients and 20 healthy individuals. Plasma levels of 138 proteins were detected in at least 75% of these subjects by iTRAQ mass spectrometry. Eighteen proteins were identified that had (i) differential levels between pre-transplant patients with end-stage heart failure and healthy individuals; and (ii) levels that returned to normal by 1 month post-transplant in patients with stable heart function after transplantation. Seventeen of the 18 markers were validated by multiple reaction monitoring mass spectrometry in a cohort of 39 heart failure patients treated with drug therapy, of which 30 had recovered heart function and 9 had not. This 17-protein biomarker panel had 93% sensitivity and 89% specificity, while the RAMP® NT-proBNP assay had the same specificity but 80% sensitivity. Performance further improved when the panel was combined with NT-proBNP, yielding a net reclassification index relative to NT-proBNP of 0.28.
We have identified potential blood biomarkers of recovered heart function by harnessing data from transplant patients. These biomarkers can lead to the development of an inexpensive protein-based blood test that could be used by physicians to monitor response to therapy in heart failure, resulting in more personalized, front-line heart failure patient management.
European Journal of Heart Failure 05/2014; 16(5). DOI:10.1002/ejhf.65 · 6.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12(-/-) but not wild-type mice to express IκBα and thus export IFN-α makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrate's gene exons and by MMP-12-mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3-infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.
Nature medicine 04/2014; 20(5). DOI:10.1038/nm.3508 · 27.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute rejection is a major complication of solid organ transplantation that prevents the long-term assimilation of the allograft. Various populations of lymphocytes are principal mediators of this process, infiltrating graft tissues and driving cell-mediated cytotoxicity. Understanding the lymphocyte-specific biology associated with rejection is therefore critical. Measuring genome-wide changes in transcript abundance in peripheral whole blood cells can deliver a comprehensive view of the status of the immune system. The heterogeneous nature of the tissue significantly affects the sensitivity and interpretability of traditional analyses, however. Experimental separation of cell types is an obvious solution, but is often impractical and, more worrying, may affect expression, leading to spurious results. Statistical deconvolution of the cell type-specific signal is an attractive alternative, but existing approaches still present some challenges, particularly in a clinical research setting. Obtaining time-matched sample composition to biologically interesting, phenotypically homogeneous cell sub-populations is costly and adds significant complexity to study design. We used a two-stage, in silico deconvolution approach that first predicts sample composition to biologically meaningful and homogeneous leukocyte sub-populations, and then performs cell type-specific differential expression analysis in these same sub-populations, from peripheral whole blood expression data. We applied this approach to a peripheral whole blood expression study of kidney allograft rejection. The patterns of differential composition uncovered are consistent with previous studies carried out using flow cytometry and provide a relevant biological context when interpreting cell type-specific differential expression results. We identified cell type-specific differential expression in a variety of leukocyte sub-populations at the time of rejection. The tissue-specificity of these differentially expressed probe-set lists is consistent with the originating tissue and their functional enrichment consistent with allograft rejection. Finally, we demonstrate that the strategy described here can be used to derive useful hypotheses by validating a cell type-specific ratio in an independent cohort using the nanoString nCounter assay.
PLoS ONE 04/2014; 9(4):e95224. DOI:10.1371/journal.pone.0095224 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples.
We compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding.
Globin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA-Seq is highly reproducible within a large dynamic range of detection and provides an accurate estimation of RNA concentration in peripheral whole blood. High-throughput sequencing is thus a promising technology for whole blood transcriptomics and biomarker discovery.
PLoS ONE 03/2014; 9(3):e91041. DOI:10.1371/journal.pone.0091041 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intimal smooth muscle cells (SMCs) contribute to the foam cell population in arterial plaque, and express lower levels of the cholesterol exporter ATP-binding cassette transporter AI (ABCA1) when compared to medial arterial SMCs. The relative contribution of SMCs to the total foam cell population and their expression of ABCA1 when compared to intimal monocyte-derived macrophages, however, are unknown. While expression of macrophage markers by SMCs following lipid loading has been described, the relevance of this phenotypic switch by SMCs in human coronary atherosclerosis has not been determined.
Human coronary artery sections from hearts explanted at the time of transplantation were processed to clearly delineate intracellular and extracellular lipids and allow co-staining for cell-specific markers. Co-staining for Oil Red O and the SMC-specific marker SM α-actin of foam cell rich lesions revealed that 50±7% (avg ± SEM, n=14 subjects) of total foam cells were SMC-derived. ABCA1 expression by intimal SMCs was significantly reduced between early and advanced atherosclerotic lesions, with no loss in ABCA1 expression by myeloid-lineage cells. Co-staining with the macrophage marker CD68 and SM α-actin revealed that 40±6% (n=15) of CD68-positive cells originated as SMCs in advanced human coronary atherosclerosis.
These findings suggest SMCs contain a much larger burden of the excess cholesterol in human coronary atherosclerosis than previously known, due in part to their relative inability to release excess cholesterol via ABCA1, when compared to myeloid-lineage cells. Our results also indicate that many cells identified as monocyte-derived macrophages in human atherosclerosis are in fact SMC-derived.
[Show abstract][Hide abstract] ABSTRACT: In this study, we explored a time course of peripheral whole blood transcriptomes from kidney transplantation patients who either experienced an acute rejection episode or did not in order to better delineate the immunological and biological processes measureable in blood leukocytes that are associated with acute renal allograft rejection. Using microarrays, we generated gene expression data from 24 acute rejectors and 24 nonrejectors. We filtered the data to obtain the most unambiguous and robustly expressing probe sets and selected a subset of patients with the clearest phenotype. We then performed a data-driven exploratory analysis using data reduction and differential gene expression analysis tools in order to reveal gene expression signatures associated with acute allograft rejection. Using a template-matching algorithm, we then expanded our analysis to include time course data, identifying genes whose expression is modulated leading up to acute rejection. We have identified molecular phenotypes associated with acute renal allograft rejection, including a significantly upregulated signature of neutrophil activation and accumulation following transplant surgery that is common to both acute rejectors and nonrejectors. Our analysis shows that this expression signature appears to stabilize over time in nonrejectors but persists in patients who go on to reject the transplanted organ. In addition, we describe an expression signature characteristic of lymphocyte activity and proliferation. This lymphocyte signature is significantly downregulated in both acute rejectors and nonrejectors following surgery; however, patients who go on to reject the organ show a persistent downregulation of this signature relative to the neutrophil signature.
Bioinformatics and biology insights 01/2014; 8:17-33. DOI:10.4137/BBI.S13376.
[Show abstract][Hide abstract] ABSTRACT: To identify novel predictors for coronary artery bypass grafting failure, we probed for associations with known clinical and biochemical risk factors for atherosclerosis. We also used microarray analysis to identify novel single nucleotide polymorphisms to better understand the genetics and pathogenesis of graft occlusion.
The present study was a nested case-control substudy of the Radial Artery Patency Study 5-year follow-up data. From 1996 to 2001, 87 patients underwent coronary artery bypass grafting. Of these, 26 patients (29.9%) had an occluded study graft (saphenous vein or radial artery) at 8.0 ± 1.1 years. The clinical parameters, late angiography, blood biomarker levels, and surgical outcomes data were included in a multivariate analysis to determine the independent predictors of graft failure.
The risk factors of graft failure were fibrinogen (odds ratio [OR], 3.94; 95% confidence interval [CI], 1.33-11.63; P = .01), creatinine (OR, 1.06; 95% CI, 1.02-1.10; P = .006), and diabetes mellitus (OR, 5.15; 95% CI, 1.08-24.59; P = .04). High-density lipoprotein (OR, 0.74; 95% CI, 0.53-1.02; P = .06) was weakly protective; however, low-density lipoprotein and total cholesterol were not predictors. We then identified the association of several human single nucleotide polymorphisms with graft failure, including mutations in glutathione-S-transferase α3. Human coronary arteries and bypass grafts demonstrated increased protein expression of glutathione-S-transferase α3, a known cardioprotective factor, in the atherosclerotic regions and surrounding adventitial tissues.
We identified diabetes as a potential clinical predictor and plasma fibrinogen, creatinine, and high-density lipoprotein as potential novel biomarkers. These might help risk stratify patients for the development of graft failure. We also demonstrated a novel association between glutathione-S-transferase α3 and graft failure.
The Journal of thoracic and cardiovascular surgery 12/2013; 148(2). DOI:10.1016/j.jtcvs.2013.10.011 · 4.17 Impact Factor