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Abraham T.,Institute for HeartLung Health | Kayra D.,Institute for HeartLung Health | McManus B.,Institute for HeartLung Health | Scott A.,University of British Columbia
Journal of Structural Biology | Year: 2012

The structural remodeling of collagens is important in several biological processes including wound healing, tendon repair and adaptation, fibrosis and morphogenesis. Multiphoton microscopy is efficient in the induction of highly specific second harmonic generation (SHG) signal from non-centrosymmetric macromolecules such as fibrillar collagens. Although the detectors in the reflection geometry have been normally employed for capturing the backward scattered SHG considering the wide range of engineered thick tissue applications, there are still questions about the generated 3D collagen structures because of the directional pattern of SHG signals. The present study dealt with an in vitro collagen-fibroblast raft or bioartificial tendon model where the stimulation of fibroblast cells induced lateral orientation of collagen Type I fibers. The SHG signals originating from 3D collagen matrix were captured simultaneously in both forward and backward scattering directions. Our structural analysis indicates that collagen fibers formed in such in vitro model systems are predominantly of uniform sizes and are aligned preferentially in the lateral direction. The criss-cross arrangements of laterally oriented fibers are evident in the initial stages of contraction but eventually those laterally oriented collagen fibers are found to be aligned in parallel to each other as well as to the fibroblasts after an extended period of contraction. Our comprehensive quantitative assessment of simultaneously captured forward and backward 3D SHG image datasets, which includes the SHG signal decay, fiber diameter, cell dimensions, colocalization profiles, the 3D voxel volumes and Fourier analysis, indicates strong correlation of structural features identified in forward and backward directions. © 2012 Elsevier Inc. Source

Hendel A.,University of British Columbia | Hendel A.,Institute for HeartLung Health | Granville D.J.,University of British Columbia | Granville D.J.,Institute for HeartLung Health
Matrix Biology | Year: 2013

Dysregulated angiogenesis contributes to the pathogenesis of chronic inflammatory diseases. Modulation of the extracellular matrix by immune-derived proteases can alter endothelial cell-matrix interactions as well as endothelial cell sprouting, migration and capillary formation. Granzyme B is a serine protease that is expressed by a variety of immune cells, and accumulates in the extracellular milieu in many chronic inflammatory disorders that are associated with dysregulated angiogenesis. Although granzyme B is known to cleave fibronectin, an essential glycoprotein in vascular morphogenesis, the role of granzyme B in modulating angiogenesis is unknown. In the present study, granzyme B cleaved both plasma fibronectin and cell-derived fibronectin, resulting in the release of multiple fibronectin fragments. Granzyme B cleavage of fibronectin resulted in a dose-dependent reduction in endothelial cell adhesion to fibronectin as well as reduced endothelial cell migration and tubular formation. These events were prevented when granzyme B activity was inhibited by a small molecule inhibitor. In summary, granzyme B-mediated cleavage of fibronectin contributes to attenuated angiogenesis through the disruption of endothelial cell - fibronectin interaction resulting in impaired endothelial cell migration and tubular formation. © 2012 Elsevier B.V. Source

Hollander Z.,University of British Columbia | Lin D.,University of British Columbia | Ng R.,University of British Columbia | Ignaszewski A.,University of British Columbia | And 8 more authors.
Transplantation | Year: 2010

Background. Acute rejection is still a significant barrier to long-term survival of the allograft. Current acute rejection diagnostic methods are not specific enough or are invasive. There have been a number of studies that have explored the blood or the biopsy to discover genomic biomarkers of acute rejection; however, none of the studies to date have used both. Methods. We analyzed endomyocardial biopsy tissue and whole blood-derived messenger RNA from 11 acute rejection and 20 nonrejection patients using Affymetrix Human Genome U133 Plus 2.0 chips. We used a novel approach and gained insight into the biology of rejection based on gene expression in the biopsy, and applied this knowledge to the blood analysis to identify novel blood biomarkers. Results. We identified probesets that are differentially expressed between acute rejection and nonrejection patients in the biopsy and blood, and developed three biomarker panels: (1) based on biopsy-only (area under the curve=0.85), (2) based on biopsy-targeted whole blood (area under the curve=0.83), and (3) based on whole blood-only (area under the curve=0.60) analyses. Conclusions. Most of the probesets replicated between biopsy and blood are regulated in opposite direction between the two sources of information. We also observed that the biopsy-targeted blood biomarker discovery approach can improve performance of the biomarker panel. The biomarker panel developed using this targeted approach is able to diagnose acute cardiac allograft rejection almost as well as the biopsy-only based biomarker panel. © 2010 by Lippincott Williams & Wilkins. Source

Singh A.,University of British Columbia | Singh A.,Institute for HeartLung Health | Cohen Freue G.V.,Institute for HeartLung Health | Oosthuizen J.L.,University of British Columbia | And 14 more authors.
Proteomics - Clinical Applications | Year: 2012

Purpose: This proteomics study was designed to determine the utility of iTRAQ MALDI-TOF/TOF technology to compare plasma samples from carefully phenotyped mild, atopic asthma subjects undergoing allergen inhalation challenge. Experimental design: Eight adult subjects with mild, allergic asthma (four early responders (ERs) and four dual responders (DRs)) participated in the allergen inhalation challenge. Blood samples were collected prior to and 2 h after the inhalation challenge. Sixteen plasma samples (two per subject), technical replicates, and pooled controls were analyzed using iTRAQ. Technical validation was performed using LC-MRM/MS. Moderated robust regression was used to determine differentially expressed proteins. Results: Although this study did not show significant differences between pre- and post-challenge samples, discriminant analysis indicated that certain proteins responded differentially to allergen challenge with respect to responder type. At pre-challenge, fibronectin was significantly elevated in DRs compared to ERs and remained significant in the multiple reaction monitoring validation. Conclusions and clinical relevance: This proof of principle demonstration has shown that iTRAQ can uncover differences in the human plasma proteome between two endotypes of asthma and merits further application of iTRAQ to larger cohorts of asthma and other respiratory diseases. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Singh A.,University of British Columbia | Singh A.,Institute for HeartLung Health | Yamamoto M.,University of British Columbia | Yamamoto M.,Institute for HeartLung Health | And 18 more authors.
PLoS ONE | Year: 2013

Some asthmatic individuals undergoing allergen inhalation challenge develop an isolated early response whereas others develop a dual response (early plus late response). In the present study we have used transcriptomics (microarrays) and metabolomics (mass spectrometry) of peripheral blood to identify molecular patterns that can discriminate allergen-induced isolated early from dual asthmatic responses. Peripheral blood was obtained prior to (pre-) and 2 hours post allergen inhalation challenge from 33 study participants. In an initial cohort of 14 participants, complete blood counts indicated significant differences in neutrophil and lymphocyte counts at pre-challenge between early and dual responders. At post-challenge, significant genes (ALOX15, FADS2 and LPCAT2) and metabolites (lysolipids) were enriched in lipid metabolism pathways. Enzymes encoding for these genes are involved in membrane biogenesis and metabolism of fatty acids into pro-inflammatory and anti-inflammatory mediators. Correlation analysis indicated a strong negative correlation between ALOX15, FADS2, and IL5RA expression with 2-arachidonoylglycerophosphocholine levels in dual responders. However, measuring arachidonic acid and docosahexaenoic acid levels in a validation cohort of 19 participants indicated that the free form of DHA (nmoles/μg of protein) was significantly (p = 0.03) different between early and dual responders after allergen challenge. Collectively these results may suggest an imbalance in lipid metabolism which dictates pro- (anti-) inflammatory and pro-resolving mechanisms. Future studies with larger sample sizes may reveal novel mechanisms and therapeutic targets of the late phase asthmatic response. © 2013 Singh et al. Source

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