[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: Vitronectin, a multifunctional glycoprotein, is involved in coagulation, inhibition of the formation of the membrane attack complex (MAC), cell adhesion and migration, wound healing, and tissue remodeling. The primary cellular source of vitronectin is hepatocytes; it is not known whether resident cells of airways produce vitronectin, even though the glycoprotein has been found in exhaled breath condensate and bronchoalveolar lavage from healthy subjects and patients with interstitial lung disease. It is also not known whether vitronectin expression is altered in subjects with asthma and COPD. In this study, bronchial tissue from 7 asthmatic, 10 COPD and 14 control subjects was obtained at autopsy and analyzed by immunohistochemistry to determine the percent area of submucosal glands occupied by vitronectin. In a separate set of experiments, quantitative colocalization analysis was performed on tracheobronchial tissue sections obtained from donor lungs (6 asthmatics, 4 COPD and 7 controls). Vitronectin RNA and protein expressions in bronchial surface epithelium were examined in 12 subjects who undertook diagnostic bronchoscopy. Vitronectin was found in the tracheobronchial epithelium from asthmatic, COPD, and control subjects, although its expression was significantly lower in the asthmatic group. Colocalization analysis of 3D confocal images indicates that vitronectin is expressed in the glandular serous epithelial cells and in respiratory surface epithelial cells other than goblet cells. Expression of the 65-kDa vitronectin isoform was lower in bronchial surface epithelium from the diseased subjects. The cause for the decreased vitronectin expression in asthma is not clear, however, the reduced concentration of vitronectin in the epithelial/submucosal layer of airways may be linked to airway remodeling.
PLoS ONE 03/2015; 10(3):e0119717. DOI:10.1371/journal.pone.0119717 · 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: Tendinopathy is often discovered late because the initial development of tendon pathology is asymptomatic. The aim of this study was to examine the potential role of mast cell involvement in early tendinopathy using a high-intensity uphill running (HIUR) exercise model. Twenty-four male Wistar rats were divided in two groups: running group (n = 12); sedentary control group (n = 12). The running-group was exposed to the HIUR exercise protocol for 7 weeks. The calcaneal tendons of both hind limbs were dissected. The right tendon was used for histologic analysis using Bonar score, immunohistochemistry, and second harmonic generation microscopy (SHGM). The left tendon was used for quantitative polymerase chain reaction (qPCR) analysis. An increased tendon cell density in the runners were observed compared to the controls (P = 0.05). Further, the intensity of immunostaining of protein kinase B, P = 0.03; 2.75 ± 0.54 vs 1.17 ± 0.53, was increased in the runners. The Bonar score (P = 0.05), and the number of mast cells (P = 0.02) were significantly higher in the runners compared to the controls. Furthermore, SHGM showed focal collagen disorganization in the runners, and reduced collagen density (P = 0.03). IL-3 mRNA levels were correlated with mast cell number in sedentary animals. The qPCR analysis showed no significant differences between the groups in the other analyzed targets. The current study demonstrates that 7-week HIUR causes structural changes in the calcaneal tendon, and further that these changes are associated with an increased mast cell density.
Scandinavian Journal of Medicine and Science in Sports 07/2013; 23(6). DOI:10.1111/sms.12089 · 2.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lung is a complex gas exchanger with interfacial area (where the gas
exchange takes place) is about the size of a tennis court. Respiratory
function is linked to the biomechanical stability of the gas exchange or
alveolar regions which directly depends on the spatial distributions of
the extracellular matrix fibers such fibrillar collagens and elastin
fibers. It is very important to visualize and quantify these fibers at
their native and inflated conditions to have correct morphometric
information on differences between control and diseased states. This can
be only achieved in the ex vivo states by imaging directly frozen lung
specimens inflated to total lung capacity. Multiphoton microscopy, which
uses ultra-short infrared laser pulses as the excitation source,
produces multiphoton excitation fluorescence (MPEF) signals from
endogenously fluorescent proteins (e.g. elastin) and induces specific
second harmonic generation (SHG) signals from non-centrosymmetric
proteins such as fibrillar collagens in fresh human lung tissues [J.
Struct. Biol. (2010)171,189-196]. Here we report for the first time 3D
image data obtained directly from thick frozen inflated lung specimens
(~0.7- 1.0 millimeter thick) visualized at -60°C without prior
fixation or staining in healthy and diseased states. Lung specimens
donated for transplantation and released for research when no
appropriate recipient was identified served as controls, and diseased
lung specimens donated for research by patients receiving lung
transplantation for very severe COPD (n=4) were prepared as previously
described [N. Engl. J. Med. (2011) 201, 1567]. Lung slices evenly spaced
between apex and base were examined using multiphoton microscopy while
maintained at -60°C using a temperature controlled cold stage with a
temperature resolution of 0.1°C. Infrared femto-second laser pulses
tuned to 880nm, dry microscopic objectives, and non-de-scanned
detectors/spectrophotometer located in the reflection geometry were used
for generating the 3D images/spectral information. We found that this
novel imaging approach can provide spatially resolved 3D images with
spectral specificities from frozen inflated lungs that are sensitive
enough to identity the micro-structural details of fibrillar collagens
and elastin fibers in alveolar walls in both healthy and diseased
Proceedings of SPIE - The International Society for Optical Engineering 02/2013; 8588. DOI:10.1117/12.2002593 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Changes in cellular cholesterol affect insulin secretion, and β-cell-specific deletion or loss-of-function mutations in the cholesterol efflux transporter ATP-binding cassette transporter A1 (ABCA1) result in impaired glucose tolerance and β-cell dysfunction. Upregulation of ABCA1 expression may therefore be beneficial for the maintenance of normal islet function in diabetes. Studies suggest that microRNA-33a (miR-33a) expression inversely correlates with ABCA1 expression in hepatocytes and macrophages. We examined whether miR-33a regulates ABCA1 expression in pancreatic islets, thereby affecting cholesterol accumulation and insulin secretion. Adenoviral miR-33a overexpression in human or mouse islets reduced ABCA1 expression, decreased glucose-stimulated insulin secretion, and increased cholesterol levels. The miR-33a-induced reduction in insulin secretion was rescued by cholesterol depletion by methyl-β-cyclodextrin or mevastatin. Inhibition of miR-33a expression in apolipoprotein E knockout islets and ABCA1 overexpression in β-cell-specific ABCA1 knockout islets rescued normal insulin secretion and reduced islet cholesterol. These findings confirm the critical role of β-cell ABCA1 in islet cholesterol homeostasis and β-cell function and highlight modulation of β-cell miR-33a expression as a means to influence insulin secretion.
[Show abstract][Hide abstract] ABSTRACT: The structural remodeling of extracellular matrix proteins in peripheral lung region is an important feature in chronic obstructive pulmonary disease (COPD). Multiphoton microscopy is capable of inducing specific second harmonic generation (SHG) signal from non-centrosymmetric structural proteins such as fibrillar collagens. In this study, SHG microscopy was used to examine structural remodeling of the fibrillar collagens in human lungs undergoing emphysematous destruction (n=2). The SHG signals originating from these diseased lung thin sections from base to apex (n=16) were captured simultaneously in both forward and backward directions. We found that the SHG images detected in the forward direction showed well-developed and well-structured thick collagen fibers while the SHG images detected in the backward direction showed striking different morphological features which included the diffused pattern of forward detected structures plus other forms of collagen structures. Comparison of these images with the well-established immunohistochemical staining indicated that the structures detected in the forward direction are primarily the thick collagen type I fibers and the structures identified in the backward direction are diffusive structures of forward detected collagen type I plus collagen type III. In conclusion, we here demonstrate the feasibility of SHG microscopy in differentiating fibrillar collagen subtypes and understanding their remodeling in diseased lung tissues.
[Show abstract][Hide abstract] ABSTRACT: Granzyme B (GZMB) is a proapoptotic serine protease that is released by cytotoxic lymphocytes. However, GZMB can also be produced by other cell types and is capable of cleaving extracellular matrix (ECM) proteins. GZMB contributes to abdominal aortic aneurysm (AAA) through an extracellular, perforin-independent mechanism involving ECM cleavage. The murine serine protease inhibitor, Serpina3n (SA3N), is an extracellular inhibitor of GZMB. In the present study, administration of SA3N was assessed using a mouse Angiotensin II-induced AAA model. Mice were injected with SA3N (0-120 μg/kg) before pump implantation. A significant dose-dependent reduction in the frequency of aortic rupture and death was observed in mice that received SA3N treatment compared with controls. Reduced degradation of the proteoglycan decorin was observed while collagen density was increased in the aortas of mice receiving SA3N treatment compared with controls. In vitro studies confirmed that decorin, which regulates collagen spacing and fibrillogenesis, is cleaved by GZMB and that its cleavage can be prevented by SA3N. In conclusion, SA3N inhibits GZMB-mediated decorin degradation leading to enhanced collagen remodelling and reinforcement of the adventitia, thereby reducing the overall rate of rupture and death in a mouse model of AAA.
[Show abstract][Hide abstract] ABSTRACT: Following tendon injury, cartilage, bone and fat metaplasia are often observed, making the optimization of tenocyte differentiation an important clinical goal. In this study we examined the effect of static and cyclic mechanical loading on the expression of genes which play a role in tenocyte differentiation and function, namely scleraxis (Scx) and Type I collagen (Col1a1), and determined the effect of varying mechanical parameters including (1) static vs dynamic load, (2) increasing strain magnitude, (3) inclusion of 10 s rest periods, and (4) increasing cycle number. Cyclic loading resulted in a greater increase of tenocyte gene expression than static loading over 3 weeks in culture. Increasing strain levels potentiated the induction of tenocyte genes. The insertion of a 10 s rest periods further enhanced tenocyte gene expression, as did increasing repetition numbers. These results suggest that mechanical signaling exerts an important influence on the expression of genes which play a role in determining the tendon phenotype. Further work is required to confirm and extend these findings in primary cells such as resident tendon progenitor/stem cells, in order to provide an improved understanding of biology from which optimized rehabilitation programs can be developed.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to determine whether administration of a mast cell inhibitor (sodium cromolyn, SC) would influence tendon repair and extracellular matrix gene expression following acute injury. CD1 mouse patellar tendons were unilaterally injured and mast cell prevalence was determined. The effect of SC injection on tendon hypercellularity, cross-sectional area, collagen organization, and expression of extracellular matrix-related genes was examined. Mast cell prevalence was markedly increased in injured patellar tendons (p = 0.009), especially at 8 weeks post-injury (p = 0.025). SC injection increased collagen organization compared to uninjected animals at 4 weeks and attenuated the development of tendon hypercellularity and tendon thickening post-injury. Expression of CTGF, ADAMTS1, and TIMP3 in injured tendon was reduced in the SC group. SC injections moderated the structural alterations of healing tendon in association with downregulation of several genes associated with tendon fibrosis. This work corroborates previous findings pointing to a role of mast cells in tendon repair.
Journal of Orthopaedic Research 05/2011; 29(5):678-83. DOI:10.1002/jor.21291 · 2.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multiphoton microscopy has become a powerful imaging method for minimally invasive evaluation of extracellular matrix (ECM) and cellular structures deep within tissues in their native environments. This technology, which uses ultra-short femto-second laser pulses as the excitation source, is efficient in multiphoton excitation fluorescence (MPEF) of endogenously fluorescent macromolecular systems and induction of highly specific second harmonic generation (SHG) signals from non-centrosymmetric macromolecules such as fibrillar collagens. Both these signals can be captured simultaneously to provide spatially resolved 3D structural organization of ECM as well as cellular morphologies in lung or airway tissue with spectral specificity and sensitivity. These imaging modalities are minimally invasive since structures deep within tissues can be visualized without the need for tissue fixation and/or sectioning. Much of the traditional histological and chemical procedures associated with conventional microscopy methods, which may alter native structure of lung tissue samples, can be circumvented to generate more accurate 3D morphological and fine structural information. In addition to outlining basic principles associated with MPEF and SHG microscopy methods, this review reports potential uses of these high resolution imaging modalities in lung structural imaging. We place special emphasis on imaging 3D structural features of airways, visualizing and quantifying ECM remodeling associated with mouse asthma model as well as the potential uses for multiphoton microscopy in in vitro airway applications.
[Show abstract][Hide abstract] ABSTRACT: This study investigated the expression of Scleraxis in a murine model of patellar tendon injury in which the central third of the patellar tendon was unilaterally injured. The presence of tendon pathology was assessed using dual photon microscopy, conventional histology and microCT. Tendon pathology was also quantified noninvasively over a 12-week period using high-frequency ultrasound and laser Doppler flowmetry. Gene expression (Scx, Tnmd, and Col1a1) was determined at defined end-points (1, 4, 8, and 12 weeks) using qPCR on RNA from individual patellar tendons on injured and uninjured sides. There was significant development of tendon pathology as gauged by ultrasound and laser Doppler over 12 weeks. Injured tendons demonstrated significant histological and microCT evidence of pathological change, and disorganized collagen with reduced density. The expression of Scx and Col1a1 was unchanged at 1 week, significantly upregulated at 4 and 8 weeks, and had returned to baseline by 12 weeks. Tnmd expression was unchanged at 1 week, and significantly increased at 4, 8, and 12 weeks. Patellar tendon injury was associated with marked increases in the expression of Scx, Tnmd, and Col1a1. Our data suggest new roles for Scleraxis in coordinating the response to injury in the pathogenesis of tendon disorders.
Journal of Orthopaedic Research 02/2011; 29(2):289-96. DOI:10.1002/jor.21220 · 2.99 Impact Factor