Jon Carthy

Wnt3a Induces Myofibroblast Differentiation by Upregulating TGF-β Signaling Through SMAD2 in a β-Catenin-Dependent Manner

Growing evidence suggests the Wnt family of secreted glycoproteins and their associated signaling pathways, linked to development, are recapitulated during wound repair and regeneration events. However, the role of the Wnt pathway in such settings remains unclear. In the current study, we treated mouse fibroblasts with 250 ng/mL of recombinant Wnt3a for 72 hours and examined its affect on cell morphology and function. Wnt3a induced a spindle-like morphology in fibroblasts characterized by the increased formation of stress fibres. Wnt3a decreased the proliferation of fibroblasts, but significantly increased cell migration as well as fibroblast-mediated contraction of a collagen lattice. Wnt3a significantly increased the expression of TGF-β and its associated signaling through SMAD2. Consistent with this, we observed significantly increased smooth muscle α-actin expression and incorporation of this contractile protein into stress fibres following Wnt3a treatment. Knockdown of β-catenin using siRNA reversed the Wnt3a-induced smooth muscle α-actin expression, suggesting these changes were dependent on canonical Wnt signaling through β-catenin. Neutralization of TGF-β with a blocking antibody significantly inhibited the Wnt3a-induced smooth muscle α-actin expression, indicating these changes were dependent on the increased TGF-β signaling. Collectively, this data strongly suggests Wnt3a promotes the formation of a myofibroblast-like phenotype in cultured fibroblasts, in part, by upregulating TGF-β signaling through SMAD2 in a β-catenin-dependent mechanism. As myofibroblasts are critical regulators of wound healing responses, these findings may have important implications for our understanding of normal and aberrant injury and repair events.

Collagen matrix remodeling in 3-dimensional cellular space resolved using second harmonic generation and multiphoton excitation fluorescence

The structural remodeling of collagen is important in biological processes such as fibrosis, developmental morphogenesis and wound repair. Highly ordered collagen macromolecules produce second harmonic generation signals without the need for any exogenous label. Conversely, the cellular components stained with exogenous labels generate multiphoton excitation fluorescence signals. Both these signals can be captured simultaneously to provide spatially resolved structural reorganization of a collagen matrix and cells. This study dealt with an in vitro collagen gel contraction model of wound repair, in which fibroblasts are seeded into a 3-dimensional type I collagen matrix. When cells are stimulated to trigger collagen contraction, we found the fibroblasts to be highly elongated as well as interconnected in 2-dimensional space, and the collagen, in the form of a visibly clear fibril structure, accumulated around the cells. In the absence of contraction, on the other hand, the cells were predominantly round in shape and no sign of collagen accumulation around the cell was evident despite the presence of the fibrillar collagen morphology in the matrix. Our data suggest second harmonic and multiphoton excitation fluorescence signals can be used in tandem to provide spatially resolved 3-dimensional structural remodeling of a collagen matrix during wound repair.

Minimally invasive imaging method based on second harmonic generation and multiphoton excitation fluorescence in translational respiratory research

For translational respiratory research including in the development of clinical diagnostic tools, a minimally invasive imaging method, which can provide both cellular and extracellular structural details with sufficient specificity, sensitivity and spatial resolution, is particularly useful. Multiphoton microscopy causes excitation of endogenously fluorescent macromolecular systems and induces highly specific second harmonic generation signals from non‐centrosymmetric macromolecules such as fibrillar collagens. Both these signals can be captured simultaneously to provide spatially resolved 3D structural organization of extracellular matrix as well as the cellular morphologies in their native states. Besides briefly discussing the fundamentals of multiphoton excitation fluorescence and harmonic generation signals and the instrumentation details, this review focuses on the specific applications of these imaging modalities in lung structural imaging, particularly morphological features of alveolar structures, visualizing and quantifying extracellular matrix remodelling accompanying emphysematous destructions as well as the IPF, detecting lung cancers and the potential use in the tissue engineering applications.

Mapping of the Wnt/β-Catenin/TCF Response Elements in the Human Versican Promoter

Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix and is considered to be crucial to several key cellular processes involved in development and disease. There is differential temporal and spatial expression of versican by multiple cell types and in different developmental and pathological timeframes. In order to fully appreciate the functional roles of versican as it relates to changing patterns of expression in development and disease, an in-depth knowledge of versicans biosynthetic processing is necessary. We have recently shown that β-catenin/T-cell factor (TCF) complex formation at the versican promoter site is essential for activation of versican transcription. The transcriptional activator β-catenin is the key mediator of the canonical Wnt signaling pathway. However, β-catenin does not itself bind DNA and thus functions via interaction with TCF/Lymphoid-enhancing factor (LEF) transcription factors. These proteins contain a high-mobility group (HMG) box that binds DNA in a sequence-specific manner. Thus, in the case of active Wnt signaling, β-catenin activates, in cooperation with proteins of the TCF/LEF family, the expression of a wide variety of target genes. The goal of this chapter is to describe the techniques used to elucidate the transcriptional control of versican by the β-catenin/TCF response elements in its promoter site and to demonstrate how this signaling may be assayed experimentally. These approaches provide insight into the transcriptional regulation of the versican gene and provide the basis for the identification of novel Wnt/β-catenin/TCF-regulated genes that are part of the signaling machinery regulating early embryogenesis, neoplasia, and cardiovascular remodeling.

Versican and CD44 in in vitro valvular interstitial cell injury and repair

BACKGROUND Versican is one of the key components of the extracellular matrix (ECM) that is expressed during injury, inflammatory, and repair processes. The current study evaluated the relationship between versican and the membrane receptor CD44 during in vitro valvular interstitial cell (VIC) injury and repair. METHODS Subconfluent, confluent, and wounded cultures of human VICs were fixed and immunostained to detect versican and the membrane receptor CD44. To examine the relationship between versican and CD44, a blocking antibody to CD44 was added to cultured VICs, and in vitro wound repair along with pericellular versican organization and stress fiber formation were examined. RESULTS Immunohistochemistry demonstrated that versican is prominent intracellularly, as well as extracellularly, in actively proliferating VICs. In contrast, versican was only localized to fibrils in the extracellular space in between cells in confluent (quiescent) cultures. Following wounding, versican expression was up-regulated, and it was secreted as ECM at the trailing edge of migrating cells. The staining for CD44 was similarly localized to the trailing edge of migrating VICs in wounded cultures. Treatment of VICs with a CD44-blocking antibody disrupted the organization of versican in the pericellular matrix and inhibited stress fiber formation in these cells. Functionally, blocking CD44 significantly inhibited VIC-mediated contraction of type I collagen gels (35.7%±0.7% vs. 23.3%±1.4% of initial gel area, P<.01). CONCLUSIONS Versican is a key component of the provisional wound repair ECM that is expressed following injury to VICs. The receptor CD44 plays an important role in organizing the provisional ECM. SUMMARY Our data suggests VICs synthesize and secrete versican following injury. These cells also up-regulate CD44, a receptor that binds versican. Blocking CD44 disrupted the organization of versican and inhibited stress fiber formation. Functionally, blocking CD44 inhibited cell-mediated contraction of a collagen matrix. Collectively, these data suggest versican expression and organization are important to valve cell injury and repair.

WNT3A induces a contractile and secretory phenotype in cultured vascular smooth muscle cells that is associated with increased gap junction communication.

Evidence suggests a role for Wnt signaling in vascular wound repair and remodeling events. Despite this, very little is known about the effect of Wnt ligands on the structure and function of vascular cells. In this study, we treated vascular smooth muscle cells with 250 ng/ml of recombinant Wnt3a for 72 h and observed changes in the cell phenotype. Our data suggest Wnt3a completely alters the phenotype of vascular smooth muscle cells. The Wnt3a-treated cells appeared larger and had increased formation of stress fibers. These cells also had increased expression of the smooth muscle contractile proteins, calponin and smooth muscle α-actin, and contracted a collagen lattice faster than control cells. The Wnt3a-treated smooth muscle cells displayed increased extracellular matrix synthesis, as measured by collagen I and III mRNA expression, along with increased expression of MMP2 and MMP9, but decreased TIMP2 levels. The Wnt3a-induced change in cell phenotype was associated with increased expression of the gap junction protein connexin 43. Consistent with this, Wnt3a-treated smooth muscle cells displayed enhanced intercellular communication, as measured by the scrape-loading dye transfer technique. The canonical Wnt antagonist, dickkopf-related protein 1, completely reversed the contractile protein and connexin 43 expression seen in the Wnt3a-treated cells, suggesting these changes were dependent on canonical Wnt signaling. Collectively, this data suggest Wnt3a promotes a contractile and secretory phenotype in vascular smooth muscle cells that is associated with increased gap junction communication.

Versican V1 Overexpression Induces a Myofibroblast-Like Phenotype in Cultured Fibroblasts

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. Conclusions Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

Versican localizes to the nucleus in proliferating mesenchymal cells

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 multifluorescent immunostaining showed strong colocalization of versican and nucleolin, suggesting a nucleolar localization of versican in nondividing 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. Multifluorescent 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. CONCLUSIONS 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.

Inhibition of vascular endothelial growth factor reduces cardiac allograft vasculopathy

BACKGROUND Cardiac allograft vasculopathy (CAV) is a leading expression of chronic organ rejection at and beyond 1 year post-transplantation. Host bone marrow (BM)-derived cell migration to the allograft has been demonstrated in earlier work. Vascular endothelial growth factor (VEGF) is endogenously overexpressed within allografts. Graft neo-angiogenesis has been proposed as a mechanism by which VEGF may contribute to CAV. Herein we assess the therapeutic effect of inhibition of VEGF expression in CAV. METHODS In 129J mice, female donor hearts were heterotopically transplanted into C57/B16 males and treated with soluble VEGF receptor 1 (sVEGFR1) or vehicle control. The effect of VEGF inhibition on BM-mediated microvascular outgrowth and endothelial cell migration and proliferation were assessed using in vitro assays of aortic ring angiogenesis, wound healing and proliferation, respectively. RESULTS At 21 days post-transplantation, treatment with sVEGFR1 significantly reduced both percent luminal narrowing (p < 0.05) and percent of vessels affected (p < 0.005). sVEGFR1 significantly reduced average wet heart weight (p < 0.05), whereas mean ventricular cross-sectional area remained similar. Treatment of aortic rings with both sVEGFR1 and VEGFR2 tyrosine phosphorylation inhibitor (Ki 8751) significantly reduced BM-mediated microvascular outgrowth length (p < 0.05) and area (p < 0.05). Treatment of human coronary artery endothelial cells with sVEGFR1 and Ki 8751 significantly reduced BM-mediated endothelial cell migration (p < 0.005) and proliferation (p < 0.05). CONCLUSIONS VEGF inhibition reduces the severity and incidence of CAV in mouse models of cardiac transplantation, while attenuating myocardial edema and neo-angiogenesis. Using this model, we provide in vitro evidence of the role of VEGF signaling in BM-mediated microvascular outgrowth and endothelial cell migration and proliferation. VEGF inhibition may represent a novel approach to CAV treatment and prevention.

Imaging of collagen matrix remodeling in three-dimensional space using second harmonic generation and two photon excitation fluorescence

Second harmonic generation (SHG), a nonlinear optical phenomenon, exhibits several in-common characteristics of twophoton excited fluorescence (TPEF) microscopy. These characteristics include identical equipment requirements from experiment to experiment and the intrinsic capability of generating 3-dimensional (D) high resolution images. Structural protein arrays that are highly ordered, such as collagen, produce strong SHG signals without the need for any exogenous label (stain). SHG and TPEF can be used together to provide information on structural rearrangements in 3D space of the collagen matrix associated with various physiological processes. In this study, we used SHG and TPEF to detect cellmediated structural reorganization of the extracellular collagen matrix in 3D space triggered by dimensional changes of embedded fibroblasts. These fibroblasts were cultured in native type I collagen gels and were stimulated to contract for a period of 24 hours. The gels were stained for cell nuclei with Hoechst and for actin with phalloidin conjugated to Alexa Fluor 488. We used non-de-scanned detectors and spectral scanning mode both in the reflection geometry for generating the 3D images and for SHG spectra, respectively. We used a tunable infrared laser with 100-fs pulses at a repetition rate of 80-MHz tuned to 800-nm for Hoechst and Alexa 488 excitations. We employed a broad range of excitation wavelengths (800 to 880-nm) with a scan interval of 10 nm to detect the SHG signal. We found that spectrally clean SHG signal peaked at 414-nm with excitation wavelength of 830-nm. The SHG spectrum has a full width half maximum (FWHM) bandwidth of 6.60-nm, which is consistent with its scaling relation to FWHM bandwidth 100-fs excitation pulses. When stimulated to contract, we found the fibroblasts to be highly elongated as well as interconnected in 2D space, and the collagen matrix, in the form of a visibly clear fibril structure, accumulated around the cells. In the absence of contraction, on the other hand, the cells were predominantly round in shape and no sign of collagen accumulation around the cell was evident despite the presence of SHG signal as well as the fibrillar collagen morphology in the collagen matrix. We here conclude that SHG in conjunction with TPEF can serve as a noninvasive method to provide spatially resolved 3D structural reorganization of collagen matrices triggered by various physiological processes.