Chemical regulators of epithelial plasticity reveal a nuclear receptor pathway controlling myofibroblast differentiation.

Plasticity in epithelial tissues relates to processes of embryonic development, tissue fibrosis and cancer progression. Pharmacological modulation of epithelial transitions during disease progression may thus be clinically useful. Using human keratinocytes and a robotic high-content imaging platform, we screened for chemical compounds that reverse transforming growth factor ß (TGF-ß)-induced epithelial-mesenchymal transition. In addition to TGF-ß receptor kinase inhibitors, we identified small molecule epithelial plasticity modulators including a naturally occurring hydroxysterol agonist of the liver X receptors (LXRs), members of the nuclear receptor transcription factor family. Endogenous and synthetic LXR agonists tested in diverse cell models blocked α-smooth muscle actin expression, myofibroblast differentiation and function. Agonist-dependent LXR activity or LXR overexpression in the absence of ligand counteracted TGF-ß-mediated myofibroblast terminal differentiation and collagen contraction. The protective effect of LXR agonists against TGF-ß-induced pro-fibrotic activity raises the possibility that anti-lipidogenic therapy may be relevant in fibrotic disorders and advanced cancer.

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.

In vitro and ex vivo vanadium antitumor activity in (TGF-ß)-induced EMT. Synergistic activity with carboplatin and correlation with tumor metastasis in cancer patients.

Epithelial to mesenchymal transition (EMT) plays a key role in tumor progression and metastasis as a crucial event for cancer cells to trigger the metastatic niche. Transforming growth factor-ß (TGF-ß) has been shown to play an important role as an EMT inducer in various stages of carcinogenesis. Previous reports had shown that antitumor vanadium inhibits the metastatic potential of tumor cells by reducing MMP-2 expression and inducing ROS-dependent apoptosis. However, the role of vanadium in (TGF-ß)-induced EMT remains unclear. In the present study, we report for the first time on the inhibitory effects of vanadium on (TGF-ß)-mediated EMT followed by down-regulation of ex vivo cancer stem cell markers. The results demonstrate blockage of (TGF-ß)-mediated EMT by vanadium and reduction in the mitochondrial potential of tumor cells linked to EMT and cancer metabolism. Furthermore, combination of vanadium and carboplatin (a) resulted in synergistic antitumor activity in ex vivo cell cultures, and (b) prompted G0/G1 cell cycle arrest and sensitization of tumor cells to carboplatin-induced apoptosis. Overall, the findings highlight the multifaceted antitumor action of vanadium and its synergistic antitumor efficacy with current chemotherapy drugs, knowledge that could be valuable for targeting cancer cell metabolism and cancer stem cell-mediated metastasis in aggressive chemoresistant tumors.

Commercially Available Preparations of Recombinant Wnt3a Contain Non-Wnt Related Activities Which May Activate TGF-ß Signaling.

The Wnt ligands are a family of secreted signaling proteins which play key roles in a number of cellular processes under physiological and pathological conditions. Wnts bind to their membrane receptors and initiate a signaling cascade which leads to the nuclear localization and transcriptional activity of ß-catenin. The development of purified recombinant Wnt ligands has greatly aided in our understanding of Wnt signaling and its functions in development and disease. In the current study, we identified non-Wnt related signaling activities which were present in commercially available preparations of recombinant Wnt3a. Specifically, we found that treatment of cultured fibroblasts with recombinant Wnt3a induced immediate activation of TGF-ß and BMP signaling and this activity appeared to be independent of the Wnt ligand itself. Therefore, while purified recombinant Wnt ligands continue to be a useful tool for studying this signaling pathway, one must exercise a degree of caution when analyzing the results of experiments that utilize purified recombinant Wnt ligands.

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.

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 ß1 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-ß 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.

Tamoxifen Inhibits TGF-ß-Mediated Activation of Myofibroblasts by Blocking Non-Smad Signaling Through ERK1/2.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine which stimulates the differentiation of fibroblasts into myofibroblasts. Myofibroblasts are critical for normal wound healing, but also accumulate pathologically in a number of chronic inflammatory conditions where they are key contributors to aberrant tissue remodeling and fibrosis, and in cancer stroma. In the current study, we identified a role for tamoxifen as a potent inhibitor of the TGF-ß-mediated activation of primary human skin and breast fibroblasts. Our data indicate that tamoxifen does not interfere with canonical Smad signaling downstream of TGF-ß but rather blocks non-Smad signaling through ERK1/2 MAP-kinase and the AP-1 transcription factor FRA2. We further demonstrate by siRNA-mediated knockdown that FRA2 is critical for the induced expression of myogenic proteins in response to TGF-ß. Functionally, TGF-ß-stimulated fibroblast-mediated contraction of collagen gels was impaired in the presence of tamoxifen. Altogether, these data demonstrate that tamoxifen prevents myofibroblast differentiation and, therefore, may provide therapeutic benefits to patients suffering from chronic inflammatory conditions or cancer.

Mapping of the Wnt/ß-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 versican's biosynthetic processing is necessary. We have recently shown that ß-catenin/T-cell factor (TCF) complex formation at the versican promoter site is essential for activation of versican transcription. The transcriptional activator ß-catenin is the key mediator of the canonical Wnt signaling pathway. However, ß-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, ß-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 ß-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/ß-catenin/TCF-regulated genes that are part of the signaling machinery regulating early embryogenesis, neoplasia, and cardiovascular remodeling.

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 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 myofibroblast differentiation by upregulating TGF-ß signaling through SMAD2 in a ß-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-ß 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 ß-catenin using siRNA reversed the Wnt3a-induced smooth muscle α-actin expression, suggesting these changes were dependent on canonical Wnt signaling through ß-catenin. Neutralization of TGF-ß with a blocking antibody significantly inhibited the Wnt3a-induced smooth muscle α-actin expression, indicating these changes were dependent on the increased TGF-ß signaling. Collectively, this data strongly suggests Wnt3a promotes the formation of a myofibroblast-like phenotype in cultured fibroblasts, in part, by upregulating TGF-ß signaling through SMAD2 in a ß-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.

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.

Versican: signaling to transcriptional control pathways.

Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix, which provides a loose and hydrated matrix during key events in development and disease. Versican participates in cell adhesion, proliferation, migration, and angiogenesis, and hence plays a central role in tissue morphogenesis and maintenance. In addition, versican contributes to the development of a number of pathologic processes including atherosclerotic vascular diseases, cancer, tendon remodeling, hair follicle cycling, central nervous system injury, and neurite outgrowth. Versican is a complex molecule consisting of modular core protein domains and glycosaminoglycan side chains, and there are various steps of synthesis and processes regulating them. Also, there is differential temporal and spatial expression of versican by multiple cell types and in different developmental and pathological time frames. 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 versican's biosynthetic processing is necessary. The goal of this review is to evaluate the current status of our knowledge regarding the transcriptional control of versican gene regulation. We will be focusing on the signal transduction pathways, promoter regions, cis-acting elements, and trans-factors that have been characterized.

Regulation of the versican promoter by the beta-catenin-T-cell factor complex in vascular smooth muscle cells.

The proteoglycan versican is pro-atherogenic and central to vascular injury and repair events. We identified the signaling pathways and promoter elements involved in regulation of versican expression in vascular smooth muscle cells. Phosphatidylinositol 3-kinase inhibitor, LY294002, significantly decreased versican-luciferase (Luc) promoter activity and endogenous mRNA levels. We further examined the roles of protein kinase B and glycogen synthase kinase (GSK)-3beta, downstream effectors of phosphatidylinositol 3-kinase, in the regulation of versican transcription. Co-transfection of dominant negative and constitutively active protein kinase B constructs with a versican-Luc construct decreased and increased promoter activity, respectively. Inhibition of GSK-3beta activity by LiCl augmented accumulation of beta-catenin and caused induction of versican-Luc activity as well as versican mRNA levels. Beta-catenin has no DNA binding domain, therefore it cannot directly induce transcription of the versican promoter. Software analysis of the versican promoter revealed two potential binding sites for T-cell factors (TCFs), proteins that confer transcriptional activation of beta-catenin. Electrophoretic mobility shift and supershift assays revealed specific binding of human TCF-4 and beta-catenin to oligonucleotides corresponding to a potential TCF binding site in the versican promoter. In addition to binding assays, we directly assessed the dependence of versican promoter activity on TCF binding sites. Site-directed mutagenesis of the TCF site located -492 bp relative to the transcription start site markedly diminished versican-Luc activity. Co-transfection of TCF-4 with versican-Luc did not increase promoter activity, but addition of beta-catenin and TCF-4 significantly stimulated basal versican promoter activity. Our findings suggest that versican transcription is predominantly mediated by the GSK-3beta pathway via the beta-catenin-TCF transcription factor complex in smooth muscle cells, wherein such regulation contributes to the normal or aberrant formation of provisional matrix in vascular injury and repair events.