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Cancer-associated fibroblasts (CAFs) are key actors in modulating the progression of many solid tumors, such as breast cancer (BC). Herein, we identify an integrin [alpha]11/PDGFR[beta]-positive CAF subset displaying tumor-promoting features in BC. In the preclinical MMTV-PyMT mouse model, integrin [alpha]11 deficiency led to a drastic reduction of tumor progression and metastasis. A clear association between integrin [alpha]11 and PDGFR[beta] was found at both transcriptional and histological levels in BC specimens. High stromal integrin [alpha]11/PDGFR[beta] expression was associated with high grades and poorer clinical outcome in human BC patients. Functional assays using 5 CAF subpopulations (1 murine, 4 human) revealed that integrin [alpha]11 promotes CAF invasion and CAF-induced tumor cell invasion upon PDGF-BB stimulation. Mechanistically, the proinvasive activity of integrin [alpha]11 relies on its ability to interact with PDGFR[beta] in a ligand-dependent manner and to promote its downstream JNK activation, leading to the production of tenascin C, a proinvasive matricellular protein. Pharmacological inhibition of PDGFR[beta] and JNK impaired tumor cell invasion induced by integrin [alpha][11.sup.+] CAFs. Collectively, our study uncovers an integrin [alpha][11.sup.+] subset of protumoral CAFs that exploits the PDGFR[beta]/JNK signaling axis to promote tumor invasiveness in BC., Introduction Breast cancer (BC) is the most common type of cancer and the second leading cause of cancer-related death in women worldwide. Despite increasing knowledge of BC biology and huge [...]

Author(s): Jan H. Lui [1]; Tomasz J. Nowakowski [1]; Alex A. Pollen [1]; Ashkan Javaherian [1]; Arnold R. Kriegstein (corresponding author) [1]; Michael C. Oldham (corresponding author) [1] Evolutionary expansion [...]

Purpose The calcium-sensitizing drug levosimendan increases myocardial contractility and, by activating K.sup.+-channels, dilates pulmonary vessels. In the acute setting, levosimendan is clinically used to treat right heart failure in pulmonary hypertension. As K.sup.+-channel activation elicits several beneficial effects in the vascular system, we hypothesized that levosimendan also attenuates the remodeling process in the monocrotaline model of rat pulmonary hypertension. Methods and results Animal subgroups received levosimendan, the K.sup.+-channel opener nicorandil, or levosimendan together with the K.sup.+-adenosine triphosphate (ATP)-sensitive potassium channel (K.sub.ATP) blocker glibenclamide. Morphometric analyses revealed that levosimendan and nicorandil attenuated the increased pulmonary vascular medial wall thickness after monocrotaline challenge. Accordingly, in vivo BrdU assays revealed that levosimendan significantly diminished proliferation of pulmonary arterial smooth muscle cells (PASMCs), and this effect was attenuated by glibenclamide. Levosimendan also reduced right ventricular hypertrophy, but this effect was not glibenclamide sensitive and not recapitulated by nicorandil. In cell culture, levosimendan had a direct inhibitory effect on the platelet-derived growth factor (PDGF)-induced proliferation of PASMCs, which however required high concentrations of the compound, pointing towards an endothelial effect. Indeed, levosimendan increased cyclic guanosine monophosphate (cGMP) in human umbilical vein endothelial cells (HUVECs) and impaired the tumor necrosis factor-[alpha] (TNF-[alpha])-induced inflammatory expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2), and monocyte chemotactic protein-1 (MCP-1). In luciferase reporter gene assays in HUVECs, levosimendan dose-dependently attenuated the TNF-[alpha]-stimulated increase of proinflammatory transcription factors activator protein 1 (AP1), hypoxia-inducible factor-1[alpha] (HIF-1[alpha]), and nuclear factor-[kappa]B (NF-[kappa]B). Conclusions Levosimendan attenuates pulmonary vascular remodeling, presumably by an antiproliferative and anti-inflammatory effect which is mediated by cellular hyperpolarization. The compound also has a direct inhibitory effect on cardiac hypertrophy, which is however K.sup.+-channel independent., Author(s): M. Revermann [sup.1] [sup.2], M. Schloss [sup.1], A. Mieth [sup.1], A. Babelova [sup.1], K. Schröder [sup.1], S. Neofitidou [sup.1], J. Buerkl [sup.1], T. Kirschning [sup.2], R. T. Schermuly [sup.3], [...]

Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell-derived [VEGFR2.sup.+]mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)-BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyhransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A-induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras-/- mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in [VEGFR2.sup.+] progenitor cells resulted in endothelial differentiationthrough the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in [VEGFR2.sup.+] progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6-9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras-Erk signaling to direct endothelial specification of [VEGFR2.sup.+] vascular progenitor cells.

Chemotaxis (i.e., directed migration) of hepatic stellate cells to areas of inflammation is a requisite event in the liver's response to injury. Previous studies of signaling pathways that regulate stellate cell migration suggest a key role for focal adhesions, but the exact function of these protein complexes in motility remains unclear. Focal adhesions attach a cell to its substrate and therefore must be regulated in a highly coordinated manner during migration. To test the hypothesis that focal adhesion turnover is an essential early event for chemotaxis in stellate cells, we employed a live-cell imaging technique in which chemotaxis was induced by locally stimulating the tips of rat stellate cell protrusions with platelet-derived growth factor-BB (PDGF). Focal adhesions were visualized with an antibody directed against vinculin, a structural component of the focal adhesion complex. PDGF triggered rapid disassembly of adhesions within 6.25 min, subsequent reassembly by 12.5 min, and continued adhesion assembly in concert with the spreading protrusion until the completion of chemotaxis. Blockade of adhesion disassembly by growing cells on fibronectin or treatment with nocodazole prevented a chemotactic response to PDGF. Augmentation of adhesion disassembly with ML-7 enhanced the chemotactic response to PDGF. These data suggest that focal adhesion disassembly is an essential early event in stellate cell chemotaxis in response to PDGF. platelet-derived growth factor; migration; microtubules; myosin light chain kinase; fibronectin

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.04.438 Byline: Jocelyn A. McDonald (a)(b), Elaine M. Pinheiro (a), Lisa Kadlec (c), Trudi Schupbach (c), Denise J. Montell (a) Keywords: Drosophila melanogaster; EGFR; PVF1; PVR; Vein; Spitz; Keren; Gurken; Border cells; Migration Abstract: Cell migration is an important feature of embryonic development as well as tumor metastasis. Border cells in the Drosophila ovary have emerged as a useful in vivo model for uncovering the molecular mechanisms that control many aspects of cell migration including guidance. It was previously shown that two receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and PDGF- and VEGF-related receptor (PVR), together contribute to border cell migration. Whereas the ligand for PVR, PVF1, is known to guide border cells, it is unclear which of the four activating EGFR ligands function in this process. We developed an assay to detect the ability of secreted factors to reroute migrating border cells in vivo and tested the activity of EGFR ligands compared to PVF1. Two ligands, Keren and Spitz, guided border cells whereas the other ligands, Gurken and Vein, did not. In addition, only Keren and Spitz were expressed at the appropriate stage in the oocyte, the target of border cell migration. Therefore, a complex combination of EGFR and PVR ligands together guide border cells to the oocyte. Author Affiliation: (a) Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA (b) Department of Molecular Genetics, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA (c) HHMI, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA Article History: Received 18 November 2005; Revised 31 March 2006; Accepted 4 April 2006

Diabetes mellitus is associated with an increased risk of vascular disease, with significant alterations in systemic endothelial progenitor cells (EPCs) and peripheral vascular function. To identify the contribution of the different vascular compartments in the diabetic impairment of vascularization, we employed streptozotocin- and control-treated 3-mo-old C57B1/6 mice in an isogeneic pinnal cardiac allograft model, revealing a significant delay in vascularization of wild-type cardiac tissue transplanted into diabetic mice. To investigate the basis of this impairment, the function of diabetic bone marrow cells was tested by transplantation of bone marrow cells isolated from diabetic and control mice into intact, unirradiated 18-mo-old C57B1/6 mice, which have impaired function of both EPCs and peripheral endothelial cells. Importantly, cells derived from control, but not diabetic, bone marrow integrated into transplanted cardiac allografts. To assess the contribution of diabetic changes in the local vasculature, diabetic mice were treated with pinnal injections of platelet-derived growth factor (PDGF)-AB, which promotes cardiac angiogenesis in wild-type mice. However, whereas PDGF-AB enhanced allograft function in control mice, the activity of the cardiac transplants in the PDGF-AB-treated diabetic mice was significantly decreased. To decipher the potential interactions between systemic bone marrow-derived cells and local vascular pathways, diabetic mice were transplanted with wild-type bone marrow cells with or without PDGF-AB pinnal pretreatment, resulting in improved allograft function and donor cell recruitment only in the combination treatment arm. Overall, these studies show that the diabetic impairment in cardiac angiogenesis can be reversed by targeting the synergism between local trophic pathways and systemic cell function. diabetes mellitus; endothelial progenitor cells; platelet-derived growth factor: aging: heart

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.07.018 Byline: Dipika Sharma, Lesya Holets, Xiaoming Zhang, William H. Kinsey Keywords: Zebrafish; Development; Epiboly; Fyn Kinase; Calcium Abbreviations: BSA, bovine serum albumin; CHAPS, 3[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate; CSK, src specific kinase; DAPI, 4'6-diamidino-2phenylindole dihydrochloride; DEPC, diethyl pyrocarbonate; DIC, differential interference contrast; EDTA, ethylenediaminetetraacetic acid; EVL, enveloping cell layer; FGF, fibroblast growth factor; HEPES, [2-hydroxyethyl]piperazine-N'-[2-ethane-sulfonic acid]; IP3, Ins 1,4,5 P.sub.3; PAGE, polyacrylamide gel electrophoresis; PTK, protein tyrosine kinase; SDS, sodium dodecyl sulfate; SH2 and SH3, Src homology domain 2 and 3, respectively; PDGF, platelet-derived growth factor; PBT, (PBS + 0.1% Tween 20); YSL, yolk syncitial layer Abstract: The function of Fyn kinase during zebrafish development through the blastula stage was investigated through the use of dominant-negative constructs designed to suppress the function of zebrafish c-Fyn. Microinjection of SH2 domain-containing fusion protein or mRNA encoding the mutated, catalytically inactive Fyn at 45 min post-insemination had no significant effect during cleavage and did not inhibit formation of the yolk syncitial layer. Smoothing of the enveloping cell layer at the midblastula transition occurred normally and expression of bon/mixer and mezzo, zygotic transcription factors indicated that activation of the zygotic genome did occur. Signaling pathways involved with axis determination such as [beta]-catenin, activin, and nodal appeared to function normally as evidenced by expression of boz, goosecoid, and mezzo. However, while formation of the yolk syncitial layer was normal, the marginal blastomeres failed to migrate toward the vegetal pole and epiboly did not occur, a phenotype similar but distinct from that resulting from suppression of c-Yes kinase. The block to development was prevented by co-injection of c-Fyn mRNA with the dominant-negative construct indicating that it was a specific effect. Injection of the dominant-negative mRNA into individual blastomeres indicated that the effect was exerted on the intrinsic ability of the individual blastomeres to respond to signals directing epiboly and not on the signals themselves. Analysis of the pattern of calcium signaling in experimental and control embryos demonstrated that the elevated [Ca.sup.2+].sub.i characteristic of the marginal blastomeres was suppressed. Together, these observations indicate that Fyn kinase plays an important role in epiboly, possibly through its effects in calcium signaling. Author Affiliation: Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160, USA Article History: Received 3 March 2005; Revised 8 July 2005; Accepted 18 July 2005

The influence of cholesterol enrichment of arterial smooth muscle cells on cell membrane expression of G-protein heterodimers and p21ras was determined through mechanistic studies of cultured smooth muscle cells. Results have shown that cholesterol enrichment significantly reduced basal MAP-kinase activity and reduced PDGF- and AlF4- induced MAP kinase activity. It was deduced that cholesterol enrichment exhibits inhibition activity in response to the reduction of the membrane p21ras content.

The mechanisms by which G-protein-agonists mimic eicosanoid synthesis and by which cholesterol modifies the process were determined through agonist studies using GTPgammaS cells. Results have shown that inhibition of the ability of cholesterol enrichment to reduce membrane-associated G-protein subunits was secondary to mevalonate, which in turn, induced the PGI2 release in cholesterol-enriched cells.

The human interleukin (IL) 6 and IL-6 receptor genes ensure interstitial pneumonia with the characteristic histopathological alterations. The transforming growth factor beta-1 and platelet-derived growth factor B genes cause fibrosis and less of alveolitis, but IL-6 and IL-6 receptor induce effective alveolitis and less of fibrosis. For the experimental observations, expression vectors are incorporated in Wistar rats to the locally overexpression humoral factors in the lungs through the trachea.

A study of the role of Ras protein in platelet-derived growth factor (PDGF)-stimulated chemotaxis using cells expressing a dominant-negative Ras to find whether Ras inhibition suppresses movement towards PDGF-BB shows that cells expressing dominant-negative Ras migrated towards the soluble fibronectin revealing that the capability of these cells to migrate is conserved. The study indicates that Ras may not be needed for fibronectin-stimulated cell motility.

Human macrophages, on exposure to hypoxia, synthesize and secrete acidic and basic fibroblast growth factor (a/bFGF) and platelet-derived growth factor that stimulate the growth of hypoxic endothelial cells (ECs). Angiogenesis is promoted in a hypoxic environment by cellular components such as macrophages and microvascular ECs. Normally, hypoxia slows EC proliferation and suppresses EC bFGF.

A novel divalent metal ion binding site present in the alpha-platelet-derived growth factor receptor (alpha-PDGFR) kinase insert (KI) domain regulates the binding of the recombinant alpha-PDGFR KI domain with the p85 N-SH2 domain. Presence of Ca2+ ions enhances this binding. In the presence of insulin, maturation is delayed upon injecting this domain into Xenopus oocytes and this reveals the compatibility of the three-dimensional structure of the KI domain with biological activity.

Translocation of glucose transporter type 4 (GLUT4) occurs in mammalian cells with the help of platelet-derived growth factor (PDGF)-stimulated activity of phosphatidylinositol (PI) 3-kinase. A study of mutant PDGF receptors confirm the role of PI3-kinase in GLUT4 translocation. In 3T3-L1 adipocytes and chinese hamster ovary cells that overexpress PDGF receptors, GLUT4 translocation is induced by PDGF and insulin.