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  • Electrophoresis and electroblotting of native collagens. 3364721

    Electrophoretic and immunoblotting techniques, while now used routinely for the biochemical characterization of many proteins, have not been used for the identification of native collagens. We present here an acidic electrophoresis system using very low percentage acrylamide gels which maintains collagen solubility and allows migration of native dermal collagens. The method gives uniform gels which can be made mechanically stable for subsequent electroblotting. The resulting nitrocellulose transfer allows immunological detection of collagens using either polyclonal or monoclonal antibodies and can be used to screen antibody specificities. The majority of murine monoclonal antibodies directed against collagen bind only to conformational epitopes on the native triple-helical collagen, and thus cannot be screened by Western blotting. This method therefore enables the electrophoretic screening of these monoclonal antibodies and provides an alternative approach for their characterization.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Hepatocytes express nerve growth factor during liver injury: evidence for paracrine regulation of hepatic stellate cell apoptosis. 14578185

    A key feature of recovery from liver fibrosis is hepatic stellate cell (HSC) apoptosis, which serves the dual function of removing the major source of neomatrix and tissue inhibitors of metalloproteinases thereby facilitating matrix degradation. The mechanisms regulating HSC apoptosis remain undefined but may include the interaction of nerve growth factor (NGF) with its receptor, p75, on HSC. In this study, by TaqMan polymerase chain reaction in situ hybridization and immunohistochemistry, we demonstrate that NGF is expressed by hepatocytes during fibrotic injury. Peak hepatocyte expression of NGF (48 hours after CCl(4) injection) coincides with maximal rate of apoptosis of HSC by terminal dUTP nick-end labeling staining. Addition of recombinant NGF to HSC in tissue culture causes a dose-dependent increase in apoptosis. NGF regulates nuclear factor (NF)-kappaB activity, reducing p50/p65 binding detected by electromobility shift assay and reduced NF-kappaB CAT reporter activities from both basal unstimulated levels and after NF-kappaB induction by tumor necrosis factor. In each case, a relative reduction in NF-kappaB binding was associated with a significant increase in caspase 3 activity. These data provide evidence that NGF is expressed during fibrotic liver injury and may regulate number of activated HSCs via induction of apoptosis.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Fibroblast α11β1 integrin regulates tensional homeostasis in fibroblast/A549 carcinoma heterospheroids. 25076207

    We have previously shown that fibroblast expression of α11β1 integrin stimulates A549 carcinoma cell growth in a xenograft tumor model. To understand the molecular mechanisms whereby a collagen receptor on fibroblast can regulate tumor growth we have used a 3D heterospheroid system composed of A549 tumor cells and fibroblasts without (α11+/+) or with a deletion (α11-/-) in integrin α11 gene. Our data show that α11-/-/A549 spheroids are larger than α11+/+/A549 spheroids, and that A549 cell number, cell migration and cell invasion in a collagen I gel are decreased in α11-/-/A549 spheroids. Gene expression profiling of differentially expressed genes in fibroblast/A549 spheroids identified CXCL5 as one molecule down-regulated in A549 cells in the absence of α11 on the fibroblasts. Blocking CXCL5 function with the CXCR2 inhibitor SB225002 reduced cell proliferation and cell migration of A549 cells within spheroids, demonstrating that the fibroblast integrin α11β1 in a 3D heterospheroid context affects carcinoma cell growth and invasion by stimulating autocrine secretion of CXCL5. We furthermore suggest that fibroblast α11β1 in fibroblast/A549 spheroids regulates interstitial fluid pressure by compacting the collagen matrix, in turn implying a role for stromal collagen receptors in regulating tensional hemostasis in tumors. In summary, blocking stromal α11β1 integrin function might thus be a stroma-targeted therapeutic strategy to increase the efficacy of chemotherapy.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • A new mechanism for pillar formation during tumor-induced intussusceptive angiogenesis: inverse sprouting. 21827961

    One of the hallmarks of intussusceptive angiogenesis is the development of intraluminal connective tissue pillars. The exact mechanism of pillar formation has not yet been elucidated. By using electron and confocal microscopy, we observed intraluminal nascent pillars that contain a collagen bundle covered by endothelial cells (ECs) in the vasculature of experimental tumors. We proposed a new mechanism for the development of these pillars. First, intraluminal endothelial bridges are formed. Second, localized dissolution of the basement membrane occurs and a bridging EC attaches to a collagen bundle in the underlying connective tissue. A pulling force is then exerted by the actin cytoskeleton of the ECs via specific attachment points, which contain vinculin, to the collagen bundle, resulting in suction and subsequent transport of the collagen bundle into and through the vessel lumen. Third, the pillar matures through the immigration of connective tissue cells and the deposition of new collagenous connective tissue. The proposed simple mechanism generates a connection between the processes of endothelial bridging and intussusceptive angiogenesis and identifies the source of the force behind pillar formation. Moreover, it ensures the rapid formation of pillars from pre-existing building blocks and the maintenance of EC polarity. To describe it, we coined the term inverse sprouting.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Intrarenal dopamine inhibits progression of diabetic nephropathy. 22688335

    The kidney has a local intrarenal dopaminergic system, and in the kidney, dopamine modulates renal hemodynamics, inhibits salt and fluid reabsorption, antagonizes the renin-angiotensin system, and inhibits oxidative stress. The current study examined the effects of alterations in the intrarenal dopaminergic system on kidney structure and function in models of type 1 diabetes. We studied catechol-O-methyl-transferase (COMT)(-/-) mice, which have increased renal dopamine production due to decreased dopamine metabolism, and renal transplantation was used to determine whether the effects seen with COMT deficiency were kidney-specific. To determine the effects of selective inhibition of intrarenal dopamine production, we used mice with proximal tubule deletion of aromatic amino acid decarboxylase (ptAADC(-/-)). Compared with wild-type diabetic mice, COMT(-/-) mice had decreased hyperfiltration, decreased macula densa cyclooxygenase-2 expression, decreased albuminuria, decreased glomerulopathy, and inhibition of expression of markers of inflammation, oxidative stress, and fibrosis. These differences were also seen in diabetic mice with a transplanted kidney from COMT(-/-) mice. In contrast, diabetic ptAADC(-/-) mice had increased nephropathy. Our study demonstrates an important role of the intrarenal dopaminergic system to modulate the development and progression of diabetic kidney injury and indicate that the decreased renal dopamine production may have important consequences in the underlying pathogenesis of diabetic nephropathy.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Type I collagen is a molecular target for inhibition of angiogenesis by endogenous thrombospondin-1. 16247480

    Three-dimensional explant cultures of muscle tissue were used to characterize secreted proteins regulated by endogenous levels of the angiogenesis modulator thrombospondin (TSP)-1. Explants from TSP1 null mice exhibit enhanced neovascularization associated with increased endothelial outgrowth but decreased outgrowth of perivascular smooth muscle cells . The absence of endogenous TSP1 did not diminish activation of latent transforming growth factor-beta and moderately decreased matrix metalloproteinase levels. However, significant changes in other secreted proteins were observed. Endogenous TSP1 decreased mRNA levels for collagens Ialpha1, Ialpha2, and IIIalpha1 and laminin alpha4 and increased collagen IValpha1 mRNA expression. Endogenous TSP1 also decreased the level of type I collagen protein produced by the vascular outgrowths. Collagens Ialpha1, Ialpha2, and IIIalpha1 are known tumor endothelial markers, suggesting that TSP1 coordinately regulates a set of extracellular matrix genes that reverse the angiogenic switch. Suppression of collagen Ialpha1 or Ialpha2 mRNAs using antisense morpholinos inhibited outgrowth in TSP1 null explants and proliferation of TSP1 null endothelial cells, indicating that type I collagen synthesis is limiting for this neovascularization response.
    Document Type:
    Reference
    Product Catalog Number:
    AB765P
    Product Catalog Name:
    Anti-Mouse Collagen Type I Antibody