Auxagen, Inc.

SAINT LOUIS, MO, United States

Auxagen, Inc.

SAINT LOUIS, MO, United States
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Tsai C.C.,WCP Pathology Laboratories Inc. | Huang S.S.,Auxagen, Inc.
Journal of Cell Science | Year: 2011

Cell-surface retention sequence (CRS) binding protein (CRSBP-1) is a membrane glycoprotein identified by its ability to bind PDGFBB and VEGF-A via their CRS motifs (clusters of basic amino acid residues). CRSBP-1 is identical to LYVE-1 and exhibits dual ligand (CRS-containing proteins and hyaluronic acid) binding activity, suggesting the importance of CRSBP-1 ligands in lymphatic function. Here, we show that CRSBP-1 ligands induce disruption of VE-cadherin-mediated intercellular adhesion and opening of intercellular junctions in lymphatic endothelial cell (LEC) monolayers as determined by immunofluorescence microscopy and Transwell permeability assay. This occurs by interaction with CRSBP-1 in the CRSBP-1-PDGFβR-β-catenin complex, resulting in tyrosine phosphorylation of the complex, dissociation of β-catenin and p120-catenin from VE-cadherin, and internalization of VEcadherin. Pretreatment of LECs with a PDGFβR kinase inhibitor abolishes ligand-stimulated tyrosine phosphorylation of VE-cadherin, halts the ligand-induced disruption of VE-cadherin intercellular adhesion and blocks the ligand-induced opening of intercellular junctions. These CRSBP-1 ligands also induce opening of lymphatic intercellular junctions that respond to PDGFβR kinase inhibitor in wild-type mice (but not in Crsbp1-null mice) as evidenced by increased transit of injected FITC-dextran and induced edema fluid from the interstitial space into lymphatic vessels. These results disclose a novel mechanism involved in the opening of lymphatic intercellular junctions. © 2011. Published by The Company of Biologists Ltd.


Chen C.-L.,Saint Louis University | Tetri L.H.,Saint Louis University | Neuschwander-Tetri B.A.,Saint Louis University | Huang S.S.,Auxagen, Inc. | Huang J.S.,Saint Louis University
Journal of Nutritional Biochemistry | Year: 2011

Dietary trans fats (TFs) have been causally linked to atherosclerosis, but the mechanism by which they cause the disease remains elusive. Suppressed transforming growth factor (TGF)-β responsiveness in aortic endothelium has been shown to play an important role in the pathogenesis of atherosclerosis in animals with hypercholesterolemia. We investigated the effects of a high TF diet on TGF-β responsiveness in aortic endothelium and integration of cholesterol in tissues. Here, we show that normal mice fed a high TF diet for 24 weeks exhibit atherosclerotic lesions and suppressed TGF-β responsiveness in aortic endothelium. The suppressed TGF-β responsiveness is evidenced by markedly reduced expression of TGF-β type I and II receptors and profoundly decreased levels of phosphorylated Smad2, an important TGF-β response indicator, in aortic endothelium. These mice exhibit greatly increased integration of cholesterol into tissue plasma membranes. These results suggest that dietary TFs cause atherosclerosis, at least in part, by suppressing TGF-β responsiveness. This effect is presumably mediated by the increased deposition of cholesterol into cellular plasma membranes in vascular tissue, as in hypercholesterolemia. © 2011 Elsevier Inc.


Hou W.-H.,Saint Louis University | Liua I.-H.,Saint Louis University | Huang S.S.,Auxagen, Inc. | Huang J.S.,Saint Louis University
FEBS Letters | Year: 2012

CRSBP-l/LYVE-1 ligands (PDGF-BB, VEGF-A 165 and hyaluronic acid) have been shown to induce opening of lymphatic intercellular junctions in vitro and in vivo by stimulating contraction of lymphatic endothelial cells (LECs). The mechanism by which CRSBP-1 ligands stimulate contraction of LECs is not understood. Here we demonstrate that CRSBP-1 is localized to the plasma membrane as well as intracellular fibrillar structures in LECs, including primary human dermal LECs and SVEC4-10 cells. CRSBP-1-associated fibrillar structures are identical to the ER network as evidenced by the co-localization of CRSBP-1 and BiP in these cells. CRSBP-1 ligands stimulate contraction of the ER network in a CRSBP-1-dependent and paclitaxel (a microtubule-stabilizing agent)-sensitive manner. These results suggest that ligand-stimulated ER contraction is associated with ligand-stimulated contraction in LECs. © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.


Huang S.S.,Auxagen, Inc. | Chen C.-L.,National Sun Yat - sen University | Huang F.W.,Dana-Farber Cancer Institute | Hou W.-H.,Saint Louis University | Huang J.S.,Saint Louis University
Journal of Cellular Biochemistry | Year: 2016

Dimethyl sulfoxide (DMSO) is used to treat many diseases/symptoms. The molecular basis of the pharmacological actions of DMSO has been unclear. We hypothesized that DMSO exerts some of these actions by enhancing TGF-β activity. Here we show that DMSO enhances TGF-β activity by ∼3-4-fold in Mv1Lu and NMuMG cells expressing Smad-dependent luciferase reporters. In Mv1Lu cells, DMSO enhances TGF-β-stimulated expression of P-Smad2 and PAI-1. It increases cell-surface expression of TGF-β receptors (TβR-I and/or TβR-II) by ∼3-4-fold without altering their cellular levels as determined by 125I-labeled TGF-β-cross-linking/Western blot analysis, suggesting the presence of large intracellular pools in these cells. Sucrose density gradient ultracentrifugation/Western blot analysis reveals that DMSO induces recruitment of TβR-II (but not TβR-I) from its intracellular pool to plasma-membrane microdomains. It induces more recruitment of TβR-II to non-lipid raft microdomains than to lipid rafts/caveolae. Mv1Lu cells transiently transfected with TβR-II-HA plasmid were treated with DMSO and analyzed by indirect immunofluoresence staining using anti-HA antibody. In these cells, TβR-II-HA is present as a vesicle-like network in the cytoplasm as well as in the plasma membrane. DMSO causes depletion of TβR-II-HA-containing vesicles from the cytoplasm and co-localization of TβR-II-HA and cveolin-1 at the plasma membrane. These results suggest that DMSO, a fusogenic substance, enhances TGF-β activity presumably by inducing fusion of cytoplasmic vesicles (containing TβR-II) and the plasma membrane, resulting in increased localization of TβR-II to non-lipid raft microdomains where canonical signaling occurs. Fusogenic activity of DMSO may play a pivotal role in its pharmacological actions involving membrane proteins with large cytoplasmic pools. J. Cell. Biochem. 117: 1568-1579, 2016. © 2015 Wiley Periodicals, Inc.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 283.57K | Year: 2016

DESCRIPTION provided by applicant Pulmonary fibrosis PF including idiopathic PF IPF is a group of progressive incurable lung diseases with high mortality rates PF is characterized by excessive deposition of extracellular matrix proteins in lung parenchyma via TGF dependent pathways Existing therapies only modestly slow the progression and likely only indirectly impact TGF mediated pathways Recent identification of the involvement of abnormal lymphangiogenesis in IPF and bleomycin induced PF has provided new insights into cellular and molecular mechanisms that may limit systemic therapeutics It is hypothesized that vascular remodeling which occurs after lung injury leads to deposition of hyaluronic acid a component of lymph and a weak lymphangiogenesis factor This results in abnormal lymphangiogenesis which produces new lymphatic vessels with functional defects lymphatic drainage defects leading to the vicious cycle of repeated injury and fibrosis Abnormal lymphangiogenesis a rate limiting step in PF offers an excellent target for future inhaled PF therapeutics The molecular basis of the role of abnormal lymphangiogenesis in the formation of PF is unknown We hypothesize that TGF a potent fibrogenic cytokine produced in lung upon injury contributes to abnormal lymphangiogenesis which occurs during vascular remodeling after lung injury and is involved in the vicious cycle of repeated injury and fibrosis injury abnormal lymphangiogenesis lymphatic drainage defects accumulation of TGF n which leads to PF This hypothesis is based on several lines of evidence TGF accumulates in lung tissues of PF including IPF TGF inhibits normal lymphangiogenesis in lung tissues TGF induces lung edema by increasing epithelial and endothelial permeability and TGF is a major cytokine which is responsible for production of fibrotic extracellular matrix This hypothesis suggests that to ameliorate PF TGF antagonists targeted to the lung draining lymphatic systems by intrapulmonary administration can inhibit not only the matrix production but the cycle of repeated injury and fibrosis In the last decade TGF binding proteins anti TGF antibodies and soluble type II TGF receptors and small molecule inhibitors which target latent TGF activation and TGF signaling have been developed and used to treat PF in animal models However the lung draining lymphatics are not specifically targeted in these treatments This will limit their efficacy in treating human patients In additio these agents have systemic and off target effects that result in narrow therapeutic windows To address the limited efficacy and off target effects we generated a novel TGF receptor antagonist with both TGF antagonist and wound healing promoting activities It can be effectively safely and affordably used for the management of PF in humans In this project we will determine the efficacy of this antagonist in treating PF by intranasal delivery which targets the lung lymphatic system and lung parenchyma in two mouse models of lung fibrotic disease We expect that the results from these proposed studies will lead to a novel effective therapy of PF in patients PUBLIC HEALTH RELEVANCE The goal of this project is to develop a novel TGF receptor antagonist which exhibits anti fibrotic and wound healing activities as a drug candidate It is expected to ameliorate and reverse lung fibrosis which currently lacks effective therapy The clinical availability of this novel and potent antagonist will benefit millions of patients worldwie


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 998.03K | Year: 2010

DESCRIPTION (provided by applicant): Every year in the United States, gt1.25 million people suffer from burns, 6.5 million have chronic skin ulcers caused by pressure, venous stasis or diabetes mellitus and 0.25 million have keloids sufficiently severe to require surgery. Burn treatment costs 1.8 billion per year in the US. The treatment of persons with chronic skin ulcers costs 13 billion per year in the US. The annual cost of diabetic peripheral neuropathy and/or neuropathic foot ulcers in the U.S. is 0.8 billion for type I diabetics and 10.1 billion for type II diabetics. Limb-sparing surgical procedures are also widely used. In spite of these large costs for the care and the treatment of diabetic foot ulcers, each year 82,000 limb amputations are still performed on US patients with diabetic ulcers because current therapy is not very effective. These surgical treatments cost about 0.3 billion per year in the US. Currently, there is no agent which has been shown to be effective for treating cutaneous wounds. As these costs suggest, there is an urgent need for developing effective agents to accelerate wound healing and reduce scarring or tissue fibrosis in patients with burn injuries, blast injuries, chronic skin ulcers, keloids and other similar disorders. New products to treat these patients will drive the market. Accumulating evidence indicates that TGF-2, a cytokine, provides an ideal target for developing novel therapeutic agents for many types of wounds including chronic wounds. TGF-2 is produced at the wound site and is responsible for recruiting inflammatory cells and fibroblasts to the wound site, inhibiting epithelial cell growth (wound re-epithelialization) and stimulating extracellular matrix synthesis by fibroblasts (fibrosis) at the wound site. In prior studies, we developed a synthetic TGF-2 peptide antagonist (termed TGF-2 peptantagonist) which is the only known synthetic TGF-2 receptor antagonist. TGF-2 peptantagonist can enhance wound healing and reduce scarring in pig skin burn/excision and rabbit skin excision wound models. However, the efficacy of the synthetic TGF-2 peptantagonist is limited by its poor solubility in aqueous solution. This project aims at developing new chemical forms of our TGF-2 peptantagonist with excellent solubility, high tissue penetration ability and potent TGF-2 antagonist activity as drug candidates for treating wounds in humans. In Phase I studies, we have developed two new TGF-2 peptantagonists with rationally engineered properties exhibit high solubility in aqueous solution and are 10-times more potent than the prototype (unmodified) TGF-2 peptantagonist in inhibiting TGF-2 activity in vitro. One of these two new TGF-2 peptantagonists has been shown to potently prevent tissue injury, accelerate wound healing and reduce scarring in several animal injury models. These promising results have provided rationales for the proposed studies in the Phase II project. We hope the clinical availability of these two novel TGF-2 peptantagonists will be commercialized by large pharmaceutical companies, eventually directly benefiting individuals with various types of wounds including normal, impaired and chronic wounds. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop synthetic TGF-2 antagonists into wound healing agents for accelerating wound healing and reducing scarring. The clinical availability of these agents would benefit millions of patients who suffer from various types of wounds including normal, impaired and chronic wounds.


Patent
Washington University in St. Louis and Auxagen, Inc. | Date: 2014-03-13

The present invention relates to adjuvant compositions, vaccine compositions, and methods of enhancing an immune response to an antigen.


PubMed | Auxagen, Inc., University of Missouri-St. Louis, Dana-Farber Cancer Institute and National Sun Yat - sen University
Type: Journal Article | Journal: Journal of cellular biochemistry | Year: 2016

Regular consumption of moderate amounts of ethanol has important health benefits on atherosclerotic cardiovascular disease (ASCVD). Overindulgence can cause many diseases, particularly alcoholic liver disease (ALD). The mechanisms by which ethanol causes both beneficial and harmful effects on human health are poorly understood. Here we demonstrate that ethanol enhances TGF--stimulated luciferase activity with a maximum of 0.5-1% (v/v) in Mv1Lu cells stably expressing a luciferase reporter gene containing Smad2-dependent elements. In Mv1Lu cells, 0.5% ethanol increases the level of P-Smad2, a canonical TGF- signaling sensor, by 2-3-fold. Ethanol (0.5%) increases cell-surface expression of the type II TGF- receptor (TR-II) by 2-3-fold from its intracellular pool, as determined by I(125) -TGF--cross-linking/Western blot analysis. Sucrose density gradient ultracentrifugation and indirect immunofluorescence staining analyses reveal that ethanol (0.5% and 1%) also displaces cell-surface TR-I and TR-II from lipid rafts/caveolae and facilitates translocation of these receptors to non-lipid raft microdomains where canonical signaling occurs. These results suggest that ethanol enhances canonical TGF- signaling by increasing non-lipid raft microdomain localization of the TGF- receptors. Since TGF- plays a protective role in ASCVD but can also cause ALD, the TGF- enhancer activity of ethanol at low and high doses appears to be responsible for both beneficial and harmful effects. Ethanol also disrupts the location of lipid raft/caveolae of other membrane proteins (e.g., neurotransmitter, growth factor/cytokine, and G protein-coupled receptors) which utilize lipid rafts/caveolae as signaling platforms. Displacement of these membrane proteins induced by ethanol may result in a variety of pathologies in nerve, heart and other tissues.


PubMed | Auxagen, Inc., University of Missouri-St. Louis, Institute for Drug Evaluation Platform and National Sun Yat - sen University
Type: | Journal: Journal of cellular biochemistry | Year: 2016

For several decades, cholesterol has been thought to cause ASCVD. Limiting dietary cholesterol intake has been recommended to reduce the risk of the disease. However, several recent epidemiological studies do not support a relationship between dietary cholesterol and/or blood cholesterol and ASCVD. Consequently, the role of cholesterol in atherogenesis is now uncertain. Much evidence indicates that TGF-, an anti-inflammatory cytokine, protects against ASCVD and that suppression of canonical TGF- signaling (Smad2-dependent) is involved in atherogenesis. We had hypothesized that cholesterol causes ASCVD by suppressing canonical TGF- signaling in vascular endothelium. To test this hypothesis, we determine the effects of cholesterol, 7-dehydrocholesterol (7-DHC; the biosynthetic precursor of cholesterol), and other sterols on canonical TGF- signaling. We use Mv1Lu cells (a model cell system for studying TGF- activity) stably expressing the Smad2-dependent luciferase reporter gene. We demonstrate that 7-DHC (but not cholesterol or other sterols) effectively suppresses the TGF--stimulated luciferase activity. We also demonstrate that 7-DHC suppresses TGF--stimulated luciferase activity by promoting lipid raft/caveolae formation and subsequently recruiting cell-surface TGF- receptors from non-lipid raft microdomains to lipid rafts/caveolae where TGF- receptors become inactive in transducing canonical signaling and undergo rapid degradation upon TGF- binding. We determine this by cell-surface


PubMed | Auxagen, Inc., University of Missouri-St. Louis, Dana-Farber Cancer Institute and National Sun Yat - sen University
Type: Journal Article | Journal: Journal of cellular biochemistry | Year: 2016

Dimethyl sulfoxide (DMSO) is used to treat many diseases/symptoms. The molecular basis of the pharmacological actions of DMSO has been unclear. We hypothesized that DMSO exerts some of these actions by enhancing TGF- activity. Here we show that DMSO enhances TGF- activity by 3-4-fold in Mv1Lu and NMuMG cells expressing Smad-dependent luciferase reporters. In Mv1Lu cells, DMSO enhances TGF--stimulated expression of P-Smad2 and PAI-1. It increases cell-surface expression of TGF- receptors (TR-I and/or TR-II) by 3-4-fold without altering their cellular levels as determined by (125) I-labeled TGF--cross-linking/Western blot analysis, suggesting the presence of large intracellular pools in these cells. Sucrose density gradient ultracentrifugation/Western blot analysis reveals that DMSO induces recruitment of TR-II (but not TR-I) from its intracellular pool to plasma-membrane microdomains. It induces more recruitment of TR-II to non-lipid raft microdomains than to lipid rafts/caveolae. Mv1Lu cells transiently transfected with TR-II-HA plasmid were treated with DMSO and analyzed by indirect immunofluoresence staining using anti-HA antibody. In these cells, TR-II-HA is present as a vesicle-like network in the cytoplasm as well as in the plasma membrane. DMSO causes depletion of TR-II-HA-containing vesicles from the cytoplasm and co-localization of TR-II-HA and cveolin-1 at the plasma membrane. These results suggest that DMSO, a fusogenic substance, enhances TGF- activity presumably by inducing fusion of cytoplasmic vesicles (containing TR-II) and the plasma membrane, resulting in increased localization of TR-II to non-lipid raft microdomains where canonical signaling occurs. Fusogenic activity of DMSO may play a pivotal role in its pharmacological actions involving membrane proteins with large cytoplasmic pools. J. Cell. Biochem. 117: 1568-1579, 2016. 2015 Wiley Periodicals, Inc.

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