Platelet-activating factor (PAF) activated phospholipase D (PLD) in WT-H cells, CHO cells stably expressing cloned guinea-pig PAF receptor. The PLD activation was found to be dependent on extracellular Ca2+, protein kinase C (PKC), and a currently unidentified protein tyrosine kinase (PTK). PTK inhibitors ST-638 and genistein inhibited PLD activation induced by PAF as well as phorbol myristate acetate, indicating that PTK acts downstream of PKC. Furthermore, activation of MAP (mitogen-activated protein) kinases, as assessed by their phosphorylation, was also dependent on Ca2+, PKC, and PTK. The correlation between PLD activity and MAP kinase activation, together with the previously observed MAP kinase activation associated with arachidonic acid release by cPLA2 [Honda et ah (1994) J. BioL Chem. 269, 2307–2315], led us to examine the involvement of MAP kinase in PLD activation. The results indicate that PLD and MAP kinases are activated through the common pathway consisting of Ca2+, PKC, and the unidentified PTK, which act in parallel, but not in a linear sequence.
Abstract: In the present study, an activation mechanism for phospholipase D (PLD) in [ 3 H]palmitic acid‐labeled pheochromocytoma PC12 cells in response to carbachol (CCh) was investigated. PLD activity was assessed by measuring the formation of [ 3 H]phosphatidylethanol ([ 3 H]PEt), the specific marker of PLD activity, in the presence of 0.5% (vol/vol) ethanol. CCh caused a rapid accumulation of [ 3 H]PEt, which reached a plateau within 1 min, in a concentration‐dependent manner. The [ 3 H]PEt formation by CCh was completely antagonized by atropine, demonstrating that the CCh effect was mediated by the muscarinic acetylcholine receptor (mAChR). A tumor promoter, phorbol 12‐myristate 13‐acetate (PMA), also caused an increase in [ 3 H]PEt content, which reached a plateau at 30–60 min after exposure, but an inactive phorbol ester, 4a‐phorbol 12,13‐didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 μM), blocked PMA‐induced [ 3 H]PEt formation by 77%, it had no effect on the CCh‐induced formation. These results suggest that mAChR‐induced PLD activation is independent of PKC, whereas PLD activation by PMA is mediated by PKC. NaF, a common GTP‐binding protein (G protein) activator, and a stable analogue of GTP, guanosine 5′‐O‐(3‐thiotriphosphate) (OTPGmS), also stimulated [ 3 H]PEt formation in intact and digitonin‐permeabilized cells, respectively. GTP, UTP, and CTP were without effect. Furthermore, guanosine 5′‐O‐(2‐thiodiphosphate) significantly inhibited CCh‐ and GTPΓS‐ induced [ 3 H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 μM) had no effect on [ 3 H]PEt formation by GTPγS. Pretreatment of cells with pertussis toxin (10–200 ng/ml) for 15 h failed to suppress CCh‐induced [ 3 H]PEt formation, although the pertussis toxin‐sensitive G protein(s) in membranes was completely ADP‐ribosylated under the same conditions. From these results, we conclude that the mechanisms of PMA‐ and CCh‐stimulated PLD activation are different from each other and that CCh‐induced PLD activation is independent of PKC and mediated, at least in part, via a pertussis toxin‐insensitive G protein.
The hyaluronan (HA)–rich extracellular matrix plays dynamic roles during tissue remodeling. Versican and serum-derived HA-associated protein (SHAP), corresponding to the heavy chains of inter-α-trypsin inhibitor, are major HA-binding molecules in remodeling processes, such as wound healing. Versican G1-domain fragment (VG1F) is generated by proteolysis and is present in either remodeling tissues or the mature dermis. However, the macrocomplex formation of VG1F has not been clarified. Therefore, we examined the VG1F-containing macrocomplex in pressure ulcers characterized by chronic refractory wounds. VG1F colocalized with SHAP-HA in specific regions of the granulation tissue but not with fibrillin-1. A unique VG1F-SHAP-HA complex was isolated from granulation tissues using gel filtration chromatography and subsequent cesium chloride–gradient ultracentrifugation under dissociating conditions. Consistent with this molecular composition, recombinant versican G1, but not versican G3, interacted with the two heavy chains of inter-α-trypsin inhibitor. The addition of recombinant VG1 in fibroblast cultures enhanced VG1F-SHAP-HA complex deposition in the pericellular extracellular matrix. Comparison with other VG1F-containing macrocomplexes, including dermal VG1F aggregates, versican-bound microfibrils, and intact versican, highlighted the tissue-specific organization of HA-rich extracellular matrix formation containing versican and SHAP. The VG1F-SHAP-HA complex was specifically detected in the edematous granulation tissues of human pressure ulcers and in inflamed stages in a mouse model of moist would healing, suggesting that the complex provides an HA-rich matrix suitable for inflammatory reactions. The hyaluronan (HA)–rich extracellular matrix plays dynamic roles during tissue remodeling. Versican and serum-derived HA-associated protein (SHAP), corresponding to the heavy chains of inter-α-trypsin inhibitor, are major HA-binding molecules in remodeling processes, such as wound healing. Versican G1-domain fragment (VG1F) is generated by proteolysis and is present in either remodeling tissues or the mature dermis. However, the macrocomplex formation of VG1F has not been clarified. Therefore, we examined the VG1F-containing macrocomplex in pressure ulcers characterized by chronic refractory wounds. VG1F colocalized with SHAP-HA in specific regions of the granulation tissue but not with fibrillin-1. A unique VG1F-SHAP-HA complex was isolated from granulation tissues using gel filtration chromatography and subsequent cesium chloride–gradient ultracentrifugation under dissociating conditions. Consistent with this molecular composition, recombinant versican G1, but not versican G3, interacted with the two heavy chains of inter-α-trypsin inhibitor. The addition of recombinant VG1 in fibroblast cultures enhanced VG1F-SHAP-HA complex deposition in the pericellular extracellular matrix. Comparison with other VG1F-containing macrocomplexes, including dermal VG1F aggregates, versican-bound microfibrils, and intact versican, highlighted the tissue-specific organization of HA-rich extracellular matrix formation containing versican and SHAP. The VG1F-SHAP-HA complex was specifically detected in the edematous granulation tissues of human pressure ulcers and in inflamed stages in a mouse model of moist would healing, suggesting that the complex provides an HA-rich matrix suitable for inflammatory reactions. Wound healing is a regulated process that occurs in four successive stages, as follows: hemostasis, inflammation, proliferation/granulation, and remodeling/maturation. During the healing of deep skin ulcers, granulation tissue transiently forms, characterized by angiogenesis and inflammatory cell infiltration; this process is essential for the subsequent remodeling process.1Eming S.A. Krieg T. Davidson J.M. Inflammation in wound repair: molecular and cellular mechanisms.J Invest Dermatol. 2007; 127: 514-525Abstract Full Text Full Text PDF PubMed Scopus (1437) Google Scholar, 2Moali C. Hulmes D.J.S. Extracellular and cell surface proteases in wound healing: new players are still emerging.Eur J Dermatol. 2009; 19: 552-564Crossref PubMed Scopus (59) Google Scholar, 3Eming S.A. Martin P. Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation.Sci Transl Med. 2014; 6: 1-16Crossref Scopus (1523) Google Scholar A pressure ulcer is a common type of chronic ulcer and a refractory wound often observed in elderly patients.3Eming S.A. Martin P. Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation.Sci Transl Med. 2014; 6: 1-16Crossref Scopus (1523) Google Scholar, 4Vande Berg J.S. Rudolph R. Pressure (decubitus) ulcer: variation in histopathology—a light and electron-microscope study.Hum Pathol. 1995; 26: 195-200Abstract Full Text PDF PubMed Scopus (44) Google Scholar Although an inflammatory stage is required for wound healing, prolonged inflammation is associated with delayed wound healing, often observed in pressure ulcers.3Eming S.A. Martin P. Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation.Sci Transl Med. 2014; 6: 1-16Crossref Scopus (1523) Google Scholar Moreover, these lesions exhibit various pathological features, such as angiogenesis and edema in stromal tissues,4Vande Berg J.S. Rudolph R. Pressure (decubitus) ulcer: variation in histopathology—a light and electron-microscope study.Hum Pathol. 1995; 26: 195-200Abstract Full Text PDF PubMed Scopus (44) Google Scholar indicative of the variable inflammatory conditions during the healing process. Therefore, the spatiotemporal regulation of the inflammatory response is critical for optimal wound healing in pressure ulcers. The hyaluronan (HA)–rich extracellular matrix (ECM) is characteristically observed in remodeling tissues, such as cancer stroma and wounds.5Yeo T.K. Brown L. Dvorak H.F. Alterations in proteoglycan synthesis common to healing wounds and tumors.Am J Pathol. 1991; 138: 1437-1450PubMed Google Scholar, 6Frenkel J.S. The role of hyaluronan in wound healing.Int Wound J. 2014; 11: 159-163Crossref PubMed Scopus (93) Google Scholar, 7Petrey A.C. de la Motte C.A. Hyaluronan, a crucial regulator of inflammation.Front Immunol. 2014; 5: 101Crossref PubMed Scopus (302) Google Scholar, 8Oksala O. Salo T. Tammi R. Hakkinen L. Jalkanen M. Inki P. Larjava H. Expression of proteoglycans and hyaluronan during wound-healing.J Histochem Cytochem. 1995; 43: 125-135Crossref PubMed Scopus (227) Google Scholar The localization of HA in the ECM is mediated by HA-binding molecules.9Day A.J. Prestwich G.D. Hyaluronan-binding proteins.J Biol Chem. 2002; 277: 4575-4579Crossref PubMed Scopus (47) Google Scholar Among them, versican, a large chondroitin sulfate proteoglycan, is a major HA-binding molecule in dermal connective tissue.10Hasegawa K. Yoneda M. Kuwabara H. Miyaishi O. Itano N. Ohno A. Zako M. Isogai Z. Versican, a major hyaluronan-binding component in the dermis, loses its hyaluronan-binding ability in solar elastosis.J Invest Dermatol. 2007; 127: 1657-1663Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar In stable tissues, such as the mature dermis, versican localizes to fibrillin-microfibrils by binding fibrillin-1, which serves as a structural molecule.10Hasegawa K. Yoneda M. Kuwabara H. Miyaishi O. Itano N. Ohno A. Zako M. Isogai Z. Versican, a major hyaluronan-binding component in the dermis, loses its hyaluronan-binding ability in solar elastosis.J Invest Dermatol. 2007; 127: 1657-1663Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 11Zimmermann D.R. Dours-Zimmermann M.T. Schubert M. Bruckner-Tuderman L. Versican is expressed in the proliferating zone in the epidermis and in association with the elastic network of the dermis.J Cell Biol. 1994; 124: 817-825Crossref PubMed Scopus (213) Google Scholar, 12Bode-Lesniewska B. Dours-Zimmermann M.T. Odermatt B.F. Briner J. Heitz P.U. Zimmermann D.R. Distribution of the large aggregating proteoglycan versican in adult human tissues.J Histochem Cytochem. 1996; 44: 303-312Crossref PubMed Scopus (162) Google Scholar, 13Isogai Z. Aspberg A. Keene D.R. Ono R.N. Reinhardt D.P. Sakai L.Y. Versican interacts with fibrillin-1 and links extracellular microfibrils to other connective tissue networks.J Biol Chem. 2002; 277: 4565-4572Crossref PubMed Scopus (171) Google Scholar, 14Murasawa Y. Watanabe K. Yoneda M. Zako M. Kimata K. Sakai L.Y. Isogai Z. Homotypic versican g1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.J Biol Chem. 2013; 288: 29170-29181Crossref PubMed Scopus (14) Google Scholar Localization is independent of elastic fibers in cancer and developing tissues.5Yeo T.K. Brown L. Dvorak H.F. Alterations in proteoglycan synthesis common to healing wounds and tumors.Am J Pathol. 1991; 138: 1437-1450PubMed Google Scholar, 15Yoneda M, Zhao M, Zhuo LS, Watanabe H, Yamada Y, Huang L, Nagasawa S, Nishimura H, Shinomura T, Isogai Z, Kimata K: Roles of inter-alpha-trypsin inhibitor and hyaluronan-binding proteoglycans in hyaluronan-rich matrix formation. New Frontiers in Medical Sciences: Redefining Hyaluronan. Edited by Abatangelo G, Weigel PH. Amsterdam: Elsevier Science, 2001. pp. 21–30Google Scholar, 16Koyama H. Hibi T. Isogai Z. Yoneda M. Fujimori M. Amano J. Kawakubo M. Kannagi R. Kimata K. Taniguchi S.I. Itano N. Hyperproduction of hyaluronan in Neu-induced mammary tumor accelerates angiogenesis through stromal cell recruitment: possible involvement of versican/PG-M.Am J Pathol. 2007; 170: 1086-1099Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 17Takahashi Y. Kuwabara H. Yoneda M. Isogai Z. Tanigawa N. Shibayama Y. Versican G1 and G3 domains are upregulated and latent transforming growth factor-beta binding protein-4 is downregulated in breast cancer stroma.Breast Cancer. 2012; 19: 46-53Crossref PubMed Scopus (16) Google Scholar These findings suggest that versican plays tissue- or stage-specific roles through its distinct ECM formation. Members of the a disintegrin-like and metalloproteinase with thrombospondin motif (ADAMTS) family of proteases, particularly ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-9, and ADAMTS-15, cleave versican at a specific site and are, therefore, classified as versicanases.18Apte S.S. A disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif (ADAMTS) superfamily: functions and mechanisms.J Biol Chem. 2009; 284: 31493-31497Crossref PubMed Scopus (375) Google Scholar Versican G1 domain–containing fragments (VG1Fs) are generated by these proteases and present in mature tissues, such as the dermis and aorta.14Murasawa Y. Watanabe K. Yoneda M. Zako M. Kimata K. Sakai L.Y. Isogai Z. Homotypic versican g1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.J Biol Chem. 2013; 288: 29170-29181Crossref PubMed Scopus (14) Google Scholar, 19Sorrell J.M. Carrino D.A. Baber M.A. Caplan A.I. Versican in human fetal skin development.Anat Embryol. 1999; 199: 45-56Crossref PubMed Scopus (42) Google Scholar, 20Sandy J.D. Westling J. Kenagy R.D. Iruela-Arispe M.L. Verscharen C. Rodriguez-Mazaneque J.C. Zimmermann D.R. Lemire J.M. Fischer J.W. Wight T.N. Clowes A.W. Versican V1 proteolysis in human aorta in vivo occurs at the Glu(441)-Ala(442) bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4.J Biol Chem. 2001; 276: 13372-13378Crossref PubMed Scopus (376) Google Scholar, 21Carrino D.A. Calabro A. Darr A.B. Dours-Zimmermann M.T. Sandy J.D. Zimmermann D.R. Sorrell J.M. Hascall V.C. Caplan A.I. Age-related differences in human skin proteoglycans.Glycobiology. 2011; 21: 257-268Crossref PubMed Scopus (45) Google Scholar VG1F forms aggregates in the dermis and recruits increased levels of HA onto microfibrils through its homotypic and HA-binding activity.14Murasawa Y. Watanabe K. Yoneda M. Zako M. Kimata K. Sakai L.Y. Isogai Z. Homotypic versican g1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.J Biol Chem. 2013; 288: 29170-29181Crossref PubMed Scopus (14) Google Scholar On the other hand, VG1F also induces apoptosis and angiogenesis through its biological activities in tissue remodeling and development.22McCulloch D.R. Nelson C.M. Dixon L.J. Silver D.L. Wylie J.D. Lindner V. Sasaki T. Cooley M.A. Argraves W.S. Apte S.S. ADAMTS metalloproteases generate active versican fragments that regulate interdigital web regression.Dev Cell. 2009; 17: 687-698Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 23Enomoto H. Nelson C.M. Somerville R.P.T. Mielke K. Dixon L.J. Powell K. Apte S.S. Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation.Development. 2010; 137: 4029-4038Crossref PubMed Scopus (107) Google Scholar However, the mechanisms of these tissue-dependent VG1F functions are not fully understood. Another HA-binding molecule, inter-α-trypsin inhibitor (IαI), covalently binds HA to generate a serum-derived HA-associated protein (SHAP)–HA complex.24Yoneda M. Suzuki S. Kimata K. Hyaluronic-acid associated with the surfaces of cultured fibroblasts is linked to a serum-derived 85-kda protein.J Biol Chem. 1990; 265: 5247-5257PubMed Google Scholar, 25Huang L. Yoneda M. Kimata K. A serum-derived hyaluronan-associated protein (SHAP) is the heavy-chain of the inter-alpha-trypsin inhibitor.J Biol Chem. 1993; 268: 26725-26730PubMed Google Scholar SHAP consists of IαI heavy chains (HCs) that bind HA via an ester linkage.25Huang L. Yoneda M. Kimata K. A serum-derived hyaluronan-associated protein (SHAP) is the heavy-chain of the inter-alpha-trypsin inhibitor.J Biol Chem. 1993; 268: 26725-26730PubMed Google Scholar, 26Zhuo L.S. Hascall V.C. Kimata K. Inter-alpha-trypsin inhibitor, a covalent protein-glycosaminoglycan-protein complex.J Biol Chem. 2004; 279: 38079-38082Crossref PubMed Scopus (178) Google Scholar, 27Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. Evidence for the covalent binding of SHAP, heavy-chains of inter-alpha-trypsin inhibitor, to hyaluronan.J Biol Chem. 1995; 270: 26657-26663Crossref PubMed Scopus (149) Google Scholar Notably, SHAP-HA is found in the synovial fluid of patients with rheumatoid arthritis who display prolonged inflammatory reactions in their joints,28Kida D. Yoneda M. Miyaura S. Ishimaru T. Yoshida Y. Ito T. Ishiguro N. Iwata H. Kimata K. The SHAP-HA complex in sera from patients with rheumatoid arthritis and osteoarthritis.J Rheumatol. 1999; 26: 1230-1238PubMed Google Scholar, 29Yingsung W. Zhuo L.S. Mörgelin M. Yoneda M. Kida D. Watanabe H. Ishiguro N. Iwata H. Kimata K. Molecular heterogeneity of the SHAP-hyaluronan complex: isolation and characterization of the complex in synovial fluid from patients with rheumatoid arthritis.J Biol Chem. 2003; 278: 32710-32718Crossref PubMed Scopus (93) Google Scholar the inflamed tissues of patients with inflammatory bowel disease,30de la Motte C.A. Hascall V.C. Drazba J. Bandyopadhyay S.K. Strong S.A. Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid:polycytidylic acid: inter-alpha-trypsin inhibitor is crucial to structure and function.Am J Pathol. 2003; 163: 121-133Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar and experimental tooth infections in canines,31Eba H. Murasawa Y. Iohara K. Isogai Z. Nakamura H. Nakamura H. Nakashima M. The anti-inflammatory effects of matrix metalloproteinase-3 on irreversible pulpitis of mature erupted teeth.PLoS One. 2012; 7: e52523Crossref PubMed Scopus (43) Google Scholar indicating its critical role in inflammation. The present study clarified the tissue-specific transient formation of a unique VG1F-SHAP-HA macrocomplex in the granulation tissue of pressure ulcers. These findings may provide new insights into the role of HA-versican–rich ECM in impaired tissue remodeling processes, such as pressure ulcers. Polyclonal antibodies (pAbs) against the VG1 domain, pAb 6084 and pAb 7080 (an antibody for synthetic peptide), have been described previously.10Hasegawa K. Yoneda M. Kuwabara H. Miyaishi O. Itano N. Ohno A. Zako M. Isogai Z. Versican, a major hyaluronan-binding component in the dermis, loses its hyaluronan-binding ability in solar elastosis.J Invest Dermatol. 2007; 127: 1657-1663Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 14Murasawa Y. Watanabe K. Yoneda M. Zako M. Kimata K. Sakai L.Y. Isogai Z. Homotypic versican g1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.J Biol Chem. 2013; 288: 29170-29181Crossref PubMed Scopus (14) Google Scholar, 32Ohno-Jinno A. Isogai Z. Yoneda M. Kasai K. Miyaishi O. Inoue Y. Kataoka T. Zhao J.S. Li H. Takeyama M. Keene D.R. Sakai L.Y. Kimata K. Iwaki M. Zako M. Versican and fibrillin-1 form a major hyaluronan-binding complex in the ciliary body.Invest Ophthalmol Vis Sci. 2008; 49: 2870-2877Crossref PubMed Scopus (22) Google Scholar The monoclonal antibody 2B1 against the human versican G3 (VG3) domain was acquired from Seikagaku Kogyo (Tokyo, Japan). An antibody recognizing the DPEAAE neoepitope generated by ADAMTS proteolysis of versican (pAb 8531) was characterized as almost equivalent to the commercially available antibody in our previous study.14Murasawa Y. Watanabe K. Yoneda M. Zako M. Kimata K. Sakai L.Y. Isogai Z. Homotypic versican g1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.J Biol Chem. 2013; 288: 29170-29181Crossref PubMed Scopus (14) Google Scholar Rabbit anti-mouse versican pAb recognizing the GAGβ domain (AB1033) was purchased from Millipore-Chemicon (Temecula, CA). pAb against a synthetic peptide (A66QNGN IKIGQDYKGR VSVP84) for the human versican A subdomain of human versican (V1; https://www.ncbi.nlm.nih.gov/protein; accession number NP_001157569) was generated by Operon Biotechnology (Tokyo, Japan). Its specificity was confirmed by Western blotting using conditioned media from normal dermal fibroblasts treated with chondroitinase ABC as an immobilized ligand (pAb 10693) (Supplemental Figure S1). Rabbit anti-mouse versican polyclonal antibody against the GAGα domain was raised against a polypeptide representing D380 to E957 of mouse versican V0 (https://www.ncbi.nlm.nih.gov/protein; accession number NP_001074718) and has been characterized previously.33Matsumoto K. Kamiya N. Suwan K. Atsumi F. Shimizu K. Shinomura T. Yamada Y. Kimata K. Watanabe H. Identification and characterization of versican/PG-M aggregates in cartilage.J Biol Chem. 2006; 281: 18257-18263Crossref PubMed Scopus (52) Google Scholar The recognition sites for pAb 6084, pAb 7080, pAb 10693, pAb 8531, AB1033, and monoclonal antibody 2B1 are shown in Figure 1. pAb 9543 (a gift from Dr. Lynn Sakai, Shriners Hospital, Portland, OR) was used as an anti–fibrillin-1 antibody. A goat pAb (clone AF2104) against tumor necrosis factor–stimulated gene-6 (TSG-6; clone AF2104) was purchased from R&D Systems (Minneapolis, MN). A goat antibody against synthetic peptide of TSG-6 (N-20, sc-21828) and a rat monoclonal antibody against TSG-6 (Q75.2.10, sc-65889) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX). A rabbit pAb against hyaluronan and proteoglycan binding link protein gene family (HAPLN)-1 (link protein, clone H93, sc-135184) and goat pAb against HAPLN-1 (K14, sc-46826; and C-14, sc-46825) were purchased from Santa Cruz Biotechnology Inc. A rabbit pAb against pentraxin 3 (13797-1-AP) was purchased from Proteintech (Rosemont, IL). A rabbit pAb against amino acid 865 to 894 of human HC2 of ITIH2 (LS-C165332) and a rabbit pAb against HC1 of human ITIH1 (LS-C121083) were purchased from LSBio (Seattle, WA). Mouse pAb against HC1 of human ITIH1 (10R-8431) was purchased from Fitzgerald Industries International, Inc. (Acton, MA). A rabbit pAb that recognizes IαI (all HCs) was purchased from Sigma-Aldrich (St. Louis, MO; anti-IαI antibodies). A goat pAb against bikunin (sc-21597) was purchased from Santa Cruz Biotechnology Inc. A mouse monoclonal antibody against the macrophage marker CD68 (STJ-6572) was purchased from St. John's Laboratory Ltd (London, UK). For the detection of HA, biotin-conjugated hyaluronan-binding protein (bHABP; Seikagaku Kogyo) was used. The following antibodies were used to detect antigen-antibody complexes: horseradish peroxidase–conjugated anti-rabbit IgG (Dako, Glostrup, Denmark), horseradish peroxidase–conjugated anti-HIS antibody (Invitrogen, Carlsbad, CA), Alexa Fluor 633–conjugated streptavidin (Invitrogen), Alexa Fluor 568–conjugated goat anti-mouse IgG (Invitrogen), Alexa Fluor 488–conjugated goat anti-rabbit IgG (Invitrogen), and 5-nm gold particle–conjugated goat anti-rabbit IgG (GE Healthcare, Little Chalfont, UK). Recombinant versican polypeptides spanning the globular domains rVN (for VG1) and rVC (for VG3) were expressed by mammalian cells and characterized as described.10Hasegawa K. Yoneda M. Kuwabara H. Miyaishi O. Itano N. Ohno A. Zako M. Isogai Z. Versican, a major hyaluronan-binding component in the dermis, loses its hyaluronan-binding ability in solar elastosis.J Invest Dermatol. 2007; 127: 1657-1663Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 14Murasawa Y. Watanabe K. Yoneda M. Zako M. Kimata K. Sakai L.Y. Isogai Z. Homotypic versican g1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.J Biol Chem. 2013; 288: 29170-29181Crossref PubMed Scopus (14) Google Scholar IαI was purified from bovine serum using heparin affinity chromatography, as described.34Salier J.P. Martin J.P. Lambin P. McPhee H. Hochstrasser K. Purification of the human-serum inter-alpha-trypsin inhibitor by zinc chelate and hydrophobic interaction chromatographies.Anal Biochem. 1980; 109: 273-283Crossref PubMed Scopus (38) Google Scholar Recombinant human HAPLN1 (2608-HP-025) and human TSG-6 (2104-TS-050) proteins were obtained from R&D Systems. A recombinant pentraxin 3 protein was purchased from Thermo Fischer Scientific (Yokohama, Japan). To prepare the SHAP-HA complex, HA (high molecular weight; Seikagaku Kogyo) was incubated with fetal bovine serum (HyClone, South Logan, UT) and then isolated using ultracentrifugation containing cesium chloride, as described.24Yoneda M. Suzuki S. Kimata K. Hyaluronic-acid associated with the surfaces of cultured fibroblasts is linked to a serum-derived 85-kda protein.J Biol Chem. 1990; 265: 5247-5257PubMed Google Scholar, 25Huang L. Yoneda M. Kimata K. A serum-derived hyaluronan-associated protein (SHAP) is the heavy-chain of the inter-alpha-trypsin inhibitor.J Biol Chem. 1993; 268: 26725-26730PubMed Google Scholar Monomeric versican was purified from normal human fibroblast-conditioned media by using sequential anion-exchange chromatography and ultracentrifugation with cesium chloride under dissociative conditions.35Isogai Z. Shinomura T. Yamakawa N. Takeuchi J. Tsuji T. Heinegård D. Kimata K. 2B1 antigen characteristically expressed on extracellular matrices of human malignant tumors is a large chondroitin sulfate proteoglycan, PG-M/versican.Cancer Res. 1996; 56: 3902-3908PubMed Google Scholar After written informed consent was obtained from the patients, wound surface protein samples from two distinct sites of four pressure ulcers were obtained using disposable plastic tongue depressors (ORAX; Emikon, Tokyo, Japan) or cotton swabs (Nihon Menbou, Tokyo, Japan) for wound smear experiments. The samples were smeared onto glass slides (Platinum; Matsunami, Osaka, Japan), air dried for 60 minutes, fixed with 10% (v/v) formalin (Wako, Osaka, Japan) in phosphate-buffered saline (PBS), and blocked with 50% Starting Block T20 Blocking Buffer (Thermo Fischer Scientific) in PBS containing 0.1% Tween-20. This study was approved by the local institutional ethics committees of the National Center for Geriatrics and Gerontology (Obu, Japan). For immunohistochemistry, formalin-fixed, paraffin-embedded sections of pressure ulcers obtained at surgical excision were prepared (n = 6). Deparaffinized sections (6 μm thick) on glass slides were treated with 0.1% saponin for 15 minutes at 22°C before blocking, and the sections were blocked with blocking buffer for 60 minutes at room temperature. Then, the sections were incubated with primary antibodies in blocking buffer for 60 minutes at room temperature, washed three times in PBS containing 0.1% Tween-20 for 10 minutes at room temperature, incubated with secondary antibodies for 60 minutes at room temperature, and washed three times in PBS containing 0.1% Tween-20 for 10 minutes. For dual immunofluorescence, tissue sections were incubated with pAb 6084, and bound antibodies were detected by using Alexa Fluor 633–conjugated anti-rabbit IgG. Probed sections were subsequently incubated with pAb 8531 preconjugated with HiLyte Fluor 555 (HiLyte Fluor 555 Labeling Kit-NH2; Dojinbo, Kumamoto, Japan) for another 60 minutes at room temperature. Sections were submerged in 20 μL of Fluoromount (Diagnostic BioSystems, Pleasanton, CA), mounted with Mount-Quick (Daido Sangyo Co, Ltd, Saitama, Japan), and then sealed with a cover glass (Iwaki, Shizuoka, Japan). This part of the study was approved by the local institutional ethics committees of Kurashiki Heisei Hospital (Kurashiki, Japan). Tissue sections were visualized using a confocal laser microscope (Carl Zeiss LSM700 or V2URGB Stitch; Carl Zeiss, Oberkochen, Germany) that excluded nonspecific fluorescence. All processes were performed as described previously.14Murasawa Y. Watanabe K. Yoneda M. Zako M. Kimata K. Sakai L.Y. Isogai Z. Homotypic versican g1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.J Biol Chem. 2013; 288: 29170-29181Crossref PubMed Scopus (14) Google Scholar Briefly, sequential dual-color images were captured, and negative controls were scanned before sample analysis. Nonspecific fluorescence was not observed in negative controls (absence of primary antibodies) using 10% laser power, 650 gain, and −0.50 offset. Subsequent samples were scanned using these standard conditions. All images were acquired using a 63× oil-immersion objective. Scanning was performed with a pinhole size of 1.0 Airy unit and eight-time line averaging. The images were stored in a 512 × 512-pixel, 12-bit tagged image file format. Wound tissues were obtained from distinct pressure ulcers using protocols approved by the ethics committees of Toki Municipal Hospital (Toki, Japan); patients provided written informed consent (n = 2). Tissue samples attached to gauze for approximately 24 hours were extracted for 72 hours at 4°C with 6 mol/L guanidine hydrochloride (GdnHCl), 50 mmol/L Tris-HCl, 1 mmol/L phenylmethylsulfonyl fluoride, and 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich), pH 7.5. Extracts were concentrated using an Amicon Ultra-4 centrifugal filter unit (50-kDa cutoff; Millipore, Burlington, MA) and subjected to molecular-sieve chromatography using Sepharose CL-2B (GE Healthcare) equilibrated and eluted with 4 mol/L Gdn, and 50 mmol/L Tris-HCl, pH 7.5, as described.14Murasawa Y. Watanabe K. Yoneda M. Zako M. Kimata K. Sakai L.Y. Isogai Z. Homotypic versican g1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.J Biol Chem. 2013; 288: 29170-29181Crossref PubMed Scopus (14) Google Scholar For further analysis, cesium chloride was added to the void volume fractions, which were then centrifuged at 140,000 × g for 48 hours at 10°C. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce/Thermo Fisher, Waltham, MA) with bovine serum albumin as the standard. Dot blot analyses were performed using 5 μL of sample, as described previously.14Murasawa Y. Watanabe K. Yoneda M. Zako M. Kimata K. Sakai L.Y. Isogai Z. Homotypic versican g1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.J Biol Chem. 2013; 288: 29170-29181Crossref PubMed Scopus (14) Google Scholar VG1F-containing aggregates from the normal dermis were characterized in our previous study.14Murasawa Y. Watanabe K. Yoneda M. Zako M. Kimata K. Sakai L.Y. Isogai Z. Homotypic versican g1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.J Biol Chem. 2013; 288: 29170-29181Crossref PubMed Scopus (14) Google Scholar Samples from the wound surfaces of separate pressure ulcers (n = 4) were processed using identical procedures to isolate VG1F-containing aggregates. Cesium chloride–gradient fractions containing VG1F aggregates from wounds were dialyzed three times against water at 4°C, rotary shadowed, and observed using the electron microscope at Hanaichi Electron Microscope (Okazaki, Japan). The fractions were spread onto glass slides (Matsunami), air dried for 60 minutes at room temperature, fixed with 10% (v/v) formalin in PBS (Wako), and subjected to immunostaining, as described in Immunochemical Analysis of Wound Surface Samples and Tissue Sections. The slides were observed using confocal microscopy, as described above (Immunochemical Analysis of Wound Surface Samples and Tissue Sections). For biochemical analyses, isolated samples (10 μg) were treated with 0.01 mg/mL trypsin (proteomics grade, T6567; Sigma-Aldrich) in 1 mL of digestion buffer (50 mmol/L NH4HCO3, pH 8.5, and 5% acetonitrile) and 0.1 μg of V8 protease (Sigma-Aldrich) at 37°C for 12 hours.14Murasawa Y. Watanabe K. Yoneda M. Zako M. Kimata K. Sakai L.Y. Isogai Z. Homotypic versican g1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.J Biol Chem. 2013; 288: 29170-29181Crossref PubMed Scopus (14) Google Scholar Some of the samples were further treated with Streptomyces hyaluronidase in 50 mmol/L acetate buffer, su
Abstract The Japanese Dermatological Association prepared guidelines focused on the treatment of skin ulcers associated with connective tissue disease/vasculitis practical in clinical settings of dermatological care. Skin ulcers associated with connective tissue diseases or vasculitis occur on the background of a wide variety of diseases including, typically, systemic sclerosis but also systemic lupus erythematosus (SLE), dermatomyositis, rheumatoid arthritis (RA), various vasculitides and antiphospholipid antibody syndrome (APS). Therefore, in preparing the present guidelines, we considered diagnostic/therapeutic approaches appropriate for each of these disorders to be necessary and developed algorithms and clinical questions for systemic sclerosis, SLE, dermatomyositis, RA, vasculitis and APS.
