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• W2123509716 abstract "The metalloprotease ADAMTS9 participates in melanoblast development and is a tumor suppressor in esophageal and nasopharyngeal cancer. ADAMTS9 null mice die before gastrulation, but, ADAMTS9+/− mice were initially thought to be normal. However, when congenic with the C57Bl/6 strain, 80% of ADAMTS9+/− mice developed spontaneous corneal neovascularization. β-Galactosidase staining enabled by a lacZ cassette targeted to the ADAMTS9 locus showed that capillary endothelial cells (ECs) in embryonic and adult tissues and in capillaries growing into heterotopic tumors expressed ADAMTS9. Heterotopic B.16-F10 melanomas elicited greater vascular induction in ADAMTS9+/− mice than in wild-type littermates, suggesting a potential inhibitory role in tumor angiogenesis. Treatment of cultured human microvascular ECs with ADAMTS9 small-interfering RNA resulted in enhanced filopodial extension, decreased cell adhesion, increased cell migration, and enhanced formation of tube-like structures on Matrigel. Conversely, overexpression of catalytically active, but not inactive, ADAMTS9 in ECs led to fewer tube-like structures, demonstrating that the proteolytic activity of ADAMTS9 was essential. However, unlike the related metalloprotease ADAMTS1, which exerts anti-angiogenic effects by cleavage of thrombospondins and sequestration of vascular endothelial growth factor165, ADAMTS9 neither cleaved thrombospondins 1 and 2, nor bound vascular endothelial growth factor165. Taken together, these data identify ADAMTS9 as a novel, constitutive, endogenous angiogenesis inhibitor that operates cell-autonomously in ECs via molecular mechanisms that are distinct from those used by ADAMTS1. The metalloprotease ADAMTS9 participates in melanoblast development and is a tumor suppressor in esophageal and nasopharyngeal cancer. ADAMTS9 null mice die before gastrulation, but, ADAMTS9+/− mice were initially thought to be normal. However, when congenic with the C57Bl/6 strain, 80% of ADAMTS9+/− mice developed spontaneous corneal neovascularization. β-Galactosidase staining enabled by a lacZ cassette targeted to the ADAMTS9 locus showed that capillary endothelial cells (ECs) in embryonic and adult tissues and in capillaries growing into heterotopic tumors expressed ADAMTS9. Heterotopic B.16-F10 melanomas elicited greater vascular induction in ADAMTS9+/− mice than in wild-type littermates, suggesting a potential inhibitory role in tumor angiogenesis. Treatment of cultured human microvascular ECs with ADAMTS9 small-interfering RNA resulted in enhanced filopodial extension, decreased cell adhesion, increased cell migration, and enhanced formation of tube-like structures on Matrigel. Conversely, overexpression of catalytically active, but not inactive, ADAMTS9 in ECs led to fewer tube-like structures, demonstrating that the proteolytic activity of ADAMTS9 was essential. However, unlike the related metalloprotease ADAMTS1, which exerts anti-angiogenic effects by cleavage of thrombospondins and sequestration of vascular endothelial growth factor165, ADAMTS9 neither cleaved thrombospondins 1 and 2, nor bound vascular endothelial growth factor165. Taken together, these data identify ADAMTS9 as a novel, constitutive, endogenous angiogenesis inhibitor that operates cell-autonomously in ECs via molecular mechanisms that are distinct from those used by ADAMTS1. Following de novo formation of the vasculature from undifferentiated mesodermal precursors during embryogenesis (vasculogenesis), endothelial cells (ECs) proliferate and migrate to generate new capillaries from pre-existing blood vessels, a process termed angiogenesis.1Folkman J Fundamental concepts of the angiogenic process.Curr Mol Med. 2003; 3: 643-651Crossref PubMed Scopus (551) Google Scholar, 2Ferrara N Kerbel RS Angiogenesis as a therapeutic target.Nature. 2005; 438: 967-974Crossref PubMed Scopus (2254) Google Scholar Angiogenesis is critical for embryonic development, subsequent organ growth, and physiological processes such as menstruation and wound healing.3Roy R Zhang B Moses MA Making the cut: protease-mediated regulation of angiogenesis.Exp Cell Res. 2006; 312: 608-622Crossref PubMed Scopus (160) Google Scholar It is a prominent feature of age-related macular degeneration and diabetic retinopathy, and tumor growth is angiogenesis-dependent.1Folkman J Fundamental concepts of the angiogenic process.Curr Mol Med. 2003; 3: 643-651Crossref PubMed Scopus (551) Google Scholar, 2Ferrara N Kerbel RS Angiogenesis as a therapeutic target.Nature. 2005; 438: 967-974Crossref PubMed Scopus (2254) Google Scholar Owing to this considerable relevance for human pathology, the fundamental mechanisms that regulate angiogenesis have been the subject of intense investigation. Proteases are considered to be key participants in angiogenesis because of their traditional roles in remodeling of the vascular basement membrane during EC sprouting and migration.4Davis GE Senger DR Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization.Circ Res. 2005; 97: 1093-1107Crossref PubMed Scopus (935) Google Scholar, 5Genis L Galvez BG Gonzalo P Arroyo AG MT1-MMP: universal or particular player in angiogenesis?.Cancer Metastasis Rev. 2006; 25: 77-86Crossref PubMed Scopus (113) Google Scholar However, proteases also influence EC behavior by shedding cell-surface receptors and growth factors, exposing cryptic adhesion sites, or releasing bioactive fragments.3Roy R Zhang B Moses MA Making the cut: protease-mediated regulation of angiogenesis.Exp Cell Res. 2006; 312: 608-622Crossref PubMed Scopus (160) Google Scholar, 6van Hinsbergh VW Engelse MA Quax PH Pericellular proteases in angiogenesis and vasculogenesis.Arterioscler Thromb Vasc Biol. 2006; 26: 716-728Crossref PubMed Scopus (333) Google Scholar Thus, a role for proteases as negative regulators of angiogenesis is also accepted.3Roy R Zhang B Moses MA Making the cut: protease-mediated regulation of angiogenesis.Exp Cell Res. 2006; 312: 608-622Crossref PubMed Scopus (160) Google Scholar, 7Hamano Y Zeisberg M Sugimoto H Lively JC Maeshima Y Yang C Hynes RO Werb Z Sudhakar A Kalluri R Physiological levels of tumstatin, a fragment of collagen IV alpha3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via alphaV beta3 integrin.Cancer Cell. 2003; 3: 589-601Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 8Heljasvaara R Nyberg P Luostarinen J Parikka M Heikkila P Rehn M Sorsa T Salo T Pihlajaniemi T Generation of biologically active endostatin fragments from human collagen XVIII by distinct matrix metalloproteases.Exp Cell Res. 2005; 307: 292-304Crossref PubMed Scopus (160) Google Scholar, 9Lee NV Sato M Annis DS Loo JA Wu L Mosher DF Iruela-Arispe ML ADAMTS1 mediates the release of antiangiogenic polypeptides from TSP1 and 2.EMBO J. 2006; 25: 5270-5283Crossref PubMed Scopus (170) Google Scholar The ADAMTS protease family contains 19 secreted mammalian metalloproteases that localize to the cell-surface and/or extracellular matrix.10Apte SS A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs: the ADAMTS family.Int J Biochem Cell Biol. 2004; 36: 981-985Crossref PubMed Scopus (213) Google Scholar, 11Porter S Clark IM Kevorkian L Edwards DR The ADAMTS metalloproteinases.Biochem J. 2005; 386: 15-27Crossref PubMed Scopus (618) Google Scholar Several ADAMTS metalloproteases have specialized physiological roles that were revealed by analysis of human genetic disorders, or naturally occurring and engineered animal mutations,12Colige A Sieron AL Li SW Schwarze U Petty E Wertelecki W Wilcox W Krakow D Cohn DH Reardon W Byers PH Lapiere CM Prockop DJ Nusgens BV Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis are caused by mutations in the procollagen I N-proteinase gene.Am J Hum Genet. 1999; 65: 308-317Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 13Levy GG Nichols WC Lian EC Foroud T McClintick JN McGee BM Yang AY Siemieniak DR Stark KR Gruppo R Sarode R Shurin SB Chandrasekaran V Stabler SP Sabio H Bouhassira EE Upshaw Jr, JD Ginsburg D Tsai HM Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura.Nature. 2001; 413: 488-494Crossref PubMed Scopus (1435) Google Scholar, 14Dagoneau N Benoist-Lasselin C Huber C Faivre L Megarbane A Alswaid A Dollfus H Alembik Y Munnich A Legeai-Mallet L Cormier-Daire V ADAMTS10 mutations in autosomal recessive Weill-Marchesani Syndrome.Am J Hum Genet. 2004; 75: 801-806Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar but the functions of other members of this family, such as ADAMTS9, are poorly understood. ADAMTS proteases share a complex modular structure, comprising a metalloprotease domain coupled to a large ancillary domain containing thrombospondin type-1 repeats (TSRs), which are the hallmark of this family.10Apte SS A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs: the ADAMTS family.Int J Biochem Cell Biol. 2004; 36: 981-985Crossref PubMed Scopus (213) Google Scholar The TSRs of ADAMTS proteases are similar to those of the anti-angiogenic molecules thrombospondin-1 (TSP-1) and TSP-2.15Kuno K Kanada N Nakashima E Fujiki F Ichimura F Matsushima K Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inflammation associated gene.J Biol Chem. 1997; 272: 556-562Crossref PubMed Scopus (438) Google Scholar, 16Vazquez F Hastings G Ortega MA Lane TF Oikemus S Lombardo M Iruela-Arispe ML METH-1, a human ortholog of ADAMTS-1, and METH-2 are members of a new family of proteins with angio-inhibitory activity.J Biol Chem. 1999; 274: 23349-23357Crossref PubMed Scopus (382) Google Scholar, 17Carpizo D Iruela-Arispe ML Endogenous regulators of angiogenesis–emphasis on proteins with thrombospondin–type I motifs.Cancer Metastasis Rev. 2000; 19: 159-165Crossref PubMed Scopus (39) Google Scholar Indeed, ADAMTS1 was previously shown to inhibit angiogenesis via both proteolytic and non-proteolytic mechanisms, ie, by cleavage of TSP-1 and TSP-2, releasing anti-angiogenic fragments, as well as by sequestration of the pro-angiogenic growth factor vascular endothelial growth factor (VEGF)165 by the TSR-containing ancillary domain.9Lee NV Sato M Annis DS Loo JA Wu L Mosher DF Iruela-Arispe ML ADAMTS1 mediates the release of antiangiogenic polypeptides from TSP1 and 2.EMBO J. 2006; 25: 5270-5283Crossref PubMed Scopus (170) Google Scholar, 18Luque A Carpizo DR Iruela-Arispe ML ADAMTS1/METH1 inhibits endothelial cell proliferation by direct binding and sequestration of VEGF165.J Biol Chem. 2003; 278: 23656-23665Crossref PubMed Scopus (210) Google Scholar Adamts1 mRNA is widely expressed during embryogenesis and in adult tissues, but its expression in the vasculature appears to be restricted to smooth muscle cells, and is not reported in capillary endothelium.19Thai SN Iruela-Arispe ML Expression of ADAMTS1 during murine development.Mech Dev. 2002; 115: 181-185Crossref PubMed Scopus (63) Google Scholar, 20Gunther W Skaftnesmo KO Arnold H Bjerkvig R Terzis AJ Distribution patterns of the anti-angiogenic protein ADAMTS-1 during rat development.Acta Histochem. 2005; 107: 121-131Crossref PubMed Scopus (19) Google Scholar Although Adamts1 null mice have significant perinatal lethality,21Mittaz L Russell DL Wilson T Brasted M Tkalcevic J Salamonsen LA Hertzog PJ Pritchard MA Adamts-1 is essential for the development and function of the urogenital system.Biol Reprod. 2004; 70: 1096-1105Crossref PubMed Scopus (148) Google Scholar, 22Lee NV Rodriguez-Manzaneque JC Thai SN Twal WO Luque A Lyons KM Argraves WS Iruela-Arispe ML Fibulin-1 acts as a cofactor for the matrix metalloprotease ADAMTS-1.J Biol Chem. 2005; 280: 34796-34804Crossref PubMed Scopus (81) Google Scholar and Adamts1−/− ovaries have fewer and larger ovarian blood vessels than normal, generalized defects in vascular development have not been reported.23Shozu M Minami N Yokoyama H Inoue M Kurihara H Matsushima K Kuno K ADAMTS-1 is involved in normal follicular development, ovulatory process and organization of the medullary vascular network in the ovary.J Mol Endocrinol. 2005; 35: 343-355Crossref PubMed Scopus (85) Google Scholar However, ADAMTS1 produced by keratinocytes and skin fibroblasts regulates the migration of EC during wound healing.24Krampert M Kuenzle S Thai SN Lee N Iruela-Arispe ML Werner S ADAMTS1 proteinase is up-regulated in wounded skin and regulates migration of fibroblasts and endothelial cells.J Biol Chem. 2005; 280: 23844-23852Crossref PubMed Scopus (63) Google Scholar ADAMTS9 is the most highly conserved member of the ADAMTS family, being similar to Caenorhabditis elegans Gon-1, which is required for nematode morphogenesis.25Blelloch R Kimble J Control of organ shape by a secreted metalloprotease in the nematode Caenorhabditis elegans.Nature. 1999; 399: 586-590Crossref PubMed Scopus (166) Google Scholar ADAMTS9 not only has an identical active site sequence as ADAMTS1, but contains 15 potentially anti-angiogenic TSRs.26Somerville RP Longpre JM Jungers KA Engle JM Ross M Evanko S Wight TN Leduc R Apte SS Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans GON-1.J Biol Chem. 2003; 278: 9503-9513Crossref PubMed Scopus (274) Google Scholar ADAMTS9 was previously identified as a tumor suppressor gene in esophageal and nasopharyngeal cancer.27Lo PH Leung AC Kwok CY Cheung WS Ko JM Yang LC Law S Wang LD Li J Stanbridge EJ Srivastava G Tang JC Tsao SW Lung ML Identification of a tumor suppressive critical region mapping to 3p14.2 in esophageal squamous cell carcinoma and studies of a candidate tumor suppressor gene, ADAMTS9.Oncogene. 2007; 26: 148-157Crossref PubMed Scopus (66) Google Scholar, 28Lung HL Lo PH Xie D Apte SS Cheung AK Cheng Y Law EW Chua D Zeng YX Tsao SW Stanbridge EJ Lung ML Characterization of a novel epigenetically silenced, growth-suppressive gene, ADAMTS9, and its association with lymph node metastases in nasopharyngeal carcinoma.Int J Cancer. 2008; 123: 401-408Crossref PubMed Scopus (53) Google Scholar Recent work demonstrated that ADAMTS9 worked cooperatively with another Gon-1-related protease, ADAMTS20, in the colonization of skin by neural crest-derived melanoblasts.29Silver DL Hou L Somerville R Young ME Apte SS Pavan WJ The secreted metalloprotease ADAMTS20 is required for melanoblast survival.PLoS Genet. 2008; 4: e1000003Crossref PubMed Scopus (91) Google Scholar Using in situ hybridization during murine development, we previously found that ADAMTS9 was expressed in capillaries.30Jungers KA Le Goff C Somerville RP Apte SS ADAMTS9 is widely expressed during mouse embryo development.Gene Expr Patterns. 2005; 5: 609-617Crossref PubMed Scopus (74) Google Scholar However, early embryonic lethality of ADAMTS9 null mice appeared to preclude analysis of its role in vascular development, as well as angiogenesis in the tumor context.29Silver DL Hou L Somerville R Young ME Apte SS Pavan WJ The secreted metalloprotease ADAMTS20 is required for melanoblast survival.PLoS Genet. 2008; 4: e1000003Crossref PubMed Scopus (91) Google Scholar We demonstrate here that when congenic with the C57Bl/6 strain, ADAMTS9+/− mice develop spontaneous corneal neovascularization within a few weeks of weaning and that heterotopic tumors in these mice attract more vasculature than wild-type mice. It is shown that ADAMTS9 acts via a cell-autonomous mechanism in microvascular endothelium and that it represents a nonredundant anti-angiogenic activity, since it does not share the mechanisms used by ADAMTS1, and differs from it in other fundamental respects. Transgenic mice with targeted inactivation of ADAMTS9 by insertion of an IRES-lacZ cassette in exon 12 (encoding TSR 1) were obtained under license from Deltagen (San Carlos, CA). The targeted allele was bred for 10 generations into inbred C57Bl/6 mice to achieve a congenic strain, and minimize the influence of genetic variation on the phenotype. For analysis of ADAMTS9 expression during wound healing, full-thickness, circular, 10-mm diameter excisional wounds were made in dorsal skin of an 8-week-old male mouse. The wound bed with surrounding skin was excised 5 days later, fixed in 4% paraformaldehyde, stained for β-galactosidase (β-gal) as described below, and embedded in paraffin. For analysis of ADAMTS9 expression during tumor growth, isogenic tumor cells (B.16-F10 melanoma and Lewis lung carcinoma) were injected subcutaneously over the flank of 8-week-old male mice. Tumors were excised with surrounding tissue after 1 week, stained for β-gal as described below, and embedded in paraffin. For comparative analysis of tumor-induced vasculature in ADAMTS9+/− and wild-type littermates, 2 × 106 B.16-F10 melanoma cells were injected (day 0) on each flank of 8-week-old mice (n = 8 for ADAMTS9+/− mice, n = 10 for wild-type controls) and tumor-induced angiogenesis was assayed on day 9 essentially as previously described.31Taylor KL Oates RK Grane R Leaman DW Borden EC Lindner DJ IFN-alpha1,8 inhibits tumor-induced angiogenesis in murine angiosarcomas.J Interferon Cytokine Res. 2006; 26: 353-361Crossref PubMed Scopus (20) Google Scholar, 32Baker DP Lin EY Lin K Pellegrini M Petter RC Chen LL Arduini RM Brickelmaier M Wen D Hess DM Chen L Grant D Whitty A Gill A Lindner DJ Pepinsky RB N-terminally PEGylated human interferon-beta-1a with improved pharmacokinetic properties and in vivo efficacy in a melanoma angiogenesis model.Bioconjug Chem. 2006; 17: 179-188Crossref PubMed Scopus (131) Google Scholar, 33Bauer JA Morrison BH Grane RW Jacobs BS Borden EC Lindner DJ IFN-alpha2b and thalidomide synergistically inhibit tumor-induced angiogenesis.J Interferon Cytokine Res. 2003; 23: 3-10Crossref PubMed Scopus (32) Google Scholar These experiments were done in a double-blind design in which the mice were assigned a code and the author performing the tumor study (D.J.L.) was not provided with the genotype. Following completion of quantitative analysis, the code was broken and the data were analyzed using a Student's t-test. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Cleveland Clinic. For routine histological analysis, mouse eyes and other tissues were fixed in 4% paraformaldehyde, followed by paraffin embedding and staining of 5- to 10-μm-thick sections with H&E. β-gal histochemistry was undertaken at different gestational ages and in adult (>3-month-old) tissues, excisional wound beds, and tumors as previously described.34McCulloch DR Goff CL Bhatt S Dixon LJ Sandy JD Apte SS Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues.Gene Expr Patterns. 2009; 9: 314-323Crossref PubMed Scopus (75) Google Scholar Immunohistochemistry of β-gal-stained paraffin sections was done using anti-PECAM-1 (rat monoclonal antibody MEC13.3, Pharmingen), anti-endomucin (rat monoclonal V.7C7)35Brachtendorf G Kuhn A Samulowitz U Knorr R Gustafsson E Potocnik AJ Fassler R Vestweber D Early expression of endomucin on endothelium of the mouse embryo and on putative hematopoietic clusters in the dorsal aorta.Dev Dyn. 2001; 222: 410-419Crossref PubMed Scopus (46) Google Scholar provided by Dr. Dietmar Vestweber, rat monoclonal anti-podoplanin antibody 8.1.1 (Developmental Studies Hybridoma Bank),36Farr AG Berry ML Kim A Nelson AJ Welch MP Aruffo A Characterization and cloning of a novel glycoprotein expressed by stromal cells in T-dependent areas of peripheral lymphoid tissues.J Exp Med. 1992; 176: 1477-1482Crossref PubMed Scopus (142) Google Scholar rabbit polyclonal anti-NG2 (Millipore Corp., Billerica, MA) and anti-smooth muscle α-actin (SMA) monoclonal antibody (Sigma-Aldrich) in a conventional indirect immunoalkaline phosphatase method. Before incubation with the anti-endomucin primary antibody, sections were treated by antigen retrieval. Briefly, slides were immersed in citrate-EDTA buffer (10 mmol/L citric acid, 2 mmol/L EDTA, 0.05% v/v Tween-20, pH 6.2) and microwaved four times for 1.5 minutes at 50% power in a domestic microwave oven. The buffer was allowed to stand for 30 seconds between each microwave cycle. After four cycles of microwaving, slides were allowed to cool for 20 to 30 minutes in citrate-EDTA buffer. Immortalized human brain microvascular ECs (HBMECs)37Callahan MK Williams KA Kivisakk P Pearce D Stins MF Ransohoff RM CXCR3 marks CD4+ memory T lymphocytes that are competent to migrate across a human brain microvascular endothelial cell layer.J Neuroimmunol. 2004; 153: 150-157Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar were routinely cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum. Clonetics microvascular ECs from human dermis (HDMECs) and heart (HHMECs) were cultured according to the supplier's instructions (Cambrex Corporation, East Rutherford, NJ). Human umbilical vein ECs (HUVECs) were cultured in M199 medium (Invitrogen, Carlsbad, CA) containing 15% fetal bovine serum, 10 units/ml heparin, and fibroblast growth factor-2 (PeproTech, Rocky Hill, NJ, 3 ng/ml). Human alveolar basal epithelial carcinoma cell line A549 was grown as recommended by the supplier (ATCC, Manassas, VA). Mammalian expression plasmids for expression of full-length ADAMTS9 or catalytically inactive mutants (Glu435Gln or Glu435Ala), for C-terminally truncated ADAMTS9 extending up to TSR 8 (designated ADAMTS9 N-L2), or its mutants containing substitution of Ala for Arg74/209/287 (Arg74/209/287Ala) or Ala for Glu435 (Glu435Ala, catalytically inactive) have been previously described.26Somerville RP Longpre JM Jungers KA Engle JM Ross M Evanko S Wight TN Leduc R Apte SS Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans GON-1.J Biol Chem. 2003; 278: 9503-9513Crossref PubMed Scopus (274) Google Scholar, 38Koo BH Longpre JM Somerville RP Alexander JP Leduc R Apte SS Cell-surface processing of pro-ADAMTS9 by furin.J Biol Chem. 2006; 281: 12485-12494Crossref PubMed Scopus (55) Google Scholar, 39Koo BH Longpre JM Somerville RP Alexander JP Leduc R Apte SS Regulation of ADAMST9 secretion and enzymatic activity by its propeptide.J Biol Chem. 2007; 282: 16146-16154Crossref PubMed Scopus (53) Google Scholar The ADAMTS1 expression plasmid was kindly provided by Dr. Luisa Iruela-Arispe. All ADAMTS9 plasmids encode a C-terminal myc tag or FLAG tag. Since ADAMTS9 N-L2 is more robustly expressed by transfected cells than full-length ADAMTS9 (which loses the C-terminal tag by proteolysis, rendering its detection difficult), and cleaves versican, indicating that proteolytic activity is preserved,39Koo BH Longpre JM Somerville RP Alexander JP Leduc R Apte SS Regulation of ADAMST9 secretion and enzymatic activity by its propeptide.J Biol Chem. 2007; 282: 16146-16154Crossref PubMed Scopus (53) Google Scholar it was used instead of full-length ADAMTS9 in several experiments. Because furin cleavage following Arg287 reduces proteolytic activity,39Koo BH Longpre JM Somerville RP Alexander JP Leduc R Apte SS Regulation of ADAMST9 secretion and enzymatic activity by its propeptide.J Biol Chem. 2007; 282: 16146-16154Crossref PubMed Scopus (53) Google Scholar the Arg74/209/287Ala mutant, which abrogates furin processing of ADAMTS9, was used to obtain enhanced activity relative to the wild-type construct. Mouse TSP-1 and TSP-2 expression plasmids were provided by Dr. Olga Stenina and Dr. Paul Bornstein, respectively. COS-1 or HEK293F cells were transfected with expression plasmids using FuGENE6 (Roche Applied Sciences, Indianapolis, IN). HBMECs and HUVECs (plated on vitronectin) were transfected using Targefect-HUVEC according to the supplier's instructions (Targeting Systems, El Cajon, CA). Four pre-designed human ADAMTS9 small interfering (si)RNAs and a negative control siRNA were purchased from Applied Biosystems/Ambion, Austin, TX. Fifty nmol/L of siRNA was added to the cells in combination with Lipofectamine 2000 (Invitrogen) for transfection, and incubated for 48 hours. Total RNA was isolated from siRNA-transfected cells using Trizol (Invitrogen), and cDNA was generated using SuperScript III First-Strand kit (Invitrogen). Primer sets were designed for reverse transcription (RT)-PCR as follows; for human ADAMTS9, forward primer 5′-CGGTTTGTAGAAGTCTTG-3′ and reverse primer 5′-CAGGTTCGTTAAGCAAAC-3′ with an expected amplicon of 622 bp; glyceraldehyde-3-phosphate dehydrogenase (Gapdh), forward primer 5′-GATTTGGTCGTATTGGGC-3′ and 5′-CGTGTTGTCATACTTCTC-3′, with an expected amplicon of 401 bp was used as a control. One pre-designed siRNA gave maximal suppression and was used in subsequent experiments. For assaying cell adhesion, HBMECs were transfected with control or ADAMTS9 siRNA and detached 48 hours later using a PBS-based cell dissociation buffer (Invitrogen); 1 × 105 cells were plated on fibronectin (Sigma-Aldrich), vitronectin, laminin-1 (Sigma-Aldrich, St. Louis, MO), or collagen IV (Sigma-Aldrich) coated 96-well culture plates in serum-free medium and allowed to adhere for 1 hour. The cells were gently washed with serum-free medium and fixed with 3.7% formaldehyde. Attached cells were stained with 0.5% Crystal violet, washed, lysed in 1% SDS, and color intensity was measured at 570 nm, with background absorbance subtracted at 630 nm. Cell spreading was investigated on uncoated or Matrigel-coated culture slides (BD Biosciences, Franklin Lakes, NJ). The cells (1 × 103) were incubated for 3 hours, and actin filaments were stained with fluorescein isothiocyanate-phalloidin (Sigma-Aldrich). Photographs were taken under an Olympus inverted fluorescence microscope. For comparison of proliferation of ADAMTS9 siRNA-treated cells with control siRNA-transfected cells, HBMEC (1 × 104) were plated in 24-well culture plates and incubated at 37°C for 48 hours. Cell numbers were obtained with a Coulter counter, as well as by the WST-1 assay (Roche Applied Science). An in vitro cell monolayer wounding assay was performed to evaluate HBMEC and A549 cell migration. The siRNA-transfected cells were cultured to confluence in six-well culture plates coated with Matrigel (100 mg/ml) in 50 mmol/L NaHCO3 and a linear scratch was made using a 200-μL pipette tip. Photographs were recorded every 10 minutes by time-lapse photomicroscopy in a temperature- and gas-controlled environment with a time-lapse inverted microscope, until closing of the scratch was observed. A tube-formation assay was performed using Matrigel as a substrate, and 150 μl/well of growth factor depleted Matrigel (9 g/L) was added to 24-well culture plates and allowed to gel for 15 minutes at 37°C. HBMECs, HDMECs, or HHMECs transfected by siRNA or expression plasmid were detached by brief treatment with 0.05% trypsin solution, and cells (5 × 104) were added to the culture plates after resuspension in medium containing 5% fetal bovine serum and VEGF165 (PeproTech, 40 mg/L). After 6 hours incubation (siRNA treatment) or 24 hours (overexpression experiments) the cells were fixed with 3.7% formaldehyde and the lengths of the tube-like structures were measured using ImageJ software (NIH). ADAMTS9 or ADAMTS1 expression plasmids were cotransfected with TSP-1 or TSP-2 into COS-1 cells or HEK293F cells. Forty-eight hours (18 hours for COS-1 cells) following transfection, cells were incubated in 293 SFM-II medium (Invitrogen) for 24 hours (HEK293F cells) or serum-free Dulbecco's modified Eagle's medium (DMEM) for 72 hours (COS-1 cells), and conditioned medium and cell-lysate was collected and subjected to reducing or non-reducing SDS-polyacrylamide gel electrophoresis. Western blotting was done using anti-FLAG M2 (Sigma-Aldrich) or anti-myc monoclonal antibody for detection of ADAMTS9, anti-ADAMTS1 monoclonal antibody (Antibody 2197, R&D systems, Minneapolis, MN), anti-TSP1 monoclonal antibody (Catalog No. SC-59887, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-mouse TSP-2 monoclonal antibody (Catalog No. 611150, BD Biosciences). For detection of cleavage of TSP-1 at the Glu311-Leu312 peptide bond, previously described guinea-pig antibodies numbered 78 and 79 that recognize the new N-terminus and C-terminus of cleaved TSP1, respectively, and antibody 80, which recognizes the uncleaved spanning peptide across the cleavage site9Lee NV Sato M Annis DS Loo JA Wu L Mosher DF Iruela-Arispe ML ADAMTS1 mediates the release of antiangiogenic polypeptides from TSP1 and 2.EMBO J. 2006; 25: 5270-5283Crossref PubMed Scopus (170) Google Scholar (kindly provided by Dr. Luisa Iruela-Arispe) were used for Western blotting. ADAMTS9 N-L2 or ADAMTS1 was immunoprecipitated from conditioned media of transfected HEK293F cells using anti-myc or anti-ADAMTS1 respectively. Protein G-sepharose beads were washed three times with PBS and then with binding buffer [HEPES (50 mmol/L), NaCl (150 mmol/L), 0.5% Nonidet P-40, 0.5% bovine serum albumin, CaCl2 (1 mmol/L), MgCl2 (1 mmol/L)]. Then, 200 ng of VEGF165 and 200 ng of heparin (Sigma-Aldrich) were added to the beads in a final volume of 500 μl of binding buffer, and incubated for 2 hours at 4°C with rotation. The bound protein was eluted with reducing SDS-sample buffer. The eluted samples were analyzed by Western blotting with anti-VEGF polyclonal antibody (Cat No. AB1442, Millipore, Billerica, MA). The presence of co-immunoprecipitated ADAMTS9 N-L2 and ADAMTS1 was verified by Western blotting using anti-myc monoclonal antibody 9E10 (Invitrogen, Carlsbad, CA) and anti-ADAMTS1 monoclonal antibody respectively. HUVECs were cultured to confluence and further incubated for 6 hours in serum-free medium" @default.
• W2123509716 created "2016-06-24" @default.
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• W2123509716 date "2010-03-01" @default.
• W2123509716 modified "2023-10-18" @default.
• W2123509716 title "ADAMTS9 Is a Cell-Autonomously Acting, Anti-Angiogenic Metalloprotease Expressed by Microvascular Endothelial Cells" @default.
• W2123509716 cites W1651336784 @default.
• W2123509716 cites W1972247867 @default.
• W2123509716 cites W1976384802 @default.
• W2123509716 cites W1977094975 @default.
• W2123509716 cites W1978709727 @default.
• W2123509716 cites W1987322459 @default.
• W2123509716 cites W1993399270 @default.
• W2123509716 cites W1995033273 @default.
• W2123509716 cites W2000307014 @default.
• W2123509716 cites W2001243726 @default.
• W2123509716 cites W2003059292 @default.
• W2123509716 cites W2007036356 @default.
• W2123509716 cites W2010264164 @default.
• W2123509716 cites W2012160572 @default.
• W2123509716 cites W2013652287 @default.
• W2123509716 cites W2016498752 @default.
• W2123509716 cites W2017759961 @default.
• W2123509716 cites W2023967584 @default.
• W2123509716 cites W2030246164 @default.
• W2123509716 cites W2031856001 @default.
• W2123509716 cites W2032175563 @default.
• W2123509716 cites W2032712201 @default.
• W2123509716 cites W2032986550 @default.
• W2123509716 cites W2033388295 @default.
• W2123509716 cites W2039871617 @default.
• W2123509716 cites W2041788236 @default.
• W2123509716 cites W2043703144 @default.
• W2123509716 cites W2049569075 @default.
• W2123509716 cites W2055395011 @default.
• W2123509716 cites W2058239698 @default.
• W2123509716 cites W2061847367 @default.
• W2123509716 cites W2061945394 @default.
• W2123509716 cites W2063482106 @default.
• W2123509716 cites W2067901911 @default.
• W2123509716 cites W2074465151 @default.
• W2123509716 cites W2081172215 @default.
• W2123509716 cites W2084740377 @default.
• W2123509716 cites W2103245619 @default.
• W2123509716 cites W2114604545 @default.
• W2123509716 cites W2129123556 @default.
• W2123509716 cites W2160229338 @default.
• W2123509716 cites W2166841658 @default.
• W2123509716 cites W65284337 @default.
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