title: Monocyte urokinase-type plasminogen activator up-regulation reduces thrombus size in a model of venous thrombosis.
abstract: BACKGROUND
  Our previous studies showed that the direct injection of an adenovirus construct expressing urokinase-type plasminogen activator (uPA) into experimental venous thrombi significantly reduces thrombus weight. The systemic use of adenovirus vectors is limited by inherent hepatic tropism and inflammatory response. As macrophages are recruited into venous thrombi, it is reasonable to speculate that these cells could be used to target the adenovirus uPA (ad-uPA) gene construct to the thrombus. The aims of this study were to determine whether macrophages transduced with ad-uPA have increased fibrinolytic activity and whether systemic injection of transduced cells could be used to target uPA expression to the thrombus and reduce its size.
  
  
  METHODS
  The effect of up-regulating uPA was examined in an immortalized macrophage cell line (MM6) and macrophages differentiated from human blood monocyte-derived macrophages (HBMMs). Cells were infected with ad-uPA or blank control virus (ad-blank). Fibrinolytic mediator expression, cell viability, and cytokine expression were measured by activity assays and enzyme-linked immunosorbent assays. Monocyte migration was measured using a modified Boyden chamber assay. A model of venous thrombosis was developed and characterized in mice with severe combined immunodeficiency (SCID). This model was used to study whether systemically administered macrophages over-expressing uPA reduced thrombus size. Uptake of HBMMs into the thrombus induced in these mice was confirmed by a combination of PKH2-labeled cell tracking and colocalization with human leukocyte antigen (HLA) by immunohistology.
  
  
  RESULTS
  Compared with ad-blank, treated HBMMs transduction with ad-uPA increased uPA production by >1000-fold (P = .003), uPA activity by 150-fold (P = .0001), and soluble uPA receptor (uPAR) by almost twofold (P = .043). Expression of plasminogen activator inhibitor (PAI-1) and PAI-2 was decreased by about twofold (P = .011) and threefold (P = .005), respectively. Up-regulation of uPA had no effect on cell viability or inflammatory cytokine production compared with ad-blank or untreated cells. Ad-uPA transduction increased the migration rate of HBMMs (about 20%, P = .03) and MM6 cells (>twofold, P = .005) compared with ad-blank treated controls. Human macrophage recruitment into the mouse thrombus was confirmed by the colocalization of HLA with the PKH2-marked cells. Systemic injection of uPA-up-regulated HBMMs reduced thrombus weight by approximately 20% compared with ad-blank (P = .038) or sham-treated controls (P = .0028).
  
  
  CONCLUSIONS
  Transduction of HBBM with ad-uPA increases their fibrinolytic activity. Systemic administration of uPA up-regulated HBBMs reduced thrombus size in an experimental model of venous thrombosis. Alternative methods of delivering fibrinolytic agents are worth exploring.
authors.affiliation: King's College London British Heart Foundation Centre, Academic Department of Surgery, Cardiovascular Division, National Institute for Health Research Biomedical Research Centre at Guy's and St Thomas' National Health Service Foundation Trust, London, United Kingdom.
authors.forename: Julia
authors.initials: J
authors.lastname: Humphries
authors.forename: James A
authors.initials: JA
authors.lastname: Gossage
authors.forename: Bijan
authors.initials: B
authors.lastname: Modarai
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Burnand
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Sisson
authors.forename: Colin
authors.initials: C
authors.lastname: Murdoch
authors.forename: Alberto
authors.initials: A
authors.lastname: Smith
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002465:Cell Movement; D002470:Cell Survival; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004195:Disease Models, Animal; D005342:Fibrinolysis; D005456:Fluorescent Dyes; D015316:Genetic Therapy; D005822:Genetic Vectors; D006680:HLA Antigens / Q000032:analysis; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology / Q000276:immunology / Q000637:transplantation*; D051379:Mice; D016513:Mice, SCID; D009930:Organic Chemicals; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D017396:Plasminogen Activator Inhibitor 2 / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D014161:Transduction, Genetic; D015854:Up-Regulation; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D020246:Venous Thrombosis / Q000097:blood / Q000201:enzymology / Q000235:genetics / Q000628:therapy*
chemical_list: D016207:Cytokines; D005456:Fluorescent Dyes; D006680:HLA Antigens; D018836:Inflammation Mediators; D009930:Organic Chemicals; D017395:Plasminogen Activator Inhibitor 1; D017396:Plasminogen Activator Inhibitor 2; D055293:Receptors, Urokinase Plasminogen Activator; C051321:SERPINE1 protein, human; C082445:PKH 2 dye; D014568:Urokinase-Type Plasminogen Activator
references.citation: J Clin Invest. 2007 Jan;117(1):89-93
references.pmid: 17200712
references.citation: Thromb Haemost. 1992 Jan 23;67(1):95-100
references.pmid: 1377417
references.citation: Arterioscler Thromb Vasc Biol. 2008 Oct;28(10):1753-9
references.pmid: 18669887
references.citation: J Vasc Surg. 1998 Jul;28(1):67-73; discussion 73-4
references.pmid: 9685132
references.citation: Surg Gynecol Obstet. 1963 May;116:593-99
references.pmid: 14023567
references.citation: J Vasc Surg. 1999 Nov;30(5):894-9
references.pmid: 10550187
references.citation: Thromb Haemost. 2003 Jan;89(1):169-76
references.pmid: 12540967
references.citation: Thromb Haemost. 2002 Jul;88(1):131-6
references.pmid: 12152653
references.citation: Cell Mol Life Sci. 2004 Nov;61(22):2840-58
references.pmid: 15558213
references.citation: J Vasc Surg. 2006 Nov;44(5):1085-90
references.pmid: 17098546
references.citation: Cancer Res. 1990 Dec 1;50(23):7450-6
references.pmid: 1701343
references.citation: Thromb Haemost. 1999 Aug;82(2):298-304
references.pmid: 10605717
references.citation: J Exp Med. 1987 Feb 1;165(2):320-39
references.pmid: 2434595
references.citation: EMBO J. 1997 May 15;16(10):2610-20
references.pmid: 9184208
references.citation: Tech Vasc Interv Radiol. 2001 Jun;4(2):111-4
references.pmid: 11981797
references.citation: J Vasc Surg. 2003 Nov;38(5):1090-8
references.pmid: 14603221
references.citation: Am J Pathol. 2002 Nov;161(5):1701-10
references.pmid: 12414517
references.citation: Thromb Haemost. 1999 Nov;82(5):1510-5
references.pmid: 10595646
references.citation: Circulation. 2003 Feb 18;107(6):869-75
references.pmid: 12591758
references.citation: Thromb Res. 1983 Jun 1;30(5):425-9
references.pmid: 6351343
references.citation: J Cell Biol. 1989 Nov;109(5):2471-9
references.pmid: 2509486
references.citation: J Clin Invest. 1994 Apr;93(4):1380-7
references.pmid: 8163642
references.citation: Thromb Haemost. 2001 Jun;85(6):1018-24
references.pmid: 11434678
references.citation: J Vasc Surg. 1995 Nov;22(5):573-9
references.pmid: 7494358
references.citation: Curr Opin Mol Ther. 2008 Oct;10(5):439-48
references.pmid: 18830919
references.citation: J Immunol. 2006 Nov 15;177(10):7303-11
references.pmid: 17082649
references.citation: J Vasc Surg. 1998 Nov;28(5):826-33
references.pmid: 9808849
references.citation: Methods Cell Biol. 1990;33:469-90
references.pmid: 2084480
references.citation: Scand J Immunol. 2000 Sep;52(3):257-63
references.pmid: 10972901
references.citation: Dis Mon. 2005 Feb-Mar;51(2-3):104-11
references.pmid: 15900261
references.citation: Am J Physiol. 1999 Sep;277(3):L573-9
references.pmid: 10484465
references.citation: Hum Gene Ther. 2002 Jan 1;13(1):129-41
references.pmid: 11779417
references.citation: Angew Chem Int Ed Engl. 2005 Feb 4;44(7):1057-1061
references.pmid: 15688429
references.citation: EMBO J. 2003 Nov 17;22(22):5994-6003
references.pmid: 14609946
references.citation: J Immunol Methods. 1997 Sep 24;207(2):157-68
references.pmid: 9368642
references.citation: Circulation. 2004 May 4;109(17):2129-35
references.pmid: 15096455
references.citation: J Vasc Surg. 2006 Mar;43(3):601-8
references.pmid: 16520180
references.citation: Hum Gene Ther. 2001 Mar 1;12(4):439-45
references.pmid: 11242535
references.citation: Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):8211-5
references.pmid: 9653166
references.citation: Thromb Haemost. 2002 Jun;87(6):1006-10
references.pmid: 12083479