Monocyte urokinase-type plasminogen activator up-regulation reduces thrombus size in a model of venous thrombosis.
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.
Humphries
Julia
J
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.
Gossage
James A
JA
Modarai
Bijan
B
Burnand
Kevin G
KG
Sisson
Thomas H
TH
Murdoch
Colin
C
Smith
Alberto
A
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*
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
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