D Chen

Critical roles for thrombin in acute and chronic inflammation

Summary.u2002 Thrombin can amplify inflammation induced by other stimuli, either through ischemia (consequent upon thrombosis), indirectly through generation of downstream mediators such as activated protein C, or directly via signals through protease activated receptors (PAR). This paper will summarize recent data from our laboratory indicating that thrombin is required to initiate CCR2‐dependent leukocyte recruitment and that it is the principal determinant of the outcome after vascular injury, via PAR‐1 activation of a distinct subset of smooth muscle cell progenitors. In both, tissue factor (TF) initiates thrombin generation and the thrombin acts locally, exemplifying that the initiation phase can generate autocrine or paracrine signalling molecules. Thrombin is an important constituent of innate immunity, able to amplify and modify responses to invading pathogens or tissue damage. With novel anti‐thrombin therapeutics and agents to target PAR, a new understanding of the importance of thrombin may allow the development of innovative anti‐inflammatory strategies.

Transferrin receptor-mediated gene transfer to the corneal endothelium.

Background. The application of gene therapy to prevent allograft rejection requires the development of noninflammatory vectors. We have therefore investigated the use of a nonviral system, transferrin-mediated lipofection, to transfer genes into the cornea with the aim of preventing corneal graft rejection. Methods. Rabbit and human corneas were cultured ex vivo and transfected with either lipofection alone or in conjunction with transferrin. The efficiency of transfection, localization, and kinetics of marker gene expression were determined. Strategies to increase gene expression, using chloroquine and EDTA, were investigated. In addition to a marker gene, a gene construct encoding viral interleukin 10 (vIL-10) was transfected and its functional effects were examined in vitro. Results. Transferrin, liposome, and DNA were demonstrated to interact with each other, forming a complex. This complex was found to deliver genes selectively to the endothelium of corneas resulting in gene expression. Treatment of corneas with chloroquine and EDTA increased the transfection efficiency eightfold and threefold, respectively. We also demonstrated that constructs encoding vIL-10 could be delivered to the endothelium. Secreted vIL-10 was shown to be functionally active by inhibition of a mixed lymphocyte reaction. Conclusions. Our data indicate that transferrin-mediated lipofection is a comparatively efficient nonviral method for delivering genes to the corneal endothelium. Its potential for use in preventing graft rejection is shown by the ability of this system to induce vIL-10 expression at secreted levels high enough to be functional.

Expression of Hirudin Fusion Proteins in Mammalian Cells A Strategy for Prevention of Intravascular Thrombosis

BACKGROUNDnIntravascular thrombosis occurs in disorders of diverse pathogeneses, including allograft and xenograft rejection. In this in vitro study, we describe an approach for tethering the specific thrombin inhibitor hirudin to plasma membranes as part of a genetic strategy for regulating intravascular coagulation.nnnMETHODS AND RESULTSnAn HLA class I leader sequence was fused with hirudin linked to domains 3 and 4 of human CD4 and intracytoplasmic sequence from either CD4 or human P-selectin. The constructs were transfected into mouse fibroblasts, Chinese hamster ovary (CHO)-K1 cells, immortalized porcine endothelial cells (IPECs), and a pituitary secretory cell line (D16/16). Thrombin binding to the hirudin fusion proteins expressed on fibroblasts and CHO-K1 cells could be blocked by an anti-hirudin monoclonal antibody and by pretreatment of thrombin with either the synthetic tripeptide thrombin inhibitor PPACK or native hirudin. Hirudin expression significantly modified the procoagulant phenotype of IPECs in human plasma, leading to prolongation of clotting times. Hirudin-CD4-P-selectin fusion proteins accumulated in adrenocorticotropic hormone-containing granules in D16/16 cells, with no cell surface expression except on activation with phorbol ester, when hirudin relocated to the outer membrane.nnnCONCLUSIONSnHirudin fusion proteins were expressed on mammalian cells, where they reduced local thrombin levels and inhibited fibrin formation. Regulated expression was achieved on activated cells by use of the cytoplasmic sequence from P-selectin. In vivo, these fusion proteins may prove useful transgenic or gene therapy agents for preventing intravascular thrombosis.

NK-cell-dependent acute xenograft rejection in the mouse heart-to-rat model

Abstract:u2002 Background:u2002 Acute humoral xenograft rejection is characterized by widespread intravascular thrombosis with a significant NK‐cell and macrophage infiltrate. Although in vitro and ex vivo data have shown that NK cells are capable of killing xenogeneic tissue, the precise role they play in vivo is still not certain. Consequently, there are few tested strategies for dealing with NK‐cell‐mediated rejection, should this prove to be a problem. One reason for this has been the lack of a relevant rodent model in which rejection by these cells can be easily studied.

Regenerative repair after endoluminal injury in mice with specific antagonism of protease activated receptors on CD34+ vascular progenitors

Tissue factor (TF) and thrombin are involved in intimal hyperplasia (IH) and remodelling following vascular injury. Because many neointimal smooth muscle cells (VSMCs) derive from circulating vascular progenitors (VPs), we investigated how thrombin influences VP phenotype and function. Following wire-induced carotid artery injury in mice, the majority of circulating VPs expressed TF, were capable of initiating clotting in vitro, and had protease-activated receptors (PAR)-1, -2, and -4. Thrombin, through PAR-1, inhibited apoptosis and caused proliferation, resulting in the outgrowth of VP coexpressing markers of activated endothelial cells and VSMCs, even in the presence of growth factors. These mixed-phenotype VPs circulated as a minority population after injury and shared a similar phenotype with many neointimal cells. Labeled CD34(+) cells, injected up to 2 weeks after injury, could be detected in the injured vessel wall, suggesting that continued recruitment may contribute to progressive IH. Finally, CD34(+) cells incubated with thrombin prior to injection promoted florid neointimal lesions, whereas those incubated with PAR antagonists inhibited IH and promoted regenerative repair characterized by the development of a quiescent endothelium. We conclude that IH after vascular injury is due to the direct actions of thrombin on mobilized VPs.

Postinjury vascular intimal hyperplasia in mice is completely inhibited by CD34+ bone marrow-derived progenitor cells expressing membrane-tethered anticoagulant fusion proteins

Summary.u2002 Background:u2002Coagulation proteins promote neointimal hyperplasia and vascular remodelling after vessel injury, but the precise mechanisms by which they act in vivo remain undetermined. Objectives:u2002This study, using an injury model in which the neointima is derived from bone marrow (BM)‐derived cells, compared inhibition of tissue factor or thrombin on either BM‐derived or existing vascular smooth muscle cells. Methods:u2002Two transgenic (Tg) mouse strains expressing membrane‐tethered tissue factor pathway inhibitor (TFPI) or hirudin (Hir) fusion proteins driven by an α smooth muscle actin (SMA) promoter were generated (α‐TFPI‐Tg and α‐Hir‐Tg) and the phenotype after wire‐induced endovascular injury was compared with that in wild‐type (WT) controls. Results:u2002WT mice developed progressive neointimal expansion, whereas injury in either Tg was followed by repair back to a preinjured state. This was also seen when WT mice were reconstituted with BM from Tg mice but not when Tgs were reconstituted with WT BM, in which injury was followed by slowly progressive neointimal expansion. Injection of CD34+ cells from Tg mice into injured WT mice resulted in the accumulation of fusion protein‐expressing cells from day 3 onwards and an absence of neointimal hyperplasia in those areas. Conclusions:u2002Neointimal development after wire‐induced endovascular injury in mice was completely inhibited when BM‐derived cells infiltrating the damaged artery expressed membrane tethered anticoagulant fusion proteins under an α‐SMA promoter. These findings enhance our understanding of the pathological role that coagulation proteins play in vascular inflammation.