Inhibition of complement component C3 reduces vein graft atherosclerosis in apolipoprotein E3-Leiden transgenic mice.

BACKGROUND: Venous bypass grafts may fail because of development of intimal hyperplasia and accelerated atherosclerosis. Inflammation plays a major role in these processes. Complement is an important part of the immune system and participates in the regulation of inflammation. The exact role of complement in the process of accelerated atherosclerosis of vein grafts has not yet been explored, however. METHODS AND RESULTS: To assess the role of complement in the development of vein graft atherosclerosis, a mouse model, in which a venous interposition was placed in the common carotid artery, was used. In this model, vein graft thickening appeared within 4 weeks. The expression of complement components was studied with the use of immunohistochemistry on sections of the thickened vein graft. C1q, C3, C9, and the regulatory proteins CD59 and complement receptor-related gene y could be detected in the lesions 4 weeks after surgery. Quantitative mRNA analysis for C1q, C3, CD59, and complement receptor-related gene y revealed expression of these molecules in the thickened vein graft, whereas C9 did not show local mRNA expression. Furthermore, interference with C3 activation with complement receptor-related gene y-Ig was associated with reduced vein graft thickening, reduced C3 and C9 deposition, and reduced inflammation as assessed by analysis of influx of inflammatory cells, such as leukocytes, T cells, and monocytes. In addition, changes in apoptosis and proliferation were observed. When C3 was inhibited by cobra venom factor, a similar reduction in vein graft thickening was observed. CONCLUSIONS: The complement cascade is involved in vein graft thickening and may be a target for therapy in vein graft failure disease.

Anti-MCP-1 gene therapy inhibits vascular smooth muscle cells proliferation and attenuates vein graft thickening both in vitro and in vivo.

OBJECTIVE: Because late vein graft failure is caused by intimal hyperplasia (IH) and accelerated atherosclerosis, and these processes are thought to be inflammation driven, influx of monocytes is one of the first phenomena seen in IH, we would like to provide direct evidence for a role of the MCP-1 pathway in the development of vein graft disease. METHODS AND RESULTS: MCP-1 expression is demonstrated in various stages of vein graft disease in a murine model in which venous interpositions are placed in the carotid arteries of hypercholesterolemic ApoE3Leiden mice and in cultured human saphenous vein (HSV) segments in which IH occurs. The functional involvement of MCP-1 in vein graft remodeling is demonstrated by blocking the MCP-1 receptor CCR-2 using 7ND-MCP-1. 7ND-MCP1 gene transfer resulted in 51% reduction in IH in the mouse model, when compared with controls. In HSV cultures neointima formation was inhibited by 53%. In addition, we demonstrate a direct inhibitory effect of 7ND-MCP-1 on the proliferation of smooth muscle cell (SMC) in HSV cultures and in SMC cell cultures. CONCLUSIONS: These data, for the first time, prove that MCP-1 has a pivotal role in vein graft thickening due to intimal hyperplasia and accelerated atherosclerosis.

Gene transfer of the urokinase-type plasminogen activator receptor-targeted matrix metalloproteinase inhibitor TIMP-1.ATF suppresses neointima formation more efficiently than tissue inhibitor of metalloproteinase-1.

Proteases of the plasminogen activator (PA) and matrix metalloproteinase (MMP) system play an important role in smooth muscle cell (SMC) migration and neointima formation after vascular injury. Inhibition of either PAs or MMPs has previously been shown to result in decreased neointima formation in vivo. To inhibit both protease systems simultaneously, a novel hybrid protein, TIMP-1.ATF, was constructed consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) domain, as MMP inhibitor, linked to the receptor-binding amino terminal fragment (ATF) of urokinase. By binding to the u-PA receptor this protein will not only anchor the TIMP-1 moiety directly to the cell surface, it will also prevent the local activation of plasminogen by blocking the binding of urokinase-type plasminogen activator (u-PA) to its receptor. Adenoviral expression of TIMP-1.ATF was used to inhibit SMC migration and neointima formation in human saphenous vein segments in vitro. SMC migration was inhibited by 65% in Ad.TIMP-1.ATF-infected cells. Infection with adenoviral vectors encoding the individual domains, Ad.TIMP-1 and Ad.ATF, reduced migration by 32% and 52%, respectively. Neointima formation in saphenous vein organ cultures infected with Ad.TIMP-1.ATF was inhibited by 72% compared with 42% reduction after Ad.TIMP-1 infection and 34% after Ad.ATF infection. These data show that binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface strongly enhances the inhibitory effect of TIMP-1 on neointima formation in human saphenous vein cultures.

The inactivation of single-chain urokinase-type plasminogen activator by thrombin on cultured human endothelial cells.

Single-chain urokinase-type plasminogen activator (scu-PA) is cleaved by thrombin, resulting in an inactive molecule called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T). There is no knowledge about cell-mediated inactivation of scu-PA. We have studied whether scu-PA bound to cultured human umbilical vein endothelial cells (HUVEC) could be inactivated by thrombin. High molecular weight scu-PA was bound to HUVEC and incubated with increasing amounts of thrombin for 30 min at 37 degrees C. Cell-bound urokinase-type plasminogen activator (u-PA) was released and levels of scu-PA, tcu-PA/T and active two-chain u-PA were measured using sensitive bioimmunoassays. Cell-bound scu-PA was efficiently inactivated by thrombin. Fifty percent inactivation of scu-PA occurred at about 0.2 nM thrombin. In the presence of monoclonal anti-urokinase receptor IgG, at least 50% of the binding of scu-PA to HUVEC was inhibited. The relative amount of tcu-PA/T that was generated by thrombin was not affected by the monoclonal antibody. These results indicated that scu-PA bound to HUVEC via the urokinase receptor can be inactivated by thrombin. The efficient inactivation of cell-bound scu-PA suggests that a cofactor for thrombin may be involved, like thrombomodulin or glycosaminoglycans. It is concluded that scu-PA bound to the urokinase receptor on a cell surface can be inactivated by thrombin, which may have profound effects on u-PA-mediated local fibrinolysis and extracellular proteolysis during processes in which thrombin is also involved.

Adenoviral expression of a urokinase receptor-targeted protease inhibitor inhibits neointima formation in murine and human blood vessels.

BACKGROUND: Smooth muscle cell migration, in addition to proliferation, contributes to a large extent to the neointima formed in humans after balloon angioplasty or bypass surgery. Plasminogen activator/plasmin-mediated proteolysis is an important mediator of this smooth muscle cell migration. Here, we report the construction of a novel hybrid protein designed to inhibit the activity of cell surface-bound plasmin, which cannot be inhibited by its natural inhibitors, such as alpha(2)-antiplasmin. This hybrid protein, consisting of the receptor-binding amino-terminal fragment of uPA (ATF), linked to the potent protease inhibitor bovine pancreas trypsin inhibitor (BPTI), can inhibit plasmin activity at the cell surface. METHODS AND RESULTS: The effect of adenovirus-mediated ATF.BPTI expression on neointima formation was tested in human saphenous vein organ cultures. Infection of human saphenous vein segments with Ad.CMV.ATF.BPTI (5x10(9) pfu/mL) resulted in 87.5+/-3.8% (mean+/-SEM, n=10) inhibition of neointima formation after 5 weeks, whereas Ad.CMV.ATF or Ad.CMV.BPTI virus had only minimal or no effect on neointima formation. The efficacy of ATF.BPTI in vivo was demonstrated in a murine model for neointima formation. Neointima formation in the femoral artery of mice, induced by placement of a polyethylene cuff, was strongly inhibited (93.9+/-2%) after infection with Ad.CMV.mATF.BPTI, a variant of ATF.BPTI able to bind specifically to murine uPA receptor; Ad.CMV.mATF and Ad.CMV.BPTI had no significant effect. CONCLUSIONS: These data provide evidence that adenoviral transfer of a hybrid protein that binds selectively to the uPA receptor and inhibits plasmin activity directly on the cell surface is a powerful approach to inhibiting neointima formation and restenosis.

Inhibition of accelerated atherosclerosis in vein grafts by placement of external stent in apoE*3-Leiden transgenic mice.

OBJECTIVE: Vein grafts fail because of the development of intimal hyperplasia and accelerated atherosclerosis. Placement of an external stent around vein grafts resulted in an inhibition of intimal hyperplasia in several animal studies. Here, we assess the effects of external stenting on accelerated atherosclerosis in early vein grafts in carotid arteries in hypercholesterolemic apolipoprotein E*3-Leiden transgenic mice. METHODS AND RESULTS: Venous interposition grafting was performed in apolipoprotein E*3-Leiden mice fed standard chow or a highly cholesterol-rich diet for 4 weeks. After engraftment, external stents with different inner diameters (0.4 or 0.8 mm) were placed. In unstented vein grafts in hypercholesterolemic mice, thickening up to 50 times the original thickness, with foam cell-rich lesions, calcification, and necrosis, was observed within 28 days. The atherosclerotic lesions observed show high morphological resemblance to atherosclerotic lesions observed in human vein grafts. In stented vein grafts in hypercholesterolemic mice, no foam cell accumulation or accelerated atherosclerosis was observed. Compared with unstented vein grafts, stenting of vein grafts in a hypercholesterolemic environment resulted in a 94% reduction of vessel wall thickening. These effects were independent of stent size. CONCLUSIONS: Extravascular stent placement results in strong inhibition of accelerated vein graft atherosclerosis in hypercholesterolemic transgenic mice and thereby provides a perspective for therapeutic intervention in vein graft diseases.

Adenoviral gene transfer of a u-PA receptor-binding plasmin inhibitor and green fluorescent protein: inhibition of migration and visualization of expression.

Smooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

Adenovirus-mediated transfer of the 39 kD receptor-associated protein increases fibrinolytic capacity.

BACKGROUND: The mesothelium has an important role in maintaining an adequate fibrinolytic capacity in the peritoneal cavity and thus in preventing the formation of fibrinous peritoneal adhesions by secreting the fibrinolytic enzyme tissue-type plasminogen activator (t-PA). The fibrinolytic activity of human mesothelial cells (HMCs) is counteracted by rapid uptake of t-PA via the low-density lipoprotein receptor-related protein (LRP). The 39 kD receptor-associated protein (RAP) is an inhibitor of binding of t-PA to LRP, but RAP itself is also rapidly degraded via LRP. METHODS: Adenovirus-mediated RAP gene transfer technology was used to evaluate the effect of prolonged overexpression of RAP on t-PA accumulation in conditioned medium of HMCs under basal and inflammatory conditions. RESULTS: Infection of HMCs with a recombinant adenovirus carrying the RAP cDNA resulted within one day in t-PA levels that were maximally twofold to threefold increased as compared with noninfected or adenovirus-beta-galactosidase-infected cells. Whereas upon prolonged incubation, t-PA levels in the conditioned medium of uninfected cells leveled off because of rapid uptake and degradation via LRP, t-PA concentrations in the medium of adenovirus-RAP-infected cells continued to increase, reaching fivefold control levels after 72 hours. The increased t-PA accumulation persisted for seven days and then slowly returned to control values over the next few weeks. In contrast, the production of a specific inhibitor of t-PA, plasminogen activator inhibitor-1 (PAI-1), was not affected by adenoviral RAP gene transfer. Northern blotting analysis showed that t-PA, PAI-1, and LRP mRNA concentrations were not changed after adenoviral infection, underlining that the elevated t-PA levels are the result of RAP-blocked uptake and degradation of t-PA rather than increased t-PA synthesis. RAP gene transfer also restored diminished fibrinolytic activity of cytokine-treated mesothelial cells. CONCLUSIONS: Adenovirus-mediated transfer of the RAP gene provides an efficient way of transiently increasing the fibrinolytic capacity of mesothelial cells.

Improved adenovirus vectors for infection of cardiovascular tissues.

To identify improved adenovirus vectors for cardiovascular gene therapy, a library of adenovirus vectors based on adenovirus serotype 5 (Ad5) but carrying fiber molecules of other human serotypes, was generated. This library was tested for efficiency of infection of human primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Based on luciferase, LacZ, or green fluorescent protein (GFP) marker gene expression, several fiber chimeric vectors were identified that displayed improved infection of these cell types. One of the viruses that performed particularly well is an Ad5 carrying the fiber of Ad16 (Ad5.Fib16), a subgroup B virus. This virus showed, on average, 8- and 64-fold-increased luciferase activities on umbilical vein ECs and SMCs, respectively, compared to the parent vector. GFP and lacZ markers showed that approximately 3-fold (ECs) and 10-fold (SMCs) more cells were transduced. Experiments performed with both cultured SMCs and organ cultures derived from different vascular origins (saphenous vein, iliac artery, left interior mammary artery, and aorta) and from different species demonstrated that Ad5.Fib16 consistently displays improved infection in primates (humans and rhesus monkeys). SMCs of the same vessels of rodents and pigs were less infectable with Ad5.Fib16 than with Ad5. This suggests that either the receptor for human Ad16 is not conserved between different species or that differences in the expression levels of the putative receptor exist. In conclusion, our results show that an Ad5-based virus carrying the fiber of Ad16 is a potent vector for the transduction of primate cardiovascular cells and tissues.

Binding of human urokinase-type plasminogen activator to its receptor: residues involved in species specificity and binding.

Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to UPAR appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild-type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated 125I-labeled mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate-treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated 125I-labeled mutant UPA could not bind to phorbol myristate acetate-treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated 125I-labeled wild-type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a very small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR could be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no effect on binding to UPAR on human ECs. The functional integrity of the growth factor domain in the non-receptor binding Asn22Tyr mutant is suggested by the fact that binding of this mutant to a murine UPAR can be restored after additional mutations in the growth factor domain, Asn27,His29,Trp30 to Arg27,Arg29,Arg30. We conclude that Asn22 and Asn27,His29,Trp30 in human UPA are key determinants in the species-specific binding of the enzyme to its receptor and that changing Asn22 into Tyr results in a UPA mutant with strongly reduced binding to UPAR.

Simultaneous detection of NOS-3 protein expression and nitric oxide production using a flow cytometer.

Nitric oxide (NO) is involved in the regulation of SMC proliferation during intimal hyperplasia as has been shown by the inhibitory effect on intimal hyperplasia of adenovirus-mediated ceNOS overexpression in injured arteries in pig. Good assays to quantify the NO-producing enzymes, i.e., NO synthases (NOS), are essential to analyze the mechanism of action of NO in this process. We have developed novel flow cytometric assays for the simultaneous detection of NOS-3 protein, using NOS-3 specific antibodies, and NO production using 4,5-diaminofluorescein-diacetate (DAF-2/DA). The presence of NOS-3 protein and NO production is demonstrated on human A549 and HepG2 cells infected with a NOS-3 adenovirus (Ad.NOS-3). A comparative study showed that the flow cytometric assays are equally sensitive as Western blot analysis, the citrulline assay, or the Sievers assay. On human endothelial and SMC, NOS-3 protein and NO production were simultaneously detected with the assays, both under basal conditions and after Ad.NOS-3transduction. Simultaneous analysis of NOS-3 protein and NO production, made possible by the here-described novel flow cytometric assays, is of significant value to those investigating NOS-3 and NO.

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of a cell surface-targeted plasmin inhibitor.

OBJECTIVE: Joint destruction in rheumatoid arthritis (RA) is a result of degradation and invasion of the articular cartilage by the pannus tissue. The present study was undertaken to examine the role of the plasminogen activation system in cartilage degradation and invasion by synovial fibroblasts and investigate a novel gene therapeutic approach using a cell surface-targeted plasmin inhibitor (ATF.BPTI). METHODS: Adenoviral vectors were used for gene transfer. The effects of ATF.BPTI gene transfer on RA synovial fibroblast-dependent cartilage degradation were studied in vitro, and cartilage invasion was studied in vivo in the SCID mouse coimplantation model. RESULTS: The results indicate that cartilage matrix degradation by rheumatoid synovial fibroblasts is plasmin mediated and depends on urokinase-type plasminogen activator for activation. Targeting plasmin inhibition to the cell surface of the fibroblasts by gene transfer of a cell surface-binding plasmin inhibitor resulted in a significant reduction of cartilage matrix degradation in vitro and of cartilage invasion in vivo. Compared with uninfected rheumatoid synovial fibroblasts, the mean +/-SEM cartilage degradation in vitro was reduced to 87.9+/-0.9% after LacZ gene transfer versus a reduction to 24.0+/-1.6% after ATF.BPTI gene transfer (P<0.0001). The mean +/- SEM in vivo cartilage invasion score was 3.1+/-0.4 in the control-transduced fibroblasts and 1.8+/-0.4 in the ATF.BPTI-transduced fibroblasts (P<0.05). CONCLUSION: These results indicate a role of the plasminogen activation system in synovial fibroblast-dependent cartilage degradation and invasion in RA, and demonstrate an effective way to inhibit this by gene transfer of a cell surface-targeted plasmin inhibitor.

Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells.

The chicken anemia virus protein apoptin induces a p53-independent, Bcl-2-insensitive type of apoptosis in various human tumor cells. Here, we show that, in vitro, apoptin fails to induce programmed cell death in normal lymphoid, dermal, epidermal, endothelial, and smooth-muscle cells. However, when normal cells are transformed they become susceptible to apoptosis by apoptin. Long-term expression of apoptin in normal human fibroblasts revealed that apoptin has no toxic or transforming activity in these cells. In normal cells, apoptin was found predominantly in the cytoplasm, whereas in transformed and malignant cells it was located in the nucleus, suggesting that the localization of apoptin is related to its activity. These properties make apoptin a potential agent for the treatment of a large number of tumors, also those lacking p53 and/or overexpressing Bcl-2.

In vivo suppression of restenosis in balloon-injured rat carotid artery by adenovirus-mediated gene transfer of the cell surface-directed plasmin inhibitor ATF.BPTI.

Injury-induced neointimal development results from migration and proliferation of vascular smooth muscle cells (SMC). Cell migration requires controlled proteolytic degradation of extracellular matrix surrounding the cell. Plasmin is a major contributor to this process by degrading various matrix proteins directly, or indirectly by activating matrix metalloproteinases. This makes it an attractive target for inhibition by gene transfer. An adenoviral vector, Ad.ATF.BPTI, was constructed encoding a hybrid protein, which consists of the aminoterminal fragment (ATF) of urokinase-type plasminogen activator (u-PA) linked to bovine pancreas trypsin inhibitor (BPTI), a potent inhibitor of plasmin. This hybrid protein binds to the u-PA receptor, thereby inhibiting plasmin activity at the cell surface, and was found to be a potent inhibitor of cell migration in vitro. Local infection with Ad.ATF.BPTI of balloon-injured rat carotid artery resulted in detectable expression of ATF.BPTI mRNA and protein in the vessel wall. Morphometric analysis of arterial cross-sections revealed that delivery of Ad.ATF.BPTI to the carotid artery wall at the time of balloon injury inhibited neointima formation by 53% (P < 0.01) at 14 days and 19% (P = NS) at 28 days after injury when compared with control vector-infected arteries. Intima/media ratios were decreased by 60% (P < 0.01) and 35% (P < 0.05) at 14 and 28 days, respectively, when compared with control vector-infected arteries. Furthermore, a small but significant increase in medial area was found in the Ad.ATF.BPTI-treated arteries at 28 days (P < 0.05). These results show that local infection of the vessel wall with Ad.ATF.BPTI reduces neointima formation, presumably by inhibiting SMC migration, thereby offering a novel therapeutic approach to inhibiting neointima development.

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3.

Matrix metalloproteinases (MMPs) are believed to be pivotal enzymes in the invasion of articular cartilage by synovial tissue in rheumatoid arthritis (RA). Here, we investigated the effects of gene transfer of tissue inhibitors of metalloproteinases (TIMPs) on the invasiveness of RA synovial fibroblasts (RASF) in vitro and in vivo. Adenoviral vectors (Ad) were used for gene transfer. The effects of AdTIMP-1 and AdTIMP-3 gene transfer on matrix invasion were investigated in vitro in a transwell system. Cartilage invasion in vivo was studied in the SCID mouse co-implantation model for 60 days. In addition, the effects of AdTIMP-1 and AdTIMP-3 on cell proliferation were investigated. A significant reduction in invasiveness was demonstrated in vitro as well as in vivo in both the AdTIMP-1- and AdTIMP-3-transduced RASF compared with untransduced SF or SF that were transduced with control vectors. in vitro, the number of invading cells was reduced to 25% (P<0.001) in the AdTIMP-1-transduced cells and to 13% (P<0.0001) in the AdTIMP-3-transduced cells (% of untransduced cells). Cell proliferation was significantly inhibited by AdTIMP-3 and, less, by AdTIMP-1. In conclusion, overexpression of TIMP-1 and TIMP-3 by Ad gene transfer results in a marked reduction of the invasiveness of RASF in vitro and in the SCID mouse model. Apart from the inhibition of MMPs, a reduction in proliferation rate may contribute to this effect. These results suggest that overexpression of TIMPs, particularly TIMP-3 at the invasive front of pannus tissue, may provide a novel therapeutic strategy for inhibiting joint destruction in RA.