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.

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 coimplantation 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% (Po0.001) in the AdTIMP-1-transduced cells and to 13% (Po0.0001) 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.

Background: Formation of deposits of the insoluble amyloid ␤-peptide is believed to be causally related with neurodegeneration in Alzheimer disease (AD). The ␤-peptide originates from a larger amyloid precursor protein (APP) by the action of proteolytic enzymes. The first proteolytic event leading to amyloid formation is the cleavage of APP by the membrane-bound aspartyl protease BACE-1, also known as memapsin-2. Inhibition of BACE-1 is thought to be a therapeutic approach to AD. Measuring BACE-1 activity in biological samples would be useful to elucidate the mechanism of AD and for development of AD drugs. Methods: We developed a sensitive and specific activity assay for BACE-1. The assay is based on a genetically engineered proenzyme that is specifically activated by BACE-

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. Conclusion. These results indicate a role of the plasminogen activation system in synovial fibroblastdependent cartilage degradation and invasion in RA, and demonstrate an effective way to inhibit this by gene transfer of a cell surface-targeted plasmin inhibitor.

SummaryAn enzyme immuno assay was developed to measure complexes of tissue-type plasminogen activator (t-PA) with C1-inhibitor in order to study the role of C1-inhibitor as an inhibitor of t-PA in plasma. In vitro experiments with melanoma and recombinant t-PA learned that purified C1-inhibitor reacts with both single chain t-PA and two chain t-PA. The rate constants ranged from 3.0 to 5.2 M-1s-1 In plasma, melanoma and recombinant two chain t-PA were hardly inhibited by C1-inhibitor, in contrast to melanoma and recombinant single chain t-PA which were inhibited to the same extent by endogenous C1-inhibitor as they were by purified C1-inhibitor. In vivo, t-PA/C1-inhibitor complex could be measured in plasma in a few cases in healthy volunteers (0.62 ± 0.43 ng/ml t-PA equivalents), after exercise (0.84 ± 0.25 ng/ml t-PA equivalents) and after a desmopressin infusion (0.26 ± 0.04 ng/ml t-PA equivalents). However, t-PA/C1-inhibitor complex was found in plasma in all cases after venous occlusion (1.7 ± 0.5 ng/ml t-PA equivalents), in peritoneal fluid from patients suffering from peritoneal inflammatory disease (2.2 ± 1.3 ng/ml t-PA equivalents) and in plasma from healthy volunteers during a t-PA infusion (27.7 ± 18.5 ng/ml t-PA equivalents at peak level). In the last case, about 8 % of the infused dose of recombinant t-PA (alteplase) was inhibited by C1-inhibitor at peak level. The half-life (t1/2α) of t-PA antigen in plasma was found not to be altered when t-PA was inhibited by C1-inhibitor (4.0 min and 4.2 min, respectively). Thus, in vivo, t-PA/C1-inhibitor complex is mostly present when t-PA escapes rapid liver clearance and accumulates in one place (e.g. during venous occlusion or in peritoneal fluid) or when it circulates in high concentrations (e.g. during t-PA infusion).