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Inhibition of angiogenesis is a promising approach for the treatment of solid tumors, inflammatory diseases and different hematological malignancies such as multiple myeloma. One of the central molecules in capillary formation during angiogenesis is the integrin alphaVbeta3 (aVb3), therefore aVb3 is a potential target molecule to inhibit angiogenesis. The aim of this study was to inhibit alphaV-mediated angiogenesis in vitro using RNA interference (RNAi) technology as well as antisense oligodeoxyribonucleotides (asON). We used synthetic small interfering RNAs (siRNA) and asON directed against the alphaV chain of aVb3 to inhibit integrin expression. Five siRNAs were selected on the basis of a systematic alignment of computer-predicted secondary structures of target mRNA and on the basis of current recommendations for siRNA oligonucleotide design. In parallel, 3 asON were examined. They had the sequence of the antisense sequence of 3 of the siRNAs molecules, respectively. SiRNAs, asON and respective control sequences were transfected into human umbilical vein endothelials cells (HUVEC) using lipofection. Following stimulation by phorbol 12-myristate 13-acetate (PMA), two siRNAs showed a dose- and time dependent inhibition of PMA-induced aV-mRNA and -protein upregulation as assessed by real-time RT-PCR and flow cytometry. At a concentration of 25 nM a 100% (SD: 4.9%) inhibition of aV upregulation was observed, whereas transfection of the respective asON sequences resulted in a 63% (SD: 6.1%) inhibition of aV upregulation at 25nM. To evaluate the anti-angiogenic potential of siRNAs in comparison to asON a cell culture model of human angiogenesis based on the co-cultivation of endothelial cells and dermal fibroblasts was used. Transfection of the most efficient siRNA sequence at a concentration of 50nM resulted in an inhibition of total length of capillary-like tubules by 48.7% (SD: 3.6%) in comparison to 21.8% (SD: 9.8%) by the respective asON sequence treated cultures. In conclusion, siRNAs can successfully be selected on the basis of computer-predicted secondary structures. In comparison with asON having the same sequence as the antisense strand of the respective siRNA the siRNA-mediated inhibition of aV expression showed a stronger inhibition of capillary tube formation in an angiogenesis in vitro assay. Therefore, siRNAs are useful tools for functional aV knock-down experiments and might be a therapeutic alternative for antagonists which bind directly to the integrins aVb3 or aVb5.

In this study, we elucidated the potential of recombinant adeno-associated virus type-2 (rAAV-2) vectors for lung cancer gene therapy. Cell lines of the three major histological subtypes of non-small cell lung cancer (NSCLC) were highly susceptible for rAAV-2 showing transduction rates between 63.4 and 98.9%. In contrast, cell lines of small cell carcinomas were resistant to rAAV-2 infection. For restoration of p53 function in p53 deficient NSCLC, a rAAV-2 vector was constructed containing wt p53 cDNA. Following transduction with rAAV-p53, cell growth of all NSCLC cell lines was significantly reduced in a dose-dependent manner between 44 and 71.7% in comparison with rAAV-GFP transduced cells. The reduction of tumor cell growth was associated with increased apoptosis. Adding cisplatin to rAAV-p53-infected cells led to a significant growth inhibition between 81 and 91% indicating a synergistic effect between cisplatin and rAAV-p53. Interestingly, the tumor cells surviving cisplatin and rAAV-p53 treatment were inhibited in their ability to form colonies as reflected by a reduction of colony growth between 57 and 90.4%. In conclusion, rAAV-2 vectors exhibit a strong tropism for NSCLC. Successful inhibition of tumor cell growth following transduction with a rAAV-p53 vector underlines the potential role of rAAV-2 in cancer gene therapy.

The integrin alpha v beta 3 plays a central role in angiogenesis. In this study, we used antisense oligodeoxyribonucleotides (ONs) directed against the alpha v subunit of alpha v beta 3 to inhibit integrin expression. Ten ON sequences, which were selected by systematic alignment of computer-predicted secondary structures of alpha v mRNA, were transfected into human umbilical vein endothelial cells (HUVECs). Following stimulation by PMA, five antisense ONs significantly inhibited alpha v mRNA and protein expression in activated HUVEC at a concentration of 0.05 mciroM with complete prevention of PMA-induced alpha v up-regulation by the most potent antisense ON. Inhibition of alpha v expression was associated with significant inhibition of migration of HUVEC by 28% and had no effect on proliferation and apoptosis. Moreover, transfection of antisense ON inhibited the formation of tube-like structures of HUVEC in Matrigel by 44%. In a cell culture model of angiogenesis consisting of a co-culture of endothelial cells with fibroblasts, transfection of antisense ONs resulted in an inhibition of tube formation of 61%. In conclusion, alpha v antisense ONs are potent inhibitors of angiogenesis in vitro. They might, therefore, be a therapeutic alternative to antagonists, which directly bind to alpha v integrins, and might be useful for the treatment of malignant tumors and hematological malignancies.

Acute graft-versus-host disease (aGVHD) is a serious complication of allogeneic peripheral blood stem cell transplantation (PBSCT). Patients with severe aGVHD not responding to treatment with steroids have a poor prognosis. We treated four patients with severe aGVHD refractory to steroids with infliximab, a chimeric human/mouse antiTNFalpha antibody. Patients (CML 2, MM 1, AML 1) developed grade III-IV GVHD at a median of 34 days (range 15-76) after myeloablative PBSCT (two), donor lymphocyte infusion for relapsed CML (one) or non-myeloablative PBSCT (one), respectively. All patients had severe intestinal involvement in addition to skin and/or liver disease and had received treatment with high-dose steroids (four) for a median of 11 days (range 5-17) in addition to CsA (four) and MMF (three). Infliximab (10 mg/kg) was given once a week until clinical improvement. In three of four patients a complete resolution of diarrhea and significant improvement of skin and liver disease were observed. Two patients received one, one patient two and one patient three infliximab infusions. At present two patients are alive >200 days after therapy, one with limited cGVHD. Two patients died, one of progressive malignant disease without GVHD and one of refractory GVHD. Infliximab is apparently an active drug for the treatment of aGVHD.