Introduction: Inflammation and angiogenesis are hallmarks of rheumatoid arthritis (RA) that contribute largely to the formation of pannus tissue and joint destruction in patients suffering from RA. We have recently shown that intravenously applied cationic liposomes target efficiently angiogenic endothelial cells in the synovial vasculature of rheumatoid joints and therefore may also serve as potent vehicles for systemic drug delivery and therapy in RA. Therefore the aim of our study was to quantify the antiangiogenic and antiinflammatory properties of EndoTAG-1^® (paclitaxel formulated in cationic liposomes) in the inflamed joints of murine models of RA and to compare the therapeutical efficacy of EndoTAG-1^® to Taxol^® (paclitaxel in Cremophor EL).

Materials and Methods: Targeting of fluorescently labelled cationic liposomes to the synovial vasculature in mice with antigen-induced arthritis (AIA) was analysed by intravital microscopy. Density of functional vessels and adhesion of fluorescently labelled platelets or leukocytes were determined after treatment with EndoTAG-1^®. Knees were subjected to clinical scoring and histopathological analysis.

Results: EndoTAG-1^® treatment of AIA mice with developing or in established disease showed a strong attenuation of the course of the disease as well as a potent anti-inflammatory effect. Histological analysis of knee sections demonstrated a dramatic reduction of the pannus and infiltration of inflammatory cells. Enrichment of EndoTAG at the synovial vasculature of AIA mice was observed when compared with healthy mice. Treatment of AIA mice with EndoTAG-1^® concomitant to disease induction showed a complete remission of the course of the disease as shown by a significant decrease of clinical scores compared to both control and Taxol^® treated groups. A complete inhibition (98%) of neo-vascularisation was observed in the synovial vasculature of mice with AIA that were treated with EndoTAG-1^® whereas Taxol^® alone showed only 50% inhibitory effect. Rolling and adhesion of platelets were reduced to 53% (paclitaxel 5%) and 98% (paclitaxel 57%), respectively.

Discussion: Our in vivo data clearly demonstrates that anti-angiogenic and anti-inflammatory activity of Endo-TAG-1^® contribute to the therapeutical efficacy of this drug in RA. Notably, therapeutic efficacy with Endo-TAG-1^® was superior to Taxol^®. This strongly suggests that systemic delivery of cationic liposomes is very well suited to enrich compounds to rheumatoid joints for therapy and could be a promising treatment option for RA.

Background: The healthy endothelium consists of quiescent endothelial cells that, under appropriate stimulation, can undergo profound changes leading to an activated phenotype. Activated endothelial cells of the synovial vasculature play a major role in the inflammatory process occurring in rheumatoid arthritis (RA) and enhanced angiogenesis contributes to the development and maintenance of RA. Thus, the endothelium can be used as an gateway for drug delivery and therapy. Cationic liposomes have been shown to target angiogenic endothelial cells in chronic inflammation in mice, also shown in tumors. They may also serve as potent vehicles for drug delivery to the synovial vasculature of rheumatoid joints.

Methods: To test whether cationic liposomes can serve as vehicles for drug delivery in RA we investigated the targeting of fluorescently labelled cationic liposomes (LipoRed) to the activated synovial vasculature of knees from arthritic mice. In a second step we used cationic liposomes carrying the compound MDG-12 (Endo-MDG-12) to study the effects of a targeted delivery of this drug to the inflamed joints in Antigen induced Arthritis (AIA). Targeting of LipoRed to the synovial vasculature was analysed by intravital microscopy (IVM) in mice with AIA. Synovial tissue was investigated at day 8 after AIA induction. Time resolved binding of liposomes was quantified at functional vessels of the microvasculature. Mice with AIA were intravenously treated with EndoMDG-12 in a therapeutic setting. Knee joints were subjected to clinical scoring and histopathology analysis.

Results: In a time dependent manner, intravenously applied LipoRed enriched more then three fold in the synovial vasculature of AIA mice when compared with healthy mice. In AIA animals maximum binding measured as relative fluorescence (Fmax=142 RFU) was already achieved 5 min after LiopoRed application (tmax) and dropped to the half maximum after 100 min (tmax/2)compared with healthy mice with a Fmax=48 RFU, tmax=15 min and tmax/2=60 min. Treatment of AiA animals with EndoMDG-12 showed a clear attenuation of the course of the disease. Analysis of the clinical score and thickness of knee joints showed a significant decrease of both parameters compared with the control group.

Conclusion: Based on our in vivo data, cationic liposomes seem to be very well suited to deliver compounds to rheumatoid joints for diagnosis and/or therapy. Furthermore, our results from animal experiments suggest that cationic liposomes like EndoMDG-12 could be a promising treatment option for RA.

Objectives: Platelets are suggested to participate in the pathogenesis of rheumatoid arthritis. We showed for the first time in vivo an increase of platelet-endothelial cell interactions in mice with Antigen-induced-Arthritis (AiA). However, underlying mechanisms are not clear so far. Therefore, the aim of the present study was to investigate first the impact of P-selectin on AiA and second the influence of platelet P-Selectin on the endothelial damage and activation of leukocytes by means of intravital microscopy.

Methods: In the first part C57/Bl6 and P-selectin deficient mice were disposed in four groups (n=7; control/AiA per strain). In the second part ex-vivo labelled platelets were transferred between different strains in AiA. The extent of AiA was assessed by knee joint swelling and by histological scoring. Platelet- and leukocyte-endothelium interactions were investigated.

Results: In arthritic P-selectin deficient mice compared to arthritic C57/Bl6 mice, platelet interaction was significantly reduced and reached the level of both control groups without AiA. In addition interaction of leukocytes in P-selectin deficient arthritic animals was significantly decreased in comparison to arthritic C57/Bl6 animals. Swelling of the knee joint and histological score was reduced in arthritic P-selectin deficient mice compared to arthritic C57/Bl6 mice. In the second part a significant increase of leukocyte-endothelial cell interaction in P-selectin deficient arthritic recipients was shown if P-selectin sufficient platelets were donated. If P-selectin deficient platelets were donated, the number of adherent leukocytes was significantly reduced.

Conclusion: We demonstrated for the first time in vivo a significant decrease of the interaction of platelets and leukocytes with the endothelium in P-selectin deficient mice with AiA and a reduction of clinical and histological symptoms of arthritis. Furthermore we showed that platelets provide storage of P-selectin for the recruitment of leukocytes in P-selectin deficient animals and therefore platelets seem to play a critical role in leukocyte-endothelial cell interaction. These findings suggest that P-Selectin is involved in the development and maintenance of arthritis and it could be at least partly responsible for the leukocyte tissue damage in AiA. Therefore, a reduced presence of P-selectin due to inhibition of platelets could be a new option for treatment of rheumatoid arthritis.

In orthopedic surgery, sterilization of bone used for reconstruction of osteoarticular defects caused by malignant tumors is carried out in different ways. At present, to devitalize tumor-bearing osteochondral segments, mainly extracorporal irradiation or autoclaving is used. Both methods have substantial disadvantages, e.g. loss of biomechanical and biological integrity of the bone. In particular integration at the autograft-host junction after reimplantation is often impaired due to alterations of the osteoinductivity following irradiation or autoclaving. As an alternative approach, high hydrostatic pressure (HHP) treatment of bone is a new technology, now being used in preclinical testing to inactivate tumor cells without alteration of biomechanical properties of bone, cartilage and tendons. The aim of this study was to investigate the influence of HHP on fibronectin (FN), vitronectin (VN), and type I collagen (col. I) as major extracellular matrix proteins of bone tissue, accountable among others for the osteoinductive properties of bone.

Fibronectin, vitronectin and type I collagen were subjected to HHP (300 and 600 MPa) prior to the coating of cell culture plates with these pre-treated proteins. Following the biological properties were measured by means of cell proliferation, adherence, and spreading of the human osteosarcoma cell line (Saos-2) and primary human osteoblast-like cells.

Up to 600 MPa all tested matrix proteins did not show any changes, regarding the biological properties adherence, spreading and proliferation.

We anticipate that, in orthopedic surgery, HHP can serve as a novel, promising methodical approach, by damaging normal and tumor cells without alteration of osteoinductive properties, thus facilitating osteointegration of the devitalized bone segment in cancer patients after reimplantation.