Intravascular adenovirus-mediated VEGF-C gene transfer reduces neointima formation in balloon-denuded rabbit aorta.

BACKGROUND: Gene transfer to the vessel wall may provide new possibilities for the treatment of vascular disorders, such as postangioplasty restenosis. In this study, we analyzed the effects of adenovirus-mediated vascular endothelial growth factor (VEGF)-C gene transfer on neointima formation after endothelial denudation in rabbits. For comparison, a second group was treated with VEGF-A adenovirus and a third group with lacZ adenovirus. Clinical-grade adenoviruses were used for the study. METHODS AND RESULTS: Aortas of cholesterol-fed New Zealand White rabbits were balloon-denuded, and gene transfer was performed 3 days later. Animals were euthanized 2 and 4 weeks after the gene transfer, and intima/media ratio (I/M), histology, and cell proliferation were analyzed. Two weeks after the gene transfer, I/M in the lacZ-transfected control group was 0. 57+/-0.04. VEGF-C gene transfer reduced I/M to 0.38+/-0.02 (P:<0.05 versus lacZ group). I/M in VEGF-A-treated animals was 0.49+/-0.17 (P:=NS). The tendency that both VEGF groups had smaller I/M persisted at the 4-week time point, when the lacZ group had an I/M of 0.73+/-0.16, the VEGF-C group 0.44+/-0.14, and the VEGF-A group 0. 63+/-0.21 (P:=NS). Expression of VEGF receptors 1, 2, and 3 was detected in the vessel wall by immunocytochemistry and in situ hybridization. As an additional control, the effect of adenovirus on cell proliferation was analyzed by performing gene transfer to intact aorta without endothelial denudation. No differences were seen in smooth muscle cell proliferation or I/M between lacZ adenovirus and 0.9% saline-treated animals. CONCLUSIONS: Adenovirus-mediated VEGF-C gene transfer may be useful for the treatment of postangioplasty restenosis and vessel wall thickening after vascular manipulations.

Distribution of hyaluronan in articular cartilage as probed by a biotinylated binding region of aggrecan.

The proportion of total tissue hyaluronan involved in interactions with aggrecan and link protein was estimated from extracts of canine knee articular cartilages using a biotinylated hyaluronan binding region-link protein complex (bHABC) of proteoglycan aggregate as a probe in an ELISA-like assay. Microscopic sections were stained with bHABC to reveal free hyaluronan in various sites and zones of the cartilages. Articular cartilage, cut into 20 microns-thick sections, was extracted with 4 M guanidinium chloride (GuCl). Aliquots of the extract (after removing GuCl) were assayed for hyaluronan, before and after papain digestion. The GuCl extraction residues were analyzed after solubilization by papain. It was found that 47-51% of total hyaluronan remained in the GuCl extraction residue, in contrast to the 8-15% of total proteoglycans. Analysis of the extract revealed that 24-50% of its hyaluronan was directly detectable with the probe, while 50-76% became available only after protease digestion. The extracellular matrix in cartilage sections was stained with the bHABC probe only in the superficial zone and the periphery of the articular surfaces, both sites known to have a relatively low proteoglycan concentration. Trypsin pretreatment of the sections enhanced the staining of the intermediate and deep zones, presumably by removing the steric obstruction caused by the chondroitin sulfate binding region of aggrecans. Enhanced matrix staining in these zones was also obtained by a limited digestion with chondroitinase ABC. The results indicate that a part of cartilage hyaluronan is free from endogenous binding proteins, such as aggrecan and link protein, but that the chondroitin sulfate-rich region of aggrecan inhibits its probing in intact tissue sections. Therefore, hyaluronan staining was more intense in cartilage areas with lower aggrecan content. A large proportion of hyaluronan resists GuCl extraction, even from 20-micrograms-thick tissue sections.

Adaptation of canine femoral head articular cartilage to long distance running exercise in young beagles.

OBJECTIVE: To study the effects of long term (one year), long distance (up to 40 km/day) running on the metabolism of articular cartilage the biosynthesis of proteoglycans was examined by in vitro labelling of anterior (weight bearing) and posterior (less weight bearing) areas of the femoral head from young beagles. METHODS: Total sulphate incorporation rates were determined and distribution of the incorporated sulphate was localised by quantitative autoradiography. Concentration and extractability of the proteoglycans were determined, and proteoglycan structures were investigated by gel filtration chromatography, agarose gel electrophoresis, and chemical determinations. RESULTS: In the less weight bearing area the amount of extractable proteoglycans was decreased (p < or = 0.02), simultaneously with an increased concentration of residual glycosaminoglycans in the tissue after 4 M GuCl extraction (p < or = 0.05). In control animals proteoglycan synthesis was most active in the deep zone of the cartilage, whereas exercise increased synthesis in the intermediate zone. There was a tendency to a lower keratan: chondroitin sulphate ratio in the running dogs. No macroscopical or microscopical signs of articular degeneration or injury were visible in any of the animals. CONCLUSION: The articular cartilage of the femoral head showed a great capacity to adapt to the increased mechanical loading. The reduced proteoglycan extractability in the less weight bearing area changed it similar to the weight bearing area, suggesting that the low extractability of proteoglycans reflects the long term loading history of articular cartilage. The congruency of the femoral head with acetabulum seems to protect the cartilage from the untoward alterations that occur in the femoral condyles subjected to a similar running programme.

Effects of long-term running exercise on canine femoral head articular cartilage.

After long-term running program (40 km/day) anterior and posterior tissue samples from canine femoral head were labeled ex vivo in the presence of 35S-SO4. Sulfate incorporation rates did not differ between runner and control groups. The statistically significant changes in runners included a decreased uronic acid concentration (p < or = 0.02) and proportion of extractable proteoglycans (p < or = 0.05) as well as increased concentration of tissue uronic acid after 4 M GuCl extraction (p < or = 0.05) in the posterior area. These results support an idea of strengthened cartilage tissue after this kind of motion and load.