selleck Sorafenib Up regulated angiogenic growth fac tors included Hgf, insulin like growth factor 1, mid kine and angiopoietin 1, whereas leptin and epidermal growth factor were decreased in arthritic paws. No difference for total Vegf a transcript levels could be detected between arthritic and control tissue, whereas its receptors Flk 1 and neuropilin 2 were increased in arthritic mouse paws. Interestingly, the expression levels of genes that play a role in inhibiting angiogenesis were also increased during CIA. Gene specific real time RT PCR for selected genes using samples from individual animals was performed to validate the observed trends in the array. Inhibitors,Modulators,Libraries For this pur pose, samples Inhibitors,Modulators,Libraries were obtained from arthritic animals on day 1, day 4, day 8 and day 12 of CIA, as well as from mice immunised with CFA IFA without CII.
To investigate pos sible alterations before the onset of clinical arthritis, we included a group of mice that were sacrificed 14 days after immunisation with collagen but without macro scopic signs of CIA. The expression Inhibitors,Modulators,Libraries of target genes was normalised to Arp expression, which displayed expression stability among the different condi tions. Figure 3 illustrates the kinetics of mRNA expression for Hgf, Igf 1, Mdk, Tnfa, Il 1b and Il 6 in arthritic tissue. Their temporal expression pattern reflected disease activity, with most prominent changes occurring at the peak phases of CIA, with mean clinical scores SEM for the analysed paw of 1 0. 00 on day 1, 1. 75 0. 25 on day 4, and 2. 14 0. 37 on day 8.
A significant increase in transcript levels could be observed on day 8 for all genes, with mRNA levels gradually decreasing on day 12, which is consis tent with the array results. We have Inhibitors,Modulators,Libraries previously reported that expression of Ang 1, Ang 2, tyrosine kinase with immunoglobulin like and EGF like domains 1 and Tie 2 increases as arthritis progressed, peaking on day 8 of disease. Of these, Ang 2 and Tie 2 showed modest changes on the array, whereas Ang 1 increased 2. 7 fold on day 8. Unexpectedly, the array identified the expression of total Vegf a mRNA as unchanged in arthritic tissue, despite strong evidence supporting a role Inhibitors,Modulators,Libraries for VEGF in both animal models of disease and human RA. Therefore, we examined the expression of dif ferent VEGF isoforms using samples from individual ani mals by gene specific RT PCR.
Transcript levels for Vegf120 and Vegf164 were significantly elevated on day 8 of arthritis compared to pre arthritic animals. However, the changes were only minor. The expression of Vegf188 did not change during the course of CIA. No significant change in the mRNA level neither for placental growth factor could be observed at the analysed time points. For the regula tion of angiogenesis, not only the expression of growth factors is important, but also the expression of the respective receptors.