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1.  Suppression of murine collagen-induced arthritis by targeted apoptosis of synovial neovasculature 
Arthritis Research  2001;3(6):357-361.
Because angiogenesis plays a major role in the perpetuation of inflammatory arthritis, we explored a method for selectively targeting and destroying new synovial blood vessels. Mice with collagen-induced arthritis were injected intravenously with phage expressing an RGD motif. In addition, the RGD peptide (RGD-4C) was covalently linked to a proapoptotic heptapeptide dimer, D(KLAKLAK)2, and was systemically administered to mice with collagen-induced arthritis. A phage displaying an RGD-containing cyclic peptide (RGD-4C) that binds selectively to the αvβ3 and αvβ5 integrins accumulated in inflamed synovium but not in normal synovium. Homing of RGD-4C phage to inflamed synovium was inhibited by co-administration of soluble RGD-4C. Intravenous injections of the RGD-4C–D(KLAKLAK)2 chimeric peptide significantly decreased clinical arthritis and increased apoptosis of synovial blood vessels, whereas treatment with vehicle or uncoupled mixture of the RGD-4C and the untargeted proapoptotic peptide had no effect. Targeted apoptosis of synovial neovasculature can induce apoptosis and suppress clinical arthritis. This form of therapy has potential utility in the treatment of inflammatory arthritis.
PMCID: PMC64846  PMID: 11714389
angiogenesis; apoptosis; collagen-induced arthritis; rheumatoid arthritis
2.  Apoptosis and p53 expression in rat adjuvant arthritis 
Arthritis Research  2000;2(3):229-235.
The kinetics of apoptosis and the apoptosis-regulating gene p53 in adjuvant arthritis (AA) were investigated to assess the value of the AA rat model for testing apoptosis-inducing therapies. Very few terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL)-positive cells were detected during the early phases of AA, but on day 23 (chronic arthritis) the percentage of TUNEL-positive cells was significantly increased. Expression of p53 in synovial tissue gradually increased from days 5-23, which was markedly higher than p53 levels in rheumatoid arthritis (RA) synovium. Significant apoptosis only occurs late in rat AA and is concordant with marked p53 overexpression, making it useful model for testing proapoptotic therapies, but rat AA is not the best model for p53 gene therapy because dramatic p53 overexpression occurs in the latter stages of the disease.
Introduction:
RA is a chronic inflammatory disorder that is characterized by inflammation and proliferation of synovial tissue. The amount of DNA fragmentation is significantly increased in rheumatoid synovium. Only low numbers of apoptotic cells are present in rheumatoid synovial tissue, however. The proportion of cells with DNA strand breaks is so great that this disparity suggests impaired apoptosis. Therefore, the development of novel therapeutic strategies that are aimed at inducing apoptosis in rheumatoid synovial tissue is an attractive goal.
Although animal models for arthritis only approximate RA, they provide a useful test system for the evaluation of apoptosis-inducing therapies. AA in rats is among the most commonly used animal models for RA. For the interpretation of such studies, it is essential to characterize the extent to which apoptosis occurs during the natural course of the disease. Therefore, we evaluated the number of apoptotic cells and the expression of p53 in various phases of AA.
Materials and methods:
In order to generate the AA rat model, Lewis rats were immunized with Mycobacterium tuberculosis in mineral oil on day 0. Paw swelling usually started around day 10. For the temporal analysis rats were sacrificed on days 0, 5 (prearthritis), 11 (onset of arthritis), 17 (accelerating arthritis), or 23 (chronic arthritis).
For the detection of apoptotic cells, the hind paws were harvested on days 0(n=6),5 (n=6), 11 (n=6), 17 (n=6), or 23 (n=4). The right ankle joints were fixed in formalin, decalcified in ethylenediaminetetra-acetic acid, embedded in paraffin, and sectioned. The TUNEL method was applied. The percentage of TUNEL-positive cells of the total inflammatory cell infiltrate was noted.
For Western blot analysis, hind paws were harvested on days 0 (n=2), 5 (n=3), 11 (n=4), 17 (n=4), or 23 (n=4). In addition, hind paws of normal rats (n=2) were studied. The right ankle joints were snap frozen and pulverized. Synovial tissue was also obtained by arthroscopy of three patients with longstanding (>5 years) RA. After protein extraction in lysis buffer, equal amounts of protein samples from lysates were pooled and examined by Western bolt analysis using anti-p53 monoclonal antibody D07, which recognizes wild-type and mutant p53 from rodents and humans.
For immunohistochemical analysis, six rats were sacrificed on day 23 after immunization and synovial tissue of the right ankle joints was snap frozen and evaluated by immunohistochemistry using anti-p53-pan. The sections were evaluated semi-quantitatively using a 0-4 scale.
The kruskal-Wallis test for several group means was used to compare the percentage of TUNEL-positive cells at different time points.
Results:
The percentages of TUNEL-positive cells were strongly dependent on the stage of the disease. Very few TUNEL-positive cells were detected in normal rats or in the early phases of AA; the number of TUNEL-positive cells was 1% or less of the total cell infiltrate, including neutrophils, from days 0-17 (Table 1). On day 23, however, the percentage of TUNEL-positive cells was significantly increased [15.8±5.1% (mean ± standard error of the mean); P=0.01]. TUNEL-positive cells were observed in the intimal lining layer and synovial sublining of the invasive front, as well as in the articular cartilage (Fig. 1).
Subsequently, we examined expression of the tumor suppressor gene p53, because this is a key regulator of apoptosis. Expression of p53 in pooled rat AA joint extracts gradually increased from day 0 (6 arbitrary units) to day 23 (173 arbitrary units), which was markedly higher than p53 levels in RA synovium (32 arbitrary units; Table 1). Overexpression of p53 protein on day 23 was confirmed by immunohistochemistry in a separate experiment in six rats with AA. Overexpression of p53 was observed in the intimal lining layer and synovial sublining in all rats on day 23. In all cases a semiquantitative score of 4 was assigned, indicating that 51% or more of the cells were positive, whereas control sections were negative.
Discussion:
The results presented here reveal that the number of TUNEL-positive cells remained very low until chronic arthritis developed. This indicates that, although there was sufficient DNA damage to cause an increment in p53 expression in the early phases, DNA strand breaks that can be detected by TUNEL assays only occurred in chronic AA. The observation that TUNEL-positive cells were nearly absent in early AA clearly indicates that only very few cells were undergoing programmed cell death. This is an important observation, which makes it possible to study the effects of apoptosis-inducing therapies in situ in early and accelerating AA. An effective therapy would obviously increase the number of TUNEL-positive cells.
There is already some overexpression of p53 in the preclinical phase and during the onset of the arthritis, with an additional increment in p53 expression during accelerating and chronic arthritis. Presumably, this is wild-type p53, because the disease duration is likely too short to allow for the development of p53 mutations. Transcription of p53 is probably increased in response to the toxic environment of the inflamed joint. The increased expression of p53 in the joints of rats with chronic AA was even greater than that observed in synovial tissue of RA patients with long-standing disease.
Overexpression of p53 and increased numbers of apoptotic cells did not occur simultaneously in this model; rather p53 overexpression preceded increased apoptosis. Activation of p53 leads to induction of cell growth arrest, allowing time for DNA repair. It appears that DNA damage is only extensive enough to induce apoptosis in the latter stages of AA. Factors other than p53 may also play an important role in the actual induction of apoptosis
Taken together, significant apoptosis only occurs late in AA and it follows marked p53 overexpression, making it a useful model for testing proapoptotic therapies. AA is not the best model for p53 gene therapy, however, because dramatic p53 overexpression occurs in the latter stages of the disease.
PMCID: PMC17810  PMID: 11056668
adjuvant arthritis; apoptosis; p53; rheumatoid arthritis

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