Introduction

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Proliferating tissues require angiogenesis to support their growth, and thus diseases such as cancer and chronic inflammation are thought to be angiogenesis dependent (Folkman, 1995; Jackson, 1996). In the case of chronic inflammation, angiogenesis may be required not only to support the proliferation but also to allow the massive cellular infiltration associated with the chronically inflamed state. Angiogenesis is normally under very tight control. In the normal adult, the majority of the vasculature is stable, with endothelial cell turnover on the order of thousands of days. Nevertheless, these quiescent cells can rapidly switch to an angiogenic phenotype under certain conditions, as, for example, in wound healing. Once a new capillary bed is established, however, the endothelium normally returns to its quiescent state.

Understanding the signals that regulate angiogenesis is key to controlling it under pathological conditions. Growth factors such as VEGF and FGF are clearly able to induce angiogenesis and, in the case of VEGF, appear to be regulated by physiological signals like hypoxia. Hypoxia is not always necessary, however; some inflammatory mediators can potently induce angiogenesis in vivo even in the absence of hypoxia. Both IL-1 and TNF- can induce angiogenesis in the normally avascular cornea (BenEzra et al., 1990; BenEzra and Maftzir, 1996; Fajardo et al., 1992). These two cytokines have numerous activities, including upregulation of other cytokines, such as IL-8; upregulation of adhesion molecule expression; stimulation of matrix metalloproteinase expression; and increased prostaglandin production (Dinarello, 1991). Many of these activities may contribute to the angiogenic activity of these cytokines. Thus, inhibition of the activity of IL-1 and TNF- could have an obvious benefit in angiogenesis-dependent inflammatory diseases. One means of inhibiting IL-1 and TNF- activity is by decreasing their production. SB 220025 is a new compound belonging to the CSAID^TM class of cytokine biosynthesis inhibitors (Cuenda et al., 1995; Lee et al., 1994), which act specifically on p38/CSBP MAP kinase to block a cascade, resulting in decreased production of IL-1 and TNF- as well as other mediators, such as IL-6 and prostaglandins (Beyaert et al., 1996; Pouliot et al., 1997). We used this compound to examine the role of these inflammatory mediators in inflammatory angiogenesis.

	Materials and Methods

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Compounds. Medroxyprogesterone was obtained from Sigma Chemical (St. Louis, MO). SB 203580 [4-(4-flourophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole)] and SB 220025 [5-(2amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole)] were synthesized by Medicinal Chemistry, SmithKline Beecham.

Measurements of kinase activity. p38 was prepared and assayed as previously described (Young et al., 1997), with the following modifications: 30-µl reactions contained 25 mM HEPES, pH 7.5, 8 mM MgCL₂, 70 nM enzyme, 0.17 mM ATP (2 µCi of ³²P), 0.2 mM Na₃VO₄, 0.67 mM T669 peptide (derived from the EGFR KRELVEPLTPSGEAPNQALLR), 0.167% dimethylsulfoxide and compounds. Reactions were incubated at 30°C for 10 min, stopped with 10 µl of 0.3 M phosphoric acid, captured on phosphocellulose filter paper (P81), washed with 75 mM phosphoric acid and counted by liquid scintillation.

LPS induction of TNF-. The method of Olivera et al. (1992) was used for LPS induction of TNF-. Balb/C mice (Charles River Labs, Wilmington, MA) were administered the test compound or vehicle (acidified tragacanth) 30 min before challenge with intraperitoneal injection of 25 µg of LPS (Escherichia coli, type W, 055:B5; Difco, Detroit, MI). After 120 min, blood was collected through exsanguination, and serum samples were used to measure TNF- levels by ELISA (see below).

Murine air pouch granuloma. This model is based on the methods of Colville-Nash et al. (1995). Female Balb/C mice (20 ± 2 g) were used, and granulomatous tissue was induced in anesthetized animals (Aerrane; Hanna Pharmaceutical Supply, Wilmington, DE) through the injection of 3 ml of air into the dorsal subcutaneous tissue on day 1, followed by the injection of 0.5 ml of 0.1% v/v croton oil (Sigma) in Freund's complete adjuvant (Sigma) on day 0. The dosing regimen was started on day 0 and continued until day 5, in conscious animals, with the compound being solubilized in 0.2 ml of N,N,dimethyl acetoacetamide (Sigma)/Cremephor El (Sigma)/saline or water (10:10:80) (saline was used for intraperitoneal injections, whereas water was used for oral dosing). On the indicated days, the animals were anesthetized and warmed to 40°C for peripheral vasodilation. A vascular cast was made by the intravenous injection of 1 ml of solution of 10% carmine red/5% gelatin solution (Sigma). The animals were chilled at 2° to 4°C for 3 hr before the removal of the granulomatous tissue.

Cytokine ELISAs. Tissue extracts were made through homogenization of granulomas in 0.5 ml of 5 mM KH₂PO₄/0.1 g of wet tissue. IL-1 levels were determined using a Cytoscreen Immunoassay Kit (BioSource International, Camarillo, CA). TNF- levels were determined using the following assay: plates were coated with hamster anti-murine TNF- antibody (Genzyme, Cambridge, MA) for 2 hr at 37°C and washed and blocked with a casein/BSA solution (5 g/liter for each) for 1 hr at 37°C, and the samples were added and incubated at 4°C overnight. Plates were washed, and the secondary antibody, rabbit anti-mouse TNF- (Genzyme), was added for 2 hr at 37°C; the plates were washed, and the tertiary antibody goat anti-rabbit peroxidase conjugate (BioSource International, Camarillo, CA) was added for 2 hr at 37°C. The plates were then washed, and substrate OPD (Sigma) was added for 20 min at room temperature. The reaction was terminated with 25 µl of 0.1 M NaF/well, the absorbance read at 460 nm. Sample values for both ELISAs are calculated using DeltaSoft ELISA analysis software (Biomettalics Inc., Princeton, NJ).

Collagen-induced arthritis. The model was described previously (Griswold et al., 1988). Briefly, male DBA/1 LacJ mice (16-18 g; Jackson Labs, Bar Harbor, ME) were primed intradermally with 0.1 ml of an emulsion consisting of equal volumes of Freund's complete adjuvant and bovine type II collagen at 2 mg/ml in 0.01 N acetic acid. After 21 days, the mice were given an intraperitoneal booster of 0.1 ml of 1 mg/ml bovine type II collagen in 0.01 N acetic acid without adjuvant. After the booster, mice were evaluated daily for incidence and severity of arthritis in their limbs. Scores of 0 to 4 for each limb were determined subjectively, in which 0 equals noninvolved and 4 equals the greatest severity of erythema and swelling. The maximum possible score for an arthritic mouse was 16 (4 points/limb). When the severity score reached 4, which was typically within 2 weeks of the collagen booster, mice were randomly assigned to vehicle or drug-treated groups, and oral dosing with SB 220025 or vehicle alone began. This day was designated day 0, and dosing continued for 10 days. Mice were evaluated, with no attention paid to their group, on days 7 and 10. All scoring was done by the same scorer as previously described (Griswold et al., 1988).

	Results

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SB 220025 selectively inhibits p38 MAP kinase. The pyrimidyl imidazole compounds as exemplified by SB 203580 have been previously demonstrated to specifically inhibit p38 (Cuenda et al., 1995; Lee et al., 1994; Young et al., 1997). SB 220025, a novel member of this structural class (fig. 1), was tested in a number of kinase assays to assess its use as a p38 inhibitor (table 1). This compound inhibited p38 phosphorylation of an EGFR peptide substrate with an IC₅₀ value of 60 nM, which is 10-fold more potent than SB 203580 (IC₅₀ = 0.6 µM; Cuenda et al., 1995). In selectivity assays, p38 inhibition by SB 220025 was >1000-fold selective over Erk (p42/p44 MAP kinase), 500-fold selective over PKA, >1000-fold selective over EGFR and 50-fold selective over PKC.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Structures of SB 203580 and SB 220025.

SB 220025 inhibits inflammatory cytokine production in vivo. To examine the in vivo efficacy of SB 220025, an acute model of LPS-induced TNF- expression was used. SB 220025 at a range of doses from 3 to 50 mg/kg was given to mice orally 30 min before challenge with LPS. Serum TNF- was measured by ELISA after 2 hr. This compound dose-dependently inhibited TNF- production with an ED₅₀ value of 7.5 mg/kg (fig. 2). Greater than 80% inhibition was obtained at 50 mg/kg. Thus, SB 220025 is an orally available, potent inhibitor of TNF- synthesis

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effect of SB 220025 on LPS induction of TNF- in vivo. Mice were pretreated with the indicated doses of SB 220025 and challenged with an intraperitoneal injection of LPS. TNF- levels in the serum were measured 2 hr after the challenge by ELISA. The vehicle control TNF- level was 4.35 ± 0.4 ng/mouse (n = 8). ED50 value was determined by regression analysis.

SB 220025 inhibits angiogenesis in the murine air pouch granuloma model. The inflammatory cytokines TNF- and IL-1 are potent inducers of angiogenesis. To test whether they are involved in the promotion of angiogenesis in chronic inflammation, we used the cytokine-suppressive p38/CSBP MAP kinase inhibitor SB 220025 in a model of inflammatory angiogenesis. The murine air pouch granuloma has been characterized as a chronic inflammatory progression with a profound angiogenic component (Colville-Nash et al., 1995). It provides a model in which modulation of angiogenesis in an inflammatory bed can be quantified. Granulomas were formed in a 3-ml dorsal subcutaneous air pouch by injection of 0.5 ml of Freund's complete adjuvant and croton oil. Within 3 days, a cohesive granulomatous tissue encased the adjuvant mixture. The granulomas were evaluated by weight, histology and vascular index (mg of carmine dye/g of dry tissue), which was used to assess the extent of angiogenesis.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Tissue dry weight and vascular index (mg of carmine dye/g of dry tissue) of day 6 granulomas after treatment with SB 220025 or medroxyprogesterone. Dosing began when granulomas were induced. Data are expressed as a percentage of vehicle-treated control animals ± S.D. Control vascular index was 5.4, and control dry weight was 0.28 g (n = 5). *Significant from control at P < .05, calculated by Duncan's multiple-range test.

Effect of SB 220025 on the time course of angiogenesis. We evaluated the effect of the p38/CSBP inhibitor at several time points to determine whether its effects would be different at the various stages of inflammatory and angiogenic progression. SB 220025 was given orally, at an intermediate dose of 30 mg/kg twice a day starting on day 0, and granulomas were evaluated on days 3, 5, 7 and 14. Granuloma size remained fairly constant and was unaffected by the SB 220025 (fig. 4). The vascular index of the control group rose gradually from day 3 to 14, whereas the vascular index of the treated group remained constant. At day 3, the compound did not cause a significant reduction in vascular index compared with control; however, at days 5, 7 and 14, the vascular index was lowered significantly by SB 220025. Thus, the p38/CSBP MAP kinase inhibitor did not affect the initial burst of angiogenesis but did prevent the increase in angiogenesis that occurs after day 3. 

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Time course of granuloma development in murine air pouch angiogenesis model. Animals were orally dosed twice daily with either vehicle or 30 mg/kg SB 220025 from day 0 until removal of granuloma tissue at days 3, 5, 7 or 14. A, Granuloma size was determined by dry weight to control for possible differences in edema. B, Vascular index (mg of carmine dye/g of dry tissue) was determined as described in the text. C, TNF- levels within granuloma were determined by ELISA on tissue homogenates. D, IL-1 levels were determined by ELISA in the same tissue homogenates as C. Data are shown as mean ± S.D. (n = 5). *Significant from control at P < .05. **Significant from control at P < .01, calculated by Duncan's multiple-range test.

Microscopic analysis angiogenesis in the granuloma. Angiogenesis in the granuloma was microscopically evaluated using cedarwood oil clearing. Figure 5 shows the vasculature of day 6 granulomas from both untreated and SB 220025-treated mice. The profound angiogenesis in the granuloma is demonstrated by the extensive vascular network in the control tissue. There was a striking reduction in the vasculature of the treated tissue. The fine capillaries seen in the control tissue were completely absent in the treated tissue, and only a few larger vessels remained visible.

View larger version (58K): [in this window] [in a new window]   Fig. 5.   Cedarwood oil histology of air pouch granuloma. Carmine dye vascular casts were made in day-6 granulomas from mice treated with SB 220025 (30 mg/kg b.i.d. p.o.) (A) or vehicle only (B). Granulomas were fixed in ethanol, cleared in cedarwood oil as described in the text, and photographed with transmitted light under 12× magnification.

Effect of SB 220025 on chronic inflammatory disease. The anti-inflammatory and antiangiogenic activities of SB 220025 suggest that it would provide an effective treatment in chronic inflammatory diseases such as rheumatoid arthritis, which has both inflammatory cytokine and angiogenic components. Thus, we tested SB 220025 in a chronic inflammatory disease model, murine collagen-induced arthritis. Mice were primed with bovine collagen, and 3 weeks later, the animals were given intraperitoneal injections of soluble collagen and monitored for the appearance of arthritis. Dosing began after arthritis was evident, usually between days 7 and 14 after collagen boost. The first day of dosing was designated day 0. Animals treated with SB 220025 (50 mg/kg p.o. b.i.d.) had no increase in severity of arthritis over 10 days, whereas the severity of arthritis in the control mice was increased at days 7 and 10 (fig. 6). Thus, the p38/CSBP MAP kinase inhibitor effectively blocked the progression of arthritis.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Collagen-induced arthritis and measurement of severity are described in the text. Dosing with SB 220025 (50 mg/kg b.i.d. p.o.) or vehicle began only when the severity score reached 4, and this was designated day 0. Data are shown as mean ± S.D. (n = 7). *Significant from control at P < .05. **Significant from control at P < .01 calculated by Student's t test.

	Discussion

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Proinflammatory cytokines such as IL-1 and TNF- have been shown to play a central role in many inflammatory processes (Dinarello, 1991). This study demonstrates the importance of IL-1 and TNF- in chronic inflammatory angiogenesis and arthritis. Angiogenesis is a normal physiological response in wound healing, but in diseases such as rheumatoid arthritis and psoriasis, it can take on a pathological role. The association between angiogenesis and chronic inflammation has led to the hypothesis that angiogenesis is induced by inflammatory events. Indeed, it has been shown that IL-1 or TNF- can induce angiogenesis in the normally avascular cornea (BenEzra et al., 1990; BenEzra and Maftzir, 1996; Fajardo et al., 1992). We evaluated the role of these cytokines in inflammatory angiogenesis in vivo by using a murine air pouch granuloma model. Both IL-1 and TNF- levels in the granuloma tissue increased sharply over the first 7 days of granuloma formation, the same time period in which angiogenesis was very active.

We modulated the activity of IL-1 and TNF- using the p38/CSBP inhibitor SB 220025, which inhibits their synthesis. This compound is more potent than the previously reported p38 inhibitor SB 203580. We observed an ED₅₀ value of 7.5 mg/kg for LPS-induced serum TNF- production, which is twice as potent a value as that reported for SB 203580 (Badger et al., 1996). SB 220025 caused a significant dose-dependent decrease in the vascular density of the granuloma, and this correlated with decreases in IL-1 and TNF- levels. The hypothesis is that decreasing IL1 and TNF- levels resulted in inhibition of angiogenesis in an inflammatory tissue bed.

When we analyzed a time course of granuloma development, we observed that the control group granuloma size, as measured by dry weight, increased dramatically from day 0 to 3 and then was steady from day 3 to 14. In contrast, the control group vascular index increased steadily from day 3 to 14. This time course is somewhat different from that reported by Colville-Nash et al. (1995), who observed a decrease in granuloma size after day 7, and biphasic vascular index, with a sharp peak at day 5 and a smaller peak at day 14. In other studies with this model, we observed a similar biphasic vascular index (data not shown); however, the dramatic maximum and minimum at days 5 and 7 appear to be very transient and have not been consistently reproducible. Thus, it is important to always run a control group for each time point in each drug study. Similar to Colville-Nash et al. (1995), we observed a regression in granuloma size, if followed beyond day 14.

In the present study, SB 220025 had no effect on granuloma size and did not inhibit angiogenesis at the 3-day time point, but it blocked the subsequent increases in vascularity most noticeably over days 5 and 7. This antiangiogenic activity correlates well with the highest levels of IL-1 and TNF- in the granuloma. These time points also represent a switch from an acute to a chronic inflammatory phenotype (Jackson et al., 1997), suggesting that the p38/CSBP MAP kinase inhibitor is most effective at inhibiting angiogenesis associated with chronic inflammation. The initial angiogenesis occurring over the first 3 days is likely to be induced by something other than IL-1 or TNF- because their levels have not yet risen. One possibility is VEGF, which has been shown to be at its highest levels early in granuloma development (Appleton et al., 1996). Moreover, at day 14 in the control group, angiogenesis was still increasing in this study, even though TNF- and IL-1 levels had dropped substantially. This time point represents another phenotypic change in the granuloma, when the chronic inflammatory phenotype gives way to a fibrotic phenotype (Jackson et al., 1997). Thus, angiogenesis may be driven by another factor or factors, such as FGF, at this stage.

Granuloma size was not decreased by inhibition of angiogenesis with SB 220025. This was not surprising because granuloma growth is not angiogenesis dependent in this air pouch model (Colville-Nash et al., 1995; Jackson et al., 1997). The model provides an in vivo system for the study of hyperangiogenesis in an inflammatory tissue but is not a model of inflammatory disease. To test the effect of SB 220025 in a model of rheumatoid arthritis, an angiogenesis-dependent chronic inflammmatory disease, we used murine collagen-induced arthritis. Using a therapeutic dosing regimen, in which dosing did not begin until there was evidence of arthritic joint disease, SB 220025 was able to prevent further increases in the severity of arthritis. Thus, an inhibitor of IL-1/TNF- synthesis and angiogenesis was a very effective treatment for arthritis. This agrees with other studies that demonstrated that TNF- antibodies (Piguet et al., 1992) were an effective treatment for collagen arthritis and that the angiogenesis inhibitor AGM-1470 was able to reduce the severity of collagen-induced arthritis in rats (Peacock et al., 1992). Interestingly, in a study of other anti-inflammatory drugs (Griswold et al., 1988), the nonsteroidal anti-inflammatory drug ibuprofen was not particularly effective in this model, further suggesting that the anticytokine and antiangiogenic properties of SB 220025 are key to its antiarthritic activity.

Although inhibition of IL-1 and TNF- synthesis is strongly implicated to be responsible for the antiangiogenic and antiarthritic activities of SB 220025, it is possible that inhibition of the synthesis of other cytokines also may be involved. Other factors, such as the inducible cyclooxygenase, IL-6, IL-8 and GM-CSF, also are regulated by p38/CSBP MAP kinase (Beyaert et al., 1996; Lee et al., 1988, 1989, 1993; Pouliot et al., 1997) and thus may be affected by SB 220025. However, IL-1 and TNF- are reported to have more potent angiogenic activities than eicosanoids and these other cytokines, and the most effective antiangiogenic activity of SB 220025 on days 3 and 5 of granuloma development correlated well with inhibition of IL-1 and TNF- synthesis. It is important to note that p38 inhibitors such as SB 203580 and SB 220025 also affect the signaling pathways of these cytokines and thus may work via inhibition of both cytokine synthesis and action (Badger et al., 1996; Cuenda et al., 1995). In addition, although it is a very selective inhibitor of p38 MAP kinase and we are unaware of any other activities that could account for its pharmacology, it is possible that SB 220025 may also inhibit an as-yet-unidentified kinase. Therefore, in vivo data should be interpreted with normal caution.

The association between inflammation and angiogenesis has long been observed, but until recently there has been little evidence to clearly demonstrate the link. This study shows that angiogenesis is dependent on inflammatory cytokines in a chronic inflammatory model. It is not clear whether inflammatory cytokines are involved in other angiogenesis dependent processes, such as tumor growth, and this remains to be tested. From our studies and others (Badger et al., 1996), it is apparent that p38/CSBP MAP kinase inhibition should provide an effective treatment for chronic proliferative inflammatory diseases.