Significant advances in the treatment of rheumatoid arthritis (RA) have been achieved in the past decades, with the increased spectrum of available therapies resulting in improved treatment outcome. However, individual responses to treatment vary. Thus, selecting and optimizing treatment for individual RA patients is challenging (1, 2). One potential approach is to monitor early treatment response. Unfortunately, this is currently limited. Measurement of serum parameters of inflammation, which may not accurately reflect disease activity within joints (3, 4). Serial radiographic assessment of bone demineralization and joint space narrowing to monitor treatment response typically begins late in the course of the disease, limiting its usefulness as a method for short-term assessment of treatment effects (5, 6). Other imaging techniques, such as magnetic resonance imaging and ultrasound, may have potential roles in the evaluation of the disease in the future (3, 4), but again typically provide information on anatomic changes that are later manifestations of RA.
Matrix-degrading enzymes, produced primarily by synovial fibroblasts and synovial macrophages, have a substantial role in the destruction of arthritic joints. These proteolytic enzymes contribute significantly to the invasion and degradation of articular structures (7–9). Several enzymes of different protease families, such as matrix metalloproteinases (MMPs) (10, 11) and cysteine proteases, e.g., cathepsin B, are involved in this process (12–14). Since matrix-degrading enzymes are highly up-regulated in RA (7–14) and effective therapies must decrease the destructive activity of these proteases, we hypothesized that these enzymes are suitable targets for in vivo imaging of early treatment response. The ability to measure such indirect biomarkers of disease activity would be useful in both the preclinical and clinical realms of RA treatment.
Previously, we developed near-infrared fluorescence (NIRF) imaging “smart” probes, which markedly increase their level of fluorescence after target interaction (15). These selective protease probes are biocompatible, and their fluorescence is quenched when administered intravenously until activated by enzymatic cleavage in the target tissue. This allows localization of signal in areas of activity with low fluorescence background in adjacent tissues that do not activate the probes and, importantly, increased signal indirectly correlates with increased proteolytic activity. Imaging probes based on this concept, with specificities for Lys–Lys bonds (such as cathepsins B, L, and S, and plasmin) and other probes with selectivity for specific proteases, such as cathepsin D or MMP-2, have been developed and characterized. They have previously been applied to imaging of enzyme activity in tumors, atherosclerosis, and inflammation, demonstrating their substantial value for noninvasive imaging of molecular markers of disease (15–18). Such molecular reporters may serve as indicators for objective assessment of treatment response, for stage and severity of disease, and prognosis, may facilitate early diagnosis, and may allow for more effective drug development.
The goals of this study were to examine whether methotrexate (MTX) treatment would alter protease activity, to determine whether protease-activated NIRF imaging probes are sensitive reporters for imaging the presence of target enzymes in arthritic joints, and to determine whether such probes could be used as molecular reporters for early monitoring of treatment response in RA. For this purpose, untreated and MTX-treated mice with collagen-induced arthritis (CIA) were injected with protease-activated imaging probes. Proteases that target the Lys–Lys cleavage site, including cathepsin B, activate probe fluorescence. Thereafter, fluorescence reflectance imaging was performed at different time points after administration, the fluorescence intensity of inflamed and healthy paws was analyzed, and fluorescence activation was examined histologically.
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At several fixed times after intravenous injection of the imaging probe, optical-reflectance NIRF imaging was performed. Figure 1B shows a raw NIRF image and Figure 1C shows a color-coded NIRF image (obtained 24 hours after probe injection) superimposed on a white light image of a mouse with arthritis that affected the right fore paw. In contrast to nonarthritic paws, arthritic paws showed high fluorescence intensity, which indicated intense enzymatic cleavage of the imaging probe in inflamed paws, which results in fluorescence activation.
To investigate fluorescence activation on the histologic level, tissue sections of arthritic and nonarthritic paws were examined for NIRF and in parallel by immunohistochemistry using antibodies specific for cathepsin B. Figure 2 shows images of serial cryostat sections of inflamed, hyperplastic synovium from a mouse with CIA 24 hours after injection of the imaging probe. Fluorescence of the probe, as seen on the NIRF image (Figure 2A), was detected in the hyperplastic synovium of inflamed joints. Most notably, the vast majority of synovial cells showed fluorescence. In the hyperplastic synovium of uninjected control mice (control for autofluorescence), no NIRF signal was found (results not shown). Serial section staining (Figure 2B) revealed that virtually all cells expressed cathepsin B protein. This helped confirm that the fluorescent signal (Figure 2A) was secondary to protease expression from cells. In Figures 2C and D, serial synovial sections stained with H&E and an isotype control of the anti–cathepsin B antibody as a negative control are shown. The isotype control, using the same staining procedure with the exception of a nonreactive isotype of the primary antibody, showed only minimal nonspecific staining. As additional negative and positive controls, we also stained mouse liver tissue, known to express cathepsin B. Using a nonspecific isotype of the primary antibody, the liver sections showed negligible nonspecific staining, whereas using the anti–cathepsin B antibody as the primary antibody resulted in intense staining throughout the liver tissue (results not shown).
Figure 2. Serial cryosections of inflamed, hyperplastic synovium from a mouse with collagen-induced arthritis. A, Near-infrared fluorescence (NIRF) image of activated protease probe. B, Anti–cathepsin B antibody stain for immunoreactive protease. C, Hematoxylin and eosin stain. D, Isotype control of antibody stain. The large structure in the upper right is bone. Almost all the cells in the hyperplastic synovium exhibited cathepsin B activity, as shown in B. Images were obtained 24 hours after injection of the NIRF probe.
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To examine whether MTX treatment altered activation of fluorescence, fluorescence reflectance imaging of untreated and MTX-treated arthritic mice was performed at different times after intravenous injection of the imaging probe. Figure 3 shows white light, raw NIRF, and color-coded NIRF images superimposed on the white light images of untreated mice (Figures 3A and C) and MTX-treated mice (Figures 3B and D) 24 hours after injection of the protease-activated probe. Fluorescence intensities of arthritic toes and paws of MTX-treated mice were found to be substantially lower than those of untreated mice.
Figure 3. White light, raw near-infrared fluorescence (NIRF), and color-coded NIRF images superimposed on white light images of mice with collagen-induced arthritis (CIA) 24 hours after injection of the probe. A, Untreated mouse with arthritis in 3 toes of the right fore paw (arrowheads). B, Methotrexate (MTX)–treated mouse with CIA in a toe of the right fore paw (arrowhead). C, Untreated mouse with arthritis in the right hind paw. D, MTX-treated mouse with CIA in the right hind paw. MTX reduced fluorescence intensity from arthritic toes and paws compared with untreated mice.
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Figure 4 shows the time course of fluorescence intensity, presented as RFI based on ROI analysis, in arthritic and nonarthritic toes (Figure 4A) and paws (Figure 4B) of untreated and treated mice after probe injection. The RFI of nonarthritic toes and paws increased only slightly with time. In contrast, the NIRF signal of arthritic toes and paws of untreated mice increased rapidly due to intense cleavage of the protease-activated probe. After 24 hours, inflamed toes and paws of untreated arthritic mice showed a 5-fold and 7-fold higher RFI than the toes and paws, respectively, of untreated healthy mice (P < 0.001). Compared with the fluorescence intensity in untreated arthritic mice, the signal measured in arthritic toes and paws of MTX-treated animals was found to be significantly lower at 12 hours and later (P < 0.001); after 6 hours, the level of significance was P < 0.001 for toes and P = 0.01 for paws. After 24 hours, inflamed toes and paws of treated arthritic mice showed a 3-fold and 2-fold lower RFI than inflamed toes and paws, respectively, of untreated arthritic mice.
Figure 4. Time course of fluorescence intensities in arthritic and nonarthritic toes and paws of mice after probe injection. A, Fluorescence from arthritic toes of untreated and methotrexate (MTX)–treated mice and healthy toes of untreated mice. B, Fluorescence from arthritic paws of untreated and MTX-treated mice and healthy paws of untreated mice. Values are the mean ± SEM relative fluorescence intensity (RFI). MTX treatment significantly decreased fluorescence at 6, 12, and 24 hours postinjection (P < 0.001 for toes and P = 0.01 for paws at 6 hours; P < 0.001 for toes and paws at 12 and 24 hours). At 24 hours, the RFI in the toes and paws of untreated arthritic mice was increased 5-fold and 7-fold, respectively, compared with healthy mice.
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Measurement of paw weights in all arthritic groups revealed no significant differences between treated and untreated groups at the time of imaging (P = 0.97 and P = 0.3 for fore paws and hind paws, respectively, between treated and untreated groups). There was no correlation between relative fluorescent signal intensity and paw weight (r values ranged between 0.01 and 0.1 for the various groups).
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The noninvasive visualization and quantitation of biologic processes on the cellular and molecular levels for use as biomarkers of disease activity is an emerging approach to therapy monitoring. In this context, the results of the present study demonstrate that protease-activated NIRF imaging “smart” probes can serve as sensitive reporters for imaging treatment response to antirheumatic drugs such as MTX. An advantage of activated probes in contradistinction to targeted probes, e.g., fluorescence-labeled antibodies with a specificity for a certain target, is that the background signal is much lower due to the very low fluorescence in the activated probe's native state, while the high fluorescence postactivation at the target site provides additional molecular fidelity of the signal. In addition, another form of signal amplification occurs using “smart” probes because one target enzyme molecule can activate multiple signal molecules (15).
The imaging technologies used in this study are inexpensive and have high throughput capability (22). The properties of near-infrared light propagation allow for the direct recording of signal after penetration through 1 cm of tissue, making human peripheral joints easily accessible via surface reflectance techniques. Additionally, 3-dimensional localization and quantification of near-infrared fluorochromes at nanomolar concentrations, after penetration of up to 10 cm of tissue, is feasible by tomographic techniques that use advanced reconstruction (23). Thus, no technologic hurdles exist that would prevent the implementation of near-infrared recording of “smart” probe activation in patients with RA.
A protease-activated imaging probe was chosen because matrix-degrading enzymes, including cysteine proteases such as cathepsin B, are highly up-regulated in patients with RA, contributing significantly to the destruction of arthritic joints (7–14). Besides directly degrading major joint components, cysteine proteases are capable of activating MMPs. Thus, cysteine proteases also contribute to a cooperative process among the proteolytic enzymes (for review, see ref. 24). In addition, expression of cathepsin B appears to be restricted predominantly to the synovial cells attached to cartilage and bone at sites of joint erosion in RA patients (12, 13). Furthermore, cysteine proteases are expressed in the synovium of RA patients at a very early stage of the disease and in established erosive RA, and are absent from normal synovium (8, 25, 26). The protein expression of cathepsin B and procathepsin L correlates with the score of chronic inflammatory activity (14). Moreover, enzymatic activity of cysteine proteases in the joints of rats with antigen-induced arthritis is positively correlated with the severity of joint destruction as well as with inflammation parameters (27), and parallels disease activity (28). Thus, cysteine proteases are suitable targets, especially for the early monitoring of treatment response.
The imaging probe used in this work cleaves Lys–Lys bonds. Cathepsin B is one protease that cleaves such bonds, and we have demonstrated here that in the CIA model there is colocalization of fluorescence imaging signal and cathepsin B expression at the histologic level. However, additional proteases, such as cathepsins L and S and plasmin, cleave the Lys–Lys bond and may contribute, in part, to signal activation in this and other disease applications. Alternate probe constructs may allow increased specificity for cathepsin B cleavage, similar to increased specificity shown for MMP-2 (16). Importantly, the probe used here more accurately reflects total available protease load in vivo than probes with alternate specificities, and may thus be a more useful clinical biomarker of disease status and response to therapy, given the heterogeneity of gene expression in the clinical setting compared with animal model systems.
MTX was selected as a proof-of-concept antirheumatic drug to evaluate the suitability of enzyme-activated NIRF imaging probes for early monitoring of treatment response. MTX is probably the most commonly used drug in the treatment of RA and serves as a gold standard (1, 2). Furthermore, studies demonstrate the ability of MTX, like other treatments, to decrease the activity of proteolytic enzymes in the synovium (29, 30). Using the smart probe paradigm, we have been able to show that MTX significantly decreases fluorescence intensity of inflamed toes and paws of mice with CIA up to 2–3-fold 24 hours after probe injection. This decrease could easily be determined by measuring changes in fluorescence activity (Figures 3 and 4). Fluorescence intensity measured at the target site in this work reflects a combination of probe delivery and activation. However, under the current experimental conditions, the dominant effect is most likely due to changes in enzyme activity, and is unlikely to reflect changes in probe delivery since paw weight measurements showed no significant differences between treated and untreated arthritic paws. Additionally, there was no correlation between fluorescent signal intensity and paw weight in all groups, also suggesting that there were no differences in leak (contributing to edema) between treated and untreated animals during the course of the experiment. The MTX dose administered to the mice exceeded the clinical dose. However, comparably high MTX doses are needed to induce a sufficient treatment response in this animal model (31).
In conclusion, noninvasive optical imaging using enzyme-activated NIRF probes such as ones that are selective for the proteases that cleave Lys–Lys bonds, e.g., cathepsin B, may be useful for evaluating the presence of target enzymes in arthritic joints and for the early monitoring of treatment response to antirheumatic drugs such as MTX. Potential clinical benefits include the rapid titration of therapy choice, therapy dosing, quantitative early evaluation of clinical outcome, and more effective drug development.