VDIPEN, a metalloproteinase-generated neoepitope, is induced and immunolocalized in articular cartilage during inflammatory arthritis (1)
Several matrix metallopro- teinases (MMPs) have been implicated in the catabolism of cartilage ECM: stromelysin (SLN; EC 3.4.24.17), collagenase (CLN; EC3.4.24.7), gelatinase A (GLN-A; EC3.4.24.24), and gelatinase B (GLN-B; EC3.4.24.35). SLN appears to be important in both inflammatory and degenerative arthritides, because it can directly degrade aggrecan and link protein, type II collagen telopeptide, and type IX collagen, is elevated in synovial fluids and cartilage ofRA and osteoarthritic (OA) patients, and may be up-regulated in synoviocytes and chondrocytes.
Generation and accumulation of this VDIPEN neoepitope, which is common to both human and murine aggrecans, can thus provide a convenient marker of MMP activity in cartilage. An antibody recognizing this COOH-terminal aggrecan neoepitope has been developed; it does not recognize the VDIPEN sequence when it is an integral part of the peptide VDIPENFFGVG.
SLN digestion of unfixed mouse paw cryosections induces intense anti-VDIPEN staining within the GAG-rich pericellular (PC) matrix of chondrocytes and at the articular cartilage surface; moderate VDIPEN labeling is also induced in the interterritorial zones of the AC. Preincubation of anti-VDIPEN IgG with 250 ng/ml YTGEDFVDIPEN peptide completely blocked this labeling, whereas similar quantities of the spanning peptide (YTGEDFVDIPENFFGV) failed to inhibit staining.
A single 50-kD aggrecan GI fragment was detected with this anti-VDIPEN antibody in Western blots of SLN-digested mouse paw extracts. The anti-VDIPEN antibody did not detect any other fragments in this digested sample, or in extracts of untreated control paws, indicating that its ability to detect the 50-kD aggrecan segment generated by SLN is specific.
Most 10-, 20-, and 28-d CIA mice exhibited VDIPEN staining in their hind paw AC, whereas vehicle-injected controls were negative. However,the patterns of DIPEN induction and GAG depletion differed markedly between 10 and 20 d.
Intense VDIPEN immunostaining was widespread at the articular surfaces of various tarsal and metatarsal joints of stage 2 paws at 20 d. VDIPEN epitope appeared to be induced and heavily concentrated in the pericellular matrices of AC chondrocytes and at the eroding outer surface of the articular cartilage. Moderate VDIPEN labeling was also present in the interterritorial zones of AC. Endogenous VDIPEN staining was completely blocked by pre- incubating anti-VDIPEN IgG with YTGEDFVDIPEN but not by pretreatment with YTGEDFVDIPENFFGV; nonimmune rabbit IgG did not generate any immuno-peroxidase labeling.
Quantities of endogenous VDIPEN labeling were expressed as a percent of the maximum anti-VDIPEN signal generated by exhaustive SLN digestion of normal AC, and GAG depletion was normalized to the GAG content of untreated AC. Although the mean staining density observed in VDIPEN-positive foci at 10 d in CIA was 21% of the SLN-treated maximum, it increased to 70% of the maximum at 20 d. GAG levels within corresponding AC regions were 95% of control values at day 10 but decreased to 38% of normal at 20 d. Similar levels of VDIPEN induction and GAG depletion were observed in paw AC of mice with PGIA for 1-5 mo.
There is a possibility that additional enzymes could also be participating in aggrecan cleavage in CIA and PGIA. Fragments consistent with another aggrecan cleavage site in the interglobular domain between Glu373 and Ala374 have been reported to accumulate in joint fluids of patients with inflammatory and noninflammatory joint diseases. The induction of an aggrecan neoepitope at this strategic location suggests that cytokines generated during inflammatory arthritis stimulate chondrocytes to synthesize and secrete MMPs, which initiate destruction of the extracellular matrix by cleaving aggrecan. There- fore, the early appearance of VDIPEN labeling in PC matrices is a highly sensitive indicator of the commencement of joint pathology initiated by chondrocytes.