Serum biomarker levels for musculoskeletal disease in two- and three-year-old racing Thoroughbred horses: A prospective study of 130 horses.

REASON FOR PERFORMING STUDY: Biomarkers have shown some in vivo promise for the detection of musculoskeletal injuries, but further study to assess biomarker levels in clinical orthopaedic disease is required. OBJECTIVE: To assess 7 serum biomarkers for the detection of musculoskeletal injuries. METHODS: Two- and 3-year-old racehorses were entered into the study (n = 238). Exit criteria were lack of training for >30 days, or completion of 10 study months. Data from horses with solitary musculoskeletal injuries and completion of >2 months were analysed. Musculoskeletal injury was considered intra-articular fragmentation (IAF), tendon or ligamentous injury (TL), stress fractures (SF) and dorsal metacarpal disease (DMD). Monthly lameness examination and serum collection were performed. Serum was analysed for glycosaminoglycan (GAG), type I and II collagen degradation (C1, 2C), type II collagen synthesis (CPII), type II collagen degradation (Col CEQ), aggrecan synthesis (CS846), osteocalcin (OC) as a marker of bone formation and (C-terminal telopeptide of type I collagen) CTX as a marker of bone degradation. RESULTS: Of the 238 horses 59 injured and 71 uninjured control horses met the analysis criteria. Based on injury no significant differences in the proportions were observed for age, gender or lesion type, although a higher proportion of injuries occurred at the beginning of the study. Of injured horses, 16 (27%) sustained an IAF, 17 (29%) a TL injury, 7 (12%) SF and 19 (32%) were diagnosed with DMD. There were significant changes seen in biomarkers based on the injury incurred when longitudinal samples were assessed. Furthermore, based on the serum biomarkers collected prior to injury, horses could be correctly classified as injured or uninjured 73.8% of the time. CONCLUSIONS: A unique biomarker pattern occurred before each injury and this was beneficial in classifying horses as injured or uninjured. POTENTIAL RELEVANCE: Biomarkers have the potential to be used as a screening aid prior to musculoskeletal injury.

Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.

OBJECTIVE: To discriminate between changes in biomarkers with exercise compared to changes in biomarkers with osteoarthritis (OA) in exercising horses. METHOD: Sixteen, 2-year-old horses were randomly assigned either to an exercise-alone (n=8) or OA-affected (also exercised) (n=8) group. All horses had both mid-carpal joints arthroscoped and OA induced in one mid-carpal joint in the OA-affected joints of OA-affected horses. Two weeks after surgery all horses commenced a strenuous exercise program on a high-speed treadmill. Clinical outcomes and synovial fluid and serum biomarkers, were evaluated weekly. Synovial and serum biomarkers evaluated were epitope CS846 (CS846), epitope CPII (CPII), glycosaminoglycans (GAGs), epitope Col CEQ (Col CEQ) (a marker of type II collagen degradation), type I and II collagen degradation fragments (C1,2C), osteocalcin, C-terminal of bone type I collagen (CTX1), type I collagen (Col I) and (synovial fluid only of cartilage) prostaglandin E2 (PGE2) levels. Horses were euthanized at day 91 and their joints assessed grossly, histopathologically, and histochemically. RESULTS: Exercise induced a significant increase in synovial fluid CS846, CPII, GAG, Col CEQ, C1,2C, osteocalcin and Col I concentrations. There was a significant increase in synovial fluid CS846, CPII, Col CEQ, C1,2C, osteocalcin, Col I and PGE2 concentrations in OA-affected joints compared to exercise-alone joints. The concentration of serum CS846, CPII, GAG, osteocalcin, C1,2C and Col I increased with exercise. For each of these biomarkers there was also a statistically significant increase in serum biomarker levels in OA-affected horses compared to exercise-alone horses. CONCLUSIONS: Six synovial fluid and serum biomarkers were useful in separating early experimental OA from exercise alone but synovial fluid CTX1 and serum Col CEQ and CTX1 were not.

Joint dependent concentrations of bone alkaline phosphatase in serum and synovial fluids of horses with osteochondral injury: an analytical and clinical validation.

OBJECTIVES: Validate use of a commercially available immunoassay for measurement of bone alkaline phosphatase (BAP) in equine serum and synovial fluid (SF), and investigate the effects of osteochondral (OC) injury in horses on BAP concentrations in serum and SF. METHODS: SF was collected from 37 joints of 34 Thoroughbred (TB) racehorses undergoing arthroscopic surgery for the removal of OC fragments from either the carpal joints (n=18) or the metacarpo-/metatarsophalangeal (MP) joints (n=19). SF was also obtained from 52 joints of 16 normal TB horses, collected bilaterally from carpal joints of 10 horses (n=40), and MP joints of six horses (n=12). Blood was obtained from all 50 horses. A commercially available immunoassay was validated and subsequently used to determine equine serum and SF BAP concentrations. Correlations to radiographic and arthroscopic scores were assessed. RESULTS: BAP concentrations were significantly lower in serum from horses with OC injury in their carpal or MP joints than in serum from normal horses. SF BAP concentrations in normal and OC injured carpal joints were significantly higher than MP joints. BAP concentrations were significantly higher in SF from OC injured carpal joints than normal. BAP concentrations were affected by joint sampled, with age having a significant interaction. Concentrations of BAP in the serum (<30U/L), SF (>22U/L) and a ratio of SF to serum > or = 0.5 were predictive of OC injury. Radiographic and arthroscopic scores significantly correlated with serum BAP concentrations, and SF:serum BAP correlated with arthroscopic scores. CONCLUSIONS: Determination of serum and SF BAP concentrations may be beneficial in the investigation of early joint injury. Joint and injury dependent differences in BAP concentrations allowed the estimation of predictive value for identifying OC injury.

Early post-traumatic osteoarthritis-like changes in human articular cartilage following rupture of the anterior cruciate ligament.

OBJECTIVE: Injury to the anterior cruciate ligament (ACL) frequently leads to post-traumatic osteoarthritis (OA). In this study we determined whether early degenerative changes characteristic of idiopathic OA are induced in articular cartilage following ACL injury. METHODS: A small sample of femoral articular cartilage was removed at surgery, as part of ACL reconstruction, from a total of 50 patients with ACL injuries. Of these, 28 underwent surgery less than 1 year post-injury. Control cartilages were obtained from the same site from 21 persons at autopsy. All cartilages were examined for molecular changes. The content of type II collagen, its cleavage by collagenases and its denaturation were determined by immunoassay. The total content of glycosaminoglycan (GAG), which is principally aggrecan, was measured colorimetrically. Data were expressed per unit DNA (GAG and collagen content) or as a percentage of total collagen cleaved or denatured. Other cartilages from the same site (8 controls, 12 less than 1 year and 8 more than 1 year post-injury) were frozen sectioned and examined histologically to determine by Mankin grading cartilage degeneration. RESULTS: Histological analyses revealed that control subjects exhibited staining for proteoglycan, which was reduced in some patients following ACL rupture. Degeneration of the articular surface was sometimes observed 1 year after ACL rupture. Although the Mankin grade increased with time after rupture these changes were not significant. Immunoassays, however, revealed an increase in GAG content within 1 year which was maintained after 1 year although no longer significant. No changes in total type II collagen content were observed during the period of study. However, there were significant increases in the denaturation and cleavage of type II collagen less than and more than 1 year post-ACL rupture. Total type II collagen content was directly correlated with GAG content in all three groups, with the significance being weakest at more than 1 year. After 1 year an inverse correlation was observed between total type II collagen content and collagen cleavage as well as denaturation. CONCLUSIONS: These observations reveal that joint instability resulting from ACL injury rapidly results in degenerative changes characteristic of those seen in idiopathic OA at arthroplasty and in experimental OA following ACL surgery. These changes may contribute to the development of post-traumatic OA that is commonly observed following ACL injury. The observations support and extend conclusions from other studies on human and animal articular cartilage and synovial fluids post-ACL injury that have revealed a rapid onset of damage to type II collagen and an initial increase in proteoglycan content characteristic of experimental OA post-ACL injury. This study provides direct evidence for the rapid development of degenerative changes characteristic of OA following ACL injury.

The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone.

The osteocyte is the terminally differentiated state of the osteogenic mesenchymal progenitor immobilized in the bone matrix. Despite their numerical prominence, little is known about osteocytes and their formation. Osteocytes are physically separated in the bone matrix but seemingly compensate for their seclusion from other cells by maintaining an elaborate network of cell processes through which they interact with other osteocytes and bone-lining cells at the periosteal and endosteal surfaces of the bone. This highly organized architecture suggests that osteocytes make an active contribution to the structure and maintenance of their environment rather than passively submitting to random embedding during bone growth or repair. The most abundant matrix protein in the osteocyte environment is type-I collagen and we demonstrate here that, in the mouse, osteocyte phenotype and the formation of osteocyte processes is highly dependent on continuous cleavage of type-I collagen. This collagenolytic activity and formation of osteocyte processes is dependent on matrix metalloproteinase activity. Specifically, a deficiency of membrane type-1 matrix metalloproteinase leads to disruption of collagen cleavage in osteocytes and ultimately to the loss of formation of osteocyte processes. Osteocytogenesis is thus an active invasive process requiring cleavage of collagen for maintenance of the osteocyte phenotype.

Significant exercise-related changes in the serum levels of two biomarkers of collagen metabolism in young horses.

OBJECTIVE: To identify metabolic biomarkers that can be evaluated in serum for monitoring the effects of exercise on skeletal development in mammals. DESIGN: Sera of foals from three groups (box-stall rest, pasture and training) were serially collected over the first 5 months of life and assayed for eight biomarkers of cartilage and bone metabolism. Sub-populations from each group were sampled for an additional 6 months of identical exercise. RESULTS: When expressed as a percentage of baseline values, lower serum levels of the carboxy-terminal propeptides of type II collagen (CPII), and higher serum levels of the cross-linked telopeptide fragments of degraded type I collagen (CTx1) were found in the trained foals compared to the other groups. Significant differences disappeared in those foals sampled during an additional 6 months of identical exercise. The most significant correlations were between serum biomarkers of bone metabolism, being positive between anabolic markers and negative between anabolic and catabolic markers. Serum levels of CTx1 and CPII significantly increased with age in all groups throughout the study. CONCLUSIONS: We have identified two markers of collagen metabolism, CPII and CTx1, as potential serum indicators of the exercise effects on the developing skeletal system in horses. Forced exercise during the first months postpartum appeared to have a negative effect on collagen turnover when compared to levels in pastured foals. Routine monitoring of collagen biomarkers in sera of exercising young mammals may allow for the early detection of abnormalities in skeletal tissue metabolism and for subsequent intervention before permanent damage occurs.

Elevation of a collagenase generated type II collagen neoepitope and proteoglycan epitopes in synovial fluid following induction of joint instability in the dog.

CLINICAL RELEVANCE: Measurement of markers of cartilage pathology in synovial fluid may provide clinical rheumatologists and osteoarthritis (OA) researchers important information for early diagnosis of OA as well as a method for monitoring disease progression and response to treatment. This study demonstrates the value of this approach in an established model of OA (cranial cruciate ligament rupture) at a point distant enough from the original surgical manipulation so as to have little to no effect on the marker concentrations. OBJECTIVE: The objective of this study was to determine whether measurement of markers of cartilage collagen cleavage and proteoglycan turnover in synovial fluid from a canine model could be used to detect cartilage changes following the onset of joint instability during the development of OA. DESIGN: A model of joint instability that develops OA was created in 18 mature dogs using monopolar radiofrequency energy (MRFE). MRFE was arthroscopically applied to one cranial cruciate ligament (CCL) while the contralateral CCL was sham treated. The treated CCLs ruptured approximately 8 weeks (55 +/- 1.6 days) after MRFE treatment. Synovial fluid was collected at time zero prior to MRFE treatment, 4 weeks after MRFE treatment, and at 4, 8, and 16 weeks after CCL rupture. Synovial fluid concentrations of the neoepitope COL2-3/4C long (type II collagen cleavage by collagenase) and epitopes 3B3(-) (proteoglycan aggrecan sulfation) and 846 (associated with aggrecan synthesis) were analyzed. RESULTS: Compared to sham treated joints, the synovial fluid concentrations of COL2-3/4C long and 3B3(-) were significantly increased 2.2 fold and 2.9 fold, respectively, in joints with MRFE treated CCLs following CCL rupture. Concentrations of the 846 epitope in synovial fluid showed a trend toward an increase, which was not significant, after CCL rupture. CONCLUSIONS: Concentrations of the collagenase-cleaved type II collagen neoepitope and 3B3(-) epitope in synovial fluid were significantly increased by 4 weeks and remained elevated for at least 16 weeks after CCL rupture. This suggests that in dogs the COL2-3/4C long neoepitope and 3B3(-) epitope are sensitive markers for changes in joint cartilage turnover in joints that are developing OA.

Excessive degradation of type II collagen in articular cartilage in equine osteochondrosis.

Articular osteochondrosis (OCD) occurs in both man and animals. The etiology remains to be determined. Studies of OCD lesions in animals may provide clues as to its pathogenesis. The aim of our study was to determine whether there was evidence for increased degradation namely proteoglycan (PG) release and type II collagen cleavage in articular cartilage harvested from OCD lesions. We examined ex vivo explants at post-mortem from equine OCD lesions and macroscopically normal site and age matched cartilage. These were cultured over a 10 day period in serum-free medium. Type II collagen cleavage was measured in articular cartilage and media using an Elisa assay to detect the COL2-3/4C(short) epitope, which is generated on cleavage of the triple helix of type II collagen by collagenases. PG release was measured by a dye-binding assay. Cumulative release of PG and COL2-3/4C(short) and their contents in cartilage at the end of the culture period were determined. In OCD lesions there was a significant increase in type II collagen cleavage by collagenase but no evidence for increase of PG degradation. These findings point to a selective increase in type II collagen cleavage by collagenases, in OCD lesions of the kind observed in osteoarthritis. Further work is needed to determine whether changes represent primary or secondary events in the pathogenesis of OCD.

Synovial fluid gelatinase concentrations and matrix metalloproteinase and cytokine expression in naturally occurring joint disease in horses.

OBJECTIVES: To determine concentrations of matrix metalloproteinase (MMP)-2 and -9 in synovial fluid; and mRNA expression of MMP-1, -13, and -3; interleukin[IL]-1alpha and beta; and tumor necrosis factor (TNF)-alpha in synovial membrane and articular cartilage from horses with naturally occurring joint disease. SAMPLE POPULATION: Synovial fluid (n = 76), synovial membrane (59), and articular cartilage (45) from 5 clinically normal horses and 55 horses with joint disease categorized as traumatic (acute [AT] or chronic [CT]), osteochondritis dissecans (OCD), or septic (S). PROCEDURE: Synovial fluid gelatinase concentrations were analyzed, using zymography. Synovial membrane and articular cartilage mRNA expression for MMP-1, -3, and -13, IL-1alpha and beta, TNF-alpha, type-II collagen, and aggrecan were analyzed, using quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Synovial fluid pro-MMP-2 concentration was significantly higher in diseased joints than normal joints. Septic joints had significantly higher concentrations of pro and active MMP-9. Stromelysin-1 was expressed in > or = 80% of synovial membrane and articular cartilage samples and was strongly influenced by age. Collagenases were rarely expressed, with MMP-13 expressed only in diseased joints. Interleukin-1beta expression was significantly higher in all OCD samples and was influenced by age. Tumor necrosis factor-alpha expression was significantly higher in cartilage from joints with AT and OCD. There was no correlation between MMP or cytokines and type-II collagen or aggrecan expression. CONCLUSIONS AND CLINICAL RELEVANCE: Matrix metalloproteinase-2 and -3 are abundant in naturally occurring joint disease and normal joints. Interleukin-1beta and TNF-alpha may be important in the pathogenesis of OCD. Age affects MMP and IL-1beta concentrations.

Use of an antineoepitope antibody for identification of type-II collagen degradation in equine articular cartilage.

OBJECTIVE: To develop an antibody that specifically recognizes collagenase-cleaved type-II collagen in equine articular cartilage. SAMPLE POPULATION: Cartilage specimens from horses euthanatized for problems unrelated to the musculoskeletal system. PROCEDURE: A peptide was synthesized representing the carboxy- (C-) terminus (neoepitope) of the equine type-II collagen fragment created by mammalian collagenases. This peptide was used to produce a polyclonal antibody, characterized by western analysis for reactivity to native and collagenase-cleaved equine collagens. The antibody was evaluated as an antineoepitope antibody by ELISA, using peptides +/- an amino acid at the C-terminus of the immunizing peptide. Collagen cleavage was assayed from equine articular cartilage cultured with interleukin-1 (IL-1), +/- a synthetic MMP inhibitor, BAY 12-9566. Cartilage specimens from osteoarthritic and nonarthritic joints were compared for antibody staining. RESULTS: An antibody, 234CEQ, recognized only collagenase-generated 3/4-length fragments of equine type-II collagen. This was a true antineoepitope antibody, as altering the C-terminus of the immunizing peptide significantly decreased competition for binding in an inhibition ELISA. The IL-1-induced release of type-II collagen fragments from articular cartilage was prevented with the MMP inhibitor. Cartilage from an osteoarthritic joint of a horse had increased staining with the 234CEQ antibody, compared with normal articular cartilage. CONCLUSIONS AND CLINICAL RELEVANCE: We generated an antineoepitope antibody recognizing collagenase-cleaved type-II collagen of horses. This antibody detects increases in type-II collagen cleavage in diseased equine articular cartilage. The 234CEQ antibody has the potential to aid in the early diagnosis of arthritis and to monitor treatment responses.

Type II collagen degradation in articular cartilage fibrillation after anterior cruciate ligament transection in rats.

OBJECTIVE: To investigate the kinetics of early cartilage changes in mechanically induced osteoarthritis (OA) and the association of these changes with damage to the type II collagen network. METHODS: Experimental OA was induced by anterior cruciate ligament transsection in the rat knee joint (ACLT-OA). Animals were sacrificed after 2, 7, 14, 28 and 70 days. Knee joints were evaluated using routine histology and immunohistochemistry for denatured (unwound) type II collagen to detect collagen damage. An antibody recognizing the collagenase cleavage site in type II collagen was used to study the role of collagenase in this process. RESULTS: The first changes of the articular cartilage after anterior cruciate ligament transection occurred in the superficial zone. These changes included loss of superficial chondrocytes, swelling of the remaining chondrocytes and superficial fibrillation. The swelling of the chondrocytes did not result from a change towards the hypertrophic phenotype, since these cells did not stain for type X collagen. A marked increase in denatured type II collagen staining was present in the fibrillated areas. Staining of the collagenase cleavage site showed the same distribution as denatured collagen but was clearly less intense. Collagen damage could never be detected before fibrillation occurred and was not present in non-fibrillated areas. CONCLUSIONS: These results indicate that in this model cartilage degeneration starts at the articular surface and that this degeneration is associated with a localized expression of type II collagen degradation products.

Comparison of the degradation of type II collagen and proteoglycan in nasal and articular cartilages induced by interleukin-1 and the selective inhibition of type II collagen cleavage by collagenase.

OBJECTIVE: To compare interleukin-1alpha (IL-1alpha)-induced degradation of nasal and articular cartilages in terms of proteoglycan loss and type II collagen cleavage, denaturation, and release; to examine the temporal relationship of these changes; and to investigate the effects of an inhibitor of collagenase 2 and collagenase 3 on these catabolic processes. METHODS: Discs of mature bovine nasal and articular cartilages were cultured with or without human IL-1alpha (5 ng/ml) with or without RS102,481, a selective synthetic inhibitor of collagenase 2 and collagenase 3 (matrix metalloproteinase 8 [MMP-8] and MMP-13, respectively) but not of collagenase 1 (MMP-1). Immunoassays were used to measure collagenase-generated type II collagen cleavage neoepitope (antibody COL2-3/4C(short)) and denaturation (antibody COL2-3/4m), as well as total type II collagen content (antibody COL2-3/4m) in articular cartilage and culture media. A colorimetric assay was used to measure total proteoglycan concentration (principally of aggrecan) as sulfated glycosaminoglycans (sGAG). RESULTS: IL-1alpha initially induced a decrease in tissue proteoglycan content in nasal cartilage. A progressive loss of proteoglycan was noted during culture in articular cartilages, irrespective of the presence of IL-1alpha. In both cartilages, proteoglycan loss was followed by IL-1alpha-induced cleavage of type II collagen by collagenase, which was often reflected by increased denaturation. The inhibitor RS102,481 had no clear effect on the reduction in proteoglycan content (measured by sGAG) and collagen denaturation in either cartilage, but at 10 nM it inhibited the enhanced cleavage of type II collagen, partially in nasal cartilage and completely in articular cartilage. CONCLUSION: IL-1alpha-induced cleavage and denaturation of type II collagen is observed in both hyaline cartilages and is secondary to proteoglycan loss. It probably involves different collagenases, since there is no evidence of a rate-limiting role for collagenase 1 in articular cartilage, unlike the case for nasal cartilage. Inhibitors of this kind may be of value in the treatment of cartilage damage in arthritis. Also, the ability to detect the release of type II collagen collagenase-generated fragments from degraded cartilage offers the potential to monitor cartilage collagen damage and its control in vivo.

Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1).

OBJECTIVE: To examine whether type II collagen cleavage by collagenase and loss of proteoglycan are excessive in human osteoarthritic (OA) articular cartilage compared with nonarthritic articular cartilage, and whether this can be inhibited by a selective synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1 [MMP-1]). METHODS: Articular cartilage samples were obtained during surgery from 11 patients with OA and at autopsy from 5 adults without arthritis. The articular cartilage samples were cultured in serum-free medium. A collagenase-generated neoepitope, which reflects cleavage of type II collagen, and proteoglycan glycosaminoglycan (GAG), which predominantly reflects aggrecan release, were assayed in culture media. In addition, cultures were performed using either of 2 synthetic MMP inhibitors, both of which inhibited collagenase 2 (MMP-8) and collagenase 3 (MMP-13), but one of which spared collagenase 1. Cultures were also biolabeled with 3H-proline in the presence and absence of these inhibitors to measure collagen synthesis (as tritiated hydroxyproline) and incorporation in articular cartilage. RESULTS: As a group, cleavage of type II collagen by collagenase was significantly increased in OA cartilage samples. In contrast, proteoglycan (GAG) release was not increased. This release of a collagenase-generated epitope was inhibited by both MMP inhibitors in 2 of 5 nonarthritic samples and in 9 of 11 OA cartilage samples. The inhibitor that spared collagenase 1 was generally more effective and inhibited release from 4 of 5 nonarthritic cartilage samples and the same OA cartilage samples. Group analyses revealed that the inhibition of collagenase neoepitope release by both inhibitors was significant in the OA patient cartilage, but not in the nonarthritic cartilage. Proteoglycan loss was unaffected by either inhibitor. Newly synthesized collagen (predominantly, type II) exhibited increased incorporation in OA cartilage, but only in the presence of the inhibitor that arrested collagenase 1 activity. CONCLUSION: These results further indicate that the digestion of type II collagen by collagenase is selectively increased in OA cartilage, and that this can be inhibited in the majority of cases by a synthetic inhibitor that can inhibit collagenases 2 and 3, but not collagenase 1. The results also suggest that in OA, newly synthesized collagen is digested, but in a different manner than that of resident molecules. Proteoglycan release was not increased in OA cartilage and was unaffected by these inhibitors. Inhibitors of this kind may be of value in preventing damage to type II collagen in human arthritic articular cartilage.

Changes in collagen turnover in early acute respiratory distress syndrome.

Pulmonary fibrosis is a well-recognized feature of acute respiratory distress syndrome (ARDS). Using immunoassays of bronchoalveolar lavage (BAL), fluid we investigated the synthesis of type I procollagen (PICP) and type I/II collagen degradation products (COL2-3/4C(short) neoepitope) in patients with ARDS, acute lung injury (ALI), subjects with risk factors for ARDS (At Risk), and healthy/ventilated control subjects. PICP was measured by ELISA as a marker of type I procollagen synthesis. COL2-3/4C(short) neoepitope was measured by an inhibition ELISA as a marker of collagenase degradation of type I/II collagen. BAL was performed initially within 48 h of ventilation (Day 1) and then subsequently on Day 4. Dilution of epithelial lining fluid (ELF) was corrected for by plasma urea comparison. Increased PICP levels were observed in the ELF from ARDS and ALI subjects on Day 1 compared with subjects At Risk (median values, 124.9 and 95.0 ng/ml versus 38.0 ng/ml, respectively, p < 0.0005). By contrast, the levels of COL2-3/4C(short) neoepitope were significantly reduced in the subjects with ARDS versus the At Risk subjects (13.22 ng/ml versus 32.33 ng/ml, p < 0.0005). This translated into a greatly increased PICP:COL2-3/4C(short) ratio in the subjects with ARDS (p < 0.0001). There was a significant decline in the PICP level in the subjects with ARDS between Days 1 and 4 (n = 15, p < 0.05). Linear regression analysis showed a significant association between PICP and lung injury score in the subjects with ARDS (p = 0.01). Our data suggests an early shift in balance between type I collagen synthesis and degradation by collagenase. The resultant increase in type I collagen would favor matrix deposition and the development of pulmonary fibrosis in the lungs of subjects with ARDS.

Type II collagen degradation in spontaneous osteoarthritis in C57Bl/6 and BALB/c mice.

OBJECTIVE: Degradation of type II collagen during osteoarthritis (OA) is thought to be the key process leading to cartilage destruction. In this study, we investigated whether OA is characterized by either a generalized breakdown of the collagenous network or a localized process. Furthermore, we determined if collagen degradation was linked to cell death. METHODS: Two mouse strains that develop spontaneous OA, C57Bl/6 and BALB/c mice, were examined. Type II collagen degradation in type II collagen-induced arthritis was also examined for comparison. Immunolocalization with the COL2-3/4m and COL2-3/4C antibodies was used to demonstrate denatured type II collagen and the collagenase cleavage site in type II collagen, respectively. RESULTS: Both the C57Bl/6 and the BALB/c mice developed OA changes, although clear compartmental differences existed between the two strains. In both strains, type II collagen degradation was clearly present at sites of degeneration, but was absent from intact articular cartilage. Collagen degradation was absent from areas with cell death. CONCLUSION: These results indicate that type II collagen degradation in spontaneous murine OA is associated with degeneration and is a localized, instead of a generalized, process.

Secretory leukocyte protease inhibitor suppresses the inflammation and joint damage of bacterial cell wall-induced arthritis.

Disruption of the balance between proteases and protease inhibitors is often associated with pathologic tissue destruction. To explore the therapeutic potential of secretory leukocyte protease inhibitor (SLPI) in erosive joint diseases, we cloned, sequenced, and expressed active rat SLPI, which shares the protease-reactive site found in human SLPI. In a rat streptococcal cell wall (SCW)-induced model of inflammatory erosive polyarthritis, endogenous SLPI was unexpectedly upregulated at both mRNA and protein levels in inflamed joint tissues. Systemic delivery of purified recombinant rat SLPI inhibited joint inflammation and cartilage and bone destruction. Inflammatory pathways as reflected by circulating tumor necrosis factor alpha and nuclear factor kappaB activation and cartilage resorption detected by circulating levels of type II collagen collagenase-generated cleavage products were all diminished by SLPI treatment in acute and chronic arthritis, indicating that the action of SLPI may extend beyond inhibition of serine proteases.

Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques.

BACKGROUND: Several recent studies attempted to classify plaques as those prone to cause clinical manifestations (vulnerable, atheromatous plaques) or those less frequently associated with acute thrombotic complication (stable, fibrous plaques). Defining the cellular and molecular mechanisms that underlie these morphological features remains a challenge. Because interstitial forms of collagen determine the biomechanical strength of the atherosclerotic lesion, this study investigated expression of the collagen-degrading matrix metalloproteinase (MMP) interstitial collagenase-3 (MMP-13) and the previously studied MMP-1 in human atheroma and used a novel technique to test the hypothesis that collagenolysis in atheromatous lesions exceeds that in fibrous human atherosclerotic lesions. METHODS AND RESULTS: Human carotid atherosclerotic plaques, similar in size, were separated by conventional morphological characteristics into fibrous (n=10) and atheromatous (n=10) lesions. Immunohistochemical and Western blot analysis demonstrated increased levels of MMP-1 and MMP-13 in atheromatous versus fibrous plaques. In addition, collagenase-cleaved type I collagen, demonstrated by a novel cleavage-specific antibody, colocalized with MMP-1- and MMP-13-positive macrophages. Macrophages, rather than endothelial or smooth muscle cells, expressed MMP-13 and MMP-1 on stimulation in vitro. Furthermore, Western blot analysis demonstrated loss of interstitial collagen type I and increased collagenolysis in atheromatous versus fibrous lesions. Finally, atheromatous plaques contained higher levels of proinflammatory cytokines, activators of MMPs. CONCLUSIONS: This report demonstrates that atheromatous rather than fibrous plaques might be prone to rupture due to increased collagenolysis associated with macrophages, probably mediated by the interstitial collagenases MMP-1 and MMP-13.

Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage.

We demonstrate the direct involvement of increased collagenase activity in the cleavage of type II collagen in osteoarthritic human femoral condylar cartilage by developing and using antibodies reactive to carboxy-terminal (COL2-3/4C(short)) and amino-terminal (COL2-1/4N1) neoepitopes generated by cleavage of native human type II collagen by collagenase matrix metalloproteinase (MMP)-1 (collagenase-1), MMP-8 (collagenase-2), and MMP-13 (collagenase-3). A secondary cleavage followed the initial cleavage produced by these recombinant collagenases. This generated neoepitope COL2-1/4N2. There was significantly more COL2-3/4C(short) neoepitope in osteoarthritis (OA) compared to adult nonarthritic cartilages as determined by immunoassay of cartilage extracts. A synthetic preferential inhibitor of MMP-13 significantly reduced the unstimulated release in culture of neoepitope COL2-3/4C(short) from human osteoarthritic cartilage explants. These data suggest that collagenase(s) produced by chondrocytes is (are) involved in the cleavage and denaturation of type II collagen in articular cartilage, that this is increased in OA, and that MMP-13 may play a significant role in this process.