George R. Dodge

Immunohistochemical detection and immunochemical analysis of type II collagen degradation in human normal, rheumatoid, and osteoarthritic articular cartilages and in explants of bovine articular cartilage cultured with interleukin 1.

Articular cartilage destruction and loss of function in arthritic diseases involves proteolytic degradation of the connective tissue matrix. We have investigated the degradation of cartilage collagen by developing immunochemical methods that permit the identification and analysis of type II collagen degradation in situ. Previously, a technique to specifically identify type II collagen degradation in situ in articular cartilage did not exist. These methods utilize a polyclonal antiserum (R181) that specifically reacts with unwound alpha-chains and CNBr-derived peptides, alpha 1(II)CB11 and alpha 1(II)CB8, of human and bovine type II collagens. The experimental approach is based on the fact that when fibrillar collagens are cleaved the helical collagen molecule unwinds, exposing hidden epitopes. Here we demonstrate the use of R181 in studying type II collagen degradation in bovine articular cartilage that has been cultured with or without IL-1 and in human normal, rheumatoid, and osteoarthritic articular cartilages. Compared to cartilages either freshly isolated or cultured without IL-1, bovine cartilage cultured with IL-1 for 3-5 d showed an increase in both pericellular and intercellular immunohistochemical staining. Extracts of these cartilages contained type II collagen alpha chains that were increased in amount after culture with IL-1 for 11 d. In addition, culture with IL-1 resulted in the appearance of alpha chain fragments of lower molecular weight. All human arthritic tissues examined showed areas of pronounced pericellular and territorial staining for collagen degradation as compared with non-diseased tissues, indicating that chondrocytes are responsible in part for this degradation as compared with non-diseased tissues. In most cases rheumatoid cartilage was stained most intensely at the articular surface and in the deep and mid-zones, whereas osteoarthritic cartilage usually stained more in the superficial and mid-zones, but less intensely. Distinct patterns of sites of collagen degradation reflect differences in collagen destruction in these diseases, suggesting possible different sources of chondrocyte activation. These experiments demonstrate the application of immunological methods to detect collagen degradation and demonstrate an increase of collagen degradation in human arthritides and in IL-1-treated viable bovine cartilage.

Transcatheter delivery of c-myc antisense oligomers reduces neointimal formation in a porcine model of coronary artery balloon injury.

BackgroundSmooth muscle cell proliferation and extracellular matrix accumulation are the principal mechanisms leading to vascular restenosis. We have previously demonstrated the growth-inhibitory effect of antisense oligomers targeting the c-myc proto-oncogene in human smooth muscle cells. The goal of this study was to investigate whether c-myc antisense oligomers reduce neointimal formation in balloon-denuded porcine coronary arteries. Methods and ResultsFirst, type I collagen synthesis, which reflects synthetic function, was markedly reduced following c-myc antisense oligomers in porcine vascular smooth muscle cells independent of the growth inhibition. These effects in vitro provided the rationale for assessing c-myc antisense oligomers in the prevention of neointima in vivo. Second, the efficiency of single transcatheter delivery of oligomers into denuded porcine coronary arteries was determined. Despite rapid plasma clearance following local delivery, oligomers persisted at the site of injection for at least 3 days, exceeding by severalfold their concentration in peripheral organs. Third, morphometric analyses were carried out in balloon-denuded coronary arteries at 1 month after transcatheter c-myc an-V tisense oligomer administration. Maximal neointimal area was reduced from 0.80±0.17 mm2 in the control group (n=12) to 0.24±0.06 mm2 in the antisense-treated group (n= 13, P<.01). Likewise, a significant reduction in maximal neointimal thickness was observed in the antisense-treated group (P<.01). These changes in vascular remodeling following denuding injury resulted in an increase in residual lumen from 64±6% in the control group to 81±5% in the antisense-treated group (P<.05). Conclusions(1) Single transcatheter administration al-lowed for endoluminal delivery of oligomers to the site of coronary arterial injury. (2) C-myc antisense oligomers reduced the formation of neointima in denuded coronary arteries, implying a therapeutic potential of this approach for the prevention of coronary restenosis. (3) It is postulated that the c-myc proto-oncogene is involved in the process of vascular remodeling, regulating smooth muscle cell proliferation and extracellular matrix synthesis.

Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes

OBJECTIVEnThe functional integrity of articular cartilage is determined by a balance between chondrocyte biosynthesis of extracellular matrix and its degradation. In osteoarthritis (OA), the balance is disturbed by an increase in matrix degradative enzymes and a decrease in biosynthesis of constitutive extracellular matrix molecules, such as collagen type II and aggrecan. In this study, we examined the effects of the sulfate salt of glucosamine (GS) on the mRNA and protein levels of the proteoglycan aggrecan and on the activity of matrix metalloproteinase (MMP)-3 in cultured human OA articular chondrocytes.nnnDESIGNnFreshly isolated chondrocytes were obtained from knee cartilage of patients with OA. Levels of aggrecan and MMP-3 were determined in culture media by employing Western blots after incubation with GS at concentrations ranging from 0.2 to 200 microM. Zymography (casein) was performed to confirm that effects observed at the protein level were reflected at the level of enzymatic activity. Northern hybridizations were used to examine effects of GS on levels of aggrecan and MMP-3 mRNA. Glycosaminoglycan (GAG) assays were performed on the cell layers to determine levels of cell-associated GAG component of proteoglycans.nnnRESULTSnTreatment of OA chondrocytes with GS (1.0-150 microM) resulted in a dose-dependent increase in aggrecan core protein levels, which reached 120% at 150 microM GS. These effects appeared to be due to increased expression of the corresponding gene as indicated by an increase in aggrecan mRNA levels in response to GS. MMP-3 levels decreased (18-65%) as determined by Western blots. Reduction of MMP-3 protein was accompanied by a parallel reduction in enzymatic activity. GS caused a dose-dependent increase (25-140%) in cell-associated GAG content. Chondrocytes obtained from 40% of OA patients failed to respond to GS.nnnCONCLUSIONSnThe results indicate that GS can stimulate mRNA and protein levels of aggrecan core protein and, at the same time, inhibit production and enzymatic activity of matrix-degrading MMP-3 in chondrocytes from OA articular cartilage. These results provide a cogent molecular mechanism to support clinical observations suggesting that GS may have a beneficial effect in the prevention of articular cartilage loss in some patients with OA.

Quantification of cartilage biomechanical and biochemical properties via T1ρ magnetic resonance imaging

The aim of this study is to develop T1ρ as an MR marker of the compositional and functional condition of cartilage. Specifically, we investigate the correlation of changes in cartilage biomechanical and biochemical properties with T1ρ relaxation rate in a cytokine‐induced model of degeneration. Bovine cartilage explants were cultured with 30 ng/mL of interleukin‐1β to mimic the cartilage degradation of early osteoarthritis. The average rate of T1ρ relaxation was calculated from T1ρ maps acquired on a 4.7 T research scanner. Stress‐relaxation biomechanical tests were conducted with a confined compression apparatus to measure uniaxial aggregate modulus (HA) and hydraulic permeability (k0) using linear biphasic theory. Proteoglycan, collagen, and water content were measured via biochemical assays. Average T1ρ relaxation rate was strongly correlated with proteoglycan content (R2 = 0.926), HA (R2 = 0.828), and log10 k0 (R2 = 0.862). Results of this study demonstrate that T1ρ MRI can detect changes in proteoglycan content and biomechanical properties of cartilage in a physiologically relevant model of cartilage degeneration. The T1ρ technique can potentially be used to noninvasively and quantitatively assess the biochemical and biomechanical characteristics of articular cartilage in humans during the progression of osteoarthritis. Magn Reson Med, 2005.

Correlation of T1ρ with fixed charge density in cartilage

To establish the specificity of T1ρ with respect to fixed charge density (FCD) as a measure of proteoglycan (PG) content in cartilage during the onset of osteoarthritis (OA).

The extracellular matrix of cartilage in the growth plate before and during calcification: Changes in composition and degradation of type II collagen

SummaryCalcification occurs in the extracellular matrix of the hypertrophic zone of the growth plate when the extra-cellular matrix volume is reduced to a minimum and alkaline phosphatase content is maximal. The present study shows that significant quantitative and qualitative changes occur in the composition and structure of macromolecules in the extracellular matrix before and during calcification in the proximal tibial growth plate of the bovine fetus. These were detected in part by using microchemical and microimmuno-chemical analyses of sequential transverse frozen sections at chemical analyses of sequential transverse frozen sections at defined sites throughout the growth plate. Concentrations of matrix molecules in the extracellular matrix have not previously been determined biochemically. They were measured per unit matrix volume by using combined immunochemical/chemical-histomorphometric analyses. The concentrations within the extracellular matrix of the C-propeptide of type II collagen, aggregating proteoglycan (aggrecan), and hyaluronic acid all progressively increased in the maturing and hypertrophic zones, being maximal (or near maximal) at the time of initiation of mineralization. These results for proteoglycan are contrary to some earlier reports of a loss of proteoglycan prior to mineralization which measured the tissue content of proteoglycan rather than that present in the extracellular matrix, the volume of which is progressively reduced as the growth plate matures. The C-propeptide data provides a quantitative confirmation of previous immunohistochemical studies. Total collagen concentration (measured as hydroxyproline) in the extracellular matrix initially increased through the proliferating and maturing zones but then rapidly decreased in the hypertrophic zone. Immunohistochemical studies revealed that this is associated with the unwinding of the triple helix of type II collagen (previously shown to result from cleavage) which starts in pericellular sites in the zone of maturation (when type X collagen is first synthesized) and then extends throughout the hypertrophic zone. The significance of these matrix changes in the development and mineralization of the growth plate is discussed.

Direct evidence for active metalloproteinases mediating matrix degradation in interleukin 1-stimulated human articular cartilage.

When adult human articular cartilage was maintained in organ culture in the presence of interleukin 1 beta, increased destruction of the extracellular matrix was observed, as judged by increased type II collagen degradation in situ determined immunohistochemically and the increased release of proteoglycan into the culture medium. Concomitant with these changes was the increased release of latent metalloproteinases into the culture medium. Culture of cartilage in the presence of a peptidylhydroxamate metalloproteinase inhibitor indicated a key role for the active forms of these enzymes in situ, since it produced a marked reduction in both proteoglycan release and collagen degradation. This compound had no detectable cytotoxic effects in organ culture and did not reduce the secretion of the metalloproteinases. The results of this study provide direct evidence that the latent metalloproteinase precursors, whose release is greatly stimulated by interleukin 1, are indeed activated to some degree and participate in cartilage matrix degradation.

Differential MHC Class II-Mediated Presentation of Rheumatoid Arthritis Autoantigens by Human Dendritic Cells and Macrophages

Rheumatoid arthritis is characterized by synovial joint infiltration of activated CD4+ T cells and MHC class II+ APC, and is linked to specific HLA-DR alleles. Candidate autoantigens in synovial fluid and cartilage include type II collagen (CII) and cartilage gp39 (HCgp39). Using preparations of native Ag and T cells derived from Ag-immunized DR4-transgenic mice, we determined that human ex vivo differentiated DR4+ dendritic cells (DC) and macrophages (Mφ) can mediate MHC class II presentation of CII or HCgp39 epitopes. The form of the Ag (soluble, partially degraded, or particulate) delivered to the APC influenced its presentation by DC and Mφ. DC efficiently presented partially degraded, but not native CII α-chains, while Mφ presentation was most efficient after phagocytosis of bead-conjugated CII. Both DC and Mφ presented soluble HCgp39, and activated Mφ from some donors presented epitopes derived from endogenously synthesized HCgp39. When synovial fluid from rheumatoid arthritis patients was used as a source of Ag, DC presentation of HCgp39 and CII epitopes was efficient, indicating that synovial fluid contains soluble forms of CII and HCgp39 amenable to internalization, processing, and presentation. These data support the hypothesis that CII and HCgp39 are autoantigens and that their class II-mediated presentation by DC and Mφ to T cells in vivo has a critical role in the pathogenesis of human rheumatoid arthritis.

Dose-dependent effects of corticosteroids on the expression of matrix- related genes in normal and cytokine-treated articular chondrocytes

Abstract:Objective and Design: To assess the effects of glucocorticoids on the expression of multiple matrix-related genes in normal and cytokine-treated cultured equine articular chondrocytes in a phenotypically correct suspension culture.¶Material or Subjects: Articular cartilage harvested from the joints of 15 foals, 7 yearling horses, and 16 adult horses.¶Treatment: Glucocorticoids (dexamethasone, prednisolone, triamcinolone) at 10–10 to 10–4 M.¶Methods: Equine articular chondrocytes maintained in suspension cultures were treated with glucocorticoids with and without human recombinant interleukin 1-β (IL1-β) and tumor necrosis factor-α (TNF-α). Northern blots of total RNA from the treated cells were probed with equine specific cDNA probes for a number of cartilage matrix-related genes. Zymography, Western blotting, and fluorography were also performed to study the effects on protein synthesis.nResults: The glucocorticoids, dexamethasone, triamcinolone, and prednisolone, markedly decreased MMP1, MMP3, MMP13, TIMP1, and ferritin steady-state mRNA levels. There were no qualitative differences seen among the tested corticosteroids although dexamethasone and triamcinolone appeared to be slightly more potent than prednisolone. The effects of the glucocorticoids on MMP transcription occurred consistently at lower doses than those required to similarly downregulate type II collagen and aggrecan. Link protein and fibronectin mRNA were increased by the glucocorticoids, and biglycan and decorin were minimally affected. Fluorography of [14-C] proline-labeled media demonstrated that the decrease in type II collagen transcription (mRNA levels) was paralleled at the protein level. Zymography and Western blotting confirmed the decrease in functional metalloproteinases found in chondrocyte cultures following glucocorticoid treatment.¶Conclusions: The effects of glucocorticoids are complex inasmuch as they differentially affect numerous genes involved in the composition of cartilage matrix and the degradation of that matrix. This study provides new insight into the effects of glucocorticoids on the regulation of extracellular matrix and matrix-related genes by demonstrating that low doses of glucocorticoids can inhibit the degradative metalloproteinases with minimal negative effects on the transcription of extracellular matrix genes.

Sodium magnetic resonance imaging of proteoglycan depletion in an in vivo model of osteoarthritis1

RATIONALE AND OBJECTIVESnThe aim of the study was to investigate the feasibility of using sodium magnetic resonance imaging (MRI) as a noninvasive quantitative technique for measuring proteoglycan (PG) content in an in vivo porcine model of osteoarthritis (OA).nnnMATERIALS AND METHODSnBiochemical conditions similar to those of OA were created by an intra-articular injection of recombinant porcine interleukin-1beta (IL-1beta) into the knee joint of pigs (n = 6) before performing MRI. The contralateral knee joint was given a saline injection to serve as an internal control. Sodium MRI data were acquired on a 4-T clinical MR scanner and used to compute quantitative sodium and fixed charge density (FCD) maps based on a previously established methodology. In vivo FCD maps were compared with FCD maps obtained using ex vivo patellae harvested from the specimens. The tissue and joint fluid were subjected to histologic and immunohistochemical analyses as independent measurements of IL-1beta activity and PG loss.nnnRESULTSnThe average FCD of IL-1beta-treated patellae was measured to be 49% lower than that of saline-treated patellae, indicating a loss of PG content. These results were supported by histologic and immunochemical findings, most notably a reduction in staining for PG and an increase in matrix metalloproteinases in the synovial fluid.nnnCONCLUSIONnSodium MRI can serve as a quantitative method to measure in vivo changes in PG content in an animal model of OA. The use of a noninvasive quantitative in vivo PG measurement technique such as sodium MRI on an animal model would aid greatly in efforts to monitor the efficacy of treatments for OA. Furthermore, these results indicate that early degenerative events could be detected noninvasively in vivo in humans with PG-depleting diseases such as OA.