Biomedical subjects

D D Dean

Publications and source records attributed to D D Dean.

At least 145 records · Page 8Linked to original sources

PubMed1992

Matrix vesicles contain metalloproteinases that degrade proteoglycans.

This study explored whether extracellular matrix processing enzymes are present in matrix vesicles produced by rat costochondral resting zone and growth zone chondrocytes in culture. It was found that there was a differential distribution of enzyme activities related to the cartilage zone from which the cells were isolated. There was a 3-fold enrichment of total and active acid metalloproteinase in growth zone chondrocyte (GC) matrix vesicles whereas no enrichment in enzyme activity was observed in resting zone chondrocyte (RC) matrix vesicles. Total and active neutral metalloproteinase were similarly enriched 2-fold in GC matrix vesicles. TIMP, plasminogen activator and beta-glucuronidase activities were highest in the plasma membranes of both cell types. No collagenase, lysozyme, or hyaluronidase activity was found. The data indicate that matrix vesicles are selectively enriched in enzymes that degrade proteoglycans. The highest concentrations of these enzymes are found in matrix vesicles produced by growth zone chondrocytes, suggesting that this may be a mechanism by which the more differentiated cell modulates the matrix for calcification.

Animals↗

PubMed1992

Preliminary observations of chondral abrasion in a canine model.

Articular cartilage repair was followed for one year in skeletally mature dogs after destabilisation by anterior cruciate ligament transection of the stifle joint (CT), abrasion of the inferior medial condyle (ABR) to bleeding bone, or anterior cruciate transection followed by chondral abrasion (CT/ABR). ABR animals formed repair cartilage at the abrasion site (ABR and CT/ABR) at six months as determined by arthroscopy and at necropsy. CT and CT/ABR animals had an additional cartilage ulcer on the superior aspect of the medial condyle. The abraded site extended in CT/ABR condyles. Repair cartilage (ABR and CT/ABR) contained reduced amounts of proteoglycan as seen by histological loss of safranin O staining and reduced uronic acid content. Fibrocartilage was suggested by histological appearance, hypocellularity, and a higher hydroxyproline content. In contrast with ABR animals, the repair cartilage in the CT/ABR animals contained near normal amounts of hydroxyproline. Collagen profiles of abrasion site repair cartilage in ABR animals had more types I and V collagens, similar amounts of type VI collagen, and decreased amounts of types II, IX, and XI collagens than CT/ABR animals. The results of this study are consistent with abrasion chondroplasty leading to a repair cartilage. Despite extended ulcers, repair cartilage from the destabilised joint (CT/ABR) animals was more hyaline-like in its hydroxyproline content and collagen composition than repair cartilage from the stable joint (ABR animals). In these models additional measures appear to be needed as the defects induced by abrasion chondroplasty did not form a functional hyaline cartilage.

Animals↗

PubMed1991

Amelioration of lapine osteoarthritis by treatment with glycosaminoglycan-peptide association complex (Rumalon).

The chondroprotective potential of glycosaminoglycan-peptide association complex (GP-C) was examined in the medial meniscectomy model of lapine osteoarthritis (OA). Prophylactic treatment with increasing doses of intramuscular GP-C (0.05-0.5 ml/kg) caused a significant reduction in OA lesion area and histologic scores, and the effect on disease activity appeared to be dose related. The DNA and uronic acid contents of OA tissue were unaffected by prophylactic treatment with GP-C. However, levels of hydroxyproline in OA cartilage increased to near control levels with prophylactic treatment. Cartilage levels of active and total metalloproteinases that digest proteoglycans were elevated in rabbits with OA; prophylactic treatment with low-dose GP-C (0.05 ml/kg) produced a significant reduction in active, but not total, enzyme. Cartilage levels of tissue inhibitor of metalloproteinases in animals with OA were comparable with control levels, but rose with increasing doses of GP-C. We also investigated GP-C as a therapeutic treatment in animals that had already developed OA lesions. Carbon black retention and histologic score returned to near-normal after therapeutic treatment with GP-C. Uronic acid and hydroxyproline levels were decreased in OA cartilage. Therapeutic treatment with GP-C had no statistically significant effect on uronic acid levels, but was associated with increased hydroxyproline content in the cartilage. The changes in metalloproteinase and metalloproteinase inhibitor were similar to those found in the studies of prophylactic treatment. The findings in this animal model may help explain some of the beneficial effects of GP-C in human OA.

Animals↗

PubMed1991

Proteinase-mediated cartilage degradation in osteoarthritis.

Osteoarthritis (OA) is a disease of complex etiology that results in articular cartilage breakdown. Current experimental evidence strongly suggests that proteinases may be involved in this loss of cartilage matrix. The present review summarizes the evidence for implicating proteinases in the etiopathogenesis of OA and suggests that blockade of proteinase activity may provide a rational basis for new therapeutic agents.

Cartilage↗

PubMed1990

Changes in active and latent collagenase in human placenta around the time of parturition.

High levels of collagenase are present in cervical extracts and in the circulation in women at parturition. This study examines the content of collagenase in human placentas at term, the possible contribution to circulating enzyme, and the changes that occur at parturition. Active and latent forms of collagenase are detectable in placentas with apparent relative molecular mass of 60,000 and 65,000 d, respectively. Gel-filtration chromatography was used to identify the presence of excess tissue inhibitor of metalloproteinase as the major collagenase inhibitor in the extracellular matrix of human placentas. After the onset of labor, there was a significant increase in total collagenase activity. Inactivation of the tissue inhibitor of metalloproteinase by reduction of placental extracts with dithiothreitol and alkylation with iodoacetamide resulted in a twelvefold to seventeenfold increase in collagenase activity. Umbilical cord collagenase levels were significantly lower than those in maternal circulation. The possibility of circulating collagenase originating from placenta in labor is discussed.

Chromatography, Gel↗

PubMed1990

Osteoarthritis research: animal models.

Although limited in scope and detail, the current review has sought to highlight the contribution of animal models in the study of OA. Further use of these and other models should provide information that may lead to methods for the early detection and successful treatment of human OA.

Animals↗

PubMed1990

Production of collagenase and tissue inhibitor of metalloproteinases (TIMP) by rat growth plates in culture.

Growth plate cartilage from normal and vitamin D-phosphate deficient (-VDP) rats was cultured to study the production of collagenase and tissue inhibitor of metalloproteinases (TIMP) in vitro. All tissues secreted latent collagenase into the medium at a constant rate during the 5 days in culture. Microdissected-VDP growth plates, containing predominatly hypertrophic cells, released up to 8-fold more collagenase into the medium than either intact-VDP or normal growth plates. TIMP was also secreted during the culture, but its rate of production was not as dependent on tissue type as collagenase. The tissue level of collagenase and TIMP before culture was compared with that found in conditioned medium and remnant tissue after culture. During the 5 day culture period microdissected-VDP growth plates, containing predominatly hypertrophic cells, produced 3-times more collagenase/microgram DNA over the starting level than either intact-VDP or normal growth plates. TIMP was never found in tissues after they had been cultured, but was present in all tissues before culture except those containing predominatly hypertrophic cells. The amount of TIMP required to block collagenase was calculated. Growth plates in culture produced enough TIMP to block all collagenase found in the medium and remnant tissue, while extracts of uncultured intact -VDP growth plates, and those divided to contain hypertrophic cells, had an excess of collagenase over TIMP. The results suggest that hypertrophic cells produce far more collagenase than other cells in the growth plate, but all cell types have about the same capacity to synthesize TIMP. As a result, increased collagenase synthesis by hypertrophic cells may surpass increases in TIMP synthesis and lead to collagen removal. This would allow for thinning of the longitudinal septa and expansion of the hypertrophic cells.

Animals↗

PubMed1989

Prophylactic treatment of canine osteoarthritis with glycosaminoglycan polysulfuric acid ester.

The prophylactic effect of glycosaminoglycan polysulfuric acid ester (GAGPS) on cartilage lesions was studied using the Pond-Nuki model of canine osteoarthritis. Starting 2 days after anterior cruciate transection, GAGPS or saline was administered intraarticularly twice weekly for 4 weeks. After 4 weeks, gross and histologic medial femoral condylar lesions had developed to a lesser degree in GAGPS-treated dogs than in saline-treated dogs. The uronic acid and hydroxyproline levels in cartilage were significantly higher in the GAGPS-treated dogs than in the saline-treated dogs. Levels of active and latent collagenase in the cartilage of GAGPS-treated dogs were lower than in the cartilage of saline-treated dogs. With GAGPS treatment, swelling of the cartilage, an indicator of collagen network integrity, remained near control levels. Although increased synthesis of proteoglycan and collagen may account for some of these results, we propose that one mechanism of action of GAGPS is its ability to decrease collagen degradation, either by decreasing the synthesis of collagenase or by directly inhibiting the production of collagenase in cartilage.

Animals↗

PubMed1989

Therapeutic treatment of canine osteoarthritis with glycosaminoglycan polysulfuric acid ester.

The therapeutic effect of glycosaminoglycan polysulfuric acid ester (GAGPS) was studied using the Pond-Nuki model of canine osteoarthritis. The clinical setting was simulated by permitting 4 weeks ambulation without treatment, following anterior cruciate transection. Animals were then injected with GAGPS, 4 mg/kg intramuscularly, twice weekly during weeks 4-8. Control animals received intramuscular saline. The study was terminated 4 weeks after completion of the GAGPS or saline regimen (i.e., 12 weeks postoperatively). Cartilage from the medial femoral condyle was analyzed for collagen integrity (swelling properties), hydroxyproline, uronic acid, active and total proteoglycan (PG)-degrading metalloproteinase, PG-degrading serine proteinase, and histopathology (Mankin score). Condylar cartilage from animals treated with GAGPS demonstrated less cartilage swelling, less total and active metalloproteinase, and lower histopathologic scores than were found in cartilage from saline-treated animals. GAGPS was able to suppress PG-degrading enzyme activity and maintain a more normal-appearing cartilage. It is proposed that GAGPS suppressed PG breakdown by decreasing synthesis of metalloproteinase or by directly inhibiting metalloproteinase in cartilage, rather than by increasing synthesis of PG by chondrocytes.

Animals↗

PubMed1989

The role of ovarian proteases and their inhibitors in ovulation.

To assess the role of inhibitors of proteolytic enzymes, such as plasminogen activator (PA) and collagenase in the ovulatory process, inhibitor activity and mRNA levels were examined in periovulatory rat and human ovaries. In the rat, immature animals received 20 IU of pregnant mare serum gonadotropin (PMSG) followed 52 h later by 10 IU of hCG. Ovaries were removed at intervals from 0 to 20 h after human chorionic gonadotropin (hCG) administration. Inhibitor activity for metalloproteinases, such as collagenase, increased from 60.5 +/- 4.1 inhibitor units/ovary at 0 h (i.e., time of hCG treatment) to a maximum of 218.2 +/- 11.4 units/ovary at 8 h after hCG before decreasing at 12 h (time of ovulation) and 20 h (122.2 +/- 7.9 and 71.6 +/- 8.1 units/ovary, respectively). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in our in vitro fertilization program. Metalloproteinase inhibitor activity was evaluated in follicular fluid as well as the levels of PA and PA inhibitor (PAI) mRNA by Northern analysis. Increasing metalloproteinase inhibitor activity was positively correlated with follicular levels of estradiol (p less than 0.001) and progesterone (p less than 0.02, N = 26). Chromatographic separation of follicular fluid resulted in two peaks of metalloproteinase inhibitor activity. The large molecular weight (MW) inhibitor had an approximate size of 700 kilodaltons (kDa) and may represent alpha 2-macroglobulin, a serum-derived inhibitor. The small MW inhibitor shared many of the characteristics of tissue-derived inhibitors of metalloproteinases. Partial purification of the small MW inhibitor by Concanavalin A-Sepharose and Heparin-Sepharose chromatography demonstrated the inhibitor to be a glycoprotein with an approximate MW = 28-29 K. Northern analysis of human granulosa cell total RNA from preovulatory follicles showed little or no detectable tissue-type PA or urokinase-type PA mRNA. In contrast, two species of PA inhibitor type-1 mRNA were detected in relative abundance. The present findings demonstrate the presence of proteolytic inhibitors in periovulatory ovaries of the rat and human. These ovarian inhibitors may play a role in regulating connective tissue remodeling during follicular rupture.

Animals↗

PubMed1989

Association of collagenase and tissue inhibitor of metalloproteinases (TIMP) with hypertrophic cell enlargement in the growth plate.

In the transition from proliferating to hypertrophic cell zones in the growth plate, there is an increased in chondrocyte cell volume and a corresponding decrease in collagen content to allow for cell enlargement. To substantiate our hypothesis that collagenase is responsible for these changes, growth plates from rats treated with bisphosphonate (HEBP) were compared histologically and biochemically with growth plates from normal and vitamin D and phosphate deficient (-VDP) rats. HEBP-treated rats developed an expanded hypertrophic cell zone (HCZ) characterized by the presence of two distinct populations of hypertrophic cells. The proximal hypertrophic cells were only 2-fold enlarged compared to the proliferating cells, whereas 1/6 of the distal hypertrophic cells were enlarged almost 5-fold and appeared morphologically identical with hypertrophic cells from normal and -VDP rats. The HEBP growth plates were divided into cross-sectional thirds and analyzed for active and latent collagenase. The juxta-metaphyseal (lower 1/3) cartilage contained 100% of the fully enlarged hypertrophic cells and appeared identical to those found in normal and -VDP growth plates, along with 81% of the active and 77% of the total collagenase. Collagenase and tissue inhibitor of metalloproteinases (TIMP) were measured in extracts of similarly divided tissues. The presence of true collagenas was confirmed by using [3H]-telopeptide-free collagen. TIMP levels were inversely related to the presence of active collagenase and cellular hypertrophy. Substantial levels of latent collagenase were found in the extracellular fluid at sites of active collagenolysis, but not in the fluid phase surrounding the 2-fold enlarged hypertrophic cells. It is postulated that increased amounts of active collagenase and insufficient levels of TIMP may account for the reduced collagen content seen in the lower HCZ of both -VDP and HEBP rickets. Unlike active collagenase, which remains localized by binding to collagen, latent enzyme is probably restricted in its mobility throughout the extracellular space by diffusion, itself, or the interstices of the extracellular matrix.

Animals↗

PubMed1989

Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage.

Cartilage specimens from tibial plateaus, obtained from 13 osteoarthritic (OA) patients and seven controls, were selected from three regions: zone A, center of fibrillated area; zone B, area adjacent to fibrillation, and zone C, remote region of plateau. Acid and neutral metalloproteinases and tissue inhibitor of metalloproteinase (TIMP) were extracted with 2 M guanidine. Methods were developed to selectively destroy either proteinases or TIMP to prevent cross-reaction during assay. Acid and neutral proteinases were elevated approximately 150% in OA; TIMP was elevated approximately 50%. A positive correlation (r = 0.50) was found between acid and neutral proteinase activities in OA, but not in controls. Both proteinases were elevated two-to threefold in zones A, B, and C. However, the self-active form of the acid metalloproteinase was elevated only in zones A and B (200%); it correlated well with the Mankin scores, whereas the total activities did not. TIMP was elevated (50%) only in zones A and B. Both the proteinase levels and the Mankin score were elevated to a greater extent in the medial, than in the lateral, compartment. Titration of TIMP against the two metalloproteinases indicates that there is a small excess of inhibitor over enzymes in normal cartilage. In OA, TIMP does not increase to the same extent as the proteinases; the resultant excess of proteinases over TIMP may contribute to cartilage breakdown.

Adult↗

PubMed1989

Hypertrophic chondrocytes produce immunoreactive collagenase in vivo.

A monospecific, polyclonal antibody to neutral collagenase purified from rat myometrial cells was used to examine decalcified sections of normal and rachitic rat long bones, including epiphyses, for presence of immunoreactive collagenase in situ. Reactive antigen was uniformly present in hypertrophic chondrocytes of articular and epiphyseal plate cartilage. Preincubation of antibody with excess homogeneously purified antigen blocked the staining, indicating specificity of the staining for the antigen. Reaction was present in the borders of the enlarging lacunae, suggesting functional importance of this enzyme in the process of lacunar enlargement prior to mineralization. No detectable enzyme was observed in osteoblasts, osteocytes, or osteoclasts. No difference between expression of collagenase by bone cells in rachitic or normal bone was seen. Thus, at the level of sensitivity of this procedure, neutral collagenase appears to function in mature rat bone only in enlarging chondrocyte lacunae.

Animals↗

PubMed1988

Elevated tissue levels of collagenase during dilation of uterine cervix in human parturition.

Seven biopsy specimens from the cervix and 17 from the lower uterine segment were obtained in 24 women at term (37 to 42 weeks). Collagenase was extracted and assayed on telopeptide-free [3H]collagen; typical collagen cleavage products were found on sodium dodecyl sulfate-gel electrophoresis. There was no significant difference between collagenase levels in the cervix and in the lower uterine segment in women not in labor and with the cervix closed. Levels of active and latent collagenase in 11 such specimens were 0.14 +/- 0.03 and 0.64 +/- 0.90 U/gm wet weight, respectively (mean +/- SEM; 1 U = 1 micrograms collagen digested per minute at 30 degrees C). Thirteen women at term in active labor with cervical dilation of 4 to 8 cm exhibited a thirteenfold increase in mean collagenase activity in the lower uterine segment. Active and latent collagenase increased to 2.06 +/- 0.92 and 8.64 +/- 2.87 U/gm, respectively. This is the first direct evidence that interstitial collagenase increases markedly during cervical dilation in human parturition.

Cervix Uteri↗

PubMed1988

Future therapeutic trends in osteoarthritis.

Since cartilage contains no nerve endings, symptoms of osteoarthritis (OA) are indirect. Therapy of OA, to date, has been directed at the symptoms of pain, signs of inflammation and loss of function. A major advance has been surgical joint replacement. This benefit however, is often limited by the joint area involved and to the survival of the endoprosthesis. Orthopedic new approaches to therapy of OA include removal of abnormal tissue to stimulate repair (e.g., burring, abrasion) and grafting (e.g., osteochondral grafts, perichondrium, periosteum) to the subchondral bone. Controlled activity (e.g., passive motion) has been studied alone and with the above. Can a medication retard or reverse the degradative process of OA? Several medications are being examined for potential "chondroprotective" characteristics. Some of these agents are not new: oversulfated glycosaminoglycans (Arteparon) derived from cartilage and glycosaminoglycan peptides (Rumalon) derived from cartilage and bone marrow extracts may be prototypes of this approach to therapy. Other agents demonstrating potential benefit in retarding cartilage degradation may include non-steroidal anti-inflammatory agents, tiaprofenic acid, sodium pentosan sulfate and low dose corticosteroids. This concept of "chondroprotection" provides us a new approach to a disease in need of a new approach.

Animals↗

PubMed1987

High levels of serum collagenase in premature labor--a potential biochemical marker.

An improved sensitive assay for collagenase, which uses [3H]telopeptide-free collagen as a substrate, was used to measure changes in serum collagenase levels in 96 women and ten men (18-35 years old). Both latent and active forms of collagenase were detected in serum by molecular sieve chromatography; these forms had a relative molecular weight (Mr) of 65,000 and 45,000, respectively. Only latent collagenase was detected in crude serum after destroying inhibitors by treatment with 3 M potassium thiocyanate. Collagenase levels in males were lower than in nongravid females (34 +/- 5 versus 53 +/- 5 U/dL; mean +/- SEM; 1 unit = 1 microgram collagen digested per minute at 30C). During pregnancy there was no significant change in serum collagenase levels until the onset of spontaneous labor in full-term pregnancies (37-42 weeks), at which point there was a 66% increase over the nongravid level to a value of 88 +/- 5 U/dL. There was a further rise at one day postpartum, and high levels continued for at least four days. Women in premature labor (24-36 weeks) exhibited an eightfold increase in the level of serum collagenase to 405 +/- 110 U/dL; 16 of 17 patients in this group had collagenase levels above the 95th percentile for women at 16-40 weeks but not in labor. This evaluation of serum collagenase may provide a key for detecting premature labor. It is suggested that the increase in serum collagenase arises from the lower uterine segment and cervix.

Cervix Uteri↗

PubMed1987

Levels of metalloproteases and tissue inhibitor of metalloproteases in human osteoarthritic cartilage.

Human articular cartilage contains 2 distinct metalloproteases which degrade proteoglycan. One protease acts optimally at pH 5.3 and the other at pH 7.2. In addition, cartilage contains a tissue inhibitor of metalloproteases (TIMP) that inhibits both proteases. Methods have been developed for the estimation of metalloproteases and TIMP in extracts of cartilage prepared in buffered 2 M guanidine-HCl. In osteoarthritic cartilage, levels of the 2 metalloproteases increase 3-fold or more, while the level of TIMP remains constant. It is postulated that a balance is maintained between inhibitor and metalloprotease levels in normal cartilage and that in osteoarthritis increased secretion of proteases upsets this balance and results in degradation of the extracellular matrix.

Cartilage, Articular↗

PubMed1987

The effect of glycosaminoglycan polysulfuric acid ester on articular cartilage in experimental arthritis: effects on collagenolytic enzyme activity and cartilage swelling properties.

Some of the effects of the semi-synthetic glycosaminoglycan polysulfuric acid ester (GAGPE) were investigated on the osteoarthritis-like lesions in the Pond-Nuki dog model, in respect to histological grade of anatomical lesions, collagenolytic enzyme activity, and a parameter of collagen network integrity. Prophylactic treatment by intra-articular injections twice weekly for 4 weeks caused amelioration of canine cartilage erosions. Preliminary evidence for suppression of collagenolytic enzyme activity, as well as protection of a tight collagen network studied in the canine cartilage was obtained.

Animals↗