Pathological changes and expression of lysine oxidases and matrix metalloproteinases -1, -2, and -3 in ligaments of patients with haemophilic arthritis.

INTRODUCTION: Studying the pathological changes of ligaments in patients with haemophilic arthritis (HA) has important significance for guiding the release of ligaments during total knee arthroplasty (TKA) and exploring interventions to prevent ligament lesions. AIM: This study was conducted to show the pathological changes and investigate the lysine oxidase (LOX) and matrix metalloproteinase (MMP)-1, -2, and -3 levels in the ligaments of patients with HA compared with those of patients with osteoarthritis (OA). METHODS: Ligaments obtained during the TKA were stained with Masson trichrome, Verhoeff-Van Gieson and haematoxylin and eosin to show the basic pathological changes. Collagen I, elastin, LOXs and MMP-1, -2, and -3 expression levels were detected via western blot. LOX and MMP-1, -2, and -3 mRNA expression levels were analysed via quantitative real-time PCR. RESULTS: Compared with OA ligaments, HA ligaments were constructed more loosely with wider gaps, more breaks, haemocytodeposition and local hypertrophy among the fibres. LOXs and MMP mRNA expression levels were upregulated in the HA tissues, which was consistent with the western blot results. Collagen I and elastin levels were also higher in patients with HA. CONCLUSIONS: The metabolism of the ligaments in patients with HA is more complex than in those with OA, and the ligaments of patients with HA have stronger healing and destruction processes. This pathology is related to iron overload and imbalanced inflammatory factors due to repeated intra-articular bleeding.

Expression of Heparanase in uterosacral ligaments of women with or without uterine prolapse.

OBJECTIVE: Pelvic organ prolapse (POP) is a global health problem for which the pathophysiological mechanism remains unclear. The loss of extracellular matrix proteins is considered an important molecular basis for this pathology. Heparanase is a heparin sulfate degrading endoglycosidase that has an important role in various biological processes and is a key component of extracellular matrix. The aim of this study was to compare expression of Heparanase in connective tissue of uterosacral ligaments in women with or without uterine prolapse. STUDY DESIGN: Thirty-nine women who underwent hysterectomy for benign reasons were enrolled in the study. Twenty-three women with uterine prolapse (stage ≥3) who underwent vaginal hysterectomy (VH) - POP group, were compared to sixteen women without uterine prolapse who underwent abdominal hysterectomy (stage <2) - control group. Uterosacral ligaments (USL) biopsies were obtained from all uterine specimens near their origin. All tissue samples were analyzed by immunohistochemistry and tested for the presence of Heparanase using antiheparanse antibody 733. RESULTS: Heparanse positive staining was more common in the connective tissue of uterosacral ligaments in women with uterine prolapse. Positive staining was seen in 17/23 (73.9 %) women with uterine prolapse compared to 4/16 (25 %) without uterine prolapse (p = 0.003). On multivariate logistic regression analysis, positive staining displayed a trend for an independent association with POP, after controlling for menopausal status and parity (OR 13.57, 95 %CI 0.82-224.4, p = 0.06). CONCLUSION: Heparanase expression is more common in the connective tissue of uterosacral ligaments in women with uterine prolapse compared to women with no prolapse.

Transforming growth factor ß1 and extracellular matrix protease expression in the uterosacral ligaments of patients with and without pelvic organ prolapse.

OBJECTIVES: This study was undertaken to evaluate the expression of transforming growth factor ß1 (TGF-ß1) and matrix metalloproteinase 9 (MMP-9), key regulators of the extracellular matrix composition, in the uterosacral ligaments (USLs) of women with pelvic organ prolapse (POP) compared with controls. METHODS: Under an institutional review board approval, USL samples were obtained from women undergoing vaginal hysterectomy for stage 2 or greater POP (cases, n = 21) and from women without POP undergoing vaginal hysterectomy for benign indications (controls, n = 19). Hematoxylin and eosin and trichrome staining were performed on the USL sections, and the distribution of smooth muscle and fibrous tissue were quantified. Immunohistochemical staining was performed using anti-TGF-ß1 and anti-MMP-9 antibodies. The expressions of TGF-ß1 and MMP-9 were evaluated by the pathologist, who was blinded to all clinical data. RESULTS: Transforming growth factor ß1 expression positively correlated with MMP-9 expression (R = 0.4, P = 0.01). The expressions of TGF-ß1 and MMP-9 were similar in subjects with POP versus controls. There was a significant increase in fibrous tissue (P = 0.008) and a corresponding decrease in smooth muscle (P = 0.03), associated with increasing age. The TGF-ß1 expression, but not MMP-9 expression, also significantly increased with age (P = 0.02). DISCUSSION: Although our study uncovered age-related alterations in USL composition and TGF-ß1 expression, there was no difference in the expression of TGF-ß1 or MMP-9 in the subjects with POP versus controls.

Matrix metalloproteinases in a sea urchin ligament with adaptable mechanical properties.

Mutable collagenous tissues (MCTs) of echinoderms show reversible changes in tensile properties (mutability) that are initiated and modulated by the nervous system via the activities of cells known as juxtaligamental cells. The molecular mechanism underpinning this mechanical adaptability has still to be elucidated. Adaptable connective tissues are also present in mammals, most notably in the uterine cervix, in which changes in stiffness result partly from changes in the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). There have been no attempts to assess the potential involvement of MMPs in the echinoderm mutability phenomenon, apart from studies dealing with a process whose relationship to the latter is uncertain. In this investigation we used the compass depressor ligaments (CDLs) of the sea-urchin Paracentrotus lividus. The effect of a synthetic MMP inhibitor - galardin - on the biomechanical properties of CDLs in different mechanical states ("standard", "compliant" and "stiff") was evaluated by dynamic mechanical analysis, and the presence of MMPs in normal and galardin-treated CDLs was determined semi-quantitatively by gelatin zymography. Galardin reversibly increased the stiffness and storage modulus of CDLs in all three states, although its effect was significantly lower in stiff than in standard or compliant CDLs. Gelatin zymography revealed a progressive increase in total gelatinolytic activity between the compliant, standard and stiff states, which was possibly due primarily to higher molecular weight components resulting from the inhibition and degradation of MMPs. Galardin caused no change in the gelatinolytic activity of stiff CDLs, a pronounced and statistically significant reduction in that of standard CDLs, and a pronounced, but not statistically significant, reduction in that of compliant CDLs. Our results provide evidence that MMPs may contribute to the variable tensility of the CDLs, in the light of which we provide an updated hypothesis for the regulatory mechanism controlling MCT mutability.

Effects of mechanical strain on human mesenchymal stem cells and ligament fibroblasts in a textured poly(L-lactide) scaffold for ligament tissue engineering.

The purpose of this study was to prove the effect of cyclic uniaxial intermittent strain on the mRNA expression of ligament-specific marker genes in human mesenchymal stem cells (MSC) and anterior cruciate ligament-derived fibroblasts (ACL-fibroblasts) seeded onto a novel textured poly(L-lactide) scaffold (PLA scaffold). Cell-seeded scaffolds were mechanically stimulated by cyclic uniaxial stretching. The expression of ligament matrix gene markers: collagen types I and III, fibronectin, tenascin C and decorin, as well as the proteolytic enzymes matrix metalloproteinase MMP-1 and MMP-2 and their tissue specific inhibitors TIMP-1 and TIMP-2 was investigated by analysing the mRNA expression using reverse transcriptase polymerase chain reaction and related to the static control. In ACL-fibroblasts seeded on PLA, mechanical load induced up-regulation of collagen types I and III, fibronectin and tenascin C. No effect of mechanical stimulation on the expression of ligament marker genes was found in undifferentiated MSC seeded on PLA. The results indicated that the new textured PLA scaffold could transfer the mechanical load to the ACL-fibroblasts and improved their ligament phenotype. This scaffold might be suitable as a cell-carrying component of ACL prostheses.

Gene expression and immunoreactivity of elastolytic enzymes in the uterosacral ligaments from women with uterine prolapse.

Altered elastin metabolism has been documented in pelvic tissues from women with pelvic floor dysfunction. This study was conducted to quantify the expression of elastolytic enzymes in uterine cervix and uterosacral ligaments from women with uterine prolapse compared to asymptomatic normal controls. Paired tissues of uterosacral ligament and cervical tissues were obtained from 27 women with uterine prolapse and 14 normal controls. Steady state of matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinase 2 (TIMP-2), neutrophil elastase, α-1 antitrypsin immunoreactivity, and messenger RNA (mRNA) expression were analyzed by immunohistochemistry and real-time polymerase chain reaction, respectively. When compared with controls, women with uterine prolapse had a significantly greater level of MMP-2 immunoreactivity and mRNA expression, but less TIMP-2 and α-1 antitrypsin immunoreactivity and mRNA expression in their uterosacral ligaments. However, neutrophil elastase mRNA expression was similar between uterine prolapse and control tissue. Our results showed that there was a close relationship between expressions of MMP-2, TIMP-2, and α-1 antitrypsin in uterosacral ligament and the occurrence of uterine prolapse.

Optimizing an intermittent stretch paradigm using ERK1/2 phosphorylation results in increased collagen synthesis in engineered ligaments.

Dynamic mechanical input is believed to play a critical role in the development of functional musculoskeletal tissues. To study this phenomenon, cyclic uniaxial mechanical stretch was applied to engineered ligaments using a custom-built bioreactor and the effects of different stretch frequency, amplitude, and duration were determined. Stretch acutely increased the phosphorylation of p38 (3.5±0.74-fold), S6K1 (3.9±0.19-fold), and ERK1/2 (2.45±0.32-fold). The phosphorylation of ERK1/2 was dependent on time, rather than on frequency or amplitude, within these constructs. ERK1/2 phosphorylation was similar following stretch at frequencies from 0.1 to 1 Hz and amplitudes from 2.5% to 15%, whereas phosphorylation reached maximal levels at 10 min of stretch and returned toward basal within 60 min of stretch. Following a single 10-min bout of cyclic stretch, the cells remained refractory to a second stretch for up to 6 h. Using the phosphorylation of ERK1/2 as a guide, the optimum stretch paradigm was hypothesized to be 10 min of stretch at 2.5% of resting length repeated every 6 h. Consistent with this hypothesis, 7 days of stretch using this optimized intermittent stretch program increased the collagen content of the grafts more than a continuous stretch program (CTL=3.1%±0.44%; CONT=4.8%±0.30%; and INT=5.9%±0.56%). These results suggest that short infrequent bouts of loading are optimal for improving engineered tendon and ligament physiology.

Gubernaculum as icebreaker: do matrix metalloproteinases in rodent gubernaculum and inguinal fat pad permit testicular descent?

PURPOSE: Cryptorchidism is the most common male congenital abnormality. The rodent gubernaculum steers the testis from abdomen to scrotum postnatally by eversion and migration through the developing inguinal fat pad (IFP). We hypothesize that extracellular matrix remodeling in/around the gubernaculum is necessary for eversion and migration and is permitted by timed IFP maturation and aimed to examine regional development and matrix metalloproteinase (MMP) content. METHODS: Embryonic day 19 (E19) and postnatal days 0 and 2 (P0, P2) wild-type Sprague-Dawley rats (n = 10) were prepared for histologic examination (trichrome) and immunohistochemistry (membrane-type MMP-1 [MT1-MMP], MMP2) and analyzed using light/confocal microscopy. RESULTS: At E19, IFP contained fibroblasts and immature cells in an extensive collagenous extracellular matrix. Cells in the gubernaculum base were cytoplasmic-MT1-MMP-positive (inactive). At P0, the gubernaculum had everted, and adjacent cells were membranous-MT1-MMP-positive (active). At P2, the gubernaculum was migrating through the IFP, and adjacent cells were membranous-MT1-MMP-positive. Adipocyte maturation began cranially in the IFP and proceeded in a craniocaudal gradient until more uniformly mature at P2. CONCLUSION: The MT1-MMP-positive cells may remodel the gubernaculum for eversion and provide the collagenolysis necessary for migration, like an icebreaking ship, through the IFP, which matures to permit migration through collagen-rich tissue. Disruption of these processes may cause cryptorchidism.

Increased matrix metalloproteinases-1,-9 in the uterosacral ligaments and vaginal tissue from women with pelvic organ prolapse.

OBJECTIVES: To investigate the possible association of increased matrix metalloproteinases (MMPs)-1,-9 with pelvic organ prolapse (POP) and to evaluate whether inflammatory processes contribute to its development. STUDY DESIGN: Forty women who underwent hysterectomy, 20 with POP grade 2 and above, and 20 without POP, participated in the study. Biopsies from the uterosacral ligaments and vaginal mucosa were obtained from each woman. Each biopsy was sectioned and stained for MMP-1 and MMP-9 by immunohistochemical methods and with hematoxylin and eosin (H&E). MMP-1,-9 expressions were evaluated on the immunostained slides. H&E stained sections were examined for possible inflammatory changes. RESULTS: A higher stromal (extra-cellular) expression of MMPs-1,-9 was found in POP cases compared with controls in vaginal biopsies (MMP-1: p=0.004; MMP-9: p=0.042) as well as in uterosacral ligament biopsies (MMP-1: p=0.011; MMP-9: p=0.015). Increased intracellular expression of both MMPs was also demonstrated in fibroblasts in biopsies of women with POP (p<0.001 for all). Most of these differences persisted after controlling for age. The degree of inflammatory changes reflected by the number of lymphocytes, plasma cells and capillary-sized blood vessels per 10 high power fields, was similar in specimens obtained from women with and without POP. CONCLUSIONS: The expression of MMPs-1,-9 appears to be increased in tissues from women with POP. This supports an association, although not a causal relation, between increased MMPs-1,-9 and POP. Inflammation does not seem to play an important role in the pathogenesis of POP.

Expression of matrix metalloproteinase-1 in uterosacral ligaments tissue of women with genital prolapse.

Collagen metabolism is altered in the pelvic organ tissues of women with genital prolapse. The aim of this study was to compare collagen metabolism by measuring matrix metalloproteinase-1 (MMP-1) expression in uterosacral ligament tissues of postmenopausal women with and without genital prolapse. Uterosacral ligament tissues were obtained at the time of abdominal or vaginal surgery from twenty-four patients with pelvic organ prolapse (POP) and 21 women who underwent gynecologic surgery for benign indications. The tissue samples were analyzed by immunohistochemistry. There were no differences in age, BMI and parity between two groups. The patients with genital prolapse demonstrated significantly higher occurences of MMP-1 expression compared to controls. These findings indicate that increased MMP-1 expression in uterosacral ligaments is associated with genital prolapse. Our data are consistent with the theory that increased collagen breakdown may play an important role in the onset and development of pelvic organ prolapse (POP).

Immunoexpression of constitutive and inducible cyclooxygenase isoforms in distinguishing and accessory structures of synovial joints in rat foetuses.

Joint formation is a developmental process regulated by various factors including bone morphogenetic proteins, transforming and growth factors, etc. Recently, a high expression of cyclooxygenase (COX) isoforms in the foetal cartilaginous elements was also revealed. On the other hand, various joint and skeletal abnormalities were seen in laboratory animal and human offspring, exposed in utero to several COX inhibitors. Immunoexpression of constitutive (COX-1) and inducible (COX-2) cyclooxygenase isoforms was evaluated in various articular structures of untreated and unfamiliar 21-day-old male rat foetuses. Both COX isoforms were detected in the articular cartilage and joint capsule, as well as in the intra-articular disc of the temporomandibular joint and meniscus of the knee joint. COX-1 immunostaining was revealed in the anterior and posterior cruciate ligament of the knee joint and the labrum of the hip and shoulder, whereas COX-2 immunoreactivity in those structures was not found. It could be concluded that both constitutive and inducible COX isoforms are physiologically expressed in various structures of synovial joints in rat foetuses at the end of prenatal development.

Decreased endopelvic fascia elastin content in uterine prolapse.

BACKGROUND: Genital prolapse is a debilitating manifestation of pelvic floor dysfunction. The cause of this condition has not been elucidated. The purpose of this study was to determine elastin content and RNA expression of related enzymes of elastin synthesis in uterosacral ligament biopsies from women with severe prolapse, and controls with normal pelvic support. METHODS: Biopsies were taken from the uterosacral ligament tissue of 31 women with Grade III or greater prolapse and 29 women with normal pelvic support. Elastin content was assessed by measuring desmosine using radioimmunoassay, and quantitative real time PCR was performed to quantify mRNA levels of lysyl oxidase (LOX), lysyl oxidase like-1 (LOXL1), LOXL2 and fibulin-5 (FIB-5). RESULTS: The mean desmosine concentration found in uterosacral ligaments of women with prolapse (n =26) was 103.3+/-59.3 pmolD/mgP compared to controls (n =29) 120.5+/-47.4 pmolD/mgP (p =0.1943). In the subgroup of subjects with complete procidentia (n =8), mean desmosine concentration was 50.6+/-25.8 and 127.1+/-42.2 pmolD/mgP in age-matched controls (n =12) (p <0.05). In tissue from subjects with more than 2 vaginal deliveries (n =18), the mean desmosine concentration was 99.9+/-60.7 and 133.0+/-44.0 pmolD/mgP in controls (n =17) (p <0.05). Expression of LOX, LOXL1 and LOXL2 decreased 8.2-fold+/-3.4, 5.0-fold+/-1.7 and 15.2-fold+/-5.2, respectively (mean+/-SD) in cases versus controls (p<0.05). Expression of FIB-5 was increased 3.1-fold+/-0.7 compared to controls (p<0.05). CONCLUSIONS: Significantly decreased desmosine content was measured in the uterosacral ligament tissue from women with prolapse versus controls in women with parity >2 and in women with complete procidentia. Suppression of mRNA for LOX and two LOX isoenzymes was correspondingly present. These results suggest that altered elastin metabolism is present in women with uterine prolapse.

Increased expression of matrix metalloproteinase 2 in uterosacral ligaments is associated with pelvic organ prolapse.

The uterosacral ligaments are an important part of the pelvic support system and connective tissue alterations are thought to contribute to the development of pelvic organ prolapse (POP). The objective of this study was to compare the expression of matrix metalloproteinases (MMPs) 1 and 2 in these ligaments in women with and without POP. We analyzed the tissue samples obtained from left and/or right uterosacral ligaments of 17 women with POP and 18 controls by immunohistochemistry. There was no difference in MMP-1 expression between women with POP and those without. In contrast, the MMP-2 expression was significantly related to the presence of POP (p=0.004) rather than to age or parity. There was no difference in MMP-1 and MMP-2 expression between left and right uterosacral ligaments in women with POP compared to controls. Our findings strongly indicate that increased MMP-2 expression in uterosacral ligaments is associated with POP.

Effects of stimulus with proinflammatory mediators on nitric oxide production and matrix metalloproteinase activity in explants of cranial cruciate ligaments obtained from dogs.

OBJECTIVE: To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators. SAMPLE POPULATION: Tissue specimens obtained from 7 healthy adult Beagles that were (mean +/- SD) 4.5 +/- 0.5 years old and weighed 12.5 +/- 0.8 kg. PROCEDURE: The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS). RESULTS: All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and non-stimulated cultures. CONCLUSIONS AND CLINICAL RELEVANCE: Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP.

Evaluation of tartrate-resistant acid phosphatase and cathepsin K in ruptured cranial cruciate ligaments in dogs.

OBJECTIVE: To determine localization of tartrate-resistant acid phosphatase (TRAP) and cathepsin K in ruptured and healthy cranial cruciate ligaments (CCL) in dogs. ANIMALS: 30 dogs with ruptured CCL, 8 aged dogs without ruptured CCL, and 9 young dogs without ruptured CCL. PROCEDURE: The CCL was examined histologically and cells containing TRAP and cathepsin K were identified histochemically and immunohistochemically, respectively. RESULTS: Cathepsin K and TRAP were detected within the same cells, principally within the epiligamentous region and to a lesser extent in the core region of ruptured CCL. Numbers of cells containing TRAP and cathepsin K were significantly greater in ruptured CCL, compared with CCL from young or aged dogs, and numbers of such cells were greater in CCL from aged dogs, compared with those of young dogs. In aged dogs, small numbers of cells containing TRAP and cathepsin K were seen in intact CCL associated with ligament fascicles in which there was chondroid transformation of ligament fibroblasts and disruption of the extracellular matrix. CONCLUSIONS AND CLINICAL RELEVANCE: Ruptured CCL contain greater numbers of cells with the proteinases TRAP and cathepsin K than CCL from healthy, young, or aged dogs. Results suggest that cell-signaling pathways that regulate expression of these proteinases may form part of the mechanism that leads to upregulation of collagenolytic ligament remodeling and progressive structural failure of the CCL over time.

Expression of connexin 26 and Na,K-ATPase in the developing mouse cochlear lateral wall: functional implications.

The immunohistochemical localization of connexin 26 (a gap junction protein) and Na,K-ATPase in the mouse cochlear lateral wall was studied at different ages between 0 and 30 days after birth (DAB). Connexin 26-like immunoreactivity was sparsely distributed among the connective tissue cells just lateral to the future marginal cells of the stria vascularis on 0 DAB. In the mice of 3-6 DAB, connexin 26 was observed in the strial basal cell area, and was increased in its distribution density on 10 DAB. Connexin 26 was sparsely distributed among the fibrocytes in the spiral ligament and the suprastrial zone on 10 DAB, and its distribution density increased rapidly in the mouse on 12 DAB. The immunohistochemical distribution reached the adult pattern in the cochlear lateral wall on 15 DAB. Weak Na, K-ATPase-like immunoreactivity was observed in the epithelial cells, corresponding to the future strial marginal cells, on 0 DAB. Its staining intensity was enhanced with the increase of age, and reached the adult pattern on 10 DAB. In contrast, Na,K-ATPase-like immunoreactivity in the type II fibrocytes and suprastrial fibrocytes was first detected on 12 DAB, and reached the mature level on 15 DAB. It is well known that the endolymphatic potential (EP) reaches the adult level 2 weeks after birth. The expression patterns of connexin 26 and Na,K-ATPase in the fibrocytes of the spiral ligament and the suprastrial zone coincided with the rapid growth and maturation of EP. These findings may suggest a role for the gap junctional communications and Na,K-ATPase activity of the fibrocytes within the cochlear lateral wall in the generation and maturation of EP.

Doxycycline and chemically modified tetracyclines inhibit gelatinase A (MMP-2) gene expression in human skin keratinocytes.

The mechanism of tetracycline-induced inhibition of matrix metalloproteinases (MMP) was studied by measuring the MMP secretion and MMP-2 mRNA levels in unkeratinizing periodontal ligament epithelial cells and skin keratinocytes cultured in the presence of doxycycline or chemically modified tetracyclines (CMT) lacking antimicrobial activity. Doxycycline, CMT-1, and CMT-8 exerted a direct dose-dependent inhibition of porcine periodontal ligament epithelial cell medium MMP activity as assayed by gelatin enzymography. Both the 92-kDa (MMP-9) and 72-kDa (MMP-2) gelatinases were inhibited by the tetracyclines added to the conditioned medium. Culturing the cells in the presence of the tetracyclines required considerably smaller concentrations to reduce the secreted MMP activity. The drugs were not toxic to the epithelial cells at concentrations from 4 to 250 micrograms/mL up to 24 h of culture. Tetracycline effects on the MMP-2 mRNA levels were studied in human skin keratinocytes using Northern hybridization analysis with a specific cDNA probe. A marked inhibition in the MMP-2 gene expression was observed by 6 h with 5 micrograms/mL of doxycycline, CMT-1 or CMT-8. Doxycycline inhibition was somewhat stronger than the two other tetracyclines. After 24 h of culture with 50 micrograms/mL of the drugs, the total RNA levels also decreased by 33 to 40%. The 72-kDa gelatinase activity in culture medium of the keratinocytes followed roughly the pattern of inhibition of the gene expression. We conclude that doxycycline and the chemically modified tetracyclines, in addition to inhibiting the MMP activity may also reduce the enzyme expression at the transcriptional level.

Absence of 5 alpha-reductase activity in the gubernaculum during descent of the fetal pig testis.

To evaluate the hypothesis that testicular descent is mediated by the action of high local concentrations of dihydrotestosterone in the gubernaculum, testis, or epididymis, we obtained tissues from 30 male pig fetuses between 63 and 101 days of gestation. We assayed the 5 alpha-reductase activity in homogenates of pooled tissue by following the conversion of (3H)testosterone to (3H)dihydrotestosterone. The 5 alpha-reductase activity in the urethra and prostate increased prior to and during, but decreased after, the period of testicular descent. The 5 alpha-reductase activity in the gubernaculum remained constant throughout gestation and was not significantly higher than the background activity found in umbilical cord, testis plus epididymis, striated thigh muscle and heat inactivated samples of prostate and urethra. Prior to testicular descent, the 5 alpha-reductase activity was approximately 60 to 300 times higher in the urethra than in the gubernaculum, and approximately 20 to 55 times higher in the prostate than in the gubernaculum. These findings indicate that local conversion of testosterone to dihydrotestosterone in the gubernaculum or epididymis does not play a role in the mediation of testicular descent in the pig fetus.

Inhibitors of collagenase in ligaments and tendons of rabbits immobilized for 4 weeks.

Our laboratories have previously measured the capacity for collagenase production by normal and immobilized rabbit periarticular connective tissues (PCT), i.e., anterior cruciate ligaments (ACL), medial collateral ligament (MCL) and patellar tendon (PT). Rabbit limbs were immobilized for 4 weeks and then the tissues were cultured for 4 days. Collagenase and inhibitor (TIMP) were measured in the medium. Collagenase activity was significantly decreased in the medium from all experimental tissues, relative to the levels in contralateral control tissues. On the other hand there was no significant change in inhibitor levels in the three experimental tissues. These results support the idea that collagenase is decreased due to decreased enzyme expression and not to increased inhibitor production.

Neutral endopeptidase from nuchal ligament of fetal calves.

The nuchal ligament of unborn calves contains a neutral endopeptidase that is biochemically and immunologically similar to the neutral endopeptidase (NEP), or enkephalinase, from human kidney. Enzymatic activity was inhibited more than 90% by phosphoramidon (1 microM). The specific activity in membrane fractions, as determined by hydrolysis of the dansylated substrate, DAPGN, was similar in tissue from fetuses of gestational ages ranging from 100 to 280 days. NEP activity in adult ligament tissue, however, was less than 10% of that in fetal tissue. Fibroblasts dissociated from ligament tissue by collagenase displayed less NEP activity than did preparations of intact ligament, and activity was even lower in cultured cells. By contrast, fibroblasts cultured from fetal calf lungs had NEP activity comparable to that in the ligament tissue. When ligament fibroblasts were cultured on subcellular matrices derived from fetal lung fibroblasts the NEP activity increased relative to those cultured on plastic alone. These studies confirm the presence of neutral endopeptidase (NEP) in the nuchal ligament of the fetal calf. The consistent activity through a range of gestational ages and the influence of the subcellular matrix suggest that this enzyme might be involved in growth of the ligament during fetal life.