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Biomedical subjects

D E Woolley

Publications and source records attributed to D E Woolley.

At least 55 records · Page 3Linked to original sources
PubMed1995

Activation of precursors for matrix metalloproteinases 1 (interstitial collagenase) and 3 (stromelysin) by rat mast-cell proteinases I and II.

Histological studies have previously demonstrated an association between mast-cell activation/degranulation and areas of connective-tissue lysis in vivo; in addition, mast-cell extracts have been shown to activate latent forms of collagenase and stromelysin. In the present study we have examined the potential roles of rat mast-cell proteinase (RMCP) I and RMCP II as activators of the precursors of matrix metalloproteinase (MMP)-1 (interstitial collagenase), MMP-2 (gelatinase A) and MMP-3 (stromelysin 1). Both RMCPs I and II activated proMMP-3 by converting the 57 kDa precursor into a 45 kDa polypeptide. The N-terminal amino acid of 45 kDa MMP-3 activated by RMCP II was identified as Phe83. By contrast, only RMCP II activated the 52 kDa proMMP-1 by converting it into a 41 kDa protein and generating the new N-termini, namely Gln80 and Val82. The collagenolytic activity which resulted from this cleavage was only 35% of the full activity, but this could not be augmented by subsequent treatment with MMP-3, the latter being a crucial enzyme for the generation of the fully active MMP-1 with Phe81 at the N-terminus, in conjunction with other serine proteinases. Thus RMCP II activates proMMP-1 via a mechanism different from that reported for the stepwise processing by combinations of other trypsin-like enzymes and MMP-3. ProMMP-2 (pro-gelatinase A) was not activated by either RMCP I or RMCP II, despite processing to smaller products.

Amino Acid Sequence↗
PubMed1995

Comparative studies of collagenase and stromelysin-1 expression by rheumatoid synoviocytes in vitro.

Matrix metalloproteinases such as collagenase and stromelysin are recognised as important cartilage-degrading enzymes in the pathophysiology of rheumatoid arthritis. Synovial fibroblasts and macrophages are the major cellular components of rheumatoid synovium, but the regulation and relative expression of collagenase and stromelysin by these two cell types remains uncertain. Using in vitro cultures of adherent rheumatoid synovial cells we have examined the coordinate or separate expression of collagenase and stromelysin-1 by dual immunolocalisation and Western blotting techniques. Synovial fibroblasts, when activated by macrophage-derived products in primary culture or by interleukin-1/phorbol myristate acetate in subcultures, released significant quantities of collagenase and stromelysin in their inactive, precursor forms. The ratio of released procollagenase: prostromelysin varied between different synovial cell preparations. Dual immunolocalisation studies demonstrated both coordinate and separate expression of the two enzymes by single cells. Approximately 80% of the activated fibroblasts, especially those with stellate morphology, showed co-expression of both enzymes. By contrast synovial macrophages had a modest or negligible capacity to elaborate either enzyme under the same in vitro conditions. In many fibroblastic cells both collagenase and stromelysin were co-localised to the perinuclear Golgi region and the same cytoplasmic compartments. Vesicular structures appear to provide intracellular transport for both enzymes to sites of secretion. Both enzymes showed preferential pericellular binding to a collagenous substratum rather than any association with the plasma membrane/cell surface.

Arthritis, Rheumatoid↗
PubMed1995

Mast cell and eosinophil distribution and activation in human endometrium throughout the menstrual cycle.

Tryptase and chymase immunolocalization techniques have been used to examine the distribution, activation, and tryptase/chymase phenotype of mast cells (MCs) in 107 endometrial specimens that represented every day of the human menstrual cycle. MCs were identified in the endometrium in all stages of the menstrual cycle; similar MC numbers were observed for the functionalis, basalis, and muscularis. Extensive MC activation/degranulation, as judged by extracellular tryptase, was a common feature of the functionalis in specimens sampled just prior to and during menstruation. MC activation was also prominent in the functionalis at times coincident with recognized stromal edema. MCs of the functionalis did not contain chymase; all stained for tryptase and represent the MCT phenotype. By contrast, the basalis and muscularis showed a proportion of MCs containing both tryptase and chymase, MCTC. One important function for extracellular MC tryptase and chymase is their ability to activate precursor forms of the matrix metalloproteinases, enzymes recognized as instrumental in stromal degradation. Quantitative analysis of MC numbers, expressed relative to stromal cell numbers/mm2, indicated no major changes during the menstrual cycle, although changes in MC morphology, granule content, and activation/degranulation were recognized for specific stages. Eosinophils, detected with monoclonal antibodies EG1 and EG2, were absent from extravascular sites between Days 5 and 26 but showed local accumulations just prior to and during menstruation. Since MCs and eosinophils between them contain a variety of potent mediators, it seems likely that both cell types assume important functional roles in relation to tissue and vascular remodeling associated with endometrial physiology.

Cell Count↗
PubMed1995

Mast cells, cytokines, and metalloproteinases at the rheumatoid lesion: dual immunolocalisation studies.

OBJECTIVES: To examine the distribution and activation of mast cells (MCs) in the rheumatoid lesion (cartilage-pannus junctions demonstrating cartilage erosion), and to determine whether or not their tissue distribution is related to that for tumour necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), stromelysin-1, and collagenase. METHODS: Immunolocalisation of MC-tryptase was used to identify MCs and their states of degranulation in 35 specimens of cartilage-pannus junctions. Dual immunolocalisation techniques using alkaline phosphatase and peroxidase conjugated antibodies were used to compare the distributions of MCs with the proinflammatory cytokines TNF alpha and IL-1, and the cartilage or matrix degrading enzymes stromelysin-1 and collagenase. RESULTS: Stromelysin-1, TNF alpha, and IL-1 beta were especially prominent at the cartilage-pannus junctions, albeit with patchy distributions. Extracellular MC tryptase, indicative of MC activation/degranulation, was commonly observed at sites of cartilage erosion, and was often associated with the microenvironmental expression of TNF alpha, IL-1 beta, stromelysin-1, and collagenase. Such observations were often associated with localised sites of tissue oedema and stromal disruption. CONCLUSION: MC activation was frequently associated with proinflammatory cytokine and metalloproteinase expression by neighbouring cells, thereby suggesting an important contributory role for the MC in mediating matrix degradation and oedematous changes within microfoci of the rheumatoid lesion.

Aged↗
PubMed1995

Distribution, activation and tryptase/chymase phenotype of mast cells in the rheumatoid lesion.

OBJECTIVE: To determine the distribution, activation, and tryptase/chymase phenotype of mast cells (MCs) in the rheumatoid lesion. METHODS: MC tryptase and chymase were studied by immunohistochemistry using monoclonal antibodies and examination by brightfield, interference, and fluorescent microscopy. Thirty four specimens of cartilage-pannus junction and 26 specimens of rheumatoid synovium, all derived from knee surgery, were examined. RESULTS: MCs were identified in all specimens examined, but their distribution and local concentrations varied, both within and between specimens. As a proportion of total synovial cells, there were more MCs in fibrous synovial tissues than in those with active inflammatory cell infiltrations; MCs usually showed a peripheral distribution around lymphocytic/mononuclear cell infiltrations. Most cartilage-pannus specimens demonstrated local concentrations of MCs at, or close to, sites of cartilage erosion, a significant proportion of which showed extracellular tryptase indicative of MC degranulation. MC degranulation was often associated with localised oedema and disruption of the stromal matrix. Two MC phenotypes were identified: one population contained tryptase alone (MCT) whilst another contained both tryptase and chymase (MCTC). The ratio MCT:MCTC approximated 8:1. CONCLUSIONS: This histological study demonstrated that local concentrations of MCs and their activation/degranulation are commonly observed in the rheumatoid lesion, and especially at sites of cartilage erosion. Such observations add weight to the concept that MCs contribute to the processes of inflammation, matrix degradation and tissue remodelling.

Aged↗
PubMed1995

Matrix metalloproteinases and their tissue inhibitors at the ovine trophoblast-uterine interface.

Trophoblast invasiveness in ruminants is limited to fusion of migrating binucleate cells with uterine epithelium, but considerable tissue remodelling and angiogenesis occurs within the endometrium at implantation. Such processes are elsewhere associated with an altered balance of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). ProMMPs-1, -2, -3 are secreted by cultured ovine endometrial stromal, but not epithelial, cells and expression of proMMP-1 and proMMP-3 is inhibited by interferon tau, the dominant preimplantation trophoblast product, independently of its effects on prostaglandin production. Messenger RNA for TIMP-1 and TIMP-2 is expressed in endometrium throughout the oestrous cycle and early pregnancy. Both TIMP-1 mRNA (0.9 kb transcript) and TIMP-2 mRNA (1.0 kb transcript) expression increase at day 12 through to day 20, suggesting a role in preventing trophoblast invasion. Expression of the 3.5 kb transcript for TIMP-2 decreases from day 16 and is undetectable on day 20. Uterine flushings from both nonpregnant and pregnant ewes on day 16 after oestrus contain proMMP-2, possibly transudated from plasma, and proMMP-9, but concentrations are higher in pregnancy. These enzymes are also detected in conditioned medium following culture of trophoblasts at days 16-20. Whether the trophoblast MMPs have a role in implantation in sheep remains to be established. The production of MMPs and TIMPs in the endometrium and the changes associated with implantation events suggest that they contribute to the marked edometrial remodelling associated with early placentation.

Animals↗
PubMed1994

Catabolism of intact fibrillin microfibrils by neutrophil elastase, chymotrypsin and trypsin.

We present ultrastructural and biochemical evidence for the turnover of intact fibrillin microfibrils by the serine proteinases, neutrophil elastase, chymotrypsin and trypsin. Rotary shadowing electron microscopy revealed that serine proteinase treatment of intact microfibrils isolated from foetal bovine skin resulted in extensive degradation. Microfibrils were destroyed by neutrophil elastase and effectively disrupted by chymotrypsin and trypsin, with no morphologically identifiable arrays remaining. Evidence of defined fibrillin degradation products was obtained by Western blotting of these enzyme-treated fibrillin assemblies. Fibrillin immunoprecipitated from dermal fibroblast culture medium was also comprehensively degraded by these enzymes. These observations demonstrate that serine proteinases are potent effectors for the physiological and pathological catabolism of microfibrils, and suggest a key role in elastic fibre degradation.

Animals↗
PubMed1994

Mast cell proteinases activate precursor forms of collagenase and stromelysin, but not of gelatinases A and B.

Mast cell activation in vivo is often associated with areas of oedema and connective-tissue degradation. Tryptase and chymase are the major serine proteinases released by mast cells, but they appear to have little activity on most components of the extracellular matrix. The matrix metalloproteinases (MMP) are purported to degrade almost all connective tissue elements and are secreted by cells in the form of inactive precursors. Since the mechanisms of MMP activation in vivo are poorly understood we have examined the potential of mast cell proteinases to activate the precursor forms of human collagenase (MMP-1), stromelysin (MMP-3), gelatinase A (MMP-2) and gelatinase B (MMP-9). Mast cell proteinases prepared from purified dog mastocytoma cells were shown to process and activate purified precursor forms of both MMP-1 and MMP-3. Using antipain and chymostatin, inhibitors for tryptase and chymase, respectively, it was demonstrated that both pMMP-1 and pMMP-3 were effectively processed and activated by the chymase component. By contrast, tryptase activated only pMMP-3. The mast cell proteinases were unable to process or activate purified precursor forms of MMP-2 and MMP-9. However, MMP-3 previously activated by mast cell proteinases was shown to activate pMMP-9, but not pMMP-2. Since we have no evidence that mast cells express these four metalloenzymes, the release of mast cell serine proteinases following activation/degranulation could contribute to local metalloproteinase activation and subsequent matrix degradation.

Amino Acid Sequence↗
PubMed1994

Distribution of gelatinase B (MMP-9) and type IV collagen in colorectal carcinoma.

Gelatinase B (92 kD, matrix metalloproteinase-9, MMP-9), an enzyme capable of degrading several connective tissue components, was demonstrated by immunolocalization in all specimens of colorectal carcinoma (n = 40), but its distribution between specimens was variable. MMP-9 expression was more frequently observed in advanced tumour stages and was especially prevalent at the side and deep margins of the tumours, and ulceration sites. MMP-9 staining was observed for transformed epithelial cells, macrophages and neutrophils, but seldom for vascular or stromal cells. By contrast, the enzyme was absent from epithelial cells of normal mucosal tissue. Immunostaining of type IV collagen, the major structural component of basement membranes, revealed a general depletion or loss of these structures both within the tumours and at the tumour margins. Dual immunolocalization of MMP-9 and type IV collagen demonstrated that MMP-9 expression at specific sites in the tumour was often inversely related to the distribution of type IV collagen. MMP-9 expression was most pronounced at the invasive tumour margins and in microfoci where tumour cells were in close proximity to inflammatory cells. Such observations support the concept that localized proteolytic and collagenolytic activities contribute to the invasive properties of colorectal tumours.

Aged↗
PubMed1994

Comparison of the effects of propofol and thiopental on the pattern of maximal electroshock seizures in the rat.

The anticonvulsant effects of propofol and thiopental (thiopentone) were determined by measuring the durations of the various phases of maximal electroshock seizures in the rat. Five min. after intraperitoneal administration of subanaesthetic (6.25, 12.5 and 25 mg/kg) and 50 mg/kg doses of propofol, the 2 highest doses abolished both tonic hindlimb extensor phases (full and partial extension) in all rats and decreased the duration of the total tonic phases of the seizure. Although the lowest dose produced no effect, the 12.5 mg/kg dose decreased the duration of both the full and partial tonic extensor phases and increased the duration of tonic flexion, showing that even this low dose had anticonvulsant activity. Subanesthetic doses of thiopental (5, 10 and 20 mg/kg) produced similar changes in the maximal electroshock seizures except that even the lowest dose also significantly decreased the duration of total extension and total tonus. Postseizure depression was prolonged only by the highest dose of propofol. Thus, even low doses of either propofol or thiopental, that produced only minimal behavioural effects, had marked anticonvulsant effects against electrically induced convulsions in the rat. No evidence of enhanced convulsant maximal electroshock seizures patterns was observed at any dose.

Animals↗
PubMed1994

Rat fibrosarcoma as a source of stable co-polymer of type I and type III collagens.

During conventional procedures for the extraction of collagen from rat fibrosarcoma it was observed that a significant fraction of the acid-soluble collagen remained insoluble when subsequently fractionated at neutral pH. Analysis of this collagenous component, using amino acid analysis, cyanogen bromide peptide mapping, proteinase susceptibility and the action of denaturing and reducing agents, revealed different properties when compared to other collagen species. The results indicate that the tumour collagen fraction is probably a stable complex of type I and III collagens. This native collagen heteropolymer represented approximately 18% of the total collagen from rat fibrosarcoma and as such provides a unique natural source of this collagen complex for further analytical studies.

Amino Acids↗
PubMed1994

Matrix metalloproteinase production by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase-A, gelatinase-B, and stromelysin-1 and their differential regulation by interleukin-1 alpha and tumor necrosis factor-alpha.

Matrix metalloproteinases (MMPs) together degrade virtually all the components of the extracellular matrix and are likely to play a role in remodeling of endometrial tissue during the normal menstrual cycle. Primary cultures of human endometrial stromal cells secreted a number of MMPs. MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) were measured in culture medium by specific enzyme assays. Production of the enzymes did not correlate with the time of the menstrual cycle at which the tissue was collected. Identities of MMP-1 and MMP-3 were confirmed by Western blots, by comparison of mol wt with those of purified enzymes on casein zymography, and by inhibition of these activities with EDTA and 1,10-phenanthroline. Northern analysis demonstrated specific messenger ribonucleic acid for pro-MMP-1 and pro-MMP-3 in phorbol myristate acetate-stimulated stromal cells. Two gelatinases were detected by gelatin zymography: MMP-2 (gelatinase-A) was present in two forms (72 and 67 kilodaltons), and MMP-9 (gelatinase-B) was present as a homodimer with a mol wt of approximately 180 kilodaltons. MMP-9, but not MMP-2, secretion was stimulated by phorbol myristate acetate. All enzymes could be activated in vitro by (4-aminophenyl)mercuric acetate. Both interleukin-1 alpha and tumor necrosis factor-alpha stimulated the secretion of MMP-1, MMP-3, and MMP-9, but not MMP-2, from the cells in a concentration-dependent manner. MMP production by endometrial stromal cells has a potentially important role in the processes of menstruation and implantation.

Blotting, Western↗
PubMed1993

Metalloproteinase and TIMP expression by the human breast carcinoma cell line 8701-BC.

It is widely accepted that collagenolytic enzymes are required to facilitate the invasion and spread of tumour cells into host tissues. Immunohistochemical, zymographic and PCR analyses have produced evidence that the recently established human mammary carcinoma cell line, 8701-BC, expresses several metalloproteinases (MMP-1, -2, -9 and -10) and their tissue inhibitors (TIMP-1 and -2). Application of these different techniques has led to several observations, both complementary and dissimilar. Whereas PCR analysis showed that mRNA was detected for each of the proteins, the immunolocalization study demonstrated that MMP-1, MMP-2, MMP-9 and TIMP-1 production was restricted to only a proportion of the tumour cells, with no evidence of MMP-3 or TIMP-2 synthesis. Such observations suggested phenotypic heterogeneity within the cell line, which was further examined by use of the tumour cell clones BC-3A and BC-61 derived from the parental 8701-BC line. Comparative studies using zymography and PCR analysis demonstrated differences in MMP-2 and MMP-10 expression between the 3 cultures. The data indicate that the 8701-BC cell line retains an inherent capacity for metalloproteinase and TIMP expression, with the production of both interstitial collagenase (MMP-1) and the 2 basement-membrane-degrading enzymes (MMP-2 and MMP-9) representing an aggressive collagenolytic phenotype. The concomitant production of TIMP-1 by these cell cultures, and the apparent phenotypic heterogeneity displayed by these lines, suggest that metalloproteinase dysregulation may represent an important feature of clonal heterogeneity. Although the 8701-BC and BC-61 cells were much more invasive than those of the BC-3A clone, as judged by the penetration of "Matrigel", it has not yet been possible to relate this invasive potential to the metalloproteinase and TIMP profiles reported here for each cell line.

Breast Neoplasms↗
PubMed1993

Differential expression of gelatinase B (MMP-9) and stromelysin-1 (MMP-3) by rheumatoid synovial cells in vitro and in vivo.

Primary cultures of adherent rheumatoid synovial cells (ASC) are comprised of variable proportions of fibroblasts, macrophages and stellate cells (activated fibroblasts). These cultures were shown to produce the metalloproteinases stromelysin-1 (MMP-3), gelatinase A (MMP-2) and gelatinase B (MMP-9) by Western blotting and zymography techniques. Immunolocalisation studies showed that MMP-3 was mainly produced by the fibroblastic cells whereas MMP-9 was restricted to macrophages (CD68 positive). Subcultured synovial fibroblasts, devoid of macrophages, did not produce MMP-9 as judged by zymography and immunolocalisation; but when stimulated with phorbol myristate acetate and interleukin-1 alpha both MMP-9 and MMP-3 were co-expressed. These 'activated' fibroblasts assumed a dendritic or stellate morphology, which in localisation studies was usually associated with enhanced enzyme production. Immunolocalisation studies of rheumatoid synovial tissue showed that relatively few cells were positive for MMP-3 and MMP-9. Localisation of MMP-9 corresponded to a proportion of macrophages positive for the CD68 marker throughout the synovial tissue. MMP-3 localisation was not associated with the macrophage marker, but was observed in both the synovial lining layer and deeper stromal locations. Widespread distribution of both enzymes was not observed in fresh tissues, but this increased in tissues subjected to short-term explant cultures. Thus, both in vitro and in vivo studies indicated that synovial fibroblasts or B-cells are effective producers of MMP-3 whereas macrophages elaborate MMP-9, observations that demonstrated different metalloproteinase phenotypes under similar environmental conditions.

Arthritis, Rheumatoid↗
PubMed1993

Effects of hypertonic and isotonic fluid infusion on the flash evoked potential in rats: hemorrhage, resuscitation, and hypernatremia.

In resuscitation from hemorrhagic shock, very small volumes of hypertonic saline (HS) improve blood pressure while reducing intracranial pressure and edema formation. The effects of hypertonic resuscitation fluids and hypernatremia on electrophysiologic brain function have not been studied. The present study was done in two parts. First we examined the effects of hemorrhagic shock and resuscitation with either 7.5% HS or lactated Ringer's (LR) solution on the flash evoked potential (FEP). Rats were bled to a mean arterial pressure (MAP) of 35 mm Hg for 1 hour, then resuscitated with HS (n = 10) or LR (n = 10) to a MAP of 80 mm Hg for another hour. Resuscitation required 3.8 +/- 0.5 mL/kg HS and 42.9 +/- 7.5 mL/kg LR (p < 0.05). During hemorrhage, FEP latencies increased and amplitudes decreased. During resuscitation, these variables returned toward baseline values. There were no significant differences between groups, although HS tended to restore the FEP better than LR. We next examined the effects on the FEP of hypernatremia and hyperosmolarity produced by two different hyperosmotic fluids. Over a 1-hour period, 16 mL/kg HS (n = 8), 16 mL/kg IsoSal (4.5% saline, 5.9% glucose, 6.4% mixed amino acids; n = 8), or 40 mL/kg LR (n = 8) was infused into normovolemic rats. Plasma sodium levels increased in both hyperosmotic groups (baseline = 145.2 +/- 0.7 mEq/L; after infusion, HS = 202.4 +/- 9.8 mEq/L, IsoSal = 163.3 +/- 4.2 mEq/L; p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Production of matrix metalloproteinase 1 (interstitial collagenase) and matrix metalloproteinase 2 (gelatinase A: 72 kDa gelatinase) by ovine endometrial cells in vitro: different regulation and preferential expression by stromal fibroblasts.

Ovine endometrial cells (epithelial plus stromal), prepared from ovariectomized ewes treated with oestrogen and progesterone to mimic the luteal phase of the oestrous cycle were maintained in serum-free medium for 48 h in the presence or absence of phorbol myristate acetate (PMA, 100 nmol l-1), a known stimulus for production of matrix metalloproteinases (MMP) in other cells. Matrix metalloproteinase-1 (MMP-1, interstitial collagenase) and matrix metalloproteinase-2 (MMP-2, gelatinase A) activities were expressed by the cells in the absence of PMA; most were in the latent form and required activation by (4-aminophenyl) mercuric acetate (APMA). Exposure to PMA over 48 h resulted in a significant increase in MMP-1 activity but only a modest and nonsignificant increase in MMP-2 activity. Gelatin zymography demonstrated that proMMP-2 (72 kDa) was produced by both PMA-treated and untreated cells and an active form of 67 kDa was also present. Immunolocalization of MMP-1 and MMP-2 was seen within the cells following treatment with monensin. Highly purified epithelial and stromal cells were similarly cultured and analysis of the conditioned medium showed that MMP-1 and MMP-2 were produced predominantly by stromal rather than epithelial cells. Thus, both MMP-1, which degrades interstitial collagens, and MMP-2, an important enzyme for degradation of type IV and V collagens, are synthesized and released by ovine endometrial stromal cells in culture, but MMP-1 is produced primarily upon stimulation, whereas MMP-2 production is constitutive.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗