Effects of fibrinolytic inhibitors on chondrogenesis of bone-marrow derived mesenchymal stem cells in fibrin gels.

The objective of this study was to examine the effect of two fibrinolytic inhibitors, aprotinin and aminohexanoic acid, on chondrogenesis of rabbit bone marrow mesenchymal stem cells (BM-MSCs). Rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand White rabbits. Cell-fibrin constructs were made by mixing a cell-fibrinogen (10(7) cells/ml; 40 mg/ml fibrinogen) solution with a thrombin (5 IU/ml) solution and then divided into four groups: aprotinin control, aprotinin + transforming growth factor beta (TGF-beta), aminohexanoic acid control, and aminohexanoic acid + TGF-beta. Each of these groups was further treated with three different concentrations of inhibitors and the TGF-beta groups were treated with 10 ng/ml of TGF-beta1. The chondrogenic gene expressions, DNA content, and glycosaminoglycan content of samples were analyzed after 14 days of culture. The aprotinin groups exhibited significantly higher levels of aggrecan gene expression and glycosaminoglycan content than the aminohexanoic acid groups. However, inhibitor neither influenced gene expression of type II collagen nor proliferation (i.e., DNA content) of BM-MSCs. These findings suggest that fibrinolytic inhibitors used to control degradation of fibrin clot may influence TGF-beta-induced chondrogenesis of BM-MSCs.

Induction of expression of c-fos and c-myc protooncogenes by basic calcium phosphate crystal: effect of beta-interferon.

Basic calcium phosphate (BCP) crystals control the traverse of cells from the G0-G1 to S-phase of the cell cycle and initiate proliferation by rendering fibroblasts competent to respond to insulin-like growth factors in plasma. The present study examines whether BCP crystals induce transcription of the protooncogenes c-fos and c-myc and the effect of beta-interferon (IFN-beta) on protooncogene transcription as well as BCP crystal-induced DNA synthesis. Stimulation of density-arrested BALB/c-3T3 cells with either BCP crystal or platelet-derived growth factor (PDGF) results in maximal accumulation of c-fos mRNA at 30 min after stimulation. Induction of c-myc transcription by BCP crystal or PDGF occurs within 1 h and is maximal at around 3 h after stimulation. Simultaneous addition of IFN-beta with either BCP crystals or PDGF had little effect on c-fos induction but delayed both c-myc message accumulation and entry into S phase. The delay in c-myc message induction after IFN-beta treatment cannot account for the observed delay in the onset of DNA synthesis, since IFN-beta can be added at up to 6 h after stimulation with either PDGF or BCP crystals, and a similar delay in the onset of DNA synthesis is still observed.

The role of cyclic-3',5'-adenosine monophosphate in prostaglandin-mediated inhibition of basic calcium phosphate crystal-induced mitogenesis and collagenase induction in cultured human fibroblasts.

Synovial fluid basic calcium phosphate (BCP) crystals are associated with severe destructive arthropathies characterised by synovial proliferation and non-inflammatory degradation of intra-articular collagenous structures. BCP crystals stimulate fibroblast and chondrocyte mitogenesis, metalloprotease secretion and prostaglandin production. As a tissue protective effect of prostaglandins has been suggested, we recently studied the effect of PGE1 on BCP crystal-induced mitogenesis and collagenase mRNA accumulation in human fibroblasts (HF). We demonstrated a dose-dependent inhibition of BCP crystal-induced mitogenesis and collagenase mRNA accumulation. The mechanism of PGE1 inhibition of BCP crystal-induced mitogenesis and collagenase mRNA accumulation was therefore explored. PGE1 (100 ng/ml) increased HF intracellular cAMP 40-fold over control. BCP alone caused no such change but inhibited the PGE1-induced increase in intracellular cAMP by at least 60%. The PGE1-induced increase in intracellular cAMP was also blocked by the adenyl cyclase inhibitor, 2',5'-dideoxyadenosine (ddA) (10 microM) and ddA reversed the PGE1-mediated inhibition of BCP crystal-induced mitogenesis. Dibutyryl cAMP also inhibited BCP crystal-induced mitogenesis in a concentration-dependent manner. Agents which increase intracellular cAMP levels such as the adenyl cyclase activator forskolin and the phosphodiesterase, inhibitor 3-isobutyl-1-methylxanthine (IBMX) mimicked the effect of PGE1 on HF collagenase mRNA levels. PGE1 inhibits the biologic effects of BCP crystals through the cAMP signal transduction pathway and such inhibition may have significant therapeutic implications.

Effect of substrate concentration on inhibition of prostaglandin synthetase of bull seminal vesicles by anti-inflammatory drugs and fenamic acid analogs.

Although microsomes of bull seminal vesicle synthesize prostaglandins F2alpha, E2 and D2 from arachidonic acid under suitable assay conditions, prostaglandin E2 is the only significant product at either low concentration of arachidonic acid or high concentration of microsomes. Studies of inhibition of prostaglandin synthesis in vitro by anti-inflammatory drugs at both high (1 mM) and low (1 muM) concentrations of arachidonic acid, suggest three distinct mechanisms of inhibition. Benzydamine and flazalone are non-competitive or weakly competitive with arachidonic acid and, at high concentrations of arachidonic acid, they augment sythesis of prostaglandin E2 while inhibiting production of prostaglandins F2alpha and D2. Niflumic acid and the arylacetic acids naproxen and ibuprofen are competitive inhibiting all products equally, but with 100-500-fold greater potency at the low substrate concentration. The fenamic acids, indomethacin, aspirin, and phenylbutazone also inhibit equally all prostaglandin products, but are only 20--50 times more potent at the low substrate concentration. Studies with analogs of the fenamic acids indicate that the diphenylamine protion of their structure is essential for inhibition of prostaglandin synthesis, whereas the o-carboxyl and m-alkul substitutents greatly enhance inhibitory potency.

Specific inhibition of basic calcium phosphate and calcium pyrophosphate crystal-induction of metalloproteinase synthesis by phosphocitrate.

Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystal deposition diseases are a group of heterogeneous arthritides which are a significant source of morbidity in the elderly. Both crystals induced mitogenesis and metalloproteinase (MP) synthesis and secretion by fibroblasts and chondrocytes which may promote degradation of intra-articular tissue. We have previously shown that phosphocitrate (PC), an inhibitor of hydroxyapatite crystallization, specifically blocks BCP crystal-induced mitogenesis in 3T3 cells. This led us to examine the effect of PC on BCP and CPPD crystal induction of MP synthesis in human fibroblasts. PC (10(-3) to 10(-4) M) specifically inhibited the crystal-induced collagenase and stromelysin mRNA accumulation while having no effect on epidermal growth factor-induced or basal levels of mRNA for both enzymes. Western blots (collagenase) of conditioned media confirmed that PC blocked crystal-induced proteinase secretion as well. Moreover, PC (10(-3) M) also blocked the crystal induction of c-fos and c-jun. Since FOS and JUN proteins form a transacting activator (AP-1) for expression of collagenase and stromelysin genes, PC may block the synthesis of both enzymes by inhibiting the transcription of c-fos and c-jun.

Inhibition of neutral endopeptidase 3.4.24.11 in conscious dogs with pacing induced heart failure.

OBJECTIVE: The effects of a selective neutral endopeptidase inhibitor, SQ 28,603 (N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-beta-alanine), were determined in an experimental model of heart failure. METHODS: The symptoms of heart failure were induced by rapid ventricular pacing for one or three weeks in dogs with surgically implanted catheters for measurement of atrial pressures and mean arterial pressure and with ultrasonic flow probes for determination of cardiac output and renal blood flow. RESULTS: Inhibition of neutral endopeptidase by 10, 30, or 100 mumol.kg-1 SQ 28,603 given intravenously increased sodium excretion, cyclic GMP excretion, and plasma concentrations of atrial natriuretic peptide in a dose related manner in conscious dogs paced for one week. SQ 28,603 (100 mumol.kg-1) stimulated similar natriuretic and cyclic GMP responses in dogs paced for three weeks although baseline glomerular filtration rate was reduced. Because the natriuresis was maintained despite the smaller filtered sodium load, the increase in fractional sodium excretion was significantly greater after three weeks of pacing (from 0.5(0.2) to 3.7(0.7)%) than after one week of tachycardia (from 0.1(0.0) to 2.0(0.3)%). By contrast, SQ 28,603 (100 mumol.kg-1) did not affect renal, haemodynamic, or hormonal variables in normal conscious dogs where baseline atrial natriuretic peptide (18(3) fmol.ml-1) was lower than in the paced animals (104(10) fmol.ml-1). CONCLUSIONS: Inhibition of neutral endopeptidase in dogs with pacing induced heart failure protected endogenous atrial natriuretic peptide from degradation and stimulated sustained natriuresis, presumably via a tubular mechanism.

Stimulation of fibroblast biosynthetic activity by serum of patients with pretibial myxedema.

Skin fibroblasts from the shoulder and lower extremities of normal individuals, as well as from patients with pretibial myxedema (PTM) were grown in culture. When cells reached the monolayer stage, they were labeled with 3H-glucosamine and tested for hyaluronic acid synthesis in the presence of either serum from PTM patients or normal human serum. All the fibroblasts from the pretibial area synthesized 2 to 3 times more hyaluronic acid when incubated with PTM sera than when incubated in normal human serum. Fibroblasts cultured from skin of the back or prepuce did not respond to PTM sera. This heat-stable, protease-sensitive, and dialyzable, fibroblast-stimulating factor is not a 7S gamma-globulin. The enhanced sensitivity to PTM sera exhibited by fibroblasts from the lower extremities may explain why the lesions in this disease are restricted primarily to that area.

Induction of stromelysin-1 and collagenase synthesis in fibrochondrocytes by tumor necrosis factor-alpha.

Stromelysin-1 and collagenase mRNA levels were assayed in fibrochondrocytes by Northern blot analysis at 0, 2, 4, 8 and 24 h after stimulation with tissue necrosis factor-alpha (TNF-alpha). Peak collagenase mRNA levels occurred 24 h after stimulation and were increased nine-fold over the level at time 0. Stromelysin-1 mRNA levels peaked 8 h after stimulation, with a five-fold increase over the level at time 0. A TNF-alpha dose-related response to both collagenase and stromelysin-1 mRNA accumulation was also demonstrated. Confirmation of the presence of secreted metallo-proteinases in the conditioned media was established by immunoprecipitation of stromelysin-1 and Western blotting of collagenase. Both enzymes were secreted in latent forms. Consistent with stromelysin-1 activity, substrate gels demonstrated a doublet of caseinase activity with molecular masses at 57 kDa and 59 kDa in TNF-alpha stimulated samples. Collagenase assays of conditioned media also demonstrated a significant increase in collagenase activity after stimulation by TNF-alpha. While epidermal growth factor had a minimal effect on stromelysin-1 and collagenase expression, transforming growth factor-beta, and insulin-like growth factor-1 did not induce either enzyme activity.

Identification of collagen subtypes in synovial fluid sediments from arthritic patients.

Collagen fibers in synovial fluid sediment were described a decade ago. Since then, tissue-specific collagen molecules (types) have been characterized. Techniques were devised to identify the collagen types in joint fluid sediment. Collagens were found in 12 of 17 pellets prepared from fluid aspirates from 17 knee joints of patients with various forms of arthritis. Collagen types I and III and polypeptide chains A and B (basement membrane collagen) were specifically identified in four of seven fluids from patients with active systemic lupus erythematosus (SLE) and in a single fluid from a patient with severe septic arthritis. This "collagen profile" was identical to that of rheumatoid synovium. Type II collagen, characteristic of hyaline articular cartilage, was found in two of six fluids from osteoarthritic joints. The presence of sufficient collagen (about 5 micrograms) to permit typing was correlated with roentgenographic evidence of joint space narrowing; the presence of the "synovial" collagen profile was correlated with decreased joint fluid pH.

Dual metalloprotease inhibitors: mercaptoacetyl-based fused heterocyclic dipeptide mimetics as inhibitors of angiotensin-converting enzyme and neutral endopeptidase.

A series of 7,6- and 7,5-fused bicyclic thiazepinones and oxazepinones were generated and incorporated as conformationally restricted dipeptide surrogates in mercaptoacyl dipeptides. These compounds are potent inhibitors of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) both in vitro and in vivo. Compound 1a, a 7,6-fused bicyclic thiazepinone, demonstrated excellent blood pressure lowering in a variety of animal models characterized by various levels of plasma renin activity and significantly potentiated urinary sodium, ANP, and cGMP excretion in a cynomolgus monkey assay. On the basis of its potency and duration of action, compound 1a (BMS-186716) was advanced into clinical development for the treatment of hypertension and congestive heart failure.

Development and design of specific inhibitors of angiotensin-converting enzyme.

Captopril is a remarkably effective new antihypertensive drug designed and developed as a potent and specific inhibitor of angiotensin-converting enzyme, a zinc metallopeptidase that participates in the synthesis of a hypertensive peptide, angiotensin II, and in the degradation of a hypotensive peptide, bradykinin. Earlier studies with a snake venom peptide (teprotride or SQ 20881) that could be administered only by injection demonstrated that specific inhibitors of angiotensin-converting enzyme could be highly effective as antihypertensive drugs, and helped to clarify the specificity and mechanism of action of the enzyme. A hypothetical model of the active center of angiotensin-converting enzyme based on its presumed analogy to the well characterized zinc metallopeptidase carboxypeptidase A was used to guide logical sequential improvements of a weakly active prototype inhibitor that led eventually to the highly optimized structure of captopril. The hypothetical working model of the active site of angiotensin-converting enzyme used to develop captopril continues to provide a firm basis for development of new types of specific inhibitors of this biologically important enzyme.

In vitro testing for the risk of arachnoiditis from myelographic contrast media.

The risk of arachnoiditis from aqueous myelographic contrast media has been assayed reliably only in experimental animals. The effect of contrast media on protein and collagen production by fibroblasts in vitro was studied. Iocarmate, metrizamide, and iopamidol added to the culture medium caused cells to produce more protein and collagen. The degree to which the contrast medium stimulated collagen production correlated with the risk of arachnoiditis from the intrathecal use of the contrast medium. In vitro testing appears to be an effective assay for arachnoiditis.

Growth of osteoblasts on porous calcium phosphate ceramic: an in vitro model for biocompatibility study.

Biomaterial implantation in animals is commonly used for biocompatibility studies as well as examination of long-term interaction between tissue and the test material. An in vitro cell culture model is proposed as an alternative which will save animal lives and reduce the pain and discomfort of animals used for such studies. In this study the biomaterial was matched to the cell types typical of the implant site of the particular material: porous calcium phosphate ceramic, used as dental and orthopaedic implants, with periosteal fibroblasts, osteoblasts and chondrocytes. All three cell types attached on to the ceramic and formed multicellular layers. Numbers of periosteal fibroblasts, osteoblasts and chondrocytes increased 29-, 23- and 17-fold, respectively, during the 10 wk period. Osteoblasts retained their phenotypic expression by producing only Type I collagen. Parathyroid hormone (PTH, 50 nM) suppressed the alkaline phosphatase activity of osteoblasts by over 50% and increased cAMP by more than 10-fold over control cultures.

Solubilization of hydroxyapatite crystals by murine bone cells, macrophages and fibroblasts.

Calcium phosphate ceramic is widely used as implant material. It is made up of hydroxyapatite, beta-tricalcium phosphate or various combinations of both. In the present study, we use an in vitro model to examine the role of cell-mediated resorption of calcium phosphate ceramic implant material. We compare the abilities of two sequential enzymatic released populations of bone cells from murine calvaria (Population II and Population V), macrophages and dermal fibroblasts to solubilize 45Ca-labelled hydroxyapatite crystals. These crystals were incubated with each of the cell types for 24 h in the presence or absence of parathyroid hormone, prostaglandin E2, calcitonin, and 1,25-dihydroxyvitamin D3. The amount of cell-mediated hydroxyapatite solubilization was determined by measuring the radioactivity in an aliquot of the supernatant after centrifugation. Using dermal fibroblasts as a baseline, relative abilities of macrophages, Population II and Population V to degrade crystals were 10.5, 5 and 2 times that of fibroblasts. Crystal-cell contact was required. While none of the bone resorption agents tested had any effect on this process, crystal dissolution by bone cells was inhibited by two lysosomotropic agents, NH4Cl and chloroquine.

Mechanisms of connective tissue damage by crystals containing calcium.

From available clinical, radiographic, and synovial fluid findings, coupled with in vivo radiolabelled crystal turnover data and in vitro experimental data, a hypothesis has been formulated relative to the pathogenesis of BCP crystal deposition diseases (Fig. 2). Synovial lining cells phagocytose BCP crystals and particulate collagens in the joint fluid. During and/or after internalization these cells are stimulated in a variety of ways: 1) protease synthesis and secretion is relentlessly stimulated, which may damage joint tissues producing clinically evident loss of collagenous tissues including cartilage, bone, and tendon, and which may release additional amounts of crystals and particulate collagens into the synovial fluid, completing a vicious cycle; 2) PGE2 production is greatly augmented; 3) DNA synthesis is stimulated as a result of increased inositol phospholipid turnover and intracellular crystal dissolution. The increased number of synovial cells also augments the total local generation of proteases and prostenoids. Mechanical factors such as trauma or joint overuse also contribute to the pathogenesis of joint destruction as discussed in the article on the clinical aspects of BCP crystal deposition.

The role of crystals in osteoarthritis.

The deposition of calcium-containing crystals in articular tissues is probably an under-recognized event. Clinical observations indicate that an exaggerated and uniquely distributed cartilage degeneration is associated with these deposits. Measurements of putative markers of cartilage breakdown suggest that the presence of these crystals magnifies the degenerative process. In vitro studies indicate two potential mechanisms by which crystals cause degeneration. These involve the stimulation of mitogenesis in synovial fibroblasts and the secretion of proteases by cells that phagocytose these crystals. Approaches that might ameliorate the degenerative process may ensue from new information about how crystals form and how they exert their biologic effects.

Ductal carcinoma in situ arising in mammary hamartoma.

Hamartoma of the breast is an uncommon lesion. Although it can possess characteristic radiological features, the pathological appearance is not distinctive. Hamartoma is generally considered benign, but four cases have been reported with ductal and lobular carcinoma arising in hamartomas. This report describes further cases of hamartoma from which ductal carcinoma in situ arose, with one showing early invasion. In both cases, the tumours were within the hamartomas and were adequately excised during lumpectomies of the hamartomas, and the patients were well afterwards. This report emphasises the importance of adequate sampling of mammary hamartoma.

Hamartoma of the breast: a clinicopathological review.

AIMS: To review 25 cases of breast hamartoma and discuss the pathological criteria, and the usefulness of imaging modalities, fine needle aspiration cytology (FNAC), and needle core biopsy in the diagnosis. METHODS: The hamartomas were assessed for interlobular fibrotic stroma, stromal adipose tissue content, pseudo-angiomatous stromal hyperplasia, and epithelial changes (hyperplasia, adenosis or apocrine metaplasia, and cyst formation). All imagings, previous FNACs, and biopsies were also reviewed. RESULTS: Imaging (mammography, ultrasound, and magnetic resonance imaging) was performed in 18 cases, and mostly showed encapsulated masses with a heterogeneous appearance. Microscopically, all hamartomas demonstrated good demarcation with fibrous tissue condensation. Adipose tissue was noted in all cases (5-90%; mean, 31%), and interlobular fibrosis in 21 cases. Benign epithelial hyperplasia occurred in 10 cases, and pseudo-angiomatous stromal hyperplasia or cystic ducts in eight cases each. Apocrine metaplasia, calcification, stromal giant cells, and adenosis occurred in four cases or less. Two cases showed coexisting ductal carcinoma in situ limited to within the hamartoma. Needle core biopsies (four cases) and FNAC (14 cases) were largely insufficient, inconclusive, or non-specific. CONCLUSIONS: Hamartomas do not possess specific diagnostic histological features. The role of FNAC and needle core biopsy in making the diagnosis is limited, and requires clinical and radiological correlation to avoid underdiagnosis.

Rat brain enkephalinase: characterization of the active site using mercaptopropanoyl amino acid inhibitors, and comparison with angiotensin-converting enzyme.

Over fifty mercaptopropanoyl amino acids and related derivatives were synthesized to define the steric, electronic and stereochemical requirements for binding to the active site of enkephalinase (ENKASE), and also for their ability to inhibit angiotensin-converting enzyme (ACE). In this way the character of ENKASE and ACE active sites were compared.

Purification and characterization of enkephalinase, angiotensin converting enzyme, and a third peptidyldipeptidase from rat brain.

Three distinct peptidyldipeptidases (exopeptidases releasing carboxyl terminal dipeptide residues) can be solubilized from nerve terminal membrane fractions from whole rat brain or striatum, and separated by ion exchange chromatography. Brain angiotensin-converting enzyme (PDP-1) cleaves Hip-His-Leu, but not 80 nM [3H-Tyr1, Leu5]-enkephalin, and is markedly inhibited by several specific inhibitors such as captopril, teprotide, and MK-422. Enkephalinase (PDP-2) cleaves 80 nM [3H-Tyr1, Leu5]-enkephalin, but not Hip-His-Leu; it is not inhibited by any of the standard competitive inhibitors of angiotensin-converting enzyme (all analogs of carboxyl-terminal peptide sequences Phe-Ala-Pro or Ala-Pro), but is strongly inhibited by captopril analogs such as thiorphan (Phe-Gly analog). A third peptidyldipeptidase (PDP-3) cleaves Hip-His-Leu, but not 80 nM [3H-Tyr1, Leu5]-enkephalin; it is inhibited by dipeptide analog inhibitors such as captopril and thiorphan, but not by longer peptides such as teprotide or tripeptide analog inhibitors such as MK-422. Both PDP-2 (enkephalinase) and PDP-3 are apparently present in nerve terminal membranes predominantly as inactive proenzyme precursors, which elute from DEAE-cellulose at high salt concentration, and are activated very slowly by a process involving one or more trypsin-like enzymes. Rechromatography of activated PDP-2 and PDP-3 achieves a nearly complete separation of the two enzymes, both markedly purified, since each is much less acidic than its proenzyme precursor. Purified enkephalinase does not appear to have any significant endopeptidase activity. It cleaves Hip-Phe-Arg 200 times more effectively than Hip-Phe-Arg-NH2, and appears to be quite selective for cleaving the terminal dipeptide residue, Phe-Arg, from bradykinin, with no release of the second dipeptide and no cleavage of the Gly4-Phe5 interior peptide bond.