Human Synovia Contains Trefoil Factor Family (TFF) Peptides 1-3 Although Synovial Membrane Only Produces TFF3: Implications in Osteoarthritis and Rheumatoid Arthritis.

OBJECTIVE: Trefoil factor family peptide 3 (TFF3) has been shown to support catabolic functions in cases of osteoarthritis (OA). As in joint physiology and diseases such as OA, the synovial membrane (SM) of the joint capsule also plays a central role. We analyze the ability of SM to produce TFF compare healthy SM and its secretion product synovial fluid (SF) with SM and SF from patients suffering from OA or rheumatoid arthritis (RA). METHODS: Real-time PCR and ELISA were used to measure the expression of TFFs in healthy SM and SM from patients suffering from OA or RA. For tissue localization, we investigated TFF1-3 in differently aged human SM of healthy donors by means of immunohistochemistry, real-time PCR and Western blot. RESULTS: Only TFF3 but not TFF1 and -2 was expressed in SM from healthy donors as well as cases of OA or RA on protein and mRNA level. In contrast, all three TFFs were detected in all samples of SF on the protein level. No significant changes were observed for TFF1 at all. TFF2 was significantly upregulated in RA samples in comparison to OA samples. TFF3 protein was significantly downregulated in OA samples in comparison to healthy samples and cases of RA significantly upregulated compared to OA. In contrast, in SM TFF3 protein was not significantly regulated. CONCLUSION: The data demonstrate the production of TFF3 in SM. Unexpectedly, SF contains all three known TFF peptides. As neither articular cartilage nor SM produce TFF1 and TFF2, we speculate that these originate with high probability from blood serum.

Evaluation of surfactant proteins A, B, C, and D in articular cartilage, synovial membrane and synovial fluid of healthy as well as patients with osteoarthritis and rheumatoid arthritis.

OBJECTIVE: Surfactant Proteins (SPs) are well known from lung and form, along with phospholipids, a surface-active-layer at the liquid-air-interface of the alveolar lining. They play a major protective role by lowering surface tension, activating innate and adaptive immune defense at the lung mucosal interface, especially during infection. We analyzed the regulation of SPs in human and mouse articular chondrocytes, synoviocytes, and synovial fluid under healthy and inflammatory conditions, as well as in tissues of patients suffering from osteoarthritis and rheumatoid arthritis. METHODS: Immunohistochemistry, RT-PCR, qRT-PCR, ELISA, Western blotting were performed in cell cultures and tissue samples to determine localization, regulation, and concentration of SPs. RESULTS: All four SPs, were expressed by healthy human and mouse articular chondrocytes and synoviocytes and were also present in synovial fluid. Treatment with inflammatory mediators like IL-1ß and TNF-α led to short-term upregulation of individual SPs in vitro. In tissues from patients with osteoarthritis and rheumatoid arthritis, protein levels of all four SPs increased significantly compared to the controls used. CONCLUSION: These results show the distribution and amount of SPs in tissues of articular joints. They are produced by chondrocytes and synoviocytes and occur in measurable amounts in synovial fluid. All four SPs seem to be differently regulated under pathologic conditions. Their physiological functions in lowering surface tension and immune defense need further elucidation and make them potential candidates for therapeutic intervention.

Expression and Localization of Lung Surfactant Proteins in Human Testis.

BACKGROUND: Surfactant proteins (SPs) have been described in various tissues and fluids including tissues of the nasolacrimal apparatus, airways and digestive tract. Human testis have a glandular function as a part of the reproductive and the endocrine system, but no data are available on SPs in human testis and prostate under healthy and pathologic conditions. OBJECTIVE: The aim of the study was the detection and characterization of the surfactant proteins A, B, C and D (SP-A, SP-B, SP-C, SP-D) in human testis. Additionally tissue samples affected by testicular cancer were investigated. RESULTS: Surfactant proteins A, B, C and D were detected using RT-PCR in healthy testis. By means of Western blot analysis, these SPs were detected at the protein level in normal testis, seminoma and seminal fluid, but not in spermatozoa. Expression of SPs was weaker in seminoma compared to normal testicular tissue. SPs were localized in combination with vimentin immunohistochemically in cells of Sertoli and Leydig. CONCLUSION: Surfactant proteins seem to be inherent part of the human testis. By means of physicochemical properties the proteins appear to play a role during immunological and rheological process of the testicular tissue. The presence of SP-B and SP-C in cells of Sertoli correlates with their function of fluid secretion and may support transportation of spermatozoa. In seminoma the expression of all SP's was generally weaker compared to normal germ cells. This could lead to a reduction of immunomodulatory and rheology processes in the germ cell tumor.

Articular cartilage chondrocytes express aromatase and use enzymes involved in estrogen metabolism.

INTRODUCTION: Sex hormones, especially estrogens, have been implicated in articular cartilage metabolism and the pathogenesis of postmenopausal osteoarthritis. The conversion by aromatase (CYP19A1) of androstenedione into estrone (E1) and of testosterone into 17ß-estradiol (E2) plays a key role in the endogenous synthesis of estrogens in tissue. METHODS: We analyzed the expression of aromatase (CYP19A1) in immortalized C-28/I2 and T/C-28a2 chondrocytes, as well as in cultured primary human articular chondrocytes and human articular cartilage tissue, by means of RT-PCR, Western blotting and immunohistochemistry. By means of quantitative RT-PCR and enzyme-linked immunosorbent assay, we also determined whether the aromatase inhibitor letrozole influences estrogen metabolism of cultured chondrocytes in immortalized C-28/I2 chondrocytes. RESULTS: Aromatase mRNA was detected in both immortalized chondrocyte cell lines, in cultured primary human chondrocytes, and in human articular cartilage tissue. By means of Western blot analysis, aromatase was detected at the protein level in articular cartilage taken from various patients of both sexes and different ages. Cultured primary human articular chondrocytes, C-28/I2 and T/C-28a2, and human articular cartilage tissue reacted with antibodies for aromatase. Incubation of C-28/I2 chondrocytes with 10⁻¹¹ M to 10⁻7M letrozole as an aromatase inhibitor revealed significantly increased amounts of the mRNAs of the enzyme cytochrome P4501A1 (CYP1A1), which is involved in the catagen estrogen metabolism, and of the estrogen receptors ER-α and ER-ß. Concomitantly, synthesis of estrone (E1) was significantly downregulated after incubation with letrozole. CONCLUSIONS: We demonstrate that human articular cartilage expresses aromatase at the mRNA and protein levels. Blocking of estrone synthesis by the aromatase inhibitor letrozole is counteracted by an increase in ER-α and ER-ß. In addition, CYP1A1, an enzyme involved in catabolic estrogen metabolism, is upregulated. This suggests that articular chondrocytes use ERs functionally. The role of endogenous synthesized estrogens in articular cartilage health remains to be elucidated.

Variation in the hypothenar muscles and its impact on ulnar tunnel syndrome.

Compression of the ulnar nerve at Guyon's canal can be caused not only by tumor-like structures, a fibrotic arch, a ganglion, lipoma, aneurysm or thrombosis but also by anomalous hypothenar muscles which are reviewed here. For the search of relevant papers, PubMed and crucial anatomical textbooks were consulted. The abductor digiti minimi is the most variable hypothenar muscle. It can possess one to three muscle bellies. Additional heads can arise from the flexor retinaculum, the palmaris longus tendon, the pronator quadratus tendon or the deep fascia of the palmar side of the forearm. Our own case of an aberrant abductor digiti minimi appearing like connective tissue and originating in the antebrachial fascia is included here. Hematoxylin and eosin staining revealed that macroscopically non-muscle-like tissue contained skeletal muscle tissue. The muscle itself resembled other described cases. In addition, at the flexor digiti minimi accessory heads with origin from the flexor retinaculum, the antebrachial fascia or the long flexor muscles of the forearm can be detected. By contrast, the opponens digiti minimi mostly lacks variations and is sometimes missing. In our opinion, this is due to its hidden location. However, in few cases an additional head can arise from the lower arm aponeurosis. Furthermore, additional (fourth) hypothenar muscles might be expressed. These muscles are characterized by origins in the forearm and insertions on the head of the 5th metacarpal bone or on the 5th proximal phalanx. It must be noted that accessory hypothenar muscles might look like connective tissue at first glance. Often their origin extends to the antebrachial fascia. This can be explained by the phylogenetic fact that all intrinsic muscles of the hand are derived from muscle masses that originated in the forearm. In the opinion of several authors, ulnar nerve compression mostly is evoked by hyper trophied variant hypothenar muscles due to overuse as for example in carpenters. In some rare cases, an aberrant hypothenar muscle can also evoke median nerve compression.

C-28/I2 and T/C-28a2 chondrocytes as well as human primary articular chondrocytes express sex hormone and insulin receptors--Useful cells in study of cartilage metabolism.

Sex hormones and insulin have been implicated in articular cartilage metabolism. To supplement previous findings on the regulation of matrix synthesis with 17ß-estradiol and insulin and to find a possible model to study cartilage metabolism in vitro, we evaluated the expression of estrogen receptors α and ß (ERα, ERß), androgen receptor (AR) and insulin receptor (IR), in immortalized C-28/I2 and T/C-28a2 chondrocytes and in human primary articular cartilage cells. Chondrocytes were treated with increasing concentrations of 17ß-estradiol, dihydrotestosterone or insulin and analyzed by means of RT-PCR and Western blotting. Both cell lines as well as human articular chondrocytes expressed ER α and ß, AR and IR at mRNA and protein levels. In immortalized C-28/I2 chondrocytes, we showed that increasing concentrations of 17ß-estradiol diminished the 95kDa band of IR. Since 17ß-estradiol suppresses insulin-induced proline incorporation and type II collagen synthesis, as we have previously demonstrated, our findings give the first clue that 17ß-estradiol may have negative effects on cartilage anabolism triggered by insulin during hormonal imbalance. Compared to chondrocytes cultured without hormones, immunostaining for ERα/ß, AR and IR was decreased in both cell lines after incubation of cells with the receptor-specific hormones. It can be assumed that C-28/I2 and T/C-28a2 chondrocytes interact with the respective hormones. Our findings provide a reproducible model for investigating sex hormone and insulin receptors, which are present in low concentrations in articular chondrocytes, in the tissue-specific context of cartilage metabolism.

17ß-estradiol reduces expression of MMP-1, -3, and -13 in human primary articular chondrocytes from female patients cultured in a three dimensional alginate system.

Clinical observations have suggested a relationship between osteoarthritis and a changed sex-hormone metabolism, especially in menopausal women. This study analyzes the effect of 17ß-estradiol on expression of matrix metalloproteinases-1, -3, -13 (MMP-1, -3, -13) and tissue inhibitors of metalloproteinases-1, -2 (TIMP-1, -2) in articular chondrocytes. An imbalance of matrix metalloproteinases (MMPs) specialized on degradation of articular cartilage matrix over the respective inhibitors of these enzymes (TIMPs) that leads to matrix destruction was postulated in the pathogenesis of osteoarthritis. Primary human articular chondrocytes from patients of both genders were cultured in alginate beads at 5% O(2) to which 10(-11)M-10(-5)M 17ß-estradiol had been added and analyzed by means of immunohistochemistry, immunocytochemistry and real-time RT-PCR. Since articular chondrocytes in vivo are adapted to a low oxygen tension, culture was performed at 5% O(2). Immunohistochemical staining in articular cartilage tissue from patients and immunocytochemical staining in articular chondrocytes cultured in alginate beads was positive for type II collagen, estrogen receptor α, MMP-1, and -13. It was negative for type I collagen, MMP-3, TIMP-1 and -2. Using real-time RT-PCR, it was demonstrated that physiological and supraphysiological doses of 17ß-estradiol suppress mRNA levels of MMP-3 and -13 significantly in articular chondrocytes of female patients. A significant suppressing effect was also seen in MMP-1 mRNA after a high dose of 10(-5)M 17ß-estradiol. Furthermore, high doses of this hormone led to tendentially lower TIMP-1 levels whereas the TIMP-2 mRNA level was not influenced. In male patients, only incubations with high doses (10(-5)M) of 17ß-estradiol were followed by a tendency to suppressed MMP-1 and TIMP-1 levels while TIMP-2 mRNA level was decreased significantly. There was no effect on MMP-13 expression of cells from male patients. Taken together, application of 17ß-estradiol in physiological doses will improve the imbalance between the amounts of MMPs and TIMPs in articular chondrocytes from female patients. Downregulation of TIMP-2 by 17ß-estradiol in male patients would not be articular cartilage protective.

Trefoil factor 3 is induced during degenerative and inflammatory joint disease, activates matrix metalloproteinases, and enhances apoptosis of articular cartilage chondrocytes.

OBJECTIVE: Trefoil factor 3 (TFF3, also known as intestinal trefoil factor) is a member of a family of protease-resistant peptides containing a highly conserved motif with 6 cysteine residues. Recent studies have shown that TFF3 is expressed in injured cornea, where it plays a role in corneal wound healing, but not in healthy cornea. Since cartilage and cornea have similar matrix properties, we undertook the present study to investigate whether TFF3 could induce anabolic functions in diseased articular cartilage. METHODS: We used reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry to measure the expression of TFF3 in healthy articular cartilage, osteoarthritis (OA)-affected articular cartilage, and septic arthritis-affected articular cartilage and to assess the effects of cytokines, bacterial products, and bacterial supernatants on TFF3 production. The effects of TFF3 on matrix metalloproteinase (MMP) production were measured by enzyme-linked immunosorbent assay, and effects on chondrocyte apoptosis were studied by caspase assay and annexin V assay. RESULTS: Trefoil factors were not expressed in healthy human articular cartilage, but expression of TFF3 was highly up-regulated in the cartilage of patients with OA. These findings were confirmed in animal models of OA and septic arthritis, as well as in tumor necrosis factor alpha- and interleukin-1beta-treated primary human articular chondrocytes, revealing induction of Tff3/TFF3 under inflammatory conditions. Application of the recombinant TFF3 protein to cultured chondrocytes resulted in increased production of cartilage-degrading MMPs and increased chondrocyte apoptosis. CONCLUSION: In this study using articular cartilage as a model, we demonstrated that TFF3 supports catabolic functions in diseased articular cartilage. These findings widen our knowledge of the functional spectrum of TFF peptides and demonstrate that TFF3 is a multifunctional trefoil factor with the ability to link inflammation with tissue remodeling processes in articular cartilage. Moreover, our data suggest that TFF3 is a factor in the pathogenesis of OA and septic arthritis.

Extracellular matrix changes in knee joint cartilage following bone-active drug treatment.

Certain drugs or treatments that are known to affect bone quality or integrity might have side effects on the extracellular matrix of articular cartilage. We investigated the effects of vitamin D and calcium deficiency, estrogen deficiency, and hypercortisolism alone or in combination with bisphosphonates or sodium fluoride in an animal model, viz., the Göttingen miniature pig (n=29). The articular cartilage from knee joints was analyzed for its content of glycosaminoglycans (GAGs, as macromolecules responsible for the elasticity of articular cartilage) by a spectrometric method with dimethylene blue chloride. In cryo- or paraffin sections, alkaline phosphatase (AP, as an enzyme indicating mineralization or reorganization of articular cartilage matrix) was localized by enzyme histochemistry, and positive cells were counted, whereas differently sulfated GAGs were stained histochemically. A significant decrease in GAG content was measured in ovariectomized and long-term glucocorticoid-treated animals compared with untreated animals. In the glucocorticoid/sodium fluoride group, GAGs were significantly diminished, and significantly fewer AP-positive chondrocytes were counted compared with the control. GAG content was slightly higher, and significantly more AP-positive chondrocytes were counted in short-term glucocorticoid-treated animals then in the control group. GAGs, as part of proteoglycans, are responsible for the water-storage capacity that gives articular cartilage its unique property of elasticity. Thus, ovariectomy and long-term glucocorticoid therapy, especially when combined with sodium fluoride, have detrimental effects on this tissue.

Influence of 17beta-estradiol and insulin on type II collagen and protein synthesis of articular chondrocytes.

Clinical observations have suggested a relationship between osteoarthritis and a changed estrogen metabolism in menopausal women. Type II collagen is one main structural protein of articular cartilage matrix and its synthesis is increased by insulin in growth plate cartilage. Therefore, it was investigated if [(3)H]-proline incorporation and type II collagen synthesis (immunocytochemistry, ELISA) in female bovine articular chondrocytes are affected by 17beta-estradiol and/or insulin. Articular chondrocytes were cultured in monolayers at 5% O(2) in medium containing serum for 5-9 days, followed by application of 10(-13) to 10(-9) M estradiol or 5 microg/ml insulin during a serum-free culture phase of 2-3 days. Immunostaining for type II collagen was strong in the serum-free culture phase whereas it was negative for type I collagen, indicating that cells did not dedifferentiate to fibroblast-like cells during culture in serum-free medium. Whereas insulin raised the proline incorporation and the type II collagen synthesis significantly, physiological doses of estradiol did not show significant effects. The stimulating effect of insulin on the [(3)H]-proline incorporation or the type II collagen synthesis was significantly suppressed after preincubation of cells with 10(-11) to 10(-9) M estradiol resembling an unfavorable effect for articular cartilage. The suppression was reversed if cells were incubated with 10(-11) to 10(-7) M tamoxifen or ICI 182,780 combined with 10(-11) or 10(-9) M estradiol followed by incubation with 5 microg/ml insulin, indicating an estrogen receptor-mediated process. Because the articular cartilage of diabetic patients is biomechanically less stable, further experiments are needed to clarify the role of estradiol and insulin in the metabolism of articular chondrocytes.

Androgen receptors and gender-specific distribution of alkaline phosphatase in human thyroid cartilage.

The degree of mineralization in human thyroid cartilage is gender specific. Until now, laryngeal tissue was tested for sexual hormone receptors by the use of radiolabelled hormones only without exact localization of the receptors. In this study immediately frozen cartilage specimens from seven male and one female patient who underwent laryngectomy were used for immunolocalization of sexual hormone receptors. Additionally, serum sexual hormone levels were measured by means of radioimmunoassay. Alkaline phosphatase was localized enzymohistochemically in another cohort of six male and four female cartilage specimens from laryngectomies and autopsies. Chondrocytes in thyroid cartilage from both sexes reacted with antibodies to the androgen receptor. The low serum testosterone levels, which varied between 1.5 and 3.9 ng/ml, did not correlate with insufficient mineralization of thyroid cartilage in men (r=0.363, P=0.432). Chondrocytes did not react with antibodies to the estrogen receptor alpha and the progesterone receptor in both sexes. Expression of alkaline phosphatase started about the middle of the second decade. Some chondrocytes near the mineralization front were positive for androgen receptor and alkaline phosphatase, other chondrocytes were negative for both. Our results suggest the involvement of androgen receptor positive chondrocytes in thyroid cartilage mineralization, probably by a testosterone-linked stimulation of alkaline phosphatase.

Estradiol protects cultured articular chondrocytes from oxygen-radical-induced damage.

Osteoarthritis (OA) is aggravated in menopausal women possibly because of changed serum estrogen levels. Estradiol has been postulated to affect oxidative stress induced by reactive oxygen species (ROS) in articular chondrocytes. We generated ROS in cultured bovine articular chondrocytes by incubating them with combined Fe2SO4, vitamin C, and hydrogen peroxide. The release of thiobarbituric-acid-reactive substances (TBARS, lipid peroxidation) and lactate dehydrogenase (LDH, membrane damage) was measured photometrically. Various estradiol doses and vitamin E, serving as control with an established anti-oxidative capacity, were applied either upon each exchange of medium and during radical production (strategy 1) or only during radical production (strategy 2). In chondrocytes incubated according to strategy 1, the production of TBARS and LDH release were significantly suppressed by 10(-10)-10(-4) M estradiol or by vitamin E. Under strategy 2, the production of TBARS was significantly suppressed at estradiol concentrations higher than 10(-6) M, whereas LDH release was inhibited at concentrations of 10(-6)-10(-4) M. Vitamin E showed no significant effects. As repeated application of estradiol and vitamin E produced the best results, estradiol, like vitamin E, was speculated to accumulate in the plasma membrane and to decrease membrane fluidity resulting in protection against lipid peroxidation (non-genomic effect). Thus, in contrast to the neuroprotective effect of 17beta-estradiol in supraphysiological doses reported recently, the anti-oxidative potential of estradiol appears to protect articular chondrocytes from ROS-induced damage when the hormone is given repeatedly in a physiological range. Decreased estradiol levels may therefore contribute to menopausal OA in the long term.

Topographical and histological examination of osteophytes taken from arthrotic femoral heads.

Until now it is not known whether osteophytes of the femoral head develop because of pathological joint alterations or arise from normal remodeling processes secondary to osteoarthrosis. Firstly, we analysed the topographical localization of osteophytes. We then compared the extracellular matrix components of macroscopically normal cartilage from the margin of osteophytes with osteophytic cartilage from weight bearing and non-weight bearing zones by histochemical staining of low and heavily sulfated glycosaminoglycans. For examination 65 femoral heads were taken during endoprosthetic hip surgery. Osteophytes from different locations and macroscopically normal cartilage from the margin of osteophytes were excised, decalcified and embedded in paraplast. A lateral or medial localization of osteophytes (47 cases) was more common than a ventral or dorsal position (18 cases). Histochemical staining for low and heavily sulfated glycosaminoglycans from normal cartilage at the rim of osteophytes was stronger in the unmineralized cartilaginous zones compared to the mineralized cartilaginous zone. Weight bearing zones of osteophytic cartilage, on the other hand, showed an even distribution of the two differently sulfated glycosaminoglycans. Surprisingly, non-weight bearing zones of osteophytic cartilage showed a weaker staining for low and especially for heavily sulfated glycosaminoglycans in the superficial cartilage layer than in the deep cartilage layer. Altogether, osteophytic cartilage can be regarded as a reparative phenomenon for two reasons: Firstly, osteophytes arise very often at the weight bearing lateral and medial femoral head. Secondly, despite local differences in osteophytic cartilage, the same types of glycosaminoglycans are synthesized as in normal cartilage at the margin of osteophytes.

The effect of estrogens and dietary calcium deficiency on the extracellular matrix of articular cartilage in Göttingen miniature pigs.

Clinical observations have suggested that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis (OA). However, positive and negative associations between the incidence of OA and serum estrogen concentrations have been reported. In contrast to this, osteoporosis is regarded as a disease with a strong estrogen-dependent component. Moreover, there is an interaction between estrogen and calcium deficiency: calcium supplementation potentiates the effect of estrogen therapy. The present study was designed to investigate how estrogen deficiency affects the articular cartilage depending on calcium supply. The distribution of different types of glycosaminoglycans and collagens can be used as an indicator for extracellular matrix changes induced by estrogen deficiency. Different levels of dietary calcium were therefore fed to intact and ovariectomized Göttingen miniature pigs for one year before articular cartilage was harvested. The histochemical staining for heavy sulfated glycosaminoglycans in the extracellular matrix of ovariectomized miniature pigs, especially of those fed with a low calcium diet, was stronger in comparison to intact animals. In intact animals type II-collagen was immunodetected in all zones of unmineralized and mineralized articular cartilage, while immunostaining for this protein was negative to weak in the deep radiated fiber zone of ovariectomized minipigs. These results suggest that the synthesis of heavy sulfated glycosaminoglycans and immunohistochemically detectable type II-collagen is possibly influenced by estrogen deficiency. In conclusion, under estrogen deficiency, the extracellular matrix of articular cartilage underwent similar changes to those observed in physiologically aging cartilage where keratan sulfate is increased as a heavy sulfated glycosaminoglycan.