Psoralen and Bakuchiol Ameliorate M-CSF Plus RANKL-Induced Osteoclast Differentiation and Bone Resorption Via Inhibition of AKT and AP-1 Pathways in Vitro.

BACKGROUND/AIMS: Psoralen and bakuchiol are the main active compounds found in the traditional Chinese medicine Psoralea corylifolia L., and have been used to treat osteoporosis. This study aims to investigate the anti-osteoporosis effects of these two compounds using osteoclasts precursor differentiation and bone absorption assays in vitro. METHODS: Primary mouse osteoclasts precursor cells were induced by M-CSF (macrophage colony stimulating factor) plus RANKL (receptor activator of nuclear factor kappa-B ligand) in vitro. TRACP (tartrate-resistant acid phosphatase) enzyme activity and toluidine blue staining were used to observe the effects of psoralen and bakuchiol on osteoclast differentiation and bone resorption, respectively. Gelatin zymography was used to assess MMP (matrix metalloproteinase) activity, and ELISA was performed to measure cathepsin K activity. Western blotting analysis for expression of phosphorylated AKT, ERK, NF-kB, and c-jun; and immunofluorescence analysis for c-jun and p65 nuclear translocation in induced osteoclasts were then used to determine the mechanism of anti-bone resorption of psoralen and bakuchiol. RESULTS: Mature osteoclasts were induced by M-CSF plus RANKL from primary bone marrow macrophages in vitro. Both psoralen and bakuchiol significantly inhibited TRACP enzyme activity and slightly decreased the number of TRACP+ multinuclear osteoclasts induced by M-CSF plus RANKL. Bakuchiol significantly decreased bone lacunae area and attenuated MMP-2 activity induced by M-CSF plus RANKL in osteoclasts. Both psoralen and bakuchiol significantly decreased the expression and nuclear translocation of phosphorylated c-jun stimulated by M-CSF plus RANKL, but no significant effect on p65 translocation was observed in osteoclasts. Additionally, bakuchiol significantly attenuated the increased of M-CSF plus RANKL-induced phosphorylation of AKT in osteoclasts. CONCLUSIONS: Psoralen and bakuchiol ameliorated M-CSF plus RANKL-induced osteoclast differentiation and bone resorption via inhibition of AKT and AP-1 pathways activation in vitro.

Effects of angiotensin II type I receptor blocker losartan on orthodontic tooth movement.

INTRODUCTION: Drugs that block the renin-angiotensin system (RAS) are widely used for treating hypertension, heart and kidney failure, and the harmful effects of diabetes. Components of the RAS have been identified in various organs, but little is known of their effects on bone remodeling. The aim of this study was to evaluate whether the blockage of the RAS influences strain-induced bone remodeling in a model of orthodontic tooth movement. METHODS: An orthodontic appliance was placed in C57BL6/J mice that were randomly divided into 2 groups: vehicle-treated mice (VH) and mice treated with losartan (an angiotensin II receptor blocker). Orthodontic tooth movement and the number of tartrate-resistant acid phosphatase-positive cells were determined by histopathologic analysis. The expression of mediators involved in bone remodeling was evaluated by quantitative real-time polymerase chain reaction. Blood pressure was measured before and during the experimental period. RESULTS: Orthodontic tooth movement and tartrate-resistant acid phosphatase-positive cells were significantly reduced in the losartan group compared with the VH group. mRNA levels of osteoclast markers (RANK, RANKL, cathepsin K, and metalloproteinase 13) were lower in the losartan mice than in the VH group, whereas the expressions of osteoblast markers and negative regulators of bone resorption (periostin, dentin matrix protein, alkaline phosphatase, collagen 1A1, semaphorin 3A3, metalloproteinase 2, and osteoprotegerin) were higher in the VH group. CONCLUSIONS: Blockage of the RAS system decreases osteoclast differentiation and activity and, consequently, results in decreased strain-induced bone remodeling in orthodontic tooth movement.

Characterization of multinucleated giant cells in synovium and subchondral bone in knee osteoarthritis and rheumatoid arthritis.

BACKGROUND: Multinucleated giant cells have been noticed in diverse arthritic conditions since their first description in rheumatoid synovium. However, their role in the pathogenesis of osteoarthritis (OA) or rheumatoid arthritis (RA) still remains broadly unknown. We aimed to study the presence and characteristics of multinucleated giant cells (MGC) both in synovium and in subchondral bone tissues of patients with OA or RA. METHODS: Knee synovial and subchondral bone samples were from age-matched patients undergoing total joint replacement for OA or RA, or non-arthritic post mortem (PM) controls. OA synovium was stratified by histological inflammation grade using index tissue sections. Synovitis was assessed by Krenn score. Histological studies employed specific antibodies against macrophage markers or cathepsin K, or TRAP enzymatic assay. RESULTS: Inflamed OA and RA synovia displayed more multinucleated giant cells than did non-inflamed OA and PM synovia. There was a significant association between MGC numbers and synovitis severity. A TRAP negative/cathepsin K negative Langhans-like subtype was predominant in OA, whereas both Langhans-like and TRAP-positive/cathepsin K-negative foreign-body-like subtypes were most commonly detected in RA. Plasma-like and foam-like subtypes also were observed in OA and RA synovia, and the latter was found surrounding adipocytes. TRAP positive/cathepsin K positive osteoclasts were only identified adjacent to subchondral bone surfaces. TRAP positive osteoclasts were significantly increased in subchondral bone in OA and RA compared to PM controls. CONCLUSIONS: Multinucleated giant cells are associated with synovitis severity, and subchondral osteoclast numbers are increased in OA, as well as in RA. Further research targeting multinucleated giant cells is warranted to elucidate their contributions to the symptoms and joint damage associated with arthritis.

Augmented LPS responsiveness in type 1 diabetes-derived osteoclasts.

Bone abnormalities are frequent co-morbidities of type 1 diabetes (T1D) and are principally mediated by osteoblasts and osteoclasts which in turn are regulated by immunologic mediators. While decreased skeletal health in T1D involves alterations in osteoblast maturation and function, the effect of altered immune function on osteoclasts in T1D-associated bone and joint pathologies is less understood. Here T1D-associated osteoclast-specific differentiation and function in the presence and absence of inflammatory mediators was characterized utilizing bone marrow-derived osteoclasts (BM-OCs) isolated from non-obese diabetic (NOD) mice, a model for spontaneous autoimmune diabetes with pathology similar to individuals with T1D. Differentiation and osteoclast-mediated bone resorption were evaluated along with cathepsin K, MMP-9, and immune soluble mediator expression. The effect of lipopolysaccharide (LPS), a pro-inflammatory cytokine cocktail, and NOD-derived conditioned supernatants on BM-OC function was also determined. Although NOD BM-OCs cultures contained smaller osteoclasts, they resorbed more bone concomitant with increased cathepsin K, MMP-9, and pro-osteoclastogenic mediator expression. NOD BM-OCs also displayed an inhibition of LPS-induced deactivation that was not a result of soluble mediators produced by NOD BM-OCs, although a pro-inflammatory milieu did enhance NOD BM-OCs bone resorption. Together these data indicate that osteoclasts from a T1D mouse model hyper-respond to RANK-L resulting in excessive bone degradation via enhanced cathepsin K and MMP-9 secretion concomitant with an increased expression of pro-osteoclastic soluble mediators. Our data also suggest that inhibition of LPS-induced deactivation in NOD-derived BM-OC cultures is most likely due to NOD osteoclast responsiveness rather than LPS-induced expression of soluble mediators.

Chemical introduction of the green fluorescence: imaging of cysteine cathepsins by an irreversibly locked GFP fluorophore.

An activity-based probe, containing an irreversibly locked GFP-like fluorophore, was synthesized and evaluated as an inhibitor of human cathepsins and, as exemplified with cathepsin K, it proved to be suitable for ex vivo imaging and quantification of cysteine cathepsins by SDS-PAGE.

Mechanical strain modulates extracellular matrix degradation and byproducts in an isoform-specific manner.

Many studies have shown that mechanical forces can alter collagen degradation by proteases, and this mechanochemical effect may potentially serve an important role in determining extracellular matrix content and organization in load-bearing tissues. However, it is not yet known whether mechano-sensitive degradation depends on particular protease isoforms, nor is it yet known whether particular degradation byproducts can be altered by mechanical loading. In this study, we tested the hypothesis that different types of proteases exhibit different sensitivities to mechanical loading both in degradation rates and byproducts. Decellularized porcine pericardium samples were treated with human recombinant matrix metalloproteinases-1, -8, -9, cathepsin K, or a protease-free control while subjected to different levels of strain in a planar, biaxial mechanical tester. Tissue degradation was monitored by tracking the decay in mechanical stresses during displacement control tests, and byproducts were assessed by mass spectrometry analysis of the sample supernatant after degradation. Our key finding shows that cathepsin K-mediated degradation of collagenous tissue was enhanced with increasing strain, while MMP1-, MMP8-, and MMP9-mediated degradation were first decreased and then increased by strain. Degradation induced changes in tissue mechanical properties, and proteomic analysis revealed strain-sensitive degradome signatures with different ECM byproducts released at low vs. high strains. This evidence suggests a potentially new type of mechanobiology wherein mechanical forces alter the degradation products that can provide important signaling feedback functions during tissue remodeling.

Cathepsin K expression in the spectrum of perivascular epithelioid cell (PEC) lesions of the kidney.

The perivascular epithelioid cell (PEC) is a unique cell type coexpressing contractile proteins (mainly α-smooth muscle actin), melanocytic markers, including microphthalmia-associated transcription factor (MITF), and estrogen and progesterone receptors. It is constantly present in a group of tumors called PEComas. Renal PEComas include the common angiomyolipoma as well as less common lesions such as microscopic angiomyolipoma, intraglomerular lesions, angiomyolipoma with epithelial cysts, epithelioid angiomyolipoma, oncocytoma-like angiomyolipoma and lymphangioleiomyomatosis of the renal sinus. It has been demonstrated that most of these lesions are determined by mutations affecting genes of the tuberous sclerosis complex, tuberous sclerosis 1 (TSC1) and tuberous sclerosis 2 (TSC2), with eventual deregulation of the RHEB/MTOR/RPS6KB2 pathway, and it has been observed that some PEComas regressed during sirolimus therapy, an MTOR inhibitor. Recently, overexpression of MITF has been related to the expression of the papain-like cysteine protease cathepsin K in osteoclasts where it has inhibited MTOR. The aim of this study is to evaluate cathepsin K immunohistochemically in the entire spectrum of PEComa lesions in the kidney. The study population consisted of 84 renal PEComa lesions, including 5 composed predominantly of fat (lipoma-like angiomyolipoma), 15 almost exclusively composed of spindle-shaped smooth muscle cells (leiomyoma-like angiomyolipoma) and 31 common angiomyolipomas composed of a mixture of fat, spindle and epithelioid smooth muscle cells, and abnormal thick-walled blood vessels, 15 microscopic angiomyolipomas, 5 intraglomerular lesions, 2 oncocytoma-like angiomyolipomas, 8 epithelioid angiomyolipomas, 2 angiomyolipomas with epithelial cysts and 1 example of lymphangioleiomyomatosis of the renal sinus. In all of the renal PEComas, cathepsin K was found to be constantly and strongly expressed and seems to be a more powerful marker than other commonly used markers for their identification, especially to confirm the diagnosis on needle biopsies.

Cathepsin K is Superior to HMB45 for the Diagnosis of Pulmonary Lymphangioleiomyomatosis.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare cystic lung disease affecting predominantly young women. Classified as a low-grade malignant soft tissue neoplasm from the family of perivascular epithelioid cell (PEC) tumors or PEComas, it is characterized by a proliferation of abnormal smooth muscle-like cells (LAM cells), coexpressing myogenic and melanocytic markers, with HMB45 as the gold-standard immunohistochemical diagnostic marker. Cathepsin K, a papain-like cysteine protease with high matrix degrading activity, is commonly used in the pathologic diagnosis of other PEComa tumors, but there are few data regarding its expression in pulmonary LAM. This study compares the sensitivity of cathepsin K with that of HMB45 as immunohistochemical diagnostic markers for pulmonary LAM. Twenty-one (n=21) specimens of pulmonary LAM were retrieved from the archives of the Department of Pathology of the Cleveland Clinic. All cases were evaluated for protein expression of HMB45 and cathepsin K, on consecutive sections of formalin-fixed, paraffin-embedded tissue. The intensity and the total area of the immunostaining were quantified using an Aperio Scan Scope and analyzed with imaging software (Spectrum). Statistical analysis was performed using GraphPad software. The probability of a positive stained lesion on a transbronchial biopsy for each antibody was calculated. The percentage of LAM cells expressing cathepsin K was significantly higher than for HMB45 and overall expression was statistically significantly higher (P=0.0116). Our findings conclude that cathepsin K is a significantly more sensitive immunohistochemical marker than HMB45 in diagnosing pulmonary LAM.

Differential expression of cathepsin K in neoplasms harboring TFE3 gene fusions.

Cathepsin K is a protease whose expression is driven by microphthalmia transcription factor (MITF) in osteoclasts. TFE3 and TFEB are members of the same transcription factor subfamily as MITF and all three have overlapping transcriptional targets. We have shown that all t(6;11) renal cell carcinomas, which harbor an Alpha-TFEB gene fusion, as well as a subset of the Xp11 translocation renal carcinomas, which harbor various TFE3 gene fusions, express cathepsin K, while no other common renal carcinoma does. We have hypothesized that overexpression of TFEB or certain TFE3 fusion proteins function like MITF in these neoplasms, and thus activate cathepsin K expression. However, the expression of cathepsin K in specific genetic subtypes of Xp11 translocation carcinomas, as well as alveolar soft part sarcoma, which harbors the same ASPSCR1-TFE3 gene fusion as some Xp11 translocation carcinomas, has not been addressed. We performed immunohistochemistry for cathepsin K on 14 genetically confirmed t(X;1)(p11;q21) carcinomas, harboring the PRCC-TFE3 gene fusion; eight genetically confirmed t(X;17)(p11;q25) carcinomas, harboring the ASPSCR1-TFE3 gene fusion; and 18 alveolar soft part sarcomas (12 genetically confirmed), harboring the identical ASPSCR1-TFE3 gene fusion. All 18 alveolar soft part sarcomas expressed cathepsin K. In contrast, all eight ASPSCR1-TFE3 carcinomas were completely negative for cathepsin K. However, 12 of 14 PRCC-TFE3 carcinomas expressed cathepsin K. Expression of cathepsin K distinguishes alveolar soft part sarcoma from the ASPSCR1-TFE3 carcinoma, harboring the same gene fusion. The latter can be useful diagnostically, especially when alveolar soft part sarcoma presents in an unusual site (such as bone) or with clear cell morphology, which raises the differential diagnosis of metastatic ASPSCR1-TFE3 renal cell carcinoma. The difference in expression of cathepsin K between the PRCC-TFE3 and ASPSCR1-TFE3 carcinomas, together with the observed clinical differences between these subtypes of Xp11 translocation carcinomas, suggests the possibility of functional differences between these two related fusion proteins.

Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues.

Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications. Here, we detail a method of multiplex cathepsin zymography to detect and distinguish the activity of mature cathepsins K, L, S, and V by exploiting differences in individual cathepsin substrate preferences, pH effects, and electrophoretic mobility under non-reducing conditions. Specific identification of cathepsins K, L, S, and V in one cell/tissue extract was obtained with cathepsin K (37 kDa), V (35 kDa), S (25 kDa), and L (20 kDa) under non-reducing conditions. Cathepsin K activity disappeared and V remained when incubated at pH 4 instead of 6. Application of this antibody free, species independent, and medium-throughput method was demonstrated with primary human monocyte-derived macrophages and osteoclasts, endothelial cells stimulated with inflammatory cytokines, and normal and cancer lung tissues, which identified elevated cathepsin V in lung cancer.

Effect of cetirizine, a histamine (H(1)) receptor antagonist, on bone modeling during orthodontic tooth movement in rats.

INTRODUCTION: Histamine (H(1)) receptor antagonists are widely used drugs for treatment of allergic conditions. Although histamine was shown to be involved in bone remodeling, the aim of this study was to determine the effects of cetirizine, an H(1) receptor antagonist, on bone modeling processes during orthodontic tooth movement. METHODS: We used 3 groups of Wistar rats: control group (n = 16), appliance-only group (n = 16) and cetirizine group (n = 16). Each animal of the last 2 groups was fitted with a superelastic closed-coil spring appliance and treated daily with saline solution or cetirizine. Tooth movement was measured weekly from day 0 to day 42. Gene expression levels for bone turnover markers cathepsin K and osteocalcin were determined by means of real-time polymerase chain reaction. Histologic samples were analyzed by using histomorphometry. RESULTS: Cetirizine decreased the amount of tooth movement from day 28 onward (P <0.01), and it also decreased osteoclast volume density (P <0.001). An increase in alveolar bone volume density was observed in the cetirizine group (P <0.01) compared with the appliance-only group. No statistically significant differences were observed in osteoclast activity, osteoblast volume density, and osteoblast activity between the cetirizine and the appliance-only groups. CONCLUSIONS: Cetirizine influences bone modeling, mainly by inhibiting bone resorption. Therefore, H(1) receptor antagonists could interfere with orthodontic treatment.

Cathepsin K (Clone EPR19992) Demonstrates Uniformly Positive Immunoreactivity in Renal Oncocytoma, Chromophobe Renal Cell Carcinoma, and Distal Tubules.

Introduction. Cathepsin K is overexpressed in several tumors associated with microphthalmia transcription factor (MiTF) family or mechanistic target of rapamycin (mTOR) upregulation. Among renal neoplasms, MiTF translocation renal cell carcinoma (RCC), perivascular epithelioid cell neoplasms (PEComa), and eosinophilic solid and cystic RCC have demonstrated Cathepsin K immunoreactivity. In this study, we demonstrate a uniform Cathepsin K expression in oncocytoma, chromophobe RCC (CHRCC), and distal tubules. Design. We stained 13 oncocytomas, 13 CHRCC, 14 clear cell RCC (CCRCC), 9 papillary RCC (PRCC), 9 PEComas, and 5 MiTF RCC. Additionally, we assessed immunoreactivity for Cathepsin K in non-neoplastic renal parenchyma. Immunolabeling was performed on regularly charged slides from formalin-fixed paraffin-embedded tissue with monoclonal anti-rabbit antibodies to human Cathepsin K (clone EPR19992, Abcam). Results. All oncocytomas demonstrated diffuse strong cytoplasmic immunolabeling. CHRCC demonstrated uniform less intense immunolabeling in all cases with membranous accentuation. The assessment of the non-neoplastic renal parenchyma in all cases showed strong cytoplasmic immunoreaction in distal tubules and proximal tubules stained faintly. Mesangial cells were not immunoreactive. All MiTF RCC and PEComas were immunoreactive for Cathepsin K, whereas CCRCC and PRCC were negative in all cases. Conclusions. In this study, we expand the spectrum of renal neoplasms reactive with a particular clone of Cathepsin K (EPR19992). Distal tubules are strongly immunoreactive for Cathepsin K. Our conclusions need to be taken into consideration when differential diagnosis includes MiTF RCC or PEComa and this Cathepsin K clone is included in the immunohistochemical panel. This newer antibody clone was not tested in prior publications, potentially explaining the difference in conclusions.

Uterine leiomyosarcomas with osteoclast-like giant cells associated with high expression of RUNX2 and RANKL.

Uterine leiomyosarcoma (ULMS) with osteoclast-like giant cells (OLGCs) has been reported as a rare phenomenon in ULMS, and its clinico-pathological features and tumorigenesis remain unclear. We recently reported high expression of receptor activator of nuclear factor κB ligand (RANKL) in ULMS with OLGCs. As osteoblasts produce RANKL, in this study, we analyzed the expression of Runt-related transcription factor 2 (RUNX2), a critical transcription factor for osteoblasts, and osteoclast-related proteins in three cases of ULMS with OLGCs as well as five conventional ULMSs and nine leiomyomas. Immunohistochemistry and real-time reverse transcription quantitative polymerase chain reaction analyses showed high expression of RUNX2 and RANKL in ULMS with OLGCs. In these cases, macrophages expressed receptor activator of nuclear factor κB (RANK), and OLGCs expressed osteoclast-related proteins (nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and cathepsin K). Accumulation sites of cathepsin K-positive OLGCs showed hemorrhagic appearance and degraded type IV collagen. We reviewed reported cases of ULMS with OLGCs, including ours, and found that they presented an aggressive course even at stage I. Furthermore, metastatic lesions showed similar histological features to those of OLGC association in ULMS. Here, we show that tumor cells in ULMS with OLGCs highly express RUNX2 and RANKL and that osteoclastic differentiation of macrophages occurs in the tumor tissue.

Detection of femtomole quantities of mature cathepsin K with zymography.

Cathepsin K, the most potent mammalian collagenase, has been implicated in osteoporosis, cancer metastasis, atherosclerosis, and arthritis. Although procathepsin K is stable and readily detected, the active mature cathepsin K eludes detection by in vitro methods due to its shorter half-life and inactivation at neutral pH. We describe, for the first time, reliable detection, visualization, and quantification of mature cathepsin K to femtomole resolution using gelatin zymography. The specificity of the method was validated with cathepsin K knockdown using small interfering RNA (siRNA) transfection of human monocyte-derived macrophages, and enzymatic activity confirmed with benzyloxycarbonyl-glycine-proline-arginine-7-amino-4-methylcoumarin (Z-GPR-AMC) substrate hydrolysis was fit to a computational model of enzyme kinetics. Furthermore, cathepsin K zymography was used to show that murine osteoclasts secrete more cathepsin K than is stored intracellularly, and this was opposite to the behavior of the macrophages from which they were differentiated. In summary, this inexpensive, species-independent, antibody-free protocol describes a sensitive method with broad potential to elucidate previously undetectable cathepsin K activity.

In vitro osteoclastogenesis on textile chitosan scaffold.

Textile chitosan fibre scaffolds were evaluated in terms of interaction with osteoclast-like cells, derived from human primary monocytes. Part of the scaffolds was further modified by coating with fibrillar collagen type I in order to make the surface biocompatible. Monocytes were cultured directly on the scaffolds in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL) for up to 18 days. Confocal laser scanning microscopy (CLSM) as well as scanning electron microscopy (SEM) revealed the formation of multinuclear osteoclast-like cells on both the raw chitosan fibres and the collagen-coated scaffolds. The modified surface supported the osteoclastogenesis. Differentiation towards the osteoclastic lineage was confirmed by the microscopic detection of cathepsin K, tartrate resistant acid phosphatase (TRAP), acidic compartments using 3-(2,4-dinitroanillino)-3'-amino-N-methyldipropylamine (DAMP), immunological detection of TRAP isoform 5b, and analysis of gene expression of the osteoclastic markers TRAP, cathepsin K, vitronectin receptor, and calcitonin receptor using reverse transcription-polymerase chain reaction (RT-PCR). The feature of the collagen-coated but also of the raw chitosan fibre scaffolds to support attachment and differentiation of human monocytes facilitates cell-induced material resorption--one main requirement for successful bone tissue engineering.

Cathepsin-K is a sensitive immunohistochemical marker for detection of micro-granulomas in hypersensitivity pneumonitis.

UNLABELLED: Hypersensitivity Pneumonitis (HP) is an interstitial lung disease that occurs upon exposure to a variety of inhaled organic antigens. The presence of small non-caseating granulomas and isolated giant cells is not specific, but is considered a relevant histological feature for HP. The detection of granulomas is widely considered as easy on standard histological stains, but microgranuloma detection can be difficult and/or time consuming, especially in chronic HP cases. Cathepsin K (Cath-K) is a potent cysteine protease expressed at high levels in activated macrophages (osteoclasts, and epithelioid cells in granulomas), but is not expressed in resident macrophages thus representing a promising marker to rapidly detect and quantitatively evaluate microgranulomas in interstitial lung diseases. We analyzed the expression of Cath-K by immunohistochemistry in 22 subacute and chronic HP cases, using semi-quantitative scores. Control samples included normal lung tissue, and a variety of interstitial lung diseases: 3 Wegener's granulomatosis, 3 sarcoidosis, 3 tuberculosis, 1 berylliosis, 20 idiopathic pulmonary fibrosis (IPF), 2 Langerhans' cell histiocytosis, 5 nonspecific-interstitial pneumonia (NSIP), 5 cryptogenic organising-pneumonia (COP), 2 Airway-Centered Interstitial Fibrosis (ACIF), 5 desquamative interstitial pneumonia (DIP), 3 respiratory bronchiolitis interstitial lung disease (RB-ILD). Intense expression of Cath-K was demonstrated in epithelioid and giant cells in all cases containing granulomas (HP, sarcoidosis, Wegener's granulomatosis, berylliosis, tuberculosis). Among HP cases 19/22 (86.3%) contained granulomas that could be semiquantitatively evaluated. In all HP and control cases alveolar macrophages did not express Cath-K, including cases characterised by large collections of alveolar macrophages such as DIP and RB-ILD. CONCLUSIONS: Cath-K represents a sensitive and specific marker to detect and quantitate granulomatous reactions in interstitial lung diseases, and is particularly useful in chronic HP cases.

Expression of elastolytic cathepsins in human skin and their involvement in age-dependent elastin degradation.

BACKGROUND: Skin ageing is associated with structure-functional changes in the extracellular matrix, which is in part caused by proteolytic degradation. Since cysteine cathepsins are major matrix protein-degrading proteases, we investigated the age-dependent expression of elastolytic cathepsins K, S, and V in human skin, their in vitro impact on the integrity of the elastic fibre network, their cleavage specificities, and the release of bioactive peptides. METHODS: Cathepsin-mediated degradation of human skin elastin samples was assessed from young to very old human donors using immunohistochemical and biochemical assays, scanning electron microscopy, and mass spectrometry. RESULTS: Elastin samples derived from patients between 10 and 86 years of age were analysed and showed an age-dependent deterioration of the fibre structure from a dense network of thinner fibrils into a beaded and porous mesh. Reduced levels of cathepsins K, S, and V were observed in aged skin with a predominant epidermal expression. Cathepsin V was the most potent elastase followed by cathepsin K and S. Biomechanical analysis of degraded elastin fibres corroborated the destructive activity of cathepsins. Mass spectrometric determination of the cleavage sites in elastin revealed that all three cathepsins predominantly cleaved in hydrophobic domains. The degradation of elastin was efficiently inhibited by an ectosteric inhibitor. Furthermore, the degradation of elastin fibres resulted in the release of bioactive peptides, which have previously been associated with various pathologies. CONCLUSION: Cathepsins are powerful elastin-degrading enzymes and capable of generating a multitude of elastokines. They may represent a viable target for intervention strategies to reduce skin ageing.

Role for cathepsin K in emphysema in smoke-exposed guinea pigs.

The protease-antiprotease imbalance in the lung plays an important role in the pathogenesis of smoke-induced emphysema. The aim of this study was to characterize the proteolytic responses leading to emphysema formation in the guinea pig smoke exposure model. Guinea pigs were exposed to cigarette smoke for 1, 2, 4, 8, and 12 weeks. Age-matched guinea pigs exposed to room air served as controls. Cigarette smoke induced inflammation after 4 weeks and generated emphysematous changes in the guinea pigs after 12 weeks of smoke exposure. Increased phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases was demonstrated post cigarette smoke exposure. A decrease in elastin and collagen and the loss of type III collagen were observed in the alveolar wall of smoke-exposed guinea pigs. Interestingly, no change was seen in the expression of collagenolytic matrix metalloproteinases. Furthermore, the authors observed a 3-fold increase in cathepsin K activity in the lungs of smoke-exposed guinea pigs. The significance of this finding was supported by human studies that demonstrate increased expression of cathepsin K in the lungs of patients with emphysema. Elevation of cathepsin K in guinea pig lungs after smoke exposure likely constitutes a critical event leading to the disruption of lung extracellular matrix in this model.

Gliclazide reduced oxidative stress, inflammation, and bone loss in an experimental periodontal disease model.

OBJECTIVE: The aim of this study was to evaluate the effects of gliclazide on oxidative stress, inflammation, and bone loss in an experimental periodontal disease model. MATERIAL AND METHODS: Male albino Wistar rats were divided into no ligature, ligature, and ligature with 1, 5, and 10 mg/kg gliclazide groups. Maxillae were fixed and scanned using micro-computed tomography to quantify linear and bone volume/tissue volume (BV/TV) and volumetric bone loss. Histopathological, immunohistochemical and immunofluorescence analyses were conducted to examine matrix metalloproteinase-2 (MMP-2), cyclooxygenase 2 (COX-2), cathepsin K, members of the receptor activator of the nuclear factor kappa-Β ligand (RANKL), receptor activator of nuclear factor kappa-Β (RANK), osteoprotegerin (OPG) pathway, macrophage migration inhibitory factor (MIF), superoxide dismutase-1 (SOD-1), glutathione peroxidase-1 (GPx-1), NFKB p 50 (Cytoplasm), NFKB p50 NLS (nuclear localization signal), PI3 kinase and AKT staining. Myeloperoxidase activity, malondialdehyde and glutathione levels, while interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) levels were evaluated by spectroscopic ultraviolet-visible analysis. A quantitative reverse transcription polymerase chain reaction was used to quantify the gene expression of the nuclear factor kappa B p50 subunit (NF-κB p50), phosphoinositide 3-kinase (PI3k), protein kinase B (AKT), and F4/80. RESULTS: Micro-computed tomography showed that the 1 mg/kg gliclazide treatment reduced linear bone loss compared to the ligature, 5 mg/kg gliclazide, and 10 mg/kg gliclazide treatments. All concentrations of gliclazide increased bone volume/tissue volume (BV/TV) compared to the ligature group. Treatment with 1 mg/kg gliclazide reduced myeloperoxidase activity, malondialdehyde, IL-1ß, and TNF-α levels (p≤0.05), and resulted in weak staining for COX-2, cathepsin k, MMP-2, RANK, RANKL, SOD-1, GPx-1,MIF and PI3k. In addition, down-regulation of NF-κB p50, PI3k, AKT, and F4/80 were observed, and OPG staining was strong after the 1 mg/kg gliclazide treatment. CONCLUSIONS: This treatment decreased neutrophil and macrophage migration, decreased the inflammatory response, and decreased bone loss in rats with ligature-induced periodontitis.

Periarteriolar Glioblastoma Stem Cell Niches Express Bone Marrow Hematopoietic Stem Cell Niche Proteins.

In glioblastoma, a fraction of malignant cells consists of therapy-resistant glioblastoma stem cells (GSCs) residing in protective niches that recapitulate hematopoietic stem cell (HSC) niches in bone marrow. We have previously shown that HSC niche proteins stromal cell-derived factor-1α (SDF-1α), C-X-C chemokine receptor type 4 (CXCR4), osteopontin (OPN), and cathepsin K (CatK) are expressed in hypoxic GSC niches around arterioles in five human glioblastoma samples. In HSC niches, HSCs are retained by binding of SDF-1α and OPN to their receptors CXCR4 and CD44, respectively. Protease CatK cleaves SDF-1α to release HSCs out of niches. The aim of the present study was to reproduce the immunohistochemical localization of these GSC markers in 16 human glioblastoma samples with the addition of three novel markers. Furthermore, we assessed the type of blood vessels associated with GSC niches. In total, we found seven GSC niches containing CD133-positive and nestin-positive GSCs as a single-cell layer exclusively around the tunica adventitia of 2% of the CD31-positive and SMA-positive arterioles and not around capillaries and venules. Niches expressed SDF-1α, CXCR4, CatK, OPN, CD44, hypoxia-inducible factor-1α, and vascular endothelial growth factor. In conclusion, we show that GSC niches are present around arterioles and express bone marrow HSC niche proteins.