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 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.

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.

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.

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.

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

Abstract 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-1β) 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-1β, 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.

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.

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.

Angiomyomatous hamartoma of lymph nodes, revisited: clinicopathologic study of 21 cases, emphasizing its distinction from lymphangioleiomyomatosis of lymph nodes.

Angiomyomatous hamartoma of lymph nodes (AMH-LN) is an uncommon benign proliferation of smooth muscle, blood vessels, collagenous stroma, and adipocytes, most commonly affecting inguinal LN. A similar constellation of cell types constitutes various members of the perivascular epithelioid cell tumor (PEComa) family, including lymphangioleiomyomatosis (LAM), which can involve LN in women. Because some LN-LAM patients have tuberous sclerosis complex and/or other PEComa family lesions, it is clinically relevant to distinguish LN-LAM from AMH-LN. Given their similar features, however, the possibility that AMH-LN is a morphologic variant of LN-LAM merits inquiry. The dual melanocytic and myoid immunophenotype distinguishes the PEComa family from its mimics. Cathepsin K has recently emerged as a more sensitive marker for the PEComa family than HMB-45, which can be weak and focal, but cathepsin K has not been studied in AMH-LN. This study evaluated 21 AMH-LNs for clinical, morphologic, and immunophenotypic features of LN-LAM. None (0/21) had tuberous sclerosis complex or PEComas. Thirteen (62%) were male, unlike LN-LAM, which is restricted to women. All cases exhibited intraparenchymal proliferation of variable-sized, thick-walled blood vessels within collagenous stroma containing a sparse to focally cellular population of haphazardly distributed smooth muscle cells. Admixed adipocytes were commonly present. None exhibited classical features of LN-LAM such as subcapsular localization, extranodal extension, intralymphatic growth, compact nests, branching lymphatic channels, plump cell shape, or foamy/clear cytoplasm. None exhibited any staining for cathepsin K, HMB-45, or microphthalmia transcription factor. There is no clinical, morphologic, or immunohistochemical evidence to suggest that AMH-LN is a variant of LN-LAM.

[Overexpression of Klotho inhibits differentiation of RAW264.7 cells into osteoclasts].

Objective To explore the effect of overexpression of Klotho (KL) on the differentiation of RAW264.7 cells intoosteoclasts induced by soluble receptor activator of nuclear factor-κB ligand ( sRANKL). Methods The RAW264.7 cells were divided into Ad-KL group, Ad-GFP group and control group. Inverted microscope was used to observe KL transfected cells. Quantitative real-time PCR (qRT-PCR) and Western blotting were used to detect mRNA and protein expression level of KL, respectively. Tartrate-resistant acidphosphatase (TRAP) staining was applied to observe the osteoclast differentiation and maturation. The qRT-PCR and Western blotting were performed to assess mRNA or protein expression levels of TRAP and cathepsin K (CTSK). Results Klotho gene was transfected into RAW264.7 cells successfully. Compared with Ad-GFP group and control group, KL mRNA and protein expression levels of Ad-KL group increased significantly. TRAP staining showed that the number and volume of TRAP-positive cells in Ad-KL group were less than those in Ad-GFP group and control group. Compared with Ad-GFP group and control group, TRAP and CTSK mRNA and protein expression levels were reduced remarkably in Ad-KL group. Conclusion Overexpression of Klotho inhibits the differentiation of RAW264.7 cells into osteoclasts.

Multiplex Cathepsin Zymography to Detect Amounts of Active Cathepsins K, L, S, and V.

Cysteine cathepsins are powerful proteases that can degrade other proteins, among which are the extracellular matrix proteins collagen and elastin. Multiplex cathepsin zymography is an assay that can quantify the amount of active cathepsins in a cell or tissue preparation. This method works for measuring the amounts of active cathepsins K, L, S, and V in a cell or tissue preparation without requiring the use of antibodies for specific identification which tremendously reduces cost. This chapter will demonstrate the utility and interpretation of this method with mammalian cells and tissue to quantify amounts of active cathepsins K, L, S, and V without complicating signals of the procathepsin. Multiplex cathepsin zymography has many advantages: (1) it separates cathepsins K, L, S, and V by electrophoretic migration distance, (2) allows visual confirmation of cathepsin identity, (3) does not detect procathepsins, and (4) can be quantified with densitometry.

Xp11 Translocation Renal Cell Carcinomas (RCCs) With RBM10-TFE3 Gene Fusion Demonstrating Melanotic Features and Overlapping Morphology With t(6;11) RCC: Interest and Diagnostic Pitfall in Detecting a Paracentric Inversion of TFE3.

Xp11 translocation renal cell carcinomas (RCC) are characterized by several different translocations involving the TFE3 gene. Tumors with different specific gene fusions may have different clinicopathologic manifestations. Only 3 RBM10-TFE3 RCCs have been reported to date. Here, we added 4 cases of this rare type of tumors with clinicopathologic, immunohistochemical, molecular, and ultrastructural analyses. Most tumors had similar patterns with mixed architectures as follows: acinar, tubular and papillary patterns of epithelioid cells combined with sheets of small cells with "pseudorosette-like" architectures, mimicking the typical morphology of t(6;11) RCC. Cytoplasmic vacuolization, nuclear groove, and psammoma bodies were observed in most cases. Immunohistochemically, all 4 cases demonstrated moderate to strong immunoreactivity for TFE3, Cathepsin K, CD10, Ksp-cadherin, E-cadherin, P504S, RCC marker, PAX8 and vimentin, whereas negativity for TFEB, HMB45, and CK7. CKpan and Melan-A were at least focally expressed. The antibody to Ki-67 showed labeling of 3% to 8% (mean, 5%) of tumor cell nuclei. ;Of interest, several immunostainings demonstrated expression discrepancy in different histology patterns. RBM10-TFE3 fusion transcripts were identified in all cases by reverse transcription-polymerase chain reaction. By fluorescence in situ hybridization, all 4 cases showed unusual split signals with a distance <1 signal diameter (co-localized or subtle split signals) and usually had false-negative results. We also observed ultrastructures, including melanin pigment, nuclear groove, numerous glycogens, mitochondrion with areas of high electron density material, basement membrane material, and cell junctions with poor development. All 4 patients were alive with no evidence of recurrent disease. Our report adds to the known data regarding RBM10-TFE3 RCC.

RBM10-TFE3 Renal Cell Carcinoma: A Potential Diagnostic Pitfall Due to Cryptic Intrachromosomal Xp11.2 Inversion Resulting in False-negative TFE3 FISH.

Xp11 translocation renal cell carcinoma (RCC) are defined by chromosome translocations involving the Xp11 breakpoint which results in one of a variety of TFE3 gene fusions. TFE3 break-apart florescence in situ hybridization (FISH) assays are generally preferred to TFE3 immunohistochemistry (IHC) as a means of confirming the diagnosis in archival material, as FISH is less sensitive to the variable fixation which can result in false positive or false negative IHC. Prompted by a case report in the cytogenetics literature, we identify 3 cases of Xp11 translocation RCC characterized by a subtle chromosomal inversion involving the short arm of the X chromosome, resulting in an RBM10-TFE3 gene fusion. TFE3 rearrangement was not detected by conventional TFE3 break-apart FISH, but was suggested by strong diffuse TFE3 immunoreactivity in a clean background. We then developed novel fosmid probes to detect the RBM10-TFE3 gene fusion in archival material. These cases validate RBM10-TFE3 as a recurrent gene fusion in Xp11 translocation RCC, illustrate a source of false-negative TFE3 break-apart FISH, and highlight the complementary role of TFE3 IHC and TFE3 FISH.

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.

Effects of combined ovariectomy with dexamethasone on rat lumbar vertebrae.

OBJECTIVE: This study investigated the effects of combined ovariectomy with dexamethasone treatment on rat lumbar vertebrae in comparison with osteoporosis induced via ovariectomy or dexamethasone alone, and analysis of the associated molecular mechanism. METHODS: Sixty-two female Sprague-Dawley rats (3 months' old) were randomly divided into five treatment groups: an untreated baseline (BL) group; those receiving a sham operation (SHAM); those receiving a dexamethasone injection alone (DEXA); those undergoing bilateral ovariectomy (OVX); and those subjected to both ovariectomy and dexamethasone injection (OVX-DEXA). Animals in the BL group were euthanized at the beginning of the experiment, whereas animals in the remaining groups were euthanized at the end of the first month (M1), second month (M2), or third month (M3). Bone mineral density, bone microarchitecture, biomechanical properties of vertebrae, and serum levels of estrogen, amino-terminal propeptide of type I collagen (PINP), and ß-C-telopeptide of type I collagen (ß-CTX) were measured. In addition, we examined biglycan, runt-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), lipoprotein receptor-related protein-5 (LRP-5), cathepsin K (CTSK), and sclerostin mRNA expression. RESULTS: Bone mineral content and bone mineral density were markedly lower in the OVX-DEXA group compared with the OVX group at all time points examined. The relative bone surface (BS/TV, mm(-1), relative bone volume (BV/TV,%), and trabecular number (Tb.N, 1/mm) were markedly lower in the OVX-DEXA group compared with the remaining groups, whereas trabecular separation (Tb.Sp, mm) was markedly higher in the OVX-DEXA group compared with the remaining groups at M2 or M3. The OVX-DEXA group showed lower compressive strength and lower stiffness compared with the other groups at M2 and M3. Compressive displacement and energy absorption capacity were also markedly lower in the OVX-DEXA group compared with the OVX group at M3. Estradiol levels were markedly lower in the OVX-DEXA group compared with the other groups. Biglycan, runt-related transcription factor 2, osteoprotegerin, and lipoprotein receptor-related protein-5 were down-regulated in the DEXA, OVX, and OVX-DEXA groups compared with the BL and SHAM groups, whereas cathepsin K and sclerostin were up-regulated in the OVX-DEXA group compared with the DEXA and OVX groups. CONCLUSIONS: Ovariectomy combined with dexamethasone induced more serious osteoporosis in the rat lumbar spine than either ovariectomy or dexamethasone alone. The combined effect may be due to a combination of suppressed bone formation and increased bone resorption related to an estradiol deficit.

Catepsina K como biomarcador de afectación ósea en la enfermedad de Gaucher tipo 1 / Cathepsin K as a biomarker of bone involvement in type 1 Gaucher disease

Fundamento y objetivo: La enfermedad de Gaucher es un trastorno hereditario, que se origina como consecuencia del déficit de la actividad β-glucocerebrosidasa ácida, responsable de la degradación de glucosilceramida hasta ceramida y glucosa. Aunque el trastorno de base es fundamentalmente hematológico, el hueso es la segunda estructura más frecuentemente afectada. La catepsina K (CATK) es una enzima implicada en el proceso de remodelado óseo, habiéndose propuesto que la determinación de sus concentraciones séricas podría aportar información complementaria a la de otros biomarcadores. Pacientes y métodos: Se realizó un estudio en 20 controles sanos y 20 pacientes con enfermedad de Gaucher tipo 1, de las comunidades autónomas de Andalucía y Extremadura. Se determinaron como biomarcadores de remodelado óseo la bone alkaline phosphatase (B-ALP, «fosfatasa alcalina ósea»), el amino-terminal propeptide of procollagen type 1 (P1NP, «propéptido aminoterminal del procolágeno 1»), la β-Cross Laps, carboxy-terminal telopeptide of collagen type 1 (CTx, «fracción β del colágeno tipo 1») y CATK por técnicas de electroquimioluminiscencia y enzimoinmunoanálisis. Resultados: Existe un incremento en los niveles de CATK y las ratios CATK/P1NP y CATK/B-ALP en los pacientes con Gaucher tipo 1 respecto a la media obtenida en el grupo control. Por otro lado, considerando la existencia o no de manifestaciones óseas en el grupo de pacientes, la CATK y la ratio CATK/P1NP muestran niveles medios superiores en aquellos pacientes con daño óseo respecto a los que no lo presentan. Conclusiones: Aunque los estudios radiológicos constituyen el gold-standard para el seguimiento de enfermedad ósea en pacientes con enfermedad de Gaucher tipo 1, debe considerarse la utilidad de la CATK como posible indicador de daño óseo en estos pacientes. Asimismo, este parámetro puede utilizarse en la monitorización del tratamiento de la enfermedad ósea (AU)

Background and objective: Gaucher disease is an inherited disorder caused by deficit of acid β-glucocerebrosidase, responsible for the degradation of glucosylceramide to ceramide and glucose. Although the disorder is primarily hematologic, bone is the second most commonly affected structure. Cathepsin K (CATK) is an enzyme involved in bone remodelling process. It has been proposed that determination of its serum concentrations may provide additional information to other biomarkers. Patients and methods: The study included 20 control subjects and 20 Gaucher type 1 patients from Andalusia and Extremadura regions. We analyzed the biomarkers of bone remodelling: the bone alkaline phosphatase (B-ALP), the N-terminal propeptide of type 1 procollagen (P1NP), the β carboxyterminal telopeptide of type 1 collagen (CTx) and the CATK through electrochemiluminescence and immunoassay techniques. Results: There is an increase in levels of CATK, CATK/P1NP and CATK/B-ALP ratios in type 1 Gaucher patients compared to the control group. Considering the existence of skeletal manifestations in the patient group, the CATK and CATK/P1NP ratio showed higher levels in patients with bone damage compared to those without it. Conclusions: Although imaging studies are the gold standard for monitoring bone disease in type 1 Gaucher patients, the utility of CATK should be considered as a possible indicator of bone damage in these patients. Furthermore, this parameter can be used in the monitoring of the treatment of bone pathology (AU)

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.

A controlled, cross-sectional exploratory study on markers for the plasminogen system and inflammation in crevicular fluid samples from healthy, mucositis and peri-implantitis sites.

PURPOSE: To investigate expression of gene markers for the plasminogen system, inflammation, and bone resorption/remodelling in peri-implant crevicular fluid samples from healthy subjects, subjects with mucositis and subjects with peri-implantitis. A possible inhibitory effect of suppuration on the analysis of gene expression in samples from subjects with peri-implantitis was also analysed. MATERIALS AND METHODS: Peri-implant crevicular fluid (PICF) was sampled from 25 healthy subjects (H), 25 subjects with mucositis (M) and 25 subjects with peri-implantitis (P) using paper points and suction tips. The samples were analysed by quantitative polymerase chain reaction (qPCR). The following biomarkers associated with the plasminogen system, inflammation and bone resorption/ remodelling were investigated: interleukin-1 beta (IL-1ß), interleukin 8 (IL-8), tissue plasminogen activator (tPA), plasminogen activator inhibitor 2 (PAI-2), tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CatK). RESULTS: IL-1ß and IL-8 were significantly upregulated in the P group, and tPA and PAI-2 were significantly upregulated in the M group. These four genetic markers were oppositely regulated in samples from the subjects in the mucositis compared with the peri-implantitis group. TRAP and CatK showed no differences between the groups. The presence of suppuration did not have a detectable effect on gene analysis in samples from subjects with peri-implantitis. CONCLUSIONS: Markers for the plasminogen system and inflammation could be used to distinguish between mucositis and peri-implantitis. The results suggested that the plasminogen system was sufficiently upregulated allowing for resolution of inflammation and healing at the inflamed implant site in subjects with mucositis, whereas such upregulation was insufficient resulting in impaired healing and prolonged inflammation in subjects with peri-implantitis. The combination of tissue inflammation and low levels of tPA was a strong predictor of marginal bone loss in this study. It may be an interesting candidate for the unambiguous diagnosis of mucositis and peri-implantitis independent of radiographs and could possibly constitute a powerful future tool for rapid assessment of the periimplant tissue condition and the effect of subject treatment.