CatK Expression Research Articles

Cathepsin K Expression is Upregulated Following Micropulse Vibration Stimulation of Osteoclasts Cultured in Vitro

Aim: To evaluate osteoclast inhibition following micropulse vibration and the effects of micropulse vibration on the expression of IL-1β, TNF-α, and RANKL in osteoblasts and osteoclasts and that of cathepsin K (CatK) in osteoclasts cultivated in vitro. Materials and methods: Primary murine osteoblasts and osteoclasts were cultured in vitro and stimulated with an AcceleDent Aura device for 20 min per day for 0 to 96 hours, with microvibrations of 0.25 N/30 Hz. Subsequently, the levels of IL-1β, TNF-α, and RANKL in the supernatants of the culture medium of both types of cells were measured by enzyme-linked immunosorbent assays (ELISAs). In addition, the level of CatK in osteoclasts was evaluated by flow cytometry. Results: Microvibrations significantly upregulated IL-1β and TNF-α expression and downregulated RANKL expression in osteoblasts compared with the corresponding expression in the control group. Compared to those in the control group, osteoclasts treated with microvibrations showed significant downregulation of IL-1β, TNF-α, and RANKL expression. CatK levels in stimulated osteoclasts were increased compared to those in the control group. Conclusion: The type of microvibration applied in this study inhibits osteoclastogenesis and upregulates the expression of CatK, an enzyme that induces bone matrix degradation in osteoclasts cultured in vitro, which stimulates bone formation by osteoblasts and accelerates bone mineralization.

Potential Role of Odanacatib in the Treatment of Postmenopausal Intervertebral Disc Degeneration

Odanacatib (ODN), a cathepsin K (CatK) inhibitor, is widely used for the treatment of postmenopausal osteoporosis. However, whether ODN is efficient in delaying intervertebral disc degeneration (IVDD) remaining unknown. We aimed to explore the effect of ODN in the postmenopausal IVDD using an ovariectomized (OVX) mouse model. We collected the disc tissues from female spinal fracture patients and analyzed the CatK expression in different estrogen levels. Besides, we injected ranged concentration of OND subcutaneously in the OVX mice and tested the disc height index (DHI), aggrecan positive area, collagen I, collagen II, inflammatory factors, and apoptosis-related gene expression comparing to control. The intervertebral disc degraded and the CatK gene expression decreased in the disc lacking of estrogen. OVX method increased the collagen I, MMP-3, MMP-13, caspase-3, and caspase-8 expression, but reduced DHI, and the content of aggrecan and collagen II, indicating an IVDD tendency. However, the ODN treatment could suppress MMP-3, MMP-13, caspase-3, and caspase-8 and protect the stability of extracellular matrix (ECM). ODN is a potential drug to delay the IVDD by suppressing apoptosis and ECM degradation.

Cathepsin-K inhibition enhances anti-cancerous activity within oral squamous cell carcinoma cells: Uncloaking the potency of new K21 formulation

BackgroundThe ability of cancer cells to be invasive and metastasize depend on several factors, of which the action of protease activity takes center stage in disease progression. Purpose/objectiveTo analyze function of new K21 molecule in the invasive process of oral squamous cell carcinoma (OSCC) cell line. Materials & methodsThe Fusobacterium (ATCC 23726) streaks were made, and pellets were resuspended in Cal27 (ATCC CRL-2095) OSCC cell line spheroid cell microplate. Cells were seeded and Lysotracker staining performed for CathepsinK red channel. Cell and morphology were evaluated using Transmission Electron microscopy. Thiobarbituric acid assay was performed. OSCC was analyzed for Mic60. Raman spectra were collected from the cancer cell line. L929 dermal fibroblast cells were used for Scratch Assay. ELISA muti arrays were used for cytokines and matrix molecules. Internalization ability of fibroblast cells were also analyzed. Structure of K21 as a surfactant molecule with best docked poses were presented. ResultsDecrease in lysosomal staining was observed after 15 and 30 min of 0.1% treatment. Tumor clusters were associated with cell membrane destruction in K21 primed cells. There was functional silencing of Mic60 via K21, especially with 1% concentration with reduced cell migration and invasiveness. Raman intensity differences were seen at 700 cm−1, 1200 cm−1 and 1600 cm−1 regions. EVs were detected within presence of fibroblast cells amongst K21 groups. Wound area and wound closure showed the progress of wound healing. ConclusionOver expression of CatK can be reduced by a newly developed targeted K21 based drug delivery system leading to reduced migration and adhesion of oral squamous cell carcinoma cells. The K21 drug formulation can have great potential for cancer therapies due to targeting and cytotoxicity effects.

Differential protein expression of osteoclastogenic factors in odontogenic cysts and tumors.

The osteolytic activity of odontogenic cysts and tumors is directly associated with their growth and aggressiveness. The influence of proteins expressed by epithelial and mesenchymal cells on this biological event differs between indolent cystic lesions, aggressive cystic lesions, and odontogenic tumors. The objective of this study was to compare the immunohistochemical expression of factors that stimulate (receptor activator of nuclear factor kappa-Β ligand - RANKL, cathepsin K - CatK and matrix metallopeptidase 8 - MMP-8) and inhibit (osteoprotegerin - OPG) osteoclastogenesis between dentigerous cyst (DC), glandular odontogenic cyst (GOC), odontogenic keratocyst (OKC), and ameloblastoma (AB). Paraffin-embedded sections of nine DCs, nine GOCs, 20 OKCs, 21 ABs, and four dental follicles (DFs) were subjected to immunohistochemistry. Immunoreactivity was analyzed semiquantitatively and quantitatively in epithelium and connective tissue, respectively. The proteins were immunoexpressed in epithelial and mesenchymal cells of all lesions studied. The expression of RANKL and CatK was higher in OKC, AB, and GOC (p<0.005). Higher expression of OPG was found in DF and DC compared to the other markers (p<0.005). MMP-8 expression was high in GOC and OKC. This study demonstrated the differential expression of factors that inhibit and stimulate bone resorption during the development of DC, GOC, OKC, and AB. Higher expression of RANKL and CatK was observed in more aggressive lesions. OPG appears to be one of the molecules responsible for the slower growth of DC.

Differential Roles of Cysteinyl Cathepsins in TGF-β Signaling and Tissue Fibrosis.

SummaryTransforming growth factor beta (TGF-β) signaling contributes to tissue fibrosis. Here we demonstrate that TGF-β enhances CatS and CatK expression but reduces CatB and CatL expression in mouse kidney tubular epithelial cells (TECs). CatS- and CatK deficiency reduces TEC nuclear membrane importer importin-β expression, Smad-2/3 activation, and extracellular matrix (ECM) production. Yet CatB- and CatL-deficiency displays the opposite observations with reduced nuclear membrane exporter RanBP3 expression. CatS and CatK form immunocomplexes with the importin-β and RanBP3 more effectively than do CatB and CatL. On the plasma membrane, CatS and CatK preferentially form immunocomplexes with and activate TGF-β receptor-2, whereas CatB and CatL form immunocomplexes with and inactivate TGF-β receptor-1. Unilateral ureteral obstruction-induced renal injury tests differential cathepsin activities in TGF-β signaling and tissue fibrosis. CatB- or CatL-deficiency exacerbates fibrosis, whereas CatS- or CatK-deficiency protects kidneys from fibrosis. These cathepsins exert different effects in the TGF-β signaling cascade independent of their proteolytic properties.

Targeting cathepsin K diminishes prostate cancer establishment and growth in murine bone

BackgroundThe processes of prostate cancer (PCa) invasion and metastasis are facilitated by proteolytic cascade involving multiple proteases, such as matrix metalloproteinases, serine proteases and cysteine proteases including cathepsin K (CatK). CatK is predominantly secreted by osteoclasts and specifically degrades collagen I leading to bone destruction. PCa and breast cancer preferentially metastasize to the bone. Importantly, CatK expression level is greater in PCa bone metastatic sites compared to primary tumor and normal prostate tissues. However, the underlying mechanism of CatK during PCa metastases into the bone remains to be elucidated. We investigated the functional role of CatK during the PCa establishment and growth process in the murine bone.MethodsCatK mRNA expression was validated by RT-PCR, protein expression by immunoblotting in PCa LNCaP, C4-2B, and PC3 cells as well as in PCa tissues. Its protein production was measured using ELISA assay. The effect of both knockdowns via siRNA and CatK inhibitor was compared in regard to PCa cell invasion. We further studied the dose-dependent CatK inhibitor effect on conditioned media-induced bone resorption. In setting up an animal model, C4-2B cells were injected into the tibiae of SCID mice. The animals treated with either vehicle or CatK inhibitor for 8 weeks at the time of tumor cell injection (tumor establishment model; protocol I) or 4 weeks after tumor cell injection (tumor progression model; protocol II) were applied to histological and histomorphometric analyses.ResultsWe confirmed CatK expression in PCa LNCaP, C4-2B, and PC3 cells as well as in PCa tissues. Furthermore, we observed the inhibitory effects of a selective CatK inhibitor on PCa cell invasion. The CatK inhibitor dose-dependently inhibited PCa-conditioned media-induced bone resorption. Upon injection of C4-2B cells into the tibiae of SCID mice, the selective CatK inhibitor significantly prevented the tumor establishment in protocol I, and reduced the tumor growth in bone in protocol II. It also decreased serum PSA levels in both animal models. The inhibitory effects of the CatK inhibitor were enhanced in combination with zoledronic acid (ZA).ConclusionThe selective CatK inhibitor may prevent the establishment and progression of PCa in bone, thus making it a novel therapeutic approach for advanced PCa.

Differential Roles of Cysteinyl Cathepsins in TGF-β Signaling and Tissue Fibrosis

TGF-β signaling, Smad activation, and extracellular matrix (ECM) production contribute to tissue fibrosis. Here we demonstrate that TGF-β enhances the expression of cysteinyl cathepsins CatS and CatK, but reduces CatB and CatL expression in mouse kidney tubular epithelial cells (TECs). TECs from Ctss-/- and Ctsk-/- mice show reduced expression of nuclear membrane importer protein importin-β, Smad-2/3 activation, and ECM production, yet those from Ctsb-/- and Ctsl-/- mice display increased importin-I² expression, Smad-2/3 activation, and ECM production, but reduced nuclear membrane exporter RanBP3 expression. CatS and CatK form immunocomplexes with the nuclear membrane importin-β and RanBP3 more effectively than do CatB and CatL. On the plasma membrane, CatS and CatK preferentially form immunocomplexes with and activate TGF-β receptor-2, while in contrast CatB and CatL form immunocomplexes with and inactivate TGF-β receptor-1. Unilateral ureteral obstruction-induced renal injury tests differential cathepsin activities in TGF-β signaling and tissue fibrosis. TECs in fibrotic tissues exhibit decreased CatB and CatL, but increased CatS and CatK activities. CatB- or CatL-deficiency exacerbates fibrosis, while CatS- or CatK-deficiency protects kidneys from fibrosis. These results shed novel mechanistic insight into how these closely related cysteinyl cathepsins can exert opposing effects in the TGF-β signaling cascade independent of their proteolytic properties. Funding: This work is supported by grants from the National Institute of Health [HL080472 to PL, HL123568 and HL60942 to GPS], and the RRM Charitable Fund to PL. Declaration of Interest: The authors have declared no conflicts of interest. Ethical Approval: All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and were approved by the Harvard Medical School Standing Committee on Animals (protocol #03759).

Cathepsin K-deficiency impairs mouse cardiac function after myocardial infarction

BackgroundExtracellular matrix metabolism and cardiac cell death participate centrally in myocardial infarction (MI). This study tested the roles of collagenolytic cathepsin K (CatK) in post-MI left ventricular remodeling. Methods and resultsPatients with acute MI had higher plasma CatK levels (20.49 ± 7.07 pmol/L, n = 26) than those in subjects with stable angina pectoris (8.34 ± 1.66 pmol/L, n = 28, P = .01) or those without coronary heart disease (6.63 ± 0.84 pmol/L, n = 93, P = .01). CatK protein expression increases in mouse hearts at 7 and 28 days post-MI. Immunofluorescent staining localized CatK expression in cardiomyocytes, endothelial cells, fibroblasts, macrophages, and CD4+ T cells in infarcted mouse hearts at 7 days post-MI. To probe the direct participation of CatK in MI, we produced experimental MI in CatK-deficient mice (Ctsk−/−) and their wild-type (Ctsk+/+) littermates. CatK-deficiency yielded worsened cardiac function at 7 and 28 days post-MI, compared to Ctsk+/+ littermates (fractional shortening percentage: 5.01 ± 0.68 vs. 8.62 ± 1.04, P < .01, 7 days post-MI; 4.32 ± 0.52 vs. 7.60 ± 0.82, P < .01, 28 days post-MI). At 7 days post-MI, hearts from Ctsk−/− mice contained less CatK-specific type-I collagen fragments (10.37 ± 1.91 vs. 4.60 ± 0.49 ng/mg tissue extract, P = .003) and more fibrosis (1.67 ± 0.93 vs. 0.69 ± 0.20 type-III collagen positive area percentage, P = .01; 14.25 ± 4.12 vs. 6.59 ± 0.79 α-smooth muscle actin-positive area percentage, P = .016; and 0.82 ± 0.06 vs. 0.31 ± 0.08 CD90-positive area percentage, P = .008) than those of Ctsk+/+ mice. Immunostaining demonstrated that CatK-deficiency yielded elevated cardiac cell death but reduced cardiac cell proliferation. In vitro studies supported a role of CatK in cardiomyocyte survival. ConclusionPlasma CatK levels are increased in MI patients. Heart CatK expression is also elevated post-MI, but CatK-deficiency impairs post-MI cardiac function in mice by increasing myocardial fibrosis and cardiomyocyte death.

Disheveled 2 around the implant at an early stage of hyperlipidemic rats

This study aims to investigate the expression of disheveled 2 (Dvl2) around the implant of hyperlipidemic rats at an early stage after the implantation. A total of 24 Wistar rats were divided equally into the experimental group fed with high-fat diet group and control group fed with a normal diet. After 8 weeks, the serum lipid levels were detected, and rats received implants in the femur metaphysis. Rats were sacrificed at 1, 3, and 5 days after implantation, and the bones around implants were obtained. Methylene blue-acid fuchsin staining was performed to observe the implant-bone interface. Real-time polymerase chain reaction was performed on runt-related transcription factor 2 (Runx2), cathepsin K (CatK), and Dvl2. Dvl2 Western blot or immunoprecipitation, phosphorylation, and ubiquitination were also conducted. In the experimental group, less osteoblasts, lower expression of Runx2 and Dvl2, and lower Dvl2 phosphorylation (P<0.05) than those of the control group were observed; furthermore, the CatK expression and Dvl2 ubiquitination were higher than those in the control group (P<0.05). Hyperlipidemia may suppress bone remodeling around the implant at an early stage by Dvl2 down-regulation, phosphorylation, and up-regulated ubiquitination.

Human acellular amniotic membrane: A potential osteoinductive biomaterial for bone regeneration.

Human acellular amniotic membrane is an acellular, naturally extracellular matrix material with various bioactive factors, which applied in tissue engineering in clinic. Several studies have applied human acellular amniotic membrane in skin and ocular surface tissue engineering to enhance tissue regeneration. However, the application of human acellular amniotic membrane in bone tissue engineering was rarely investigated. The aim of the current study was to investigate the osteoinductivity, angiogenesis and the early molecular changes of human acellular amniotic membrane to bone regeneration. Our results showed that human acellular amniotic membrane with excellent biocompatibility was beneficial for bone marrow mesenchymal stem cells proliferation and osteogenic differentiation. In rat femoral defect model, the existence of human acellular amniotic membrane significantly improved bone regeneration in the defects. The gene expression of CXCR-4, MCP-1, OC and CatK which were connected with cells recruitment and bone remodeling, was enhanced in the defects implanted with human acellular amniotic membrane. The results of this study suggest that human acellular amniotic membrane is an osteoinductive biomaterial for bone regeneration.

Cathepsin K Deficiency Ameliorates Systemic Lupus Erythematosus-like Manifestations in Faslpr Mice.

Cysteinyl cathepsin K (CatK) is expressed in osteoclasts to mediate bone resorption, but is also inducible under inflammatory conditions. Faslpr mice on a C57BL/6 background develop spontaneous systemic lupus erythematosus-like manifestations. Although normal mouse kidneys expressed negligible CatK, those from Faslpr mice showed elevated CatK expression in the glomeruli and tubulointerstitial space. Faslpr mice also showed elevated serum CatK levels. CatK deficiency in Faslpr mice reduced all tested kidney pathologies, including glomerulus and tubulointerstitial scores, glomerulus complement C3 and IgG deposition, chemokine expression and macrophage infiltration, and serum autoantibodies. CatK contributed to Faslpr mouse autoimmunity and pathology in part by its activity in TLR-7 proteolytic processing and consequent regulatory T (Treg) cell biology. Elevated TLR7 expression and proteolytic processing in Faslpr mouse kidneys and Tregs showed significantly reduced levels in CatK-deficient mice, leading to increased spleen and kidney Treg content. Purified CD4+CD25highFoxp3+ Tregs from CatK-deficient mice doubled their immunosuppressive activity against T effector cells, compared with those from CatK-sufficient mice. In Faslpr mice, repopulation of purified Tregs from CatK-sufficient mice reduced spleen sizes, autoantibody titers, and glomerulus C3 and IgG deposition, and increased splenic and kidney Treg contents. Tregs from CatK-deficient mice had significantly more potency than CatK-sufficient Tregs in reducing spleen sizes, serum autoantibody titers, and glomerulus C3 deposition, and in increasing splenic and kidney Treg content. This study established a possible role of CatK in TLR7 proteolytic activation, Treg immunosuppressive activity, and lupus autoimmunity and pathology.

Increased expression and activation of cathepsin K in human osteoarthritic cartilage and synovial tissues.

Few studies have analyzed Cathepsin K (CatK) expression in human osteoarthritic tissues. We investigated CatK expression and activation in human articular cartilage using clinical specimens. Human osteoarthritic cartilage was obtained during surgery of total hip arthroplasty (n = 10), and control cartilage was from that of femoral head replacement for femoral neck fracture (n = 10). CatB, CatK, CatL, CatS, and Cystatin C (CysC) expressions were evaluated immunohistochemically and by real-time PCR. Intracellular CatK protein was quantified by ELISA. Intracellular CatK activity was also investigated. Osteoarthritis (OA) chondrocytes were strongly stained with CatK, particularly in the superficial layer and more damaged areas. CatB, CatL, CatS, and CysC were weakly stained. CatK mRNA expression was significantly higher in OA group compared to that in control group (p = 0.043), whereas those of CatB, CatL, CatS, and CysC did not differ significantly. Mean CatK concentration (4.83 pmol/g protein) in OA chondrocytes was higher than that (3.91 pmol/g protein) in control chondrocytes (p = 0.001). CatK was enzymatically more activated in OA chondrocytes as compared with control chondrocytes. This study, for the first time, revealed increased CatK expression and activation in human OA cartilage, suggesting possible crucial roles for it in the pathogenesis of osteoarthritic change in articular cartilage.

Overexpression of cystatin C in synovium does not reduce synovitis or cartilage degradation in established osteoarthritis.

IntroductionCathepsin K (catK) expression is increased in cartilage, bone and synovium during osteoarthritis (OA). To study the role of catK expression and elevated cathepsin activity in the synovium on cartilage destruction in established OA, we overexpressed cystatin C (cysC), a natural cysteine protease inhibitor, in the synovium of rabbit OA joints.MethodsThe ability of cysC to inhibit activity of cathepsins in rabbit OA synovium lysates was tested in vitro using protease activity assay. In vivo, the tissue localization of recombinant adeno-associated virus (rAAV) with LacZ gene after intra-articular injection was determined by β-galactosidase staining of rabbit joints 4 weeks later. To inhibit cathepsin activity in the synovium, a rAAV2-encoding cysC was delivered intra-articularly into rabbit joints 4 weeks after OA was induced by anterior cruciate ligament transection (ACLT). Seven weeks postinjection, endogenous catK and cysC levels as well as the vector-derived cysC expression in the synovium of normal and OA joints were examined by RNA quantification. Synovial cathepsin activity and catK, catB and catL protein levels were determined by activity and Western blot analyses, respectively. Synovitis and cartilage degradation were evaluated by histopathological scoring.ResultsIn vitro, the ability of cysC to efficiently inhibit activity of purified catK and OA-induced cathepsins in rabbit synovial lysates was demonstrated. In vivo, the intra-articular delivery of rAAV2/LacZ showed transduction of mostly synovium. Induction of OA in rabbit joints resulted in fourfold increase in catK mRNA compared to sham controls while no change was detected in endogenous cysC mRNA levels in the synovium. Protein levels for catK, catB and catL were also increased in the synovium with a concomitant fourfold increase in cathepsin activity. Joints treated with rAAV2/cysC showed both detection of vector genomes and vector-derived cysC transcripts in the synovium. Production of functional cysC by the vector was demonstrated by complete block of cathepsin activity in the synovium. However, this did not decrease synovitis, bone sclerosis or progression of cartilage degradation.ConclusionsIncreased production of natural cathepsin inhibitor, cysC, in OA synovium does not alleviate synovitis or cartilage pathology during a preexisting OA.

Efficacy of a cathepsin K inhibitor in a preclinical model for prevention and treatment of breast cancer bone metastasis.

Cathepsin K (CatK) is essential for osteoclast-mediated bone resorption. CatK expression is also detected in breast cancer cells that metastasize to bone. Here, the CatK inhibitor L-235 dosed in prevention (10, 30, and 100 mg/kg, p.o., b.i.d.) or treatment regimen (30 mg/kg) was compared with the bisphosphonate zoledronic acid (ZOL, 7.5 μg/kg/wk, s.c.) in the intratibial injection model of MDA-MB-231 breast carcinoma in nude rats. Progression of osteolysis, skeletal tumor burden, and local metastasis was evaluated by radiography through 42 days and ex vivo μCT and histology. IHC and RT-PCR confirmed the increases in CatK protein and mRNA levels in human breast cancer primary and metastatic tumors. In the experimental model of breast cancer bone metastasis, L-235 dosed in preventive mode resulted in a dose-related reduction of osteolysis of 72%, 75%, and 87% respectively, compared with ZOL by 86% versus intact. Similarly, L-235 significantly reduced intratibial tumor volume by 29%, 40%, and 63%, respectively, compared with 56% by ZOL versus vehicle. Efficacy of L-235 and ZOL on reduction of osteolytic lesions and tumor burden was comparable in treatment versus preventive regimens. All L-235 doses inhibited cortical disruption and extraskeletal tumor growth to a level comparable with ZOL. Assessment of local metastasis demonstrated that treatment with the CatK inhibitor was more effective than ZOL in reducing breast cancer invasion. These data support the role of CatK in breast cancer skeletal growth and metastasis and CatK inhibitors may represent a novel oral therapy for treatment of metastatic breast cancer.

Ultraviolet A-induced cathepsin K expression is mediated via MAPK/AP-1 pathway in human dermal fibroblasts.

BackgroundCathepsin K (CatK), a cysteine protease with the potent elastolytic activity, plays a predominant role in intracellular elastin degradation in human dermal fibroblasts (HDFs), and contributes to solar elastosis. In previous studies, CatK expression was downregulated in photoaged skin and fibroblasts, but upregulated in acute UVA-irradiated skin and fibroblasts. The underlying mechanisms regulating UVA-induced CatK expression remain elusive.ObjectiveThis study investigates mechanisms involved in the regulation of UVA-induced CatK expression in HDFs.MethodsPrimary HDFs were exposed to UVA. Cell proliferation was analyzed using a colorimetric assay of relative cell number. Quantitative real-time RT-PCR and Western blot were performed to detect CatK expression in HDFs on three consecutive days after 10 J/cm2 UVA irradiation, or cells treated with increasing UVA doses. UVA-activated MAPK/AP-1 pathway was examined by Western blot. Effects of inhibition of MAPK pathway and knockdown of Jun and Fos on UVA-induced CatK expression were also measured by real-time RT-PCR and Western blot.ResultsUVA significantly increased CatK mRNA and protein expression in a dose-dependent manner. UVA-induced CatK expression occurred along with UVA-activated phosphorylation of JNK, p38 and Jun, UVA-increased expression of Fos. Inactivation of JNK and p38MAPK pathways both remarkably decreased UVA-induced CatK expression, which was suppressed more by inhibition of JNK pathway. Furthermore, knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatK expression.ConclusionUVA is capable of increasing CatK expression in HDFs, most likely by activation of MAPK pathway and of AP-1, which has been shown to be the case for matrix metalloproteinases. As current strategies for selecting anti-photoaging agents focus on their ability to decrease MMPs' expression through inhibiting UV- activated MAPK pathway, future strategies should also consider their effect on CatK expression.

Cathepsin K-mediated Notch1 activation contributes to neovascularization in response to hypoxia.

Cysteine proteases play important roles in pathobiology. Here we reveal that cathepsin K (CatK) has a role in ischaemia-induced neovascularization. Femoral artery ligation-induced ischaemia in mice increases CatK expression and activity, and CatK-deficient mice show impaired functional recovery following hindlimb ischaemia. CatK deficiency reduces the levels of cleaved Notch1 (c-Notch1), Hes1 Hey1, Hey2, vascular endothelial growth factor, Flt-1 and phospho-Akt proteins of the ischaemic muscles. In endothelial cells, silencing of CatK mimicked, whereas CatK overexpression enhanced, the levels of c-Notch1 and the expression of Notch downstream signalling molecules, suggesting CatK contributes to Notch1 processing and activates downstream signalling. Moreover, CatK knockdown leads to defective endothelial cell invasion, proliferation and tube formation, and CatK deficiency is associated with decreased circulating endothelial progenitor cells-like CD31(+)/c-Kit(+) cells in mice following hindlimb ischaemia. Transplantation of bone marrow-derived mononuclear cells from CatK(+/+) mice restores the impairment of neovascularization in CatK(-/-) mice. We conclude that CatK may be a potential therapeutic target for ischaemic disease.

Cathepsin K overexpression modifies lung development in newborn mice

Cathepsin K (CatK), contributes to the development of chronic lung disease in newborn infants, but the impact of CatK for the lungs may be multifaceted. We have previously demonstrated that low level of CatK is associated with newborn lung injury and CatK deficiency aggravates lung injury in hyperoxia-exposed newborn mice. Thus, we hypothesized that sustained/higher expression of CatK could ameliorate hyperoxia-induced injury and restrain the development of pulmonary fibrosis. We studied the lungs of newborn wild-type (WT) and CatK overexpressing transgenic mice (TG) that were exposed to hyperoxia or room air for 7 or 14 days after birth. Fourfold pulmonary overexpression of CatK did not affect the growth or lung weight in room-air bred TG mice. The distal airspaces in TG mice were, however, enlarged on postnatal days (PN) 7 and 14, the latter together with increased apoptosis, compared with WT controls. Survival rate was normal and no respiratory distress was observed in air-bred TG mice. Hyperoxia inhibited alveolarization and increased collagen accumulation in WT mice. In TG mice, hyperoxia for 1 week did not aggravate the lung injury, and the lung morphology and already enlarged alveoli remained unchanged in TG mice at PN7. Prolonged hyperoxic exposure caused significant lung injury and mortality similarly in both group of mice, and only few mice survived until PN14. In summary, CatK overexpression slightly enlarges distal airways in infant mice, but hyperoxic environment is initially better tolerated when compared to WT mice. These findings suggest multifaceted role for CatK in lung development and newborn lung injury.

Cathepsin K Activity Controls Injury-Related Vascular Repair in Mice

Cathepsin K (CatK) is one of the most potent mammalian collagenases. We showed previously the increased expression of CatK in human and animal atherosclerotic lesions. Here, we hypothesized that ablation of CatK mitigates injury-induced neointimal hyperplasia. Male wild-type (CatK(+/+)) and CatK-deficient (CatK(-/-)) mice underwent ligation or a combination of ligation and polyethylene cuff-replacement injuries to the right common carotid artery just proximal to its bifurcation, and they were then processed for morphological and biochemical studies at specific time points. On operative day 28, CatK(-/-) significantly reduced neointimal formation and neovessel formation in both single- and combination-injured arteries compared with the Cat K(+/+) mice. At early time points, CatK(-/-) reduced the lesion macrophage contents and medial smooth muscle cell proliferation, the mRNA levels of monocyte chemoattractant protein-1, toll-like receptor-2, toll-like receptor-4, chemokine ligand-12, and the gelatinolytic activity related to matrix metalloproteinase-2/-9. An aorta-explant assay revealed that smooth muscle cell movement was impaired in the CatK(-/-) mice compared with the CatK(+/+) mice. In addition, the smooth muscle cells and macrophages from CatK(-/-) mice had less invasive ability through a reconstituted basement membrane barrier. This vasculoprotective effect was mimicked by Cat inhibition with trans-epoxysuccinyl-L-leucylamido-{4-guanidino} butane (E64d). These results demonstrate an essential role of CatK in neointimal lesion formation in response to injury, possibly via the reduction of toll-like receptor-2/-4-mediated inflammation and smooth muscle cell proliferation, suggesting a novel therapeutic strategy for the control of endovascular treatment-related restenosis by regulating CatK activity.

Abstract 9629: Cathepsin K-Mediated Noich1 Activation Contributes to Neovascularization in Response to Hypoxia

Background: Cysteine proteases play important roles in human pathobiology. This report shows the participation of cathepsin K (CatK) in ischemia-induced neovascularisation. Methods and Results: Left femoral artery ligation-induced ischemia in mice showed increased CatK expression and activity. CatK-deficient young and aged mice showed impaired functional recovery following hind ischemia. These mice also showed reduced levels of cleaved Notch1 (c-Notch1), vascular endothelial growth factor (VEGF), Flt-1 and phospho-Akt proteins of the ischemic muscles. In vitro , CatK knockdown reduced the levels of c-Notch1, VEGF and Flt1, essential molecules for angiogenesis and vasculogenesis. Together, the effects of CatK knockdown led to defective endothelial cell invasion, proliferation and tube formation. Recombinant human CatK degrades gama-secretase substrate in time- and dose dependent manners, suggesting CatK is involved in Notch1 activation. This was reinforced by the finding that CatK inhibition impairs Notch1 activation in response to hypoxia with no change in known gama-secretase activity.CatK-deficiency decreased circulating EPC-like CD31+/c-Kit+cells in mice of both ages. Transplantation of bone-marrow-derived mononuclear cells from CatK+/+ mice restored neovascularisation in CatK -/- mice. Conclusions: CatK activity controls neovascularisation via partial modulation of Notch1 signaling activation in response to hypoxia. Thus, CatK has a critical role in vascular formation and represents a new therapeutic target for vascular disease.

Mechanisms With Clinical Implications for Atrial Fibrillation–Associated Remodeling: Cathepsin K Expression, Regulation, and Therapeutic Target and Biomarker

BackgroundThe cysteine protease cathepsin K (CatK) has been implicated in the pathogenesis of cardiovascular disease. We sought to determine the link between atrial fibrillation (AF) and plasma CatK levels and to investigate the expression of and therapeutic target for CatK in vivo and in vitro.Methods and ResultsPlasma CatK and extracellular matrix protein peptides (intact procollagen type I of N‐terminal propeptide; carboxyl‐terminal telopeptide of type I collagen [ICTP]) were measured in 209 consecutive patients with AF (paroxysmal AF, 146; persistent AF, 63) and 112 control subjects. In addition, the regulation of CatK expression was investigated in vivo and vitro. Patients with AF had higher plasma CatK and ICTP levels than did control subjects. Patients with persistent AF had higher levels of plasma CatK and ICTP than did patients with paroxysmal AF. CatK was correlated with ICTP concentration and left atrial diameter in all subjects. In rabbits, superoxide production, CatK activity, fibrosis, and the levels of atrial tissue angiotensin II, angiotensin type 1 receptor, gp91phox, phospho‐p38 mitogen‐activated protein kinase, and CatK were greater in those with tachypacing‐induced AF than in controls, and these changes were reversed with angiotensin type 1 receptor antagonist. Olmesartan and mitogen‐activated protein kinase inhibitor decreased the CatK expression induced by angiotensin II in rat neonatal myocytes.ConclusionsThese data indicated that increased plasma CatK levels are linked with the presence of AF. Angiotensin type 1 receptor antagonist appears to be effective in alleviating atrial fibrosis in a rabbit AF model, partly reducing angiotensin type 1 receptor‐p38mitogen‐activated protein kinase‐dependent and ‐independent CatK activation, thus preventing AF.