chondromodulin-I Research Articles

T Cells Directed against the Metastatic Driver Chondromodulin-1 in Ewing Sarcoma: Comparative Engineering with CRISPR/Cas9 vs. Retroviral Gene Transfer for Adoptive Transfer.

Ewing sarcoma (EwS) is a highly malignant sarcoma of bone and soft tissue with early metastatic spread and an age peak in early puberty. The prognosis in advanced stages is still dismal, and the long-term effects of established therapies are severe. Efficacious targeted therapies are urgently needed. Our previous work has provided preliminary safety and efficacy data utilizing T cell receptor (TCR) transgenic T cells, generated by retroviral gene transfer, targeting HLA-restricted peptides on the tumor cell derived from metastatic drivers. Here, we compared T cells engineered with either CRISPR/Cas9 or retroviral gene transfer. Firstly, we confirmed the feasibility of the orthotopic replacement of the endogenous TCR by CRISPR/Cas9 with a TCR targeting our canonical metastatic driver chondromodulin-1 (CHM1). CRISPR/Cas9-engineered T cell products specifically recognized and killed HLA-A*02:01+ EwS cell lines. The efficiency of retroviral transduction was higher compared to CRISPR/Cas9 gene editing. Both engineered T cell products specifically recognized tumor cells and elicited cytotoxicity, with CRISPR/Cas9 engineered T cells providing prolonged cytotoxic activity. In conclusion, T cells engineered with CRISPR/Cas9 could be feasible for immunotherapy of EwS and may have the advantage of more prolonged cytotoxic activity, as compared to T cells engineered with retroviral gene transfer.

Identification of marker genes to monitor residual iPSCs in iPSC-derived products

BackgroundEngineered tissues and cell therapies based on human induced pluripotent stem cells (iPSCs) represent a promising approach for novel medicines. However, iPSC-derived cells and tissues may contain residual undifferentiated iPSCs that could lead to teratoma formation after implantation into patients. As a consequence, highly sensitive and specific methods for detecting residual undifferentiated iPSCs are indispensable for safety evaluations of iPSC-based therapies. The present study provides an approach for identifying potential marker genes for iPSC impurities in iPSC-derived cells using RNA sequencing data from iPSCs and various differentiated cell types. MethodsIdentifying iPSC marker genes for each cell type individually provided a larger and more specific set of potential marker genes than considering all cell types in the analysis. Thus, the authors focused on identifying markers for iPSC impurities in iPSC-derived cardiomyocytes (iCMs) and validated the selected genes by reverse transcription quantitative polymerase chain reaction. The sensitivity of the candidate genes was determined by spiking different amounts of iPSCs into iCMs and their performance was compared with the previously suggested marker lin-28 homolog A (LIN28A). ResultsEmbryonic stem cell-related gene (ESRG), long intergenic non-protein coding RNA 678 (LINC00678), CaM kinase-like vesicle-associated (CAMKV), indoleamine 2,3-dioxygenase 1 (IDO1), chondromodulin (CNMD), LINE1-type transposase domain containing 1 (L1DT1), LIN28A, lymphocyte-specific protein tyrosine kinase (LCK), vertebrae development-associated (VRTN) and zinc finger and SCAN domain containing 10 (ZSCAN10) detected contaminant iPSCs among iCMs with a limit of detection that ranged from 0.001% to 0.1% depending on the gene and iCM batch used. ConclusionsUsing the example of iCMs, the authors provide a strategy for identifying a set of highly specific and sensitive markers that can be used for quality assessment of iPSC-derived products.

Robust detection of undifferentiated iPSC among differentiated cells

Recent progress in human induced pluripotent stem cells (iPSC) technologies suggest that iPSC application in regenerative medicine is a closer reality. Numerous challenges prevent iPSC application in the development of numerous tissues and for the treatment of various diseases. A key concern in therapeutic applications is the safety of the cell products to be transplanted into patients. Here, we present novel method for detecting residual undifferentiated iPSCs amongst directed differentiated cells of all three germ lineages. Marker genes, which are expressed specifically and highly in undifferentiated iPSC, were selected from single cell RNA sequence data to perform robust and sensitive detection of residual undifferentiated cells in differentiated cell products. ESRG (Embryonic Stem Cell Related), CNMD (Chondromodulin), and SFRP2 (Secreted Frizzled Related Protein 2) were well-correlated with the actual amounts of residual undifferentiated cells and could be used to detect residual cells in a highly sensitive manner using qPCR. In addition, such markers could be used to detect residual undifferentiated cells from various differentiated cells, including hepatic cells and pancreatic cells for the endodermal lineage, endothelial cells and mesenchymal cells for the mesodermal lineage, and neural cells for the ectodermal lineage. Our method facilitates robust validation and could enhance the safety of the cell products through the exclusion of undifferentiated iPSC.

Dual Roles of Ascidian Chondromodulin-1: Promoting Cell Proliferation Whilst Suppressing the Growth of Tumor Cells.

Chondromodulin-1 (ChM-1) is an extracellular matrix protein that plays crucial roles in tumor cell growth and angiogenesis in vertebrates and humans. ChM-1 is highly expressed in the invertebrate Ciona savignyi, a marine ascidian chosen as a model. The effect of the recombinant Ciona mature ChM-1 peptide (Cs-mChM-1) on cell proliferation, migration and angiogenesis was evaluated on cultured cells. The results revealed that low concentrations of Cs-mChM-1 (12.5 nM) promoted osteoblastic cell (MC3T3-E1) growth and protected cells from H2O2-induced damage. However, a higher concentration of Cs-mChM-1 (i.e., 500 nM) not only suppressed both growth and migration of tumor cells, including human cervical cancer (HeLa) cells and human neuroblastoma (SH-SY5Y) cells, but also significantly inhibited proliferation and angiogenesis of human umbilical vein endothelial cells (HUVECs). The expression levels of cyclinD1 and mitogen-activated protein kinase 1 (MAPK1) were slightly increased in Cs-mChM-1 treated MC3T3-E1 cells, whereas these genes decreased in treated HeLa cells, SH-SY5Y cells and HUVECs. This result indicates that Cs-mChM-1 modifies cell behavior by regulating cell cycle and cell adhesion. Thus, the present results reveal that recombinant peptides of ChM-1 from invertebrates can play a dual role in cell proliferation and migration of different cell types. The inhibition effects on tumor cell growth and angiogenesis indicate potential pharmaceutical applications for recombinant Cs-mChM-1.

Abstract B03: The endochondral bone protein CHM1 sustains an undifferentiated, invasive phenotype promoting lung metastasis in Ewing sarcoma

Abstract Background: Ewing sarcoma (ES), an osteogenic malignancy that mainly affects children and young adults, is characterized by early metastasis to lung and bone. In the clinical setting, prognosis for patients with metastatic ES at diagnosis is clearly worse than for those without metastases (5-year survival > 30%). Hence, there is an urgent need to understand the fundamental molecular mechanisms of ES differentiation, invasion, and metastasis to possibly identify novel therapeutic strategies to prevent metastasis. The purpose of this study was to shed further light into the function of Chondromodulin 1 (CHM1) on ES pathogenesis, especially on metastasis, and at best to establish new therapeutic targets. Material and Methods: Expression of CHM1 was analyzed using microarrays and its function was examined by RNA interference (RNAi). To analyze resulting changes qRT-PCR, ELISA, FACS, IHC, proliferation and invasion assays, as well as a xeno-transplant model in immune deficient mice were applied. Results: In this study, we investigated the role of the BRICHOS chaperon domain containing endochondral bone protein chondromodulin I (CHM1) in ES pathogenesis. CHM1 is significantly overexpressed in ES and ChIP data demonstrate CHM1 to be directly bound by EWS-FLI1. Using RNA interference we demonstrate that CHM1 enhanced contact-dependent as well as independent proliferation and the invasive potential of ES cells in vitro. This invasiveness was in part mediated via CHM1-regulated MMP9 expression. In a xenograft mouse model CHM1 was essential for the establishment of lung metastases, which is in line with the observed increased CHM1 expression in patient specimens with ES lung metastases. Mechanistically, CHM1 promoted chondrogenic differentiation capacity of ES cells but suppressed endothelial differentiation. Further, CHM1 suppressed the number of TRAP+ osteoclasts in an orthotopic model of tumor growth in line with suppression of osteolytic genes such as HIF1A, IL6, JAG1, and VEGF, indicating that CHM1-blocked osteomimicry might play a role in homing, colonization, and invasion into bone tissues. Conclusions: Our results suggest that CHM1 is an important player suppressing endothelial differentiation capacity and seems essential for the invasive and metastatic capacities of ES. Citation Format: Kristina von Heyking, Julia Calzada-Wack, Stefanie Göllner, Oxana Schmidt, Tim Hensel, David Schirmer, Annette Fasan, Carsten Müller-Tidow, Poul Sorensen, Stefan Burdach, Günther H.S. Richter. The endochondral bone protein CHM1 sustains an undifferentiated, invasive phenotype promoting lung metastasis in Ewing sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B03.

Chondromodulin‑I suppresses tumorigenesis of human osteosarcoma cells.

Osteosarcoma is the most common type of bone cancer, and accounts for ~3% of cancers that occurring in children. Chondromodulin‑I (ChM-I) is a 25kDa glycoprotein that is expressed mainly in cartilage. ChM-I demonstrates anti‑angiogenic activity and has been suggested to inhibit endothelial cells from invading cartilage, and then has been shown to be an inhibitor of tumorigenesis. However, it remains unclear if ChM‑I has any direct anti‑tumorigenesis role on osteosarcoma. Therefore, the present study aimed to identify whether ChM‑I has any direct inhibit effect on human osteosarcoma cells. A bromodeoxyuridine incorporation assay was performed on the Saos‑2 human osteosarcoma cell line treated with or without recombinant human ChM‑I, to evaluate its impact on DNA synthesis. An adenovirus carrier for the expression of ChM‑I was constructed and transfected into tumor cells invitro to evaluate the effect of ChM‑I on tumor cells. Additionally, ChM‑I was knocked down by using small interfering RNA to downregulate the expression of ChM‑I. Cell invasion, migration and cell‑colony formation assays, and xenograft tumor experiments were performed to evaluate the effects of ChM‑I on tumor cells invitro and invivo. The results demonstrated that ChM‑I could suppress DNA synthesis of human osteosarcoma cells, and it also exerted an inhibitory effect on the proliferation and colony formation abilities of human osteosarcoma cells. In addition, ChM‑I inhibited cell invasion and migration invitro and suppressed osteosarcoma cell growth significantly invivo. In conclusion, ChM‑I directly suppressed the proliferation and growth of osteosarcoma cells in an anchorage‑independent manner, and may therefore be a promising drug for the treatment of osteosarcoma.

Proteomic analysis of chondromodulin-I-induced differentiation of mesenchymal stem cells into chondrocytes

To identify novel proteins that might help clarify the molecular mechanisms underlying chondromodulin-I (ChM-I) induction of mesenchymal stem cells (MSCs) differentiate into chondrocytes. MSCs are triggered to differentiate into chondrocytes, which are recognized as important factors in cartilage tissue engineering. ChM-I is a glycoprotein that stimulates the growth of chondrocytes and inhibits angiogenesis in vitro. In this study, the proteomic approach was used to evaluate protein changes between undifferentiated MSCs and ChM-I-transfected MSCs. The expression of the protein spots was analyzed using two-dimensional gel electrophoresis. Then, 14 protein spots were identified between MSCs and ChM-I-transfected MSCs. 309 proteins were identified using mass spectrometry (MS). The differentially regulated proteins were categorized and annotated using Protein Analysis Through Evolutionary Relationships (PANTHER) analysis with the aid of the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool. These proteins are included in a variety of metabolic pathways and signal transduction pathways, such as focal adhesion, glycolysis, actin cytoskeleton regulation, and ribosome. These results demonstrate novel information about the molecular mechanism by which ChM-I induce MSCs to differentiate into chondrocytes. These results also provide a solid foundation for the development of tissue-engineered cartilage.

Chondromodulin‑I expression and correlation with angiogenesis in human osteoarthritic cartilage.

The present study aimed to evaluate the expression and localization of chondromodulin‑I (ChM‑I) in human osteoarthritic cartilage and its correlation with vascular invasion during osteoarthritis (OA) progression. Osteochondral specimens were collected from patients with OA, as well as from young and aged donors without joint diseases. The grade and the number of vascular channels terminating in non‑calcified cartilage of these collected specimens were assessed by Safranin‑O/Fast green staining. ChM‑I expression in articular cartilage was examined by immunohistochemistry, western blotting and reverse transcription‑quantitative polymerase chain reaction analyses. ChM‑I protein and mRNA levels in articular cartilage appeared to be consistent between the normal young and aged groups (P>0.05). In mildly degene-rated cartilage, ChM‑I expression decreased in the extracellular matrix (ECM) of the superficial zone and in the cytoplasm of the superficial and middle zone compared with normal cartilage (P<0.05). In moderately degenerated cartilage, ChM‑I protein expression was reduced in the ECM of all zones of articular cartilage, but the immunostaining intensity in the cytoplasm was increased. In severely degenerated cartilage, ChM‑I expression was detected primarily in the cytoplasm of the cluster‑forming chondrocytes. The density of vascular channels was correlated with the ChM‑I expression levels in cartilage ECM. ChM‑I expression was reduced in OA cartilage matrix, compared with normal cartilage (both young and aged), and correlated with angiogenesis, indicating that loss of ChM‑I may promote angiogenesis in OA cartilage. Expression of ChM‑I protein in the cytoplasm was decreased in mildly degenerated cartilage, whereas ChM‑I expression increased in moderately degenerated cartilage accompanied by chondrocyte proliferation. These findings suggested that attenuation of ChM‑I in the cartilage ECM may be due to decreased expression of ChM‑I in cytoplasm of early stage OA and increased depletion of ChM‑I in the ECM of advanced stage OA.

Lysosome-associated membrane glycoprotein 1 predicts fratricide amongst T cell receptor transgenic CD8+ T cells directed against tumor-associated antigens.

AimAutologous as well as allogeneic CD8+ T cells transduced with tumor antigen specific T cell receptors (TCR) may cause significant tumor lysis upon adoptive transfer. Besides unpredictable life-threatening off-target effects, these TCRs may unexpectedly commit fratricide. We hypothesized lysosome-associated membrane glycoprotein 1 (LAMP1, CD107a) to be a marker for fratricide in TCR transgenic CD8+ T cells.MethodsWe identified HLA-A*02:01/peptide-restricted T cells directed against ADRB3295. After TCR identification, we generated HLA-A*02:01/peptide restricted TCR transgenic T cells by retroviral transduction and tested T cell expansion rates as well as A*02:01/peptide recognition and ES killing in ELISpot and xCELLigence assays. Expansion arrest was analyzed via Annexin and CD107a staining. Results were compared to CHM1319-TCR transgenic T cells.ResultsBeta-3-adrenergic receptor (ADRB3) as well as chondromodulin-1 (CHM1) are over-expressed in Ewing Sarcoma (ES) but not on T cells. TCR transgenic T cells demonstrated HLA-A*02:01/ADRB3295 mediated ES recognition and killing in ELISpot and xCELLigence assays. 24h after TCR transduction, CD107a expression correlated with low expansion rates due to apoptosis of ADRB3 specific T cells in contrast to CHM1 specific transgenic T cells. Amino-acid exchange scans clearly indicated the cross-reactive potential of HLA-A*02:01/ADRB3295- and HLA-A*02:01/CHM1319-TCR transgenic T cells. Comparison of peptide motive binding affinities revealed extended fratricide among ADRB3295 specific TCR transgenic T cells in contrast to CHM1319.ConclusionAmino-acid exchange scans alone predict TCR cross-reactivity with little specificity and thus require additional assessment of potentially cross-reactive HLA-A*02:01 binding candidates. CD107a positivity is a marker for fratricide of CD8+ TCR transgenic T cells.

Chondromodulin-1 ameliorates osteoarthritis progression by inhibiting HIF-2α activity

Hypoxia is known to stabilize hypoxia-inducible factor (HIF) and initiate angiogenic signaling cascade. However, cartilage living in hypoxia environment can maintain avascularity. It is well known that abrogation of avascularity is related to cartilage degradation in osteoarthritis (OA). The aims of present study were to investigate the role of chondromodulin-1 (ChM-1), an endogenously anti-angiogenic protein in cartilage, during chondrocyte maturation and OA progression, as well as to explore the molecular mechanisms underlying the function of ChM-1 with a focus on HIF-2α pathway. Angiogenic-related markers were evaluated in OA cartilage and different stages of chondrocyte differentiation. Chondrocytes transfected with ChM-1 lentivirus or siRNA was treated with tumor necrosis factor (TNF-α) to investigate the role of ChM-1 in chondrocyte hypertrophic changes. Invivo study was conducted by using a surgical induced OA rat model with intra-articular injection of lentivirus ChM-1 (LV-ChM-1) or mock lentivirus (LV-GFP) control. Transcriptional activity of HIF-2α was determined by chromatin immunoprecipitation (ChIP) assay to unveil the mechanisms of ChM-1. Majority angiogenic factors increased in severe OA cartilage, while anti-angiogenic factors including ChM-1 decreased. ChM-1 expression was strongly related with chondrocyte differentiation and chondrogenesis invitro. ChM-1 overexpression protected chondrocytes from TNF-α induced hypertrophy, and intra-articular injection of LV-ChM-1 delayed OA progression. ChM-1 delayed HIF-2α nuclear translocation at early time-points and decreased transcriptional activity of HIF-2α on collagen type Х α1 (COL10A1), vascular endothelial growth factor A (VEGFA) and matrix metallopeptidase-13 (MMP-13). ChM-1 maintains cartilage homeostasis by inhibiting HIF-2α induced catabolic activity and regulation of ChM-1 in cartilage may be a promising therapeutic strategy for OA.

Transfection of chondromodulin I into human breast cancer cells and its effect on the inhibition of cancer cell growth.

Breast cancer affects one in every eight women, and has been associated with higher rates of female mortality than any other cancer type, with the exception of lung cancer. It has been reported that chondromodulinI (ChM-I) was able to suppress tumor angiogenesis and growth invivo. In order to investigate the antitumor action of ChM‑I on human breast cancer cells, a plasmid expressing ChM‑I was constructed and transfected into human breast cancer cells using an adenoviral vector. Reverse transcription‑polymerase chain reaction detected ChM‑I expression in human breast cancer cell lines, whereas no expression was detected in the control groups. In order to assess the effect of ChM‑I on human breast cancer cells, cell counting kit‑8 (CCK‑8) and colony formation analyses were used to detect tumor cell proliferation, and the proliferation of ChM‑I‑transfected cells was significantly reduced, as compared with the control. In addition, the mRNA expression levels of cell cycle‑associated genes in ChM‑I‑transfected cells were significantly decreased, as compared with the control, which suggested that ChM‑I transfection was able to inhibit the expression of genes associated with the cell cycle. The results of the present study indicated that ChM‑I was able to inhibit the growth of breast cancer cells; thus suggesting that ChM-I may have potential clinical applications in the treatment of breast cancer.

Pulsed ultrasound regulates angiogenesis and vascular invasion of the osteochondral junction in arthritic knee cartilage

Objective To observe the effect of low-intensity pulsed ultrasound (LIPUS) on angiogenesis and vascular invasion in the osteochondral junctions of rabbits with osteoarthritis. Methods Eighteen 2 to 2.5 kg New Zealand rabbits were randomly divided into an early control group (EC), an early osteoarthritis group (EO) and an osteoarthritis plus LIPUS treatment group (ET), each of 6. The rabbits of the EO and ET groups received anterior cruciate ligament resection at their left hind limbs, while those of the EC group were sham-operated. The ET group was exposed to LIPUS radiation four weeks later when osteoarthritis had been established, while the EO and EC groups were exposed to fake LIPUS radiation. After four weeks of treatment the rabbits were sacrificed and pathological changes in the articular surface of the femoral condyle were assessed using Safranin O-fast green cartilage staining. Western blotting was used to examine the expression of integrin β1, p-FAK, p-JNK, p-p38, VEGF and chondromodulin-1(ChM-1). Results Compared with the EC group, significantly more angiogenesis and vascular invasion was observed in the osteochondral junctions of the EO and ET groups, with more vessels at the osteochondral junction and marked disorder of the tidal line broken up by vessels. The expression of integrin β1, p-FAK, VEGF, p-p38 and p-JNK was significantly higher in the EO group than in the EC group, while ChM-1 expression was significantly lower. Compared with the EO group, the expression of integrin β1, p-FAK and ChM-1 in the ET group was significantly higher, while that of VEGF, p38 and p-JNK was significantly lower. Conclusions LIPUS inhibited angiogenesis and vascular invasion in the osteochondral junctions of the osteoarthritic rabbits through activating an integrin β1-FAK-p38/JNK-VEGF/ChM-1 pathway. Key words: Osteoarthritis; Angiogenesis; Vascular endothelial growth factor; Ultrasound; Integrin β1

IL-1β-regulating angiogenic factors expression in perforated temporomandibular disk cells via NF-κB pathway.

A high density of blood vessels is observed in the perforated disks of temporomandibular joint (TMJ), but the underlying mechanism is unknown. This study aimed to explore the regulation of disk angiogenesis in the perforated disks. Expressions of vascular endothelial growth factor (VEGF), angiogenin-1 (Ang-1), chondromodulin-1 (ChM-1), and thrombospondins-1 (TSP-1) were compared between healthy and perforated TMJ disk cells with or without interleukin-1β (IL-1β) incubation. The tube formation, cell migration, and expressions of matrix-metalloproteinases (MMPs) in human umbilical vein endothelial cell line (HUV-EC-C) were investigated in conditional media of disk cells. Western blot was performed to determine protein level of VEGF, Ang-1, ChM-1 and TSP-1 in IL-1β-induced disk cells cultured by NF-κB- or P38-specific pathway inhibitors, respectively. Conditional media from perforated disk cells induced more tube formation, cell migration, and MMPs' expression in HUV-EC-C. Expressions of VEGF and Ang-1 were significantly higher, and ChM-1 and TSP-1 were lower in perforated disks compared to healthy disks. The VEGFA concentration was 291.1 ± 36.09 pg/ml in perforated disk cell conditioned media, markedly larger than that in NDCCM (144.9 ± 33.69 pg/ml). IL-1β induced VEGF through NF-κB signaling pathway and Ang-1 through p38 MAPK pathway, while repressed expression of ChM-1 and TSP-1 was through NF-κB pathway. Blockade of each pathway markedly restrained inducing effect of cultural media on HUV-EC-C tube formation and migration. Perforated disk cells secreted more angiogenic factors which might induced via NF-κB pathway.

Chondromodulin-1 functions as a tumor suppressor in gastric adenocarcinoma.

Chondromodulin-1 (ChM1) is a cartilage-specific glycoprotein that stimulates the growth of chondrocytes and inhibits the tube formation of endothelial cells. Endogenously, ChM1 is expressed in the cartilage and is an anti-angiogenic factor. ChM1 has been reported to suppress the proliferation of multiple human tumor cells in an anchorage-independent manner. However, the role of ChM1 in carcinogenesis of gastric cancer remains unknown. By quantitative RT-PCR and western blotting we examined the expression of ChM1 in gastric cancer tissue and normal gastric tissue. In vitro we investigated the functional and mechanistic roles of ChM1 in the inhibition of gastric cancer cell aggressiveness. We observed that ChM1 expression was remarkably downregulated in gastric cancer cell lines compared with the immortal normal gastric epithelial cell line GES-1. Importantly, ChM1 was frequently downregulated in gastric cancer tissue compared with normal gastric tissue. Low ChM1 mRNA expression was associated with higher clinical stages, higher lymph node metastasis, and poorer prognosis of patients. Functional assays in vitro showed that ectopic expression of ChM1 was able to inhibit gastric tumor cell proliferation by arresting the cell cycle. Overall, our findings indicate that ChM1 is a potential tumor suppressor in gastric cancer, suggesting that it may be useful as a biomarker for the treatment and prognosis of gastric cancer.

Chondromodulin-1 ameliorates osteoarthritis progression in vitro and in vivo

Purpose: Articular cartilage is an avascular tissue by nature, in which resident chondrocytes maintain a stable phenotype that is resistant to hypertrophy and angiogenesis throughout life. Hypoxia is known to stabilize hypoxia-inducible factor-α (mainly HIF-1α and HIF-2α) and initiate an angiogenic signalling cascade. However, normal cartilage maintains avascularity under hypoxic conditions; and the abrogation of avascularity in cartilage is related to several joint diseases such as osteoarthritis (OA), indicating the important role of anti-angiogenic factors in normal cartilage homeostasis. Chondromodulin-1 (ChM-1) is an anti-angiogenic protein endogenously expressed in cartilage and has recently been shown to stabilize the chondrocyte phenotype during cartilage tissue repair via an unknown mechanism. These findings suggested that anti-angiogenic factors had a critical role in maintaining the physiological functions of chondrocytes and prevent hypertrophy. Our aims in the present study were to clarify the role of ChM-1 during chondrocyte maturation, OA development and therapy, in an animal model, as well as to explore the mechanistic pathways of ChM-1 with a focus of the regulation of the HIF pathways. Methods: ChM-1 expression during chondrocyte maturation was investigated in chondrogenic culture conditions in cells derived from cartilage and bone marrow. Lentivirus vectors carrying ChM-1 complementary DNA (LV-ChM-1) and ChM-1 siRNA were constructed to respectively over-express and down-regulate ChM-1. Human OA cartilage samples were collected and subjected to gene profiling of angiogenic and anti-angiogenic cytokines using Human Angiogenesis PCR Arrays (Qiagen, Australia). Tumor necrosis factor alpha (TNFα) was applied to mimic the inflammatory environment in OA. Articular cartilage cells (ACCs) over-expressing or under-expressing ChM-1 were exposed to TNFα, and the chondrogenic and hypertrophic markers (RUNX2, COL10, MMP13, ALP and VEGF) were assayed by qRT-PCR and western blot. To evaluate the effect of ChM-1 in OA development, an OA model was created in rats by surgical sectioning of the meniscus. For the mechanistic study, intracellular protein levels, nuclear accumulation and transcriptional activity of HIF-2α with ChM overexpression were evaluated by western blot and Chromatin-Immunoprecipitation (ChIP) assay, respectively. Results: Compared with healthy cartilage, the expression of angiogenic factors was significantly upregulated and anti-angiogenic factors were suppressed in OA cartilage (Figure 1A). ChM-1 expression was strongly correlated with chondrogenesis in cells derived from both cartilage and bone marrow. In OA cartilage, the Angiogenesis PCR Array revealed decreased gene expression in the majority of anti-angiogenic factors, whereas the gene expression of angiogenic-related cytokines increased (Figure 1B). ACCs over-expressing ChM-1 appeared to protect chondrocytes from TNFα induced hypertrophy, and was commensurate with decreased gene and protein level of MMP13, COL10 and VEGF; whereas siRNA-decreased ChM-1 levels resulted in the loss of chondrogenic marker expression and chondrocytes undergoing hypertrophy and becoming more sensitive to inflammatory cytokines. LV-ChM-1 induced over-expression of ChM-1 in the rat model resulted in delayed OA development. It was noted that ChM-1 delayed HIF-2α nuclear translocation at an early time-point and decreased the transcriptional activity of HIF-2α on VEGF and MMP13. Conclusions: These findings demonstrate that ChM-1 is a potent anti-angiogenic factor that is essential to maintain the chondrocyte phenotype and prevent these cells from undergoing hypertrophy during OA development by inhibition of the HIF-2α mediated angiogenic pathway. Thus, this study shows that anti-angiogenic factors such as ChM-1 are essential for the maintenance of chondrocyte homeostasis, prevent hypoxia induced angiogenesis in cartilage, and protect chondrocyte from inflammatory cytokines induced hypotrophy.

Chondrogenic differentiation of ChM-I gene transfected rat bone marrow-derived mesenchymal stem cells on 3-dimensional poly (L-lactic acid) scaffold for cartilage engineering.

We have explored the role of Chondromodulin-I (ChM-I) in chondrogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) in 3-dimensional (3D) scaffold for cartilage tissue engineering. BMSCs of Sprague Dawley (SD) rats were cultured on poly-(L-lactic acid) [PLLA] scaffolds with different pore sizes (80-200 μm, 200-450 μm) with or without surface modification by chitosan. Cell viability, proliferation, and morphology were measured using confocal microscope and the CCK-8 method. Untransfected BMSCs, BMSCs expressing pcDNA3.1(+), BMSCs expressing plasmid pcDNA3.1 (+)/ChM-I were cultured on 3D scaffolds in standard growth medium or transforming growth factor-β1 (TGF-β1) supplemented chondrogenic induction medium in vitro for 3 weeks and the expression of collagen type II was determined. Cell-scaffolds constructs were implanted subcutaneously for 3 months in vivo. BMSCs had a higher viability and proliferation in PLLA scaffolds of pore size 200-450 μm than that of 80-200 μm, and surface modification with chitosan did not enhance cell attachment. The ChM-I gene enhanced chondrogenesis and increased collagen type II synthesis. Immunohistochemistry from in vivo study showed enhanced cartilage regeneration in BMSCs expressing pcDNA3.1 (+)/ChM-I on 3D PLLA scaffolds. It also demonstrated that TGF-β1 might promote chondrogenesis of rat BMSCs by synergizing with the ChM-I gene. ChM-I could be beneficial to future applications in cartilage repair.

The N-Terminal Cleavage of Chondromodulin-I in Growth-Plate Cartilage at the Hypertrophic and Calcified Zones during Bone Development

Chondromodulin-I (ChM-I) is a 20–25 kDa anti-angiogenic glycoprotein in cartilage matrix. In the present study, we identified a novel 14-kDa species of ChM-I by immunoblotting, and purified it by immunoprecipitation with a newly raised monoclonal antibody against ChM-I. The N-terminal amino acid sequencing indicated that it was an N-terminal truncated form of ChM-I generated by the proteolytic cleavage at Asp37-Asp38. This 14-kDa ChM-I was shown by the modified Boyden chamber assay to have very little inhibitory activity on the VEGF-A-induced migration of vascular endothelial cells in contrast to the intact 20–25 kDa form of ChM-I (ID50 = 8 nM). Immunohistochemistry suggested that 20–25 kDa ChM-I was exclusively localized in the avascular zones, i.e. the resting, proliferating, and prehypertrophic zones, of the cartilaginous molds of developing long bone, whereas the 14-kDa form of ChM-I was found in hypertrophic and calcified zones. Immunoblotting demonstrated that mature growth-plate chondrocytes isolated from rat costal cartilage actively secrete ChM-I almost exclusively as the intact 20–25 kDa form into the medium in primary culture. Taken together, our results suggest that intact 20–25 kDa ChM-I is stored as a component of extracellular matrix in the avascular cartilage zones, but it is inactivated by a single N-terminal proteolytic cleavage in the hypertrophic zone of growth-plate cartilage.

The role of angiogenetic factors in the pathogenesis and the progression of cardiac valve disease

Recently, the aging of population and the changing dietary habit to western-style have led to increase in atherosclerotic diseases, such as myocardial infarction and cerebral infarction, in our country. Aortic valve diseases, whose mechanism is similar to atherosclerosis, are also increasing. The medication proved to be effective does not exist, and surgical management is major in treatment of these diseases. Although development of safer medication is expected, little is known about their molecular mechanism of the pathogenesis and the progression. The balance of angiogenetic and angioinhibitory factors is important for normal developing and homeostasis in many organs. The tissue of cardiac valves is mostly avascular like cartilages and tendons, although the heart is the organ having abundance of vessels. We focused on angiogenetitic and angioinhibitory factors expressing in the cartilages and tendons, and found Chondromodulin- I, Tenomodulin and Periostin have crucial roles in degeneration of the cardiac valve by controlling angiogenesis and production of Matrix metalloproteinase protein (MMP) . Now, we review the mechanism of cardiac valve disease focusing on the role of angiogenetic factors.

Chondromodulin‐I derived from the inner meniscus prevents endothelial cell proliferation

The meniscus is a fibrocartilaginous tissue that plays an important role in controlling complex biomechanics of the knee. A perimeniscal capillary plexus supplies the outer meniscus, whereas the inner meniscus is composed of avascular tissue. Anti-angiogenic molecules, such as chondromodulin-I (ChM-I) and endostatin, have pivotal roles in preserving the avascularity of cartilage. However, the anti-angiogenic role of ChM-I is unclear in the meniscus. We hypothesized that the inner meniscus might maintain its avascular feature by expressing ChM-I. Immunohistochemical analyses revealed that ChM-I was mainly detected in the inner and superficial zones of the meniscus. On the other hand, endostatin distribution was similar between the inner and outer meniscus. In Western blot, ChM-I was detected only in the inner meniscus, whereas endostatin was equally observed in both inner and outer menisci. In addition, ChM-I concentration of the inner meniscus-derived conditioned medium was higher than that of the outer meniscus-derived medium. ChM-I removal from the inner meniscus-derived medium and functional blocking of ChM-I significantly increased endothelial cell proliferation. In this study, we demonstrated that the inner meniscus contained larger amounts of ChM-I, and that the inner meniscus-derived ChM-I inhibited endothelial cell proliferation. Our results suggest that ChM-I may be a key anti-angiogenic factor for maintaining the avascularity of the inner meniscus.

Synthetic disulfide‐bridged cyclic peptides mimic the anti‐angiogenic actions of chondromodulin‐I

Chondromodulin-I (ChM-I) is a 25-kDa glycoprotein in cartilage matrix that inhibits angiogenesis. It contains two distinctive structural domains: the N-terminal third of the molecule is a hydrophilic domain that contains O-linked and N-linked oligosaccharide chains, and the C-terminal two-thirds is a hydrophobic domain that contains all of the cysteine residues. In the present study, we have attempted to further uncover the structural requirements for ChM-I to exert anti-angiogenic activity by monitoring its inhibition of the vascular endothelial growth factor (VEGF)-A-induced migration of HUVEC in vitro. Site-directed mutagenesis experiments revealed that the cyclic structure formed by the disulfide bridge between Cys83 and Cys99 in human ChM-I is indispensable for its anti-angiogenic function. Moreover, the C-terminal hydrophobic tail (from Trp111 to Val120) was found to play an important role in ensuring the effectiveness of ChM-I activity on HUVEC. A synthetic cyclic peptide corresponding to the ChM-I region between Ile82 to Arg100 also inhibited the migration of HUVEC, while replacing the Cys83 and Cys99 residues in this peptide with Ser completely negated this inhibitory activity. An additional synthetic cyclic peptide harboring the hydrophobic C-terminal tail of ChM-I clearly mimicked the inhibitory action of this protein on the migration of HUVEC and successfully inhibited tumor angiogenesis and growth in a xenograft mouse model of human chondrosarcoma.