Structure and function of aggrecan

Aggrecan is the major proteoglycan in the articular cartilage. This molecule is important in the proper functioning of articular cartilage because it provides a hydrated gel structure (via its interaction with hyaluronan and link protein) that endows the cartilage with load-bearing properties. It is also crucial in chondroskeletal morphogenesis during development. Aggrecan is a multimodular molecule expressed by chondrocytes. Its core protein is composed of three globular domains (G1, G2, and G3) and a large extended region (CS) between G2 and G3 for glycosaminoglycan chain attachment. G1 comprises the amino terminus of the core protein. This domain has the same structural motif as link protein. Functionally, the G1 domain interacts with hyaluronan acid and link protein, forming stable ternary complexes in the extracellular matrix. G2 is homologous to the tandem repeats of G1 and of link protein and is involved in product processing. G3 makes up the carboxyl terminus of the core protein. It enhances glycosaminoglycan modification and product secretion. Aggrecan plays an important role in mediating chondrocyte-chondrocyte and chondrocyte-matrix interactions through its ability to bind hyaluronan.

Effects of Basic Fibroblast Growth Factor and Insulin-like Growth Factor on Cultured Cartilage Cells from Skate Raja porasa

Effects of basic fibroblast growth factor (bFGF) and insulin like growth factor II (IGF II) on cartilage cells from proboscis of skate, Raja porasa Günther, were investigated in this study. The cartilage cells were cultured in 20% FBS supplemented MEM medium at 24℃. Twelve hours after culture initiation, the cartilage cells were treated with bFGF and IGF II at different concentration combinations. It was found that 20 ng/ml of bFGF or 80 ng/ml of IGF II was enough to have obvious stimulating effect on the growth and division of skate cartilage cells. Test of bFGF and IGF II together, revealed that 20 ng/ml of bFGF and 80 ng/ml of IGF II together had the best stimulating effect on the growth and division of skate cartilage cells. The cartilage cells cultured could form a monolayer at day 7.

T-2 toxin-induced apoptosis involving Fas,p53,Bcl-xL,Bcl-2,Bax and caspase-3 signaling pathways in human chondrocytes

Objective:To investigate the effects of T-2 toxin on expressions of Fas,p53,Bcl-xL,Bcl-2,Bax and caspase-3 on human chondrocytes.Methods:Human chondrocytes were treated with T-2 toxin(1～20 ng/ml)for 5 d.Fas,p53 and other apoptosis-related proteins such as Bax,Bcl-2,Bcl-xL,caspase-3 were determined by Western blot analysis and their mRNA expressions were determined by reverse transcriptase-polymerase chain reaction(RT-PCR).Results:Increases in Fas,p53 and the pro-apoptotic factor Bax protein and mRNA expressions and a decrease of the anti-apoptotic factor Bcl-xL were observed in a dose-dependent manner after exposures to 1～20 ng/ml T-2 toxin,while the expression of the anti-apoptotic factor Bcl-2 was unchanged.Meanwhile,T-2 toxin could also up-regulate the expressions of both pro-caspase-3 and caspase-3 in a dose-dependent manner.Conclusion:These data suggest a possible underlying molecular mechanism for T-2 toxin to induce the apoptosis sig- naling pathway in human chondrocytes by regulation of apoptosis-related proteins.

Increased expression of chondroitin sulphate proteoglycans in rat hepatocellular carcinoma tissues

AIM:To investigate the expression of chondroitin sulphate proteoglycans(CSPGs)in rat liver tissues of hepatocellular carcinoma(HCC).METHODS:Thirty male Sprague Dawley rats were randomly divided into two groups:control group(n=10) and HCC model group(n=20).Rats in the HCC model groups were intragastrically administrated with 0.2%(w/v)N-diethylnitrosamine(DEN)every 5 d for 16 wk,whereas 0.9%(w/v)normal saline was administered to rats in the control group.After 16 wk from the initiation of experiment,all rats were killed and livers were collected and fixed in 4%(w/v)paraformaldehyde.All tissues were embedded in paraffin and sectioned.Histological staining(hematoxylin and eosin and Toluidine blue)was performed to demonstrate the onset of HCC and the content of sulphated glycosaminoglycan(sGAG).Immunohistochemical staining was performed to investigate the expression of chondroitin sulphate(CS)/dermatan sulphate(DS)-GAG,heparan sulphate(HS)-GAG,keratan sulphate(KS)-GAG in liver tissues.Furthermore,expression and distribution of CSPG family members,including aggrecan,versican,biglycan and decorin in liver tissues,were also immunohistochemically determined.RESULTS:After 16 wk administration of DEN,malignant nodules were observed on the surface of livers from the HCC model group,and their hepatic lobule structures appeared largely disrupted under microscope.Toluidine blue staining demonstrated that there was an significant increase in sGAG content in HCC tissues when compared with that in the normal liver tissues from the control group[0.37±0.05 integrated optical density per stained area(IOD/area)and 0.21± 0.01 IOD/area,P<0.05].Immunohistochemical studies demonstrated that this increased sGAG in HCC tissues was induced by an elevated expression of CS/DS(0.28±0.02 IOD/area and 0.18±0.02 IOD/area,P< 0.05)and HS(0.30±0.03 IOD/area and 0.17±0.02 IOD/area,P<0.01)but not KS GAGs in HCC tissues.Further studies thereby were performed to investigate the expression and distribution of several CSPG components in HCC tissues,including aggrecan,versican,biglycan and decorin.Interestingly,there was a distinct distribution pattern for these CSPG components between HCC tissues and the normal tissues.Positive staining of aggrecan,biglycan and decorin was localized in hepatic membrane and/or pericellular matrix in normal liver tissues;however,their expression was mainly observed in the cytoplasm,cell membranes in hepatoma cells and/or pericellular matrix within HCC tissues.Semi-quantitative analysis indicated that there was a higher level of expression of aggrecan(0.43± 0.01 and 0.35±0.03,P<0.05),biglycan(0.32±0.01 and 0.25±0.01,P<0.001)and decorin(0.29±0.01 and 0.26±0.01,P<0.05)in HCC tissues compared with that in the normal liver tissues.Very weak versican positive staining was observed in hepatocytes near central vein in normal liver tissues;however there was an intensive versican distribution in fibrosis septa between the hepatoma nodules.Semi-quantitative analysis indicated that the positive rate of versican in hepatoma tissues from the HCC model group was much higher than that in the control group(33.61%and 21.28%,P <0.05).There was no positive staining in lumican and keratocan,two major KSPGs,in either normal or HCC liver tissues.CONCLUSION:CSPGs play important roles in the onset and progression of HCC,and may provide potential therapeutic targets and clinical biomarkers for this prevalent tumor in humans.

Promotion of the articular cartilage proteoglycan degradation by T-2 toxin and selenium protective effect

Objective: To identify the relationship between T-2 toxin and Kashin-Beck disease (KBD),the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in vitro. Methods: Chondrocytes were isolated from human articular cartilage and cultured in vitro. Hyaluronic acid (HA),soluble CD44 (sCD44),IL-1β and TNF-α levels in super-natants were measured by enzyme-linked immunosorbent assay (ELISA). CD44 content in chondrocyte membrane was deter-mined by flow cytometry (FCM). CD44,hyaluronic acid synthetase-2 (HAS-2) and aggrecanases mRNA levels in chondrocytes were determined using reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemical method was used to investigate expressions of BC-13,3-B-3(-) and 2-B-6 epitopes in the cartilage reconstructed in vitro. Results: T-2 toxin inhibited CD44,HAS-2,and aggrecan mRNA expressions,but promoted aggrecanase-2 mRNA expression. Meanwhile,CD44 expression was found to be the lowest in the chondrocytes cultured with T-2 toxin and the highest in control plus selenium group. In addition,ELISA results indicated that there were higher sCD44,IL-1β and TNF-α levels in T-2 toxin group. Similarly,higher HA levels were also observed in T-2 toxin group using radioimmunoprecipitation assay (RIPA). Furthermore,using monoclonal antibodies BC-13,3-B-3 and 2-B-6,strong positive immunostaining was found in the reconstructed cartilage cultured with T-2 toxin,whereas no positive staining or very weak staining was observed in the cartilage cultured without T-2 toxin. Selenium could partly inhibit the effects of T-2 toxin above. Conclusion: T-2 toxin could inhibit aggrecan synthesis,promote aggrecanases and pro-inflammatory cytokines production,and consequently induce aggrecan degradation in chondrocytes. These will perturb metabolism balance between aggrecan synthesis and degradation in cartilage,inducing aggrecan loss in the end,which may be the initiation of the cartilage degradation.

Hyaluronidase Proof for Endothelial Glycocalyx as Partaker of Microcirculation Disturbances

Covalent modification of bovine testicular hyaluronidase with chondroitin sulphate led to changes in the pattern of glycation of native and modified enzyme in its reaction with neutral saccharides and N-acetylhexosamines. Thus, mono- and di-saccharides inactivated the native hyaluronidase to a greater extent than the chondroitin sulfate-modified enzyme. N-acetylhexosamine, on the opposite, inactivated the modified hyaluronidase to a greater extent than the native one. These properties made it possible to use native and modified hyaluronidase as an informative research system for in vivo measurement of the predominant type of saccharide agents in the circulation. The proposed approach was experimentally substantiated by obtained results of the study on these interactions of hyaluronidase derivatives with hyaluronan fragments and their mixture. In a model of post-ischemic perfusion of the rat limb, the effect of hyaluronidase derivatives and their components on restoration of the microcirculation were tracked using laser Doppler flowmetry. Native hyaluronidase accelerated the restoration of initial level of microcirculation, but modified enzyme was markedly inhibited by glycocalyx degradation products. N-acetylhexosamine was positioned at the reducing terminal of these products as a natural label for these glycocalyx fragments. These and other data obtained under various experimental conditions supported the participation of endothelial glycocalyx in microcirculation disturbances.

Chondrogenic differentiation of rat bone marrow mesenchymal stem cells induced by puerarin and tetrandrine

Objective: This study aims to clarify the effect of the active components puerarin and tetrandrine on the chondrogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods: Using network pharmacology, protein targets of puerarin and tetrandrine were predicted, and a database of cartilage formation targets was established. The protein target information related to disease was then collected, and the drug-targeting network was constructed by analyzing the protein–protein interactions. Genes related to chondrogenesis induced by puerarin and tetrandrine and chondroblast differentiation signaling pathways were searched. Finally, potential drug-and disease-related genes,as well as proteins, were screened and verified using real-time RT-PCR and western blotting.Results: Network pharmacological studies have shown that puerarin and tetrandrine are involved in BMSCs cartilage differentiation. The experimental results showed that puerarin and tetrandrine could regulate the expression of cartilage differentiation-related genes and proteins. Puerarin increased the protein expression of COL2 A1, COL10 A1, MMP13, and SOX-9,as well as the gene expression of Col2 a1, Mmp13, Tgfb1, and Sox-9. Tetrandrine increased the protein expression of COL2 A1,COL10 A1, MMP13, and SOX-9, as well as the gene expression of Col10 a1, Tgfb1, Sox-9, and Acan. The combination of puerarin and tetrandrine increased the protein expression of COL2 A1, COL10 A1, MMP13, and SOX-9 and the gene expression of Col2 a1,Col10 a1, Sox-9, and Acan.Conclusions: Puerarin, tetrandrine, and their combination can promote the proliferation of BMSCs and induce their differentiation into chondrocytes, and they are thus expected to be inducers of chondrogenic differentiation. These results suggest that puerarin and tetrandrine have potential therapeutic effects on osteoarthritis.

Tumor-targeted delivery of siRNA by surface-modified LPC nanoparticles

With increasing knowledge of the molecular mechanisms of endogenous RNA interference,systemic delivery of small interfering RNA（siRNA） via targeted nanoparticles has emerged as a potential strategy for cancer gene therapy.In this study,a novel formulation[liposome-protamine-chondroitin sulfate nanoparticles（LPC-NP）]was developed for siRNA delivery by self-assembling with charge-charge interaction.The LPC-NP was further modified by DSPE-PEG_（2000） and DSPE-PEG_（2000）-T7 by the post-insertion method.T7,a transferrin-like seven-amino acid peptide,is a targeting ligand for transferrin receptor-overexpressed MCF-7 breast cancer cells.The particle size and zeta potential of LPC-NP were approximately 90 nm and ＋35 mV,respectively. It was shown that PEG modification could significantly decrease aggregation of LPC-NP in serum,and T7 peptide modified LPC-NP could significantly increase the cellular uptake and the gene-silencing effect of siRNA.In vitro cytotoxicity assay exhibited that significant cell growth inhibition was achieved in MCF-7 cells after the delivery of anti-EGFR siRNA.Our encouraging results suggested that T7-modified LPC-NP might be a promising carrier for RNAi-based tumor therapy.

An AIEE polyelectrolyte as a light-up fluorescent probe for heparin sensing in full detection range

Polyelectrolyte PTPEPyH, containing tetraphenylethene (TPE) and pyridinium, was synthesized. Its optical properties were investigated with spectroscopies and the results showed that there were two emission-enhanced stages in the interaction between positive-charged PTPEPyH and negative-charged biomacromolecule heparin. The mechanism was very different due to the reduced non-radiative energy loss and the change of surroundings. The polyelectrolyte PTPEPyH, compared with ChS and HA, also showed high selectivity for heparin in the buffer solution and could be used as a potential bio-probe for heparin quantification in the clinical full range.

Direct protective effect of interleukin-10 on articular chondrocytes in vitro

Abstract:Objective To assess whether interleukin-10 (IL-10) is chondroprotective in vitro. Methods Chondrocytes were isolated from femoral cartilage of rats (7-10 days) by digestion with collagenase Ⅱ. The first passage cells were grown in 24- well plates with DMEM, supplemented with 10% fetal bovine serum, for 2-4 days. The cells were then cultured in 0.1% fetal bovine serum DMEM medium, and given respectively interleukin-1 (IL-1) 100?μ/ml, IL-1 100?μ/ml+recombinant murine interleukin-10 (rmIL-10) 20?ng/ml, rmIL-10 20?ng/ml, and cultured for 48 hours. Scanning electron morphology and immunohistochemical study of nitric oxide synthase 2 and matric metalloproteinase 3 mRNA in situ hybridization were performed. Cell proliferation and morphology were observed under inverted microscope from the beginning of cell culture for three weeks. Results IL-1 stimulated granule production in the cytoplasma of chondrocytes, and the cells died in the second and third weeks of culture. IL-10 antagonized IL-1, protected the cells from death and maintained chondrocyte proliferation. Scanning electron morphology showed that IL-1 stimulated the formation of numerous microvilli on the cell surface, while thin and less numerous microvilli were found in cultures with IL-10. Immunohistochemical study and in situ hybridization showed that IL-10 inhibited NOS2 and MMP3 expression.Conclusion IL-10 not only inhibits the synthesis of inflammatory cytokines, but also directly protects chondrocytes by antagonizing IL-1.

The expression of N-cadherin,fibronectin during chondrogenic differentiation of MSC induced by TGF-β_1

To investigate the role of N-cadherin and fibronectin during chondrogenesis. Methods - lmmunohistochemical method and autibody induced changes of aggregation of cells were used to assay the expressions of N-cadherin and fibronectin during cell differentiation. Results： The N-cadherin was present in the area of the cell nodular area in the 24 hours group after adding chondrogenic revulsant, then there was a down-regulating trend. Fibronectin was expressed in 48 and 72 hours groups after adding chondrogenic revulsant, and showed to be negative afterward. The antibody against fibronectin or Ncadherin could inhibit the formation of cellular nodule markedly. Conclusions： Cell adhesion factors play an important role during cell differentiation. TGF-β1 stimulates chondrogenesis via transition from an initial N-cadherin-contributing stage to a succedent fibronectin-contributing stage during the process of chondrogenesis in MSCs. Further study is needed to evaluate whether or not it can promote chondrogenesis by transfecting cDNA of CAMs to MSCs.

Effects of cold-damp and hot-damp environment on VEGF and IL-1 expression in joint cartilage cells in adjuvant arthritis in rats

OBJECTIVE:To study the effects of environmental factors on the degree of injury and expression of vascular endothelial growth factor(VEGF) and interleukin-1(IL-1) in cartilage cells of the joint in a rat model of adjuvant arthritis(AA).METHODS:SD rats aged 10 months were randomly divided into 4 groups that varied by temperature and humidity housing conditions and induction of AA:a control group,a model group,a cold-damp group,and a hot-damp group.All groups except the control group were induced with AA.After 4 w,VEGF and IL-1 expression in cartilage cells of ankle joints of hind limbs were observed.RESULTS:Mean area,optical density,and numbers of VEGF-and IL-1-positive cells in the model group,the cold-damp group,and the hot-damp group were significantly higher than that of the control group(all P<0.05).Optical density and positive cell numbers in the cold-damp group and the hot-damp group were significantly higher than that of the model group(all P<0.05).Optical density and positive cell numbers in the hot-damp group were significantly higher than that of the cold-damp group.Bone in the hot-damp and cold-damp groups was severely injured.CONCLUSION:Environmental factors such as high humidity combined with either high or low temperature increase the severity of damage and expression of VEGF and IL-1 in cartilage cells of joints in rats induced with AA.

Green fluorescent protein as marker in chondrocytes overexpressing human insulin-like growth factor-l for repair of articular cartilage defects in rabbits

Objective： To label the primary articular chondrocytes overexpressing human insulin-like growth factor （ IdGF-1 ） with green fluorescent protein （GFP） for repair of articular cartilage defects in rabbits. Methods： GFP cDNA was inserted into PeDNA3.1- hlGF-1 to label the expression vector. The recombinnnt vector, pcGI, a mammalian expression vector with multiple cloning sites under two respective cytomegalovirus promoters/enhancers, was transfected into the primary articular chondrocytes with the help of lipofectamine. After the positive cell clones were selected by G418, G418- resistant chondrocytes were cultured in medium for 4 weeks. The stable expression of hlGF-1 in the articular chondrocytes was determined by in situ hybridization and immunocytochemical analysis and the GFP was confirmed under a fluorescence microscope. Methyl thiazolyl tetrazolium （MTT） and flow cytometer methods were employed to determine the effect of transfection on proliferation of chondrocytes. Gray value was used to analyze quantitatively the expression of type lI collagen. Results： The expression of hlGF-1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hlGF-1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks. After the transfectton of IGF-1, the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of type lI collagen. Conclusions： The hlGF-1 eukaryotic expression vector containing GFP marker gene has been successfully constructed. GFP, which can be visualized in real time and in situ, is stably expressed in articular chondrocytes overexpressing hlGF-1. The labeled articular chondrocytes overexpressing hlGF-1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.