| BackgroundOsteoarthritis(OA)has become the leading cause of disability in the elderly,severe joint pain and dysfunction is the major clinical symptoms.At present,the effect of treatment of OA though take anti-inflammatory,analgesic is limited,which can not change the course of the disease.The reason for the lack of effective treatment of OA,mainly due to its complex pathogenesis is not clear.Angiogenesis in cartilage and the destruction of the barrier between the articular cartilage and bone,which aggravate the inflammatory response,is the important factors that leading clinical symptoms and disease progression.However,the relationship between the angiogenesis of OA cartilage and the degeneration in osteoarthritis is not clear.Normal cartilage without blood vessels,which is the result lead by the balance between the angiogenic factors and the anti-angiogenic factors.Angiogenesis in OA cartilage mainly due to that the balance is broken.Vascular endothelial growth factor(VEGF)is considered to be a key factor in angiogenesis.The expression of VEGF in the superficial,middle and deep layer of OA cartilage was up-regulated,while angiogenesis was mainly occured in deep cartilage,which was related to the decreased expression of anti-angiogenic factors.This suggests that the reduction of anti-angiogenic factors in OA cartilage is an important cause leading to vascular ingrowth.Chondromodulin-I(ChM-I)is a kind of cartilage endogenous and anti-angiogenic glycoprotein,which stimulate proliferation of chondrocyte and inhibit the proliferation of vascular endothelial cells and VEGF-induced cytoskeleton and falsification Foot extension,thus affecting the vascular endothelial cell chemotaxis and tubular formation.ChM-I expression in normal cartilage,is an important factor to maintain cartilage tissue without vascular condition.At present,the expression of ChM-I in cartilage of OA patients and the relationship with the angiogenesis in cartilage and its regulation mechanism are unclear. yinyang1(YY1)is a member of the GL1-Krüpp1 family of zinc-finger transcription factors,widely found in human,mouse and Xenopus laevis,and plays a dual role in promoting or inhibiting gene transcription.YY1 expression was upregulated in various tumor tissues and nasal polyps,which promote angiogenesis.Recently,the study of transcription factor in OA microarray data analysis,the results suggest that transcription factor YY1 is one of the important transcription factors in the OA regulatory network,which is related to the origin of OA.IL-1? can increase the abundance and activity of YY1 in cardiomyocytes.IL-1? is also the main inflammatory factor in OA cartilage.The expression of YY1 in OA cartilage and its mechanism are not clear.YYl can bind to the promoter of ChM-I and inhibit its expression.YY1 / ChM-I signaling pathway can regulate the expression of ChM-I in bone marrow mesenchymal stem cells and normal chondrocytes.It is unclear whether this signal pathway participates in the regulation of angiogenesis in OA cartilage.ObjectiveIn summary,we hypothesized that IL-1? up-regulates YY1 expression in OA chondrocytes,and then YY1 binds to the promoter region of ChM-I,leading to down-regulation of ChM-I expression,thereby attenuating cartilage resistance to angiogenesis.In this study,we will test the above hypothesis from human histopathology and in vitro cytology.Methods1.To observe the changes of angiogenesis and expression of ChM-I and YY1 in articular cartilage of patients with OA and the relationship between them.We collected articular cartilage specimens from knee of OA patients.Pathological sections,HE staining,solid green / safranine O staining,grading.2.Immunohistochemical staining was performed on ChM-I and YY1.Total RNA and protein were extracted from cartilage samples.The expression levels of ChM-I and YY1 in articular cartilage of OA patients were detected by real-time polymerase chain reaction(RT-PCR)and Western blot.3.Normal human chondrocytes were extracted and cultured in vitro,which was confirmed with toluidine blue staining and Collagen II.Stimulated with IL-1?,the expression of ChM-I and YY1 in human chondrocytes was detected by RT-qPCR and Western blot.4.The short hairpin RNA(shRNA)of YY1 was used to transfect human chondrocytes.To explore the effect of the interference of YY1 gene to IL 1?-induced ChM-I and YY1 expression in chondrocytes.5.Human umbilical vein endothelial cells(HUVEC)were treated with conditioned medium from each experimental group.The migration,invasion and tubulointerstitial changes of HUVEC were observed.Results1.Relationship between angiogenesis and degenerationof the cartilage in OA : angiogenesis occurs mainly in the cartilage calcification layer.Morphological changes in the calcified layer of the cartilage(thickness,tidal line,line of adhesion,number of chondrocytes)are related to the area of vessels channel.2.ChM-I expression in OA cartilage: in the mild degenerated cartilage,the immunostaining intensity of Chm-I in the chondrocytes cytoplasm of the surface of decreased significantly.The staining intensity of ChM-I in the intermediate layer was weakened.In the deep region,ChM-I immunostaining was reduced.ChM-I protein and mRNA level decreased compared to the normal cartilage.In moderate degenerated cartilage,ChM-I immunostaining in the cytoplasm and extracellular matrix of superficial chondrocytes was decreased.In the middle region,ChM-I immunostaining intensity was stronger than that in mild degenerative cartilage.In the deep region,ChM-I immunostaining was significantly decreased.ChM-I protein levels and mRNA expression was reduced compared to mild degenerated cartilage.In severe degenerative cartilage,ChM-I immunostaining was significantly attenuated in all regions of the extracellular matrix.Immunostaining of ChM-I occurs predominantly in the extracellular matrix of clusters of proliferating cell populations.ChM-I mRNA expression showed no significant changes compared with moderate degenerated cartilage.YY1 expression in OA cartilage: in mild degenerated cartilage,the immunostaining intensity of YY1 in the surface of the cartilage was increased.In the middle region,the immunostaining intensity of YY1 was slightly enhanced.In the deep region,the immunostaining intensity of YY1 increased.YY1 protein and mRNA was increased contrast with normal cartilage.In the moderate degenerated cartilage,immunostaining of YY1 was stronger than that of the surface layer of mild degenerated cartilage.In the middle region,the immunostaining intensity of YY1 increased.In the deep region,the immunostaining intensity of YY1 was enhanced in the mild degeneration.YY1 protein and mRNA of the degree of mild degeneration of cartilage increased.In the soft degeneration,the immunostaining intensity of YY1 was significantly increased in the cell clusters of the articular surface,and the immunostaining intensity of YY1 in the deep region decreased significantly.YY1 protein and mRNA levels were not significantly higher than moderate degeneration cartilage.Relationship among ChM-I,YY1 and angiogenesis: vascular density was related to the immunostaining intensity of ChM-I in the extracellular matrix but not with the immunostaining intensity and mRNA levels of ChM-I in chondrocytes.Vascular density was related to the immunostaining intensity and mRNA levels of YY1 in the extracellular matrix.3.The phenotype of chondrocytes was maintained in the cultured chondrocytes from human normal cartilage,both toluidine blue and collagen II was positive.IL-1? down-regulated the expression of ChM-I in human chondrocytes.As the expression of ChM-I was down-regulated by the stimulation of IL-1? in human chondrocytes make clear,and then to detect the expression of YY1 in chondrocytes stimulated by IL-1? was significantly increased by western blot.4.The transfection efficiency of shRNA YY1 on human chondrocytes was 91%,the level of YY1 mRNA and protein was downregulated 63% and 61%,respectively.Inhibit the expression of YY1,then the downregulated expression of ChM-I in the human chondrocytes stimulated by IL-1? was rebounded significantly,which suggested that transcription factors YY1 participate in the process of down-regulation of ChM-I mediated IL-1?.This is reported in the literature that YY1 can directly bind to the promoter region of ChM-I and inhibit the transcription of ChM-I.5.HUVEC was treated with conditioned medium in each group.The results showed that the conditioned media from IL-1?-stimulated normal human chondrocyte could significantly reduce the width of HUVEC scars and reduce the number of invasive cells and tube formation.And YY1 shRNA interference can make the above experimental results appear the opposite change.The results suggest that IL-1?-induced human chondrocytes can promote the migration,invasion and tubulogenesis of HUVEC in vitro,while YY1 shRNA can inhibit these abilities.DiscussionsThis study found that angiogenesis in OA cartilage occurs mainly in the cartilage calcification layer,the thickness of calcification layer,the roughness of tidemark and cement line,the number of chondrocytes and the area of vascular in the calcification layer are related to the degree of degeneration.The thickness of the calcified layer of 2nd grade is thinner than that of the 1st grade,which indicates that the morphological changes of the calcified layer are affected by different factors in different stages of degeneration.On the one hand,subchondral bone through the osteogenic reaction thinning calcification.We found that the roughness of the cement lines increased with the degree,which is closely related to the thickness of subchondral bone,and bone sleeve was formated around the vascular channel.On the other hand,calcified layer was thickened by subchondral bone through the infiltration.Our study shows that the area of vascular have a greater impact on the calcified cartilage.Both of these effects are related to angiogenesis.This study found that ChM-I in normal cartilage is high expression,suggesting that ChM-I may be of great significance on maintaining normal cartilage-free vascular environment.In addition,we found that the density of vascular channel in osteoarthritic cartilage grows up with the degree of degeneration,while the expression of ChM-I in extracellular matrix decreased.This suggests that a decrease in ChM-I protein during the course of osteoarthritis may reduce the ability of cartilage to inhibit angiogenesis.Our results suggest that the cause of ChM-I decline in different stages of osteoarthritis may be different.In the early stage of osteoarthritis,ChM-I expression may be associated with fibrosis of cartilage.Because of mild degenerated osteoarthritis,fibrosis was found in the surface of the cartilage,while ChM-I expression decreased in this region.In moderate to severe degenerative cartilage,there are more chondrocyte clones.ChM-I expression was elevated in the cytoplasm of clonal chondrocytes in the middle region.The level of ChM-I mRNA was also up-regulated.In moderate-to-severe degenerative cartilage,the ChM-I expression was down-regulated in all regions,although ChM-I expression was upregulated in the middle region.Therefore,we hypothesized that ChM-I degradation in cartilage of late osteoarthritis may be related to its degradation and consumption.This study is the first time to show the expression of YY1 in osteoarthritic cartilage.The expression of YY1 was detected in normal cartilage tissues and the proliferation cells of osteoarthritic cartilage,and the expression of YY1 was up-regulated.Which may be related to the promotion of YYl cell proliferation.In addition,this experiment showed that there was no expression of YY1 in deep cartilage of severe osteoarthritis,which may be related to the apoptosis of deep degeneration cartilage cells.The expression of YY1 in chondrocytes of OA patients was correlated with the expression of ChM-I and angiogenesis.This correlation was further confirmed by in vitro experiments.In vitro experiments showed that human chondrocytes were stimulated by IL-1? to promote the migration,invasion and tubulogenesis of HUVEC.This may be due to a decrease in ChM-I,as it was found that IL-1? stimulated human chondrocytes resulting in a decrease in ChM-I expression.While IL-1?-induced ChM-I decline may be mediated by YY1.It was found that the expression of YY1 was up-regulated in human chondrocytes stimulated by IL-1?,contrary to the change of ChM-I.We further used gene interference to inhibite the expression of YY1 in human chondrocytes by shRNA,which suppress the down-regulation of ChM-I expression mediated by IL-1?.Moreover,inhibition of YY1 in chondrocytes resulted in better inhibition of HUVEC cell migration,invasion and formation of tubules by conditioned media.ConclusionThe morphology of calcified layer of articular cartilage in the early OA was changed,this was connected with the pathological changes of OA;vascular ingrowth is an important cause of the morphological changes of the calcified layer;and the expression of ChM-I and YY1 protein in chondrocytes was closely related to the angiogenesis of OA cartilage;in vitro,stimulated human chondrocytes by IL-1? in vitro,which can upregulate the expression of YY1 and inhibite the expression of ChM-I,and reducte the ability of cartilage cells againsting HUVEC proliferation,migration and invasion and tube formation,while the above situation can be obviously alleviated when the the expression of YY1 was inhibited by the gene interference in human chondrocytes.In conclusion,the inflammatory mediators(IL-1?)of OA cartilage reduced the expression of ChM-I and the ability of anti-angiogenesis of chondrocytes through the YY1 / ChM-I signaling pathway,leading to the angiogenesis in cartilage calcified layer. |
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