Study On The Mechanisms Of Cartilage Degenerantion And Matrix Degradation In Human Osteoarthritis

Osteoarthritis(OA)is the most common worldwide age-related and post-traumatic degenerative joint disorder.According to the statistics of the Centers for Disease Control and Prevention,arthritis affects an estimated 26.0%of women and 19.1%of men who are diagnosed by a doctor in the United States and OA is present in 80%of the population by age 65.OA is mainly characterized by a cartilage homeostasis disorder with subsequent inflammation and degradation that results in chronic physical disability and progressive irreversible dysfunction.Despite accumulating reports on chondrocytes that have identified strategies to predict and modify O A progression,effective measures of disease-modifying OA diagnoses and outcomes are still lacking.A better understanding of the pathophysiology of inflammation and the mechanisms underlying the role of chondrocytes in the process leading to OA is of critical importance.METHODS:In Part 1,We performed unbiased transcriptome-wide scRNA-seq analysis,computational analysis and histological assays on 1464 chondrocytes from 10 OA patients undergoing knee arthroplasty surgery.We investigated the relationship between transcriptional programs of the OA landscape and clinical outcome using severity index analysis and correspondence analysis.In Part 2,The physiological involvement of HPIP in cartilage formation was analyzed in Col2a1-CreERT2;HPIPf/f and HPIPf/f mice.The function of HPIP in OA was examined in Col2a1-CreERT2;HPIPf/f and HPIPf/f mice using an OA model created by anterior cruciate ligament transection(ACLT).The role of HPIP as a candidate for OA therapy was evaluated using the intra-articular injection of adeno-associated virus(AAV)carrying HPIP-specific short hairpin RNA(shRNA).HPIP expression in OA patients was elected.RNA-sequencing(RNA-seq)and chromatin immunoprecipitation sequencing(ChIP-seq)assays were performed to delineate the underlying 14 mechanisms.In Part 3,we examined the potential CTS-mediated miRNA molecules using nicroarray analysis and created OA mouse model created by anterior cruciate ligament transection.Histology assay and ?CT and molecular experiment were also performed to investigate the mechanisms of cryptotanshinone-mediated cartilage degeneration and matrix degradation in human OA.RESULTS:1)We identified seven molecularly defined populations of chondrocytes in human OA cartilage,including three novel phenotypes with distinct functions.We present gene expression profiles and transcriptional networks among chondrocytes at different OA stages at a single-cell resolution.We found a potential transition among proliferative chondrocytes,prehypertrophic chondrocytes and hypertrophic chondrocytes(HTCs),and defined a new subdivision within HTCs.We revealed novel markers for cartilage progenitor cells(CPCs)and demonstrated a relationship between CPCs and fibrocartilage chondrocytes using computational analysis.Notably,we derived predictive targets with respect to clinical outcomes and clarified the role of different cell types for the early diagnosis and treatment of OA.2)Elevated HPIP levels were found in OA patients.Col2a1-CreERT2/HPIPf/f mice exhibited obvious skeletal abnormalities compared with their HPIPf/f littermates.HPIP deficiency in mice protected against developing O A induced by anterior cruciate ligament transection.Moreover,intra-articular injection of adeno-associated virus(AAV)carrying HPIP-specific short hairpin RNA(shRNA)in vivo attenuated OA histological signs when administered after injury.Notably,in vitro RNA-sequencing(RNA-seq)and chromatin immunoprecipitation sequencing(ChIP-seq)profiles identified that HPIP modulates OA cartilage degeneration through transcriptional activation of WNT target genes.Mechanistically,HPIP promotes the transcription of Wnt targets by interacting with lymphoid enhancer binding factor 1(LEF1).Furthermore,HPIP potentiates the transcriptional activity of LEF1 and acetylates histone H3 lysine 56 in the promoters of WNT signaling pathway targets.3)We found that CTS significantly promoted miR-106a-5p expression in chondrocytes.Using the OA mouse model created by anterior cruciate ligament transection,we revealed that intra-articular injection of miR-106a-5p agomir attenuated OA.In addition,miR-106a-5p inhibited GLI-similar 3(GLIS3)production by directly targeting the 3'-untranslated region.CTS promoted miR-106a-5p expression through recruitment of a member of the paired box(PAX)family of transcription factors,PAX5,to the miR-106a-5p promoter.Inhibition of PAX5 mimicked the effect of miR-106a-5p and abolished the CTS ability to regulate miR-106a-5p expression.In OA patients,miR-106-5p is down-regulated which is accompanied by down-regulation of PAX5 and up-regulation of GLIS3.CONCLUSIONS:Our results provide new insights into chondrocyte taxonomy and present potential clues for effective and functional manipulation of human OA cartilage regeneration that could lead to improved health;that HPIP is an attractive target in the regulatory network for developing O A therapy and that these novel functions of HPIP will likely lead to new avenues of OA treatment;the PAX5/miR-106a-5p/GLIS3 axis acts as a novel pleiotropic regulator in CTS-mediated OA cartilage protection,suggesting that miR-106a-5p and PAX5 activation and GLIS3 inhibition might be useful and attractive for therapeutic strategies to treat OA patients.