Brachyury promotes extracellular matrix synthesis through transcriptional regulation of Smad3 in nucleus pulposus.

Metabolic dysfunction of the extracellular matrix (ECM) is one of the primary causes of intervertebral disc degeneration (IVDD). Previous studies have demonstrated that the transcription factor Brachyury (Bry) has the potential to promote the synthesis of collagen II and aggrecan, while the specific mechanism is still unknown. In this study, we used a lipopolysaccharide (LPS)-induced model of nucleus pulposus cell (NPC) degeneration and a rat acupuncture IVDD model to elucidate the precise mechanism through which Bry affects collagen II and aggrecan synthesis in vitro and in vivo. First, we confirmed Bry expression decreased in degenerated human nucleus pulposus (NP) cells (NPCs). Knockdown of Bry exacerbated the decrease in collagen II and aggrecan expression in the lipopolysaccharide (LPS)-induced NPCs degeneration in vitro model. Bioinformatic analysis indicated that Smad3 may participate in the regulatory pathway of ECM synthesis regulated by Bry. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter gene assays demonstrated that Bry enhances the transcription of Smad3 by interacting with a specific motif on the promoter region. In addition, Western blot and reverse transcription-qPCR assays demonstrated that Smad3 positively regulates the expression of aggrecan and collagen II in NPCs. The following rescue experiments revealed that Bry-mediated regulation of ECM synthesis is partially dependent on Smad3 phosphorylation. Finally, the findings from the in vivo rat acupuncture-induced IVDD model were consistent with those obtained from in vitro assays. In conclusion, this study reveals that Bry positively regulates the synthesis of collagen II and aggrecan in NP through transcriptional activation of Smad3.NEW & NOTEWORTHY Mechanically, in the nucleus, Bry enhances the transcription of Smad3, leading to increased expression of Smad3 protein levels; in the cytoplasm, elevated substrate levels further lead to an increase in the phosphorylation of Smad3, thereby regulating collagen II and aggrecan expression. Further in vivo experiments provide additional evidence that Bry can alleviate IVDD through this mechanism.

Lack of Syndecan-1 promotes the pathogenesis of experimental rheumatoid arthritis.

Syndecan-1 (Sdc-1), a transmembrane heparan sulfate protein, is implicated in several pathophysiological processes including rheumatoid arthritis (RA). The exact role of Syndican-1 in this autoimmune disease is still undetermined. This study explores the involvement level of Sdc-1 in the development of RA in a collagen II-induced arthritis mice model. RA was induced in two mice strains (wild-type BALB/c group and Sdc-1 knockout) by collagen II. Mice underwent regular clinical observations and scoring. After sacrifice, leg biopsies were taken from mice for histological examination, using a variety of stains. In addition, proteins were extracted, and molecular assessment of TNF-α was performed using the western blot technique. In the Sdc-1 knockout group, clinical scoring results showed a significantly more severe experimental RA; histology showed a significant increase in bone erosion, cartilage destruction, inflammation, and less granulated mast cells than the wild-type. In addition, molecular assessment of TNF-α showed more increase in expression in the Sdc-1 knockout models compared to the wild-type. Data suggest that lack of Sdc-1 enhances the inflammatory characteristics in RA. However, more molecular studies and investigations are needed to determine its exact role and possible mechanisms involved.

Ophthalmic manifestations of Czech dysplasia.

Czech dysplasia is an autosomal dominant type 2 collagenopathy that is caused by heterozygosity for the recurrent p.(Arg275Cys) COL2A1 variant. Affected individuals usually present with skeletal abnormalities such as metatarsal hypoplasia of the third and fourth toes and early-onset arthropathy, as well as hearing loss. To date, no ophthalmic findings have been reported in patients with Czech dysplasia even though COL2A1 has been implicated in other ocular conditions such as type 1 Stickler syndrome. For the first time, we report the ocular findings in four families with Czech dysplasia, including type 1 vitreous anomaly, hypoplastic vitreous, retinal tears, and significant refractive error. These novel ocular findings expand the phenotype associated with Czech dysplasia and may aid clinicians as an additional diagnostic feature. Patients with congenital abnormalities of vitreous gel architecture have an increased risk of retinal detachment, and as such, patients may benefit from prophylaxis. Considering that many of the patients did not report any ocular symptoms, vitreous phenotyping is of key importance in identifying the need for counseling with regard to prophylaxis.

Osteophyte Cartilage as a Potential Source for Minced Cartilage Implantation: A Novel Approach for Articular Cartilage Repair in Osteoarthritis.

Osteoarthritis (OA) is a common joint disorder characterized by cartilage degeneration, often leading to pain and functional impairment. Minced cartilage implantation (MCI) has emerged as a promising one-step alternative for large cartilage defects. However, the source of chondrocytes for MCI remains a challenge, particularly in advanced OA, as normal cartilage is scarce. We performed in vitro studies to evaluate the feasibility of MCI using osteophyte cartilage, which is present in patients with advanced OA. Osteophyte and articular cartilage samples were obtained from 22 patients who underwent total knee arthroplasty. Chondrocyte migration and proliferation were assessed using cartilage fragment/atelocollagen composites to compare the characteristics and regenerative potential of osteophytes and articular cartilage. Histological analysis revealed differences in cartilage composition between osteophytes and articular cartilage, with higher expression of type X collagen and increased chondrocyte proliferation in the osteophyte cartilage. Gene expression analysis identified distinct gene expression profiles between osteophytes and articular cartilage; the expression levels of COL2A1, ACAN, and SOX9 were not significantly different. Chondrocytes derived from osteophyte cartilage exhibit enhanced proliferation, and glycosaminoglycan production is increased in both osteophytes and articular cartilage. Osteophyte cartilage may serve as a viable alternative source of MCI for treating large cartilage defects in OA.

Whole-Transcriptome Sequencing of Knee Joint Cartilage from Kashin-Beck Disease and Osteoarthritis Patients.

The aim of this study was to provide a comprehensive understanding of similarities and differences in mRNAs, lncRNAs, and circRNAs within cartilage for Kashin-Beck disease (KBD) compared to osteoarthritis (OA). We conducted a comparison of the expression profiles of mRNAs, lncRNAs, and circRNAs via whole-transcriptome sequencing in eight KBD and ten OA individuals. To facilitate functional annotation-enriched analysis for differentially expressed (DE) genes, DE lncRNAs, and DE circRNAs, we employed bioinformatic analysis utilizing Gene Ontology (GO) and KEGG. Additionally, using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), we validated the expression levels of four cartilage-related genes in chondrocytes. We identified a total of 43 DE mRNAs, 1451 DE lncRNAs, and 305 DE circRNAs in KBD cartilage tissue compared to OA (q value < 0.05; |log2FC| > 1). We also performed competing endogenous RNA network analysis, which identified a total of 65 lncRNA-mRNA interactions and 4714 miRNA-circRNA interactions. In particular, we observed that circRNA12218 had binding sites for three miRNAs targeting ACAN, while circRNA12487 had binding sites for seven miRNAs targeting COL2A1. Our results add a novel set of genes and non-coding RNAs that could potentially serve as candidate diagnostic biomarkers or therapeutic targets for KBD patients.

A single cell transcriptional atlas of early synovial joint development.

Synovial joint development begins with the formation of the interzone, a region of condensed mesenchymal cells at the site of the prospective joint. Recently, lineage-tracing strategies have revealed that Gdf5-lineage cells native to and from outside the interzone contribute to most, if not all, of the major joint components. However, there is limited knowledge of the specific transcriptional and signaling programs that regulate interzone formation and fate diversification of synovial joint constituents. To address this, we have performed single cell RNA-Seq analysis of 7329 synovial joint progenitor cells from the developing murine knee joint from E12.5 to E15.5. By using a combination of computational analytics, in situ hybridization and in vitro characterization of prospectively isolated populations, we have identified the transcriptional profiles of the major developmental paths for joint progenitors. Our freely available single cell transcriptional atlas will serve as a resource for the community to uncover transcriptional programs and cell interactions that regulate synovial joint development.

Blocking TXNIP reduced IL-1ß Induced chondrocyte cell inflammation.

Osteoarthritis (OA) is the most common joint disease in the elderly and is characterized by progressive and irreversible degeneration of articular cartilage, particularly cartilage loss and callus formation. This study would like to investigate the important role and the molecular mechanism of OA progression following interleukin 1ß (IL-1ß)-induced chondrocyte injury regulated by TXNIP. In this study, high-purity mouse chondrocytes were obtained by enzymatic two-step digestion for primary culture. Toluidine blue staining and type II collagen immunofluorescence were used to identify cells through histochemical staining after slide mounting. The relative expression of TXNIP was detected by immunohistochemical staining and qRT-PCR.Aiming at the shRNA sequence of the TXNIP gene, the shRNA expression vector was constructed and packaged with lentivirus to form the lentiviral vector shTXNIP. After inhibiting the expression of TXNIP by transfecting shTXNIP into normal mouse chondrocytes, the CCK-8 kit was used for detecting its effect on cell proliferation after transfection, and the effect on chondrocyte apoptosis was detected by flow cytometry. The staining kit was used to detect the effect of TXNIP knockout on chondrocyte aging, and the differential expression of TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen genes in chondrocytes was detected by RT-PCR and Western-bolt. Western blot was used to detect the expression of upstream-related protein P-ERK, downstream-related protein NLRP3 and Caspase1 after inflammatory injury of mouse articular chondrocytes. Results showed that the expression level of TXNIP in chondrocytes induced by different concentrations of il-1ß was proportional to the concentration. After silencing TXNIP by shRNA, cell proliferation increased, chondrocyte apoptosis was weakened, and chondrocyte aging was weakened. The differential expression of genes such as TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen and the differential expression of protein levels were relatively decreased. In addition, the expression of the upstream-related protein P-ERK did not change much when TXNIP was silenced, and the expression levels of the downstream-related proteins NLRP3 and Caspase1 were slightly reduced. In conclusion, silencing TXNIP can inhibit il-1ß-induced chondrocyte apoptosis and aging, and has a positive effect on cell proliferation. However, this study has not clarified the molecular mechanism involved in TXNIP and the process of its signaling expression pathway.

Two major genes associated with autoimmune arthritis, Ncf1 and Fcgr2b, additively protect mice by strengthening T cell tolerance.

A breach of T cell tolerance is considered as a major step in the pathogenesis of rheumatoid arthritis. In collagen-induced arthritis (CIA) model, immunization with type II collagen (COL2) leads to arthritis in mice through T cells responding to the immunodominant COL2259-273 peptide. T cells could escape from thymus negative selection because endogenous COL2259-273 peptide only weakly binds to the major histocompatibility complex class II (MHCII) molecule Aq. To investigate the regulation of T cell tolerance, we used a new mouse strain BQ.Col2266E with homozygous D266E mutations in the Col2 gene leading to a replacement of the endogenous aspartic acid (D) to glutamic acid (E) at position 266 of the COL2259-273 peptide, resulting in stronger binding to Aq. We also established BQ.Col2264R mice carrying an additional K264R mutation changed the lysine (K) at position 264 to eliminate the major TCR recognition site. The BQ.Col2266E mice were fully resistant to CIA, while the BQ.Col2264R mice developed severe arthritis. Furthermore, we studied two of the most important non-MHCII genes associated with CIA, i.e., Ncf1 and Fcgr2b. Deficiency of either gene induced arthritis in BQ.Col2266E mice, and the downstream effects differ as Ncf1 deficiency reduced Tregs and was likely to decrease expression of autoimmune regulator (AIRE) while Fcgr2b did not. In conclusion, the new human-mimicking mouse model has strong T cell tolerance to COL2, which can be broken by deficiency of Fcgr2b or Ncf1, allowing activation of autoreactive T cells and development of arthritis.

CD44 mediates hyaluronan to promote the differentiation of human amniotic mesenchymal stem cells into chondrocytes.

OBJECTIVES: CD44 is the major receptor for hyaluronan (HA), but its effect on HA-induced differentiation of human amnion mesenchymal stem cells into chondrocytes is unclear. This study aimed to investigate the effects and mechanisms of CD44 in HA-induced chondrogenesis. METHODS: Immunocytochemistry and toluidine blue staining were used to assess the secretion of type II collagen and aggrecan, respectively. qRT-PCR and western blotting were performed to evaluate the expression of key genes and proteins. RESULTS: The expression of aggrecan and type II collagen was downregulated after using the anti-CD44 antibody (A3D8). The transcriptional levels of chondrocytes­associated genes SRY­box transcription factor 9, aggrecan, and collagen type II alpha 1 chain were also decreased. Thus, CD44 may mediate HA-induced differentiation of hAMSCs into chondrocytes. Further investigation indicated that expression of phosphorylated (p)­Erk1/2 and p­Smad2 decreased following CD44 inhibition. The changes in the expression of p-Erk1/2 and p-Smad2 were consistent after using the ERK1/2 inhibitor (U0126) and agonist (EGF), respectively. After administering the p-Smad2 inhibitor, the expression levels of p-ERK1/2 and p-Smad2 appeared downregulated. The results showed crosstalk between Erk1/2 and Smad2. Moreover, inhibition of p-Erk1/2 and p-Smad2 significantly reduced the accumulation of aggrecan and type II collagen. CONCLUSION: These data indicate that CD44 mediates HA-induced differentiation of hAMSCs into chondrocytes by regulating Erk1/2 and Smad2 signaling.

Hypoxia Preconditioned Serum (HPS) Promotes Proliferation and Chondrogenic Phenotype of Chondrocytes In Vitro.

Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects remains challenging in terms of maintaining chondrogenic phenotype during in vitro chondrocyte expansion. Growth factor supplementation has been found supportive in improving ACI outcomes by promoting chondrocyte redifferentiation. Here, we analysed the chondrogenic growth factor concentrations in the human blood-derived secretome of Hypoxia Preconditioned Serum (HPS) and assessed the effect of HPS-10% and HPS-40% on human articular chondrocytes from osteoarthritic cartilage at different time points compared to normal fresh serum (NS-10% and NS-40%) and FCS-10% culture conditions. In HPS, the concentrations of TGF-beta1, IGF-1, bFGF, PDGF-BB and G-CSF were found to be higher than in NS. Chondrocyte proliferation was promoted with higher doses of HPS (HPS-40% vs. HPS-10%) and longer stimulation (4 vs. 2 days) compared to FCS-10%. On day 4, immunostaining of the HPS-10%-treated chondrocytes showed increased levels of collagen type II compared to the other conditions. The promotion of the chondrogenic phenotype was validated with quantitative real-time PCR for the expression of collagen type II (COL2A1), collagen type I (COL1A1), SOX9 and matrix metalloproteinase 13 (MMP13). We demonstrated the highest differentiation index (COL2A1/COL1A1) in HPS-10%-treated chondrocytes on day 4. In parallel, the expression of differentiation marker SOX9 was elevated on day 4, with HPS-10% higher than NS-10/40% and FCS-10%. The expression of the cartilage remodelling marker MMP13 was comparable across all culture conditions. These findings implicate the potential of HPS-10% to improve conventional FCS-based ACI culture protocols by promoting the proliferation and chondrogenic phenotype of chondrocytes during in vitro expansion.

A Model of Type II Collagen-Induced Spondylitis and Arthritis in F1 Hybrid Male Mice.

In this study, we tested a new model of ankylosing spondylitis in order to determine its histological and radiological features needed to investigate peripheral arthritis, spondylitis, and formation of the new bone tissues. F1 hybrid male mice (BALB/c×DBA/1), a progeny of spondylitis-susceptible BALB/c male mice and rheumatoid arthritis-susceptible DBA/1 female mice, were immunized intraperitoneally with bovine type II collagen (CII) mixed with adjuvant dimethyldioctadecylammonium bromide. Radiological and histological studies were performed at the peak of swelling, redness, and stiffness. The incidence of peripheral arthritis and spondylitis induced by CII in F1 hybrid mice were 66 and 62%, respectively. X-ray examination revealed bone erosion and spondylitis in the peripheral joints, as well as the formation of new bone tissues in the coccygeal vertebrae and between LIII and LIV vertebrae. The histological study showed lymphocyte and plasma cell infiltration, capillary dilation, congestion, and endochondral ossification of the lumbar vertebrae. This novel model of CII-induced spondylitis in F1 hybrid mice provoked axial and peripheral arthritides inducing chronic inflammation. In this model, the formation of new bone tissue in the stiff spine is characterized by endochondral ossification. The advanced model is an additional and valuable tool for investigation of the autoimmune reactions in spondylitis.

A Novel missense mutation of COL2A1 gene in a large family with stickler syndrome type I.

Stickler syndrome type I (STL1, MIM 108300) is characterized by ocular, auditory, skeletal and orofacial manifestations. Nonsyndromic ocular STL1 (MIM 609508) characterized by predominantly ocular features is a subgroup of STL1, and it is inherited in an autosomal dominant manner. In this study, a novel variant c.T100>C (p.Cys34Arg) in COL2A1 related to a large nonsyndromic ocular STL1 family was identified through Exome sequencing (ES). Bioinformatics analysis indicated that the variant site was highly conserved and the pathogenic mechanism of this variant may involve in affected structure of chordin-like cysteine-rich (CR) repeats of ColIIA. Minigene assay indicated that this variant did not change alternative splicing of exon2 of COL2A1. Moreover, the nonsyndromic ocular STL1 family with 16 affected members showed phenotype variability and certain male gender trend. None of the family members had hearing loss. Our findings would expand the knowledge of the COL2A1 mutation spectrum, and phenotype variability associated with nonsyndromic ocular STL1. Search for genetic modifiers and related molecular pathways leading to the phenotype variation warrants further studies.

CDC5L promotes early chondrocyte differentiation and proliferation by modulating pre-mRNA splicing of SOX9, COL2A1, and WEE1.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is a common pathological condition that causes intractable myelopathy and radiculopathy, mainly the result of an endochondral ossification-like process. Our previous genome-wide association study identified six susceptibility loci for OPLL, including the cell division cycle 5-like (CDC5L) gene region. Here, we found CDC5L to be expressed in type II collagen-producing chondrocyte-like fibroblasts in human OPLL specimens, as well as in differentiating ATDC5 chondrocytes. Cdc5l siRNA transfection in murine chondrocytes decreased the expression of the early chondrogenic genes Sox9 and Col2a1, diminished the cartilage matrix production, and enhanced the expression of parathyroid-hormone-related protein (a resting chondrocyte marker). We also showed that Cdc5l shRNA suppressed the growth of cultured murine embryonal metatarsal cartilage rudiments and that Cdc5l knockdown suppressed the growth of ATDC5 cells. Fluorescence-activated cell sorting analysis revealed that the G2/M cell cycle transition was blocked; our data showed that Cdc5l siRNA transfection enhanced expression of Wee1, an inhibitor of the G2/M transition. Cdc5l siRNA also decreased the pre-mRNA splicing efficiency of Sox9 and Col2a1 genes in both ATDC5 cells and primary chondrocytes; conversely, loss of Cdc5l resulted in enhanced splicing of Wee1 pre-mRNA. Finally, an RNA-binding protein immunoprecipitation assay revealed that Cdc5l bound directly to these target gene transcripts. Overall, we conclude that Cdc5l promotes both early chondrogenesis and cartilage growth and may play a role in the etiology of OPLL, at least in part by fine-tuning the pre-mRNA splicing of chondrogenic genes and Wee1, thus initiating the endochondral ossification process.

Combining stretching and gallic acid to decrease inflammation indices and promote extracellular matrix production in osteoarthritic human articular chondrocytes.

Osteoarthritis (OA) patients undergo cartilage degradation and experience painful joint swelling. OA symptoms are caused by inflammatory molecules and the upregulation of catabolic genes leading to the breakdown of cartilage extracellular matrix (ECM). Here, we investigate the effects of gallic acid (GA) and mechanical stretching on the expression of anabolic and catabolic genes and restoring ECM production by osteoarthritic human articular chondrocytes (hAChs) cultured in monolayers. hAChs were seeded onto conventional plates or silicone chambers with or without 100 µM GA. A 5% cyclic tensile strain (CTS) was applied to the silicone chambers and the deposition of collagen and glycosaminoglycan, and gene expressions of collagen types II (COL2A1), XI (COL11A2), I (COL1A1), and X (COL10A1), and matrix metalloproteinases (MMP-1 and MMP-13) as inflammation markers, were quantified. CTS and GA acted synergistically to promote the deposition of collagen and glycosaminoglycan in the ECM by 14- and 7-fold, respectively. Furthermore, the synergistic stimuli selectively upregulated the expression of cartilage-specific proteins, COL11A2 by 7-fold, and COL2A1 by 47-fold, and, in contrast, downregulated the expression of MMP-1 by 2.5-fold and MMP-13 by 125-fold. GA supplementation with CTS is a promising approach for restoring osteoarthritic hAChs ECM production ability making them suitable for complex tissue engineering applications.

Low-intensity photobiomodulation at 632.8 nm increases tgfß3, col2a1, and sox9 gene expression in rat bone marrow mesenchymal stem cells in vitro.

The high incidence of cartilage destructions, as well as the social and economic importance of this pathology attracted great interest to the problem. At the present time, some data are available about the 632.8 nm low-intensity laser photobiomodulation positive effect on the cartilage tissue proliferation. The effect of this wavelength laser irradiation on the mesenchimal stem cell (MSC) differentiation in the chondrogenic direction was studied. The main aim of this work was to assess the low-intensity photobiomodulation effect on chondrogenesis. In this experiment, the cell model was used to compare the photobiomodulation and cytokine Tgfß3 (transforming growth factor ß 3) effects. Bone marrow MSCs were isolated from Wistar rats and cultured for the third passage. Chondrogenic effects of low-intensity He-Ne laser photobiomodulation and cytokine Tgfß3 (10 ng/µL) were analyzed and compared after 21 days. The radiation source was the standard LGN-208 helium-neon (He-Ne) laser (632.8 nm, 1.7 mWt). Irradiation was performed cyclically for 15 min with 45-min pauses. The increase of the responsible for chondrogenesis (col2a1, tgfß3, and sox9) main gene expression under the photobiomodulation at 632.8 nm was evaluated in comparison with Tgfß3 effect. The tgfß3, col2a1, and sox9 gene expression increase was obtained in two experimental groups: using the laser photobiomodulation and cytokine Tgfß3 effect. Gene expression levels of tgfß3, col2a1, and sox9 were measured using real-time polymerase chain reaction (RT-PCR) according to the -ΔΔCt method. It was found that the responsible for chondrogenesis genes expression (tgfß3, col2a1, sox9) increased under the action of specific laser photobiomodulation during the observation period (from 0 to 21 days). The chondrogenic differentiation effect under the laser irradiation is less significant than Tgfß3 cytokine effect.

Therapeutic Targets and Emerging Treatments in Advanced Chondrosarcoma.

Due to resistance to standard anticancer agents, it is difficult to control the disease progression in patients with metastatic or unresectable chondrosarcoma. Novel therapeutic approaches, such as molecule-targeting drugs and immunotherapy, are required to improve clinical outcomes in patients with advanced chondrosarcoma. Recent studies have suggested several promising biomarkers and therapeutic targets for chondrosarcoma, including IDH1/2 and COL2A1. Several molecule-targeting agents and immunotherapies have shown favorable antitumor activity in clinical studies in patients with advanced chondrosarcomas. This review summarizes recent basic studies on biomarkers and molecular targets and recent clinical studies on the treatment of chondrosarcomas.

Hearing Outcomes in Stickler Syndrome: Variation Due to COL2A1 and COL11A1.

OBJECTIVES: Stickler syndrome (SS) is a heterogeneous inherited connective tissue disorder, often due to a mutation in COL2A1 or COL11A1. Mutations in these genes cause collagen abnormalities affecting ocular, auditory, orofacial, and skeletal systems, including hearing loss, micrognathia, and cleft palate. Understanding the variability of hearing phenotypes based on genetic mutation has a significant impact on treatment and long-term care. DESIGN: A retrospective chart review of pediatric patients with a confirmed diagnosis of SS between January 2003 and December 2018 at a tertiary pediatric hospital was performed. Patients were excluded if they did not have genetic evaluation, craniofacial/ear, nose, and throat evaluation, and/or audiologic testing. Charts were reviewed for the following information: age, race, sex, SS diagnosis, genetic variant of SS, and audiological testing data. RESULTS: There were 29 confirmed patients with SS who met criteria, 16 with type I (COL2A1) and 13 with type II (COL11A1). Of the 13 patients with type II, 12 (92%) demonstrated hearing loss, ranging in severity from mild to severe. In type I, 25% of patients had mild or resolved hearing loss. CONCLUSION: Results suggest that patients with type II SS are more likely to have congenital hearing loss than type I. Data also suggest that the COL11A1 mutation shows consistently more severe hearing loss than the COL2A1 mutation.

Environment-Sensing Aryl Hydrocarbon Receptor Inhibits the Chondrogenic Fate of Modulated Smooth Muscle Cells in Atherosclerotic Lesions.

BACKGROUND: Smooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. METHODS: We combined RNA-sequencing, chromatin immunoprecipitation followed by sequencing, assay for transposase-accessible chromatin using sequencing, and in vitro assays in human coronary artery SMCs, with single-cell RNA-sequencing, histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease. RESULTS: Genomic studies coupled with functional assays in cultured human coronary artery SMCs revealed that AHR modulates the human coronary artery SMC phenotype and suppresses ossification in these cells. Lineage-tracing and activity-tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMCs in the atherosclerotic lesion cap. Furthermore, single-cell RNA-sequencing studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMCs expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term "chondromyocytes," were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMCs in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout in comparison with wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses. CONCLUSIONS: Overall, we conclude that AHR promotes the maintenance of lesion cap integrity and diminishes the disease-related SMC-to-chondromyocyte transition in atherosclerotic tissues.

Do biomarkers allow a differentiation between osteonecrosis of the femoral head and osteoarthritis of the hip? - a biochemical, histological and gene expression analysis.

OBJECTIVE: Osteonecrosis of the femoral head (ONFH) is a devastating disease of the hip joint. Its early diagnosis is crucial to increase the chances of joint preserving, yet difficult due to similarities with osteoarthritis (OA) of the hip in its clinical appearance. The purpose of this study was to enhance the understanding of ONFH and its pathologic processes in contrast to OA and to identify serum biomarkers helping to improve the diagnosis of the disease. DESIGN: Bone and bone marrow samples were collected from 24 patients diagnosed with OA and 25 patients with ONFH during total hip replacement surgery. RNA was isolated, histological examination, determination of free reactive oxygen species as well as gene expression and biomarker analysis were performed. RESULTS: Histological analysis revealed differences in the structural and cellular pattern between the groups. Gene expression analysis revealed a significant upregulation for the genes ASPN, COL1A1, COL2A1 and IL6 and a significant downregulation for HIF1A in ONFH compared to OA group. Analysis of serum biomarkers showed significant differences between the groups for asporin and adiponectin. A final logistical regression model including the parameters adiponectin, asporin and HIF 1α was overall significant, explained 34.5 % of variance and classified 74.5 % of the cases correctly. CONCLUSION: The combination of adiponectin, asporin and HIF 1α as serum biomarkers revealed a classification accuracy of 74.5 %. The information provided in this study may help to enhance the understanding of pathologic processes in ONFH and to elaborate further aspects of prediction and treatment.

Zinc finger protein-440 promotes cartilage degenerative mechanisms in human facet and knee osteoarthritis chondrocytes.

OBJECTIVES: To investigate the role of zinc finger protein 440 (ZNF440) in the pathophysiology of cartilage degeneration during facet joint (FJ) and knee osteoarthritis (OA). METHODS: Expression of ZNF440 in FJ and knee cartilage was determined by immunohistochemistry, quantitative (q)PCR, and Western blotting (WB). Human chondrocytes isolated from FJ and knee OA cartilage were cultured and transduced with ZNF440 or control plasmid, or transfected with ZNF440 or control small interfering RNA (siRNA), with/without interleukin (IL)-1ß. Gene and protein levels of catabolic, anabolic and apoptosis markers were determined by qPCR or WB, respectively. In silico analyses were performed to determine compounds with potential to inhibit expression of ZNF440. RESULTS: ZNF440 expression was increased in both FJ and knee OA cartilage compared to control cartilage. In vitro, overexpression of ZNF440 significantly increased expression of MMP13 and PARP p85, and decreased expression of COL2A1. Knockdown of ZNF440 with siRNA partially reversed the catabolic and cell death phenotype of human knee and FJ OA chondrocytes stimulated with IL-1ß. In silico analysis followed by validation assays identified scriptaid as a compound with potential to downregulate the expression of ZNF440. Validation experiments showed that scriptaid reduced the expression of ZNF440 in OA chondrocytes and concomitantly reduced the expression of MMP13 and PARP p85 in human knee OA chondrocytes overexpressing ZNF440. CONCLUSIONS: The expression of ZNF440 is significantly increased in human FJ and knee OA cartilage and may regulate cartilage degenerative mechanisms. Furthermore, scriptaid reduces the expression of ZNF440 and inhibits its destructive effects in OA chondrocytes.