Small RNAseq between young and old normal equine chondrocytes
ABSTRACT: This project aims to identify differentially expressed small non coding RNAs between young and old normal chondrocytes isolated from the metacarpophalangeal joint of 10 horses; 5 young and 5 old.
Project description:Small RNA isolated from synovial fluid of the metacarpophalangeal joints of horses. Horses either had minimal signs of osteoarthritis based on macroscopic and microscopic joint scoring or early (mild) osteoarthritis. Differential expression of small non-coding RNAs was undertaken.
Project description:Recent studies have shown that mesenchymal stem cells (MSCs) can play a restorative role against degenerative joint diseases in horses. The purpose of this study was to investigate whether fetal bone marrow-derived cells (BMC)-derived nanoparticles (BMC-NPs) can stimulate the survival of equine chondrocytes. Equine fetal BMCs were isolated and characterized, and the role of BMC-NPs s in equine chondrocytes undergoing inflammatory cell death was examined. BMCs have several characteristics, such as the potential to differentiate into chondrocytes and osteocytes. Additionally, BMCs expressed immunoregulatory genes in response to treatment with tumor necrosis factor-alpha (TNF-?) and Interleukin 1 beta (IL-1?). We found that BMC-NPs were taken up by equine chondrocytes. Functionally, BMC-NPs promoted the growth of chondrocytes, and reduced apoptosis induced by inflammatory cytokines. Furthermore, we observed that BMC-NPs upregulated the phosphorylation of protein kinase B (Akt) in the presence of IL-1?, and reduced the phosphorylation of TNF-?-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the chondrocytes. Cumulatively, our study demonstrated that equine fetal BMC-NPs have the potential to stimulate the survival of chondrocytes damaged by inflammatory cytokines. Thus, BMC-NPs may become an alternative cell-free allogenic therapeutic for degenerative joint diseases in horses.
Project description:The present study reports for the first time the presence of giant crystals in mitochondria of equine chondrocytes. These structures show dark contrast in TEM images as well as a granular substructure of regularly aligned 1-2 nm small units. Different zone axes of the crystalline structure were analysed by means of Fourier transformation of lattice-resolution TEM images proving the crystalline nature of the structure. Elemental analysis reveals a high content of nitrogen referring to protein. The outer shape of the crystals is geometrical with an up to hexagonal profile in cross sections. It is elongated, spanning a length of several micrometres through the whole cell. In some chondrocytes, several crystals were found, sometimes combined in a single mitochondrion. Crystals were preferentially aligned along the long axis of the cells, thus appearing in the same orientation as the chondrocytes in the tissue. Although no similar structures have been found in the cartilage of any other species investigated, they have been found in cartilage repair tissue formed within a mechanically stimulated equine chondrocyte construct. Crystals were mainly located in superficial regions of cartilage, especially in joint regions of well-developed superficial layers, more often in yearlings than in adult horses. These results indicate that intramitochondrial crystals are related to the high mechanical stress in the horse joint and potentially also to the increased metabolic activity of immature individuals.
Project description:Messenger RNA (mRNA) decay rates control not only gene expression levels, but also responsiveness to altered transcriptional input. We undertook this study to examine transcriptome-wide posttranscriptional regulation in both normal and osteoarthritic (OA) human articular chondrocytes.Human articular chondrocytes were isolated from normal or OA tissue. Equine articular chondrocytes were isolated from young or old horses at a commercial abattoir. RNA decay was measured across the transcriptome in human cells by microarray analysis following an actinomycin D chase. Messenger RNA levels in samples were confirmed using quantitative reverse transcription-polymerase chain reaction.Examination of total mRNA expression levels demonstrated significant differences in the expression of transcripts between normal and OA chondrocytes. Interestingly, almost no difference was observed in total mRNA expression between chondrocytes from intact OA cartilage and those from fibrillated OA cartilage. Decay analysis revealed a set of rapidly turned over transcripts associated with transcriptional control and programmed cell death that were common to all chondrocytes and contained binding sites for abundant cartilage microRNAs. Many transcripts exhibited altered mRNA half-lives in human OA chondrocytes compared to normal cells. Specific transcripts whose decay rates were altered were generally less stable in these pathologic cells. Examination of selected genes in chondrocytes from young and old healthy horses did not identify any change in mRNA turnover.This is the first investigation into the "posttranscriptome" of the chondrocyte. It identifies a set of short-lived chondrocyte mRNAs likely to be highly responsive to altered transcriptional input as well as mRNAs whose decay rates are affected in OA chondrocytes.
Project description:Cartilage injury often precipitates osteoarthritis which has driven research to bolster repair in cartilage impact damage. Autologous chondrocytes transduced with rAAV5-IGF-I were evaluated in chondral defects in a well-established large animal model. Cartilage was harvested from the talus of 24 horses; chondrocytes were isolated and stored frozen. Twenty million cells were cultured and transduced with 10(5) AAV vg/cell prior to implantation. Chondrocytes from eight horses were transduced with rAAV5-IGF-I, chondrocytes from eight horses with rAAV5-GFP, and chondrocytes from eight horses were not transduced. A 15?mm full-thickness chondral defect was created arthroscopically in the lateral trochlear ridge of the femur in both femoropatellar joints. Treated defects were filled with naive or gene-enhanced chondrocytes, in fibrin vehicle. Control defects in the opposite limb received fibrin alone. rAAV5-IGF-I transduced chondrocytes resulted in significantly better healing at 8 week arthroscopy and 8 month necropsy examination when compared to controls. At 8 months, defects implanted with cells expressing IGF-I had better histological scores compared to control defects and defects repaired with naive chondrocytes. This included increased chondrocyte predominance and collagen type II, both features of hyaline-like repair tissue. The equine model closely approximates human cartilage healing, indicating AAV-mediated genetic modification of chondrocytes may be clinically beneficial to humans.
Project description:Osteochondrosis is a failure of the endochondral ossification that affects developing joints in humans and several animal species. It is a localized idiopathic joint disorder characterized by focal chondronecrosis and growing cartilage retention, which can lead to the formation of fissures, subchondral bone cysts, or intra-articular fragments. Osteochondrosis is a complex multifactorial disease associated with extracellular matrix alterations and failure in chondrocyte differentiation, mainly due to genetic, biochemical, and nutritional factors, as well as traumas. This study describes the main proteomic alterations occurring in chondrocytes isolated from osteochondrotic cartilage fragments. A comparative analysis performed on equine osteochondrotic and healthy chondrocytes showed 26 protein species as differentially represented. In particular, quantitative changes in the extracellular matrix, cytoskeletal and chaperone proteins, and in cell adhesion and signaling molecules were observed in osteochondrotic cells, compared to healthy controls. Functional group analysis annotated most of these proteins in "growth plate and cartilage development", while others were included in "glycolysis and gluconeogenesis", "positive regulation of protein import", "cell-cell adhesion mediator activity", and "mitochondrion nucleoid". These results may help to clarify some chondrocyte functional alterations that may play a significant role in determining the onset and progression of equine osteochondrosis and, being related, of human juvenile osteochondrosis.
Project description:Ageing is a leading risk factor predisposing cartilage to osteoarthritis. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs (sncRNAs). RNA from young and old chondrocytes from macroscopically normal equine metacarpophalangeal joints was extracted and subjected to small RNA sequencing (RNA-seq). Differential expression analysis was performed in R using package DESeq2. For transfer RNA (tRNA) fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. Selected microRNA (miRNAs or miRs) and small nucleolar RNA (snoRNA) findings were validated using real-time quantitative Polymerase Chain Reaction (qRT-PCR) in an extended cohort of equine chondrocytes. tRNA fragments were further investigated in low- and high-grade OA human cartilage tissue. In total, 83 sncRNAs were differentially expressed between young and old equine chondrocytes, including miRNAs, snoRNAs, small nuclear RNAs (snRNAs), and tRNAs. qRT-PCR analysis confirmed findings. tRNA fragment analysis revealed that tRNA halves (tiRNAs), tiRNA-5035-GluCTC and tiRNA-5031-GluCTC-1 were reduced in both high grade OA human cartilage and old equine chondrocytes. For the first time, we have measured the effect of ageing on the expression of sncRNAs in equine chondrocytes. Changes were detected in a number of different sncRNA species. This study supports a role for sncRNAs in ageing cartilage and their potential involvement in age-related cartilage diseases.
Project description:Osteoarthritis (OA) is the most common form of joint disease in middle-aged and older individuals. Previous studies have shown that over-expression of matrix-degrading proteinases and proinflammatory cytokines is associated with osteoarthritic cartilage degradation. However, it remains unclear which transcription factors regulate the expression of these cartilage-degrading molecules in articular chondrocytes. This study demonstrated that mice lacking Nfat1, a member of the nuclear factor of activated T cells (NFAT) transcription factors, exhibited normal skeletal development but displayed loss of type II collagen (collagen-2) and aggrecan with over-expression of specific matrix-degrading proteinases and proinflammatory cytokines in young adult articular cartilage of load-bearing joints. These initial changes are followed by articular chondrocyte proliferation/clustering, progressive articular surface destruction, periarticular chondro-osteophyte formation and exposure of thickened subchondral bone, all of which resemble human OA. Forced expression of Nfat1 delivered with lentiviral vectors in cultured 3 month-old primary Nfat1 knockout (Nfat1(-/-)) articular chondrocytes partially or completely rescued the abnormal catabolic and anabolic activities of Nfat1(-/-) articular chondrocytes. These new findings revealed a previously unrecognized critical role of Nfat1 in maintaining the physiological function of differentiated adult articular chondrocytes through regulating the expression of specific matrix-degrading proteinases and proinflammatory cytokines. Nfat1 deficiency causes OA due to an imbalance between the catabolic and anabolic activities of adult articular chondrocytes, leading to articular cartilage degradation and failed repair activities in and around articular cartilage. These results may provide new insights into the aetiology, pathogenesis and potential therapeutic strategies for osteoarthritis.
Project description:The aim of this work was to develop an equine metacarpophalangeal joint model that induces osteoarthritis that is not primarily mediated by instability or inflammation. The study involved six Standardbred horses. Standardized cartilage surface damage or "grooves" were created arthroscopically on the distal dorsal aspect of the lateral and medial metacarpal condyles of a randomly chosen limb. The contralateral limb was sham operated. After 2 weeks of stall rest, horses were trotted 30 minutes every other day for 8 weeks, then evaluated for lameness and radiographed. Synovial fluid was analyzed for cytology and biomarkers. At 10 weeks post-surgery, horses were euthanized for macroscopic and histologic joint evaluation. Arthroscopic grooving allowed precise and identical damage to the cartilage of all animals. Under the controlled exercise regime, this osteoarthritis groove model displayed significant radiographic, macroscopic, and microscopic degenerative and reactive changes. Histology demonstrated consistent surgically induced grooves limited to non-calcified cartilage and accompanied by secondary adjacent cartilage lesions, chondrocyte necrosis, chondrocyte clusters, cartilage matrix softening, fissuring, mild subchondral bone inflammation, edema, and osteoblastic margination. Synovial fluid biochemistry and cytology demonstrated significantly elevated total protein without an increase in prostaglandin E2, neutrophils, or chondrocytes. This equine metacarpophalangeal groove model demonstrated that standardized non-calcified cartilage damage accompanied by exercise triggered altered osteochondral morphology and cartilage degeneration with minimal or inefficient repair and little inflammatory response. This model, if validated, would allow for assessment of disease processes and the effects of therapy.
Project description:Degenerative joint disease (DJD) is a major cause of reduced athletic function and retirement in equine performers. For this reason, regenerative therapies for DJD have gained increasing interest. Platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) were isolated from a 6-year-old donor horse. MSCs were either used in their native state or after chondrogenic induction. In an initial study, 20 horses with naturally occurring DJD in the fetlock joint were divided in 4 groups and injected with the following: 1) PRP; 2) MSCs; 3) MSCs and PRP; or 4) chondrogenic induced MSCs and PRP. The horses were then evaluated by means of a clinical scoring system after 6 weeks (T1), 12 weeks (T2), 6 months (T3) and 12 months (T4) post injection. In a second study, 30 horses with the same medical background were randomly assigned to one of the two combination therapies and evaluated at T1. The protein expression profile of native MSCs was found to be negative for major histocompatibility (MHC) II and p63, low in MHC I and positive for Ki67, collagen type II (Col II) and Vimentin. Chondrogenic induction resulted in increased mRNA expression of aggrecan, Col II and cartilage oligomeric matrix protein (COMP) as well as in increased protein expression of p63 and glycosaminoglycan, but in decreased protein expression of Ki67. The combined use of PRP and MSCs significantly improved the functionality and sustainability of damaged joints from 6 weeks until 12 months after treatment, compared to PRP treatment alone. The highest short-term clinical evolution scores were obtained with chondrogenic induced MSCs and PRP. This study reports successful in vitro chondrogenic induction of equine MSCs. In vivo application of (induced) MSCs together with PRP in horses suffering from DJD in the fetlock joint resulted in a significant clinical improvement until 12 months after treatment.