Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
(N/A)
Non Technical Summary
Osteoarthritis (OA) is the most common cause of chronic lameness in horses and places a significant economic and welfare burden on the equine industry due to the cost of treatment and loss of use of affected animals. Post-traumatic osteoarthritis (PTOA) affects equine athletes across breeds and disciplines and can lead to shortened careers and reduced quality of life. While the precise risk of a horse developing PTOA after an injury (either a single event or repetitive use), data from human studies suggests that it may be as high as 50% even with surgical intervention. Unfortunately, PTOA is usually only diagnosed after permanent cartilage damage has occurred and there are no available treatments that can halt or reverse the disease. Ideally, treatment for PTOA would be started before irreversible joint damage has occurred. However, the development of early diagnostic tests and new treatments for PTOA relies on our understanding of the earliest triggering factors for the disease and how it progresses in its early stages. The objective of this study is to identify these early triggering factors by measuring changes in gene and protein expression in a model of joint injury that is specifically designed to mimic naturally occurring disease in horses. A major benefit of the "fetlock chip" model that we will use is that the horses can be evaluated at multiple time points during the study, providing a "video" of disease-related changes in the joints rather than just a "snapshot" of disease. Further, this model does not require sacrificing the subjects, and therefore all of the measurements that will be used could be applied to clinical patients in the future.Twelve horses will be enrolled in the study. Each horse will have a small bone chip created arthroscopically in one front fetlock (metacarpophalangeal) joint. The other front fetlock will be surgically explored but no chip will be made, serving as a control and allowing us to use fewer horses while still obtaining good data for comparisons. A small sample of joint fluid and a sample of the tissue lining the joint will be taken at the time of surgery for baseline measurements of gene and protein expression. After surgery, the horses will undergo an exercise program. Weekly lameness exams will be performed, and additional samples of joint fluid will be collected every 8 weeks. A "second-look" surgery will be performed at 16 weeks after injury; the joints will be photographed and another sample of the tissue lining each joint will be collected. The horses will then undergo another period of exercise with clinical evaluations and joint fluid collection. Finally, 32 weeks after injury, a third arthroscopic surgery will be performed at which point the bone chip will be removed. Changes in gene expression (from the tissue lining the joint collected at each surgery) and protein expression (from the joint fluid) will be evaluated over time within the injured joints and will also be compared between the injured and non-injured joints. We will determine which, if any, gene and protein changes predict clinical changes such as lameness or joint swelling over the course of the study.At the end of this project, we expect to have identified some of the earliest changes in gene and protein expression after a joint injury, which will fill a critical gap in our understanding of how PTOA begins in equine athletes. In addition to insights about the disease, we expect to identify genes and proteins that could serve as targets for the development of early diagnostic tests or for new treatments. In the future, we will verify our results in horses with naturally occurring PTOA as we move towards clinical applications of this work.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Goals / Objectives
Post-traumatic osteoarthritis (PTOA) is a significant cause of wastage among equine athletes, representing a major economic and welfare burden to the equine industry. Unfortunately, a major challenge in managing this PTOA is that by the time clinical signs are apparent, it is relatively late in the course of disease and irreversible cartilage damage has already occurred. Further, existing pharmacologic treatments cannot halt the progression of PTOA. It should be possible to detect changes in the joint before they become irreversible since the disease has a known instigator (trauma). However, a crucial knowledge gap is that the proximate cause and precise timing of the joint transitioning from a state of healing to a state of degeneration is not understood.Our long-term goal is to identify diagnostic markers of PTOA that can be detected prior to the development of permanent joint damage. We have developed a novel non-destabilization osteochondral fragment model in the horse that recapitulates early naturally-occurring PTOA, reliably inducing disease with repeatable mild joint pathology (superficial cartilage wear lines, synovial subintimal fibrosis) and clinical signs (mild joint effusion and gait asymmetry). This non-terminal model is uniquely suited to evaluate the onset and early trajectory of PTOA in the same individuals over a long time-course, mimicking natural disease. The objective of this proposal is to identify the earliest morphological and molecular events that occur in the joint after induction of PTOA in our equine model, focusing on the role of the synovium, and to correlate these events with synovial fluid protein signatures. Our central hypothesis is that the synovium is critical in the transition from an anabolic to a catabolic state in early PTOA. Based on our preliminary data, we expect that this role involves gene pathways for extracellular matrix (ECM) turnover, angiogenesis, and growth factor signaling. This work aims to establish whether altered synovial tissue gene expression secondary to surgical induction of PTOA results in measurable changes in protein secretion into the synovial fluid and further that these molecular changes correlate with clinical signs in a study cohort of healthy adult horses subjected to our PTOA model protocol. Sham-operated joints will serve as within-subject controls.
Project Methods
Efforts: The study cohort will be comprised of 12 skeletally mature mixed breed horses (males [castrated] and females, 4-7 years of age, 450-550kg) who are systemically healthy and free from pre-existing lameness. Based on our previous work and data from young human athletes, sex differences are not expected, but can be accounted for in statistical analyses. An osteochondral fragment will be created in one randomly chosen metacarpophalangeal joint (MCPJ) at the first surgery, while the other MCPJ will serve as a sham-operated control. Horses will be evaluated weekly for joint effusion, range of motion, and gait asymmetry throughout the study period. Synovial tissue biopsies will be taken from the dorsomedial aspect of each jointat the time of surgery for gene expression analysis and histopathology. Horses will be rested for two weeks after surgery, then exercised on a treadmill 5 days a week for 14 weeks. Synovial fluid samples will be collected every 8 weeks. Clinical assessments, consisting of lameness exam, gait analysis, goniometer measurements, and joint effusion scoring, will be performed weekly. Synovial fluid samples will be collected every 8 weeks for quantitative proteomics analysis. All analyses will be performed on both injured and sham-operated limbs. At 16 weeks after fragment creation, a second arthroscopic procedure will be performed, and synovial biopsies will be collected from all horses from both injured and sham-operated joints. The second half of the study protocol is identical to the first half. Fragments will be removed from all horses at the time of the third surgery, 32 weeks after creation. Again, synovial biopsies will be collected from all horses at 32 weeks, from both injured and sham-operated joints. In this model, creation of the fragment mimics the inciting injury for naturally occurring PTOA. We expect progression of disease in the second half of the study protocol. This may be reflected in more persistent clinical signs (lameness, effusion), more severe changes on histopathology, and/or a primarily catabolic gene and protein expression profile.Clinical Data Analysis: For evaluation of changes in outcome measures over time, a repeated measure analysis of variance (ANOVA) with Tukey's correction for multiple comparisons will be used. A student's t-test or ANOVA (or their nonparametric equivalents) will be used to compare outcome measures between injured and sham joints at each time point. Correlation between clinical and morphological parameters and quantified gene or protein expression (either absolute expression or fold-change between injured and uninjured joints) will be determined graphically and using a Pearson or Spearman correlation test, as appropriate for data type.Transcriptomics: Reads will be quasi-mapped to NCBI's EquCab3.0 reference genome using Salmon. Surrogate variable analysis (SVA) will be performed to remove unwanted sources of biologic or technical variation. Pairwise comparisons for differential expression (DE) analysis will be made between baseline, sham-operated joints and injured joints at each time point. Multiple testing correction will be performed using the false discovery rate (FDR) method, with FDR <0.05. Functional annotation and gene ontology enrichment testing will be performed in PANTHER. Pathway analysis of DE genes will be performed using the Reactome Pathway Knowledgebase with FDR < 0.05. Ten genes considered to be of most clinical significance after differential expression and pathway analysis of RNAseq data will be validated using qPCR. In addition to simple correlation analysis, gene expression data will also be used in a series of transcriptomic prediction models to assess the contribution of differentially expressed (DE) gene sets to variability in measured clinical outcomes.Proteomics: Protein identification will be performed using MaxQuant (v1.6.0.16) software referencing the Uniprot Equus caballus and Bos taurus databases. Quantitation will be done in MaxQuant using the reporter ion MS3. After normalization, volcano plots comparing relative protein abundances will be generated in Perseus with FDR <0.05. Annotation of known or predicted functions for the expressed proteins will be performed using UniProtKB/Swiss-Prot (www.uniprot.org). PANTHER will be used to extract GO terms from the data, and STRING (https://string-db.org) will be employed to visualize protein-protein interactions.Evaluation: Major research project milestones include completion of data collection from each group within the study cohort and analysis of each component of study data (clinical, morphological, gene expression, proteomics). Attainment of these milestones will be indicated by 1) completion of research work, 2) presentation at scientific meetings, and 3) publication in the scientific literature.