Molecular and genetic basis of cartilage repair and post-traumatic osteoarthritis in mice

The aim of this project is to study molecular and genetic basis of cartilage regeneration and post-traumatic osteoarthritis (PTOA) in crosses of the LG/J (large) and SM/J (small) recombinant inbred mouse strains. These two inbred strains have obvious differences in body size and growth, and are known to differ in a wide-range of other traits and have been maintained by brother-sister mating for over 200 generations. Their intercross is a model for studying the genetics of complex traits segregating many interacting genes of small effect. Most pertinent to my project, LG/J has remarkable regeneration abilities quite distinct from other inbred strains. Ear punches in LG/J heal without scarring and replace all damaged tissues. As studies of wound healing have indicate substantial genetic variation in ability to heal, differences in ear tissue repair have been mapped to 19 different locations across the genome with strong effects on chromosomes 9 and 11. Mapping of quantitative trait loci (QTLs) to 10 cM genomic intervals for cartilage regeneration and PTOA is in progress. Utilizing whole genome sequences for SM/J at 14X coverage and LG/J at 21X coverage that document 4.4 million single nucleotide polymorphisms (SNPs) between these strains and enhance our ability to follow-up mapping results with sequence analysis.
Our previous studies have indicated a positive correlation between articular cartilage regeneration and protection from PTOA. Our search is aided by an additional findings that articular cartilage repair is positively correlated with regeneration of ear wound tissues. We can now leverage this finding to help identify gene variants that are in common between cartilage repair and protection from PTOA: that is, the identification of gene variants that can be used to predict which mice will be susceptible to PTOA. In addition to the analysis of gene loci, we can use these phenotyped strains to gather further information on molecular mechanisms of cartilage regeneration and PTOA. Therefore, we developed the hypothesis that comparison of gene expression in healer and non-healer strains will establish the molecular pathways that are correlated with repair and protection from PTOA. We already know that the expression of genes involved in the Wnt (Axin2, Wnt16) and DNA repair (Xrcc2, Pcna) pathways have high expression in healer strains. These results indicate that molecular differences in joint tissues exist. To further this analysis, we will use laser capture microdissection to dissect out growth plate, subchondral bone and cartilage from joint sections for RNA and microRNA analyses to compare gene expression differences in healer and non-healer strains. Via SNP analysis of each strain, we will be able to track the gene variant allele from the parental strains to the recombinant inbred lines. As the SNP map is very dense (75 SNPs per cM), we will be able to locate regulatory regions that may affect expression levels of specific genes. Finally the mRNA levels will be mapped across in all strains studied.
To test the gene variants, we will monitor intrinsic functional differences of chondrocytes and mesenchymal stem cells in healer and non-healer strains. The hypothesis is that the innate functional differences in chondrocytes and mesenchymal stem cells (MSCs) account for their varying abilities to migrate, be recruited, differentiate and express specific molecules to promote regeneration of damaged articular cartilage and protect from getting PTOA. The rationale is that there is some evidence for distinct differences in cell cycle properties between healer and non-healer strains. The cellular basis for the differences in regeneration is not completely understood. A study that has attempted to compare cells from healer and non-healer strains suggests that highly regenerative cells are hyperproliferative with increased apoptosis. The combined effects of increased proliferation and apoptosis may allow the organism to eliminate old cells and keep the cell turnover rate high. Healer and non-healer strains will be used to study chondrocytes and MSCs for functional parameters in cell culture. Distribution of ear wound healing in the healer and non-healer strains suggests that these strains are likely to be fixed for all 18 healing alleles or non-healing alleles, respectively, thus encompassing the range of functional differences in healing. Thus, the major goal would be to identify innate differences in properties of chondrocytes and MSCs between healers and non-healers at the molecular, functional, and mechanistic level that could contribute to their differential pathology after joint injury. Questions addressed will be: (1) Are cells intrinsically different in activity due to differences in RNA expression? (2) Do cells from healers and non-healers respond differently to stimuli (cytokines or dynamic compression)? (3) Are there differences in cell recruitment to the injury? (4) Are there differences in the ability of MSCs to migrate to the wound and differentiate into chondrocytes?

Figure 1: Time-course of articular cartilage regeneration: (A) A time-course analysis of articular cartilage regeneration in LGXSM-6 and LGXSM-33 lines showed a significant difference in regeneration only at 16-weeks post-surgery. (B–I) Representative sagittal sections of full-thickness articular cartilage lesions form LGXSM-6 (B–E) and LGXSM-33 (F–I) lines stained with toluidine blue for proteoglycan at 4-, 8-, 12- and 16-weeks post-surgery are shown. There was a significant proteoglycan deposition in LGXSM-6 (healer) compared to LGXSM-33 (non-healer) at 16-weeks time-point. At all other time-points, no significant regenerative response was observed. Asterisk (*) indicates statistical significance (p<0.011); arrow indicates site of defect; AC = articular cartilage, SB = subchondral bone, GP = growth plate, BMC = bone marrow cavity, bar (all panels) = 0.1 mm
Transcriptome signatures in knee joint tissues predict osteoarthritis in patients with meniscus injury

While OA encompasses all knee joint tissues, the focus of research has been on the articular cartilage over the past several decades. Undoubtedly, the work on articular cartilage has advanced our understanding of the liaison between cartilage degeneration and OA, but this bias towards cartilage-centric research may hamper a more comprehensive of the impact of OA on the joint as a whole. Work on the knee meniscus, central to knee joint congruence as well as other aspects of its physiology, has largely been ignored until recently. While the role of the intact meniscus in OA is unclear, meniscus injury certainly plays a vital role in OA onset and progression. In contrast to previous research focused on the biomechanics and gross structural and functional aspects of meniscus, we pioneered the work studying the molecular biology of the meniscus. This work has led to the identification of transcript level changes in meniscus reflecting the molecular phenotype, in relation to age, obesity, sex, injury pattern, and status of articular cartilage. As these patient related factors have been implicated in OA, their relationship to meniscus injury is a segue to the interaction of meniscus injury and OA. Another significant aspect of our approach is evidence that the molecular status of the meniscus at injury can potentially predict patients that will develop OA. It means that the interaction between molecular signatures in articular cartilage and known genetic risk alleles can tell us who will go on to develop OA immediately after a meniscus injury has occurred!
While transcriptome profiling in conjunction with OA risk-alleles suggests a molecular “pre-OA” phenotype in some patients, the ability to predict a population at risk for OA can only be assessed by future clinical outcomes. At the molecular level, studies would be undertaken to appreciate the differences between normal (healthy) and injured tissues from patients with meniscus injury, anterior cruciate ligament (ACL) injury or both. Meniscus, ACL and articular cartilage tissues will be compared for their crosstalk and potential role in joint homeostasis. Differentially expressed gene transcripts will be prioritized based on their known role in a pathway and in vitro assays will be used to validate the function of individual genes. Despite advancements in the science and understanding of OA pathogenesis, efforts are needed for the selection of appropriate therapeutic candidates. The molecules identified from these high throughput analyses can be used as therapeutic candidates or targets.

Figure 2: Heat map view of z-scored expression profiles of gene transcripts differentially expressed between healthy and OA cartilage. Hierarchical clustering separated the study patients into three distinct clusters. The list of gene transcripts used for cluster analysis was generated in a prior study by Karlsson et al. (Osteoarthritis Cartilage. 2010;18:581–92) that looked at the transcriptome-wide RNA expression of healthy and OA cartilage. This list represented all the expressed gene transcripts differentially expressed between healthy and OA cartilage at >15-fold difference.
Emerging role of periostin in extracellular matrix synthesis and degradation in human cartilage

We have identified that periostin is expressed in ligament tissues after acute injury and is absent in chronic ligament injuries. Periostin (encoded by POSTN gene) is a secreted matricellular protein expressed in a wide-range of collagen-rich fibrous connective tissues, suggested to influence collagen fibrillogenesis. Conversely, lost or reduced levels of periostin result in substantial damage to connective tissues. Now our focus is on studying the crosstalk between periostin and collagen genes and the potential implications in matrix synthesis and tissue healing. This study has the potential to advance our understanding of tissue injury and healing. The role of periostin in the tissue and cells of the injured tissues could be a key to enabling ligament repair as an alternative treatment approach to the current gold standard of ligament reconstruction. By defining that role, and investigating how manipulation of periostin influences extracellular matrix and healing of the injured ligament tissue, this approach will advance our understanding of the potential for biologic healing in the injured tissues. Furthermore, what this study learns about periostin may have applications to other clinical healing environments, such as rotator cuff healing, bone healing and skin wound healing. Our ongoing efforts of examining human knee tissues at transcriptome level will yield tremendous insights into predicting early molecular manifestations of osteoarthritis before the onset of clinical disease.

Figure 3: Periostin expression in cartilage. Normal and OA cartilage was obtained from patients without and with OA and periostin expression was measured by immunostaining. Periostin was highly expressed in OA cartilage.
Translational strategies

Several modes of therapy have been advocated for OA, including the systemic and intra-articular administration of pharmacological agents. The systemic mode has several side effects. In addition, the patient and disease characteristics that dictate who will and who will not respond to a given mode of therapy remain unknown. Of the several current efforts surrounding the treatment of OA, gene therapy has merit, but it has been limited by concerns about safety, efficacy, and cost-effectiveness. Gene therapy has been the subject of conflicting levels of attention but has recently been undergoing a resurgence. Advancements in gene therapy posit how a gene therapy application can be made safer and how the problems associated with gene transfer and expression can be solved, thereby enabling the development of strategies to overcome a variety of barriers to gene therapy application. Viral vectors from which the viral genes have been removed also provide a renewable source of gene expression. The viral promoter is replaced by the endogenous (species-specific) promoter upstream of the therapeutic gene. Although this approach has been used in gene therapy trials, it has potential limitations, such as immunogenicity, insertional mutageneses, persistence and sustainability of transgene expression, and tissue/cell specificity. A number of strategies have been made to overcome several problems related to adenovirus gene therapy. For example, adenovirus serotype 5 represents a number of unique attributes that make it suitable for gene therapy application. To circumvent the problem of cell specificity and tissue refraction, bispecific molecular adapters have allowed for the modification of adenovirus tropism for targeted gene transfer. The discovery of unconventional immunoglobulins derived from the serum of camels and alpacas provides compatibility with the cytosolic biosynthesis of adenovirus capsid proteins, thus allowing for target-cell specificity and ultimately making possible their use for adenovirus-mediated gene therapy for a particular tissue in the joint. Similarly, to circumvent the broad negative effects of pre-existing immunity to common human serotypes of adenoviruses, researchers have developed vectors based on chimpanzee-derived adenoviruses for gene therapy application. Although this may sound a bit too ambitious, if such a therapy can at least delay joint replacement (the only available surgical option for end-stage OA) by a decade, this would exert a significant impact on quality of life. At least, these study would provide a platform to test the therapeutic efficacy of genes in vitro as well as in a mouse model of PTOA which we have developed over the past few years.

Bottom line: The information obtained from genetic mouse strains, animal models that reflect common sport injuries in humans, high-throughput technologies for the discovery of new and novel genes in tissues obtained from patients with various injury phenotypes, and proof-of-concept studies involving gene therapy places my lab in a unique position for translational application and will lead the road to the precision medicine for PTOA.

Figure 4: An overview of research vision: Schematic showing how gene therapy can be used to test the therapeutic genes obtained from transcriptome analysis (patient) and genetic analysis (mouse). This also shows how animal models can be used to test the feasibility and efficacy of the genes of interest as well as the applicability of gene therapy.
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