Graduate Thesis Or Dissertation


Molecular Signaling Patterns of the Articular Cartilage and Marginal Transition Zone in Foals with Osteochondrosis Public Deposited

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  • Osteochondrosis (OC) is a developmental orthopedic disease that results from a disruption to normal endochondral ossification in the articular-epiphyseal growth complex. In mammals, the hedgehog pathway and growth factor signaling are essential to this process. Previous studies in yearling horses with OC have shown upregulation of Indian hedgehog and insulin-like growth factor-I (IGF-1), and a downregulation of the hedgehog transcription factor, glioma associated oncogene (Gli1). Further histological evidence has shown that OC develops from necrosis of blood vessels lining the cartilage canals at the osteochondral junction, leading to localized failure of endochondral ossification. However, more information on cell specific regulation at these locations is needed. The first aim of the present study was to characterize differences in gene expression patterns of growth factor signaling and hedgehog-associated regulators in chondrocytes surrounding cartilage canals and along the osteochondral junction in both normal and diseased cartilage. The objective was to identify gene expression patterns of hedgehog-associated regulators and growth factors in chondrocytes surrounding cartilage canals and along the osteochondral junction in OC affected horses ≤6 months of age. We hypothesized that OC samples would have a general upregulation of gene expression of hedgehog signaling and growth factors, with a downregulation of hedgehog transcription factor gli1. Archived osteochondral samples from the lateral femoral trochlear ridge of 9 foals aged 1-6 months (OC=2 males, 2 females; Normal=5 females) were used. Chondrocyte samples were harvested from the osteochondral junction and surrounding cartilage canals using laser capture microdissection. cDNA from laser-captured microdissection samples was used to establish gene expression. To maximize the amount of cDNA available from these limited samples, logarithmic pre-amplification with equine specific primers and the Taqman PreAmp Master Mix kit (Applied Biosystems, Foster city, CA) was performed. Quantitative Real time (qPCR) of diluted pre-amplified samples (1:20) was then performed using equine specific primers and Taqman probes and the real-time PCR system and software (Applied Biosystems, Foster City, CA). Genes evaluated included glioma associated proteins (Gli1, Gli3), patched 1 and 2 (Ptc1, Ptc2), smoothened (Smo), parathyroid hormone-related peptide receptor 1 (PTHrP-R1), transforming growth factor- β (TGF- β), insulin-like growth factor-I α (IGF-I α), and platelet-derived growth factor- β (PDGF- β). Gene expression was calculated using a relative standard curve and 18S ribosomal RNA as the housekeeping gene to normalize data. Statistical analysis comparing gene expression of OC-affected and normal control LCM samples was performed using a Mann-Whitney U test (p≤0.05). In OC samples, there was significantly lower gene expression of Gli1 (p=0.016) and a trend for lower gene expression of Smo (p=0.055) and Ptc1 (p=0.095) in chondrocytes adjacent to the cartilage canals compared to normal control samples. Conversely, there was significantly higher expression of IGF-I α in OC samples compared to normal controls in chondrocytes surrounding the cartilage canals (p=0.0317). No significant difference in gene expression was found for Gli3, Smo, PTHrP-R1, TGF- β or PDGF- β in chondrocytes surrounding cartilage canals. In chondrocytes along the osteochondral junction, there was no difference in gene expression for any of the genes examined. The second aim of the study was to characterize the protein expression of matrix and molecular regulators in the marginal transitional zone of foals having OC compared to normal foals. The marginal transitional zone is peripherally located within the diarthrodial joint, and represents the interface of articular cartilage, periosteum, and the fibrous joint capsule. Several families of proteins with known roles in cartilage and bone development were investigated, including matrix molecules, Wnt signaling, apoptotic factors and paracrine cell signaling molecules. Archived osteochondral samples, harvested from the lateral femoral trochlear ridge of 15 foals aged 1-6 months (OC=4 males, 3 females; Normal=1 male, 7 females) were used from a previous study, including 6 intact males and 9 females. Immunohistochemistry was performed on all osteochondral samples, using 1:20 dilution of primary antibodies, including rabbit a-human polyclonal (Ihh, PTH-rP, VEGF, PDGF, β-catenin, Wnt 11, Lubricin, Caspase-10, Fas, Thymosin β10) or mouse a- human monoclonal (MMP-3, MMP-13, Dkk-1, Wnt 4, Sox9, Collagen type IIB, Osteocalcin, Cytochrome C). Our results demonstrate differential protein expression in the marginal transitional zone from the lateral femoral trochlear ridge of foals affected by OC. Alterations in protein expression of OC-affected foals mainly involve components of extracellular matrix homeostasis and canonical Wnt signaling. Matrix expression of collagen type IIB and lubricin are decreased and MMP-3 expression is increased in OC-affected marginal transitional zone samples. Canonical Wnt signaling is inhibited in OC-affected marginal transitional zone samples, based on increased Dickkopf-1 and decreased β-catenin protein expression. Most apoptotic and paracrine signaling proteins are not altered in OC-affected marginal transitional zone samples.
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