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The role of mammalian high temperature requirement protease A1 (HTRA1) in somatic stem cell differentiation and mineralized matrix formation remains controversial, having been demonstrated to impart either anti- or pro-osteogenic effects, depending on the in vitro cell model used. The aim of this study was therefore to further evaluate the role of HTRA1 in regulating the differentiation potential and lineage commitment of murine mesenchymal stem cells in vitro, and to assess its influence on bone structure and regeneration in vivo. Our results demonstrated that short hairpin RNA-mediated ablation of Htra1 in the murine mesenchymal cell line C3H10T1/2 increased the expression of several osteogenic gene markers, and significantly enhanced matrix mineralization in response to BMP-2 stimulation. These effects were concomitant with decreases in the expression of chondrogenic gene markers, and increases in adipogenic gene expression and lipid accrual. Despite the profound effects of loss-of-function of HTRA1 on this in vitro osteochondral model, these were not reproduced in vivo, where bone microarchitecture and regeneration in 16-week-old Htra1-knockout mice remained unaltered as compared to wild-type controls. By comparison, analysis of femurs from 52-week-old mice revealed that bone structure was better preserved in Htra1-knockout mice than age-matched wild-type controls. These findings therefore provide additional insights into the role played by HTRA1 in regulating mesenchymal stem cell differentiation, and offer opportunities for improving our understanding of how this multifunctional protease may act to influence bone quality.

Although several tendon-selective genes exist, they are also expressed in other musculoskeletal tissues. As cell and tissue engineering is reliant on specific molecular markers to discriminate between cell types, tendon-specific genes need to be identified. In order to accomplish this, we have used RNA sequencing (RNA-seq) to compare gene expression between tendon, bone, cartilage and ligament from horses. We identified several tendon-selective gene markers, and established eyes absent homolog 2 (EYA2) and a G-protein regulated inducer of neurite outgrowth 3 (GPRIN3) as specific tendon markers using RT-qPCR. Equine tendon cells cultured as three-dimensional spheroids expressed significantly greater levels of EYA2 than GPRIN3, and stained positively for EYA2 using immunohistochemistry. EYA2 was also found in fibroblast-like cells within the tendon tissue matrix and in cells localized to the vascular endothelium. In summary, we have identified EYA2 and GPRIN3 as specific molecular markers of equine tendon as compared to bone, cartilage and ligament, and provide evidence for the use of EYA2 as an additional marker for tendon cells in vitro.

Two main features common to all solid tumors are tissue hypoxia and inflammation, both of which cause tumor progression, metastasis, therapy resistance and increased mortality. Chronic inflammation is associated with increased cancer risk, as demonstrated for inflammatory bowel disease patients developing colon cancer. However, the interplay between hypoxia and inflammation on the molecular level remains to be elucidated. We found that MC-38 mouse colon cancer cells contain functional hypoxic (HIF-1α) and inflammatory (p65/RelA) signaling pathways. In contrast to cells of the myeloid lineage, HIF-1α levels remained unaffected in MC-38 cells treated with LPS, and hypoxia failed to induce NF-κB. A similar regulation of canonical HIF and NF-κB target genes confirmed these results. RNA deep sequencing of HIF-1α and p65/RelA knock-down cells revealed that a surprisingly large fraction of HIF target genes required p65/RelA for hypoxic regulation and a number of p65/RelA target genes required HIF-1α for proinflammatory regulation, respectively. Hypoxia attenuated the inflammatory response to LPS by inhibiting nuclear translocation of p65/RelA independently of HIF-1α, which was associated with enhanced IκBα levels and decreased IKKβ phosphorylation. These data demonstrate that the interaction between hypoxic and inflammatory signaling pathways needs to be considered when designing cancer therapies targeting HIF or NF-κB.

Summary Impaired osteogenic differentiation of resident bone marrow stromal cells (BMSCs) of aged patients with low turnover osteoporosis is considered to be one of the major contributing factors in the development of structural and mechanical deficiencies associated with osteoporotic bone. As such, therapies designed to counteract these effects are currently being pursued with an aim to restoring the stem cell niche and enhancing bone quality. In the current thesis, evidence is provided which supports the use of autologous adipose-derived stromal cells (ASCs) as a means by which to combat age-related bone loss. Initial studies were undertaken to compare the differentiation potential of ASCs from osteoporotic SAMP6 and non-osteoporotic SAMR1 mice in 2D in vitro culture systems. Their ability to differentiate towards osteoblast and adipocyte cell lineages was evaluated by histological, biochemical and molecular techniques. In contrast to BMSCs, the differentiation potential of ASCs was found to be comparable between both strains. Further in vitro studies were performed to assess the osteogenic potential of SAMP6-derived ASCs cultured under 3D conditions using long-term silk fibroin scaffold cultures and short-term scaffold free microtissue spheroid (ASC-MT) cultures. In both cases, ASCs underwent efficient osteogenesis resulting in mineralized tissue formation. In the final study, ASCs and ASC-MT were evaluated for their ability to enhance trabecular bone quality following a single intratibial injection in SAMP6 mice using micro-CT, histological and molecular based analyses. Both ASCs and ASC-MT could be identified within treated bones after 42 days and moreover, resulted in significant improvements in several parameters of trabecular bone quality. Furthermore, human ASCs harvested from osteoporotic patients were also demonstrated as having a positive influence on the osteogenic differentiation potential of dysfunctional osteoporotic patient-derived BMSCs in vitro. I