WISP1/CCN4 Function: The CCN family is named from its founding members (Cyr61, CTGF, Nov) and consists of six members now known as Cyr61/CCN1, CTGF/CCN2, Nov/CCN3 WISP1/CCN4, WISP2/CCN5 and WISP3/CCN6. They have diverse functions including regulation of differentiation, proliferation and cell migration. All six CCN members are found in the skeleton but with unique locations. For example, during normal skeletal development, CCN2 and 3 are highly expressed in cartilage, while CCN4 is largely confined to newly forming bone. Considering that many of the CCN family members bind and regulate TGF-beta, it is not surprising that they are being considered as potential therapeutic targets for diseases such as fibrosis, cancer and osteoarthritis in which TGF-beta plays a crucial role in tissue pathology. This year we continued to investigate the role of Wisp1 in cartilage homeostasis and published a book chapter describing methods used to analyze Wisp1 function using both gain and loss of function approaches. Biglycan Function: The small leucine-rich proteoglycan (SLRP) family is composed of 17 members sub-divided into classes (I-V) based on their amino acid sequence and genomic organization. All members of the SLRP family (excluding asporin) have extensive post-translational glycosylation on a relatively small protein core backbone composed of repeat structures rich in leucine. For years, evidence has been mounting about the importance of SLRPs in skeletal function. We have focused on the SRLP, biglycan (Bgn), because of its high level of expression in bones and teeth. Taken together, our work highlights the fact that Bgn is not needed for bone development but, rather, appears to play a role in skeletal aging. This has been demonstrated using mice unable to make bgn that are found to acquire early onset osteoporosis (osteopenia/low bone mass), osteoarthritis and ectopic bone in their tendons. We previously identified Bgn as a potential regulator of angiogenesis during fracture healing. As mentioned earlier, Bgn is a member SLRP family and is abundant in mineralized tissue. Bgn-deficient (KO) mice have defective bone formation and mineralization, which may, in part, be caused by changes in the expression and hierarchical structure of other important matrix components of bone such as type I collagen. Indeed, mouse models have shown that the absence of Bgn leads to abnormal collagen fibril shape and character, which could be one of the foundations for the mineralized tissue defects observed in the Bgn-KO mice. The cellular and molecular basis for the bone abnormalities found in the absence of Bgn appears to be from defects in osteogenic progenitors that have a reduced ability to undergo osteogenesis in vitro. Several factors have been implicated in modulating the Bgn-osteogensis regulatory axis, including TGF-beta, BMP-2 and Wnt signaling. Recently we found that Bgn has strong binding affinity for the angiogenic factor, VEGF; however, it did not potentiate the effect of VEGF in a human umbilical vein endothelial cell (HUVEC) vessel forming assay. This observation led us to consider that there were yet unidentified factors that work with Bgn to control vessel formation. Our attention was drawn to endostatin, a 20 kDa C-terminal fragment of type XVIII collagen that has potent antiangiogenic activities, and appears to regulate angiogenesis in multiple ways. The goal of this investigation was to determine if endostatin could be a new partner for Bgn, and to deepen our mechanistic understanding of Bgn's role in regulating angiogenesis during fracture healing. By infusing barium sulfate (BaSO4) into WT and Bgn-KO mice we discovered the positive effect of Bgn in modulating angiogenesis during fracture healing. Using micro-computed tomography angiography we found a significant decrease in the vessel size and volume among other parameters in the fractured Bgn-KO bones compared to WT controls. To further understand the mechanistic basis for this, we explored the relationship between Bgn and the anti-angiogenic protein endostatin. Immunohistochemistry (IHC) showed co-localization of Bgn and endostatin in regions of bone formation, with increased endostatin staining in Bgn-KO compared to Wt at 14 days post-fracture. To further elucidate the relationship between Bgn and endostatin, an endothelial cell tube formation assay was used. This study showed that endothelial cells treated with endostatin had significantly decreased vessel length and vessel branches compared to untreated cells, while cells treated with endostatin and Bgn at a 1:1 molar ratio had vessel length and vessel branches comparable to untreated cells. This indicated that Bgn was able to mitigate the inhibitory effect of endostatin on endothelial cell growth. In summary, these results suggest that Bgn is needed for proper blood vessel formation during fracture healing, and one mechanism by which Bgn impacts angiogenesis is through inhibition of endostatin. The burden associated with fractures necessitates a thorough understanding of the fracture healing process to optimize treatment. Crucial to this process is the formation of new blood vessels to deliver the components required for the fracture to heal. In this study, Bgn was shown to play a role in the process of angiogenesis, and that effect appears to be partially mediated through endostatin suppression. Although its role in angiogenesis has been shown, questions remain on how Bgn mediates its effect on this process. SLRPs and the temporomandibular joint (TMJ) The temporomandibular joint (TMJ) is a unique structure that is critical for speaking and chewing. TMJ osteoarthritis (OA) is the most prevalent and debilitating type of TMJ disorder, which causes permanent tissue loss and joint dysfunction. There are no standard diagnostic tests or treatments for this degenerative joint disease due to the limited understanding of the cellular and molecular mechanisms underlying TMJ biology and TMJ OA pathology. Therefore, comprehensive mechanistic studies would potentially contribute toward the development of diagnostic biomarkers and clinical therapy. In our current studies we used bgn and fmod-deficient (DKO) mice, which acquire early onset TMJ OA to further study the mechanisms causing this pathology. TMJ-OA is a complex disease that affects both cartilage and subchondral bone. It is accompanied by loss of extracellular matrix (ECM) and may be controlled by Bone Morphogenetic Protein 2 (BMP-2). We analyzed the effect of BMP-2 in both cartilage and subchondral bone in a TMJ-OA animal model that is deficient in Bgn and Fmod (DKO). Whole mandibles were dissected from 3 week-old wild-type (Wt) and DKO mice, and incubated with and without BMP-2 for 2 days using an explant culture system. Condyle growth was measured by CT and the expression levels of cartilage and bone related genes were analyzed using real time (RT)-PCR or by immunohistochemistry (IHC) from condyles that contained an intact cartilage/subchondral bone interface. Osteoclast activity was estimated by TRAP staining and by TRAP, Rankl, AdamTs4 mRNA expression levels. Our results showed that most parameters examined were slightly up-regulated in WT samples treated with BMP-2 and this up-regulation was significantly enhanced in the DKO mice. The up-regulation of both catabolic and anabolic agents did not appear to positively affect the overall growth of DKO condyles compared to WT controls. In summary, the up-regulation of both anabolic and catabolic genes in the WT and DKO TMJs treated with BMP-2 suggests that BMP increases matrix turnover in the condyle and, further, that Bgn and Fmod could have protective roles in regulating this process.
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