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. Last year we described the development and use of Wisp1-KO mouse to investigate the functions of Wisp1 in mineralized tissues. With aging, Wisp1-KO mice had shorter bones accompanied by a reduced width of their growth plates compared to WT mice. This observation led us to test the possibility that Wisp1 could control chondrogenesis. To understand the role of Wisp1 in chondrogenesis, human bone marrow stromal cells (hBMSCs) were transduced with Wisp1 adenovirus (to increase Wisp1) or siRNA to WISP1 (to decrease Wisp1) in the presence or absence of transforming growth factor-beta 3 (TGF-beta 3). Overexpression of Wisp1 enhanced TGF-beta 3-induced SMAD2/3 phosphorylation and chondrogenesis of hBMSCs in an in vitro assay using a micromass culture model. In the reverse situation, knockdown of WISP1 inhibited the TGF-beta 3-induced SMAD2/3 phosphorylation and synthesis of cartilage matrix in micromass cultures of hBMSCs. Immunoprecipitationwestern blot analysis showed that Wisp1 bound to TGF-beta 3 and regulated the ability of TGF-beta 3 to bind to hBMSCs. In vivo analysis confirmed there was a significant decrease in the gene expression levels of chondrocyte markers in cartilage samples from Wisp1-KO mice, compared to those from wild type (WT) control. In order to investigate the regenerative properties of the articular cartilage in Wisp1-KO mice, articular cartilage defects were surgically performed in the knee joints of young mice, and the results showed that the cartilage was partially repaired in WT mice, but not in Wisp1-KO mice. In conclusion, these results show, for the first time, that Wisp1 has a positive influence on chondrogenic differentiation by modulating the effects of TGF-beta 3. 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. The annual incidence of adult bone fractures has been estimated at 9.1 to 36 per thousand per year, with 5-10% of fractures complicated by non-union or delayed union. Non-union, the failure of a broken bone to heal, causes significant morbidity and is the result of impaired healing. Poor vascularity of the fracture zone disrupts healing and has been identified as a risk factor for non-unions. By studying the mechanisms controlling angiogenesis in fracture healing, a framework can be formed, which would allow for the development of therapies to improve fracture site vascularity and decrease non-unions. 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) endothelial 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 Bgns 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.
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