Background on the SLRPs The 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. Research performed in the MBBTS indicates that the underlying mechanisms are based on the paradigm that bgn serves as a """"""""fine-tune"""""""" regulator of key growth factors including TGF-beta, BMP-2 and wnt-3a. Each of these growth factors are well known to have profound effects on skeletal function and bgn's location at the cell surface makes it well suited for such regulatory functions. The regulation of extracellular growth factors by bgn subsequently influences skeletal progenitor cell fate ultimately influencing bone, cartilage and tendon cell differentiation and subsequently tissue formation. In the course of our studies we suspected compensation in the loss of function models and to test this concept, mice were created that were deficient in both bgn and fibromodulin (fmod) or bgn and lumican (lum), which are SLRPs that are co-expressed with bgn in many tissues of the musculoskeletal system. Closer analysis of these doubly deficient mice uncovered strong synergy in function in numerous tissues in the craniofacial complex including the stroma of the cornea and the periodontal ligament and surrounding alveolar bone. SLRPs and Bone Healing Previous work from our group showed that mice deficient in bgn acquire age-related osteopenia (low bone mass). Osteopenia contributes to fracture and to edentulism (tooth loss), both of which can be significant health burdens.
One aim of our research program is to determine exactly how the ECM, and in particular bgn, contributes to skeletal function. Understanding how ECM components such as bgn control bone formation and bone loss could help in the development of new ways to increase bone mass and improve skeletal function in cases such as osteoporosis and fracture healing. All of our work to date on bgn suggests that it has an important role in regulating bone formation. Healing broken bones involves a complex sequence of events including hematoma formation, inflammation, callus formation, neovascularization, osteoblastic callus mineralization, and ultimately osteoclastic remodeling of new bone. This process results in the formation of new lamellar bone in the fractured area. To further understand the role that bgn plays in this kind of bone formation, we created fractures in the femurs of 6-week-old male wild type (WT) and bgn-deficient mice using a custom-made standardized fracture device, and analyzed the process of healing. The formation of a callus around the fracture site was then examined at both 7 and 14 days post-fracture in WT and bgn-deficient mice by immunohistochemistry IHC) using antibodies against bgn. Our studies howed that bgn is expressed in the fracture callus of WT mice, localizing around woven bone and cartilage. Micro-computed tomography (Micro-CT) analysis of the region surrounding the fracture line further indicated that the bgn-deficient mice have a smaller callus with a bone volume and total mineral density (TMD) approximately half that of the WT mice. Histology of the same region also showed the presence of less cartilage and woven bone in the bgn-deficient mice compared to WT mice. Picrosirus Red staining of the callus visualized under polarized light indicated there was less fibrillar collagen in the bgn-deficient mice, a finding confirmed by immunohistochemistry using antibodies against type I collagen. The reduction in collagen content could be one basis for the reduced total mineral density observed in the healing bones of the bgn-deficient mice. The next goal of this study was to understand how bgn controls new bone formation in the context of fracture healing. Our histological analysis revealed that that the smaller callus in the bgn-deficient bones was accompanied by the ingrowth of fewer vessels. Quantitative RT-PCR of the callus at 7-days post-fracture was then carried out to measure osteogenic and angiogenic factors, including vascular endothelial growth factor (VEGF), a protein previously shown to regulate angiogenesis. Considering bgn is rife on the surfaces of cells in both bone and cartilage, we predicted that it could bind to VEGF and regulate its activities there. To test this possibility, we are examining the ability of bgn to bind to VEGF by solid phase binding assays using purified bgn core protein devoid of GAG chains. The outcome of this work showed the bgn core protein bound to VEGF in a dose dependent fashion. To test the possibility that bgn can regulate VEGF functions, we tested whether exogenous bgn affects VEGF-induced signaling. The inability of bgn to directly enhance VEGF-induced signaling suggested that bgn has a unique role in regulating vessel formation, potentially related to VEGF storage or stabilization in the matrix. Taken together, these results suggest that bgn has a regulatory role in the process of bone formation during fracture healing, and further, that regulation of angiogenesis could be the molecular basis.The final stage of this experiment, which is ongoing, will be to determine whether the ectopic application of bgn could accelerate bone healing. Considering our previous work showing that bgn can regulate bmp2/4-induced osteoblast differentiation, it is likely that it could be capable of enhancing bmp2-induced osteogenesis, a process known to be critical in fracture healing. In summary, we predict that bgn could accelerate the bone healing process after fracture and thus provide a foundation for future clinical development of its use in skeletal repair.
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