The Impact of Syndecan-1 on Angiogenesis during Bone Aging

Arras Christian, Timmen Melanie, Bixel M. Gabriele, Arras Philipp, Stange Richard

Abstract

Objectives: Bone is a highly vascularized organ with many different pathways that effect bone regeneration andremodeling. Recently, we demonstrated that the heparan sulfate proteoglycan Syndecan-1 (Sdc1)plays a role in the interaction between osteoclasts and osteoblasts. Furthermore, Sdc1 is well knownas an important player in vascularization of tissues during several modification processes e.g. themultiple myeloma.Since osteogenesis and angiogenesis are closely related in bone, we expected Sdc1 to have aninfluence on vessel structure during aging. Therefore, angiogenesis of metaphyseal bone in youngmice with a high active bone remodeling was compared to aged mice in which bone resorptionoverbalances bone formation and the influence of Syndecan-1 deficiency was characterized.Methods: AnimalsGrowth plates of 36 mice were investigated (C57BL/6 (WT) and Sdc1-/-, 12 mice/timepoint, 4, 12 and18 months, 6 males and 6 females). Both femura of each mouse were dissected, cryoprotected andembedded.SectioningAt -23°C the embedded bones were sectioned into 80 μm thick slices such that the 3D network of thevascularization of the bone could be visualized.StainingFor each bone two slices from the periphery and two slices from the center of the bone were stainedwith Endomucin antibody (sc-65495, Santa Cruz Biotechnology) and DAPI as counter staining.Imaging & Quantification3D imaging was performed with Olympus IX83 fluorescence and 2-photone microscopy. The number ofbulbs which build the borderline of the blood vessels at the growth plate in bone representsangiogenic activity which was investigated at the interfaces of the growth plate. For the first time, weused a custom made software to quantify vessel number semi-automatically, reproducible andinvestigator independent.Results and Conclusion: We verified our custom-made software using slices of WT mice and showed that there is no variationin the number of vascular bulbs related to the width of the growth plate in periphery versus thecentral zone of bones in all age groups. This indicates a homogeneously distributed angiogenesisthroughout this area. Furthermore, in both, WT and Sdc1 deficient mice the number of bulbsdecreased significantly with age. However, comparing WT and Sdc1 knockout mice a highly significantdecrease in the active sprouting angiogenesis in 4 months and 12 months old Sdc1-/- mice was foundwhereas 18 months old mice showed no significant differences.We could demonstrate for the first time, that Syndecan-1 has a significant impact on active sproutingangiogenesis during osteogenesis and bone aging. As mice age, the impact of Sdc1 on angiogenesis isdecreased and overall angiogenesis is reduced during aging. Using 3D imaging and semi-automatedquantification of angiogenesis in long bones, we could extend the possibility of research in this field.