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  • Gardiquimod In bone PGE exerts both anabolic and catabolic


    In bone, PGE2 exerts both anabolic and catabolic effects [6], [7], [8], [9]. Administration of PGE2 to mice lacking each of the four prostanoid receptors identified EP4 as the primary mediator of PGE2-induced bone formation [10]. While the role of EP1 in osteoblastic differentiation and bone metabolism is not as well defined, selective EP1 agonists have been shown to stimulate the proliferation of osteoblast progenitors, but impair osteoblastic differentiation [11]. Consistent with these findings, we previously demonstrated that loss of EP1 accelerates osteoblastic differentiation and fracture repair [12]. Although there is evidence supporting a role for PGE2/EP1 signaling in osteoblastic differentiation and bone regeneration, the effects of EP1 receptor signaling on bone Gardiquimod during two critical phases, growth and aging, are poorly understood. EP1 has been implicated in other aging-related pathologies including neurodegenerative disease [13] and hemin-mediated neurotoxicity [14]. In the present study we examined the hypothesis that EP1 acts as a negative regulator of bone formation and skeletal growth, while loss of EP1 promotes maintenance of bone during aging. We report that mice maintain increased bone mineral density and stronger cortical and trabecular bone biomechanical properties with aging. The trabecular bone of the mice is also resistant to and protective against aging-induced ovariectomy-induced bone loss. The altered bone properties of the mice result mainly from an increased bone formation rate. In vitro studies confirmed that the EP1 receptor acts to inhibit bone marrow osteoprogenitor cell differentiation and mineralization.
    Methods Experimental Animals: All animal studies were conducted with the approval of the University Committee on Animal Resources at the University of Rochester. Wild type C57BL/6J (WT) mice were purchased from Jackson Laboratories (Bar Harbor, ME) at 4-weeks of age. (KO) mice [15] are on a C57Bl/6J genetic background and were generously provided Dr. Matthew Breyer (Vanderbilt University). Female mice were used for ovariectomy experiments, while male mice were used for aging experiments. Histology and histomorphometric analysis: Femurs were collected from and WT mice at 2-months, 6-months, 1-year of age, or 8-weeks after OVX or sham surgery. Specimens were fixed in 10% neutral buffered formalin, decalcified for 21days in 10% EDTA (pH7.2), processed and embedded in paraffin. Three-micron sections were taken from three different levels (30μm lateral to the mid-shaft of the femur, mid-shaft of the femur, and 30μm medial to the femur mid-shaft), and stained for TRAP+ osteoclasts; sections were counterstained with methyl green. At least five specimens per genotype per age were used for histomorphometric and histological analyses. Micro-computed tomography (μCT): Femurs and vertebrae were harvested and scanned at 10.5μM resolution using a Scanco VivaCT 40 (Scanco Medical AG). Trabecular bone volume fraction bone volume (BV)/total volume (TV), number (Tb.N.), thickness (Tb.Th), and spacing (Tb.Sp) were measured in the distal metaphyseal region of the femur and the trabecular region of the L4 vertebrae. The femur trabecular bone region of interest (ROI) began 210 microns proximal to the last remnant of growth plate interrupting the trabecular space, and spanned 1060.5 microns proximally (101 10.5μm slices). Cortical BMD, polar moment of inertia (pMOI), area (Ct. Ar) and thickness (Ct. Th) were measured in the mid-diaphyseal region of the femur. Biomechanical testing: 3-point bending: Mechanical properties were assessed by three-point bending as we have previously described [16]. Briefly, femurs were cleaned of soft tissue and mounted on two supports spaced 8mm apart on an Instron 8800 device (Instron). The femurs were loaded at a displacement rate of 0.10mm/s with data points recorded every 0.01s by Bluehill software (Instron). The maximum load at failure, energy to max, and stiffness were calculated from force versus deformation data.