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, 25 (3), 606-16

Fluorescent Risedronate Analogues Reveal Bisphosphonate Uptake by Bone Marrow Monocytes and Localization Around Osteocytes in Vivo

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Fluorescent Risedronate Analogues Reveal Bisphosphonate Uptake by Bone Marrow Monocytes and Localization Around Osteocytes in Vivo

Anke J Roelofs et al. J Bone Miner Res.

Abstract

Bisphosphonates are effective antiresorptive agents owing to their bone-targeting property and ability to inhibit osteoclasts. It remains unclear, however, whether any non-osteoclast cells are directly affected by these drugs in vivo. Two fluorescent risedronate analogues, carboxyfluorescein-labeled risedronate (FAM-RIS) and Alexa Fluor 647-labeled risedronate (AF647-RIS), were used to address this question. Twenty-four hours after injection into 3-month-old mice, fluorescent risedronate analogues were bound to bone surfaces. More detailed analysis revealed labeling of vascular channel walls within cortical bone. Furthermore, fluorescent risedronate analogues were present in osteocytic lacunae in close proximity to vascular channels and localized to the lacunae of newly embedded osteocytes close to the bone surface. Following injection into newborn rabbits, intracellular uptake of fluorescently labeled risedronate was detected in osteoclasts, and the active analogue FAM-RIS caused accumulation of unprenylated Rap1A in these cells. In addition, CD14(high) bone marrow monocytes showed relatively high levels of uptake of fluorescently labeled risedronate, which correlated with selective accumulation of unprenylated Rap1A in CD14(+) cells, as well as osteoclasts, following treatment with risedronate in vivo. Similar results were obtained when either rabbit or human bone marrow cells were treated with fluorescent risedronate analogues in vitro. These findings suggest that the capacity of different cell types to endocytose bisphosphonate is a major determinant for the degree of cellular drug uptake in vitro as well as in vivo. In conclusion, this study shows that in addition to bone-resorbing osteoclasts, bisphosphonates may exert direct effects on bone marrow monocytes in vivo.

Figures

Fig. 1
Fig. 1
Growth of HAP on CAP in the presence of fluorescent RIS analogues in vitro. Constant composition (CC) crystal growth experiments were conducted in the presence of 1.0 µM BP in order to assess inhibitory potency of fluorescent RIS analogues (AF647-RIS, FAM-RIS) compared with the parent molecule (RIS). Experimental conditions were δHAP (relative supersaturation) = 9.00, pH 7.40 at 37.0°C, and IS (ionic strength) = 0.15 M.
Fig. 2
Fig. 2
Detection of AF647-RIS in whole tissues. Three month old mice were subcutaneously injected with AF647-RIS (0.9 mg/kg), and the tibia, femur, kidneys, liver, and spleen were dissected either 1 (A, B) or 7 days after injection (C, D). Following fixation overnight, bones and organs were scanned on a LI-COR Odyssey Infrared Imager (LI-COR Biosciences) using a 680 nm laser to detect the presence of AF647-RIS. (A, C) Near-infrared fluorescence images of tissues from control and AF647-RIS-treated mice. (B, D) Average fluorescence intensity was quantified using Odyssey Version 2.1 software (LI-COR Biosciences) and is expressed as mean ± SD (1 day: n = 4; 7 days: n = 3). a: p < .001 compared with control; b: p < .05 compared with control.
Fig. 3
Fig. 3
Histologic analysis of FAM-RIS and AF647-RIS binding to bone surfaces in vivo. Three month old mice were injected with 0.5 mg/kg (A) or 1 mg/kg (B, C) FAM-RIS or 0.9 mg/kg AF647-RIS (D, E) and sacrificed 24 hours later. Tibiae were fixed in formaldehyde, embedded in methyl methacrylate, and sectioned longitudinally (A–D) or cross-sectionally through the midshaft (E). (A) Series of xy confocal microscopy images of a 2 µm section were acquired on an LSM710 META system using a 20× objective. Scans were tiled using ZEN software to generate a high-resolution overview image of FAM-RIS (black) labeling of bone surfaces. Bar = 500 µm. (B, C) 2 µm sections were counterstained with the nuclear dye TO-PRO-3 and wheatgerm-agglutinin-Alexa Fluor 594 (C) prior to analysis by confocal microscopy. Green: FAM-RIS; blue: TO-PRO-3; red: wheatgerm-agglutinin-Alexa Fluor 594. Arrows indicate the presence of FAM-RIS around osteocytes. Bar = 10 µm. (D) xy image, acquired directly from the MMA-embedded tibial block just below the block surface, showing AF647-RIS labeling (black) within cortical bone near the periosteal surface. Top image: Detector gain optimized for detection of AF647-RIS around vascular channels (arrows). Bottom image: Detector gain optimized for detection of AF647-RIS around osteocytic lacunae surrounding the vascular channels (arrows). Bar = 50 µm. (E) Quantification of AF647-RIS labeling of osteocyte lacunar walls. The mean fluorescence intensity (corrected for tissue autofluorescence) and the percentage of positively labeled osteocytic lacunae are expressed as a function of the distance to the nearest labeled bone or vascular channel surface (data binned into 10 µm increments). Data shown are the mean ± SEM of four mice (0 to 70 µm) or three mice (70 to 100 µm).
Fig. 4
Fig. 4
FAM-RIS and AF647-RIS uptake by rabbit osteoclasts in vivo. Newborn rabbits were injected with 0.5 mg/kg FAM-RIS or 0.9 mg/kg AF647-RIS (A, B) or with 3 mg/kg FAM-RIS or the molar equivalent dose of 1.2 mg/kg RIS (C) or vehicle and sacrificed 24 hours later. (A) Confocal microscopic image of a longitudinal section through an ulna from a FAM-RIS-treated (green) rabbit counterstained with the nuclear dye TO-PRO-3 (blue). Tissue autofluorescence shown in red channel. Bar = 10 µm. (B, C) Bone marrow cells were isolated from the long bones by scraping out the marrow, and osteoclasts were isolated using anti-VNR magnetic beads. (B) Isolated osteoclasts were left to adhere on glass coverslips for 4 hours, fixed in formaldehyde, and counterstained with TO-PRO-3 (FAM-RIS and vehicle) or sytox green (AF647-RIS) prior to analysis of intracellular drug uptake using confocal microscopy. i: Osteoclast from vehicle-treated rabbit. ii: Osteoclast from FAM-RIS-treated rabbit. Green: FAM-RIS; blue: TO-PRO-3; red: Magnetic bead autofluorescence. iii: Osteoclast from AF647-RIS-treated rabbit. Green: AF647-RIS; blue: sytox green. Bar = 10 µm. (C) Isolated VNR-positive and VNR-negative cells were lysed and analyzed for the presence of unprenylated Rap1A by Western blotting using an antibody that binds with high affinity to the unprenylated form of the protein (uRap1A) and an antibody that detects total Rap1.
Fig. 5
Fig. 5
FAM-RIS and AF647-RIS uptake by rabbit bone marrow cells in vitro. Bone marrow cells isolated from the long bones of newborn rabbits by scraping out the marrow (A–C, E) or isolated from a bone marrow aspirate of a patient undergoing total hip replacement surgery (D) were treated in vitro with vehicle only; with 10 nM, 100 nM, or 1 µM AF647-RIS (A–D) or FAM-RIS (A, B); or with 10 µg/mL FITC-dextran (E) for 24 hours. Cells then were stained with anti-CD14-APC for FAM-RIS- and FITC-dextran-treated cells or with anti-CD14-FITC for AF647-RIS-treated cells. (A) Representative flow cytometry profiles showing AF647-RIS uptake. (B) Quantification of flow cytometry results of rabbit bone marrow cells, shown as mean fluorescence intensity expressed as mean ± SD (n = 3). (C) Confocal microscopic image (1 µm optical section) showing intracellular uptake of AF647-RIS by CD14+ cells. Bar = 10 µm. (D) Quantification of AF647-RIS uptake by human bone marrow cells by flow cytometry. Results are shown as mean fluorescence intensity and expressed as mean ± SD (three replicates). (E) Quantification of flow cytometry analysis of FITC-dextran uptake, shown as mean fluorescence intensity and expressed as mean ± SD (n = 3). a: p < .001; b: p < .01 versus control and versus respective CD14neg/low cells; c: p < .05 versus control.
Fig. 6
Fig. 6
AF647-RIS and RIS uptake by rabbit bone marrow cells in vivo. Newborn rabbits were subcutaneously injected with 0.9 mg/kg AF647-RIS (A–C) or 5 mg/kg RIS (D) or vehicle and sacrificed 24 houre (A–D) or 7 days later (B). Bone marrow cells then were isolated from the long bones by scraping out the marrow. (A–C) Cells were stained with anti-CD14-FITC and analyzed by flow cytometry and confocal microscopy. (A) Representative flow cytometry profiles showing detectable uptake of AF647-RIS by some bone marrow cells in vivo, a subset of which is CD14high. (B) Quantification of flow cytometry results of bone marrow cells analyzed 24 hours (left) and 7 days (right) after administration of AF647-RIS. Results are shown as mean fluorescence intensity and expressed as mean ± SD (n = 3). a: p ≤ .01; b: p < 0.001 versus control and versus CD14neg/low cells. (C) 1 µm optical section showing AF647-RIS uptake by a CD14+ cell analyzed following flow cytometric sorting of CD14high AF647-RIS+ cells (green: AF647-RIS; blue: CD14-FITC; red: sytox orange; bar = 10 µm). (D) Osteoclasts were isolated using anti-VNR magnetic bead separation, followed by isolation of monocytes from the VNR-negative fraction by anti-CD14 magnetic beads. Cells were lysed, and unprenylated Rap1A and total Rap1 were detected by SDS-PAGE and Western blotting. Red: Unprenylated Rap1A (uRap1A); green: Rap1.

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