Silencing P2Y12 and P2Y13 receptors rehabilitates the ADP-induced P2Y1-mediated osteogenic commitment of post-menopausal mesenchymal stromal cells

Figures

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  Fig. 1. Pattern of ADP-sensitive P2Y receptors immunoreactivity in cultured BM-MSCs from young and Pm women. (Panel a) presents representative immunofluorescence confocal micrographs of BM-MSCs from a young female (37 years old) and a Pm woman (67 years old) grown for 7 and 21 days in an osteogenic-inducing medium stained against P2Y1, P2Y12 and P2Y13 receptors (green). Blue dots represent nuclei stained with DAPI. The scale bar is 50 μm. In (panel b), ordinates represent the fluorescence intensity per cell (arbitrary units, a.u.) of the indicated immunotarget as a function of the number of days in culture (days 7 and 21). Scatter dot plots (with mean ± SD) represent pooled data from a total of 105–120 cells analysed from 1 young woman (37 years old) and a total of 584–708 cells analysed from 6 Pm women (73 ± 6 years old). **P < 0.01 and ****P < 0.0001 (non-parametric Kruskal-Wallis with Dunn’s multiple comparison test) represent significant differences. Data in panel c) shows the (i) P2Y12/P2Y1 and (ii) P2Y13/P2Y1 density ratios at culture day 7 and 21. Symbols (mean ± SD) represent pooled data from 9 Pm women (73 ± 5 years old) and 1 young female (37 years old). The dashed horizontal…

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  Fig. 2. Tonic activation of ADP-sensitive P2Y receptors is impaired in Pm BM-MSCs. BM-MSCs were allowed to grow in an osteoblast-inducing medium for 21, 28 and 35 days, either in the absence or presence of selective P2Y1 (MRS 2179, 0.3 µM), P2Y12 (AR-C66096, 0.1 µM) and P2Y13 (MRS 2211, 10 µM) receptor antagonists. Panels (a) and (b) show, for (a) young and (b) Pm women BM-MSCs, the ALP activity (in nmol/min/MTT) and extracellular matrix mineralization (in µm2). Scatter dot plots (with mean ± SD) represent pooled data from 1 young woman (37 years old) and 2 to 5 Pm women (74 ± 5 years old); four to sixteen replicates were performed per individual. *P < 0.05, ***P < 0.001 and ****P < 0.0001 (parametric ordinary one-way ANOVA with uncorrected Fisher’s LSD test for young woman results and non-parametric Kruskal-Wallis test with uncorrected Dunn’s test for Pm women) represent significant differences. Right hand-side images show typical micrographs displaying bone nodule formation (red-brownish spots) in BM-MSC cultures from a young female (37 years old) and a Pm woman (71 years old). Scale bar is 50 μm

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  Fig. 3. Selective activation of the P2Y1 receptor promotes BM-MSCs growth and osteogenic differentiation in a younger female, but not in Pm women. BM-MSCs were allowed to grow in an osteoblast-inducing medium for 7, 14, 21, 28, and 35 days, either in the absence or presence of the selective P2Y1 receptor agonist (MRS 2365, 0.1 µM). Panels (a) and (b) show, for (a) young and (b) Pm women BM-MSCs, the growth/viability (MTT assay), ALP activity (in nmol/min/MTT), and extracellular matrix mineralization (in µm2) for the indicated experimental groups. Scatter dot plots (with mean ± SD) represent pooled data from 1 young woman (37 years old) and 4 to 6 Pm women (72 ± 6 years old); four to sixteen replicates were performed per individual. *P < 0.05 and ***P < 0.001 (parametric ordinary one-way ANOVA with uncorrected Fisher’s LSD test and unpaired t-test for young woman results; non-parametric Kruskal-Wallis test with uncorrected Dunn’s test and two-tailed Mann Whitney test for Pm women) represent significant differences. Right hand-side images show typical micrographs displaying bone nodule formation (red-brownish spots) in BM-MSC cultures from a young female (37 years old) and a Pm woman (71…

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  Fig. 4. Blockage of P2Y12 and P2Y13 receptors partially rehabilitates the osteogenic potential of the P2Y1 receptor in Pm BM-MSCs. BM-MSCs were allowed to grow in an osteoblast-inducing medium for 21, 28, and 35 days, either in the presence of the selective P2Y1 receptor agonist (MRS 2365, 0.1 µM), applied alone or combined with the P2Y12 (AR-C66096, 0.1 µM) or P2Y13 (MRS 2211, 10 µM) selective antagonists. Panels (a) and (b) show the relative variation from the control situation (Δ = drug–CTR) in the (a) ALP activity (nmol/min/MTT) and (b) the extracellular matrix mineralization (µm2). Zero represents the identity between treated cells and ALP activity or total mineralized cell area obtained in non-treated (CTR) cells (horizontal dash line); control values are represented in Fig. 3b. Scatter dot plots (with mean ± SD) represent pooled data from 4 to 6 Pm women (72 ± 6 years old); four to eight replicates were performed per individual. *P < 0.05, **P < 0.01, and ****P < 0.0001 (non-parametric Kruskal-Wallis test with uncorrected Dunn’s test) represent significant differences. Right hand-side images show typical micrographs displaying bone nodule formation (red-brownish spots) in BM-MSC…

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  Fig. 5. Transient P2Y12 or P2Y13 gene silencing using a short hairpin RNA partially rescues the P2Y1-mediated osteogenic differentiation in BM-MSCs from Pm women. Cells were exposed for 24 h to lenti-shRNAs designed to silence P2Y12 or P2Y13 receptors (or exposed to a scramble sequence) and then allowed to grow for 7, 21, and 35 days in an osteogenic-inducing medium. Panels (a) and (b) present representative immunofluorescence micrographs of Pm BM-MSCs from a Pm woman (83 years old) stained against (a) P2Y12 and (b) P2Y13 receptors (green). Blue dots represent nuclei stained with DAPI. The scale bar is 50 μm. In scatter dot plots (with mean ± SD), ordinates represent the fluorescence intensity per cell (arbitrary units, a.u.) of the indicated immunotarget as a function of the number of days in culture (day 7 and 21) from 6 Pm women (75 ± 6 years old) at the indicated experimental conditions. *P < 0.05 (RM one-way ANOVA with the Geisser-Greenhouse correction and uncorrected Fisher’s LSD test) represents significant differences. Panel c) presents representative immunofluorescence micrographs of BM-MSCs from a Pm woman (74 years old) stained against the P2Y1 receptor (green) after exposure…

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  Fig. 6. The P2Y1-induced [Ca2+]i responses are compromised in Pm BM-MSC unless the negative interplay with overexpressed P2Y13 receptors in cholesterol-rich lipid raft/caveolae microdomains is abrogated with MβC. Panel (a) relates to microplate calcium measurements. (a.i.) Shows the concentration-response curve of [Ca2+]i oscillations produced by MRS 2365 (0.01-10 µM) in Pm BM-MSCs allowed to grow for 21 days. The concentration-response curve was fitted using the least squares method by non-linear regression function: log(drug) versus response assuming that data share best-fit values from top and bottom and a hill slope equal to 1. Right hand-side graphs show fluorescence [Ca2+]i transients in response to MRS 2365 (0.1 µM) either alone or in the presence of MβC (2 mM) with or without the selective P2Y13 receptor antagonist, MRS 2211 (10 µM). *P < 0.05 and ****P < 0.0001 (multiple unpaired t-tests) represent significant differences. (aii) Comparison of the magnitude of MRS 2365 (0.1 µM)-induces [Ca2+]i rises in Pm BM-MSC cultures in the absence or presence of MβC (2 mM), either alone or applied with the selective P2Y13 receptor antagonist, MRS 2211 (10 µM). [Ca2+]i transients were…