Fig 1: PARP Inhibitors Decrease Calcification of hVSMCs In Vitro(A) qRT-PCR analysis of hVSMCs and MC3T3 cells in vitro shows expression of PARP1 and PARP2 enzymes in control cultures. hVSMC cultures under calcifying conditions show increased expression of PARP2 (n = 3).(B) Slot blot of hVSMC and MC3T3 cells under calcifying conditions showing increased PAR.(C) The o-cresolphthalein assay (n = 3) showed a time-dependent increase in mineralization, which was inhibited in a dose-dependent manner by the PARP inhibitor PJ-34 (0.5, 1.5, and 10 uM).(D) PARP activity was reduced by PJ34 treatment in a dose-dependent manner (n = 4) in hVSMC cultures. See Figures S6A and S6B for data on bVSMC model.(E) The PARG inhibitor DEA (0.1 mM) increased calcification of hVSMCs treated with high Ca/P media quantified by o-cresolphthalein. (n = 6). Mean ± SEM, Student’s t test, **p < 0.01, ****p < 0.0001.(F) PARP inhibitors can block calcification of hVSMCs (n = 3) and MC3T3 (n = 6) cells. PJ = PJ-34, Min, minocycline; Ola, Olaparib; Ruc, Rucaparib; Nir, Niraparib; Vel, Veliparib; all are at 3 µM. See Table S2 for enzyme inhibitor assay details and Figure S6C for details on minocycline as a PARP1/2 inhibitor.Graphs in (A), (C), (D), and (F) show mean ± SEM. Statistical significance was tested with one-way ANOVA with Dunnett’s post hoc tests. *p between p < 0.05, **p < 0.01, ****p < 0.0001.(G) Alizarin Red S staining of mineral in hVSMC cultures in the absence and presence of PARP inhibitors PJ-34 (3 µM) and minocycline (3 µM).(H) Slot blot showing increased PAR under calcifying conditions and its inhibition by minocycline (3 µ?).
Fig 2: The PARP2 Inhibitor Minocycline Inhibits Biomineralization In Vivo(A) Quantification of calcium content in rat arteries under control (n = 4), CKD (n = 14), and CKD minocycline-treated rats (n = 14). Calcium is significantly reduced in the 50 mg/kg treated group. See Figures S7A and S7B for overview and background data for the in vivo experiments.(B) Immunohistochemistry of rat aorta from control, CKD, and CKD minocycline-treated rats showing mineral (VK), PAR, DNA damage (?H2AX), and VSMCs (alpha smooth muscle [aSM] actin). Arrows in row b indicate areas of extracellular PAR, and arrows in row c indicate cells positive for ?H2AX. Scale bar, 500 µm.(C) Quantification of the mineralized area of the aorta in control (n = 4), CKD (n = 14), and CKD minocycline-treated (n = 14) rats.(D) Quantification of ?H2AX staining in the rat aorta from control (n = 4), CKD (n = 14), and CKD minocycline-treated rats (n = 14).(E) Quantification of DNA damage in control (n = 4) and uncalcified arteries from the CKD minocycline-treated group (n = 6).(F) Quantification of the total number of smooth muscle cells per unit area in the aorta control (n = 4), CKD (n = 14), and CKD minocycline-treated (n = 14) rats.(G) Electron microscopy assessment of bones from the treated animals. (top) SEM images (scale bars, 500 µm), below expansions of the indicated areas of the SEM images (scale bars, 100 µm), and (bottom) TEM images showing details of the collagen fibril calcification (scale bars, 500 nm).(H) Effect of minocycline on bone remodeling. The area fraction (in %) of “solid bone” in the cortical area of the bone cross section is decreased in minocycline-treated CKD animals.All data were tested for normality using Shapiro-Wilk test. (A, C, D, F, and H) Mean ± SEM. Statistical significance was determined by Kurskall-Wallis test followed by Mann-Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001. (E) Mean ± SEM. Statistical significance was determined by unpaired Student’s t test, *p < 0.05. See Figure S7C for details of the quantification.
Supplier Page from Abcam for Recombinant human PARP2 protein (GST tag N-Terminus)