Journal: bioRxiv

Article Title: A complex osteoporotic milieu is associated with arterial stiffening and PDGF-BB-mediated calcification of human smooth muscle cells

doi: 10.1101/2025.10.25.684499

Figure Lengend Snippet: Flow cytometry analysis of (A) total granulocytes, (B) basophils, (C) eosinophils, (D) neutrophils, and platelets in the peripheral blood of non-osteoporotic frail controls (CON-F; n=16), osteopenic frail individuals (OPE; n=16), and osteoporotic frail individuals (OPO; n=28 out of 38). Differences in immune cell subsets were assessed using a Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test vs. CON-F if data were normally distributed. Non-normal data were analyzed with a Kruskal-Wallis test and Dunn’s multiple comparison. § indicates incomplete immune cell subset data. (E) Sera screening of non-osteoporotic middle-aged controls (CON-M), non-osteoporotic healthy older controls (CON-H), CON-F, OPE, and OPO individuals using bead-based multiplex assays. Clustered expression levels are given as median Z-scores. Factors with significantly different expression levels or distinct trends with p<0.1 in OPE or OPO individuals vs. CON-M, CON-H, and CON-F include (G) sCD40L, (H) PDGF-BB, (I) OPN, and (J) SDF-1. Differences between serum factors were assessed using two-way ANOVAs with Dunnett’s post-test (p<0.05) vs. CON-H, CON-M, or CON-F. Only significant differences (p<0.05) and distinct trends with p<0.1 are shown. (K) Spearman correlation of the significantly different cellular and serum factors in non-osteoporotic controls (CON: CON-M, CON-H, CON-F) and (L) in osteopenic and osteoporotic individuals (low BMD: OPE, OPO). (M) Differences between correlation coefficients of CON and low BMD were assessed using Fisher’s Z-test; * p<0.05, ** p<0.001, *** p<0.0001.

Article Snippet: The role of PDGF-BB was further assessed using osteogenic medium containing sex-mixed participant-derived sera pools supplemented with either 25 μg/ml neutralizing polyclonal human PDGF antibody (αPDGF; Cat# AB-20, RRID:AB_354273; R&D Systems ® ) dissolved in PBS, or 50 μM chebulinic acid (CheA; Cat# SMB00271, Sigma-Aldrich or Cat# A16208, AdooQ ® BioScience, USA), dissolved in ddH 2 O and briefly heated up to 70 °C to prepare a 1 mM stock.

Techniques: Flow Cytometry, Comparison, Multiplex Assay, Expressing

Journal: bioRxiv

Article Title: A complex osteoporotic milieu is associated with arterial stiffening and PDGF-BB-mediated calcification of human smooth muscle cells

doi: 10.1101/2025.10.25.684499

Figure Lengend Snippet: SMC undergoing osteogenic differentiation (OM - osteogenic medium) in an in vitro model of SMC calcification were stimulated with recombinant PDGF-BB, sCD40L, OPN, or SDF-1α. Deposited calcified matrix was stained with alizarin red. (A ) Representative images of the deposited calcified matrix. (B-E) Quantified staining, normalized to cell count. A paired, two-tailed t-test was used to assess significant differences (p<0.05) vs. OM without supplements for each time point. (F) Phosphate levels in the supernatant during osteogenic differentiation with the respective recombinant factors. Statistical analysis at each time point was performed using a mixed-effects model with Geisser-Greenhouse correction and Dunnett’s post-test vs. OM without supplements (p<0.05); no significant differences were detected. (G) SMC proliferation in expansion medium (EM; dashed line) after stimulation with the respective recombinant factors. Differences in proliferation were assessed at each time point using two-way ANOVA with Geisser-Greenhouse correction and Dunnett’s post-test vs. EM without supplements (p<0.05); no significant differences were detected. (H) Gene expression analysis of SMC after four days of osteogenic differentiation with the respective recombinant factors. Paired, two-tailed t-tests vs. OM without supplements were used to assess significant differences (p<0.05) across independent experiments.

Article Snippet: The role of PDGF-BB was further assessed using osteogenic medium containing sex-mixed participant-derived sera pools supplemented with either 25 μg/ml neutralizing polyclonal human PDGF antibody (αPDGF; Cat# AB-20, RRID:AB_354273; R&D Systems ® ) dissolved in PBS, or 50 μM chebulinic acid (CheA; Cat# SMB00271, Sigma-Aldrich or Cat# A16208, AdooQ ® BioScience, USA), dissolved in ddH 2 O and briefly heated up to 70 °C to prepare a 1 mM stock.

Techniques: In Vitro, Recombinant, Staining, Cell Counting, Two Tailed Test, Gene Expression

Journal: bioRxiv

Article Title: A complex osteoporotic milieu is associated with arterial stiffening and PDGF-BB-mediated calcification of human smooth muscle cells

doi: 10.1101/2025.10.25.684499

Figure Lengend Snippet: (A) SMC undergoing osteogenic differentiation were stimulated with sex-mixed sera pools of non-osteoporotic healthy older controls (CON-H) or osteoporotic frail individuals with (OPO + ) or without confounders of vascular calcification (OPO - ). PDGF-BB or its downstream signaling was inhibited in the respective sera pools using either 25 µg/ml neutralizing polyclonal PDGF antibody (αPDGF) or 50 µM chebulinic acid (CheA). Deposited calcified matrix was stained with alizarin red, quantified, and normalized to the cell count. A one-way ANOVA with Geisser-Greenhouse correction and Dunnett’s post-test was used to assess significant differences vs. CON-H and the respective osteoporotic group without inhibitor for each time point. (B) Representative images of deposited calcified matrix stained with alizarin red. (C) Phosphate levels determined in the supernatant during osteogenic differentiation. A two-way ANOVA with Geisser-Greenhouse correction and Dunnett’s post-test was used to assess significant differences vs. CON-H for each time point. (D) Cell counts determined by Hoechst staining after stimulation with the respective sera pools supplemented with αPDGF or CheA. A one-way ANOVA with Geisser-Greenhouse correction and Dunnett’s post-test was used to assess significant differences vs. CON-H and the respective osteoporotic group without inhibitor for each time point. Only significant differences (p<0.05) and distinct trends with p<0.1 are shown.

Article Snippet: The role of PDGF-BB was further assessed using osteogenic medium containing sex-mixed participant-derived sera pools supplemented with either 25 μg/ml neutralizing polyclonal human PDGF antibody (αPDGF; Cat# AB-20, RRID:AB_354273; R&D Systems ® ) dissolved in PBS, or 50 μM chebulinic acid (CheA; Cat# SMB00271, Sigma-Aldrich or Cat# A16208, AdooQ ® BioScience, USA), dissolved in ddH 2 O and briefly heated up to 70 °C to prepare a 1 mM stock.

Techniques: Staining, Cell Counting