Fig 1: CPPs induce endothelial activation and endothelial-to-mesenchymal transition. (A) HCAECs and HITAECs were cultured in the presence of PBS, MPPs, CPP-P, or CPP-S (100 µL of particles per well of a 6-well plate, OD650 = 0.08–0.10, for CPP-P and CPP-S: equal to 50 µg calcium or 1.2 × 105 OsteoSense 680EX-positive PKH67-negative events) for 4 h, and total RNA was extracted with the following expression profiling for the indicated genes (n = 3 wells per group). Heat map shows differentially expressed genes (fold change > 2) between groups. (B) HCAECs and HITAECs were cultured in the presence of PBS, MPPs, CPP-P, or CPP-S (100 µL of particles per well of a 6-well plate, OD650 = 0.08–0.10, for CPP-P and CPP-S: equal to 50 µg calcium or 1.2 × 105 OsteoSense 680EX-positive PKH67-negative events) for 4 h. Conditioned media were profiled for interleukin-6 and -8 using an enzyme-linked immunosorbent assay (n = 6 wells per group). Each dot represents one well of the culture plate. Whiskers indicate the range, box bounds indicate the 25th–75th percentiles, and centre lines indicate the median. p-values are provided above the boxes (statistically significant differences are marked by asterisks), Kruskal–Wallis test with post hoc false discovery rate correction by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. (C) Conditioned media and cell lysate from HCAECs and HITAECs from the experiment in (B) were profiled using a cytokine array. Note the increase in interleukin-8 in both the supernatant and cell lysate as well as the higher contents of macrophage migration inhibitory factor (MIF) and chemokine (C-X-C motif) ligand 1 (CXCL1) in the supernatant. Numbers indicate densitometry relative to the PBS group. (D) HCAECs and HITAECs were cultured in the flow system for 24 h with subsequent treatment with PBS, MPPs, CPP-P, or CPP-S (500 µL of particles per flow system unit, OD650 = 0.08–0.10, for CPP-P and CPP-S: equal to 250 µg calcium or 0.6 × 106 OsteoSense 680EX-positive PKH67-negative events) for 4 h and incubation with CellTracker Green CMFDA labelled peripheral blood mononuclear cells (PBMCs) for 1 h. Representative images. Note the considerable adhesion of PBMCs to CPP-P and CPP-S-treated cells. (E) Semi-quantitative image analysis of the experiment in (D). Each dot represents one image (n = 40 images per group). Whiskers indicate the 25th–75th percentiles, and centre lines indicate the median. p-values are provided above boxes (statistically significant differences are marked by asterisks), Kruskal–Wallis test with post hoc false discovery rate correction by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. (F) HCAECs and HITAECs were treated with PBS or CPP-P (1000 µL of particles per T-75 flask, OD650 = 0.08–0.10, equal to 500 µg calcium or 1.2 × 106 OsteoSense 680EX-positive PKH67-negative events) for 4 h with further immunoblotting for proteins reflective of endothelial activation and endothelial-to-mesenchymal transition. Note increases in VCAM1 and ICAM1, cell adhesion molecules mediating leukocyte attachment, and Snail and Slug, the key transcription factors regulating endothelial-to-mesenchymal transition. ß-tubulin was used as the loading control. Numbers indicate densitometry relative to the PBS group. HCAECs—human coronary artery endothelial cells, HITAECs—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPPs—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, OD650—optical density at 650 nm wavelength, CMFDA—5-chloromethylfluorescein diacetate, VCAM1—vascular cell adhesion molecule 1, ICAM1—intercellular cell adhesion molecule 1, KDR—kinase insert domain receptor, VE-cadherin—vascular endothelial cadherin, and N-cadherin—neural cadherin.
Fig 2: The levels of 40 adipokines of fibroblast line a FIB2 and b FIB85 are depicted in these graphs. These data were calculated via two independent Human Adipokine Arrays each (Catalog #ARY024, R&D Systems, Minneapolis, Minneapolis). Abbreviations are as follows: TNF superfamily member 13b (BAFF/BlyS/TNFSF13B), bone morphogenetic protein 4 (BMP4), complement factor 9 (complement factor), dipeptidyl peptidase 4 (DPPIV/CD26), S100 calcium binding protein A12 (EN-RAGE), fibroblast growth factor 2 (FGF basic), fibroblast growth factor 19 (FGF-19), hepatocyte growth factor (HGF), intercellular adhesion molecule 1 (ICAM-I/CD54), insulin-like growth factor binding protein 2 (IGFBP-2), insulin-like growth factor binding protein 3 (IGFBP-3), insulin-like growth factor binding protein 4 (IGFBP-4), insulin-like growth factor binding protein 6 (IGFBP-6), insulin-like growth factor binding protein 7 (IGFBP-rp1/IGFBP-7), interleukin 1 Beta (IL-1beta/IL-1F2), interleukin 6 (IL-6), interleukin 8 (8CXCL8/IL-8), interleukin 10 (IL-10), interleukin 11 (IL-11), laryngeal adductor paralysis (LAP), transforming growth factor beta 1 (TGF-beta1), LIF interleukin 6 family cytokine (LIF), C-C motif chemokine ligand 2 (CCL2/MCP-1), colony-stimulating factor 1 (M-CSF), macrophage migration inhibitory factor (MIF), delta-like non-canonical notch ligand 1 (Pref-1/DLK-1/FA1), advanced glycosylation end-product specific receptor (RAGE), C-C motif chemokine ligand 5 (CCL5/RANTES), TIMP metallopeptidase inhibitor 1 (TIMP-1), TIMP metallopeptidase inhibitor 3 (TIMP-3), tumor necrosis factor (TNF-alpha), VEGF (vascular endothelial growth factor A)
Fig 3: These graphs depict the levels of 39 obesity-related molecules that accompany a a low VEGF (vascular endothelial growth factor A) and b a high VEGF expression. These data were evaluated via a Human Adipokine Array (Catalog #ARY024, R&D Systems, Minneapolis, Minneapolis). Abbreviations are as follows: TNF superfamily member 13b (BAFF/BlyS/TNFSF13B), bone morphogenetic protein 4 (BMP4), complement factor 9 (complement factor), dipeptidyl peptidase 4 (DPPIV/CD26), S100 calcium binding protein A12 (EN-RAGE), fibroblast growth factor 2 (FGF basic), fibroblast growth factor 19 (FGF-19), hepatocyte growth factor (HGF), intercellular adhesion molecule 1 (ICAM-I/CD54), insulin-like growth factor binding protein 2 (IGFBP-2), insulin-like growth factor binding protein 3 (IGFBP-3), insulin-like growth factor binding protein 4 (IGFBP-4), insulin-like growth factor binding protein 6 (IGFBP-6), insulin-like growth factor binding protein 7 (IGFBP-rp1/IGFBP-7), interleukin 1 Beta (IL-1beta/IL-1F2), interleukin 6 (IL-6), interleukin 8 (8CXCL8/IL-8), interleukin 10 (IL-10), interleukin 11 (IL-11), laryngeal adductor paralysis (LAP), transforming growth factor beta 1 (TGF-beta1), LIF interleukin 6 family cytokine (LIF), C-C motif chemokine ligand 2 (CCL2/MCP-1), colony-stimulating factor 1 (M-CSF), macrophage migration inhibitory factor (MIF), delta-like non-canonical notch ligand 1 (Pref-1/DLK-1/FA1), advanced glycosylation end-product specific receptor (RAGE), C-C motif chemokine ligand 5 (CCL5/RANTES), TIMP metallopeptidase inhibitor 1 (TIMP-1), TIMP metallopeptidase inhibitor 3 (TIMP-3), tumor necrosis factor (TNF-alpha)
Fig 4: This heatmap shows the relative levels (in arbitrary pixel values) of 14 proteins involved in angiogenesis, as evaluated by two independent Human Adipokine Arrays (Catalog #ARY024, R&D Systems, Minneapolis, MN, USA). These arrays were performed on seven vascular endothelial growth factorlow adipose-derived stroma cell samples: PAT5, FAT4, FAT3, POL, PER, TAD, KRA. Standard deviation of all measurements was below 12%. FGF basic fibroblast growth factor 2, FGF-19 fibroblast growth factor 19, HGF hepatocyte growth factor, ICAM-I/CD54 intercellular adhesion molecule 1, IGFBP‑2 insulin like growth factor binding protein 2, IGFBP‑3 insulin like growth factor binding protein 3, TIMP‑1 tissue inhibitor of metalloproteinases 1, TIMP‑3 tissue inhibitor of metalloproteinases 3, TNF-alpha tumor necrosis factor, VEGF vascular endothelial growth factor
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