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. 2023 Feb 2;43(3):443–455. doi: 10.1161/ATVBAHA.122.318420

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© 2023 The Authors.

Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

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Figure 2. — Exposure of secondary calciprotein particles (CPPs) to human umbilical vein endothelial cells (HUVECs) for 24 hours reduces NO bioavailability and eNOS (endothelial nitric oxide synthase) mRNA expression. A, Nitrite production decreased significantly in HUVECs exposed to both 50 and 100 μg/mL secondary CPPs. B, eNOS mRNA expression was only significantly reduced after 24 hours of exposure to 100 μg/mL secondary CPPs. C, Representative protein blots showing phospho-eNOS (Ser1177), total eNOS and GAPDH (of both the P-eNOS and total eNOS blots). Molecular weight standards are indicated on the left side of the immunoblots in kilodaltons (kDa) based on the marker shown in Figure S7A and S7B. D and E, eNOS total protein expression and eNOS protein phosphorylation (Ser1177-activation site) were not affected in HUVECs after 24 hours of secondary CPP exposure. F, Low-temperature SDS-PAGE blots showing non-boiled/intact (indicated by symbol N) dimer/monomer eNOS expression, and boiled/denatured (indicated by symbol B) eNOS total monomer expression. Molecular weight standards of the marker (m) are indicated on the left side of the immunoblots in kilodaltons (kDa). G and H, eNOS uncoupling ratios (dimer/monomer ratio) and total eNOS monomer expression were not affected after 24-hour secondary CPP exposure to HUVECs. Data are presented as means±SEM with individual data points and normalized to the experimental control. Significant differences are indicated for P <0.05.