Endostatin

To examine the effect of riociguat on biomarkers, patient plasma was collected at baseline and week 16. Plasma from age- and sex-matched healthy controls was also obtained. The biomarkers were measured by enzyme-linked immunosorbent assays (ELISA) and included total vascular endothelial growth factor (VEGF, R&D Systems), tissue plasminogen activator (tPA, Abcam), soluble E-selectin (sE-selectin, R&D Systems), basic fibroblast growth factor (bFGF, R&D Systems), vascular cell adhesion molecule 1 (VCAM-1, RayBiotech), soluble intracellular adhesion molecule 1 (sICAM-1, RayBiotech), N-terminal propeptide of type I collagen (PINP, MyBioSource), matrix metalloproteinase 12 (MMP12, RayBiotech), CXCL4 (R&D Systems), cyclic guanylyl cyclase (cGMP, Cayman Chemical), endostatin (R&D Systems), and soluble fms-like tyrosine kinase 1 (sFLT1, R&D Systems).

Hydrogel scaffolds (HS) were prepared as published by Deponti et al. [43 (link)] with some modifications. Briefly, after being trypsinized (0.05% trypsin EDTA, EuroClone, Pavia, Italy) at P3, the cells (80 × 10⁶ cells/mL) were re-suspended in a solution named A containing fibrinogen bovine plasma (113.2 mg/mL, Aigma-Aldrich, St. Louis, MA, USA), aprotinin (3.24 mg/mL, Sigma-Aldrich, St. Louis, MA, USA), tranexamic acid (30 mg/mL, Sigma-Aldrich, St. Louis, MA, USA) in PBS. The scaffolds were assembled in a 96-well plate: first, 150 µL of solution A was placed in a well, secondly, 150 µL of solution B (thrombin, 13.7 mg/mL in PBS, Millipore, Temecula, CA, USA) was added. The scaffolds polymerized after 30 min under a sterile hood at room temperature, and then they were removed from wells and immersed for 9 days or for 21 days in different concentrations of exogenous endostatin (R&D System, MN, USA) dissolved in culture medium: (i) 10 ng/mL (E10), (ii) 100 ng/mL (E100), (iii) 200 ng/mL (E200), (iv) no endostatin added as negative control (UNT). Four replicates for each treatment and time points were maintained in vitro, changing culture medium twice a week. At the end of each time point, samples were stored at −80 °C for biochemical and molecular analyses or fixed in 4% buffered formalin (Bio-Optica, Milan, Italy) for morphological analysis.