Platelet-derived growth factor–BB, VEGF-A, and TNF-α plasma levels were determined by ELISA (Human PDGF-BB Quantikine ELISA Kit #DBB00, Quantikine VEGF ELISA Kit; R&D Systems Europe, Abingdon, UK; #DVE00, and Human TNF-α Quantikine ELISA Kit DTA00C; R&D Systems Europe) as described by the manufacturer. Briefly, 100 mL of assay diluents (RD1X for PDGF-BB, RD1W for VEGF, and RD1F for TNF- α) was added to each well of 96-well polystyrene microplates, then 100 mL of standard or samples (EDTA-plasma) was added to each well, mixed by gently tapping the plate frame for 1 minute, and incubated for 2 hours at room temperature. Afterwards, washing with wash buffer (400 mL) was performed three times, followed by addition of 200 mL of polyclonal antibody conjugate to each well, incubation for 2 hours at room temperature and washing again with wash buffer three times. Subsequently, 200 mL of substrate solution was added to each well, incubated for 25 minutes at room temperature and finally, 25 mL of stop solution was added to each well. The concentration was determined by an ELISA reader at 450 nm. For TNF-α measurements the minimum detectable dose (MDD) defined by the manufacturer was 1.6 pg/mL. The percentage of TNF-α results falling below the limit of sensitivity is stated for each study subgroup, and calculations were based on observations above the detection limits. All ELISA readings of PDGF-BB and VEGF measurements were within the respective detection limits.