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The supernatants were collected at the end of the study period, and frozen at ??70?��C. To examine interactions between VEGF and BMP9 with reference to ET-1 production, the HMVEC-LBl and HUVEC were exposed to VEGF-121 (40?ng/mL) and/or BMP9 (2.5?ng/mL, R&D Systems, Minneapolis, MN ) in serum-free medium for 7?h. The supernatants were then collected and frozen as above. In another experiment, HMVEC-LBl were exposed to VEGF-121 (40?ng/mL) in serum-free medium for 7?h, with or without the VEGF receptor antagonist SU5416 (0�C10?��M). The supernatant was collected and frozen. In all these experiments, the supernatant was subsequently thawed and immunoreactive ET-1 levels were measured by ELISA (Enzo Life Sciences, Ann Arbor, Michigan) and expressed as pg/mL. Data are expressed as mean?��?SD. To compare groups, analysis-of-variance was performed and followed, where appropriate by the Tukey�CKramer multiple comparison test. Using Western blotting, in the HMVEC-LBl and in serum-free medium, there was no evidence of background VEGF receptor-2 phosphorylation. Addition of the VEGF receptor blockers, (E)-FeCP-oxindole and SU5416, did not affect the background phosphorylation. Addition of VEGF caused rapid phosphorylation as detected after 5?min, and this was sustained although slightly diminished at 10?min. Attempted blockade with (E)-FeCP-oxindole at the manufacturer's recommended dose had no effect on the phosphorylation. However, SU5416 blocked the effects of VEGF, completely preventing VEGFR2 phosphorylation. In both HMVEC-LBl and HUVEC, exposure to VEGF significantly decreased ET-1 production, as assessed by ET-1 levels in the supernatant, after 7?h. This reduction occurred in a concentration-dependent manner, with the greatest effect in each case seen at VEGF 40?ng/mL. At that concentration, ET-1 levels were decreased by 63% in HMVEC-LBl and by 48% in HUVEC. Within the range studied, the dose response was more evident in HMVEC-LBl. In HMVEC-LBl not exposed to VEGF, and in serum-free medium, addition of the VEGF receptor blocker, SU5416, significantly increased basal ET-1 production, by up to 21% at 3?ng/mL, p?