Erythropoietin (EPO)-induced arterial hypertension may result from vasoconstriction as well as vascular remodeling. We have shown previously in in vitro-studies on cultured porcine EC a rise in intracellular calcium and enhanced endothelin (ET)-1 synthesis when exposed to rHuEPO. Since the effects on cell calcium and ET-1 synthesis require the presence of EPO receptors and since cell proliferation involves stimulation of mitogenactivated protein (MAP) kinases, in the present study we

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... d in endothelial cells (3rd to 5th subcultures) derived from porcine aortic tissue (porcine EC) and from human umbilical venous endothelium (HU-VEC) characteristics of EPO receptors and -in the absence and presence of a polyclonal EPO receptor (EPOr) antibody (Biogenesis) -the effects of rHuEPO (Epoetin beta) at doses of 0.1 to 100 U/ml on preproET-1 mRNA expression and ET-1 synthesis determined by RT-PCR and ELISA, respectively, as well as its effects on ERK, JNK, and p38 MAP kinase phosphorylation using Western blot analysis (EGF, 10 mg/ml, served as positive control). EPO receptors were found on porcine EC with Bmax of 323 ± 11 fmol/mg protein and Kd of 24 ± 2 nmol/l. When incubated in the presence of 1% FCS for 2, 4, and 12 h spontaneous in vitro-synthesis of ET-1 by porcine EC was time-dependent reaching 6.37 ± 0.64 pmol/mg protein at 24 h incubation. In the absence of FCS 10 and 100 U/ml rHuEPO dose-dependently increased ET-1 concentration in the incubation medium from 0.37 ± 0.05 (without rHuEPO) to 0.48 ± 0.09 (n.s.) and 0.67 ± 0.17 pmol/mg protein (p < 0.05), respectively, after 4 h incubation and from 0.80 ± 0.17 (without rHuEPO) to 2.02 ± 0.26 and 2.14 ± 0.45 pmol/mg protein (p < 0.01), respectively, after 12 h incubation. HUVEC showed basal ET-1 synthesis of 0.84 ± 0.06 pmol/mg protein/4h without effects of 100 U/ml rHuEPO (0.91 ± 0.05 pmol/mg protein/4h). No definite effects of EPOr antibody were observed under the present experimental conditions. After 4 h incubation 10 and 100 U/ml rHuEPO had no effect on preproET-1 mRNA (preproET-1/GAPDH mRNA ratio 0.96 ± 0.08 (n = 5) and 0.75 ± 0.09 (n = 6), respectively, vs. 0.96 ± 0.05 (n = 5) without rHuEPO). While rHuEPO, 0.1 to 10 U/ml, had no effects on JNK or p38 MAP kinases in HUVEC, it stimulated phosphorylation of ERK 1/2 (p-ERK) in porcine EC dose-and time-dependently starting at 5 minutes, reaching a maximum at 10 minutes, and returning to control values at 60 minutes of incubation. We conclude that in porcine EC, but not in HUVEC, 1st, EPO receptors are present, 2nd, that acute rHuEPO-induced ET-1 release contributes to the vasoconstrictor action of EPO, and 3rd, that rHuEPO results in phosphorylation of ERK 1/2 which probably contributes to long-term vascular proliferation and remodeling. Free Communication June 12 Background: Circulating bone marrow-derived endothelial progenitor cells (EPCs) promote vascular reparative processes and neo-angiogenesis. We tested the hypothesis that erythropoietin (EPO) stimulates proliferation and differentiation of EPCs in vitro. Methods and Results: Peripheral mononuclear cells were isolated from healthy volunteers and cultured under selective culture conditions in order to isolate EPCs. We performed fluorescent chemical detection to verify that attached cells after 7 days in culture are EPCs. Attached cells were stained for their uptake of acetylated low density lipoprotein (acLDL) and the binding of Ulex europaeus agglutinin I (UEA-I). Dual positive cells