Endothelin-1 expression in vascular adventitial fibroblasts

Endothelin-1 expression in vascular adventitial fibroblasts. Am J Physiol Heart Circ Physiol 290: H700-H708, 2006. First published August 19, 2005; doi:10.1152/ajpheart.00326.2005.-Endothelial cells are a major source of endothelin (ET)-1, but the possibility that vascular adventitial fibroblasts generate ET-1 has not been explored. We hypothesized that aortic adventitial fibroblasts have the ability to produce ET-1, which may contribute to extracellular matrix synthesis. Vascular adventitial fibroblasts were isolated from mouse aorta and incubated with various concentrations of angiotensin II (ANG II). mRNA levels of preproET-1 and type I procollagen were detected with relative RT-PCR. ET-1 levels in culture medium were measured with ELISA. Protein levels of procollagen were detected with Western blotting. ANG II ( 10 and 100 nM, 1 mu M) induced a time- and concentration-dependent increase in preproET-1 mRNA levels (P < 0.05). Induction of preproET-1 mRNA was accompanied by release of immunoreactive peptide ET-1 (P < 0.05). ANG II-evoked increases in preproET-1 mRNA expression and ET-1 release were blocked by losartan (100 mu M), an AT(1) receptor antagonist, but not PD-123319 ( 100 mu M), an AT(2) receptor antagonist. To further confirm our findings, we cloned and then sequenced vascular fibroblast preproET-1 bidirectionally with T7 and M13 reverse sequencing primers. Their nucleotide sequences were identical to preproET-1 cDNA from mouse vascular endothelial cells ( accession no. AB081657). Moreover, ANG II- induced type I procollagen mRNA and protein expression were inhibited by BQ-123 ( 10 mu M), an ETA receptor inhibitor, but not BQ-788 (10 mu M), an ETB receptor inhibitor, suggesting a significant role of adventitial ET-1 in regulation of extracellular matrix synthesis. The results demonstrate that vascular adventitial fibroblasts are able to synthesize and release ET-1 in response to ANG II.