A vascular bed–specific pathway regulates cardiac expression of endothelial nitric oxide synthase
Pascale V. Guillot, Jason Guan, Lixin Liu, Jan A. Kuivenhoven, Robert D. Rosenberg, William C. Sessa, William C. Aird
Pascale V. Guillot, Jason Guan, Lixin Liu, Jan A. Kuivenhoven, Robert D. Rosenberg, William C. Sessa, William C. Aird
View: Text | PDF
Article

A vascular bed–specific pathway regulates cardiac expression of endothelial nitric oxide synthase

Abstract

The endothelial nitric oxide synthase (eNOS) gene is induced by a variety of extracellular signals under both in vitro and in vivo conditions. To gain insight into the mechanisms underlying environmental regulation of eNos expression, transgenic mice were generated with the 1,600-bp 5′ flanking region of the human eNos promoter coupled to the coding region of the LacZ gene. In multiple independent lines of mice, transgene expression was detected within the endothelium of the brain, heart, skeletal muscle, and aorta. β-galactosidase activity was consistently absent in the vascular beds of the liver, kidney, and spleen. In stable transfection assays of murine endothelial progenitor cells, the 1,600-bp promoter region was selectively induced by conditioned media from cardiac myocytes, skeletal myocytes, and brain astrocytes. Cardiac myocyte–mediated induction was partly abrogated by neutralizing anti–platelet-derived growth factor (PDGF) antibodies. In addition, promoter activity was upregulated by PDGF-AB. Analysis of promoter deletions revealed that a PDGF response element lies between –744 and –1,600 relative to the start site of transcription, whereas a PDGF-independent cardiac myocyte response element is present within the first 166 bp of the 5′ flanking region. Taken together, these results suggest that the eNos gene is regulated in the cardiac endothelium by both a PDGF-dependent and PDGF-independent microvascular bed–specific signaling pathway.

Authors

Pascale V. Guillot, Jason Guan, Lixin Liu, Jan A. Kuivenhoven, Robert D. Rosenberg, William C. Sessa, William C. Aird

×

Figure 4

Options: View larger image (or click on image) Download as PowerPoint
Expression of the 1600eNOS promoter is induced by myocyte-derived signal...
Expression of the 1600eNOS promoter is induced by myocyte-derived signaling pathways in vitro. MEEP cells were stably transfected with 1600eNOS LUC, 744eNOS LUC, and 166 eNOS LUC as described in Methods. Cells were starved in 0.2% FCS for 24 h and then incubated for 4 h in conditioned medium from HEK, HepG2, cardiac myocytes (MYO), skeletal myoctes (SKM), or brain astrocytes (AST) in the absence (a) or presence (b) of anti-PDGF antibodies, or in media supplemented with PDGF, VEGF, bFGF, and EGF at concentrations of 10 ng/ml (b). The results show the mean and SD of luciferase light units obtained in triplicate from one representative experiment. The 1600eNOS LUC construct was induced 4.8-fold by cardiac myocyte–conditioned medium, 4.3-fold by skeletal muscle–conditioned medium, 3-fold by astrocyte-conditioned medium, 2.4-fold by PDGF-AB, and 3-fold by cardiac myocyte–conditioned medium containing anti-PDGF antibodies (b). The 744eNOS LUC construct was induced 2.2-fold, and the 166eNOS LUC construct 2.7-fold, by myocyte-conditioned medium (c and d, respectively). *P < 0.05 vs. basal activity; #P < 0.05 vs. cardiac myocyte–conditioned medium. bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; MEEP, mouse embryonic endothelial progenitor; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor.