A vascular bed-specific pathway

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. beta-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.

Figure 1

Whole mount microphotographs of the 1600eNOSLacZ. Incubation of the organs with the X-Gal substrate reveals LacZ staining in the adult heart (a) and aorta (b). LacZ staining in the heart represents β-galactosidase activity within microvascular and macrovascular endothelial cells (see Fig. 2). The X-Gal reaction product in the aorta is accentuated in endothelial cells surrounding the ostia of two intercostal tributaries (b, arrows). eNOS, endothelial nitric oxide synthase.

Figure 2

The 1600eNOSLacZ transgene is expressed in blood vessels of the heart, brain, and skeletal muscle. LacZ staining of 9-μm tissue sections from 1600eNOS LacZ line 338-8 reveals reporter gene activity in the endothelial lining of cardiac microvessels (a, d), coronary arteries (b), skeletal muscle (e), and brain (f). There is no detectable β-galactosidase activity in cardiac myocytes (a), liver (g), kidney (h), and lung (i). Similar LacZ staining pattern in cardiac microvessels from transgenic line 338-41 can be seen in c. Scale bar: 60 μm.

Figure 3

β-galactosidase activity correlates with LacZ mRNA levels. RT-PCR analysis of LacZ, and RT-PCR and Western blot analyses of endogenous eNOS in 1600eNOS LacZ mouse tissues, reveal the presence of detectable β-galactosidase transcripts within the heart, brain, skeletal (sk) muscle, and to a lesser extent the lung, whereas eNOS transcripts and protein are present in all organs. For RT-PCR, each lane represents an RT-PCR analysis from identical DNA template. β-actin is included as a control for RNA levels. For Western blot analysis, the 140-kDa control eNOS is a positive control derived from human aortic endothelial cell lysates. RT-PCR, reverse transcriptase-PCR.

Figure 4

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.

Figure 5

eNOS RNA levels are increased by PDGF. BAECs were starved in 0.5% FCS for 24 h and then incubated with either VEGF, EGF, bFGF, or PDGF-AB at 10 ng/ml for 4 h. Total RNA (5 μg) was subjected to agarose electrophoresis. (a) Autoradiogram with an eNOS cDNA probe. (b) The ethidium-stained 28S ribosomal band. BAECs, bovine aortic endothelial cells.