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-plateletderived 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. J. Clin. Invest. 103:799-805 (1999).Methods eNOS reporter gene constructs. The various human eNOS promoter constructs were derived from F1 LUC (7). To generate the transgenic construct 1600eNOSlacZ, the KpnI-NotI promoter fragment (-1,600 to +22 bp) was isolated from the F1 LUC vector and inserted into a plasmid-containing LacZ cDNA coupled to the SV40 poly(A) tail (gift from J. Rossant, Mount Sinai Hospital, Toronto, Canada). For stable transfections, the F1 LUC was digested with SmaI, releasing the -744 to -1,600 promoter fragment. The remaining vector was religated, resulting in 744eNOS LUC. The generation of the 166eNOS LUC (also referred to as F6 LUC) has been described previously (7).Generation and analysis of transgenic mice. The 1600eNOSlacZ plasmid was digested with KpnI and NotI to release the Bluescript vector (Stratagene, La Jolla, California, USA), and the fragment consisting of the 1,600-bp eNOS promoter fragment coupled to LacZ was isolated on agarose gel. The DNA was resuspended in injection buffer and injected into fertilized mouse eggs as described previously (16). Transgenic founders were identified by Southern blot analysis using a [ 32 P]dCTP-labeled DNA probe containing LacZ coding sequence. The founders were bred to wild-type FVB mice to generate stable transgenic lines. Tissues were harvested from adult F1 offspring heterozygous for the transgene and processed for LacZ staining as described previously (16). For reverse transcriptase-PCR (RT-PCR), total RNA was harvested from various FVB mouse organs using a guanidinium thiocyanate phenol-chloro...

The Cell-specific Expression of Endothelial Nitric-oxide Synthase

“…Interestingly, expression was uniform across multiple founders and was not significantly affected by the genomic integration site. This can be contrasted with the variability in expression observed across founders with eNOS reporter transgenes containing smaller 5Ј-flanking sequences (39,53). We report here that the methylation status of our transgene mirrors the methylation pattern observed in vascular endothelial cells.…”

Endothelial nitric oxide synthase activity is linked to its presence at cell–cell contacts

“…In addition, the expression and regulation of eNOS have been suggested to depend on the vascular bed from which the cells are derived [42][43][44]. This could also account for the contrasting data.…”

Vascular Biology in Genetically Altered Mice

“…57,[141][142][143][144][145][146] To target endothelial expression of eNOS in transgenic mice, 5Ј flanking sequences from the preproendothelin-1 gene 57 were used, and studies of the eNOS promoter region itself reveal that 1600-bp of the human eNOS 5Ј flanking region will direct expression of the LacZ gene to the vascular endothelium. 147 The smooth muscle ␣-actin and SM22␣ 148 -150 promoters have been used to efficiently target smooth muscle cells in transgenic mice. With these tools comes the opportunity to specifically generate models of vascular disease, to test potentially therapeutic agents, and to overexpress wild-type or dominant-negative mutants of receptors or intracellular signaling intermediates, antisense molecules, or reporter genes, among others.…”