In vivo expression profile of an endothelial nitric oxide synthase promoter-reporter transgene

Abstract

Endothelium-derived nitric oxide (NO) is primarily attributable to constitutive expression of the endothelial nitric oxide synthase (eNOS) gene. Although a more comprehensive understanding of transcriptional regulation of eNOS is emerging with respect to in vitro regulatory pathways, their relevance in vivo warrants assessment. In this regard, promoter-reporter insertional transgenic murine lines were created containing 5,200 bp of the native murine eNOS promoter directing transcription of nuclear-localized beta-galactosidase. Examination of beta-galactosidase expression in heart, lung, kidney, liver, spleen, and brain of adult mice demonstrated robust signal in large and medium-sized blood vessels. Small arterioles, capillaries, and venules of the microvasculature were notably negative, with the exception of the vasa recta of the medullary circulation of the kidney, which was strongly positive. Only in the brain was the reporter expressed in non-endothelial cell types, such as the CA1 region of the hippocampus. Epithelial cells of the bronchi, bronchioles, and alveoli were scored as negative, as was renal tubular epithelium. Cardiac myocytes, skeletal muscle, and smooth muscle of both vascular and nonvascular sources failed to demonstrate beta-galactosidase staining. Expression was uniform across multiple founders and was not significantly affected by genomic integration site. These transgenic eNOS promoter-reporter lines will be a valuable resource for ongoing studies addressing the regulated expression of eNOS in vivo in both health and disease.

An Alternative Promoter of the Human Neuronal Nitric Oxide Synthase Gene Is Expressed Specifically in Leydig Cells

“…2 In contrast to the Leydig cell-restricted expression of the TnNOS promoter, we found that expression of the nuclear-localized Ϫ5200/ϩ28 Mu eNOSprom nls LacZ was active exclusively in the nuclei of vascular endothelium of mediumand large-sized arteries (Figure 4). Expression patterns were comparable in three independent 1, 5, and 10 copy number founders.…”

“…The nNOS (NOS1) isoform is expressed from a very complex human genomic locus spanning 240 kb at 12q24.2. 1 In contrast to the restricted endothelial cell-specific expression of the endothelial NOS (eNOS), 2 the nNOS isoform is constitutively expressed in a series of diverse cell types and tissues. 3 For instance, nNOS plays a fundamental role in the regulation of male sexual function.…”

Relative Reduction of Endothelial Nitric-Oxide Synthase Expression and Transcription in Atherosclerosis-Prone Regions of the Mouse Aorta and in an in Vitro Model of Disturbed Flow

“…51,73,74 Constitutive expression is modulated by many factors, including laminar shear stress, cytokines, oxidized LDL, hypoxia, estrogen, and cell proliferation; and expression levels are regulated by transcription, mRNA stability, and epigenetics. [75][76][77][78] To determine whether shear stress-induced modulation of eNOS expression involves regulation of transcription, HAECs were exposed to uniform laminar shear stress, and the rate of transcription was determined by measuring hnRNA levels.…”

“…51 Expression of the nuclear-localized ␤-galactosidase reporter in these transgenic mice reflects eNOS promoter activity because previous studies demonstrated that expression of the reporter was highly comparable with endogenous eNOS, 51 which suggests that the transgenic promoter faithfully reflects transcriptional activity of endogenous eNOS. Histochemical staining revealed that nuclear localized expression of ␤-galactosidase was markedly diminished in the LC ( Figure 5, A and B).…”

“…C57BL/6 mice, 129 ϫ 1/SvJ, 129, C3SW-H2b/SnJ congenic strain (C3H), B6.129P2-Nos3 tm1Unc /J (eNOS Ϫ/Ϫ ) strain (Jackson Laboratory, Bar Harbor, ME), and eNOSpromoter-␤-galactosidase-reporter transgenic mice 51 in a mixed C57BL/6-SJL strain were used at an age of 2 to 6 months. Mice were fed a standard laboratory chow diet, bred and housed at the University Health Network Animal Facility, and maintained in accordance with guidelines of the Canadian Council on Animal Care.…”