Functional Expression of Endothelial Nitric Oxide Synthase Fused to Green Fluorescent Protein in Transgenic Mice

Haperen, Rien van; Cheng, Caroline; Mees, Barend; Deel, Elza van; Waard, Monique de; Damme, Luc C.A. van; Gent, Dik C. van; Aken, Thijs van; Krams, Rob; Duncker, Dirk J.; Crom, Rini de

doi:10.1016/s0002-9440(10)63524-9

Cited by 61 publications

(75 citation statements)

References 46 publications

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“…Finally, we examined the subcellular localization of eNOS in isolated carotid arteries using en face imaging. As shown in Figure 4F, in WT carotid arteries, a majority of eNOS was localized in a distinct perinuclear crescent mainly downstream from the flow axis; this pattern was similar for Cav-1 KO and Cav-1 RC mice and consistent with previous studies examining in situ eNOS localization (30,31).…”

Section: Figuresupporting

confidence: 89%

“…Moreover, basal eNOS phosphorylation on serine 1176 in mice, a key regulatory site of phosphorylation by many kinases including Akt, AMP kinase, and cAMP-dependent protein kinase, was reduced in extracts prepared from Cav-1 KO vessels, suggesting that flow activation of upstream kinases may be impaired. An alternative possible mechanism was that perhaps the loss of caveolae results in eNOS mislocalization as suggested by some (35) but not all investigators (16,36); however, this is not the case, based on similar en face imaging of eNOS in vessels from WT, Cav-1 KO, and Cav-1 RC as seen in intact endothelium of humans (37) and of mice expressing the functional reporter eNOS-GFP as a transgene (30,31). Two very recent studies propose different mechanosensory complexes, both linked to the integrin/src/PI3K pathways, that may regulate shear signalling.…”

Section: Discussionmentioning

confidence: 99%

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Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels

Bergaya²,

Murata³

et al. 2006

Journal of Clinical Investigation

338

306

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Caveolae in endothelial cells have been implicated as plasma membrane microdomains that sense or transduce hemodynamic changes into biochemical signals that regulate vascular function. Therefore we compared longand short-term flow-mediated mechanotransduction in vessels from WT mice, caveolin-1 knockout (Cav-1 KO) mice, and Cav-1 KO mice reconstituted with a transgene expressing Cav-1 specifically in endothelial cells (Cav-1 RC mice). Arterial remodeling during chronic changes in flow and shear stress were initially examined in these mice. Ligation of the left external carotid for 14 days to lower blood flow in the common carotid artery reduced the lumen diameter of carotid arteries from WT and Cav-1 RC mice. In Cav-1 KO mice, the decrease in blood flow did not reduce the lumen diameter but paradoxically increased wall thickness and cellular proliferation. In addition, in isolated pressurized carotid arteries, flow-mediated dilation was markedly reduced in Cav-1 KO arteries compared with those of WT mice. This impairment in response to flow was rescued by reconstituting Cav-1 into the endothelium. In conclusion, these results showed that endothelial Cav-1 and caveolae are necessary for both rapid and long-term mechanotransduction in intact blood vessels.

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Section: Figuresupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels

Bergaya²,

Murata³

et al. 2006

Journal of Clinical Investigation

338

306

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“…More recently, transgenesis of the entire human eNOS gene encoding an eNOS-green fluorescent protein recapitulated imaging and functional studies of eNOS-green fluorescent protein in cultured cells (43) and unequivocally documented the bimodal distribution of eNOS into the Golgi complex and plasma membrane of endothelial cells lining intact blood vessels (46). However, the presence of eNOS in both locations has complicated the process of identifying the individual roles played by each pool of eNOS and has made it difficult to determine whether the Golgi pool is merely a biosynthetic precursor pool of enzyme in route to the plasma membrane or an active pool of enzyme.…”

Section: Discussionmentioning

confidence: 99%

Targeting of Endothelial Nitric-oxide Synthase to the Cytoplasmic Face of the Golgi Complex or Plasma Membrane Regulates Akt- Versus Calcium-dependent Mechanisms for Nitric Oxide Release

Fulton

Babbitt

Zoellner

et al. 2004

Journal of Biological Chemistry

126

125

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The heterogeneous localization of endothelial nitricoxide synthase (eNOS) on the Golgi complex versus the plasma membrane has made it difficult to dissect the regulation of each pool of enzyme. Here, we generated fusion proteins that specifically target the plasma membrane or cytoplasmic aspects of the Golgi complex and have assessed eNOS activation. Plasma membrane-targeted eNOS constructs were constitutively active, phosphorylated, and responsive to transmembrane calcium fluxes, yet were insensitive to further activation by Aktmediated phosphorylation. In contrast, cis-Golgi complex-targeted eNOS behaved similarly to wild-type eNOS and was less sensitive to calcium-dependent activation and highly responsive to Akt-dependent phosphorylation compared with plasma membrane versions. In plasma membrane-and Golgi complex-targeted constructs, Ser 1179 is critical for NO production. This study provides clear evidence for functional roles of plasma membrane-and Golgi complex-localized eNOS and supports the concept that proteins thought to be regulated and to function exclusively in the plasma membrane of cells can indeed signal and be regulated in internal Golgi membranes.Within the vascular endothelium, nitric oxide (NO) is generated via the enzyme endothelial nitric-oxide synthase (eNOS). 1

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“…Interestingly, eNOS protein localization in endothelium and signaling to eNOS in Cav-1 KO is rescued in Cav-1 RC mice Next, we used whole-mount immunofl uorescent staining to examine the infl uence of Cav-1 KO and RC on endothelial NO synthase (eNOS) localization in intact endothelium lining intrapulmonary arteries. eNOS is typically colocalized on the Golgi complex and in Cav-1 -positive plasmalemmal microdomains in vivo ( 11,17,18 ). Fig.…”

Section: Generation Of Tg Mice Expressing Ec Cav-1 On a Cav-1 -Defi Cmentioning

confidence: 98%

Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice

Murata¹,

Lin²,

Huang³

et al. 2007

J Cell Biol

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Caveolin-1 (Cav-1) is the principal structural component of caveolae organelles in smooth muscle cells, adipocytes, fi broblasts, epithelial cells, and endothelial cells (ECs). Cav-1 -defi cient (Cav-1 knockout [KO]) mice are viable and show increases of nitric oxide (NO) production in vasculature, cardiomyopathy, and pulmonary dysfunction. In this study, we generated EC-specifi c Cav-1 -reconstituted (Cav-1 RC) mice and reexamined vascular, cardiac, and pulmonary phenotypes. Cav-1 KO pulmonary arteries had decreased smooth muscle contractility and increased endothelial NO synthase activation and hypotension; the latter two effects were rescued completely in Cav-1 RC mice. Cav-1 KO mice exhibited myocardial hypertrophy, pulmonary hypertension, and alveolar cell hyperproliferation caused by constitutive activation of p42/44 mitogen-activated protein kinase and Akt. Interestingly, in Cav-1 RC mice, cardiac hypertrophy and pulmonary hypertension were completely rescued, whereas alveolar hyperplasia was partially recovered because of the lack of rescue of Cav-1 in bronchiolar epithelial cells. These results provide clear physiological evidence supporting the important role of cell type -specifi c Cav-1 expression governing multiple phenotypes in the vasculature, heart, and lung. 2374 REGULATION OF PARENCHYMAL FUNCTIONS BY ENDOTHELIAL CAVEOLIN-1 | Murata et al.WT arteries under resting and Ach-stimulated conditions (3 M for 3 min). Again, EC-specifi c Cav-1 RC suppressed this increase in cGMP. In all of the strains, the sensitivity of the soluble guanylyl cyclase was normal because the increases in cGMP production in response to the NO donor sodium nitroprusside (1 M for 3 min) were similar. channels; these eff ects are rescued by breeding global Cav-1 KO to EC-specifi c Cav-1 transgenic (TG) mice ( 15 ) to reconstitute Cav-1 (called Cav-1 RC) back into the endothelium ( 11,16 ). We show that EC-specifi c Cav-1 reconstitution corrects the abnormal vasomotion in vessels and the cardiac and pulmonary abnormalities observed in Cav-1 KO mice, demonstrating that the diverse cardiovascular phenotypes in these mice are attributable to caveolae in the endothelium and not to other cell types that express Cav-1. RESULTS Generation of TG mice expressing EC Cav-1 on a Cav-1 -defi cient backgroundTo assess the eff ects of Cav-1/caveolae on vascular, lung, and cardiac phenotypes, mice defi cient in Cav-1 were bred to TG mice expressing Cav-1 in ECs alone, as previously reported ( 11,16 ). Fig. 1 A documents Cav-1 protein expression in intrapulmonary arteries isolated from these mice. WT mice demonstrate the presence of Cav-1 ( Fig. 1 A , left column, green channel) predominantly in the endothelium and lower levels in smooth muscle ( n ϭ 5; Fig. 1 A, top, Cav-1 alone; middle, labeling of smooth muscle ␣ -actin [red] and nuclei [DAPI, blue]; bottom, merged images), whereas Cav-1 KO mice (middle column) lack Cav-1 immunostaining in both layers ( n ϭ 5). Reconstitution of Cav-1 into the endothelium generated by crossing Cav-1 KO...

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Functional Expression of Endothelial Nitric Oxide Synthase Fused to Green Fluorescent Protein in Transgenic Mice

Cited by 61 publications

References 46 publications

Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels

Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels

Targeting of Endothelial Nitric-oxide Synthase to the Cytoplasmic Face of the Golgi Complex or Plasma Membrane Regulates Akt- Versus Calcium-dependent Mechanisms for Nitric Oxide Release

Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice

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