Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: Enhancement by angiotensin II

Superoxide anion (O2−) plays a key role in the endogenous suppression of endothelium-derived nitric oxide (NO) bioactivity and has been implicated in the development of hypertension. In previous studies, we found that O2− is produced predominantly in the adventitia of isolated rabbit aorta and acts as a barrier to NO. In the present studies, we characterize the enzyme responsible for O2− production in the adventitia and show that this enzyme is a constitutively active NADPH oxidase with similar composition as the phagocyte NADPH oxidase. Constitutive O2−-generating activity was localized to aortic adventitial fibroblasts and was enhanced by the potent vasoconstrictor angiotensin II. Immunohistochemistry of aortic sections demonstrated the presence of p22phox, gp91phox, p47phox, and p67phox localized exclusively in rabbit aortic adventitia, coincident with the site of staining for O2− production. Furthermore, immunodepletion of p67phox from adventitial fibroblast particulates resulted in the loss of NADPH oxidase activity, which could be restored by the addition of recombinant p67phox. Further study into the regulation of this adventitial source of O2− is important in elucidating the mechanisms regulating the bioactivity of NO and may contribute to our understanding of the pathogenesis of hypertension.

Long term relative survival after surgery for abdominal aortic aneurysm in Western Australia: population based study

Objective: To determine the long term relative survival of all patients who had surgery for abdominal aortic aneurysm in Western Australia during 1985-94.

Hypoxia enhances stimulus-dependent induction of E-selectin on aortic endothelial cells.

In many diseases, tissue hypoxia occurs in conjunction with other inflammatory processes. Since previous studies have demonstrated a role for leukocytes in ischemia/reperfusion injury, we hypothesized that endothelial hypoxia may "superinduce" expression of an important leukocyte adhesion molecule, E-selectin (ELAM-1, CD62E). Bovine aortic endothelial monolayers were exposed to hypoxia in the presence or absence of tumor-necrosis factor alpha (TNF-alpha) or lipopolysaccharide (LPS). Cell surface E-selectin was quantitated by whole cell ELISA or by immunoprecipitation using polyclonal anti-E-selectin sera. Endothelial mRNA levels were assessed using ribonuclease protection assays. Hypoxia alone did not induce endothelial E-selectin expression. However, enhanced induction of E-selectin was observed with the combination of hypoxia and TNF-alpha (270% increase over normoxia and TNF-alpha) or hypoxia and LPS (190% increase over normoxia and LPS). These studies revealed that a mechanism for such enhancement may be hypoxia-elicited decrements in endothelial intracellular levels of cAMP (<50% compared with normoxia). Addition of forskolin and isobutyl-methyl-xanthine during hypoxia resulted in reversal of cAMP decreases and a loss of enhanced E-selectin surface expression with the combination of TNF-alpha and hypoxia. We conclude that endothelial hypoxia may provide a novel signal for superinduction of E-selectin during states of inflammation.

cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.

Identification of endothelin 1 and big endothelin 1 in secretory vesicles isolated from bovine aortic endothelial cells.

Vesicles containing endothelin 1 (ET-1) were isolated from bovine aortic endothelial cells (BAECs) by fractionation of homogenates on sucrose density gradients by ultracentrifugation. The vesicles were localized at the 1.0/1.2 M sucrose interface using a specific anti-ET-1-(16-21) RIA. Identification of ET-1 and big ET-1 in this fraction was confirmed by HPLC analysis combined with RIA. Morphological examination of the ET-1-enriched fraction by electron microscopy identified clusters of vesicles approximately 100 nm in diameter. Immunostaining of ultrathin cryosections prepared from the vesicle fraction for ET-1 or big ET-1 showed clusters of 15-nm gold particles attached to or within vesicles. Immunofluorescence staining of whole BAECs using a specific ET-1-(16-21) IgG purified by affinity chromatography revealed punctate granulation of the cell cytoplasm viewed under light microscopy. This distinct pattern of staining was shown by confocal light microscopy to be intracellular. Immunofluorescence staining of whole cells with a polyclonal antiserum for big ET-1-(22-39) showed a defined perinuclear localization of precursor molecule. Hence, several different approaches have demonstrated that ET-1 and big ET-1 are localized within intracellular vesicles in BAECs, suggesting that these subcellular compartments are an important site for processing of big ET-1 by endothelin-converting enzyme.