We conclude that LOX-1 is regulated by Ang II in vitro and in vivo, that induction of LOX-1 is mediated by the AT(1) receptor, and that repression of LOX-1 by long-term ACE inhibitor treatment may contribute to the antiatherosclerotic potential of this therapy.
The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (·O 2 − ) formation. In this study, we analysed the effect of laminar shear stress on ·O 2 − formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription-polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress in a cone-and-plate viscometer for up to 24 h. Short-term application of shear stress transiently induced ·O 2 − formation. This was inhibited by NAD(P)H oxidase inhibitor gp91ds-tat, but NAD(P)H oxidase subunit expression was unchanged. Long-term arterial laminar shear stress (30 dyne cm −2 , 24 h) down-regulated ·O 2 − formation, and mRNA and protein expression of NAD(P)H oxidase subunits Nox2/gp91 phox and p47 phox . In parallel, endothelial NO formation and eNOS, but not Cu/Zn SOD, protein expression was increased. Down-regulation of ·O 2 − formation, gp91 phox and p47 phox expression by long-term laminar shear stress was blocked by L-NAME. NO donor DETA-NO down-regulates ·O 2 − formation, gp91 phox and p47 phox expression in static cultures. In conclusion, our data suggest a transient activation of ·O 2 − formation by short-term shear stress, followed by a down-regulation of endothelial NAD(P)H oxidase in response to long-term laminar shear stress. NO-mediated down-regulation by shear stress preferentially affects the gp91 phox /p47 phox -containing NAD(P)H oxidase complex. This mechanism might contribute to the regulation of endothelial NO/·O 2 − balance and the vasoprotective potential of physiological levels of laminar shear stress.
The LOX-1 receptor, identified on endothelial cells, mediates the uptake of oxidized low-density lipoprotein (oxLDL). The oxLDL-dependent LOX-1 activation causes endothelial cell apoptosis. We here investigated the presence of LOX-1 in granulosa cells from patients under in vitro fertilization therapy. We were interested in the oxLDL-dependent LOX-1 receptor biology, in particular in the induction of apoptosis. In the human ovary, LOX-1 was localized in regressing antral follicles. In granulosa cell cultures, oxLDL-induced mRNA expression of LOX-1 in a time- and dose-dependent manner. The LOX-1 inhibitors (anti-LOX-1 antibody and kappa-carrageenan) abrogated the up-regulation of LOX-1. The oxLDL (100 microg/ml) treatment caused the autophagy form of programmed cell death: 1) reorganization of the actin cytoskeleton at the 6-h time point; 2) uptake of YO-PRO, a marker for the early step of programmed cell death, before propidium iodide staining to signify necrosis; 3) absence of apoptotic bodies and cleaved caspase-3; 4) abundant vacuole formation at the ultrastructural level; and 5) decrease of the autophagosome marker protein MAP LC3-I at the 6-h time point indicative of autophagosome formation. We conclude that follicular atresia is not under the exclusive control of apoptosis. The LOX-1-dependent autophagy represents an alternate form of programmed cell death. Obese women with high blood levels of oxLDL may display an increased rate of autophagic granulosa cell death.
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