The observations by Fielding et al. that, in human fibroblasts, high levels of cellular free cholesterol (FC) induce caveolin gene transcription [31,32] led us to examine whether a similar increase in caveolin abundance in endothelial cells could account for a reduction in NO production. Accordingly, we exposed endothelial cells to low-density lipoprotein (LDL)-cholesterol and found that both caveolin abundance and caveolin-eNOS complex formation were increased  (Fig. 11.4). Furthermore, NO release (under basal and stimulated conditions) was inhibited, thereby providing some insights for a new pathogenic mechanism linking hyper-cholesterolemia and endothelial dysfunction. A defect in the NO pathway is, indeed, viewed as a hallmark of endothelial dysfunction characterized by an impaired endothelium-dependent vasodilation and the unopposed influence of thrombogenic and proliferative factors on the vessel wall.
In parallel to these observations that cholesterol could lead to eNOS inhibition through the induction of caveolin expression, Blair et al.  documented that
Fig. 11.4 Reversible lipoprotein-dependent regulation of caveolin-eNOS interaction. High levels of native LDL stimulate caveolin transcription and thereby promote the interaction of eNOS with the increased caveolin pool (left). The resulting inhibition of eNOS activity can be reversed by statins that, by inhibiting the endogenous synthesis of cholesterol in endothelial cells (as well as indirectly by reducing circulatory LDL-choles-terol), negatively impact on caveolin expres
Fig. 11.4 Reversible lipoprotein-dependent regulation of caveolin-eNOS interaction. High levels of native LDL stimulate caveolin transcription and thereby promote the interaction of eNOS with the increased caveolin pool (left). The resulting inhibition of eNOS activity can be reversed by statins that, by inhibiting the endogenous synthesis of cholesterol in endothelial cells (as well as indirectly by reducing circulatory LDL-choles-terol), negatively impact on caveolin expres sion. The interaction of oxidized LDL (oxLDL) with the CD36 receptor located in caveolae leads to a marked depletion of caveolar cholesterol and to the translocation of caveolin and eNOS to intracellular compartments (see right), wherein basal and agonist-stimulated NO production are dramatically reduced. Conversely, HDL, through caveolar SRBI binding, provisions cholesterol esters to the cell, thereby reversing the deleterious effects of oxLDL.
oxidized LDL (oxLDL) caused the translocation of both eNOS and caveolin from caveolae to intracellular membranes (Fig. 11.4). These authors further documented that oxLDL, through class B CD36 receptor binding , act as acceptors of cholesterol, leading to marked depletion of caveolae cholesterol and redistribution of caveolin and eNOS (but not of other caveolar residents such as PKCa and gang-lioside GM1). They also showed that when examining the pattern of eNOS activation upon exposure to acetylcholine, the dose-response curve was shifted to the right by 100-fold. A recent study by the Lisanti's group also documented that the loss of caveolin-1 (as observed in caveolin knockout mice) resulted in a dramatic down-regulation of CD36 and, importantly, conferred a significant protection against atherosclerosis in double apoE/caveolin knockout mice . This latter study confirmed the findings of Kincer et al., who showed that in apoE/CD36 double knockout mice - in contrast to apoE knockout mice - the acetylcholine-evoked reduction in blood pressure is conserved (as well as the eNOS localization to caveolae) .
Whether such processes of native and oxLDL-dependent regulation of the cav-eolin-eNOS interaction are reversible was also addressed. Our group showed, both in vitro and in vivo, that hydroxymethylglutaryl coenzyme A (HMGCoA) reductase inhibitors (statins) could reduce caveolin abundance [38,39]. In cultured endothe-lial cells, the reduction in caveolin abundance obtained with atorvastatin was associated with a restoration of basal and agonist-stimulated eNOS activity (see Fig. 11.4) . In dyslipidemic, apolipoprotein (apo) E-/- mice, the alterations in heart rate and blood pressure variabilities were corrected by chronic treatment with rosuvastatin . Our findings also highlighted the therapeutic potential of statins in diseases other than hypercholesterolemia, such as hypertension or heart failure. Indeed, we showed that statins could decrease caveolin abundance in endothelial cells and in apoE-/- mice, independently of the extracellular and plasma load in LDL-cholesterol, respectively [38,39].
As for the oxLDL-mediated CD36-dependent alteration in caveolin-eNOS biology, the reversibility of the phenomenon was documented with high-density lipoprotein (HDL) that prevented both the depletion of caveolar cholesterol and the eNOS displacement from caveolae . Amazingly, the provision of cholesterol esters by HDL binding to the scavenger receptor BI (and not the inhibition of cholesterol transfer from caveolae to oxLDL) was found to account for the correction of eNOS mislocalization and the restoration of the acetylcholine-induced activation of eNOS  (see Fig. 11.4). Of note, the eNOS stimulation by HDL was recently shown to involve Src-mediated signaling .
The conclusion to be drawn from the above studies is that both native LDL in excess and oxLDL contribute to the change in NO production, and therefore account for the many functional defects associated with NO deficiency. Although the oxidative stress paradigm is well established as a trigger of the atherosclerotic process, endothelial dysfunction occurs before the appearance of any ultrastructural change in the vessel wall. It may therefore be postulated that chronologically, chronic elevations in serum LDL-cholesterol and then lipoprotein oxidation contribute to caveolin-dependent alteration in NO signaling. Importantly, both phe nomena appear reversible. Physiologically, the beneficial effects of HDL are clearly emphasized by several studies described herein. Moreover, the pharmacologically pleiotropic effects of statins can be expected (interestingly) at doses that do not necessarily require any reduction in LDL correction.
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