Angiogenesis and CaveolineNOS Interaction

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In 1999, Lisanti and colleagues documented that caveolin-1 was down-regulated by angiogenic growth factors in subconfluent endothelial cells, but not when these cells were confluent [42]. These authors further showed that, in contrast, caveolin-1 protein levels were up-regulated during endothelial cell differentiation and that expression of caveolin-1 in confluent endothelial cells stimulated endothelial tube formation [43]. Although, the original observation that growth factor exposure can down-regulate caveolin expression in endothelial cells remains a matter of debate, the second set of data (e.g., the role of caveolin in endothelial differentiation/tube reorganization) was verified by other investigators (but only for the link between a reduction in caveolin abundance and the inhibition of angiogenesis). Indeed, Grif-foni et al. found, using caveolin antisense technology, that a reduction in caveolin abundance reduced vessel formation in the chorioallantoic membrane assay [44].

Consecutively, we found that statins could stimulate tube formation from macro-vascular endothelial cells cultured on Matrigel® through a decrease in caveolin abundance (and in its inhibitory interaction with eNOS) [27]. In microvascular endothelial cells, statins only marginally decreased the abundance of caveolin protein (that amounted to almost 10-fold the pool of caveolin in macrovascular endothelial cells), and therefore did not impact on the regulation of eNOS activity. In those endothelial cells, however, statins were shown to stimulate angiogenesis through eNOS phosphorylation on Ser1177 - a process which is facilitated by the recruitment of the chaperone protein hsp90 that acts as an adaptor between eNOS and the kinase. Still, caveolin overexpression or cell loading with caveolin scaffolding domain-derived peptides prevented the ability not only of statins but also of vascular endothelial growth factor (VEGF) to stimulate in-vitro angiogenesis (Fig. 11.5). These observations were further confirmed in a mouse model of angiogenesis (in tumors). In-vivo lipofection of a caveolin plasmid led, indeed, to the inhibition of both NO-mediated vasodilation and angiogenesis in tumors [45] (Fig. 11.5).

Paradoxically, we found that in aortic endothelial cells isolated from caveolin-1-deficient mice (Cav-/-) (as well as Cav+/- mice), VEGF-induced NO production and endothelial tube formation were dramatically decreased when compared with Cav+/+ endothelial cells (see Fig. 11.5). The VEGFR-2 mistargeting (due to the lack of caveolin in Cav-/- mice) was identified as the cause of the incapacity of VEGF to activate the downstream signaling cascades, including eNOS and ERK activation. This led to dramatic consequences in a model of adaptive angiogenesis obtained after femoral artery resection [46]. In fact, contrary to Cav+/+ mice, both Cav-/- and

Fig. 11.5 Model of the regulation of angioge-nesis by caveolin-eNOS interaction. Experimentally based relationship between the abundance of caveolin and the NO-dependent angiogenesis. Note the position of the "normal" or "physiological" (100% expression) caveolin phenotype within the bell-shaped pattern, emphasizing the anti-angiogenic effects of increasing caveolin

Fig. 11.5 Model of the regulation of angioge-nesis by caveolin-eNOS interaction. Experimentally based relationship between the abundance of caveolin and the NO-dependent angiogenesis. Note the position of the "normal" or "physiological" (100% expression) caveolin phenotype within the bell-shaped pattern, emphasizing the anti-angiogenic effects of increasing caveolin abundance by recombinant (rec.) caveolin expression, caveolin scaffolding domain (CSD) peptides or LDL-cholesterol exposure as well as the pro-angiogenic effects of reducing caveolin abundance (upon statins) until a given threshold from which anti-angiogenic effects will be observed (similar to those obtained with caveolin overexpression).

Cav+/- mice failed to recover a functional vasculature, as authenticated by laser Doppler evaluation of the ischemic tissue perfusion and histochemical analyses. These data recapitulate the findings of Woodman et al., who found in Cav-/- mice a dramatic reduction in both vessel infiltration and density in tumor models of angiogenesis [47]. Interestingly, in aortic endothelial cells isolated from Cav-/-mice, we further documented that recombinant caveolin expression in endothelial cells helped to redirect the VEGFR-2 in caveolar membranes and to restore the VEGF/NO and VEGF/ERK signaling cascades. Amazingly, however, when (too-) elevated levels of recombinant caveolin were reached, VEGF exposure failed to activate ERK and eNOS [46], these findings being in good agreement with the experiments of caveolin transfection described above [45] (Fig. 11.5).

A model integrating the "compartmentalizing" effect of caveolin (e.g., receptor-effector coupling is either prevented or promoted when/where caveolin is down- or up-regulated) and the "inhibitory" hypothesis (e. g., inhibition proportional to caveolin levels) is described in Figure 11.5.

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