Abstract-3-Hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase inhibitors or statins exert direct beneficial effects on the endothelium in part through an increase in nitric oxide (NO) production. Here, we examined whether posttranslational modifications of the endothelial NO synthase (eNOS) could account for the proangiogenic effects of statins. We used endothelial cells (ECs) isolated from cardiac microvasculature, aorta, and umbilical veins, as well as dissected microvessels and aortic rings, that were cultured on reconstituted basement membrane matrix (Matrigel). Tube or precapillary formation was evaluated after statin treatment, in parallel with immunoblotting and immunoprecipitation experiments. Atorvastatin stimulated NO-dependent angiogenesis from both isolated and outgrowing (vessel-derived) ECs, independently of changes in eNOS expression. We found that in macro-but not microvascular ECs, atorvastatin stabilized tube formation through a decrease in caveolin abundance and its inhibitory interaction with eNOS. We also identified the chaperone protein hsp90 as a key target for the proangiogenic effects of statins. Using geldanamycin, an inhibitor of hsp90 function, and overexpression of recombinant hsp90, we documented that the statin-induced phosphorylation of eNOS on Ser1177 was directly dependent on the ability of hsp90 to recruit Akt in the eNOS complex. Finally, we showed that statin promoted the tyrosine phosphorylation of hsp90 and the direct interaction of hsp90 with Akt, which further potentiated the NO-dependent angiogenic processes. Our study provides new mechanistic insights into the NO-mediated angiogenic effects of statins and underscores the potential of these drugs and other modulators of hsp90 and caveolin abundance to promote neovascularization in disease states associated or not with atherosclerosis. ( Key Words: statin Ⅲ angiogenesis Ⅲ nitric oxide Ⅲ hsp90 Ⅲ caveolin I t is now established that statins, in addition to their ability to improve serum lipid profile, 1 exert beneficial effects on endothelial function through an increase in nitric oxide (NO) production and/or bioavailability. 2 A major area in which such effects of statins on peripheral cells would be valuable is therapeutic angiogenesis, eg, the development of neovascularization in ischemic tissues. NO has, indeed, been identified as a downstream mediator of various growth factors initiating the angiogenic signaling cascade in endothelial cells (ECs). 3 Recently, Kureishi et al 4 reported that simvastatin could promote angiogenesis in ischemic limbs of normocholesterolemic rabbits. These authors further documented that statins could acutely induce Akt-dependent endothelial NO synthase (eNOS) phosphorylation in cultured ECs and therefore proposed that the Akt activation of eNOS could account for the long-term beneficial effects of simvastatin observed in vivo. Although the reversal of this effect by mevalonate suggested an inhibitory effect of the statin on the isoprenoid synthesis and downstream isoprenylation/activatio...

Vascular endothelial growth factor (VEGF) exerts its angiogenic effects partly through the activation of endothelial nitric-oxide synthase (eNOS). Association with heat shock protein 90 (hsp90) and phosphorylation by Akt were recently shown to separately activate eNOS upon VEGF stimulation in endothelial cells. Here, we examined the interplay between these different mechanisms in VEGF-exposed endothelial cells. We documented that hsp90 binding to eNOS is, in fact, the crucial event triggering the transition from the Ca 2؉ -dependent activation of eNOS to the phosphorylation-mediated potentiation of its activity by VEGF. Accordingly, we showed that early VEGF stimulation first leads to the Ca 2؉ /calmodulin disruption of the caveolin-eNOS complex and promotes the association between eNOS and hsp90. eNOS-bound hsp90 can then recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn can phosphorylate eNOS. Further experiments in transfected COS cells expressing either wild-type or S1177A mutant eNOS led us to identify the serine 1177 as the critical residue for the hsp90-dependent Akt-mediated activation of eNOS. Finally, we documented that although the VEGF-induced phosphorylation of eNOS leads to a sustained production of NO independently of a maintained increase in [Ca 2؉ ] i , this late stage of eNOS activation is strictly conditional on the initial VEGFinduced Ca 2؉ -dependent stimulation of the enzyme. These data establish the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggest new ways of regulating the production of NO in response to this cytokine through the ubiquitous chaperone protein, hsp90. Nitric oxide (NO)1 contributes to the cardiovascular homeostasis through its profound effects on blood pressure, vascular remodeling, platelet aggregation, and angiogenesis (1). Under normal conditions, the endothelial isoform of NO synthase (eNOS) expressed both in endothelial cells (EC) and cardiac myocytes is the major source of NO in the cardiovascular system (2). eNOS was originally identified as a particulate enzyme, associated primarily with the plasma membrane and with intracellular organelles such as the Golgi complex (3). Recent findings suggest that trafficking of eNOS from plasmalemmal to intracellular structures is part of a physiological cycle highly sensitive to the state of cell activation (4 -8). The dynamic trafficking of eNOS between cell surface and intracellular compartments is supported in part by its close interaction with the scaffold protein caveolin (4). In addition to regulating eNOS subcellular localization, this stable interaction with caveolin leads to the inhibition of the enzyme activity in basal conditions. Upon increases in intracellular calcium ([Ca 2ϩ ] i ), Ca 2ϩ /calmodulin (CaM) displaces the inhibitory binding of caveolin to eNOS and allows enzyme activation (9). Similarly, increasing vascular flow and pressure in an in situ artery perfusion model rapidly leads to activation of caveolar eNOS with apparent eNOS ...

Tumors may evade immune responses at multiple levels, including through a defect in the lymphocyte-vessel wall interactions. The angiogenic nature of endothelial cells (EC) lining tumor blood vessels may account for such anergy. In this study, we examined whether mechanisms other than down-regulation of adhesion molecules could be involved, particularly signaling pathways dependent on the caveolae platforms. To mimic the influence of the tumor microenvironment, EC were exposed to TNF-α and the proangiogenic vascular endothelial growth factor (VEGF). We identified a dramatic inhibition of lymphocyte adhesion on activated EC following either short or long VEGF pretreatments. We further documented that VEGF did not influence the abundance of major adhesion molecules, but was associated with a defect in ICAM-1 and VCAM-1 clustering at the EC surface. We also found that overexpression of the caveolar structural protein, caveolin-1, overcame the VEGF-mediated inhibition of adhesion and restored ICAM-1 clustering. Conversely, EC transduction with a caveolin-1 small interfering RNA reduced the TNF-α-dependent increase in adhesion. Finally, we identified VEGF-induced NO production by the endothelial NO synthase as the main target of the changes in caveolin-1 abundance. We found that the NO synthase inhibitor N-nitro-l-arginine methyl ester could reverse the inhibitory effects of VEGF on lymphocyte adhesion and EC cytoskeleton rearrangement. Symmetrically, a NO donor was shown to prevent the ICAM clustering-mediated lymphocyte adhesion, thereby recapitulating the effects of VEGF. In conclusion, this study provides new insights on the mechanisms leading to the tumor EC anergy vs immune cells and opens new perspectives for the use of antiangiogenic strategies as adjuvant approaches to cancer immunotherapy.

The ultimate goal of radiotherapy is to induce irreversible damages in genetically unstable, fast-growing cancer cells while minimizing the cytotoxic effects on host tissues. The satus of the tumor vasculature is particular because it is located within the tumor but mostly arises from host cells. The aim of this study was to characterize the effects of low-dose irradiation on the function of endothelial cells lining tumor vessels. Using isolated arterioles mounted on a pressure myograph, we first documented that the nitric oxide (NO)-mediated vasorelaxation that was defective in tumor vessels was completely restored following local tumor irradiation. Immunoblot analyses revealed that this was attributable to an increase in the abundance of the endothelial NO synthase while the expression of its physiological inhibitor, caveolin-1, was reduced. We further showed that the potentiation of the NO-dependent pathway induced a marked increase in tumor blood flow and oxygenation that determined the higher sensitivity of the tumor to further irradiation. Finally, we documented that the NO-mediated effects of irradiation on the tumor vasculature increased the delivery and expression of a reporter gene into the tumor. Thus, low-dose irradiation of endothelial cells within a tumor is a key determinant of the effectiveness of radiotherapy and may offer a new strategy to increase gene and/or drug delivery to the tumor.

In tumors, caveolin-1, the structural protein of caveolae, constitutes a key switch through its function as a tumor suppressor and a promoter of metastases. In endothelial cells (EC), caveolin is also known to directly interact with the endothelial nitric oxide synthase (eNOS) and thereby to modulate nitric oxide (NO)-mediated processes including vasodilation and angiogenesis. In this study, we examined whether the modulation of the stoichiometry of the caveolin/eNOS complex in EC lining tumor blood vessels could affect the tumor vasculature and consecutively tumor growth. For this purpose, we used cationic lipids, which are delivery systems effective at targeting tumor vs. normal vascular networks. We first documented that in vitro caveolin transfection led to the inhibition of both VEGF-induced EC migration and tube formation on Matrigel. The DNA-lipocomplex was then administered through the tail vein of tumor-bearing mice. The direct interaction between recombinant caveolin and native eNOS was validated in coimmunoprecipitation experiments from tumor extracts. A dramatic tumor growth delay was observed in mice transfected with caveolin- vs. sham-transfected animals. Using laser Doppler imaging and microprobes, we found that in the early time after lipofection (e.g., when macroscopic effects on the integrity of the tumor vasculature were not detectable), caveolin expression impaired NO-dependent tumor blood flow. At later stages post-transfection, a decrease in tumor microvessel density in the central core of caveolin-transfected tumors was also documented. In conclusion, our study reveals that by exploiting the exquisite regulatory interaction between eNOS and caveolin and the propensity of cationic lipids to target EC lining tumor blood vessels, caveolin plasmid delivery appears to be a safe and efficient way to block neoangiogenesis and vascular function in solid tumors, independently of any direct effects on tumor cells.

These data demonstrate that chronic hyperglycemia is associated with functional and structural changes in the peritoneum that parallel with selective regulation of NOS isoforms and AGE deposits. The alterations are prevented by insulin treatment, which suggests that adequate control of diabetes can preserve PM integrity in diabetic patients prior to PD.

We conclude that Hsp90 is capable of inducing angiogenesis and arteriogenesis via nitric oxide (NO) in a rabbit model of chronic ischemia. Our findings describe the capillary level as an initial site of Hsp90-cDNA-induced neovascularization, followed by growth of larger conductance vessels, resulting in an improved hindlimb perfusion.

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