Julie DeWever

Caveolin-1 Expression Is Critical for Vascular Endothelial Growth Factor–Induced Ischemic Hindlimb Collateralization and Nitric Oxide–Mediated Angiogenesis

Nitric oxide (NO) is a powerful angiogenic mediator acting downstream of vascular endothelial growth factor (VEGF). Both the endothelial NO synthase (eNOS) and the VEGFR-2 receptor colocalize in caveolae. Because the structural protein of these signaling platforms, caveolin, also represses eNOS activity, changes in its abundance are likely to influence the angiogenic process in various ways. In this study, we used mice deficient for the caveolin-1 gene (Cav−/−) to examine the impact of caveolae suppression in a model of adaptive angiogenesis obtained after femoral artery resection. Evaluation of the ischemic tissue perfusion and histochemical analyses revealed that contrary to Cav+/+ mice, Cav−/− mice failed to recover a functional vasculature and actually lost part of the ligated limbs, thereby recapitulating the effects of the NOS inhibitor l-NAME administered to operated Cav+/+ mice. We also isolated endothelial cells (ECs) from Cav−/− aorta and showed that on VEGF stimulation, NO production and endothelial tube formation were dramatically abrogated when compared with Cav+/+ ECs. The Ser1177 eNOS phosphorylation and Thr495 dephosphorylation but also the ERK phosphorylation were similarly altered in VEGF-treated Cav−/− ECs. Interestingly, caveolin transfection in Cav−/− ECs redirected the VEGFR-2 in caveolar membranes and restored the VEGF-induced ERK and eNOS activation. However, when high levels of recombinant caveolin were reached, VEGF exposure failed to activate ERK and eNOS. These results emphasize the critical role of caveolae in ensuring the coupling between VEGFR-2 stimulation and downstream mediators of angiogenesis. This study also provides new insights to understand the paradoxical roles of caveolin (eg, repressing basal enzyme activity but facilitating activation on agonist stimulation) in cardiovascular pathophysiology.

Effects of Vascular Endothelial Growth Factor on the Lymphocyte-Endothelium Interactions: Identification of Caveolin-1 and Nitric Oxide as Control Points of Endothelial Cell Anergy

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.

Caveolin Plays a Central Role in Endothelial Progenitor Cell Mobilization and Homing in SDF-1–Driven Postischemic Vasculogenesis

When neovascularization is triggered in ischemic tissues, angiogenesis but also (postnatal) vasculogenesis is induced, the latter requiring the mobilization of endothelial progenitor cells (EPC) from the bone marrow. Caveolin, the structural protein of caveolae, was recently reported to directly influence the angiogenic process through the regulation of the vascular endothelial growth factor (VEGF)/nitric oxide pathway. In this study, using caveolin-1 null mice (Cav−/−), we examined whether caveolin was also involved in the EPC recruitment in a model of ischemic hindlimb. Intravenous infusion of Sca-1+ Lin− progenitor cells, but not bone marrow transplantation, rescued the defective neovascularization in Cav−/− mice, suggesting a defect in progenitor mobilization. The adhesion of Cav−/− EPC to bone marrow stromal cells indeed appeared to be resistant to the otherwise mobilizing SDF-1 (Stromal cell–Derived Factor-1) exposure because of a defect in the internalization of the SDF-1 cognate receptor CXCR4. Symmetrically, the attachment of Cav−/− EPC to SDF-1–presenting endothelial cells was significantly increased. Finally, EPC transduction with caveolin small interfering RNA reproduced this advantage in vitro and, importantly, led to a more extensive rescue of the ischemic hindlimb after intravenous infusion (versus sham-transfected EPC). These results underline the critical role of caveolin in ensuring the caveolae-mediated endocytosis of CXCR4, regulating both the SDF-1–mediated mobilization and peripheral homing of progenitor cells in response to ischemia. In particular, a transient reduction in caveolin expression was shown to therapeutically increase the engraftment of progenitor cells.

Antitumor effects of in vivo caveolin gene delivery are associated with the inhibition of the proangiogenic and vasodilatory effects of nitric oxide

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.

Decrease in Tumor Cell Oxygen Consumption after Treatment with Vandetanib (ZACTIMA™; ZD6474) and its Effect on Response to Radiotherapy

Abstract We investigated the early effects of vandetanib (ZACTIMA™; ZD6474), an inhibitor of VEGFR-dependent angiogenesis, on tumor oxygenation and on the possible consequences of combining vandetanib with radiotherapy. Tumor oxygenation, perfusion, cellular consumption of oxygen, and radiation sensitivity were studied in transplantable liver tumors after daily doses of vandetanib (25 mg kg−1 i.p.). Measurements of oxygenation (pO2) and tumor cell oxygen consumption were carried out using electron paramagnetic resonance (EPR), and perfusion parameters were assessed by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Regrowth delay assays were performed after treatment with vandetanib alone, radiation alone or a combination of both treatments. Vandetanib induced an early increase in tumor oxygenation that did not correlate with remodeling of the tumor vasculature or with changes in tumor perfusion. A decrease in tumor cell oxygen consumption was observed that could have been responsible for this increase in tumor oxygenation. Consistent with this increase in tumor oxygenation, we found that vandetanib potentiated the tumor response to radiotherapy. Our results confirm that treatment with an inhibitor of VEGFR signaling reduces oxygen consumption rate by tumor cells. The observation that vandetanib causes an early increase in tumor oxygenation has implications for the timing and sequencing of treatment with VEGF signaling inhibitors in combination with radiation.

Molecular electron paramagnetic resonance imaging of melanin in melanomas : a proof-of-concept

The incidence of malignant melanoma is increasing at an alarming rate. As the clinical outcome of the disease strongly depends on the localization of the lesion, early detection at the initial stages of development is critical. Here, we suggest spatial characterization of melanoma based on the presence of endogenous stable free radicals in melanin pigments. Taking into account the abundance of these naturally occurring free radicals in proliferating melanocytes and their localization pattern, we hypothesized that electron paramagnetic resonance (EPR) imaging could be a unique tool for mapping melanomas with high sensitivity and high resolution. The potential of EPR to image melanoma samples was demonstrated in vitro in animal and human samples. Using EPR systems operating at low frequency, we were also able to record in vivo EPR spectra and images from the melanin present in a subcutaneous melanoma implanted in a mouse. In addition to the proof‐of‐concept and the achievement of providing the first non‐invasive image of an endogenous radical, this technology may represent a key advance in improving the diagnosis of suspected melanoma lesions. Copyright