Hypoxia is a common feature in tumors associated with an increased resistance of tumor cells to therapies. In addition to O 2 diffusion-limited hypoxia, another form of tumor hypoxia characterized by fluctuating changes in pO 2 within the disorganized tumor vascular network is described. Here, we postulated that this form of intermittent hypoxia promotes endothelial cell survival, thereby extending the concept of hypoxia-driven resistance to the tumor vasculature. We found that endothelial cell exposure to cycles of hypoxia reoxyge-

Background-The therapeutic effects of nonspecific ␤-blockers are limited by vasoconstriction, thus justifying the interest in molecules with ancillary vasodilating properties. Nebivolol is a selective ␤ 1 -adrenoreceptor antagonist that releases nitric oxide (NO) through incompletely characterized mechanisms. We identified endothelial ␤ 3 -adrenoreceptors in human coronary microarteries that mediate endothelium-and NO-dependent relaxation and hypothesized that nebivolol activates these ␤ 3 -adrenoreceptors. Methods and Results-Nebivolol dose-dependently relaxed rodent coronary resistance microarteries studied by videomicroscopy (10 mol/L, Ϫ86Ϯ6% of prostaglandin F2␣ contraction); this was sensitive to NO synthase (NOS) inhibition, unaffected by the ␤ 1-2 -blocker nadolol, and prevented by the ␤ 1-2-3 -blocker bupranolol (PϽ0.05; nϭ3 to 8). Importantly, nebivolol failed to relax microarteries from ␤ 3 -adrenoreceptor-deficient mice. Nebivolol (10 mol/L) also relaxed human coronary microvessels (Ϫ71Ϯ5% of KCl contraction); this was dependent on a functional endothelium and NO synthase but insensitive to ␤ 1-2 -blockade (all PϽ0.05). In a mouse aortic ring assay of neoangiogenesis, nebivolol induced neocapillary tube formation in rings from wild-type but not ␤ 3 -adrenoreceptor-or endothelial NOS-deficient mice. In cultured endothelial cells, 10 mol/L nebivolol increased NO release by 200% as measured by electron paramagnetic spin trapping, which was also reversed by NOS inhibition. In parallel, endothelial NOS was dephosphorylated on threonine 495 , and fura-2 calcium fluorescence increased by 91.8Ϯ23.7%; this effect was unaffected by ␤ 1-2 -blockade but abrogated by ␤ 1-2-3 -blockade (all PϽ0.05). Conclusions-Nebivolol dilates human and rodent coronary resistance microarteries through an agonist effect on endothelial ␤ 3 -adrenoreceptors to release NO and promote neoangiogenesis. These properties may prove particularly beneficial for the treatment of ischemic and cardiac failure diseases through preservation of coronary reserve.

Background-In endothelial cells, caveolin-1, the structural protein of caveolae, acts as a scaffolding protein to cluster lipids and signaling molecules within caveolae and, in some instances, regulates the activity of proteins targeted to caveolae. Specifically, different putative mediators of the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation are located in caveolae and/or regulated by the structural protein caveolin-1, such as potassium channels, calcium regulatory proteins, and connexin 43, a molecular component of gap junctions. Methods and Results-Comparing relaxation in vessels from caveolin-1 knockout mice and their wild-type littermates, we observed a complete absence of EDHF-mediated vasodilation in isolated mesenteric arteries from caveolin-1 knockout mice. The absence of caveolin-1 is associated with an impairment of calcium homeostasis in endothelial cells, notably, a decreased activity of Ca 2ϩ -permeable TRPV4 cation channels that participate in nitric oxide-and EDHF-mediated relaxation. Moreover, morphological characterization of caveolin-1 knockout and wild-type arteries showed fewer gap junctions in vessels from knockout animals associated with a lower expression of connexins 37, 40, and 43 and altered myoendothelial communication. Finally, we showed that TRPV4 channels and connexins colocalize with caveolin-1 in the caveolar compartment of the plasma membrane. Conclusions-We demonstrated that expression of caveolin-1 is required for EDHF-related relaxation by modulating membrane location and activity of TRPV4 channels and connexins, which are both implicated at different steps in the EDHF-signaling pathway.

Objective-We investigated the impact of hypoxia-reoxygenation on endothelial relaxation and aimed to clarify the role of transient receptor potential cation channels V4 (TRPV4) and gap junctions in the protective effect associated with hypoxic preconditioning on the vascular function. Methods and Results-By mimicking ischemia-reperfusion in C57BL/6 male mice in vivo, we documented a reduced NOmediated relaxation and an increased endothelium-derived hyperpolarization (EDH[F])-mediated relaxation. Hypoxic preconditioning, however, restored NO relaxation and further improved the EDH(F) response. We also examined specifically 2 major effectors of the EDH(F) pathway, transient receptor potential cation channels V4 and connexins. We found that in endothelial cells, expression and activity of transient receptor potential cation channels V4 were increased by hypoxic stimuli independently of preconditioning which was interestingly associated with an increase of structural caveolar component caveolin-1 at membrane locations. Gap junctions, however, seemed to directly support EDH(F)-driven preconditioning as connexin 40 and connexin 43 expression increased and as in vivo carbenoxolone treatment completely inhibited the EDH(F) pathway and significantly reduced the protection afforded by preconditioning for the concomitant NO-mediated relaxation. Conclusion-Our

Purpose: The biological status of nitrite recently evolved from an inactive end product of nitric oxide catabolism to the largest intravascular and tissue storage of nitric oxide (NO). Although low partial O 2 pressure favors enzymatic reconversion of nitrite into NO, low pH supports a nonenzymatic pathway. Because hypoxia and acidity are characteristics of the tumor microenvironment, we examined whether nitrite injection could preferentially lead to NO production in tumors and influence response to treatments. Experimental Design: The effects of nitrite were evaluated on arteriole vasorelaxation, tumor cell respiration and tumor blood flow, oxygenation, and response to radiotherapy. Results: We first showed that a small drop in pH (-0.6 pH unit) favored the production of bioactive NO from nitrite by documenting a higher cyclic guanosine 3 ¶,5 ¶-monophosphated ependent arteriole vasorelaxation. We then documented that an i.v. bolus injection of nitrite to tumor-bearing mice led to a transient increase in partial O 2 pressure in tumor but not in healthy tissues. Blood flow measurements failed to reveal an effect of nitrite on tumor perfusion, but we found that O 2 consumption by nitrite-exposed tumor cells was decreased at acidic pH. Finally, we showed that low dose of nitrite could sensitize tumors to radiotherapy, leading to a significant growth delay and an increase in mouse survival (versus irradiation alone). Conclusions: This study identified low pH condition (encountered in many tumors) as an exquisite environment that favors tumor-selective production of NO in response to nitrite systemic injection.This work opens new perspectives for the use of nitrite as a safe and clinically applicable radiosensitizing modality.