Objective Intercellular conduction of electrical signals underlies spreading vasodilation of resistance arteries. Small and intermediate-conductance Ca2+ activated K+ channels (SKCa/IKCa) of endothelial cells serve a dual function by initiating hyperpolarization and modulating electrical conduction. We tested the hypothesis that the regulation of electrical signaling by SKCa/IKCa is altered with advancing age. Approach and Results Intact endothelial tubes (60 μm wide; 1-3 mm long) were freshly isolated from male C57BL/6 mouse (Young: 4-6 months; Intermediate: 12-14 months; Old: 24-26 months) superior epigastric arteries. Using dual intracellular microelectrodes, current was injected (±0.1-3 nA) at site 1 while recording membrane potential (Vm) at site 2 (separation distance: 50-2000 μm). Across age groups, greatest differences were observed between Young and Old. Resting Vm in Old (−38±1 mV) was more negative (P<0.05) than Young (−30±1 mV). Maximal hyperpolarization to both direct (NS309) and indirect (acetylcholine) activation of SKCa/IKCa was sustained (ΔVm ~ −40 mV) with age. The length constant (λ) for electrical conduction was reduced (P<0.05) from 1630±80 µm (Young) to 1320±80 μm (Old). Inhibiting SKCa/IKCa with apamin + charybdotoxin or scavenging H2O2 with catalase improved electrical conduction (P<0.05) in Old. Exogenous H2O2 (200 μM) in Young evoked hyperpolarization and impaired electrical conduction; these effects were blocked by apamin + charybdotoxin. Conclusions Enhanced current loss through KCa activation impairs electrical conduction along the endothelium of resistance arteries with aging. Attenuating the spatial domain of electrical signaling will restrict the spread of vasodilation and thereby contribute to blood flow limitations associated with advanced age.

Key pointsr Calcium signalling in endothelial cells of resistance arteries is integral to blood flow regulation.Oxidative stress and endothelial dysfunction can prevail during advanced age and we questioned how calcium signalling may be affected.r Intact endothelium was freshly isolated from superior epigastric arteries of Young (ß4 months) and Old (ß24 months) male C57BL/6 mice. Under resting conditions, with no difference in intracellular calcium levels, hydrogen peroxide (H 2 O 2 ) availability was ß1/3 greater in endothelium of Old mice while vascular catalase activity was reduced by nearly half. r Microvascular adaptation to advanced age may protect endothelial cells during elevated oxidative stress to preserve functional viability of the intima. Abstract Endothelial cell Ca2+ signalling is integral to blood flow control in the resistance vasculature yet little is known of how its regulation may be affected by advancing age. We tested the hypothesis that advanced age protects microvascular endothelium by attenuating aberrant Ca 2+ signalling during oxidative stress. Intact endothelial tubes (width, ß60 μm; length, ß1000 μm) were isolated from superior epigastric arteries of Young (3-4 months) and Old (24-26 months) male C57BL/6 mice and loaded with Fura-2 dye to monitor [Ca 2+ ] i . At rest there was no difference in [Ca 2+ ] i between age groups. Compared to Young, the [Ca 2+ ] i response to maximal stimulation with acetylcholine (3 μM, 2 min) was ß25% greater in Old, confirming signalling integrity with advanced age. Basal H 2 O 2 availability was ß33% greater in Old while vascular catalase activity was reduced by half. Transient exposure to elevated H 2 O 2 (200 μM, 20 min) progressively increased [Ca 2+ ] i to ß4-fold greater levels in endothelium of Young versus Old. With no difference between age groups at rest, Mn 2+ quench of Fura-2 fluorescence revealed 2-fold greater Ca 2+ influx in Young during elevated H 2 O 2 ; this effect was attenuated by ß75% using ruthenium red (5 μM) as a broad-spectrum inhibitor of transient receptor potential channels. Prolonged exposure to H 2 O 2 (200 μM, 60 min) induced ß7-fold greater cell death in endothelium of Young versus Old. Thus, microvascular endothelium can adapt to advanced age by reducing Ca 2+ influx during elevated oxidative stress. Protection from cell death during oxidative stress will sustain endothelial integrity during ageing.

Notch signaling is important for tumor angiogenesis induced by vascular endothelial growth factor A. Blockade of the Notch ligand Dll4 inhibits tumor growth in a paradoxical way. Dll4 inhibition increases endothelial cell sprouting, but vessels show reduced perfusion. The reason for this lack of perfusion is not currently understood. Here we report that inhibition of Notch signaling in endothelial cell using an inducible binary transgenic system limits VEGFAdriven tumor growth and causes endothelial dysfunction. Neither excessive endothelial cell sprouting nor defects of pericyte abundance accompanied the inhibition of tumor growth and functional vasculature. However, biochemical and functional analysis revealed that endothelial nitric oxide production is decreased by Notch inhibition. Treatment with the soluble guanylate cyclase activator BAY41-2272, a vasorelaxing agent that acts downstream of endothelial nitric oxide synthase (eNOS) by directly activating its soluble guanylyl cyclase receptor, rescued blood vessel function and tumor growth. We show that reduction in nitric oxide signaling is an early alteration induced by Notch inhibition and suggest that lack of functional vessels observed with Notch inhibition is secondary to inhibition of nitric oxide signaling. Coculture and tumor growth assays reveal that Notch-mediated nitric oxide production in endothelial cell requires VEGFA signaling. Together, our data support that eNOS inhibition is responsible for the tumor growth and vascular function defects induced by endothelial Notch inhibition. This study uncovers a novel mechanism of nitric oxide production in endothelial cells in tumors, with implications for understanding the peculiar character of tumor blood vessels. Cancer Res; 74(9); 2402-11. Ó2014 AACR.