The measurement of vascular function in isolated vessels has revealed important insights into the structural, functional, and biomechanical features of the normal and diseased cardiovascular system and has provided a molecular understanding of the cells that constitutes arteries and veins and their interaction. Further, this approach has allowed the discovery of vital pharmacological treatments for cardiovascular diseases. However, the expansion of the vascular physiology field has also brought new concerns over scientific rigor and reproducibility. Therefore, it is appropriate to set guidelines for the best practices of evaluating vascular function in isolated vessels. These guidelines are a comprehensive document detailing the best practices and pitfalls for the assessment of function in large and small arteries and veins. Herein, we bring together experts in the field of vascular physiology with the purpose of developing guidelines for evaluating ex vivo vascular function. By using this document, vascular physiologists will have consistency among methodological approaches, producing more reliable and reproducible results.

Clinical studies suggest that sleep apnea causes systemic hypertension. In addition, patients with sleep apnea have elevated plasma levels of endothelin-1 (ET-1). We hypothesized that the intermittent hypoxia/hypercapnia (IH) associated with sleep apnea causes hypertension by increasing ET-1 production. To test this hypothesis, rats with arterial and venous catheters were placed in Plexiglas chambers. IH rat chambers were flushed with an N₂-CO₂mixture for 90 seconds to achieve hypoxia/hypercapnia (5% O₂–5% CO₂) followed by 90 seconds of compressed air to achieve normoxia (21% O₂–0% CO₂). Control rat chambers were flushed with 90 seconds of air-air cycles. Cycles for both groups were repeated 8 hours per day for 11 days. Resting mean arterial pressure (MAP) and heart rate were recorded daily before the start of exposure. After 11 days, MAP was significantly elevated in IH rats compared with initial MAP (109±5 mm Hg initial, 139±11 mm Hg day 11) and compared with air-air rats (110±4 mm Hg). On day 11, cumulative doses ofPD145065(a nonselective ET-receptor antagonist) were administered intravenously to the rats breathing room air.PD145065caused a dose-dependent decrease in MAP in IH rats but did not alter MAP in air-air rats. Plasma ET-1 measured by radioimmunoassay was significantly increased on days 5 and 11 in the IH rats compared with day 1 and compared with air-air rats. There was no significant change in plasma ET-1 over time in air-air rats. We conclude that IH exposure increases both MAP and plasma ET-1 and that the increased ET-1 may contribute to the hypertension.

In clinical studies, sleep apnea is associated with hypertension, oxidative stress, and increased circulating endothelin-1 (ET-1). We previously developed a model of sleep apnea by exposing rats to eucapnic intermittent hypoxia (IH-C) during sleep, which increases both blood pressure and plasma levels of ET-1. Because similar protocols in mice increase tissue and plasma markers of oxidative stress, we hypothesized that IH-C generation of reactive oxygen species (ROS) contributes to the development of ET-1-dependent hypertension in IH-C rats. To test this, male Sprague-Dawley rats were instrumented with indwelling blood pressure telemeters and drank either plain water or water containing the superoxide dismutase mimetic, Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, 1 mM). Mean arterial pressure (MAP) and heart rate (HR) were recorded for 3 control days and 14 treatment days with rats exposed 7 h/day to IH-C or air/air cycling (Sham). On day 14, MAP in IH-C rats treated with Tempol (107 ± 2.29 mmHg) was significantly lower than in untreated IH-C rats (118 ± 9 mmHg, P < 0.05). Tempol did not affect blood pressure in sham-operated rats (Tempol = 101 ± 3, water = 101 ± 2 mmHg). Immunoreactive ET-1 was greater in plasma from IH-C rats compared with plasma from sham-operated rats but was not different from Sham in Tempol-treated IH-C rats. Small mesenteric arteries from IH-C rats but not Tempol-treated IH-C rats had increased superoxide levels as measured by ferric cytochrome c reduction, lucigenin signaling, and dihydroethidium fluorescence. The data show that IH-C increases ET-1 production and vascular ROS levels and that scavenging superoxide prevents both. Thus oxidative stress appears to contribute to increases in ET-1 production and elevated arterial pressure in this rat model of sleep apnea-induced hypertension.

We reported previously that simulating sleep apnea in rats by exposing them 7 hours per day to intermittent hypoxia/hypercapnia (IH) elevates plasma endothelin-1 and causes hypertension, which is reversed by an endothelin-1 antagonist. We hypothesized that in this model of sleep apnea–induced hypertension, vascular sensitivity to endothelin-1 is increased in combination with the elevated plasma endothelin-1 to cause the endothelin-1–dependent hypertension. In small mesenteric arteries with endothelial function disabled by passing air through the lumen, diameter and vessel wall [Ca ²⁺ ] were recorded simultaneously. IH arteries demonstrated increased constrictor sensitivity to endothelin-1 (percentage max constriction 100±0% IH versus 80±10% Sham; P <0.05). This was accompanied by increased calcium sensitivity of IH arteries. In contrast, constrictor sensitivity and increases in vessel wall [Ca ²⁺ ] to KCl and phenylephrine were not different between IH and Sham arteries. We have shown previously that endothelin-1 constriction in mesenteric arteries is mediated by endothelin A receptors. In the current study, the selective increase in endothelin-1 constriction in IH resistance arteries was accompanied by increased expression of endothelin A receptor expression (densitometry units 271±23 IH versus 158±25 Sham; P <0.05). Thus, IH hypertension appears to cause alterations in signaling components unique to endothelin-1 at the receptor level and in postreceptor signaling that increases calcium sensitivity during endothelin A activation. Future studies will determine the specific changes in vascular smooth muscle signaling in IH hypertension causing this augmented contractile phenotype.

We have previously shown that hydrogen sulfide (H₂S) reduces myogenic tone and causes relaxation of phenylephrine (PE)-constricted mesenteric arteries. This effect of H₂S to cause vasodilation and vascular smooth muscle cell (VSMC) hyperpolarization was mediated by large-conductance Ca²⁺-activated potassium channels (BK_Ca). Ca²⁺ sparks are ryanodine receptor (RyR)-mediated Ca²⁺-release events that activate BK_Ca channels in VSMCs to cause membrane hyperpolarization and vasodilation. We hypothesized that H₂S activates Ca²⁺ sparks in small mesenteric arteries. Ca²⁺ sparks were measured using confocal microscopy in rat mesenteric arteries loaded with the Ca²⁺ indicator fluo-4. VSMC membrane potential ( E_m) was measured in isolated arteries using sharp microelectrodes. In PE-constricted arteries, the H₂S donor NaHS caused vasodilation that was inhibited by ryanodine (RyR blocker), abluminal or luminal iberiotoxin (IbTx, BK_Ca blocker), endothelial cell (EC) disruption, and sulfaphenazole [cytochrome P-450 2C (Cyp2C) inhibitor]. The H₂S donor NaHS (10 μmol/l) increased Ca²⁺ sparks but only in the presence of intact EC and this was blocked by sulfaphenazole or luminal IbTx. Inhibiting cystathionine γ-lyase (CSE)-derived H₂S with β-cyano-l-alanine (BCA) also reduced VSMC Ca²⁺ spark frequency in mesenteric arteries, as did EC disruption. However, excess CSE substrate homocysteine did not affect spark activity. NaHS hyperpolarized VSMC E_m in PE-depolarized mesenteric arteries with intact EC and also hyperpolarized EC E _m in arteries cut open to expose the lumen. This hyperpolarization was prevented by ryanodine, sulfaphenazole, and abluminal or luminal IbTx. BCA reduced IbTx-sensitive K⁺ currents in freshly dispersed mesenteric ECs. These results suggest that H₂S increases Ca²⁺ spark activity in mesenteric artery VSMC through activation of endothelial BK_Ca channels and Cyp2C, a novel vasodilatory pathway for this emerging signaling molecule.

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