Perturbation of chemical coupling by an endothelial Cx40 mutant attenuates endothelium-dependent vasodilation by KCa channels and elevates blood pressure in mice.

Mutant forms of connexin40 (Cx40) exist in the human population and predispose carriers to atrial fibrillation. Since endothelial expression of Cx40 is important for electrical and chemical communication within the arterial wall, carriers of mutant Cx40 proteins may be predisposed to peripheral arterial dysfunction and dysregulation of blood pressure. We have therefore studied mice expressing either a chemically dysfunctional mutant, Cx40T202S, or wild-type Cx40, with native Cx40, specifically in the endothelium. Blood pressure was measured by telemetry under normal conditions and during cardiovascular stress induced by locomotor activity, phenylephrine or nitric oxide blockade (N(É·)-nitro-L-arginine methyl ester hydroxide, L-NAME). Blood pressure of Cx40T202STg mice was significantly elevated at night when compared with wild-type or Cx40Tg mice, without change in mean heart rate, pulse pressure or locomotor activity. Analysis over 24 h showed that blood pressure of Cx40T202STg mice was significantly elevated at rest and additionally during locomotor activity. In contrast, neither plasma renin concentration nor pressor responses to phenylephrine or L-NAME were altered, the latter indicating that nitric oxide bioavailability was normal. In isolated, pressurised mesenteric arteries, hyperpolarisation and vasodilation evoked by SKA-31, the selective modulator of SKCa and IKCa channels, was significantly reduced in Cx40T202STg mice, due to attenuation of the SKCa component. Acetylcholine-induced ascending vasodilation in vivo was also significantly attenuated in cremaster muscle arterioles of Cx40T202STg mice, compared to wild-type and Cx40Tg mice. We conclude that endothelial expression of the chemically dysfunctional Cx40T202S reduces peripheral vasodilator capacity mediated by SKCa-dependent hyperpolarisation and also increases blood pressure.

Polymorphism in endothelial connexin40 enhances sensitivity to intraluminal pressure and increases arterial stiffness.

OBJECTIVE: To determine whether impairment of endothelial connexin40 (Cx40), an effect that can occur in hypertension and aging, contributes to the arterial dysfunction and stiffening in these conditions. APPROACH AND RESULTS: A new transgenic mouse strain, expressing a mutant Cx40, (Cx40T202S), specifically in the vascular endothelium, has been developed and characterized. This mutation produces nonfunctional hemichannels, whereas gap junctions containing the mutant are electrically, but not chemically, patent. Mesenteric resistance arteries from Cx40T202S mice showed increased sensitivity of the myogenic response to intraluminal pressure in vitro, compared with wild-type mice, whereas transgenic mice overexpressing native Cx40 (Cx40Tg) showed reduced sensitivity. In control and Cx40Tg mice, the sensitivity to pressure of myogenic constriction was modulated by both NO and endothelium-derived hyperpolarization; however, the endothelium-derived hyperpolarization component was absent in Cx40T202S arteries. Analysis of passive mechanical properties revealed that arterial stiffness was enhanced in vessels from Cx40T202S mice, but not in wild-type or Cx40Tg mice. CONCLUSIONS: Introduction of a mutant form of Cx40 in the endogenous endothelial Cx40 population prevents endothelium-derived hyperpolarization activation during myogenic constriction, enhancing sensitivity to intraluminal pressure and increasing arterial stiffness. We conclude that genetic polymorphisms in endothelial Cx40 can contribute to the pathogenesis of arterial disease.

Spreading vasodilatation in the murine microcirculation: attenuation by oxidative stress-induced change in electromechanical coupling.

Regulation of blood flow in microcirculatory networks depends on spread of local vasodilatation to encompass upstream arteries; a process mediated by endothelial conduction of hyperpolarization. Given that endothelial coupling is reduced in hypertension, we used hypertensive Cx40ko mice, in which endothelial coupling is attenuated, to investigate the contribution of the renin-angiotensin system and reduced endothelial cell coupling to conducted vasodilatation of cremaster arterioles in vivo. When the endothelium was disrupted by light dye treatment, conducted vasodilatation, following ionophoresis of acetylcholine, was abolished beyond the site of endothelial damage. In the absence of Cx40, sparse immunohistochemical staining was found for Cx37 in the endothelium, and endothelial, myoendothelial and smooth muscle gap junctions were identified by electron microscopy. Hyperpolarization decayed more rapidly in arterioles from Cx40ko than wild-type mice. This was accompanied by a shift in the threshold potential defining the linear relationship between voltage and diameter, increased T-type calcium channel expression and increased contribution of T-type (3 µmol l(-1) NNC 55-0396), relative to L-type (1 µmol l(-1) nifedipine), channels to vascular tone. The change in electromechanical coupling was reversed by inhibition of the renin-angiotensin system (candesartan, 1.0 mg kg(-1) day(-1) for 2 weeks) or by acute treatment with the superoxide scavenger tempol (1 mmol l(-1)). Candesartan and tempol treatments also significantly improved conducted vasodilatation. We conclude that conducted vasodilatation in Cx40ko mice requires the endothelium, and attenuation results from both a reduction in endothelial coupling and an angiotensin II-induced increase in oxidative stress. We suggest that during cardiovascular disease, the ability of microvascular networks to maintain tissue integrity may be compromised due to oxidative stress-induced changes in electromechanical coupling.

Non-linear relationship between hyperpolarisation and relaxation enables long distance propagation of vasodilatation.

Blood flow is adjusted to tissue demand through rapidly ascending vasodilatations resulting from conduction of hyperpolarisation through vascular gap junctions. We investigated how these dilatations can spread without attenuation if mediated by an electrical signal. Cremaster muscle arterioles were studied in vivo by simultaneously measuring membrane potential and vessel diameter. Focal application of acetylcholine elicited hyperpolarisations which decayed passively with distance from the local site,while dilatation spread upstream without attenuation. Analysis of simultaneous recordings at the local site revealed that hyperpolarisation and dilatation were only linearly related over a restricted voltage range to a threshold potential, beyond which dilatation was maximal. Experimental data could be simulated in a computational model with electrotonic decay of hyperpolarisation but imposition of this threshold. The model was tested by reducing the amplitude of the local hyperpolarisation which led to entry into the linear range closer to the local site and decay of dilatation. Serial section electron microscopy and light dye treatment confirmed that the spread of dilatation occurred through the endothelium and that the two cell layers were tightly coupled. Generality of the mechanism was demonstrated by applying the model to the attenuated propagation of dilatation found in larger arteries.We conclude that long distance spread of locally initiated dilatations is not due to a regenerative electrical phenomenon, but rather a restricted linear relationship between voltage and vessel tone, which minimises the impact of electrotonic decay of voltage. Disease-related alterations in endothelial coupling or ion channel expression could therefore decrease the ability to adjust blood flow to meet metabolic demand.

Enalapril treatment alters the contribution of epoxyeicosatrienoic acids but not gap junctions to endothelium-derived hyperpolarizing factor activity in mesenteric arteries of spontaneously hypertensive rats.

Reduction in endothelium-derived hyperpolarizing factor (EDHF)-mediated dilatory function in large, elastic arteries during hypertension is reversed after blood pressure normalization. We investigated whether similar mechanisms occurred in smaller mesenteric resistance arteries from aged Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs), and SHRs treated with the angiotensin-converting enzyme inhibitor, enalapril, using immunohistochemistry, serial-section electron microscopy, electrophysiology and wire myography. Unlike the superior mesenteric artery, EDHF relaxations in muscular mesenteric arteries were not reduced in SHRs, although morphological differences were found in the endothelium and smooth muscle. In WKY rats, SHRs and enalapril-treated SHRs, relaxations were mediated by small-, large-, and intermediate-conductance calcium-activated potassium channels, which were distributed in the endothelium, smooth muscle, and both layers, respectively. However, only WKY hyperpolarizations and relaxations were sensitive to gap junction blockers, and these arteries expressed more endothelial and myoendothelial gap junctions than arteries from SHRs. Responses in WKY rats, but not SHRs, were also reduced by inhibitors of epoxyeicosatrienoic acids (EETs), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) and miconazole, although sensitivity to EET regioisomers was endothelium-independent in all rats. Enalapril treatment of SHRs reduced blood pressure and restored sensitivity to 14,15-EEZE, but not to gap junction blockers, and failed to reverse the morphological changes. In conclusion, the mechanisms underlying EDHF in muscular mesenteric arteries differ between WKY rats and SHRs, with gap junctions and EETs involved only in WKY rats. However, reduction of blood pressure in SHRs with enalapril restored a role for EETs, but not gap junctions, without reversing morphological changes, suggesting a differential control of chemical and structural alterations.

Loss of functional endothelial connexin40 results in exercise-induced hypertension in mice.

During activity, coordinated vasodilation of microcirculatory networks with upstream supply vessels increases blood flow to skeletal and cardiac muscles and reduces peripheral resistance. Endothelial dysfunction in humans attenuates activity-dependent vasodilation, resulting in exercise-induced hypertension in otherwise normotensive individuals. Underpinning activity-dependent hyperemia is an ascending vasodilation in which the endothelial gap junction protein, connexin (Cx)40, plays an essential role. Because exercise-induced hypertension is proposed as a forerunner to clinical hypertension, we hypothesized that endothelial disruption of Cx40 function in mice may create an animal model of this condition. To this end, we created mice in which a mutant Cx40T152A was expressed alongside wildtype Cx40 selectively in the endothelium. Expression of the Cx40T152A transgene in Xenopus oocytes and mouse coronary endothelial cells in vitro impaired both electric and chemical conductance and acted as a dominant-negative against wildtype Cx40, Cx43, and Cx45, but not Cx37. Endothelial expression of Cx40T152A in Cx40T152ATg mice attenuated ascending vasodilation, without effect on radial coupling through myoendothelial gap junctions. Using radiotelemetry, Cx40T152ATg mice showed an activity-dependent increase in blood pressure, which was significantly greater than in wildtype mice, but significantly less than in chronically hypertensive, Cx40knockout mice. The increase in heart rate with activity was also greater than in wildtype or Cx40knockout mice. We conclude that the endothelial Cx40T152A mutation attenuates activity-dependent vasodilation, producing a model of exercise-induced hypertension. These data highlight the importance of endothelial coupling through Cx40 in regulating blood pressure during activity.

Regulation of endothelial-specific transgene expression by the LacI repressor protein in vivo.

Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2) with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacI(R), and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacI(R) protein.

Chronic deficit in nitric oxide elicits oxidative stress and augments T-type calcium-channel contribution to vascular tone of rodent arteries and arterioles.

AIMS: As cardiovascular disease is characterized by reduced nitric oxide bioavailability, our aim was to determine the impact of this change on the mechanism underlying vascular tone of pressurized arteries in vitro and in vivo. METHODS AND RESULTS: We used pressurized cerebral and mesenteric arteries in vitro and skeletal muscle arterioles in vivo to study the contribution of L-type (1 µmol/L nifedipine) and T-type (1 µmol/L mibefradil, 3 µmol/L NNC 55-0396) calcium channels to vascular tone, following acute or chronic inhibition of nitric oxide. Acute inhibition with l-NAME (10 µmol/L) significantly increased the T-type, but not the L-type, channel contribution to vascular tone in vitro and in vivo, and altered the smooth muscle expression of the Cav3.1 and Cav3.2 T-type channels. In pressurized mesenteric arteries of Cav3.1ko and Cav3.2ko mice, acutely treated with l-NAME, the contribution of T-type channels relative to L-type channels was significantly reduced, compared with arteries from wild-type mice.Chronic l-NAME treatment (40 mg/kg/day; 14-18 days) increased blood pressure, vascular superoxide, and the contribution of T-type channels to vascular tone in vivo. The latter was reversed by acute scavenging of superoxide with tempol (1 mmol/L), or inhibition of NADPH oxidase with apocynin (500 µmol/L) or DPI (5 µmol/L). CONCLUSION: We conclude that nitric oxide deficit produces a significant increase in the contribution of Cav3.1 and Cav3.2 T-type calcium channels to vascular tone, by regulating the bioavailability of reactive oxygen species produced by NADPH oxidase. Our data provide evidence for a novel causal link between nitric oxide deficit, oxidative stress, and T-type calcium channel function.