Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries.

Background: Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease.

Endothelin-1 in Health and Disease.

Discovered almost 40 years ago, the potent vasoconstrictor peptide endothelin-1 (ET-1) has a wide range of roles both physiologically and pathologically. In recent years, there has been a focus on the contribution of ET-1 to disease. This has led to the development of various ET receptor antagonists, some of which are approved for the treatment of pulmonary arterial hypertension, while clinical trials for other diseases have been numerous yet, for the most part, unsuccessful.

Tracking endothelium-dependent NO release in pressurized arteries.

Endothelial cell (EC) dysfunction is an early hallmark of cardiovascular disease associated with the reduced bioavailability of nitric oxide (NO) resulting in over-constriction of arteries. Despite the clear need to assess NO availability, current techniques do not reliably allow this in intact arteries. Confocal fluorescence microscopy was used to compare two NO-sensitive fluorescent dyes (NO-dyes), CuFL2E and DAR-4M AM, in both cell-free chambers and isolated, intact arteries.

Signaling and structures underpinning conducted vasodilation in human and porcine intramyocardial coronary arteries.

Background: Adequate blood flow into coronary micro-arteries is essential for myocardial function. Here we assess the mechanisms responsible for amplifying blood flow into myogenically-contracting human and porcine intramyocardial micro-arteries using endothelium-dependent and -independent vasodilators.

Methods: Human and porcine atrial and ventricular small intramyocardial coronary arteries (IMCAs) were studied with pressure myography and imaged using confocal microscopy and serial section/3-D reconstruction EM.

Endothelium-Dependent Hyperpolarization: The Evolution of Myoendothelial Microdomains.

Endothelium-derived hyperpolarizing factor (EDHF) was envisaged as a chemical entity causing vasodilation by hyperpolarizing vascular smooth muscle (VSM) cells and distinct from nitric oxide (NO) ([aka endothelium-derived relaxing factor (EDRF)]) and prostacyclin. The search for an identity for EDHF unraveled the complexity of signaling within small arteries. Hyperpolarization originates within endothelial cells (ECs), spreading to the VSM by 2 branches, 1 chemical and 1 electrical, with the relative contribution varying with artery location, branch order, and prevailing profile of VSM activation.

High spatial and temporal resolution Ca imaging of myocardial strips from human, pig and rat.

Ca handling within cardiac myocytes underpins coordinated contractile function within the beating heart. This protocol enables high spatial and temporal Ca imaging of ex vivo multicellular myocardial strips. The endocardial surface is retained, and strips of 150-300-µm thickness are dissected, loaded with Ca indicators and mounted within 1.

Human coronary microvascular contractile dysfunction associates with viable synthetic smooth muscle cells.

Aims: Coronary microvascular smooth muscle cells (SMCs) respond to luminal pressure by developing myogenic tone (MT), a process integral to the regulation of microvascular perfusion. The cellular mechanisms underlying poor myogenic reactivity in patients with heart valve disease are unknown and form the focus of this study.

Methods And Results: Intramyocardial coronary micro-arteries (IMCAs) isolated from human and pig right atrial (RA) appendage and left ventricular (LV) biopsies were studied using pressure myography combined with confocal microscopy.

Phospholemman Phosphorylation Regulates Vascular Tone, Blood Pressure, and Hypertension in Mice and Humans.

Background: Although it has long been recognized that smooth muscle Na/K ATPase modulates vascular tone and blood pressure (BP), the role of its accessory protein phospholemman has not been characterized. The aim of this study was to test the hypothesis that phospholemman phosphorylation regulates vascular tone in vitro and that this mechanism plays an important role in modulation of vascular function and BP in experimental models in vivo and in humans.

Methods: In mouse studies, phospholemman knock-in mice (PLM; phospholemman [FXYD1] in which the 3 phosphorylation sites on serines 63, 68, and 69 are mutated to alanines), in which phospholemman is rendered unphosphorylatable, were used to assess the role of phospholemman phosphorylation in vitro in aortic and mesenteric vessels using wire myography and membrane potential measurements.

Endothelial Nitric Oxide Suppresses Action-Potential-Like Transient Spikes and Vasospasm in Small Resistance Arteries.

Endothelial dysfunction in small arteries is a ubiquitous, early feature of cardiovascular disease, including hypertension. Dysfunction reflects reduced bioavailability of endothelium-derived nitric oxide (NO) and depressed endothelium-dependent hyperpolarization that enhances vasoreactivity. We measured smooth muscle membrane potential and tension, smooth muscle calcium, and used real-time quantitative polymerase chain reaction in small arteries and isolated tubes of endothelium to investigate how dysfunction enhances vasoreactivity.

Intrinsic regulation of microvascular tone by myoendothelial feedback circuits.

The endothelium is an important regulator of arterial vascular tone, acting to release nitric oxide (NO) and open Ca-activated K (K) channels to relax vascular smooth muscle cells (VSMCs). While agonists acting at endothelial cell (EC) receptors are widely used to assess the ability of the endothelium to reduce vascular tone, the intrinsic EC-dependent mechanisms are less well characterized. In small resistance arteries and arterioles, the presence of heterocellular gap junctions termed myoendothelial gap junctions (MEGJs) allows the passage of not only current, but small molecules including Ca and inositol trisphosphate (IP).

Smooth muscle gap-junctions allow propagation of intercellular Ca waves and vasoconstriction due to Ca based action potentials in rat mesenteric resistance arteries.

The role of vascular gap junctions in the conduction of intercellular Ca and vasoconstriction along small resistance arteries is not entirely understood. Some depolarizing agents trigger conducted vasoconstriction while others only evoke a local depolarization. Here we use a novel technique to investigate the temporal and spatial relationship between intercellular Ca signals generated by smooth muscle action potentials (APs) and vasoconstriction in mesenteric resistance arteries (MA).

VEGF-A inhibits agonist-mediated Ca responses and activation of IK channels in mouse resistance artery endothelial cells.

Key Points: Prolonged exposure to vascular endothelial growth factor A (VEGF-A) inhibits agonist-mediated endothelial cell Ca release and subsequent activation of intermediate conductance Ca -activated K (IK ) channels, which underpins vasodilatation as a result of endothelium-dependent hyperpolarization (EDH) in mouse resistance arteries. Signalling via mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) downstream of VEGF-A was required to attenuate endothelial cell Ca responses and the EDH-vasodilatation mediated by IK activation. VEGF-A exposure did not modify vasodilatation as a result of the direct activation of IK channels, nor the pattern of expression of inositol 1,4,5-trisphosphate receptor 1 within endothelial cells of resistance arteries.

Hyperglycaemia disrupts conducted vasodilation in the resistance vasculature of db/db mice.

Vascular dysfunction in small resistance arteries is observed during chronic elevations in blood glucose. Hyperglycaemia-associated effects on endothelium-dependent vasodilation have been well characterized, but effects on conducted vasodilation in the resistance vasculature are not known. Small mesenteric arteries were isolated from healthy and diabetic db/db mice, which were used as a model of chronic hyperglycaemia.

Vasorelaxation to the Nitroxyl Donor Isopropylamine NONOate in Resistance Arteries Does Not Require Perivascular Calcitonin Gene-Related Peptide.

Nitroxyl (HNO) donors offer considerable therapeutic potential for the treatment of hypertension-related cardiovascular disorders, particularly heart failure, as they combine an inotropic action with peripheral vasodilation. Angeli's salt is the only HNO donor whose mechanism has been studied in depth, and recently, Angeli's salt vasodilation was suggested to be indirect and caused by calcitonin gene-related peptide (CGRP) released from perivascular nerves after HNO activates TRPA1 (transient receptor potential cation channel subfamily A member 1) channels. We investigated resistance artery vasorelaxation to the HNO donor, isopropylamine NONOate (IPA/NO), one of the structures providing a template for therapeutic development.

Voltage-dependent Ca entry into smooth muscle during contraction promotes endothelium-mediated feedback vasodilation in arterioles.

Vascular smooth muscle contraction is suppressed by feedback dilation mediated by the endothelium. In skeletal muscle arterioles, this feedback can be activated by Ca signals passing from smooth muscle through gap junctions to endothelial cells, which protrude through holes in the internal elastic lamina to make contact with vascular smooth muscle cells. Although hypothetically either Ca or inositol trisphosphate (IP) may provide the intercellular signal, it is generally thought that IP diffusion is responsible.

Direct visualization of the arterial wall water permeability barrier using CARS microscopy.

The artery wall is equipped with a water permeation barrier that allows blood to flow at high pressure without significant water leak. The precise location of this barrier is unknown despite its importance in vascular function and its contribution to many vascular complications when it is compromised. Herein we map the water permeability in intact arteries, using coherent anti-Stokes Raman scattering (CARS) microscopy and isotopic perfusion experiments.

Endothelial-smooth muscle cell interactions in the regulation of vascular tone in skeletal muscle.

The SMCs of skeletal muscle arterioles are intricately sensitive to changes in membrane potential. Upon increasing luminal pressure, the SMCs depolarize, thereby opening VDCCs, which leads to contraction. Mechanisms that oppose this myogenic tone can involve voltage-dependent and independent dilator pathways, and can be endothelium-dependent or independent.

Isolated Human Pulmonary Artery Structure and Function Pre- and Post-Cardiopulmonary Bypass Surgery.

Background: Pulmonary dysfunction is a known complication after cardiac surgery using cardiopulmonary bypass, ranging from subclinical functional changes to prolonged postoperative ventilation, acute lung injury, and acute respiratory distress syndrome. Whether human pulmonary arterial function is compromised is unknown. The aim of the present study was to compare the structure and function of isolated and cannulated human pulmonary arteries obtained from lung biopsies after the chest was opened (pre-cardiopulmonary bypass) to those obtained at the end of cardiopulmonary bypass (post-cardiopulmonary bypass) from patients undergoing coronary artery bypass graft surgery.

Linking hyperpolarization to endothelial cell calcium events in arterioles.

Our understanding of the relationship between EC membrane potential and Ca(2+) entry has been shaped historically by data from cells in culture. Membrane hyperpolarization was associated with raised cytoplasmic [Ca(2+) ] ascribed to the increase in the inward electrochemical gradient for Ca(2+) , as ECs are generally thought to lack VGCC. Ca(2+) influx was assumed to reflect the presence of an undefined Ca(2+) "leak" channel, although the original research articles with isolated ECs did not elucidate which Ca(2+) influx channel was involved or indeed if a transporter might contribute.

Low intravascular pressure activates endothelial cell TRPV4 channels, local Ca2+ events, and IKCa channels, reducing arteriolar tone.

Endothelial cell (EC) Ca(2+)-activated K channels (SK(Ca) and IK(Ca) channels) generate hyperpolarization that passes to the adjacent smooth muscle cells causing vasodilation. IK(Ca) channels focused within EC projections toward the smooth muscle cells are activated by spontaneous Ca(2+) events (Ca(2+) puffs/pulsars). We now show that transient receptor potential, vanilloid 4 channels (TRPV4 channels) also cluster within this microdomain and are selectively activated at low intravascular pressure.

Measurement of changes in endothelial and smooth muscle Ca²⁺ in pressurized arteries.

The use of single- and dual-wavelength Ca(2+)-sensitive fluorescent dyes to monitor changes in endothelial and/or smooth muscle intracellular Ca(2+) levels has provided information linking Ca(2+) events to changes in arterial function. Here we describe the in vitro techniques used to selectively load Ca(2+) indicators into either the endothelium or the smooth muscle of cannulated rat cremaster arteries. These vessels normally develop spontaneous myogenic tone that is largely unaffected by the loading of Ca2+ indicators or the subsequent imaging procedures.

Spatial distribution and mechanical function of elastin in resistance arteries: a role in bearing longitudinal stress.

Objective: Despite the role that extracellular matrix (ECM) plays in vascular signaling, little is known of the complex structural arrangement between specific ECM proteins and vascular smooth muscle cells. Our objective was to examine the hypothesis that adventitial elastin fibers are dominant in vessels subject to longitudinal stretch.

Methods And Results: Cremaster muscle arterioles were isolated, allowed to develop spontaneous tone, and compared with small cerebral arteries.