Pooneh Bagher

Regulation of blood flow in the microcirculation: role of conducted vasodilation

This review is concerned with understanding how vasodilation initiated from local sites in the tissue can spread to encompass multiple branches of the resistance vasculature. Within tissues, arteriolar networks control the distribution and magnitude of capillary perfusion. Vasodilation arising from the microcirculation can ‘ascend’ into feed arteries that control blood flow into arteriolar networks. Thus distal segments of the resistance network signal proximal segments to dilate and thereby increase total oxygen supply to parenchymal cells. August Krogh proposed that innervation of capillaries provided the mechanism for a spreading vasodilatory response. With greater understanding of the ultrastructural organization of resistance networks, an alternative explanation has emerged: Electrical signalling from cell to cell along the vessel wall through gap junctions. Hyperpolarization originates from ion channel activation at the site of stimulation with the endothelium serving as the predominant cellular pathway for signal conduction along the vessel wall. As hyperpolarization travels, it is transmitted into surrounding smooth muscle cells through myoendothelial coupling to promote relaxation. Conducted vasodilation (CVD) encompasses greater distances than can be explained by passive decay and understanding such behaviour is the focus of current research efforts. In the context of athletic performance, the ability of vasodilation to ascend into feed arteries is essential to achieving peak levels of muscle blood flow. CVD is tempered by sympathetic neuroeffector signalling when governing muscle blood flow at rest and during exercise. Impairment of conduction during ageing and in diseased states can limit physical work capacity by restricting muscle blood flow.

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

Endothelial cell (EC) Ca2+-activated K channels (SKCa and IKCa channels) generate hyperpolarization that passes to the adjacent smooth muscle cells causing vasodilation. IKCa channels focused within EC projections toward the smooth muscle cells are activated by spontaneous Ca2+ events (Ca2+ 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. In arterioles pressurized to 80 mmHg, ECs generated low-frequency (∼2 min−1) inositol 1,4,5-trisphosphate receptor-based Ca2+ events. Decreasing intraluminal pressure below 50 mmHg increased the frequency of EC Ca2+ events twofold to threefold, an effect blocked with the TRPV4 antagonist RN1734. These discrete events represent both TRPV4-sparklet- and nonsparklet-evoked Ca2+ increases, which on occasion led to intracellular Ca2+ waves. The concurrent vasodilation associated with increases in Ca2+ event frequency was inhibited, and basal myogenic tone was increased, by either RN1734 or TRAM-34 (IKCa channel blocker), but not by apamin (SKCa channel blocker). These data show that intraluminal pressure influences an endothelial microdomain inversely to alter Ca2+ event frequency; at low pressures the consequence is activation of EC IKCa channels and vasodilation, reducing the myogenic tone that underpins tissue blood-flow autoregulation.

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation. The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem. The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light. However curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope. While shown to be a valuable preparation to study the physiology of the microcirculation in rats and hamsters, the cremaster muscle in mice has proven particularly useful in dissecting cellular pathways involved in regulating microvascular function and real-time imaging of intercellular signaling. The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal. It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a genetically encoded fluorescent calcium indicator molecule. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of calcium signaling in the arteriolar endothelium.

Evidence for impaired neurovascular transmission in a murine model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a muscle-wasting disease caused by mutations in the dystrophin gene. Little is known about how blood flow control is affected in arteriolar networks supplying dystrophic muscle. We tested the hypothesis that mdx mice, a murine model for DMD, exhibit defects in arteriolar vasomotor control. The cremaster muscle was prepared for intravital microscopy in pentobarbital sodium-anesthetized mdx and C57BL/10 control mice (n ≥ 5 per group). Spontaneous vasomotor tone increased similarly with arteriolar branch order in both mdx and C57BL/10 mice [pooled values: first order (1A), 6%; second order (2A), 56%; and third order (3A), 61%] with no difference in maximal diameters between groups measured during equilibration with topical 10 μM sodium nitroprusside (pooled values: 1A, 70 ± 3 μm; 2A, 31 ± 3 μm; and 3A, 19 ± 3 μm). Concentration-response curves to acetylcholine (ACh) and norepinephrine added to the superfusion solution did not differ between mdx and C57BL/10 mice, nor did constriction to elevated (21%) oxygen. In response to local stimulation from a micropipette, conducted vasodilation to ACh and conducted vasoconstriction to KCl were also not different between groups; however, constriction decayed with distance (P < 0.05) whereas dilation did not. Remarkably, arteriolar constriction to perivascular nerve stimulation (PNS) at 2, 4, and 8 Hz was reduced by ∼25-30% in mdx mice compared with C57BL/10 mice (P < 0.05). With intact arteriolar reactivity to agonists, attenuated constriction to perivascular nerve stimulation indicates impaired neurovascular transmission in arterioles controlling blood flow in mdx mice.

Voltage-dependent Ca2+ entry into smooth muscle during contraction promotes endothelium-mediated feedback vasodilation in arterioles

Ca2+ entry into vascular smooth muscle activates Ca2+ signaling in the endothelium to protect tissue blood flow. Ca2+, the intercellular signal in arterioles Vasoconstriction must be balanced with vasodilation, particularly in the arterioles that supply tissues with blood. Endothelial cells protrude through holes in the internal elastic lamina in arterioles to make contact with vascular smooth muscle cells. Gap junctions are present at these sites where endothelial cells meet vascular smooth muscle cells. IP3 has been thought to be a signal that passes through these gap junctions to endothelial cells to mediate vasodilation. However, Garland et al. showed that it was Ca2+, rather than IP3, that entered vascular smooth muscle cells through voltage-gated Ca2+ channels, subsequently passed through gap junctions into endothelial cells, and initiated vasodilation mediated by endothelial cells. The magnitude of these Ca2+ signals in endothelial cells depended on IP3 receptors. These results resolve a long-standing controversy over how vascular smooth muscle cells communicate with endothelial cells to trigger feedback vasodilation. Vascular smooth muscle contraction is suppressed by feedback dilation mediated by the endothelium. In skeletal muscle arterioles, this feedback can be activated by Ca2+ 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 Ca2+ or inositol trisphosphate (IP3) may provide the intercellular signal, it is generally thought that IP3 diffusion is responsible. We provide evidence that Ca2+ entry through L-type voltage-dependent Ca2+ channels (VDCCs) in vascular smooth muscle can pass to the endothelium through positions aligned with holes in the internal elastic lamina in amounts sufficient to activate endothelial cell Ca2+ signaling. In endothelial cells in which IP3 receptors (IP3Rs) were blocked, VDCC-driven Ca2+ events were transient and localized to the endothelium that protrudes through the internal elastic lamina to contact vascular smooth muscle cells. In endothelial cells in which IP3Rs were not blocked, VDCC-driven Ca2+ events in endothelial cells were amplified to form propagating waves. These waves activated voltage-insensitive, intermediate-conductance, Ca2+-activated K+ (IKCa) channels, thereby providing feedback that effectively suppressed vasoconstriction and enabled cycles of constriction and dilation called vasomotion. Thus, agonists that stimulate vascular smooth muscle depolarization provide Ca2+ to endothelial cells to activate a feedback circuit that protects tissue blood flow.

Intravital Macrozoom Imaging and Automated Analysis of Endothelial Cell Calcium Signals Coincident with Arteriolar Dilation in Cx40BAC-GCaMP2 Transgenic Mice

Please cite this paper as: Bagher, Davis and Segal (2011). Intravital Macrozoom Imaging and Automated Analysis of Endothelial Cell Calcium Signals Coincident with Arteriolar Dilation in Cx40BAC‐GCaMP2 Transgenic Mice. Microcirculation 18(4), 331–338.

β1-Integrin Is Essential for Vasoregulation and Smooth Muscle Survival In Vivo

Objective—Integrins contribute to vascular morphogenesis through regulation of adhesion and assembly of the extracellular matrix. However, the role of &bgr;1-integrin in the mature vascular wall is less clear. Approach and Results—We sought to determine the function of &bgr;1-integrin in mature smooth muscle cells in vivo using a loss of function approach by crossing a tamoxifen-inducible sm22&agr;Cre line to a floxed &bgr;1-integrin transgenic line. Adult mice lacking smooth muscle &bgr;1-integrin survived only 10 weeks post induction. The deletion of &bgr;1-integrin resulted in profound loss of vasomotor control. Histological analysis revealed progressive fibrosis in arteries with associated apoptosis of smooth muscle cells, which was not rescued by adventitial stem cells. Smooth muscle cell apoptosis was detected in arteries with dead cells replaced primarily by collagen. Despite the catastrophic effects on vascular smooth muscle, the deleted visceral smooth muscle remained viable with the exception of a short portion of the colon, indicating that vascular but not visceral smooth muscle is particularly sensitive to changes in &bgr;1-integrin. Conclusions—This study reveals an essential function of &bgr;1-integrin in the maintenance of vasomotor control and highlights a critical role for &bgr;1-integrin in vascular, but not visceral, smooth muscle survival.

Regulation of Myoendothelial Junction Formation: Bridging the Gap

See related article, pages 1092–1102 In this issue of Circulation Research , Heberlein et al1 provide exciting new insight into the actions of plasminogen activator (PA) inhibitor (PAI)-1 by illuminating its role in governing the ability of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) to communicate with each other through specialized contacts defined as myoendothelial junctions (MEJs). In the resistance vasculature, MEJs are cellular extensions through the internal elastic lamina (IEL), whereby ECs make physical contact with adjacent VSMCs.2 At points of cell–cell contact, the presence of gap junction channels enables electrical coupling and intercellular diffusion of small solutes (<1 kDa) that govern vasomotor control. Cell–cell signaling through gap junction channels can be regulated acutely (eg, through phosphorylation of key serine residues on connexin subunits), and their protein complexes undergo hourly turnover in the plasma membrane. Such properties imply that the nature of cell–cell signaling through MEJs is under dynamic regulation. Despite implications that the MEJ is itself a dynamic structure, little is known about how MEJ formation and regression are regulated. New findings presented by Heberlein et al1 illustrate how dynamic MEJ formation and signaling may be enhanced or impaired via PAI-1 regulation of the PA system. Plasminogen is synthesized in the liver and released into the circulation as a zymogen of the serine protease, plasmin. The conversion of plasminogen to active plasmin is mediated by tissue (t)-PA and urokinase-type (u)-PA (ie, urokinase). Whereas t-PA is involved primarily in the dissolution of fibrin in the circulation, u-PA binds to the urokinase receptor (uPAR) (ie, CD87) on cell membranes, thereby localizing proteolytic activity to the vicinity of binding. In this manner, uPAR is integral to cell migration and adhesion through breakdown of the extracellular matrix (ECM).3,4 PAI-1, a ≈45-kDa glycoprotein, is a serine …

Scaffolding Builds to Reduce Blood Pressure

Disruption of a signaling microdomain in endothelial cell projections contributes to hypertension. Endothelial cells provide vasodilator signals to reduce blood pressure. In the small resistance arteries and arterioles, which determine the distribution and pressure of blood, the major signal is hyperpolarization reflecting the endothelial activity of calcium-activated potassium channels (KCa). In this issue of Science Signaling, Sonkusare et al. report that the scaffold protein AKAP150 is required for the kinase PKC and the calcium channel TRPV4 to enable receptor-mediated relaxation signaling. This scaffold enhances TRPV4 gating cooperativity and markedly amplifies the Ca2+ signal, which ultimately activates (mainly) IKCa channels. Normally restricted to tiny endothelial projections, AKAP150 localization and associated signaling is disrupted in a model of hypertension, thereby diminishing hyperpolarization and vasodilation.