Paola Corti

Interaction between alk1 and blood flow in the development of arteriovenous malformations.

Arteriovenous malformations (AVMs) are fragile direct connections between arteries and veins that arise during times of active angiogenesis. To understand the etiology of AVMs and the role of blood flow in their development, we analyzed AVM development in zebrafish embryos harboring a mutation in activin receptor-like kinase I (alk1), which encodes a TGFβ family type I receptor implicated in the human vascular disorder hereditary hemorrhagic telangiectasia type 2 (HHT2). Our analyses demonstrate that increases in arterial caliber, which stem in part from increased cell number and in part from decreased cell density, precede AVM development, and that AVMs represent enlargement and stabilization of normally transient arteriovenous connections. Whereas initial increases in endothelial cell number are independent of blood flow, later increases, as well as AVMs, are dependent on flow. Furthermore, we demonstrate that alk1 expression requires blood flow, and despite normal levels of shear stress, some flow-responsive genes are dysregulated in alk1 mutant arterial endothelial cells. Taken together, our results suggest that Alk1 plays a role in transducing hemodynamic forces into a biochemical signal required to limit nascent vessel caliber, and support a novel two-step model for HHT-associated AVM development in which pathological arterial enlargement and consequent altered blood flow precipitate a flow-dependent adaptive response involving retention of normally transient arteriovenous connections, thereby generating AVMs.

Analysis of early embryonic great-vessel microcirculation in zebrafish using high-speed confocal μPIV.

In the developing cardiovascular system, hemodynamic vascular loading is critical for angiogenesis and cardiovascular adaptation. Normal zebrafish embryos with transgenically-labeled endothelial and red blood cells provide an excellent in vivo model for studying the fluid-flow induced vascular loading. To characterize the developmental hemodynamics of early embryonic great-vessel microcirculation in the zebrafish embryo, two complementary studies (experimental and numerical) are presented. Quantitative comparison of the wall shear stress (WSS) at the first aortic arch (AA1) of wild-type zebrafish embryos during two consecutive developmental stages is presented, using time-resolved confocal micro-particle image velocimetry (μPIV). Analysis showed that there was significant WSS difference between 32 and 48 h post-fertilization (hpf) wild-type embryos, which correlates with normal arch morphogenesis. The vascular distensibility of the arch wall at systole and the acceleration/deceleration rates of time-lapse phase-averaged streamwise blood flow curves were also analyzed. To estimate the influence of a novel intermittent red-blood cell (RBC) loading on the endothelium, a numerical two-phase, volume of fluid (VOF) flow model was further developed with realistic in vivo conditions. These studies showed that near-wall effects and cell clustering increased WSS augmentation at a minimum of 15% when the distance of RBC from arch vessel wall was less than 3 μm or when RBC cell-to-cell distance was less than 3 μm. When compared to a smooth wall, the WSS augmentation increased by a factor of ~1.4 due to the roughness of the wall created by the endothelial cell profile. These results quantitatively highlight the contribution of individual RBC flow patterns on endothelial WSS in great-vessel microcirculation and will benefit the quantitative understanding of mechanotransduction in embryonic great vessel biology, including arteriovenous malformations (AVM).

Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells

Significance Hemoglobin is generally assumed to be the only globin in vertebrate RBCs. In addition to its foremost role as an oxygen carrier, mammalian hemoglobin can also operate as a nitrite reductase, producing the signaling molecule nitric oxide (NO) from nitrite. Over the last 15 yr, several novel globins have been identified with six-coordinate heme geometry and uncertain physiological functions. Here we report that a six-coordinate globin of ancient origin, named Globin X, is present in fish RBCs. We establish that Globin X is a fast nitrite reductase, and this activity can regulate NO production from fish RBCs and modulate platelet activation. Thus we provide evidence that the ancestral globins in blood were efficient nitrite reductases. The discovery of novel globins in diverse organisms has stimulated intense interest in their evolved function, beyond oxygen binding. Globin X (GbX) is a protein found in fish, amphibians, and reptiles that diverged from a common ancestor of mammalian hemoglobins and myoglobins. Like mammalian neuroglobin, GbX was first designated as a neuronal globin in fish and exhibits six-coordinate heme geometry, suggesting a role in intracellular electron transfer reactions rather than oxygen binding. Here, we report that GbX to our knowledge is the first six-coordinate globin and the first globin protein apart from hemoglobin, found in vertebrate RBCs. GbX is present in fish erythrocytes and exhibits a nitrite reduction rate up to 200-fold faster than human hemoglobin and up to 50-fold higher than neuroglobin or cytoglobin. Deoxygenated GbX reduces nitrite to form nitric oxide (NO) and potently inhibits platelet activation in vitro, to a greater extent than hemoglobin. Fish RBCs also reduce nitrite to NO and inhibit platelet activation to a greater extent than human RBCs, whereas GbX knockdown inhibits this nitrite-dependent NO signaling. The description of a novel, six-coordinate globin in RBCs with dominant electron transfer and nitrite reduction functionality provides new insights into the evolved signaling properties of ancestral heme-globins.

Efficient Reduction of Vertebrate Cytoglobins by the Cytochrome b5/Cytochrome b5 Reductase/NADH System

Cytoglobin is a heme-containing protein ubiquitous in mammalian tissues. Unlike the evolutionarily related proteins hemoglobin and myoglobin, cytoglobin shows a six-coordinated heme binding, with the heme iron coordinated by two histidine side chains. Cytoglobin is involved in cytoprotection pathways through yet undefined mechanisms, and it has recently been demonstrated that cytoglobin has redox signaling properties via nitric oxide (NO) and nitrite metabolism. The reduced, ferrous cytoglobin can bind oxygen and will react with NO in a dioxygenation reaction to form nitrate, which dampens NO signaling. When deoxygenated, cytoglobin can bind nitrite and reduce it to NO. This oxidoreductase activity could be catalytic if an effective reduction system exists to regenerate the reduced heme species. The nature of the physiological cytoglobin reducing system is unknown, although it has been proposed that ascorbate and cytochrome b5 could fulfill this role. Here we describe that physiological concentrations of cytochrome b5 and cytochrome b5 reductase can reduce human and fish cytoglobins at rates up to 250-fold higher than those reported for their known physiological substrates, hemoglobin and myoglobin, and up to 100-fold faster than 5 mM ascorbate. These data suggest that the cytochrome b5/cytochrome b5 reductase system is a viable reductant for cytoglobin in vivo, allowing for catalytic oxidoreductase activity.

Is Nitrite the Circulating Endocrine Effector of Remote Ischemic Preconditioning

Nitric oxide (NO) is a highly diffusible, free radical signaling molecule that is produced by the endothelial NO synthase (eNOS) enzyme, which converts l-arginine and molecular oxygen into l-citrulline and NO.1,2 NO diffuses from the endothelium to the smooth muscle where it binds with high affinity to the heme group of soluble guanylate cyclase, which in turn catalyzes the conversion of GTP to cGMP.3 NO signaling is largely paracrine, with potential endocrine effects limited by its radical nature and extremely high reactivity with other heme-containing proteins such as hemoglobin and myoglobin.4 When NO encounters oxyhemoglobin in blood or oxymyoglobin in cardiomyocytes, it reacts at rates near the diffusion limit to form nitrate and methemoglobin (dioxygenation reaction).5,6 It will also react with the deoxyhemes of these proteins to form iron–nitrosyl complexes, which can release NO but inefficiently via the oxidative denitrosylation reaction.7 These 2 reactions, dioxygenation and iron nitrosylation, prevent NO from forming in the endothelium and diffusing to distant organ targets, such as the heart, intestine, kidney, brain, or liver. Article, see p 1601 Despite the strict paracrine limitations imposed by this chemistry, several studies suggested that endocrine NO signaling is possible. The Kubes’ group showed that NO delivered by inhalation to cats could improve blood flow and limit inflammation in the cat intestine subjected to ischemia–reperfusion (I/R) injury8; Cannon et al6 later showed that this was possible in the human circulation. Many subsequent studies have shown that inhaled NO could rescue distal organs from I/R injury and infarction. In fact, upregulation of eNOS selectively in the heart could rescue the liver from I/R injury.9 However, free NO cannot account for these effects based on the short half-life of NO in blood, on the order …

Evidence mounts that red cells and deoxyhemoglobin can reduce nitrite to bioactive NO to mediate intravascular endocrine NO signaling: commentary on "Anti-platelet effects of dietary nitrate in healthy volunteers: involvement of cGMP and influence of sex".

It is now appreciated that two parallel systems regulate nitric oxide (NO) formation and signaling in biological systems and the vasculature. The first is the canonical endothelial NO synthase (eNOS) system, which catalyzes a five-electron oxidation of L-arginine to form NO and citrulline [30]. The second is the nitrate–nitrite–NO pathway, a more primitive, oxygen-independent and reductive pathway, clearly of great importance in bacteria and plants and only more recently found to regulate mammalian control of blood pressure, energetics, and hypoxic vasodilation [6,11,12,25,26,29]. Now, as described in the current study by Velmurugan and colleagues [40], this pathway may also inhibit platelet activation, an effect thought to be primarily regulated by NO produced by eNOS [9,23,24,36]. Although mammals do not possess nitrate reductase enzymes, the mouth commensal flora provides the necessary symbiosis, with bacterial nitrate reductase enzymes converting nitrate in food and concentrated in saliva into nitrite, which is swallowed and systemically absorbed [2,3,27]. There is cross talk between both NO synthesis pathways. Nitrite in the blood and tissue derives equally from nitrate reduction by oral bacteria and from intravascular oxidation of NO formed from eNOS [35,44]. Thus eNOS regulates blood flow via endothelial NO formation and paracrine signaling to smooth muscle but also contributes to endocrine NO formation via oxidation to nitrite and transport to distal sites for regional reduction to NO [4,8,12]. Interestingly, both systems also generate superoxide, as the NO synthase system can become uncoupled in settings of subcellular L-arginine and tetrahydrobiopterin deficiency or eNOS glutathionylation (oxidation), and then exhibits an oxidase activity, forming superoxide rather than NO [5]. The enzymes that have been shown to reduce nitrite to NO, such as the molybdopterin family members xanthine oxidase and aldehyde oxidase [21,22,28,42,45] and the heme globin family, hemoglobin, myoglobin, and neuroglobin [6,10,17,38,39], can all form superoxide as well. The fact that these systems can generate NO, superoxide, or both provides for a diversified signaling profile from NO, superoxide, peroxynitrite, nitrogen dioxide, and dinitrogen trioxide [1,13,41]. Nitrite itself is uniquely positioned as a relatively stable reactive nitrogen species (half-life in humans of approximately 40 min) but is readily reduced to NO, oxidized to nitrogen dioxide and dinitrogen trioxide, and protonated to form nitrous acid–nitrosonium cation [41]. Thus nitrite may contribute to redox signaling via NO activation of soluble guanylyl cyclase, as well as targeting protein cysteine and tyrosine residues to regulate posttranslational signaling. Although it has been known for almost 3 decades that oral nitrate can be reduced in the mouth to form nitrite, which is then converted in the stomach to NO to regulate mucosal blood flow, mucus production, and mucosal host defense via NO-mediated bacterial killing [2,3,27], a role for nitrite in the systemic formation of NO and systemic vasomotor function was only more recently appreciated [6,12,29]. It is now increasingly clear that nitrite, derived from dietary nitrate and eNOS, regulates blood pressure, blood flow, hypoxic vasodilation, and the cellular resilience to ischemia and reperfusion cytotoxicity in the heart and liver [6,7,12,18,42,43]. More recently, a role for this pathway in the control of mitochondrial proton leak and respiratory efficiency has been described [19,20]. The current study by Velmurugan and colleagues [40] explores a new chapter in the role of the nitrate–nitrite–NO pathway in cardiovascular function, by identifying an effect of dietary nitrate supplementation on platelet activation; importantly this effect requires red cells for bioconversion of nitrite to NO (Fig. 1). More specifically, the study summarizes the results of two randomized placebo-controlled crossover studies, each in 24 healthy subjects, assessing the acute effects of dietary nitrate or potassium nitrate capsules on platelet reactivity. Oral nitrate in beet root juice or as a pure chemical raised circulating nitrate and nitrite levels, inhibited ex vivo platelet aggregation responses to ADP and collagen, and reduced platelet P-selectin expression and elevated platelet cGMP levels. Interestingly, these effects were observed in vivo (inhibition of platelets after oral nitrate supplementation) and in vitro with exposure of nitrite to whole blood, but not to platelet-rich plasma, suggesting a role for the red cells in the conversion of nitrite to signaling NO. These findings thus further promote a physiological role for oral nitrate in the regulation of cardiovascular function, vasomotor control, energetics, and hemostasis, via formation of circulating nitrite.

In VIVO hemodynamic performance of wild-type vs. Mutant zebrafish embryos using high-speed confocal micro-PIV

In developing cardiovascular systems, definite performance comparison between disease and healthy hemodynamics requires quantitative tools to support advanced microscopy. Mutations in the activin receptor-like kinase 1 (ALK1) gene are responsible for the autosomal dominant vascular disease, hereditary hemorrhagic telangiectasia type 2 (HHT2), characterized by high flow arteriovenous malformations (AVMs) [1]. Recent studies show that the zebrafish mutant violet beauregrade (vbg), which harbors a mutation in alk1, develops an abnormal circulation with dilated cranial vessels and AVMs [2]. Quantitative understanding of mechanical influences on the alk1 mutant phenotype will aid treatment of HHT2 patients. Inspired by earlier studies that demonstrate the capability of using confocal micro-PIV technique to quantify biofluid dynamics in vivo [3], primarily in major vessels (dorsal aorta, vitelline veins), the present study focused on secondary branching great vessels of zebrafish embryos where microcirculation flow regimes are different. Furthermore, confocal microscopy, essentially being an imaging modality, requires rigorous validation efforts with respect to the gold standard measurement protocols (such as PIV) and synthetic scan data. Another objective of this work was to document the intra-species differences of wall shear stress (WSS) and flow physics during embryonic development in aortic arch systems of zebrafish [4].Copyright