Editorial Ceramide Signaling in the Coronary Microcirculation A Double-Edged Sword? Brian R. Weil, John M. Canty Jr
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nder basal conditions, ≈90% of the resistance to blood flow in the heart occurs in the small arteries and arterioles that make up the coronary microcirculation.1 Microvascular resistance is primarily determined by vessel density and lumen diameter. The latter is highly dynamic in the healthy heart and facilitates large changes in flow to meet increases in myocardial oxygen consumption during stress. Variations in the vascular tone of coronary microvessels results from vasodilator and vasoconstrictor signals that arise in response to physical forces, neurohormonal, and endothelium-derived substances, as well as metabolic mediators released from cardiac myocytes. Of these, vasodilation from increased shear stress or flow-induced dilation provides an important means of communication among various segments of the vasculature to regulate changes in local perfusion more accurately.2 An increasing number of studies have demonstrated that coronary microvascular dysfunction can contribute to precipitating myocardial ischemia and ventricular dysfunction in the absence of coronary artery disease (CAD).3,4 Of equal importance, impaired flow-induced responses predict cardiovascular events in patients with established cardiovascular disease, as well as in asymptomatic subjects.5
Article, see p 525 Although flow-induced dilation requires an intact endothelium, the biochemical mediators of dilation vary by organ, species, vessel size, age, and disease status. For example, in juvenile porcine coronary resistance vessels, nitric oxide (NO) mediates flow-induced dilation,6 yet flow-induced dilation is mediated by a hyperpolarizing factor in epicardial conduit arteries.7 In humans, prostaglandins mediate flow-induced dilation of coronary arterioles from children,2 whereas NO is the primary mediator of flow-induced dilation in normal adults. The presence of CAD or cardiovascular risk factors increases oxidative stress, reduces NO bioavailability, and diminishes the role of NO.8 Despite this, flow-induced dilation in humans can be maintained by mechanisms that compensate for the loss of NO. Miura et al9 demonstrated that hydrogen peroxide The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. From the Departments of Medicine, Biomedical Engineering, and Physiology and Biophysics, The Veterans Affairs Western New York Healthcare System and the Clinical and Translational Research Center at the University at Buffalo. Correspondence to John M. Canty Jr, MD, Division of Cardiovascular Medicine, Clinical Translational Research Center, University at Buffalo, Suite 7030, 875 Ellicott St, Buffalo, NY 14203. E-mail [email protected] (Circ Res. 2014;115:475-477.) © 2014 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.114.304589
(H2O2), an endothelium-derived hyperpolarizing factor, is released in response to shear stress and preserves flow-induced arteriolar vasodilation in coronary arterioles from patients with CAD. Subsequent studies demonstrated that H2O2 is produced in the mitochondria10 and elicits vasodilation via the activation of BKCa channels on vascular smooth muscle.11 Collectively, these studies support a paradigm where aging and disease herald a switch in the mechanism of coronary arteriolar flow-induced vasodilation from endothelium-derived NO to endothelium-derived H2O2.2 This transition may be beneficial in maintaining flow-induced dilation and in regulating local myocardial blood flow, but it likely comes at a cost. NO has anti-inflammatory, antiproliferative, and antithrombotic effects that protect against the development of vascular dysfunction and atherosclerotic disease.12 In contrast, H2O2 is a reactive oxygen intermediate involved in pathophysiological processes, such as inflammation, ischemia/reperfusion injury, and atherosclerosis.2,13 Because of these divergent cellular actions, manipulating the transition in mediators of flow-induced dilation between NO and H2O2 could afford a potential therapeutic target for the treatment of cardiovascular disease. In the present issue of Circulation Research, Freed et al14 shed new light on the mechanisms underlying plasticity of the biochemical mediators of flow-induced vasodilation in the human coronary microcirculation. In a series of well-conceived experiments using adipose and atrial resistance arterioles isolated from healthy and diseased human subjects, they demonstrate that ceramide, a sphingolipid, plays a key role in affecting the transition of flow-induced dilation from NO to H2O2 in CAD. Consistent with studies supporting the role of NO in normal animals and humans, the l-arginine analogue L-NAME abolished flow-induced vasodilation of adipose arterioles from healthy adults. Interestingly, overnight in vitro incubation of arterioles with exogenous ceramide did not alter the magnitude of flow-induced dilation but switched the mediator from NO to H2O2. In contrast to healthy adults, flow-induced dilation of adipose arterioles obtained from patients with chronic CAD was mediated by H2O2. Overnight incubation of these vessels with an inhibitor of the ceramide-producing enzyme, neutral sphingomyelinase, re-established NO as the mechanism of flow-induced vasodilation, a finding also observed in atrial arterioles from patients with CAD. The data indicate that ceramide plays a central role in rapidly switching the mediator of flow-induced dilation from NO to H2O2 that can be manipulated in a bidirectional fashion. These findings provide novel mechanistic insight into how the transition between NO and H2O2 occurs in CAD. The fact that they were made in human arterioles is significant in light of the species-related variability in flow-induced vasodilation
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476 Circulation Research August 15, 2014 and underscores their translational relevance in advancing our understanding of human pathophysiology. The brief (16–20 hours) time-frame required to switch from NO to H2O2 in ceramide-treated vessels (and back to NO with sphingomyelinase inhibition) is particularly interesting. As the authors point out, understanding the role of ceramide signaling in the rapid switching of vasodilator mechanisms warrants further investigation because this may identify new therapeutic approaches to treat microvascular disease. The results add to the growing body of evidence implicating ceramide accumulation as a critical regulator of cardiovascular pathophysiology.15,16 Ceramide and other sphingolipids were previously thought to serve a structural role as integral components of biological membranes simply. More recently, it has become clear that sphingolipids are also involved in signal transduction and regulating various cellular processes. Although intracellular ceramide can be formed de novo in the endoplasmic reticulum, the primary source is hydrolysis of sphingomyelin by sphingomyelinases.17 In the cardiovascular system, in vitro studies have shown that ceramide accumulation occurs in several pathophysiological conditions. For example, tumor necrosis factor-α and oxidized low-density lipoprotein increase ceramide in endothelium and vascular smooth muscle, whereas ischemia/reperfusion injury and doxorubicin-induced cardiotoxicity increase ceramide in cardiomyocytes.18 Ceramide has also received attention for its role in apoptotic signaling, as well as modulation of cell proliferation, migration, and adhesion, with divergent actions depending on cell type.17,19,20 It is important to note that the switch to H2O2 as a mechanism of flow-induced vasodilation in isolated human arterioles9,11,14 is not seen in animal models of atherosclerosis. Kuo et al21 observed impaired endothelium-dependent vasodilation to several pharmacological agonists, as well as shear stress, in arterioles isolated from pigs with diet-induced atherosclerosis. The impairment was NO dependent and was not associated with compensatory upregulation of alternative vasodilator pathways because flow-induced dilation was completely abolished in these vessels. Although endothelium-derived hyperpolarizing factor compensation to maintain acetylcholine-induced vasodilation was observed in isolated carotid artery segments of hypercholesterolemic rabbits,22 evidence of a disease-mediated or risk factor–mediated compensatory switch in microcirculatory vessels is lacking. It is unclear whether this is because the plasticity of vasodilatory mechanisms in disease states is unique to humans or because young animals, with potentially different mechanisms of vasomotor regulation than their older counterparts, are typically studied. Regardless, this conundrum may make it difficult to pursue further investigation of the role of ceramide in regulating flow-induced vasodilation in experimental models of atherosclerosis, emphasizing the importance of conducting future studies of the human coronary circulation. From a translational perspective, the observation that neutral sphingomyelinase inhibition restores NO as the mediator of flow-induced vasodilation in arterioles from patients with CAD is particularly exciting because it suggests that manipulation of ceramide biosynthesis could provide a new approach to restore endothelial function in patients with vascular disease. Although this may be difficult because of the plethora of
signaling actions attributed to ceramide and related sphingolipids, pharmacological approaches to manipulate tissue ceramide levels have been investigated.19 One approach may involve restoration of tissue glutathione concentrations. Reduced glutathione levels increase neutral sphingomyelinase activity and ceramide accumulation.16,23 Administration of the glutathione precursors N-acetylcysteine and α-lipoic acid reduce sphingomyelinase activity and improve endothelium-dependent vasodilation of isolated aortic rings from aged rats.18,24 Additional studies will be necessary to test whether restoration of glutathione concentrations in human coronary arterioles can restore NO as the mediator of flow-induced vasodilation. How do these in vitro observations relate to integrative coronary circulatory control? The shift from NO to H2O2 and preservation of flow-induced vasodilation in isolated human coronary arterioles in vitro contrasts with impaired coronary flow responses in patients with CAD risk factors and no epicardial coronary disease. In other words, the compensatory upregulation of H2O2-mediated dilation seems to be insufficient to maintain flow reserve in patients in vivo. It is possible that the compensatory response only occurs in a specific segment of the coronary microcirculation, whereas impaired flow-mediated responses in smaller arterioles or proximal coronary vessels persist. An alternative possibility is that structural alterations of coronary resistance arteries and arterioles impair minimal coronary vascular resistance in situ. Recent studies have shown that the coronary microcirculation can undergo a variety of structural changes in different pathophysiologic states.25 Structural alterations (Figure) may increase minimum
A
B
Figure. Schematic representation of potential ceramidemediated structural and functional effects on the coronary microcirculation. In healthy subjects (A), an increase in flow through a coronary arteriole elicits flow-induced dilation mediated by endothelium-derived nitric oxide. Ceramide accumulation occurs with the development of coronary artery disease (B), promoting a switch in the mediator of flow-induced dilation from nitric oxide to hydrogen peroxide. In addition, ceramide signaling elicits proliferation of vascular smooth muscle cells, contributing to arteriolar structural remodeling characterized by increased wall thickness and reduced lumen diameter. Although the compensatory upregulation of hydrogen peroxide–mediated vasodilation preserves the relative magnitude of flow-induced dilation, structural changes limit the absolute dilatory capacity of the vessel, resulting in an increased minimum coronary vascular resistance that limits coronary flow reserve.
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Weil and Canty Ceramide Signaling in Coronary Arterioles 477 vascular resistance of the microcirculation because of a reduction in luminal diameter (ie, inward remodeling). Importantly, even a small reduction in lumen diameter (which may be difficult to quantify) would significantly increase microcirculatory resistance because this varies with the fourth power of vessel diameter. Structural remodeling of resistance arteries is supported by Freed et al.14 Representative images of arterioles from patients with CAD show hypertrophic inward remodeling characterized by an increased wall thickness and a concomitant reduction in luminal diameter. Interestingly, ceramide accumulation occurred in the thickened medial layer of these vessels, indicating potentially important effects on vascular smooth muscle proliferation. This possibility is also supported by data from Auge et al,26 implicating ceramide signaling in smooth muscle cell proliferation after exposure to oxidized low-density lipoprotein. Finally, Ohanian et al27 recently observed age-related hypertrophic remodeling in sheep resistance arteries that was accompanied by an increase in sphingomyelinase activity and accumulation of long-chain ceramides. On the basis of these considerations, ceramide signaling in the microcirculation may be a 2-edged sword, with maintenance of flow-induced dilation via upregulation of H2O2 counteracted by adverse structural remodeling of coronary microvessels. The demonstration that short-term ceramide exposure rapidly switches the mediator of flow-induced vasodilation from NO to H2O2 by Freed et al14 advances our understanding of the human microcirculation in health and disease. Although ceramide-induced upregulation of H2O2 preserves flow-induced vasodilation, it may produce chronic structural changes in microcirculatory vessels. The finding that short-term inhibition of the ceramide-producing enzyme, neutral sphingomyelinase, reverts the mediator of flow-induced dilation back to NO in arterioles from patients with CAD is encouraging and may set the stage for new therapeutic opportunities to restore endothelial vasomotor function in patients.
Sources of Funding This study was supported by the National Heart Lung and Blood Institute (HL-055324, HL-061610, and F32HL-114335) and the Albert and Elizabeth Rekate Fund in Cardiovascular Medicine.
Disclosures None.
References 1. Canty Jr JM. Coronary blood flow and myocardial ischemia. In: Bonow RO, Mann DL, Zipes DP, Libby P, eds. Braunwald’s Heart Disease. 9th ed. Philadelphia, PA: Elsevier; 2012:1049–1075. 2. Beyer AM, Gutterman DD. Regulation of the human coronary microcirculation. J Mol Cell Cardiol. 2012;52:814–821. 3. Camici PG, Crea F. Coronary microvascular dysfunction. N Engl J Med. 2007;356:830–840. 4. Marzilli M, Merz CN, Boden WE, Bonow RO, Capozza PG, Chilian WM, DeMaria AN, Guarini G, Huqi A, Morrone D, Patel MR, Weintraub WS. Obstructive coronary atherosclerosis and ischemic heart disease: an elusive link! J Am Coll Cardiol. 2012;60:951–956. 5. Thijssen DH, Black MA, Pyke KE, Padilla J, Atkinson G, Harris RA, Parker B, Widlansky ME, Tschakovsky ME, Green DJ. Assessment of
flow-mediated dilation in humans: a methodological and physiological guideline. Am J Physiol Heart Circ Physiol. 2011;300:H2–12. 6. Kuo L, Davis MJ, Chilian WM. Endothelium-dependent, flowinduced dilation of isolated coronary arterioles. Am J Physiol. 1990;259:H1063–H1070. 7. Dube S, Canty JM Jr. Shear stress-induced vasodilation in porcine coronary conduit arteries is independent of nitric oxide release. Am J Physiol Heart Circ Physiol. 2001;280:H2581–H2590. 8. Kuo L, Davis MJ, Cannon MS, Chilian WM. Pathophysiological consequences of atherosclerosis extend into the coronary microcirculation. Restoration of endothelium-dependent responses by L-arginine. Circ Res. 1992;70:465–476. 9. Miura H, Bosnjak JJ, Ning G, Saito T, Miura M, Gutterman DD. Role for hydrogen peroxide in flow-induced dilation of human coronary arterioles. Circ Res. 2003;92:e31–e40. 10. Liu Y, Zhao H, Li H, Kalyanaraman B, Nicolosi AC, Gutterman DD. Mitochondrial sources of H2O2 generation play a key role in flow-mediated dilation in human coronary resistance arteries. Circ Res. 2003;93:573–580. 11. Liu Y, Bubolz AH, Mendoza S, Zhang DX, Gutterman DD. H2O2 is the transferrable factor mediating flow-induced dilation in human coronary arterioles. Circ Res. 2011;108:566–573. 12. Félétou M, Köhler R, Vanhoutte PM. Nitric oxide: orchestrator of endothelium-dependent responses. Ann Med. 2012;44:694–716. 13. Nathan CF. Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes. J Clin Invest. 1987;80:1550–1560. 14. Freed JK, Beyer AM, LoGiudice JA, Hockenberry JC, Gutterman DD. Ceramide changes the mediator of flow-induced vasodilation from nitric oxide to hydrogen peroxide in the human microcirculation. Circ Res. 2014;115:525–532. 15. Baranowski M, Górski J. Heart sphingolipids in health and disease. Adv Exp Med Biol. 2011;721:41–56. 16. Pavoine C, Pecker F. Sphingomyelinases: their regulation and roles in cardiovascular pathophysiology. Cardiovasc Res. 2009;82:175–183. 17. Levade T, Augé N, Veldman RJ, Cuvillier O, Nègre-Salvayre A, Salvayre R. Sphingolipid mediators in cardiovascular cell biology and pathology. Circ Res. 2001;89:957–968. 18. Adamy C, Mulder P, Khouzami L, et al. Neutral sphingomyelinase inhibition participates to the benefits of N-acetylcysteine treatment in post-myocardial infarction failing heart rats. J Mol Cell Cardiol. 2007;43:344–53. 19. Kolesnick R. The therapeutic potential of modulating the ceramide/ sphingomyelin pathway. J Clin Invest. 2002;110:3–8. 20. Bismuth J, Lin P, Yao Q, Chen C. Ceramide: a common pathway for atherosclerosis? Atherosclerosis. 2008;196:497–504. 21. Kuo L, Chilian WM, Davis MJ, Laughlin MH. Endotoxin impairs flow-induced vasodilation of porcine coronary arterioles. Am J Physiol. 1992;262:H1838–H1845. 22. Najibi S, Cowan CL, Palacino JJ, Cohen RA. Enhanced role of potassium channels in relaxations to acetylcholine in hypercholesterolemic rabbit carotid artery. Am J Physiol. 1994;266:H2061–H2067. 23. Liu B, Andrieu-Abadie N, Levade T, Zhang P, Obeid LM, Hannun YA. Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death. J Biol Chem. 1998;273:11313–11320. 24. Smith AR, Visioli F, Frei B, Hagen TM. Lipoic acid significantly restores, in rats, the age-related decline in vasomotion. Br J Pharmacol. 2008;153:1615–1622. 25. Martinez-Lemus LA, Hill MA, Meininger GA. The plastic nature of the vascular wall: a continuum of remodeling events contributing to control of arteriolar diameter and structure. Physiology (Bethesda). 2009;24:45–57. 26. Auge N, Garcia V, Maupas-Schwalm F, Levade T, Salvayre R, NegreSalvayre A. Oxidized LDL-induced smooth muscle cell proliferation involves the EGF receptor/PI-3 kinase/Akt and the sphingolipid signaling pathways. Arterioscler Thromb Vasc Biol. 2002;22:1990–1995. 27. Ohanian J, Liao A, Forman SP, Ohanian V. Age-related remodeling of small arteries is accompanied by increased sphingomyelinase activity and accumulation of long-chain ceramides. Physiol Rep. 2014;2:e12015. Key Words: Editorials
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Ceramide Changes the Mediator of Flow-Induced Vasodilation from Nitric Oxide to Hydrogen Peroxide in the Human Microcirculation Julie K. Freed1, Andreas M. Beyer2, John A LoGiudice3, Joseph C. Hockenberry2, David D. Gutterman2,4 1
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Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; Department of Medicine – Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; 3Department of Plastic Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and; 4VA Medical Center, Milwaukee, WI. Running title: Ceramide Changes Mediator of FID
Subject codes: [91] Oxidant stress [95] Endothelium/vascular type/nitric oxide [96] Mechanisms of atherosclerosis/growth factors [97] Other vascular biology Address for Correspondence: Dr. David D. Gutterman Department of Medicine Medical College of Wisconsin Milwaukee, WI 53226 Tel: 414-955-8495 [email protected]
In May 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.87 days.
DOI: 10.1161/CIRCRESAHA.115.303881
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ABSTRACT Rationale: Mitochondrial-derived hydrogen peroxide (H2O2) regulates flow-induced dilation (FID) in microvessels from patients with coronary artery disease (CAD). The relationship between ceramide, an independent risk factor for CAD and a known inducer of mitochondrial reactive oxygen species (ROS), and FID is unknown. Objective: We examined the hypothesis that exogenous ceramide induces a switch in the mediator of FID from nitric oxide (NO) to H2O2. Methods and Results: Internal diameter changes of resistance arterioles from human adipose and atrial tissue were measured by videomicroscopy. Mitochondrial H2O2 production was assayed in arterioles using MitoPeroxy Yellow 1 (Mito PY1). PEG-catalase, rotenone, and Mito-TEMPO, impaired FID in healthy adipose arterioles pre-treated with ceramide whereas Nω-Nitro-L-arginine methyl ester (L-NAME) had no effect. Mitochondrial H2O2 production was induced in response to flow in healthy adipose vessels pretreated with ceramide and this was abolished in the presence of PEG-catalase. Immunohistochemistry demonstrated ceramide accumulation in arterioles from both healthy and CAD patients. L-NAME reduced vasodilation to flow in adipose as well as atrial vessels from patients with CAD incubated with GW4869, a neutral sphingomyelinase (NSmase) inhibitor, whereas PEG-catalase had no effect. Conclusion: Our data indicate that ceramide has an integral role in the transition of the mediator of FID from NO to mitochondrial-derived H2O2 and that inhibition of ceramide production can revert the mechanism of dilation back to NO. Ceramide may be an important target for preventing and treating vascular dysfunction associated with atherosclerosis.
Keywords: Ceramide, shear stress, mitochondria, reactive oxygen species, nitric oxide, endothelial dysfunction, sphingomyelinase
Nonstandard Abbreviations and Acronyms: EDHF endothelial-derived hyperpolarizing factor ETC electron transport chain FID flow-induced dilation H2O2 hydrogen peroxide L-NAME Nω-nitro-ʟ-arginine mitoPY1 mito peroxy yellow 1 NSmase neutral sphingomyelinase NO nitric oxide nonCAD non-coronary artery disease ROS reactive oxygen species
DOI: 10.1161/CIRCRESAHA.115.303881
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INTRODUCTION Vasodilation to shear stress (flow-induced dilation; FID), is an endothelium-dependent process important for maintaining vascular homeostasis. Impaired FID is a powerful predictor of future cardiovascular events.1-3 Increased shear during flow stimulates endothelial release of vasoactive substances including nitric oxide (NO), prostacyclin, and endothelial derived hyperpolarizing factors (EDHFs).4-6 We have previously shown that risk factors for, or the presence of coronary artery disease (CAD), evokes a transition in the endothelial mediator of FID from NO to mitochondrial-derived hydrogen peroxide (H2O2).7-9 Although both factors elicit smooth muscle relaxation, the non-vasomotor effects of each are generally opposite with NO stimulating anti-inflammatory, anti-thrombotic, and anti-proliferative pathways, compared to the pro-inflammatory, pro-thrombotic, and pro-atherogenic nature of H2O2. 10 The mechanism by which this transition occurs with the onset of disease represents a major gap in our understanding of microvascular control. Accumulating evidence reveals that a class of bioactive metabolites, known as sphingolipids, play an essential role in cardiovascular pathophysiology. Their function is diverse and ranges from modulating cell proliferation and angiogenesis to atherosclerosis and cell death. Ceramide, a prototypical sphingolipid product of sphingomyelinase is produced in endothelial cells, found in human plasma, and is a risk factor for atherosclerosis.11-13 Furthermore ceramide is known to stimulate mitochondrial ROS production.12 Therefore we tested whether ceramide might play a role in the switch from NO to H2O2 in the mechanism of FID in the human microcirculation. We addressed this question by examining (1) whether exogenous ceramide can convert the mediator of FID from NO to H2O2 in healthy non-CAD arterioles, (2) whether ceramide induces mitochondrial production of H2O2, and (3) whether inhibition of ceramide production restores NO as the mediator of dilation in arterioles from patients with chronic CAD.
METHODS Tissue acquisition. Fresh human adipose tissue (visceral, subcutaneous, peritoneal) and right atrial appendages from patients undergoing surgical procedures were obtained as discarded surgical specimens. Tissues were placed in icecold HEPES buffer or cardioplegia solution for adipose and atrial tissue, respectively. De-identified patient demographic data was collected using the Generic Clinical Research Database (GCRD) at the Medical College of Wisconsin. All protocols were approved by the local Institutional Review Board at the Medical College of Wisconsin and the Zablocki VA Medical Center. Measurement of FID by videomicroscopy. Briefly, isolated arterioles were cannulated on glass micropipettes and secured in an organ chamber with circulating Kreb’s buffer. Following equilibration, endothelin-1 was added to assess viability and to preconstrict the vessels by 30-50% of their passive diameter. Internal diameters were measured at steadystate before and during intraluminal flow at pressure gradients of 5 to 100cmH2O. The following inhibitors were added to the bath 30 minutes prior to initiation of flow; nitric oxide synthase (NOS) inhibitor Nωnitro-ʟ-arginine (L-NAME; 10-4 mol/L), H2O2 scavenger polyethylene glycol-catalase (PEG-Catalase; 500U/mL), mitochondrial antioxidant Mito-TEMPO (10-5 mol/L), or electron transport chain (ETC) complex I inhibitor rotenone (10-6 mol/L ). C-2 Ceramide (10-5 mol/L ) or the neutral sphingomyelinase inhibitor GW4869 (4x10-6 mol/L) was incubated with arterioles from healthy patients and patients with CAD, respectively, for 16-20hrs prior to the experiment. Papaverine, (10-4 mol/L ) an endotheliumindependent vasodilator, was added at the end of each experiment to determine the vessel’s maximal diameter, then the direction of maximal flow was reversed in the presence of maximal dilation, to confirm matched impedance between pipettes.
DOI: 10.1161/CIRCRESAHA.115.303881
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Fluorescence detection of mitochondrial H2O2. Mito Peroxy Yellow 1 (Mito PY1) was used to evaluate mitochondrial-derived H2O2 in vessels during flow. Following cannulation in a warmed chamber (37oC) containing HEPES buffer (pH 7.4), arterioles were perfused intraluminally with Mito PY1 (10-5 mol/L for 1 hour). Vessels pre-treated with vehicle or ceramide (10-5 mol/L, 16-20hrs) were then exposed to either no flow (remained pressurized at 80cmH2O, 0 gradient) or flow at a pressure gradient of 100cmH2O in the presence or absence of PEG-catalase (500U/mL). Changes in mitochondrial H2O2 fluorescence probed by MitoPY1 were examined under fluorescence microscopy equipped with a krypton/argon laser fluorescent microscope (model TE 200 Nikon Eclipse) using an excitation wavelength of 488nm and measured emission between 530-590nm. Baseline measurements of fluorescence were obtained in the absence of flow and every min during 5 min of intraluminal flow. Images were then analyzed for fluorescence intensity in arbitrary units using Metamorph (Universal Imaging Corp) subtracting background fluorescence. Relative average fluorescence intensity was normalized for surface area and presented as percent change from baseline (prior to initiation of flow). All vessels used for comparison on a given day were obtained from the same patient and studied using the same imaging parameters. Immunohistochemistry. Immunohistochemistry was performed to visualize ceramide expression in human arterioles from discarded adipose tissue as previously described.9 Briefly, isolated arterioles (~200µm in diameter) were fixed in zinc formalin buffer for 24-72hrs and processed for paraffin embedding. Samples were sectioned on a HMS355 microtome at 4µm. Immunolabeling was performed using a mouse anti-human monoclonal antibody against ceramide. Immunostaining was performed using a Leica Bond MAX Immunostainer. Slides were deparaffinized and subject to heat-induced epitope retrieval (HIER) for 10 minutes at pH 6.0. The primary antibody was optimal at 1:200 using the Bond Refine-HRP detection system. Slides were scanned with a NanoZoomer HT slide scanner (Hamamatsu, Japan). Staining was quantified within the vessel wall using Metamorph (Universal Imaging Corp) and reported as average area percent or the total stained area divided by the total wall area (µm2). Materials. C-2 Ceramide, (Cayman Chemical, Ann Arbor, Michigan), and GW4869 (Sigma-Aldrich, Saint Louis, MO) were prepared in DMSO. Mitochondria Peroxy Yellow 1 (MitoPY1) was obtained from Tocris Bioscience (Bristol, UK). Mito-TEMPO was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY). All other chemicals were purchased from Sigma-Aldrich and prepared in distilled water with the exception of endothelin-1 which was prepared in 1% bovine serum albumin (BSA). Vehicle control studies indicated that the final concentration of DMSO had no effect on basal tone or function of the arterioles. The ceramide primary antibody was purchased from Enzo Life Sciences (Farmingdale, NY). Statistical analyses. Data are expressed as means +/- SEM. Flow-induced dilation is expressed as a percentage of maximal relaxation from endothelin‐1 constriction, with 100% representing full relaxation to the maximal diameter obtained by the addition of papaverine. To compare flow–response relationships, a 2-way ANOVA was used with flow gradient and treatment as parameters. When a significant difference was observed between curves (p
U
nder basal conditions, ≈90% of the resistance to blood flow in the heart occurs in the small arteries and arterioles that make up the coronary microcirculation.1 Microvascular resistance is primarily determined by vessel density and lumen diameter. The latter is highly dynamic in the healthy heart and facilitates large changes in flow to meet increases in myocardial oxygen consumption during stress. Variations in the vascular tone of coronary microvessels results from vasodilator and vasoconstrictor signals that arise in response to physical forces, neurohormonal, and endothelium-derived substances, as well as metabolic mediators released from cardiac myocytes. Of these, vasodilation from increased shear stress or flow-induced dilation provides an important means of communication among various segments of the vasculature to regulate changes in local perfusion more accurately.2 An increasing number of studies have demonstrated that coronary microvascular dysfunction can contribute to precipitating myocardial ischemia and ventricular dysfunction in the absence of coronary artery disease (CAD).3,4 Of equal importance, impaired flow-induced responses predict cardiovascular events in patients with established cardiovascular disease, as well as in asymptomatic subjects.5
Article, see p 525 Although flow-induced dilation requires an intact endothelium, the biochemical mediators of dilation vary by organ, species, vessel size, age, and disease status. For example, in juvenile porcine coronary resistance vessels, nitric oxide (NO) mediates flow-induced dilation,6 yet flow-induced dilation is mediated by a hyperpolarizing factor in epicardial conduit arteries.7 In humans, prostaglandins mediate flow-induced dilation of coronary arterioles from children,2 whereas NO is the primary mediator of flow-induced dilation in normal adults. The presence of CAD or cardiovascular risk factors increases oxidative stress, reduces NO bioavailability, and diminishes the role of NO.8 Despite this, flow-induced dilation in humans can be maintained by mechanisms that compensate for the loss of NO. Miura et al9 demonstrated that hydrogen peroxide The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. From the Departments of Medicine, Biomedical Engineering, and Physiology and Biophysics, The Veterans Affairs Western New York Healthcare System and the Clinical and Translational Research Center at the University at Buffalo. Correspondence to John M. Canty Jr, MD, Division of Cardiovascular Medicine, Clinical Translational Research Center, University at Buffalo, Suite 7030, 875 Ellicott St, Buffalo, NY 14203. E-mail [email protected] (Circ Res. 2014;115:475-477.) © 2014 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.114.304589
(H2O2), an endothelium-derived hyperpolarizing factor, is released in response to shear stress and preserves flow-induced arteriolar vasodilation in coronary arterioles from patients with CAD. Subsequent studies demonstrated that H2O2 is produced in the mitochondria10 and elicits vasodilation via the activation of BKCa channels on vascular smooth muscle.11 Collectively, these studies support a paradigm where aging and disease herald a switch in the mechanism of coronary arteriolar flow-induced vasodilation from endothelium-derived NO to endothelium-derived H2O2.2 This transition may be beneficial in maintaining flow-induced dilation and in regulating local myocardial blood flow, but it likely comes at a cost. NO has anti-inflammatory, antiproliferative, and antithrombotic effects that protect against the development of vascular dysfunction and atherosclerotic disease.12 In contrast, H2O2 is a reactive oxygen intermediate involved in pathophysiological processes, such as inflammation, ischemia/reperfusion injury, and atherosclerosis.2,13 Because of these divergent cellular actions, manipulating the transition in mediators of flow-induced dilation between NO and H2O2 could afford a potential therapeutic target for the treatment of cardiovascular disease. In the present issue of Circulation Research, Freed et al14 shed new light on the mechanisms underlying plasticity of the biochemical mediators of flow-induced vasodilation in the human coronary microcirculation. In a series of well-conceived experiments using adipose and atrial resistance arterioles isolated from healthy and diseased human subjects, they demonstrate that ceramide, a sphingolipid, plays a key role in affecting the transition of flow-induced dilation from NO to H2O2 in CAD. Consistent with studies supporting the role of NO in normal animals and humans, the l-arginine analogue L-NAME abolished flow-induced vasodilation of adipose arterioles from healthy adults. Interestingly, overnight in vitro incubation of arterioles with exogenous ceramide did not alter the magnitude of flow-induced dilation but switched the mediator from NO to H2O2. In contrast to healthy adults, flow-induced dilation of adipose arterioles obtained from patients with chronic CAD was mediated by H2O2. Overnight incubation of these vessels with an inhibitor of the ceramide-producing enzyme, neutral sphingomyelinase, re-established NO as the mechanism of flow-induced vasodilation, a finding also observed in atrial arterioles from patients with CAD. The data indicate that ceramide plays a central role in rapidly switching the mediator of flow-induced dilation from NO to H2O2 that can be manipulated in a bidirectional fashion. These findings provide novel mechanistic insight into how the transition between NO and H2O2 occurs in CAD. The fact that they were made in human arterioles is significant in light of the species-related variability in flow-induced vasodilation
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476 Circulation Research August 15, 2014 and underscores their translational relevance in advancing our understanding of human pathophysiology. The brief (16–20 hours) time-frame required to switch from NO to H2O2 in ceramide-treated vessels (and back to NO with sphingomyelinase inhibition) is particularly interesting. As the authors point out, understanding the role of ceramide signaling in the rapid switching of vasodilator mechanisms warrants further investigation because this may identify new therapeutic approaches to treat microvascular disease. The results add to the growing body of evidence implicating ceramide accumulation as a critical regulator of cardiovascular pathophysiology.15,16 Ceramide and other sphingolipids were previously thought to serve a structural role as integral components of biological membranes simply. More recently, it has become clear that sphingolipids are also involved in signal transduction and regulating various cellular processes. Although intracellular ceramide can be formed de novo in the endoplasmic reticulum, the primary source is hydrolysis of sphingomyelin by sphingomyelinases.17 In the cardiovascular system, in vitro studies have shown that ceramide accumulation occurs in several pathophysiological conditions. For example, tumor necrosis factor-α and oxidized low-density lipoprotein increase ceramide in endothelium and vascular smooth muscle, whereas ischemia/reperfusion injury and doxorubicin-induced cardiotoxicity increase ceramide in cardiomyocytes.18 Ceramide has also received attention for its role in apoptotic signaling, as well as modulation of cell proliferation, migration, and adhesion, with divergent actions depending on cell type.17,19,20 It is important to note that the switch to H2O2 as a mechanism of flow-induced vasodilation in isolated human arterioles9,11,14 is not seen in animal models of atherosclerosis. Kuo et al21 observed impaired endothelium-dependent vasodilation to several pharmacological agonists, as well as shear stress, in arterioles isolated from pigs with diet-induced atherosclerosis. The impairment was NO dependent and was not associated with compensatory upregulation of alternative vasodilator pathways because flow-induced dilation was completely abolished in these vessels. Although endothelium-derived hyperpolarizing factor compensation to maintain acetylcholine-induced vasodilation was observed in isolated carotid artery segments of hypercholesterolemic rabbits,22 evidence of a disease-mediated or risk factor–mediated compensatory switch in microcirculatory vessels is lacking. It is unclear whether this is because the plasticity of vasodilatory mechanisms in disease states is unique to humans or because young animals, with potentially different mechanisms of vasomotor regulation than their older counterparts, are typically studied. Regardless, this conundrum may make it difficult to pursue further investigation of the role of ceramide in regulating flow-induced vasodilation in experimental models of atherosclerosis, emphasizing the importance of conducting future studies of the human coronary circulation. From a translational perspective, the observation that neutral sphingomyelinase inhibition restores NO as the mediator of flow-induced vasodilation in arterioles from patients with CAD is particularly exciting because it suggests that manipulation of ceramide biosynthesis could provide a new approach to restore endothelial function in patients with vascular disease. Although this may be difficult because of the plethora of
signaling actions attributed to ceramide and related sphingolipids, pharmacological approaches to manipulate tissue ceramide levels have been investigated.19 One approach may involve restoration of tissue glutathione concentrations. Reduced glutathione levels increase neutral sphingomyelinase activity and ceramide accumulation.16,23 Administration of the glutathione precursors N-acetylcysteine and α-lipoic acid reduce sphingomyelinase activity and improve endothelium-dependent vasodilation of isolated aortic rings from aged rats.18,24 Additional studies will be necessary to test whether restoration of glutathione concentrations in human coronary arterioles can restore NO as the mediator of flow-induced vasodilation. How do these in vitro observations relate to integrative coronary circulatory control? The shift from NO to H2O2 and preservation of flow-induced vasodilation in isolated human coronary arterioles in vitro contrasts with impaired coronary flow responses in patients with CAD risk factors and no epicardial coronary disease. In other words, the compensatory upregulation of H2O2-mediated dilation seems to be insufficient to maintain flow reserve in patients in vivo. It is possible that the compensatory response only occurs in a specific segment of the coronary microcirculation, whereas impaired flow-mediated responses in smaller arterioles or proximal coronary vessels persist. An alternative possibility is that structural alterations of coronary resistance arteries and arterioles impair minimal coronary vascular resistance in situ. Recent studies have shown that the coronary microcirculation can undergo a variety of structural changes in different pathophysiologic states.25 Structural alterations (Figure) may increase minimum
A
B
Figure. Schematic representation of potential ceramidemediated structural and functional effects on the coronary microcirculation. In healthy subjects (A), an increase in flow through a coronary arteriole elicits flow-induced dilation mediated by endothelium-derived nitric oxide. Ceramide accumulation occurs with the development of coronary artery disease (B), promoting a switch in the mediator of flow-induced dilation from nitric oxide to hydrogen peroxide. In addition, ceramide signaling elicits proliferation of vascular smooth muscle cells, contributing to arteriolar structural remodeling characterized by increased wall thickness and reduced lumen diameter. Although the compensatory upregulation of hydrogen peroxide–mediated vasodilation preserves the relative magnitude of flow-induced dilation, structural changes limit the absolute dilatory capacity of the vessel, resulting in an increased minimum coronary vascular resistance that limits coronary flow reserve.
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Weil and Canty Ceramide Signaling in Coronary Arterioles 477 vascular resistance of the microcirculation because of a reduction in luminal diameter (ie, inward remodeling). Importantly, even a small reduction in lumen diameter (which may be difficult to quantify) would significantly increase microcirculatory resistance because this varies with the fourth power of vessel diameter. Structural remodeling of resistance arteries is supported by Freed et al.14 Representative images of arterioles from patients with CAD show hypertrophic inward remodeling characterized by an increased wall thickness and a concomitant reduction in luminal diameter. Interestingly, ceramide accumulation occurred in the thickened medial layer of these vessels, indicating potentially important effects on vascular smooth muscle proliferation. This possibility is also supported by data from Auge et al,26 implicating ceramide signaling in smooth muscle cell proliferation after exposure to oxidized low-density lipoprotein. Finally, Ohanian et al27 recently observed age-related hypertrophic remodeling in sheep resistance arteries that was accompanied by an increase in sphingomyelinase activity and accumulation of long-chain ceramides. On the basis of these considerations, ceramide signaling in the microcirculation may be a 2-edged sword, with maintenance of flow-induced dilation via upregulation of H2O2 counteracted by adverse structural remodeling of coronary microvessels. The demonstration that short-term ceramide exposure rapidly switches the mediator of flow-induced vasodilation from NO to H2O2 by Freed et al14 advances our understanding of the human microcirculation in health and disease. Although ceramide-induced upregulation of H2O2 preserves flow-induced vasodilation, it may produce chronic structural changes in microcirculatory vessels. The finding that short-term inhibition of the ceramide-producing enzyme, neutral sphingomyelinase, reverts the mediator of flow-induced dilation back to NO in arterioles from patients with CAD is encouraging and may set the stage for new therapeutic opportunities to restore endothelial vasomotor function in patients.
Sources of Funding This study was supported by the National Heart Lung and Blood Institute (HL-055324, HL-061610, and F32HL-114335) and the Albert and Elizabeth Rekate Fund in Cardiovascular Medicine.
Disclosures None.
References 1. Canty Jr JM. Coronary blood flow and myocardial ischemia. In: Bonow RO, Mann DL, Zipes DP, Libby P, eds. Braunwald’s Heart Disease. 9th ed. Philadelphia, PA: Elsevier; 2012:1049–1075. 2. Beyer AM, Gutterman DD. Regulation of the human coronary microcirculation. J Mol Cell Cardiol. 2012;52:814–821. 3. Camici PG, Crea F. Coronary microvascular dysfunction. N Engl J Med. 2007;356:830–840. 4. Marzilli M, Merz CN, Boden WE, Bonow RO, Capozza PG, Chilian WM, DeMaria AN, Guarini G, Huqi A, Morrone D, Patel MR, Weintraub WS. Obstructive coronary atherosclerosis and ischemic heart disease: an elusive link! J Am Coll Cardiol. 2012;60:951–956. 5. Thijssen DH, Black MA, Pyke KE, Padilla J, Atkinson G, Harris RA, Parker B, Widlansky ME, Tschakovsky ME, Green DJ. Assessment of
flow-mediated dilation in humans: a methodological and physiological guideline. Am J Physiol Heart Circ Physiol. 2011;300:H2–12. 6. Kuo L, Davis MJ, Chilian WM. Endothelium-dependent, flowinduced dilation of isolated coronary arterioles. Am J Physiol. 1990;259:H1063–H1070. 7. Dube S, Canty JM Jr. Shear stress-induced vasodilation in porcine coronary conduit arteries is independent of nitric oxide release. Am J Physiol Heart Circ Physiol. 2001;280:H2581–H2590. 8. Kuo L, Davis MJ, Cannon MS, Chilian WM. Pathophysiological consequences of atherosclerosis extend into the coronary microcirculation. Restoration of endothelium-dependent responses by L-arginine. Circ Res. 1992;70:465–476. 9. Miura H, Bosnjak JJ, Ning G, Saito T, Miura M, Gutterman DD. Role for hydrogen peroxide in flow-induced dilation of human coronary arterioles. Circ Res. 2003;92:e31–e40. 10. Liu Y, Zhao H, Li H, Kalyanaraman B, Nicolosi AC, Gutterman DD. Mitochondrial sources of H2O2 generation play a key role in flow-mediated dilation in human coronary resistance arteries. Circ Res. 2003;93:573–580. 11. Liu Y, Bubolz AH, Mendoza S, Zhang DX, Gutterman DD. H2O2 is the transferrable factor mediating flow-induced dilation in human coronary arterioles. Circ Res. 2011;108:566–573. 12. Félétou M, Köhler R, Vanhoutte PM. Nitric oxide: orchestrator of endothelium-dependent responses. Ann Med. 2012;44:694–716. 13. Nathan CF. Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes. J Clin Invest. 1987;80:1550–1560. 14. Freed JK, Beyer AM, LoGiudice JA, Hockenberry JC, Gutterman DD. Ceramide changes the mediator of flow-induced vasodilation from nitric oxide to hydrogen peroxide in the human microcirculation. Circ Res. 2014;115:525–532. 15. Baranowski M, Górski J. Heart sphingolipids in health and disease. Adv Exp Med Biol. 2011;721:41–56. 16. Pavoine C, Pecker F. Sphingomyelinases: their regulation and roles in cardiovascular pathophysiology. Cardiovasc Res. 2009;82:175–183. 17. Levade T, Augé N, Veldman RJ, Cuvillier O, Nègre-Salvayre A, Salvayre R. Sphingolipid mediators in cardiovascular cell biology and pathology. Circ Res. 2001;89:957–968. 18. Adamy C, Mulder P, Khouzami L, et al. Neutral sphingomyelinase inhibition participates to the benefits of N-acetylcysteine treatment in post-myocardial infarction failing heart rats. J Mol Cell Cardiol. 2007;43:344–53. 19. Kolesnick R. The therapeutic potential of modulating the ceramide/ sphingomyelin pathway. J Clin Invest. 2002;110:3–8. 20. Bismuth J, Lin P, Yao Q, Chen C. Ceramide: a common pathway for atherosclerosis? Atherosclerosis. 2008;196:497–504. 21. Kuo L, Chilian WM, Davis MJ, Laughlin MH. Endotoxin impairs flow-induced vasodilation of porcine coronary arterioles. Am J Physiol. 1992;262:H1838–H1845. 22. Najibi S, Cowan CL, Palacino JJ, Cohen RA. Enhanced role of potassium channels in relaxations to acetylcholine in hypercholesterolemic rabbit carotid artery. Am J Physiol. 1994;266:H2061–H2067. 23. Liu B, Andrieu-Abadie N, Levade T, Zhang P, Obeid LM, Hannun YA. Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death. J Biol Chem. 1998;273:11313–11320. 24. Smith AR, Visioli F, Frei B, Hagen TM. Lipoic acid significantly restores, in rats, the age-related decline in vasomotion. Br J Pharmacol. 2008;153:1615–1622. 25. Martinez-Lemus LA, Hill MA, Meininger GA. The plastic nature of the vascular wall: a continuum of remodeling events contributing to control of arteriolar diameter and structure. Physiology (Bethesda). 2009;24:45–57. 26. Auge N, Garcia V, Maupas-Schwalm F, Levade T, Salvayre R, NegreSalvayre A. Oxidized LDL-induced smooth muscle cell proliferation involves the EGF receptor/PI-3 kinase/Akt and the sphingolipid signaling pathways. Arterioscler Thromb Vasc Biol. 2002;22:1990–1995. 27. Ohanian J, Liao A, Forman SP, Ohanian V. Age-related remodeling of small arteries is accompanied by increased sphingomyelinase activity and accumulation of long-chain ceramides. Physiol Rep. 2014;2:e12015. Key Words: Editorials
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Ceramide Changes the Mediator of Flow-Induced Vasodilation from Nitric Oxide to Hydrogen Peroxide in the Human Microcirculation Julie K. Freed1, Andreas M. Beyer2, John A LoGiudice3, Joseph C. Hockenberry2, David D. Gutterman2,4 1
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Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; Department of Medicine – Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; 3Department of Plastic Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and; 4VA Medical Center, Milwaukee, WI. Running title: Ceramide Changes Mediator of FID
Subject codes: [91] Oxidant stress [95] Endothelium/vascular type/nitric oxide [96] Mechanisms of atherosclerosis/growth factors [97] Other vascular biology Address for Correspondence: Dr. David D. Gutterman Department of Medicine Medical College of Wisconsin Milwaukee, WI 53226 Tel: 414-955-8495 [email protected]
In May 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.87 days.
DOI: 10.1161/CIRCRESAHA.115.303881
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ABSTRACT Rationale: Mitochondrial-derived hydrogen peroxide (H2O2) regulates flow-induced dilation (FID) in microvessels from patients with coronary artery disease (CAD). The relationship between ceramide, an independent risk factor for CAD and a known inducer of mitochondrial reactive oxygen species (ROS), and FID is unknown. Objective: We examined the hypothesis that exogenous ceramide induces a switch in the mediator of FID from nitric oxide (NO) to H2O2. Methods and Results: Internal diameter changes of resistance arterioles from human adipose and atrial tissue were measured by videomicroscopy. Mitochondrial H2O2 production was assayed in arterioles using MitoPeroxy Yellow 1 (Mito PY1). PEG-catalase, rotenone, and Mito-TEMPO, impaired FID in healthy adipose arterioles pre-treated with ceramide whereas Nω-Nitro-L-arginine methyl ester (L-NAME) had no effect. Mitochondrial H2O2 production was induced in response to flow in healthy adipose vessels pretreated with ceramide and this was abolished in the presence of PEG-catalase. Immunohistochemistry demonstrated ceramide accumulation in arterioles from both healthy and CAD patients. L-NAME reduced vasodilation to flow in adipose as well as atrial vessels from patients with CAD incubated with GW4869, a neutral sphingomyelinase (NSmase) inhibitor, whereas PEG-catalase had no effect. Conclusion: Our data indicate that ceramide has an integral role in the transition of the mediator of FID from NO to mitochondrial-derived H2O2 and that inhibition of ceramide production can revert the mechanism of dilation back to NO. Ceramide may be an important target for preventing and treating vascular dysfunction associated with atherosclerosis.
Keywords: Ceramide, shear stress, mitochondria, reactive oxygen species, nitric oxide, endothelial dysfunction, sphingomyelinase
Nonstandard Abbreviations and Acronyms: EDHF endothelial-derived hyperpolarizing factor ETC electron transport chain FID flow-induced dilation H2O2 hydrogen peroxide L-NAME Nω-nitro-ʟ-arginine mitoPY1 mito peroxy yellow 1 NSmase neutral sphingomyelinase NO nitric oxide nonCAD non-coronary artery disease ROS reactive oxygen species
DOI: 10.1161/CIRCRESAHA.115.303881
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INTRODUCTION Vasodilation to shear stress (flow-induced dilation; FID), is an endothelium-dependent process important for maintaining vascular homeostasis. Impaired FID is a powerful predictor of future cardiovascular events.1-3 Increased shear during flow stimulates endothelial release of vasoactive substances including nitric oxide (NO), prostacyclin, and endothelial derived hyperpolarizing factors (EDHFs).4-6 We have previously shown that risk factors for, or the presence of coronary artery disease (CAD), evokes a transition in the endothelial mediator of FID from NO to mitochondrial-derived hydrogen peroxide (H2O2).7-9 Although both factors elicit smooth muscle relaxation, the non-vasomotor effects of each are generally opposite with NO stimulating anti-inflammatory, anti-thrombotic, and anti-proliferative pathways, compared to the pro-inflammatory, pro-thrombotic, and pro-atherogenic nature of H2O2. 10 The mechanism by which this transition occurs with the onset of disease represents a major gap in our understanding of microvascular control. Accumulating evidence reveals that a class of bioactive metabolites, known as sphingolipids, play an essential role in cardiovascular pathophysiology. Their function is diverse and ranges from modulating cell proliferation and angiogenesis to atherosclerosis and cell death. Ceramide, a prototypical sphingolipid product of sphingomyelinase is produced in endothelial cells, found in human plasma, and is a risk factor for atherosclerosis.11-13 Furthermore ceramide is known to stimulate mitochondrial ROS production.12 Therefore we tested whether ceramide might play a role in the switch from NO to H2O2 in the mechanism of FID in the human microcirculation. We addressed this question by examining (1) whether exogenous ceramide can convert the mediator of FID from NO to H2O2 in healthy non-CAD arterioles, (2) whether ceramide induces mitochondrial production of H2O2, and (3) whether inhibition of ceramide production restores NO as the mediator of dilation in arterioles from patients with chronic CAD.
METHODS Tissue acquisition. Fresh human adipose tissue (visceral, subcutaneous, peritoneal) and right atrial appendages from patients undergoing surgical procedures were obtained as discarded surgical specimens. Tissues were placed in icecold HEPES buffer or cardioplegia solution for adipose and atrial tissue, respectively. De-identified patient demographic data was collected using the Generic Clinical Research Database (GCRD) at the Medical College of Wisconsin. All protocols were approved by the local Institutional Review Board at the Medical College of Wisconsin and the Zablocki VA Medical Center. Measurement of FID by videomicroscopy. Briefly, isolated arterioles were cannulated on glass micropipettes and secured in an organ chamber with circulating Kreb’s buffer. Following equilibration, endothelin-1 was added to assess viability and to preconstrict the vessels by 30-50% of their passive diameter. Internal diameters were measured at steadystate before and during intraluminal flow at pressure gradients of 5 to 100cmH2O. The following inhibitors were added to the bath 30 minutes prior to initiation of flow; nitric oxide synthase (NOS) inhibitor Nωnitro-ʟ-arginine (L-NAME; 10-4 mol/L), H2O2 scavenger polyethylene glycol-catalase (PEG-Catalase; 500U/mL), mitochondrial antioxidant Mito-TEMPO (10-5 mol/L), or electron transport chain (ETC) complex I inhibitor rotenone (10-6 mol/L ). C-2 Ceramide (10-5 mol/L ) or the neutral sphingomyelinase inhibitor GW4869 (4x10-6 mol/L) was incubated with arterioles from healthy patients and patients with CAD, respectively, for 16-20hrs prior to the experiment. Papaverine, (10-4 mol/L ) an endotheliumindependent vasodilator, was added at the end of each experiment to determine the vessel’s maximal diameter, then the direction of maximal flow was reversed in the presence of maximal dilation, to confirm matched impedance between pipettes.
DOI: 10.1161/CIRCRESAHA.115.303881
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Fluorescence detection of mitochondrial H2O2. Mito Peroxy Yellow 1 (Mito PY1) was used to evaluate mitochondrial-derived H2O2 in vessels during flow. Following cannulation in a warmed chamber (37oC) containing HEPES buffer (pH 7.4), arterioles were perfused intraluminally with Mito PY1 (10-5 mol/L for 1 hour). Vessels pre-treated with vehicle or ceramide (10-5 mol/L, 16-20hrs) were then exposed to either no flow (remained pressurized at 80cmH2O, 0 gradient) or flow at a pressure gradient of 100cmH2O in the presence or absence of PEG-catalase (500U/mL). Changes in mitochondrial H2O2 fluorescence probed by MitoPY1 were examined under fluorescence microscopy equipped with a krypton/argon laser fluorescent microscope (model TE 200 Nikon Eclipse) using an excitation wavelength of 488nm and measured emission between 530-590nm. Baseline measurements of fluorescence were obtained in the absence of flow and every min during 5 min of intraluminal flow. Images were then analyzed for fluorescence intensity in arbitrary units using Metamorph (Universal Imaging Corp) subtracting background fluorescence. Relative average fluorescence intensity was normalized for surface area and presented as percent change from baseline (prior to initiation of flow). All vessels used for comparison on a given day were obtained from the same patient and studied using the same imaging parameters. Immunohistochemistry. Immunohistochemistry was performed to visualize ceramide expression in human arterioles from discarded adipose tissue as previously described.9 Briefly, isolated arterioles (~200µm in diameter) were fixed in zinc formalin buffer for 24-72hrs and processed for paraffin embedding. Samples were sectioned on a HMS355 microtome at 4µm. Immunolabeling was performed using a mouse anti-human monoclonal antibody against ceramide. Immunostaining was performed using a Leica Bond MAX Immunostainer. Slides were deparaffinized and subject to heat-induced epitope retrieval (HIER) for 10 minutes at pH 6.0. The primary antibody was optimal at 1:200 using the Bond Refine-HRP detection system. Slides were scanned with a NanoZoomer HT slide scanner (Hamamatsu, Japan). Staining was quantified within the vessel wall using Metamorph (Universal Imaging Corp) and reported as average area percent or the total stained area divided by the total wall area (µm2). Materials. C-2 Ceramide, (Cayman Chemical, Ann Arbor, Michigan), and GW4869 (Sigma-Aldrich, Saint Louis, MO) were prepared in DMSO. Mitochondria Peroxy Yellow 1 (MitoPY1) was obtained from Tocris Bioscience (Bristol, UK). Mito-TEMPO was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY). All other chemicals were purchased from Sigma-Aldrich and prepared in distilled water with the exception of endothelin-1 which was prepared in 1% bovine serum albumin (BSA). Vehicle control studies indicated that the final concentration of DMSO had no effect on basal tone or function of the arterioles. The ceramide primary antibody was purchased from Enzo Life Sciences (Farmingdale, NY). Statistical analyses. Data are expressed as means +/- SEM. Flow-induced dilation is expressed as a percentage of maximal relaxation from endothelin‐1 constriction, with 100% representing full relaxation to the maximal diameter obtained by the addition of papaverine. To compare flow–response relationships, a 2-way ANOVA was used with flow gradient and treatment as parameters. When a significant difference was observed between curves (p
