Classification of CMD according to the involvement of pathogenic mechanisms and the clinical setting.
\r\n\t
",isbn:"978-1-83969-452-3",printIsbn:"978-1-83969-451-6",pdfIsbn:"978-1-83969-453-0",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,hash:"a6e1a11c05ff8853c529750ddfac6c11",bookSignature:"Dr. René Mauricio Barría",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10734.jpg",keywords:"Neonatal Intensive Unit, Neonatal Diagnostic Techniques, Neonatal Nurses, Neonatologists, Newborn Diseases, Premature Diseases, Breast Feeding, Kangaroo-Mother Care Method, Neonatal Survival, Limit of Viability, Minimal Handling, Neonatal Stress",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"February 5th 2021",dateEndSecondStepPublish:"March 5th 2021",dateEndThirdStepPublish:"May 4th 2021",dateEndFourthStepPublish:"July 23rd 2021",dateEndFifthStepPublish:"September 21st 2021",remainingDaysToSecondStep:"4 days",secondStepPassed:!1,currentStepOfPublishingProcess:2,editedByType:null,kuFlag:!1,biosketch:"The principal investigator and academic expert in epidemiological methods and evidence-based health with an emphasis on children's health. His research interests lie in the areas of Maternal-Child Health, Neonatal Care, and Environmental Health. From 2010 until 2017 he was Director of the Evidence-Based Health Office and currently serves as Director of the Nursing Institute at the Universidad Austral de Chile.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"88861",title:"Dr.",name:"R. Mauricio",middleName:null,surname:"Barría",slug:"r.-mauricio-barria",fullName:"R. Mauricio Barría",profilePictureURL:"https://mts.intechopen.com/storage/users/88861/images/system/88861.jpg",biography:"R. Mauricio Barría, DrPH, is a Principal Investigator and Associate Professor at the Faculty of Medicine at Universidad Austral de Chile. He was trained as an epidemiologist and received his MSc in Clinical Epidemiology from Universidad de la Frontera in Temuco, Chile, and his DrPH from Universidad de Chile in Santiago, Chile. His research interests lie in the areas of Maternal-Child Health, Neonatal Care and Environmental Health. He is skilled in epidemiological studies designs with special interest in cohort studies and clinical trials. Since 2010 until 2017 he was Director of the Evidence-Based Health Office and currently serves as Director of the Nursing Institute at the Universidad Austral de Chile. He has published several articles related to the care and health of the newborn and is a reviewer of several international journals.",institutionString:"Austral University of Chile",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"4",totalChapterViews:"0",totalEditedBooks:"4",institution:{name:"Austral University of Chile",institutionURL:null,country:{name:"Chile"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"16",title:"Medicine",slug:"medicine"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"345821",firstName:"Darko",lastName:"Hrvojic",middleName:null,title:"Mr.",imageUrl:"//cdnintech.com/web/frontend/www/assets/author.svg",email:"darko@intechopen.com",biography:null}},relatedBooks:[{type:"book",id:"6550",title:"Cohort Studies in Health Sciences",subtitle:null,isOpenForSubmission:!1,hash:"01df5aba4fff1a84b37a2fdafa809660",slug:"cohort-studies-in-health-sciences",bookSignature:"R. 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These demands are physiologically met through an extensive and unique vascular network, which is usually known as coronary circulation. The coronary circulation includes larger arteries, also known as coronary arteries, smaller vessels (with a diameter below 100 μm), such as arterioles, capillaries and venules, that together form the coronary microcirculation and larger epicardial veins [1].
\nHistorically, the large epicardial arteries were considered the coronary circulation. Nowadays, the scientific reports suggest the coronary circulation is characterized by an extreme complexity in terms of morphology but also physiology. Moreover, the theory that the coronary circulation involves the larger epicardial arteries is no longer acceptable given the extensive vascular network present in the myocardium.
\nCoronary artery disease (CAD) is usually associated with larger epicardial coronary arteries. However, previous studies have shown an important link between microcirculatory dysfunction and cardiovascular disease. In fact, pathological changes in smaller vessels have been detected prior to clinical manifestations of cardiovascular disease [1]. Moreover, microcirculatory dysfunction may even be a risk indicator for metabolic syndrome and associated cardiovascular disease [1, 2].
\nThis review aimed to explore the (a) morphology, with particular interest on the anatomical and histological aspects; (b) physiology, providing an insight on the several endothelium-dependent and endothelium-independent regulatory mechanisms; and (c) pathophysiology of the cardiac microcirculation, with a special focus on the changes in the regulatory mechanisms, on the atherogenesis and on the correlation to the systemic cardiovascular disease.
\nBased on the morphology and function, the coronary circulation involves several types of vessels as follows (from the larger arteries to the largest veins): epicardial arteries or coronary arteries, small arteries or intramural arteries, arterioles, capillaries, venules and epicardial veins. These vessels may be grouped according to their size into (a) coronary macrocirculation, referring to vessels with diameter higher than 100 μm (which includes the coronary arteries, the intramural arteries and the epicardial veins) and (b) coronary microcirculation, for vessels with a diameter lower than 100 μm, where the arterioles, capillaries and venules may be included.
\nThe arterioles are smaller arteries that originate from the intramural arteries and run parallel to the myocardial fibres [3–5]. These vessels are characterized by a marked decrease in blood pressure, contributing to the blood flow resistance, along their length and by an increasing responsiveness to metabolites, for example hydrogen peroxide, adenosine, among others [3, 4, 6]. Therefore, arterioles represent the main metabolic regulation component of the myocardial blood flow and aim at controlling the blood flow to the capillary network [3, 4, 7].
\nThe anatomy of these vessels varies along their length: the proximal and middle portions tend to present similar characteristics to the larger arteries, although with a thick tunica media [several layers of vascular smooth muscle cells (VSMCs)], while the distal portions, also termed terminal or precapillary arterioles, may present a thinner tunica media (one to two layers of VSMCs) or not even present any VSMC layer, which is replaced by small unique cells that will be explored in the following subsection, the pericytes (Figure 1) [8]. Moreover, the internal elastic membrane, in the tunica intima, may not be present [6]. The tunica adventitia is usually thinner in these vessels [6].
\nVascular network and capillary neurovascular unit. As presented in the figure, the larger arteries (with a well-defined smooth muscle layer that may vary in size) present morphological differences to the capillaries, which do not present smooth muscle layer, being substituted by pericytes. The capillary neurovascular unit then includes the endothelium, basal lamina and pericytes, which are surrounded by neuron terminals. Moreover, the vascular network also includes other cell types, such as fibroblasts, collateral blood vessels, among others. Adapted from Zhang et al. [9] and prepared using Servier Medical Art (http://www.servier.com/).
The connection between the arterial and the venous systems is fundamentally achieved by a capillary network placed amid the arterioles and the venules (Figure 1).
\nThe capillaries, with a diameter lower than 10 μm (average of 5.7 μm), are microscopic vessels that present numerous anastomotic loops (connections between the arterial and the venous systems), playing a crucial role in the exchange of nutrients and oxygen between the blood and the myocardium [5]. The capillary density may average up to 3500/mm2 in the healthy myocardium and seems to vary from the subendocardium, which presents a higher oxygen-transport, to the subepicardium [5, 10, 11].
\nThese vessels present structural differences to other vessels as the wall is essentially composed of two layers: an inner layer, the endothelium, and its basal lamina (Figure 1) [6]. In the inner layer, the endothelial cell junctions may be smaller, forming intercellular clefts, or larger, creating intercellular gaps.
\nAccording to their morphology, capillaries may be classified into three main categories: (a) continuous capillaries, (b) fenestrated capillaries and (c) discontinuous capillaries [6]. In the coronary microcirculation, the continuous capillaries are the most prevalent type. These vessels are commonly found in muscle, lung and central nervous system and are characterized by the presence of numerous pinocytotic vesicles and the absence of fenestrations [6]. These fenestrations, present in the fenestrated capillaries, are microscopic pores (80–100 nm in diameter) that allow the rapid diffusion of smaller molecules or proteins, which is particularly important in some tissues, such as the intestine and endocrine glands [6]. The discontinuous capillaries, also known as sinusoidal capillaries or sinusoids, present a higher diameter than other capillaries as well as an irregular shape and may be found in the liver, among other tissues [6].
\nEmbedded in the basal membrane of capillaries, between the endothelium and the parenchyma, small contractile cells called pericytes may be found (Figure 1) [12, 13].
\nPericytes may vary morphologically and physiologically depending on the vascular bed and on the position in the vascular bed itself [13]. Nevertheless, they generally extend processes along and around capillaries [12, 13]. In the central nervous system and kidneys, pericytes play an important role in angiogenesis, regulation of the endothelium, among other functions [12, 13]. These cells seem to be particularly relevant in the central nervous system where the regional blood flow regulation is of crucial importance [13]. These pericytes may also present contractile properties [12, 13]. Several proteins have been suggested to confer contractility to pericytes, such as α-smooth muscle actin and tropomyosin [13]. However, previous studies suggest that the contractile mechanisms differ from the VSMCs [13].
\nAlthough the role of pericytes in coronary physiology is not yet fully understood, the high number of these cells in cardiac capillaries and the similar characteristics to the central nervous system pericytes indicates these cells may play an important role in the regulation of the vessel diameter as well as permeability [12].
\nIn the capillary network, other structures may be found such as capillary sinuses, which consist of reservoir-like spaces that could behave as micropumps [8].
\nAfter the exchange of nutrients and oxygen at the capillary level, the deoxygenated blood, containing metabolic products, proceeds to the venules, which present numerous intercommunications, through confluence of capillaries and postcapillary vessels [5]. Although the coronary circulation has been extensively studied over the years, little is known about the intramural venous system. Nevertheless, previous studies have suggested a larger venous network comparatively to the arterial network in the myocardium [5]. In fact, the existence of two veins per artery has been suggested [5].
\nThe venules usually present a diameter ranging from 10 to 50 μm and similar anatomical characteristics to the arterioles [8]. The proximal venules, that is postcapillary venules, usually exhibit only two layers: an inner layer, the endothelium and an outer layer, the basal membrane [6, 8]. The endothelium of the venules seems to be highly responsive to vasoactive agents, namely histamine and 5-hydroxytryptamine, commonly known as serotonin [6]. As well as in terminal arterioles and capillaries, pericytes may also be found in the venular wall in a particularly higher extent than in arterioles or capillaries [6, 8].
\nThe distal venules are morphologically different relatively to the postcapillary venules, as they may present a thin tunica media (one to two layers of VSMCs) and a thin tunica adventitia on the outer side of the vessel [6]. The absence of pericytes is a key characteristic of these distal venules [6]. These venules initially course parallel to the muscle fibres, accompanying the arterioles and capillaries, then changing their position and configuration to meet the larger coronary veins [5].
\nIn healthy conditions, the myocardial blood supply is fundamentally provided through the normal coronary circulation. However, in the presence of cardiac disease, such as chronic cardiac disease or regional ischemic injuries, the myocardial perfusion may be compromised [4, 5]. Compensatory circulatory communications named arteriovenous anastomoses or shunts seem to play a key role in the preservation of the myocardial perfusion in these situations [4, 5, 14]. This collateral circulation links directly the arteries or arterioles to the veins or venules, bypassing the capillary bed [14]. The arteriole of these shunts frequently presents morphological particularities: a thicker tunica media with a higher content in VSMCs, a more developed tunica adventitia, forming a capsule of connective tissue, abundant innervation and are frequently coiled [6].
\nThe direct communication between the heart chamber and the coronary circulation is generally referred to Thebesian vessels [5]. These vessels were first described by Thebesius in 1708 [15] and involve the communication between the heart chamber and the capillaries and venules, referring to a venular connection [5, 16, 17]. These veins usually present a diameter of 200–400 μm and are more frequent in the right ventricle [5]. This type of chamber-vessel communication was later studied by Wearn et al. [18] who further described and defined this and other types of vessels, namely the arteriosinusoidal vessels and the arterioluminal vessels [5, 16, 18]. The arteriosinusoidal vessels provide a communication between a heart chamber and a myocardial sinusoid and are irregularly shaped short branches (diameter from 50 to 350 μm) composed of just an endothelial layer [5, 18]. The arterioluminal vessels are smaller vessels (diameter from 40 to 200 μm) that provide a direct communication to a heart chamber (more frequently the left ventricle), presenting a morphology similar to arterioles [5, 18]. Although previous studies have demonstrated the presence of these special vessels, their clinical significance is still debatable [5].
\nThe physiologic behaviour of the coronary circulation is inherently linked to a balance between the blood supply and the metabolic demand of the heart [19]. Furthermore, the physiological responses in the microcirculation seem to depend on the vessel size and type and appear to vary within the microcirculation itself and from those in the macrocirculation [19–21]. Physiologically, the coronary microcirculation is able to respond to a wide range of stimuli, such as growth and physical exercise, through adaptive processes, essential to the maintenance of its physiology [19]. In fact, vessels present a high adaptation ability and may undergo both acute and chronic adjustments. The acute adjustments involve changes in the vascular smooth muscle tone, while the chronic adjustments involve wall structure changes [19].
\nThe vascular tone is defined as the ratio between baseline and maximal vessel diameter and is determined by the vascular smooth muscle function [19]. In turn, this is regulated by several mechanisms, such as (a) the myogenic tone, which is an intrinsic property of the VSMC, (b) the metabolic control exerted by adjacent cells, (c) the endothelial function responding to changes in the shear stress and (d) autonomic innervation and circulating factors, such as hormones [19].
\nThe myogenic tone is produced by the response of the VSMCs to changes in transmural pressure that leads to stretching of the vessel wall [19, 22, 23]. This relation seems to be linear, that is increasing transmural pressure leads to higher vasoconstriction (reduction in the lumen diameter) [19]. The mechanism underlying this response appears to involve the opening of ion channels, namely nonspecific cation channels, with an increase in intracellular sodium and calcium and consequently the depolarization of the VSMCs [19]. Several receptors have been implicated in the myogenic response, such as (a) integrins [24], (b) transient-receptor potential channels (TRPs) [25, 26] and (c) G protein-coupled receptors [27].
\nThe coronary blood flow is intrinsically linked with metabolic demands of the myocardium, namely of oxygen. At rest, the myocardial oxygen extraction averages 60–70%, which leads to the coronary venous pO2 of about 20 mmHg [28]. During physical exercise, several mechanisms of adaptation are triggered in the myocardium, pO2 seems to be kept constant, which highlights the role of several pathways, namely the myocardial aerobic metabolism [28]. This energy production is generally dependent on mitochondrial oxidative phosphorylation pathways [19]. Among several metabolites produced in these intracellular pathways, carbon dioxide (CO2) and reactive oxygen species (ROS) seem to play an important role in physiological conditions [19, 28].
\nAs previously mentioned, CO2 production is linked to metabolic demands and therefore dependent on the myocardium oxygen consumption [19, 28]. This metabolite results from two main metabolic pathways: (a) the pyruvate dehydrogenase reaction and (b) the citric acid cycle [28]. The pyruvate dehydrogenase reaction converts pyruvate into acetyl-CoA, which is a substrate for the production of citrate, according to the following reaction:
After the conversion of oxaloacetate into citrate, the citric acid cycle involves several reactions in chain. Some of them also involve the production of CO2, such as the production of α-ketoglutarate (reaction 2) and succinyl-CoA (reaction 3).
The increased production of CO2 may also induce a decrease in pH due to the increase in proton concentration, as presented in the following reaction:
This change in pH seems to promote the coronary vasodilation [28–30].
\nAs can be seen in Figure 2, the metabolic production of ROS also plays an important role in the metabolic regulation of the coronary blood flow involving a feedforward mechanism [19, 28]. Among the several ROS, hydrogen peroxide (H2O2) seems to be one of the most important metabolites being considered a feedforward vasodilator [31]. H2O2 results from the conversion of superoxide anions (O2⋅−) by the superoxide dismutase (SOD) [28]. In turn, the superoxide anions result from the reduction in O2 by electrons released from mitochondrial complexes (I and III) [28]. This pathway may be stimulated by shear stress in human coronary resistance arteries [32].
\nThe vasodilator properties of H2O2 have long been studied, but the precise underlying mechanisms are not yet fully established [28]. Previous studies suggested H2O2 behaves as an endothelium-derived hyperpolarizing factor (EDHF) [34, 35], as described in the following subsection. However, other previous studies suggested the mechanism may involve the stimulation of the nitric oxide (NO) production or be mediated by the guanylyl cyclase in human coronary arterioles [36]. These pathways will be further discussed in the following subsections.
\nOther studies have suggested additional mechanisms involved in the metabolic regulation exerted by H2O2 on the coronary blood flow. The involvement of oxidation of thiol groups as a pathway of coronary metabolic dilation in isolated coronary arterioles has been previously proposed [37]. The thiol groups are involved in many pathophysiological mechanisms and play a key role in the biological protection against oxidative injuries [38, 39]. This oxidation process promotes modifications in the protein conformation and includes the conversion of protein-bound thiols (-SH) into sulfenic (SO−, reaction 5), sulphinic (SOO−) and sulphonic (SOOO−) acids as well as disulphide bridges (S-S, reaction 6) [37, 39].
\nFeedforward reactive oxygen species-dependent metabolic regulation of coronary blood flow. The increased metabolic demands of the myocardium trigger an increase in mitochondrial metabolism and flux, through the electron transport chain (ETC), increasing the production of O2⋅− and subsequently of H2O2 by manganese SOD (MnSOD). H2O2 then diffuses to the VSMC and activates voltage-dependent K+ (KV) channels promoting the hyperpolarization of the VSMCs and thus the vasodilation in the coronary microcirculation. Adapted from Muller-Delp [33] and prepared using Servier Medical Art (http://www.servier.com/).
Furthermore, these modifications in the redox state of the cell may also affect the hyperpolarization mediated by thiol-dependent voltage-dependent K+ (KV) channels, which will be further explored in the following subsection [40].
\nOther metabolic vasodilators may also be involved, such as adenosine (which concentration is dependent on the metabolism) and potassium ions, which will be explored further below.
\nThe endothelial function plays a crucial role in the vascular physiology, especially in the regulation of the vascular tone. The endothelium is responsible for the production of a number of different vasoactive substances, such as: (a) endothelium-derived contracting factors (EDCFs), such as endothelin, prostanoids and 20-hydroxyeicosatetraenoic acid (20-HETE) and (b) endothelium-derived relaxing factors (EDRFs), such as NO, prostaglandins (e.g. prostacyclin) and EDHFs, for example H2O2 and epoxyeicosatrienoic acids (EETs) [35, 41–47].
\nVasoconstrictors. The stimulation of receptors in the endothelial cell membrane may trigger the production of several EDCFs, namely prostanoids and endothelin, particularly endothelin-1, among others (Figure 3) [35, 41]. The prostanoids are vasoactive substances that result from the arachidonic acid pathway. Following the stimulation of specific membrane receptors, such as muscarinic receptors for acetylcholine and purinergic (P2Y) receptors for adenosine triphosphate (ATP), the increase in intracellular Ca2+ promotes the production of arachidonic acid from membrane phospholipids by phospholipase A2 [41]. The arachidonic acid is then converted by the endothelial cyclooxygenase-1 (COX-1) to endoperoxides and ultimately to prostanoids, namely thromboxane A2 (TXA2) and prostaglandins, such as prostacyclin (PGI2) [41]. Additionally, the COX-1 activity might also promote the production of ROS [41]. Those vasoactive substances (i.e. TXA2 and prostaglandins) may then diffuse to the smooth muscle layer where they activate thromboxane-prostanoid (TP) receptors, promoting the contraction of the VSMCs [41].
\nMultitude of pathways involved in the endothelium-dependent contraction. Abbreviations: 5-HT, 5-hydrotryptamine; ACE, angiotensin-converting enzyme; ACh, acetylcholine; ADP, adenosine diphosphate; AT-I, angiotensin-I; AT1, angiotensin receptor; AT-II, angiotensin-II; ATG, angiotensinogen; BDK, bradykinin; COX, cyclooxygenases; ECE, endothelin-converting enzyme; ET-1, endothelin-1; ETA, endothelin receptor A; ETB, endothelin receptor B; NO, nitric oxide; eNOS, endothelial nitric oxide synthase; NOX, NADPH oxidase; PGs, prostaglandins; PLA2, phospholipase A2; ROS, reactive oxygen species; TGFβ1, transforming growth factor; Thr, thrombin; TP, thromboxane-prostanoid receptor; TXA2, thromboxane A2; VSMC, vascular smooth muscle cell; XO, xanthine oxidase. Adapted from Virdis et al. [49] and prepared using Servier Medical Art (http://www.servier.com/).
Endothelin (ET) is considered a major vasoactive substance in the EDCF family and a major vascular function regulator. In fact, this term refers to a group of peptides synthesized by the endothelin-converting enzyme (ECE) that may mediate vasoconstriction through the stimulation of receptors, namely ETA and ETB receptors, in the VSMC membrane [48, 49]. Among the several peptides, ET-1 is the most known, and its vasoactive properties have been extensively researched. This peptide promotes a long-lasting vasoconstriction essential to the vessel tone control in coronary arterioles, as reduction ET-1 induces an elevation of coronary blood flow in increased demand situations, that is increased metabolism [50, 51].
\n20-HETE is a metabolite that results from the conversion of arachidonic acid by the 4A and 4F families of cytochrome P450 mono-oxygenases (CYP), particularly in the VSMCs but also in the endothelial cells [35]. This metabolite seems to play an important role in the regulation of the vascular tone, behaving as a potent endogenous vasoconstrictor in several vascular tissues, namely in the brain and in the heart [35, 52].
\nVasodilators. NO is the most researched EDRF worldwide and is produced in the endothelial cells by the endothelial nitric oxide synthase (eNOS). This constitutive enzyme converts L-arginine to L-citrulline and requires several cofactors, such as calcium, calmodulin, 3,4-tetrahydrobiopterin (BH4) and nicotinamide adenine dinucleotide phosphate (NADPH) [53]. The NO-mediated vasodilation primarily involves the conversion of guanosine triphosphate (GTP) to cGMP by soluble guanylyl cyclase (solGC) [34]. However, other mechanisms may also be involved in the NO-mediated vasodilation, namely the hyperpolarization of the VSMCs [34, 54], which will be further explored below. The production of NO may be regulated by several mechanisms, which have been previously explored and published [55]. In addition to the stimulation of receptors on the endothelial cell membrane, the eNOS-mediated production of NO may also be stimulated by shear forces exerted by the blood flow on the vessel wall, as explored further below.
\nPathways involved in the endothelium-dependent and endothelium-independent relaxation of the VSMC. Stimulation of the endothelial cells by acetylcholine (ACh) or other agents (e.g. bradykinin and shear stress) results in the formation and release of an EDRF identified as nitric oxide (NO). Substances such as adenosine, nitroprusside (NP), H+, CO2 and K+ can be produced in the parenchymal tissue and elicit vasodilation by direct action on vascular smooth muscle. Adapted from Koeppen et al. [58] and prepared using Servier Medical Art (http://www.servier.com/).
Although NO is considered the major pathway of endothelium-mediated vasodilation in the systemic circulation, multiple pathways may be involved in this physiological response, such as the prostaglandins-induced vasodilation (Figure 4). The prostaglandins are constitutively produced by cyclooxygenases (COX) [34]. The main substrate of these enzymes is arachidonic acid, which is converted from diacylglycerol or phospholipids, respectively, by phospholipase A2 and phospholipase C [34]. Several prostaglandins are produced by COX, although the main vasoactive prostaglandin produced in the endothelium is PGI2 [35, 56, 57]. Similarly to NO, PGI2 may diffuse from the endothelial cells to the VSMCs where they activate their (IP) receptors and trigger the conversion of ATP into cyclic adenosine monophosphate (cAMP) by adenylyl cyclase (AC) [34, 57]. This activation promotes the hyperpolarization of the VSMCs and hence the vasodilation [34, 57]. However, these prostaglandins, namely PGI2, may also elicit vasoconstriction in disease, as previously discussed [35, 41].
\nSeveral vasoactive substances have been included in the EDHFs family, such as H2O2, carbon dioxide (CO2), hydrogen sulphide (H2S), C-natriuretic peptide (CNP), EETs, potassium ion (K+), among others [34, 35, 54, 59]. Previous studies suggested these factors play a key role in the VSMC hyperpolarization in smaller vessels rather than in larger ones [19, 34].
\nHyperpolarization of the VSMC. Abbreviations: ACh, acetylcholine; BK, bradykinin; BKCa, large conductance Ca2+-activated K+ channels; CaV, voltage-activated Ca2+ channels; Cx, connexin; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; IKCa, intermediate conductance Ca2+-activated K+ channels; KIR, inwardly rectifying K+ channels; NO, nitric oxide; PE, phenylephrine; RyR, ryanodine receptor; SKCa, small conductance Ca2+-activated K+ channels; SP, substance P, TRPC1, transient receptor potential canonical channel 1; TRPV4, transient receptor potential vanilloid channel 4; VSMC, vascular smooth muscle cell. Adapted from Félétou et al. [35] and prepared using Servier Medical Art (http://www.servier.com/).
The hyperpolarization of the VSMC may involve several ionic channels, such as the voltage-activated Ca2+ (CaV) channels, which regulate the intracellular Ca2+ concentration, the KV channels and the Ca2+-activated K+ (KCa) channels [35, 40]. The KCa channels may be subdivided into small (SKCa or KCa 2.3 isoform), intermediate (IKCa or KCa 3.1 isoform) and large (BKCa) conductance Ca2+-activated K+ channels, which are located in specific cellular and subcellular sites [35]. The hyperpolarization of the VSMCs may be triggered directly, through receptors on the VSMC membrane, or indirectly, through the hyperpolarization of the endothelial cells [35].
\nAs can be seen in Figure 5, the direct hyperpolarization may be promoted through the stimulation of BKCa channels on discrete locations of the VSMC layer, that is smooth muscle plasmerosome, associated with the TRP canonical channel 1 (TRPC1) and the TRP vanilloid channel 4 (TRPV4). These signal complexes promote (a) the influx of Ca2+, which is then stored through ryanodine receptor (RyR) on the endoplasmic reticulum and (b) the efflux of K+, contributing to the formation of a potassium cloud in the intercellular space, which functions as a negative-feedback mechanism. This ionic cloud may activate inwardly rectifying K+ (KIR) channels and Na+/K+-ATPase promoting the influx of K+ to the VSMC, thus leading to the hyperpolarization and vasodilation. This hyperpolarization also inhibits the Ca2+ influx through CaV channels that may be stimulated by the binding of noradrenaline or phenylephrine to the adrenergic receptors on the membrane of VSMCs. The stimulation of these receptors leads to the increase in the intracellular Ca2+ concentration triggering the depolarization of the VSMC. Furthermore, this increase in intracellular Ca2+ may subsequently activate KV and BKCa channels, which then promote the efflux of K+ ions to the intercellular space, thus controlling the ionic balance and contributing to the formation of the potassium cloud [35, 57].
\nMoreover, the VSMCs may be indirectly hyperpolarized through the hyperpolarization of the endothelial cells [35]. Following activation of endothelial receptors and action of shear stress, the increased intracellular calcium in the endothelial cell triggers the opening of SKCa (located at the homocellular endothelial gap junctions and caveolin-rich domains) and IKCa channels (preferentially located at the myoendothelial gap junctions or MEJ) leading to K+ efflux and consequently to the hyperpolarization of the endothelial cell [35]. In turn, this may ultimately lead to the hyperpolarization of the VSMCs by direct electric coupling through MEJs, which consist of a cell-cell contact resulting from the projection of an endothelial cell or a VSMC through the internal elastic membrane (Figure 5) [35, 60]. These contacts are essentially established through connexins (Cx), namely Cx40 and Cx37 [35, 61]. Particularly, at the level of the MEJs, the IKCa channels may be activated directly or through the generation of Ca2+ pulsars, contributing further to the potassium cloud in the intercellular space, eventually promoting the activation of KIR channels and Na+/K+-ATPase involved in the hyperpolarization of the VSMCs. The influx of Ca2+ from the intercellular space to the VSMC, through CaV channels may be detected by Ca2+-sensing receptors (CaSR), which may activate IK1 gene, involved in the hyperpolarization of the VSMC [35, 57].
\nBesides the hyperpolarization of the VSMCs, the EDHFs, particularly H2O2, may also promote vasodilation through other mechanisms, namely by stimulating the production of prostaglandin E2 in the endothelial cell, thus promoting the endothelium-dependent vasodilation [62].
\nThe relative importance of each pathway is still unestablished, but it has been proposed to depend for example on the activation state of the VSMCs, the density of MEJs and the expression of KIR and Na+/K+-ATPase [57].
\nAs previously mentioned, in addition to the stimulation of receptors on the endothelial cell membrane, other factors may modulate the endothelial function, namely the forces exerted by the blood flow on the vessel wall. There are two major forces: (a) one perpendicular to the wall and (b) another parallel to the wall, known as wall shear stress that results from the friction of blood flow on the endothelial cells [63]. These shear forces trigger several pathways, such as (a) production, release and binding of bradykinin to endothelial cell membrane receptors and (b) bradykinin-independent pathways, namely the activation of the Akt phosphorylation pathway and the ROS-mediated hyperpolarization of the VSMC. The production and release of bradykinin, which may bind to its Gq-coupled endothelial receptors, increases the activity of eNOS thus promoting the synthesis of NO [64]. The activation of the Akt phosphorylation pathway also promotes the production of NO by eNOS [64, 65]. In human coronary arterioles, the shear forces exerted on the vessel wall may also promote the ROS-mediated hyperpolarization of the VSMC through two main mechanisms: one involving the EETs and other involving the direct stimulation of ROS production (Figure 6). First, the shear stress may induce the production of EETs by triggering the cleavage of arachidonic acid from the cellular membrane by phospholipases. The arachidonic acid then works as a substrate to CYP for the production of EETs, which may activate the TRPV4 channels promoting an increase in intracellular Ca2+, thus stimulating the mitochondrial production of O2⋅−. The production of ROS may also result from the direct stimulation of TRPV4 channels and NADPH oxidases. The O2⋅− produced through both these mechanisms is then dismutated to H2O2, which diffuses to the VSMCs to oxidize cysteine residues of protein kinase G 1α (PKG1α), activating this enzyme. The activation of this enzyme promotes the opening of BKCa channels and the hyperpolarization of the VSMCs resulting in vasodilation of coronary arterioles [32, 34, 66, 67].
\nFlow-mediated dilation in the human coronary arterioles. Abbreviations: AA, arachidonic acid; BKCa, large conductance Ca2+-activated K+ channels; cGMP, cyclic guanosine monophosphate; CuZnSOD, copper-zinc superoxide dismutase; CYP, cytochrome P450; CYS 42, cysteine residue; EETs, epoxyeicosatrienoic acids; GC, guanylyl cyclase; GTP; guanosine triphosphate; H2O2, hydrogen peroxide; KCa, Ca2+-activated K+ channels; MnSOD, manganese superoxide dismutase; NADPH, nicotinamide adenine dinucleotide phosphate; NOX, NADPH oxidase; O2⋅−, superoxide anion; PKG1α, protein kinase G 1α; PLA2, phospholipase A2; PLs, phospholipids; TRPV4, transient receptor potential vanilloid channel 4. Adapted from Durand et al. [34] and prepared using Servier Medical Art (http://www.servier.com/).
Previous studies showed the sensitivity to these pathways of vasodilation increases with decreasing vessel diameter thus assuming a particularly important role in the coronary microcirculation [19]. Previous studies have also suggested the relative weight of these pathways changes from childhood to adulthood and between healthy and pathological conditions. In a preliminary study with human-isolated arterioles, Zinkevich et al. [68] proposed the flow-mediated dilation (FMD) in infants was exclusively COX-dependent, that is mediated by prostaglandins, while in adulthood the main pathway involved the NO. However, in the presence of coronary artery disease (CAD), both these mechanisms seem to remain as secondary pathways as the EDHF-mediated vasodilation (especially by H2O2) gains importance, serving as backup mechanisms in disease [66]. In fact, low response to shear forces and high mechanical stress seem to predispose to vascular dysfunction and disease [63].
\nThe heart is a highly organized organ where several cells may be found, namely endothelial cells and cardiomyocytes. Therefore, the physiological mechanisms depend on the communication between the several types of cells. Until today, many endothelial-derived cardio-active factors have been identified and characterized (Figure 7). The cardiac modulator effects of some of these factors, such as NO, PGI2, ET-1 and neuregulin-1 (NRG-1), have been previously acknowledged. Other factors, namely Dickkopf-3 (DKK3), periostin, thrombospondin-1 (TSP-1), follistatin (FST), apelin and connective tissue growth factor (CTGF), also appear to modulate the cardiomyocyte function, though with little evidence so far. These cardio-active factors seem to be interdependent (additive, synergistic or inhibitory) as their modulator effects may be exerted on the same target cell [69].
\nCommunication between endothelial cells and cardiomyocytes. Abbreviations: CTGF, connective tissue growth factor; DKK3, Dickkopf-3; ET-1, endothelin; FST, follistatin; NO, nitric oxide; NRG-1, neuregulin-1; PGI-2, prostacyclin; TSP-1, thrombospondin. Adapted from Lim et al. [69].
The previously explored pathways are nowadays considered the major pathways of regulation of the vessel tone. However, other mechanisms may also come into play, such as the autonomic nervous system and circulating factors.
\nThe innervation of the coronary circulation by the sympathetic and the parasympathetic divisions of the autonomic nervous system have been previously shown [70]. The endothelial production of vasoactive substances, namely NO, may be influenced by the stimulation of specific receptors in the endothelial cell membrane, such as muscarinic receptors for acetylcholine [41, 70]. Furthermore, the coronary circulation may also be regulated through adrenergic receptors (i.e. α- and β-adrenergic receptors) in both the endothelial cell and the VSMC membranes [70]. In general, the stimulation of the α-adrenergic stimulation seems to induce vasoconstriction, with the exception for the α2 receptors which seem to elicit vasodilation. Moreover, the stimulation of β-adrenergic receptors generally induces vasodilation with β2 receptors being the main population in the coronary microcirculation [19, 70, 71]. This autonomic innervation provides a mechanism for vessel tone regulation, particularly important during exercise. However, the role of the parasympathetic innervation remains debatable in the human coronary microcirculation [70].
\nMoreover, several circulating factors may also modulate the coronary blood flow through the regulation of the vessel tone, such as angiotensin II and other hormones (e.g. cortisol and tiroxine, among others), adipokines (particularly adiponectin) and growth factors among many others [19, 72].
\nAs previously discussed, the coronary microcirculation plays a key role in the myocardial perfusion. Therefore, the presence of functional and/or structural abnormalities of this circulatory pathway may impair the myocardial perfusion and be involved alone as the main mechanism of myocardial ischaemia. These abnormalities are normally designated as coronary microvascular dysfunction (CMD) [3]. The CMD may be assessed by several methods, though one of the most used methods is through the determination of the coronary flow reserve (CFR), which represents an integrated measure of coronary blood flow in both the macro- and microcirculation. The CFR involves the maximal vasodilation of a vessel in response to an endothelium-independent vasodilator, such as adenosine, thus reflecting the ratio of hyperaemic to baseline blood flow. This ratio may be measured through several methods, namely echocardiography and positron emission tomography (PET) [3].
\nCMD type | \nClinical setting | \nPathogenic mechanisms | \n
---|---|---|
In the absence of myocardial disease or obstructive CAD | \nCardiovascular risk factors (e.g. ageing, arterial hypertension, smoking, diabetes) Microvascular angina | \nEndothelial dysfunction VSMC dysfunction Vascular wall remodeling | \n
In the presence of myocardial disease | \nCardiomyopathies (e.g. HCM, DCM) Aortic stenosis | \nVascular wall remodeling VSMC dysfunction Extramural compression Luminal obstruction | \n
In the presence of obstructive CAD | \nAcute coronary syndrome AMI | \nEndothelial dysfunction VSMC dysfunction Luminal obstruction | \n
Iatrogenic microembolization | \nCoronary reperfusion procedures (e.g. PCI) Revascularization (i.e. CABG) | \nLuminal obstruction Autonomic dysfunction | \n
Classification of CMD according to the involvement of pathogenic mechanisms and the clinical setting.
Abbreviations: AMI, acute myocardial infarction; CABG, coronary artery bypass grafting; DCM, dilated cardiomyopathy; HCM, hypertrophic cardiomyopathy; PCI, percutaneous coronary intervention; VSMC, vascular smooth muscle cell. Adapted from Crea et al. [20].
CMD may present several pathogenic underlying mechanisms, depending on the source of the abnormality, namely structural and functional (Table 1), which will be discussed in this section. According to the underlying clinical setting, the CMD may also be classified into four types: type 1, in the absence of cardiomyopathies or obstructive CAD; type 2, in the presence of cardiomyopathies; type 3, in the presence of CAD; and type 4, iatrogenic [3, 20, 73, 74].
\nThe most common functional abnormalities are the dysfunction of the endothelial cells and/or the VSMCs, involving cardiovascular risk factors or cardiomyopathies, and the autonomic nervous system dysfunction, secondary to coronary reperfusion procedures.
\nAs presented in Table 1, the traditional cardiovascular risk factors (i.e. ageing, gender, obesity, smoking, hypertension, dyslipidaemia and diabetes) may impair the endothelial function by several mechanisms, namely increased production of EDCFs and/or decreased production of EDRFs [3, 73]. Furthermore, this impairment may also contribute to the dysfunction of the VSMCs, which may also result from structural changes, derived from cardiomyopathies or arterial hypertension, described further below.
\nAgeing is considered as one of the major cardiovascular risk factors that may influence the endothelial function. This influence seems to primarily involve both functional and structural changes, which will be discussed further below [42, 75]. Several mechanisms have been identified to mediate these changes and have been previously reviewed [55, 64, 76, 77]. In fact, the imbalance between vasoconstriction and vasodilation seems to be a key mechanism underlying ageing-induced vascular dysfunction.
\nGender-associated differences related to hormones (i.e. oestrogens) have been previously described for the vascular reactivity in several vascular beds [42, 65]. The stimulation of G protein-coupled receptors by these hormones seems to promote an increased production of EDRFs, especially NO, which could elucidate the lower incidence of coronary disease and atherosclerosis in premenopausal women compared to men of the same age and postmenopausal women. In fact, impaired expression of eNOS was previously reported in postmenopausal women and suggested as a gender-specific risk factor in coronary surgery [78]. Moreover, Muir et al. [79] also showed differences in the endothelium-dependent vasodilation between males and females. In the coronary circulation, oestrogens seem to promote a decreased vascular tone, which promotes a reduced blood flow resistance and thus a higher coronary blood flow [80].
\nObesity has also been considered an important cardiovascular risk factor; thus, a healthy diet and the regular practice of exercise have been proposed as important preventive measures. The influence of obesity in the vasoreactivity involves several mechanisms, namely an impaired regulation of vascular tone, a systemic chronic inflammation, induced by adipokines, which are involved in CMD, an altered lipidic profile (i.e. dyslipidaemia) and increased incidence of atherosclerosis and vascular oxidative stress [55, 80–82].
\nCigarette smoking has been widely recognized as a major cardiovascular risk factor that induces endothelial dysfunction. In the peripheral circulation, this effect primarily involves the decreased production of EDRFs, namely NO, mainly through the impairment of eNOS activity [79, 83]. Interestingly, this downregulation of NO-mediated vasodilation seems to be exposure-dependent [84]. The ability to induce endothelial dysfunction may also manifest in the coronary circulation, particularly in long-term smokers, independently of the presence of atherosclerotic plaques [85]. Moreover, Kaufmann et al. [86] found CMD in asymptomatic smokers in the absence of CAD. These patients presented a reduction in 21% of the CFR, which could be restored with the short-term administration of vitamin C. These findings suggested that the smoking-associated CMD may involve an increase in oxidative stress in the coronary microcirculation [86].
\nSimilarly to smoking, arterial hypertension is also considered a major cardiovascular risk factor. Previous studies suggested the increased production of EDCFs as the main mechanism underlying the hypertension-induced endothelial dysfunction [41]. This effect is primarily triggered by an increase in intracellular Ca2+, stimulating a higher production of COX-derived prostanoids (e.g. TXA2 and PGI2) and ROS, namely O2⋅− [41]. The prostanoids may then diffuse to the VSMCs activating TP receptors, with subsequent influx of Ca2+, creating the conditions to a predominant vasoconstriction [41]. The ROS may also influence the vascular function since they may react with NO, reducing its availability, or even stimulate the influx of Ca2+ [41].
\nThe vascular effects of dyslipidaemia, namely hypercholesterolaemia, are dependent on the degree of atherogenesis. In fact, the accumulation and oxidation of low-density lipoproteins (LDLs) are considered major steps in the development of the chronic inflammatory process that is atherosclerosis [87]. The accumulation of LDL in the subendothelial matrix depends on the circulating LDL levels as the LDLs diffuse from the lumen to the vessel wall through endothelial cell junctions [87]. Once in the subendothelial matrix, the LDLs may undergo oxidation by reacting with endothelium-derived ROS, producing oxidized LDLs [87]. These proinflammatory factors may mediate several effects on the vessel wall, mainly the impairment of NO-mediated vasodilation and the atherogenesis [64]. In fact, oxidized LDLs may (a) trigger the influx of asymmetric dimethyl-
Diabetes is a known cardiovascular risk factor responsible for several effects on the cardiac and peripheral vascular systems, which intermediate an increased morbidity and mortality [92]. In spite of the array of mechanisms involved, the relation between diabetes and CMD is not yet fully understood [93]. Previous studies suggested several mechanisms involved in the diabetes-induced vascular dysfunction, namely (a) impaired production of NO, due to BH4 deficiency, increased arginase activity, increased ADMA influx or downregulation of the Akt phosphorylation pathway; (b) increased production of EDCFs, namely endothelin; and (c) other NO-independent mechanisms, such as hyperglycaemia [55]. Chronic hyperglycaemia has been previously suggested to play a key role in the diabetes-related CMD, as several mechanisms may be involved [55], namely the endothelial-protein glycation through the formation of advanced glycation end-products (AGEs) and subsequent stimulation of the respective receptors (RAGE). In fact, previous studies suggested that diabetic patients (both type 1 and type 2) present a marked reduction in the endothelium-dependent and endothelium-independent coronary vasodilation [94].
\nThe impairment of the vasodilator response of the coronary microcirculation may also be present in patients with angina-like chest pain but without evidence of obstructive CAD or myocardial disease. This situation is usually known as microvascular angina or coronary syndrome X. The literature seems to be contradictory as some studies suggest no changes in the coronary blood flow and in the CFR [73], while others showed the presence of CMD through impairment of the endothelium-dependent and endothelium-independent vasodilation [95], reduction in the coronary blood flow and CFR [96] and evidence of myocardial ischaemia [97, 98]. However, the precise pathogenic mechanisms involved in these changes are not yet completely understood as this situation seems to be multifactorial [99].
\nThe autonomic nervous system dysfunction, following acute myocardial infarction (AMI) and/or coronary reperfusion procedures, may also contribute to the CMD. In fact, increased coronary vasoconstriction has been previously shown, after AMI and successful percutaneous coronary angioplasty, both at the site of stenosis and distal to it, suggesting abnormal coronary vasodilator response [73, 100].
\nAfter AMI, the CMD may result from autonomic dysfunction and luminal obstruction, discussed further below. The autonomic dysfunction in the AMI-associated CMD involves increased sympathetic activation with increased vasoconstriction. These findings were confirmed by Gregorini et al. [101], who showed this impaired autonomic function might be reverted with α-blockers, such as phentolamine (nonselective α-blocker) and urapidil (α1-selective blocker), which may improve the recovery of myocardial perfusion after coronary stenting in patients with AMI [101–103]. Autonomic dysfunction secondary to percutaneous coronary angioplasty was also showed by Gregorini et al. [104] who linked the left ventricular macro- and microcirculatory dysfunction in patients with transient ischaemia. In this study, phentolamine and urapidil were similarly used to block the α-adrenergic neurotransmission and propranolol (nonselective β-blocker) and the β-adrenergic neurotransmission, and the results showed that the increased coronary vasoconstriction, secondary to percutaneous coronary angioplasty, may be prevented with α-adrenergic receptor antagonists as no effect was demonstrated for the β-adrenergic blockade. Moreover, this study suggested that CFR may still be decreased for 7 days to 3 months after the procedure [104]. In a similar study, Kozàkovà et al. [105] confirmed the potential usefulness of urapidil to improve the left ventricular function in the angioplasty follow-up. Moreover, persistent yet reversible CMD after coronary revascularization was also previously showed [106].
\nIn addition to functional abnormalities, structural abnormalities, namely vascular remodelling, vascular rarefaction, perivascular fibrosis, luminal obstruction and infiltration of the myocardium and vascular wall, may also contribute to CMD [3, 73].
\nThe remodelling of the vessel wall involves persistent modifications which may result from several remodelling signals, such as (a) the wall shear stress (which mechanisms have been previously discussed), (b) the circumferential wall stress (resulting from the stretch of the smooth muscle layer) and (c) specific metabolic signals [19, 107]. As mentioned above, the physiologic metabolic control of coronary blood flow mainly involves CO2 and ROS. In a pathological setting (i.e. myocardial ischaemia) however, the metabolic control may involve several mediators, such as oxygen, adenosine, prostaglandins, nitric oxide and protons [28]. In the presence of myocardial ischaemia, the decreased pO2 is detected by (a) the cardiomyocytes triggering the production of adenosine (which promotes the VSMC hyperpolarization through its receptors A2A and A2B) and NO, by (b) the endothelium, inducing the production of prostaglandins and by (c) the VSMCs, where KATP and CaV channels are activated leading to the hyperpolarization of these cells and to the vasodilation [28]. These remodelling signals promote, on one hand, the increase in the luminal diameter and, on the other hand, the VSMC plasticity and matrix remodelling, inducing the wall thickening [19, 108]. However, in the presence of certain factors, such as ageing, arterial hypertension and cardiomyopathies, these mechanisms of adaptation may be impaired leading to pathogenic changes.
\nIn addition to the functional changes, ageing may induce these structural modifications, namely the proliferation of VSMCs and increase the inflammation status in the vascular wall, which is linked to atherogenesis, leading to the remodelling of the vessel wall and to the decrease in the luminal diameter [75].
\nOther situations may also contribute to the vascular remodelling, especially arterial hypertension and cardiomyopathies. Previous studies have suggested that arterial hypertension may promote the thickening of the smooth muscle layer, by stimulating the proliferation of VSMCs and collagen fibres [3, 73].
\nFurthermore, cardiomyopathies (especially hypertrophic cardiomyopathy) may also contribute to the vascular remodelling. According to Maron et al. [109], “cardiomyopathies are a heterogeneous group of diseases of the myocardium associated with mechanical and/or electrical dysfunction that usually (but not invariably) exhibit inappropriate ventricular hypertrophy or dilatation and are due to a variety of causes that frequently are genetic. Cardiomyopathies either are confined to the heart or are part of generalized systemic disorders, often leading to cardiovascular death or progressive heart failure-related disability”. The cardiomyopathies are usually classified into primary and secondary, based on the American Heart Association classification [109]. On the basis of the management of cardiomyopathy with a morphofunctional phenotype, the European Society of Cardiology proposed in 2008 the classification of cardiomyopathies into the hypertrophic (HCM), dilated (DCM), restrictive (RCM), arrhytmogenic right ventricular (ARVC) and unclassified varieties [110]. Each of these groups was subdivided into familial or genetic and nonfamilial or nongenetic forms [110]. In 2014, another classification was proposed by the World Heart Federation, involving a descriptive genotype-phenotype nosology system, the MOGE(S) classification [111]. Vascular remodelling in coronary arterioles has been previously associated with both HCM and DCM [73]. Similarly to arterial hypertension, the remodelling of these vessels also involves the thickening of both the smooth muscle layer and the intimal layer. These morphological changes may contribute to the CMD associated with HCM, as patients with this cardiomyopathy showed a marked decrease in vasodilator response in the endocardium, proportional to the degree of hypertrophy [3, 73]. Relatively to DCM, the degree of CMD may be considered an independent prognostic factor for cardiac events [73, 112, 113].
\nThe coronary microvascular function may also be influenced by modifications in the vascular density, particularly by vascular rarefaction, that is the reduction in the number of microcirculatory vessels, which may also be recognized as hypotrophic remodelling [19]. The presence of arterial hypertension and the extravascular compression, observed in aortic stenosis and cardiomyopathies, induces vascular rarefaction leading to the reduction in the CFR. In addition to the vascular rarefaction, both situations may also induce perivascular fibrosis promoting structural modifications of the vessel wall [73, 114].
\nCMD may also be characterized by luminal obstruction originated from (a) obstructive CAD or (b) iatrogenic microembolization [73].
\nAccording to the mechanisms underlying the associated CMD as well as the clinical findings, the obstructive CAD may be divided into stable CAD, unstable CAD and AMI [73]. Changes in the CFR were previously showed in patients with both stable CAD and acute coronary syndromes.
\nIn patients with stable CAD, the CMD distal to a coronary stenosis seems to be triggered through two main pathways: (a) increased prearteriolar and arteriolar constriction, increasing the blood flow resistance and decreasing the myocardial perfusion and (b) impaired prearteriolar dilation in the presence of increased myocardial oxygen demands [73]. Although in the presence of coronary stenosis (for example during exercise), the transmural myocardial perfusion tends to be redistributed, with an increase in the subendocardial perfusion. The impairment of this mechanism in patients with stable CAD may lead to increased microvascular vasoconstriction, which might promote a critical stenosis and thus capillary derecruitment distal to the stenosis, ultimately contributing to CMD [73].
\nSimilarly to stable CAD, unstable CAD (i.e. acute coronary syndromes without ST-segment elevation) may also involve a CMD distal to a critical stenosis which might play a role in the severity of the myocardial ischaemia. In addition to the mechanisms described for stable CAD, this type of acute coronary syndromes also involves other mechanisms, such as thrombogenesis [73]. In fact, Marzilli et al. [115] suggested that the blockade of the platelet glycoprotein IIb/IIIa receptor with abciximab might improve the microvascular function in patients with unstable CAD. Moreover, the inflammation status may also come into play as suggested by previous studies that showed a direct relation between CMD and the systemic levels of C-reactive protein, a marker of inflammation independent of the cardiovascular risk factors [116, 117]. Both of these factors may contribute to the luminal obstruction observed in patients with unstable CAD.
\nLuminal obstruction is a key characteristic of the AMI. Early after a myocardial infarction, patients may present a marked reduction in the CFR that could significantly impair the contractility of the myocardium in the infarction region [73]. In addition to the autonomic dysfunction (previously explored), this impaired myocardial contractility might also be reverted with α-blockers [73, 101]. Even after reperfusion procedures, the CMD involving luminal obstruction in the stenotic and poststenotic areas may be responsible for the failure of the reperfusion, situation usually known as “no-reflow” phenomenon. This phenomenon is characterized by the lack of morphological and functional integrity in the microcirculation, in spite of successful reperfusion procedures [12, 73, 118, 119] and is associated with clinically significant decreased prognosis. The pathogenesis of the “no-reflow” phenomenon seems to be multifactorial; thus, a classification has been previously proposed which divides this phenomenon into (a) structural and (b) functional types. The structural type involves irreversible changes in the wall of the microvessels, while the functional type includes morphologically intact yet functionally compromised microvessels. The functional changes include impairment of the endothelium-dependent vasodilation, autonomic nervous system dysfunction and extravascular compression due to interstitial oedema, among others [73, 120, 121]. Recently, O’Farrell et al. [12] proposed a key role of pericytes in the pathogenesis of this phenomenon (Figure 8), suggesting that these cells irreversibly constrict the coronary microcirculation impeding the adequate reperfusion after AMI.
\nRole of pericytes in healthy and ischaemic microvessels: (a) normal blood flow in coronary arterioles and capillaries covered with pericytes; (b) in ischaemia, the pericytes may constrict the coronary microcirculation compromising the coronary blood flow and leading to coronary microvascular dysfunction; (c) after reperfusion procedures, the coronary microvascular dysfunction may block the re-establishment of the normal blood flow, situation usually known as no-reflow phenomenon.
As previously mentioned, CMD may also be originated from iatrogenic microembolization after coronary reperfusion procedures or coronary artery bypass grafting. During or after these procedures, plaque rupture may occur thus releasing plaque content into the blood which in turn might lead to luminal obstruction in the microcirculation [73].
\nIn addition to the previous explored structural changes, the infiltration of the vessel wall with metabolic deposits may also be found. This infiltration is commonly found in infiltrative diseases, such as Anderson-Fabry disease and other metabolic disorders. The Anderson-Fabry disease involves a genetically linked (X chromosome) deficiency of lysosomal α-galactosidase A, which leads to damages in several organs, namely the heart, through the deposition of glycosphingolipid in cardiomyocytes and in the vascular wall [73]. In turn, this infiltration promotes the hypertrophy and fibrosis of cardiomyocytes as well as CMD and perivascular fibrosis [73]. In fact, Elliott et al. [122] demonstrated these patients present a marked decrease in CFR, confirming the presence of CMD in the pathogenesis of the cardiomyopathy induced by this disease.
\nThis review provides an insight on the morphology, physiology and pathophysiology of the cardiac microcirculation. As discussed, the heart is one of the most nutrient and oxygen demanding organs as this demand needs to be satisfied with an adequate vascularization of the myocardium through an extensive macro- and microvascular network. Although most of the cardiac diseases, such as the acute coronary syndrome, are commonly associated with the coronary macrocirculation (i.e. epicardial coronary arteries), the coronary microcirculation also seems to play a key role in the coronary pathophysiology. This role involves both molecular and clinical aspects that should not be overlooked and that constitute potential diagnostic and therapeutic targets, particularly important in the early pathogenesis of these diseases.
\nTo maintain hemostasis, new blood cells must be constantly generated to replace those lost through injury, disease, or age. Hematopoiesis, is the process where hematopoietic stem cells (HSC) differentiate into mature blood cells and is tightly regulated by the bone marrow (BM) micro-environment (or stem cell niche; reviewed in [1]), signal transduction pathways (reviewed in [2]), cytokines (reviewed in [3]), transcription factors (reviewed in [4]), epigenetics, (reviewed in [5]) and metabolic pathways (reviewed in [6]). HSCs are rare, constituting only 0.001% of peripheral blood (PB) and 0.05% of BM cells, but are responsible for producing a lifetime supply of blood cells. HSCs are cells that able to durably self-renew whilst also being multipotent. This differentiation is generally considered to occur via several intermediate progenitor cells, ultimately terminating in the specific mature blood cell through a process termed fate restriction or lineage commitment.
\nThe compartmentalization of HSC, their progenitors and terminally differentiated blood cells, into different stages of differentiation, is traditionally based on the expression of cell surface proteins (Figure 1). The recent emergence of single cell technologies such as fluorescent in situ hybridization, high-throughput single-cell quantitative PCR, single cell mass spectrometry and mass cytometry however, have led to re-analysis of these models of hematopoietic differentiation [7]. Discrete progenitor cell populations, as determined by cell surface markers, have been shown to consist of heterogenous populations with different fates [8]. Recently, a study by Velten et al., 2017, using a combination of single cell technologies and xenotransplantation as functional validation, proposed that early hematopoiesis consists of, a cellular continuum of low-primed undifferentiated (CLOUD) hematopoietic stem progenitor cells (HSPC), with simultaneous lineage gene expression for multiple fates [9]. This study suggested that early discrete stable progenitors do not exist, with any lineage determination occurring further downstream than originally presumed.
\nHuman hematopoiesis. Schematic diagram showing classical model of hematopoietic lineage commitment, with phenotypical cell surface markers (red), transcription factors determining differentiation (green box) and growth factors involved in myelopoiesis (blue). Hematopoietic stem cell (HSC), cluster of differentiation (CD), hematopoietic progenitor cell (HPC), common myeloid progenitor (CMP), common lymphoid progenitor (CLP), interleukin (IL), granulocyte macrophage (GM) colony-stimulating-factor (CSF), stem cell factor (SCF), thrombopoietin (TPO), erythropoietin (EPO), granulocyte myeloid progenitor (GMP), runt-related transcription factor 1 (RUNX1), transcription factor stem cell leukemia (SCL), ccaat enhancer binding proteins (C/EBP), friend of GATA protein 1 (FOG-1).
Regardless of provenance, leukemogenesis is characterized by a block in differentiation and an accumulation of immature white blood cell blasts with a rapid increase in these blasts, characteristic of the acute leukemias. Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are heterogenous diseases with a block in lymphoid or myeloid differentiation, respectively. They occur due to one or more genetic insults. Whilst ALL is predominantly a disease of children (80%), with a greater than 90% 5 y survival rate [10], in adults long term survival stands at only 30–40% [11]. AML in contrast is primarily a disease of the elderly, and like adult ALL it’s 5 y survival rate is around 30%, however this falls in the over 60’s to a particularly bleak 10% [12]. In ALL, recent advances for example in the use of tyrosine kinase inhibitors and CAR-T cell therapy, have started to suggest improvements to overall survival [10]. However, in patients fit enough to tolerate chemotherapy, the standard treatment for AML since 1973 has been a seven-day continuous intravenous infusion of cytarabine (Ara-C) (100–200 mg/m2) and 3 daily doses of daunorubicin (45–90 mg/m2), sometimes followed by allogeneic or autologous stem cell transplantation, and despite some recent advances (reviewed in [13, 14]), current treatments appear to have reached their efficacious limits and new therapies are required.
\nOne potential therapeutic opportunity involves exploiting the metabolic differences that exist between malignant and non-malignant cells [15]. Differences that, in AML at least, appear exacerbated by cellular levels of reactive oxygen species (ROS) [16].
\nROS is the collective term for several oxygen containing free radicals and other reactive molecules, such as hydrogen peroxide (H2O2). Physiologically, ROS are initially generated via the univalent reduction of molecular oxygen which generates superoxide (O2•−). Superoxide (t1/2 = 1 μs) subsequently dismutates to H2O2 (t1/2 = 1 ms) [17], either spontaneously or via the catalytic action of the enzyme superoxide dismutase (SOD), or reacts with other ROS molecules, forming a variety of other ROS (Figure 2). Functionally, ROS is important in innate immunity, protein folding in the endoplasmic reticulum and as a cell signalling molecule involved in cellular proliferation, survival, differentiation and gene expression [18].
\nFormation of reactive oxygen species (ROS). Diatomic oxygen (O2) is univalently reduced by peroxisomes (PO), xanthine oxidase (XO), the electron transport chain (ETC), or NADPH oxidase (NOX) to generate superoxide (O2\n•−). PO may also reduce O2 directly to form H2O2. O2\n•− may then dismutate to H2O2 either spontaneously or through the enzymatic action of superoxide dismutase (SOD). Hydroxyl radicals (OH•) may then be formed from H2O2 via the formation of hypochlorous radical (HOCl) in the PO, or via Fenton chemistry. Reactive nitrogen species (RNS) may also be formed through the reaction of nitric oxide radical (NO•) with O2\n•−.
There are several sources of cellular ROS, including the mitochondria, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOX), the cytochrome P450 enzymes, peroxisomes and the metabolic enzyme xanthine oxidase (XO).
\nGeneration of ROS by the mitochondria is primarily a function of ‘electron leakage’ from the electron transport chain (ETC), however, mitochondrial ROS may also be generated as a result of numerous enzymes including monoamine oxidase, cytochrome b5 reductase, glycerol-3-phosphate dehydrogenase, aconitase, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (reviewed in [19]). Mitochondrial ROS production resulting from the ETC generates O2\n•−, and is thought to occur as result of one of three mechanisms. The first mechanism is a consequence of a high NADH/NAD+ ratio, and results from oxygen interacting with fully reduced FMN. Mitochondrial ROS generated by this mechanism has been observed due to mitochondrial mutation, physiological damage such as ischemia or aging, and only small amounts of ROS are thought to be generated via these mechanisms in normally respiring cells [20]. The second mechanism occurs when there is a high level of reduced co-enzyme Q (CoQH2) in complex II, which in the presence of a high proton motive force generated by the proton pump, force electrons back into complex I in a process known as reverse electron transport (RET). Whilst RET generated ROS has also been implicated in diseases such as ischemia, it is now also thought to be involved as a cell signalling molecule in metabolic adaptation, myeloid differentiation and response to bacterial infection [21]. The third mechanism of ROS generation by the ETC occurs at complex III and has also been implicated in ROS signalling. The formation of O2\n•− occurs at the ubiquinol oxidation centre (Qo) site of the cytochrome bc1 complex, in which fully oxidized CoQ supports formation of O2\n•−, through the transfer of electrons from reduced heme b1 to molecular oxygen [22]. Generation of O2\n•− by complex I and II occurs exclusively in the mitochondrial matrix, whereas O2\n•− generated by complex III also occurs in the intermembrane space. O2\n•− generated in the mitochondrial matrix is rapidly converted to H2O2 by mitochondrial SOD (Mn-SOD), whereas O2\n•− generated in the intermembrane space travels through the outer mitochondrial membrane prior to conversion to H2O2 by cytosolic SOD (Cu/Zn-SOD).
\nWhilst mitochondrial oxidative phosphorylation is a major source of intracellular ROS, the main source of extracellular ROS involves the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOX). The NOX family of enzymes comprise of seven members, NOX1–5 and dual oxidase (DUOX) 1 and 2. NOX enzymes are transmembrane proteins that transfer electrons from NADPH to molecular oxygen, generating O2\n•− (or H2O2), which can then be converted to other forms of ROS. Different NOX isoforms share conserved structural features comprising of six helical transmembrane domains (TM) (with helix III and helix V containing two heme-binding histidines), and a C-terminus cytosolic domain (DH), which allows binding of FAD and NADPH (Figure 3). Difficulties in obtaining suitable levels of NOX proteins mean that to date relatively little crystal structure data is available. However, a recently published report [23], has elucidated the structure of the TM and DH domains (common to all NOX isoforms) of Cylindrospermum stagnale NOX5 in complex with FAD. In this structure, the six transmembrane helices of TM domain form a pyramidal shape with the base on the cytosolic side, a N-terminus α-helix runs parallel to the cytosolic side of the membrane and the heme groups sit in cavities formed by helices II–V, so that one is positioned near the cytosolic side of the membrane (heme 1) and the other on the outer side (heme 2). The DH domain, located on the cytosolic side, contains two pockets, one for FAD binding and one for NADPH binding. The FAD is positioned so that the flavin is in direct contact with heme 1 of the TM, to promote interdomain electron transfer. The proposed mechanism of electron transfer then involves NADPH donating its electron to FAD, which in turn donates an electron to heme 1 and then to heme 2 via Trp378 (equating to Phe 215 in human NOX2, Phe 200 in human NOX4 and Val 362 in human NOX5) of the loop between helix II and III of the TM, before reduction of molecular oxygen, via a final electron transfer step generates O2\n•− (Figure 3).
\nGeneration of superoxide (O2\n•−) by NADPH Oxidase (NOX). Schematic diagram showing the major structural features of NOX2, it’s activation by phosphorylation (P) of p67phoxand p47phox and the assembly of the major subunits of the NOX complex, and the generation of superoxide via electron transfer from NADPH to flavin adenine dinucleotide (FAD) to heme groups to diatomic oxygen. Guanosine triphosphate (GTP), guanosine diphosphate (GDP), homology domain (DH), RAS-related C3 botulinum toxin substrate 2 (Rac2).
From a metabolic perspective, one source of NOX2 activation results when cells experience intermittent hypoxia. Under this condition activation of the metabolic enzyme XO, an enzyme important in the catabolism of purines and a major source of cellular ROS, occurs [24]. XO activation leads to increased ROS, which induces Ca2+ activation of protein kinase C, an enzyme important in cell signalling, migration of p47phox and p67phox to the cell membrane, resulting in activation of the NOX2 complex (Figure 3). Finally it is important to note, from a cell signalling perspective, that extracellular H2O2 (which is rapidly formed from O2\n•−) is readily transported across the cell membrane via the transmembrane water permeable channel protein family of aquaporins [25, 26].
\nROS has been implicated in both HSC quiescence and hematopoietic differentiation. HSC reside in the bone marrow and their quiescence is known to be negatively regulated by ROS. Forkhead box O (FOXO) transcription factors are involved in cell-cycle arrest and apoptosis and are activated in response to oxidative stress whereupon they translocate to the nucleus [27]. Translocation of FOXO4 to the nucleus has been shown to be a function of redox signalling, where oxidation of cys-239 by ROS mediates the formation of disulphide bonds with nuclear import receptor transportin-1, which in turn allows nuclear localization [28]. FOXO deactivation occurs as a result of phosphorylation in response to activation of the regulatory cell cycle PI3K/AKT/mTOR pathway, resulting in their export from the nucleus and subsequent degradation in the cytoplasm [29]. Studies in murine HSC have shown that deletion of FOXO3a, which upregulates transcription of Mn-SOD [30], results in decreased HSC renewal [31] which is mediated by the tumor suppressor protein ataxia-telangiectasia mutated (ATM) and is accompanied by elevated ROS levels and myeloid lineage expansion [32]. Deletion of ATM in mice resulted in BM failure which was restored following treatment with antioxidants [33]. In a different study, isolation of murine HSC into ROS high and ROS low populations showed that the ROS low population maintained self-renewal capacity following serial transplantations, whilst the self-renewal capacity of the ROS high population was exhausted following the third serial transplantation. Treatment of the ROS high HSC with the antioxidant N-acetyl cysteine (NAC), the p38 inhibitor SB203508 or rapamycin (a mTOR inhibitor), restored self-renewal activity [34]. Interestingly, the ROS high population in this study also exhibited a decreased ability to adhere to cells containing calcium sensing receptors, whilst NOX generated ROS has additionally been implicated in osteoclast differentiation in human mesenchymal cells, further emphasizing a potential regulatory role of ROS, in the BM niche [35].
\nWhilst these increased ROS levels are associated with HSC losing quiescence, it has also been shown, in the human megakaryocytic cell line MO7e, that hematopoietic cytokines, such as granulocyte macrophage-colony stimulating factor, interleukin-3, stem cell factor and thrombopoietin all increase ROS levels [36]. In megakaryopoiesis, ROS has been shown to increase platelet production and maturation in the chronic myeloid leukemia (CML) cell line MEG-01 and primary human megakaryocytes [37], which in murine models is mediated by the transcription factor NF-E2 [38]. Following lineage commitment, megakaryocyte progenitors undergo endomitosis (chromosomal replication in the absence of cell division), which in murine cells is potentially mediated by NOX1-derived ROS [39]. In human HSC, NOX-derived ROS has also been shown to be crucial for megakaryocyte differentiation via activation of ERK, AKT and JAK2 signalling pathways [40], whilst another study revealed the importance of cytochrome P450 2E1-generated ROS in megakaryocyte differentiation in human HSC [41]. As noted above, increased ROS in HSC has been associated with expanded myelopoiesis. Interestingly, a recent study using murine CMP, showed that higher levels of ROS impeded megakaryopoiesis, instead directing differentiation of CMP into GMP [42]. Finally, ROS has also been shown to induce differentiation of the promonocytic cell line, U937, into macrophages [43], and the differentiation of primary human monocytes into dendritic cells [44].
\nOne of the first studies implicating ROS in carcinogenesis was performed in mice subcutaneously injected with C3H mouse fibroblasts, that had been previously cultured in vitro with neutrophils stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated or unstimulated or with the ROS generating enzyme XO and hypoxanthine. In this study approximately 20% of mice treated with these cells developed tumors within 13–22 weeks compared to none of the control mice [45]. In 1991, analysis of H2O2 production in human melanoma, colon, pancreatic, neuroblastoma, breast and ovarian cancer cell lines, revealed constitutively active H2O2 production over a 4 h period, generating H2O2 levels similar to those observed in TPA stimulated neutrophils, suggesting increased ROS production may be a feature of transformation [46]. Later, studies in patients with liver disease suggested ROS plays a part in hepatocarcinogenesis [47], and levels of Cu/Zn-SOD are significantly lower in hepatoma tissue than normal human liver tissue [48]. Further, homozygous deletion of Cu/Zn-SOD in mice results in decreased lifespan, with 70% developing hepatocarcinoma or benign nodular hyperplasia [49], whilst homozygous deletion of Mn-SOD in mice is lethal within two weeks of birth [50]. In the same study, heterozygous deletion of Mn-SOD resulted in increased incidence of hemangioma and adenocarcinoma and significant increases in the incidence of lymphoma. Currently, elevated ROS levels have been reported in many solid tumors and the role they play in tumorigenesis is complex and multifaceted (reviewed in [51]).
\nIn leukemia, a study which collected blood samples from ALL and CML patients samples and compared them with normal blood samples showed elevated levels of ROS in both ALL and CML patients [52], whilst elevated levels of NOX generated ROS, are observed, alongside increased proliferation in both AML models and AML patient samples when compared with healthy controls [53]. Reactions of ROS with DNA can generate numerous oxidised bases, including 8-hydroxy-2-deoxyguanosine (8-OHdG) which causes G:C to T:A DNA transversions (reviewed in [54]). Increased levels of 8-OHdG have been observed in patients with breast cancer [55], gastric carcinomas [56], lung cancer [57] and colorectal cancer [58]. In leukemia, a study of 116 Chinese children with either ALL or AML revealed significantly elevated levels of 8-OHdG, whilst 8-OHdG levels were also significantly elevated in relapsed AML adult patients [59].
\nAs a signalling molecule, ROS can lead to hyperactivation of the PI3K pathway, a common feature of many cancers, resulting in increased cell survival, VEGF production, secretion of MMP (reviewed in [60]) and inactivation of FOXO [32]. In AML, constitutive activation of the PI3K/AKT pathway is frequently observed [61, 62], however the role of FOXO is less clear. A recent study revealed that FOXO1 expression in osteoblasts mediated β-catenin initiated AML [63], whilst a study of AML patient samples showed that 40% exhibited FOXO activation, that upon inhibition resulted in myeloid differentiation and AML cell death [64]. Additionally, in both CML and AML the BCR-ABL fusion protein and FMS-like tyrosine kinase receptor 3 internal tandem duplications (FLT3-ITD) have been shown to lead to phosphorylation of AKT resulting in increased activation of NOX, and increased ROS production (reviewed in [65]), which may in turn reinforce PI3K/AKT activation.
\nBroadly defined, cellular metabolism involves a series of catabolic or anabolic chemical reactions which generate or use energy as part of this process. In chemotrophs this energy is obtained through the oxidation of nutrients, with the energy typically stored in the form of ATP. Whilst in higher organisms a plethora of enzymatically catalyzed metabolic reactions occur, which are all part of different interconnecting metabolic pathways with multitudinous feedback mechanisms. These pathways are evolutionarily highly conserved with the citric acid cycle, for example, essentially a feature in all terrestrial life. There are three main classes of molecules involved in metabolism; carbohydrates, proteins and lipids that are either catabolized to generate energy or energy stores or used by anabolic pathways in the synthesis of, for example, nucleotides and structural molecules such as cell membranes. In mammals, a triumvirate of glycolysis, citric acid cycle and the ETC are central to the generation of ATP, with glycolysis and the citric acid cycle contributing 2 ATP molecules each and the ETC generating up to 34 ATP molecules in a process collectively termed aerobic respiration (reviewed in [66]).
\nGiven the skew towards ATP production in the ETC, Otto Warburg’s observation in 1956 that aerobic glycolysis was a hallmark feature of cancer cells [15], was initially attributed to being the result of defective mitochondria in malignant cells, and initially raised little interest. However, this hypothesis is now known in most cases to be incorrect (reviewed in [67]) and instead, it has been shown that mitochondrial respiration is often necessary in tumorigenesis [68]. However, given its ubiquity and despite its inefficiency when compared with ETC, it is clear that the phenomenon of increased aerobic glycolysis (eponymously titled ‘The Warburg Effect’), must offer cancer cells some competitive advantage, although its exact ontology remains unclear. One hypothesis contends that whilst inefficient, aerobic glycolysis generates ATP at a rate 10–100 times faster than oxidative phosphorylation, therefore supplying cancer cells with energy at a faster rate. This increased glycolytic flux could then, potentially generate more nucleotides, amino acids and lipids for biosynthesis as well as generating the reducing agent NADPH, to deal with the increased levels of ROS common in many cancer cells [69]. Alternatively, increases in excreted lactate as a result of aerobic glycolysis would likely generate a more acidic microenvironment, breaking down stromal membrane structures and potentially increasing cancer cell motility and metastasis [70].
\nIt has been shown that activation of the tumor suppressor protein ATM by ROS promotes glucose-6-phosphate dehydrogenase (G-6-PD) activity, the first step of the pentose phosphate pathway (PPP), which in turn generates NADPH [71]. Given that major cellular antioxidant systems, ultimately rely on NADPH to provide their reducing power, it is perhaps not surprising that ROS in both normal and aberrant cellular processes is inextricably linked with metabolism. In the cytosol, NADPH is primarily generated through the PPP, whilst a number of mechanisms exist for mitochondrial NADPH generation [72], which include the serine synthesis pathway (SSP) (via the folate cycle) [73] and the action of the citric acid cycle enzyme isocitrate dehydrogenase (IDH). IDH1 and IDH2 are commonly mutated in AML [74], although in this context NADPH is consumed, and the D-2-hydroxyglutarate generated leads to stabilization of the hypoxia regulator, hypoxia inducible factor alpha (HIF-1α) [75].
\nHIF-1α as a target of ROS is controversial [76], however it is overexpressed in many cancers where it induces expression of numerous glycolytic genes. The ROS regulated transcription factor nuclear-related factor 2 (NRF2) has also been shown to modulate metabolism in lung cancer cell lines, through the upregulation of enzymes involved in the NADPH production, notably G-6-PD, IDH1 and malic enzyme 1 [77] and high NRF2 levels have previously been reported in AML [78]. Furthermore, the tumor suppressor protein TP53 is also important in regulating metabolism. Homozygous deletion of TP53 in mice results in decreased oxygen consumption arising from decreased mitochondrial respiration [79]. TP53 expression has been shown to inhibit, both glucose transporter (GLUT) 1 and 4 and the glycolytic enzyme phosphoglycerate mutase (PGAM) (reviewed in [80]) leading to decreased glycolysis and potentially increased metabolism via the PPP and SSP. Finally, TP53 also upregulates the apoptosis regulator (TIGAR) an enzyme which has an active domain similar to 6-Phosphofructo-2-kinase/fructoste-2,6-bisphosphatase (PFKFB). TIGAR catalyzes the reaction of fructose-2,6-bisphosphate (F-2,6-BP) to fructose-6-phosphate (F-6-P), which inhibits glycolysis, redirects metabolites into the PPP, generating NADPH [81].
\nChanges of cellular ROS levels in both normal signalling as well cell signalling following cellular transformation result in changes in numerous signalling pathways controlling multiple cellular functions including growth, proliferation and differentiation. A number of these signalling pathways, exercise regulatory control over various metabolic pathways, which in turn modulate ROS levels via several feedback mechanisms (Figure 4). In leukemia, mutations in the RAS gene are present in about 15% of hematological malignancies [82]. RAS activates the PI3K/AKT/mTOR pathway which promotes nucleotide biosynthesis and lipid synthesis (reviewed in [83]) as well as HIF-1α, which upregulate glycolysis via the activation of numerous glycolytic genes. In addition to HIF-1α, other ROS activated transcription factors are important in metabolic regulation such as STAT3, which has been shown to promote glycolysis in hepatocellular carcinoma cell lines [84], FOXO3A, which inhibits glycolysis via activation of tuberous sclerosis 1 protein [85] and NF-κB which was shown to upregulate GLUT3 in mouse embryonic fibroblasts [86].
\nRegulation of metabolic pathways. Schematic illustration outlining some of the regulatory mechanism involved in glycolysis and other key metabolic pathways. Transcription factors are in pink and signalling pathways in blue. Reactive oxygen species (ROS), forkhead box O (FOXO), pyruvate kinase muscle 2 (PKM2), signal transducer and activator of transcription (STAT), nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), glucose transporter (GLUT) hypoxia inducible factor-1 alpha (HIF-1α), tumour suppressor protein 53 (TP53), glycogen synthase kinase 3β (GSK-3β), isocitrate dehydrogenase (IDH), succinate dehydrogenase (SDH), fumarate hydratase (FH), protein kinase B (AKT), mammalian target of rapamycin (mTOR), phosphoinositide 3-kinase (PI3K), synthesis of cytochrome c oxidase 2 (SCO2) and prolyl-hydroxylase domain (PHD).
Nuclear localization of the glycolytic enzyme pyruvate kinase muscle 2 (PKM2) is also ROS mediated, where it acts as a co-factor in the activation of the transcription factor, c-MYC. RAS also activates c-MYC which is overexpressed in greater than 50% of human cancers and c-MYC has been shown to activate glycolysis via the upregulation of GLUT, the glycolytic enzymes hexokinase (HK), phosphoglucose isomerase (PGI), phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), PKM2, as well as lactate dehydrogenase A (LDHA), pyruvate dehydrogenase kinase 1 (PDK1) and PFKFB3 (reviewed in [87]). Increased glutaminolysis is also a target of c-MYC, which upregulates the glutamine transporter ASCT2 and a key enzyme glutaminase. Additionally, c-MYC was shown to upregulate both phosphoglycerate dehydrogenase (PHGDH) which catalyzes the first step of the SSP, serine hydroxymethyltransferase, part of the folate cycle as well as several genes involved in fatty acid metabolism and the citric acid cycle (reviewed in [67]). In contrast TP53 is known to inhibit glycolysis through inhibition of GLUT1, GLUT4 and PGAM and through activation of TIGAR and synthesis of cytochrome c oxidase 2 (SCO2). Inhibition of glycolysis also occurs due to the regulatory role of miRNA. For example, miR-195-5p inhibits GLUT3, miR-143 inhibits HK2 and miR-155 inhibits HIF-1α. Furthermore, TP53 induces miR-34a which suppresses HK1, HK2, GPI and PDK1, as well as sirtuin 1, which activates FOXO1, NF-κB and in a positive feedback loop TP53 (reviewed in [80]).
\nGiven the role that ROS plays in regulating metabolism, it is not surprising that expression of nearly all enzymes associated with glycolysis have been shown to be altered in solid tumors, a pattern also observed in leukemia. In ALL, micro-array analysis showed significant upregulation of PFK as well as the glucose transporters GLUT1 and GLUT4 in pediatric B-ALL samples [88], whilst deletion of GLUT1 in primary human B-ALL cells suppressed leukemic progression in vivo [89]. In AML, upregulation of GLUT1 mRNA [90] and the fructose transporter GLUT5 [91] have also been reported to be associated with poor outcome in AML patients. Furthermore, NOX generated ROS has previously been reported to modulate cellular glucose uptake through increased GLUT1 activity, in leukemic cell lines [92]. In Philadelphia+ ALL (Ph+ALL) GLUT5 has been found to be upregulated at both the mRNA and protein level [93]. Song et al have identified HK2 overexpression as a feature of AML patients who failed to show remission [90], whilst decreased proliferation in the AML cell line, KG-1, was observed upon knock-down of PGI with shRNA [94]. The HK inhibitors 2-deoxy-D-glucose and 3-bromopyruvate have both been shown to be cytotoxic in AML patient samples harboring a FLT3-ITD mutation both alone and in combination with sorafenib [90, 95]. In chronic lymphocytic leukemia (CLL), a study by Ryland et al., 2013 showed increased expression of glyceraldehyde phosphate dehydrogenase (GAPDH) in CLL patients compared to healthy controls [96]. Proteomic studies revealed elevated levels of aldolase A (ALDO(A)), ALDO(C) and enolase 1 (ENO1) in the chemoresistant leukemia cell line K562/A02 when compared with parental K562 cells and in the case of ENO1 this was confirmed by western blot [97]. Elevated levels of ENO2 have also been reported in patients with ALL where it is associated with lower overall survival [98], whilst PGAM is upregulated in both AML and CML patient samples [99]. LDH is a tetramer which exists as five isoforms, comprising of two subunits LDHA and LDHB in different combinations and encoded by the LDHA and LDHB genes [100], with LDHA strongly catalyzing pyruvate to lactate and LDHB preferentially catalyzing the reverse reaction. In B-ALL, mRNA expression levels of LDHB were shown to be decreased [88], suggesting increased lactate production, whilst more recently increased serum levels of LDH were found in patients with B-ALL in conjunction with increased levels of total oxidant status and decreased total anti-oxidant status [101]. Another recent study involving 204 patients with acute leukemia’s also reported that LDH plasma levels were significantly elevated compared to healthy controls and were also increased in relapse patients compared to those in complete remission [102]. Recently, it was shown that ROS dependent proliferative increases observed in hematopoietic models [103] were also accompanied by increased glucose uptake and expression of the regulatory glycolytic enzyme PFKFB3 [53], whilst downregulation of this enzyme suppressed growth both in vivo and in vitro [16]. This study also reported that metabolomic analysis comparing AML patient samples with high/low levels of ROS, which showed significantly elevated levels of glucose, glucose-6-phosphate (G-6-P) and F-6-P in the ROS high patients. Another metabolomic study involving serum from 400 AML patients compared with 446 healthy controls, identified elevated levels of the glycolytic intermediates 3-phosphoglycerate (3-PG), pyruvate and lactate as conferring a poor prognosis for survival [104]. Interestingly, a recent study showed that the bromodomain and extra-terminal protein inhibitor JQ1, which has shown promise in ALL by targeting c-myc, downregulates expression of HK2, PKM2 and LDHA both at the transcriptional and protein level [105].
\nThe citric acid cycle is a series of metabolic reactions involving oxidation/reduction reactions, which generate nicotinamide adenine dinucleotide (NAD)H and flavin adenine dinucleotide (FAD)H via the transfer of hydride ions, thus providing electrons for the ETC which is a major source of cellular ROS (reviewed in [106]). Mutations of IDH, which catalyzes the decarboxylation of isocitrate to alpha-ketoglutarate are frequently reported in AML (reviewed in [107]). Characterization of the inhibitor AG-221, which has been shown to inhibit mutant IDH2 in AML cells in vitro and in vivo and is currently undergoing phase I/II clinical trials [108], as is the IDH1 inhibitor, AG-120 [109]. A metabolomic study which examined a cohort of 183 patients with de novo AML matched with 232 healthy controls showed significant differences in citrate levels between AML patients and controls [110]. In pediatric ALL a recent metabolomic study revealed increased metabolites of glycolysis, the citric acid cycle and the PPP in patients testing positive for measurable residual disease compared to those testing negative [111]. Interestingly use of nicotinamide phosphoribosyltransferase (NAMPT) inhibitors on ALL cell lines and patient samples showed cytotoxicity in vitro. NAMPT is a key enzyme in the synthesis of the oxidizing agent NAD+, in both glycolysis and the citric acid cycle.
\nThe SSP branches from the glycolytic pathway at the glycolytic intermediate 3-PG, where it is converted into 3-phosphohydroxypyruvate by the enzyme PHGDH, followed by conversion to phosphoserine by phosphoserine aminotransferase 1 and finally to serine by the action of the enzyme phosphoserine phosphatase (reviewed in [73]). Regulation of the SSP is achieved through 2-phosphoglycerate (2-PG) which activates PHGDH whilst serine activates the tetrameric form of PKM2 leading to increased glycolysis and decreased levels of 2-PG. Importantly serine can enter the folate cycle, which provides another route for the generation of NADPH, which has been shown to contribute to tumor growth in vivo [112]. Whilst overexpression of PHGDH has been reported in melanomas, colorectal and breast cancers, little has been published from a leukemia perspective. Knock-down of PHGDH has been shown to inhibit the growth of the leukemia cell line, HL-60 [113], and in multiple myeloma increased expression of PHGDH led to increased SSP activity and antioxidant capacity in cells resistant to treatment with the proteasome inhibitor bortezomib [114].
\nThe PPP generate nucleotides for biosynthesis and is a major source of cellular NADPH, an important cellular antioxidant. The first step involves the dehydrogenation of G-6-P to 6-phosphogluconolactone (6-PG) catalyzed by G-6-PD and the conversion of NADP+ to [115]. Gluconolactonase catalyzes the hydrolysis of 6-PG to 6-phosphogluconate, which is then catalyzed by 6-phosphogluconate dehydrogenase (6-PGD) to ribulose-5-phosphate (Ru-5-P) alongside the generation of a second NADPH. Ru-5-P can then be converted into ribose-5-phosphate (R-5-P) by the enzymatic action of ribulose-5-phosphate isomerase. R-5-P can then be used in the synthesis of nucleotides. Alternatively, where redox homeostasis and not nucleotide synthesis is the major requirement of the cell Ru-5-P can be catalyzed by ribulose-5-phosphate epimerase, into xyulose-5-phosphate (X-5-P) and via a series of further metabolic reactions back into the glycolytic intermediates F-6-P and glyceraldehyde-3-phosphate. G-6-PD is the rate limiting step of the PPP and is regulated by the NADP+/NADPH ratio, RAS/PI3K signalling and phosphorylation by Src, whilst 6-PGD is inhibited by 3-PG [99]. In cancer, aberrant RAS signalling or activation of Src can promote activation of the PPP. In AML, a recent study showed upregulation of G-6-PD mRNA in approximately 60% of patients, although it was not correlated with overall survival or relapse [116]. Targeting of xenograft mice injected with the leukemic cell line K562, with the antimalarial drug dihydroartemisinin and the 6-PGD inhibitor Physicon resulted in decreased tumor growth, whilst primary leukemia cells isolated from the PB of AML patients showed significantly decreased viability, with no toxicity observed in hematopoietic cells isolated from healthy individuals [117]. A metabolomic study comparing primary AML samples with either high or low levels of ROS, have also shown increased levels of the PPP metabolites sedoheptulose-7-phosphate and Ru-5-P in the samples with higher ROS levels [16]. Another study, using both AML cell lines and patient material, showed increased glucose metabolism and increased flux through the PPP, alongside increased G-6-PD mRNA expression [118]. Importantly, this study showed that use of the G-6-PD inhibitor 6-aminonictoinamide (6-AN) in AML cell lines induced both in vitro and in vivo cytotoxicity, and induced apoptosis in primary AML cells but not normal HPCs. In B-ALL, redirection of carbon from the glycolytic pathway to the PPP by the serine/threonine-protein phosphatase 2A (PP2A), has been shown to occur to combat cellular oxidative stress. Synergistic inhibition of G-6-PD by 6-AN and PP2A inhibitor LB100 induced cell death in patient derived Ph+ALL [119].
\nLipid metabolism has also been shown to be dysregulated in both solid tumors and hematological malignancies (reviewed in [120]). Increased fatty acid oxidation (FAO) allows cancer cells to overcome metabolic and oxidative stress through the generation of ATP and NADPH. Significant changes to lipid metabolite levels are seen in AML patient samples with either high levels or low levels of ROS [16], whilst suppression of NOX2 has also been shown to increase FAO [121]. Furthermore, inhibition of the FAO using Avocatin B results in decreased NADPH levels and ROS dependent cell death in primary human AML samples but not normal mononuclear cells [122]. In ALL, use of L-asparaginase has been shown to increase FAO activity as a metabolic escape mechanism, however use of the FAO inhibitor etomoxir in combination with L-asparaginase has been shown to increase sensitivity of both leukemic cell lines and patient samples [123].
\nIn the last twenty years, it has become increasingly clear that ROS play a significant role in cellular signalling, particularly pathways associated with growth, differentiation and survival, whilst its roles in HSC quiescence and normal hematopoiesis have started to be delineated. In many cancers including hematological malignancies, ROS levels have been shown to be elevated, leading to aberrant signalling in these pathways. Previously, arguments for both the use of anti-oxidant and pro-oxidant treatments in leukemia have been made (reviewed in [124]). Despite the transformation of survival rates in patients with acute promyelocytic leukemia using arsenic trioxide [125] cancer cells often upregulate the production of antioxidants, and downregulate pro-apoptotic pathways such as TP53, as a response to high ROS, allowing them to escape apoptosis. In addition, it has been shown that both cancer stem cells [126, 127] and leukemic stem cells [128] exhibit low ROS levels, suggesting that even if treatment with pro-oxidants eliminates the bulk of cancer cells, cancer/leukemic stem cells may survive and relapse occur. Conversely, studies involving the use of antioxidants in treatment and epidemiological studies of antioxidant use, have shown mixed results (reviewed in [129, 130]). Increasingly it is becoming apparent that increased levels of ROS are leading to changes in signalling pathways directly or indirectly controlling metabolism, as a mechanism for managing oxidative stress. Whilst, it has long been known that cancer cells exhibit greatly altered metabolism, only recently have the purposes behind this altered metabolism, started to be elucidated. Consequently, synergistic treatments involving the use of metabolic inhibitors, alongside classical treatments for leukemias are being explored. Future work, elucidating the intricate mechanisms governing the interplay between ROS and metabolism, alongside new and more specific metabolic inhibitors provide much promise for the future treatment of leukemia.
\nWe are grateful to Blood Cancer UK for programmatic funding and to Tenovus Cancer Care for funding Andrew Robinson. We are grateful to Wellcome ISSF for funding aspects of ROS research. We are grateful for support from the NCRI AML trials cell bank and the AML patients for providing primary samples used in several of our studies.
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