Structural and functional alterations in epicardial coronary artery vessels are the most frequent and important causes of myocardial ischemia and angina symptoms. In recent years, however, it has become evident that abnormalities of coronary microcirculation can also cause an impairment of coronary blood flow (CBF) and result in myocardial ischemia.1,2 While large epicardial coronary vessels (>500 μm in diameter) have only a conductive function of blood flow, with pressure loss being trivial along their course, coronary microcirculation is the principal site of coronary resistance, about 20–30% being localized in prearterioles (artery vessels of 100–500 μm in diameter) and about 40–50% in arterioles (vessels of <100 μm in diameter) (Fig. 1).3 Of note, coronary microcirculation represents more than 90% of the whole coronary circulation and is the place where most of the regulation of CBF occurs.2
Coronary microvascular dysfunction (CMVD) can occur either in the context of specific cardiac or systemic disease (secondary CMVD) or independently of any other disease (primary CMVD).2 The distinction can be relevant, as secondary CMVD may present distinct pathogenetic mechanisms, in some way related to the underlying disease, whereas the mechanisms of primary CMVD remain largely to be elucidated. Moreover, although CMVD in patients with primary stable microvascular angina (MVA) seems usually associated with a good prognosis4,5 and its prognostic value in those with primary unstable MVA2 is under scrutiny,6,7 the detection of secondary CMVD has been shown to be of prognostic value, at least in some specific cardiac diseases.8,9
Accordingly, the identification and characterization of CMVD can be important not only for progression in the knowledge of coronary pathophysiology but also because it can be potentially relevant for the therapeutic approach to symptomatic patients and for risk stratification in specific clinical settings. A classification of CMVD according to the clinical context in which it can occur has recently been suggested and is shown in Table 1.1
In contrast to the study of epicardial vessels, the assessment of coronary microcirculation cannot be done by coronary angiography, as small coronary arteries have dimensions below the resolution of angiographic techniques. Thus, structural abnormalities of these vessels might only be assessed on endomyocardial biopsy samples;10,11 the latter, however, are not ethically and clinically justified in most patients and can be unfruitful.
In contrast, several methods have been proposed for the investigation of the functional state of coronary microcirculation.12 The assessment of coronary microvascular (CMV) function, however, presents several issues. First, as coronary microvessels cannot be visualized by imaging techniques, CMVD can only be identified indirectly through measurements of changes in CBF and in coronary vascular resistance in response to appropriate challenges. Second, CMVD can be difficult to demonstrate when it involves only small regions of the coronary circulation. Third, small coronary arteries are influenced by several vasodilator and vasoconstrictor substances; thus, it can be difficult to achieve full assessment of coronary microcirculation in individual patients. Finally, the physiologic state of coronary microcirculation depends on a great number of factors (e.g. heart rate, blood pressure, inotropic state of the myocardium, ventricular mass, etc.), which suggests prudence in the interpretation of interindividual differences in the parameters measured to assess CMV function. Despite these limitations, if attention is paid in guaranteeing standard conditions and accurate measurements during the tests, sufficiently reliable information can be obtained on the functional state of coronary microcirculation.
Although there is clear-cut evidence that CMVD plays a pathophysiological and clinical role also in patients with obstructive CAD,13 the discrimination between the effects of macrovascular and microvascular coronary abnormalities on CBF impairment is difficult to achieve. Thus, at present, the assessment of CMVD in clinical practice can mainly concern patients in whom coronary angiography shows normal or near-normal epicardial coronary arteries.
Several methods have been proposed to assess CMV function in these patients,14 including invasive gold standard methods. However, in recent years technological advances have led to the development of several noninvasive imaging techniques that allow sufficiently reliable measures of CMVD. The aim of this review is to summarize the state of the art about the methods proposed to investigate CMV function and compare their strengths and weaknesses for their application in clinical practice.
Vasoactive stimuli for assessment of coronary microvascular function
The most frequent method used to assess CMV function is to measure the vasodilator capacity of the coronary microcirculation in response to dilator stimuli. Typically, the stimulus can be administered at doses presumed to achieve maximal vasodilation and reduction of coronary resistances. The vasodilator capacity is then usually measured as the ratio of CBF during maximal vasodilation over basal CBF, which corresponds to the coronary flow reserve (CFR).
Vasodilator stimuli can directly assess the relaxing response of vascular smooth muscle cells (SMCs) (endothelium-independent vasodilation), or they can assess the vasodilator response mediated by endothelial release of relaxing factors, mainly nitric oxide (endothelium-dependent vasodilation), or both.
A complete characterization of CMV function would also require, however, at least in some patients, the assessment of CMV response to vasoconstrictor stimuli. In this case, however, only tests carried out during an invasive study can be considered reliable, as the exclusion of significant vasoconstriction of epicardial vessels during the test can only be obtained by angiography. The main vasodilator and vasoconstrictor stimuli that have been utilized in clinical studies are summarized in Table 2.
The most widely used substance to assess CFR is adenosine. Adenosine is a major physiologic regulator of CBF. It is usually released by myocardial cells during increased oxygen consumption, following ATP degradation, and induces arteriolar dilatation by directly acting on SMC A2 receptors. To assess CFR, adenosine is administered at a dose of 0.14 μg/kg per min, which has been suggested to achieve maximal vasodilation.15 Possible side effects include bradycardia, due to atrioventricular or sinoatrial node blockade, and bronchoconstriction, both mediated by A1 receptors. A relevant advantage of adenosine is its very short half-life (10 s), which allows rapid regression of side effects and repetition of the test during the same session.
A frequently used substance to assess endothelium-independent CMV dilation is also dipyridamole, which has similar effects to adenosine; indeed, it acts by inhibiting adenosine degradation by adenosine deaminase in the tissues.16 A CFR less than 2.5 by either adenosine or dipyridamole is usually considered as diagnostic of impairment of CMVD, independent of the technique used to assess basal and hyperaemic CBF. Due to some significant variability in healthy individuals, however, a CFR less than 2.0 might be more a more specific and diagnostic value.
Acetylcholine is the most widely used substance to assess endothelium-dependent CMV dilation.17,18 This substance, however, can only be used during invasive procedures, as it requires intracoronary infusion. Furthermore, acetylcholine is not the ideal drug to assess endothelium-mediated vasodilation because it can also act directly on SMCs, inducing vasoconstriction.
Recently, the cold pressor test (CPT)19 has been used to assess endothelium-dependent coronary vasodilator function. CPT is a simple test that can be applied noninvasively. The test consists in putting a hand of the patient in ice water for 90–120 s. Cold and peripheral pain induce sympathetic stimulation that slightly increases heart rate and blood pressure; the consequent enhancement of myocardial oxygen demand increases arteriolar CBF and flow-mediated dilation of prearteriolar vessels. Stimulation of endothelial α-adrenergic receptors also leads to the release of nitric oxide, contributing to dilation of resistance arteries. Also in this case, however, a direct vasodilator effect through stimulation of SMC β2-receptors or an increased response of α-adrenergic-mediated constriction of SMCs might complicate the interpretation of the response to CPT. CPT is generally safe and well tolerated; rare side effects include the induction of vasovagal reflexes (with orthostatic syndrome and/or bradycardia). The normal increase of CBF in response to CPT has poorly been investigated, and significantly variable results have been reported in different studies, although it is lower than that seen with adenosine.19
The assessment of CMV function should be performed under strictly standardized conditions. It should be performed by skilled operators, preferably during the same period of the day (i.e. always in the morning or in the afternoon), after appropriate withdrawal of all potential vasoactive substances (including drugs, caffeine, alcohol and tobacco), and in a fasting state. All these conditions can be difficult to achieve when invasive methods are used, whereas they might more easily be followed with noninvasive methods. Basic hemodynamic parameters (e.g. heart rate and blood pressure) should be carefully recorded throughout the test.
Transthoracic Doppler echocardiography
Transthoracic Doppler echocardiography (TTDE) allows the measurement of CBF velocity, which is taken as an indirect measure of CBF (Fig. 2). CMV function can be usually assessed in the left anterior descending (LAD) coronary artery, which is easier to visualize compared with other coronary arteries; in several patients, however, the test can also be performed in the left circumflex coronary artery and in the posterior interventricular descending coronary artery (PDA).20,21
The arteries are visualized using color Doppler flow mapping guidance, with a velocity range of 12–16 cm/s. A high-frequency transducer (5–7 MHz) is used to visualize the mid-distal LAD, which is close to the chest. For the PDA and the circumflex arteries, which are deeper, a low-frequency transducer (3.5 MHz) is used instead.
CBF in the mid-distal part of the LAD artery is identified in a parasternal view, with the patient in the left lateral decubitus. CBF in the PDA artery, on the contrary, can be visualized from a modified apical two-chamber view, whereas CBF in the distal part of the circumflex artery (or its marginal branches) can be searched on the basal and mid-portion of the left ventricular lateral wall in the apical four-chamber view.20,21
CBF velocity is measured by pulsed wave Doppler echocardiography, using a sample volume of 3–4 mm placed on the color signal in the artery, with the incident angle kept as small as possible (below 40°). Diastolic CBF velocity measurements are performed offline by contouring the spectral Doppler signals, using the integrated software package of the ultrasound system. CBF velocity is measured at baseline and during vasodilator stimuli, and is usually determined as an average of three consecutive cardiac cycles (Fig. 2). CFR is measured as the ratio of hyperemic diastolic peak flow velocity during maximal vasodilator stimuli (mainly adenosine) to basal flow velocity. It is important to stress that during the administration of the vasodilator stimulus, the probe must be kept in the same position as at baseline, and machine settings, including size of sample volume and velocity scale, maintained constant.
Validation studies and normal values
Reproducibility studies have shown that both intraobserver and interobserver variability of TTDE–CFR performed by skilled operators does not exceed 5%.21 Interestingly, the few studies that assessed intraindividual variability of TTDE results have shown high reproducibility of measures, with a coefficient of variation not exceeding 6%.22,23
Several studies have investigated the reliability of TTDE in assessing CMV dilator function by comparing the results with those obtained with the gold standard method of intracoronary Doppler flow wire (ICDW) recording24–29 (Table 3). These validation studies, usually performed in patients undergoing coronary angiography for suspected CAD, have shown that TTDE is feasible in the majority of patients and show a high agreement with CFR obtained with ICDW recording, with correlation coefficients ranging from 85 to 97% and consistent concordance by Bland–Altman analysis.24–26
In most validation studies, CFR was assessed in the LAD coronary artery, in which TTDE allows valuable measurements of CBF velocity in 78–91% of the patients.24–26 The fewer studies performed in other arteries have shown that a reliable test could be obtained in 70–81% and 43–72% in the right and the circumflex coronary artery, respectively.27–29
In a small study on 10 healthy volunteers, CFR assessed by TTDE was compared with that obtained by PET, showing again a high agreement with the reference method.30
Accordingly, validation studies have shown good reliability in identifying patients with reduced CFR. This is usually identified by values lower than 2.0, which are unlikely to be detectable in apparently healthy individuals (Fig. 3).31 In these studies, average CFR values in response to adenosine ranged from 2.92 to 4.531–37 (Table 4), likely in relation to some differences among studies in age, sex and/or presence of cardiovascular risk factors.
TTDE has been used to assess CMV dilation in several clinical conditions known to be characterized by CMV dysfunction, showing results comparable to those found in the same groups of patients with other, more standardized methods. Although studies directly validating TTDE in these clinical conditions are lacking, the concordant results with studies using other methods further support the reliability of this simple noninvasive tool.
A significant reduction of CFR, in particular, has been documented with TTDE in patients with cardiovascular risk factors,38,39 those with idiopathic dilated cardiomyopathy (IDCM)40 or hypertrophic cardiomyopathy,41 and those with MVA31,42 or takotsubo syndrome.43
Importantly, TTDE has also been used to document the changes of CMV dilator function in response to drug therapy, in particular in patients with hypertension.38,39
Of note, a relation between reduced CFR assessed by TTDE and clinical outcome has been observed in preliminary studies in patients with dilated or hypertrophic cardiomyopathy.40,41 The utility of CMV function assessed by TTDE in predicting events in the different clinical settings, however, needs confirmation in larger studies.
Advantages and limitations
TTDE presents several advantages compared with other methods used for assessment of CMV function and CFR. It is totally noninvasive, easily available at bedside, not time consuming, cheap, and suitable for serial measurements to follow CMV function over time or check the effects of therapeutic interventions.
However, some important limitations of the method should be acknowledged. First, at present, there is sufficient evidence of its reliability only when CMV function is assessed in the LAD artery. Second, a valuable echographic window cannot be obtained in all patients. Third, the test requires appropriate experience by the operator.
Myocardial contrast echocardiography
Myocardial contrast echocardiography (MCE) is a noninvasive imaging technique, which exploits properties of intravenously administered microbubbles, which act as a contrast agent, to assess myocardial blood flow (MBF). On standard echocardiography, indeed, blood flow cannot usually be detected because red blood cells are very poor backscatters due to small dimensions and mild differences in acoustic impedance as compared to serum. Visualization of microbubbles is based on the peculiar ultrasound–bubble interaction. Because their dimension is smaller than the wavelength used in diagnostic echocardiography, microbubbles, such as red blood cells, would be unable to reflect transmitted ultrasound beam. However, they scatter ultrasound waves, producing a signal reliably detected by echocardiographic probes.44
Microbubbles are constituted by high molecular weight gases (nitrogen or fluorocarbon gas) encapsulated in a shell of phospholipids or albumin, and act as intravascular agents, as they do not cross the endothelial layer.44 When injected in a peripheral vein, microbubbles mix with blood and reach the right cardiac chambers. Then, due to their small sizes (1.1–8 μm), they freely pass throughout pulmonary capillaries and reach the left ventricular cavity and the coronary circulation, depicting the distribution of MBF.
When ultrasound energy, expressed as a mechanical index, is kept low within the diagnostic range, the scatter signal of microbubbles enhances detection of blood flow within the left ventricular cavity, but it is still too weak to be differentiated from the signal generated by the myocardial walls. By increasing the mechanical index, however, microbubbles begin to resonate, producing cycles of compression and rarefaction; this results in signals of low amplitude at a frequency (second harmonics) two times higher than the fundamental one, which can be detected by the probe and can therefore be processed to enable visualization of the myocardial blood pool.
Similarly to microspheres in the experimental setting, the signal derived from microbubbles is proportional to their concentration in the blood compartment. During continuous intravenous infusion, the signal is related to myocardial blood volume (MBV) and MBF. Accordingly, myocardial regions supplied by dysfunctional or damaged microcirculation appear as completely or partially not opacified (i.e. display perfusion defects).
Myocardial perfusion by MCE can be assessed by a semiquantitative method based on a scoring system, which grades each of 16 or 17 myocardial segments as 1, 2 or 3 when normal, reduced/delayed or absent opacification is seen, respectively. Furthermore, perfusion defects can be quantitated by measuring their endocardial length or transmural area and expressing them as a percentage of total endocardial border length or total myocardial wall area, respectively.
By plotting the curve of microbubble intensity over time, a quantitative measure of MBF can also be calculated as follows: y(t) = A (1 − e−βt), where y is the signal intensity, A is the plateau intensity (i.e. MBV), β reflects the rate of rise of intensity from microbubbles (hence, blood velocity), and t represents the pulsing interval. A reliable measure of MBF is provided by A × β.45 Measures of MBF by this method were found to be comparable to those obtained by PET.46 Of note, some software automatically encodes different grades of myocardial perfusion into various colors, thus providing a map of left ventricular perfusion.
To evaluate CMV dilator function by MCE, the contrast agent is given intravenously through an antecubital vein access, by continuous slow infusion (at the rate of 0.8–1 ml/min, depending on the concentration necessary to achieve the best compromise between clear visualization and avoidance of attenuation artifacts), which allows a better assessment of myocardial perfusion compared with bolus administration, which is instead usually sufficient for intracardiac shunt detection and intracavitary endocardial border delineation.
In the clinical setting, MCE has often been used to assess the state of coronary microcirculation in patients with acute myocardial infarction (AMI) undergoing successful recanalization of the infarct-related artery44,47–63 (Table 5).
The assessment of myocardial perfusion after successful percutaneous coronary intervention (PCI) aims at establishing the real effectiveness of the intervention. Indeed, it is now clear that an optimal recanalization of the infarct-related coronary artery does not always translate into an effective restoration of CBF at the microvascular level.64 This phenomenon, known as ‘no-reflow’, is caused by microvascular obstruction, which is the consequence of a complex series of events that include distal microembolization and ischemia/reperfusion injury of coronary microcirculation, with leukocyte plugging, red blood cell aggregates and vasospasm.65 When assessed by MCE, no-reflow appears as a black myocardial area localized within the dysfunctional segments (see above) (Fig. 4).
Thanks to the ability to easily repeat the test over time, MCE has contributed to clarify some important pathophysiological aspects of no-reflow. In particular, it has been demonstrated that no-reflow is a process characterized partly by structural and irreversible damage of coronary microcirculation and partly by functional abnormalities that may resolve over time. Indeed, about 50% of AMI patients exhibiting no-reflow after PCI show improvement in myocardial perfusion at 1 month follow-up, suggesting a relevant functional component of the microvascular damage. In the remaining 50% of the patients, however, no improvement is shown at follow-up, suggesting sustained, irreversible microvascular damage. Importantly, the persistence of perfusion defects predicts postinfarct left ventricular remodeling52 and a higher incidence of major adverse events,66 whereas recovery of myocardial perfusion parallels improvement of contractile function,51,67 reverse left ventricular remodeling68 and better outcome.
Importantly, a high agreement has been shown between MCE and cardiovascular magnetic resonance, as well as Tc-99m-sestamibi single-photon emission computed tomography (MIBI-SPECT),69 in detecting and quantifying the extent of no-reflow. Interestingly, quantitative measures of no-reflow by MCE appeared the most reliable predictors of postischemic left ventricular remodeling in a study.58
MCE has recently provided pathophysiological characterization of the takotsubo syndrome. Clinical presentation of this syndrome resembles that of an acute coronary syndrome, often with severe impairment of left ventricular function and ST-segment elevation.70 These patients, however, show normal coronary arteries at angiography, thus suggesting that microvascular abnormalities might be involved in the pathogenesis of the syndrome. MCE assessment of myocardial perfusion during the acute phase has indeed revealed large transmural perfusion defects within the dysfunctional area. Interestingly, adenosine administration during this phase produced a transient improvement of myocardial perfusion and function, thus suggesting that an intense constriction of coronary microcirculation is responsible for the perfusion defect and the severe myocardial dysfunction. Accordingly, at 1-week follow-up, normalization of myocardial perfusion was paralleled by recovery of left ventricular function63 (Fig. 5).
Finally, MCE has also contributed to confirm CMVD in patients with the stable form of MVA. Indeed, CFR assessed by both MCE and TTDE was significantly reduced in these patients as compared with controls, with a high correlation between the two methods.71
Advantages and limitations
Similar to TTDE, MCE is easy to perform and user-friendly for operators, with a favorable cost–effectiveness ratio. However, its wide application in clinical practice has been limited by safety issues following the report of some major adverse events occurring in a few critically ill patients after contrast administration, which urged the Food and Drug Administration and European Medicines Agency (EMEA) to forbid use of microbubbles in the cardiovascular arena.72 This black box warning was removed in April 2008, after excluding a strict cause–effect relation between microbubble injection and adverse clinical events. Nevertheless, EMEA warnings remain on the use of microbubbles in the acute phase of coronary syndromes, which may still limit the clinical application of MCE, although it continues to be safely applied in the research setting.
PET is a radionuclide technique that allows reproduction, using specific tracers, of tissue images that reflect specific functional aspects of a specific tissue.
Imaging with PET offers unrivalled sensitivity and specificity for studying noninvasively human pathophysiology in vivo. Cardiological research with PET has flourished over the past 20 years, but it is only more recently that cardiology has begun to benefit from the maximum efficiency of which PET is capable. From the physical point of view, scanning of the heart is more challenging because of greater complication in the correction for photon attenuation and scattered radiation and because of heart and respiratory motion.
The success of PET is founded on the properties of positron-emitting isotopes. Their short physical half-lives make it possible to administer a tracer dose high enough to obtain useful data, but such that the radiation burden to the patient is acceptably low. Positron emitters do not exist in nature and must be produced artificially by means of a particle accelerator (generally a cyclotron). Production of isotopes with the shortest half-lives has to be carried out in the vicinity of the scanner and necessitates the installation of cyclotron and radiochemistry facilities. However, 18F compounds can be delivered from a relatively remote site of production.
Positrons are emitted with a continuous range of energies up to a maximum, characteristic of each particular isotope. The positron is successively slowed down by Coulomb interaction with atomic electrons and annihilates with an electron when its energy has been reduced to close to zero, resulting in a pair of photons flying off in opposite directions with energy of 511 keV. Positrons emitted from a tracer injected into the body are not measured directly, but indirectly from the photons emitted when the positron annihilates with an electron. Detectors placed on both sides of the active volume are connected in a so-called coincidence circuit so that if both detectors record an event within a very short interval (about 10−8 s), it is assumed that a positron annihilation has taken place.73
PET has been shown to be a reliable tool to quantify MBF.74,75 The two tracers most widely used to measure MBF by PET are oxygen-15 labeled water (H215O)76–78 and nitrogen-13 labeled ammonia 13NH3.79–8113NH3 is administered intravenously as a bolus, whereas H215O can be given either intravenously, as a bolus injection or as a slow infusion, or by inhalation of oxygen-15 labeled carbon dioxide (C15O2), which is converted to H215O by carbonic anhydrase in the lungs.
Rubidium-82 (82Rb)82,83 was initially used for quantification of MBF. PET studies with 82Rb, however, are limited by the significant dependence of its myocardial extraction on flow rate and on myocardial metabolic state, which can make quantification of regional MBF less accurate, particularly during hyperemia and in metabolically impaired myocardium. In addition, the high positron energy of this radionuclide results in relatively poor image quality and a reduced spatial resolution.
Whereas PET cameras measuring MBF usually work in two-dimensional mode, the new generation of PET systems now available work in three-dimensional mode, with potential benefits, particularly in efficiency.84
PET studies have significantly contributed to characterize CMVD in several clinical settings. Relevant information has, in particular, been acquired in patients with cardiovascular risk factors. Smoking has been shown by PET to reduce adenosine-induced hyperemia by 17% and CFR by 21% compared with nonsmoking (P < 0.05). Interestingly, administration of the antioxidant vitamin C normalized coronary microcirculatory function, lending support to the hypothesis that the damaging effect of smoking can be explained, at least in part, by an increased oxidative stress (Fig. 6).85,86
PET studies have also confirmed the reduction of CFR in asymptomatic patients with hypercholesterolemia and shown how CMV function can be normalized by cholesterol-lowering strategies.86–88 Importantly, a relation has been found between CFR and low-density lipoprotein cholesterol subfraction, at least in those with high total cholesterol,89 supporting a direct pathogenic role of this subfraction in the development of CMVD.
Abnormal CFR has been demonstrated in several PET studies in patients with essential hypertension and angiographically normal coronary arteries.90–92 Importantly, these studies have clarified that the impairment of CFR in hypertensive patients is largely independent of the presence and degree of left ventricular hypertrophy,93 suggesting that CMVD is mainly a consequence of vascular remodeling or functional alterations of endothelial cells and/or SMCs.94,95 In addition, the abnormality of hyperemic MBF was found to be regionally heterogeneous in some patients, whereas the whole myocardium was involved in others.96
A PET study97 has also demonstrated a markedly impaired coronary microvascular vasodilation in response to adenosine and to CPT in young patients with uncomplicated diabetes, showing similar results in type 1 and type 2 diabetes, thus suggesting that hyperglycemia, rather than insulin sensitivity might be a major determinant of CMVD.
Clinically relevant results from PET studies have been obtained about the role of CMVD in patients with hypertrophic cardiomyopathy (HCM). Several studies98–101 have indeed demonstrated a marked impairment of the vasodilator response to dipyridamole, which usually diffusely involves the left ventricle, in agreement with the evidence at autopsy of widespread remodeling of intramural coronary arterioles.102,103 Accordingly, symptoms and signs of myocardial ischemia are often found in these patients despite the absence of epicardial artery lesions. Importantly, myocardial ischemia caused by CMVD in these patients might contribute to the most relevant complications of HCM, including ventricular arrhythmias and progressive impairment of systolic left ventricular function.102,103 Of note, a recent study has documented that the severity of CMVD, assessed by PET, was an independent predictor of long-term clinical deterioration and death from cardiovascular causes in these patients (Fig. 7).9
Also in patients with IDCM, an impaired MBF at rest and an impaired CFR104,105 have been demonstrated by PET studies. The impaired CMV dilator capacity has been shown to be an independent predictor of an increased risk of death and progression of heart failure (Fig. 8).8
Finally, PET has significantly contributed to characterize CMV function in patients with MVA. Although data have not always been concordant,106 several studies have shown a reduction in CMV dilation in response to both endothelium-independent and endothelium-dependent stimuli.107–110 Moreover, two studies have suggested the presence of intramyocardial heterogeneity in the impairment of CMV dysfunction, supporting the hypothesis of a nonuniform CMV alteration that might explain the difficulty in detecting left ventricular dysfunction during angina and ST-segment changes in these patients.109,110
Advantages and limitations
The most important advantage of PET in assessing CMV function is that it allows a reliable quantitative measure of MBF, both at rest and during vasoactive stimuli, thus allowing us to establish whether an impairment of vasodilator reserve is actually related to an impaired increase of MBF (i.e. to true CMVD), rather than to a basal increase in MBF, as can be seen in the hyperadrenergic state. A further merit of PET is the possibility to assess both global and regional CMV function.
Some limitations, however, need to be highlighted. First, the technique is expensive, time consuming and can only be performed in highly specialized centers. Accordingly, the technique is poorly available and poorly suitable for serial assessment of CMV function. Its resolution is less than optimal (about 5 mm), making detection of small areas of MBF abnormality difficult. Finally, the potential risk of radiation exposure should be considered, although the dose of radiation to which patients are exposed during MBF assessment with PET can be considered to be negligible.73
Cardiovascular magnetic resonance
MRI uses the property of resonance of nuclei of atoms exposed to a magnetic field to reconstruct images of the body. The application of powerful magnetic and radio frequency fields induces the nuclei to produce a rotating magnetic field that is detected by a scanner and used to reconstruct images of the scanned section. Magnetic fields of different nuclei rotate at different speeds, thus allowing accurate discrimination of different structures. Administration of paramagnetic contrast agents may improve discrimination of body tissues and organs.
The assessment of myocardial perfusion by cardiac magnetic resonance (CMR) is based on the changes in myocardial signal intensity of gadolinium, an extracellular contrast agent.111 After intravenous gadolinium injection, an ECG-gated acquisition of first-pass CMR images is obtained for the next 30–60 s, with the patients holding their breath to avoid interference motion with CMR image acquisition. Quantitative measurements of MBF (in units of ml/min per g of tissue) at rest and during hyperemia can be determined based on intensity curves of regions of interest.111
CMR has been used to assess CMVD in several clinical settings. In the Multi-Ethnic Study of Atherosclerosis, perfusion defects have been assessed in asymptomatic individuals with cardiovascular risk factors, although individuals selected also had evidence of coronary artery calcification.112
CMVD has also been documented by CMR in revascularized patients with coronary artery disease,113 those with takotsubo disease,114 and those with hypertrophic115 or dilated116 cardiomyopathy.
Significant CMV abnormalities have also been documented by CMR in patients with MVA. Panting et al.117 first showed an impairment of subendocardial CBF in response to adenosine. Lanza et al.118 have subsequently shown significant perfusion defects during dobutamine stress test in 56% of MVA patients, which correlated with a reduction of CBF response to adenosine as assessed by TTDE in the LAD coronary artery (Fig. 9).119
Finally, CMR has become the method of choice to identify and characterize areas of microvascular obstruction (‘no-reflow’) in patients with AMI undergoing successful recanalization of the infarct-related coronary artery. These areas can indeed be identified as hypoenhanced zones, suggesting CMV obstruction, in the context of the infarcted regions,120 and correlated with a worse clinical outcome121 (Fig. 10).122
Advantages and limitations
CMR perfusion imaging is characterized by high spatial resolution, lack of risk related to ionizing radiation and lack of attenuation-related problems (e.g. for breast shadow). Furthermore, the same examination allows assessment of not only perfusion but also global and regional myocardial contractility, as well as tissue morphology.119,123 Moreover, CMR allows an accurate evaluation of subendocardial and subepicardial perfusion, as well as of regional coronary resistance, in combination with the estimated diastolic perfusion time.122–124 Finally, by means of specific acquisition protocols, it is possible to visualize areas of postischemic edema of the left ventricle.123
Some important limitations, however, need to be acknowledged. To achieve an adequate signal-to-noise ratio of myocardial tissue enhancement, relatively large doses of gadolinium are needed. This affects the signal processing for MBF quantification, an issue that has only partially been resolved by using methods for nonlinear myocardial signal intensity correction.125 Concerns about toxicity of gadolinium-based contrast agents have also recently been highlighted, particularly in patients with chronic renal failure, in whom their use is associated with nephrogenic systemic fibrosis.126 Accordingly, gadolinium should not be used in patients with a glomerular filtration rate of 30 ml/min or less.
Postprocessing requires manual definition of myocardial regions of interest as well as of subendocardial and subepicardial areas, which can be time consuming and introduce some bias. Furthermore, during first pass, a fraction of gadolinium may diffuse into the interstitium; accordingly, myocardial signal intensity may depend not only on coronary perfusion but also on tissue blood volume, size of the extravascular compartment, and degree of capillary permeability, which can complicate quantitative assessment of MBF;127,128 to overcome these issues, however, specific models of signal analysis have been proposed and validated.129,130
Finally, some general conditions may preclude assessment of myocardial perfusion by CMR, including claustrophobia, arrhythmias and implanted devices.
Several invasive techniques have been proposed for the assessment of CMV function, although this is now predominantly assessed by intracoronary recording of CBF by Doppler and pressure wires.
This method is based on the indicator-dilution (Fick's) principle.131 Briefly, cold saline is infused into the coronary sinus at a known temperature and rate and blood temperature is measured downstream. The dilution of the tracer is indicated by the reduction in blood temperature, which is proportional to CBF and can therefore be calculated. This method only allows measurement of the whole CBF. However, if the procedure is performed in the great cardiac vein, CBF in the anterior ventricular wall (almost exclusively perfused by the LAD coronary artery) can be measured.131
Importantly, intracoronary thermodilution can now be performed using intracoronary wires that incorporate thermal sensors. In this case, a 3-ml bolus of room-temperature saline is injected into a coronary artery; the entry of saline is registered by a shaft thermistor over the wire while a distal sensor captures the resulting temperature shift. The mean transit time (mTT) of the indicator is calculated, which is inversely proportional to CBF. CFR, in this case, is calculated as the ratio between the resting mTT and hyperemic mTT.132,133
Thermodilution methods present some drawbacks. Stable positioning of the catheter, especially in the coronary sinus, can be difficult and quantification of flow requires accurate recording of temperature and volume of the indicator at the injection and the distal site, which can often be problematic and display relatively high variability, also due to frequent incomplete mixing of saline and blood.
Gas washout method
This method134 is also based on the indicator-dilution principle, but uses an inert gas as a tracer (usually argon or xenon). Again, the change in the coronary sinus of the gas concentration from the site of injection to that recorded downstream allows calculation of CBF.
The gas, as a radioactive tracer, can also be inhaled at a known concentration over a 5-min period. Analyses of gas concentrations by gas chromatography in arterial (coronary) and coronary sinus blood samples allow calculation of CBF, as this is proportional to tissue concentration of the gas divided by its mean artery-coronary sinus difference. Alternatively, the gas can be injected directly into the coronary arteries. These methods can also allow assessment of regional CBF, based on the scintigraphic detection of activity of the radio-labeled gas. However, the latter approach has major limitations in several clinical conditions.
Intracoronary Doppler flow wire
Intracoronary Doppler wires allow direct measurement of CBF velocity in single epicardial arteries, based on the Doppler effect, which establishes that the velocity and direction of blood flow can be derived by determining the frequency shift resulting from the emitted and the returning ultrasound waves, according to the Doppler equation.135
Intracoronary Doppler wires can incorporate a pressure sensor that allows measurement of intracoronary blood pressure. The product of CBF velocity and the section area of the vessel gives a measure of CBF.
Although CBF velocity measurements from Doppler signals are robust, determination of the cross-sectional area of the vessel for calculations of CBF remains problematic even with quantitative coronary angiography. However, this issue is of limited relevance in the case of normal coronary arteries, due to the usually limited variations of epicardial vessels during administration of vasodilator stimuli for the assessment of CFR.
Technical problems may also impair the correct acquisition of the signal, including guiding catheter obstruction to flow, poor zeroing/calibration and signal loss. Additionally, suboptimal guide catheter engagement may result in inadequate delivery of vasodilator agents, thus limiting the accuracy of CFR. Technical artifacts of signal acquisition must be recognized and then minimized to obtain reliable flow data.
Other invasive indexes of coronary microvascular function
Some specific characteristics of the Doppler velocity waveform have also been used to deduce information about microvascular injury. In particular, the deceleration time of diastolic flow (DDT) reflects the compliance of the microvascular compartment. Rapid flow deceleration can indeed be regarded as a sign of increased coronary microvascular resistance, suggesting (pre)-arteriolar constriction.136
Measurement of coronary resistance can more easily be added to that of CBF during invasive tests to better characterize CMV function. Coronary resistance can be measured at rest and at peak hyperemia (hyperemic microvascular resistance), thus obtaining a measure of its reduction during maximal vasodilation.137,138 It can be calculated using the formula:
where MAP is the mean aortic pressure and RAP the right atrial pressure. However, the calculation can in most cases be reliably simplified as the ratio of coronary DBP to average peak flow velocity.
Coronary resistance has also been proposed to be estimated by the index of microvascular resistance, defined as the product of coronary pressure by the hyperemic mTT (which is inversely correlated to absolute flow, as measured simultaneously with the coronary pressure wire).137–139
To circumvent the issue of cross-sectional area imaging, a similar index of resistance has been introduced, the velocity-based index of CMVR, which is calculated as the ratio between central arterial pressure and Doppler CBF velocity and expressed in mmHg/cm per s.140
An increased corrected Thrombolysis in Myocardial Infarction frame count of contrast agent during coronary angiography141 and a higher blush grade142 have both been considered as a rough, but sufficiently reliable, expression of increased coronary microvascular constriction. These methods provide a semiquantitative categorization of CBF, with the implicit assumption that, in the absence of significant stenosis, a slow flow in epicardial vessels and a lower opacification of myocardium, respectively, would reflect an impairment of coronary microcirculation.
Finally, a direct assessment of CMV function during catheterization might also be obtained by the use of a first-pass distribution analysis (FPA) technique. This method measures the absolute CBF on angiographic images through densitometric analysis of spatial and temporal aspects of the contrast propagation through the myocardium.143,144 The FPA technique would be an easy-to-implement method, which might be also done in conjunction with routine coronary angiography. The method, however, has until now been applied and validated only in animal models, and, therefore, its reliability in humans needs to be addressed before it can be recommended for use in clinical practice.
Invasive methods have until recently been the only kind of tests that could be used to reliably assess CMV function, and they have given an outstanding contribution in identifying and characterizing CMVD in several clinical settings. In particular, the ICDW technique has become the most widely used invasive method to explore the state of coronary microcirculation. Of note, invasive studies allow the investigation not only of CMV dilator function but also of CMV response to constrictor stimuli.
Invasive methods have been used to assess CMV function in patients with the no-reflow phenomenon after primary coronary revascularization in the acute phase of myocardial infarction, showing a dramatic reduction in CFR, which has also been shown to predict a worse clinical prognosis.145–148 Intracoronary Doppler studies have also contributed to document CMVD in patients with either dilated149 or hypertrophic cardiomyopathy,150 as well as in patients with takotsubo disease.151
An impairment of both endothelium-independent and endothelium-dependent CMV dilation in patients with MVA has also been demonstrated by invasive studies152–155 (Fig. 11), whereas some other reports have documented an increased constrictor response to vasoactive stimuli in these patients.156
Finally, intracoronary Doppler recordings have given a crucial contribution in characterizing the impairment of CMV function detectable in the territory of stenotic epicardial coronary arteries treated by PCIs.157
Advantages and limitations
When performed carefully, invasive methods allow the most accurate assessment of CMV dilator function. They indeed allow intracoronary injection of pharmachological stimuli, such as adenosine, with the possibility to achieve maximal CMV dilatation with appropriate doses without affecting blood pressure, which might instead be reduced with the systemic administration of the vasodilator substance, with consequences on myocardial perfusion and the correct assessment of CFR. Furthermore, only invasive methods allow a reliable assessment of CMV response to vasoconstrictor stimuli, as they may be integrated with angiography to exclude significant epicardial vasoconstriction.
The most reliable invasive methods to investigate CMV function are based on intracoronary Doppler recording. An advantage of this method is that it allows a separate assessment of CBF in the different coronary artery territories. The use of intracoronary thermodilution also seems reliable and data about CFR are comparable to those of intracoronary Doppler measures.132
Invasive methods, however, have inevitable limitations. They are more expensive compared with most noninvasive methods; they are also time consuming, which is often incompatible with the busy routine activity of most cath-labs, cannot easily been repeated serially, and portend a possible increase in serious adverse events.
In this article, we have briefly reviewed the available methods of investigating CMV function in patients without any significant obstruction of epicardial coronary arteries. A comparative summary of these methods is shown in Table 6.
The optimal method for investigation of CMV function should be largely available, easy to perform, not expensive, without risks, reliable and reproducible. None of the available methods, however, fulfill all these ideal characteristics.
Invasive methods, in particular intracoronary Doppler recording, remain the most reliable for CMV function assessment, as they allow direct measurements and control for several potentially confounding variables. Furthermore, only invasive techniques allow the reliable assessment of the response of coronary microcirculation to constrictor stimuli. However, costs, prolongation of invasive procedures and potential risks make these methods unsuitable for clinical routine use, and even for most research studies that require serial measurements.
Sophisticated and technologically advanced noninvasive methods, such as PET and CMR, are now available to assess CMV function. These methods appear very reliable, with potential large margins for further improvement in the near future, and they might be considered as reference tests for most research studies. However, low availability, high costs and some potential risks significantly limit their routine use for CMV studies.
A promising method is represented by contrast echocardiography, which is easier to perform and more widely available. Further studies, however, seem necessary to exclude some appreciable risks related to contrast agents and to better establish the reliability and reproducibility of quantitative measurements of CBF.
Finally, TTDE is the most simple, widely available, cheap and safe technique, but it presents significant limitations due to some technical difficulties and to the fact that it is, to a large extent, operator-dependent. Furthermore, it may limit the examination to the LAD coronary artery territory only in most cases. Yet, in clinical practice, it might constitute the first screening test to identify patients with significant impairment of CMV function, followed, as a second step, by CMR in patients in whom it is inconclusive or in whom a more accurate assessment of CMV function is required, with MCE or PET being possible alternatives in selected cases and/or in experienced centers.
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