Endothelial Progenitor Cells: An Appraisal of Relevant Data from Bench to Bedside.

The mobilization of endothelial progenitor cells (EPCs) into circulation from bone marrow is well known to be present in several clinical settings, including acute coronary syndrome, heart failure, diabetes and peripheral vascular disease. The aim of this review was to explore the current literature focusing on the great opportunity that EPCs can have in terms of regenerative medicine.

Influence of depression and anxiety on circulating endothelial progenitor cells in patients with acute coronary syndromes.

OBJECTIVES: Circulating endothelial progenitor cells (EPCs) are related to endothelial function and progression of coronary artery disease. There is evidence of decreased numbers of circulating EPCs in patients with a current episode of major depression. We investigated the relationships between the level of circulating EPCs and depression and anxiety in patients with acute coronary syndrome (ACS). METHODS: Patients with ACS admitted to three Cardiology Intensive Care Units were evaluated by the SCID-I to determine the presence of lifetime and/or current mood and anxiety disorders according to DSM-IV criteria. The EPCs were defined as CD133(+) CD34(+) KDR(+) and evaluated by flow cytometry. All patients underwent standardized cardiological and psychopathological evaluations. Parametric and nonparametric statistical tests were performed where appropriate. RESULTS: Out of 111 ACS patients, 57 were found to have a DSM-IV lifetime or current mood or anxiety disorder at the time of the inclusion in the study. The ACS group with mood or anxiety disorders showed a significant decrease in circulating EPC number compared with ACS patients without affective disorders. In addition, EPC levels correlated negatively with severity of depression and anxiety at index ACS episode. CONCLUSIONS: The current study indicates that EPCs circulate in decreased numbers in ACS patients with depression or anxiety and, therefore, contribute to explore new perspectives in the pathophysiology of the association between cardiovascular disorders and affective disorders.

Circulating endothelial progenitor cells and large artery structure and function in young subjects with uncomplicated type 1 diabetes.

BACKGROUND: Carotid intima-media thickness (IMT), indices of large artery stiffness and measures of endothelium function may be used as markers of early atherosclerosis in type 1 diabetes mellitus (T1DM). The aim of the present study was to compare the indices of large artery structure and function as well as endothelial function and regenerating capacity between adolescents with T1DM and healthy control of similar age. In addition, the associations of different vascular measures with endothelial progenitor cells (EPCs), glyco-metabolic control and serum levels of advanced glycation endproducts (AGEs), soluble receptors for AGEs (sRAGE) and adiponectin were evaluated. METHODS: Sixteen uncomplicated young T1DM patients (mean age 18 ± 2 years, history of disease 11 ± 5 years, HbA1c 7.7 ± 1.1%) and 26 controls (mean age 19 ± 2 years) were studied. A radiofrequency-based ultrasound system (Esaote MyLab 70) was used to measure carotid IMT and wave speed (WS, index of local stiffness), applanation tonometry (PulsePen) was applied to obtain central pulse pressure (PP) and augmentation index (AIx), and carotid-femoral pulse wave velocity (PWV, Complior) was used as index of aortic stiffness. Peripheral endothelium-dependent vasodilation was determined as reactive hyperemia index (RHI, EndoPAT). Circulating EPCs, glycometabolic profile, AGEs (autofluorescence method), sRAGE and adiponectin were also measured. RESULTS: After adjusting for age, sex and blood pressure, T1DM adolescents had significantly higher carotid IMT (456 ± 7 vs. 395 ± 63 µm, p < 0.005), carotid WS (p < 0.005), PWV (p = 0.01), AIx (p < 0.0001) and central PP (p < 0.01) and lower EPCs (p = 0.02) as compared to controls. RHI was reduced only in diabetic patients with HbA1c ≥7.5% (p < 0.05). In the overall population, EPCs were an independent determinant of carotid IMT (together with adiponectin), while fasting plasma glucose was an independent determinant of carotid WS, AIx and central PP. CONCLUSIONS: Our findings suggest that young subjects with relatively long-lasting T1DM have a generalized preclinical involvement of large artery structure and function, as well as a blunted endothelium regenerating capacity. Hyperglycemia and suboptimal chronic glycemic control seem to deteriorate the functional arterial characteristics, such as large arteries stiffness, wave reflection and peripheral endothelium-dependent vasodilation, whereas an impaired endothelium regenerating capacity and adiponectin levels seem to influence arterial structure.

Carotid artery intima-media thickness: normal and percentile values in the Italian population (camp study).

AIMS: Carotid intima-media thickness (IMT) is one of the best non-invasive parameters for evaluating previous vascular lesions and could be used to identify a preclinical stage of the atherosclerotic process. The aim of our research was to develop an epidemiological study of the normal mean values of IMT of the common carotid artery, adjusted for age and sex, in the Italian population. METHODS AND RESULTS: In this multicenter study, a total of 1017 patients (596 males, mean age: 58.5 + 13.2 years) were enrolled at four different Italian centers. Inclusion criteria were the absence of cardiovascular risk factors or presence of not more than one. Patients underwent two-dimensional echo-color Doppler scanning of the carotid arteries, adopting a high-definition vascular echographic apparatus and a 11-3 MHz linear electronic probe. The arithmetical mean of the IMT value was calculated. Data obtained from this study show the carotid IMT changes in relation to age and sex. In particular, it grows higher with increasing age, and is always higher in men than in women. CONCLUSION: In relation to the percentile distribution of the values in the population analyzed, the normal range of m-IMT could be established just on the basis of the patient's age and sex. In this way, the ultrasound scan operator can rely on a simple reference scheme. This will help to refine the use of carotid ultrasound as an excellent tool for detecting asymptomatic carotid alterations and patients at high risk for cerebral and cardiovascular disease.

Methods for evaluating endothelial function: a position statement from the European Society of Cardiology Working Group on Peripheral Circulation.

The endothelium holds a pivotal role in cardiovascular health and disease. Assessment of its function was until recently limited to experimental designs due to its location. The advent of novel techniques has facilitated testing on a more detailed basis, with focus on distinct pathways. This review presents available in-vivo and ex-vivo methods for evaluating endothelial function with special focus on more recent ones. The diagnostic modalities covered include assessment of epicardial and microvascular coronary endothelial function, local vasodilation by venous occlusion plethysmography and flow-mediated dilatation, arterial pulse wave analysis and pulse amplitude tonometry, microvascular blood flow by laser Doppler flowmetry, biochemical markers and bioassays, measurement of endothelial-derived microparticles and progenitor cells, and glycocalyx measurements. Insights and practical information on the theoretical basis, methodological aspects, and clinical application in various disease states are discussed. The ability of these methods to detect endothelial dysfunction before overt cardiovascular disease manifests make them attractive clinical tools for prevention and rehabilitation.

Fibrin as a scaffold for cardiac tissue engineering.

Fibrin is a natural biopolymer with many interesting properties, such as biocompatibility, bioresorbability, ease of processing, ability to be tailored to modify the conditions of polymerization, and potential for incorporation of both cells and cell mediators. Moreover, the fibrin network has a nanometric fibrous structure, mimicking extracellular matrix, and it can also be used in autologous applications. Therefore, fibrin has found many applications in tissue engineering, combined with cells, growth factors, or drugs. Because a major limitation of cardiac cell therapy is low cell engraftment, the use of biodegradable scaffolds for specific homing and in situ cell retention is desirable. Thus, fibrin-based injectable cardiac tissue engineering may enhance cell therapy efficacy. Fibrin-based biomaterials can also be used for engineering heart valves or cardiac patches. The aim of this review is to show cardiac bioengineering uses of fibrin, both as a cell delivery vehicle and as an implantable biomaterial.

Coronary artery anomalies and their clinical relevance.

Coronary artery anomalies (CAAs) represent one of the most confusing topic in cardiology and affect approximately 1% of the general population. Although some anomalies seem to be only anatomical curiosities, others may sometimes have fatal consequences. This review describes the anatomical characteristics of main CAAs and focuses on the pathophysiological mechanisms by which CAAs may cause a pathological state. The last section describes these therapeutical options of this congenital disorders.

[Asymptomatic Takayasu's arteritis: an incidental diagnosis]. / Arterite asintomatica di Takayasu: una diagnosi occasionale.

We present the case of a 56 years-old female patient that was admitted to our Unit after an incidental observation of bilateral absence of the radial pulses, with impossibility of brachial arterial pressure measurement. The patient reported being completely asymptomatic in occasion of the episode, thus like previously and later on to it. We diagnosed the patient being affected of Takayasu arteritis and adeguate therapy had been undertaken.

Oxidative stress in response to high glucose levels in endothelial cells and in endothelial progenitor cells: evidence for differential glutathione peroxidase-1 expression.

Endothelial cells and endothelial progenitor cells (EPCs) play a key role in the pathogenesis of vascular disease. Both cell types are affected by the oxidative stress but their susceptibility may be different. This study aimed to investigate the antioxidative enzymes activated in EPCs after high constant glucose exposure as compared to endothelial cells (HUVECs). Both cells were incubated in the presence of normal (5mM) and high constant (25mM) d-glucose, as well as l-glucose as osmotic control for 48 and 96h. After a 48-hour exposure to high d-glucose, cell viability was significantly decreased both in EPCs and HUVECs as compared with normal d-glucose (p<0.01). However, after 96h there was no difference between EPCs grown on normal or high d-glucose, while HUVEC viability was affected by high d-glucose at 96h too (p<0.001). High d-glucose exposure induced a significant increase in reactive oxygen species (ROS) production in both cell types at 48h; however, after 96h, a significant decrease in ROS production (p<0.01) and a parallel marked increase in glutathione peroxidase type 1 (GPx-1) expression (p<0.01) and activity (p<0.01) were observed in EPCs compared to HUVECs. These data suggest that EPCs have a well-adaptive response to oxidative stress induced by constant and sustained high glucose exposure. This resistance to high glucose levels might be due to increased expression and activity of glutathione peroxidase allowing better cell survival.

Assessment of residual viability by enoximone echocardiography in patients with previous myocardial infarction correlation with positron emission tomographic studies and functional follow-up.

BACKGROUND: The aim of this study was to evaluate enoximone echocardiography (EE) for the identification of residual myocardial viability in postinfarction patients. Findings obtained during EE were compared with those acquired by myocardial uptake of fluorine-18 fluorodeoxyglucose ((18)F-FDG) positron emission tomography (PET) and functional follow-up results. METHODS: Twenty-five patients underwent EE and PET (18)F-FDG studies. An asynergic segment was considered as having contractile enhancement when the wall motion score decreased by > or = 1 grade during EE and was defined as viable if (18)F-FDG uptake score was > or = 2 grade on PET. RESULTS: Of 293 dysfunctional segments at baseline, 139 (47%) were viable by PET criteria; 117 (40%) had contractile enhancement induced by enoximone (P = 0.07). Agreement between EE and PET was found in 75% of involved segments (K = 0.46, P < 0.001). The majority of discrepancies (65%, P < 0.01) were mainly due to discordant segments in which PET revealed evidence of (18)F-FDG uptake but EE showed no change in wall motion. In 179 revascularized segments, negative predictive value for functional recovery of both tests reached the same value (89% for both), whereas positive predictive value was 82% for EE and 68% for PET, respectively (P < 0.05). Sensitivity was 85% for EE and 88% for PET (P = ns); specificity was 87% and 70%, respectively (P < 0.01). CONCLUSIONS: EE yields a fair concordance with PET study. Compared with PET, despite a similar negative accuracy, EE shows a greater specificity for prediction of function recovery after revascularization. (Echocardiography 2010;27:544-551).

An uncommon clinical condition: chronic thrombosis of the inferior vena cava. A case report and review of literature.

The lifetime incidence of deep vein thrombosis (DVT) is approximately 0.1% in general population and even more uncommon in subjects below 40 years of age. Thrombosis of the inferior vena cava (IVC) is an exceptionally rare clinical condition, with etiological factors similar to lower limb DVT. We present a case of post-traumatic chronic obstruction of the IVC in 41 years-old man, caused by a prolonged squatted position, while he was working as a bricklayer. We visited the patient fifteen years after the onset of the first clinical setting showing a severe post thrombotic syndrome, as a consequence of the already diagnosticated thrombosis, involving predominantly the right inferior leg. Thrombophilia screening tests showed patient to be a heterozygous carrier of methylenetetrahydrofolate reductase (MTHFR) gene mutation. Computed tomography (CT) scan confirmed the thrombotic obstruction of the infrahepatic IVC, both common iliac veins, right external and internal iliac veins, with multiple collateral pathways. Because of thrombosis extension, inherited prothrombotic condition and the young age of the patient, we decided to continue life-long oral anticoagulant therapy.

Human peripheral blood endothelial progenitor cells synthesize and express functionally active tissue factor.

INTRODUCTION: Endothelial progenitor cells are circulating cells able to home to sites of vascular damage and to contribute to the revascularization of ischemic areas. We evaluated whether endothelial progenitor cells synthesize tissue factor, a procoagulant protein also involved in angiogenesis. MATERIALS AND METHODS: Endothelial progenitor cells were obtained from the peripheral blood mononuclear fraction of normal donors and cultured in endothelial medium supplemented with specific growth factors. The procoagulant activity expressed by cells disrupted by freeze-thaw cycles was assessed by a one stage clotting assay. Tissue factor mRNA expression was evaluated by RT-PCR. RESULTS: Endothelial progenitor cells do not express procoagulant activity in baseline conditions. However, lipopolysaccharide induces the expression of procoagulant activity. The effect is dose-dependent and reaches statistical significance at 100 ng/mL lipopolysaccharide. Inhibition with an anti-tissue factor antibody and amplification of cDNA with primers based on the tissue factor sequence confirm the identity of this activity with tissue factor. The kinetics of tissue factor expression by endothelial progenitor cells is identical to that of human umbilical vein endothelial cells showing maximal activity within 4 hours, and then decreasing; in contrast, tissue factor expression by mononuclear cells lasts for longer times. Both 5,6-dichloro-beta D-ribofuranosyl-benzimidazole and cycloheximide prevented the expression of procoagulant activity. Stimulation of endothelial progenitor cells with tumor necrosis factor-alpha did not elicit any detectable procoagulant activity. CONCLUSIONS: Endothelial progenitor cells can be stimulated by lipopolysaccharide to synthesize tissue factor. This protein might be involved in thrombotic phenomena and might contribute to endothelial progenitor cells related neovascularization.

Arterial stiffness and ventricular stiffness: a couple of diseases or a coupling disease? A review from the cardiologist's point of view.

The assessment of arterial stiffness, a common feature of ageing, exacerbated by many common disorders such as hypertension, diabetes mellitus, or renal diseases, has become an attractive tool for identifying structural and functional abnormalities of the arteries in the preclinical stages of the atherosclerotic disease. Arterial stiffness has been recognized as an important pathophysiological determinant of systolic blood pressure and pulse pressure increases and therefore the cause of cardiovascular complications, demonstrating also an independent predictive value for cardiovascular events. Although there are many techniques and indices currently available, their large clinical application is limited by a lack of standardization, with important difficulties when one try effectively to measure, quantify, and compare. Moreover, information on the 'heart-vessel coupling disease', in which combined stiffness of both heart and arteries interact to limit cardiovascular performance and its possible implications in different clinical conditions, is still not well known. We overviewed main methods and indices used to estimate arterial stiffness and aimed to provide an insight into the knowledge of the ventricular-arterial coupling from the cardiologist's point of view.

Transvenous removal of pacing and implantable cardiac defibrillating leads using single sheath mechanical dilatation and multiple venous approaches: high success rate and safety in more than 2000 leads.

AIMS: The aim of the present study was to describe a 10 years single-centre experience in pacing and defibrillating leads removal using an effective and safe modified mechanical dilatation technique. METHODS AND RESULTS: We developed a single mechanical dilating sheath extraction technique with multiple venous entry site approaches. We performed a venous entry site approach (VEA) in cases of exposed leads and an alternative transvenous femoral approach (TFA) combined with an internal transjugular approach (ITA) in the presence of very tight binding sites causing failure of VEA extraction or in cases of free-floating leads. We attempted to remove 2062 leads [1825 pacing and 237 implantable cardiac defibrillating (ICD) leads; 1989 exposed at the venous entry site and 73 free-floating] in 1193 consecutive patients. The VEA was effective in 1799 leads, the TFA in 28, and the ITA in 205; in the overall population, we completely removed 2032 leads (98.4%), partially removed 18 (0.9%), and failed to remove 12 leads (0.6%). Major complications were observed in eight patients (0.7%), causing three deaths (0.3%). CONCLUSION: Mechanical single sheath extraction technique with multiple venous entry site approaches is effective, safe, and with a good cost effective profile for pacing and ICD leads removal.

The prostacyclin analogue iloprost increases circulating endothelial progenitor cells in patients with critical limb ischemia.

Patients with critical limb ischemia (CLI) have low levels of endothelial progenitor cells (EPC). Iloprost has been demonstrated to stimulate vascular endothelial growth factor (VEGF) and promote angiogenesis. We investigated the effects of iloprost on EPC levels in vivo in CLI patients. Twenty-three patients with stage III and IV CLI were treated with iloprost for four weeks, improving clinical and instrumental parameters. Mononuclear cells isolated from peripheral blood were cultured to obtain "early" EPC, evaluated counting adherent cells with double positivity for acetylated low-density lipoprotein uptake and Ulex Europaeus lectin at flow cytometry. These cells also co-expressed the monocyte markers CD14 and CD45. Iloprost increased EPC number in the whole patient population: pre-treatment median: 13,812/ml; range: 1,263-83,648/ml; post-treatment median: 23,739/ml; range: 3,385-99,251/ml; p = 0.035, irrespective of age, sex, disease stage or atherosclerosis risk factors. In conclusion, iloprost increases EPC number in peripheral blood in vivo. Such an effect may have therapeutic relevance.

Expression, pharmacology, and functional role of somatostatin receptor subtypes 1 and 2 in human macrophages.

Somatostatin (SRIF)-14 is recognized as an important mediator between the nervous and the immune system, although the functional role of its receptors (sst(1)-sst(5)) is poorly understood in humans. In our study, we demonstrate that human macrophages, differentiated from PBMC-derived monocytes, express sst(1) and sst(2) mRNAs. sst(1) and sst(2) are mostly localized at the cell surface and display active binding sites. In particular, sst(1)/sst(2) activation results in a weak internalization of sst(1), and the sst(2) internalization appears more efficient. At the functional level, the activation of SRIF receptors by the multiligand analogs SOM230 and KE108, but not by SRIF-14 or cortistatin-14, reduces macrophage viability. Their effects are mimicked by the selective activation of sst(1) and sst(2) using CH-275 and SMS 201-995/L-779,976, respectively. Further, sst(1)- and sst(2)-mediated effects are reversed by the sst(1) antagonist SRA-880 or the sst(2) antagonist CYN 154806, respectively. CH-275, SMS 201-995, and L-779,976, but not SRIF-14, decrease mRNA expression and secretion of the MCP-1. In addition, SRIF-14, CH-275, SMS 201-995, and L-779,976 decrease IL-8 secretion, and they do not affect IL-8 mRNA expression. In contrast, SRIF-14 and sst(1)/sst(2) agonists do not affect the secretion of matrix metalloproteinase-9. Collectively, our results suggest that the SRIF system, through sst(1) and sst(2), exerts mainly an immunosuppressive effect in human macrophages and may, therefore, represent a therapeutic window that can be exploited for the development of new strategies in pharmacological therapy of inflammation.

Exposure to passive smoking: a test to predict endothelial dysfunction and atherosclerotic lesions.

Acute exposure to environmental tobacco smoke (ETS) is considered to adversely influence atherogenesis. The aim of this article was to assess whether brachial ultrasonography in subjects with endothelial dysfunction after ETS exposure is associated with atherosclerotic lesions. Never smoker healthy volunteers (n = 18) and subjects with a previous myocardial infarction (MI; n = 10) were studied. Healthy volunteers were 12 men (66%) and 6 women (34%) with a mean age of 34 +/- 9 years. Post-MI subjects were men with a mean age of 53.8 +/- 4.8 years. After assessing endothelial function (by brachial ultrasonography) at rest, study subjects underwent brachial ultrasonography twice: in a smoke-free environment and then in the same environment polluted by cigarette combustion (35 ppm carbon monoxide concentration). Carboxyhemoglobin concentration was measured before and after ETS exposure. Baseline brachial-artery diameter, diameter during reactive hyperemia, and diameter after sublingual nitroglycerin (GTN) administration (endothelium-independent vasodilator) were measured at rest and in both smoke-free and smoking environments. Each study subject acted as their own control. No comparison was made between the two groups. A strong correlation between ETS exposure and endothelial dysfunction was observed in both groups. Post-MI subjects also showed endothelium-independent vasodilation worsening, which is usually due to arterial wall alterations. After ETS exposure, mean flow-mediated vasodilation after GTN was significantly (P < .01) reduced only in post-MI subjects (P < .01). Carboxyhemoglobin concentration increased in both groups (P < .01). ETS exposure may be an effective test to identify endothelial dysfunction and arterial wall alterations by using brachial ultrasonography.

Left ventricular function in normotensive young adults with well-controlled type 1 diabetes mellitus.

The aim of this study was to determine whether early myocardial structural and functional systolic and diastolic alterations in asymptomatic and uncomplicated patients with type 1 diabetes mellitus (DM) could be detected using the new highly sensitive echocardiographic techniques of integrated backscatter and color Doppler myocardial imaging. Forty asymptomatic and uncomplicated patients with type 1 DM and 40 gender- and age-matched normal controls were studied. All patients were analyzed by conventional and new echocardiographic techniques (integrated backscatter and color Doppler myocardial imaging). Patients with DM showed reduced systolic function compared with controls, evidenced by significantly lower peak strain, strain rates, and cyclic variation indexes at the septum (p <0.0001, <0.01, and <0.001, respectively) and at the posterior wall level (p <0.0001, <0.0001, and <0.001, respectively). On receiver-operating characteristic curve analysis, systolic strain and the cyclic variation index showed the highest discriminating power for separating patients with DM and control subjects. Neither structural or ultrastructural nor diastolic functional abnormalities were detected. On univariate regression analysis, a significant inverse correlation was found for DM duration with conventional (E/A ratio) and unconventional (tissue Doppler imaging E/A ratio) indexes of diastolic function, in the absence of any correlation for systolic function. In conclusion, highly sensitive ultrasonic techniques demonstrate evidence of left ventricular systolic dysfunction in the early stage of type 1 DM, in the absence of ultrastructural and left ventricular diastolic functional abnormalities.

Polymorphic analysis of the matrix metalloproteinase-9 gene and susceptibility to sporadic abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) has a multifactorial aetiology and the importance of genetic components is getting increasing interest. Alteration in the structure of the vascular extracellular matrix has been described in AAA. Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins which alter the vessel wall stability. We evaluated two different polymorphisms, a CA repeat and a cytosine to thymidine transition in the promoter sequence of MMP-9 gene for frequency in 146 patients with AAA. We compared the results with those of 156 healthy subjects. No difference was found in the allelic distribution of either polymorphisms. We therefore found no evidence that MMP-9 is a marker of susceptibility for AAA.

Early textural and functional alterations of left ventricular myocardium in mild hypothyroidism.

The aim of the present study was to evaluate cardiac function and texture in patients with subclinical hypothyroidism (sHT) both by conventional and new ultrasonic intramyocardial tissue techniques. sHT was characterized by normal serum free tetraiodotironine and free triiodotironine levels and slightly increased serum TSH level. Twenty-four patients affected by sHT and 24 sex- and age-matched healthy volunteers were studied. All subjects were submitted to conventional two-dimensional (2D)-color Doppler echocardiography, pulsed wave tissue Doppler imaging (PWTDI) for the analysis of the diastolic function, color Doppler myocardial imaging (CDMI) for the analysis of regional strain and strain-rate and integrated backscatter (IBS) for the evaluation of intrinsic contractility and tissue characterization. The results of the present study were: (a) the detection in sHT subjects of a lower cyclic variation index (CVI) indicating an altered myocardial intrinsic contractility; (b) a higher ultrasonic myocardial reflectivity indicating an altered myocardial texture; (c) the detection of lower systolic strain and strain-rate indicating an alteration of myocardial regional deformability; (d) an initial impairment of left ventricular diastolic function indicated by a decrease of peak E mitral flow velocity and an increase of peak A mitral flow velocity. All parameters studied with conventional 2D-echo in sHT patients were comparable with controls, except for a mild alteration in diastolic function. A significant correlation among systo-diastolic modifications detected by CDMI and IBS and serum TSH levels were found. The CVI at septum, the PWDTI S-peak wave and the systolic strain at septum were inversely related to the serum TSH levels. In conclusion, the new intramyocardial ultrasonic techniques confirm and extend the previous knowledge on the effect of the sHT on the heart, allowing the detection of early ultrastructural and regional functional systolic and diastolic abnormalities.