Single-Cell RNA Sequencing Reveals Metabolic Stress-Dependent Activation of Cardiac Macrophages in a Model of Dyslipidemia-Induced Diastolic Dysfunction.

BACKGROUND: Metabolic distress is often associated with heart failure with preserved ejection fraction (HFpEF) and represents a therapeutic challenge. Metabolism-induced systemic inflammation links comorbidities with HFpEF. How metabolic changes affect myocardial inflammation in the context of HFpEF is not known. METHODS: We found that ApoE knockout mice fed a Western diet recapitulate many features of HFpEF. Single-cell RNA sequencing was used for expression analysis of CD45+ cardiac cells to evaluate the involvement of inflammation in diastolic dysfunction. We focused bioinformatics analysis on macrophages, obtaining high-resolution identification of subsets of these cells in the heart, enabling us to study the outcomes of metabolic distress on the cardiac macrophage infiltrate and to identify a macrophage-to-cardiomyocyte regulatory axis. To test whether a clinically relevant sodium glucose cotransporter-2 inhibitor could ameliorate the cardiac immune infiltrate profile in our model, mice were randomized to receive the sodium glucose cotransporter-2 inhibitor dapagliflozin or vehicle for 8 weeks. RESULTS: ApoE knockout mice fed a Western diet presented with reduced diastolic function, reduced exercise tolerance, and increased pulmonary congestion associated with cardiac lipid overload and reduced polyunsaturated fatty acids. The main immune cell types infiltrating the heart included 4 subpopulations of resident and monocyte-derived macrophages, determining a proinflammatory profile exclusively in ApoE knockout- Western diet mice. Lipid overload had a direct effect on inflammatory gene activation in macrophages, mediated through endoplasmic reticulum stress pathways. Investigation of the macrophage-to-cardiomyocyte regulatory axis revealed the potential effects on cardiomyocytes of multiple inflammatory cytokines secreted by macrophages, affecting pathways such as hypertrophy, fibrosis, and autophagy. Finally, we describe an anti-inflammatory effect of sodium glucose cotransporter-2 inhibitor in this model. CONCLUSIONS: Using single-cell RNA sequencing , in a model of diastolic dysfunction driven by hyperlipidemia, we have determined the effects of metabolic distress on cardiac inflammatory cells, in particular on macrophages, and suggest sodium glucose cotransporter-2 inhibitors as potential therapeutic agents for the targeting of a specific phenotype of HFpEF.

Pathophysiological basis of the cardiological benefits of SGLT-2 inhibitors: a narrative review.

In recent years, GLP-1 receptor agonists (GLP-1RA), and SGLT-2 inhibitors (SGLT-2i) have become available, which have become valuable additions to therapy for type 2 diabetes as they are associated with low risk for hypoglycemia and cardiovascular benefits. Indeed, SGLT-2i have emerged as a promising class of agents to treat heart failure (HF). By inhibiting SGLT-2, these agents lead to excretion of glucose in urine with subsequent lowering of plasma glucose, although it is becoming clear that the observed benefits in HF cannot be explained by glucose-lowering alone. In fact, multiple mechanisms have been proposed to explain the cardiovascular and renal benefits of SGLT-2i, including hemodynamic, anti-inflammatory, anti-fibrotic, antioxidant, and metabolic effects. Herein, we review the available evidence on the pathophysiology of the cardiological benefits of SGLT-2i. In diabetic heart disease, in both clinical and animal models, the effect of SGLT-2i have been shown to improve diastolic function, which is even more evident in HF with preserved ejection fraction. The probable pathogenic mechanisms likely involve damage from free radicals, apoptosis, and inflammation, and therefore fibrosis, many of which have been shown to be improved by SGLT-2i. While the effects on systolic function in models of diabetic heart disease and HF with preserved ejection fraction is limited and contrasting, it is a key element in patients with HF and reduced ejection fraction both with and without diabetes. The significant improvement in systolic function appears to lead to subsequent structural remodeling of the heart with a reduction in left ventricle volume and a consequent reduction in pulmonary pressure. While the effects on cardiac metabolism and inflammation appear to be consolidated, greater efforts are still warranted to further define the entity to which these mechanisms contribute to the cardiovascular benefits of SGLT-2i.

Nebivolol versus placebo in patients undergoing anthracyclines (CONTROL Trial): rationale and study design.

AIMS: Anthracyclines are the chemotherapeutic agents most frequently associated with cardiotoxicity, while remaining widely used. Different neurohormonal blockers have been tested as a primary prevention strategy to prevent or attenuate the onset of cardiotoxicity, with mixed results. However, prior studies were often limited by a nonblinded design and an assessment of cardiac function based only on echocardiographic imaging. Moreover, on the basis of an improved mechanistic understanding of anthracycline cardiotoxicity mechanisms, new therapeutic strategies have been proposed. Among cardioprotective drugs, nebivolol might be able to prevent the cardiotoxic effects of anthracyclines, through its protective properties towards the myocardium, endothelium, and cardiac mitochondria. This study aims to evaluate the cardioprotective effects of the beta blocker nebivolol in a prospective, placebo-controlled, superiority randomized trial in patients with breast cancer or diffuse large B cell lymphoma (DLBCL) who have a normal cardiac function and will receive anthracyclines as part of their first-line chemotherapy programme. METHODS: The CONTROL trial is a randomized, placebo-controlled, double-blinded, superiority trial. Patients with breast cancer or a DLBCL, with a normal cardiac function as assessed by echocardiography, scheduled for treatment with anthracyclines as part of their first-line chemotherapy programme will be randomized 1 : 1 to nebivolol 5 mg once daily (o.d.) or placebo. Patients will be examined with cardiological assessment, echocardiography and cardiac biomarkers at baseline, 1 month, 6 months and 12 months. A cardiac magnetic resonance (CMR) assessment will be performed at baseline and at 12 months. The primary end point is defined as left ventricular ejection fraction reduction assessed by CMR at 12 months of follow-up. CONCLUSION: The CONTROL trial is designed to provide evidence to assess the cardioprotective role of nebivolol in patients undergoing chemotherapy with anthracyclines. CLINICAL TRIAL REGISTRATION: The study is registered in the EudraCT registry (number: 2017-004618-24) and in the ClinicalTrials.gov registry (identifier: NCT05728632).

Solving the Riddle of Sudden Cardiac Death in Hypertrophic Cardiomyopathy: The Added Role of Cardiac Magnetic Resonance.

Cardiac magnetic resonance (CMR) has been recently implemented in clinical practice to refine the daunting task of establishing the risk of sudden cardiac death (SCD) in patients with hypertrophic cardiomyopathy (HCM). We present an exemplificative case highlighting the practical clinical utility of this imaging modality in a 24-year-old man newly diagnosed with an apical HCM. CMR was essential in unmasking a high risk of SCD, which appeared low-intermediate after traditional risk assessment. A discussion examines the essential role of CMR in guiding the patient's therapy and underlines the added value of CMR, including novel and potential CMR parameters, compared to traditional imaging assessment for SCD risk stratification.

Atrial arrhythmias and heart failure: A "modern view" of an old paradox.

BACKGROUND: Heart failure (HF) and atrial arrhythmias (AAs) are two clinical conditions that characterize the daily clinical practice of cardiologists. In this perspective review, we analyze the shared etiopathogenetic pathways of atrial arrhythmias, which are the most common cause of atrial arrhythmias-induced cardiomyopathy (AACM) and HF. HYPOTHESIS: The aim is to explore the pathophysiology of these two conditions considering them as a "unicum", allowing the definition of a cardiovascular continuum where it is possible to predict the factors and to identify the patient phenotype most at risk to develop HF due to atrial arrhythmias. METHODS: Potentially eligible articles, identified from the Electronic database (PubMed), and related references were used for a literature search that was conducted between January 2022 and January 2023. Search strategies were designed to identify articles that reported atrial arrhythmias in association with heart failure and vice versa. For the search we used the following keywords: atrial arrhythmias, atrial fibrillation, heart failure, arrhythmia-induced cardiomyopathy, tachycardiomyopathy. We identified 620 articles through the electronic database search. Out of the 620 total articles we removed 320 duplicates, thus selecting 300 eligible articles. About 150 titles/abstracts were excluded for the following reasons: no original available data, no mention of atrial arrhythmias and heart failure crosstalk, very low quality analysis or evidence. We excluded also non-English articles. When multiple articles were published on the same topic, the articles with the most complete set of data were considered. We preferentially included all papers that could provide the best evidence in the field. As a result, the present review article is based on a final number of 104 references. RESULTS: While the pathophysiology of AACM and Heart Failure with reduced ejection fraction (HFrEF) has been studied in detail over the years, the causal link between atrial arrhythmias and heart failure with Preserved Ejection Fraction (HFpEF) has been often subject of interest. HFpEF is strictly related to AAs, which has always been considered significant risk factor. In this review we described the pathophysiological links between atrial fibrillation and heart failure. Furthermore, we illustrated and discussed the preclinical and clinical predicting factors of AF and HFpEF, and the corresponding targets of the available therapeutic agents. Finally, we outlined the patient phenotype at risk of developing AF and HFpEF (Central Illustration). CONCLUSIONS: In this review, we underline how these two clinical conditions (AF and HFpEF) represent a "unicum" and, therefore, should be considered as a single disease that can manifest itself in the same phenotype of patients but at different times. Furthermore, considering that today we have few therapeutic strategies to treat these patients, it would be good to make an early diagnosis in the initial stages of the disease or intervene even before the development of signs and symptoms of HF. This is possible only by paying greater attention to patients with predisposing factors and carrying out a targeted screening with the correct diagnostic methods. A systemic approach aimed at improving the immuno-metabolic profile of these patients by lowering the body mass index, threatening the predisposing factors, lowering the mean heart rate and reducing the sympathetic nervous system activation is the key strategy to reduce the clinical impact of this disease.

Time-dependent effect of GLP-1 receptor agonists on cardiovascular benefits: a real-world study.

BACKGROUND: Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) have shown cardiovascular benefits in cardiovascular outcome trials in type 2 diabetes mellitus. However, the most convincing evidence was obtained in subjects with established cardiovascular (CV) disease. We analyzed the determinants of GLP-1 RA-mediated CV protection in a real-world population of persons with type 2 diabetes with and without a history of CV events with long-term follow-up. METHODS: Retrospective cohort study of 550 individuals with type 2 diabetes (395 in primary CV prevention, 155 in secondary CV prevention), followed at a single center after the first prescription of a GLP-1 RA between 2009 and 2019. CV and metabolic outcomes were assessed. RESULTS: Median duration of follow-up was 5.0 years (0.25-10.8) in primary prevention and 3.6 years (0-10.3) in secondary prevention, with a median duration of treatment of 3.2 years (0-10.8) and 2.5 years (0-10.3) respectively. In the multivariable Cox regression model considering GLP-1 RA treatment as a time-dependent covariate, in the primary prevention group, changes in BMI and glycated hemoglobin did not have an impact on MACE risk, while age at the time of GLP-1 initiation (HR 1.08, 95% CI 1.03-1.14, p = 0.001) and GLP-1 RA cessation by time (HR 3.40, 95% CI 1.82-6.32, p < 0.001) increased the risk of MACE. Regarding the secondary prevention group, only GLP-1 RA cessation by time (HR 2.71, 95% CI 1.46-5.01, p = 0.002) increased the risk of MACE. With respect to those who withdrew treatment, subjects who continued the GLP-1 RA had significantly greater weight loss and lower glycated hemoglobin levels during follow-up. CONCLUSIONS: In this real-world type 2 diabetes population, discontinuation of GLP-1 RA treatment was associated to a higher risk of major cardiovascular events, in both subjects with and without a history of CV events.

Biochemical Efficacy of Sodium-Glucose Cotransporter 2 Inhibitors by Cardiovascular Risk Profile and Volume Status in a Real-World Diabetic Population.

ABSTRACT: Despite large-scale randomized clinical trials (RCTs) highlighting a consistent prognostic benefit of sodium-glucose cotransporter 2 inhibitors (SGLT2is) both in diabetic patients at high cardiovascular risk and in those with heart failure, there is relative paucity of data on their biochemical effects in a real-world setting. We performed a retrospective analysis on consecutive diabetic patients who were prescribed a SGLT2i in a tertiary referral center and completed at least 1 year of treatment. Changes in glycated hemoglobin, weight, and hematocrit were compared across 2 cardiovascular risk categories, defined through the inclusion criteria of 3 large RCTs. Of the 459 patients screened, 312 completed 1 year of treatment (68.0%), 92 interrupted the treatment prematurely (20.0%), and 55 were lost to follow-up (12.0%). The most common cause of drug discontinuation was genital or urinary tract infections (9.4%). At 1 year, reduction in glycated hemoglobin concentration (-0.7 ± 1.5%, P < 0.001) and body weight (2.4 ± 4.6 kg, P < 0.001) was comparable between patients at high versus low cardiovascular risk, while hematocrit increase (2.3 ± 3.3%, P < 0.001) was more marked in patients with high cardiovascular risk and low baseline hematocrit. In a real-world population of diabetic patients, SGLT2is were well-tolerated at 1 year and led to improved glycemic control and weight loss. Hematocrit increase was more consistent in patients with high cardiovascular risk and signs of fluid overload, indicating euvolemic restoration as a potential cardioprotective mechanism mediated by these compounds.

Myocardial hypoxic stress mediates functional cardiac extracellular vesicle release.

AIMS: Increased shedding of extracellular vesicles (EVs)-small, lipid bilayer-delimited particles with a role in paracrine signalling-has been associated with human pathologies, e.g. atherosclerosis, but whether this is true for cardiac diseases is unknown. METHODS AND RESULTS: Here, we used the surface antigen CD172a as a specific marker of cardiomyocyte (CM)-derived EVs; the CM origin of CD172a+ EVs was supported by their content of cardiac-specific proteins and heart-enriched microRNAs. We found that patients with aortic stenosis, ischaemic heart disease, or cardiomyopathy had higher circulating CD172a+ cardiac EV counts than did healthy subjects. Cellular stress was a major determinant of EV release from CMs, with hypoxia increasing shedding in in vitro and in vivo experiments. At the functional level, EVs isolated from the supernatant of CMs derived from human-induced pluripotent stem cells and cultured in a hypoxic atmosphere elicited a positive inotropic response in unstressed CMs, an effect we found to be dependent on an increase in the number of EVs expressing ceramide on their surface. Of potential clinical relevance, aortic stenosis patients with the highest counts of circulating cardiac CD172a+ EVs had a more favourable prognosis for transcatheter aortic valve replacement than those with lower counts. CONCLUSION: We identified circulating CD172a+ EVs as cardiac derived, showing their release and function and providing evidence for their prognostic potential in aortic stenosis patients.

Early detection of elevated cardiac biomarkers to optimise risk stratification in patients with COVID-19.

OBJECTIVE: Risk stratification is crucial to optimise treatment strategies in patients with COVID-19. We aimed to evaluate the impact on mortality of an early assessment of cardiac biomarkers in patients with COVID-19. METHODS: Humanitas Clinical and Research Hospital (Rozzano-Milan, Lombardy, Italy) is a tertiary centre that has been converted to the management of COVID-19. Patients with confirmed COVID-19 were entered in a dedicated database for cohort observational analyses. Outcomes were stratified according to elevated levels (ie, above the upper level of normal) of high-sensitivity cardiac troponin I (hs-TnI), B-type natriuretic peptide (BNP) or both measured within 24 hours after hospital admission. The primary outcome was all-cause mortality. RESULTS: A total of 397 consecutive patients with COVID-19 were included up to 1 April 2020. At the time of hospital admission, 208 patients (52.4%) had normal values for cardiac biomarkers, 90 (22.7%) had elevated both hs-TnI and BNP, 59 (14.9%) had elevated only BNP and 40 (10.1%) had elevated only hs-TnI. The rate of mortality was higher in patients with elevated hs-TnI (22.5%, OR 4.35, 95% CI 1.72 to 11.04), BNP (33.9%, OR 7.37, 95% CI 3.53 to 16.75) or both (55.6%, OR 18.75, 95% CI 9.32 to 37.71) as compared with those without elevated cardiac biomarkers (6.25%). A multivariate analysis identified concomitant elevation of both hs-TnI and BNP as a strong independent predictor of all-cause mortality (OR 3.24, 95% CI 1.06 to 9.93). CONCLUSIONS: An early detection of elevated hs-TnI and BNP predicts mortality in patients with COVID-19. Cardiac biomarkers should be systematically assessed in patients with COVID-19 at the time of hospital admission in order to optimise risk stratification.

Monotherapy with a P2Y12 inhibitor or aspirin for secondary prevention in patients with established atherosclerosis: a systematic review and meta-analysis.

BACKGROUND: Antiplatelet therapy is recommended among patients with established atherosclerosis. We compared monotherapy with a P2Y12 inhibitor versus aspirin for secondary prevention. METHODS: In this systematic review and meta-analysis, all randomised trials comparing P2Y12 inhibitor with aspirin monotherapy for secondary prevention in patients with cerebrovascular, coronary, or peripheral artery disease were evaluated for inclusion. On Dec 18, 2019, we searched PubMed, Embase, BioMedCentral, Google Scholar, and the Cochrane Central Register of Controlled Trials. Additionally, we reviewed references from identified articles and searched abstracts from 2017 to 2019 presented at relevant scientific meetings. Data about year of publication, inclusion and exclusion criteria, sample size, baseline patients' features including the baseline condition determining study inclusion (ie, cerebrovascular, coronary, or peripheral artery disease), P2Y12 inhibitor type and dosage, aspirin dosage, endpoint definitions, effect estimates, follow-up duration, and percentage of patients lost to follow-up were collected. Odds ratios (ORs) and 95% CIs were used as metric of choice for treatment effects with random-effects models. Co-primary endpoints were myocardial infarction and stroke. Key secondary endpoints were all-cause death and vascular death. Heterogeneity was assessed with the I2 index. This study is registered with PROSPERO (CRD42018115037). FINDINGS: A total of nine randomised trials were identified and included in this study, and 42 108 patients randomly allocated to a P2Y12 inhibitor (n=21 043) or aspirin (n=21 065) were included in our analyses. Patients who received a P2Y12 inhibitor had a borderline reduction for the risk of myocardial infarction compared with those who received aspirin (OR 0·81 [95% CI 0·66-0·99]; I2=10·9%). Risks of stroke (OR 0·93 [0·82-1·06]; I2=34·5%), all-cause death (OR 0·98 [0·89-1·08]; I2=0%), and vascular death (OR 0·97 [0·86-1·09]; I2=0%) did not differ between patients who received a P2Y12 inhibitor and those who received aspirin. Similarly, the risk of major bleeding (OR 0·90 [0·74-1·10]; I2=3·9%) did not differ between patients who received a P2Y12 inhibitor and those who received aspirin. The number needed to treat to prevent one myocardial infarction with P2Y12 inhibitor monotherapy was 244 patients. Findings were consistent regardless of the type of P2Y12 inhibitor used. INTERPRETATION: Compared with aspirin monotherapy, P2Y12 inhibitor monotherapy is associated with a risk reduction for myocardial infarction and a comparable risk of stroke in the setting of secondary prevention. The benefit of P2Y12 inhibitor monotherapy is of debatable clinical relevance, in view of the high number needed to treat to prevent a myocardial infarction and the absence of any effect on all-cause and vascular mortality. FUNDING: Italian Ministry of Education.

Single-Cell Sequencing of Mouse Heart Immune Infiltrate in Pressure Overload-Driven Heart Failure Reveals Extent of Immune Activation.

BACKGROUND: Inflammation is a key component of cardiac disease, with macrophages and T lymphocytes mediating essential roles in the progression to heart failure. Nonetheless, little insight exists on other immune subsets involved in the cardiotoxic response. METHODS: Here, we used single-cell RNA sequencing to map the cardiac immune composition in the standard murine nonischemic, pressure-overload heart failure model. By focusing our analysis on CD45+ cells, we obtained a higher resolution identification of the immune cell subsets in the heart, at early and late stages of disease and in controls. We then integrated our findings using multiparameter flow cytometry, immunohistochemistry, and tissue clarification immunofluorescence in mouse and human. RESULTS: We found that most major immune cell subpopulations, including macrophages, B cells, T cells and regulatory T cells, dendritic cells, Natural Killer cells, neutrophils, and mast cells are present in both healthy and diseased hearts. Most cell subsets are found within the myocardium, whereas mast cells are found also in the epicardium. Upon induction of pressure overload, immune activation occurs across the entire range of immune cell types. Activation led to upregulation of key subset-specific molecules, such as oncostatin M in proinflammatory macrophages and PD-1 in regulatory T cells, that may help explain clinical findings such as the refractivity of patients with heart failure to anti-tumor necrosis factor therapy and cardiac toxicity during anti-PD-1 cancer immunotherapy, respectively. CONCLUSIONS: Despite the absence of infectious agents or an autoimmune trigger, induction of disease leads to immune activation that involves far more cell types than previously thought, including neutrophils, B cells, Natural Killer cells, and mast cells. This opens up the field of cardioimmunology to further investigation by using toolkits that have already been developed to study the aforementioned immune subsets. The subset-specific molecules that mediate their activation may thus become useful targets for the diagnostics or therapy of heart failure.

UHRF1 epigenetically orchestrates smooth muscle cell plasticity in arterial disease.

Adult vascular smooth muscle cells (VSMCs) dedifferentiate in response to extracellular cues such as vascular damage and inflammation. Dedifferentiated VSMCs are proliferative, migratory, less contractile, and can contribute to vascular repair as well as to cardiovascular pathologies such as intimal hyperplasia/restenosis in coronary artery and arterial aneurysm. We here demonstrate the role of ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) as an epigenetic master regulator of VSMC plasticity. UHRF1 expression correlated with the development of vascular pathologies associated with modulation of noncoding RNAs, such as microRNAs. miR-145 - pivotal in regulating VSMC plasticity, which is reduced in vascular diseases - was found to control Uhrf1 mRNA translation. In turn, UHRF1 triggered VSMC proliferation, directly repressing promoters of cell-cycle inhibitor genes (including p21 and p27) and key prodifferentiation genes via the methylation of DNA and histones. Local vascular viral delivery of Uhrf1 shRNAs or Uhrf1 VSMC-specific deletion prevented intimal hyperplasia in mouse carotid artery and decreased vessel damage in a mouse model of aortic aneurysm. Our study demonstrates the fundamental role of Uhrf1 in regulating VSMC phenotype by promoting proliferation and dedifferentiation. UHRF1 targeting may hold therapeutic potential in vascular pathologies.

The Synergy stent in high-bleeding risk patients: why design matters.

Antithrombotic management after percutaneous coronary intervention is based on dual antiplatelet therapy (DAPT), that has unequivocally shown to reduce the risk of recurrent ischemic events at cost of an important risk of bleeding. In order to balance ischemic and bleeding risks, DAPT duration should be based on patients and lesions features as well as stent type. Based on these considerations, patients at high bleeding risk (HBR) undergoing PCI represent a challenging subgroup. The Synergy stent (Boston Scientific, Marlborough, MA, USA) is a new-generation everolimus-eluting stent with a biodegradable polymer coating, presenting several features that have the potential to reduce acute and long-term thrombogenicity and the subsequent need for DAPT. The aim of this manuscript is to review the evidence about the impact of stent design on the optimal antithrombotic strategy in HBR patients and the potential role of the Synergy BP-EES in this relevant population.

Unmet Needs in the Pathogenesis and Treatment of Cardiovascular Comorbidities in Chronic Inflammatory Diseases.

The developments that have taken place in recent decades in the diagnosis and therapy of a number of diseases have led to improvements in prognosis and life expectancy. As a consequence, there has been an increase in the number of patients affected by chronic diseases and who can face new pathologies during their lifetime. The prevalence of chronic heart failure, for example, is approximately 1-2% of the adult population in developed countries, rising to ≥10% among people >70 years of age; in 2015, more than 85 million people in Europe were living with some sort of cardiovascular disease (CVD) (Lubrano and Balzan World J Exp Med 5:21-32, 5; Takahashi et al. Circ J 72:867-72, 8; Kaptoge et al. Lancet 375:132-40, 9). Chronic disease can become, in turn, a major risk factor for other diseases. Furthermore, several new drugs have entered clinical practice whose adverse effects on multiple organs are still to be evaluated. All this necessarily involves a multidisciplinary vision of medicine, where the physician must view the patient as a whole and where collaboration between the various specialists plays a key role. An example of what has been said so far is the relationship between CVD and chronic inflammatory diseases (CIDs). Patients with chronic CVD may develop a CID within their lifetime, and, vice versa, a CID can be a risk factor for the development of CVD. Moreover, drugs used for the treatment of CIDs may have side effects involving the cardiovascular system and thus may be contraindicated. The purpose of this paper is to investigate the close relationship between these two groups of diseases and to provide recommendations on the diagnostic approach and treatments in light of the most recent scientific data available.