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Background In the last few years the role of gut microbiome in human health has been reported. Growing evidence indicate that it is linked to intestinatal diseases, such as the irritable bowel syndrome (IBS), and atherosclerotic disorders. The effect on lipid metabolism seems to be one of the potential mechanisms by which gut microbiome might favour the atherosclerotic process. IBS is a functional bowel disorder defined by the presence of recurrent episodes of abdominal pain associated with altered bowel habits. The severity of psychological and gastrointestinal symptoms in IBS has been linked with a distinct intestinal microbiota signature. In this retrospective study the effect of a novel nutraceutical compound, namely Triobiotix, on gastrointestinal symptoms in IBS patients has been evaluated. Effects on lipid profile have bee also recorded. Methods Triobiotix is a nutraceutical consisting of: Maltodextrin; mineralized extract of Lithothamnion (Lithothamnion calcareum (Pallas) Areschoug, thallus dry extract); Bioecolians® gluco-oligosaccharides; Ferment mix (corn starch, Bifidobacterium animalis ssp. Lactis BLC1 (DSM 17741), Lactobacillus acidophilus LA3 (DSM 17742), Lactobacillus rhamnosus IMC 501 (DSM 16104), Lactobacillus paracasei Streptococc5 501 IMC102 SP4 (DSM 19385); short-chain fructo-oligosaccharides powder. The database of 40 Italian General Practitioners (GPs) were analyzed. At total of 587 patients with IBS and on Triobiotix were identified. Of these in only 535, a complete first and second control data was available. The primary endpoint of these analysis was to assess whether Triobiotix resulted in a reduction in pain and meteorism, thus resulting in a lesser intensity of the main gastrointestinal symptoms. Secondary endpoints were to look for significative changes in lab values of total cholesterol, triglycerides levels and glycaemia. Results Treatment with Triobiotix for four weeks resulted in a reduction in the frequency and intensity of bloating, abdominal pain, and tenesmus. Unexpectedly, at the T1 control, mean cholesterol values were significatively reduced compared to the T0 evaluation. Triglycerides levels were also significantly decreased (186.37±66.4 vs. 176.13±67.9; p < 0.05). Conclusions Our analysis showed an unexpected effect of this combination of micronutrients on lipid profile beyond IBS symptoms. However, further studies are needed to confirm this evidence and to evaluate the particular compound responsible of this effect. A goal of LDL-C less of 115mg/dL is desirable for patients at low to moderate CVR, a significative portion of population is far from this target. The discovery of novel micronutrients combinations with cholesterol lowering effects could be of great importance for the management of patients with low-moderate dyslipidaemia.

Image focus A 42-year-old woman was carried to emergency room lamenting sudden memory loss. Her husband witnessed the onset of the episode. No recent head trauma was reported, blood pressure was under control. Anterograde and retrograde memory loss was present and confirmed by a neurologist. No other neurological signs were elicitable. Blood exams and toxicological analysis were negative. ECG, chest radiography, and cerebral TC showed no abnormalities. At physical examination a carotid bruit could be appreciated. We performed an eco-color-Doppler of carotid artery that showed a left common and internal carotid artery dissection (figure 1). The diagnosis of ICA dissection was confirmed with a contrast CT. The coexistence of an aortic dissection was excluded with the same imaging technique. The anterograde amnesia resolved 15 h after the admission. A neurologist confirmed that the patient experimented an episode of transient global amnesia (TGA). After a vascular surgery consultation, a conservative treatment was deemed appropriate. After a 6 month follow up the patient didn't complain of any other neurological symptom or sign. Transient global amnesia (TGA) is characterized by reversible anterograde and retrograde amnesia of sudden onset. This amnesic syndrome resolves spontaneously within 24 h. Annual incidence rate for all ages of spontaneous ICA dissection is approximately of 2.6 per 100 000 inhabitants, however data are extremely variable among different studies. TGA has never been associated with a spontaneous ICA dissection. In order to not miss a spontaneous ICA dissection as a rare ethology of TGA, we suggest performing an eco-color-Doppler of the epi-aortic vessels in patient affected by such amnesic syndrome.

A 39 year old man presented to the emergency department with dyspnoea on exertion, chest pain and fatigue. Few years before, the patient was stabbed in the left chest thus developing Pneumothorax and mild pericardial effusion successfully treated with chest tube drainage and medical therapy. Physical examination revelead giugular vein distension and lower extremity edema. The echocardiographic evaluation showed typical signs of constrictive pericarditis which was confirmed by cardiac catheterization. Furthermore, because of clinical worsening and the absence of ongoing inflammation on PET-TC, the patient was referred to surgery. Pericardiectomy was successfully performed with rapid symptoms relief and clinical benefits. Histologic examination of pericardium showed the presence of thick fibrotic tissue with clusters of squamous epithelial cells with no cellular atypias or malignancy. Therefore, we hypothesize that following the chest stabbing wound, squamous and epitheloid cells have invaded the pericardial space producing a fibrotic reaction responsable of the development of the constriction. Furthermore multimodality imaging has been essential for diagnosis and management.

Image focus A 65-year-old women was scheduled for coronary angiography during the pre-surgery evaluation because of an abdominal aortic aneurysm (AAA) detected at ultrasound (Figure). The patient referred worsening dyspnoea and asthenia in the last few months, associated with atypical chest pain. Because of these symptoms and taking into account that AAA patients are at very high cardiovascular risk, exclusion of coronary artery disease seemed appropriate. At admission, patient showed enlarged lymph nodes at cervical and inguinal region. In the suspicion of a malignant cancer, a contrast enhanced computed tomography was scheduled, showing an increase in para-aortic, inguinal, cervical and axillary lymph nodes size in absence of the AAA (Figure). After blood tests and an haematologic consultation, a final diagnosis of chronic myeloid leukaemia was made. The Echo-colour Doppler is the first line imaging test for the diagnosis of AAA. However, in some cases it might be not sufficient. Para-aortic lymphadenopathy should be considered as a possible pitfall in the differential diagnosis of abdominal aortic aneurysm.

Beyond the Intima Media Thickness assessment: the measurement of overall echogenicity (GSM) of Carotid Intima Media Complex shows a positive correlation with Arterial Stiffness of hypertensive patients. According to the latest ESC guidelines on CVD prevention (2021) arterial stiffness still has a role in predicting future CVD risk and defining patients’ CVD risk level. However, significative measurement difficulties make a widespread evaluation of the AS problematic, especially in the ambulatorial outpatient setting. The IMT measurement is no longer recommended in the CVD risk assessment due to lack of standardization in measurement protocol and low power to reclassify patients toward higher CV risk. Understanding the correlation between the structure and function of the large arteries can lead to an optimization of cardiovascular disease prevention strategies. At present, indices of arterial stiffness have not been associated with qualitative morphological characteristics of intima-media complex. The IM GSM is a relatively simple measurement that can be acquired during a carotid color-Doppler ultrasound exam. The aim of this study was to investigate the relationship between indices of arterial stiffness and echogenicity of intima media complex (IM GSM) of the common carotid arteries in subjects with arterial hypertension. In our study population of 421 hypertensive patients, subjects with IM GSM > 30 showed a significantly increased arterial stiffness and LVMI (P < 0.001). A weak positive correlation was found between IM GSM and SBP (r =0.35, < 0.001), and duration of hypertension (r = 0.31, < 0.01). Our study suggests that increased stiffness observed in hypertensives is associated with qualitative changes in carotid arterial wall.

Introduction: Mammal cardiomyocytes (CMs) lose their proliferative capacity early after birth and switch to an adult phenotype, characterized by organized sarcomeres. The mechanisms that control this switch remain largely elusive. Among the changes experienced by the heart at birth is a sudden increase in mechanical load. Hypothesis: This study tests the hypothesis that variations in mechanical loading may regulate both the proliferation and the maturation of cardiomyocytes. Methods: Engineered heart tissues (EHTs) were generated using neonatal rat heart cells, with a modified protocol allowing to alter mechanical load. Unloading is obtained by reducing the distance between the silicon posts that anchor the extremities of the EHT, while overloading is induced by changing Young’s module of posts using metal braces. Adult heart unloading in vivo was achieved by heterotopically transplanting a mouse heart into the neck of syngeneic recipient mice, which were injected with EdU twice per week. Both hearts, native and transplanted, were harvested at day 30. Results: In line with our hypothesis, mechanical unloading of developed EHTs significantly increased the percentage of EdU-, Ki67- and pH3-positive CMs at 72 hours. In contrast, increasing afterload induced CM maturation, characterized by increased cross-sectional area and force of contraction, associated with reduced proliferation. Consistently, heterotopically transplanted hearts showed a significant increase in the the number of proliferating CMs in both ventricles, which were almost absent in native hearts. Conclusions: Our data indicate that mechanical unloading induce CM proliferation in both rat EHTs and an in vivo in a mouse model of heterotopic heart transplantation. These results support our initial hypothesis and indicate that mechanical loading is a master regulator of cardiomyocytes proliferation and maturation. Ongoing experiments aim at identifying the molecular mechanisms that regulate this process.

Aims Adult mammals, including humans, fail to regenerate the majority of the lost cardiomyocytes (CMs) that are replaced with scar tissue after injury. This lack of regenerative response is due to the loss of proliferative capacity of adult CMs which in mice occurs 7 days after birth. An in vitro model that recapitulates these changes has not been developed yet. Using rat engineered heart tissues (rEHTs) we have developed a custom-made cryoinjury system to test the hypothesis that maturation of CMs in EHTs regulates the proliferative response of CMs after injury. Methods rEHT were generated using neonatal rat heart cells. A discrete lesion was produced on the mid-section of mature (Day 18) and immature (Day 6) EHTs using a custom-made system based on liquid nitrogen and a 23G needle and medium was supplemented with EdU for 48 h. Results Cryoinjury in mature EHTs produces a localized injury, preserving their residual contractile activity that does not recover over time. We observed a significant increase of EdU+CMs post injury (6.3 ± 1.9% vs. 10.1 ± 1.6%) without significant changes in Ki67+ and pH3+ CMs suggesting that cryoinjury in mature rEHTs induces DNA synthesis but not CM proliferation. Injury in mature EHTs induced also significant proliferation and activation of fibroblasts with collagen deposition. Interestingly, cryoinjury performed in immature EHTs stimulated a significant proliferative response in CMs Conclusions Similar to adult rodents, we show that cryoinjury induces DNA synthesis in CMs without proliferative response and contractile recovery. On the other hand, cryoinjury in immature EHTs leads to CMs proliferation. Moreover, mature EHT fibroblast response to injury retraces the activation progression of cardiac fibroblast after infarction characterized by proliferation, increase of activation markers, increase of collagen deposition suggesting EHTs as a novel model to investigate the biology of cardiac regeneration upon injury.

Aims T-lymphocytes plays an important role in the pathophysiology of acute coronary syndromes. T-cell activation in vitro by pro-inflammatory cytokines may lead to functional tissue factor (TF) expression, indicating a possible contribution of immunity to thrombosis. Oxidized low-density lipoproteins (oxLDLs) are found abundantly in atherosclerotic plaques. We aimed at evaluating the effects of oxLDLs on TF expression in T cells and the role of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Methods and results CD3+ cells were isolated from healthy volunteers. Gene, protein, and surface expression of TF, as well as of LOX-1, were assessed at different time-points after oxLDL stimulation. To determine whether oxLDL-induced TF was LOX-1 dependent, T cells were pre-incubated with an LOX-1 inhibiting peptide (L-RBP) or with an anti-LOX-1 blocking antibody. To exclude that TF expression was mediated by reactive oxygen species (ROS) generation, oxLDL-stimulated T cells were pre-incubated with superoxide dismutase + catalase or with 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), an intracellular free radical scavenger. Finally, to determine if the observed findings in vitro may have a biological relevance, the presence of CD3+/TF+/LOX-1+ cells was evaluated by immunofluorescence in human carotid atherosclerotic lesions. oxLDLs induced functionally active TF expression in T cells in a dose- and time-dependent manner, independently on ROS generation. No effect was observed in native LDL-treated T cells. LOX-1 expression was also induced by oxLDLs in a time- and dose-dependent manner. Pre-incubation with L-RBP or anti-LOX-1 antibody almost completely inhibited oxLDL-mediated TF expression. Interestingly, human carotid plaques showed significant infiltration of CD3+ cells (mainly CD8+ cells), some of which were positive for both TF and LOX-1. Conclusion oxLDLs induce functional TF expression in T-lymphocytes in vitro via interaction of oxLDLs with LOX-1. Human carotid atherosclerotic plaques contain CD3+/CD8+cells that express both TF and LOX-1, indicating that also in patients these mechanisms may play an important role.

Background Heart failure with preserved ejection fraction (HFpEF) is a prevalent clinical condition in the aging population. HFpEF lacks specific therapies with a significant impact on survival, different therapeutic approaches have indeed failed, indicating a specific need in understanding the pathophysiology of the disease. Cardiac hypertrophy is one of the main features of HFpEF that contributes to impair diastolic function. Reducing ventricular stiffness associated with cardiomyocyte hypertrophy may indeed result in improved diastolic filling. Growth differentiation factor 11 (Gdf11), a TGF-β family factor, has been identified as a circulating factor able to reduce cardiac hypertrophy. Purpose Similar to myostatin (Gdf8), Gdf11 promotes the activation of atrophy pathways that induce ubiquitination of sarcomeric proteins. Gdf11 and Gdf8 activate both type I and II Tgf-β receptors, specifically by interacting with Acvr2a and Acvr2b (type II) and Alk 4/5/7 (type I), they activate Smad 3/4 pathway. Our data indicate a specific and more potent effect of Gdf11 in reducing cardiomyocytes size that is not recapitulated by Gdf8. Understanding the specific effect of Gdf11 on cardiomyocytes is crucial to develop therapeutic strategies to target the hypertrophic phenotype. Methods To investigate the effects of specific type I receptor KO on Smad signaling cascade we performed our preliminary experiment on HL-1 cells, a cardiac muscle cell line carrying a doxycycline inducible Cas9 transgene. Cell sensitivity of HL-1 to Gdf11 and Gdf8 was tested by performing a dose-response curve using a luciferase reporter for Smad 3/4 pathway activation (CAGA12). Selective Tgf-β type I receptor KO was induced using, for each receptor, two sgRNAs that have been designed to cleave the receptor coding sequence creating INDEL mutations and disrupt proper translation of the protein and confirmed by western blotting. Smad 3/4 activity was measured using a CAGA12-luciferase assay on HL-1-Cas9 transduced cells. Results Our data in HL-1 cells confirm a more potent effect of Gdf11 in activating Smad 3/4 pathway when compared to Gdf8 (Fig. 1A). Selective Alk4 and Alk5 KO induced a similar reduction in Smad 3/4 activation for both Gdf11 and Gdf8. Interestingly, Alk7 KO significantly reduced Gdf11 signaling that was not recapitulated when using Gdf8, suggesting that Alk7 receptor is crucial for Gdf11-dependent Smad 3/4 activation in HL-1 cells (Fig. 1B). Figure 1 Conclusions Our preliminary results indicate that part of Gdf11 cardiac specificity when compared to Gdf8 may reside in the usage of Alk7 for signaling. Alk7 has proposed as a protective factor for pathological cardiac hypertrophy by negatively regulating Mek-Erk1/2 signalling. Our results are now under investigation in vitro using mouse neonatal cardiomyocytes expressing Cas-9 and in vivo using cardiac-specific-Cas9-expressing C57 transgenic mice, in resting condition and after induction of pathological hypertrophy.

Background Matrix metalloproteases (MMPs) have been implicated in the pathogenesis of acute coronary syndromes (ACS). However, little is known about the mechanisms responsible for MMP expression in ACS. C-reactive protein (CRP) not only is an independent risk factor for cardiovascular events, but also may exert direct pro-atherosclerotic effects. Therefore, we aimed at determining whether CRP might induce MMP-9 in two different experimental conditions: 1) smooth muscle cells (SMCs) in vitro, and 2) patients with ACS. Methods and results Effects of increasing concentrations of CRP on MMP-9 expression were evaluated in vitro in human SMCs. TIMP-1 protein expression, the selective inhibitor of MMP-9, was also evaluated. CRP dose-dependently induced MMP-9 expression in SMCs by promoting MMP-mRNA transcription, as well as MMP-9 secretion. In contrast, no differences were found for TIMP-1 protein expression. In vivo, MMP-9 and CRP levels were measured in blood samples obtained from the aorta (Ao) and the coronary sinus (Cs) of patients with normal coronary arteries (controls, n=21), stable angina (n=24), and ACS (n=30). Both MMP-9 and CRP plasma levels were significantly increased across the coronary circulation only in patients with ACS. Interestingly, a significant correlation between MMP-9 and CRP plasma levels was found. Conclusions CRP induced MMP-9 expression and activity in human SMCs in culture; patients presenting with ACS have increased transcoronary plasma levels of MMP-9 and CRP with a significant correlation between these two markers. This may explain the heightened risk of coronary events in subjects with elevated levels of CRP.

High plasma levels of low density lipoproteins (LDLs) represent one of the major risk factors for cardiovascular disease, as shown by many epidemiological studies. On the other hand, randomized trials designed to address the clinical impact of lipid lowering interventions, have clearly shown that reduction in LDL plasma levels lead to a significant decrease in major cardiovascular events. Based on these observations, pharmacological modulation of LDLs has been highly investigated. Statins, alone or in combination, represent the most powerful agents to date available to reach the LDLs levels suggested by the current guidelines. However, in some patients the recommended LDL reduction is difficult to be achieved because of genetic background (familial hypercholesterolemia), side effects (statin intolerance), or simply because of a non-sufficient response. In the last few years, our understanding of the basic mechanisms involved in the lipoprotein metabolism has progressed significantly. The crucial role of proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged. The main characterized function of PCSK9 relates to the binding to LDL-C receptors (LDLR) in hepatocytes. However, PCSK9 does not interfere with the binding between LDL and its own receptor, but with the ability of the latest to return to the surface of the hepatocyte and bind new LDL molecules. Based on these observations, blocking PCSK-9 may reduce the LDLR clearance, thus increasing the ability of LDLR to remove circulating LDLs. Pharmacological inhibition of this protein has been proposed as new therapeutic approach. The clinical evidence available to date seem to fully support this hypothesis.

Aims To investigate whether selected matrix metalloproteinases (MMPs) are released in the coronary circulation of patients with acute coronary syndrome (ACS), whether this release is related to platelet activation, and whether it contributes to sustained platelet activation. Methods and results Blood from the aorta (Ao) and the coronary sinus (Cs) was obtained from 21 controls (non-cardiac chest pain), 24 stable angina (SA), and 30 ACS patients, before performing percutaneous transluminal coronary angioplasty. Selected MMPs, some platelet activation- and atheroma-related markers, and the platelet activation-potentiating activity of plasma were measured. Total MMP-2, active MMP-2, and MMP-9 were released in the coronary circulation of patients with ACS, but not of those with SA or controls. Similarly, transcoronary gradients of b-thromboglobulin (b-TG) and platelet factor 4, two platelet-specific proteins, and of soluble CD40L and secretory phospholipase A2 (sPLA2), markers of inflammation and platelet activation, were higher in ACS patients than in the other groups. In contrast, plasma monocyte chemoattractant protein-1, a platelet-unrelated marker of atherogenesis, was not increased in the Cs compared with Ao in any of the groups. Transcoronary gradients of both b-TG and sPLA2 correlated with those of total and active MMP-2 in ACS, but not in controls or SA. Plasma from the Cs of ACS patients potentiated platelet activation, an effect suppressed by the specific MMP-2-inhibitor, tissue inhibitor of MMP-2 (TIMP-2). Conclusion Matrix metalloproteinase-2 is released in the coronary circulation of ACS patients, derives in part from activated platelets, and may contribute to sustained intracoronary platelet activation.

Background C-reactive protein (CRP) plasma levels correlate with cardiovascular events. Although a direct role for CRP in atherothrombosis has been suggested, at the moment little is known about its involvement in the pathophysiology of acute coronary syndromes (ACS). Thus, the aim of this study was to determine whether CRP is produced in the culprit lesion and released within the coronary circulation of patients with ACS and whether it may affect coronary endothelial function. Methods Blood samples were simultaneously obtained from the aorta (Ao) and the coronary sinus (CS) of patients with normal coronary artery (n=16), stable angina (n=30), and ACS (n=29) for later measurement of plasma CRP levels. Endothelium-dependent and -independent coronary vasodilation were evaluated by means of a Doppler Flow Wire in response to the increasing intracoronary doses of acetylcholine and adenosine, respectively. Results CRP plasma levels were significantly higher across the coronary circulation only in ACS patients with the culprit lesion located in the left coronary artery, while no differences between CS and Ao CRP plasma levels were observed in all other groups. Transcardiac CRP levels were correlated with impairment in coronary endothelium-dependent vasodilation. In six additional patients (SA=3 and ACS=3), subjected to coronary atherectomy, real-time quantitative PCR revealed presence of CRP mRNA only in unstable plaques. Conclusions Thus, CRP is produced and released within the coronary circulation of patients with ACS; this is associated with impairment of endothelial function, suggesting a new pathophysiological link between CRP and ACS.

Background. The mechanisms by which regenerative therapies improve cardiac function are incompletely defined. Numerous laboratories have demonstrated that the stem cell chemoattractant Stromal cell derived factor-1 (SDF-1) improves cardiac function after myocardial infarction (MI). This study used a genetic fate-mapping approach to ask the question: Is the beneficial effect of SDF-1 delivery due to cardiomyocyte regeneration, increased capillary density, or both? Methods. We used a genetic fate-mapping system that allows “pulse-chase” studies of cardiomyocyte turnover in mice. We crossed an inducible cardiomyocyte-specific Mer-Cre-Mer transgenic mouse with Z/EG reporter mice. Using a high-efficiency 4-OH-tamoxifen protocol, we then induced Cre recombination and expression of GFP in cardiomyocytes only. SDF-1(S4V), a protease-resistant form of SDF-1, was delivered with nanofibers after coronary ligation. Results. The percentage of pulse-labeled GFP+ cardiomyocytes was 81±4% (n=6) in sham-operated mice, as anticipated with this system. GFP+ cardiomyocytes were 60±5% and 69±4% (n=11) respectively at MI border and MI remote areas of the control nanofiber group, consistent with our previous study and suggesting a significant stem/precursor cell contribution to cardiomyocyte replacement after injury (P Conclusion. Using genetic “pulse-chase” fate mapping, these data indicate that the stem cell chemoattractant SDF-1 does not induce measurable adult mammalian cardiomyocyte replacement by stem/precursor cells following injury. However, SDF-1 significantly increases myocardial capillary density, suggesting that increased angiogenesis but not cardiogenesis is responsible for the beneficial effects of SDF-1 on cardiac function.

OBJECTIVES The aim of the present study was to test the hypothesis that retrovirus-mediated in vivo tissue factor pathway inhibitor {TFPI) gene transfer to the arterial wall would efficiently inhibit thrombosis without causing significant changes in systemic hemostatic variables. BACKGROUND Acute coronary syndromes (unstable angina and acute myocardial infarction) are usually caused by atherosclerotic plaque rupture, with consequent activation of the coagulation cascade and circulating platelets. Tissue Factor (TF) exposure represents an early event in this pathophysiologic sequence, leading to activation of the extrinsic coagulation pathway and thrombin formation. Tissue factor pathway inhibitor is a naturally occurring inhibitor of the extrinsic pathway. METHODS In the present study, the gene coding for rabbit TFPI was inserted in a retroviral vector under control of a tetracycline-inducible promoter. Replication-defective, infectious, recombinant retroviruses were used to transfect rabbit carotid arteries with either TFPI or a reporter gene-green fluorescent protein (GFP). RESULTS Retroviral-mediated arterial gene transfer of TFPI resulted in potent inhibition of intravascular thrombus formation in stenotic and injured rabbit carotid arteries, whereas transfection of the contralateral carotid artery with GFP had no effect on thrombosis. No significant changes in systemic hemostatic variables {prothrombin time and partial thromboplastin time) were observed when thrombosis was inhibited. CONCLUSIONS These data suggest that retroviral-mediated transfection of the arterial wall with TFPI might represent an attractive approach for the treatment of thrombotic disorders. (J Am Coll Cardiol 2001;38:569-76) (C) 2001 by the American College of Cardiology.