Platelet-derived exosomes from septic shock patients induce myocardial dysfunction

Abstract Introduction Mechanisms underlying inotropic failure in septic shock are incompletely understood. We previously identified the presence of exosomes in the plasma of septic shock patients. These exosomes are released mainly by platelets, produce superoxide, and induce apoptosis in vascular cells by a redox-dependent pathway. We hypothesized that circulating platelet-derived exosomes could contribute to inotropic dysfunction of sepsis. Methods We collected blood samples from 55 patients with septic shock and 12 healthy volunteers for exosome separation. Exosomes from septic patients and healthy individuals were investigated concerning their myocardial depressant effect in isolated heart and papillary muscle preparations. Results Exosomes from the plasma of septic patients significantly decreased positive and negative derivatives of left ventricular pressure in isolated rabbit hearts or developed tension and its first positive derivative in papillary muscles. Exosomes from healthy individuals decreased these variables non-significantly. In hearts from rabbits previously exposed to endotoxin, septic exosomes decreased positive and negative derivatives of ventricular pressure. This negative inotropic effect was fully reversible upon withdrawal of exosomes. Nitric oxide (NO) production from exosomes derived from septic shock patients was demonstrated by fluorescence. Also, there was an increase in myocardial nitrate content after exposure to septic exosomes. Conclusion Circulating platelet-derived exosomes from septic patients induced myocardial dysfunction in isolated heart and papillary muscle preparations, a phenomenon enhanced by previous in vivo exposure to lipopolysaccharide. The generation of NO by septic exosomes and the increased myocardial nitrate content after incubation with exosomes from septic patients suggest an NO-dependent mechanism that may contribute to myocardial dysfunction of sepsis.

Association between UCP2 A55V polymorphism and risk of cardiovascular events in patients with multi-vessel coronary arterial disease

Background UCP2 (uncoupling protein 2) plays an important role in cardiovascular diseases and recent studies have suggested that the A55V polymorphism can cause UCP2 dysfunction. The main aim was to investigate the association of A55V polymorphism with cardiovascular events in a group of 611 patients enrolled in the Medical, Angioplasty or Surgery Study II (MASS II), a randomized trial comparing treatments for patients with coronary artery disease and preserved left ventricular function. Methods The participants of the MASS II were genotyped for the A55V polymorphism using allele-specific PCR assay. Survival curves were calculated with the Kaplan–Meier method and evaluated with the log-rank statistic. The relationship between baseline variables and the composite end-point of cardiac death, acute myocardial infarction (AMI), refractory angina requiring revascularization and cerebrovascular accident were assessed using a Cox proportional hazards survival model. Results There were no significant differences for baseline variables according genotypes. After 2 years of follow-up, dysglycemic patients harboring the VV genotype had higher occurrence of AMI (p=0.026), Death+AMI (p=0.033), new revascularization intervention (p=0.009) and combined events (p=0.037) as compared with patients carrying other genotypes. This association was not evident in normoglycemic patients. Conclusions These findings support the hypothesis that A55V polymorphism is associated with UCP2 functional alterations that increase the risk of cardiovascular events in patients with previous coronary artery disease and dysglycemia.

Postmortem diagnosis of acute myocardial infarction in patients with acute respiratory failure: demographics, etiologic and pulmonary histologic analysis

OBJECTIVES: Acute respiratory failure is present in 5% of patients with acute myocardial infarction and is responsible for 20% to 30% of the fatal post-acute myocardial infarction. The role of inflammation associated with pulmonary edema as a cause of acute respiratory failure post-acute myocardial infarction remains to be determined. We aimed to describe the demographics, etiologic data and histological pulmonary findings obtained through autopsies of patients who died during the period from 1990 to 2008 due to acute respiratory failure with no diagnosis of acute myocardial infarction during life. METHODS: This study considers 4,223 autopsies of patients who died of acute respiratory failure that was not preceded by any particular diagnosis while they were alive. The diagnosis of acute myocardial infarction was given in 218 (4.63%) patients. The age, sex and major associated diseases were recorded for each patient. Pulmonary histopathology was categorized as follows: diffuse alveolar damage, pulmonary edema, alveolar hemorrhage and lymphoplasmacytic interstitial pneumonia. The odds ratio of acute myocardial infarction associated with specific histopathology was determined by logistic regression. RESULTS: In total, 147 men were included in the study. The mean age at the time of death was 64 years. Pulmonary histopathology revealed pulmonary edema as well as the presence of diffuse alveolar damage in 72.9% of patients. Bacterial bronchopneumonia was present in 11.9% of patients, systemic arterial hypertension in 10.1% and dilated cardiomyopathy in 6.9%. A multivariate analysis demonstrated a significant positive association between acute myocardial infarction with diffuse alveolar damage and pulmonary edema. CONCLUSIONS: For the first time, we demonstrated that in autopsies of patients with acute respiratory failure as the cause of death, 5% were diagnosed with acute myocardial infarction. Pulmonary histology revealed a significant inflammatory response, which has not previously been reported.

The effect of beta-blockade on myocardial remodelling in Chagas' cardiomyopathy

OBJECTIVE: Chagas' disease has spread throughout Latin America because of the high rate of migration among these countries. Approximately 30% of Chagas' patients will develop cardiomyopathy, and 10% of these will develop severe cardiac damage leading to heart failure. Beta-blockade improves symptoms and survival in heart failure patients; however, its efficacy has not been well established in Chagas' disease. We evaluated the role of carvedilol in cardiac remodeling and mortality in a Chagas' cardiomyopathy animal model. METHODS: We studied Trypanosoma cruzi infection in 55 Syrian hamsters that were divided into three groups: control (15), infected (20), and infected + carvedilol (20). Animals underwent echocardiography, electrocardiography, and morphometry for collagen evaluation in ventricles stained with picrosirius red. RESULTS: The left ventricular diastolic diameter did not change between groups, although it was slightly larger in infected groups, as was left ventricular systolic diameter. Fractional shortening also did not change between groups, although it was slightly lower in infected groups. Collagen accumulation in the interstitial myocardial space was significantly higher in infected groups and was not attenuated by carvedilol. The same response was observed in the perivascular space. The survival curve showed significantly better survival in the control group compared with the infected groups; but no benefit of carvedilol was observed during the study. However, in the acute phase (up to 100 days of infection), carvedilol did reduce mortality. CONCLUSION: Carvedilol did not attenuate cardiac remodeling or mortality in this model of Chagas' cardiomyopathy. The treatment did improve survival in the acute phase of the disease.

Early Diagnosis of Myocardial Dysfunction in Patients With Hematological Malignancies Submitted to Chemotherapy. Preliminary Results.

Early Diagnosis of Miocardial Dysfunction in Patients with Hematological Malignancies Submitted to Chemotherapy. Preliminary Background: Considering the current diagnostic improvements and tl1erapeutic approaches, patients witl 1 cancer can now be healed or keep the disease under control, still, the chemotherapy may cause heart damage, evolving to Congestive Heart Failure. Recognition of those changes increases the chances of control the endpoints; hence, new parameters of cardiac and fluid mechanics analysis have been used to assess the myocardial function, pursuing an earlier diagnosis of the cardiac alterations. This study aimed to detect early cardiac dysfunction consequently to chemotherapy in patients with hematological malignancies (HM). Methods: Patients with leukemia and lymphoma, submitted to chemotherapy, without knowing heart diseases were studied. Healthy volunteers served as the control group. Conventional 2DE parameters of myocardial function were analyzed. The peak global longitudinal, circumferential and radial left ventricular (LV) strain were deternined by 2D and 3D speckle tracking (STE); peak area strain measured by 3D STE and LV torsionn, twisting rate, recoil / recoil rate assessed by 2D STE. The LV vortex formation time (VFT) during the rapid diastolic filling was estimated by the 2D mitral valve (MV) planimetry and Pulsed Doppler LV inflow by: VFT- 4(1-β) / π x α3 x LVEF Where 1- β is the E wave contribution to the LV stroke volume and α3 is a volumetric variable related to the MV area. The statistical level was settled on 5%. Results: See Table. Conclusion: Despite the differences between the two groups concerning the LVESV, LVEF and E´, those parameters still are in the normal range when considering the patients submitted to chemotherapy; thus, in the clinical setting, they are not so noticeable. The 3D GLS was smaller among the patients, oppositely to the 2D GLS, suggesting that the former variable is more accurate to assess tlhe LV systolic function. The VFT is a dimensionless measure of the optimal vortex development inside the LV chamber; reflecting the efficiency of the diastolic filling and, consequently, blood ejection. This index showed to be diminished in patients with HM submitted to chemotherapy, indicating an impairment of the in1pulse and thrust, hence appearing to be a very early marker of diastolic and systolic dysfunction in this group.

Acute inhibition of excessive mitochondrial fission after myocardial infarction prevents long-term cardiac dysfunction

BACKGROUND: Ischemia and reperfusion (IR) injury remains a major cause of morbidity and mortality and multiple molecular and cellular pathways have been implicated in this injury. We determined whether acute inhibition of excessive mitochondrial fission at the onset of reperfusion improves mitochondrial dysfunction and cardiac contractility postmyocardial infarction in rats. METHODS AND RESULTS: We used a selective inhibitor of the fission machinery, P110, which we have recently designed. P110 treatment inhibited the interaction of fission proteins Fis1/Drp1, decreased mitochondrial fission, and improved bioenergetics in three different rat models of IR, including primary cardiomyocytes, ex vivo heart model, and an in vivo myocardial infarction model. Drp1 transiently bound to the mitochondria following IR injury and P110 treatment blocked this Drp1 mitochondrial association. Compared with control treatment, P110 (1 μmol/L) decreased infarct size by 28 ± 2% and increased adenosine triphosphate levels by 70+1% after IR relative to control IR in the ex vivo model. Intraperitoneal injection of P110 (0.5 mg/kg) at the onset of reperfusion in an in vivo model resulted in improved mitochondrial oxygen consumption by 68% when measured 3 weeks after ischemic injury, improved cardiac fractional shortening by 35%, reduced mitochondrial H2O2 uncoupling state by 70%, and improved overall mitochondrial functions. CONCLUSIONS: Together, we show that excessive mitochondrial fission at reperfusion contributes to long-term cardiac dysfunction in rats and that acute inhibition of excessive mitochondrial fission at the onset of reperfusion is sufficient to result in long-term benefits as evidenced by inhibiting cardiac dysfunction 3 weeks after acute myocardial infarction.

Stem Cells as a therapy for myocardial infarction in animal models

Advances in stem cell biology have challenged the notion that infarcted myocardium is irreparable. The pluripotent ability of stem cells to differentiate into specialized cell lines began to garner intense interest within cardiology when it was shown in animal models that intramyocardial injection of bone marrow stem cells (MSCs), or the mobilization of bone marrow stem cells with spontaneous homing to myocardium, could improve cardiac function and survival after induced myocardial infarction (MI) [1, 2]. Furthermore, the existence of stem cells in myocardium has been identified in animal heart [3, 4], and intense research is under way in an attempt to clarify their potential clinical application for patients with myocardial infarction. To date, in order to identify the best one, different kinds of stem cells have been studied; these have been derived from embryo or adult tissues (i.e. bone marrow, heart, peripheral blood etc.). Currently, three different biologic therapies for cardiovascular diseases are under investigation: cell therapy, gene therapy and the more recent “tissue-engineering” therapy . During my Ph.D. course, first I focalised my study on the isolation and characterization of Cardiac Stem Cells (CSCs) in wild-type and transgenic mice and for this purpose I attended, for more than one year, the Cardiovascular Research Institute of the New York Medical College, in Valhalla (NY, USA) under the direction of Doctor Piero Anversa. During this period I learnt different Immunohistochemical and Biomolecular techniques, useful for investigating the regenerative potential of stem cells. Then, during the next two years, I studied the new approach of cardiac regenerative medicine based on “tissue-engineering” in order to investigate a new strategy to regenerate the infracted myocardium. Tissue-engineering is a promising approach that makes possible the creation of new functional tissue to replace lost or failing tissue. This new discipline combines isolated functioning cells and biodegradable 3-dimensional (3D) polymeric scaffolds. The scaffold temporarily provides the biomechanical support for the cells until they produce their own extracellular matrix. Because tissue-engineering constructs contain living cells, they may have the potential for growth and cellular self-repair and remodeling. In the present study, I examined whether the tissue-engineering strategy within hyaluron-based scaffolds would result in the formation of alternative cardiac tissue that could replace the scar and improve cardiac function after MI in syngeneic heterotopic rat hearts. Rat hearts were explanted, subjected to left coronary descending artery occlusion, and then grafted into the abdomen (aorta-aorta anastomosis) of receiving syngeneic rat. After 2 weeks, a pouch of 3 mm2 was made in the thickness of the ventricular wall at the level of the post-infarction scar. The hyaluronic scaffold, previously engineered for 3 weeks with rat MSCs, was introduced into the pouch and the myocardial edges sutured with few stitches. Two weeks later we evaluated the cardiac function by M-Mode echocardiography and the myocardial morphology by microscope analysis. We chose bone marrow-derived mensenchymal stem cells (MSCs) because they have shown great signaling and regenerative properties when delivered to heart tissue following a myocardial infarction (MI). However, while the object of cell transplantation is to improve ventricular function, cardiac cell transplantation has had limited success because of poor graft viability and low cell retention, that’s why we decided to combine MSCs with a biopolimeric scaffold. At the end of the experiments we observed that the hyaluronan fibres had not been substantially degraded 2 weeks after heart-transplantation. Most MSCs had migrated to the surrounding infarcted area where they were especially found close to small-sized vessels. Scar tissue was moderated in the engrafted region and the thickness of the corresponding ventricular wall was comparable to that of the non-infarcted remote area. Also, the left ventricular shortening fraction, evaluated by M-Mode echocardiography, was found a little bit increased when compared to that measured just before construct transplantation. Therefore, this study suggests that post-infarction myocardial remodelling can be favourably affected by the grafting of MSCs delivered through a hyaluron-based scaffold

Automated Segmentation and Non-Rigid Registration for the Quantitative Evaluation of Myocardial Perfusion from Magnetic Resonance Images

Myocardial perfusion quantification by means of Contrast-Enhanced Cardiac Magnetic Resonance images relies on time consuming frame-by-frame manual tracing of regions of interest. In this Thesis, a novel automated technique for myocardial segmentation and non-rigid registration as a basis for perfusion quantification is presented. The proposed technique is based on three steps: reference frame selection, myocardial segmentation and non-rigid registration. In the first step, the reference frame in which both endo- and epicardial segmentation will be performed is chosen. Endocardial segmentation is achieved by means of a statistical region-based level-set technique followed by a curvature-based regularization motion. Epicardial segmentation is achieved by means of an edge-based level-set technique followed again by a regularization motion. To take into account the changes in position, size and shape of myocardium throughout the sequence due to out of plane respiratory motion, a non-rigid registration algorithm is required. The proposed non-rigid registration scheme consists in a novel multiscale extension of the normalized cross-correlation algorithm in combination with level-set methods. The myocardium is then divided into standard segments. Contrast enhancement curves are computed measuring the mean pixel intensity of each segment over time, and perfusion indices are extracted from each curve. The overall approach has been tested on synthetic and real datasets. For validation purposes, the sequences have been manually traced by an experienced interpreter, and contrast enhancement curves as well as perfusion indices have been computed. Comparisons between automatically extracted and manually obtained contours and enhancement curves showed high inter-technique agreement. Comparisons of perfusion indices computed using both approaches against quantitative coronary angiography and visual interpretation demonstrated that the two technique have similar diagnostic accuracy. In conclusion, the proposed technique allows fast, automated and accurate measurement of intra-myocardial contrast dynamics, and may thus address the strong clinical need for quantitative evaluation of myocardial perfusion.

Adipose-derived stem cells and tissue revascularization: enhancing islet survival and performance for diabetes care.

Pancreatic islet transplantation represents a fascinating procedure that, at the moment, can be considered as alternative to standard insulin treatment or pancreas transplantation only for selected categories of patients with type 1 diabetes mellitus. Among the factors responsible for leading to poor islet engraftment, hypoxia plays an important role. Mesenchymal stem cells (MSCs) were recently used in animal models of islet transplantation not only to reduce allograft rejection, but also to promote revascularization. Currently adipose tissue represents a novel and good source of MSCs. Moreover, the capability of adipose-derived stem cells (ASCs) to improve islet graft revascularization was recently reported after hybrid transplantation in mice. Within this context, we have previously shown that hyaluronan esters of butyric and retinoic acids can significantly enhance the rescuing potential of human MSCs. Here we evaluated whether ex vivo preconditioning of human ASCs (hASCs) with a mixture of hyaluronic (HA), butyric (BU), and retinoic (RA) acids may result in optimization of graft revascularization after islet/stem cell intrahepatic cotransplantation in syngeneic diabetic rats. We demonstrated that hASCs exposed to the mixture of molecules are able to increase the secretion of vascular endothelial growth factor (VEGF), as well as the transcription of angiogenic genes, including VEGF, KDR (kinase insert domain receptor), and hepatocyte growth factor (HGF). Rats transplanted with islets cocultured with preconditioned hASCs exhibited a better glycemic control than rats transplanted with an equal volume of islets and control hASCs. Cotransplantation with preconditioned hASCs was also associated with enhanced islet revascularization in vivo, as highlighted by graft morphological analysis. The observed increase in islet graft revascularization and function suggests that our method of stem cell preconditioning may represent a novel strategy to remarkably improve the efficacy of islets-hMSCs cotransplantation.

The cardio-renal axis. Non ST-segment elevation myocardial infarction risk stratification according to renal dysfunction

Background: Chronic kidney disease (CKD) is one of the strongest risk factor for myocardial infarction (MI) and mortality. The aim of this study was to assess the association between renal dysfunction severity, short-term outcomes and the use of in-hospital evidence-based therapies among patients with non–ST-segment elevation myocardial infarction (NSTEMI). Methods: We examined data on 320 patients presenting with NSTEMI to Maggiore’s Emergency Department from 1st Jan 2010 to 31st December 2011. The study patients were classified into two groups according to their baseline glomerular filtration rate (GFR): renal dysfunction (RD) (GFR<60) and non-RD (GFR≥60 ml/min). Patients were then classified into four groups according to their CKD stage (GFR≥60, GFR 59-30, GFR 29-15, GFR <15). Results: Of the 320 patients, 155 (48,4%) had a GFR<60 ml/min at baseline. Compared with patients with a GFR≥60 ml/min, this group was, more likely to be female, to have hypertension, a previous myocardial infarction, stroke or TIA, had higher levels of uric acid and C-reactive protein. They were less likely to receive immediate (first 24 hours) evidence-based therapies. The GFR of RD patients treated appropriately increases on average by 5.5 ml/min/1.73 m2. The length of stay (mean, SD) increased with increasing CKD stage, respectively 5,3 (4,1), 7.0 (6.1), 7.8 (7.0), 9.2 (5.8) (global p <.0001). Females had on average a longer hospitalization than males, regardless of RD. In hospital mortality was higher in RD group (3,25%). Conclusions: The in-hospital mortality not was statically difference among the patients with a GFR value ≥60 ml/min, and patients with a GFR value <60 ml/min. The length of stay increased with increasing CKD stages. Despite patients with RD have more comorbidities then without RD less frequently receive guideline –recommended therapy. The GFR of RD patients treated appropriately improves during hospitalization, but not a level as we expected.

Genetic and environmental factors associated with cardiovascular diseases and acute myocardial infarction

Acute myocardial infarction (AMI) is a multifactorial disease with a complex pathogenesis where lifestyle, individual genetic background and environmental risk factors are involved. Altered inflammatory responses seems to be implicated in the pathogenesis of atherosclerosis. To understand which genes may predispose to increased risk of cardiovascular disease gene polymorphism of immune regulatory genes, and clinical events from the Offs of parents with an early AMI were investigated. Genetics data from Offs were compared with those obtained from healthy subjects and an independent cohort of patients with clinical sporadic AMI. Rates of clinical events during a 24 years follow up from Offs and from an independent Italian population survey were also evaluated. This study showed that a genetic signature consisting of the concomitant presence of the CC genotype of VEGF, the A allele of IL-10 and the A allele of IFN-γ was indeed present in the Offs population. During the 24-year follow-up, Offs with a positive familiarity in spite of a relatively young age showed an increased prevalence of diabetes, ischemic heart disease and stroke. In these patients with the genetic signature the EBV and HHV-6 herpes virus were also investigated and founded. These findings reinforce the notion that subjects with a familial history of AMI are at risk of an accelerated aging of cardiovascular system resulting in cardiovascular events. These data suggest that selected genes with immune regulatory functions and envoronmental factors are part of the complex genetic background contributing to familiarity for cardiovascular diseases.N

Evidence-based polytherapies and long-term mortality after acute myocardial infarction in very old subjects compared with elderly and adults: a nested case-control study

Background: Clinical trials have demonstrated that selected secondary prevention medications for patients after acute myocardial infarction (AMI) reduce mortality. Yet, these medications are generally underprescribed in daily practice, and older people are often absent from drug trials. Objectives: To examine the relationship between adherence to evidence-based (EB) drugs and post-AMI mortality, focusing on the effects of single therapy and polytherapy in very old patients (≥80 years) compared with elderly and adults (<80 years). Methods: Patients hospitalised for AMI between 01/01/2008 and 30/06/2011 and resident in the Local Health Authority of Bologna were followed up until 31/12/2011. Medication adherence was calculated as the proportion of days covered for filled prescriptions of angiotensin-converting enzyme inhibitors (ACEIs)/angiotensin receptor blockers (ARBs), β-blockers, antiplatelet drugs, and statins. We adopted a risk set sampling method, and the adjusted relationship between medication adherence (PDC≥75%) and mortality was investigated using conditional multiple logistic regression. Results: The study population comprised 4861 patients. During a median follow-up of 2.8 years, 1116 deaths (23.0%) were observed. Adherence to the 4 EB drugs was 7.1%, while nonadherence to any of the drugs was 19.7%. For both patients aged ≥80 years and those aged <80 years, rate ratios of death linearly decreased as the number of EB drugs taken increased. There was a significant inverse relationship between adherence to each of 4 medications and mortality, although its magnitude was higher for ACEIs/ARBs (adj. rate ratio=0.60, 95%CI=0.52–0.69) and statins (0.60, 0.50–0.72), and lower for β-blockers (0.75, 0.61–0.92) and antiplatelet drugs (0.73, 0.63–0.84). Conclusions: The beneficial effect of EB polytherapy on long-term mortality following AMI is evident also in nontrial older populations. Given that adherence to combination therapies is largely suboptimal, the implementation of strategies and initiatives to increase the use of post-AMI secondary preventive medications in old patients is crucial.