Digital Theses Archive


Tesi etd-05102018-144548

Type of thesis
Molecular and epigenetic biomarkers in pediatric patients with heart failure: effect of Ventricular Assist Device implantation.
Scientific disciplinary sector
SCIENZE MEDICHE - Translational Medicine
relatore Prof. PASSINO, CLAUDIO
  • Heart failure; pediatric patients
  • Ventricular assist device
Exam session start date
Background: Heart Failure (HF) is defined in children as a progressive syndrome caused by genetic, circulatory, neurohormonal and molecular abnormalities that results in characteristic signs and symptoms such as edema, respiratory distress, growth failure and exercise intolerance. Over recent years, the use of Ventricular Assist Devices (VADs) as a bridge to transplant for HF children has steadily increased due to improvements in device technology, broadened indications for VAD placement, and improvements in post-implantation care. Several studies reported that VAD therapy could at least partially reverse some of the pathological processes that occur during HF and, in some cases, may even lead to restoration of myocardial function. The use of circulating biomarkers as tool for monitoring the remodelling mechanisms modified by VAD, that is also crucial for the clinical management of adult patients with HF, is unknown in pediatric setting.<br><br>Aim: The aim of this PhD project was to examine baseline levels and time-course changes up to one month in biohumoral markers representing the key pathways of HF patho-physiology in pediatric patients who underwent VAD placement. In particular, cardiac and inflammatory biomarkers, clinically recommended for adult patients, and emerging biomarkers such as circulatory miRNAs (c-miRNAs) were evaluated.<br>Specifically, in order to better understand both the possible clinical and patho-physiological role of biomarkers of cardiac stress, injury, fibrosis and inflammation, in HF pediatric patients:<br>1) the baseline levels of biomarkers from HF pediatric patients were compared with those from a control group of healthy children; <br>2) biomarker levels during postoperatively time-course after VAD implant from HF pediatric patients were compared with those from adult patients with HF.<br>The biohumoral characterization of the HF children group was completed evaluating the miRNAs profile of serum samples at baseline and during time-course after VAD implant. In particular, our purpose was: <br>1) to sequence c-miRNAs in pediatric patients with HF; <br>2) to evaluate the effects of VAD on the c-miRNAs levels; <br>3) to identify the potential targets of selected c-miRNA; <br>4) to in vitro validate miRNA targets. <br><br>Methods: Blood samples were collected from 12 children with HF [22 (6.5-144) months, 6 males, 17 (12.5-19) LVEF%] before and at 4 hrs, 1, 3, 7, 14 and 30 days after VAD implant. The levels of cardiac (NT-proBNP, cTnI, sST2 and Gal-3) and inflammatory (IL-6, IL-8, IL-10 and TNFα) biomarkers were measured in HF children at baseline and during time course using dedicated immunoassays. The circulating levels of cardiac and inflammatory biomarkers from HF pediatric patients were compared with those from a group of 106 healthy children [17.47 (1.8-122) months, 65 males] and with those from a group of adult patients with HF [n=7; 52.36(48.4-60.6) years, 7 males, 24 (18.75-27.25) LVEF%] supported with VAD. Plasma samples from adult patients, were obtained at the same time points of pediatric population. c-miRNA profile was determined in serum sample from HF children by NGS at the moment of VAD implant and after 30 days of treatment. Selected c-miRNAs were validated by Real-Time PCR and their changes were assessed in blood samples collected during time-course after VAD implant. Putative miRNA targets were selected using miRWalk database. Validation of selected miRNA targets was performed using an in vitro cellular model.<br><br>Results: At baseline, plasma levels of NT-proBNP, hs-cTnI, sST2, were significantly higher in HF children compared with healthy children [NT-proBNP (ng/L): 7266.5 (4428.5-9172) vs 96.7 (38.7-255.97); hs-cTnI (ng/L): 33.6 (10.25-427.3) vs (4.1 (2.6-13.8) ; sST2 (ng/mL): 36.88 (23.88-95) vs 18.8 (12.3-27.27); HF vs controls, p&lt;0.005]. No difference was observed for cytokines and Gal-3 between the two groups. <br>During the post-operative time-course after VAD implant, circulating levels of all biomarkers increased up to 1 day after VAD and then decreased at pre-VAD levels in 1 month in both adults and children. Circulating levels of only NT-proBNP decreased significantly after 30 days Post-VAD treatment compared to pre-VAD values in pediatric patients. During time-course, NT-proBNP and sST2 levels were significantly higher in children than adults, while IL-6 was lower.<br>After NGS analysis, a total of 169 c-miRNAs were detected in serum samples of HF pediatric patients. N=13 c-miRNAs were simultaneously modulated at 30 days after VAD implant compared to pre-VAD. Among them, only N=6 c-miRNAs (miR-483-3p; miR-409-3p; miR-485-3p; miR-432-5p; miR-150-3p and miR-375) were validated by Real-Time-PCR. In silico analysis revealed that the putative targets of selected c-miRNAs were involved in the hemostatic process with a synergistic effect. The in vitro study confirmed a regulatory effect of miR-409-3p towards coagulation factor VII(F7) and thrombin (FII) (p=0.05 transfected cells vs control). <br><br>Conclusions: Abnormal levels of NT-proBNP, cTnI and sST2 were observed in HF children compared with the healthy group, while no differences were observed for cytokines, suggesting the involvement of cardiac but not inflammatory biomarkers in mechanisms of remodeling in HF children. Circulating levels of cardiac and inflammatory biomarkers were differently modified by VAD implant in children compared to adults, indicating that different molecular pathways may underlie HF patho-physiology of the two populations. The selected and validated c-miRNAs modified by VAD implant are involved in the regulation of hemostasis event, a well known adverse event for this group of patients. Taken together, all these results could suggest that circulating biomarkers, at molecular and epigenetic levels, could be useful to delineate a specific and targeted therapeutic intervention, thus helping to address a personalized management for HF pediatric patients.<br>