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Treatment of chronic leg ulcers remains a major challenge and it is a substantial financial burden on individuals, families, caregivers, and health care system. There is increasing evidence on using of autologous Platelet-rich-plasma in wound repair but limited clinical data are available on the efficacy and safety of the use of umbilical cord blood platelet gel (CBPG). In our pilot study, for the first time, we aimed to evaluated the safety and efficacy of the use of umbilical CBPG combined with a hydrogel dressing in 10 patients with chronic venous ulcers (VU). The protocol consisted of application of umbilical cord blood platelet-rich plasma (PRP) combined with a Carboxymethyl cellulose (CMC)-based hydrogel dressing once a week for 4 weeks. The 80% of patients after 4 weeks of treatment had a significantly decrease in wound size. Moreover, we obtained an improvement in terms of mean Wound Bed Score (WBS), numeric rating scale (NRS) value and the EQ-5D index score. This pilot study showed that the topically therapeutic administration of umbilical CBPG associated with a CMC-based hydrogel dressing has the potential to accelerate the healing of chronic lesions without adverse reaction. However, additional studies with larger sample size and longer follow-up periods are required to confirm our findings.

Introduction: Bicuspid Aortic Valve (BAV), the most common congenital heart defect, is a major cause of aortic regurgitation or stenosis requiring valve replacement and thoracic aortic aneurysms predisposing to acute aortic dissections (TAD). The spectrum of BAV ranges from severe early onset valve and aortic complications to sporadic late onset disease. Hypothesis: Early onset BAV (EBAV) cases with valve or aortic complications that require intervention prior to age 30 are enriched for rare genetic variants that cause BAV and TAD. Methods: We performed whole exome sequencing of 147 EBAV cases in 141 families who were enrolled in the UTHealth Bicuspid Aortic Valve Research Registry. Candidate variants in the EBAV cohort (26% female, mean age 18, 44% with TAD) were compared to unselected controls from the Genome Aggregation Database (gnoMAD) and the Database of Genotypes and Phenotypes (dbGAP). We considered variants with minor allele frequencies (MAF) < 1%, Combined Annotation Dependent Depletion (CADD) scores > 25, and damaging (Polyphen-2) or deleterious (SIFT) functional prediction scores. Genomic copy number variants (CNVs) were detected using CoNIFER and prioritized when deletions involved genes with probability of loss intolerance (pLI) > 0.9. Variants were validated using quantitative PCR or Sanger sequencing. Results: We identified 6 rare variants of USP10 in 6 EBAV families (4% of cohort): 4 CNVs (2 duplications and 2 deletions) that are rare in dbGAP controls (4 in 15,414) and 2 deleterious rare missense variants (MAF-5 in gnoMAD). Two of the 4 CNVs were de novo events in trios. In contrast, rare deleterious variants of the known causal BAV genes NOTCH1 (1), ROBO4 (1), GATA4 (1), GATA5 (1), and SMAD6 (4) were found in 7 total families. USP10 encodes a ubiquitin peptidase that is required for endothelial Notch signaling during vascular development. Conclusions: We identified rare and de novo variants of USP10 that implicate USP10 as a new candidate gene for BAV.

Platelets contain abundant growth factors and cytokines that have a positive influence on the migration and proliferation of different cell types by modulating its physiopathological processes. As it is known that human umbilical cord blood platelet lysate (UCB‐PL) contains a supraphysiological concentration of growth factors, in the present study, we investigated its effectiveness in wound‐healing processes. Human UCB‐PL was obtained by the freeze/thaw of platelet concentrate (1.1 × 10(9) platelets/L), and its effect was evaluated on human or mouse endothelial cells, monocytes, fibroblasts, and keratinocytes in different concentrations. Human UCB‐PL was observed to have high levels of pro‐angiogenic growth factor than peripheral blood platelet‐rich plasma. Among the cell lines, different concentrations of human UCB‐PL were necessary to influence their viability and proliferation. For L929 cells, 5% of total volume was necessary, while for human umbilical vein endothelial cell, it was 10%. Cell migration on monocytes was increased with respect to the positive control, and scratch closure on keratinocytes was increased with respect to serum‐free medium with only 10% of human UCB‐PL. We concluded that the human UCB‐PL may be useful to produce a large amount of standard platelet concentrates sufficient for several clinical‐scale expansions avoiding inter‐individual variability, which can also be used as a functional tool for clinical regenerative application for wound healing.

Background Ascending thoracic aortic aneurysm (ATAA) is a major cause of morbidity and mortality worldwide. The pathogenesis of medial degeneration of the aorta remains undefined. High-throughput secretome analysis by mass spectrometry may be useful to elucidate the molecular mechanisms involved in aneurysm formation as well as to identify biomarkers for early diagnosis or targets of therapy. The purpose of the present study was to analyze the secreted/released proteins from ATAA specimens of both tricuspid aortic valve (TAV) and bicuspid aortic valve (BAV) patients. Methods Aortic specimens were collected from patients undergoing elective surgery and requiring graft replacement of the ascending aorta. Each sample of the ascending aortic aneurysm, 4 BAV (3 males; aged 53.5 ± 11.4 years) and 4 TAV (1 male; 78 ± 7.5 years), was incubated for 24 h in serum-free medium. Released proteins were digested with trypsin. Peptide mixtures were fractioned by nano-high performance liquid chromatography and analyzed by mass spectrometry. Following identification of differentially expressed proteins, quantitative real time polymerase chain reaction (qRT-PCR) analysis was performed. Results The comparison between the proteins released from BAV and TAV aneurysmatic tissues showed significantly diverging expression fingerprints in the two groups of patients. Bioinformatics analysis revealed 38 differentially released proteins; in particular 7 proteins were down-regulated while 31 were up-regulated in BAV with respect to TAV. Most of the proteins that were up-released in BAV were related to the activation of transforming growth factor (TGF)-β signaling. Latent TGF-β binding protein 4 (LTBP4) exhibited one of the highest significant under-expressions (10-fold change) in BAV secretomes with respect to TAV. qRT-PCR analysis validated this significant difference at LTBP4 gene level (BAV: 1.03 ± 0.9 vs TAV: 3.6 ± 3.2; p Conclusion Hypothesis-free secretome profiling clearly showed diverging expression fingerprints in the ATAA of TAV and BAV patients, confirming the crucial role of TGF-β signaling in modulating ATAA development in bicuspid patients.

Compelling evidence has shown that microRNAs (miRs) are involved in the pathophysiology of BAV-associated aortopathy. The purpose of this study was to assess the biological role as well as the circulating expression of two miRs (miR-424-3p and miR-3688-3p) that have been previously identified as significantly dysregulated in thoracic aortic aneurysm specimens of BAV patients. Bioinformatic tools were used to predict miR gene targets followed by functional validation transfecting synthetic miR mimics and negative controls into human aortic smooth muscle cells (HASMCs). Levels of miRs and target genes were evaluated by qRT-PCR. The circulating miR expression profile analysis was assessed on plasma samples collected from a cohort of 72 patients with aortopathy including 39 BAV (33 males; 58 ± 13 years) and 33 TAV patients (26 males; 67 ± 9 years). Computational analysis revealed that SMAD7 and YAP1 were potential targets of miR-424-3p and miR-3688-3p, respectively. Transfection with mimics confirmed a significantly decreased gene expression of SMAD7 and YAP1 compared to mimic negative control (p = 0.04 and p = 0.0005, respectively) or blank control (p = 0.01 and p = 0.0007, respectively). Overexpression of miR-3688-3p also significantly upregulated pro-apoptotic caspase-3 gene expression compared to mimic negative control (p = 0.02) or blank control (p = 0.01). Furthermore, a significant down-regulation of the circulating miR-424-3p was observed in BAV compared to TAV patients (p = 0.001). In multiple linear regression analysis, the aortic valve morphology (β = − 0.29, p = 0.04) and the presence of aortic stenosis (β = − 0.28, p = 0.03) had a significant effect on the miR-424-3p expression. In conclusion, our study demonstrated that miR-424-3p and miR-3688-3p directly targeted SMAD7 and YAP1 in HASMCs, pivotal genes of the TGF-β and Hippo-signaling pathways. Circulating miR-424-3p was also found to be significantly decreased in BAV patients when compared to TAV patients, especially in patients with aortic stenosis. Further large studies of well-characterized BAV patient cohorts are needed to define the clinical significance of the miR-424-3p.

The development of small-caliber grafts still represents a challenge in the field of vascular prostheses. Among other factors, the mechanical properties mismatch between natural vessels and artificial devices limits the efficacy of state-of-the-art materials. In this paper, a novel nanocomposite graft with an internal diameter of 6 mm is proposed. The device is obtained through spray deposition using a semi-interpenetrating polymeric network combining poly(ether)urethane and polydimethilsyloxane. The inclusion of BaTiO3 nanoparticles endows the scaffold with piezoelectric properties, which may be exploited in the future to trigger beneficial biological effects. Graft characterization demonstrated a good nanoparticle dispersion and an overall porosity that was not influenced by the presence of nanoparticles. Graft mechanical properties resembled (or even ameliorated) the ones of natural vessels: both doped and non-doped samples showed a Young's modulus of ∼700 kPa in the radial direction and ∼900 kPa in the longitudinal direction, an ultimate tensile strength of ∼1 MPa, a strain to failure of ∼700%, a suture retention force of ∼1.7 N and a flexural rigidity of ∼2.5 × 10−5 N m2. The two grafts differed in terms of burst strength that resulted ∼800 kPa for the control non-doped samples and ∼1100 kPa for the doped ones. The graft doped with BaTiO3 nanoparticles showed a d33 coefficient of 1.91 pm/V, almost double than the non-doped control. The device resulted highly stable, with a mass loss smaller than 2% over 3 months and an excellent biocompatibility.

The valve replacement therapy is the standard treatment for severe heart valve diseases. Nowadays, two types of commercial prosthesis are available: mechanical and biological, but both of them have severe limitations. Moreover, alternative therapeutic approach for valve replacement, based on minimally invasive techniques (MIAVR), motivates the search for new valve materials. In this study a polyurethane-based self-expandable tri-leaflets heart valve compatible with MIAVR procedure is proposed. The device is based on the development, fabrication and characterization of three different elements: the leaflets, the polymeric stent for supporting the leaflets, and the external metallic stent for anchoring the valve to the native aortic root. The polymeric stent and the valve leaflets were fabricated using a thermoplastic silicone-polycarbonate-urethane using 3D printing and spray technology while the external metallic stent was made in nickel titanium (Nitinol) to obtain a self-expandable valve after the crimping process. The three elements were assembled in the completed device and tested by crimping, fatigue and fluid-dynamic test. The novel polymeric valve proposed showed promising results about valve crimping capabilities, durability and fluid dynamic performances. This approach could offer advantages such as low cost and to produce a tailor-made device basing on patient's imaging data. Moreover, the selected biomaterial offers the potential to have a device that could need of permanent anticoagulation and lack of calcification.

The present study examined the effects of a bilayered fibrin/poly(ether)urethane scaffold loaded with platelet lysate by a combination of electrospinning and spray, phase-inversion method for wound healing. In particular, the poly(ether)urethane layer was obtained using by a spray phase-inversion method and the fibrin fibers network were loaded with platelet lysate by electrospinning. The kinetics release and the bioactivity of growth factors released from platelet lysate-scaffold were investigated by ELISA and cell proliferation test using mouse fibroblasts, respectively. The in-vitro experiments demonstrated that a bilayered fibrin/poly(ether)urethane scaffold loaded with platelet lysate provides a sustained release of bioactive platelet-derived growth factors. The effect of a bilayered fibrin/poly(ether)urethane scaffold loaded with platelet lysate on wound healing in diabetic mouse (db/db) was also investigated. The application of the scaffold on full-thickness skin wounds significantly accelerated wound closure at day 14 post-surgery when compared to scaffold without platelet lysates or commercially available polyurethane film, and at the same level of growth factor-loaded scaffold. Histological analysis demonstrated an increased re-epithelialization and collagen deposition in platelet lysate and growth factor loaded scaffolds. The ability of bilayered fibrin/poly(ether)urethane scaffold loaded with platelet lysate to promote in-vivo wound healing suggests its usefulness in clinical treatment of diabetic ulcers.

Resumen Introduccion y objetivos Los polimorfismos de nucleotido unico situados en un lugar de union de microacidos ribonucleicos (miARN) pueden tener diferentes efectos en la expresion genica, y ello puede influir en el riesgo de enfermedad. Este estudio tiene como objetivo evaluar la asociacion existente entre los polimorfismos de nucleotido unico y los haplotipos presentes en la region 3’UTR del gen GATA4 y el riesgo de cardiopatia congenita. Metodos Se utilizaron algoritmos de bioinformatica para analizar los polimorfismos de nucleotido unico en los presuntos lugares de union de miARN en la region 3’UTR del gen GATA4 y para calcular la diferencia de energia de hibridacion libre (ΔFE, kcal/mol) para cada alelo de tipo natural (wild-type) en comparacion con cada variante alelica. Resultados Formaron la poblacion de estudio 146 pacientes caucasicos (73 varones; edad, 6,68 ± 7,79 anos) y 265 recien nacidos sanos (147 varones). Se considero que la suma de todos los ΔFE predecia la importancia biologica de los polimorfismos de nucleotido unico al unirse a mas miARN. A continuacion se determino el genotipo de los 4 polimorfismos (+1158 C > T, + 1256 A > T, + 1355 G > A, +1521 C > G) que tenian el valor predicho de ΔFE total mas alto (9,91, 14,85, 11,03 y 21,66 kcal/mol respectivamente) en un estudio de casos y controles (146 pacientes y 250 controles). Al aplicar una correccion por multiplicidad de pruebas, tan solo el alelo +1158 T mostro una diferencia significativa entre los pacientes y los controles. El analisis de los haplotipos puso de manifiesto que el haplotipo T-T-G-C (mas infrecuente en los pacientes con cardiopatias congenitas que en los controles) se asociaba a una disminucion del riesgo significativa (p = 0,03), mientras que el haplotipo muy infrecuente C-A-A-C, que se daba de manera muy poco comun en los controles (0,3%) en comparacion con los pacientes con la enfermedad (2,4%), se asociaba a un aumento de 4 veces en el riesgo de enfermedad (p = 0,04). Conclusiones Las variantes frecuentes de la region 3’UTR del gen GATA4 interaccionan de manera conjunta y con ello afectan a la susceptibilidad a la cardiopatia congenita, probablemente mediante la alteracion de la regulacion postranscripcional de los miARN.

Recent experimental studies showed that ablation of the aryl hydrocarbon receptor (AhR) as well as its activation by exogenous ligands disrupt the molecular networks involved in heart formation and function, leading to congenital heart disease (CHD). However, no evidence is available about the role of AhR in humans. We assessed the prevalence of a functional AhR genetic variant (p.Arg554Lys) in CHD patients as well as its joint effects with parental exposure. A total of 128 CHD patients (76 males; age 6.2 ± 6.7 years) and 274 controls (160 males; age at birth) were genotyped for the AhR polymorphism by using the TaqMan® Drug Metabolism Genotyping assay. Both case and control parents completed a structured questionnaire on demographic, lifestyle and preconception exposures. Genotype (p = 0.001) and allele (p

The molecular mechanisms underlying thoracic aortic aneurysm (TAA) in patients with bicuspid aortic valve (BAV) are incompletely characterized. MicroRNAs (miRNAs) may play a major role in the different pathogenesis of aortopathy. We sought to employ next-generation sequencing to analyze the entire miRNome in TAA tissue from patients with BAV and tricuspid aortic valve (TAV). In the discovery stage, small RNA sequencing was performed using the Illumina MiSeq platform in 13 TAA tissue samples (seven patients with BAV and six with TAV). Gene ontology (GO) and KEGG pathway analysis were used to identify key pathways and biological functions. Validation analysis was performed by qRT-PCR in an independent cohort of 30 patients with BAV (26 males; 59.5 ± 12 years) and 30 patients with TAV (16 males; 68.5 ± 9.5 years). Bioinformatic analysis identified a total of 489 known mature miRNAs and five novel miRNAs. Compared to TAV samples, 12 known miRNAs were found to be differentially expressed in BAV, including two up-regulated and 10 down-regulated (FDR-adjusted p-value ≤ 0.05 and fold change ≥ 1.5). GO and KEGG pathway enrichment analysis (FDR-adjusted p-value < 0.05) identified different target genes and pathways linked to BAV and aneurysm formation, including Hippo signaling pathway, ErbB signaling, TGF-beta signaling and focal adhesion. Validation analysis of selected miRNAs confirmed the significant down-regulation of miR-424-3p (p = 0.01) and miR-3688-3p (p = 0.03) in BAV patients as compared to TAV patients. Our study provided the first in-depth screening of the whole miRNome in TAA specimens and identified specific dysregulated miRNAs in BAV patients.

Congenital heart disease (CHD) is a genetically heterogeneous disease. Targeted next-generation sequencing (NGS) offers a unique opportunity to sequence multiple genes at lower cost and effort compared to Sanger sequencing. We tested a targeted NGS of a specific gene panel in a relatively large population of non-syndromic CHD patients. The patient cohort comprised 68 CHD patients (45 males; 8.3 ± 1.7 years). Amplicon libraries for 16 CHD-strictly related genes were generated using a TruSeq® Custom Amplicon kit (Illumina, CA) and sequenced using the Illumina MiSeq platform. Sequence data were processed through the MiSeq Reporter and wANNOVAR softwares. After applying stringent filtering criteria, 20 missense variants in 9 genes were predicted to be damaging and were validated by Sanger sequencing with 100% concordance. Fourteen variants were present in public databases with very rare allele frequency, of which four variants (p.Arg25Cys in NKX2-5, p.Val763Ile in ZFPM2, p.Arg1398Gln and Gly1826Asp in MYH6) have been previously linked to CHD or cardiomyopathy. The remaining six variants in four genes (GATA4, NKX2-5, NOTCH1, TBX1) were novel mutations, currently not found in public databases, and absent in 200 control alleles of healthy subjects. Four patients (5.8%) carried two missense variants (1 compound heterozygote in the same gene and 3 double heterozygotes in different genes), with possibly synergistic deleterious effects. Targeted NGS is a powerful and efficient tool to detect DNA sequence variants in multiple genes, providing the opportunity for discovery of the co-occurrence of two or more missense rare variants.

Congenital heart diseases (CHDs) are recognized as the most common type of birth malformations. Although recent advances in pre- and neonatal diagnosis as well as in surgical procedures have reduced the morbidity and mortality for many CHD, the etiology for CHD remains undefined. In non-syndromic and isolated (without a familial history or a Mendelian inheritance) forms of CHDs, a multifactorial pathogenesis with interplay between inherited and non-inherited causes is recognized. In this paper, we discuss the current knowledge of the potential molecular mechanisms, mediating abnormal cardiac development in non-syndromic and isolated CHD, including mutations in cardiac transcription factors, the role of somatic mutations and epigenetic alterations as well as the influence of gene-environment interactions. In the near future, the advent of high-throughput genomic technologies with the integration of system biology will expand our understanding of isolated, non-syndromic CHDs for their prevention, early diagnosis and therapy.

Bicuspid Aortic Valve (BAV), the most common adult congenital heart defect, is a major cause of aortic insufficiency or stenosis requiring valve replacement and thoracic aortic aneurysms predisposing to acute aortic dissections. The spectrum of BAV ranges from early onset valve and aortic

We report the clinical presentation and genetic screening of 2 patients with thoracic aortic aneurysms. A novel TGFBR2 mutation in the 5'untranslated region (c.-59C>T) was identified in a 31-year-old man with a Stanford type A aortic dissection. Bioinformatics tools showed that c.-59C>T variant was predicted to affect exonic splicing enhancer, as validated by quantitative real-time RT-PCR, revealing a sixfold increase of TGFBR2 mRNA in aneurysmal aortic tissue collected during surgery. A previously described missense mutation, p.E239K, in the SMAD3 gene was identified in a 60-year-old man who presented with diffuse vasculopathy. These findings suggest that the features of aneurysmal disease extending beyond the ascending aorta may help to target SMAD3 genetic screening and that alterations in the core splicing machinery can contribute to aneurysmal disease.

Somatic mutations and dysregulation by microRNAs (miRNAs) may have a pivotal role in the Congenital Heart Defects (CHDs). The purpose of the study was to assess both somatic and germline mutations in the GATA4 and NKX2.5 genes as well as to identify 3'UTR single nucleotide polymorphisms (SNPs) in the miRNA target sites. We enrolled 30 patients (13 males; 13.4±8.3 years) with non-syndromic CHD. GATA4 and NKX2.5 genes were screened in cardiac tissue of sporadic and in blood samples of familial cases. Computational methods were used to detect putative miRNAs in the 3'UTR region and to assess the Minimum Free Energy of hybridization (MFE, kcal/mol). Difference of MFEs (ΔMFE) ≥4 kcal/mol between alleles was considered biologically relevant on miRNA binding. The sum of all ΔMFEs (|ΔMFEtot|=∑|ΔMFE|) was calculated in order to predict the biological importance of SNPs binding more miRNAs. No evidence of novel GATA4 and NKX2.5 mutations was found both in sporadic and familial patients. Bioinformatic analysis revealed 27 putative miRNAs binding to identified SNPs in the 3'UTR of GATA4. ΔMFE ≥4 kcal/mol between alleles was obtained for the +354A>C (miR-4299), +587A>G (miR-604), +1355G>A (miR-548v, miR-139-5p) and +1521C>G (miR-583, miR-3125, miR-3928) SNPs. The +1521C>G SNP showed the highest ΔMFEtot (21.66 kcal/mol). Luciferase reporter assays indicated that miR-583 was dose-dependently effective in regulating +1521 C allele compared with +1521 G allele. Based on the analysis of 100 CHD cases and 204 healthy newborns, the +1521 G allele was also associated with a lower risk of CHD (OR=0.5, 95% CI 0.3-0.9, p=0.03), likely due to the relatively low binding of the miRNA and high levels of protein. These results suggest that common SNPs in the 3'UTR of GATA4 alter miRNA gene regulation contributing to the pathogenesis of CHDs.

Congenital heart defects (CHDs) are the most prevalent of all birth defects and the leading cause of death in the first year of life. The molecular causes of most CHDs remain largely unknown. The LIM homeodomain transcriptor factor ISL1 is a marker for undifferentiated cardiac progenitor cells that give rise to both the right ventricle and the inflow and outflow tracts, which are affected by several cardiovascular malformations. Contradictory findings about the role of the ISL1 rs1017 single-nucleotide polymorphism in increasing the risk of CHD have been reported. In this study, we aimed to investigate whether the ISL1 rs1017 genetic polymorphism conferred susceptibility to CHD in the white population. In a case-control study design, 309 patients with CHD (197 men [age 21.3 ± 25.2]) and 500 healthy controls (272 men [age 15.7 ± 21.3]) were genotyped for the ISL1 rs1017 polymorphism. No significant difference in the genotype and variant allele distributions was found between patients and controls. In addition, the ISL1 rs1017 polymorphism was not associated with the risk of CHD neither overall (p = 0.7) nor stratifying the population by sex and CHD classification. In conclusion, ISL1 common variant rs1017 is not associated with increased genetic risk of CHD in the white population.

Background N-acetylcysteine (NAC) is considered a promising radio-protector for its antioxidant and anticarcinogenic properties. We examined the ability of NAC to confer protection against radiation-induced chromosomal DNA damage during cardiac catheterization procedures. Methods Sixty-five patients (52 males, age 64.4±11.9years) undergoing invasive cardiovascular procedures (peripheral transluminal angioplasty, n =45; cardiac resynchronization therapy, n =15 and ablation therapy n =5) were enrolled: 35 patients (26 males, age 63.4±11.1years) received the standard hydration protocol consisting of intravenous isotonic saline for 12h after catheterization (Group I), and 30 patients (26 males, age 65.5±12.9years) received a clinically driven double intravenous dose of NAC (6mg/kg/h diluted in 250mL of NaCl 0.9%) for 1h before and a standard dose (6mg/kg/h diluted in 500mL of NaCl 0.9%) for 12h following catheterization (Group II). Micronucleus assay (MN) was performed as biomarker of chromosomal DNA damage before, 2 and 24h after the radiation exposure. Dose-area product (DAP; Gy cm 2 ) was assessed as physical measure of radiation load. Results DAP was higher in NAC-treated patients (I=54.7±23.6 vs II=126.2±79.2Gy cm 2 , p =0.0001). MN frequency was 13.7±4.7‰ at baseline and showed a significant rise at 2h (18.0±6.8 p =0.01) and 24h (17.6±5.9, p =0.03) in the Group I. There was no significant increase of MN in the Group II (13.7±7.0, 15.5±6.0 and 14.9±6.3 for baseline, 2h and 24h respectively, p =0.4). Conclusion NAC treatment given to prevent contrast-induced nephropathy may also reduce DNA damage induced by ionizing radiation exposure during cardiac catheterization procedures.

ObjectiveThe angiotensin-converting enzyme (ACE) is highly expressed in the aneurysmal vascular wall, in both animal models and human disease. Genetic variations in ACE could be crucial in determining the risk of thoracic aortic aneurysm (TAA). The aim of the present study was to examine the role of ACE insertion/deletion polymorphism on the risk of TAA in patients with bicuspid aortic valves or tricuspid aortic valves.MethodsWe enrolled 216 patients (158 men; age, 58.9 ± 14.9 years) with TAA, associated with bicuspid aortic valves (n = 105) and tricuspid aortic valves (n = 111) compared with 312 patients (252 men; age, 54.6 ± 11.0 years) with angiographically proven coronary artery disease and 300 healthy controls (91 men; age, 40.4 ± 10.5 years).ResultsThe genotype distribution of ACE insertion/deletion was significantly different between the patients with TAA compared with both the control group (P = .0005) and the coronary artery disease group (P = .03). The genotypes were not different between the control group and the coronary artery disease group (P = .3). Compared with the controls, both the bicuspid aortic valve patients (P = .0008) and tricuspid aortic valve patients (P

The GSTP1 gene, highly expressed early in fetal life, is the most abundant phase 2 xenobiotic metabolism enzyme in a human placenta. Fetal inherited GSTP1 Ile105Val polymorphism may modify the metabolism and excretion of xenobiotics from fetal tissue and increase the risk of congenital heart disease (CHD). This study aimed to analyze the joint effects of GSTP1 genetic polymorphism (Ile105Val) and maternal environmental exposure on CHD risk. Within a case-control design, a total of 190 children with CHD (104 boys age 4 ± 5.6 years) and 190 healthy children (114 newborn boys) were genotyped for the GSTP1 Ile105Val polymorphism. Mothers completed a structured questionnaire on the demographics as well as the preconceptional and lifestyle exposures. A higher frequency of mothers of children with CHD (38 %) reported a positive history of exposure to toxicants (occupational and environmental) than mothers of healthy children (23 %) (p = 0.0013). Logistic regression analysis showed that maternal occupational and environmental exposures increased the risk of CHD (odds ratio, 2.6; 95 % confidence interval, 1.6-4.2; p < 0.0001). No significant differences in Ile105Val genotype frequencies were observed between the children with CHD and the healthy children (p = 0.9). Furthermore, case-control analysis showed no evidence of significant interaction between the maternal exposures and GSTP1 polymorphism. Maternal exposure to toxicants increased the risk of children with CHD. However, fetal GSTP1 Ile105Val polymorphism did not increase the risk of CHD.

Background/objectives Children with congenital heart disease are exposed by repeated imaging to ionizing radiation, which may have important implications for lifetime health risks. Leukocyte telomere length (LTL), a reliable biomarker of genomic instability, is associated with increased risk of cancer and cardiovascular disease. We investigated LTL in grown-up patients with CHD (GUCHs) and a positive history of medical radiation exposure as well as the influence of functional polymorphisms of genes involved in DNA repair. Methods A group of 50 GUCH patients (26 males; age 25.2±9.0years) and 50 healthy age/gender-matched subjects (20 males; 27.0±3.1years) were enrolled. In GUCH patients, the cumulative exposure was estimated as effective dose (ED) in milliSievert. LTL was measured by quantitative RT-PCR. X-ray repair cross complementing-1 (XRCC1) and X-ray repair cross complementing-3 (XRCC3) SNPs (XRCC1Arg399Gln, XRCC1Arg194Tr and XRCC3 Thr241Met) were evaluated. Results GUCHs showed significantly shorter LTL compared with controls (1.0±0.3 vs 1.3±0.4, p=0.001). A significant inverse correlation between LTL and cumulative radiological ED was observed (r=−0.34, p=0.03). Patients with Thr/Met XRCC3 or Met/Met XRCC3 genotypes were significantly associated with a significantly shorter LTL compared with wild-type genotype (p=0.01 for Thr/Met and p=0.008 for Met/Met). Carriers of XRCC1 194Trp and XRCC3 241Met alleles presented a significant interaction with cumulative radiation dose exposure for LTL (both p interaction =0.02). Conclusions GUCH patients have LTL shortening, suggesting evidence of early biological aging. Common SNPs in DNA repair genes modify the effects of medical exposure to radiation LTL-related degenerative diseases.

Background: The seventh Committee on "Biological effects of Ionizing Radiation" (BEIR VII, 2006) underlines "the need of studies of infants who are exposed to diagnostic radiation because catheters have been placed in their hearts". Objective: To determine the Lifetime Attributable Risk (LAR) of cancer associated with the estimated cumulative radiological dose in 59 children (42 males, age=2.8±3.2 years) with complex CHD, and to assess chromosomal DNA damage after cardiac catheterization procedures. Methods: In all patients, the cumulative exposure was estimated as effective dose in milliSievert (mSv), and LAR cancer was determined from BEIR VII report. In a subset of 18 patients (13 males, age: 5±2± 5.7 years) micronucleus (MN) as biomarker of DNA damage and long-term risk predictor of cancer was assayed before and 2 hours after catheterization procedures. Dose–area product (DAP; Gy cm2) was assessed as measure of patient dose. Results: The median life time cumulative effective dose was 7.7 mSv per patient (range 4.6-41.2 mSv). Cardiac catheterization procedures and computed tomography were responsible for 95% of the total effective dose. For a 1-year-old child, the LAR cancer was 1 in 382 (25 th -75 th percentiles 1 in 531-1 in 187) and 1 in 156 (25 th -75 th percentiles 1 in 239-1 in 83) for male and female patients, respectively. Median MN values increased significantly 2 hours after procedure. when compared to baseline (pre= 6 ‰ vs post= 9 ‰ p=0.02). The median of DAP values was 20 Gy cm 2 (range of 1-277 Gy cm 2 ). Conclusion: CHD children are exposed to a significant cumulative dose. Both indirect cancer risk estimations and direct DNA data emphasize the need for strict radiation dose optimization in children.

Cardiac diagnostic or therapeutic testing is an essential tool for diagnosis and treatment of cardiovascular disease, but it also involves considerable exposure to ionizing radiation. Every exposure produces a corresponding increase in cancer risk, and risks are highest for radiation exposure during infancy and adolescence. Recent studies on chromosomal biomarkers corroborate the current radioprotection assumption showing that even modest radiation load due to cardiac catheter-based fluoroscopic procedures can damage the DNA of the cell. In this article, we review the biological and clinical risks of cardiac imaging employing ionizing radiation. We also discuss the perspectives offered by the use of molecular biomarkers in order to better assess the long-term development of health effects.

Introduction and objectives Single-nucleotide polymorphisms within a microRNA binding site can have different effects on gene expression, influencing the risk of disease. This study aimed to evaluate the association between single-nucleotide polymorphisms and haplotypes in the 3’UTR of the GATA4 gene and congenital heart disease risk. Methods Bioinformatics algorithms were used to analyze single-nucleotide polymorphisms in putative microRNA-binding sites of GATA4 3’UTR and to calculate the difference in free energy of hybridization (ΔFE, kcal/mol) for each wild-type vs the variant allele. Results The study population comprised 146 Caucasian patients (73 males; 6.68 ± 7.79 years) and a 265 healthy newborn participants (147 males). The sum of all |ΔFE| was considered to predict the biological importance of single-nucleotide polymorphisms binding more microRNAs. Next, the 4 polymorphisms (+1158 C > T, +1256 A > T, +1355 G > A, +1521 C > G) with the highest predicted |ΔFE tot | (9.91, 14.85, 11.03, 21.66 kcal/mol, respectively) were genotyped in a case-control study (146 patients and 250 controls). Applying a correction for multiple testing only the +1158 T allele was found to be associated with a reduced risk showing significant difference between patients and controls. Haplotype analysis showed that the T-T-G-C haplotype (more uncommon in congenital heart diseases than in controls) was associated with a significantly decreased risk ( P = .03), while the rare C-A-A-C haplotype, which was very uncommon in controls (0.3%) compared with the disease (2.4%), was associated with a 4-fold increased risk of disease ( P = .04). Conclusions Common variants in 3’UTR of the GATA4 gene jointly interact, affecting the congenital heart disease susceptibility, probably by altering microRNA posttranscriptional regulation.

Several single-nucleotide polymorphisms (SNPs) have been recognized as associated with ischemic heart disease (IHD) although the optimal set of risk genotypes has not be identified. This study aimed to examine whether identified high-risk SNPs are associated with early onset of IHD. In the GENOCOR study, 44 high-risk SNPs were genotyped in 114 patients with early onset of IHD (46.2 ± 5.1 years) and 384 patients with late onset of IHD (60.7 ± 5.9 years). The associations between individual SNPs and early onset IHD were assessed. A multilocus genetic risk score (GRS) for each associated risk genetic markers was constructed by summing the number of risk alleles. The SNPs significantly associated with IHD were: -482C > T of Apolipoprotein C III gene (ApoC3, p = 0.02); 1171 5A > 6A of Matrix metalloproteinase 3 stromelisine I gene (p = 0.01); G98T of Selectin E gene (p = 0.05); C/G of 9p21.3 locus (p = 0.01). Likelihood ratio test showed a strong interaction for increasing risk of early IHD between the presence of ApoC3 and 9p21.3 locus with hypertriglyceridemia (p = 0.0008, 0.0011) as well as between 9p21.3 locus and smoking (p = 0.0010) after correction for multiple testing. The OR for premature IHD for GRS unit was 1.3 (95% CI 1.1-1.6, p = 0.001). Patients in the top tertile of GRS were estimated to have a 3.2-fold (95% CI 1.5-6.8; p = 0.001) increased risk of early IHD compared with those in the bottom tertile. The results show that currently identified high-risk SNPs confer an additive biomarker for cardiovascular events. GRS may provide important incremental information on the genetic component of IHD.

Bicuspid aortic valve (BAV) is the most common congenital cardiac malformation, affecting 1-2% of the general population. BAV is frequently associated with significant aortic pathology (valve regurgitation, endocarditis, severe aortic stenosis, aortic aneurysm and dissection) accounting for considerable morbidity and mortality. Despite its clinical relevance, the pathogenesis of BAV disease and its complications remain largely unknown. The purpose of this paper is to discuss and summarize our current understanding of BAV in terms of epidemiology, genetics and clinics.

Congenital heart defects (CHDs) are the most prevalent of all birth malformations arising from the complex interplay of environmental exposures and genes. Modifiable environmental risk factors are still largely unknown, especially for paternal exposure. The aim of the present study was to examine the association between the environmental exposures of both parents and CHD risk and to explore the modification effect of metabolizing gene polymorphisms in children who lack the genetic capacity to produce the glutathione S-transferase (GST) GSTM1 and GSTT1 enzymes. A total of 330 parents of a child with CHD and 330 parents of a child without any congenital malformations were compared in terms of lifestyle habits and toxicant exposure. GST gene polymorphisms were investigated in 180 patients with CHD (104 males, age 4.9 ± 5.8 years). Paternal smoking (≥15 cigarettes/day) was significantly associated with CHD risk (odds ratio [OR] 2.1, 95% confidence interval [CI] 1.3 to 3.5, p = 0.002). Both maternal (OR 2.6, 95% CI 1.6 to 4.2, p

Patients with non ischemic-dilated cardiomyopathy (DCM) are characterized by an activation of the adenosinergic system and reduced coronary flow reserve (CFR) evaluated by transthoracic Doppler echocardiography during vasodilator adenosinergic stress (dipyridamole administration). The aim of this study was to assess whether genetic polymorphisms (263CT and 1976CT) of the A2(A) receptor gene affect CFR response in patients with DCM.We enrolled a group of 80 patients with DCM (55 male; age, 62±10.3 years) and 162 healthy volunteers (55 male; age, 45.1±9.5 years). Doppler-derived CFR (high-dose dipyridamole coronary diastolic peak flow velocity to resting coronary peak flow velocity ratio) of distal left anterior descending artery was determined in DCM. A2(A) receptor genotyping was determined in all patients by polymerase chain reaction-restriction fragment length polymorphism analysis. The expression of A2(A) protein and mRNA was also assessed in healthy controls.The genotype distribution of the 263CT (P=0.5) and 1976CT (P=0.8) polymorphisms was not significantly different between patients and controls. Patients with 1976TT genotype had significantly lower CFR value than 1976CC patients (2.3±0.7, 2.0±0.5 and 1.9±0.4, P0.05 for CC, CT and TT genotypes, respectively). Controls who were heterozygous (P=0.02) or homozygous (P=0.001) for the T1976 allele showed a significant increase in A2(A) receptor protein.These data demonstrate that A2(A) 1976CT polymorphism is associated with a blunted coronary vasodilatory response in patients with DCM, and support a direct consequences of this single nucleotide polymorphism for protein expression. Additional studies are needed to better define the functional role of this genetic variant as well as to clarify the potential clinical impact of genetics during pharmacological stress cardiac imaging.

Congenital heart diseases--the most prevalent and fatal birth defects worldwide--result from a combination of genetic predisposition and environmental factors. Currently, there is a growing body of epidemiological literature on parental and occupational prenatal risk factors. We report a case of a 4-months-old infant whose mother was professionally exposed to the cardiovascular catheterization laboratory during the first 4 weeks of pregnancy. The total radiation dose recorded by dosimeters under apron was 4220 microSiviert equivalent to 210 chest X-rays, suggesting the hypothesis of maternal ionising radiation induced-teratogenic effect.

Objective: It has been suggested that atherosclerotic mechanisms are involved in the pathogenesis of aortic valve stenosis (AVS). We hypothesised that low levels of the soluble receptor for advanced glycation end-products (sRAGE) might be associated with AVS due to its clinical and pathological associations with atherosclerosis. Methods: We enrolled 75 consecutive patients with severe AVS scheduled for surgical aortic valve replacement and 39 controls without AVS matched for age and gender. Besides the traditional risk factors, we evaluated plasma levels of sRAGE, C-reactive protein (CRP) and IL-6. All patients underwent transthoracic echocardiography, carotid arteries ultrasound scan and coronary angiography. The aortic and coronary calcium by multislice computed tomography was assessed in AVS patients. Results: The values of sRAGE were significantly lower (pConclusion: Since sRAGE could exert antiatherogenic effects by preventing inflammatory responses mediated by cell surface RAGE activation, low levels in AVS patients indicate that ligand-RAGE axis could contribute to pathogenesis of AVS., This article may be used for non-commercial purposes in accordance with CC BY-NC-ND license terms and conditions.

The bicuspid aortic valve (BAV) and specific systemic autoimmune diseases are associated with cardiovascular manifestation, including aortic aneurysm. We reported a case of 64 year-old patient with BAV and a history of ankylosing spondylitis (AS) and systemic lupus erythematosus (SLE), and who developed ascending thoracic aortic aneurysm. The patient presented also the homozygosity for genetic variants of MMP9, ACE, MTHFR and PAI-1 genes. Gene-environmental interactions may represent an additional pathogenetic dimension in the still challenging management of the abnormalities of the aortic wall, including dilatation, aneurysm and dissection.

A 52-year old female cardiologist with 16 year radiation exposure in Cath Lab as an interventional cardiologist developed a multifocal papillary thyroid carcinoma. Dosimetric (below apron) cumulative exposure totalled 56 mSv, corresponding to 2,800 chest radiographs. The patient also carried genetic polymorphisms of genes (XRCC1 and XRCC3) involved in DNA repair, increasing the cancer risk after ionizing radiation exposure. Dose optimization and diligent radioprotection are essential to minimize cancer risk in professionally exposed cardiologist. Good dosimetric practice is essential to establish a legally plausible cause-effect relationship between exposure and damage.

Interventional cardiologists working in high-volume cardiac catheterization laboratory are exposed to significant occupational radiation risks. Common single-nucleotide polymorphisms (SNPs) in DNA repair genes are thought to modify the effects of low-dose radiation exposure on DNA damage, the main initiating event in the development of cancer and hereditary disease. The aim of this study was to determine the relationship between XRCC1 (Arg194Trp and Arg399Gln), OGG1 (Ser326Cys), APE1 (Asp148Glu) and XRCC3 (Thr241Met) SNPs and chromosomal DNA damage. We enrolled 77 subjects: 40 interventional cardiologists (27 male, 41.3+/-9.4 years and 13 female, 37.8+/-8.4 years) and 37 clinical cardiologists (26 male, 39.4+/-9.5 years and 11 female, 35.0+/-9.8 years) without radiation exposure as the control group. Micronucleus (MN) assay was performed as biomarker of chromosomal DNA damage and an early predictor of cancer. MN frequency was significantly higher in interventional cardiologists than in clinical physicians (19.7+/-7.8 per thousand vs. 13.5+/-6.3 per thousand, p=0.0003). Within the exposed group, individuals carrying a XRCC3 Met241 allele had higher frequency than homozygous XRCC3 Thr241 (21.2+/-7.8 per thousand vs. 16.6+/-7.1 per thousand, p=0.03). Individuals with two or more risk alleles showed a higher MN frequency when compared to subjects with one or no risk allele (18.4+/-6.6 per thousand vs. 14.4+/-6.1 per thousand, p=0.02). An interactive effect was found between smoking, exposure10 years and the presence of the two or more risk alleles on the MN frequency (F=6.3, p=0.02). XRCC3 241Met alleles, particularly in combination with multiple risk alleles of DNA repair genes, contribute to chromosomal DNA damage levels in interventional cardiologists.

Background The purpose of our study was to investigate the potential contribution of germline mutations in NOTCH1, GATA5 and TGFBR1 and TGFBR2 genes in a cohort of Italian patients with familial Bicuspid Aortic Valve (BAV). Methods All the coding exons including adjacent intronic as well as 5′ and 3′ untranslated (UTR) sequences of NOTCH1, GATA5, TGFBR1 and TGFBR2 genes were screened by direct gene sequencing in 11 index patients (8 males; age = 42 ± 19 years) with familial BAV defined as two or more affected members. Results Two novel mutations, a missense and a nonsense mutation (Exon 5, p.P284L; Exon 26, p.Y1619X), were found in the NOTCH1 gene in two unrelated families. The mutations segregated with the disease in these families, and they were not found on 200 unrelated chromosomes from ethnically matched controls. No pathogenetic mutation was identified in GATA5, TGFBR1 and TGFBR2 genes. Conclusions Two novel NOTCH1 mutations were identified in two Italian families with BAV, highlighting the role of a NOTCH1 signaling pathway in BAV and its aortic complications. These findings are of relevance for genetic counseling and clinical care of families presenting with BAV. Future studies are needed in order to unravel the still largely unknown genetics of BAV.