Ventricular Septal Defects: Molecular Pathways and Animal Models.

Ventricular septation is a complex process which involves the major genes of cardiac development, acting on myocardial cells from first and second heart fields, and on mesenchymal cells from endocardial cushions. These genes, coding for transcription factors, interact with each other, and their differential expression conditions the severity of the phenotype. In this chapter, we will describe the formation of the ventricular septum in the normal heart, as well as the molecular mechanisms leading to the four main anatomic types of ventricular septal defects: outlet, inlet, muscular, and central perimembranous, resulting from failure of development of the different parts of the ventricular septum. Experiments on animal models, particularly transgenic mouse lines, have helped us to decipher the molecular determinants of ventricular septation. However, a precise description of the anatomic phenotypes found in these models is mandatory to achieve a better comprehension of the complex mechanisms responsible for the various types of VSDs.

Identification of Six Pathogenic Genes for Tibetan Familial Ventricular Septal Defect by Whole Exome Sequencing.

INTRODUCTION: Ventricular septal defect (VSD) is the most common congenital heart malformation in children. This study aimed to investigate potential pathogenic genes associated with Tibetan familial VSD. METHODS: Whole genomic DNA was extracted from eight Tibetan children with VSD and their healthy parents (a total of 16 individuals). Whole-exome sequencing was performed using the Illumina HiSeq platform. After filtration, detection, and annotation, single nucleotide variations and insertion-deletion markers were examined. Comparative evaluations using the Sorting Intolerant from Tolerant, PolyPhen V2, Mutation Taster, and Combined Annotation Dependent Depletion databases were conducted to predict harmful mutant genes associated with the etiology of Tibetan familial VSD. RESULTS: A total of six missense mutations in genetic disease-causing genes associated with the development of Tibetan familial VSD were identified: activin A receptor type II-like 1 (c.652 C > T: p.R218 W), ATPase cation transporting 13A2 (c.1363 C > T: p.R455 W), endoplasmic reticulum aminopeptidase 1 (c.481 G > A: p.G161 R), MRI1 (c.629 G > A: p.R210Q), tumor necrosis factor receptor-associated protein 1 (c.224 G > A: p.R75H), and FBN2 (c.2260 G > A: p.G754S). The Human Gene Mutation Database confirmed activin A receptor type II-like 1, MRI1, and tumor necrosis factor receptor-associated protein 1 as pathogenic mutations, while FBN2 was classified as a probable pathogenic mutation. CONCLUSIONS: This novel study directly screens genetic variations associated with Tibetan familial VSD using whole-exome sequencing, providing new insights into the pathogenesis of VSD.

SOX7 deficiency causes ventricular septal defects through its effects on endocardial-to-mesenchymal transition and the expression of Wnt4 and Bmp2.

SOX7 is a transcription factor-encoding gene located in a region on chromosome 8p23.1 that is recurrently deleted in individuals with ventricular septal defects (VSDs). We have previously shown that Sox7-/- embryos die of heart failure around E11.5. Here, we demonstrate that these embryos have hypocellular endocardial cushions with severely reduced numbers of mesenchymal cells. Ablation of Sox7 in the endocardium also resulted in hypocellular endocardial cushions, and we observed VSDs in rare E15.5 Sox7flox/-;Tie2-Cre and Sox7flox/flox;Tie2-Cre embryos that survived to E15.5. In atrioventricular explant studies, we showed that SOX7 deficiency leads to a severe reduction in endocardial-to-mesenchymal transition (EndMT). RNA-seq studies performed on E9.5 Sox7-/- heart tubes revealed severely reduced Wnt4 transcript levels. Wnt4 is expressed in the endocardium and promotes EndMT by acting in a paracrine manner to increase the expression of Bmp2 in the myocardium. Both WNT4 and BMP2 have been previously implicated in the development of VSDs in individuals with 46,XX sex reversal with dysgenesis of kidney, adrenals and lungs (SERKAL) syndrome and in individuals with short stature, facial dysmorphism and skeletal anomalies with or without cardiac anomalies 1 (SSFSC1) syndrome, respectively. We now show that Sox7 and Wnt4 interact genetically in the development of VSDs through their additive effects on endocardial cushion development with Sox7+/-;Wnt4+/- double heterozygous embryos having hypocellular endocardial cushions and perimembranous and muscular VSDs not seen in their Sox7+/- and Wnt4+/- littermates. These results provide additional evidence that SOX7, WNT4 and BMP2 function in the same pathway during mammalian septal development and that their deficiency can contribute to the development of VSDs in humans.

High-risk genes involved in common septal defects of congenital heart disease.

The septation defect is one of the main categories of congenital heart disease (CHD). They can affect the septation of the atria leading to atrial septal defect (ASD), septation of ventricles leading to ventricular septal defect (VSD), and formation of the central part of the heart leading to atrioventricular septal defect (AVSD). Disruption of critical genetic factors involved in the proper development of the heart structure leads to CHD manifestation. Because of this, to identify the high-risk genes involved in common septal defects, a comprehensive search of the literature with the help of databases and the WebGestalt analysis tool was performed. The high-risk genes identified in the analysis were checked in 16 Indian whole-exome sequenced samples, including 13 VSD and three Tetralogy of Fallot for in silico validation. This data revealed three variations in GATA4, i.e., c.C1223A at exon 6: c.C602A and c.C1220A at exon 7; and one variation in MYH6, i.e., c.G3883C at exon 28 in two VSD cases. This study supports previously published studies that suggested GATA4 and MYH6 as the high-risk genes responsible for septal defects. Thus, this study contributes to a better understanding of the genes involved in heart development by identifying the high-risk genes and interacting proteins in the pathway.

Identification of variants of ISL1 gene promoter and cellular functions in isolated ventricular septal defects.

Ventricular septal defects (VSDs) are the most common congenital heart defects (CHDs). Studies have documented that ISL1 has a crucial impact on cardiac growth, but the role of variants in the ISL1 gene promoter in patients with VSD has not been explored. In 400 subjects (200 patients with isolated and sporadic VSDs: 200 healthy controls), we investigated the ISL1 gene promoter variant and performed cellular functional experiments by using the dual-luciferase reporter assay to verify the impact on gene expression. In the ISL1 promoter, five variants were found only in patients with VSD by sequencing. Cellular functional experiments demonstrated that three variants decreased the transcriptional activity of the ISL1 promoter (P < 0.05). Further analysis with the online JASPAR database demonstrated that a cluster of putative binding sites for transcription factors may be altered by these variants, possibly resulting in change of ISL1 protein expression and VSD formation. Our study has, for the first time, identified novel variants in the ISL1 gene promoter region in the Han Chinese patients with isolated and sporadic VSD. In addition, the cellular functional experiments, electrophoretic mobility shift assay, and bioinformatic analysis have demonstrated that these variants significantly alter the expression of the ISL1 gene and affect the binding of transcription factors, likely resulting in VSD. Therefore, this study may provide new insights into the role of the gene promoter region for a better understanding of genetic basis of the formation of CHDs and may promote further investigations on mechanism of the formation of CHDs.

Genetic analysis of the CITED2 gene promoter in isolated and sporadic congenital ventricular septal defects.

Ventricular septal defect (VSD) is the most common congenital heart defect. Previous studies have reported genetic variations in the encoding region of CITED2 highly associated with cardiac malformation but the role of CITED2 gene promoter variations in VSD patients has not yet been explored. We investigated the variation of CITED2 gene promoter and its impacts on gene promoter activity in the DNA of paediatric VSD patients. A total of seven variations were identified by Sanger sequencing in the CITED2 gene promoter region in 400 subjects, including 200 isolated and sporadic VSD patients and 200 healthy controls. Using dual-luciferase reporter assay, we found four of the 7 variations identified significantly decreased the transcriptional activity of the CITED2 gene promoter in HEK-293 cells (P < .05). Further, a bioinformatic analysis with the JASPAR databases was performed and a cluster of putative binding sites for transcription factors was created or disrupted by these variations, leading to low expression of CITED2 protein and development of VSD. Our study for the first time demonstrates genetic variations in the CITED2 gene promoter in the Han Chinese population and the role of these variations in the development of VSD, providing new insights into the aetiology of CHD.

MicroRNA-29c affects zebrafish cardiac development via targeting Wnt4.

As a single cardiac malformation, ventricular septal defect (VSD) is the most common form of congenital heart disease. However, the precise molecular mechanisms underlying VSD are not completely understood. Numerous microRNAs (miRs/miRNAs) are associated with ventricular septal defects. miR-29c inhibits the proliferation and promotes the apoptosis and differentiation of P19 embryonal carcinoma cells, possibly via suppressing Wnt4 signaling. However, to the best of our knowledge, no in vivo studies have been published to determine whether overexpression of miR-29c leads to developmental abnormalities. The present study was designed to observe the effect of miRNA-29c on cardiac development and its possible mechanism in vivo. Zebrafish embryos were microinjected with different doses (1, 1.6 and 2 µmol) miR-29c mimics or negative controls, and hatchability, mortality and cardiac malformation were subsequently observed. The results showed that in zebrafish embryos, miR-29c overexpression attenuated heart development in a dose-dependent manner, manifested by heart rate slowdown, pericardial edema and heart looping disorder. Further experiments showed that overexpression of miR-29c was associated with the Wnt4/β-catenin signaling pathway to regulate zebrafish embryonic heart development. In conclusion, the present results demonstrated that miR-29c regulated the lateral development and cardiac circulation of zebrafish embryo by targeting Wnt4.

Protein biomarkers and risk scores in pulmonary arterial hypertension associated with ventricular septal defect: integration of multi-omics and validation.

The molecular mechanisms underlying pulmonary arterial hypertension (PAH) in congenital ventricular septal defects (VSD) are unclear. We aimed to reveal molecular pathways and potential biomarkers by multi-omics analysis in VSD-PAH. Plasma from 160 children, including 120 VSD patients with/without PAH and 40 healthy children was studied by integrated proteomics, metabolomics, and bioinformatics analyses. Proteomics identified 107 differential proteins (DPs) between patients with/without PAH including significantly increased adiponectin (ADIPO), dopamine ß-hydroxylase (DBH), alanyl membrane aminopeptidase (ANPEP), transferrin receptor 1, and glycoprotein Ib platelet α-subunit and decreased guanine nucleotide-binding protein Gs in VSD-PAH. Metabolomics discovered 191 differential metabolites between patients with/without PAH, including elevation of serotonin, taurine, creatine, sarcosine, and 2-oxobutanoate, and decrease of vanillylmandelic acid, 3,4-dihydroxymandelate, 15-keto-prostaglandin F2α, fructose 6-phosphate, l-glutamine, dehydroascorbate, hydroxypyruvate, threonine, l-cystine, and 1-aminocyclopropane-1-carboxylate. The DPs were validated in a new cohort of patients (n = 80). Integrated analyses identified key pathways, including cAMP, ECM receptor interaction, AMPK, hypoxia-inducible factor 1, PI3K-Akt signaling pathways, and amino acid metabolisms. Increased plasma protein levels of DBH, ADIPO, and ANPEP were found to be independently associated with the occurrence of PAH, with a new total risk score from these three proteins developed for clinical diagnosis. In this integrated multi-omics analysis in VSD-PAH patients, we have, for the first time, found that VSD-PAH patients present important differential proteins, metabolites, and key pathways. We have developed a total risk score (based on the plasma concentration of DBH, ANPEP, and ADIPO) as a predictor of development of PAH in CHD-VSD patients. Therefore, these proteins may be used as biomarkers, and the new total risk score has significant clinical implications in the diagnosis of PAH.

Dysregulation of Notch signaling in cardiac mesenchymal cells of patients with tetralogy of Fallot.

BACKGROUND: Tetralogy of Fallot (TF) is a severe congenital defect of heart development. Fine-tuned sequential activation of Notch signaling genes is responsible for proper heart chamber development. Mutations in Notch genes have been associated with TF. The aim of this study was to analyze the activity of the Notch pathway in cardiac mesenchymal cells derived from ventricular tissue of TF patients. METHODS: Cardiac mesenchymal cells were isolated from 42 TF patients and from 14 patients with ventricular septal defects (VSDs), used as a comparison group. The Notch pathway was analyzed by estimating the expression of Notch-related genes by qPCR. Differentiation and proliferation capacity of the cells was estimated. RESULTS: The TF-derived cells demonstrated a dysregulated pattern of Notch-related gene expression comparing to VSD-derived cells. Correlation of Notch signaling activation level by HEY1/HES1 expression level with proliferation and cardiogenic-like differentiation of cardiac mesenchymal cells was observed but not with clinical parameters nor with the age of the patients. CONCLUSIONS: The data suggest a contribution of dysregulated Notch signaling to the pathogenesis of tetralogy of Fallot and importance of Notch signaling level for the functional state of cardiac mesenchymal cells, which could be critical considering these cells for potential cell therapy approaches.

Growth and Clinical Characteristics of Children with Floating-Harbor Syndrome: Analysis of Current Original Data and a Review of the Literature.

BACKGROUND: Floating-Harbor syndrome (FHS) is a rare condition characterized by dysmorphic facial features, short stature, and expressive language delay. OBJECTIVE: The aim of this study was to describe a cohort of patients with FHS and review the literature about the response to recombinant human growth hormone (rhGH) therapy. METHODS: Anthropometric and laboratory data from 7 patients with FHS were described. The molecular diagnosis was established by multigene analysis. Moreover, we reviewed the literature concerning patients with FHS treated with rhGH. RESULTS: All 7 patients were born small for gestational age. At first evaluation, 6 patients had a height standard deviation score (SDS) ≤-2 and 1 had short stature in relation to their target height. Bone age was usually delayed, which rapidly advanced during puberty. Nonspecific skeletal abnormalities were frequently noticed, and normal to elevated plasma IGF-I levels were observed in all except 1 patient with growth hormone deficiency. Information about 20 patients with FHS treated with rhGH was analyzed (4 from our cohort and 16 from the literature). The median height changes during the treatment period (approx. 2.9 years) were 1.1 SDS (range from -0.4 to 3.1). Nontreated patients had an adult height SDS of -4.1 ± 1.2 (n = 10) versus -2.6 ± 0.8 SDS (n = 7, p 0.012) for treated patients. CONCLUSION: We observed a laboratory profile compatible with IGF-1 insensitivity in some patients with FHS. Nevertheless, our study suggests that children with FHS may be considered as candidates for rhGH therapy. Further studies are necessary to establish the real benefit and safety of rhGH therapy in these patients.

Uric acid and sphingomyelin enhance autophagy in iPS cell-originated cardiomyocytes through lncRNA MEG3/miR-7-5p/EGFR axis.

This study aimed to determine the metabolites associated with ventricular septal defect (VSD) and the underlying mechanisms. Blood samples and thymus tissues were collected from VSD patients to perform LC-MS-based metabolomics assay and generate iPS cell-derived cardiomyocytes, respectively. VSD rat model was used in vivo study. RT-PCR, western blotting, immunohistochemistry, luciferase activity assay, GFP-LC3 adenovirus and GFP and RFP tfLC3 assay, and transmission electron microscopy were performed to investigate the underlying mechanisms. The metabolites uric acid (UA) and sphingomyelin (SM) increased in the serum of VSD patients, along with enhanced autophagy. The combination of UA and SM treatment could promote autophagy and inhibit EGFR and AKT3 expressions. Overexpression of EGFR and AKT3 suppressed autophagy in UA and SM-treated cardiomyocytes, respectively. Also, lncRNA MEG3 knockdown and overexpression could enhance and inhibit autophagy in UA and SM-treated cardiomyocytes, respectively, through targeting miR-7-5p. Moreover, miR-7-5p mimics and inhibitors promoted and inhibited autophagy in UA and SM-treated cardiomyocytes, respectively, via target EGFR. In VSD rat model, upregulation of MEG3 could reverse high level of autophagy and decrease serum UA and SM. In conclusion, UA and SM are essential VSD-associated metabolic biomarkers and MEG3/miR-7-5p/EGFR axis is critical to the regulation of autophagy in cardiomyocytes.

The cardiac proteome in patients with congenital ventricular septal defect: A comparative study between right atria and right ventricles.

Right ventricle (RV) remodelling occurs in neonatal patients born with ventricular septal defect (VSD). The presence of a defect between the two ventricles allows for shunting of blood from the left to right side. The resulting RV hypertrophy leads to molecular remodelling which has thus far been largely investigated using right atrial (RA) tissue. In this study we used proteomic and phosphoproteomic analysis in order to determine any difference between the proteomes for RA and RV. Samples were therefore taken from the RA and RV of five infants (0.34 ±â€¯0.05 years, mean ±â€¯SEM) with VSD who were undergoing cardiac surgery to repair the defect. Significant differences in protein expression between RV and RA were seen. 150 protein accession numbers were identified which were significantly lower in the atria, whereas none were significantly higher in the atria compared to the ventricle. 19 phosphorylation sites (representing 19 phosphoproteins) were also lower in RA. This work has identified differences in the proteome between RA and RV which reflect differences in contractile activity and metabolism. As such, caution should be used when drawing conclusions based on analysis of the RA and extrapolating to the hypertrophied RV. SIGNIFICANCE: RV hypertrophy occurs in neonatal patients born with VSD. Very little is known about how the atria responds to RV hypertrophy, especially at the protein level. Access to tissue from age-matched groups of patients is very rare, and we are in the unique position of being able to get tissue from both the atria and ventricle during reparative surgery of these infants. Our findings will be beneficial to future research into heart chamber malformations in congenital heart defects.

Salivary interleukin-6 levels in children with early childhood caries and ventricular septal defect.

BACKGROUND: The efficacy of dental treatment on the levels of inflammatory markers in ventricular septal defect (VSD) has not been fully explored. AIMS: The aim of this study was to assess the level of salivary interleukin-6 (IL-6) in children with early childhood caries (ECC) and VSD and to compare its levels before and after comprehensive full mouth rehabilitation. METHODS: Saliva samples were collected from children with ECC and VSD before dental treatment and 1-month posttreatment. The salivary IL-6 levels were analyzed using the enzyme-linked immunosorbent assay method. Gingival index (GI) was also timely recorded during the study period. Oral health awareness sessions were conducted for children and their parents. The Fisher's Least Significant Difference test was used for intergroup comparison of salivary IL-6 levels, while the paired t-test compared the values of GI before and after treatment. RESULTS: The mean salivary IL-6 levels 1 month after treatment had reduced (P < 0.000). There was a reduction in GI score (P = 0.002). CONCLUSIONS: When children having VSD and ECC are dentally rehabilitated and followed up periodically, the level of IL-6 is minimized, hence improving the quality of life of the affected children.

Anatomy of the ventricular septal defect in congenital heart defects: a random association?

BACKGROUND: A ventricular septal defect (VSD) is an integral part of most congenital heart defects (CHD). To determine the prevalence of VSD in various types of CHD and the distribution of their anatomic types. METHODS: We reviewed 1178 heart specimens with CHD from the anatomic collection of the French Reference Centre for Complex Congenital Heart Defects. During the morphologic study a special attention was paid to the localisation of the VSD viewed from the right ventricular side. The VSDs were classified as muscular, central perimembranous, outlet located between the two limbs of the septal band, and inlet. The specimens were classified according to the 9 categories and 23 subcategories of the anatomic and clinical classification of CHD1 (ACC-CHD). RESULTS: Ventricular septum was almost always intact in anomalies of pulmonary veins (4/73, 5%), Ebstein anomaly (3/21, 14%), and double-inlet right ventricle (DIRV, 1/10, 10%). There was always a VSD in tetralogy of Fallot and variants (TOF, 123 cases) and common arterial trunk (CAT, 55 cases), always of the outlet type. There was almost always a VSD in double inlet left ventricle (33/34, 97%, always muscular), congenitally corrected transposition of great arteries (ccTGA, 23/24, 96%), interrupted aortic arch (IAA, 25/27, 93%), and double outlet right ventricle (DORV, 92/106, 87%). A VSD was found in 68% of aortic coarctation (CoA, 43/63), 62% of heterotaxy syndromes (21/34), 54% of transposition of the great arteries (TGA, 104/194). The VSD was located between the two limbs of the septal band in 100% of TOF and CAT, 80% of IAA, 77% of DORV, 82% of DD. The VSD was of the inlet type in 17% of cc TGA and in 71% of heterotaxy syndromes. In TGA, the VSD was outlet in 40%, central perimembranous in 25%, muscular in 25%, inlet in 10%. In CoA, the VSD was outlet in 44%, central perimembranous in 35%, muscular in 21%. In the 10% hearts with isolated VSD, the distribution was outlet in 44%, central perimembranous in 36%, muscular in 18%, and inlet in 2%. CONCLUSION: The anatomic distribution of VSD is similar in isolated VSD, CoA and TGA, while the VSD is predominantly outlet in outflow tract defects except TGA. This reinforces the allegedly different mechanisms in TGA and cardiac neural crest defects. This anatomic approach could provide new insights in the grouping and aetiology of CHD.

MicroRNA expression, target genes, and signaling pathways in infants with a ventricular septal defect.

This study aimed to systematically investigate the relationship between miRNA expression and the occurrence of ventricular septal defect (VSD), and characterize the miRNA target genes and pathways that can lead to VSD. The miRNAs that were differentially expressed in blood samples from VSD and normal infants were screened and validated by implementing miRNA microarrays and qRT-PCR. The target genes regulated by differentially expressed miRNAs were predicted using three target gene databases. The functions and signaling pathways of the target genes were enriched using the GO database and KEGG database, respectively. The transcription and protein expression of specific target genes in critical pathways were compared in the VSD and normal control groups using qRT-PCR and western blotting, respectively. Compared with the normal control group, the VSD group had 22 differentially expressed miRNAs; 19 were downregulated and three were upregulated. The 10,677 predicted target genes participated in many biological functions related to cardiac development and morphogenesis. Four target genes (mGLUR, Gq, PLC, and PKC) were involved in the PKC pathway and four (ECM, FAK, PI3 K, and PDK1) were involved in the PI3 K-Akt pathway. The transcription and protein expression of these eight target genes were significantly upregulated in the VSD group. The 22 miRNAs that were dysregulated in the VSD group were mainly downregulated, which may result in the dysregulation of several key genes and biological functions related to cardiac development. These effects could also be exerted via the upregulation of eight specific target genes, the subsequent over-activation of the PKC and PI3 K-Akt pathways, and the eventual abnormal cardiac development and VSD.

Molecular mechanisms of Ellis­van Creveld gene variations in ventricular septal defect.

The Ellis-van Creveld (EVC) gene is associated with various congenital heart diseases. However, studies on EVC gene variations in ventricular septal defect (VSD) and the underlying molecular mechanisms are sparse. The present study detected 11 single­nucleotide polymorphisms (SNPs) in 65 patients with VSD and 210 control patients from the Chinese Han population. Of the identified SNPs only the c.1727G>A SNP site was positively associated with the development of VSD (P<0.007). A known mutation, c.343C>G, was also identified, which causes a leucine to valine substitution at amino acid 115 of the EVC protein (p.L115V). The results of functional prediction indicated that c.343C>G may be a pathogenic mutation. In addition, in NIH3T3 mouse embryonic fibroblast cells, the EVC c.343C>G mutation significantly decreased cell proliferation and increased apoptosis. Further investigation demonstrated that in NIH3T3 cells, overexpression of EVC c.343C>G mutation reduced the binding between EVC and smoothened, which further downregulated the activity of the hedgehog (Hh) signaling pathway and the expression of downstream cyclin D1 and B­cell lymphoma 2 proteins with SAG. The c.1727G>A SNP of the EVC gene increased VSD susceptibility in patients from the Chinese Han population. The molecular mechanism underlying the development of VSD induced by the EVC c.343C>G mutation may be due to a reduction in the anti­apoptotic and proliferative abilities of cardiomyocytes via downregulation of Hh pathway activity. The results of the present study may provide novel targets for the diagnosis and treatment of patients with VSD.

The stromal cell-derived factor 1/C-X-C chemokine receptor type 4 axis is important in neutrophil migration caused by cardiopulmonary bypass in children.

OBJECTIVES: Acute lung injury caused by cardiopulmonary bypass (CPB) is characterized by massive neutrophil migration to the lungs. Neutrophil migration may be closely related to stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor Type 4 (CXCR4) axis activation, which plays an essential role in modulating the trafficking of neutrophils. We investigated the changes in the expression of SDF-1/CXCR4 axis components before and after CPB as well as the role of the axis in driving the migration of neutrophils in patients with congenital heart disease. METHODS: Fifteen children undergoing elective open-heart surgery under CPB (CPB group) and 15 children undergoing minimally invasive ultrasound-guided closure of a ventricular septal defect (control group) were enrolled in this non-randomized clinical trial. Neutrophil CXCR4 expression was evaluated using quantitative reverse transcription polymerase chain reaction and flow cytometry. An enzyme-linked immunosorbent assay was used to measure plasma SDF-1 levels. The migration characteristics of neutrophils under 8 different combinations designated A-H were assayed with and without a specific CXCR4 antagonist, AMD3100, to evaluate the functional significance of the SDF-1/CXCR4 axis. RESULTS: Both CXCR4 gene and protein expressions were elevated in the CPB group compared with the control group after CPB (0.81 ± 0.55 vs 1.76 ± 1.32; P < 0.05, 1.96 ± 0.86 vs 2.65 ± 0.79; P < 0.05), and plasma SDF-1 levels were also increased in the former compared with the latter (197.84 ± 19.96 pg/ml vs 539.13 ± 99.83 pg/ml; P < 0.05). The in vitro experiments showed that plasma isolated post-CPB exhibited the strongest chemotactic effect on neutrophils. The CPB group showed a higher chemotaxis index, which serves as a marker for the effects of plasma on neutrophils, than that for the control group after CPB (37.38 ± 9.39 vs 13.61 ± 2.59; P < 0.05). In addition, the CXCR4 antagonist AMD3100 significantly abrogated the increase in neutrophil migration in the CPB group. CONCLUSIONS: Exposure to CPB, which activates the SDF-1/CXCR4 axis, using an antagonist to prevent neutrophil trafficking, may be a beneficial therapy for the related complications.

Influence of high-flow modified ultrafiltration on brain oxygenation and perfusion during surgery for children with ventricular septal defects: a pilot study.

BACKGROUND: Modified ultrafiltration (MUF) can be performed in infants with ventricular septal defects (VSDs) after cardiopulmonary bypass (CPB) to reduce haemodilution and its potential adverse effects. High-flow MUF might reduce ultrafiltration duration and hasten the necessary correction of haemodilution during CPB. However, its influence on brain oxygenation remains controversial. OBJECTIVE: This non-randomized, prospective, pilot study aimed to investigate the influence of high-flow MUF on brain oxygenation in infants with VSDs. METHODS: High-flow MUF (≥20 mL/kg/min) was performed in twenty infants. Brain oxygen saturation (rSO2) and tissue haemoglobin index (tHI) were non-invasively and continuously measured intraoperatively using near-infrared spectroscopy (NIRS). Transcranial Doppler non-invasively detected the mean flow velocity of the middle cerebral artery (Vmean). RESULTS: rSO2 increased significantly during MUF, as did tHI, Vmean, mean arterial pressure and haematocrit (all p<0.05). No correlation was found between changes in rSO2 and changes in other parameters (all p≥0.05). CONCLUSION: In infants with ventricular septal defects managed with CPB during VSDs repair, high-flow MUF did not reduce brain oxygenation.

Effectiveness of the critical congenital heart disease screening program for early diagnosis of cardiac abnormalities in newborn infants.

OBJECTIVES: To evaluate the effectiveness of critical congenital heart disease (CCHD) screening program for early diagnosis of cardiac anomalies in newborn infants.  Methods: This is a hospital-based prospective cross-sectional study conducted in the Pediatric and Neonatology Department, King Fahad Hospital at  Albaha, Saudi Arabia, between February 2016 and February 2017. Results: We screened 2961 (95.4%) of 3103 patients in a nursery unit; 142 (4.6%) patients were not screened. The test was positive in 114 (3.9%) patients and negative in 2847 (96.1%). There were 94 (3.2%) false positives and 20 (0.7%) true positives. Critical cardiac defects were diagnosed in 7 (0.2%) patients of all screened infants, and severe pulmonary hypertension was diagnosed in 13 (0.4%) patients. True negative results were found in 2841(96%) patients, and no cardiac defect was diagnosed, whereas false negative results were seen in 6 (0.2%) patients diagnosed with ventricular septal defect. The sensitivity was 77%, and the specificity was very high at 97%, with a positive predictive value of 18%, and a negative predictive value of 99.8% (95% confidence interval 13.78-19.18, p=0.0001). Conclusion: Pulse oximetry was found to be easy, safe, sensitive, and highly specific for diagnosis of CCHD.

BMP2 expression in the endocardial lineage is required for AV endocardial cushion maturation and remodeling.

Distal outgrowth, maturation and remodeling of the endocardial cushion mesenchyme in the atrioventricular (AV) canal are the essential morphogenetic events during four-chambered heart formation. Mesenchymalized AV endocardial cushions give rise to the AV valves and the membranous ventricular septum (VS). Failure of these processes results in several human congenital heart defects. Despite this clinical relevance, the mechanisms governing how mesenchymalized AV endocardial cushions mature and remodel into the membranous VS and AV valves have only begun to be elucidated. The role of BMP signaling in the myocardial and secondary heart forming lineage has been well studied; however, little is known about the role of BMP2 expression in the endocardial lineage. To fill this knowledge gap, we generated Bmp2 endocardial lineage-specific conditional knockouts (referred to as Bmp2 cKOEndo) by crossing conditionally-targeted Bmp2flox/flox mice with a Cre-driver line, Nfatc1Cre, wherein Cre-mediated recombination was restricted to the endocardial cells and their mesenchymal progeny. Bmp2 cKOEndo mouse embryos did not exhibit failure or delay in the initial AV endocardial cushion formation at embryonic day (ED) 9.5-11.5; however, significant reductions in AV cushion size were detected in Bmp2 cKOEndo mouse embryos when compared to control embryos at ED13.5 and ED16.5. Moreover, deletion of Bmp2 from the endocardial lineage consistently resulted in membranous ventricular septal defects (VSDs), and mitral valve deficiencies, as evidenced by the absence of stratification of mitral valves at birth. Muscular VSDs were not found in Bmp2 cKOEndo mouse hearts. To understand the underlying morphogenetic mechanisms leading to a decrease in cushion size, cell proliferation and cell death were examined for AV endocardial cushions. Phospho-histone H3 analyses for cell proliferation and TUNEL assays for apoptotic cell death did not reveal significant differences between control and Bmp2 cKOEndo in AV endocardial cushions. However, mRNA expression of the extracellular matrix components, versican, Has2, collagen 9a1, and periostin was significantly reduced in Bmp2 cKOEndo AV cushions. Expression of transcription factors implicated in the cardiac valvulogenesis, Snail2, Twist1 and Sox9, was also significantly reduced in Bmp2 cKOEndo AV cushions. These data provide evidence that BMP2 expression in the endocardial lineage is essential for the distal outgrowth, maturation and remodeling of AV endocardial cushions into the normal membranous VS and the stratified AV valves.