The extent of the raphe in bicuspid aortic valves is associated with aortic regurgitation and aortic root dilatation.

BACKGROUND: The clinical course of bicuspid aortic valves (BAVs) is variable. Data on predictors of aortopathy and valvular dysfunction mainly focus on valve morphology. AIM: To determine whether the presence and extent of the raphe (fusion site of valve leaflets) is associated with the degree of aortopathy and valvular dysfunction in patients with isolated BAV and associated aortic coarctation (CoA). METHODS: Valve morphology and aortic dimensions of 255 BAV patients were evaluated retrospectively by echocardiography. RESULTS: BAVs with a complete raphe had a significantly higher prevalence of valve dysfunction (especially aortic regurgitation) than BAVs with incomplete raphes (82.9 vs. 66.7 %, p = 0.01). Type 1A BAVs (fusion of right and left coronary leaflets) and complete raphe had larger aortic sinus diameters compared with the rest of the population (37.74 vs. 36.01, p = 0.031). Patients with CoA and type 1A BAV had significantly less valve regurgitation (13.6 vs. 55.8 %, p < 0.001) and smaller diameters of the ascending aorta (33.7 vs. 37.8 mm, p < 0.001) and aortic arch (25.8 vs. 30.2 mm, p < 0.001) than patients with isolated BAV. CONCLUSIONS: Type 1A BAV with complete raphe is associated with more aortic regurgitation and root dilatation. The majority of CoA patients have incomplete raphes, associated with smaller aortic root diameters and less valve regurgitation.

Development of major aorto-pulmonary collateral arteries in vegf120/120 isoform mouse embryos with tetralogy of fallot.

The degree of right ventricular outflow tract obstruction, pulmonary stenosis (PS) and the development of major aorto-pulmonary collateral arteries (MAPCAs) in patients with tetralogy of Fallot (TOF) is related to clinical outcome. Vegf120/120 mutant mouse embryos develop TOF with various degrees of PS, comparable to humans. We aimed to study the ontogeny of the development of MAPCAs in this mouse model. The development of the right ventricular outflow tract, pulmonary arteries, and ductus arteriosus (DA) and formation of MAPCAs were studied in both wild type as well as Vegf120/120 mice from embryonic day 10.5 until day 19.5. Of the 49 Vegf120/120 embryos, 35 embryos (71%) had ventral displacement of the outflow tract and a subaortic ventricular septal defect. A time-related development in severity of PS to pulmonary atresia (PA) was observed. From embryonic day 12.5, hypoplasia of the DA was seen in 13 (37%) and absent DA in 12 (37%) of these embryos. The 3 (6%) embryos with PA and absent DA developed MAPCAs, after day 15.5. In all, the MAPCAs arose from both subclavian arteries, running posterior in the thoracic cavity, along the vagal nerve. The MAPCAs connected the pulmonary arteries at the site of the hilus. A time-related development of PS to PA can lead, in combination with absent DA, to the development of MAPCAs later in embryonic life as an alternative route for pulmonary perfusion in this mouse model. This finding contributes to a better understanding of the consecutive morphological changes in the development toward MAPCAs in humans.

Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia.

Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event - leading to the preferential expression of VEGF-A165b over VEGF165a - prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy.

Normal and abnormal development of the aortic wall and valve: correlation with clinical entities.

Dilation of the wall of the thoracic aorta can be found in patients with a tricuspid (TAV) as well as a bicuspid aortic valve (BAV) with and without a syndromic component. BAV is the most common congenital cardiovascular malformation, with a population prevalence of 0.5-2 %. The clinical course is often characterised by aneurysm formation and in some cases dissection. The non-dilated aortic wall is less well differentiated in all BAV as compared with TAV, thereby conferring inherent developmental susceptibility. Furthermore, a turbulent flow, caused by the inappropriate opening of the bicuspid valve, could accelerate the degenerative process in the aortic wall. However, not all patients with bicuspidy develop clinical complications during their life. We postulate that the increased vulnerability for aortic complications in a subset of patients with BAV is caused by a defect in the early development of the aorta and aortic valve. This review discusses histological and molecular genetic aspects of the normal and abnormal development of the aortic wall and semilunar valves. Aortopathy associated with BAV could be the result of a shared developmental defect during embryogenesis.

Differential and linear insertion of atrioventricular valves: a useful tool?

OBJECTIVE: The differential insertion of the atrioventricular valves is the ultrasonographic representation of the more apical attachment of the tricuspid valve to the septum with respect to the mitral valve. A linear insertion is present when both valves form a linear continuum and has been suggested as a marker for atrioventricular septal defects (AVSDs). The objective of this study was to evaluate the anatomical substratum of differential and linear insertions of the atrioventricular valves in normal fetal hearts and fetal hearts with an AVSD. METHODS: The extent and position of the fibrous skeleton and attachment of the atrioventricular valves to the septum were studied in histological sections of 17 normal hearts and four hearts with an AVSD from 10 + 0 weeks' gestation to 3 days postpartum with various immunohistochemical tissue markers. In addition, spatiotemporal image correlation (STIC) volumes of 10 normal hearts and STIC volumes of eight hearts with an AVSD at 13 + 6 to 35 + 5 weeks' gestation were examined. RESULTS: The differential insertion of the atrioventricular valves was visible in normal hearts in the four-chamber plane immediately beneath the aorta, but nearer the diaphragm a linear insertion was found. In hearts with an AVSD, a linear appearance was observed in the four-chamber plane immediately beneath the aorta. Towards the diaphragm, however, first a differential insertion and, more caudally, a linear insertion was found. CONCLUSIONS: Both differential and linear insertions can be found in normal fetal hearts and fetal hearts with AVSD, depending on the plane in which the four-chamber view is visualized. Therefore, measurement of the differential insertion is likely to be useful only in experienced hands.

Expression of Id2 in the second heart field and cardiac defects in Id2 knock-out mice.

The inhibitor of differentiation Id2 is expressed in mesoderm of the second heart field, which contributes myocardial and mesenchymal cells to the primary heart tube. The role of Id2 in cardiac development is insufficiently known. Heart development was studied in sequential developmental stages in Id2 wildtype and knockout mouse embryos. Expression patterns of Id2, MLC-2a, Nkx2.5, HCN4, and WT-1 were analyzed. Id2 is expressed in myocardial progenitor cells at the inflow and outflow tract, in the endocardial and epicardial lineage, and in neural crest cells. Id2 knockout embryos show severe cardiac defects including abnormal orientation of systemic and pulmonary drainage, abnormal myocardialization of systemic and pulmonary veins, hypoplasia of the sinoatrial node, large interatrial communications, ventricular septal defects, double outlet right ventricle, and myocardial hypoplasia. Our results indicate a role for Id2 in the second heart field contribution at both the arterial and the venous poles of the heart.

Functional plasticity of the developing cardiovascular system: examples from different vertebrates.

Technical advances that have made it possible to perform physiological measurements on very small organisms, including those in embryonic and larval stages, have resulted in the formation of the discipline of developmental physiology. The transparency and size of developing organisms in some areas permit insights into physiological processes that cannot be obtained with opaque, adult organisms. On the other hand, it is widely accepted that without eggs, there are no chickens, so physiological adaptations during early life are just as important to species survival as those manifested by adults. Physiological adaptations of early developmental stages, however, are not always the same as patterns known in adults; they often follow their own rules. The adaptability of early developmental stages demonstrates that development is not stereotyped and a phenotype is not just the result of genetic information and the expression of a certain series of genes. Environmental factors influence phenotype production, and this in turn results in flexibility and plasticity in physiological processes. This article comprises exemplary studies presented at the Fourth International Conference in Africa for Comparative Physiology and Biochemistry (Maasai Mara, Kenya, 2008). It includes a brief introduction into technical advances, discusses the developing cardiovascular system of various vertebrates, and demonstrates the flexibility and plasticity of early developmental stages. Fluid forces, oxygen availability, ionic homeostasis, and the chemical environment (including, e.g., hormone concentrations or cholesterol levels) all contribute to the shaping and performance of the cardiovascular system.

Cell tracking using iron oxide fails to distinguish dead from living transplanted cells in the infarcted heart.

Recently, debate has arisen about the usefulness of cell tracking using iron oxide-labeled cells. Two important issues in determining the usefulness of cell tracking with MRI are generally overlooked; first, the effect of graft rejection in immunocompetent models, and second, the necessity for careful histological confirmation of the fate of the labeled cells in the presence of iron oxide. Therefore, both iron oxide-labeled living as well as dead epicardium-derived cells (EPDCs) were investigated in ischemic myocardium of immunodeficient non-obese diabetic (NOD)/acid: non-obese diabetic severe combined immunodeficient (NOD/scid) mice with 9.4T MRI until 6 weeks after surgery, at which time immunohistochemical analysis was performed. In both groups, voids on MRI scans were observed that did not change in number, size, or localization over time. Based on MRI, no distinction could be made between living and dead injected cells. Prussian blue staining confirmed that the hypointense spots on MRI corresponded to iron-loaded cells. However, in the dead-EPDC recipients, all iron-positive cells appeared to be macrophages, while the living-EPDC recipients also contained engrafted iron-loaded EPDCs. Iron labeling is inadequate for determining the fate of transplanted cells in the immunodeficient host, since dead cells produce an MRI signal indistinguishable from incorporated living cells.

Left ventricular function in the post-infarct failing mouse heart by magnetic resonance imaging and conductance catheter: a comparative analysis.

AIM: Murine myocardial infarction (MI) models are increasingly used in heart failure studies. Magnetic resonance imaging (MRI) and pressure-volume loops by conductance catheter (CC) enable physiological phenotyping. We performed a comparative analysis of MRI vs. CC to assess left ventricular (LV) function in the failing mouse heart. METHODS: MI was created by LAD ligation. MRI (day 14) and CC (day 15) were used to determine LV end-diastolic volume (EDV), end-systolic volume (ESV) and ejection fraction (EF). RESULTS: Pooled data yielded moderate-to-strong linear correlations: EDV: R = 0.61; ESV: R = 0.72; EF: R = 0.81. We analysed three groups, no MI (sham, n = 10), small MI (<30% of LV, n = 14) and large MI (>30%, n = 20). Volumes and EF were consistently lower by CC than by MRI, but group differences were evident for both techniques. Receiver-operating characteristic analysis indicated good sensitivity and specificity for both techniques, with superior results for MRI. CONCLUSIONS: CC and MRI are highly valuable for evaluation of LV volume and function. MRI is recommended for longitudinal studies, accurate absolute volumes and anatomical information. Unique features of CC are its online signal with high temporal resolution, and advanced analysis of LV function and energetics.

Development of the cardiac conduction system and the possible relation to predilection sites of arrhythmogenesis.

The cardiac conduction system (CCS) encompasses a complex system responsible for the coordinated contraction of the heart. In the developing heart, as well as in the adult heart, tissues of the (putative) CCS are characterized by different properties than the surrounding working myocardium, which can be observed on a histological level, as well as by the expression patterns of several immunohistological and molecular markers. In recent years, many markers have been studied that have helped to elucidate the processes involved in CCS development. It has become clear that multiple genes, cells and their interactions are involved in this complex process. In this article, an overview of the current knowledge of CCS development is supplied. Furthermore, several controversies regarding conduction system development are discussed, as well as the possible significance of embryologic development of the CCS for the development of arrhythmias later in life.

Preservation of left ventricular function and attenuation of remodeling after transplantation of human epicardium-derived cells into the infarcted mouse heart.

BACKGROUND: Proper development of compact myocardium, coronary vessels, and Purkinje fibers depends on the presence of epicardium-derived cells (EPDCs) in embryonic myocardium. We hypothesized that adult human EPDCs might partly reactivate their embryonic program when transplanted into ischemic myocardium and improve cardiac performance after myocardial infarction. METHODS AND RESULTS: EPDCs were isolated from human adult atrial tissue. Myocardial infarction was created in immunodeficient mice, followed by intramyocardial injection of 4x10(5) enhanced green fluorescent protein-labeled EPDCs (2-week survival, n=22; 6-week survival, n=15) or culture medium (n=24 and n=18, respectively). Left ventricular function was assessed with a 9.4T animal MRI unit. Ejection fraction was similar between groups on day 2 but was significantly higher in the EPDC-injected group at 2 weeks (short term), as well as after long-term survival at 6 weeks. End-systolic and end-diastolic volumes were significantly smaller in the EPDC-injected group than in the medium-injected group at all ages evaluated. At 2 weeks, vascularization was significantly increased in the EPDC-treated group, as was wall thickness, a development that might be explained by augmented DNA-damage repair activity in the infarcted area. Immunohistochemical analysis showed massive engraftment of injected EPDCs at 2 weeks, with expression of alpha-smooth muscle actin, von Willebrand factor, sarcoplasmic reticulum Ca2+-ATPase, and voltage-gated sodium channel (alpha-subunit; SCN5a). EPDCs were negative for cardiomyocyte markers. At 6-weeks survival, wall thickness was still increased, but only a few EPDCs could be detected. CONCLUSIONS: After transplantation into ischemic myocardium, adult human EPDCs preserve cardiac function and attenuate ventricular remodeling. Autologous human EPDCs are promising candidates for clinical application in infarcted hearts.

Epicardium-derived cells in cardiogenesis and cardiac regeneration.

During cardiogenesis, the epicardium grows from the proepicardial organ to form the outermost layer of the early heart. Part of the epicardium undergoes epithelial-mesenchymal transformation, and migrates into the myocardium. These epicardium-derived cells differentiate into interstitial fibroblasts, coronary smooth muscle cells, and perivascular fibroblasts. Moreover, epicardium-derived cells are important regulators of formation of the compact myocardium, the coronary vasculature, and the Purkinje fiber network, thus being essential for proper cardiac development. The fibrous structures of the heart such as the fibrous heart skeleton and the semilunar and atrioventricular valves also depend on a contribution of these cells during development. We hypothesise that the essential properties of epicardium-derived cells can be recapitulated in adult diseased myocardium. These cells can therefore be considered as a novel source of adult stem cells useful in clinical cardiac regeneration therapy.

Maternal hyperhomocysteinaemia is a risk factor for congenital heart disease.

OBJECTIVE: To investigate the inter-relation between mother and infant homocysteine, folate and vitamin B12 status and the risk of a child with congenital heart disease (CHD). DESIGN: Case-control study. SETTING: Erasmus MC, University Medical Centre, Rotterdam, the Netherlands. POPULATION: Participants were 149 case-mothers and their children with CHD (n = 151) and 183 control-mothers with their children (n = 175). METHODS: Approximately 17 months after the index-pregnancy maternal fasting, children's random venous blood samples were drawn to measure plasma total homocysteine, serum and red blood cell (RBC) folate, and serum vitamin B12 concentrations. Data were compared between cases and controls using the Mann-Whitney U test. The biochemical parameters were dichotomised according to the cutoff value of the 10th percentile of vitamin concentrations and the 90th percentile of homocysteine concentrations based on control data. Risk estimates for the association between CHD and the biochemical parameters were estimated in a logistic regression model. MAIN OUTCOME MEASURES: Medians (minimum-maximum) and odds ratios (OR) (95% confidence intervals [CI]). RESULTS: The OR (95% CI) of having a child with CHD was 2.9 (1.4-6.0) for maternal hyperhomocysteinaemia (>14.3 micromol/l). This finding is substantiated by a significant concentration-dependent risk (Ptrend = 0.004). Hyperhomocysteinaemic case-mothers showed significantly lower serum folate and vitamin B12 concentrations than normohomocysteinaemic case-mothers. Serum and RBC folate concentrations were significantly higher in case-children than that in control-children. CONCLUSIONS: Maternal hyperhomocysteinaemia is associated with an increased risk of CHD, partially due to low folate and vitamin B12 status. The folate status of children warrants further investigation.

Morphogenetic differences of secundum atrial septal defects.

It is generally considered that the development of secundum atrial septal defect (ASDII) or oval fossa defect is the result of excessive resorption of the embryological atrial septum primum, but this does not seem to explain all defects. We investigated 58 postmortem hearts with an ASDII and 22 normal hearts from patients ranging in age from 1 day to 49 years. The different structures of the oval fossa were examined. In 86% of the specimens, the defects were the result of a malformation of the valvula foraminis ovalis or embryological atrial septum primum, and in 14% an absent superior limbus (septum secundum) was the cause of the interatrial communication. The "septum primum" ASDs were divided into four subgroups based on the degree of deficiency of the septum primum and position of the ostium secundum within the septum primum. We conclude that the morphogenesis of ASDII is variable and both septum primum and septum secundum defects occur, which may be relevant in view of genetic studies that may lead to further differentiation of patients with and without genetically determined ASDIIs.

Potential involvement of vascular endothelial growth factor in pathophysiology of Turner syndrome.

Vascular endothelial growth factor (VEGF) is a specific growth factor for endothelium but plays also a role in the signaling involved in embryonic endocardial-to-mesenchymal transformation of the endocardial cushions. Furthermore, VEGF is the major vascular permeability factor in both fetal and postnatal life. Overexpression of VEGF during fetal life is associated with fetal hydrops and abnormal endocardial cushion development and therefore with congenital heart defects. Cases of prenatal cervical hygroma like in Turner syndrome show both hydrops and cardiac defects. We hypothesize that excess VEGF formed in the wall of the distended jugular sacs (cervical hygroma's) results in other abnormal features characteristic for Turner syndrome such as short stature and gonadal dysgenesis. This implicates that if excess VEGF could be limited prenatally, the phenotypical expression of Turner syndrome can possibly be reduced.

Development of the right ventricular inflow tract and moderator band: a possible morphological and functional explanation for Mahaim tachycardia.

Atriofascicular accessory bundles with AV-node like conduction properties can sustain atrioventricular (AV) re-entrant tachycardia (Mahaim tachycardia). During early embryogenesis, the AV canal is situated above the primitive left ventricle (LV), and a right AV connection has not been achieved yet. We studied the formation of the right ventricular (RV) inflow tract in relation to the developing cardiac conduction system and hypothesized a morphological explanation for functional atriofascicular bypass tracts. Analysis of lacZ-expression during sequential stages of cardiogenesis was performed in CCS-lacZ transgenic mice (E9.5 to 15.5). Embryos were stained for beta-galactosidase activity and the myocardial marker HHF35. At early stages CCS-lacZ expression was observed in a ring surrounding the AV canal, which connected at the inner curvature to the primary fold. The first sign of formation of the (CCS-lacZ negative) RV inlet component was a groove in the CCS-lacZ positive tissue of the primary fold. Outgrowth of the RV inlet tract resulted in division of the primary fold in a septal part, the trabecula septomarginalis and a lateral part, the moderator band, which extended laterally up to the right AV ring. Electrophysiological measurements in embryonic hearts (E15.5) in which the right atrium (RA) and RV were isolated from the left atrium (LA) and LV supported the functionality of this AV-connection via the moderator band, by demonstrating sequential atrial and ventricular activation in both RA/RV and LA/LV preparations. In conclusion, our observations may provide a possible morphological and functional explanation for atriofascicular accessory pathways via the moderator band, underlying Mahaim tachycardia.