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In fetal aortic stenosis (AS), it remains challenging to predict left ventricular development over the course of pregnancy. Myocardial organization, differentiation and fibrosis could be potential biomarkers relevant for biventricular outcome. We present four cases of fetal AS with varying degrees of severity and associate myocardial deformation on fetal ultrasound with postmortem histopathological characteristics. During routine fetal echocardiography, speckle tracking recordings of the cardiac four-chamber view were performed to assess myocardial strain as parameter for myocardial deformation. After pregnancy termination, postmortem cardiac specimens were examined using immunohistochemical labeling (IHC) of key markers for myocardial organization, differentiation and fibrosis and compared to normal fetal hearts. Two cases with critical AS presented extremely decreased left ventricular (LV) strain on fetal ultrasound. IHC showed overt endocardial fibro-elastosis, which correlated with pathological fibrosis patterns in the myocardium and extremely disturbed cardiomyocyte organization. The LV in severe AS showed mildly reduced myocardial strain and less severe disorganization of the cardiomyocytes. In conclusion, the degree of reduction in myocardial deformation corresponded with high extent to the amount of pathological fibrosis patterns and cardiomyocyte disorganization. Myocardial deformation on fetal ultrasound seems to hold promise as a potential biomarker for left ventricular structural damage in AS.

Background. MRI-detected inflammation around the metacarpophalangeal (MCP-)joints is prevalent in RA and poses a markedly increased risk of RA-development when present in arthralgia patients. Such inflammation is called ‘peritendinitis’, since anatomy literature reports no presence of a tenosynovial sheath at these tendons. However, the presence or absence of a synovial sheath at these extensor tendons has never been studied. Methods. A macroscopy and microscopy study of extensor-tendons at the MCP-joints of two embalmed human hands was performed. Routine histology was performed with Haematoxylin-Eosin staining. Results. We found evidence for the presence of synovial lining around extensor-tendons at MCP-joints. A delimited space surrounding the extensor digitorum tendon was observed, which was lined with an epithelium representing fibroblast-like synoviocytes. Conclusion. Contrast-enhancement around extensor tendons at MCP joints observed on MRI in RA represents tenosynovitis and thus inflammation of synovial tissue. Further studies with larger numbers of specimen are warranted to study anatomic variants. In addition, the molecular composition of the tenosynovium remains to be characterized.

Introduction Past research has demonstrated that the urethral tonus is mainly under sympathetic control. Since 5 years, a beta 3-adrenoceptor (ADRB3) agonist is available in the treatment of overactive bladder syndrome. The presence of ADRB3 within the human urethra has not been demonstrated to date. Presence of ADRB3 in the urethra could influence urethral tonus. The aim of this study is to investigate the presence of ADRB3 in the human female urethra. Material and Methods We performed anatomical studies in five female specimens. Three specimens were obtained from the body donation program, two from female patients with muscle-invasive bladder cancer, where radical resection of bladder and urethra was performed. The urethra up till the bladder neck was separated from the rest of the bladder and freshly obtained for this study. For demonstrating ADRB3 expression, we used rabbit polyclonal anti-human ADRB3 LS-A4198. Results Expression of ADBR3 was demonstrated in the epithelial layer of all urethral parts, except at the level of the meatus. The level of ADRB3 expression was highest in the mid urethra. There was no direct contact between ADRB3 and nerve tissue. ADRB3 expression was also demonstrated in the stratified muscle layer at the level of the external urethral sphincter. Conclusions This is the first study to demonstrate the expression of ADRB3 in the human female urethra. There is an absence of a direct connection between ADRB3 and nerve tissue.

Background/Introduction Bicuspid aortic valve (BAV) is associated with ascending aorta aneurysms and dissections. Presently, genetic factors and pathological flow patterns are considered responsible for aneurysm formation in BAV. Despite, indication for preventive surgery is presently only defined by vessel diameter which is poor marker for dissection, as it does not take other processes responsible for the vulnerability of the aorta into account. Purpose Inflammation is not considered a player in BAV aortopathy. We introduce a quantitative immunohistochemistry (IHC) approach to sensitively look at the potential role of both the innate and adaptive immune system in BAV aortopathy. Methods Dilated (n=8), non-dilated (n=14) and dissected (n=4) BAV ascending aortas were collected during surgery or from post-mortem donors. Median time from symptoms to surgery for dissections was just over 4 hours. Tissue was stained with a novel 8-colour IHC technique allowing for simultaneous visualization of 6 markers per slide, completed with DAPI nuclear counter-stain and elastin fiber autofluorescence. One panel focused on the adaptive immune system (identifying B cells and classic dendritic cells type 2 (cDC2s) and phenotyping T cells), and the other on the innate immune system (assessing macrophage polarization and neutrophil extravasation). All cells were identified and comprehensively phenotyped using automated quantitative analysis. Results Aneurysm formation was associated with an organized and consistent increase of lymphocytes in the adventitia. B cell follicles and helper T cell expansion were identified, suggestive of a targeted adaptive immune response (Fig. 1a). Only dissected aortas showed a statistically significant increase of helper T (p=0.3) and cDC2s (p=0.3) in the media, when compared to non-dilated and dilated samples (Fig. 1b). The short time between dissection symptoms and surgery suggests these cells were present before the dissection occurred. Furthermore, aneurysms and dissections are associated with a shift in macrophage phenotype to the more aggressive M1-like subset. In summary, we found that a progression of aggressive immune cells in the adventitia and media was correlated to a progression in disease state; from normal to dilated to dissected. Conclusions Aorta dilatation in patients with BAV is associated with an expansion of B and helper T cells in the adventitial compartment without changes in the media. This result might indicate an antigen-driven adaptive immune response. Only dissections show an increase in helper T cells and cDC2s in the media, together with polarization of macrophages to a more M1-like phenotype. We hypothesize that antigen-specific helper T cells expand in the adventitia, migrate to the media, and then potentiate macrophages which can eventually lead to tissue degeneration. These associations could shine light on the final step in the deterioration of the aorta towards a dissection. Figure 1. Microscopy results Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): TTW-NWO open technology grant (STW-14716), ERC-2014-StG-336454-CoNQUeST

Background In a normal neonatal heart the ductus arteriosus forms the direct continuity of the pulmonary trunk to the descending aorta being the main channel for the prenatal blood flow. The pulmonary arteries originate separately from the pulmonary trunk. In congenital heart malformations with pulmonary stenosis to atresia there is often an abnormal lateral DA to left pulmonary artery connection combined with a localised narrowing (pulmonary ductal coarctation / PDC) or even interruption. We investigated embryonic remodelling and pathogenesis of this area Material and methods Normal development was studied in WntCre reporter mice (E10.0-12.5) for neural crest cells and Nkx2.5 immunostaining for second heart field cells. Data were compared to stage matched human embryos including histopathology of neonatal specimen, and a VEGF120/120 mutant mouse strain developing pulmonary atresia. Results Normal mouse and human embryos showed that, in the early embryo, the mid-pharyngeal endothelial plexus, partly covered by second heart field, connected side-ways to the 6 th pharyngeal arch artery (PAA). The ventral segment of this PAA effectively formed the proximal pulmonary artery. The origin of the cells encasing the endothelial tubes differed significantly. The dorsal 6 th PAA segment (future DA on the left side) was solely surrounded by neural crest cells. The ventral segment had a dual outer lining with neural crest cells laterally and second heart field cells medially, while the distal pulmonary artery was covered by neither of these cells. The asymmetric contribution of second heart field to the future pulmonary trunk on the left side of the aortic sac (so-called pulmonary push) was evident. During remodelling the ventral segment of the 6 th PAA became incorporated into the pulmonary trunk leading to a separate connection of the left and right pulmonary arteries to the latter. The VEGF120/120 embryos showed a severely stunted pulmonary push and later on a variety of vascular anomalies in the DA to pulmonary artery connections. Conclusion Side-way connection of the DA to the left pulmonary artery is a congenital anomaly. The primary problem is a stunted development of the second heart field-derived pulmonary push leading to pulmonary stenosis/atresia and a subsequent lack of proper incorporation of the ventral segment of the 6 th PAA into the aortic sac. Clinically, the aberrant smooth muscle tissue of the DA should be addressed to prohibit development of severe PDC or even interruption of the left pulmonary artery.

Patients with a congenital bicuspid aortic valve (BAV), a valve with two instead of three aortic leaflets, have an increased risk of developing thoracic aneurysms and aortic dissection. The mechanisms underlying BAV-associated aortopathy are poorly understood. This study examined BAV-associated aortopathy in Nos3−/− mice, a model with congenital BAV formation. A combination of histological examination and in-vivo ultrasound imaging was used to investigate aortic dilation and dissections in Nos3−/− mice. Moreover, cell lineage analysis and single cell RNA sequencing were used to observe the molecular anomalies within vascular smooth muscle cells (VSMCs) of Nos3−/− mice. Spontaneous aortic dissections was found in ascending aortas located at the sinotubular junction in ∼13% of Nos3−/− mice. Moreover, Nos3−/−mice were prone to develop aortic dilations in the mid- and distal-ascending aorta during early adulthood. Lower volumes of elastic fibres were found within vessel walls of the ascending aortas of Nos3−/−mice as well as incomplete coverage of the aortic inner media by neural crest (NCC)-derived VSMCs. VSMCs of Nos3−/− showed downregulation of 15 genes of which 7 were associated with aortic aneurysms and dissections in the human population. Elastin mRNA was most markedly downregulated, followed by Fibulin-5 expression, both primary components of elastic fibres. This study demonstrates that disrupted endothelial mediated NO signalling in mice causes next to a congenital BAV also aortic dilation and dissection as a consequence of inhibited elastic fibre formation in VSMCs within the ascending aorta of Nos3−/−mice.

Background: Patients with a bicuspid aortic valve (BAV) have an increased risk for aortic dilation and dissection. In this study, we provide a histological stratification of the developing aorta in the tricuspid aortic valve (TAV) and the BAV populations as a reference for future studies on aortopathy and related syndromes. Methods: Non-dilated TAV and BAV ascending aortic wall samples were collected, including 60 TAV (embryonic&ndash, 70 years) and 32 BAV specimens (fetal&ndash, 72 years, categorized in eight age groups. Results: In TAV, intimal development starts in the neonatal phase. After birth, the thickness of the medial layer increases significantly by increase of elastic lamellae up to and including the &ldquo, young child&rdquo, phase stabilizing afterwards. The BAV shows already prenatal intimal thickening becoming significantly thinner after birth subsequently stabilizing. In BAV, increase in elastic lamellae is seen between the young child and the adolescent phases, stabilizing afterwards. Conclusions: Vascular development in TAV is described in three phases: maturation, stabilization, and degeneration. For BAV, the development can be described in two phases: maturation (already prenatally) and degeneration. After birth, the development of the aorta is characterized by degeneration, leading to weakening of the ascending aortic wall and increasing the risk of aortopathy.

Aims Electroanatomical voltage mapping (EAVM) is an important diagnostic tool for fibrosis identification and risk stratification in non-ischaemic cardiomyopathy (NICM); currently, distinct cut-offs are applied. We aimed to evaluate the performance of EAVM to detect fibrosis by integration with whole heart histology and to identify the fibrosis pattern in NICM patients with ventricular tachycardias (VTs). Methods and results Eight patients with NICM and VT underwent EAVM prior to death or heart transplantation. EAVM data was projected onto slices of the entire heart. Pattern, architecture, and amount of fibrosis were assessed in transmural biopsies corresponding to EAVM sites. Fibrosis pattern in NICM biopsies (n = 507) was highly variable and not limited to mid-wall/sub-epicardium. Fibrosis architecture was rarely compact, but typically patchy and/or diffuse. In NICM, biopsies without abnormal fibrosis unipolar voltage (UV) and bipolar voltage (BV) showed a linear association with wall thickness (WT). The amount of viable myocardium showed a linear association with both UV and BV. Accordingly, any cut-off to delineate fibrosis performed poorly. An equation was generated calculating the amount of fibrosis at any location, given WT and UV or BV. Conclusion Considering the linear relationships between WT, amount of fibrosis and both UV and BV, the search for any distinct voltage cut-off to identify fibrosis in NICM is futile. The amount of fibrosis can be calculated, if WT and voltages are known. Fibrosis pattern and architecture are different from ischaemic cardiomyopathy and findings on ischaemic substrates may not be applicable to NICM.

Pulmonary atresia with intact ventricular septum is a severe cardiac malformation that is ductus dependent and needs immediate postnatal care. The phenotype is characterized by three levels of pathology that are considered important for diagnosis and the choice of cardiac surgery. The pathology concerns an hypoplasia and hypertrophy of the right ventricle in which there is a variation in the involvement of the inlet, trabecular and outflow part of the right ventricle. There is also a variable degree of tricuspid valve pathology including hypoplasia and stenosis. The pulmonary valve is atretic which may have been preceded, in the fetal stage, by pulmonary stenosis. This latter phenomenon may be triggered by the severe coronary anomalies that are found in about 30% of the patients. This coronary anomaly is characterized by ventriculo-coronary arterial communications which is accompanied by sometimes severe main coronary artery pathology including interruptions of the main branches. In the latter situation the coronary myocardial perfusion becomes right ventricular dependent. Current cardiac developmental data show that the right ventricular myocardium is derived from the second heart field. Its anterior part contributes this myocardium to the outflow tract and is also employed by the neural crest cells to enter the heart. The posterior second heart field provides the epicardium derived cells (EPDC) that are important for myocardial wall compaction and formation of the coronary vasculature. We hypothesize that pulmonary atresia with intact ventricular septum without ventriculo-coronary arterial communications is primarily based on anterior SHF directed outflow tract septation anomalies, while pulmonary atresia with intact ventricular septum with ventriculo-coronary arterial communications has an additional major problem related to epicardium derived contribution. Developmentally they can therefore be considered as two different diseases that might need separate treatment protocols.

Objective: The hypothesis that severe fetal hydrops is caused by an excess of vascular endothelial growth factor (VEGF), mainly produced in the fetal heart, is tested. Methods: Immunohistochemical VEGF-stained postmortem biopsies from the right ventricle and right atrium of 8 hydropic fetuses were compared to those of 8 nonhydropic fetuses. The endocardium, myocardium, epicardium, endothelium, and vascular smooth muscle cells were scored on intensity of VEGF-staining. The Mann-Witney test was used to test for significancy (p < 0.05) of the differences in staining. Increased vascularization as a result of VEGF was measured in both groups by standard randomization count. Results: The endocardium, epicardium and endothelium of the coronary vessels showed significantly (p < 0.05) more intense VEGF-staining in the hydrops group than in the control group. The atria showed more intense staining than the ventricles in both groups. The hydropic fetuses showed a significantly increased number of coronary vessels in the myocardium. These vessels contained more blood cells than the coronary vessels in nonhydropic fetuses. Conclusion: The fetal heart appears to be a major source of excess VEGF in fetal hydrops.

BACKGROUND: Maternal diabetes (MD) is a risk factor for offspring to develop cardiovascular anomalies; this is of growing clinical concern since the number of women in childbearing age with compromised glucose homeostasis is increasing. Hyperglycemia abrogates cardiovascular development in vitro; however, a link to cardiovascular defects in diabetic offspring remains to be investigated. METHODS: We have studied cardiovascular development in offspring of MD rats by examining serial histological sections of GD 12.0-18.0 offspring. Development of pharyngeal arch artery malformations was analyzed and related to intracardiac anomalies. RESULTS: Pharyngeal arch artery and intracardiac defects were present in 27 of 37 MD GD 13.0-18.0 offspring. Early sixth arch arteries showed abrogated arteriogenesis, whereas fourth arch artery defects developed as a result of abnormal remodeling. Morphometrical analysis showed increased apoptosis in regressing artery segments and reduced apoptosis in persisting artery segments. Double outlet right ventricle with infundibular stenosis (tetralogy of Fallot) was predominantly found in combination with sixth artery defects and pulmonary atresia. As confirmed by morphometric analysis and three-dimensional (3D)-reconstructions, outflow tract defects coincided with endocardial cushion hypoplasia. Cases with teratology of Fallot additionally showed a shorter outflow tract. No relation with apoptosis or disturbed neural crest cell migration was found. CONCLUSIONS: Our data uniquely demonstrate mechanistic differences involved in the development of sixth and fourth artery anomalies. Whereas increased apoptosis induces fourth artery anomalies, pulmonary outflow obstruction abrogates sixth artery differentiation independent of apoptosis. The model presented allows analysis of diabetic conditions on cardiovascular development in vivo, essential for elucidating this teratology.

Background: Pregestational diabetes is a major risk factor of congenital heart defects (CHDs). Glutathione is depleted and reactive oxygen species (ROS) production is elevated in diabetes. In the present study, we aimed to examine whether treatment with N-acetylcysteine (NAC), which increases glutathione synthesis and inhibits ROS production, prevents CHDs induced by pregestational diabetes.Methods: Female mice were treated with streptozotocin (STZ) to induce pregestational diabetes prior to breeding with normal males to produce offspring. Some diabetic mice were treated with N-acetylcysteine (NAC) in drinking water from E0.5 to the end of gestation or harvesting of the embryos. CHDs were identified by histology. ROS levels, cell proliferation and gene expression in the fetal heart were analyzed.Results: Our data show that pregestational diabetes resulted in CHDs in 58% of the offspring, including ventricular septal defect (VSD), atrial septal defect (ASD), atrioventricular septal defects (AVSD), transposition of great arteries (TGA), double outlet right ventricle (DORV) and tetralogy of Fallot (TOF). Treatment with NAC in drinking water in pregestational diabetic mice completely eliminated the incidence of AVSD, TGA, TOF and significantly diminished the incidence of ASD and VSD. Furthermore, pregestational diabetes increased ROS, impaired cell proliferation, and altered Gata4, Gata5 and Vegf-a expression in the fetal heart of diabetic offspring, which were all prevented by NAC treatment.Conclusions: Treatment with NAC increases GSH levels, decreases ROS levels in the fetal heart and prevents the development of CHDs in the offspring of pregestational diabetes. Our study suggests that NAC may have therapeutic potential in the prevention of CHDs induced by pregestational diabetes. © 2014 Moazzen et al.; licensee BioMed Central Ltd.

Background —Transforming growth factor-β 2 (TGF-β 2 ) is a member of a family of growth factors with the potential to modify multiple processes. Mice deficient in the TGF-β 2 gene die around birth and show a variety of defects of different organs, including the heart. Methods and Results —We studied the hearts of TGF-β 2 –null mouse embryos from 11.5 to 18.5 days of gestation to analyze the types of defects and determine which processes of cardiac morphogenesis are affected by the absence of TGF-β 2 . Analysis of serial sections revealed malformations of the outflow tract (typically a double-outlet right ventricle) in 87.5%. There was 1 case of common arterial trunk. Abnormal thickening of the semilunar valves was seen in 4.2%. Associated malformations of the atrioventricular (AV) canal were found in 62.5% and were composed of perimembranous inlet ventricular septal defects (37.5%), AV valve thickening (33.3%), overriding tricuspid valve (25.0%), and complete AV septal defects (4.2%). Anomalies of the aorta and its branches were seen in 33.3%. Immunohistochemical staining showed failure of myocardialization of the mesenchyme of the atrial septum and the ventricular outflow tract as well as deficient valve differentiation. Morphometry documented this to be associated with absence of the normal decrease of total endocardial cushion volume in the older stages. Apoptosis in TGF-β 2 –knockout mice was increased, although regional distribution was normal. Conclusions —TGF-β 2 –knockout mice exhibited characteristic cardiovascular anomalies comparable to malformations seen in the human population.

Using various microscopical techniques we studied the development of the atrioventricular valves in human hearts between 5 and 19 weeks of development. Within the atrioventricular cushions two different layers could be recognized that remained present in all ages studied. The atrial layer, being present at the side of the atrioventricular orifice, was positive for laminin while the ventricular layer, that was connected to the myocardium, was positive for fibronectin and collagen III. Fate-mapping of these two layers, morphometrics, and scanning electron microscopy, supplemented with in vivo labeling of cushion tissue in chicken hearts have lead to new insights in the process of valve development. The cushions became freely movable prevalvular leaflets by delamination of ventricular myocardium underneath the cushion tissue. This myocardium gradually retracted towards annulus and papillary muscles and finally disappeared, resulting in fibrous, non-myocardial valves. The atrial layer of the cushions remained present as a jelly-like surface on the valve leaflets while the ventricular layer of the cushions became the compact fibrous tissue of the leaflets and the chords. Chordal development was first visible at 10 weeks of development when gaps were formed in the ventricular layer of the cushions on top of the papillary muscles. These gaps enlarged into the interchordal spaces while the cushion tissue in between the gaps lengthened to form the chords. We conclude that the leaflets as well as the chords of the atrioventricular valves are derived from atrioventricular cushion tissue. Myocardium is only important for loosening of the leaflets while keeping connection with the developing papillary muscles. Errors in delamination or retraction of myocardium or remodeling of cushion tissue into chords form the basis for various congenital valve anomalies.

Background Neoaortic root dilatation is observed after the arterial switch operation for transposition of the great arteries. Although structural differences in the vessel wall of these patients may be of influence, we hypothesize that a histomorphologic difference in composition and embedding of the fibrous annulus in transposition of the great arteries may play a role in neoaortic root dilatation. Methods Two normal human hearts and two unoperated human hearts with transposition of the great arteries, 1 day postnatal, were studied. Histologic sections stained for collagen, myocardium, and elastin were prepared, and three-dimensional reconstructions of the outflow tracts were made to enable comparison of the morphologic structures between the normal hearts and those with transposition of the great arteries. Results The amount of collagen in the arterial roots was diminished in hearts with transposition of the great arteries compared with the normal hearts. In addition, the anchorage and embedding of both arterial roots in the myocardium was less extensive in transposition of the great arteries. The changed position of the arteries in the malformed hearts results in less support for the roots from the surrounding atrioventricular myocardium. Conclusions The combination of the observed histomorphologic differences in amount of collagen and myocardial support may be an explanation for the neoaortic root dilatation observed after the arterial switch operation. The developmental background of the observed deficient fibrous annulus formation may originate from an epicardial problem.

Objectives: To understand papillary muscle malformations, such as in parachute mitral valves or parachute-like asymmetric mitral valves, we studied the development of papillary muscles. Methods: Normal human hearts at between 5 and 19 weeks of development were studied with immunohistochemistry, three-dimensional reconstructions, and gross inspection. Scanning electron microscopy was used to study human and rat hearts. Results: In embryonic hearts a prominent horseshoe-shaped myocardial ridge runs from the anterior wall through the apex to the posterior wall of the left ventricle. In the atrioventricular region this ridge is continuous with atrial myocardium and covered with cushion tissue. The anterior and posterior parts of the trabecular ridge enlarge and loosen their connections with the atrial myocardium. Their lateral sides gradually delaminate from the left ventricular wall, and the continuity between the two parts is incorporated in the apical trabecular network. In this way the anterior and posterior parts of the ridge transform into the anterolateral and the posteromedial papillary muscles, respectively. Simultaneously, the cushions remodel into valve leaflets and chordae. Only the chordal part of the cushions remains attached to the developing papillary muscles. Conclusions: Disturbed delamination of the anterior or posterior part of the trabecular ridge from the ventricular wall, combined with underdevelopment of chordae, seems to be the cause of asymmetric mitral valves. Parachute valves, however, develop when the connection between the posterior and anterior part of the ridge condenses to form one single papillary muscle. Thus parachute valves and parachute-like asymmetric mitral valves originate in different ways. (J Thorac Cardiovasc Surg 1998; 116:36-46)