Nynke M.S. van den Akker

Cardiac malformations and myocardial abnormalities in podoplanin knockout mouse embryos: Correlation with abnormal epicardial development.

Epicardium and epicardium‐derived cells have been shown to be necessary for myocardial differentiation. To elucidate the function of podoplanin in epicardial development and myocardial differentiation, we analyzed podoplanin knockout mouse embryos between embryonic day (E) 9.5 and E15.5 using immunohistochemical differentiation markers, morphometry, and three‐dimensional reconstructions. Podoplanin null mice have an increased embryonic lethality, possibly of cardiac origin. Our study reveals impairment in the development of the proepicardial organ, epicardial adhesion, and spreading and migration of the epicardium‐derived cells. Mutant embryos show a hypoplastic and perforated compact and septal myocardium, hypoplastic atrioventricular cushions resulting in atrioventricular valve abnormalities, as well as coronary artery abnormalities. The epicardial pathology is correlated with reduced epithelial–mesenchymal transformation caused by up‐regulation of E‐cadherin, normally down‐regulated by podoplanin. Our results demonstrate a role for podoplanin in normal cardiac development based on epicardial–myocardial interaction. Abnormal epicardial differentiation and reduced epithelial–mesenchymal transformation result in deficient epicardium‐derived cells leading to myocardial pathology and cardiac anomalies. Developmental Dynamics 237:847–857, 2008.

The Alternatively Spliced Anti-Angiogenic Family of VEGF Isoforms VEGFxxxb in Human Kidney Development

Background/Aim: Vascular endothelial growth factor (VEGF), required for renal development, is generated by alternative splicing of 8 exons to produce two families, pro-angiogenic VEGFxxx, formed by proximal splicing in exon 8 (exon 8a), and anti-angiogenic VEGFxxxb, generated by distal splicing in exon 8 (exon 8b). VEGF165b, the first described exon 8b-containing isoform, antagonises VEGF165 and is anti-angiogenic in vivo. Methods: Using VEGFxxxb-specific antibodies, we investigated its expression quantitatively and qualitatively in developing kidney, and measured the effect of VEGF165b on renal endothelial and epithelial cells. Results: VEGFxxxb formed 45% of total VEGF protein in adult renal cortex, and VEGF165b does not increase glomerular endothelial cell permeability, it inhibits migration, and is cytoprotective for podocytes. During renal development, VEGFxxxb was expressed in the condensed vesicles of the metanephros, epithelial cells of the comma-shaped bodies, invading endothelial cells and epithelial cells of the S-shaped body, and in the immature podocytes. Expression reduced as the glomerulus matured. Conclusion: These results show that the anti-angiogenic VEGFxxxb isoforms are highly expressed in adult and developing renal cortex, and suggest that the VEGFxxxb family plays a role in glomerular maturation and podocyte protection by regulating the pro-angiogenic pro-permeability properties of VEGFxxx isoforms.

PDGF-B signaling is important for murine cardiac development: Its role in developing atrioventricular valves, coronaries, and cardiac innervation

We hypothesized that PDGF‐B/PDGFR‐β‐signaling is important in the cardiac contribution of epicardium‐derived cells and cardiac neural crest, cell lineages crucial for heart development. We analyzed hearts of different embryonic stages of both Pdgf‐b−/− and Pdgfr‐β−/− mouse embryos for structural aberrations with an established causal relation to defective contribution of these cell lineages. Immunohistochemical staining for αSMA, periostin, ephrinB2, EphB4, VEGFR‐2, Dll1, and NCAM was performed on wild‐type and knockout embryos. We observed that knockout embryos showed perimembranous and muscular ventricular septal defects, maldevelopment of the atrioventricular cushions and valves, impaired coronary arteriogenesis, and hypoplasia of the myocardium and cardiac nerves. The abnormalities correspond with models in which epicardial development is impaired and with neuronal neural crest–related innervation deficits. This implies a role for PDGF‐B/PDGFR‐β‐signaling specifically in the contribution of these cell lineages to cardiac development. Developmental Dynamics 237:494–503, 2008.

Platelet-derived growth factors in the developing avian heart and maturating coronary vasculature.

Platelet‐derived growth factors (PDGFs) are important in embryonic development. To elucidate their role in avian heart and coronary development, we investigated protein expression patterns of PDGF‐A, PDGF‐B, and the receptors PDGFR‐α and PDGFR‐β using immunohistochemistry on sections of pro‐epicardial quail–chicken chimeras of Hamburger and Hamilton (HH) 28–HH35. PDGF‐A and PDGFR‐α were expressed in the atrial septum, sinus venosus, and throughout the myocardium, with PDGFR‐α retreating to the trabeculae at later stages. Additionally, PDGF‐A and PDGFR‐α were present in outflow tract cushion mesenchyme and myocardium, respectively. Small cardiac nerves and (sub)epicardial cells expressed PDGF‐B and PDGFR‐β. Furthermore, endothelial cells expressed PDGF‐B, while vascular smooth muscle cells and interstitial epicardium‐derived cells expressed PDGFR‐β, indicating a role in coronary maturation. PDGF‐B is also present in ventricular septal development, in the absence of any PDGFR. Epicardium‐derived cells in the atrioventricular cushions expressed PDGFR‐β. We conclude that all four proteins are involved in myocardial development, whereas PDGF‐B and PDGFR‐β are specifically important in coronary maturation. Developmental Dynamics 233:1579–1588, 2005.

Periostin expression by epicardium-derived cells is involved in the development of the atrioventricular valves and fibrous heart skeleton

The epicardium is embryologically formed by outgrowth of proepicardial cells over the naked heart tube. Epicardium-derived cells (EPDCs) migrate into the myocardium, contributing to myocardial architecture, valve development, and the coronary vasculature. Defective EPDC formation causes valve malformations, myocardial thinning, and coronary defects. In the atrioventricular (AV) valves and the fibrous heart skeleton isolating atrial from ventricular myocardium, EPDCs colocalize with periostin, a matrix molecule involved in remodeling. We investigated whether proepicardial outgrowth inhibition affected periostin expression and how this related to development of the AV valves and fibrous heart skeleton. Periostin expression by epicardium and EPDCs was confirmed in vitro in primary cultures of human and quail EPDCs. Disturbing EPDC formation in quail embryos reduced periostin expression in the endocardial cushions and AV junction. Disturbed fibrous tissue development resulted in AV myocardial connections reflected by preexcitation electrocardiographic patterns. We conclude that EPDCs are local producers of periostin. Disturbance of EPDC formation results in decreased cardiac periostin levels and hampers the development of fibrous tissue in AV junction and the developing AV valves. The resulting cardiac anomalies might link to Wolff-Parkinson White syndrome with persistent AV myocardial connections.

Abnormal lymphatic development in trisomy 16 mouse embryos precedes nuchal edema

Ultrasound measurement of increased nuchal translucency is a method of risk assessment for heart malformations and trisomy 21 in human pregnancy. The developmental background of this nuchal edema is still not sufficiently understood. We have studied the process in trisomy 16 mice that show nuchal edema and heart malformations. We used trisomy 16 and wild‐type (WT) embryos from embryonic day (E) 12.5 to E18.5. In WT embryos at E13, bilateral jugular lymphatic sacs are visible that share a lymphatic–venous membrane with the jugular vein. We could not in any case discern a valve between these vessels. At E14 in the TS16 embryos, the lymphatic sacs become enlarged showing abnormally thickened endothelium, specifically at the site of the membrane. In these embryos, severe edema develops in the nuchal region. There is a very close colocalisation of the nerves with the vascular structures. The start of reorganization of the jugular lymphatic sac to a lymph node is observed in both wild‐type and TS16 but is diminished in the latter. In conclusion, abnormal size and structure of the jugular lymphatic sacs coincides with the development of nuchal edema. A disturbance of lymphangiogenesis might be the basis for increased nuchal translucency that is often observed in diseased human fetuses. Developmental Dynamics 230:378–384, 2004.

Platelet‐derived growth factor is involved in the differentiation of second heart field‐derived cardiac structures in chicken embryos

For the establishment of a fully functional septated heart, addition of myocardium from second heart field‐derived structures is important. Platelet‐derived growth factors (PDGFs) are known for their role in cardiovascular development. In this study, we aim to elucidate this role of PDGF‐A, PDGF‐C, and their receptor PDGFR‐α. We analyzed the expression patterns of PDGF‐A, ‐C, and their receptor PDGFR‐α during avian heart development. A spatiotemporal pattern of ligands was seen with colocalization of the PDGFR‐α. This was found in second heart field‐derived myocardium as well as the proepicardial organ (PEO) and epicardium. Mechanical inhibition of epicardial outgrowth as well as chemical disturbance of PDGFR‐α support a functional role of the ligands and the receptor in cardiac development. Developmental Dynamics 238:2658–2669, 2009.