Daniel G. M. Molin

VEGF : A modifier of the del22q11 (DiGeorge) syndrome?

Hemizygous deletion of chromosome 22q11 (del22q11) causes thymic, parathyroid, craniofacial and life-threatening cardiovascular birth defects in 1 in 4,000 infants. The del22q11 syndrome is likely caused by haploinsufficiency of TBX1, but its variable expressivity indicates the involvement of additional modifiers. Here, we report that absence of the Vegf164 isoform caused birth defects in mice, reminiscent of those found in del22q11 patients. The close correlation of birth and vascular defects indicated that vascular dysgenesis may pathogenetically contribute to the birth defects. Vegf interacted with Tbx1, as Tbx1 expression was reduced in Vegf164-deficient embryos and knocked-down vegf levels enhanced the pharyngeal arch artery defects induced by tbx1 knockdown in zebrafish. Moreover, initial evidence suggested that a VEGF promoter haplotype was associated with an increased risk for cardiovascular birth defects in del22q11 individuals. These genetic data in mouse, fish and human indicate that VEGF is a modifier of cardiovascular birth defects in the del22q11 syndrome.

Altered apoptosis pattern during pharyngeal arch artery remodelling is associated with aortic arch malformations in Tgfβ2 knock-out mice

OBJECTIVE The morphogenetic process underlying the remodelling of the embryonic mammalian pharyngeal arch artery system is unknown. Within this process, the right sixth, carotid ducts and the distal part of the dorsal aorta (right alpha-segment) regress. In order to unravel the underlying mechanism we studied the role of apoptosis in the normal regression of pharyngeal arch artery segments and in a mouse model that develops aortic arch malformations. METHODS Normal remodelling was studied in wild-type Swiss (CPBS) and altered remodelling in the Tgfbeta2-/- compared to the Tgfbeta2+/+ (Swiss/Bl6) strain using immunohistochemistry and morphometric analysis. RESULTS During normal remodelling, apoptosis occurs in the mesenchyme surrounding pharyngeal arch arteries before regression starts. With the onset of regression, apoptosis spreads from the mesenchyme to the media. Morphometric evaluation confirms the increase in apoptosis in the actin-positive media of the disappearing segments. In Tgfbeta2-/-, aberrant apoptosis was found in both fourth arch arteries, whereas the right dorsal aorta lacks apoptosis associated with normal regression. Fourth arch hypoplasia is the main arch abnormality. In the most severe case, the fourth arch is interrupted and the right dorsal aorta alpha-segment persists, giving rise to aortic arch interruption type-B and an aberrant right subclavian artery. CONCLUSIONS We have shown for the first time that specific vascular apoptosis patterns accompany normal regression and that the incidence of apoptosis is selectively altered in the case of arch artery abnormalities in Tgfbeta2 knock-out mice.

Expression of transient receptor potential mRNA isoforms and Ca(2+) influx in differentiating human stem cells and platelets.

Store-regulated Ca(2+) entry (SOCE) is an important mechanism of elevating cytosolic [Ca(2+)]i in platelets, though the Ca(2+) influx channels involved are still unclear. We screened human platelets and their precursor cells (human stem cells and megakaryocytes) for the presence of candidate influx channels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34(+)) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61(+)/CD42b(low)) and mature (CD61(+)/CD42b(high)) megakaryocytes as well as platelets expressed in addition unspliced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet lineage, since immature (CD14(+)/CD61(-)/CD42b(-)) and mature monocytes expressed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A, 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell line, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem cells to mature megakaryocytes and platelets was accompanied by increased influx of extracellular Ca(2+) after pretreatment of the cells with thapsigargin or thrombin. Expression of new Trp isoforms in the differentiated cells is thus accompanied by increased SOCE.

Transforming Growth Factor β–SMAD2 Signaling Regulates Aortic Arch Innervation and Development

Aortic arch interruptions in humans and animal models are mainly caused by aberrant development of the fourth pharyngeal arch artery. Little is known about the maturation of this vessel during normal and abnormal development, which is the subject of this study. Tgf&bgr;2 knockout mice that present with fourth artery defects have been associated with defective neural crest cell migration. In this study, we concentrated on pharyngeal arch artery development during developmental days 12.5 to 18.5, focusing on neural crest cell migration using a Wnt1-Cre by R26R neural crest cell reporter mouse. Fourth arch artery maturation was studied with antibodies directed against smooth muscle &agr;-actin and neural NCAM-1 and RMO-270. For diminished transforming growth factor &bgr; (TGF-&bgr;) signaling, SMAD2 and fibronectin have been analyzed. Neural crest migration and differentiation into smooth muscle cells is unaltered in mutants, regardless of the cardiovascular defect found; however, innervation of the fourth arch artery is affected. Absent staining for nuclear SMAD2, NCAM-1, and RMO-270 in the fourth artery in mutant coincides with severe defects of this segment. Likewise, fibronectin expression is diminished in these cases. From these data we conclude the following: (1) neural crest cell migration is not a common denominator in cardiovascular defects of Tgf&bgr;2−/− mice; (2) fourth arch artery maturation is a complex process involving innervation; and (3) TGF-&bgr;2 depletion diminishes SMAD2-signaling in the fourth arch artery and coincides with reduced vascular NCAM-1 expression and neural innervation of this artery. We hypothesize that disturbed maturation of the fourth pharyngeal arch artery, and especially abrogated vascular innervation, will result in fourth arch interruptions.

Feed-forward signaling by membrane-bound ligand receptor circuit: the case of NOTCH DELTA-like 4 ligand in endothelial cells.

The DELTA like-4 ligand (DLL4) belongs to the highly conserved NOTCH family and is specifically expressed in the endothelium. DLL4 regulates crucial processes in vascular growth, including endothelial cell (EC) sprouting and arterial specification. Its expression is increased by VEGF-A. In the present study, we show that VEGF-induced DLL4 expression depends on NOTCH activation. VEGF-induced DLL4 expression was prevented by the blockage of NOTCH signaling with γ-secretase or ADAM inhibitors in human cardiac microvascular ECs. Similar to VEGF-A, recombinant DLL4 itself stimulated NOTCH signaling and resulted in up-regulation of DLL4, suggesting a positive feed-forward mechanism. These effects were abrogated by NOTCH inhibitors but not by inhibition of VEGF signaling. NOTCH activation alone suffices to induce DLL4 expression as illustrated by the positive effect of NOTCH intracellular domain (NICD)-1 or -4 overexpression. To discriminate between NICD/RBP-Jκ and FOXC2-regulated DLL4 expression, DLL4 promoter activity was assessed in promoter deletion experiments. NICD induced promoter activity was dependent on RBP-Jκ site but independent of the FOXC2 binding site. Accordingly, constitutively active FOXC2 did not affect DLL4 expression. The notion that the positive feed-forward mechanism might propagate NOTCH activation to neighboring ECs was supported by our observation that DLL4-eGFP-transfected ECs induced DLL4 expression in nontransfected cells in their vicinity. In summary, our data provide evidence for a mechanism by which VEGF or ligand-induced NOTCH signaling up-regulates DLL4 through a positive feed-forward mechanism. By this mechanism, DLL4 could propagate its own expression and enable synchronization of NOTCH expression and signaling between ECs.

Targeting of ICAM-1 on vascular endothelium under static and shear stress conditions using a liposomal Gd-based MRI contrast agent

BackgroundThe upregulation of intercellular adhesion molecule-1 (ICAM-1) on the endothelium of blood vessels in response to pro-inflammatory stimuli is of major importance for the regulation of local inflammation in cardiovascular diseases such as atherosclerosis, myocardial infarction and stroke. In vivo molecular imaging of ICAM-1 will improve diagnosis and follow-up of patients by non-invasive monitoring of the progression of inflammation.ResultsA paramagnetic liposomal contrast agent functionalized with anti-ICAM-1 antibodies for multimodal magnetic resonance imaging (MRI) and fluorescence imaging of endothelial ICAM-1 expression is presented. The ICAM-1-targeted liposomes were extensively characterized in terms of size, morphology, relaxivity and the ability for binding to ICAM-1-expressing endothelial cells in vitro. ICAM-1-targeted liposomes exhibited strong binding to endothelial cells that depended on both the ICAM-1 expression level and the concentration of liposomes. The liposomes had a high longitudinal and transversal relaxivity, which enabled differentiation between basal and upregulated levels of ICAM-1 expression by MRI. The liposome affinity for ICAM-1 was preserved in the competing presence of leukocytes and under physiological flow conditions.ConclusionThis liposomal contrast agent displays great potential for in vivo MRI of inflammation-related ICAM-1 expression.

Soluble Jagged-1 Inhibits Neointima Formation by Attenuating Notch-Herp2 Signaling

Objective—Notch has been implicated in neointima formation as reflected by increased Notch/Jagged expression on vascular injury and the promigratory effect of Notch signaling on smooth muscle cells. Soluble Jagged-1 (sJag1) has been shown to inhibit Notch signaling in vitro; however, its capacity to suppress neointima formation remains unknown. Methods and Results—Balloon injury of rat carotid arteries induced Notch1, Notch3, and Jagged-1 expression at days 3 and 14 postinjury. Notch signaling was activated as shown by increased expression of the Notch target gene Herp2. Adenoviral sJag1 (Ad-sJag1) transfection reduced neointima formation in carotid artery and enhanced reendothelialization, whereas adenoviral full-length Jagged-1 (Ad-Fl-Jag1) or LacZ had no effect. Injury-induced Herp2 expression was absent in vessels treated with Ad-sJag1. Consistently, Herp2 expression was reduced in Ad-sJag1-infected or recombinant sJag1 –treated coronary artery smooth muscle cells (CASMCs). Ad-sJag1 had no effect on human umbilical endothelial cell behavior, but it significantly reduced proliferation and migration of CASMCs. Overexpression of Herp2 in sJag1-treated CASMCs rescued the migratory and proliferative capacity in vitro. Conclusion—Our results demonstrate that sJag1 can inhibit neointima formation after balloon injury by decreasing smooth muscle cell proliferation and migration through interference with Notch-Herp2 signaling.

A Chicken Model for DGCR6 as a Modifier Gene in the DiGeorge Critical Region

DGCR6 is the most centromeric gene in the human DiGeorge critical region and is the only gene in the region with a second functional copy on a repeat localized more distally on chromosome 22. We isolated the chicken ortholog of DGCR6 and showed an embryonic expression pattern that is initially broad but becomes gradually restricted to neural crest cell derivatives of the cardiovasculature. Retrovirus based gene transduction was used to deliver sense and antisense messages to premigrating neural crest cells in vivo. Embryos in which DGCR6 expression was attenuated revealed cardiovascular anomalies reminiscent of those found in DiGeorge syndrome. Moreover, the expression profiles of three other genes from the DiGeorge critical region, TBX-1, UFD1L, and HIRA, were shown to be altered in this model. TBX-1 and UFD1L levels were increased, whereas HIRA was decreased in the hearts and pharyngeal arches of embryos treated with antisense or partial sense constructs, but not with sense constructs for DGCR6. The expression changes were transient and followed the normal DGCR6 expression profile. These data show that neural crest cells might have a role in the distribution of modulator signals to the heart and pharyngeal arches. Moreover, it shows a repressor function for DGCR6 on the expression of TBX-1 and UFD1L. For the first time, Di-George syndrome is shown to be a contiguous gene syndrome in which not only several genes from the critical region, but also different cell types within the embryo, interact in the development of the phenotype.

Cellular decisions in cardiac outflow tract and coronary development: An act by VEGF and NOTCH

Congenital cardiac abnormalities are, due to their relatively high frequency and severe impact on quality of life, an important focus in cardiovascular research. Recently, various human studies have revealed a high coincidence of VEGF and NOTCH polymorphisms with cardiovascular outflow tract anomalies, such as bicuspid aortic valves and Tetralogy of Fallot, next to predisposition for cardiovascular pathologies, including atherosclerosis and aortic valve calcification. This genetic association between VEGF/NOTCH mutations and congenital cardiovascular defects in humans has been supported by substantial proof from animal models, revealing interaction of both pathways in cellular processes that are crucial for cardiac development. This review focuses on the role of VEGF and NOTCH signaling and their interplay in cardiogenesis with special interest to coronary and outflow tract development. An overview of the association between congenital malformations and VEGF/NOTCH polymorphisms in humans will be discussed along with their potential mechanisms and processes as revealed by transgenic mouse models. The molecular and cellular interaction of VEGF and subsequent Notch-signaling in these processes will be highlighted.

Electrosensitive Polyacrylic Acid/Fibrin Hydrogel Facilitates Cell Seeding and Alignment

Three-dimensional cell culture and conditioning is an effective means to guide cell distribution and patterning for tissue engineered constructs such as vascular grafts. Polyacrylic acid is known as an electroresponsive polymer, capable of transforming environmental stimuli like electrical energy to mechanical forces. In this study, we developed an electrosensitive and biocompatible hydrogel-based smart device composed of acrylic acid and fibrin as a tissue engineered construct to mechanically stimulate cells. Structural properties of the hydrogel were assessed by FTIR-ATR, scanning electron microscopy, prosimetry, and swelling measurement. Distribution and alignment of porcine smooth muscle cells (pSMCs) seeded on the surface of lyophilized hydrogels were evaluated and quantified by two-photon laser scanning microscopy. Smooth muscle cell tissue constructs exposed to 2 h of pulsatile electrical stimulation showed significantly enhanced cell penetration and alignment due to dynamic changes produced by alternative swelling and deswelling, in comparison with static samples. On the basis of the results, this hydrogel under electrical stimulation works as a mechanical pump, which can direct SMC alignment and facilitate infiltration and distribution of cells throughout the structure.