A novel role for VEGF in endocardial cushion formation and its potential contribution to congenital heart defects

Development ◽  
2001 ◽  
Vol 128 (9) ◽  
pp. 1531-1538 ◽  
Author(s):  
Y. Dor ◽  
T.D. Camenisch ◽  
A. Itin ◽  
G.I. Fishman ◽  
J.A. McDonald ◽  
...  
Keyword(s):  
Growth Factor ◽  
Heart Development ◽  
Heart Defects ◽  
Negative Regulator ◽  
Congenital Defects ◽  
Endocardial Cushion ◽  
Potential Contribution ◽  
Vegf Receptor ◽  
Endocardial Cushions ◽  
Mesenchymal Transformation

Normal cardiovascular development is exquisitely dependent on the correct dosage of the angiogenic growth factor and vascular morphogen vascular endothelial growth factor (VEGF). However, cardiac expression of VEGF is also robustly augmented during hypoxic insults, potentially mediating the well-established teratogenic effects of hypoxia on heart development. We report that during normal heart morphogenesis VEGF is specifically upregulated in the atrioventricular (AV) field of the heart tube soon after the onset of endocardial cushion formation (i.e. the endocardium-derived structures that build the heart septa and valves). To model hypoxia-dependent induction of VEGF in vivo, we conditionally induced VEGF expression in the myocardium using a tetracycline-regulated transgenic system. Premature induction of myocardial VEGF in E9.5 embryos to levels comparable with those induced by hypoxia prevented formation of endocardial cushions. When added to explanted embryonic AV tissue, VEGF fully inhibited endocardial-to-mesenchymal transformation. Transformation was also abrogated in AV explants subjected to experimental hypoxia but fully restored in the presence of an inhibitory soluble VEGF receptor 1 chimeric protein. Together, these results suggest a novel developmental role for VEGF as a negative regulator of endocardial-to-mesenchymal transformation that underlies the formation of endocardial cushions. Moreover, ischemia-induced VEGF may be the molecular link between hypoxia and congenital defects in heart septation.

Prenatal ethanol exposure impairs the conduction delay at the atrioventricular junction in the looping heart

2021 ◽  
Author(s):  
Shan Ling ◽  
Michael W Jenkins ◽  
Michiko Watanabe ◽  
Stephanie M Ford ◽  
Andrew M Rollins
Keyword(s):  
Heart Development ◽  
Molecular Mechanisms ◽  
Early Stage ◽  
Heart Defects ◽  
Ethanol Exposure ◽  
Cardiac Conduction ◽  
Conduction Delay ◽  
Prenatal Ethanol Exposure ◽  
Endocardial Cushions ◽  
Atrioventricular Junction

The etiology of ethanol-related congenital heart defects has been the focus of much study, but most research has concentrated on cellular and molecular mechanisms. We have shown with optical coherence tomography (OCT) that ethanol exposure led to increased retrograde flow and smaller atrioventricular (AV) cushions compared to controls. Since AV cushions play a role in patterning the conduction delay at the atrioventricular junction (AVJ), this study aims to investigate whether ethanol exposure alters the AVJ conduction in early looping hearts and whether this alteration is related to the decreased cushion size. Quail embryos were exposed to a single dose of ethanol at gastrulation, and Hamburger-Hamilton stage 19 - 20 hearts were dissected for imaging. Cardiac conduction was measured using an optical mapping microscope and we imaged the endocardial cushions using OCT. Our results showed that, compared with controls, ethanol-exposed embryos exhibited abnormally fast AVJ conduction and reduced cushion size. However, this increased conduction velocity (CV) did not strictly correlate with decreased cushion volume and thickness. By matching the CV map to the cushion size map, we found that the slowest conduction location was consistently at the atrial side of the AVJ, which had the thinner cushions, not at the thickest cushion location at the ventricular side as expected. Our findings reveal regional differences in the AVJ myocardium even at this early stage in heart development. These findings reveal the early steps leading to the heterogeneity and complexity of conduction at the mature AVJ, a site where arrhythmias can be initiated.

scholarly journals β-Catenin is required for endothelial-mesenchymal transformation during heart cushion development in the mouse

2004 ◽  
Vol 166 (3) ◽  
pp. 359-367 ◽  
Author(s):  
Stefan Liebner ◽  
Anna Cattelino ◽  
Radiosa Gallini ◽  
Noemi Rudini ◽  
Monica Iurlaro ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Heart Development ◽  
Transcriptional Activity ◽  
Ex Vivo ◽  
Smooth Muscle Actin ◽  
Muscle Actin ◽  
Endocardial Cushions ◽  
Smad Phosphorylation ◽  
Mesenchymal Transformation

During heart development endocardial cells within the atrio-ventricular (AV) region undergo TGFβ-dependent epithelial-mesenchymal transformation (EMT) and invade the underlying cardiac jelly. This process gives rise to the endocardial cushions from which AV valves and part of the septum originate. In this paper we show that in mouse embryos and in AV explants TGFβ induction of endocardial EMT is strongly inhibited in mice deficient for endothelial β-catenin, leading to a lack of heart cushion formation. Using a Wnt-signaling reporter mouse strain, we demonstrated in vivo and ex vivo that EMT in heart cushion is accompanied by activation of β-catenin/TCF/Lef transcriptional activity. In cultured endothelial cells, TGFβ2 induces α-smooth muscle actin (αSMA) expression. This process was strongly reduced in β-catenin null cells, although TGFβ2 induced smad phosphorylation was unchanged. These data demonstrate an involvement of β-catenin/TCF/Lef transcriptional activity in heart cushion formation, and suggest an interaction between TGFβ and Wnt-signaling pathways in the induction of endothelial-mesenchymal transformation.

scholarly journals Extracellular Fibrillar Structure of Latent TGFβ Binding Protein-1: Role in TGFβ-dependent Endothelial-Mesenchymal Transformation during Endocardial Cushion Tissue Formation in Mouse Embryonic Heart

1997 ◽  
Vol 136 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Yuji Nakajima ◽  
Kohei Miyazono ◽  
Mitsuyasu Kato ◽  
Masao Takase ◽  
Toshiyuki Yamagishi ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Binding Protein ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Embryonic Heart ◽  
Fibrillar Structure ◽  
Endocardial Cushion ◽  
Tissue Formation ◽  
Mesenchymal Transformation

Transforming growth factor-β (TGFβ) is a dimeric peptide growth factor which regulates cellular differentiation and proliferation during development. Most cells secrete TGFβ as a large latent TGFβ complex containing mature TGFβ, latency associated peptide, and latent TGFβ-binding protein (LTBP)-1. The biological role of LTBP-1 in development remains unclear. Using a polyclonal antiserum specific for LTBP-1 (Ab39) and three-dimensional collagen gel culture assay of embryonic heart, we examined the tissue distribution of LTBP-1 and its functional role during the formation of endocardial cushion tissue in the mouse embryonic heart. Mature TGFβ protein was required at the onset of the endothelial-mesenchymal transformation to initiate endocardial cushion tissue formation. Double antibody staining showed that LTBP-1 colocalized with TGFβ1 as an extracellular fibrillar structure surrounding the endocardial cushion mesenchymal cells. Immunogold electronmicroscopy showed that LTBP-1 localized to 40–100 nm extracellular fibrillar structure and 5–10-nm microfibrils. The anti–LTBP-1 antiserum (Ab39) inhibited the endothelial-mesenchymal transformation in atrio-ventricular endocardial cells cocultured with associated myocardium on a three-dimensional collagen gel lattice. This inhibitory effect was reversed by administration of mature TGFβ proteins in culture. These results suggest that LTBP-1 exists as an extracellular fibrillar structure and plays a role in the storage of TGFβ as a large latent TGFβ complex.

scholarly journals Vascular Pool of Releasable Soluble VEGF Receptor-1 (sFLT1) in Women With Previous Preeclampsia and Uncomplicated Pregnancy

2014 ◽  
Vol 99 (3) ◽  
pp. 978-987 ◽  
Author(s):  
Tracey L. Weissgerber ◽  
Augustine Rajakumar ◽  
Ashley C. Myerski ◽  
Lia R. Edmunds ◽  
Robert W. Powers ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Whole Blood ◽  
Placental Growth Factor ◽  
Potential Contribution ◽  
Vegf Receptor ◽  
Vascular Endothelial ◽  
Uncomplicated Pregnancy ◽  
Heparin Plasma ◽  
Endothelial Growth Factor

Context: Research examining the source of excess soluble fms-like tyrosine kinase 1 (sFLT1) in preeclampsia has focused on the placenta. The potential contribution of the releasable store of sFLT1 in the systemic vasculature is unknown. Objective: We asked whether the nonplacental releasable store of sFLT1 is larger in women with previous preeclampsia than in women with a previous uncomplicated pregnancy. Design: We administered heparin to nulligravid women and to women with previous preeclampsia or a previous uncomplicated pregnancy. We compared post-heparin sFLT1 concentrations with those observed in uncomplicated pregnancy and preeclampsia. Setting: The study was performed at Magee-Womens Hospital. Patients: Participants included nulligravidas (n = 8), women 6–24 months postpartum (previous uncomplicated pregnancy, n = 16; previous preeclampsia, n = 15), and pregnant women (uncomplicated pregnancy, n = 30; preeclampsia, n = 25). Intervention: Nonpregnant women received an unfractionated heparin bolus. Main Outcome Measures: Pre- and post-heparin plasma sFLT1, placental growth factor, and vascular endothelial growth factor were measured. Results: In nonpregnant women, heparin increased plasma sFLT1 by 250-fold (P < .01), increased placental growth factor by 7-fold (P < .01), and decreased free vascular endothelial growth factor (P < .01). These changes did not differ between nulligravidas, women with previous preeclampsia, and women with a previous uncomplicated pregnancy. Post-heparin sFLT1 in nonpregnant women was higher than sFLT1 in uncomplicated pregnancy, but lower than sFLT1 in preeclampsia. Baseline and post-heparin sFLT1 were positively correlated (r2 = 0.19; P < .01). Heparin increased the concentration of the 100-kDa sFLT1 isoform. Adding heparin to whole blood or plasma did not increase sFLT1. Conclusions: Nonpregnant women have a significant vascular store of releasable sFLT1. The size of this store does not differ between women with previous preeclampsia vs women with previous uncomplicated pregnancy.

Vascular Endothelial Growth Factor (VEGF) is Released from Platelets during Blood Clotting: Implications for Measurement of Circulating VEGF Levels in Clinical Disease

1998 ◽  
Vol 94 (4) ◽  
pp. 395-404 ◽  
Author(s):  
Nicholas J. A. Webb ◽  
Martyn J. Bottomley ◽  
Carolyn J. Watson ◽  
Paul E. C. Brenchley
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Elevated Serum ◽  
Potential Contribution ◽  
Vegf Receptor ◽  
Vascular Endothelial ◽  
Disease States ◽  
Endothelial Growth Factor ◽  
Vegf Release ◽  
Vegf Level

1. Dysregulated vascular endothelial growth factor (VEGF) expression has been reported in several pathological states based upon evidence of elevated serum VEGF levels. Using two immunoassays for VEGF, this study determines normal plasma and serum VEGF ranges, determines which are more likely to reflect circulating VEGF levels and investigates a potential contribution of VEGF from platelets to VEGF levels detected in serum. 2. The presence of soluble VEGF receptor, sflt-1, at a molar excess of 7:1 significantly reduced measured VEGF levels in both assays. Serum VEGF levels were higher than plasma levels in children [(mean ± S.E.M.) 306.1 ± 39.4 versus 107.4 ± 24.9 pg/ml, P < 0.0001] and adults (249.4 ± 46.4 versus 76.1 ± 10.7 pg/ml, P < 0.0001). Serum VEGF increased with clotting time (P = 0.0005 t0 compared with 2 h samples); plasma VEGF levels were not affected by time between sampling and centrifugation. 3. Calcium-induced clotting of platelet-rich but not platelet-poor plasma induced VEGF release with a proportional response between platelet count and VEGF level and isolated platelets released significant quantities of VEGF upon incubation with thrombin. Reverse transcriptase—PCR studies confirmed that platelets express VEGF121 and VEGF165 mRNA. 4. These data suggest that plasma is the preferred medium to measure VEGF levels; a significant and highly variable platelet-mediated secretion of VEGF during the clotting process invalidates the use of serum as an indicator of circulating VEGF levels in disease states.

scholarly journals Elevated glucose inhibits VEGF-A–mediated endocardial cushion formation

2003 ◽  
Vol 160 (4) ◽  
pp. 605-615 ◽  
Author(s):  
Josephine M. Enciso ◽  
Dita Gratzinger ◽  
Todd D. Camenisch ◽  
Sandra Canosa ◽  
Emese Pinter ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
High Glucose ◽  
Matrix Metalloproteinase 2 ◽  
Migration And Invasion ◽  
Endocardial Cushion ◽  
Vegf Receptor ◽  
Mmp Inhibitor ◽  
Elevated Glucose ◽  
Endocardial Cell ◽  
Mesenchymal Transformation

Atrioventricular (AV) septal defects resulting from aberrant endocardial cushion (EC) formation are observed at increased rates in infants of diabetic mothers. EC formation occurs via an epithelial-mesenchymal transformation (EMT), involving transformation of endocardial cells into mesenchymal cells, migration, and invasion into extracellular matrix. Here, we report that elevated glucose inhibits EMT by reducing myocardial vascular endothelial growth factor A (VEGF-A). This effect is reversed with exogenous recombinant mouse VEGF-A165, whereas addition of soluble VEGF receptor-1 blocks EMT. We show that disruption of EMT is associated with persistence of platelet endothelial cell adhesion molecule-1 (PECAM-1) and decreased matrix metalloproteinase-2 (MMP-2) expression. These findings correlate with retention of a nontransformed endocardial sheet and lack of invasion. The MMP inhibitor GM6001 blocks invasion, whereas explants from PECAM-1 deficient mice exhibit MMP-2 induction and normal EMT in high glucose. PECAM-1–negative endothelial cells are highly motile and express more MMP-2 than do PECAM-1–positive endothelial cells. During EMT, loss of PECAM-1 similarly promotes single cell motility and MMP-2 expression. Our findings suggest that high glucose-induced inhibition of AV cushion morphogenesis results from decreased myocardial VEGF-A expression and is, in part, mediated by persistent endocardial cell PECAM-1 expression and failure to up-regulate MMP-2 expression.

scholarly journals Rare mutations of ADAM17 from TOFs induce hypertrophy in human embryonic stem cell-derived cardiomyocytes via HB-EGF signaling

2019 ◽  
Vol 133 (2) ◽  
pp. 225-238 ◽  
Author(s):  
Yifang Xie ◽  
Anyun Ma ◽  
Boshi Wang ◽  
Rui Peng ◽  
Yingchun Jing ◽  
...  
Keyword(s):  
Growth Factor ◽  
Heart Development ◽  
Heart Defects ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Heparin Binding ◽  
Loss Of Function ◽  
Knock Out ◽  
Critical Pathways

Abstract Tetralogy of Fallot (TOF) is the most common cyanotic form of congenital heart defects (CHDs). The right ventricular hypertrophy is associated with the survival rate of patients with repaired TOF. However, very little is known concerning its genetic etiology. Based on mouse model studies, a disintergrin and metalloprotease 10/17 (ADAM10 and ADAM17) are the key enzymes for the NOTCH and ErbB pathways, which are critical pathways for heart development. Mutations in these two genes have not been previously reported in human TOF patients. In this study, we sequenced ADAM10 and ADAM17 in a Han Chinese CHD cohort comprised of 80 TOF patients, 286 other CHD patients, and 480 matched healthy controls. Three missense variants of ADAM17 were only identified in 80 TOF patients, two of which (Y42D and L659P) are novel and not found in the Exome Aggregation Consortium (ExAC) database. Point mutation knock-in (KI) and ADAM17 knock-out (KO) human embryonic stem cells (hESCs) were generated by CRISPR/Cas9 and programmed to differentiate into cardiomyocytes (CMs). Y42D or L659P KI cells or complete KO cells all developed hypertrophy with disorganized sarcomeres. RNA-seq results showed that phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt), which is downstream of epidermal growth factor receptor (EGFR) signaling, was affected in both ADAM17 KO and KI hESC-CMs. In vitro experiments showed that these two mutations are loss-of-function mutations in shedding heparin-binding EGF-like growth factor (HB-EGF) but not NOTCH signaling. Our results revealed that CM hypertrophy in TOF could be the result of mutations in ADAM17 which affects HB-EGF/ErbB signaling.

scholarly journals Lack of endothelial cell survivin causes embryonic defects in angiogenesis, cardiogenesis, and neural tube closure

Blood ◽  
2007 ◽  
Vol 109 (11) ◽  
pp. 4742-4752 ◽  
Author(s):  
Femke Zwerts ◽  
Florea Lupu ◽  
Astrid De Vriese ◽  
Saskia Pollefeyt ◽  
Lieve Moons ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Neural Tube ◽  
Heart Development ◽  
Neural Tube Closure ◽  
Endocardial Cushions ◽  
Vascular Integrity ◽  
Apoptosis Protein ◽  
Mesenchymal Transformation ◽  
Tube Closure

Abstract We explored the physiologic role of endothelial cell apoptosis during development by generating mouse embryos lacking the inhibitor of apoptosis protein (IAP) survivin in endothelium. This was accomplished by intercrossing survivinlox/lox mice with mice expressing cre recombinase under the control of the endothelial cell specific tie1 promoter (tie1-cre mice). Lack of endothelial cell survivin resulted in embryonic lethality. Mutant embryos had prominent and diffuse hemorrhages from embryonic day 9.5 (E9.5) and died before E13.5. Heart development was strikingly abnormal. Survivin-null endocardial lineage cells could not support normal epithelial-mesenchymal transformation (EMT), resulting in hypoplastic endocardial cushions and in utero heart failure. In addition, 30% of mutant embryos had neural tube closure defects (NTDs) that were not caused by bleeding or growth retardation, but were likely due to alterations in the release of soluble factors from endothelial cells that otherwise support neural stem cell proliferation and neurulation. Thus, regulation of endothelial cell survival, and maintenance of vascular integrity by survivin are crucial for normal embryonic angiogenesis, cardiogenesis, and neurogenesis.

Folic acid prevents functional and structural heart defects induced by prenatal ethanol exposure

2021 ◽  
Author(s):  
Stephanie M Ford ◽  
Cameron J Pedersen ◽  
Matthew R Ford ◽  
Jun W Kim ◽  
Ganga H Karunamuni ◽  
...  
Keyword(s):  
Blood Flow ◽  
Folic Acid ◽  
Heart Diseases ◽  
Prenatal Alcohol Exposure ◽  
Heart Defects ◽  
Alcohol Exposure ◽  
Ethanol Exposure ◽  
Endocardial Cushion ◽  
Prenatal Ethanol Exposure ◽  
Endocardial Cushions

Increased regurgitant blood flow has been linked to endocardial cushion defects and resultant congenital heart diseases (CHDs). Prenatal alcohol exposure (PAE) has been shown to alter early blood flow resulting in abnormal endocardial cushions and CHDs. Compounds, including folic acid (FA), mitigate PAE effects and prevent CHDs, but few studies have assessed their effects on blood flow. We modeled binge drinking in quail embryos at gastrulation. Embryos were exposed to ethanol alone, FA (3.2 μg/egg) alone, and the two simultaneously. We quantified in cardiac looping stages (equivalent to 4 weeks of human gestation) regurgitant blood flow with Doppler optical coherence tomography (OCT) and endocardial cushion volumes using OCT imaging. Incidences of abnormal body curvature and heart rates were also measured. Embryos exposed to ethanol showed significantly increased regurgitant blood flow compared to controls, while embryos given FA with ethanol had significantly reduced regurgitant blood flow but did not return to control levels. Ethanol exposure led to significantly smaller, abnormal endocardial cushions and the addition of FA improved their size, but they remained smaller than controls. Abnormal body curvatures after PAE were reduced in incidence but not fully prevented by FA. FA supplementation partially alleviated PAE induced abnormal cardiovascular function and morphology. Normal blood flow and endocardial cushions are both required to produce a healthy four-chambered heart. These findings support that FA supplementation should begin early in pregnancy to prevent heart as well as neural tube defects. Investigations into the efficacy of combinations of compounds to prevent PAE-induced defects is warranted.

scholarly journals Pigment epithelium-derived factor downregulates vascular endothelial growth factor (VEGF) expression and inhibits VEGF–VEGF receptor 2 binding in diabetic retinopathy

2006 ◽  
Vol 37 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Sarah X Zhang ◽  
Joshua J Wang ◽  
Guoquan Gao ◽  
Kyoungmin Parke ◽  
Jian-xing Ma
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Pigment Epithelium ◽  
Negative Regulator ◽  
Vegf Receptor ◽  
Vascular Endothelial ◽  
Vegf Expression ◽  
Angiogenic Inhibitor ◽  
Pigment Epithelium Derived Factor ◽  
Endothelial Growth Factor

It has been shown that the balance between vascular endothelial growth factor (VEGF), a major angiogenic stimulator, and pigment epithelium-derived factor (PEDF), a potent angiogenic inhibitor, is critical for the regulation of vascular permeability and angiogenesis. However, the regulation of the balance is largely unclear. The present study demonstrated that there is a reciprocal interaction between VEGF and PEDF in the retina. PEDF significantly decreased VEGF expression in both retinal capillary endothelial cells (RCEC) and Müller cells. This PEDF effect was confirmed in the retina of rats with oxygen-induced retinopathy. Silencing of the PEDF gene by siRNA in Müller cells resulted in a significant upregulation of VEGF expression at both the RNA and protein levels, suggesting that PEDF is an endogenous negative regulator of VEGF. The further study of the mechanism showed that PEDF inhibited hypoxia-induced increases in VEGF promoter activity, HIF-1 nuclear translocation and mitogen activated protein kinase phosphorylation. These results suggest that PEDF inhibits VEGF expression at the transcriptional level. In addition, PEDF effectively inhibited VEGF binding to RCEC. Moreover, in vitro receptor-binding assay demonstrated that PEDF competed with VEGF for binding to VEGF receptor 2, which may represent a new mechanism for PEDF activity. On the other hand, VEGF significantly downregulated PEDF expression in RCEC, but not in retinal Müller cells, suggesting a VEGF receptor-mediated process. These results suggest that the reciprocal regulation between VEGF and PEDF may play a role in angiogenic control. The decrease in PEDF levels in the retina is at least partially responsible for the increase in VEGF expression and subsequent vascular leakage and neovascularization in diabetes.

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