Josephine Enciso

Elevated glucose inhibits VEGF-A-mediated endocardial cushion formation: modulation by PECAM-1 and MMP-2

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.

A null mutation of Hhex results in abnormal cardiac development,defective vasculogenesis and elevated Vegfa levels

The homeobox gene Hhex has recently been shown to be essential for normal liver, thyroid and forebrain development. Hhex–/– mice die by mid-gestation (E14.5) and the cause of their early demise remains unclear. Because Hhex is expressed in the developing blood islands at E7.0 in the endothelium of the developing vasculature and heart at E9.0-9.5, and in the ventral foregut endoderm at E8.5-9.0, it has been postulated to play a critical role in heart and vascular development. We show here, for the first time, that a null mutation of Hhex results in striking abnormalities of cardiac and vascular development which include: (1) defective vasculogenesis, (2) hypoplasia of the right ventricle, (3) overabundant endocardial cushions accompanied by ventricular septal defects, outflow tract abnormalities and atrio-ventricular (AV) valve dysplasia and (4) aberrant development of the compact myocardium. The dramatic enlargement of the endocardial cushions in the absence of Hhex is due to decreased apoptosis and dysregulated epithelial-mesenchymal transformation (EMT). Interestingly, vascular endothelial growth factor A (Vegfa) levels in the hearts of Hhex–/– mice were elevated as much as three-fold between E9.5 and E11.5, and treatment of cultured Hhex–/– AV explants with truncated soluble Vegfa receptor 1, sFlt-1, an inhibitor of Vegf signaling, completely abolished the excessive epithelial-mesenchymal transformation seen in the absence of Hhex. Therefore, Hhex expression in the ventral foregut endoderm and/or the endothelium is necessary for normal cardiovascular development in vivo, and one function of Hhex is to repress Vegfa levels during development.

Endothelial deletion of murine Jag1 leads to valve calcification and congenital heart defects associated with Alagille syndrome

The Notch signaling pathway is an important contributor to the development and homeostasis of the cardiovascular system. Not surprisingly, mutations in Notch receptors and ligands have been linked to a variety of hereditary diseases that impact both the heart and the vasculature. In particular, mutations in the gene encoding the human Notch ligand jagged 1 result in a multisystem autosomal dominant disorder called Alagille syndrome, which includes tetralogy of Fallot among its more severe cardiac pathologies. Jagged 1 is expressed throughout the developing embryo, particularly in endothelial cells. Here, we demonstrate that endothelial-specific deletion of Jag1 leads to cardiovascular defects in both embryonic and adult mice that are reminiscent of those in Alagille syndrome. Mutant mice display right ventricular hypertrophy, overriding aorta, ventricular septal defects, coronary vessel abnormalities and valve defects. Examination of mid-gestational embryos revealed that the loss of Jag1, similar to the loss of Notch1, disrupts endothelial-to-mesenchymal transition during endocardial cushion formation. Furthermore, adult mutant mice exhibit cardiac valve calcifications associated with abnormal matrix remodeling and induction of bone morphogenesis. This work shows that the endothelium is responsible for the wide spectrum of cardiac phenotypes displayed in Alagille Syndrome and it demonstrates a crucial role for Jag1 in valve morphogenesis.

Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy

Nitric oxide (NO) has been demonstrated to mediate events during ovulation, pregnancy, blastocyst invasion and preimplantation embryogenesis. However, less is known about the role of NO during postimplantation development. Therefore, in this study, we explored the effects of NO during vascular development of the murine yolk sac, which begins shortly after implantation. Establishment of the vitelline circulation is crucial for normal embryonic growth and development. Moreover, functional inactivation of the endodermal layer of the yolk sac by environmental insults or genetic manipulations during this period leads to embryonic defects/lethality, as this structure is vital for transport, metabolism and induction of vascular development. In this study, we describe the temporally/spatially regulated distribution of nitric oxide synthase (NOS) isoforms during the three stages of yolk sac vascular development (blood island formation, primary capillary plexus formation and vessel maturation/remodeling) and found NOS expression patterns were diametrically opposed. To pharmacologically manipulate vascular development, an established in vitro system of whole murine embryo culture was employed. During blood island formation, the endoderm produced NO and inhibition of NO (L-NMMA) at this stage resulted in developmental arrest at the primary plexus stage and vasculopathy. Furthermore, administration of a NO donor did not cause abnormal vascular development; however, exogenous NO correlated with increased eNOS and decreased iNOS protein levels. Additionally, a known environmental insult (high glucose) that produces reactive oxygen species (ROS) and induces vasculopathy also altered eNOS/iNOS distribution and induced NO production during yolk sac vascular development. However, administration of a NO donor rescued the high glucose induced vasculopathy, restored the eNOS/iNOS distribution and decreased ROS production. These data suggest that NO acts as an endoderm-derived factor that modulates normal yolk sac vascular development, and decreased NO bioavailability and NO-mediated sequela may underlie high glucose induced vasculopathy.

Progesterone Receptor in the Vascular Endothelium Triggers Physiological Uterine Permeability Preimplantation

Vascular permeability is frequently associated with inflammation and is triggered by a cohort of secreted permeability factors such as vascular endothelial growth factor (VEGF). Here, we show that the physiological vascular permeability that precedes implantation is directly controlled by progesterone receptor (PR) and is independent of VEGF. Global or endothelial-specific deletion of PR blocks physiological vascular permeability in the uterus, whereas misexpression of PR in the endothelium of other organs results in ectopic vascular leakage. Integration of an endothelial genome-wide transcriptional profile with chromatin immunoprecipitation sequencing revealed that PR induces an NR4A1 (Nur77/TR3)-dependent transcriptional program that broadly regulates vascular permeability in response to progesterone. Silencing of NR4A1 blocks PR-mediated permeability responses, indicating a direct link between PR and NR4A1. This program triggers concurrent suppression of several junctional proteins and leads to an effective, timely, and venous-specific regulation of vascular barrier function that is critical for embryo implantation.

Maternal Diabetes: Effects on Embryonic Vascular Development—A Vascular Endothelial Growth Factor-A-mediated Process

Major congenital malformations, many of which result from abnormal cardiovascular patterning, remain the leading cause in infant mortality and morbidity. Targeted mutations of several genes (including VEGF and VEGF receptors) and certain teratogenic agents (including excess α-D-glucose) give rise to embryonic lethal phenotypes associated with failure in the formation of a functional vitelline circulation and aberrant organogenesis. Our work to date has demonstrated that yolk sac vasculopathy and failure of endocardial cushion epithelial-mesenchymal transformation occurs in hyperglycemic conditions in murine whole conceptus culture and in embryos from streptozotocin-induced diabetic mice. These cardiovascular abnormalities are associated with changes in expression and phosphorylation state of adhesion molecules such as platelet endothelial growth factor-1 and expression of growth factors such as vascular endothelial growth factor (VEGF-A). Further understanding of the effects of maternal diabetes on yolk sac and embryonic vasculogenesis/angiogenesis and organogenesis may lead to novel approaches in treating and preventing major birth defects.

Endothelial cell migration during murine yolk sac vascular remodeling occurs by means of a rac1 and FAK activation pathway in vivo

The molecular mechanism(s) controlling cell migration during vascular morphogenesis in vivo remain largely undefined. To address this within a physiological context, we used retinaldehyde dehydrogenase‐2 (Raldh2) null mouse embryos and demonstrate that retinoic acid (RA) deficiency results in abnormal yolk sac vascular remodeling due to decreased Rac1 activation, increased RhoA activation, and increased focal adhesions. Vinculin was increased in Raldh2−/− yolk sacs, and molecular events important for focal adhesion turnover, FAK phosphorylation (Tyr397) and FAK‐paxillin association, were decreased. RA‐rescue of vascular remodeling down‐regulated vinculin and restored FAK phosphorylation (Tyr397) and FAK‐paxillin association. Furthermore, vascular rescue with vascular endothelial growth factor‐A, Indian hedgehog, and basic fibroblast growth factor restored FAK phosphorylation (Tyr397) in the endothelium of Raldh2−/− yolk sacs. Our results provide new insights into the regulation of endothelial cell migration during vascular remodeling in vivo by adding the Rac1 and FAK activation pathway as a critical mediator of focal adhesion formation and turnover during vascular remodeling. Developmental Dynamics 239:2570–2583, 2010.

Understanding Abnormalities in Vascular Specification and Remodeling

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Nutrient regulation of tumor and vascular endothelial cell proliferation.

Specific bioactive dietary components, such as the steroid receptor superfamily ligands vitamins A and D, have been studied extensively as potential cancer preventive and therapeutic agents due to their ability to regulate key processes in a variety of cell types which are dysregulated in neoplastic transformation namely, proliferation and differentiation. Alteration of one or more factors that regulate cell cycle control has been described as a predisposing event for early tumor development. In addition to tumor cell proliferation, the viability, growth and metastasis of solid tumors are also dependent on the vascularization of the tumor and establishment of blood flow. Both vitamins A and D exhibit anti-angiogenic properties which further strengthen their role as potential targets for the prevention and treatment of cancer. This review focuses on the role of vitamins A and D in preventing early tumor initiation and progression via control of the cell cycle in both tumor and vascular endothelial cells.