Proteomic Alterations Associated with Biomechanical Dysfunction are Early Processes in the Emilin1 Deficient Mouse Model of Aortic Valve Disease.

Aortic valve (AV) disease involves stiffening of the AV cusp with progression characterized by inflammation, fibrosis, and calcification. Here, we examine the relationship between biomechanical valve function and proteomic changes before and after the development of AV pathology in the Emilin1-/- mouse model of latent AV disease. Biomechanical studies were performed to quantify tissue stiffness at the macro (micropipette) and micro (atomic force microscopy (AFM)) levels. Micropipette studies showed that the Emilin1-/- AV annulus and cusp regions demonstrated increased stiffness only after the onset of AV disease. AFM studies showed that the Emilin1-/- cusp stiffens before the onset of AV disease and worsens with the onset of disease. Proteomes from AV cusps were investigated to identify protein functions, pathways, and interaction network alterations that occur with age- and genotype-related valve stiffening. Protein alterations due to Emilin1 deficiency, including changes in pathways and functions, preceded biomechanical aberrations, resulting in marked depletion of extracellular matrix (ECM) proteins interacting with TGFB1, including latent transforming growth factor beta 3 (LTBP3), fibulin 5 (FBLN5), and cartilage intermediate layer protein 1 (CILP1). This study identifies proteomic dysregulation is associated with biomechanical dysfunction as early pathogenic processes in the Emilin1-/- model of AV disease.

Inner cell mass-specific expression of a cell adhesion molecule (PECAM-1/CD31) in the mouse blastocyst.

Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1 or CD31) is thought to be a vascular-specific protein, but its function has not been clearly defined. Here, we demonstrate by using confocal immunofluorescence microscopy that PECAM-1 is first detected in the mouse blastocyst, which contains no vascular cells, and its expression is restricted to the pluripotent inner cell mass (ICM) cells. Expression is localized to cell-cell borders of the ICM and is detected at the very first signs of blastocoel formation. Consistent with these observations is that embryonic transcripts of PECAM-1 mRNA, as detected by RT-PCR, greatly increase during the morula-to-blastocyst transition and seven of the eight known alternatively spliced isoforms of PECAM-1 are expressed in the blastocyst. The synthesis of PECAM-1 is independent of compaction, cytokinesis, and DNA replication, as it is detected in embryos that are chronologically at the blastocyst stage following culture of 8-cell embryos in Ca2+-free medium, or medium containing cytochalasin D or aphidicolin, respectively. By the late blastocyst stage, PECAM-1 expression is restricted to the pluripotent epiblast, at which point it has a mutually exclusive expression pattern to that of type IV collagen, a basement membrane marker. The reduction in PECAM-1 transcripts in retinoic acid-induced differentiation of F9 teratocarcinoma cells, a model of epiblast-to-primitive endoderm differentiation, confirmed the epiblast-specific expression of PECAM-1. By the egg cylinder stage of development, at which point the epiblast is no longer pluripotent, PECAM-1 is not detected. This ICM-specific pattern of expression suggests a novel developmental role of PECAM-1 that is independent of its function in vascular ontogeny.

Optimization of adenoviral vector-mediated gene transfer to pulmonary arteries in newborn swine.

Efficient pulmonary vascular gene transfer in neonates would aid in understanding the pathophysiology of, and ultimately allow the development of specific therapies for, pulmonary vascular diseases. The purpose of this study was to optimize efficiency, and evaluate the duration, of catheter-based adenoviral vector-mediated pulmonary artery gene transfer in newborn pigs. An adenovirus vector encoding LacZ was infused via percutaneously placed catheters that were advanced to segmental pulmonary arteries under fluoroscopic guidance. Optimal viral dose and duration of expression were determined by histochemical evaluation of gene transfer efficiency 72 hr, 2 weeks, and 1 month after gene delivery. The effect of protamine on the efficiency of gene transfer was studied by assaying transgene protein in lung at 72 hr. The optimal viral dose was 2 x 10(10) plaque-forming units (PFU). Seventy-two hours after infusion, expression predominated in medium-sized artery endothelial cells, 40% of which expressed beta-galactosidase. At 2 weeks, the distribution of expression had changed such that the majority of transduced cells were seen not in arteries but in gas exchange units of lung. No histochemical evidence of transgene expression was seen 1 month after virus infusion. The addition of protamine to virus infusate resulted in a fivefold increase in transgene protein product in lung tissue assayed 72 hr after gene transfer. Adenoviral vector-mediated gene transfer in neonatal swine results in high-efficiency transduction of arterial endothelial cells. However, the time course of gene transfer is not significantly prolonged compared with the adult. The addition of protamine results in a significant improvement in transduction efficiency, permitting lower doses of virus.

Molecular determinants of neural crest migration.

Normal septation of the cardiac outflow tract requires migration of neural crest cells from the posterior rhombencephalon to the branchial arches and developing conotruncal endocardial cushions. Proper migration of these cells is mediated by a variety of molecular cues. Adhesion molecules, such as integrins, are involved in the interaction of neural crest cells with the extracellular matrix, while cadherins allow neural crest cells to interact with each other during their migration. Pax3 appears to be important for proliferation of neural crest precursors, and connexin-43-mediated gap junction communication influences the rate of migration. Endothelin and its receptors are required for normal postmigratory differentiation. Platelet-derived growth factor and retinoic acid have roles in neural crest migration and differentiation as well. Finally, the similarity between the cardiovascular malformations seen in the DiGeorge and 22q11 deletion syndromes and animal models of neural crest deficiency has led to the examination of the role of genes located near or within the DiGeorge critical region in neural crest migration.

Advances in understanding the molecular regulation of cardiac development.

Although impressive progress has been made in the diagnosis and treatment of congenital heart disease, there has been an explosion of new information about the basic molecular mechanisms that control normal heart development and subsequent congenital cardiovascular malformations. Since the advent of targeted null mutations in mice (gene "knockouts"), it has become increasingly evident that defects in the heart and vascular system frequently result from gene alterations and that these defects are often responsible for in utero demise. New genes have been discovered that control looping of the heart, distinguish arteries from veins, and direct formation of the semilunar valve and atrioventricular valves. A pivotal role for several genes expressed by the cardiac neural crest document the importance of these cells in aortic arch selection, in addition to their role in aorticopulmonary septation. In addition, myocardial and endothelial progenitor cells have been isolated from bone marrow stromal cells, and human embryonic stem cells have been successfully isolated, paving the way for developmental approaches to tissue engineering and organ regeneration. Finally, the first successful attempt at in utero manipulation of genes that might palliate certain forms of congenital heart disease has been presented. These recent advances are detailed in this article.

The expression of Jagged1 in the developing mammalian heart correlates with cardiovascular disease in Alagille syndrome.

The establishment of the cardiovascular system represents an early, critical event essential for normal embryonic development, and defects in cardiovascular development are a frequent cause of both in utero and neonatal demise. Congenital cardio-vascular malformations, the most frequent birth defect, can occur as isolated events, but are frequently presented clinically within the context of a constellation of defects that involve multiple organs and that define a specific syndrome. In addition, defects can be a primary effect of gene mutations or result from secondary effects of altered cardiac physiology. Alagille syndrome (AGS) is an autosomal dominant disorder characterized by developmental abnormalities of the heart, liver, eye, skeleton and kidney. Congenital heart defects, the majority of which affect the right-sided or pulmonary circulation, contribute significantly to mortality in AGS patients. Recently, mutations in Jagged1 ( JAG1 ), a conserved gene of the Notch intercellular signaling pathway, have been found to cause AGS. In order to begin to delineate the role of JAG1 in normal heart development we have studied the expression pattern of JAG1 in both the murine and human embryonic heart and vascular system. Here, we demonstrate that JAG1 is expressed in the developing heart and multiple associated vascular structures in a pattern that correlates with the congenital cardiovascular defects observed in AGS. These data are consistent with an important role for JAG1 and Notch signaling in early mammalian cardiac development.

Tetralogy of Fallot with anomalous coronary artery: double outflow technique.

A new technique to repair tetralogy of Fallot with an anomalous coronary artery crossing the right ventricular outflow tract is described, together with intermediate term follow-up. Using a pedicled flap of the anterior pulmonary artery wall as the floor, and a vascular or prosthetic patch as the roof, a composite conduit with the potential for growth is constructed. Together with the native outflow tract, this provides unobstructed egress from the right ventricle to the branch pulmonary arteries. Since 1990, 4 infants aged 2-weeks to 6-months have undergone primary repair using this technique. Intermediate term follow-up shows adequate durability of the repair.

Adenoviral-mediated expression of antisense RNA to fibroblast growth factors disrupts murine vascular development.

Fibroblast growth factors (FGFs) are expressed in the developing embryo and are postulated to regulate embryonic and vascular growth. The goal of this study was to elucidate the role of basic fibroblast growth factor (FGF-2) in early murine embryonic cardiovascular development in the mouse embryo. Gestation day 7.5 embryos were harvested and placed in culture, and 12 hr later replication-defective adenovirus (0.5 x 10(6) plaque forming units) encoding either beta-galactosidase or antisense FGF-2 RNA was injected into the sinus venosus of the cultured embryos. Embryos receiving only replication-defective adenovirus expressing the beta-galactosidase gene continued to develop normally over the next 12 hr. In contrast, those receiving adenovirus encoding antisense FGF-2 RNA displayed marked morphogenetic abnormalities, including cessation of growth and abnormal yolk sac vascular development, even though the embryonic hearts continued to beat. Abnormal development of the yolk sac vasculature was confirmed by microangiography and by histologic examination. Coinjection of virus carrying FGF-2 cDNA in the sense orientation reversed the effects of antisense FGF-2 RNA expression. These results confirm the efficacy of the replication-defective adenovirus for targeting gene expression to the developing vasculature and provide evidence for a critical role of FGF in the normal vascular assembly in the early embryo. Cessation of embryonic growth on expression of antisense FGF-2 RNA was most likely attributable to failure of efficient circulation of the early embryonic blood cells from the yolk sac into the embryo.

Increased endothelial cell expression of platelet-endothelial cell adhesion molecule-1 during hyperoxic lung injury.

Lung injury is a frequent consequence of oxygen (O2) therapy administered to newborns and adults with respiratory distress. Acute exposure to hyperoxia results in a well-described pathophysiologic response in the lungs. Because inflammation is an important component of pulmonary O2 toxicity, we have an interest in identifying the inflammatory mediators that increase during hyperoxia. Platelet-endothelial cell adhesion molecule-1 (PECAM-1), a member of the immunoglobulin superfamily that is expressed at the junctions between endothelial cells, is essential to the transendothelial migration of leukocytes. We hypothesized that increased expression of PECAM-1 occurs in pulmonary endothelial cells during hyperoxic lung injury. Adult mice were exposed to 100% O2 for up to 96 h. We analyzed PECAM-1 expression by RNA blot hybridization, in situ hybridization, and immunohistochemistry. A increase in PECAM-1 mRNA was seen as soon as 2 d of hyperoxia relative to unexposed control mice. PECAM-1 mRNA and protein were found in endothelial cells of both large and small arteries. The expression of PECAM-1 in capillary vessels was further confirmed using in situ hybridization at the electron microscope level. This increase in PECAM-1 expression coincided with the appearance of leukocytes in lung tissue. These observations suggest that PECAM-1 expression is a relatively early step in the inflammation cascade, and intervention at this phase may be critical to the prevention of further damage.

Noninvasive, in utero imaging of mouse embryonic heart development with 40-MHz echocardiography.

BACKGROUND: The increasing number of transgenic and targeted mutant mice with embryonic cardiac defects has resulted in the need for noninvasive techniques to examine cardiac structure and function in early mouse embryos. We report the first use of a novel 40-MHz ultrasound imaging system in the study of mouse cardiac development in utero. METHODS AND RESULTS: Transabdominal scans of mouse embryos staged between 8.5 and 13.5 days of gestation (E8.5 to E13.5) were obtained in anesthetized mice. Atrial and ventricular contractions could be discerned from E9.5, and changes in cardiac morphology were observed from E9.5 to E13.5. Hyperechoic streaming patterns delineated flow through the umbilical, vitelline, and other major blood vessels. Diastolic and systolic ventricular areas were determined by planimetry of the epicardial borders, and fractional area change was measured as an index of contractile function. Significant increases in ventricular size were documented at each stage between E10.5 and E13.5, and the ability to perform serial imaging studies over 3 days of embryonic development is described. Finally, the detection of vascular cell adhesion molecule 1 (VCAM-1) homozygous null mutant embryos demonstrates the first example of noninvasive, in utero analysis of cardiac structure and function in a targeted mouse mutant. CONCLUSIONS: We used 40-MHz echocardiography to identify key elements of the early mouse embryonic cardiovascular system and for noninvasive dimensional analysis of developing cardiac ventricles. The ability to perform serial measurements and to detect mutant embryos with cardiac defects highlights the usefulness of the technique for investigating normal and abnormal cardiovascular development.

The transcription factor NF-ATc is essential for cardiac valve formation.

Nuclear factor of activated T cells (NF-AT) is the name of a family of four related transcription factors that may be needed for cytokine gene expression in activated lymphocytes. Here we report that mice with a targeted disruption of the NF-ATc gene show an unexpected and dramatic defect in cardiac morphogenesis, with selective absence of the aortic and pulmonary valves, leading to death in utero from congestive heart failure at days 13.5-17.5 of gestation. In contrast, tricuspid and mitral valve morphogenesis is normal. NF-ATc is the first transcription factor known to be expressed only in the endothelial cells of the heart. As in T cells, nuclear translocation of NF-ATc in cardiac endothelial cells is controlled by the calcium-regulated phosphatase calcineurin: NF-ATc remains cytoplasmic in normal embryos cultured with cyclosporin A, an inhibitor of calcineurin. Abnormal development of the cardiac valves and septae is the most frequent form of birth defect, yet few molecular regulators of valve formation are known. Our results indicate that NF-ATc may play a critical role in signal-transduction processes required for normal cardiac valve formation.

Cre-mediated generation of a VCAM-1 null allele in transgenic mice.

A conditional null allele for VCAM-1 was generated in mice through a one step ES cell selection procedure by flanking the proximal promoter and exons 1 and 2 with loxP sites. The ES cells were used to create chimeric mice, which were then used to produce mice homozygous for the VCAM-1 conditional null, or floxed allele. Although the PGKneo cassette was retained in the promoter, the homozygous mice produced levels of VCAM-1 transcripts similar to that seen in wild-type mice. Homozygous VCAMflox/flox mice were mated to transgenic lines of mice expressing the cre gene under control of the murine platelet endothelial cell adhesion molecule-1 (PECAM-1) promoter. Surprisingly, the VCAMflox allele in all tissues examined from mice that inherited the cre-transgene had underwent complete excision of the floxed VCAM-1 sequences. The 'deleted' VCAM-1 allele (VCAMdel) was stably inherited, even in those mice that did not inherit the cre transgene, indicating the recombination occurs at an early stage of development prior to germ cell development. Thus the cre mice can be used for ubiquitous gene rearrangement in vivo. The data also suggest a novel simplified strategy for using the Cre/loxP system in vivo, in which a single ES cell and line of mice can be used to create mice carrying either a null or conditional null allele.

Fibronectins are essential for heart and blood vessel morphogenesis but are dispensable for initial specification of precursor cells.

The underlying mechanisms of lethal cardiovascular defects associated with the fibronectin-null (FN.null) mutation in mouse embryos were investigated by lineage analysis of myocardial, endocardial, and endothelial cells. A wide variation in phenotype was observed on two genetic backgrounds. In the less severe class (C57/BL6 background), FN.null embryos display a defective heart. Myocardial cells express the specific marker MF-20 and are correctly localized in the anterior trunk region, but myocardial organization is disrupted, resulting in a bulbous heart tube. Endocardial cells express the specific marker platelet-endothelial cell adhesion molecule-1 (PECAM-1) and are localized within the myocardium, but the endocardium appears collapsed. Endothelial cells of two vascular beds are specified, but the aortae are distended and lack contact with the surrounding mesenchyme, while no vessels form in the yolk sac. Defects in the more severe class suggest that FNs are essential earlier in development on the 129/Sv background. Myocardial and endocardial cells are specified, but morphogenesis of the myocardium and endocardium does not occur. Aortic endothelial cells are specified and localized normally, but remain scattered. Yolk sac endothelial cells resemble those of the less severe class. We conclude that FNs are essential for organization of heart and blood vessels, but are dispensable for cellular specification in the appropriate regions within the embryo.

A region of mouse chromosome 16 is syntenic to the DiGeorge, velocardiofacial syndrome minimal critical region.

DGS and VCFS, haploinsufficiencies characterized by multiple craniofacial and cardiac abnormalities, are associated with a microdeletion of chromosome 22q11.2. Here we document synteny between a 150-kb region on mouse chromosome 16 and the most commonly deleted portion of 22q11.2. Seven genes, all of which are transcribed in the early mouse embryo, have been identified. Of particular interest are two serine/threonine kinase genes and a novel goosecoid-like homeobox gene (Gscl). Comparative sequence analysis of a 38-kb segment reveals similarities in gene content, order, exon composition, and transcriptional direction. Therefore, if deletion of these genes results in DGS/VCFS in humans, then haploinsufficiencies involving this region of chromosome 16 should recapitulate the developmental field defects characteristic of this syndrome.

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1/CD31): A Multifunctional Vascular Cell Adhesion Molecule.

PECAM-1/CD31 is a member of the immunoglobulin gene superfamily found on platelets, leukocytes, and endothelial cells, where it concentrates at cell-cell borders. It has been shown to both mediate cell-cell adhesion through homophilic and heterophilic interactions and to transduce intracellular signals that upregulate the function of integrins on leukocytes. Its cellular distribution and ability to mediate adhesive and signaling phenomena suggested that PECAM-1 was a multifunctional vascular cell adhesion molecule involved in leukocyte-endothelial and endothelial-endothelial interactions. These initial suggestions have been largely confirmed as recent studies have implicated PECAM-1 in the inflammatory process and in the formation of blood vessels. As our understanding of the molecular and functional properties of PECAM-1 grows, new insights will be gained that may have therapeutic implications for cardiovascular development and disease. (Trends Cardiovasc Med 1997;7:203-210). © 1997, Elsevier Science Inc.

Adenovirus-mediated gene transfer during initial organogenesis in the mammalian embryo is promoter-dependent and tissue-specific.

Replication-defective adenoviruses have received increasing attention as vectors for exogenous gene administration in a variety of experimental and pathological conditions. However, little information exists about their utility for in utero gene therapy, and no information exists concerning their efficacy for gene delivery during initial organogenesis in the mammalian embryo. To evaluate the feasibility of using these vectors for exogenous gene transduction during the initial stages of organogenesis in the mammal, we injected an adenovirus vector carrying the bacterial beta-galactosidase (lacZ) gene under the control of either the cytomegalovirus (CMV) promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) into early, post-gastrulation, mouse embryos, and evaluated expression following 36-48 h in culture. These studies suggest that adenovirus-mediated gene delivery may provide an efficient method of gene transduction during critical developmental stages with no detectable adverse effects on normal development during early morphogenesis. In addition, the type of promoter used had a significant effect on the tissue distribution of gene expression.

Relationship of the extracellular matrix to coronary neovascularization during development.

The main goal of this study was to determine the temporal and spatial relationship of several components of the extracellular matrix (ECM) to coronary vascularization during prenatal and early postnatal development. Rat microvessels were visualized by immunolabeling for platelet endothelial cell adhesion molecule (PECAM-1), and by exposure to the lectin from Griffonia simplicifolia I. Coronary vasculogenesis, which first occurs in gestation day 13 (E13) hearts, was preceded by the deposition of fibronectin. The onset of laminin immunoreactivity in basement membranes coincided with tube formation and was followed by the appearance of collagen IV. Discontinuous collagen IV staining of basement membranes typified early tube formation but progressed to completely encircle capillaries. Sparse staining of collagen I and III was observed in prenatal hearts, but increased after birth. Staining for both molecules was limited mainly to the adventitia of vessels larger than capillaries, and as a component of septa and the epicardium. To determine the effects of loading conditions on key ECM molecules relating to neovascularization, avascular E12 rat hearts were grafted to the anterior eye chamber of adult hosts. In these hearts, which are hemodynamically unloaded, the appearance and distribution of ECM components were similar to hearts developing in utero. It was concluded that during heart development: (1) fibronectin may provide a primary scaffolding for the migration of primordial endothelial cells/angioblasts; (2) tube formation coincides with lamin deposition and is closely followed by the appearance of collagen IV; (3) collagens I and III are not related to tube formation in the prenatal heart; and (4) the relationship of the ECM to vessel formation is not notably altered in the absence of a ventricular load. Furthermore the early onset of PECAM-1 immunoreactivity suggests that it is a useful endothelial marker and may play a role in tube formation.