Thomas H. Rosenquist - US grants

Chicken embryos which undergo ablation f cardiac neural crest manifest characteristic heart defects. This stud will test the hypothesis that the elastic arteries in these embryos will also have an associated defect, in the following parameters: (1) the composition, deployment and alignment of the elastic matrix of the vessels; (2) the function of the vessels; and (3) the ability of the vessels to respond to stimulation of elastogenesis. These hypotheses will be tested by integrated analyses of structure and function in control and experimental embryos ranging from stage 28 (septation) to stage 46(hatching). The time of occurrence and distribution of tropoelastins and other elastinrelated proteins will be determined by immunohistochemistry. The alignment of the elastic matrix will be determined by measurement of induced birefringence. Functional capacity of the vessels will be determined by measurement of their active and passive length=tension relationships. Response to elastogenic stimulation will be measured in vessels in vitro. This integrated study of structure, function and experimental embryology will establish the basic biology of normal vessel development; and abnormal vessel development that accompanies heart anomalies. These data may then provide a basis for rational design of a prognosis and treatment plan for the pulmonary artery defect that accompanies cyanotic congenital heart disease in man; which may be fatal; and for which there is n cure.

Atherosclerosis occurs regularly at certain sites in the vessels of man and some experimental models, while other sites are spared. The bases of susceptibility and resistance may lie in the morphogenesis of the vessels. Vessels whose smooth muscle originates in the neural ectoderm are relat- ively spared, while those whose smooth muscle arises from local mesoderm are relatively susceptible; hybrid areas at the ectodermal-mesodermal interface are most susceptible. The long-term goal of this study is to determine the relationship between vessel morphogenesis and the development of atherosclerosis, using a well-described avian model. Properties that are known to be associated with atherogenesis will be compared during embryogenesis in the vessels of atherosclerosis-susceptible and resistant avian strains. Then, to test the nature of the interaction between heterogeneous vessels the ectodermal vessel primordia will be transplanted from the neural crest of atherosclerosis-prone donor embryos onto resistant recipients; and conversely. Finally, the smooth muscle cell growth-promoting capacity of ectodermal vessels will be determined. The studies proposed here will provide new data on the origin, development, structure, composition, and growth-promoting capacities of vessels of differing origins. It will be the first integrated and comprehensive study of the comparative developmental biology of genetically resistant and susceptible vessel walls. Ultimately these studies should be useful to the clinician as the relationship between developmental factors and susceptibility to atherosclerosis becomes more clearly defined.

Supplemental folic acid is associated with significant decreases in conotruncal, orofacial, and neural tube defects. The amino acid homocysteine increases with folate deficiency, and elevated homocysteine per se appears to be a risk factor for these defects. Our recent studies have led to the hypothesis that homocysteine may perturb neural crest and neural tube development by acting as an antagonist for the N-methyl-D-aspartate glutamate receptor (NMDAR). Indeed, some of the best known risk factors for neural crest and neural tube abnormalities also are NMDAR antagonist. The present proposal will test the hypothesis that homocysteine induces conotruncal and related defects by acting as an NMDAR antagonist. Both the chicken and the mouse embryo models will be employed in these experiments. There are three aims of this proposal: Aim 1, to test the hypotheses that NMDAR agonists will rescue homocysteine-treated embryos, and conversely, that exogenous NMDAR antagonists will interact synergistically with homocysteine to exacerbate the disruption of normal development. Aim 2, to test the hypothesis that homocysteine and exogenous NMDAR antagonists disrupt the expression of key genes during hypothesis that homocysteine and exogenous NMDAR antagonists disrupt the expression of key genes during neural crest migration and neural tube closure. Aim 2, to determine the effect of homocysteine and exogenous NMDAR antagonists on neural crest cell functions. This proposal offers the first unifying hypothesis regarding a mechanism for a set of important risk factors for abnormal development that includes therapeutic and recreational drugs, environmental contaminants, and low folate. A common mechanism of actions would show that these factors may interact in previous unsuspected ways. This proposal will begin to explore the role of gene/environment interactions in the induction of these abnormalities. Although folate supplements will result in fewer abnormalities, the most effective and comprehensive prevention strategies will come through a thorough understanding of the mechanisms that underlie abnormal development..

homocysteine; NMDA receptors; pathologic process; inhibitor /antagonist; cell growth regulation; vascular smooth muscle; atherosclerosis; atherosclerotic plaque; growth factor receptors; mitogens; growth factor; laboratory rabbit;

The central theme and overall objective of this program is to determine the biological mechanisms whereby folic acid insufficiency and hyperhomocysteinemia may contribute to abnormal development of the heart. This program project is designed to provide maximum focus upon this theme, and to optimize scientific and intellectual synergy among members of the research team. Discovery of the cellular mechanisms that provide this protection is the objective of the research program proposed here. Two hypotheses will be tested: Hyperhomocysteinemia that results from folic acid insufficiency may induce abnormal development of the conotruncal region of the heart, as well as other neural crest and neural tube derivatives, by inhibiting the function of N-methyl-D-aspartate receptors (NMDA). Folate insufficiency also may induce abnormal development by a direct effect upon the growth and differentiation of neural crest and neural tube cells directly, for example, by limiting the availability of methyl groups for gene methylation. A principle objective of this research program is to sort out the biological effects of low folate from those of hyperhomocysteinemia; and to determine how these two mechanisms may interact. It is inferred that they converge upon processes that are especially critical to the cardiac neural crest, other regions of the neural crest, and the neural tube. Project 1 will examine the teratogenic interaction of homocysteine with other NMDA antagonists, and will determine the degree to which embryos can be rescued by NMDA activation. Project 2 will investigate the impact of impaired folate binding and transport on the development of the heart, as well as other neural crest and neural tube derivatives, using transgenic mouse embryos models made for this purpose. Project 3 will concentrate upon the relative roles of hyperhomocysteinemia and the NMDA on the one hand, and folate insufficiency on the other, as they impact on neural crest cell migration and differentiation. Project 4 will test the elements of each of these hypotheses in a population-based study.

The biological basis for well-known relationships among conotruncal and neural tube defects is now generally assumed to be found in the common origin of their primordial cells. However, there is no consensus about the mechanism that leads to these defects. A high level of protection of protection is offered for conotruncal, other neural crest, and neural tube defects by folic acid supplementation, implying that an unnamed process of extraordinary importance is sensitively dependent upon an adequate supply of folic acid. For the present proposal, we will test the hypothesis that homocysteine is a teratogen for the conotruncus and other derivatives of the neuroepithelium; and that folic acid supplementation provides protection for embryos by reducing the concentration of homocysteine can induce abnormal development of the conotruncus and other neural crest/neural tube derivatives by acting as an NMDA receptor (NMDA) antagonist. By this mechanism, homocysteine may interact with other NMDA antagonists to exacerbate the teratogenic effect; conversely, it may be predicted that activation of the NMDA would rescue embryos exposed to homocysteine and related compounds. The following specific aims will test this hypothesis. Aim 1, to determine how exogenous NMDA antagonists may interact with homocysteine to exacerbate the disruption of normal development. Aim 2, to measure the degree to which activation of the NMDA may rescue embryos that are treated with homocysteine and related compounds. Aim 3, to analyze changes in gene expression in embryos treated with homocysteine and other NMDA antagonists. SIGNIFICANCE. This proposal offers the first unifying hypothesis regarding a mechanism for a large set of risk factors for abnormal development whose common effect is to inhibit the function of the NMDA. These may include therapeutic drugs, recreation drugs, environmental pollutants, and sequelae of malnutrition. A common mechanism of action would permit these factors to interact in previously unsuspected ways, potentially to exacerbate their respective effects. Effective and comprehensive prevention strategies may be achieved through understanding of such interactive mechanisms.