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

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.

Regulation of cardiac cushion development by hyaluronan.

Hyaluronan is an extracellular matrix component implicated in expansion of the extracellular space, organization of supramolecular architecture, cell motility, proliferation, tumour metastases and wound healing. Hyaluronan is highly expressed in the developing heart but it is only a minor component of the mature heart. The loss of hyaluronan synthase-2 (Has2) results in embryonic lethality with a phenotype remarkably similar to that of the versican-deficient heart defect mouse. Has2-deficient embryos lack hyaluronan-containing cardiac jelly, and at embryonic day 9.5 show arrested development, with an apparent absence of the right ventricle and underdevelopment of the conustruncus segment, and pericardial effusion consistent with heart failure. Cardiac cushions are totally absent, and endocardial cell migration over collagen gels is not detectable in Has2-deficient atrioventricular (AV) canal explants. Endothelial to mesenchymal transformation is also defective in AV explants from Has2-null embryos. The normal phenotype is restored in AV canal explants from Has2-deficient embryos by co-culture with wild type AV canal explants, with conditioned media from wild type AV explants or with exogenous hyaluronan. These results provide evidence for a direct role for hyaluronan during endocardial cushion and AV canal morphogenesis.

Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme.

We identified hyaluronan synthase-2 (Has2) as a likely source of hyaluronan (HA) during embryonic development, and we used gene targeting to study its function in vivo. Has2(-/-) embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5-10). Heart explants from Has2(-/-) embryos lack the characteristic transformation of cardiac endothelial cells into mesenchyme, an essential developmental event that depends on receptor-mediated intracellular signaling. This defect is reproduced by expression of a dominant-negative Ras in wild-type heart explants, and is reversed in Has2(-/-) explants by gene rescue, by administering exogenous HA, or by expressing activated Ras. Conversely, transformation in Has2(-/-) explants mediated by exogenous HA is inhibited by dominant-negative Ras. Collectively, our results demonstrate the importance of HA in mammalian embryogenesis and the pivotal role of Has2 during mammalian development. They also reveal a previously unrecognized pathway for cell migration and invasion that is HA-dependent and involves Ras activation.

A novel receptor tyrosine kinase, Mer, inhibits TNF-alpha production and lipopolysaccharide-induced endotoxic shock.

The regulation of monocyte function and the inhibition of TNF-alpha production during bacterial sepsis are critical in attenuating adverse host responses to endotoxemia. To study the function of a novel receptor tyrosine kinase, mer, that is expressed in monocytes, we generated mice (merkd) that lack the signaling tyrosine kinase domain. Upon LPS challenge, merkd animals died of endotoxic shock (15/17, 88.2%), whereas control wild-type mice survived (1/15, 6.7% died). Susceptible merkd mice exhibited edema, leukocyte infiltration, and signs of endotoxic shock that correlated with higher levels of TNF-alpha found in the serum of merkd mice as compared with wild-type control animals. Death due to LPS-induced endotoxic shock in merkd mice was blocked by administration of anti-TNF-alpha Ab, suggesting that overproduction of this cytokine was principally responsible for the heightened suseptibility. The increase in TNF-alpha production appeared to be the result of a substantial increase in the LPS-dependent activation of NF-kappa B nuclear translocation resulting in greater TNF-alpha production by macrophages from merkd mice. Thus, Mer receptor tyrosine kinase signaling participates in a novel inhibitory pathway in macrophages important for regulating TNF-alpha secretion and attenuating endotoxic shock.

Expression of an evolutionarily conserved function associated molecule on sheep, horse and cattle natural killer cells.

Natural killer (NK) cells are large granular lymphocytes that lyse a wide variety of transformed and virally-infected target cells without prior exposure to antigen, and without restriction by major histocompatibility complex antigens. Although NK cells have been identified in a variety of mammalian species, how NK cells recognize antigen and trigger lysis is unknown. Recently, monoclonal antibodies made against NK-like cells from teleost fish were shown to react with NK cells from humans and rats, and to inhibit their cytolytic activity. The role of this apparently evolutionarily conserved function-associated molecule (FAM) has been further investigated utilizing a variety of domesticated farm animal species. It was observed that the anti-FAM mAb reacted specifically with peripheral blood lymphocytes isolated from sheep, horses and cattle. Further, the anti-FAM mAb inhibited NK cell lytic activity in each of these species. Finally, the anti-FAM mAb was found to inhibit conjugate formation between NK and target cells, implying that the FAM was involved in antigen recognition by NK cells in each of these species. In conclusion, it appears that NK cell function is mediated by an evolutionarily conserved FAM in a wide variety of species.

Expression of a novel function-associated molecule on cells mediating spontaneous cytolysis in swine.

In the present study, we have analyzed the effects of the anti-FAM mAb 5C6, on endogenous NK cell and lymphokine-activated killer (LAK) cell activity of swine. We report that the anti-FAM mAb 5C6 binds to fresh and cultured lymphocytes with spontaneous cytotoxic activity from swine peripheral blood. In conjunction, the anti-FAM mAb 5C6 inhibits swine cytolysis against K562 target cells. Inhibition was found to be mediated by blocking of conjugate formation. The level of spontaneous killing and mAb inhibition was correlated with the number of swine cells staining positive with the mAb 5C6 as assessed by flow cytometry.