Differences in hyaluronic acid-mediated functions and signaling in arterial, microvessel, and vein-derived human endothelial cells

Hyaluronic acid (HA), a nonsulfated glycosaminoglycan, regulates cell adhesion and migration. Small HA fragments (3-25 disaccharide units) induce neovascularization. We investigated the effect of HA and a HA fragment (10-15 disaccharide units, F1) on primary human endothelial cells (ECs). Human pulmonary ECs (HPAEC) and lung microvessel ECs (HMVEC-L) bound HA (K(d) approximately 1 and 2.3 nm, respectively) and expressed 17,780 and 16,690 HA binding sites, respectively. Both ECs showed HA-mediated cell adhesion; however, HMVEC-L was 1.5-fold better. Human umbilical vein ECs neither bound HA nor showed HA-mediated adhesion. All three ECs expressed CD44 ( approximately 110 kDa). The expression of receptor for HA-mediated motility (RHAMM) (approximately 80 kDa) was the highest in HMVEC-L, followed by HPAEC and human umbilical vein ECs. RHAMM, not CD44, bound HA in all three ECs. F1 was better than HA and stimulated a 2. 5- and 1.8-fold mitogenic response in HMVEC-L and HPAEC, respectively. Both HA and F1 induced tyrosine phosphorylation of p125(FAK), paxillin, and p42/44 ERK in HMVEC-L and HPAEC, which was blocked by an anti-RHAMM antibody. These results demonstrate that RHAMM is the functional HA receptor in primary human ECs. Heterogeneity exists among primary human ECs of different vascular origins, with respect to functional HA receptor expression and function.     

Cytoskeletal changes correlated with the loss of neuronal polarity in axotomized lamprey central neurons

Axotomy within 500 μm of the soma (close axotomy) causes identified neurons (anterior bulbar cells or ABCs) in the lamprey hindbrain to lose their normal polarity and regenerate axons ectopically from dendritic tips, while axotomy at more distal sites (distant axotomy) results in orthotopic axonal regeneration from the axon stump. We performed immunocytochemical, electron microscopic and in situ hybridization analyses comparing ABCs subjected to close and distant axotomy to elucidate the mechanism by which neuronal polarity is lost. We show that polarity loss in ABCs is selectively and invariably preceded and accompained by the following cellular changes: (1) a loss of many dendritic microtubules and their replacement with neurofilaments, (2) a loss of immunostaining for acetylated tubulin in the soma and proximal dendrites, and (3) an increase of immunostaining for phosphorylated neurofilaments in the distal dendrites. We also show that these changes do not depend on either the upregulation or spatial redistribution of neurofilament message, and thus must involve changes in the routing of neurofilament protein within axotomized ABCs. We conclude that close axotomy causes dendrites to undergo axonlike changes in the mechanisms that govern the somatofugal transport of neurofilament protein, and suggest that these changes require the reorganization of dendritic microtubules. We also suggest that the bulbous morphology and lack of f-actin in the tips of all regenerating sprouts supports the possibility that axonal regeneration in the lamprey CNS does not involve actin-mediated "pulling" of growth cones, but depends instead on the generation of internal extrusive forces.     

Inactivation of soybean lipoxygenase 1 by 12-iodo-cis-9-octadecenoic acid

12-Iodo-cis-9-octadecenoic acid (12-IODE) is a time-dependent, irreversible inactivator of soybean lipoxygenase 1. The rate of inactivation is independent of 12-IODE concentration above 20 microM and is half-maximal at about 4 microM. Inactivation by 12-IODE requires lipid hydroperoxide, which must be present even after the initial oxidation of the iron in the enzyme from ferrous to ferric. Inactivation by 12-IODE is also dependent on O2. These findings suggest that 12-IODE is converted by the enzyme into a more reactive species, which is responsible for inactivation. No inactivation has been detected with 12-iodooctadecanoic acid, 12-bromo-cis-9-octadecenoic acid, 12-iodo-trans-9-octadecenoic acid, or a mixture of stereoisomers of 9,11-octadecadienoic acid.     

Differentiation and visualization of diverse cellular phenotypic responses in primary high-content screening

High-throughput screening, based on subcellular imaging, has become a powerful tool in lead discovery. Through the generation of high-quality images, not only the specific target signal can be analyzed but also phenotypic changes of the whole cell are recorded. Yet analysis strategies for the exploration of high-content screening results, in a manner that is independent from predefined control phenotypes, are largely missing. The approach presented here is based on a well-established modeling technique, self-organizing maps (SOMs), which uses multiparametric results to group treatments that create similar morphological effects. This report describes a novel visualization of the SOM clustering by using an image of the cells from each node, with the most representative cell highlighted to deploy the phenotype described by each node. The approach has the potential to identify both expected hits and novel cellular phenotypes. Moreover, different chemotypes, which cause the same phenotypic effects, are identified, thus facilitating "scaffold hopping."     

PULMONARY HEART DISEASE—A Panel Discussion

Abruptio placentae, in its severe form, causes the most hazardous type of third trimester bleeding. The severe grade may be accompanied by systemic effects, some of which are potentially lethal. A knowledge of these, as well as a system of grading the severity in terms of maternal risk, is essential to an understanding of therapy. Cases should be graded in severity from I to III on the basis of clinical factors. A delay in delivery, in Grade III, may result in an increased incidence of serious maternal complications. In Grade II, immediate cesarean section has reduced the fetal mortality rate.In managing Grade III premature separation of the placenta, the following steps should be carried out: (1) Laboratory study, including blood cross-matching and determination of plasma fibrinogen; (2) vaginal examination to confirm the diagnosis and to rupture of the membranes; (3) indicated therapy of systemic effects with fresh whole blood and fibrinogen, before considering any operative delivery; (4) election of a mode of delivery which will terminate the pregnancy in less than about six hours after onset of separation; this will frequently be cesarean section; (5) careful attention to postpartum care to avoid shock and renal failure.In Grade II, the same principles of therapy obtain. If the fetal heart tones are present, however, and vaginal delivery is not imminent, immediate cesarean section is justified. Complete conservatism, with vaginal delivery, is recommended in Grade I.