Adventitia-derived nitric oxide in rat aortas exposed to endotoxin: cell origin and functional consequences

The role of adventitial cells in bacterial lipopolysaccharide (LPS)-induced vascular nitric oxide (NO) overproduction has been largely ignored. In rat aortas exposed to LPS in vitro or in vivo, it was found that adventitia contained the major part of NO synthase (NOS)-2 protein (Western blot and immunohistochemistry) and generated the largest amount of NO (electron paramagnetic resonance spin trapping). NOS-2 immunoreactive cells were mainly resident macrophages at an early stage (5 h, in vitro or in vivo) and fibroblasts at a later stage (20 h, in vitro). Adventitial NOS-2 activity largely accounted for 1) the relaxing effect of L-arginine in rings exposed to LPS in vivo, 2) generation of an "NO store" revealed by N-acetylcysteine-induced relaxation, and 3) formation of protein-bound dinitrosyl iron complexes in the medial layer of aortic rings exposed to LPS in vitro. In conclusion, the adventitia is a powerful source of NO triggered by LPS in the rat aorta. This novel source of NO has an important impact on smooth muscle function and might be implicated in various inflammatory diseases.     

Osmotic fragility of the erythrocyte membrane: characterization by modeling of the transmittance curve as a function of the NaCl concentration

The theoretical extinction of blood suspensions submitted to a slow dialysis is analyzed in terms of their NaCl concentration. The model involves two adjustable parameters, chi and K, related to swelling and hemolysis. During swelling, the erythrocyte volume is supposed to vary linearly with the saline concentration. During hemolysis, an exponential decay of the hemoglobin concentration in the erythrocyte is used. The theoretical transmittance curves are consistent with the measurements carried out at a wavelength of 0.808 microm on native and incubated blood samples. Chi and K are relevant parameters to characterize quantitatively the fragility of the erythrocyte membrane. The effect of a non ideal character of the hemoglobin solutions and of normal distributions of chi and K is also discussed.     

Nic1p, a relative of bacterial transition metal permeases in Schizosaccharomyces pombe, provides nickel ion for urease biosynthesis

The Schizosaccharomyces pombe genome sequencing project identified an open reading frame (O74869 and O74912, named Nic1p in the present study) with significant similarity to members of a family of bacterial transition metal permeases. These uptake systems transport Ni(2+) ion with extremely high affinity across the bacterial cytoplasmic membrane, but they differ in selectivity toward divalent transition metal cations. An S. pombe mutant harboring an interrupted nic1 allele (nic1-1) was strongly impaired in (63)Ni(2+) uptake in the presence of a high molar ratio of Mg(2+) relative to Ni(2+), conditions that reflect the natural situation. Under these conditions, the nic1-1 mutant contained only background activities of the nickel-dependent cytoplasmic enzyme urease and could not catabolize urea. Among a series of divalent transition metal cations tested (Cd(2+), Co(2+), Cu(2+), Mn(2+), and Zn(2+)), only Co(2+) caused considerable inhibition of Nic1p-mediated Ni(2+) uptake. On the other hand, experiments with (57)Co(2+) (at nm concentrations) did not show significant differences in Co(2+) uptake between the nic1-1 mutant and the parental strain. Our data suggest that Nic1p acts as a plasma-membrane nickel transporter in fission yeast, a finding that invites searches for isologous counterparts in higher eukaryotes.     

Monocytes induce reversible focal changes in vascular endothelial cadherin complex during transendothelial migration under flow

The vascular endothelial cell cadherin complex (VE-cadherin, alpha-, beta-, and gamma-catenin, and p120/p100) localizes to adherens junctions surrounding vascular endothelial cells and may play a critical role in the transendothelial migration of circulating blood leukocytes. Previously, we have reported that neutrophil adhesion to human umbilical vein endothelial cell (HUVEC) monolayers, under static conditions, results in a dramatic loss of the VE-cadherin complex. Subsequent studies by us and others (Moll, T., E. Dejana, and D. Vestweber. 1998. J. Cell Biol. 140:403-407) suggested that this phenomenon might reflect degradation by neutrophil proteases released during specimen preparation. We postulated that some form of disruption of the VE-cadherin complex might, nonetheless, be a physiological process during leukocyte transmigration. In the present study, the findings demonstrate a specific, localized effect of migrating leukocytes on the VE-cadherin complex in cytokine-activated HUVEC monolayers. Monocytes and in vitro differentiated U937 cells induce focal loss in the staining of VE-cadherin, alpha-catenin, beta-catenin, and plakoglobin during transendothelial migration under physiological flow conditions. These events are inhibited by antibodies that prevent transendothelial migration and are reversed following transmigration. Together, these data suggest that an endothelial-dependent step of transient and focal disruption of the VE-cadherin complex occurs during leukocyte transmigration.     

Inducible NO synthase activity in blood vessels and heart: new insight into cell origin and consequences

Induction of the inducible form of nitric oxide synthase (iNOS) in the vascular and cardiac tissue by several inflammatory stimuli may result in the production of large amounts of nitric oxide (NO) for a sustained period. Recent data obtained in the rat aorta in which iNOS was induced by lipopolysaccharide (LPS) have demonstrated that adventitial cells represent the main site of NO production. Adventitial-derived NO can exert an immediate down-regulatory effect on smooth muscle contraction (via activation of the cyclic GMP pathway) but may also initiate longer lasting effects through the formation of NO stores within the medial layer. One candidate for such NO stores are dinitrosyl non-heme iron complexes. Low molecular weight thiols interact with preformed NO stores and promote vasorelaxation by a cyclic GMP-independent mechanism involving the activation of potassium channels. In the heart, the induction of iNOS is involved in delayed protection against ischemia-reperfusion-induced functional damages. Recent data obtained with monophosphoryl lipid A, a non-toxin derivative of LPS, strongly suggest that iNOS-derived NO in the rat heart does not act as an immediate mediator of the cardioprotection but rather as a trigger of long-term protective mechanisms. Thus, the present data reveal the important role of adventitial cells as a site of iNOS expression and activity in intact blood vessels. The induction of adaptive mechanisms in the heart and the formation of releasable NO stores in blood vessels are examples of long-term consequences of iNOS induction. These new information are relevant for a better understanding of the circumstances in which NO overproduction by iNOS may play either a beneficial or deleterious role in these tissues.     

Suppression of tumor growth and metastasis in Mgat5-deficient mice

Golgi beta1,6N-acetylglucosaminyltransferase V (MGAT5) is required in the biosynthesis of beta1,6GlcNAc-branched N-linked glycans attached to cell surface and secreted glycoproteins. Amounts of MGAT5 glycan products are commonly increased in malignancies, and correlate with disease progression. To study the functions of these N-glycans in development and disease, we generated mice deficient in Mgat5 by targeted gene mutation. These Mgat5-/- mice lacked Mgat5 products and appeared normal, but differed in their responses to certain extrinsic conditions. Mammary tumor growth and metastases induced by the polyomavirus middle T oncogene was considerably less in Mgat5-/- mice than in transgenic littermates expressing Mgat5. Furthermore, Mgat5 glycan products stimulated membrane ruffling and phosphatidylinositol 3 kinase-protein kinase B activation, fueling a positive feedback loop that amplified oncogene signaling and tumor growth in vivo. Our results indicate that inhibitors of MGAT5 might be useful in the treatment of malignancies by targeting their dependency on focal adhesion signaling for growth and metastasis.