scholarly journals The Endothelium-Dependent Effects of Thimerosal on Mouse Pial Arterioles in vivo: Evidence for Control of Microvascular Events by EDRF as Well as Prostaglandins

1992 ◽  
Vol 12 (4) ◽  
pp. 703-706 ◽  
Author(s):  
William I. Rosenblum ◽  
Hiroyuki Nishimura ◽  
Earl F. Ellis ◽  
Guy H. Nelson
Keyword(s):  
Platelet Adhesion ◽  
Endothelial Damage ◽  
Cyclooxygenase Inhibitors ◽  
Prostaglandin E ◽  
Cranial Window ◽  
Pial Arterioles ◽  
A Site ◽  
Acting In Concert

Thimerosal causes synthesis and/or release of both endothelium-derived relaxing factor (EDRF) and prostaglandins from conductance vessels in vitro. We tested its effects and mechanism of action on mouse pial arterioles in vivo using intravital microscopic techniques. Topical thimerosal dilated pial arterioles. This effect was eliminated by endothelial injury produced by a laser/Evans blue technique. Dilation was also eliminated by topical l-NMMA, a reported inhibitor of EDRF synthesis. Topical thimerosal also reduced the incidence of platelet adhesion/aggregation (“capture”) at a site of minimal endothelial damage. This effect was eliminated by l-NMMA pretreatment. The ability of thimerosal to dilate arterioles was eliminated not only by treatments thought to eliminate synthesis/release of EDRF, but also by cyclooxygenase inhibitors. However, inhibition of platelet adhesion/aggregation was not affected by cyclooxygenase inhibition. Thimerosal significantly increased production of prostaglandin E2 recovered from a closed cranial window. We conclude that the dilating effects of thimerosal on diameter require two endothelium-derived agents: EDRF and one or more prostaglandins acting in concert. However, the inhibiting effect of thimerosal on local platelet adhesion/aggregation appears to be caused only by an increase in EDRF at the injured site.

Abstract WMP83: Extracellular Vimentin Contributes to Von Willebrand Factor (VWF) String Formation in the Cerebrovasculature Following Endothelial Activation

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Sung-Ha Hong ◽  
Titilope Ishola ◽  
Qi Da ◽  
Miguel A Cruz ◽  
Sean P Marrelli
Keyword(s):  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Endothelial Activation ◽  
Histamine Stimulation ◽  
Cranial Window ◽  
Endothelial Surface ◽  
String Formation ◽  
A2 Domain

Introduction: VWF strings can form within the vascular lumen following endothelial activation and have been implicated in platelet adhesion and worsened outcome following stroke. However, the molecular interactions facilitating VWF string anchorage to the endothelial surface are currently unknown. Here we examined the novel role of endothelial vimentin in mediating the anchorage of VWF strings within the cerebrovasculature. We hypothesize that VWF released from activated endothelium remains anchored at the luminal surface (i.e. VWF strings) through direct interaction with extracellular vimentin. Methods: Cultured endothelial cell (EC) experiments were performed with human umbilical vein EC (HUVECs) and human brain microvascular EC (HBMVECs). EC were stimulated with histamine (10 uM) under flow conditions. Specific protein interactions were probed with recombinant vimentin and A2 domain of VWF. Mouse middle cerebral and superior cerebellar arteries from WT and vimentin KO mice were set up in a pressurized artery chamber, stimulated with histamine, and then processed for VWF immunofluorescence to quantify VWF strings. VWF string formation was further evaluated by cranial window preparation using labeled platelets to detect real-time in vivo string formation. Results: In cultured EC, histamine stimulation promoted the production of long VWF strings that were significantly attenuated in the presence of recombinant vimentin or A2 domain of VWF. In pressurized cerebral arteries, histamine stimulation promoted VWF strings that aligned along the luminal endothelial surface. VWF string formation was reduced 2.9 fold in arteries from vimentin knockout mice (P=0.02). Histamine stimulation in the cranial window produced platelet-adherent strings in the pial microvasculature. Conclusions: These studies provide in vitro and in vivo evidence for a novel interaction between vimentin and the A2 domain of VWF at the endothelial surface which contributes to the anchorage of VWF strings in the microvasculature. This vimentin/VWF interaction critically regulates VWF-mediated platelet adhesion at the surface of activated endothelium and could thus provide a novel therapeutic target in the treatment of acute ischemic stroke.

Endothelium-dependent L-Arg- and L-NMMA-sensitive mechanisms regulate tone of brain microvessels

1990 ◽  
Vol 259 (5) ◽  
pp. H1396-H1401 ◽  
Author(s):  
W. I. Rosenblum ◽  
H. Nishimura ◽  
G. H. Nelson
Keyword(s):  
Endothelial Damage ◽  
Blue Dye ◽  
Dependent Manner ◽  
Splitting Technique ◽  
Resistance Vessels ◽  
Helium Neon Laser ◽  
Pial Arterioles ◽  
Helium Neon

Pial arterioles on the surface of the mouse brain were observed via television microscopy and measured with an image-splitting technique. The vessels were dilated by L-arginine (L-Arg) in concentrations as low as 10(-5) M and were constricted in dose-dependent manner by NG-monomethyl-L-arginine (L-NMMA). Both the dilation and the constriction were abolished by endothelial damage. This damage was produced over a short segment of endothelium by a well-established technique that involves exposing the endothelium to a helium-neon laser in the presence of intravascular Evans blue dye. In arterioles that were responsive to 10(-5) M L-Arg, five other L-amino acids, also at 10(-5) M, failed to have any effect. The data provide direct evidence for the endothelium-dependent nature of the responses to L-Arg and L-NMMA in vivo in a defined segment of the cerebral vasculature. L-NMMA inhibited dilation by either L-Arg or acetylcholine. The data are consistent with data from in vitro studies and from studies demonstrating that L-NMMA acutely raises blood pressure. From all these earlier studies it has been hypothesized that there is a continuously acting, endothelium-dependent, L-Arg-dependent, and L-NMMA-inhibitable mechanism tending to relax blood vessels. The mediator of this mechanism is thought to be the endothelium-dependent relaxing factor for acetylcholine. Our data suggest that this mechanism is acting in the resistance vessels of the brain in vivo.

Pharmacological coupling and functional role for CGRP receptors in the vasodilation of rat pial arterioles

1996 ◽  
Vol 270 (1) ◽  
pp. H317-H323 ◽  
Author(s):  
K. W. Hong ◽  
S. E. Yoo ◽  
S. S. Yu ◽  
J. Y. Lee ◽  
B. Y. Rhim
Keyword(s):  
Channel Blocker ◽  
K Channel ◽  
Receptor Subtype ◽  
Camp Production ◽  
K Channels ◽  
Cranial Window ◽  
Pial Arterioles ◽  
Vasodilator Action

In this study, we investigated the signal transduction underlying the vasodilator action of calcitonin gene-related peptide (CGRP) in the rat pial arterioles. In an in vivo experiment, changes in pial arterial diameters (20.2 +/- 1.9 microns) were observed under suffusion with mock cerebrospinal fluid containing CGRP (10(-9)-10(-7) M) directly through a closed cranial window. Changes in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in response to CGRP and levcromakalim were measured in the pial arterioles in an in vitro experiment. CGRP-induced vasodilation and cAMP production were significantly inhibited by specific CGRP antibody serum and CGRP-(8-37) fragment, suggesting involvement of the CGRP1 receptor subtype. Vasodilation and increase in cAMP production evoked by CGRP were inhibited not only by glibenclamide (ATP-sensitive K+ channel blocker) but also by charybdotoxin (large-conductance Ca(2+)-activated K+ channel blocker), but this was not the case for the isoproterenol-induced vasodilation and cAMP production. These findings implicate the ATP-sensitive K+ channels and the large-conductance Ca(2+)-activated K+ channels in the CGRP receptor-coupled cAMP production for vasodilation. Further study is required to identify whether the cAMP-dependent K+ channel activation is related to CGRP-induced vasorelaxation of the rat pial arterioles.

EFFECTS OF SULFINPYRAZONE AND ITS METABOLITE G25671 ON PLATELET ACTIVATION AND DESENSITIZATION AND ON BRONCHOCONSTRICTION INDUCED BY THE PROSTAGLANDIN ENDOPEROXIDE ANALOGUE U46619

1987 ◽  
Author(s):  
M Hatmi ◽  
A Del Maschio ◽  
J Lefort ◽  
G De Gaetano ◽  
B B Varqaftiq ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Activation ◽  
Ex Vivo ◽  
Oral Treatment ◽  
Human Platelets ◽  
Cyclooxygenase Inhibitors ◽  
Before And After ◽  
A Site

In previous studies we have found (Br. 3. Pharmac. 85, 849, 1985) that a) human platelets pre-exposed to arachidonic acid or to the endoperoxide analogue, U46619 and then washed and resuspended, fail to respond to a second challenge by both arachidonic acid and U46619; b) desensitization by arachidonic acid and U46619 occurs at a site sensitive to endoperoxides / thromboxane (Tx) receptor antagonists; c) the desensitizing effects of U46619 are direct, whereas those of arachidonic acid are mediated by a cyclooxygenase-dependent metabolite. Sulfinpyrazone (100 μM) and its thioether metabolite G25671 (50 μM) are known to suppress arachidonic acid-induced platelet aggregation and TxB2 formation (Eur. 3. Pharmac, 101, 209, 1984). We now demonstrate that the presence of sulfinpyrazone or G25671 during platelet exposure to arachidonic acid or U46619 prevents desensitization. Platelet activation by the endoperoxide analogue U46619 is also prevented by sulfinpyrazone or G25671 (0.3-1 mM). The threshold aggregating concentrations of arachidonic acid and U46619 in healthy subjects before and after oral treatment with sulfinpyrazone were elevated by 2-3 fold and a good correlation between ex vivo and in vitro findings was established. We finally examined the actions of sulfinpyrazone and G25671 on the bronchoconstriction in vivo and parenchymal lung strip contraction in vitro induced by U46619. Neither drug had any preventive effect.Our results demonstrate that sulfinpyrazone and its metabolite G25671 are not only cyclooxygenase inhibitors but can also act as endoperoxide/Tx antagonists and indicate clearly that antagonism of U46619 by both drugs is selective for platelets.

Comparison of in vivo and in vitro prostaglandin E production by squamous cell carcinoma of the head and neck

1994 ◽  
Vol 111 (3) ◽  
pp. 189-196 ◽  
Author(s):  
C SNYDERMAN ◽  
I KLAPAN ◽  
M MILANOVICH ◽  
D HEO ◽  
R WAGNER ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Head And Neck ◽  
Squamous Cell ◽  
Prostaglandin E

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

scholarly journals A potential role for the COOH-terminal domain in the lateral packing of type III intermediate filaments.

1991 ◽  
Vol 114 (4) ◽  
pp. 773-786 ◽  
Author(s):  
P D Kouklis ◽  
T Papamarcaki ◽  
A Merdes ◽  
S D Georgatos
Keyword(s):  
Potential Role ◽  
Type Iii ◽  
Filament Assembly ◽  
Filament Bundles ◽  
Self Association ◽  
Specific Association ◽  
A Site ◽  
Idiotypic Antibody

To identify sites of self-association in type III intermediate filament (IF) proteins, we have taken an "anti-idiotypic antibody" approach. A mAb (anti-Ct), recognizing a similar feature near the end of the rod domain of vimentin, desmin, and peripherin (epsilon site or epsilon epitope), was characterized. Anti-idiotypic antibodies, generated by immunizing rabbits with purified anti-Ct, recognize a site (presumably "complementary" to the epsilon epitope) common among vimentin, desmin, and peripherin (beta site or beta epitope). The beta epitope is represented in a synthetic peptide (PII) modeled after the 30 COOH-terminal residues of peripherin, as seen by comparative immunoblotting assays. Consistent with the idea of an association between the epsilon and the beta site, PII binds in vitro to intact IF proteins and fragments containing the epsilon epitope, but not to IF proteins that do not react with anti-Ct. Microinjection experiments conducted in vivo and filament reconstitution assays carried out in vitro further demonstrate that "uncoupling" of this site-specific association (by competition with PII or anti-Ct) interferes with normal IF architecture, resulting in the formation of filaments and filament bundles with diameters much greater than that of the normal IFs. These thick fibers are very similar to the ones observed previously when a derivative of desmin missing 27 COOH-terminal residues was assembled in vitro (Kaufmann, E., K. Weber, and N. Geisler. 1985. J. Mol. Biol. 185:733-742). As a molecular explanation, we propose here that the epsilon and the beta sites of type III IF proteins are "complementary" and associate during filament assembly. As a result of this association, we further postulate the formation of a surface-exposed "loop" or "hairpin" structure that may sterically prevent inappropriate filament-filament aggregation and regulate filament thickness.

scholarly journals In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease.

1990 ◽  
Vol 10 (8) ◽  
pp. 3868-3872 ◽  
Author(s):  
C M Shumard ◽  
C Torres ◽  
D C Eichler
Keyword(s):  
18S Rrna ◽  
Precise Determination ◽  
In Vivo Studies ◽  
Rrna Sequence ◽  
S1 Nuclease ◽  
In Vitro Processing ◽  
Rrna Maturation ◽  
A Site

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

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