Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion

The effect of flow on the adhesion of polymorphonuclear leukocytes (PMNL) to vascular endothelium was investigated using a parallel plate chamber with a well-defined flow field. Washed PMNL were perfused over a monolayer of primary human umbilical vein endothelial cells (HUVEC) pretreated with formyl-methionyl-leucyl-phenylalanine (FMLP, 1 X 10(-7) mol/L) for five minutes. In other experiments HUVEC were pretreated with interleukin 1 (IL1,2 U/mL) for four hours. PMNL adhesion to stimulated and control HUVEC was measured over a physiologic range of wall shear stresses. PMNL adhesion to nylon-coated surface was also studied. At a wall shear stress of 0.98 dynes/cm2,283 +/- 37.3 PMNL/mm2 (mean +/- SEM) adhered to FMLP-treated HUVEC while 195 +/- 20.3 PMNL/mm2 adhered to control HUVEC. At 1.96 dynes/cm2, 68 +/- 14.1 PMNL/mm2 adhered to FMLP-treated HUVEC and 42 +/- 6.0 PMNL/mm2 adhered to control HUVEC. At 3.92 dynes/cm2, virtually no PMNL adherence was noted on either control or FMLP-treated HUVEC. On IL 1-treated HUVEC at 1.96 dynes/cm2, 371 +/- 25.8 PMNL/mm2 adhered while 28 +/- 2.9 PMNL/mm2 adhered to control HUVEC. PMNL adhesion to IL 1-treated and control HUVEC dropped to 10.2 +/- 3.8 and 6.8 +/- 3.5 PMNL/mm2, respectively, at 3.01 dynes/cm2. The effect of flow on PMNL adhesion appears to be an important factor in determining the outcome of the PMNL/HUVEC adhesive interaction under these experimental conditions.     

Effect of venous shear stress on CD18-mediated neutrophil adhesion to cultured endothelium

The CD11/CD18 family of glycoproteins has been identified as a mediator of a number of adhesive interactions crucial to inflammatory responses. Using a monoclonal antibody (MoAb) against CD18 (TS1/18), the role of these molecules in polymorphonuclear neutrophil (PMNL) adhesion to cultured primary human umbilical vein endothelial cells (HUVEC) was examined under venous flow conditions. Incubation of PMNL with TS1/18 (anti-CD18) did not inhibit PMNL adhesion to interleukin-1 (IL-1)-treated HUVEC at 2.0 dynes/cm2 (TS1/18-treated 305 +/- 58 PMNL/mm2 v 334 +/- 63 PMNL/mm2 on control). Furthermore, incubation of HUVEC with R6.5.D6, an MoAb against intercellular adhesion molecule-1 (ICAM-1) did not significantly inhibit PMNL adhesion to IL-1-treated HUVEC at 2.0 dynes/cm2 (P greater than .3). In contrast to the lack of inhibition of adhesion under conditions of flow, incubation of PMNL with TS1/18 reduced PMNL adherence in static adhesion assays. PMNL migration beneath HUVEC monolayers has been shown to be stimulated by 4-hour IL-1 treatment. TS1/18 and R6.5.D6 significantly inhibited migration of PMNL beneath IL-1-treated HUVEC monolayers under flow conditions by slightly more than 80% (P less than .005). In flow experiments with CD18-deficient PMNL, virtually no transendothelial migration was observed. The effect of FMLP (10(-8) mol/L) on PMNL adhesion to untreated HUVEC at wall shear stresses ranging from 0.25 to 2.0 dynes/cm2 was also investigated. FMLP had little effect on PMNL adherence at shear stresses above 0.5 dynes/cm2 (P greater than .45). In response to FMLP exposure at lower wall shear stresses, PMNL adherence to untreated HUVEC increased 6.9-fold at 0.5 dynes/cm2 (P less than .001). At 0.25 dynes/cm2, FMLP stimulation increased PMNL adherence to untreated HUVEC 6.5-fold compared with controls (P less than .005), and FMLP failed to make CD18-deficient PMNL more adherent. In experiments with PMNL pretreated with TS1/18 (anti-CD18), there was a 67% inhibition of FMLP-stimulated adhesion at 0.5 dynes/cm2 (P less than .025). The upper threshold of CD18-mediated PMNL adhesion appears to be between 0.5 and 1.0 dyne/cm2. Above these wall shear stresses, the initial attachment of PMNL to cultured endothelium was mediated almost exclusively by CD18-independent mechanisms. By simulating some of the flow parameters in the microcirculation with well-characterized shear forces, PMNL adhesion by CD18-independent and dependent mechanisms can be differentiated. These data also indicate that CD18 is an important mediator of transendothelial migration by PMNL, which have attached to the endothelium by a CD18-independent mechanism.     

Decreased levels of cytochrome P450 2E1-derived eicosanoids sensitize renal arteries to constrictor agonists in spontaneously hypertensive rats

We compared renal interlobar arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) in terms of cytochrome P450 (CYP) 4A and CYP2E1 protein expression; levels of 20-HETE, 19-HETE, and 18-HETE; and responsiveness to phenylephrine in the absence and presence of N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 30 mumol/L), a CYP4A inhibitor. Relative to data in WKY, arteries of SHR exhibited diminished (P<0.05) CYP2E1 and levels of 19-HETE (66.7+/-6.0 versus 44.9+/-2.8 pmol/mg) and 18-HETE (13.8+/-1.6 versus 7.9+/-0.5 pmol/mg), whereas CYP4A and 20-HETE levels (99.3+/-9.1 versus 98.9+/-12.8 pmol/mg) were unchanged. Phenylephrine contracted vascular rings of SHR and WKY; the R(max) was similar in both strains, but SHR vessels were more sensitive as denoted by the lower (P<0.05) EC50 (0.28+/-0.07 versus 0.71+/-0.12 mumol/L). DDMS decreased 20-HETE and, to a lesser extent, 19-HETE, while increasing (P<0.05) the EC50 for phenylephrine by 475% and 54% in vessels of SHR and WKY, respectively. The desensitizing effect of DDMS was reversed by 20-HETE. Notably, the minimal concentration of 20-HETE that decreased the EC50 for phenylephrine in DDMS-treated vessels was smaller in SHR (0.1 micromol/L) than WKY (10 micromol/L), and the sensitizing effect of 20-HETE was blunted (P<0.05) by the (R) stereoisomers of 19-HETE and 18-HETE. We conclude that the increased sensitivity to phenylephrine in arteries of SHR is attributable to a vasoregulatory imbalance produced by a deficit in vascular CYP2E1-derived products, most likely 19(R)-HETE and 18(R)-HETE, which condition amplification of the sensitizing action of 20-HETE.     

Effect of anti TNFalpha therapy on arterial diameter and wall shear stress and HDL cholesterol

It has been recently hypothesized that both TNFalpha and anti TNFalpha treatment have a stimulating effect on nitric oxide synthesis and release. Moreover, an in vitro experiment has demonstrated that HDL-cholesterol binds TNFalpha. Aims of our study were to investigate wall shear stress of peripheral arteries and endothelial function of brachial artery in subjects with Rheumatoid Arthritis (RA) at baseline and after infliximab. Moreover, we evaluated the effect of anti TNFalpha therapy on lipid profile. Ten patients with RA received infliximab therapy at weeks 0, 2 and 6. Lipids and vascular parameters were measured before and the day after each infusion. After the first treatment, FMD increased (3.7 +/- 1.9% versus 17.5 +/- 2.9%, P <0.01) and common carotid and brachial artery diameters decreased (5.9 +/- 0.2 mm versus 5.5 +/- 0.2 mm; 3.5 +/- 0.4 mm versus 3.1 +/- 0.4 mm, respectively, P <0.005). Common carotid and brachial artery wall shear stress increased (21.1 +/- 1.1 dynes/cm2 versus 23.9 +/- 1.4 dynes/cm2; 42.0 +/- 4.7 dynes/cm2 versus 51.6 +/- 5.7 dynes/cm2, P <0.01). Similar results were observed after the second and third infusion. All these parameters returned to pre-treatment level at the following infusion. HDL-cholesterol and apolipoprotein AI significantly decreased after each treatment (1st treatment: 1.4 +/- 0.05 mmol/L versus 1.2 +/- 0.06 mmol/L, P <0.01; 1.73 +/- 0.05 g/L versus 1.57 +/- 0.02 g/L, P <0.03). The present data show vasoconstriction and an increase of wall shear stress in studied arteries after infliximab. HDL cholesterol is reduced by treatment and does not seem to influence FMD.     

Modulation of arachidonic acid metabolism by olsalazine and other aminosalicylates in leukocytes.

We investigated the action of the new aminosalicylate olsalazine (disodium azodisalicylate) on arachidonic acid metabolism in comparison with 5-aminosalicylic acid (5-ASA) and sulphasalazine (SASP) by in vitro incubation of cellular homogenates from human polymorphonuclear (PMNL) and mononuclear (MNL) leukocytes with 14C-labelled arachidonic acid. Olsalazine reduced the synthesis of leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (5-HETE), 11-HETE, 12-HETE, and 15-HETE in PMNL and MNL slightly less than SASP. 5-ASA was significantly less inhibitory than olsalazine and SASP on the formation of lipoxygenase products in PMNL and on LTB4 synthesis in MNL. In contrast, in MNL the formation of 5-HETE was unaffected, and the production of 11-HETE, 12-HETE, and 15-HETE was even slightly activated by 5-ASA. Total prostaglandin synthesis was dose-dependently reduced by the aminosalicylates (SASP greater than olsalazine greater than 5-ASA), but only SASP markedly altered the prostaglandin (PG) profile, with an increase in PGE2 and PGF2 alpha at the expense of other cyclooxygenase products. It may be concluded that olsalazine resembled SASP with regard to the inhibition of the lipoxygenase but had effects intermediate between the other salicylates on cyclooxygenase. Furthermore, the alteration of the prostaglandin profile by SASP points to an overlying cofactor effect of this drug.     

The functional expression of tissue factor by fibroblasts and endothelial cells under flow conditions

The expression of tissue factor (TF) by a variety of vascular cell types under physiologic flow conditions is critical to factor X activation and in vivo clotting. Therefore, in a parallel-plate flow chamber (volume 40 microL) we mounted monolayers of human embryonic fibroblasts (FBs) or interleukin-1 alpha (IL-1 alpha) (5 U/mL x 4 hours)-stimulated human umbilical vein endothelial cells (ECs). Inflow buffer contained 10 nmol/L factor VIIa, 100 nmol/L factor X, and 2.0 mmol/L CaCl. With FBs, production of factor Xa (product of outflow concentration of factor Xa-and flow rate) increased 200-fold over the range of shear stress from 0 to 2.7 dynes/cm2. Production values (mean +/- SE (N)) were 7.93 +/- 0.024 (6), 312 +/- 7.3 (6), 688 +/- 33.1 (8), 1,033 +/- 119 (6), and 1,601 +/- 183 (7) fmol/cm2.minute at shear stresses of 0, 0.27, 0.68, 1.35, and 2.7 dynes/cm2, respectively. Further experiments at 0.68 dynes/cm2 indicated that factor Xa production increased with factor X concentration over the range from 3 to 100 nmol/L, but changed little from 300 to 1,000 nmol/L. With ECs, production was 0.13 +/- 0.86 (6), 8.17 +/- 1.65 (13), and 1.66 +/- 1.66 (5) fmol/cm2.minute at 0, 0.68, and 2.7 dynes/cm2, respectively. However, in the presence of an antibody directed against tissue factor pathway inhibitor (TFPI) production with ECs was augmented to 16.46 +/- 0.80 (8), 149.8 +/- 18.6 (8), and 48.9 +/- 10.3 (10), respectively, at these same shear stresses. Control experiments with factor VIIa, factor X, or both absent confirm for both cell types the specificity of the reaction for the TF pathway. Similarly, specificity for TF itself is shown by the virtual absence of factor Xa generation in the presence of the monoclonal antibody HTF1-7B8 directed against human TF. We conclude that ECs, even when activated, are normally unable to generate significant quantities of factor Xa in the presence of factors X and VIIa. However, significant quantities of factor Xa are possible in the presence of an inhibitor of TFPI. On the other hand, production of factor Xa by fibroblasts is markedly augmented by shear stress, yet independent of the availability of substrate factor X above an inflow concentration of 100 nmol/L. The latter suggests a direct effect of flow on the fibroblast monolayers, not substrate limitation by convective diffusion.     

Effect of extracellular magnesium on platelet activation and intracellular calcium mobilization.

A dose-dependent effect of magnesium on the inhibition of platelet aggregation and release of ATP from dense granules was observed in human platelets (in whole blood, platelet-rich plasma, or washed platelets) against various aggregation agents (ADP, U46619, collagen, or thrombin). The synthesis and release of the proaggregatory cyclooxygenase (CO) and lipoxygenase (LO) products, thromboxane A2 (TXA2) and 12-hydroxyeicosatetraenoic acid (12-HETE), respectively, in platelets were also inhibited by Mg in a dose-dependent manner (IC50 4 to 6 mmol/L). These Mg-mediated activities were further enhanced when platelets were preincubated with insulin (100 microU/mL). The effect of extracellular Mg on the change of intracellular calcium concentration ([Ca2+]i) was assessed using Fura-2/AM loaded cells in the presence or absence of extracellular Ca. Thrombin-stimulated influx of Ca ions decreased from 194 +/- 30 nmol/L to 156 +/- 21 nmol/L in the presence of 5 mmol/L Mg and to 111 +/- 16 nmol/L in 10 mmol/L Mg. However, the intracellular Ca release (as determined in the presence of 5 mmol/L EGTA) was not affected by Mg. The intracellular Ca-dependent protein kinase C and myosin light chain kinase activities on the phosphorylation of endogenous p47 and p20 proteins studied after 2 min of thrombin addition decreased only 10 to 25% in the presence of 5 to 10 mmol/L Mg. Similar results were obtained when EGTA was added prior to the initiation of protein phosphorylation. We conclude that Mg can dose dependently inhibit a wide variety of agonists on platelet aggregation. Furthermore, insulin can potentiate the inhibitory effects of Mg on platelet activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The synthetic RGDS peptide inhibits the binding of fibrinogen lacking intact alpha chain carboxyterminal sequences to human blood platelets

The alpha chain 572-574 Arg-Gly-Asp sequence of fibrinogen appears to play only a minor role in platelet aggregation based on the ability of fibrinogen preparations lacking alpha chain carboxyterminal segments to support platelet aggregation, but synthetic Arg-Gly-Asp-Ser (RGDS) peptides are capable of inhibiting platelet aggregation and fibrinogen binding. The present study thus examined the ability of RGDS peptides to inhibit platelet interactions with a plasmic degradation product of fibrinogen (8D-50) that resembles an intermediate fragment X. Gel- filtered, human blood platelets suspended in 0.01 mol/L HEPES-buffered modified Tyrode's solution, pH 7.5, were stimulated with 20 mumol/L adenosine diphosphate and the binding of 125I-labeled 8D-50 or intact fibrinogen (0.01 to 0.6 mg/mL) assessed in the presence of 0 to 117 mumol/L RGDS. The data revealed that RGDS decreased the apparent affinity of 8D-50 and intact fibrinogen for platelets but did not affect the maximum number of binding sites. RGDS thus appears to be a competitive inhibitor not only of intact fibrinogen (Ki = 12 +/- 2 mumol/L) but also of 8D-50 (Ki = 15 +/- 3 mumol/L) (mean +/- SD, n = 3).     

Stimulation of leukotriene biosynthesis in human blood leukocytes by platelet-derived 12-hydroperoxy-icosatetraenoic acid.

Addition of arachidonic acid to suspensions of human blood leukocytes induces the synthesis of small amounts only of the C-5 lipoxygenase products as demonstrated by HPLC. However, the coincubation of blood platelets with the leukocytes always resulted in an activation of the C-5 lipoxygenase and formation of (5S)-5-hydroxy-6,8,11,14-icosatetraenoic acid, (5S,12S)-5,12-dihydroxy-6,8,10,14-icosatetraenoic acid, and leukotriene B4 from exogenous arachidonic acid. It was found that the activation of arachidonic acid metabolism in leukocytes was caused by a labile compound because the synthesis of the C-5 lipoxygenase products did not occur when platelets were preincubated for 1 min or more with the substrate prior to the addition of the leukocytes. The use of cyclooxygenase inhibitors did not suppress the activation of the leukocytes by the platelets. However, the addition of 5,8,11,14-icosatetraynoic acid, an inhibitor of cyclooxygenase and C-12 and C-15 lipoxygenases, completely suppressed the formation of leukotrienes, although this substance is not an inhibitor of the C-5 lipoxygenase in human leukocytes. This indicated that a product of the C-12 lipoxygenase was likely the mediator of the stimulatory effect of platelets on leukocyte arachidonic acid metabolism. The finding that the direct addition of (12S)-12-hydroperoxy-5,8,10,14-icosatetraenoic acid, but not of the corresponding hydroxy derivative, could activate the leukocyte's C-5 lipoxygenase confirmed this hypothesis. These data demonstrate that an interaction between C-12 and C-5 lipoxygenases can promote the formation of leukotrienes and support the possibility of a cooperation between platelets and leukocytes in inflammation and hypersensitivity reactions. Furthermore, the finding provides a new interest for the platelet C-12 lipoxygenase.     

Relative antithrombotic effects of monoclonal antibodies targeting different platelet glycoprotein-adhesive molecule interactions in nonhuman primates

The relative antithrombotic effectiveness of targeting glycoprotein (GP) Ib-dependent versus GPIIb-IIIa-dependent platelet interactions has been determined in baboons by measuring thrombus formation after infusing comparable antihemostatic doses of anti-von Willebrand factor (vWF) monoclonal antibody (MoAb) BB3-BD5, anti-GPIb MoAb AP1, and anti- GPIIb-IIIa MoAb LJ-CP8 under conditions of arterial and venous flow (shear rates of 750 to 1,000 seconds-1 and 100 seconds-1, respectively). Thrombus formation was quantified as 111In-platelet deposition and 125I-fibrin accumulation on segments of collagen-coated tubing interposed in chronic exteriorized arteriovenous (AV) shunts for 40 minutes. In vitro, anti-vWF MoAb BB3 BD5 (IgG) and anti-GPIb MoAb AP1 [IgG or F(ab)2 fragments] inhibited ristocetin-induced platelet aggregation (IC50 50 nmol/L and 1 mumol/L, respectively), but neither of these MoAbs blocked platelet aggregation induced by adenosine diphosphate (ADP) (P > .5). Conversely, anti-GPIIb-IIIa MoAb LJ-CP8 inhibited platelet aggregation induced by ADP (IC50 1 mumol/L, but failed to block ristocetin-induced platelet aggregation (P > .5). In vivo, the intravenous infusion of anti-vWF MoAb BB3 BD5 or anti-GPIIb- IIIa MoAb LJ-CP8 into baboons at doses that abolished corresponding agonist-induced aggregation ex vivo (bolus injections of 0.5 mg/kg and 10 mg/kg, respectively) prolonged template bleeding times from baseline values of 4.0 +/- 0.3 minutes to > 27 +/- 4 minutes, and to > 26 +/- 4 minutes, respectively (P < .001 in both cases), without affecting the peripheral platelet count (P > .5). However, injection of anti-GPIb MoAb AP1 [10 mg/kg as IgG or 1 mg/kg as F(ab)2 fragments] produced immediate irreversible thrombocytopenia (< 40,000 platelets/microL). Anti-GPIIb-IIIa MoAb LJ-CP8 abolished platelet deposition and fibrin accumulation on collagen segments under both arterial and venous flow conditions (P < .01 in all cases), whereas MoAb BB3 BD5 produced minimal inhibition of platelet deposition and no decrease in fibrin accumulation at arterial shear rates and undetectable antithrombotic outcomes at low shear. Thus, inhibiting GPIIb-IIIa-dependent platelet recruitment abrogates both thrombus formation and platelet hemostatic function at both venous and arterial shear rates. By contrast, interfering with GPIb-vWF-dependent platelet interactions abolishes platelet hemostatic function without producing corresponding antithrombotic effects.     

Vascular CO counterbalances the sensitizing influence of 20-HETE on agonist-induced vasoconstriction

We examined the influence of interactions between CO and 20-hydroxyeicosatetraenoic acid (20-HETE) on vascular reactivity to phenylephrine and vasopressin. Renal interlobar arteries incubated in Krebs buffer released CO at a rate that is decreased (from 125.0+/-15.2 to 46.3+/-8.8 pmol/mg protein per hour, P<0.05) by the heme oxygenase inhibitor chromium mesoporphyrin (CrMP; 30 micromol/L). The level of 20-HETE in vessels was not affected by CrMP (74.3+/-6.1 versus 72.5+/-16.2 pmol/mg protein), but was decreased (P<0.05) by CO (1 micromol/L; 33.2+/-7.9 pmol/mg protein) or the cytochrome P450-4A inhibitor N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 30 micromol/L; 11.4+/-3.3 pmol/mg protein). Phenylephrine elicited development of isometric tension in vascular rings mounted on a wire-myograph (EC(50), 0.29+/-0.02 micromol/L; R(max), 3.78+/-0.19 mN/mm). The sensitivity to phenylephrine was decreased (P<0.05) by CO (1 micromol/L; EC(50), 0.60+/-0.04 micromol/L) or DDMS (EC(50), 0.71+/-0.12 micromol/L) and increased (P<0.05) by 20-HETE (10 micromol/L; EC(50), 0.08+/-0.02 micromol/L) or CrMP (EC(50), 0.11+/-0.02 micromol/L). Notably, neither CO nor CrMP changed the sensitivity to phenylephrine in vessels treated with DDMS. Refractoriness to CO and CrMP in such a setting was eliminated by inclusion of 20-HETE (1 micromol/L) in the bathing buffer. The aforementioned interventions affected the vascular reactivity to vasopressin in a similar manner. These data indicate that the reactivity of renal arteries to phenylephrine and vasopressin is reciprocally influenced by CO and 20-HETE of vascular origin and that CO desensitizes the vascular smooth muscle to constrictor agonists by interfering with the sensitizing influence of 20-HETE.     

p47phox-dependent NADPH oxidase regulates flow-induced vascular remodeling

Chronic alterations in blood flow elicit an adaptive response that tends to normalize shear stress, involving nitric oxide (NO) and matrix metalloproteinases (MMPs). To evaluate the role of NADPH oxidase in this process, we developed a new model of mouse arteriovenous fistula (AVF) connecting the right common carotid artery (RCCA) with the jugular vein, which does not affect blood pressure. Mice deficient for gp91phox and p47phox subunits of NADPH and wild-type controls were used. AVF greatly increased RCCA blood flow (0.78+/-0.12 to 4.71+/-0.78 mL/min; P<0.01), producing an abrupt rise in shear stress (35+/-1 to 261+/-17 dynes/cm2; P<0.01) within 24 hours. RCCA diameter (460+/-14 microm) gradually enlarged 1 and 3 weeks after AVF (534+/-14 microm and 627+/-19 microm; P<0.01), reducing shear stress (173+/-13 and 106+/-10 dynes/cm2, respectively). In gp91phox(-/-) mice, changes in RCCA caliber and shear stress matched controls. However, p47phox(-/-) mouse RCCAs enlarged only marginally, such that shear stress remained high (199+/-8 dynes/cm2 at 3 weeks). Likewise, remodeling was minimal in endothelial NO synthase (eNOS)(-/-) mice. In both control and gp91phox(-/-) animals, reactive oxygen species (ROS) production and MMP induction was enhanced by AVF, whereas in p47phox(-/-) and eNOS(-/-) mice such response was negligible. Similarly, nitrotyrosine staining, indicating peroxynitrite formation, was more pronounced in control and gp91phox(-/-) mice than in p47phox(-/-) and eNOS(-/-) mice. Hence, shear stress induces vascular NADPH oxidase comprising p47phox but not gp91phox. Generated ROS interact with NO to produce peroxynitrite, which in turn activates MMPs, facilitating vessel remodeling. Our study provides the first evidence that ROS play a fundamental role in flow-induced vascular enlargement.     

Leukotriene production by fresh human bone marrow cells: evidence of altered lipoxygenase activity in chronic myelocytic leukemia

The metabolism of arachidonic acid through the lipoxygenase pathway was studied in suspensions of fresh human bone marrow cells from eight patients with chronic myelocytic leukemia (CML) and 10 normal controls. After the cells were incubated with the calcium ionophore A23187 and arachidonic acid, a technique including reverse- and straight-phase high-pressure liquid chromatography (HPLC) was employed to isolate and detect different lipoxygenase-mediated compounds. The detected compounds included leukotriene B4 (LTB4), with its two major nonenzymatic isomers 6-trans-LTB4 and 12-epi-6-trans-LTB4 5S,12S-DHETE, and the monohydroxy eicosatetraenoic acids 5-HETE, 12-HETE, and 15-HETE. The pattern of lipoxygenase-mediated products from the bone marrows was similar to that previously described from human peripheral blood. Of eight bone marrow samples from CML patients, five expressed values above 600 ng LTB4/10(8) nucleated cells, as compared to only one out of 10 controls. In contrast, the CML patients produced significantly lower amounts of both the double-dioxygenation product 5S,12S-DHETE (56.8 +/- 16.0 ng [mean +/- SE] versus 146.1 +/- 31.3 ng; p less than 0.05) and the monohydroxy acid 12-HETE (965 +/- 351 ng versus 4390 +/- 1801 ng; p less than 0.05), indicating a 12-lipoxygenase deficiency. The present results show that leukotrienes are formed by human bone marrow cells and further suggest the existence of altered lipoxygenase activity in CML.     

Endogenous and exogenous adenosine inhibit granulocyte aggregation without altering the associated rise in intracellular calcium concentration

The effects of adenosine, adenosine deaminase (ADA), and an irreversible ADA inhibitor 2'-deoxycoformycin (DCF) on granulocyte aggregation in response to four different stimuli: the synthetic chemotaxin N-formyl-met-leu-phe (FMLP), zymosan-activated plasma (ZAP), the calcium ionophore A23187, and phorbol myristate acetate (PMA) were studied. Adenosine inhibited granulocyte aggregation in response to 10(- 7) mol/L FMLP in a dose-dependent fashion; inhibition in the presence of 1 mumol/L adenosine was 25% +/- 3% (SD) and was 50% (the maximal inhibition observed) with 1 mmol/L adenosine. Quantitatively similar results were obtained when ZAP or A23187 was used as the aggregant but the response to PMA was not affected. ADA not only reversed the inhibition due to adenosine but actually augmented the aggregation to FMLP by 118% +/- 9%. Similar results were obtained with ZAP and A23187 but not with PMA. These effects of ADA depended on its enzymatic activity as they could be blocked by preincubation with DCF. Fluorescent measurement of intracellular calcium in fura-2 loaded granulocyte suspensions established that neither adenosine nor ADA affected subsequent FMLP-stimulated calcium responses. Adenosine, therefore, may inhibit granulocyte responsiveness by blocking signal transduction at a point after calcium entry/mobilization but before activation of protein kinase C. Furthermore, the augmentation of responses seen with ADA suggests that endogenous adenosine may be a physiologic autocrine regulator of granulocyte function.     

Wall shear stress is lower in the carotid artery responsible for a unilateral ischemic stroke

According to the focal nature of atherosclerosis, ischemic stroke is frequently unilateral. Atherosclerotic plaques are favoured by local hemodynamic factors as low wall shear stress and/or elevated circumferential wall tension. Aim of the present study was to investigate the possible association between hemodynamic forces and cerebrovascular disease. Common carotid mean wall shear stress and circumferential wall tension, Peterson's elastic modulus, and blood flow were measured in 25 patients with a recent unilateral large-artery stroke presenting non-stenotic plaques of the carotid arteries (large-artery group), and in 10 patients affected by a recent unilateral cardioembolic stroke without carotid plaques (cardioembolic group). In the large-artery group, atherosclerosis was slightly more evident in the side of cerebral ischemia. All hemodynamic factors were more unfavourable at the affected side in this group (shear stress: 6.2+/-3.0 versus 8.6+/-4.0 dynes/cm2, p<0.0001; wall tension: (7.3+/-1.3)x10(4) versus (6.6+/-1.3)x10(4) dynes/cm, p<0.00001; Peterson's modulus: (16.8+/-11.9)x10(5) versus (12.4+/-5.7)x10(5) dynes/cm2, p=0.06). No difference was detectable in blood flow and in cardioembolic group. The present data demonstrate an unfavourable hemodynamic profile in the common carotid artery supplying the area of a single large-artery stroke and might help to explain the frequent one-sidedness of this disease.     

Inflammatory mediators promote strong sickle cell adherence to endothelium under venular flow conditions

Adherence of sickle erythrocytes to endothelium in venules is thought to initiate or propagate vaso-occlusive episodes. Because of blood shear forces with normal microvascular flow, adherence in post-capillary venules requires binding via high-affinity receptor-mediated pathways. Microvascular flow in sickle patients is episodic, even in asymptomatic patients, so adherence may also occur at low shear not requiring high-affinity binding. Sickle cell binding to endothelium was quantified under flow or static incubation with unusually large vWF, thrombospondin, alpha(4)beta(1)/VCAM-1 or alpha(4)beta(1)/fibronectin (FN). Adherence under flow at 0.5 dyne/cm(2) shear stress leads to the greatest number of adherent sickle cells. Adherence under flow at 1.0 dyne/cm(2) leads to the strongest adherence. Static incubation conditions promote weak adherence of low numbers of sickle cells to endothelium. Following attachment at 1.0 dyne/cm(2), adherence strength was 2.5 +/- 0.1 or 2.6 +/- 0.2 dynes/cm(2) for alpha(4)beta(1)/VCAM-1 or alpha(4)beta(1)/FN pathways, a level 50% greater than adherence strength mediated by thrombospondin or ULvWF (1.7 +/- 0.08 or 1.6 +/- 0.07 dynes/cm(2), respectively). Sickle cell adhesion promoted by simultaneous activation of alpha(4)beta(1)/VCAM-1 and alpha(4)beta(1)/FN pathways is the strongest at 6.2 +/- 0.2 dynes/cm(2) and adherent red cells resist detachment shear stresses up to 10 dynes/cm(2). These data demonstrate that sickle cell adhesion to endothelium is regulated both by receptor/ligand affinity and flow conditions. Thus, both microvascular flow conditions and receptor-ligand interactions may regulate sickle cell adherence in vivo.     

Abdominal aortic hemodynamic conditions in healthy subjects aged 50-70 at rest and during lower limb exercise: in vivo quantification using MRI

The prevalence of atherosclerosis in the abdominal aorta increases with age and is hypothesized to be related to adverse hemodynamic conditions including flow recirculation and low wall shear stress. Exercise has been shown to modulate these adverse conditions observed in the infrarenal aorta of healthy young subjects at rest. A custom magnetic resonance (MR)-compatible stationary cycle, an open MRI, and custom image processing software were used to quantify hemodynamic conditions in the abdominal aorta at rest and during cycling exercise in healthy subjects aged 50-70 years. The subjects increased their heart rate from 63+/-8 bpm at rest to 95+/-12 bpm during cycling exercise. Supraceliac blood flow increased from 2.3+/-0.4 to 6.0+/-1.4 l/min (P<0.001) and infrarenal flow increased from 0.9+/-0.3 to 4.9+/-1.7 l/min (P<0.001) from rest to exercise. Wall shear stress increased from 2.0+/-0.7 to 7.3+/-2.4 dynes/cm(2) at the supraceliac level (P<0.001) and 1.4+/-0.8 to 16.5+/-5.1 dynes/cm(2) at the infrarenal level (P<0.001) from rest to exercise. Flow and shear oscillations present at rest were eliminated during exercise. At rest, these older subjects experienced lower mean wall shear stress at the supraceliac level of the aorta and greater oscillations in wall shear stress as compared to a group of younger subjects (23.6+/-2.2 years). Compared to the younger subjects, the older subjects also experienced greater increases in mean wall shear stress and greater decreases in wall shear stress oscillations from rest to exercise.     

Global analysis of shear stress-responsive genes in vascular endothelial cells

DNA microarray gene expression analysis was conducted in human umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (HCAECs) exposed to laminar or turbulent shear stress. Approximately 3% of the total 5600 gene in HUVECs and HCAECs increased their expression more than two-fold or decreased it to less than half the static control in response to an arterial level of laminar shear stress (15 dynes/cm(2) for 24 hours). The proportions of shear-stress-responsive genes decreased to around 2% under the venous level of laminar shear stress (1.5 dynes/cm(2)) in both cell lines. Turbulent shear stress of 1.5 dynes/cm(2) altered the expression of 1.1% of all genes in the HCAECs. Laminar shear stress, but not turbulent shear stress, decreased the expression of a number of genes involved in DNA synthesis and the cell cycle in both HUVECs and HCAECs. Clustering analysis showed a variety of temporal profiles of gene expression in HUVECs exposed to laminar shear stress of 15 dynes/cm(2) for 3, 6, 12, 24, and 48 hours. Turbulent shear stress affected expression of many genes that play a role in vascular remodeling, including genes encoding plasminogen activators and their inhibitor, endothelin-1, transforming growth factor-beta, collagen type IV, and ephrin A1.     

Role of lipoxygenase metabolites of arachidonic acid in enhanced pulmonary artery contractions of female rabbits

Pulmonary arterial hypertension is characterized by elevated pulmonary artery pressure and vascular resistance. In women the incidence is 4-fold greater than that in men. Studies suggest that sustained vasoconstriction is a factor in increased vascular resistance. Possible vasoconstrictor mediators include arachidonic acid-derived lipoxygenase (LO) metabolites. Our studies in rabbits showed enhanced endothelium-dependent contractions to arachidonic acid in pulmonary arteries from females compared with males. Because treatment with a nonspecific LO inhibitor reduced contractions in females but not males, the present study identified which LO isoform contributes to sex-specific pulmonary artery vasoconstriction. The 15- and 5- but not 12-LO protein expressions were greater in females. Basal and A23187-stimulated release of 15-, 5-, and 12-hydroxyeicosatetraenoic acids (HETEs) from females and males were measured by liquid chromatography/mass spectrometry. Only 15-HETE synthesis was greater in females compared with males under both basal and stimulated conditions. Vascular contractions to 15-HETE were enhanced in females compared with males (maximal contraction: 44±6%versus 25±3%). The specific 15-LO inhibitor PD146176 (12 μmol/L) decreased arachidonic acid-induced contractions in females (maximal contraction: 93±4% versus 57±10%). If male pulmonary arteries were incubated with estrogen (1 μmol/L, 18 hours), protein expression of 15-LO and 15-HETE production increased. Mechanisms to explain the increased incidence of pulmonary hypertension in women are not known. Results suggest that the 15-LO pathway is different between females and males and is regulated by estrogen. Understanding this novel sex-specific mechanism may provide insight into the increased incidence of pulmonary hypertension in females.