Characterization of alpha1-adrenoceptor subtypes mediating vasoconstriction in human umbilical vein.
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1. The present study attempted to characterize pharmacologically the subtypes of alpha-adrenoceptors mediating contractions in human umbilical vein (HUV). 2. HUV rings were mounted in isolated organ baths and cumulative concentration-response curves were constructed for the alpha-adrenoceptor agonists phenylephrine and adrenaline. Adrenaline was more potent than phenylephrine (pD2=7.29 and 6.04 respectively). 3. Isoproterenol exhibited no agonism on KCl pre-contracted HUV rings. Propranolol (1 microM) and rauwolscine (0.1 microM) did not affect the concentration-response curves to adrenaline. These results demonstrate the lack of involvement of functional beta-or alpha2-adrenoceptors in adrenaline-induced vasoconstriction. 4. The non subtype selective alpha1-adrenoceptor antagonist prazosin was evaluated on phenylephrine and adrenaline concentration-response curves. The effects of the competitive alpha1A and alpha1D-adrenoceptor antagonists, 5-methyl urapidil and BMY 7378 and the irreversible alpha1B selective compound chloroethylclonidine (CEC) were also evaluated on adrenaline concentration-response curves. 5. The potencies of prazosin against responses mediated by adrenaline (pA2= 10.87) and phenylephrine (pA2= 10.70) indicate the involvement of prazosin-sensitive functional alpha1-adrenoceptor subtype in vasoconstriction of the HUV. 6. The potencies of 5-methyl urapidil (pA2 = 6.70) and BMY 7378 (pA2= 7.34) were not consistent with the activation of an alpha1A- or alpha1D-adrenoceptor population. 7. Exposure to a relatively low CEC concentration (3 microM) abolished the maximum response to adrenaline suggesting that this response was mediated by an alpha1B-adrenoceptor subtype. 8. We conclude that HUV express a prazosin-sensitive functional alpha1-adrenoceptor resembling the alpha1B-subtype according with the low pA2 values for both 5-methyl urapidil and BMY 7378 and the high sensitivity to CEC. (+info)
Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome.
(10/3475)
The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins. (+info)
Protective effect of flavonoids on endothelial cells against linoleic acid hydroperoxide-induced toxicity.
(11/3475)
The protective effect of flavonoids against linoleic acid hydroperoxide (LOOH)-induced cytotoxicity was examined by using cultured endothelial cells. When the cells were incubated with both LOOH and flavonoids, most flavonols protected the cells from injury by LOOH. Flavones bearing an ortho-dihydroxy structure also showed a protective effect against the cytotoxicity of LOOH. However, flavanones had no effect. The structure-activity relationship revealed the presence of either the ortho-di-hydroxy structure in the B ring of the flavonoids or 3-hydroxyl and 4-oxo groups in the C ring to be important for the protective activities. The interaction between flavonoids and a-tocopherol was also examined in this system. Flavonoids that were protective against LOOH-induced cytotoxicity had at least an additive effect on the action of alpha-tocopherol against LOOH-induced damage. (+info)
Escherichia coli and Porphyromonas gingivalis lipopolysaccharide interactions with CD14: implications for myeloid and nonmyeloid cell activation.
(12/3475)
Porphyromonas gingivalis, a gram-negative bacterium, is an etiologic agent for adult periodontitis. Lipopolysaccharide (LPS) released from this bacterium can react with numerous host cell types. P. gingivalis LPS stimulates tumor necrosis factor alpha and interleukin-1beta secretion from monocytes (myeloid) but does not elicit E-selectin expression from human endothelial cells (nonmyeloid). In contrast, Escherichia coli LPS facilitates expression of these inflammatory mediators through CD14-dependent pathways on both myeloid and nonmyeloid cells. LPS binding studies have revealed that although P. gingivalis and E. coli LPSs bind to CD14 differently, this fact does not adequately explain the lack of endothelial cell activation by P. gingivalis LPS. Rather, LPS binding site and blocking monoclonal antibody epitope mapping studies have suggested that CD14 presents a charged surface that captures different microbial ligands by electrostatic interactions. We propose that human endothelial cells do not respond to P. gingivalis LPS because of their inability to "recognize" CD14-P. gingivalis LPS complexes. (+info)
Identification and characterization of endothelial glycoprotein Ib using viper venom proteins modulating cell adhesion.
(13/3475)
The expression and function of a glycoprotein Ib (GPIb) complex on human umbilical vein endothelial cells (HUVECs) is still a matter of controversy. We characterized HUVEC GPIb using viper venom proteins: alboaggregins A and B, echicetin, botrocetin, and echistatin. Echicetin is an antagonist, and alboaggregins act as agonists of the platelet GPIb complex. Botrocetin is a venom protein that alters von Willebrand factor (vWF) conformation and increases its binding affinity for the GPIb complex. Echistatin is a disintegrin that blocks alphavbeta3. Echistatin, but not echicetin, inhibited the adhesion to vWF of Chinese hamster ovary (CHO) cells transfected with alphavbeta3. We found the following: (1) Binding of monoclonal antibodies against GPIbalpha to HUVECs was moderately increased after stimulation with cytokines and phorbol ester. Echicetin demonstrated an inhibitory effect. (2) Both echicetin and echistatin, an alphavbeta3 antagonist, inhibited the adhesion of HUVECs to immobilized vWF in a dose-dependent manner. The inhibitory effect was additive when both proteins were used together. (3) Botrocetin potentiated the adhesion of HUVECs to vWF, and this effect was completely abolished by echicetin, but not by echistatin. (4) CHO cells expressing GPIbalphabeta/IX adhered to vWF (in the presence of botrocetin) and to alboaggregins; GPIbalpha was required for this reaction. Echicetin, but not echistatin, inhibited the adhesion of cells transfected with GPIbalphabeta/IX to immobilized vWF. (5) HUVECs adhered strongly to immobilized vWF and alboaggregins with extensive spreading, which was inhibited by LJ1b1, a monoclonal antibody against GPIb. The purified alphavbeta3 receptor did not interact with the alboaggregins, thereby excluding the contribution of alphavbeta3 in inducing HUVEC spreading on alboaggregins. In conclusion, our data confirm the presence of a functional GPIb complex expressed on HUVECs in low density. This complex may mediate HUVEC adhesion and spreading on immobilized vWF and alboaggregins. (+info)
Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo.
(14/3475)
Hematopoietic and endothelial cell lineages share common progenitors. Accordingly, cytokines formerly thought to be specific for the hematopoietic system have been shown to affect several functions in endothelial cells, including angiogenesis. In this study, we investigated the angiogenic potential of erythropoietin (Epo), the main hormone regulating proliferation, differentiation, and survival of erythroid cells. Epo receptors (EpoRs) have been identified in the human EA.hy926 endothelial cell line by Western blot analysis. Also, recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2) phosphorylation, cell proliferation, and matrix metalloproteinase-2 (MMP-2) production in EA.hy926 cells and significantly enhances their differentiation into vascular structures when seeded on Matrigel. In vivo, rHuEpo induces a potent angiogenic response in the chick embryo chorioallantoic membrane (CAM). Accordingly, endothelial cells of the CAM vasculature express EpoRs, as shown by immunostaining with an anti-EpoR antibody. The angiogenic response of CAM blood vessels to rHuEpo was comparable to that elicited by the prototypic angiogenic cytokine basic fibroblast growth factor (FGF2), it occurred in the absence of a significant mononuclear cell infiltrate, and it was not mimicked by endothelin-1 (ET-1) treatment. Taken together, these data demonstrate the ability of Epo to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo and thus act as a bona fide direct angiogenic factor. (+info)
Distinct biological effects of macrophage inflammatory protein-1alpha and stroma-derived factor-1alpha on CD34+ hemopoietic cells.
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Chemokines are important regulators of both hemopoietic progenitor cell (HPC) proliferation and adhesion to extracellular matrix molecules. Here, we compared the biological effects of the CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) with those of the CXC chemokine stroma-derived factor-1alpha (SDF-1alpha) on immunomagnetically purified CD34+ cells from leukapheresis products (LP CD34+). In particular, studies on chemokine-induced alterations of LP CD34+ cell attachment to fibronectin-coated plastic surfaces, proliferation of these cells in colony-forming cell (CFC) assays and intracellular calcium mobilization were performed. MIP-1alpha but not SDF-1alpha was found to increase the adhesion of LP CD34+ cells to fibronectin in a dose-dependent manner. Both chemokines elicited growth-suppressive effects on LP CD34+ cells in CFC assays. While MIP-1alpha reduced the number of granulomonocytic (CFC-GM) and erythroid (BFU-E) colonies to the same extent, SDF-1alpha showed a significantly greater inhibitory effect on CFC-GM than BFU-E. Finally, we demonstrated that SDF-1alpha but not MIP-1alpha triggers increases in intracellular calcium in LP CD34+ cells. The SDF-1alpha-induced calcium response was rapid and concentration-dependent, with a maximal stimulation observed at > or = 15 ng/ml. In conclusion, our data suggest distinct biological properties of SDF-1alpha and MIP-1alpha in terms of modulation of LP CD34+ cell adhesion to fibronectin and intracellular calcium levels. However, comparable growth-suppressive effects on HPC proliferation were observed, indicating that this feature may be independent of chemokine-induced calcium responses. (+info)
High-density lipoproteins differentially modulate cytokine-induced expression of E-selectin and cyclooxygenase-2.
(16/3475)
Atherogenesis is a multifactorial chronic inflammatory disease in which low plasma levels of HDLs are a strong predictor of the condition. Although the mechanism of protection by HDLs is not precisely known, HDLs have been shown to influence many of the events involved in the development of atherosclerosis. Previously we have shown that HDLs inhibited the cytokine-induced expression of adhesion molecules (E-selectin, VCAM-1, and ICAM-1) by endothelial cells (ECs). As the complete transcriptional regulation of all 3 genes requires the NF-kappaB family of transcription factors, we examined the effect of HDLs on activation of NF-kappaB. We also investigated the effect of HDLs on 2 other cytokine-induced genes, granulocyte-macrophage colony-stimulating factor (GM-CSF) and cyclooxygenase (Cox-2; prostaglandin H2 synthase, EC 0.1.14.99.1). E-selectin expression in response to tumor necrosis factor-alpha (TNFalpha) was, as expected, inhibited in ECs that had been preincubated with HDLs. However, the level of secretion of GM-CSF in the same cultures was no different from control. In a similar manner, although HDLs had no effect on steady-state mRNA levels of GM-CSF, the levels of E-selectin were significantly inhibited by HDLs. In transient cotransfection experiments we found that HDLs inhibited the cytokine-induced expression of a reporter gene driven by the E-selectin proximal promoter (-383 to 80) but had no effect on the expression of a reporter gene driven under the control of the proximal promoter of GM-CSF (-627 to 28). As would be predicted from this differential response, HDLs did not influence the nuclear translocation or DNA binding of NF-kappaB, or alter the kinetics of degradation and resynthesis of the inhibitory protein IkappaBalpha. We found that HDLs synergized with cytokine to enhance the expression of Cox-2 and induce the synthesis of its main EC product, prostacyclin (PGI2), a potent inhibitor of platelet and leukocyte functions. In conclusion, HDL induces an antiinflammatory phenotype in cytokine-induced ECs, synergizing with cytokine to induce elevation of Cox-2 in addition to inhibiting adhesion molecule expression. Our studies show that these differential effects are mediated in a manner that is likely to be independent of NF-kappaB per se. (+info)