Methods and Findings in Experimental and Clinical Pharmacology
Vol. 25, Suppl. A, 2003
ISSN 0379-0355
Copyright 2003 Prous Science, S.A.
CCC: 0379-0355/2003
http://www.prous.com

Chemokines Involved in Angiotensin-II-Induced Leukocyte-Endothelial Cell Interaction and Modulation by both Natural and Semisynthetic Products

M.A. Blázquez, L. Milián, T. Mateo, P.J. Jose and M.J. Sanz

Pharmacology Department, University of Valencia, Valencia, Spain

Before describing the results obtained in the present study, we would like to acknowledge Almirall-Prodesfarma laboratories and the Spanish Society of Pharmacology for giving us the 2002 Pharmacology Award. Thanks to this award we were able to start this research project during a very difficult time for our team. What follows is the outcome of this opportunity.

Angiotensin II (Ang II) is implicated in the development of cardiac ischemic disorders where prominent neutrophil accumulation occurs. In this study we found that i.p. administration of Ang II (1 nM) in rats induced significant neutrophil recruitment within 4 h, preceded by CINC/KC and MIP-2 release. Studies performed using intravital microscopy within the rat mesenteric microcirculation revealed that pretreatment of the animals with a CXCR2 receptor antagonist resulted in the total abolition of acute (1 h) Ang II-induced leukocyte-endothelial cell interactions. Since CINC/KC and MIP-2 are functional rat homologues of human interleukin-8 (IL-8), we also found that Ang II induced IL-8 synthesis and release in human umbilical vein endothelial cells (HUVECs) after 4-, 24- and 48-h stimulation. When HUVECs were pretreated with IL-1 to promote IL-8 storage in Weibel-Palade bodies, Ang II caused a rapid (1 h) release of IL-8. Thus, Ang II provokes rapid neutrophil recruitment mediated through the release of CXC chemokines, and it might be a stimulus for the infiltration of neutrophils observed in acute myocardial infarction. Figure 1 illustrates the possible mechanism of Ang II-induced neutrophil accumulation.

Ang II also plays a critical role in the development of vascular lesions associated with atherosclerosis and increased expression of different CC chemokines has been detected in atherosclerotic plaques. We encountered mononuclear leukocyte recruitment after 8- and 24-h Ang II (1 nM) i.p. injection. The maximal RANTES and MCP-1 levels were detected after 4-h Ang II exposure. In addition, the administration of Met-RANTES, an antagonist of CCR1 and CCR5 receptors, inhibited Ang II-induced leukocyte-endothelial cell interactions 4 h later. Furthermore, Ang II also induced MCP-1 mRNA expression in HUVECs and a concentration-dependent release of this chemokine. Conversely, Ang II was unable to provoke eotaxin-1, -2 and MDC generation. Thus, mononuclear leukocyte infiltration is elicited by Ang II and is in part mediated by CC chemokines production. These responses may contribute to the onset of the atherosclerotic lesion when Ang II plasma levels are elevated.

We also evaluated the ability of, and mechanisms involved in, the antiinflammatory activity displayed by two phenanthrene alkaloids (uvariopsine and stephenanthrine) isolated from Dennettia Tripetala. In vivo, both alkaloids inhibited Ang II-induced leukocyte-endothelial cell interactions and P-selectin upregulation in a concentration-dependent manner. These alkaloids also diminished the generation of reactive oxygen species (ROS) in HUVECs stimulated with Ang II, in fMLP-stimulated PMNs and in the hypoxanthine/xanthine oxidase system (HX-XOD). In addition, uvariopsine and stephenanthrine inhibited PAF-induced elevation of intracellular calcium, superoxide anion and hydrogen peroxide. Thus, these alkaloids have a potential therapeutic interest for the control of leukocyte recruitment occurring in cardiovascular disease states in which Ang-II is involved.

Finally, based on the effects exerted by stephenanthrine and uvariopsine, we synthesized some aporphine and phenanthrene alkaloids from natural boldine. All alkaloids inhibited in a concentration-dependent manner the generation of ROS in different systems. Structure-activity relationship studies of these compounds revealed that phenolic alkaloids with phenanthrene skeleton are more active than those with aporphine skeleton while those without a phenolic group displayed less activity than alkaloids with aporphine skeleton. The effect of the most active alkaloids is currently being investigated on Ang II-induced leukocyte recruitment as well as chemokine release. Therefore, phenanthrene alkaloids may constitute another class of compounds likely to be used as alternative therapy in cardiovascular inflammatory disorders.

Methods and Findings in Experimental and Clinical Pharmacology Vol. 25, Suppl. A, 2003
ISSN 0379-0355 Copyright 2003 Prous Science, S.A. CCC: 0379-0355/2003 http://www.prous.com