This study demonstrates for the first time that both ABC and ddI induce the interaction between human leukocytes and endothelial cells by activating Mac-1 in neutrophils and monocytes, but not in lymphocytes, which in turn interacts with the ICAM-1 that is present on endothelial cells.
We used an in-vitro model in which human leukocytes flow over a monolayer of human endothelial cells with a shear stress similar to that observed in vivo . This reproduces the processes that precede the formation of an inflammatory focus in vivo (rolling and adhesion) and which are critical for hemostasis and vascular cell integrity. However, an exacerbation of these interactions contributes to the vascular dysfunction and injury associated with many vascular diseases (e.g., atherosclerosis, diabetic vasculopathy, hypercholesterolemia, hypertension, ischemia–reperfusion,…) . Our dynamic experimental system has been widely used to visualize and analyze the multistep recruitment of leukocytes in these diseases; moreover, it allows the mechanisms of action implicated in this recruitment to be assessed [22,23].
In our experiments, concentrations of ABC (0.1–15 μmol/l) and ddI (5 μmol/l) that mimic those present in patients (1–8 and 3–10 μmol/l respectively) [8,24,25] induced leukocyte–endothelial cell interactions (rolling and adhesion). The magnitude of the increases obtained with both drugs was smaller than that observed after direct stimulation with proinflammatory agents such as TNF-α, interleukin-4 or RANTES [26,27]; however, it was substantially greater than that exhibited in unstimulated PMNs or PBMCs from patients with different vascular conditions [28–30]. Leukocyte accumulation induced by ABC or ddI occured simultaneously with the selective upregulation of Mac-1 on the surface of human neutrophils and monocytes. Nevertheless, the expression of other adhesion molecules in these leukocytes [CD11a/CD18 or very late antigen (VLA)-4] or in endothelial cells (E-selectin, ICAM-1 or VCAM-1) was not affected. Thus, our results suggest that ABC and ddI promote the recruitment of leukocytes before activating the endothelium, and thus before dysfunction appears which needs a much longer period of exposure (24 h) to develop .
HIV-infection itself is associated with a more pronounced adhesion of leukocytes to endothelial cells  and with an elevated cardiovascular risk . Both situations could be related to the high levels of the endothelial dysfunction markers (ICAM-1, VCAM-1 and E-selectin) that are present in these patients . Since ABC has been specifically associated with an impaired endothelial function in HIV-infected patients  and given the difference between the activation profile of adhesion molecules observed in our experiments and that of HIV patients, the effects of antiretrovirals and those of the virus could feasibly be accumulative. In other words, the virus may cause endothelial activation  and antiretrovirals could activate leukocytes. This is an interesting hypothesis, but further clinical and experimental studies would be necessary before any solid conclusion can be drawn.
Mac-1 is mobilized from intracellular secretory vesicles to the cell surface within minutes of stimulating neutrophils and monocytes. Although it may interact with ICAM-2, iC3B, factor X or fibrinogen, its main ligand, ICAM-1 is constitutively expressed on the surface of the vascular endothelium . Thus, the fact that blocking either Mac-1 or ICAM-1 significantly reduced the effects of ABC and ddI point to a role for both these molecules in the rolling and adhesion induced by these two antiretrovirals. Although their involvement in adhesion is to be expected, rolling is considered to be mediated by selectins and/or VLA-4/VCAM-1 . However, there is growing evidence of the implication of Mac-1/ICAM-1 in rolling in both the activated and resting endothelium [35,36].
ABC and ddI specifically affect PMN and PBMC. This is of relevance given that there is a substantial increase in the levels of neutrophils and monocytes during acute myocardial infarction and in the expression of Mac-1 among the two cell populations . In addition, it has been reported that Mac-1 induces the binding of neutrophils to activated cardiac myocytes , and recent evidence points to the mediation of the leukocyte engagement of platelets as the link between cellular adhesion and thrombosis by Mac-1 [36,39].
Finally, the fact that similar effects to those of ABC were observed with ddI but not with lamivudine or zidovudine suggests a relationship between the chemical structure of NRTIs and the induction of leukocyte/endothelial cell interactions. It is tempting to speculate that purine analogues such as ABC or ddI have the potential to interfere with purine-signaling pathways and to provoke cardiovascular complications with inflammatory components (such as atherosclerosis and myocardial infarction) by reducing the levels of adenosine and increasing those of proinflammatory ATP. However, it is necessary to evaluate the actions, on the one hand, of other NRTIs such as tenofovir and emtricitabine, both of which are potential alternatives to ABC for patients with an elevated risk of cardiovascular disease, and on the other hand, of NNRTIs and protease inhibitors in this and other experimental settings before a sound clinical conclusion can be established.
C.D.P. performed the research; S.O., N.A. and A.B. helped perform the research and J.V.E. and A.A. designed the research and wrote the paper. J.V.E. and A.A. contributed equally to this study.
This study has been supported by grants SAF2007-60021 from Ministerio de Educación y Cultura; GV/2007/074, GVACOMP2009-266, ACOMP2009-194 and CS2009-AP-030 from Generalitat Valenciana and CD06/04/0071 (CIBERehd) and PI081325 from Ministerio de Sanidad y Consumo. C.D.P and S.O. have been supported by grants from Ministerio de Educación y Cultura and from Fundación Juan Esplugues respectively.
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