The observed FMD was lower among HIV-infected patients when compared with controls [mean (SEM): 10 (1) and 16% (2), P = 0.03, unpaired Student's t-test]. We found a negative correlation for hsCRPs and EPCs (KDR+/CD133, P = 0.029, Spearman's rank correlation test), but not for other EPC subpopulations or microparticles. No correlations were found between microparticles and FMD (data not shown).
The major contribution of our study was the observation that among HIV-infected antiretroviral drug-naive patients, there is an imbalance between the mobilization of EPCs and microparticles derived from the endothelium, as well as impairment on endothelial function. These findings may be related to the early development of CVD in this population, as already seen in the Strategies for Management of Antiretroviral Therapy (SMART) clinical trial with patients not receiving antiretroviral therapy . In fact, the mechanisms of atherosclerosis related to classic risk factors such as hypertension, smoking or diabetes have in common endothelial dysfunction . These findings open interesting perspectives for investigation of cellular mechanisms involving infection, inflammation and apoptosis of endothelial cells and a decreased reparative capacity, owing to the reduced percentage of EPCs in plasma .
The imbalance between EMPs and EPCs cannot be explained by differences in the prevalence of the classical cardiovascular risk factors, suggesting that the HIV infection itself may be related to the alterations observed in the turnover of endothelial cells. In fact, the only cardiovascular risk factor observed in the HIV-infected group was dyslipidemia characterized mainly by low HDL-c, a typical finding in antiretroviral drug-naive patients, in which a detectable viral load and inflammation coexist .
High levels of hsCRP, a marker of inflammation, are associated with increased cardiovascular risk in non-HIV-infected population and might be a better predictor of risk than low-density lipoprotein cholesterol levels in some populations . According to the JUPITER (Justification for the Use of Statins in Primary Prevention: an Intervention Trial Evaluating Rosuvastatin) trial , hsCRP can identify individuals under risk even with relatively normal cholesterol levels. Triant et al. demonstrated in a retrospective study that high levels of hsCRP and HIV infection are associated with a two-fold increase in the risk of myocardial infarction. In our study, the average hsCRP was 1.64 and 1.18 mg/l in the HIV-infected and control groups, respectively, values which are near to those reported by Baker et al. among antiretroviral drug-naive patients. These values are below the cutoff values for hsCRP associated with cardiovascular risk, suggesting that our finding of imbalance between EMPs and EPCs could be related to the viral infection itself.
Since 1997, when Asahara et al. isolated an angioblast from human peripheral blood of adults, which differentiate in vitro into endothelial cells, new important concepts on the turnover of endothelial cells were discovered. The so-called EPC has been characterized by surface markers CD34 and vascular endothelial growth factor receptor-2 (VEGFR2 or KDR in humans). Immature subset of EPCs expresses the surface marker CD133 . The expression of CD34, VEGFR2 and CD133 is typically found on angioblasts and demonstrates the immature nature of these cells . Reduced number of EPCs has been reported in patients at risk of cardiovascular events, including acute myocardial infarction and stroke [7,8].
We have chosen HIV-infected patients not previously exposed to antiretroviral therapy in order to address the influence of HIV itself on the EPCs and microparticles. The literature has well described the influence of antiretroviral therapy on endothelial function, metabolic alterations (dyslipidemia), inflammation and the development of a high cardiovascular risk due to this exposure [1,2,33]. Currently, there are only preliminary results about EPCs and microparticles in HIV-infected patients. Papasavvas et al. evaluated EPCs in 66 HIV-infected patients (nine antiretroviral drug-naive patients) and in 50 controls. The HIV-infected patients had higher percentages of EPCs (CD34+/ KDR+) in two measures during a 1-year follow-up compared with controls. These findings are in contrast with our results, reporting lower levels of EPCs in HIV-infected antiretroviral drug-naive patients. This research did not address comorbidities that could influence the level of EPCs and microparticles, as statins use, for example, in this population. After the introduction of antiretroviral treatment, with virological control, there is a decrease in markers of the inflammatory state, such as hsCRP, preceding the development of metabolic alterations [35,36]. Thus, the use of antiretroviral treatment could affect microparticles and EPCs by multiple mechanisms. Thus, one of the most important characteristics of our study was the avoidance of such confounders, examining HIV-infected patients naive of antiretroviral therapy.
Microparticles have been defined as small vesicular structures with less than 1 μm formed from the plasma membranes of diverse cell types in response to activation, injury and/or apoptosis . Most of microparticles detected in blood originates from platelets, but other cells such as leucocytes, erythrocytes and endothelial and even malignant cells could also shed microparticles [38–40]. They express on their surface antigens of the cell from which they originated, allowing their characterization . Microparticles are released from cell membranes by triggers such as cytokines, thrombin, endotoxins, hypoxia and shear stress capable of inducing activation or apoptosis [3,12]. In healthy persons, EMP represents a minority of total microparticles. Overproduction of microparticles has been related to various physiological and pathophysiological conditions [24,40,41]. Moreover, it is unclear whether increased microparticles are cause or a consequence of vascular disease states because cardiovascular-related factors, such as metabolic disturbances, cytokines and possibly infectious agents can trigger microparticles production . There are few studies reporting the levels of microparticles among HIV-infected patients. Corrales-Medina et al. found higher number of PMP among HIV-infected patients compared with controls. In the evaluation of PMP, we found no difference between groups. It is possible that some characteristics of this cohort could explain these differences, mainly the relatively good immunity and low viral load. Conversely, we found a significant increase in the EMP in the HIV-infected group compared with control. We did not find previous studies reporting the increased number of EMP on HIV-infected patients. Furthermore, EMP has been proposed as new biomarker of endothelial dysfunction [15,41], and increased number of EMP was reported in high-risk patients with uncontrolled risk factors and CVD .
We observed that HIV-infected patients had lower FMD compared with controls. These data are corroborated by several studies that showed impairment in FMD among HIV-infected patients ranging from 5.1 to 8.8% [33,35,36,44–46]. The literature has shown that endothelial dysfunction is an independent predictor of cardiovascular events . These changes in patients not yet exposed to antiretroviral and in the absence of other classic risk factors for CVD suggest a role of HIV itself as a causative agent of endothelial dysfunction.
The mechanisms by which HIV decreases the number of EPCs and increases EMP remain to be determined. HIV replication may activate endothelial surfaces directly or via upregulation of proinflammatory cytokines [47,48]. Endothelial activation can lead to vessel damage and apoptosis with release of EMP.
Reduced numbers of EPCs independently predict future cardiovascular events, thus supporting an important role for HIV itself as a cardiovascular risk factor [7,8]. This new finding in HIV-infected antiretroviral drug-naive patients may be associated with increased cardiovascular risk in the long-term follow-up, after the introduction of antiretroviral therapy and subsequent development of metabolic alterations. It could be aggravated in the future with the need of antiretroviral therapy and subsequent development of hyperlipidemia. On the contrary, the impact of early antiretroviral therapy on the turnover of endothelial cells emerges as a promising therapy for the prevention of premature CVD in these patients.
Our results showing an imbalance between EPCs and microparticles should be considered as preliminary results based on the relatively small sample size and its transversal, case–control design. However, these findings seem important to explain the high rates of premature CVD among HIV-infected patients. Further studies with longer follow-up addressing EPCs and EMPs are needed.
Conflicts of interest
This study was supported by FAPESP (São Paulo Research Foundation) grant 2008/55223-6. E.F.R.S. is a recipient of a research grant from CAPES (Coordination for the Improvement of Higher Level – or Education – Personnel). Besides this funding disclosure, the authors have no conflicts of interest to declare.
E.F.R.S. designed the study, made the acquisition and interpretation of data (clinical and laboratory), wrote the manuscript and approved the final version for the publication.
F.A.H.F. made a contribution to the conception of the design, data interpretation, revised the manuscript and approved the final version for the publication.
C.N.F. made all the acquisition, analysis and interpretation of flow cytometry, revised the manuscript and approved the final version for the publication.
P.R.A.F. made all the statistical analyses and interpretation of data, revised the manuscript and approved the final version for the publication.
M.C.O.I. reviewed the statistical analysis and interpretation of data, revised the manuscript and approved the final version for the publication.
R.S. made all the pilot analysis and interpretation of the flow cytometry data, revised the manuscript and approved the final version for the publication.
L.M.C. made a contribution to the acquisition and interpretation of FMD data and approved the final version for the publication.
S.B.T. made a contribution to the acquisition and interpretation of clinical data and approved the final version for the publication.
D.S.L. made a contribution to the conception of the study, interpretation and analysis of data, revised the manuscript and approved the final version for the publication.
Data presented previously at the 14th International Congress on Infectious Diseases, 9–12 March 2010, Miami, Florida, USA (published in the Annals of the 14th International Congress on Infectious Diseases) and at the XXXI Congresso da Sociedade de Cardiologia do Estado de São Paulo, 29 April to 5 May 2010, Sao Paulo, Sao Paulo, Brazil (published in Suplemento Especial da Revista da Sociedade de Cardiologia do Estado de São Paulo).
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Keywords:© 2011 Lippincott Williams & Wilkins, Inc.
endothelial progenitor cells; endothelial-derived microparticles; HIV infection; inflammation; platelet-derived microparticles