Since the introduction of highly active antiretroviral therapy (ART) in 1995, HIV infection has become a chronic disease. In this scenario, cardiovascular diseases, mainly atherosclerosis, have gained importance as causes of morbidity and mortality.1 This increase in cardiovascular risk in HIV-infected patients seems to be associated to endothelial dysfunction because of ART and to traditional and new cardiovascular risk factors.2,3
In the last years, circulating endothelial progenitor cells (EPCs) have gained importance as a potential endothelial protection mechanism.4,5 EPCs are bone marrow–derived cells characterized by the expression of both hematopoietic (CD34 and CD133) and endothelial—such as KDR [a type of vascular endothelial growth factor receptor 2 (VEGFR-2)], VE-cadherin, CD31, and von Willebrand factor—surface markers that have been demonstrated of their capacity to differentiate into mature endothelium.5 It has been reported that circulating EPCs could contribute to endothelial repair by homing into sites of endothelial injury for maintaining the integrity of the blood vessels.4,5 Thus, it has been hypothesized that circulating EPC levels could determine the capacity of repairing the damaged or dysfunctional endothelium. In this sense, traditional cardiovascular risk factors, such as smoking, hyperlipidaemia, hypertension, and diabetes, which are associated with endothelial dysfunction, have been also associated with low levels of EPCs.6 Furthermore, the depletion in the number of EPCs has been associated with the occurrence of a first major cardiovascular event in patients at different risks or even in healthy subjects.5–7 Moreover, restoration of EPC number and/or their functionality is possible through current therapies for cardiovascular risk factors and other means,8–10 suggesting that they could also be a promising tool for measuring therapeutic efficacy. In small studies with HIV-infected patients, alterations in the number and/or functionality of EPCs have also been reported.11–13
Much less is known regarding smooth muscle progenitor cells (SMPCs), circulating cells that express markers of mesenchymal cells [such as endoglin (CD105) or calponin] or of the smooth muscle lineage, such as α-smooth muscle actin (α-SMA), smooth muscle-myosin heavy chain (SM-MHC), or smooth muscle-22α (SM-22α).4,14 Their role in the atherosclerotic process is still controversial, with data suggesting that SMPCs might contribute to both neointima growth and plaque stability.4,14
Hence, in this study, we have investigated in the clinical setting, the association between the levels of the circulating vascular progenitor subpopulations CD34+/KDR+, CD34+/VE-cadherin and CD14+/Endoglin+, cardiovascular, and HIV-specific risk factors and the carotid intima-media thickness (c-IMT) as a marker of early atherosclerosis in a large population of HIV-infected patients.
MATERIALS AND METHODS
The protocol of this study complies with the principles of the Helsinki declaration and was approved by the Ethics and Clinical Investigation Committee of Hospital Clínico San Carlos. Informed consent was obtained from all subjects.
Consecutive consenting 200 HIV-infected patients attending the HIV Unit of the Hospital Clínico San Carlos in Madrid, aged 18 years or older, were eligible to take part in the study. The exclusion criteria were known cardiovascular disease (previous stroke, myocardial infarction, or intermittent claudication) and/or known chronic kidney disease.
Twenty-seven nonsmoker healthy subjects, without evidence of cardiovascular disease, were evaluated as a control group.
Clinical and Laboratory Measurements
Medical records were carefully reviewed at interview, a questionnaire was completed, and a thorough physical examination was performed. Gender, age, anthropometric measurements (weight, height, and waist circumference), systolic and diastolic blood pressures (SBP/DBP), smoking habit, family history of cardiovascular disease (defined as a major cardiovascular event in one or more first-degree relatives), and accumulated time on nucleoside reverse transcriptase inhibitors (NRTIs), on non-NRTIs (NNRTIs), and on protease inhibitors (PIs) was recorded. Body mass index was calculated.
A sample of fasting venous blood was obtained to determine plasma glucose, high-density lipoprotein cholesterol (HDL), and triglycerides using standard enzymatic methods. Low-density lipoprotein cholesterol concentrations were calculated using the Friedewald equation.15 The presence of hypertension, hypercholesterolemia, and hypertriglyceridemia was defined according to the Adult Treatment Panel III criteria.16
Quantification of Circulating Vascular Progenitor Cells
Peripheral blood cells were analyzed by direct flow cytometry as previously described.17 Blood cells were stained with anti-CD34 phycoerythrin-cyanin 7 (PC7)-conjugated (mouse IgG1, Beckman Coulter Inc; Bre, CA), anti-CD3 phycoerythrin-Texas Red-x (ECD)-conjugated (mouse IgG1, Beckman Coulter), anti-KDR phycoerythrin (PE)-conjugated (mouse IgG1, R&D Systems; Abingdon, UK), anti-VE-cadherin (CD144) PE-conjugated (mouse IgG2b, R&D Systems), anti-CD14 PC7-conjugated (mouse IgG1, Beckman Coulter), and anti-endoglin (CD105) PE-conjugated (mouse IgG2b, R&D Systems). Appropriate isotype controls were used for each staining procedure. After lyse-wash procedure, cells were acquired on a FC500 flow cytometer (Beckman Coulter) and peripheral mononuclear blood cells (PMNCs) were gated after excluding cellular debris in a side scatter/forward scatter dot plot. For each sample, a minimum of 100,000 events was acquired. Progenitor cells were identified as dual CD34+/KDR+, CD34+/VE-cadherin+, or CD14+/Endoglin+ cells in the CD3 negative gate.
The instrument setup was optimized daily by analyzing polystyrene fluorescent microspheres (flow-check fluorospheres; PC7 770/488 setup kit, Beckman Coulter). The same trained operator, who was blinded to the subjects' characteristics, performed all of the tests throughout the study. Results are expressed as percentage of CD34+/KDR+, CD34+/VE-cadherin+ or CD14+/Endoglin+ cells in the PMNC gated area once excluding CD3+ cells.
In a pilot study, healthy subjects showed a median absolute number of 15 (8–22) CD34+/KDR+ cells and 22 (14–33) CD34+/VE-cadherin+ cells. These data are consistent with those previously reported using the flow cytometric analysis.18
Carotid ultrasonography was performed using an HD7 ultrasound system (Philips, Ibérica, Madrid, Spain) using a sectorial 12 MHz probe as previously reported.19 Both common carotid arteries were identified in the long axis and explored starting 1 cm below the flow divider. The images were recorded in end diastole and then analyzed by specific validated software (QLab Quantification Software, Philips). c-IMT measurement was carried out in the right carotid artery on areas free of atheroma. Carotid ultrasonographies were performed by 2 trained technicians who had previously participated into a pilot study, consisting in repeated and blinded measurements performed in 29 patients, achieving a correlation coefficient greater than 0.90.
Qualitative variables were summarized by their frequency distribution and quantitative variables by their mean and standard deviation (± SD). The continuous non-normally distributed variables were summarized by the median and interquartile range (IQR; P25–P75). The Kolmogorov–Smirnov test was used to prove Gaussian distribution. In case of qualitative variables, comparison was evaluated by the test of χ2 or by Fisher exact test in case more than 25% of the expected values were less than 5. For continuous normally distribute variables, the Student t test was used to compare between 2 groups. The Mann–Whitney U test was used for continuous not normally distributed variables. The association between continuous variables was tested using the nonparametric Spearman correlations coefficient. As the circulating vascular progenitor cells were not normally distributed, these data were log-transformed to improve their distribution for statistical testing, with back-transformed results for presentation in figures and tables. A multivariate linear regression analysis was fitted to evaluate the variables associated with the circulating vascular progenitor cells. Adjustment was with those variables which, in the univariate analyses, showed a level of statistical significance of P < 0.05 and/or were considered clinically relevant. Null hypothesis was rejected by a type I error minor than 0.05 (P < 0.05). Statistical analyses were performed using the SPSS 17.0 statistical package.
Compared with healthy subjects (61% men, 40 ± 8 years), HIV-infected patients showed decreased number of CD34+/KDR+ [0.08% (0.04%–0.14%) vs 0.02% (0.01%–0.03%), P < 0.01] and CD34+/VE-cadherin+ cells [0.07% (0.04%–0.11%) vs 0.03% (0.02%–0.05%), P < 0.01]. Circulating CD14+/Endoglin+ cells tended to be lower in HIV-infected patients than in healthy subjects [3.2% (1.7%–5.1%) vs 2.1% (1.2%–3.7%), P < 0.067], although without reaching statistical significance.
Vascular Progenitor Cell Levels in HIV-Infected Patients
Twenty HIV-infected patients were ART-naive and 180 were currently being treated with ART. Most patients on ART (83.1%) had undetectable viral load (<50 copies/mL). ART-treated patients were older (48 ± 10 years vs 40 ± 12 years, P = 0.002) and had higher SBP (118 ± 17 mm Hg vs 109 ± 9 mm Hg, P < 0.024), cholesterol (190 ± 42 mg/dL vs 160 ± 31 mg/dL, P < 0.020), and triglycerides [135 (98–201) mg/dL vs 106 (74–131) mg/dL, P < 0.005]. There were no differences in the frequency of smoking (45% vs 44.4%, P = 0.567) or diabetes (0% vs 8.9%, P = 0.136).
Compared with healthy subjects, ART-naive patients showed lower number of CD34+/KDR+, CD34+/VE-cadherin+ and CD14+/Endoglin+ cells (Figs. 1A–C). Treated patients showed further decreased levels of CD34+/KDR+ and CD34+/VE-cadherin+ cells (Figs. 1A, B). However, CD14+/Endoglin+ cells were higher in ART-treated patients than in naive patients (Fig. 1C). The levels of progenitor subtype cells did not seem in relationship with CD4+ lymphocyte count. However, HIV viral load was positively associated with CD34+/KDR+ (r = 0.159, P = 0.029) and negatively with CD14+/Endoglin+ cell levels (r = −0.220, P = 0.003).
In our cohort of HIV-infected patients, the most frequent cardiovascular risk factor was smoking (44.5%), followed by hypercholesterolemia (37.2%), hypertriglyceridemia (22.6%), hypertension (16.5%), and diabetes mellitus (9.5%). No significant differences in the number of vascular progenitor cell subtypes were found between male and female or between patients with and without traditional cardiovascular risk factors, although patients without hypercholesterolemia, hypertriglyceridemia, hypertension, and/or diabetes mellitus tended to show higher levels of CD34+/KDR+ or CD34+/VE-cadherin+ cells (data not shown).
In a multivariate regression analysis of HIV-infected patients entering known cardiovascular risk factors (age, sex, hypertension, diabetes hypertriglyceridemia, and hypercholesterolemia) and HIV status (current ART-treatment and time HIV diagnosis), only being on ART remained as an independent factor of CD34+/KDR+ and CD14+/Endoglin+ cell levels (Table 1).
Association Between Vascular Progenitor Cell Levels and c-IMT
To evaluate the relationship between vascular progenitor cells and c-IMT, patients were classified into low vascular progenitor cell group (below 75th percentile) and high vascular progenitor cell group (equal or above 75th percentile). As shown in Table 2, there were no significant differences in gender, age, cardiovascular risk factors, and viral parameters between the 2 subgroups. However, time exposure to ART was associated to low CD34+/KDR+ and high CD14+/Endoglin+ levels.
There was no clear correlation observed between vascular progenitor cell levels and c-IMT. Patients with low CD34+/KDR+ and CD34+/VE-cadherin+ or increased CD14+/Endoglin+ cell levels tended to show higher c-IMT, although these differences did not reach statistical significance (Fig. 2).
Linear regression analysis revealed that c-IMT was significantly associated to age (r = 0.473, P < 0.001), SBP (r = 0.278, P < 0.001), glucose (r = 0.272, P < 0.001), time to HIV diagnosis (r = 0.207, P = 0.003), and accumulated exposure to NRTI (r = 0.215, P = 0.003) and PI (r = 0.163, P = 0.024).
Association Between Vascular Progenitor Cell Levels, c-IMT and the Use of ART
To further investigate the role of ART duration in vascular progenitor cell levels, the effects of NNRTI, NRTI, and PI were analyzed separately. One hundred seventy-two patients (95.6%) were currently prescribed on NRTI, 138 (76.6%) on NNRTI and 114 (63.3%) on PI. Prolonged exposure (accumulated time more than 5 years) to NRTI and PI was associated to both decreased levels of CD34+/KDR+ cells and higher c-IMT (Fig. 3). Patients taking NNRTI more than 5 years also showed this tendency although it did not reach statistical significance.
In a multivariate regression analysis entering known cardiovascular risk factors and HIV status, time of exposure to PI more than 5 years remained significantly associated with decreased CD34+/KDR+ cell levels.
Circulating progenitor cells with capacity to differentiate into endothelial or vascular smooth muscle cells (VSMCs) have been identified in human peripheral blood.5,20 Meanwhile, EPCs are currently considered key players in the protection and regeneration of endothelium, the role of SMPCs is more unclear.4,5,14 Our data demonstrate that ART exposure was the main predictor of decreased number of CD34+/KDR+ and CD34+/VE-endoglin+ cells and increased number of CD14+/Endoglin+ in HIV-infected patients after controlling by traditional cardiovascular risk factors and HIV parameters. However, these alterations of vascular progenitor cells were only partially associated with high c-IMT in our cohort of HIV-infected patients.
In this study, we have quantified the circulating levels of the 2 EPC subtypes CD34+/KDR+ and CD34+/VE-cadherin+. It is well accepted that CD34+/KDR+ cells are less mature or early circulating EPCs, whereas more mature circulating EPCs are positive for CD34/VE-cadherin.4,5 At present, CD34+/KDR+ cells are the only EPC subtype that has demonstrated to have a strong association with cardiovascular risk. Although with some discrepancies, in the general population, most of the studies have identified an association between traditional cardiovascular risk factors and low levels of CD34+/KDR+ EPCs.5–7 However, in our population of HIV-infected patients, traditional cardiovascular risk factors, such as smoking, hypertension, or hyperlipidemia were not found predictive of low EPC levels. Several mechanisms such as levels of oxidative stress, nitric oxide (NO) activity, or cytokines (such as SDF-1, VEGF, and CD40l) that have been suggested to directly influence the mobilization or half-life of EPCs can be influenced not only by cardiovascular risk factors but also by HIV infection itself.2,4,5,21,22 Importantly, elevated plasma levels of cytokines (such as SDF-1 and CD40L) have been inversely associated with circulating EPC numbers or endothelial dysfunction because of dysfunctional peripheral blood–derived angiogenic early outgrowth cells, respectively.23,24 So, it might be that HIV infection could overshadow the effects of the cardiovascular risk factor in reducing the number of EPCs in our cohort of HIV-infected patients.
Our data confirm previous studies using the same methodology reporting that uncontrolled HIV infection is associated with a reduction of CD34+/KDR+ EPCs in comparison with noninfected subjects11,12 and extend them demonstrating that the CD34+/VE-cadherin+ subtype EPC is also decreased. In addition, to our knowledge, this is one of the first studies to have compared the number of circulating EPCs between ART-naive and treated HIV-infected patients. ART-treated patients showed further decreased levels of CD34+/KDR+ and CD34+/VE-cadherin+ cells with respect to naive patients. Some drugs with beneficial cardiovascular actions, such as statins and angiotensin converting enzyme (ACE) inhibitors have been reported to increase circulating CD34+/KDR+.9 However, these EPC subtypes decreased in long-term statin-treated patients meanwhile the number of CD34+/VE-cadherin+ EPCs is maintained.25,26
Although somewhat controversial, several studies have demonstrated the presence of both resident and circulating SMPCs with potential to trans-differentiate into VSMCs in atherosclerotic lesions.14 CD14+/Endoglin+ cells from peripheral blood mononuclear cells (PBMCs) have been identified as an important source of circulating SMPCs.20 ART-treated patients showed an increment of CD14+/Endoglin+ cell levels with respect to naive patients. Surprisingly, their levels were similar to those displayed by noninfected patients.
Increased c-IMT is an early marker of atherosclerosis and a potent predictor of future vascular events.27 In the general population, circulating CD34+/KDR+ cells are associated with increased c-IMT. Consistently, previous data have demonstrated that their levels independently predict cardiovascular events in patients with different cardiovascular risk and even in healthy subjects.5–8 Our data show that HIV-infected patients with low number of CD34+/KDR+ and/or CD34+/VE-cadherin+ cells showed increased c-IMT with respect to those with high EPC number, although it did not reach statistical significance. One potential explanation could be the different impact of traditional cardiovascular risk factors and HIV parameters in c-IMT and EPC levels. In our study, older age and hyperglycemia were more important than HIV-related parameters as predictors of increased c-IMT, but ART exposure, mainly PI therapy, was the most powerful predictor of low levels of CD34+/KDR+ and CD34+/VE-cadherin+ EPCs. Numerous studies have suggested a relationship between the use of ART, mainly PIs, and the development of atherosclerosis.1 Interestingly, accumulated exposure to PIs and to NRTIs, but not to NNRTIs, was associated with increased c-IMT. Although our analysis found a modest association between low EPCs and increased c-IMT, we cannot rule out the role of EPCs as atherogenic risk factors in HIV-infected patients. Previous studies have identified that a 5% IMT difference corresponds to a relative risk of future vascular events (myocardial infarction or stroke) of 1.04–1.14.28 The precise role of SMPCs is more unclear. There is some evidence demonstrating that these cells may contribute to both vascular physiology and to the development of vascular disease. Patients with coronary artery disease (CAD) showed significantly increased CD14+/Endoglin+ cells compared with patients without CAD,20 suggesting that mobilization of these cells might promote vascular disease. However, chronic SMPC injection has demonstrated to limit atherosclerotic lesion development and stabilize the atherosclerotic plaque in an experimental model.29 In patients with acute coronary syndrome, their deficiency in SMPCs has been associated with plaque vulnerability.14 Therefore, longitudinal studies will be necessary to conclude the contribution of CD14+/Endoglin+ to the pathogenic mechanism of atherosclerosis in HIV-infected patients.
The main limitation of our study is the lack of specific cell surface markers that precisely identify the different subtypes of progenitor cells. Most of the markers used to identified EPCs or SMPCs (CD34, KDR, CD14, and α-SMA) can also be expressed by mesenchymal or hematopoietic cells.5,20 However, their combinations identify specific progenitor cells to which our conclusions could be restricted. It was not our intention to study the relationship between vascular progenitor cells and ART because of the differences in exposure times to individual drugs. Although we adjusted by HIV-associated parameters and traditional cardiovascular risk factors, unmeasured factors may also impact in the observed levels of circulating vascular progenitor cells in ART-treated HIV-infected patients.19 Therefore, our results should be interpreted with caution due to the cross-sectional nature of our study. A prospective study evaluating circulating vascular progenitor cells in HIV patients is currently in course in our laboratory to better investigate the role of ART.
In summary, our data demonstrate that ART exposure is the main predictor of circulating vascular progenitor cell levels. However, their levels were only partially associated with high c-IMT in our cohort of HIV-infected patients. ART has already been found to have proatherogenic effect, but our data first describe its relationship with vascular progenitor cells and c-IMT.
The authors would like to thank Maria Rodrigo for her work in data collection.
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