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Circulating CD34+KDR+ Endothelial Progenitor Cells Correlate with Erectile Function and Endothelial Function in Overweight Men
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Circulating CD34+KDR+ Endothelial Progenitor Cells Correlate with Erectile Function and Endothelial Function in Overweight Men Katherine Esposito, MD, PhD,* Miryam Ciotola, MD,* Maria Ida Maiorino, MD,* Francesco Giugliano, MD,† Riccardo Autorino, MD,† Marco De Sio, MD,† Emmanuele Jannini, MD,‡ Andrea Lenzi, MD,§ and Dario Giugliano, MD, PhD* *Department of Geriatrics and Metabolic Diseases, Division of Metabolic Diseases, Second University of Naples, Naples, Italy; †Division of Urology, Second University of Naples, Naples, Italy; ‡School of Sexology, University of L’Aquila, Italy; §Department of Medical Physiopathology, La Sapienza University, Rome, Italy DOI: 10.1111/j.1743-6109.2008.01042.x
ABSTRACT
Introduction. Bone marrow-derived endothelial progenitor cells (EPCs) circulate in the peripheral blood and are involved in endothelial homeostasis and repair. Aim. The aim of this study was to assess the circulating levels of different EPC phenotypes in overweight men with or without erectile dysfunction (ED). As endothelial dysfunction is considered a necessary link with ED, endothelium-dependent vasodilation and its relation with EPCs were also investigated. Methods. We studied 30, otherwise healthy, overweight subjects with symptomatic ED for at least 6 months, and 30 age- and weight-matched subjects without ED. Erectile function was assessed by completing the International Index of Erectile Function (IIEF-5), which consists of items 5, 15, 4, 2, and 7 from the full-scale IIEF-15. Main Outcome Measures. Seven subpopulations of EPCs were determined by flow cytometry on the basis of the surface expression of CD34, CD133, and KDR antigens: CD34+, CD133+, KDR+, CD34+CD133+, CD34+KDR+, CD133+KDR+, and CD34+CD133+KDR+. Endothelium-dependent flow-mediated dilation (FMD) was evaluated in the right brachial artery with a high-resolution ultrasound machine following reactive hyperemia. Results. CD34+KDR+ cell count was significantly lower in men with ED as compared with men without ED (63.1 ⫾ 4 vs. 92.4 ⫾ 6 cells/106 events, mean ⫾ standard error, P < 0.01). There was a significant direct correlation between circulating CD34+KDR+ cells and the IIEF score (r = 0.44; P = 0.01): men with the severe form of ED presented the lowest level of circulating EPC CD34+KDR+ cells. No significant correlation was found between the circulating levels of the other EPC phenotypes and the IIEF score. There was a significant correlation between CD34+KDR+ cell count and FMD (r = 0.45; P = 0.01), but not between FMD and the other phenotypes. Conclusions. Circulating levels of CD34+KDR+ EPC are reduced in overweight subjects with ED and correlate with the severity of ED. Other EPC phenotypes are not related to ED, suggesting that the CD34+KDR+ phenotype of EPCs may be preferred in future studies. Esposito K, Ciotola M, Maiorino MI, Giugliano F, Autorino R, De Sio M, Jannini E, Lenzi A, and Giugliano D. Circulating CD34+KDR+ endothelial progenitor cells correlate with erectile function and endothelial function in overweight men. J Sex Med 2009;6:107–114. Key Words. Circulating Endothelial Progenitor Cells; Erectile Dysfunction; Endothelial Dysfunction; Overweight Subjects
Introduction
E
rectile dysfunction (ED) affects 30 million men in the United States and more than 150 million men worldwide, compromising multiple aspects of a patient’s life, including overall
© 2008 International Society for Sexual Medicine
quality of life and interpersonal relationships [1]. ED is associated with hypertension, diabetes mellitus, obesity, metabolic syndrome, hyperlipidemia, smoking, alcohol, and sedentary behavior; endothelial dysfunction seems to have a key role in these associations [2,3]. ED is often the first clinical sign J Sex Med 2009;6:107–114
108 of endothelial dysfunction and may be an initial step for the potential development of vascular disease [4–7]. Cellular mechanisms of endothelial dysfunction are apoptosis and decreased regeneration of the endothelial monolayer [8]. Bone marrow-derived endothelial progenitor cells (EPCs) circulate in the peripheral blood and have been related to repair mechanisms in endothelial dysfunction because of their ability of proliferation and differentiation into endothelial cells [9–13]. Recent studies demonstrated a reduction of EPCs in patients with cardiovascular risk factors, chronic heart failure, or endothelial dysfunction [14–16]. Thus, circulating levels of EPCs could be a pathophysiological link between cardiovascular risk factors and endothelial dysfunction, suggesting a significant role of EPCs in the progression of atherosclerosis and cardiovascular disease [13,17]. EPCs show a wide heterogenic antigenic profile. Typical surface antigens to identify EPCs are CD34, CD133, and KDR [18]. In particular, circulating levels of CD34+CD133+ cells were recently shown to be decreased in patients with ED, with or without cardiovascular risk factors [19]; moreover, lower circulating levels of CD133+ cells were demonstrated to be an independent risk factor for ED in patients with coronary artery disease, while CD34+KDR+ cells showed no relation with ED [20]. Uncertainty still remains about the best antigenic phenotype to identify circulating EPCs, nor is there a general consensus on the role of EPCs in ED. The present study was designed to assess the circulating levels of different EPC phenotypes in overweight men with or without ED. As endothelial dysfunction is considered a necessary link with ED, endothelium-dependent vasodilation and its relation with EPCs were also investigated. Material and Methods
Participants We studied 30 overweight subjects with symptomatic ED from at least 6 months, manifested as a persistent inability to attain and maintain an erection sufficient to permit sexual activity [21]. Men were recruited among those attending the outpatient department of the Division of Metabolic Diseases at the Second University of Naples, Italy. To be enrolled in the study, subjects had to have a body mass index (BMI) >25 kg/m2, no clinical evidence of diabetes mellitus, any peripheral vascular disease, coronary heart disease, or psychological J Sex Med 2009;6:107–114
Esposito et al. disorder, and no previous use of drugs for ED or drugs able to increase endothelial progenitors’ number (statins, angiotensin-converting enzyme [ACE] inhibitors, and glitazones). Impaired renal function, including macrolbuminuria, pelvic trauma, prostatic disease, neuropathy, and other ED risk factors, such as use of drugs or alcohol, were also considered exclusion criteria. Erectile function was assessed by completing the International Index of Erectile Function (IIEF)-5, which consists of items 5, 15, 4, 2, and 7 from the fullscale IIEF-15 [22]. A group of 30 age- and weightmatched men without ED was recruited from the medical and paramedical staff, and served as internal reference. All subjects gave an informed consent to participate in the study that was approved by the institutional review board.
Quantification of Circulating EPCs Peripheral blood cells were analyzed for the expression of surface antigens by direct flow cytometry [23–26]. Fasting blood samples were processed after 1–2 hours. Mononuclear cells were isolated from peripheral venous blood by density centrifugation. Then, the isolated blood cells were stained for 30 minutes at 4°C in the dark with fluorescein isothiocyanate (FITC)-conjugated antihuman CD34 monoclonal antibody (mAb) (Becton Dickinson, Buccinasco, Bologna, Italy), phycoerythrin (PE)-conjugated antihuman KDR mAb (R&D Systems, Minneapolis, MN, USA), and allophycocyanin (APC)-conjugated antihuman CD133 (Miltenyi Biotec, Calderara di Reno, Bologna, Italy). Isotope immunoglobulin IgG1 and IgG2a antibody was used to discriminate between signal range and baseline fluorescence within the samples. After incubation, quantitative analysis was performed on a BD FACSCalibur cytometer, and 1,000,000 cells were acquired in each sample. A morphological gate was used to exclude granulocytes. Then, we gated CD34+ or CD133+ peripheral blood cells in the mononuclear cell fraction and examined the resulting population for the dual expression of KDR. In the two-dimensional dotplot analysis, we identified CD34+CD133+ cells. Total KDR+ mononuclear cells were identified separately. Triple-positive cells were identified by the dual expression of KDR and CD133 in the CD34+ gate. Data were processed with the use of the Macintosh CELLQuest software program (Becton Dickinson). Measures were repeated twice in two separate blood samples. The instrument setup was optimized daily by analyzing the expression of peripheral blood lymphocytes labeled with
Circulating CD34 +KDR + Cells and Erectile Function an anti-CD4 FITC/CD8 PE/CD3 PECy5/CD45 APC 4-color combination. The same trained operator, who was blinded to the subjects’ characteristics, performed all of the tests throughout the study.
Endothelium-Dependent Vasodilation Endothelium-dependent flow-mediated dilation (FMD) was evaluated in the right brachial artery with a high-resolution ultrasound machine (Aloka 5500, Aloka, Assago, Milano, Italy) and in temperature-controlled room (21–24°C). Reactive hyperemia was induced by inflation of a pneumatic cuff on the upper arm to suprasystolic pressure, followed by cuff deflation after 4.5 minutes. The brachial artery was scanned in longitudinal sections 2–8 cm above the elbow, and the arterial diameter was measured on B-mode images using ultrasonic calipers. The end-diastolic arterial diameter was measured from one media-adventitia interface to the other at the clearest section three times at baseline and every 20 seconds after reactive hyperemia. The maximum vessel diameter was defined as the average of the three consecutive maximum diameter measurements after hyperemia. Vasodilation was calculated as the percent change in diameter compared with baseline. The same experienced operator performed all the studies with an intraobserver variation below 5%. Anthropometric Measures and Laboratory Analyses Height and weight were recorded, with the participants wearing lightweight clothing and no shoes, using a Seca 200 scale (Seca, Hamburg, Germany) with attached stadiometer. BMI was calculated as weight in kilograms divided by the square of height in meters (kg/m2). Assays for Table 1
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glucose, total, low-density, and high-density lipoprotein cholesterol, and triglyceride levels were performed in the hospital’s chemistry laboratory.
Statistical Analysis Data in tables and figures are presented as mean ⫾ standard error unless stated otherwise. Statistical significance was assumed at a P level of <0.05. The results from flow cytometry were expressed as the number of cells per 106 events. Differences between two or more groups were evaluated by two-sided Student’s t-test and analysis of variance. The c2 test was used for comparing dichotomous variables. Statistical associations between two variables were assessed using simple linear-regression analyses. Multivariable regression analysis was performed to determine the association of circulating EPC levels and ED. The ED score as the dependent variable and the number of circulating EPCs (independent variable) were included as continuous variables. All analyses were performed using SPSS 12 for Windows (SPSS Inc., Chicago, IL, USA). Results
The baseline clinical and metabolic characteristics of the participants in the study are shown in Table 1. The average age was not different between the two groups, as were the anthropometric parameters including BMI and waist values. FMD was significantly lower in the subjects with ED, indicating a reduced endothelium-dependent vasodilation. Circulating EPCs are considered to be characterized by the expression of immature markers, such as CD34 and CD133, and endothelial
Clinical characteristics of the study subjects
Parameter
Subjects with ED (N = 30)
Subjects without ED (N = 30)
P value
Age (year) BMI (weight/m2) Waist circumference (cm) Glucose (mg/dL) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) Triglycerides (mmol/L) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Smoking (%) Hypertension (%) IIEF FMD (%)
46.1 ⫾ 1.8 30.4 ⫾ 0.8 98.2 ⫾ 1.4 93.4 ⫾ 1.7 189 ⫾ 7 47.6 ⫾ 1.8 102 ⫾ 11 124 ⫾ 1.4 83 ⫾ 0.9 20 20 11.1 ⫾ 1.0 5.9 ⫾ 0.6
47.5 ⫾ 1.4 30.1 ⫾ 0.7 99.3 ⫾ 1.3 95.2 ⫾ 1.3 193 ⫾ 8 46.9 ⫾ 1.6 128 ⫾ 12 128 ⫾ 1.3 82 ⫾ 0.8 20 16.6 23 ⫾ 0.2 10.5 ⫾ 0.9
0.29 0.45 0.23 0.12 0.18 0.24 0.11 0.35 0.41 0.91 0.54 <0.001 0.01
Data are presented as mean ⫾ standard error. ED = erectile dysfunction; BMI = body mass index; HDL = high-density lipoprotein; IIEF = International Index of Erectile Function; FMD = flow-mediated dilation.
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Esposito et al.
Figure 2 Correlation between CD34+KDR+ cell count and the International Index of Erectile Function score in overweight men with erectile dysfunction.
Figure 1 Circulating levels of seven progenitor cell phenotypes in overweight subjects with erectile dysfunction (ED) (gray columns) or without ED (black columns). The only significant difference was seen in the CD34 KDR phenotype (P = 0.01).
markers, such as KDR. With the use of these surface markers, seven different subpopulations with different antigenic profiles could be identified: CD34+, CD133+, KDR+, CD34+CD133+, CD133+KDR+, and CD34+KDR+, + + + CD34 CD133 KDR (Figure 1). There was no significant difference in most of the EPC phenotypes between the two groups, except for CD34+KDR+ cell count, which was significantly lower in the men with ED as compared with the men without ED (63.1 ⫾ 4 vs. 92.4 ⫾ 6, P = 0.01). Moreover, there was a significant correlation between circulating CD34+KDR+ cells and the IIEF score (r = 0.44; P = 0.01), in such a way that the men with the mild form of ED presented the highest levels of circulating EPC CD34+KDR+ cells (Figure 2). On the contrary, no significant correlation was found between the circulating levels of the other EPC phenotypes and the IIEF score (Table 2). Finally, there was a significant corJ Sex Med 2009;6:107–114
Figure 3 Correlation between CD34+KDR+ cell count and flow-mediated dilation (%) in overweight men with erectile dysfunction.
relation between CD34+KDR+ cell count and FMD (r = 0.45; P = 0.01) (Figure 3), but not between FMD and the other phenotypes (data not shown). Table 2 Linear correlation between endothelial progenitor cell count and the International Index of Erectile Function score according to the different antigenic phenotypes Antigenic phenotype
Pearson’s r
P
CD34+ KDR+ CD133+ CD34+CD133+ CD34+KDR+ CD133+KDR+ CD34+CD133+KDR+
0.05 0.12 -0.09 -0.04 0.44 0.03 0.05
0.38 0.15 0.27 0.43 0.01 0.56 0.65
Circulating CD34 +KDR + Cells and Erectile Function
Figure 4 Circulating CD34+KDR+ cells across categories of the International Index of Erectile Function (IIEF): mild erectile dysfunction (ED) (IIEF between 16 and 21, N = 7), moderate ED (IIEF between 10 and 15, N = 9), and severe ED (IIEF < 10, N = 14).
There was a decrement in circulating CD34+KDR+ cell count across categories of ED: in the subjects with mild ED (IIEF between 16 and 21, N = 7), the CD34+KDR+ cell count was 91 ⫾ 5.3; in the subjects with moderate ED (IIEF between 10 and 15, N = 9), the CD34+KDR+ cell count was 62 ⫾ 4.2; and in the subjects with severe ED (IIEF < 10, N = 14), the CD34+KDR+ cell count was 42.5 ⫾ 3.1 (Figure 4). The overall relation was significant (P for trend = 0.01). Multivariable regression analysis adjusted for age, smoking, BMI, waist, hypertension, and lipid levels identified lower levels of circulating CD34+KDR+ cell count as an independent risk factor for ED (P = 0.02); age (P = 0.01) and BMI (P = 0.02) were also independent predictors of ED. Discussion
EPCs are circulating immature cells that may play a major role in repair mechanisms of the endothelial monolayer, leading to improvement of endothelial function because of regeneration of the endothelial monolayer as well as neoangiogenesis [27]. Given that the group of EPCs shows a wide heterogeneity with functionally important subpopulations, there is no clear consensus on which antigenic profile best identifies progenitor cells with the potential to repair the endothelium. Three main antigenic profiles have been proposed: CD34 is an adhesion molecule expressed on hematopoietic stem cells and is typically considered a marker of immaturity; CD133 is a surface
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antigen of unknown function that identifies more immature progenitor cells than CD34 alone; and KDR represents type 2 vascular endothelial growth factor receptor and indicates early endothelial differentiation [26]. The present study represents the first comprehensive assessment of different phenotypes of EPCs in patients with ED. We found that overweight subjects with ED had significantly lower levels of circulating CD34+KDR+ cells than subjects with normal erectile function, with a significant correlation between the number of circulating CD34+KDR+ cells and the IIEF score, and a liner decrement in circulating CD34+KDR+ cells across categories of ED. Moreover, circulating levels of CD34+KDR+ cells showed a significant relation with FMD, indicating that this phenotype of EPCs is also implicated in endothelium-dependent vasodilation. Circulating levels of other EPC phenotypes did not show any significant correlation with the IIEF or FMD. Literature data evaluating the putative causative role of EPCs in ED are scanty and discordant. Circulating levels of CD34+CD133+ cells were recently shown to be decreased in patients with ED [19]. However, in the study of Foresta et al. [19], patients with ED, both with and without cardiovascular risk, were evaluated, and assessment of other EPC phenotypes was not performed. In patients with coronary artery disease, significantly lower levels of circulating CD133+ cells were found in patients with ED than in patients with a normal erectile function [20]; on the other hand, CD34+KDR+ cells were distributed similarly in both groups. Moreover, circulating CD133+ cells showed a direct correlation with ED, and reduced levels of circulating CD133+ cells were an independent risk factor for ED. However, circulating CD34+KDR+ cells could be profoundly reduced in patients with coronary heart disease, and this could help explain the different results [17]. In keeping with this possibility, two previous studies have also demonstrated that the number of EPCs (CD34+KDR+) is not related to overweight in men; however, these results were obtained in patients with confirmed coronary artery disease [15] or in patients with type 2 diabetes and peripheral vascular complications [28], which may have introduced some bias. On the other hand, the significance of overweight in the results obtained in our study is smoothed by the comparison with a control, BMImatched group without ED, which indicates that ED is the condition associated with low circulating CD34+KDR+ cells. J Sex Med 2009;6:107–114
112 The conceptual revolution in the field of progenitor cells implies that EPC depletion represents at the same time a pathogenic step of atherogenesis and a biomarker of cardiovascular risk. In this perspective, our results indicate that, among the different progenitor cell subtypes, the CD34+KDR+ phenotype may be that more closely linked to ED as well as to endothelial dysfunction; hence, this phenotype may be causatively associated with atherosclerosis. The relation between endothelial function, as evaluated by flowmediated brachial artery reactivity, and the number of EPCs has been previously described [14]. One plausible sequence of events may be that a decrease in circulating EPCs may impair FMD and hence open the way to subsequent ED, which is now increasingly seen as a predictor of cardiovascular disease [6,7]. The results of previous studies that indicate that the CD34+KDR+ cell level independently predicts cardiovascular events and atherosclerosis progression in patients with coronary artery disease [15,17], and that negatively correlate with values of carotid intima-media thickness [29] seem to support this interpretation. On the other hand, the possibility cannot be excluded that continuous endothelial damage or dysfunction, as a result of known and unknown risk factors, may lead to an eventual depletion or exhaustion of a presumed finite supply of EPCs. In general, data have become available indicating that alterations in EPCs may have an important causative role in the development and progression of atherosclerosis [30–33]. Recent studies further demonstrated a reduction of EPCs in patients with chronic heart failure [16] or endothelial dysfunction [34]. In subjects with the metabolic syndrome [26], circulating EPCs are synergistically decreased by clustering components of the syndrome, and their levels negatively correlate with the homeostasis model assessment value, a measure of insulin resistance. Virtually all risk factors for atherosclerosis have been associated with decreased levels of circulating EPCs, while absent or insufficient EPCs in patients with endothelial cell injury may affect the progression of cardiovascular disease, with EPCs as an independent predictor of cardiovascular outcomes [15]. In conclusion, the results of the present study demonstrate that circulating CD34+KDR+ cells are reduced in overweight subjects with ED, and correlate with the severity of ED and with endothelium-dependent FMD. A major limitation of the study relates to the limited number of subJ Sex Med 2009;6:107–114
Esposito et al. jects investigated, which needs confirmation, and the possibility that the findings cannot be extrapolated to other populations with ED. Strengths of the study are assessment of seven different EPC phenotypes, evaluation of overweight patients with ED, but without other known risk factors for cardiovascular disease, and the strong correlation we found between circulating CD34+KDR+ cells and the severity of ED or endothelial dysfunction. Our results also suggest that the CD34+KDR+ phenotype of circulating EPCs may be preferred in future studies in which EPC count is intended as a cardiovascular biomarker, and this may provide additional information beyond classic risk factors and inflammatory markers. Corresponding Author: Katherine Esposito, MD, PhD, Division of Metabolic Diseases, Second University of Naples, Policlinico Universitario, Piazza L. Miraglia 2, 80138 Naples, Italy. Tel/Fax: (39) 0815665054; E-mail: [email protected] Conflict of Interest: None declared.
Statement of Authorship
Category 1 (a) Conception and Design Katherine Esposito; Miryam Ciotola; Dario Giugliano; Maria Ida Maiorino (b) Acquisition of Data Marco De Sio; Riccardo Autorino; Francesco Giugliano (c) Analysis and Interpretation of Data Andrea Lenzi; Emmanuele Jannini
Category 2 (a) Drafting the Article Katherine Esposito; Miryam Ciotola; Maria Ida Maiorino; Francesco Giugliano (b) Revising It for Intellectual Content Marco De Sio; Riccardo Autorino; Dario Giugliano; Andrea Lenzi; Emmanuele Jannini
Category 3 (a) Final Approval of the Completed Article Katherine Esposito; Miryam Ciotola; Maria Ida Maiorino; Francesco Giugliano; Riccardo Autorino; Marco De Sio; Emmanuele Jannini; Andrea Lenzi; Dario Giugliano
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Circulating CD34+KDR+ Endothelial Progenitor Cells Correlate with Erectile Function and Endothelial Function in Overweight Men Katherine Esposito, MD, PhD,* Miryam Ciotola, MD,* Maria Ida Maiorino, MD,* Francesco Giugliano, MD,† Riccardo Autorino, MD,† Marco De Sio, MD,† Emmanuele Jannini, MD,‡ Andrea Lenzi, MD,§ and Dario Giugliano, MD, PhD* *Department of Geriatrics and Metabolic Diseases, Division of Metabolic Diseases, Second University of Naples, Naples, Italy; †Division of Urology, Second University of Naples, Naples, Italy; ‡School of Sexology, University of L’Aquila, Italy; §Department of Medical Physiopathology, La Sapienza University, Rome, Italy DOI: 10.1111/j.1743-6109.2008.01042.x
ABSTRACT
Introduction. Bone marrow-derived endothelial progenitor cells (EPCs) circulate in the peripheral blood and are involved in endothelial homeostasis and repair. Aim. The aim of this study was to assess the circulating levels of different EPC phenotypes in overweight men with or without erectile dysfunction (ED). As endothelial dysfunction is considered a necessary link with ED, endothelium-dependent vasodilation and its relation with EPCs were also investigated. Methods. We studied 30, otherwise healthy, overweight subjects with symptomatic ED for at least 6 months, and 30 age- and weight-matched subjects without ED. Erectile function was assessed by completing the International Index of Erectile Function (IIEF-5), which consists of items 5, 15, 4, 2, and 7 from the full-scale IIEF-15. Main Outcome Measures. Seven subpopulations of EPCs were determined by flow cytometry on the basis of the surface expression of CD34, CD133, and KDR antigens: CD34+, CD133+, KDR+, CD34+CD133+, CD34+KDR+, CD133+KDR+, and CD34+CD133+KDR+. Endothelium-dependent flow-mediated dilation (FMD) was evaluated in the right brachial artery with a high-resolution ultrasound machine following reactive hyperemia. Results. CD34+KDR+ cell count was significantly lower in men with ED as compared with men without ED (63.1 ⫾ 4 vs. 92.4 ⫾ 6 cells/106 events, mean ⫾ standard error, P < 0.01). There was a significant direct correlation between circulating CD34+KDR+ cells and the IIEF score (r = 0.44; P = 0.01): men with the severe form of ED presented the lowest level of circulating EPC CD34+KDR+ cells. No significant correlation was found between the circulating levels of the other EPC phenotypes and the IIEF score. There was a significant correlation between CD34+KDR+ cell count and FMD (r = 0.45; P = 0.01), but not between FMD and the other phenotypes. Conclusions. Circulating levels of CD34+KDR+ EPC are reduced in overweight subjects with ED and correlate with the severity of ED. Other EPC phenotypes are not related to ED, suggesting that the CD34+KDR+ phenotype of EPCs may be preferred in future studies. Esposito K, Ciotola M, Maiorino MI, Giugliano F, Autorino R, De Sio M, Jannini E, Lenzi A, and Giugliano D. Circulating CD34+KDR+ endothelial progenitor cells correlate with erectile function and endothelial function in overweight men. J Sex Med 2009;6:107–114. Key Words. Circulating Endothelial Progenitor Cells; Erectile Dysfunction; Endothelial Dysfunction; Overweight Subjects
Introduction
E
rectile dysfunction (ED) affects 30 million men in the United States and more than 150 million men worldwide, compromising multiple aspects of a patient’s life, including overall
© 2008 International Society for Sexual Medicine
quality of life and interpersonal relationships [1]. ED is associated with hypertension, diabetes mellitus, obesity, metabolic syndrome, hyperlipidemia, smoking, alcohol, and sedentary behavior; endothelial dysfunction seems to have a key role in these associations [2,3]. ED is often the first clinical sign J Sex Med 2009;6:107–114
108 of endothelial dysfunction and may be an initial step for the potential development of vascular disease [4–7]. Cellular mechanisms of endothelial dysfunction are apoptosis and decreased regeneration of the endothelial monolayer [8]. Bone marrow-derived endothelial progenitor cells (EPCs) circulate in the peripheral blood and have been related to repair mechanisms in endothelial dysfunction because of their ability of proliferation and differentiation into endothelial cells [9–13]. Recent studies demonstrated a reduction of EPCs in patients with cardiovascular risk factors, chronic heart failure, or endothelial dysfunction [14–16]. Thus, circulating levels of EPCs could be a pathophysiological link between cardiovascular risk factors and endothelial dysfunction, suggesting a significant role of EPCs in the progression of atherosclerosis and cardiovascular disease [13,17]. EPCs show a wide heterogenic antigenic profile. Typical surface antigens to identify EPCs are CD34, CD133, and KDR [18]. In particular, circulating levels of CD34+CD133+ cells were recently shown to be decreased in patients with ED, with or without cardiovascular risk factors [19]; moreover, lower circulating levels of CD133+ cells were demonstrated to be an independent risk factor for ED in patients with coronary artery disease, while CD34+KDR+ cells showed no relation with ED [20]. Uncertainty still remains about the best antigenic phenotype to identify circulating EPCs, nor is there a general consensus on the role of EPCs in ED. The present study was designed to assess the circulating levels of different EPC phenotypes in overweight men with or without ED. As endothelial dysfunction is considered a necessary link with ED, endothelium-dependent vasodilation and its relation with EPCs were also investigated. Material and Methods
Participants We studied 30 overweight subjects with symptomatic ED from at least 6 months, manifested as a persistent inability to attain and maintain an erection sufficient to permit sexual activity [21]. Men were recruited among those attending the outpatient department of the Division of Metabolic Diseases at the Second University of Naples, Italy. To be enrolled in the study, subjects had to have a body mass index (BMI) >25 kg/m2, no clinical evidence of diabetes mellitus, any peripheral vascular disease, coronary heart disease, or psychological J Sex Med 2009;6:107–114
Esposito et al. disorder, and no previous use of drugs for ED or drugs able to increase endothelial progenitors’ number (statins, angiotensin-converting enzyme [ACE] inhibitors, and glitazones). Impaired renal function, including macrolbuminuria, pelvic trauma, prostatic disease, neuropathy, and other ED risk factors, such as use of drugs or alcohol, were also considered exclusion criteria. Erectile function was assessed by completing the International Index of Erectile Function (IIEF)-5, which consists of items 5, 15, 4, 2, and 7 from the fullscale IIEF-15 [22]. A group of 30 age- and weightmatched men without ED was recruited from the medical and paramedical staff, and served as internal reference. All subjects gave an informed consent to participate in the study that was approved by the institutional review board.
Quantification of Circulating EPCs Peripheral blood cells were analyzed for the expression of surface antigens by direct flow cytometry [23–26]. Fasting blood samples were processed after 1–2 hours. Mononuclear cells were isolated from peripheral venous blood by density centrifugation. Then, the isolated blood cells were stained for 30 minutes at 4°C in the dark with fluorescein isothiocyanate (FITC)-conjugated antihuman CD34 monoclonal antibody (mAb) (Becton Dickinson, Buccinasco, Bologna, Italy), phycoerythrin (PE)-conjugated antihuman KDR mAb (R&D Systems, Minneapolis, MN, USA), and allophycocyanin (APC)-conjugated antihuman CD133 (Miltenyi Biotec, Calderara di Reno, Bologna, Italy). Isotope immunoglobulin IgG1 and IgG2a antibody was used to discriminate between signal range and baseline fluorescence within the samples. After incubation, quantitative analysis was performed on a BD FACSCalibur cytometer, and 1,000,000 cells were acquired in each sample. A morphological gate was used to exclude granulocytes. Then, we gated CD34+ or CD133+ peripheral blood cells in the mononuclear cell fraction and examined the resulting population for the dual expression of KDR. In the two-dimensional dotplot analysis, we identified CD34+CD133+ cells. Total KDR+ mononuclear cells were identified separately. Triple-positive cells were identified by the dual expression of KDR and CD133 in the CD34+ gate. Data were processed with the use of the Macintosh CELLQuest software program (Becton Dickinson). Measures were repeated twice in two separate blood samples. The instrument setup was optimized daily by analyzing the expression of peripheral blood lymphocytes labeled with
Circulating CD34 +KDR + Cells and Erectile Function an anti-CD4 FITC/CD8 PE/CD3 PECy5/CD45 APC 4-color combination. The same trained operator, who was blinded to the subjects’ characteristics, performed all of the tests throughout the study.
Endothelium-Dependent Vasodilation Endothelium-dependent flow-mediated dilation (FMD) was evaluated in the right brachial artery with a high-resolution ultrasound machine (Aloka 5500, Aloka, Assago, Milano, Italy) and in temperature-controlled room (21–24°C). Reactive hyperemia was induced by inflation of a pneumatic cuff on the upper arm to suprasystolic pressure, followed by cuff deflation after 4.5 minutes. The brachial artery was scanned in longitudinal sections 2–8 cm above the elbow, and the arterial diameter was measured on B-mode images using ultrasonic calipers. The end-diastolic arterial diameter was measured from one media-adventitia interface to the other at the clearest section three times at baseline and every 20 seconds after reactive hyperemia. The maximum vessel diameter was defined as the average of the three consecutive maximum diameter measurements after hyperemia. Vasodilation was calculated as the percent change in diameter compared with baseline. The same experienced operator performed all the studies with an intraobserver variation below 5%. Anthropometric Measures and Laboratory Analyses Height and weight were recorded, with the participants wearing lightweight clothing and no shoes, using a Seca 200 scale (Seca, Hamburg, Germany) with attached stadiometer. BMI was calculated as weight in kilograms divided by the square of height in meters (kg/m2). Assays for Table 1
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glucose, total, low-density, and high-density lipoprotein cholesterol, and triglyceride levels were performed in the hospital’s chemistry laboratory.
Statistical Analysis Data in tables and figures are presented as mean ⫾ standard error unless stated otherwise. Statistical significance was assumed at a P level of <0.05. The results from flow cytometry were expressed as the number of cells per 106 events. Differences between two or more groups were evaluated by two-sided Student’s t-test and analysis of variance. The c2 test was used for comparing dichotomous variables. Statistical associations between two variables were assessed using simple linear-regression analyses. Multivariable regression analysis was performed to determine the association of circulating EPC levels and ED. The ED score as the dependent variable and the number of circulating EPCs (independent variable) were included as continuous variables. All analyses were performed using SPSS 12 for Windows (SPSS Inc., Chicago, IL, USA). Results
The baseline clinical and metabolic characteristics of the participants in the study are shown in Table 1. The average age was not different between the two groups, as were the anthropometric parameters including BMI and waist values. FMD was significantly lower in the subjects with ED, indicating a reduced endothelium-dependent vasodilation. Circulating EPCs are considered to be characterized by the expression of immature markers, such as CD34 and CD133, and endothelial
Clinical characteristics of the study subjects
Parameter
Subjects with ED (N = 30)
Subjects without ED (N = 30)
P value
Age (year) BMI (weight/m2) Waist circumference (cm) Glucose (mg/dL) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) Triglycerides (mmol/L) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Smoking (%) Hypertension (%) IIEF FMD (%)
46.1 ⫾ 1.8 30.4 ⫾ 0.8 98.2 ⫾ 1.4 93.4 ⫾ 1.7 189 ⫾ 7 47.6 ⫾ 1.8 102 ⫾ 11 124 ⫾ 1.4 83 ⫾ 0.9 20 20 11.1 ⫾ 1.0 5.9 ⫾ 0.6
47.5 ⫾ 1.4 30.1 ⫾ 0.7 99.3 ⫾ 1.3 95.2 ⫾ 1.3 193 ⫾ 8 46.9 ⫾ 1.6 128 ⫾ 12 128 ⫾ 1.3 82 ⫾ 0.8 20 16.6 23 ⫾ 0.2 10.5 ⫾ 0.9
0.29 0.45 0.23 0.12 0.18 0.24 0.11 0.35 0.41 0.91 0.54 <0.001 0.01
Data are presented as mean ⫾ standard error. ED = erectile dysfunction; BMI = body mass index; HDL = high-density lipoprotein; IIEF = International Index of Erectile Function; FMD = flow-mediated dilation.
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Esposito et al.
Figure 2 Correlation between CD34+KDR+ cell count and the International Index of Erectile Function score in overweight men with erectile dysfunction.
Figure 1 Circulating levels of seven progenitor cell phenotypes in overweight subjects with erectile dysfunction (ED) (gray columns) or without ED (black columns). The only significant difference was seen in the CD34 KDR phenotype (P = 0.01).
markers, such as KDR. With the use of these surface markers, seven different subpopulations with different antigenic profiles could be identified: CD34+, CD133+, KDR+, CD34+CD133+, CD133+KDR+, and CD34+KDR+, + + + CD34 CD133 KDR (Figure 1). There was no significant difference in most of the EPC phenotypes between the two groups, except for CD34+KDR+ cell count, which was significantly lower in the men with ED as compared with the men without ED (63.1 ⫾ 4 vs. 92.4 ⫾ 6, P = 0.01). Moreover, there was a significant correlation between circulating CD34+KDR+ cells and the IIEF score (r = 0.44; P = 0.01), in such a way that the men with the mild form of ED presented the highest levels of circulating EPC CD34+KDR+ cells (Figure 2). On the contrary, no significant correlation was found between the circulating levels of the other EPC phenotypes and the IIEF score (Table 2). Finally, there was a significant corJ Sex Med 2009;6:107–114
Figure 3 Correlation between CD34+KDR+ cell count and flow-mediated dilation (%) in overweight men with erectile dysfunction.
relation between CD34+KDR+ cell count and FMD (r = 0.45; P = 0.01) (Figure 3), but not between FMD and the other phenotypes (data not shown). Table 2 Linear correlation between endothelial progenitor cell count and the International Index of Erectile Function score according to the different antigenic phenotypes Antigenic phenotype
Pearson’s r
P
CD34+ KDR+ CD133+ CD34+CD133+ CD34+KDR+ CD133+KDR+ CD34+CD133+KDR+
0.05 0.12 -0.09 -0.04 0.44 0.03 0.05
0.38 0.15 0.27 0.43 0.01 0.56 0.65
Circulating CD34 +KDR + Cells and Erectile Function
Figure 4 Circulating CD34+KDR+ cells across categories of the International Index of Erectile Function (IIEF): mild erectile dysfunction (ED) (IIEF between 16 and 21, N = 7), moderate ED (IIEF between 10 and 15, N = 9), and severe ED (IIEF < 10, N = 14).
There was a decrement in circulating CD34+KDR+ cell count across categories of ED: in the subjects with mild ED (IIEF between 16 and 21, N = 7), the CD34+KDR+ cell count was 91 ⫾ 5.3; in the subjects with moderate ED (IIEF between 10 and 15, N = 9), the CD34+KDR+ cell count was 62 ⫾ 4.2; and in the subjects with severe ED (IIEF < 10, N = 14), the CD34+KDR+ cell count was 42.5 ⫾ 3.1 (Figure 4). The overall relation was significant (P for trend = 0.01). Multivariable regression analysis adjusted for age, smoking, BMI, waist, hypertension, and lipid levels identified lower levels of circulating CD34+KDR+ cell count as an independent risk factor for ED (P = 0.02); age (P = 0.01) and BMI (P = 0.02) were also independent predictors of ED. Discussion
EPCs are circulating immature cells that may play a major role in repair mechanisms of the endothelial monolayer, leading to improvement of endothelial function because of regeneration of the endothelial monolayer as well as neoangiogenesis [27]. Given that the group of EPCs shows a wide heterogeneity with functionally important subpopulations, there is no clear consensus on which antigenic profile best identifies progenitor cells with the potential to repair the endothelium. Three main antigenic profiles have been proposed: CD34 is an adhesion molecule expressed on hematopoietic stem cells and is typically considered a marker of immaturity; CD133 is a surface
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antigen of unknown function that identifies more immature progenitor cells than CD34 alone; and KDR represents type 2 vascular endothelial growth factor receptor and indicates early endothelial differentiation [26]. The present study represents the first comprehensive assessment of different phenotypes of EPCs in patients with ED. We found that overweight subjects with ED had significantly lower levels of circulating CD34+KDR+ cells than subjects with normal erectile function, with a significant correlation between the number of circulating CD34+KDR+ cells and the IIEF score, and a liner decrement in circulating CD34+KDR+ cells across categories of ED. Moreover, circulating levels of CD34+KDR+ cells showed a significant relation with FMD, indicating that this phenotype of EPCs is also implicated in endothelium-dependent vasodilation. Circulating levels of other EPC phenotypes did not show any significant correlation with the IIEF or FMD. Literature data evaluating the putative causative role of EPCs in ED are scanty and discordant. Circulating levels of CD34+CD133+ cells were recently shown to be decreased in patients with ED [19]. However, in the study of Foresta et al. [19], patients with ED, both with and without cardiovascular risk, were evaluated, and assessment of other EPC phenotypes was not performed. In patients with coronary artery disease, significantly lower levels of circulating CD133+ cells were found in patients with ED than in patients with a normal erectile function [20]; on the other hand, CD34+KDR+ cells were distributed similarly in both groups. Moreover, circulating CD133+ cells showed a direct correlation with ED, and reduced levels of circulating CD133+ cells were an independent risk factor for ED. However, circulating CD34+KDR+ cells could be profoundly reduced in patients with coronary heart disease, and this could help explain the different results [17]. In keeping with this possibility, two previous studies have also demonstrated that the number of EPCs (CD34+KDR+) is not related to overweight in men; however, these results were obtained in patients with confirmed coronary artery disease [15] or in patients with type 2 diabetes and peripheral vascular complications [28], which may have introduced some bias. On the other hand, the significance of overweight in the results obtained in our study is smoothed by the comparison with a control, BMImatched group without ED, which indicates that ED is the condition associated with low circulating CD34+KDR+ cells. J Sex Med 2009;6:107–114
112 The conceptual revolution in the field of progenitor cells implies that EPC depletion represents at the same time a pathogenic step of atherogenesis and a biomarker of cardiovascular risk. In this perspective, our results indicate that, among the different progenitor cell subtypes, the CD34+KDR+ phenotype may be that more closely linked to ED as well as to endothelial dysfunction; hence, this phenotype may be causatively associated with atherosclerosis. The relation between endothelial function, as evaluated by flowmediated brachial artery reactivity, and the number of EPCs has been previously described [14]. One plausible sequence of events may be that a decrease in circulating EPCs may impair FMD and hence open the way to subsequent ED, which is now increasingly seen as a predictor of cardiovascular disease [6,7]. The results of previous studies that indicate that the CD34+KDR+ cell level independently predicts cardiovascular events and atherosclerosis progression in patients with coronary artery disease [15,17], and that negatively correlate with values of carotid intima-media thickness [29] seem to support this interpretation. On the other hand, the possibility cannot be excluded that continuous endothelial damage or dysfunction, as a result of known and unknown risk factors, may lead to an eventual depletion or exhaustion of a presumed finite supply of EPCs. In general, data have become available indicating that alterations in EPCs may have an important causative role in the development and progression of atherosclerosis [30–33]. Recent studies further demonstrated a reduction of EPCs in patients with chronic heart failure [16] or endothelial dysfunction [34]. In subjects with the metabolic syndrome [26], circulating EPCs are synergistically decreased by clustering components of the syndrome, and their levels negatively correlate with the homeostasis model assessment value, a measure of insulin resistance. Virtually all risk factors for atherosclerosis have been associated with decreased levels of circulating EPCs, while absent or insufficient EPCs in patients with endothelial cell injury may affect the progression of cardiovascular disease, with EPCs as an independent predictor of cardiovascular outcomes [15]. In conclusion, the results of the present study demonstrate that circulating CD34+KDR+ cells are reduced in overweight subjects with ED, and correlate with the severity of ED and with endothelium-dependent FMD. A major limitation of the study relates to the limited number of subJ Sex Med 2009;6:107–114
Esposito et al. jects investigated, which needs confirmation, and the possibility that the findings cannot be extrapolated to other populations with ED. Strengths of the study are assessment of seven different EPC phenotypes, evaluation of overweight patients with ED, but without other known risk factors for cardiovascular disease, and the strong correlation we found between circulating CD34+KDR+ cells and the severity of ED or endothelial dysfunction. Our results also suggest that the CD34+KDR+ phenotype of circulating EPCs may be preferred in future studies in which EPC count is intended as a cardiovascular biomarker, and this may provide additional information beyond classic risk factors and inflammatory markers. Corresponding Author: Katherine Esposito, MD, PhD, Division of Metabolic Diseases, Second University of Naples, Policlinico Universitario, Piazza L. Miraglia 2, 80138 Naples, Italy. Tel/Fax: (39) 0815665054; E-mail: [email protected] Conflict of Interest: None declared.
Statement of Authorship
Category 1 (a) Conception and Design Katherine Esposito; Miryam Ciotola; Dario Giugliano; Maria Ida Maiorino (b) Acquisition of Data Marco De Sio; Riccardo Autorino; Francesco Giugliano (c) Analysis and Interpretation of Data Andrea Lenzi; Emmanuele Jannini
Category 2 (a) Drafting the Article Katherine Esposito; Miryam Ciotola; Maria Ida Maiorino; Francesco Giugliano (b) Revising It for Intellectual Content Marco De Sio; Riccardo Autorino; Dario Giugliano; Andrea Lenzi; Emmanuele Jannini
Category 3 (a) Final Approval of the Completed Article Katherine Esposito; Miryam Ciotola; Maria Ida Maiorino; Francesco Giugliano; Riccardo Autorino; Marco De Sio; Emmanuele Jannini; Andrea Lenzi; Dario Giugliano
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