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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-0763
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 11 4599-4602
Copyright © 2006 by The Endocrine Society

Reduced Number of Circulating Endothelial Progenitor Cells in Hypogonadal Men

C. Foresta, N. Caretta, A. Lana, L. De Toni, A. Biagioli, A. Ferlin and A. Garolla

Department of Histology, Microbiology, and Medical Biotechnologies, Centre for Male Gamete Cryopreservation, University of Padova, 35121 Padova, Italy

Address all correspondence and requests for reprints to: Prof. Carlo Foresta, University of Padova, Department of Histology, Microbiology, and Medical Biotechnologies, Centre for Male Gamete Cryopreservation, Via Giustiniani 2, 35121 Padova, Italy. E-mail: carlo.foresta{at}unipd.it.


   Abstract
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 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Endothelial dysfunction seems to be the first step of the atherosclerotic process. In the past few years, it has been demonstrated that injured endothelial monolayer is restored by a premature pool of circulating progenitor cells (PCs) and a more mature one of circulating endothelial PCs (EPCs). Even though there is increasing evidence that estrogens play a beneficial role on EPCs and, even if debated, on the cardiovascular system, less is known about androgens.

Objective: Our objective was to evaluate the levels of circulating PCs and EPCs in men with hypogonadotropic hypogonadism (HH) and the effect of prolonged testosterone (T) replacement therapy on these cells.

Design and Setting: We conducted a prospective study on males with HH at a university andrological center.

Patients: The study included 10 young HH patients (28.6 ± 3.1 yr) and 25 age-matched controls.

Interventions: Idiopathic HH patients were treated with T gel therapy, 50 mg/d for 6 months.

Main Outcome Measures: We assessed circulating PC and EPC concentrations and immunocytochemistry for androgen receptor expression on cultured EPCs.

Results: At baseline, HH patients showed a significant reduction of both PCs and EPCs with respect to controls. T replacement therapy induced a significant increase of these cells with respect to baseline. Immunocytochemistry on cultured EPCs showed strong expression of the androgen receptor.

Conclusions: Hypotestosteronemia is associated with a low number of circulating PCs and EPCs in young HH subjects. T treatment is able to induce an increase in these cells through a possible direct effect on the bone marrow.


   Introduction
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 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
RECENTLY IT HAS been demonstrated that a reduction in plasma testosterone (T) might contribute to increased arterial stiffness, which in turn has been associated with increased cardiovascular risk (1). A positive interaction between androgens and the vessel wall has been hypothesized because 1) in animal models, T supplementation has been demonstrated to inhibit atheroma formation (2); 2) androgen withdrawal in men has been associated with decreased central arterial compliance (3); 3) T has been suggested to act as a protective factor against atherosclerosis because of its immunomodulating effects on plaque development and stability (4); 4) iv administration of T has been demonstrated to produce coronary vasodilation and to increase the angina threshold in men with coronary artery disease (5); and 5) long-term oral administration of T has been demonstrated to induce both endothelium-dependent and independent vasorelaxation (6).

Endothelium has a fundamental role in the control of vascular tone and blood flow (7). Conditions associated with a reduced endothelial function may determine an imbalance between vasodilating and vasoconstricting substances produced by or acting on the vascular wall increasing arterial stiffness (8). Cardiovascular risk factors, such as hypertension, diabetes, smoking, and hyperlipidemia, seem to affect the endothelial monolayer leading to endothelial dysfunction (7). Recently, it has been demonstrated that injured endothelial monolayer is restored by circulating progenitor cells (PCs) and circulating endothelial PCs (EPCs). EPCs are a circulating pool of cells, positive for CD34, AC133, and vascular endothelial growth factor receptor type 2 (VEGFR2), able to home into sites of endothelial injury and to repair endothelial damage; PCs are a more immature pool of circulating cells positive for CD34 and AC133 with similar characteristics. Both PCs and EPCs originate from hematopoietic stem cells of the bone marrow, migrate into peripheral circulation, home to sites of neovascularization, and differentiate into mature endothelial cells. In this way, they contribute to neovascularization and to endothelial repair (9, 10).

The effect of androgens on vascular health is a matter of debate even if recent evidence supports a protective role of these steroids on the cardiovascular system (1, 11). In particular, the possible effect of T on EPCs is not known. In this study, we evaluated PC and EPC concentrations before and after T treatment in a well-defined group of young hypogonadotropic hypogonadal (HH) men without cardiovascular risk factors.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
After approval from the local ethics committee, 10 idiopathic HH patients (27.2 yr of age; range, 23.7–32.5 yr) and 25 aged-matched controls (26.8 yr; range, 21.7–34.8 yr) gave informed consent and were enrolled in this study. Patients and controls were selected from our andrological unit. All patients were free of gonadotropin or T replacement therapy for at least 6 months. Six of 10 patients were newly diagnosed and then never treated with T. Four patients were off treatment for more than 6 months because of spontaneous dropout (im administration). All patients showed idiopathic hypogonadotropic hypogonadism. Controls were selected for normal T and gonadotropin levels (assessed by biochemical blood exams) and normal virilization and testis size (clinical examination) as well as fertility (anamnesis of offspring), referred to our andrological unit because of psychological erectile dysfunction [assessed by nocturnal penile tumescence and rigidity monitoring carried out with the RigiScan Plus Rigidity Assessment System from Osbon Medical Systems (Augusta, GA), performed on two consecutive nights] (12).

The rationale for studying only men with gonadotropin deficiency was to select a condition of severe hypotestosteronemia without other confounding risk factors. Hypergonadotropic hypogonadal patients were excluded because their testosteronemia usually is not as reduced as that in HH and because they often show normal or increased serum estrogen (E) levels. Hypergonadotropic hypogonadism was then considered a confounding condition.

Serum total T, E, FSH, and LH were evaluated by an immunoradiometric method (Adaltis, Bologna, Italy) in the control group at baseline and after 6 months of placebo treatment and in the patient group at baseline and after 6 months of T gel, 50 mg/d. Control patients were treated with placebo gel and not with T gel because of ethical implications in giving T to normal patients. During T or placebo treatment, patient and control subjects did not start any other therapy and did not change their lifestyle. Exclusion criteria, assessed by anamnesis, clinical examination, and biochemical blood exams, for both patients and controls, were diabetes, smoking, hypercholesterolemia, hypertriglyceridemia, hyperhomocysteinemia, obesity, and previous major cardiovascular events. Subjects under statin or phosphodiesterase type 5 inhibitor therapy were also excluded because these two drugs have been demonstrated to induce an increase of EPCs (statins) and PCs (phosphodiesterase type 5 inhibitors).

Blood samples for circulating EPC counts were evaluated by flow cytometry, as previously described (13). Briefly, analysis was performed on 150 µl peripheral blood incubated with fluorescein isothiocyanate-labeled monoclonal antibodies against human CD34 (Becton Dickinson, Milano, Italy), allophycocyanin-labeled monoclonal antibodies against human AC133 (Miltenyi Biotec, Bergisch Gladbach, Germany), and monoclonal antibodies against human VEGFR2 (Sigma-Aldrich, Milano, Italy). PCs are defined by CD34 and AC133 positivity, whereas EPCs are defined by CD34, AC133, and VEGFR2 positivity.

As previously described (13), before applying flow cytometry analysis on patients, control samples were studied in triplicate at different hours of the day at both endpoints, and these data confirmed the validity of the analysis (no significant variation within sample or within person was observed; data not shown).

Cell culture and immunocytochemical studies were performed to evaluate the expression of androgen receptor (AR) on EPCs. EndoCult basal medium was purchased from Stemcell Technologies (London, UK). Isolation and culture of EPCs were performed according to the manufacturer’s protocol. Briefly, a mononuclear cell suspension was obtained from peripheral blood by density separation using Ficoll-Paque PLUS (Amersham Biosciences, Milano, Italy). A total of 5 x 106 mononuclear cells were then plated on six-well fibronectin-coated plates (Becton Dickinson) and incubated for 2 d at 37 C, 5% CO2 with 95% humidity. Nonadherent cells were collected and 1 x 106 replated in triplicate on 24-well fibronectin-coated plates (Becton Dickinson) and incubated for an additional 6 d. Finally, for immunocytochemistry analysis, cells were fixed in 4% paraformaldehyde and stored at 4 C until use. Each well was fixed in 4% paraformaldehyde and rehydrated in graded ethanols. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide containing sodium azide and levamisole for 6 min and then rinsed gently in PBS for 3 min. For the staining, we used the Envision+ Dual Link System-HRP (DAB+) (DakoCytomation, Milano, Italy). Wells were immunostained with antibody F39.4, directed against the N-terminal part of the AR protein, amino acids 301–320 (kindly provided by Prof. A. O. Brinkmann). The primary antibody was serially diluted (1:400, 1:800, and 1:1600) and incubated for 30 min at room temperature. Wells were then washed in PBS for 3 min and stained with the peroxidase-labeled polymer for 30 min. After a wash in PBS, the substrate-chromogen solution was applied and incubated for 5 min. Finally, wells were washed with distilled water and counterstained with Mayer’s hematoxylin. A negative control was performed by omitting the primary antibody.

Data are expressed as median (range). Comparisons between baseline and the end of therapy and between patients and controls were performed by the Wilcoxon rank sum test for matched or unmatched pairs, respectively. P values < 0.05 were regarded as statistically significant.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Table 1Go shows hormonal, PC, and EPC concentrations in HH patients and controls at baseline and after 6 months T treatment (HH patients) and placebo treatment (controls).


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  		TABLE 1. Main baseline characteristics and outcomes of patients and controls

 
HH subjects showed, as expected, reduced basal total T, E, LH, and FSH with respect to control. In HH patients, after 6 months of T gel, 50 mg/d, we observed an increase to normal levels of E and T. After treatment, no modifications in FSH and LH levels were seen with respect to baseline.

At baseline, in HH subjects, we found a reduction of PCs and EPCs with respect to controls (P < 0.001). T gel therapy induced, after 6 months, an increase of both PCs and EPCs with respect to baseline (P < 0.005). In the control group, no significant modifications were seen at baseline and after 6 months. Immunocytochemistry on cultured EPCs showed a strong AR expression in the nucleus and the cytoplasm (Fig. 1Go).


Figure 1
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  		FIG. 1. AR immunocytochemistry in 6-d cultured EPCs. A and B, Ubiquitous expression of AR in EPC. Both nuclear and cytoplasmic staining were observed (cytoplasmic expression: inactive and newly synthesized AR; nuclear expression: active form of AR); magnification, x200 (A) and x1000 (B). C, Negative control; magnification, x200.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
In this study, we investigated the relationship between low serum T in HH patients and circulating levels of both PCs and EPCs. Interestingly we found that, when untreated, HH patients have low circulating levels of both PCs and EPCs, suggesting that T may have a role in all maturing and/or proliferating steps of their differentiation. Furthermore, T treatment induced a significant increase in the number of PCs and EPCs.

A reduced number of circulating EPCs have been detected in many pathological conditions such as myocardial infarction, myocardial ischemia, stroke, erectile dysfunction, peripheral artery disease, and diabetes (14, 15). The prognostic value associated with circulating number of EPCs has acquired a great interest. There is intriguing evidence of an inverse correlation between the number and function of EPCs and cardiovascular risk factors. Therefore, EPCs are going to be considered an independent predictor of future occurrence of cardiovascular events (14).

Hypogonadism has been related to central obesity, insulin resistance, dyslipidemia, and high fibrinogen levels (16). All these conditions are associated with a low number of circulating EPCs and negatively influence endothelial function.

The HH population analyzed had a reduced level of EPCs but was free from confounding factors known to alter circulating EPC number. These findings, taken together with the evidence of the role of T and the number of circulating EPCs on the vascular tree, may suggest inserting low serum T levels in the list of cardiovascular risk factors. Furthermore, T treatment induced a significant increase in the number of PCs and EPCs. The magnitude of the response found might be linked to the restoration of normal T levels. T may determine the increase of PCs and EPCs by itself, given the herein demonstrated wide expression of AR on EPCs, or after its conversion to 17ß-estradiol by the enzyme P-450 aromatase. Recently, a controversy has aroused around the advantages of E exogenous administration because of the results of two randomized clinical trials conducted in postmenopausal women using E and progestins. No primary (17) or secondary (18) benefits in preventing stroke or myocardial infarction were observed. Nevertheless, preclinical studies have demonstrated that E plays a protective role on the vascular wall by genomic and nongenomic pathways on both endothelial and smooth muscle cells as well as by increasing EPC number (19, 20). In our HH population, the 17ß-estradiol serum level was low at baseline and significantly increased after T treatment. We cannot exclude that the increase of EPC number after T therapy might be the consequence of T conversion to E. However, AR has been demonstrated to be widely expressed in the bone marrow and in particular on CD34-positive cells (21), which are the common precursors of many progenitor cells such as EPCs. Therefore, T may exert a direct effect on EPCs by its binding to nuclear AR (active form of AR) (Fig. 1Go). Taken together, our findings suggest a role of androgens on EPCs, but additional studies are needed to investigate where and how androgens may act to influence the proliferation and/or maturation of these cells.


   Acknowledgments
 
We thank Prof. Albert O. Brinkmann, Erasmus University, Rotterdam, The Netherlands, for kindly providing the AR antibody and Dr. Daniela Zuccarello for technical assistance.


   Footnotes
 
Disclosure statement: The authors have nothing to declare.

First Published Online August 22, 2006

Abbreviations: AR, Androgen receptor; E, estrogen; EPC, endothelial progenitor cell; HH, hypogonadotropic hypogonadism; PC, progenitor cell; T, testosterone; VEGFR2, vascular endothelial growth factor receptor type 2.

Received April 7, 2006.

Accepted August 14, 2006.


   References
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 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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  6. Kang SM, Jang Y, Kim JY, Chung N, Cho SY, Chae JS, Lee JH 2002 Effect of oral administration of testosterone on brachial arterial vasoreactivity in men with coronary artery disease. Am J Cardiol 89:862–864[CrossRef][Medline]
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