ORIGINAL RESEARCH—BASIC SCIENCE

The PDE5 Inhibitor Sildenafil Increases Circulating EndothelialProgenitor Cells and CXCR4 Expression

Carlo Foresta, MD, Luca De Toni, BS, Antonella Di Mambro, MD, Andrea Garolla, MD,Alberto Ferlin, MD, and Daniela Zuccarello, MD

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

DOI: 10.1111/j.1743-6109.2008.01014.x

A B S T R A C T

Introduction. Endothelial progenitor cells (EPC) are a specific subtype of progenitor cells that can be isolated fromcirculating mononuclear cells, able to migrate from the bone marrow to the peripheral circulation where theycontribute to vascular repair. CXC-motif chemochine receptor 4 (CXCR4) receptor seems to play a critical role inthis process.Aim. To assess the effects of sildenafil (a type 5 phosphodiesterase [PDE5] inhibitor) administration in 20 healthyyoung men.Methods. Evaluation of CXCR4 expression in circulating EPC before and 4 hours after in vivo administration of100 mg sildenafil by flow cytometry and colony-forming unit.Results. We found that sildenafil increases circulating EPC number, the relative expression of CXCR4 on these cellsand the ability to generate colonies in vitro.Conclusions. These data allow us to suppose an involvement of PDE5 in bone marrow release and peripheralhoming of EPC. Foresta C, De Toni L, Di Mambro A, Garolla A, Ferlin A, and Zuccarello D. The PDE5inhibitor sildenafil increases circulating endothelial progenitor cells and CXCR4 expression. J Sex Med2009;6:369–372.

Key Words. Endothelial Progenitor Cells; Endothelial Function; PDE5; CXCR4; Sildenafil

Introduction

E ndothelial progenitor cells (EPC) are a spe-cific subtype of progenitor cells that have been

isolated from circulating mononuclear cells, bonemarrow, and cord blood. EPC migrate from thebone marrow (BM) to the peripheral circulation,where they contribute to vascular repair [1]. CXC-motif chemokine receptor 4 (CXCR4), a memberof a large family of seven transmembrane domainreceptors coupled to heterotrimeric G-proteins[2,3], plays a key role in this process, as demon-strated by several experiments in animal models [4].Recently, we demonstrated that a treatment withtype 5 phosphodiesterase (PDE5) inhibitors is ableto improve endothelial function in patients affectedby erectile dysfunction (ED) [5–7]. PDE5 is one of

the enzymes involved in the cGMP metabolism,which hydrolyze cGMP to their respective linear5′-nucleoside monophosphate. By controllingcGMP, PDEs play critical roles in a wide range ofphysiological and pathological processes, and arepotential targets for drug intervention [8].

In order to evaluate the outcome of PDE5 inhi-bition on circulating EPC, we investigated in vivoand ex vivo the effects of sildenafil administrationin 20 young healthy males.

MethodsAfter approval from the local ethics committee, 20young healthy male volunteers (range 22–27 yeara)gave informed consent and were enrolled in thisstudy. All subjects were free from cardiovascularrisk factors (familiar history, smoke, alcohol, and

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drugs assumption). Blood samples (10 mL) werewithdrawn from all subjects before, 1–4–8–12hours after oral administration of 100 mg ofsildenafil. Circulating levels of progenitor cellswere analyzed as follows: 450 mL of peripheralblood were incubated with biotin-conjugatedmonoclonal antibodies antihuman VEGFR2(Sigma-Aldrich, Milano, Italy), washed and thenincubated with phycoerythrin conjugated-monoclonal antibody antihuman CD34 (BectonDickinson, Milano, Italy), allophycocyanin-labeled monoclonal antibody antihuman AC133(Miltenyi Biotec, Bergisch Gladbach, Germany),streptavidin-FITC (Sigma-Aldrich), and Pe-Cy5-labeled antihuman CXCR4 (Becton Dickinson).Red cells were lysed with Pharmlyse buffer (BectonDickinson), and samples were finally analyzed withFACScalibur flow cytometer (Becton Dickinson).PC, defined as CD34 and AC133 double-positivecells, and EPC, defined as CD34, AC133, andVEGFR2 triple-positive cells, were both expressedas cells/million of mononuclear cells (MNC). Per-centage of CXCR4 expressing progenitor cells(PC) and endothelial progenitor cells (EPC) werealso considered. Colony-forming unit (CFU) assaywas performed by isolating MNC from peripheralblood samples by density gradient centrifugation.5 ¥ 106 MNC/well were then seeded into afibronectin-coated 6-well/plates (Becton Dickin-son) in Endocult medium (Stem Cell Technologies,Milano, Italy). After 2 days, nonadherent cells wereharvested, and 1 ¥ 106 MNC/well recultured into afibronectin-coated 24-well/plates. After further 3days, colonies were stained with Giemsa azur’seosin methylene blue solution (Merck, Rome, Italy)and counted by two independent operators. Onlycolonies featured by central core of round cells withelongated sprouting cells were considered; datawere expressed as colonies/well. All the valuesshown are the averages of at least three independentexperiments (N = 3) performed in triplicate. Dif-ferences between data were determined by two-tailed student’s t-test after acceptance of normaldistribution with the Kolmogorov–Smirnov test. Pvalues (two sided) of less than 0.05 were consideredto be statistically significant.

Results

We observed a significant increase in circulatingPC and EPC number within 4 hours after admin-istration of sildenafil compared with basal level(Figure 1A, B). No increase in other blood cellscount was observed. Moreover, we evaluated the

percentage of CXCR4-positive PC and EPC,finding that the increase of these subpopulationsfollowed the same trend, achieving a peak at 4hours after sildenafil administration (Figure 1C).Interestingly, we observed that the number ofCFU, obtained ex vivo after 5 days of culture,increased significantly when obtained from bloodof subjects receiving sildenafil (fourth hour com-pared with basal) (Figure 1D).

Discussion

The mobilization of progenitor cells from the BMand the peripheral homing are a complex mecha-nisms not yet well clarified, even if involvement ofnitric oxide, cytokines, and chemokines has beendocumented in human and animal model [9]. It hasbeen previously demonstrated that treatment withPDE5 inhibitors is able to improve clinical param-eters of endothelial function in healthy men andpatients affected by ED [5–7]. Particularly, com-paring the time course and the magnitude of theincrease of EPC after vardenafil and sildenafiladministration, we observed a comparable effectafter 4 hours (44.1 � 16.5% of PC increase aftervardenafil compared with 58.3 � 18.9% aftersildenafil). In the present study, we found a signifi-cant increase in circulating PC and EPC aftersildenafil administration. It is possible to hypoth-esize that cGMP increase caused by PDE5-inibition may trigger the matrix metalloproteinase9 upregulation in the BM stem cell niches, favor-ing the extracellular matrix proteolysis and theconsequent release of progenitor cells from theBM niches to the systemic circulation. This phe-nomenon has already been described in vascularsmooth muscle as mediated by proteinkinase cyclicguanosine monophosphate-dependent and Raf/MEK/ERK cascade [10]. Another possible effectof PDE5 inhibition could be related to theincrease of growth factors expression (such asVEGF and FGF-2) as previously described [11].

Moreover, in order to understand the mecha-nism of the peripheral homing of the BM-derivedEPC, we further investigated the differentialexpression of CXCR4 in circulating PC and EPC,before and after sildenafil administration. Wefound that CXCR4 + PC and EPC increased sig-nificantly after 4 hour from stimulus, following thesame trend of numerical increase. An enhancedexpression of CXCR4 in CD34+ cells improvesthe chemotactic response of these cells vs. stromalderived factor-1, the specific agonist of CXCR4released by platelets during endothelium repair

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[12]. The cross-talk between SDF1 and CXCR4triggers the peripheral homing of progenitorcells in the endothelial damage site, causing theexposition of specific adhesion molecules [13].This effect could explain the improvement ofclinical endothelial function parameters afterPDE5 inhibitors administration, favoring thehoming phenomenon of the EPC into thedamaged endothelium.

Finally, our results on cultured EPC support thedata obtained in vivo, showing that these cells areable to originate colonies keeping the ability toproliferate previously acquired.

In conclusion, we demonstrated that PDE5inhibitor sildenafil is able to increase the numberof progenitor cells both in vivo and in vitro, and toenhance CXCR4 expression at 4 hours after thestimulus. These data allow supposing an involve-ment of PDE5 in BM release and peripheralhoming of PC and EPC.

Acknowledgments

We thank S. Magagna, M. Fontana, I. Bertuzzi for cellculture, and all the staff of the Centre for Male GameteCryopreservation for helpful discussion.

Corresponding Author: Carlo Foresta, MD, FullProfessor, Head of Centre for Male Gamete Cryo-preservation, Padova, Italy. Tel: 39-49-8218517; Fax:39-49-8218520; E-mail: carlo.foresta@unipd.it

Conflict of Interest: None declared

Statement of Authorship

Category 1(a) Conception and Design

Carlo Foresta; Luca De Toni(b) Acquisition of Data

Luca De Toni; Antonella Di Mambro(c) Analysis and Interpretation of Data

Andrea Garolla; Alberto Ferlin

AB

CD

Figure 1 (A–B) Circulating levels of PC (A) and EPC (B) in 20 healthy volunteers after administration of 100 mg of sildenafilvs. placebo. Data are expressed as cells per mL of blood (*P < 0.05). (C) Expression of CXCR4 in circulating PC and EPCbefore and 4 hours after in vivo administration of 100 mg sildenafil in 20 healthy volunteers. CXCR4 positively expressed aspercentage among PC (CD34+/AC133+ black bars) and EPC (CD34+/AC133+/VEGFR2+ white bars), assessed by flowcytometry (*P < 0.05). (D) Colony-forming unit assay of cultured EPC from peripheral blood after in vivo administration of100 mg sildenafil. Data are expressed as colonies per well (*P < 0.05). Experiments were performed three times in triplicate.

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Category 2(a) Drafting the Article

Daniela Zuccarello; Luca De Toni(b) Revising It for Intellectual Content

Daniela Zuccarello; Carlo Foresta

Category 3(a) Final Approval of the Completed Article

Carlo Foresta; Daniela Zuccarello

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