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(Circulation Research. 2007;100:434.)
© 2007 American Heart Association, Inc.

Clinical Research

Age-Dependent Impairment of Endothelial Progenitor Cells Is Corrected by Growth Hormone Mediated Increase of Insulin-Like Growth Factor-1

Thomas Thum, Sarah Hoeber, Sabrina Froese, Ivonne Klink, Dirk O. Stichtenoth, Paolo Galuppo, Marten Jakob, Dimitrios Tsikas, Stefan D. Anker, Philip A. Poole-Wilson, Jürgen Borlak, Georg Ertl, Johann Bauersachs

From the Universität Würzburg, Medizinische Klinik I (Kardiologie) (T.T, S.H., S.F., P.G., M.J. G.E., J. Bauersachs), Würzburg, Germany; National Heart and Lung Institute, Imperial College London, Faculty of Medicine (T.T., S.D.A., P.A.P.-W.), London, United Kingdom; Medizinische Hochschule Hannover, Institut für Klinische Pharmakologie (I.K., D.O.S., D.T.), Hannover, Germany; Fraunhofer Institut für Toxikologie und Experimentelle Medizin (ITEM) (T.T., J. Borlak), Hannover, Germany.

Correspondence to Thomas Thum, MD, Universitätsklinikum, Medizinische Klinik I, Kardiologie, Josef-Schneider-Str. 2, 97080 Würzburg, Germany. E-mail Thum_T{at}klinik.uni-wuerzburg.de or Johann Bauersachs, MD, Universitätsklinikum, Medizinische Klinik I, Kardiologie, Josef-Schneider-Str. 2, 97080 Würzburg, Germany. E-mail Bauersachs_J@medizin.uni-wuerzburg.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Aging is associated with an increased risk for atherosclerosis. A possible cause is low numbers and dysfunction of endothelial progenitor cells (EPC) which insufficiently repair damaged vascular walls. We hypothesized that decreased levels of insulin-like growth factor-1 (IGF-1) during age contribute to dysfunctional EPC. We measured the effect of growth hormone (GH), which increases endogenous IGF-1 levels, on EPC in mice and human subjects. We compared EPC number and function in healthy middle-aged male volunteers (57.4±1.4 years) before and after a 10 day treatment with recombinant GH (0.4 mg/d) with that of younger and elderly male subjects (27.5±0.9 and 74.1±0.9 years). Middle-aged and elderly subjects had lower circulating CD133⁺/VEGFR-2⁺ EPC with impaired function and increased senescence. GH treatment in middle-aged subjects elevated IGF-1 levels (126.0±7.2 ng/mL versus 241.1±13.8 ng/mL; P<0.0001), increased circulating EPC with improved colony forming and migratory capacity, enhanced incorporation into tube-like structures, and augmented endothelial nitric oxide synthase expression in EPC comparable to that of the younger group. EPC senescence was attenuated, whereas telomerase activity was increased after GH treatment. Treatment of aged mice with GH (7 days) or IGF-1 increased IGF-1 and EPC levels and improved EPC function, whereas a two day GH treatment did not alter IGF-1 or EPC levels. Ex vivo treatment of EPC from elderly individuals with IGF-1 improved function and attenuated cellular senescence. IGF-1 stimulated EPC differentiation, migratory capacity and the ability to incorporate into forming vascular networks in vitro via the IGF-1 receptor. IGF-1 increased telomerase activity, endothelial nitric oxide synthase expression, phosphorylation and activity in EPC in a phosphoinositide-3-kinase/Akt dependent manner. Small interference RNA-mediated knockdown of endothelial nitric oxide synthase in EPC abolished the IGF-1 effects. Growth hormone-mediated increase in IGF-1 reverses age-related EPC dysfunction and may be a novel therapeutic strategy against vascular disorders with impairment of EPC.

Key Words: endothelial progenitor cells • aging • insulin-like-growth-factor-1 • growth hormone

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endothelial injury and dysfunction are critical events in the pathogenesis of atherosclerosis. Understanding the mechanisms responsible for the repair of endothelial lesions and restoration of endothelial function will have important clinical implications. As resident endothelial cells infrequently proliferate in the vascular wall,¹ other sources of cellular replenishment have been postulated as mechanisms to repair endothelial lesions. Bone-marrow derived endothelial progenitor cells (EPC) circulate in the blood and contribute to formation of new blood vessels and homeostasis of the vasculature.²

Patients with reduced EPC levels are at increased risk for cardiovascular events and death.^3,4 Recent studies suggest augmentation of circulating EPC to result in improved coronary collateral development in coronary artery disease.⁵ Increasing age is associated with decreased number⁶ and impaired function of EPC,⁷ which may facilitate atherosclerotic processes. Regulation of EPC mobilization, differentiation and function is complex, but specific growth hormones and cytokines are explicitly involved.⁸ Insulin-like growth factor-1 (IGF-1) enhances migration, tube formation and angiogenesis of mature endothelial cells⁹ and increases telomerase activity.¹⁰ The effects of IGF-1 on endothelial cells are mediated at least in part via upregulation of endothelial nitric oxide synthase (eNOS) expression.¹¹

Low serum IGF-1 levels, common in the elderly, are associated with an increased risk for ischemic heart disease.¹² Restoration of IGF-1 in elderly individuals by growth hormone therapy may have significant beneficial health effects.¹³ In growth hormone deficient patients replacement therapy attenuates the increased tendency to develop endothelial dysfunction and severe atherosclerosis.¹⁴ Recently, Urbanek and coworkers have shown that IGF-1 promotes survival and proliferation of resident cardiac stem cells resulting in improved myocardial regeneration after myocardial infarction.¹⁵ Likewise, in the infarcted myocardium, IGF-1 promotes engraftment, differentiation, and function of implanted embryonic stem cells leading to improved myocardial function.¹⁶ IGF-1 transgenic mice demonstrate increased telomerase activity and preservation of cardiac resident stem cells.¹⁰ The effects of IGF-1 on EPC homeostasis and function are not known.

We investigated whether augmented IGF-1 levels in response to growth hormone treatment may restore the age-dependent decline in EPC levels and function in mice and humans. Ex vivo we tested, whether IGF-1 treatment of EPC of aged individuals would restore function and attenuate cellular senescence. In vitro studies were undertaken to identify underlying molecular mechanisms.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Clinical study
The ethical committees of the Universities of Hannover and Würzburg approved the study. Written informed consent was obtained from the volunteers. See the online data supplement available at http://circres.ahajournals.org for details.

Measurement of IGF-1 and Basic Blood Parameters
See the online data supplement available at http://circres.ahajournals.org

Isolation of Peripheral Blood Mononuclear Cells and CD34⁺, CD117⁺, CD133⁺ and CD133⁺/VEGFR2⁺ Progenitors
See the online data supplement available at http://circres.ahajournals.org

Determination of Progenitor Cells
A variety of assays was used to determine the number, differentiation, and function of EPC (see online data supplement).

Expression of the IGF-1 Receptor on EPC
IGF-1 receptor expression was determined in colony forming units (CFU) and monocytic EPC (see online data supplement).

Quantification of eNOS and IGF-1 Receptor Gene Expression of EPC
Total RNA was isolated from EPC of young, middle-aged (before and after GH treatment) and aged individuals according to the manufactures instructions (Qiagen, Germany). Details of the real-time PCR method are described in the supplemental online section.

Western Blotting Analysis
See the online data supplement available at http://circres.ahajournals.org

eNOS Activity
eNOS activity was determined as described (¹⁸ and online data supplement).

Small Interference RNA-Mediated Knockdown of eNOS in EPC
See the online data supplement.

Acidic ß-Galactosidase Staining of EPC
This was done as described with slight modifications (¹⁹; see online data supplement).

Telomerase Activity
This was done as previously described (²⁰; see online data supplement).

Mouse In Vivo Study
We examined the effects of both GH and IGF-1 treatment in aged mice. We also analyzed GH induced changes in EPC levels and function during interruption of IGF-1 receptor signaling in vivo by a blocking antibody or a small molecule inhibitor (see online data supplement).

Statistical Analysis
See the online data supplement available at http://circres.ahajournals.org

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Age-Related Decline in IGF-1 Levels, EPC Number and Function
IGF-1 plasma levels were significantly reduced in the elderly and middle-aged as compared with young men (120.8±10.5 ng/mL and 126.0±7.2 ng/mL versus 223.1±15.7 ng/mL; P<0.0001). Older individuals had significantly lower concentrations of circulating CD133⁺/VEGFR2⁺ cells (Figure 1A). EPC function was impaired with age, as determined by reduced colony forming capacity (Figure 1B), and impaired cellular migratory capacity (Figure 1C). Gene expression of eNOS was significantly reduced in EPC from aged subjects, whereas expression of the IGF-1R was unchanged (Figure 1D).

View larger version (33K): [in this window] [in a new window]   Figure 1. Circulating EPC number and function of middle-aged subjects before and after treatment with recombinant growth hormone compared with young controls and elderly individuals, as well as in aged mice. Clinical study (A-D): A, Number of circulating CD133+/VEGFR2+ cells in % of mononuclear cells and (B) number of endothelial colony forming units in young, middle-aged before and after treatment with recombinant human GH and elderly subjects. C, Migratory capacity of EPC from the various study groups. D, Gene expression of IGF-1R and eNOS relative to GAPDH in EPC derived from the various study groups. MA=middle-aged. Mouse study (E-H): E, Number of circulating sca1+/flk1+ cells in % of mononuclear cells and (F) migratory capacity of monocytic EPC of mice treated with placebo (control), IGF-1 (2 days), GH (2 or 7 days). G, Number of circulating sca1+/flk1+ cells in % of mononuclear cells and (H) migratory capacity of monocytic EPC of mice treated with placebo (control), GH (7 days), GH (7 days) + inhibitory IGF-1 receptor antibody (#MAB391, R&D Systems) or GH (7 days) + IGF-1 receptor inhibitor II (Calbiochem, Germany). n=4 to 6 per study group.

Treatment With Recombinant Human Growth Hormone of Middle-Aged Individuals Restored IGF-1 Levels, Increased EPC Number, and Improved EPC Function
A 10 day treatment of middle-aged men with GH increased IGF-1 serum levels from 126.0±7.2 ng/mL to 241.1±13.8 ng/mL (P<0.0001). CD133⁺/VEGFR2⁺ cells were 2-fold increased after GH treatment (Figure 1A). Likewise, the number of endothelial CFU was increased in treated individuals (Figure 1B) and both CD133⁺/VEGFR2⁺ cells and CFUs correlated with IGF-1 plasma levels (r=0.46, P<0.01 and r=0.60, P<0.0001). Hemoglobin (14.65±0.62g/dL versus 14.46±0.73g/dL), total erythrocyte (4.83±0.09x10⁶/µL versus 4.75±0.12x10⁶/µL) or leukocyte numbers (5.01±1.00x10³/µL versus 5.02±0.99x10³/µL) were unchanged by GH treatment. Migratory capacity of EPC was improved after GH treatment (Figure 1C). eNOS expression in isolated EPC was significantly increased by 64% after 10 day GH treatment, whereas that of the IGF-1R was basically unchanged (Figure 1D).

IGF-1 Mediates the Effects of GH Treatment on EPC Number and Function in Aged Mice
Both treatment of aged male mice with GH (7 days) or IGF-1 (2 days) increased systemic IGF-1 levels [control PBS-treated mice: 230±38 ng/mL; GH (7d): 520±207 ng/mL (P<0.001); IGF-1 (2d): 631±142 ng/mL (P<0.0001)] as well as sca1⁺/flk1⁺ EPC in peripheral blood and improved EPC function (see Figure 1, E and F). In contrast, a 2 day treatment with GH did not significantly increase IGF-1 levels (GH,2d: 243±6 ng/mL) nor EPC number or function indicating that IGF-1 is the main mediator of the observed effects of GH on EPC. Regression analysis revealed a positive correlation between IGF-1 levels and EPC number (r=0.45; P<0.05) or EPC migratory capacity (r=0.49; P<0.05). When the IGF-1 receptor was blocked either by a neutralizing antibody or a specific IGF-1 receptor inhibitor (see online data supplement), the GH-mediated effects on circulating EPC numbers (Figure 1G) and function (Figure 1H) were abolished.

IGF-1 Improves Differentiation and Function of EPC From Young and Elderly Individuals via the IGF-1 Receptor
To test whether increased IGF-1 concentrations may mediate the observed effects of GH treatment, we first investigated whether the IGF-1R is present on EPC.

We showed IGF-1R expression on various subtypes of progenitor cells, eg, early outgrowth EPC (CFU assay) and adherent dil-acLDL⁺/UEA-1⁺ EPC, which have previously shown to be of monocytic origin,²¹ but bind endothelial lectins and express endothelial proteins, such as the von Willebrand factor (vWF) (Figure 2 and 3). To test whether the IGF-1R is functionally active we performed further in vitro studies. IGF-1 treatment enhanced formation of endothelial CFU and increased formation of UEA-1⁺/dil-acLDL⁺ cells from cultured PBMC, indicating stimulation of EPC differentiation and function (Figure 4A, B). Migratory capacity of monocytic EPC was likewise improved by IGF-1 treatment (Figure 4A, B). In addition, IGF-1 stimulated migration of CD133⁺ derived and freshly isolated CD133⁺/VEGFR2⁺ EPC, whereas GH was without effect (Figure 5, A and C). The ability to incorporate into forming vascular networks on matrigel was significantly increased in IGF-1 pretreated EPC (Figure 4, A and B). In contrast, GH treatment in vitro, with the exception of minor improvement of cellular migration, did not display significant effects on EPC in the aforementioned assays (Figure 4). Inhibition of the IGF-1R by pretreatment with an inhibitory IGF-1R antibody abolished the effects of IGF-1 (Figure 4). IGF-1 treatment improved migratory capacity of EPC from elderly individuals, as compared with untreated cells (Figure 5B).

View larger version (31K): [in this window] [in a new window]   Figure 2. Expression of the insulin-like growth factor-1 receptor on human endothelial progenitor cells. Binding of the FITC-conjugated lectin UEA-1 to the surface of monocytic endothelial progenitor cells received by the adhesion related selection method (A and B) and to endothelial colony forming units17 (G and K) is shown as a positive control. Figures B and H demonstrate expression of the IGF-1 receptor on monocytic EPC and colony forming units, respectively. Figures E and L show findings when only the second rhodamin-labeled IgG antibody was used (negative control). Figures C, F, I, and M show the merged pictures. Representative pictures of a minimum of 5 experiments are shown.

View larger version (20K): [in this window] [in a new window]   Figure 3. Expression of the von Willebrand factor on human endothelial progenitor cells. Expression of the vWF in EPC including formation of typical Weibel-Palade bodies (intense green spots) (A). Figures B show findings with the second IgG antibody alone (negative control). Dil-acLDL uptake (C and D) and nuclear staining by DAPI (E and F). Merged pictures (G and H).

View larger version (30K): [in this window] [in a new window]   Figure 4. Effects of IGF-1 and recombinant human growth hormone on endothelial progenitor cell function. Colony forming capacity (CFU), differentiation of dil-acLDL+/UEA-1+ cells from peripheral blood mononuclear cells (dil-acLDL/UEA-1), migratory capacity (migration) and incorporation of EPC into vascular structures on matrigel (incorporation) is shown in EPC after 24 hour of treatment with GH (100 ng/mL), IGF-1 (100 ng/mL), and the concomitant treatment of IGF-1 with the PI3-kinase inhibitor wortmannin (100 nM) or an inhibitory IGF-1 receptor antibody (10 µg/mL). Representative pictures (A) and the statistical summary (B) of a minimum of five experiments per study group are shown. *=P<0.05 vs control; ***=P<0.0001 vs control; =P<0.05 vs IGF-1 100 ng/mL; =P<0.01 vs IGF-1 100 ng/mL; =P<0.0001 vs IGF-1 100 ng/mL.

View larger version (24K): [in this window] [in a new window]   Figure 5. Migratory capacity of CD133+-derived EPC, freshly isolated CD133+/VEGFR2+ EPC and rescue of impaired function of EPC derived from elderly subjects by IGF-1. A, CD133+ cells were isolated by MACS and cultured for 14d with EBM-2 (with supplements and 20% FCS) to induce an endothelial phenotype. Before the migration assay cells were treated for 24 hour with IGF-1 (100 ng/mL), IGF-1 and the PI3-kinase inhibitor wortmannin (100 nM), IGF-1 or an inhibitory IGF-1 receptor antibody (10 µg/mL) or GH (100 ng/mL) (n=5). B, Migratory capacity of monocytic EPC derived from elderly subjects before (left) and after (right) ex vivo treatment with IGF-1 (100 ng/mL for 24 hour). C, Migratory capacity of freshly isolated CD133+/VEGFR2+ EPC after treatment for 24 hour with IGF-1 (100 ng/mL), IGF-1 and the PI3-kinase inhibitor wortmannin (100 nM), IGF-1 or an inhibitory IGF-1 receptor antibody (10 µg/mL) or GH (100 ng/mL) (n=5).

To test whether the migratory potential of IGF-1 may apply to other kinds of stem cells we tested the effects of IGF-1 on human mesenchymal stem cells (hMSC). In contrast to the effects on EPC, IGF-1 (100 ng/mL) did not improve the migratory capacity of hMSC (89.5±10.7 versus 78.5±3.2 migrated hMSC/microscopic field; n=8; p=n.s.).

IGF-1 Increases Phosphoinositide 3-Kinase/Akt Mediated Expression and Phosphorylation of eNOS in Cultured EPC
As EPC function and differentiation is in part regulated via eNOS,²² we tested whether GH or IGF-1 treatment would impact phosphoinositide(PI)-3-kinase/Akt/eNOS signaling. Treatment of EPC with IGF-1 induced Akt phosphorylation, as well as expression and phosphorylation (Ser1177) of eNOS (Figure 6A). Functionally, IGF-1 treatment increased eNOS activity (Figure 6B). Inhibition of the PI3-kinase prevented IGF-1 mediated Akt phosphorylation, as well as eNOS expression and phosphorylation. Blocking of the PI3-kinase pathway reduced EPC differentiation and function (Figure 4, A and B). Inhibition of the IGF-1R with an inhibitory antibody completely abolished IGF-1 induced phospho-Akt/eNOS signaling (Figure 6A) and eNOS activity (Figure 6B). In contrast, GH had minor effects on eNOS expression, but did not affect Akt or eNOS phosphorylation or eNOS activity (Figure 6). Functional knockdown of eNOS by small interference RNA abolished the stimulatory IGF-1 effect on cellular migration of EPC, whereas addition of transfection media or scrambled siRNA had no significant effects (Figure 7).

View larger version (32K): [in this window] [in a new window]   Figure 6. Expression, phosphorylation, and function of eNOS in human EPC. A, Protein expression of phosphorylated Akt, eNOS, phosphorylated eNOS, and GAPDH in cultured EPC with treated with growth hormone (GH; 100 ng/mL) or IGF-1 (100 ng/mL) with or without PI3-kinase inhibition (wortmannin, 100 nM) or addition of an inhibitory IGF-1 receptor antibody (10 µg/mL). B, NOS activity as determined by measuring the conversion of L-[guanidino-15N2]arginine to 15N-nitrite in cultures of human EPC. 14N-nitrite was used as internal standard. At least 4 experiments were performed per study group. *=P<0.05 vs control; =P<0.05 vs IGF-1 (100 mg/mL).

View larger version (18K): [in this window] [in a new window]   Figure 7. siRNA mediated knockdown of eNOS in human EPC blocks stimulatory IGF-1 effects on migratory capacity. A, Detection of transfected EPC with FITC-labeled scrambled siRNA. B, Western blotting of eNOS and GAPDH in EPC 48 hour after transfection with eNOS siRNA (150 nM). C, Migratory capacity in controls and IGF-1 treated EPC. The IGF-1 group was concomitantly treated with transfection media (TM), scrambled siRNA (150 nM) or eNOS siRNA (150 nM). At least 4 experiments were performed per study group.

IGF-1 Impacts EPC Cellular Senescence and Telomerase Activity
Vascular endothelial cells with senescence-associated phenotypes are present in human atherosclerotic lesions of elderly patients.²³ Acidic ß-galactosidase can be detected in cultured EPC as a biochemical marker for the onset of cellular senescence.^19,24 Cultured EPC from healthy individuals treated with IGF-1 displayed significantly fewer ß-galactosidase positive cells than cells without the addition of IGF-1 (6.5±3.3 versus 21.5±5.3 ß-galactosidase positive cells; P<0.001) and showed enhanced telomerase activity, that was attenuated by PI3-kinase inhibition or blocking of the IGF-1R (Figure 8A). Growth hormone was without effect on telomerase activity.

View larger version (28K): [in this window] [in a new window]   Figure 8. Telomerase activity and cellular senescence of EPC in response to IGF-1. A, Telomerase activities in cultured monocytic EPC after treatment with IGF-1 (100 ng/mL), IGF-1 and the PI3-kinase inhibitor wortmannin (100 nM), IGF-1 and an inhibitory IGF-1 receptor antibody (10 µg/mL) or GH (100 ng/mL). At least 5 experiments were performed per study group. B, Detection of ß-galactosidase positive EPC derived from young or elderly subjects before and after ex vivo treatment with IGF-1 (100 ng/mL for 24 hour). C, Telomerase activities in CD133+ progenitors isolated from young, middle-aged (before and after GH treatment for 10d) and elderly subjects. D, Detection of ß-galactosidase positive EPC derived from middle-aged subjects before and after a 10d treatment with GH.

Elderly individuals displayed significantly increased number of ß-galactosidase positive EPC compared with EPC isolated from young subjects, demonstrating increased cellular senescence (Figure 8B), whereas telomerase activity in isolated progenitor cells was reduced (Figure 8C). Treating cultured EPC from aged individuals with IGF-1 reduced cellular senescence (Figure 8B). Likewise, growth hormone mediated IGF-1 increase in middle-aged subjects resulted in reduced EPC senescence (Figure 8D, E). Telomerase activity of CD133⁺-enriched progenitor cells was 2.3-fold (P<0.05) increased after treatment with GH and was comparable to that of young individuals (Figure 8C).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of the current study demonstrate that the age-related decline in EPC number and function can be restored by growth hormone mediated increase of IGF-1 levels. In vitro and in vivo studies extend and support these clinical observations. IGF-1, but not GH enhanced EPC differentiation and function involving PI3-kinase/phosphorylated Akt-mediated increase in eNOS phosphorylation and activity.

The finding of reduced amounts of circulating EPC with increasing age is in line with age-dependent reduction of EPC number in patients with coronary artery disease independent of risk factors for atherosclerosis or of cardiac function.⁶ Functional impairment of EPC from elderly subjects is related to endothelial dysfunction.⁷ Experimentally, bone marrow transplantation from young, but not old, nonatherosclerotic mice prevented atherosclerosis progression in apolipoprotein E knockout recipients, suggesting that deficient vascular repair because of increased age is a critical determinant of disease initiation and progression.²⁵ Taken together the data suggest that the age-related decline in progenitor number and function contribute to the progression of atherosclerosis. Recent studies have shown that patients with reduced circulating progenitor cells are at increased risk for cardiovascular events and death independent of other cardiovascular risk factors.^3,4

As EPC mobilization and revascularization is in part regulated by NO,^26–30 growth hormone-deficient patients with impaired systemic NO formation are expected to be at increased risk for cardiovascular disease.^31,32 Treatment of these patients with recombinant human GH results in an IGF-1-related stimulation of endothelial NO formation, decreased peripheral arterial resistance, and reversal of morphological and functional atherosclerotic changes in major arteries.^14,31,33 Low circulating IGF-1 levels are independently associated with increased risk for coronary artery disease or fatal ischemic heart disease.^12,34–36 In contrast, increased IGF-1 levels are related to reduced mortality in elderly individuals.³⁶ Thus, restoration of IGF-1 appears to provide protection from cardiovascular disease progression.^13,37

Our data provide a potential mechanistic link between IGF-1 and cardiovascular disease. The age-dependent reduction in IGF-1 levels and circulating EPC numbers, as well as impaired EPC function points to a causal relationship. Indeed, ten days of GH treatment increased IGF-1 levels and circulating EPC in middle-aged subjects to the values observed in the younger group. As impaired function of EPC from older patients with ischemic heart disease may limit their therapeutic potential for clinical cell therapy,³⁸ reversal of functional impairment of EPC in older individuals by GH treatment has important clinical implications. The opportunity exists to improve function of impaired EPC so as to optimize cell transplantation protocols.

Further evidence for a direct relationship between IGF-1 and EPC comes from the present in vivo and in vitro studies. Treatment of mice with IGF-1 improved number and function of EPC similar to a 7 day treatment with GH that significantly increases systemic IGF-1 levels, whereas a short term GH treatment did not increase IGF-1 nor EPC levels. Additionally, interruption of IGF-1/IGF-1 receptor signaling completely prevented the GH-mediated increase in EPC number and function.

Likewise, IGF-1, but not GH enhanced EPC differentiation, increased colony forming and migratory capacity, and stimulated EPC incorporation into tube-like structures. In addition, telomerase activity in EPC was increased and cellular senescence attenuated. Impaired function of EPC from aged subjects was normalized by IGF-1. In vivo, IGF-1 directly oppose endothelial dysfunction by enhancing NO production (reviewed in ³⁹). IGF-1 increases NOS activity by interacting with a tyrosine kinase membrane receptor that activates phosphoinositide 3-kinase,⁴⁰ which in turn activates the serine/threonine kinase Akt signaling pathway.⁴¹ In the heart, IGF-1 mediated activation of this pathway promoted cell growth and survival.⁴² In the present study, we demonstrate stimulation of PI3-kinase-dependent Akt phosphorylation and eNOS activity by IGF-1 in human EPC. Functional knockdown of eNOS using siRNA abolished the IGF-1 effects, underlining the pivotal role of eNOS in the stimulatory effects of IGF-1 on human EPC.

Within the heart, the IGF-1/IGF-1R system induces division of cardiac stem cells, upregulates telomerase activity thereby counteracting replicative senescence and preserves the pool of functionally active cardiac stem cells.^10,15,43 Myocardial regeneration may in part be mediated by IGF-1, and results in delayed onset of heart failure.⁴⁴ IGF-1 promotes engraftment, differentiation, and functional improvement after transfer of embryonic stem cells for myocardial restoration.¹⁶ Urbanek and coworkers found that resident cardiac stem cells express the IGF-1R,¹⁵ and our present data demonstrate expression of a functionally active IGF-1R in EPC. Besides cardiovascular progenitor cells, IGF-1R expression has been observed in erythroid,⁴⁵ osteogenic,⁴⁶ and neural⁴⁷ progenitor cells. Existence of an IGF-1R may be a general feature of progenitor cells. Targeting the IGF-1R on EPC opens a new line of therapeutic possibilities such as the use of GH or IGF-1 to restore progenitor cell function.⁸ In contrast, in human mesenchymal stem cells (hMSC) IGF-1 appears to function as a differentiation factor.⁴⁸ In line with this finding we found no stimulatory role of IGF-1 on migratory capacity of cultured hMSC.

Transgenic IGF-1 overexpression led to increased telomerase activity and preservation of functional capacity of aging cardiac stem cells.¹⁰ This is of importance as chronic oxidative stress and endogenous NO synthase inhibitors compromise telomere integrity, accelerate the onset of senescence in human endothelial cells and finally are associated with reduced EPC numbers.^19,48,49 Recent findings suggest that vascular cell senescence induced by telomere shortening may contribute to atherogenesis.²³ Our finding that augmented IGF-1 levels increase telomerase activity and prevent cellular senescence of aged and dysfunctional progenitor cells may have important clinical implications especially in diseases with increased cellular senescence, such as atherosclerosis.

Although the GH/IGF-I axis is involved in maintenance of normal function and homeostasis of diverse body functions, it also contributes to the progression of a number of common cancers (reviewed in ⁵⁰). Although there is no evidence of increased incidence of cancer in GH-deficient- or middle-aged heart failure patients during GH therapy,^51–53 future clinical trials investigating the role of GH or IGF-1 on stem cell biology should be performed with caution.

The current study identifies IGF-1 as an important regulator of EPC. Correction of age-related decline in number and function of EPC by growth hormone-mediated increase in IGF-1 may favor endothelial regeneration at sites of tissue damage and finally reduce cardiovascular events. Further prospective studies are needed that determine the effects of growth hormone mediated IGF-1 increase as a novel therapeutic strategy against vascular disorders with impaired number and function of EPC.

	Acknowledgments

 
The authors thank M. Leutke, Cand. Med. F. Fleissner, B. Beckmann, M.-T. Suchy, F.-M. Gutzki, D. Becker, and M. Darnedde for their skilful technical assistance. The advice of Professor E.-G. Brabant, Department of Endocrinology, Medizinische Hochschule Hannover, is acknowledged as is the help of Professor M. Böck and Dr A. Opitz (Department of Transfusion Medicine, University of Würzburg) with the leucapheresis procedure.

Sources of Funding

This work was supported in part by grants of the IZKF-Nachwuchsgruppe Cardiac Wounding and Healing (E-31 to T.T.), the Ernst und Berta Grimmke-Stiftung (to T.T.), a MSD SHARP & DOHME-Stipendium (Arteriosklerose/Hypertonie, to T.T.), the Novartis-Stiftung für therapeutische Forschung (to T.T. and J.B.) the Kompetenznetz Herzinsuffizienz TP 8 (to J.B.), and an institutional grant by Pharmacia (Karlsruhe, Germany, to D.O.S.).

Disclosures

None.

	Footnotes

 
Original received December 16, 2005; resubmission received June 19, 2006; revised resubmission received December 28, 2006; accepted January 4, 2007.

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