Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
Circulation Research. 2003;92:1049-1055
Published online before print April 3, 2003, doi: 10.1161/01.RES.0000070067.64040.7C
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
92/9/1049    most recent
01.RES.0000070067.64040.7Cv1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Assmus, B.
Right arrow Articles by Dimmeler, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Assmus, B.
Right arrow Articles by Dimmeler, S.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*ATORVASTATIN
*HEPTANOIC ACID
*LOVASTATIN
*PYRROLE
Medline Plus Health Information
*Stem Cells
Related Collections
Right arrow Ischemic biology - basic studies
Right arrow Smooth muscle proliferation and differentiation
(Circulation Research. 2003;92:1049.)
© 2003 American Heart Association, Inc.


Molecular Medicine

HMG-CoA Reductase Inhibitors Reduce Senescence and Increase Proliferation of Endothelial Progenitor Cells via Regulation of Cell Cycle Regulatory Genes

Birgit Assmus*, Carmen Urbich*, Alexandra Aicher, Wolf K. Hofmann, Judith Haendeler, Lothar Rössig, Ioakim Spyridopoulos, Andreas M. Zeiher, Stefanie Dimmeler

From Molecular Cardiology, Departments of Internal Medicine IV (B.A., C.U., A.A., J.H., L.R., I.S., A.M.Z., S.D.) and Hematology and Oncology (W.K.H.), University of Frankfurt, Frankfurt, Germany.

Correspondence to Stefanie Dimmeler, PhD, Molecular Cardiology, Dept of Internal Medicine IV, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany. E-mail Dimmeler{at}em.uni-frankfurt.de


*    Abstract
up arrowTop
*Abstract
down arrowIntroduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization of ischemic tissue. Ex vivo expansion of EPCs might be useful for potential clinical cell therapy of myocardial ischemia. However, cultivation of primary cells leads to cellular aging (senescence), thereby severely limiting the proliferative capacity. Therefore, we investigated whether statins might be able to prevent senescence of EPCs. EPCs were isolated from peripheral blood and characterized. After ex vivo cultivation, EPCs became senescent as determined by acidic ß-galactosidase staining. Atorvastatin or mevastatin dose-dependently inhibited the onset of EPC senescence in culture. Moreover, atorvastatin increased proliferation of EPCs as assessed by BrdU incorporation and colony-forming capacity. Whereas geranylgeranylpyrophosphate or farnesylpyrophosphate reduced the senescence inhibitory effect of atorvastatin, NO synthase inhibition, antioxidants, or Rho kinase inhibitors had no effect. To get further insights into the underlying downstream effects of statins, we measured telomerase activity and determined the expression of various cell cycle regulatory genes by using a microarray assay. Whereas telomerase activity did not change, atorvastatin modulated expression of cell cycle genes including upregulation of cyclins and downregulation of the cell cycle inhibitor p27Kip1. Taken together, statins inhibited senescence of EPCs independent of NO, reactive oxygen species, and Rho kinase, but dependent on geranylgeranylpyrophosphate. Atorvastatin-mediated prevention of EPC senescence appears to be mediated by the regulation of various cell cycle proteins. The inhibition of EPC senescence and induction of EPC proliferation by statins in vitro may importantly improve the functional activity of EPCs for potential cell therapy.


Key Words: aging • telomerase • statins • progenitor cells • angiogenesis


*    Introduction
up arrowTop
up arrowAbstract
*Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Increasing evidence suggests that endothelial progenitor cells (EPCs) play a crucial role in neovascularization of ischemic tissue.1,2 EPCs are considered to derive from hematopoietic stem cells, which are positive for CD34.3,4 Ischemia can trigger the release of bone marrow-derived CD34-positive cells into the periphery.5 In animal experiments, CD34-positive cells were shown to home to sites of ischemia and express endothelial markers like KDR.6 Moreover, injection of CD34-positive cells or cultivated EPCs enhances neovascularization, associated with an improvement of cardiac function.1 Thus, one may consider the use of EPCs for potential cell therapy to augment vascularization in patients with ischemic heart disease.7 However, due to the limited number of EPCs in the circulating blood (<0.05% of leukocytes),8,9 ex vivo expansion of EPCs appears to be necessary. Proliferation of primary human cells is limited by the capacity to divide and the onset of senescence.10 Cellular aging or senescence is characterized by cell cycle arrest and can be triggered by different pathways.11 Replicative senescence results from a count down of the intrinsic mitotic clock. The mitotic counter mechanism is reflected by telomere shortening.12 With each cell division, telomeres become shorter. This telomere shortening contributes to the finite number of cell divisions of somatic cells. To maintain a certain telomere length, the enzyme telomerase, a ribonucleoprotein with reverse transcriptase activity, adds telomeric repeats at the linear end of eukaryotic chromosomes.12 Premature senescence, in contrast, describes the induction of senescence by extrinsic factors such as oncogenic ras, DNA damage, oxidative stress or cumulative stress induced by in vitro culture.11,13 Furthermore, a senescent phenotype can also be induced by expression of cyclin-dependent kinase inhibitors (CDKIs).11 The senescence phenotype is indistinguishable, irrespective of the inducer. It is characterized by a high frequency of nuclear abnormalities, positive staining for ß-galactosidase activity at pH 6.0,11,14 and can be associated with an increase in expression of cell cycle inhibitory proteins such as p27Kip1 or p21Cip1/Waf1.15,16

In the present study, we investigated the regulation of senescence and proliferation in EPCs. Because we and others previously demonstrated that HMG-CoA reductase inhibitors increased the number of EPCs,8,17,18 we further elucidated a potential effect of statins on EPC senescence. Our data demonstrate that statins potently prevent the onset of EPC senescence and promote proliferation and colony formation ex vivo. The effects of statins appear to be largely independent on telomerase activity, but involve the transcriptional regulation of multiple cell cycle regulatory proteins.


*    Materials and Methods
up arrowTop
up arrowAbstract
up arrowIntroduction
*Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Materials
Ly294002 (Biomol), wortmannin (Alexis), mevalonate (Fluka), and LNMA (Alexis) were preincubated 30 minutes before statin stimulation. VEGF, HA1077, and Y27632 were purchased from Calbiochem, and atorvastatin was kindly donated by Goedecke/Parke-Davis, Freiburg, Germany. Mevastatin was activated as previously described.19 Geranylgeranylpyrophosphate and farnesylpyrophosphate were from Sigma.

Cell Culture
Mononuclear cells were isolated by density gradient centrifugation with Biocoll from peripheral blood of healthy human volunteers according to Vasa et al.8 Immediately after isolation, 4x106 mononuclear cells were plated on culture dishes coated with human fibronectin (Sigma) and maintained in endothelial basal medium (EBM; CellSystems, St Katharinen, Germany) supplemented with EGM SingleQuots and 20% FCS.

Colony Assay
After 4 days of culture, adherent cells were gently detached with EDTA. Cells (1x105) were seeded in methylcellulose plates (Methocult GF H4434, CellSystems) with 100 ng/mL human recombinant VEGF. Plates were studied under phase contrast microscopy, and colonies were counted after 10 days of incubation by two independent investigators.

Acidic ß-Galactosidase Staining
After 4 days of culture, adherent cells were detached and seeded in methylcellulose plates as described above. After 7 days, cells were fixed for 10 minutes in 2% formaldehyde, 0.2% glutaraldehyde in phosphate-buffered saline (PBS), and incubated for 12 hours at 37°C without CO2 with fresh ß-Gal staining solution: 1 mg/mL of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside, 5 mmol/L potassium ferrocyanide, 5 mmol/L potassium ferricyanide, and 2 mmol/L MgCl2; pH 6.0. Cells were counterstained with 4',6-diamidino-phenylindole (DAPI; 0.2 µg/mL in 10 mmol/L Tris-HCl, pH 7.0, 10 mmol/L EDTA, 100 mmol/L NaCl) for 10 minutes to count the total cell number. The absolute numbers of ß-galactosidase-positive cells were counted out of 1000 cells.

Telomerase Assay
EPCs were washed in PBS, and the pellet was lysed with 30 µL lysis buffer for 30 minutes at 4°C as previously described.20 Proteins were centrifuged for 20 minutes at 10 000g, and protein concentrations were determined in the supernatant using the Bradford assay. Telomerase activity was measured with 2 µg protein by the Telo TAGGG Telomerase PCR ELISAplus Kit according to the manufacturers instructions (Roche Molecular Biochemicals).

FACS Analysis
Adherent cells were gently scraped off using cell scrapers, washed in PBS, and incubated in PBS/1% BSA/1% mouse serum in the presence of the following antibodies. Staining of mouse anti-human VE-cadherin, (Santa Cruz Biotechnology), anti-human vWF antibody (BD Pharmingen), and KDR (Dianova) was visualized using RPE-conjugated goat anti-mouse F(ab')2 (DAKO). ICAM-1 (BD Pharmingen), {alpha}v (CD51) (Dianova), and E-selectin (CD62E) (Dianova) were used directly FITC-conjugated.

Cell Cycle Analysis
Adherent cells were incubated with BrdU (10 µmol/L) for 48 hours. Adherent cells were detached with trypsin, washed in PBS, and incubated with 20 µL anti-BrdU-FITC for 20 minutes and with 2.5 µL 7AAD for 15 minutes according to the manufacturer (Pharmingen, BrdU Flow Kit). Analysis was performed using a FACS SCAN flow cytometer and Cell Quest software (BD Biosciences).

Microarray Analysis
Gene expression profiling was performed with the gene chip expression assay. The protocol for sample preparation and microarray processing is available from Affymetrix. Data were analyzed with the software GeneSpring version 3.0 (Silicon Genetics) as previously described.21

Western Blot Analysis
EPCs were incubated with 60 µL lysis buffer as previously described.17 Proteins (50 µg/lane) were loaded onto SDS-polyacrylamide gels and blotted onto PVDF membranes. Western blots were performed using antibodies directed against cyclin F, cyclin D, E2F1, cdk4 (all Santa Cruz) or cyclin A, PCNA, p21Cip1/Waf1, and p27Kip1 (all BD Biosciences). Enhanced chemiluminescence was performed according to the instructions of the manufacturer (Amersham, Germany). Blots were reprobed with tubulin (Neomarkers).

Statistical Analysis
Data are expressed as mean±SEM from at least 3 independent experiments. Statistical analysis was performed by the two-tailed t test or ANOVA for multiple comparisons.


*    Results
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
HMG-CoA Reductase Inhibitors Prevent EPC Senescence and Increase Proliferation of EPCs
EPCs were generated from peripheral blood mononuclear cells as previously described.9,17 To assess the onset of senescence, acidic ß-galactosidase was detected as a biochemical marker for acidification typical for the onset of cellular senescence.11,14 Cultivation of EPCs resulted in an increase in acidic ß-galactosidase-positive cells after prolonged cultivation (Figure 1A). Coincubation with atorvastatin significantly inhibited the increase in ß-galactosidase-positive cells (Figures 1A and 1B). The inhibition of EPC senescence occurred dose-dependent with a maximal inhibitory effect achieved at 0.1 µmol/L (Figure 1C). Mevastatin also inhibited the onset of EPC senescence to a similar extent (Figure 1D), suggesting a class effect of statins. To characterize the phenotype of the adherent cells, we analyzed the expression of endothelial marker proteins. EPCs cultivated in the presence or absence of atorvastatin revealed a similar expression pattern of the endothelial marker proteins (Figure 1E).



View larger version (41K):
[in this window]
[in a new window]
 
Figure 1. Statins inhibit EPC senescence. A through D, Freshly isolated mononuclear cells were cultivated in EBM medium with supplements and 20% FCS in the presence or absence of atorvastatin (0.1 µmol/L). At day 4, cells were seeded in the presence or absence of atorvastatin (0.1 µmol/L) in methylcellulose for 7 days. Acidic ß-galactosidase activity was monitored in the adherent cells. Percentage of ß-galactosidase-positive cells (% senescent cells) was calculated. Data are mean±SEM, n=6; *P<0.05 vs control (A). Representative micrographs of ß-galactosidase-positive cells (senescent cells) and counterstaining with DAPI are shown (B). Dose-dependency is shown for atorvastatin (C) and mevastatin (D). Data are mean±SEM (% of control), n=5; *P<0.05 vs control. E, Expression of endothelial marker proteins was monitored after 4 days of incubation with 0.1 µmol/L atorvastatin. Representative FACS analysis is shown (n=3 to 5). Isotype controls are shown as dotted lines.

Consistent with previous findings,17,18 the number of EPCs also increased after atorvastatin incubation (data not shown). However, the absolute number of senescent EPCs was also significantly decreased to 16.2±2.3% ß-galactosidase-positive EPCs/high power field after atorvastatin treatment. These data demonstrate that the decrease in the percentage of senescent cells was not caused by a relative increase in cell number.

Having demonstrated that atorvastatin delayed the onset of senescence, we examined whether that translates into an increase in proliferation and clonal expansion. As shown by FACS analysis of BrdU-labeled cells, atorvastatin induced a 2-fold increase in the number of cells in the S-phase (Figure 2A). To investigate the clonal expansion potential of the cultivated EPCs, we further performed an outgrowth assay. For this purpose, EPCs were cultivated for 4 days in the presence or absence of atorvastatin. Then, cells were detached, and 1x105 EPCs were seeded in methylcellulose plates. As shown in Figures 2B and 2C, the number of colonies was significantly higher in EPCs that had been pretreated with atorvastatin. The endothelial phenotype of the outgrowing cells was confirmed by showing DiLDL uptake and lectin staining (Figure 2D).



View larger version (44K):
[in this window]
[in a new window]
 
Figure 2. Effects of atorvastatin on proliferation and colony forming capacity. A, Mononuclear cells were cultivated in EBM medium with supplements and 20% FCS in the presence or absence of atorvastatin (0.1 µmol/L). After 4 days of culture, proliferation of adherent cells was monitored by BrdU incorporation. Data are mean±SEM, n=6. B and C, EPCs were cultivated as described for A. At day 4, EPCs were seeded in methylcellulose plates, and colonies were counted after additional 10 days of cultivation. Data are mean±SEM, n=3; *P<0.05 vs control. Representative image is shown in C. D, EPC-derived colonies were phenotypically analyzed by DiLDL uptake and lectin binding. Representative images are shown (n=3).

Intracellular Signaling of HMG-CoA Reductase Inhibitors
Statins inhibit the HMG-CoA reductase and, thereby, prevent formation of mevalonate and the downstream products farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), which finally results in the inactivation of the Rho kinase.19 To investigate the involvement of these intermediates, we coincubated EPCs with mevalonate, GGPP, or FPP. As shown in Figure 3A, mevalonate completely reversed the inhibitory effect of atorvastatin on EPC senescence. Likewise, GGPP and FPP significantly prevented atorvastatin-induced inhibition of EPC senescence (Figure 3A). In contrast, senescence of EPCs was not prevented by two pharmacological inhibitors of the Rho kinase (Figure 3A), indicating that statins influence EPC senescence via isoprenylation, but independent of the Rho kinase.



View larger version (25K):
[in this window]
[in a new window]
 
Figure 3. Intracellular signaling of statins. A and B, Mononuclear cells were cultivated for 4 days in EBM medium with supplements and 20% FCS in the presence or absence of atorvastatin (AT; 0.1 µmol/L), mevalonate (200 µmol/L), farnesylpyrophosphate (FPP; 10 µmol/L), geranylgeranylpyrophosphate (GGPP; 10 µmol/L), the Rho kinase inhibitors HA1077 (10 µmol/L) or Y27632 (10 µmol/L), N-acetylcysteine (NAC, 200 µmol/L), the NO donor SNAP (20 µmol/L), or NG-monomethyl-L-arginine (LNMA; 1 mmol/L). Senescent, acidic ß-galactosidase-positive cells were counted. Data are mean±SEM (% of control), n=4 to 10; *P<0.05 vs AT (A) or control (B).

Recent studies demonstrate that statins stimulate the PI3K/Akt pathway,22 which is known to regulate senescence of mature endothelial cells.23 Therefore, we investigated the effect of the PI3K inhibitor Ly294002. After incubation with Ly294002 for 4 days, no attached cells were detectable in the presence or absence of statins, concomitant with an increase in apoptosis (data not shown). Thus, the PI3K pathway represents a potent survival signal in EPCs. A specific role of the PI3K pathway for EPC senescence cannot be assessed in the long term cultivation assay required to study cellular aging.

Statins are known to posttranscriptionally activate the endothelial NO synthase via the PI3K/Akt pathway and additionally reduce oxidative stress via regulation of isoprenylation.24 Because oxidative stress is well-established to contribute to cellular senescence25,26 and NO was recently demonstrated to prevent senescence of mature endothelial cells,20 we investigated the contribution of nitric oxide and oxidative stress for the regulation of EPC senescence. However, neither the antioxidant N-acetyl-cysteine (NAC) nor the NO donor S-nitrosopenicillamine (SNAP) did prevent the onset of EPC senescence (Figure 3B). Likewise, inhibition of the NO synthase by NG-mono-methyl-L-arginine (LNMA) did not reverse the inhibitory effect of atorvastatin (Figure 3B), suggesting that the regulation of EPC senescence is independent of NO and reactive oxygen species.

Regulation of Telomerase and Cell Cycle-Related Genes
Cellular senescence is critically influenced by the telomerase, which elongates telomeres, thereby counteracting telomere length reduction induced by each cell division. Therefore, we measured telomerase activity in EPCs. EPCs revealed a basal telomerase activity, albeit at a low level (Figure 4A). Incubation of EPCs with atorvastatin did not affect telomerase activity (Figure 4A). Furthermore, the localization of the catalytic subunit of the telomerase was not altered by atorvastatin incubation (data not shown). These data indicate that statins act independent of telomerase.



View larger version (8K):
[in this window]
[in a new window]
 
Figure 4. Effect of statins on telomerase activity. Freshly isolated mononuclear cells were cultivated in EBM medium with supplements and 20% FCS in the presence or absence of atorvastatin (0.1 µmol/L). After 24 hours or 4 days, adherent cells were detached and telomerase activity was measured by the TRAP assay. Data are mean±SEM, n=7 to 10.

To get further insights into the regulation of cell cycle regulatory genes, we performed a microarray, which allows for the detection of about 12 000 genes. Microarray analysis was performed after 10 hours of atorvastatin treatment, thus avoiding potential confounding effects of increased proliferation, which was observed after >24 hours. Cluster analysis of the cell cycle regulatory genes revealed that the expression of more than 10% of the analyzed cell cycle genes including cyclins and PCNA are increased after statin incubation (Figure 5A). Moreover, the expression of the cell cycle inhibitory protein p27 was reduced (Figure 5A). The modulation of the expression of some cell cycle regulatory proteins by atorvastatin was further confirmed on the protein level (Figure 5B). Thereby, atorvastatin increased the expression of cyclin A, cyclin F, and PCNA to 2.1±0.5-, 1.9±0.1-, and 1.5±0.06-fold, respectively. Consistent with a downregulation of p27 mRNA levels, protein expression was dose-dependently reduced to 53.1±5.7% after atorvastatin incubation for 24 hours, whereas expression of another cyclin-dependent kinase inhibitor, p21, was not affected (Figures 5B and 5C). Incubation with Ly294002 or wortmannin but not with Rho kinase inhibitors for 24 hours largely prevented the effect of atorvastatin on cyclin A, cyclin F, and p27 expression (Figures 5B through 5F and data not shown), suggesting that the PI3K pathway plays a major role for the modulation of expression of cell cycle regulatory genes. Controls confirmed that the short-term incubation with Ly294002 for 24 hours did not affect cell viability (apoptosis: 126±11% compared with untreated controls).



View larger version (58K):
[in this window]
[in a new window]
 
Figure 5. Regulation of cell cycle-related genes in endothelial progenitor cells. A, Isolated EPCs were incubated for 10 hours in the presence or absence of atorvastatin (0.1 µmol/L), RNA was isolated, and the differential gene expression was determined by microarray analysis. Cluster analysis of cell cycle-related genes is shown. Red color indicates high expression; blue color indicates low expression. Expression of selected genes (bold) was confirmed by Western blot analysis. B, EPCs were incubated with 0.1 µmol/L atorvastatin (AT) for 24 hours in the presence or absence of Ly294002 (10 µmol/L), and expression of cell cycle related genes was determined by Western blot. Tubulin serves as loading control. Representative blot out of 3 independent experiments is shown. C and D, EPCs were incubated with different concentrations of atorvastatin (AT). Representative immunoblots against p27Kip1 and loading control with tubulin are shown. Data are mean±SEM, n=3. E and F, EPCs were incubated with atorvastatin (AT 0.1 µmol/L) in the presence or absence of Ly294002 (10 µmol/L), wortmannin (20 nmol/L), or the Rho kinase inhibitors HA1077 (10 µmol/L) or Y27632 (10 µmol/L) for 24 hours. Representative immunoblots against p27Kip1 and loading control with tubulin are shown (n=3).


*    Discussion
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
The results of this study demonstrate that ex vivo cultivation of EPCs leads to rapid onset of EPC senescence. EPC senescence was associated with a very low proliferative capacity and profoundly impaired clonal expansion potential. Consistent with these findings, the activity of telomerase was very low in EPCs. At a first glance, these findings might be surprising, because stem or progenitor cells are viewed as highly potent regenerating cells with a high proliferation potential. However, one should keep in mind, that after mobilization of stem and progenitor cells into the circulation, these cells should preferentially home to sites, where they are needed, and should selectively contribute to cellular regeneration at these sites. The differentiation and proliferation state, therefore, may essentially be dependent on the cellular environment, which includes cell-to-cell communication and growth factor support. This may explain that senescence is rapidly increasing under ex vivo culture conditions, which lack cell-to-cell contact and potential autocrine factors required for functional differentiation of progenitor cells. Previous studies demonstrated that purified circulating hematopoietic CD34-positive progenitor cells exhibit a lower proliferation rate and reduced expression of various cell cycle stimulatory genes in comparison with CD34-positive cells, which were isolated directly from the bone marrow.27 The functional importance of the cellular environment is further supported by demonstrating that co-culturing peripheral blood CD34-positive cells with human endothelial cells transfected with various hematopoietic growth factors significantly enhanced the ex vivo expansion of progenitor cells.28 These data suggest that the cumulative trauma of in vitro culture ("culture shock") and an impaired environment trigger premature senescence of EPCs.

Our data provide first evidence that ex vivo incubation with statins not only increased the number of EPCs,17,18 but also delayed the onset of cellular senescence. The mechanisms, by which statins are preventing the onset of cellular senescence and increasing the proliferative capacity of EPCs, appear to involve the geranylgeranyl pathway, because mevalonate, GGPP, and FPP reversed the senescence inhibitory effect of statins. Inhibition of geranylgeranylation was shown to inactivate the Rho kinase.19 However, pharmacological inhibitors of the Rho kinase did not affect EPC senescence, excluding that inhibition of the Rho kinase mediates the senescence inhibitory effect of statins. Likewise, statins act independent of NO and reactive oxygen species, although NO and ROS have been shown to play a key role in regulation of senescence of mature endothelial cells.20,26

However, our data demonstrate that statins transcriptionally modulate the expression of multiple cell cycle regulatory proteins. Several cell cycle-promoting proteins are upregulated on mRNA and protein levels. These proteins included cyclins A, D, and F as well as the cofactor for the DNA polymerase PCNA. One may speculate that the increased expression is secondary to a pro-proliferative effect of statins. However, this appears unlikely, because the gene array analysis was performed after 10 hours at a time point where no increase in S-phase was detectable. Moreover, the expression of the cell cycle inhibitory protein p27 was reduced after statin treatment. Our data further suggest that the PI3K/Akt pathway essentially contributes to the statin-induced modulation of gene expression. The PI3K/Akt pathway is activated by statins22 and is known to transcriptionally and posttranscriptionally modulate cell cycle progression.29,30 The transcriptional regulation of p27 by Akt has been attributed to the functional inactivation of the FOXO subfamily of forkhead transcription factors.31 Indeed, the forkhead transcription factor AFX (FOXO4) is expressed in EPCs and is phosphorylated and, thereby, inactivated by statins (C.U., PhD, oral communication, 2002). These data may suggest that the statin-mediated downregulation of p27 is controlled by Akt-dependent inactivation of forkhead transcription factors. Importantly, downregulation of p27 has been shown to be involved in rescue of fibroblast senescence induced by PI3K inhibitors,15 providing also an explanation for the inhibition of senescence-like growth arrest in statin-treated EPCs. The signaling pathways, by which statins increase the mRNA level of cyclins and PCNA, is less clear. Although Akt has been shown to induce posttranscriptional stabilization of cyclin D via inhibition of GSK-3,32 the transcriptional regulation of cyclin mRNA expression has not been demonstrated. Further studies are required to elucidate the molecular mechanisms underlying these effects.

Taken together, statins modulate expression of various cell cycle regulatory proteins in EPCs via the PI3K pathway. The increase of cell cycle promoting proteins, which are essential for cell cycle progression, concomitant with a reduction of the cell cycle inhibitory protein p27 may facilitate cell cycle progression and, thereby, prevent the onset of replicative senescence. Although the short-term treatment of EPCs with the PI3K inhibitor Ly294002 clearly prevented the changes in protein expression, we could not proof a causal involvement of the PI3K for the senescence inhibitory effect of statins, because long term incubation of Ly294002 also induced apoptosis of EPCs. This is consistent with the finding that activation of Akt by statins prevents apoptosis of EPCs.18 Thus, activation of the PI3K/Akt pathway by statins may have multiple protective effects on EPCs, including the increase in number, the inhibition of apoptosis, the improvement of functional activity, and the prevention of senescence.


*    Acknowledgments
 
The study was supported by the DFG (Di 600/4-1 and SFB B6 to S.D.). We would like to thank Christiane Mildner-Rihm and Andrea Knau for excellent technical assistance.


*    Footnotes
 
*Both authors contributed equally to this study. Back

Received September 20, 2002; revision received February 25, 2003; accepted March 24, 2003.


*    References
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
up arrowResults
up arrowDiscussion
*References
 

  1. Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida S, Masuda H, Silver M, Ma H, Kearney M, Isner JM, Asahara T. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation. 2001; 103: 634–637.[Abstract/Free Full Text]
  2. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, Li T, Isner JM, Asahara T. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A. 2000; 97: 3422–3427.[Abstract/Free Full Text]
  3. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964–967.[Abstract/Free Full Text]
  4. Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane WJ, Williams M, Oz MC, Hicklin DJ, Witte L, Moore MA, Rafii S. Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors. Blood. 2000; 95: 952–958.[Abstract/Free Full Text]
  5. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM, Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med. 1999; 5: 434–438.[CrossRef][Medline] [Order article via Infotrieve]
  6. Crosby JR, Kaminski WE, Schatteman G, Martin PJ, Raines EW, Seifert RA, Bowen-Pope DF. Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation. Circ Res. 2000; 87: 728–730.[Abstract/Free Full Text]
  7. Assmus B, Schachinger V, Teupe C, Britten M, Lehmann R, Dobert N, Grunwald F, Aicher A, Urbich C, Martin H, Hoelzer D, Dimmeler S, Zeiher AM. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation. 2002; 106: 3009–3017.[Abstract/Free Full Text]
  8. Vasa M, Fichtlscherer S, Adler K, Mildner-Rihm C, Aicher A, Martin H, Zeiher AM, Dimmeler S. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation. 2001; 103: 2885–2890.[Abstract/Free Full Text]
  9. Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001; 89: e1–e7.[CrossRef][Medline] [Order article via Infotrieve]
  10. Haflick L. The limited in vitro lifetime of human diploid cell strains. Exp Cell Res. 1965; 37: 614–636.[CrossRef][Medline] [Order article via Infotrieve]
  11. Mathon NF, Lloyd AC. Cell senescence and cancer. Nat Rev Cancer. 2001; 1: 203–213.[CrossRef][Medline] [Order article via Infotrieve]
  12. Collins K. Mammalian telomeres and telomerase. Curr Opin Cell Biol. 2000; 12: 378–383.[CrossRef][Medline] [Order article via Infotrieve]
  13. Sherr CJ, DePinho RA. Cellular senescence: mitotic clock or culture shock? Cell. 2000; 102: 407–410.[CrossRef][Medline] [Order article via Infotrieve]
  14. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995; 92: 9363–9367.[Abstract/Free Full Text]
  15. Collado M, Medema RH, Garcia-Cao I, Dubuisson ML, Barradas M, Glassford J, Rivas C, Burgering BM, Serrano M, Lam EW. Inhibition of the phosphoinositide 3-kinase pathway induces a senescence-like arrest mediated by p27Kip1. J Biol Chem. 2000; 275: 21960–21968.[Abstract/Free Full Text]
  16. Macip S, Igarashi M, Fang L, Chen A, Pan ZQ, Lee SW, Aaronson SA. Inhibition of p21-mediated ROS accumulation can rescue p21-induced senescence. EMBO J. 2002; 21: 2180–2188.[CrossRef][Medline] [Order article via Infotrieve]
  17. Dimmeler S, Aicher A, Vasa M, Mildner-Rihm C, Adler K, Tiemann M, Rutten H, Fichtlscherer S, Martin H, Zeiher AM. HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway. J Clin Invest. 2001; 108: 391–397.[CrossRef][Medline] [Order article via Infotrieve]
  18. Llevadot J, Murasawa S, Kureishi Y, Uchida S, Masuda H, Kawamoto A, Walsh K, Isner JM, Asahara T. HMG-CoA reductase inhibitor mobilizes bone marrow-derived endothelial progenitor cells. J Clin Invest. 2001; 108: 399–405.[CrossRef][Medline] [Order article via Infotrieve]
  19. Laufs U, Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem. 1998; 273: 24266–24271.[Abstract/Free Full Text]
  20. Vasa M, Breitschopf K, Zeiher AM, Dimmeler S. Nitric oxide activates telomerase and delays endothelial cell senescence. Circ Res. 2000; 87: 540–542.[Free Full Text]
  21. Hofmann WK, de Vos S, Tsukasaki K, Wachsman W, Pinkus GS, Said JW, Koeffler HP. Altered apoptosis pathways in mantle cell lymphoma detected by oligonucleotide microarray. Blood. 2001; 98: 787–794.[Abstract/Free Full Text]
  22. Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ, Sessa WC, Walsh K. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med. 2000; 6: 1004–1010.[CrossRef][Medline] [Order article via Infotrieve]
  23. Breitschopf K, Zeiher AM, Dimmeler S. Proatherosclerotic factors induce telomerase inactivation in endothelial cells through an Akt-dependent mechanism. FEBS Lett. 2001; 493: 21–25.[CrossRef][Medline] [Order article via Infotrieve]
  24. Takemoto M, Node K, Nakagami H, Liao Y, Grimm M, Takemoto Y, Kitakaze M, Liao JK. Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy. J Clin Invest. 2001; 108: 1429–1437.[CrossRef][Medline] [Order article via Infotrieve]
  25. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature. 2000; 408: 239–247.[CrossRef][Medline] [Order article via Infotrieve]
  26. Xu D, Neville R, Finkel T. Homocysteine accelerates endothelial cell senescence. FEBS Lett. 2000; 470: 20–24.[CrossRef][Medline] [Order article via Infotrieve]
  27. Steidl U, Kronenwett R, Rohr UP, Fenk R, Kliszewski S, Maercker C, Neubert P, Aivado M, Koch J, Modlich O, Bojar H, Gattermann N, Haas R. Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow-derived and circulating human CD34+ hematopoietic stem cells. Blood. 2002; 99: 2037–2044.[Abstract/Free Full Text]
  28. Feugier P, Jo DY, Shieh JH, MacKenzie KL, Rafii S, Crystal RG, Moore MA. Ex vivo expansion of stem and progenitor cells in co-culture of mobilized peripheral blood CD34+ cells on human endothelium transfected with adenovectors expressing thrombopoietin, c-kit ligand, and Flt-3 ligand. J Hematother Stem Cell Res. 2002; 11: 127–138.[CrossRef][Medline] [Order article via Infotrieve]
  29. Rossig L, Jadidi AS, Urbich C, Badorff C, Zeiher AM, Dimmeler S. Akt-dependent phosphorylation of p21(Cip1) regulates PCNA binding and proliferation of endothelial cells. Mol Cell Biol. 2001; 21: 5644–5657.[Abstract/Free Full Text]
  30. Scheid MP, Woodgett JR. PKB/AKT: functional insights from genetic models. Nat Rev Mol Cell Biol. 2001; 2: 760–768.[CrossRef][Medline] [Order article via Infotrieve]
  31. Medema RH, Kops GJ, Bos JL, Burgering BM. AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27 kip1. Nature. 2000; 404: 782–787.[CrossRef][Medline] [Order article via Infotrieve]
  32. Diehl JA, Cheng M, Roussel MF, Sherr CJ. Glycogen synthase kinase-3ß regulates cyclin D1 proteolysis and subcellular localization. Genes Dev. 1998; 12: 3499–3511.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
Therapeutic Advances in Cardiovascular DiseaseHome page
M. Pirro, F. Bagaglia, L. Paoletti, R. Razzi, and M. R. Mannarino
Review: Hypercholesterolemia-associated endothelial progenitor cell dysfunction
Therapeutic Advances in Cardiovascular Disease, October 1, 2008; 2(5): 329 - 339.
[Abstract] [PDF]


Home page
Eur Heart JHome page
P. E. Westerweel, F. L.J. Visseren, G. R. Hajer, J. K. Olijhoek, I. E. Hoefer, P. de Bree, S. Rafii, P. A. Doevendans, and M. C. Verhaar
Endothelial progenitor cell levels in obese men with the metabolic syndrome and the effect of simvastatin monotherapy vs. simvastatin/ezetimibe combination therapy
Eur. Heart J., September 28, 2008; (2008) ehn431v1.
[Abstract] [Full Text] [PDF]


Home page
Therapeutic Advances in Cardiovascular DiseaseHome page
T. J. Povsic and P. J. Goldschmidt-Clermont
Review: Endothelial progenitor cells: markers of vascular reparative capacity
Therapeutic Advances in Cardiovascular Disease, June 1, 2008; 2(3): 199 - 213.
[Abstract] [PDF]


Home page
Am. J. Physiol. Heart Circ. Physiol.Home page
T. Fujii, M. Onimaru, Y. Yonemitsu, H. Kuwano, and K. Sueishi
Statins restore ischemic limb blood flow in diabetic microangiopathy via eNOS/NO upregulation but not via PDGF-BB expression
Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2785 - H2791.
[Abstract] [Full Text] [PDF]


Home page
Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
S. Takahashi, H. Nakamura, M. Seki, Y. Shiraishi, M. Yamamoto, M. Furuuchi, T. Nakajima, S. Tsujimura, T. Shirahata, M. Nakamura, et al.
Reversal of elastase-induced pulmonary emphysema and promotion of alveolar epithelial cell proliferation by simvastatin in mice
Am J Physiol Lung Cell Mol Physiol, May 1, 2008; 294(5): L882 - L890.
[Abstract] [Full Text] [PDF]


Home page
NeurologyHome page
S. -T. Lee, K. Chu, K. -H. Jung, D. -H. Kim, E. -H. Kim, V. N. Choe, J. -H. Kim, W. -S. Im, L. Kang, J. -E. Park, et al.
Decreased number and function of endothelial progenitor cells in patients with migraine
Neurology, April 22, 2008; 70(17): 1510 - 1517.
[Abstract] [Full Text] [PDF]


Home page
J Am Coll CardiolHome page
K. Ramasubbu, J. Estep, D. L. White, A. Deswal, and D. L. Mann
Experimental and clinical basis for the use of statins in patients with ischemic and nonischemic cardiomyopathy.
J. Am. Coll. Cardiol., January 29, 2008; 51(4): 415 - 426.
[Abstract] [Full Text] [PDF]


Home page
HeartHome page
W Wojakowski, M Kucia, M Kazmierski, M Z Ratajczak, and M Tendera
Circulating progenitor cells in stable coronary heart disease and acute coronary syndromes: relevant reparatory mechanism?
Heart, January 1, 2008; 94(1): 27 - 33.
[Abstract] [Full Text] [PDF]


Home page
DiabetesHome page
Y.-H. Chen, S.-J. Lin, F.-Y. Lin, T.-C. Wu, C.-R. Tsao, P.-H. Huang, P.-L. Liu, Y.-L. Chen, and J.-W. Chen
High Glucose Impairs Early and Late Endothelial Progenitor Cells by Modifying Nitric Oxide-Related but Not Oxidative Stress-Mediated Mechanisms
Diabetes, June 1, 2007; 56(6): 1559 - 1568.
[Abstract] [Full Text] [PDF]


Home page
J Am Coll CardiolHome page
E. Shantsila, T. Watson, and G. Y.H. Lip
Endothelial Progenitor Cells in Cardiovascular Disorders
J. Am. Coll. Cardiol., February 20, 2007; 49(7): 741 - 752.
[Abstract] [Full Text] [PDF]


Home page
Circ. Res.Home page
T. Thum, S. Hoeber, S. Froese, I. Klink, D. O. Stichtenoth, P. Galuppo, M. Jakob, D. Tsikas, S. D. Anker, P. A. Poole-Wilson, et al.
Age-Dependent Impairment of Endothelial Progenitor Cells Is Corrected by Growth Hormone Mediated Increase of Insulin-Like Growth Factor-1
Circ. Res., February 16, 2007; 100(3): 434 - 443.
[Abstract] [Full Text] [PDF]


Home page
Circ. Res.Home page
T. Minamino and I. Komuro
Vascular Cell Senescence: Contribution to Atherosclerosis
Circ. Res., January 5, 2007; 100(1): 15 - 26.
[Abstract] [Full Text] [PDF]


Home page
Arterioscler. Thromb. Vasc. Bio.Home page
J. Honold, R. Lehmann, C. Heeschen, D. H. Walter, B. Assmus, K.-I. Sasaki, H. Martin, J. Haendeler, A. M. Zeiher, and S. Dimmeler
Effects of Granulocyte Colony Stimulating Factor on Functional Activities of Endothelial Progenitor Cells in Patients With Chronic Ischemic Heart Disease
Arterioscler. Thromb. Vasc. Biol., October 1, 2006; 26(10): 2238 - 2243.
[Abstract] [Full Text] [PDF]


Home page
Ann. Thorac. Surg.Home page
T.-S. Li, M. Takahashi, R. Suzuki, T. Kobayashi, H. Ito, A. Mikamo, and K. Hamano
Pravastatin Improves Remodeling and Cardiac Function After Myocardial Infarction by an Antiinflammatory Mechanism Rather than by the Induction of Angiogenesis
Ann. Thorac. Surg., June 1, 2006; 81(6): 2217 - 2225.
[Abstract] [Full Text] [PDF]


Home page
CirculationHome page
R. S. Vasan
Biomarkers of Cardiovascular Disease: Molecular Basis and Practical Considerations
Circulation, May 16, 2006; 113(19): 2335 - 2362.
[Full Text] [PDF]


Home page
Am. J. Physiol. Heart Circ. Physiol.Home page
J. Chen and M. S. Goligorsky
Premature senescence of endothelial cells: Methusaleh's dilemma
Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1729 - H1739.
[Abstract] [Full Text] [PDF]


Home page
Am. J. Physiol. Heart Circ. Physiol.Home page
T.-S. Li, A. Furutani, M. Takahashi, M. Ohshima, S.-L. Qin, T. Kobayashi, H. Ito, and K. Hamano
Impaired potency of bone marrow mononuclear cells for inducing therapeutic angiogenesis in obese diabetic rats
Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1362 - H1369.
[Abstract] [Full Text] [PDF]


Home page
CirculationHome page
P. J. Goldschmidt-Clermont, M. A. Creager, D. W. Lorsordo, G. K.W. Lam, M. Wassef, and V. J. Dzau
Atherosclerosis 2005: Recent Discoveries and Novel Hypotheses
Circulation, November 22, 2005; 112(21): 3348 - 3353.
[Full Text] [PDF]


Home page
J Am Coll CardiolHome page
G. Kanaganayagam and M. S. Marber
ADMAring Endothelial Progenitor Cells: Accident, Association, or Antecedent
J. Am. Coll. Cardiol., November 1, 2005; 46(9): 1702 - 1704.
[Full Text] [PDF]


Home page