Rosuvastatin enhances angiogenesis via eNOS-dependent mobilization of endothelial progenitor cells
Rosuvastatin Enhances Angiogenesis via eNOS-Dependent Mobilization of Endothelial Progenitor Cells Junlan Zhou1.,Min Cheng2.,Yu-Hua Liao2,Yu Hu3,Min Wu4,Qing Wang5,Bo Qin6,Hong Wang7, Yan Zhu7,Xiu-Mei Gao7,David Goukassian8,Ting C.Zhao9,Yao-Liang Tang10,Raj Kishore1,
Gangjian Qin1*
1Feinberg Cardiovascular Research Institute,Northwestern University Feinberg School of Medicine,Chicago,Illinois,United States of America,2Department of Cardiology,Northwestern University Feinberg School of Medicine,Chicago,Illinois,United States of America,3Department of Hematology,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan,Hubei,P.R.China,4Department of Plastic Surgery,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan,Hubei,P.R.China,5Key Laboratory of Molecular Biophysics of the Ministry of Education,College of Life Science and Technology,Center for Human Genome Research,Cardio-X Institute,Huazhong University of Science and Technology,Wuhan,Hubei,P.R.China,6Weinberg College of Arts and Sciences,Northwestern,Chicago,Illinois,United States of America,7Tianjin State Key Laboratory of Modern Chinese Medicine,Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae,Ministry of Education,Tianjin University of Traditional Chinese Medicine,Tianjin,P.R.China,8CardioVascular Systems Biology, Steward St.Elizabeth’s Medical Center,Tufts University School of Medicine,Boston,Massachusetts,United States of America,9Department of Surgery,Boston University Medical School,Roger William Medical Center,Providence,Rhode Island,United States of America,10Division of Cardiovascular Disease,Cardiovascular Research Center, University of Cincinnati,Cincinnati,Ohio,United States of America
Abstract
Circulating endothelial progenitor cells(circEPCs)of bone marrow(BM)origin contribute to postnatal neovascularization and represent a potential therapeutic target for ischemic disease.Statins are beneficial for ischemia disease and have been implicated to increase neovascularization via mechanisms independent of lipid lowering.However,the effect of Statins on EPC function is not completely understood.Here we sought to investigate the effects of Rosuvastatin(Ros)on EPC mobilization and EPC-mediated neovascularization during ischemic injury.In a mouse model of surgically-induced hindlimb ischemia(HLI),treatment of mice with low dose(0.1mg/kg)but not high dose(5mg/kg)significantly increased capillary density and accelerated blood flow recovery,as compared to saline-treated group.When HLI was induced in mice that had received Tie2/LacZ BM transplantation,Ros treatment led a significantly larger amount of endothelial cells(ECs)of BM origin incorporated at ischemic sites than saline.After treatment of mice with a single low dose of Ros,circEPCs significantly increased from2h,peaked at4h,declined until8h.In a growth-factor reduced Matrigel plug-in assay,Ros treatment for5
d induced endothelial lineag
e differentiation in vivo.Interestingly,the enhanced circEPCs and post-HLI neovascularization
stimulated by Ros were blunted in mice deficient in endothelial nitric oxide synthase(eNOS),and Ros increased p-Akt/p-eNOS levels in EPCs in vitro,indicating these effects of Ros are dependent on eNOS activity.We conclude that Ros increases circEPCs and promotes their de novo differentiation through eNOS pathway.
Citation:Zhou J,Cheng M,Liao Y-H,Hu Y,Wu M,et al.(2013)Rosuvastatin Enhances Angiogenesis via eNOS-Dependent Mobilization of Endothelial Progenitor Cells.PLoS ONE8(5):e63126.doi:10.1371/journal.pone.0063126
Editor:Costanza Emanueli,University of Bristol,United Kingdom
Received December10,2012;Accepted March29,2013;Published May21,2013
Copyright:?2013Zhou et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.
Funding:This work was supported by National Institutes of Health grants(HL093439and HL113541to G.Q.)and American Heart Association grants(0430135N to G.Q.and10POST4360009to J.Z.).The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.
Competing Interests:Drs.Gangjian Qin,Raj Kishore,and Yao-Liang Tang are currently academic editors for the PLOS ONE journal,and this does not alter the authors’adherence to all the PLOS ONE policies on sharing data and materials.The authors declare that they have no conflict of interest.
*E-mail:g-qin@https://www.wendangku.net/doc/2d1374246.html,
.These authors contributed equally to this work.
Introduction
Cardiovascular ischemic disease is the leading cause of morbidity and mortality worldwide and constitutes a major health burden.Recent clinical studies suggest that infusion of bone morrow(BM)-derived endothelial progenitor cells(EPCs)aug-ments neovascularization of ischemic tissues and improve the therapeutic outcome[1–3].In animal studies,EPCs have been shown to contribute to new vessel formation and tissue recovery by direct integration into injured vasculature[4–6],mediating favorable cell-to-cell contact[7],secreting paracrine factors[8,9] and microparticles[10],and activating endogenous tissue stem/ progenitor cells[11].
Statins are inhibitors of3-hydroxyl-3-methyl coenzyme A reductase,the rate-limiting enzyme in cholesterol biosynthesis, and possess anti-inflammatory,anti-oxidant,anti-platelet and anti-fibrotic properties;thus,they are widely used in the treatment of dyslipidemia and the associated cardiovascular abnormalities[12–14].Interestingly,considerable benefits have been demonstrated in statins’clinical trials in patients with ischemic heart and peripheral disease,irrespective of the cholesterol concentration [15–18].In fact,statins have been shown to stimulate angiogenesis by upregulation of the expression and activity of endothelial nitric oxide synthase(eNOS)[19–22].eNOS is a key enzyme in the generation of nitric oxide in endothelial lineage cells,which not only contributes to angiogenesis induced by various stimuli[23]
but also plays an important role in the mobilization of BM EPCs [24–26];and studies from other laboratories suggest that statins enhance the functions of EPCs [27–29].
In this study,we have investigated the role of Rosuvastatin (Ros),a new and efficacious statin [30,31],in the regulation of EPC function and ischemic angiogenesis by the use of BM transplantation (BMT)and surgical hindlimb ischemia (HLI)model in knockout and transgenic mice.Our results indicate that Ros increases EPC mobilization and promotes neovasculogenesis through an eNOS dependent mechanism.
Materials and Methods Mice
Wild-type C57BL/6and FVB/N mice and Tie2/LacZ transgenic mice (on FVB/N background)were purchased from The Jackson Laboratories (Bar Harbor,Maine).All animal work presented in this report was approved by the Institutional Animal Care and Use Committee (IACUC)of Northwestern University and performed in the barrier facilities of the Center for Comparative Medicine of the university.
HLI Model
HLI was induced in 10-to 12-week-old male mice by surgical excision of the left femoral artery as described previously [5,32].The mice were anesthetized by inhaling Isoflurane TM delivered at 2–4%throughout the surgical procedure,and were injected subcutaneously with Metacam (1mg/kg)as analgesic immediately after the surgery and then daily for the next 2to 3days.After the surgery,the mice were treated with Ros (AstraZeneca Pharma-ceuticals,Cheshire,UK)and Simvastatin (Sigma-Aldrich)at serial doses for different time lengths.The blood flow recovery was monitored regularly with Laser Doppler perfusion imaging (LDPI)system (Moor Instruments,Wilmington,DE,USA)and expressed as perfusion ratio of ischemic/healthy (i.e.left/right)limbs.At 10min before euthanasia,a 50uL BS lectin (Vector Laboratories)was i.v.injected to facilitate identification of vasculature in the tissue sections.Then,the mice were euthanized by CO 2inhalation (primary method)and cervical dislocation (secondary method).
Matrigel Plug-in Model
The plugs were created by subcutaneous (s.c.)injection of 300m L mixture of growth factor-reduced Matrigel (BD Biosci-ences)with 26106BM mononuclear cells (MNCs)at the back of mice with a G27needle.After 5days,the plugs were excised,examined microscopically,and stained with X-gal.
Tie2/LacZ BM Transplantation (BMT)
Tie2/LacZ BMT was performed as described previously [4].Briefly,donor Tie2/LacZ mouse BM MNCs were isolated with density centrifugation.The recipients,8week old male FVB/N mice,were irradiated (9Gy),and each recipient received 26106Tie2/LacZ BM MNCs via tail vein injection.One month later,the recipient mice were subject to surgical HLI and daily Ros injection.The engraftment routinely reached 80,90%for FVB mice receiving Tie2/LacZ BM MNCs with the standardized protocol in our lab [5].
Circulating EPC (circEPC)Culture Assay
CircEPCs were evaluated with a culture assay as previously described [5].Briefly,peripheral blood (PB)MNCs were isolated from 300m L blood with Histopaque-1083(sigma)and seeded on 0.1%gelatin and 2.5m g/ml rat vitronectin (sigma)-precoated 4chamber slides containing 1ml EBM-2complete medium (EBM-2CM).EBM-2CM is EBM-2basal medium plus the cytokine cocktail of EGM-2-MV SingleQuots (Clonetics,Inc .,San Diego,California).At day 4,a 2m L of DiI-acLDL (Biomedical Technologies Inc.,Massachusetts)was added to incubate for 4h.The cells were then fixed in 1%paraformaldehyde (PFA)and counter-stained with 1%(v/v)Isolectin B4-FITC (Vector Labo-ratories,Inc.,California).The adherent cells positive for both Isolectin B4-FITC binding and DiI-acLDL uptake were consid-ered EPCs,which reflect the initial circulating EPCs present in the PB MNCs.Randomly chosen 12,20fields with 2006magnifi-cation were counted by blinded investigators under a fluorescent
microscope.
Figure 1.Ros enhances ischemic neovascularization.(A )C57BL/6male mice were rendered surgical hindlimb ischemia.Ros at low dose (0.1mg/kg),high dose (5mg/kg),or saline was injected daily,and blood flow recovery was monitored by LDPI at days 3,5,7,14,21,and 28after the surgery.n =8,*P ,0.05,**P ,0.01vs.Saline.(B )Representative micrographs of BS lectin staining (left panel ,2006original magnification)and quantification of capillary densities (right panel )in the limb tissues at day 14.n =8,**P ,0.01vs.Saline.doi:10.1371/journal.pone.0063126.g001
X-gal Staining and Immunohistochemistry
Tissues or Matrigel plugs were fixed either in 4%PFA or 100%methanol.Enzymatic staining for X-gal and immunohistochemical staining with antibodies for BS Lectin (Vector laboratories,Burlingame,CA)and b -Gal (Cell Signaling Technology,Beverly,MA)were performed as previously described [5,32,33].
Western Blotting
EPCs were isolated and cultured from BM MNCs for 7days with a standardized protocol established in our lab [5],and then treated with different concentrations of Ros for 30min.Western blotting analyses with antibodies for eNOS,phospho-eNOS (Ser1177),Akt,and phospho-Akt (Ser473)(Cell Signaling Tech-nology)were performed as previously described [34–36].Band
intensities were determined densitometrically with Image J software.
Statistical Analysis
Data are presented as average 6SEM.Unpaired Student’s t test was used for the significance of differences.P ,0.05was considered significant.
Results
Ros Enhances Neovascularization
To investigate the effect of Ros on neovascularization after ischemic injury,we induced acute ischemia in C57BL/6mice by surgically removing the left femoral artery.Ros at low dose (0.1mg/kg),high dose (5mg/kg),or Saline control was
injected
Figure 2.Ros increases BM-derived EPCs in neovascularization.BM MNCs isolated from Tie2/LacZ mice were used to transplant lethally irradiated syngeneic FVB/NJ mice.One month later,HLI was induced in the recipient mice,and Ros (0.1mg/kg)was s.c.injected daily.(A–C )At day 14after HLI,mice were injected with BS lectin and 10min later,euthanized.The ischemic tissues were stained immunofluorescently with BS lectin (FITC,green)and b -gal (PE,red)to indicate vasculature and BM-derived cells,respectively.(A )Representative images of immunofluorescent double staining.White arrows indicate BS lectin and b -gal double positive cells.4006original magnification.(B )Quantification of BM-derived EPCs incorporated in the neovasculature (i.e.,double positive cells)in the ischemic limb.(C )Quantification of capillary densities (BS lectin-FITC positive cells).n =6,*P ,0.05,**P ,0.01vs.Saline.(D )Blood flow was monitored with LDPI at days 3,5,7,10,14,and 16following HLI.*P ,0.05,**P ,0.01vs.Saline.
doi:10.1371/journal.pone.0063126.g002
subcutaneously (s.c.)daily from day 0to day 28following the surgery.The low-dose but not high-dose group of mice demonstrated more rapid blood flow recovery than the control group at days 3,7and 14(Figure 1A).Low dose Ros treatment also increased capillary density in the ischemic limb (Figure 1B).Thus,we continued our investigations of Ros at low dose for the following in vivo studies.
Ros Increases Incorporation of BM-derived EPCs at Sites of Ischemic Injury
To assess the effect of Ros on BM EPC contribution to neovascularization,we performed BM transplantation (BMT)to reconstitute the BM of lethally-irradiated WT FVB/N mice with BM MNCs from background-matched Tie2/LacZ mice.One month later,recipient mice with .90%engraftment were chosen to receive surgical HLI and treatment with Ros or saline.Ros treatment led to a significantly greater number of BM-derived ECs (as shown by BS lectin and b -gal double positive cells)incorporated at ischemic sites (Figures 2A and 2B),which were associated with an increased capillary density and accelerated
blood flow recovery in the ischemic limb (Figures 2C and 2D),although recovery of the mice on FVB/N background were generally slower than those on C57BL/6background (Figure 1A).
Ros Increases the Levels of circEPCs and Endothelial Lineage Differentiation from BM MNCs
To understand the cellular mechanism by which Ros increases EPC incorporation into neovascularization,we analyzed the effect of Ros on the level of circEPCs (i.e.,EPC mobilization).Different doses of Ros or saline were s.c.injected into WT C57BL/6mice and the amount of circEPC were evaluated 24h later with a EPC culture assay [5].CircEPC number peaked at a dose of 0.1mg/kg,being 6times higher than untreated mice and 3times higher than phVEGF treated mice (Figure 2A).Moreover,after single injection of 0.1mg/kg Ros or 0.2mg/kg Simvastatin (Sim)[37],circEPCs significantly increased from 2h,peaked at 4h,declined until 8h (Figure 2B).These results suggest that Statins mobilize EPCs quickly and in a dose dependent manner,and that the enhanced neovascularization in Ros-treated mice may be attributable,at least partly,to an increase in EPC
mobilization.
Figure 3.Ros increases circEPCs and promotes endothelial lineage differentiation.(A )C57BL/6male mice received daily s.c.injections of different doses of Ros for 7days.Twenty-four hours after last injection,PB MNCs were collected,and circEPCs were evaluated with a culture assay.One group received an intramuscular injection of 200ug pVEGF165plasmid as positive control.n =4,**P ,0.001vs.Saline.(B )The kinetics of PB circEPCs within 24h after s.c.injection of a single dose of Ros (0.1mg/kg)or Sim (0.2mg/kg).n =4,***P ,0.001vs.Saline.(C–D )Mice were s.c.injected with a mixture of 300uL growth factor-reduced Matrigel and 26106Tie2/LacZ BM MNCs and then s.c.injected with Ros (0.1mg/kg)or saline daily for 5days.The Matrigel plugs were then removed,fixed,and stained in X-gal solution.(C )Representative images.(D )b -gal positive cells were quantified and expressed as percentage of total cells,n =4,**P ,0.01vs.Saline.doi:10.1371/journal.pone.0063126.g003
Although the increase in new vessel formation could be the result of this mechanism alone,we also considered the possibility that Ros could enhance differentiation of EPCs.To test this hypothesis we performed experiments in which ,56106undiffer-entiated BM MNCs isolated from Tie2/LacZ mice were mixed in growth factor-reduced Matrigel and s.c.implanted into back-ground-matched WT mice that receive daily injection of either 0.1mg/kg Ros or saline for 5days.Since growth factor-reduced Matrigel does not provide a sufficient substrate to support the growth of new vessels,this model provided us with the opportunity to quantify EPC differentiation in vivo ,defined by the advent of Tie2driven LacZ expression in the population of unselected BM MNCs.As shown in Figures 3C and 3D,a significantly higher ratio of X-gal (+)to total cells was found in the Matrigel plugs in Ros-treated mice than in saline-treated mice.Since an equal number of BM MNCs were implanted in both groups,these data indicate that Ros enhances de novo EPC differentiation in vivo .
The Ros-induced Enhancement of EPC Mobilization and Blood Flow Recovery is dependent on eNOS pathway
Because eNOS mediates several beneficial effects of statins for vascular protection [38]and has also been shown to be essential for BM EPC mobilization [39],we investigated whether eNOS pathway is a potential molecular mechanism by which Ros mobilizes BM EPCs and enhances neovascularization.In eNOS-null (eNOS 2/2)mice,HLI surgery led to a significant amount of limb loss;however,there is no significantly difference between Ros and saline treatments (Figure 4A).The blood perfusions in the preserved hindlimbs were also similar between the two treatment groups (Figure 4B).Moreover,the increase in capillary density in the ischemic limbs and the elevated levels of circEPCs induced by Ros were also blunted in the eNOS 2/2mice (Figures 4C and 4D),indicating that these effects of Ros are dependent on a normal eNOS activity.Consistently,Ros treatment of cultured EPCs increased levels of phosphorylated Akt and eNOS (Figure 4E).
Discussion
In this study,we have demonstrated that Ros potently mobilizes EPCs,promotes EPC de novo differentiation,and significantly enhances neovascularization and blood flow recovery after ischemic limb injury.In addition,our results indicate that the beneficial effects of Ros on EPC mobilization and neovascular-ization are dependent on eNOS activity.
Similar to other statins [40–42],Ros demonstrates a biphasic effect on neovasculogenesis;however,the precise mechanism is currently not clear.Low doses of statins have been shown to enhance neovasculogenesis by activating endothelial Ras,promot-ing Akt and eNOS phosphorylation [40,43],and upregulating eNOS expression [44,45],whereas high statin doses may
decrease
Figure 4.The Ros-mediated enhancement of EPC mobilization and neovascularization is dependent on eNOS expression.(A–C )eNOS 2/2mice were rendered surgical HLI and s.c.injected daily with Ros (0.1mg/kg)or saline.(A )The rate of limb loss,n =18.(B )Blood flow assessments with LDPI in mice without limb loss at day 7,14,28,and 42after the surgery.(C )In a separate experiment,capillary density in the ischemic limb of day 14was evaluated with BS lectin,n =6.(D )eNOS 2/2mice received daily s.c.injections of Ros (0.1mg/kg)for 7d.PB MNCs were collected 24h after the last injection,and circEPCs were assayed with the culture assay,n =4.(E )EPCs were cultured from BM MNCs of WT mice for 7days,and then treated with different doses of Ros for 30min.The cells were lysed and the levels of phospho-Akt,Akt,phospho-eNOS,and eNOS were analyzed by Western blotting.Left panel,representative Western blotting.Right panel ,the levels of phospho-Akt and phospho-eNOS were normalized to Akt and eNOS,respectively,and expressed relative to the values in saline-treated group.n =4,*P ,0.05,**P ,0.01vs.Saline.doi:10.1371/journal.pone.0063126.g004
protein prenylation in ECs,inhibit cell growth,and induce apoptosis[40].These biphasic activities of statins on EC biology can potentially be explained by the properties of the biosynthetic pathways that originate from mevalonic acid[46],because in addition to cholesterol,mevalonic acid is an essential precursor for several cellular components including ubiquinone,isopentenylated transfer RNAs,and prenylated proteins.Mevalonate-derived intermediates have a higher affinity for the enzymes that catalyse non-sterol product formation than for the cholesterol biosynthetic enzymes.Therefore,low doses of statins may predominantly affect cholesterol synthesis and not interfere with the biosynthesis of non-sterol products that are required for cellular housekeeping functions,and only at higher statin doses may a significant inhibition of non-sterol product synthesis occur.
Ros is by far the most efficacious statin[31,47];nevertheless,the JUPITER study,despite reducing CVD and overall mortality, highlighted an increase in new onset diabetes in the Ros treated arm[47].More recently,the increase in the incidence of diabetes during statins trials has been confirmed by many meta-analyses of the randomized controlled trials and appears to be associated with a higher statin dosage[48,49].Our study suggests that a lower dose may be more favorable,at least in patients with diabetes or diabetes associated risk factors.
It is exciting that low dose of Ros significantly enhances EPC mobilization and recruitment to the site of neovascularization. Stem cell mobilization involves complicated adhesive interactions or cross-talks between stem cells and BM microenvironment [34,35,50,51].For example,G-CSF acts not directly on hemato-poietic stem cells(HSCs)but via receptors on cells of the BM stroma[52],while cleavage of VCAM-1with neutrophil protease was also involved in HSC mobilization[53].The kinetics of HSC mobilization with chemokines versus cytokines range from a few minutes(with chemokines)to several days(with hematopoietic growth factors)[51].The kinetics of EPC mobilization,however, varies considerably.Vascular trauma and ischemia was reported to induce rapid but transient EPC mobilization[54].Previous study from our institute demonstrated that circEPCs reached a peak at day7after surgical ischemia in rabbits[55].The
mobilization of EPCs in nude mice with Ad-VEGF injection is
rapid,peaks at2–3days while Angiopoietin-1exerts a delay EPC
mobilization as compared to VEGF,peaks at2weeks[56].Other
researchers reported that peripheral circEPC numbers increased
gradually,reached peak with4weeks treatment of statins in mouse
models and in patients with coronary diseases[29,57].Our data
further show that the kinetics of Ros-induced EPC mobilization is
more resembling to that of‘‘chemokine-type’’.Further investiga-
tions are underway to determine how high Ros doses affect EPC
mobilization and neovasculogenesis.
We found that the enhanced circEPCs and post-HLI angio-
genesis stimulated by Ros were blunted in eNOS2/2mice,
suggesting an essential role of eNOS.Because Ros upregulates
eNOS in both EPCs and ECs,it is difficult to dissect the extent to
which the angiogenic effect of the statin is dependent on EPCs.
Our attempt to reconstitute WT mouse BM with that of eNOS2/ 2mice was with low efficiency presumably due to the indispensable role of eNOS for a successful BMT.Currently,it
is not completely clear how eNOS is activated(i.e.,phosphory-
lated)by Ros.Since PI3K/Akt pathway has been shown essential
to EPC mobilization,migration,proliferation,and survival[37,57]
and our results indicate that Ros also mediates Akt phosphory-
lation,it is therefore likely that eNOS be a down-stream mediator
of Akt[58–60];this however,remain to be investigated in our
future study.
In summary,our study demonstrates that Ros at a lower dose
promotes ischemic neovascularization via eNOS-dependent EPC
mobilization.Thus,optimization of Ros dose may maximize the
effect of Ros for the prevention and treatment of ischemic disease. Author Contributions
Conceived and designed the experiments:JZ YL RK GQ.Performed the experiments:JZ MC MW BQ HW.Analyzed the data:JZ MC MW GQ. Contributed reagents/materials/analysis tools:YH QW YZ XG DG TCZ YLT RK.Wrote the paper:JZ GQ.
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