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TRANSLATIONAL AND CLINICAL RESEARCH |
aDepartment of Surgery, Vascular Biology Institute, University of Miami, Miller School of Medicine, Miami, Florida, USA;
bDivision of Vascular Surgery, Miami Veterans Administration, Miami, Florida, USA;
cBaptist Cardiac and Vascular Institute, Miami, Florida, USA
Key Words. Angiogenesis • Endothelial progenitor cells • Statin • SDF-1 • Ischemia
Correspondence: Correspondence: Hong Yu, Ph.D., Department of Surgery, University of Miami School of Medicine, 1600 NW, 10th Ave, RMSB 1018, Miami, Florida, USA. Telephone: 305-243-6477, Fax: 305-243-2810; e-mail: hyu{at}med.miami.edu
Received on September 17, 2007;
accepted for publication on February 22, 2008.
First published online in STEM CELLS EXPRESS February 28, 2008.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Statins are 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors and are primarily used to lower circulating cholesterol levels. In addition, statins have also been shown to protect against ischemic injury of the heart and stimulate angiogenesis in ischemic limbs of normocholesterolemic animals [7, 8]. The mechanism of action of statins has been demonstrated via mobilization of BM endothelial progenitor cells (EPCs) and facilitation of EPC incorporation into the neovasculature [4–6]. Statins have also been reported to enhance EPC migration, augment EPC chemotaxis, and inhibit EPC apoptosis both in vitro and in vivo [4, 9].
Stromal cell-derived factor-1 (SDF-1) is a chemotactic cytokine that enhances the homing of mobilized progenitor cells to areas of SDF-1 production. The mechanism of action of SDF-1 involves promotion of cell migration and proliferation [3, 10]. SDF-1 delivered locally, either as free protein or via plasmid-mediated gene expression, enhances EPC recruitment into ischemic tissue resulting in augmented angiogenesis [3, 11, 12]. Moreover, SDF-1 expression is up-regulated in ischemic tissues, suggesting that following induction of hind-limb ischemia, an SDF-1 gradient is established which functions to both facilitate progenitor cell mobilization into peripheral blood, and their homing to ischemic tissue [13].
We hypothesize that the combined use of a statin to mobilize BM EPCs and local over-expression of SDF-1 to augment EPC homing to ischemic muscle will result in superior angiogenesis versus use of either agent alone. In the present study we use the murine hindlimb ischemia model to determine the effects of Fluvastatin and SDF-1 on angiogenesis. Combined treatment with statin and SDF-1 was found to additively promote angiogenesis both in vitro and in vitro.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Fluvastatin was dissolved in PBS and used at 10 nM, 100 nM, and 1,000 nM in all in vitro experiments. SDF-1 protein (100 ng/ml) was used in all in vitro experiments. NIH 3T3 cells transduced retroviral vector carrying mouse SDF-1
gene (NIH 3T3/SDF-1) were used to deliver SDF-1 in vivo for all animal experiments [14]. LY294002 is a PI3-kinase inhibitor; NG-monomethyl-L-arginine (L-NMMA) is nitric oxide synthase (NOS) inhibitor; AMD3100 is CXCR4 antagonist; and NOC18 is nitric oxide (NO) donor.
Mononuclear cells (4 x 106) were isolated from rabbit peripheral blood (20 ml) [15], plated on culture dishes coated with fibronectin (0.1%), and cultured in endothelial cell basal medium-2 (EBM-2) supplemented with 20% fetal bovine serum (FBS) and Clonetics EGM-2 SingleQuots. Outgrown colonies appeared 10–14 days after onset of culture. These cells, defined as EPCs, were maintained in EBM-2 supplemented with 20% FBS and used within passage 2–5 for in vitro study. The EPC were characterized by acetylated low density lipoprotein uptake, lectin binding, and immunochemical staining for CD34, CD133, CD31, VEGFR-2, von Willebrand factor (vWF), VE-cadherin, and smooth muscle -actin.
EPCs were plated into 24-well plates (1 x 104 cells/well) and cultured for 48 hours in MCDB131 medium supplemented with 10% FBS plus the specified additives: 1) saline, 2) statin 10 nM, 3) statin 100 nM, 4) statin 1,000 nM, 5) SDF-1 (100 ng/ml), 6) SDF-1 and statin (10 nM), 7) SDF-1 and statin 100 nM, and 8) SDF-1 and statin 1,000 nM; and then counted for cell number. A modified Boyden chamber assay was performed to measure EPC migration [14].
EPC tube formation was assessed in Matrigel. EPCs (2 x 104) in a mixture of Matrigel and MCDB131 medium on a 24-well dish were cultured for 12 h. Endothelial tube formation was quantified by measuring both branch lengths and branch points.
EPCs (1 x 105 cells) in 24-well plates were cultured with serum free medium supplemented with different additives of statin and/or SDF-1 for 48 hours. The EPCs were then stained with 4',6-diamidino-2-phenylindole. The proportion of apoptotic EPCs was determined by counting pyknotic nuclei versus total nuclei.
Matrix Metalloproteinase-2 (MMP-2) and MMP-9 activity were analyzed with gelatin zymography [16]. Phosphorylated Akt and total Akt from EPCs were determined using the FACE Akt kit which detects phosphorylation at Ser473. NO production from the EPCs was indirectly determined using a NO Colorimetric Assay Kit by measuring NO2– and NO3– concentration in medium.
Male mice (C57BL/6J Huxb13) were anesthetized for the creation of ischemic hindlimb as described [14]. Fluvastatin (5 mg/kg) was injected intra-peritoneally (I.P.) into the mice daily for 7 days before femoral vessel excision. Administration of SDF-1 was achieved through I.M. injection of NIH 3T3/SDF-1 cells (106 cells in 0.1 ml) into the ischemic abductor muscle immediately after the vessels were resected [14]. The mice were divided into four groups randomly (six mice per group): 1) saline, 2) Fluvastatin, 3) NIH 3T3/SDF-1, and 4) Fluvastatin and NIH 3T3/SDF-1. Mice were sacrificed 21-day after vessel resection. Laser Doppler perfusion imaging (LDPI) was performed as described [14] to record blood flow over a course of 3 weeks postoperatively. Relative perfusion data were expressed as the ratio of the ischemic (right) to normal (left) limb blood flow. Capillary endothelium alkaline phosphatase (AP) was stained to quantify the presence of capillaries as described [14].
To demonstrate BM derived EPCs homing to ischemic muscle, eight week old wild-type recipient mice (C57BL/6J) were lethally irradiated. BM donor cells from green fluorescent protein (GFP) transgenic littermates were transplanted via retro-orbital sinus injection. After 4 weeks, the femoral vessels were resected, and statin/SDF-1 treatment was performed as described above. Mice were sacrificed 4 days after operation, and GFP cells in the ischemic muscle were observed under fluorescent microscope and confirmed with immunochemical staining. CD34+ EPCs were detected by immunoflourescence and immunochemical stainings.
Differentiation of BM cells into endothelial cells (EC) at the ischemic site was examined by intravenous injection of BM cells isolated from Tie2-lacZ mice into WT mice that underwent ischemic surgery. BM derived ECs in the muscle 7 days after surgery were identified as the blue cells after x-gal staining [17] and were confirmed by AP staining or immunochemical staining with anti-CD31 Ab.
Cell apoptosis in ischemic muscles were determined using in situ oligo ligation (ISOL) staining. The number of apoptotic cells and muscle fiber were counted in randomly selected fields to calculate the ratio of apoptotic cells per muscle fiber.
Results are expressed as mean ± standard deviation (S.D.). Statistically significant differences between groups were compared with one-way analysis of variance (ANOVA) for multi-groups or 2-tailed Student t-test for two groups only. Significance was attributed to a p value of less than 0.05.
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RESULTS |
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Capillary Density in the Ischemic Tissues
Capillary density in the recovered tissues was analyzed by staining for alkaline phosphatase (Fig. 4A). The number of capillaries in each muscle fiber increased in the mice treated with either statin alone (0.78 ± 0.06 pmf) or with NIH 3T3/SDF-1 alone (0.57 ± 0.11 pmf) in comparison with the saline treated mice (0.40 ± 0.05 pmf, p < .05). The combined administration of statin and NIH 3T3/SDF-1 resulted in the highest capillary density (1.04 ± 0.18 pmf, p < .05 vs. all other groups) (Fig. 4B).
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Effects of Statin and SDF-1 on Proliferation and Migration of EPC
To explore the mechanism of statin and SDF-1 induced enhancement of angiogenesis, their effects on EPC were studied in vitro. MNCs isolated from peripheral blood were cultured on fibronectin coated plates (supplemental online Fig. 2a). Outgrowth colonies formed after 2 weeks in culture (supplemental online Fig. 2b). The outgrowth cells were positive for Dil-Ac-LDL uptake and lectin binding. Most of the attached cells were positive for both stainings, therefore were considered as EPC (supplemental online Fig. 2d–f). They were able to be differentiated into endothelial-like cells after cultured in medium containing VEGF for 2 weeks (supplemental online Fig. 2c). After EPCs were passaged, they were stained positive for progenitor cell markers CD34 and CD133, EC markers CD31, VE-cadherin, VEGFR2, and vWF, and stained negative for the smooth muscle cell marker -actin (supplemental online Fig. 3).
Such isolated EPC were used for in vitro study of the effect of statin and SDF-1 on cells. fluvastatin in three concentrations was used to study the in vitro effect of statin on EPCs. There was a dose-dependent effect of statin on EPC proliferation and migration. Fluvastatin at low concentrations (10 nM, 100 nM, defined as statinlow) enhanced EPC proliferation (Fig. 6A) and migration (Fig. 6B), while an inhibitory effect on both proliferation and migration was observed at a higher concentration of statin (1,000 nM, defined as statinhigh). At the low concentrations, statin's effect on EPC proliferation was greater at 100 nM (13.72 ± 0.27 x 104 EPC/well) than at 10 nM (12.25 ± 0.15 x 104 EPC/well, n = 3, p < .01). EPC proliferation and migration were also enhanced in the presence of SDF-1 alone (12 ± 0.3 x 104 EPC/well with SDF-1 vs. 8 ± 0.3 x 104 EPC/well without SDF-1, p < .01, n = 3) (Fig. 6A, 6B). The proliferation and migration of EPC treated with combined statinlow and SDF-1 was significantly increased compared to treatment with either statin or SDF-1 alone. Statinhigh plus SDF-1 resulted in no enhancement of EPC proliferation or migration (Fig. 6A, 6B).
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Statin and SDF-1 Enhance NO Production in EPCs
Similar to the effect of statin and SDF-1 on EPC proliferation, statinlow and SDF-1 enhanced the NO production after cultured 4 hours (p < .05 vs. NS, Supplemental online Fig. 5). When both statinlow and SDF-1 were added, NO production was significantly higher than either one used alone. Statinhigh inhibited NO productions whether or not SDF-1 was presented (p < .05 vs. NS, SDF-1).
Combined Use of Statin and SDF-1 Promotes Tube Formation by EPCs
The presence of statin or SDF-1 individually did not significantly alter EPC tube formation versus control (p > .05; Fig. 6C, 6D). Total tube length in a field of view was 10.7 ± 1.7 mm, 11.6 ± 2.2 mm, 11.5 ± 6.6 mm, and 8.8 ± 0.9 mm for the cultures with saline, statin at 10 nM, statin at 100 nM, and SDF-1, respectively. However, when both statin (100 nM) and SDF-1 were present in the culture, a nearly 50% increase in tube formation by EPCs was observed (total lengths of tube 15.5 ± 1.9 mm, p < .01 vs. other groups). Treatment with statinhigh either in the presence or absence of SDF-1 resulted in inhibition of EPC tube formation. The branch point count methodology to measure tube formation yielded similar data (not shown).
SDF-1 and Statin Attenuate EPC Apoptosis
Nutrient deprivation was used to induce EPC apoptosis. The percentage of apoptotic cells was 7.1 ± 0.3% when EPCs were cultured in serum free media for 2 days (Fig. 6E). Addition of statinlow or SDF-1 alone to the culture significantly reduced EPC apoptosis. The percentages of apoptotic cells were 5.0 ± 0.4%, 4.6 ± 0.7%, and 4.3 ± 0.3% for culture conditions containing 10 nM statin, 100 nM statin, and SDF-1, respectively (p < .05 vs. control). Combined treatment with statinlow and SDF-1 resulted in significantly decreased apoptosis (3.2 ± 0.4%) versus treatment with either agent alone (p < .05) (Fig. 6E). Statinhigh, alone or in combination with SDF-1, resulted in significantly increased cell apoptosis (21.4 ± 4.3 and 23.2 ± 3.0, respectively, p < .01). EPC necrosis and apoptosis were confirmed by flow cytometer using propidium iodide (PI) and Annexin V double staining. More cell detachment and necrosis were observed when EPC were cultured in serum free medium with statinhigh, while less detached cells were detected when cells were cultured with statinlow (supplemental online Fig. 6).
Statin and SDF-1 Enhance Akt Phosphorylation
To explore SDF-1 and statin signaling pathways, the phosphorylation of Akt was examined. No difference in total Akt level among the culture conditions was detected (p > .05, data not shown). However, phosphorylated Akt was significantly increased when EPCs were cultured in the presence of statinlow and/or SDF-1 (Fig. 7A). Phosphorylated Akt significantly increased from 2.88 ± 0.05 in saline controls to 3.48 ± 0.15, 3.83 ± 0.19, and 4.31 ± 0.28 in cultures with 10 nM statin, 100 nM statin, and SDF-1, respectively (p < .05). When EPCs were cultured in the presence of both statinlow and SDF-1, phosphorylated Akt was further significantly increased to 5.13 ± 0.21 (10 nM statin) and 4.96 ± 0.18 (100 nM statin) (p < .05 vs. statinlow or SDF-1 alone). Statinhigh either with or without SDF-1 did not result in more Akt phosphorylaton.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Statins not only augment mobilization of progenitor cells by increasing circulating EPC originated from BM [4, 18], but also modulate their differentiation. MNC differentiation into EPCs in vitro is enhanced by the presence of statins [18]. Our data are consistent with these reports and give a new insight view of the mechanism for statin-induced EPC mobilization. We found that statin-induced activation of MMP-2 and MMP-9 in EPC. The increased MMP activity could result in degradation of extracellular matrix [30]. Progenitor cells will be such mobilized into circulation when the cellular attachment is reduced within the bone marrow niches. We show that statin alone can promote EPC proliferation and migration and can inhibit cell apoptosis in vitro. The proangiogenic effects of statin are also illustrated in vivo using a murine hind-limb ischemia model. In this model, fluvastatin treatment results in more EPC in circulation (supplemental online Fig. 1), more BM derived progenitor cells in ischemic muscle (Fig. 2 and 3), more cell proliferation (Fig. 5B), enhanced capillary formation (Fig. 4), and diminished cell apoptosis (Fig. 5D); these effects end up in improved reperfusion versus control (Fig. 1). The beneficial effects of statin on angiogenesis are independent of cholesterol since the total serum cholesterol level is not changed by Fluvastatin treatment under these experimental conditions (data not shown).
The effect of statins on EPCs was found to be concentration dependent. EPC proliferation, migration, and the inhibition of apoptosis are enhanced at low statin concentrations (10 nM and 100 nM) but are significantly inhibited at a higher statin concentration (1,000 nM) (Fig. 6 and 7). The toxic effect of statin at high concentration cannot be compensated by addition of SDF-1, indicating that statin causes apoptosis in a pathway different from the pathway that SDF-1 uses to prevent EPC apoptosis. Increased apoptosis at the higher statin concentration could explain the reversed effect of stain in angiogenesis. These findings are consistent with the reports in which statins were found to have proangiogenic effects at low therapeutic concentrations but angiostatic effects at high concentrations, the latter effect being reversible by geranylgeranyl pyrophosphate [19, 20].
SDF-1 is considered to be a key regulator of hematopoietic stem cell trafficking between BM and the peripheral circulation. As shown by Yamaguchi et al. [3], the effect of SDF-1 on neovascularization appears to result primarily from its ability to enhance the recruitment and incorporation of transplanted EPCs that have been demonstrated expressing CXCR4 [3, 12, 21, 22]. We used retrovirally transduced NIH 3T3 cells to hypersecrete functional SDF-1 in order to elevate local SDF-1 concentrations in the ischemic tissue. These altered cells were previously shown to continue to secrete high amounts of SDF-1 in ischemic muscle 21 days post injection [14]. The present data show that SDF-1 treatment alone also augments EPC proliferation and migration in vitro (Fig. 6). In vivo, SDF-1 increases homing of progenitor cell to sites of ischemia, enhances capillary formation, and reduces the rate of apoptosis, all resulting in enhanced reperfusion. The magnitude of pro-angiogenesis achieved by SDF-1 alone was similar to that achieved with statin monotherapy.
Combined statin and SDF-1 treatment significantly enhanced angiogenesis versus treatment with either agent alone. More cell proliferation and less apoptosis were observed both in vitro and in vivo, along with increased cell migration and tube formation in vitro, and enhanced progenitor cell incorporation and higher capillary density in ischemic tissue in vivo. It is interesting to note that neither statin nor SDF-1 alone promote EPC tube formation, but combined treatment results in significant EPC tube formation (Fig. 6C and 6D). These results suggest that SDF-1 and statin have different mechanisms of action with regards to the promotion of neovascularization. It is possible that each drug affects a specific subset of progenitor cells, but this remains to be determined.
The facilitative effect of both statin and SDF-1 on EPC proliferation and migration is involved with Akt phosphorylation and endothelial nitric oxide synthase (NOS) activation. The mechanism by which statins promote angiogenesis is through, at least partly, improved nitric oxide bioavailability. Statins have been reported to induce eNOS mRNA stability [23] and eNOS activity through a PI3k/Akt dependent pathway [18, 24–26]. However, neither eNOS mRNA/protein expression nor EPCs are reported to be essential for the therapeutic effect of fluvastatin on hypoxia-induced pulmonary hypertension; Fluvastatin improved eNOS phosphorylation by a mechanism independent of Akt activation [27]. Our data favor a mechanism involving Akt phosphorylation since phosphorylated Akt is increased when EPCs are cultured in the presence of statin (Fig. 7A), and statin-enhanced EPC proliferation and migration were inhibited by the PI3K/Akt inhibitor LY294002 (supplemental online Fig. 4).
The angiogenic effects of SDF-1 also involve increased production of NO [28] as NO is essential for EC migration and angiogenesis [29]. SDF-1 gene transfer has been shown to enhance eNOS activity [11]. Our in vitro data confirmed the involvement of Akt and eNOS in SDF-1 mediated cell migration. Phosphorylated Akt is increased when EPCs are cultured in the presence of SDF-1 (Fig. 7A). The facilitative effect of SDF-1 on EPC migration is blocked by both the Akt inhibitor LY294002 and the NOS inhibitor L-NMMA (supplemental online Fig. 4). In contrast, L-NMMA does not reverse the inhibitory effect of SDF-1 on apoptosis, indicating that the inhibitory effect of SDF-1 on apoptosis is not mediated through NO [14].
Our data also show that the expression of MMP-2 and MMP-9 was increased when EPCs were cultured in the presence of statin or SDF-1 (Fig. 7B and supplemental online Fig. 5). MMPs are a family of proteolytic enzymes that degrade components of the extracellular matrix (ECM). Degradation of ECM is an essential step for cell mobilization and migration. Our data indicate that the novel effect of statin and SDF-1 on migration is through enhancement of MMP-2 and MMP-9 activity, resulting in ECM degradation, thus promoting progenitor cell mobilization and migration.
Both Akt phosphorylation and expression of MMP-2 and MMP-9 in EPCs are further enhanced by combined treatment with statin and SDF-1. This result indicates that treatment of EPCs with either statin or SDF-1 as monotherapy results in a sub-maximal angiogenic response. The effects of statin partially overlap with that of SDF-1, and the combined use of two factors appears to have an optimal effect on progenitor cells.
We also observed the increased levels of BMCs in the ischemic muscle of mice treated with statin and SDF-1 at day 7 (Fig. 3) but a lag in the LDPI score (Fig. 1). This is presumably because cell homing is rapid but vessel remodeling in response to engrafted progenitor cells takes more time.
In summary, the combination of progenitor cell mobilization with statin and targeted recruitment into the ischemic bed by SDF-1 leads to improved blood flow in the ischemic limb versus treatment with either agent alone. Statin and SDF-1 therefore display synergism in promoting neovascularization. This study suggests that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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