Improvement of Endothelial Function by Systemic Transfusion of Vascular Progenitor Cells
Endothelial dysfunction is characterized by abnormalities in vasoreactivity and is a marker of the extent of atherosclerosis. Cellular repair by circulating progenitor cells of ongoing vascular injury may be essential for vascular integrity and function and may limit abnormalities in vasoreactivity. Apolipoprotein E–deficient (apoE−/−) mice were splenectomized and treated with high-cholesterol diet for 5 weeks, resulting in marked impairment of endothelium-dependent vasodilation of aortic segments as compared with wild-type mice. Intravenous transfusion of 2×107 spleen-derived mononuclear cells (MNCs) isolated from wild-type mice on 3 consecutive days restored endothelium-dependent vasodilation in the apoE−/− mice, as measured 7, 14, and 45 days after transfusion. Histological analyses of aortic tissue identified fluorescent-labeled, exogenously applied progenitor cells that expressed the endothelial cell marker CD31 in the endothelial cell layer of atherosclerotic lesions. Progenitor cell treatment led to increased vascular nitric oxide synthase activity. Transfusion of either in vitro-differentiated Dil-Ac-LDL/lectin-positive endothelial progenitor cells, CD11b-positive (monocyte marker), CD45R-positive (B-cell marker), or Sca-1–positive (stem cell marker) MNC subpopulations significantly improved endothelium-dependent vasodilation, although these treatments were not as effective as transfusion of total MNCs. Depletion of MNCs of either CD11b-positive, CD45R-positive, or Sca-1–positive cells resulted in significant attenuation of endothelium-dependent vasodilation as compared with nondepleted MNCs; however, vasoreactivity was still significantly improved as compared with saline-treated apoE−/− mice. Intravenous transfusion of spleen-derived MNCs improves endothelium-dependent vasodilation in atherosclerotic apoE−/− mice, indicating an important role of circulating progenitor cells for the repair of ongoing vascular injury. More than 1 subpopulation of the MNC fraction seems to be involved in this effect.
- endothelial progenitor cells
- apolipoprotein E–deficient mice
- endothelial function
- stem cells
- nitric oxide
Endothelial dysfunction, which is manifested by abnormalities in vasoreactivity and inflammatory processes, is a marker of the extent of the atherosclerotic process and ongoing vascular injury. The presence of severely abnormal vasomotion is associated with a high incidence of cardiovascular events in patients with hypertension or coronary heart disease.1–4 On the molecular level, dysfunctional endothelium is caused by increased oxidative stress and reduced nitric oxide (NO) bioavailability.5 On the cellular level, endothelial dysfunction is associated with increased rates of endothelial cell apoptosis.6 In this context, it has been shown that levels of circulating apoptotic endothelial cells are elevated in patients with severe hypertension and acute coronary syndromes.7,8 This raises the possibility that endothelial dysfunction and atherosclerosis are, at least in part, based on an impaired regeneration of the damaged endothelium in the presence of ongoing vascular injury.
There is increasing evidence that regeneration of the heart and the vasculature is governed by circulating premature cells.9–11 One type of these cells, endothelial progenitor cells (EPCs), has been implemented in the myocardial repair after infarction and in the propagation of angiogenesis following ischemia.12–16 Furthermore, EPCs have been identified as important source of vascular repair after injury.17,18 We have recently demonstrated that EPCs can be isolated from spleen-derived mononuclear cells (MNCs) and that intravenous transfusion of spleen-derived MNCs and EPCs enhances reendothelialization and diminishes neointima formation after vascular injury.19
Therefore, we hypothesized that systemic application of exogenous vascular progenitor cells potentially enhances regeneration processes of the diseased endothelium and thereby limits abnormalities in vasoreactivity. We tested this notion in splenectomized apolipoprotein E–deficient (apoE−/−) mice fed cholesterol-rich diet, an animal model of premature atherosclerosis prone to develop endothelial dysfunction based on a severe lipid disorder.20,21
Materials and Methods
Oil red O solution, salts, and other chemicals were purchased from Sigma. L-012 was obtained from Wako Chemicals.
Male, 12-week-old apoE−/− mice and C57BL/6J (wild-type) mice (Charles River, Sulzfeld, Germany) were used for this study. The animals were maintained in a 22°C room with a 12-hour light/dark cycle and received drinking water ad libitum. All mice were fed a high-fat, cholesterol-rich diet for 5 weeks, containing 21% fat, 19.5% casein, and 1.25% cholesterol (Ssniff, Soest, Germany). Plasma lipid concentrations were determined by routine chemical methods. Low-density lipoprotein (LDL) cholesterol was determined using the Friedewald formula. The mice were killed after the indicated treatments and tissue samples and blood were collected immediately. All animal experiments were performed in accordance with institutional guidelines and the German animal protection law.
Mice were anesthetized with 150 mg/kg body weight ketamine hydrochloride (Ketanest, Pharmacia) and 0.1 mg/kg body weight xylazine hydrochloride (Rompun 2%, Bayer). The spleen was dissected through a lateral incision of the left abdomen. Vessels were carefully ligated using 6/0 silk. After removal of the spleen, the abdomen was closed layer by layer with single sutures using 6/0 silk. Animals were allowed to recover for 7 days before further treatment was performed.
Preparation of MNCs
Spleens from wild-type mice were explanted and mechanically minced, and MNCs were isolated using a Ficoll gradient (Lympholite-M, Cedarlane). MNCs were stained with PKH-26, a fluorescent dye with long aliphatic tails for incorporation in the cell membrane that permits identification of these cells and their progeny, using the PKH26-GL Red Fluorescent Cell Linker Kit (Sigma) according to the instructions of the manufacturer. Labeled cells were counted and resuspended in 500 μL of normal saline solution for intravenous injection. In a subset of experiments, the same amount of spleen-derived MNCs was subjected to magnetic bead separation. MNCs were either separated into CD11b-positive (monocyte marker) or -negative cells, CD45R-positive (B-cell marker) or -negative cells, or Sca-1–positive (stem cell marker) or –negative cells. In brief, spleen-derived MNCs were washed, resuspended, and mixed with colloidal superparamagnetic microbeads conjugated to either monoclonal rat anti-mouse CD45R (B220), Sca-1, or CD11b (Mac-1) antibodies (MACS MicroBeads, Miltenyi Biotec). After incubation and additional washing, magnetic cell separation was performed by filling the cell suspension into a depletion column placed in the magnetic field of a magnetic bead separator (MACS Depletion Columns; MidiMACS Separator, Miltenyi). The collected effluent contained the negative MNC fraction depleted of either CD45R-, Sca-1–, or CD11b-positive cells. After taking the column out of the magnetic field, the attached CD45R-, Sca-1–, or CD11b-positive MNCs were collected in buffer. The separated MNC subpopulations were stained with PKH-26, counted, and resuspended in 500 μL of normal saline solution for intravenous injection.
Preparation of Spleen-Derived EPCs
Spleen-derived MNCs were seeded on fibronectin-coated 24-well plates in 0.5 mL of endothelial basal medium (EBM) (CellSystems) supplemented with 1 μg/mL hydrocortisone, 3 μg/mL bovine brain extract, 30 μg/mL gentamicin, 50 μg/mL amphotericin B, 10 μg/mL human endothelial growth factor (hEGF), and 20% FCS. After 4 days in culture, cells were extensively washed with normal saline. Adherent cells were stained with PKH-26, counted, and resuspended in 500 μL of normal saline solution for intravenous injection. In control experiments, these cells were identified as DiI-Ac-LDL/lectin double-positive cells displaying EPC phenotype, which showed expression of Sca-1, c-kit, and vascular endothelial growth factor (VEGF) receptor-2 (flk-1) and which formed tubular-like clusters comprising spindle-shaped cells at the periphery after cultivation in the described endothelial cell selection medium for 14 days.19
At the end of the 5-week treatment period with cholesterol-rich diet, splenectomized apoE−/− mice received PKH-26–stained, spleen-derived cells by intravenous tail vein injection on 3 consecutive days. Spleen-derived MNCs (2×107 cells per day) from wild-type mice were either used directly or the same amount of spleen-derived MNCs was subjected to further processing before transfusion, resulting in 1.5×107 CD11b-negative MNCs, 5×106 CD11b-positive MNCs, 1×107 CD45R-negative MNCs, 1×107 CD45R-positive MNCs, 1.9×107 Sca-1–negative MNCs, 3×105 Sca-1–positive MNCs, and 6×105 in vitro–differentiated EPCs. In addition, 2×107 cells per day of spleen-derived MNCs from 12-week-old apoE−/− mice fed with normal chow were used. Control animals (apoE−/− and wild-type mice) received a corresponding amount of normal saline solution without cells. After cell/saline treatment, all mice were fed normal chow, until aortas were excised 7, 14, and 45 days after the last transfusion, respectively. The treatment protocol is depicted in Figure 1.
Aortic Ring Preparations and Tension Recording
Vasodilation and vasoconstriction of isolated aortic ring preparations was determined in organ baths filled with oxygenated modified Tyrode buffer (37°C), as previously described.22 Adventitial tissue was carefully removed, and 3-mm segments of the thoracic aorta were investigated. A resting tension of 10 mN was maintained throughout the experiment. Drugs were added in increasing concentrations to obtain cumulative concentration-response curves: 20 and 40 mmol/L KCl, 1 nmol/L to 10 μmol/L phenylephrine, 10 nmol/L to 100 μmol/L carbachol (assessment of endothelium-dependent vasodilation after precontraction with phenylephrine), and 1 nmol/L to 10 μmol/L nitroglycerin (assessment of endothelium-independent vasodilation after precontraction with phenylephrine). The drug concentration was increased when vasoconstriction or -relaxation was completed. Drugs were washed out before the next substance was added.
Measurement of Vascular Reactive Oxygen Species
Reactive oxygen species (ROS) release in intact aortic segments was determined by L-012 chemiluminescence, as previously described.22 L-012 is a luminol derivate with high sensitivity for ROS that does not exert redox cycling itself.23 Aortas were carefully excised and placed in chilled, modified Krebs-HEPES buffer. Connective tissue was removed and aortas were cut into 2-mm segments. Chemiluminescence of aortic segments was assessed in scintillation vials containing Krebs-HEPES buffer with 100 μmol/L L-012 over 15 minutes in a scintillation counter (Lumat LB 9501, Berthold, Bad Wildbad, Germany) in 1-minute intervals. The vessel segments were then dried and dry weight was determined. ROS release is expressed as relative chemiluminescence per milligram of aortic tissue.
NOS Activity Assay
Vascular NOS activity was quantified by measuring the conversion of [3H]arginine to [3H]citrulline in aortic homogenates using a nitric oxide synthase assay kit (Calbiochem), as previously described.24 In brief, excised aortic segments were immersed in ice-cold homogenization buffer that contained 250 mmol/L Tris/HCl (pH 7.4), 10 mmol/L EDTA, and 10 mmol/L EGTA and were mechanically homogenized. NOS activity was determined in 10 μg of protein aliquots of the aortic homogenates after the addition of [3H]arginine, NADPH, tetrahydrobiopterin, and CaCl2. Rat cerebellum extracts, containing elevated amounts of neuronal NOS, were used as positive controls, whereas aortic lysates incubated in the presence of NG-nitro-l-arginine methyl ester (L-NAME) served as blanks. The amount of [3H]citrulline was quantified with a β-counter (Beckman).
Aortic segments were embedded in Tissue Tek OCT embedding medium (Miles), snap frozen, and stored at −80°C. For immunohistochemical analysis, samples were sectioned on a Leica cryostat (7 μm) and placed on poly-l-lysine (Sigma) coated slides. Cryosections were assessed for PKH-26–positive cells and for the endothelial cell marker CD31 (platelet endothelial cell adhesion molecule-1 [PECAM-1]) (monoclonal rat anti-mouse CD31 antibody, clone MEC13.3, BD Pharmingen) with an indirect immunofluorescent method. Cryosections were postfixed in 95% ethanol for 5 minutes. Slides were preincubated with normal horse serum (Vector Laboratories) for 30 minutes each. The primary antibody was applied for 1 hour at room temperature. Slides were then incubated with a biotin-conjugated secondary antibody (goat anti-rat, BD Pharmingen) for 30 minutes, followed by application of fluorescein isothiocyanate (FITC)-labeled Streptavidin for 30 minutes. Nuclear staining was performed using 4′,6-diamidino-2-phenylindole (DAPI) (Linaris). Isotype-specific antibodies (Santa Cruz Biotechnology) were used for negative controls. Sections were washed and mounted with fluorescent mounting medium (Dako) for fluorescent microscopic analysis. For the detection of atherosclerotic lesions, aortic segments were sectioned on the cryostat and sections were fixed with 3.7% formaldehyde for 1 hour, rinsed with deionized water, stained with oil red O working solution (0.5%) for 30 minutes, and rinsed again. Hematoxylin staining was performed according to standard protocols. All sections were examined under a Nikon E600 microscope using Lucia Measurement Version 4.6 software. For quantification of atherosclerotic plaque formation in the aortic root, lipid-staining area and total area of serial histological sections were measured. Atherosclerosis data are expressed as lipid-staining area in percentage of total surface area. The investigator who performed the histological analyses was blinded to the treatment of the respective animal group.
Data are presented as mean±SEM. Statistical analysis was performed using the ANOVA test followed by the Neuman–Keuls post hoc analysis. P<0.05 indicates statistical significance.
Effect of Transfusion of Spleen-Derived MNCs on Endothelial Function in ApoE−/− Mice
Male, 12-week-old apoE−/− mice and age-matched wild-type mice were splenectomized and fed a high-fat, high-cholesterol diet for 5 weeks. Total cholesterol, HDL, and LDL cholesterol plasma concentrations were significantly elevated in apoE−/− animals compared with wild-type mice (total cholesterol, 802±55 versus 111±11 mg/dL, P<0.05, versus wild-type; HDL cholesterol, 267±29 versus 78±11 mg/dL, P<0.05, versus wild-type; LDL cholesterol, 520±58 versus 19±2 mg/dL, P<0.05, versus wild-type; triglycerides, 76±13 versus 80±8 mg/dL). Figure 2A shows that in contrast to wild-type mice, endothelium-dependent vasodilation was profoundly impaired in apoE−/− mice, indicating endothelial dysfunction, as assessed in aortic ring preparations in response to increasing concentrations of carbachol (maximal relaxation: 53.5±2.7% versus 92.2±5.7%, P<0.05, versus wild-type). Endothelium-independent vasorelaxation induced by nitroglycerin was similar between the groups (Figure 2B).
After splenectomy and treatment with high-cholesterol diet for 5 weeks, apoE−/− mice received 3 transfusions of spleen-derived MNCs from wild-type mice (2×107 cells per day) or normal saline solution without cells intravenously on 3 consecutive days. Endothelial function of aortic segments was assessed 7 days after the last transfusion. Figure 2A shows that endothelium-dependent vasodilation was completely restored 7 days after MNC transfusion (maximal relaxation: 7-day MNCs, 89.4±3.0%, P<0.05, versus apoE−/− control). There was no effect of MNC treatment on endothelium-independent vasorelaxation (Figure 2B). Transfusion of MNCs had no effect on plasma lipid concentrations (total cholesterol, 806±31 mg/dL; HDL cholesterol, 208±18 mg/dL; LDL cholesterol, 572±25 mg/dL; triglycerides, 111±28 mg/dL).
To examine the effect of cells obtained from apoE−/− animals, spleen-derived MNCs from age-matched apoE−/− mice fed normal chow (2×107 cells per day) were transfused in splenectomized apoE−/− mice treated with high-cholesterol diet for 5 weeks. As displayed in Figure 2A, transfusion of apoE−/−-derived MNCs improved endothelium-dependent vasodilation after 7 days but was less effective than transfusion of MNCs obtained from wild-type animals (maximal relaxation: 75.0±3.4%; P<0.05 versus apoE−/− control and versus wild-type MNCs). Endothelium-independent vasorelaxation was not altered (Figure 2B).
Duration of the Effect of Transfusion of Spleen-Derived MNCs on Endothelial Function
After splenectomy and treatment with high-cholesterol diet for 5 weeks, apoE−/− mice received 3 transfusions of spleen-derived MNCs from wild-type mice (2×107 cells per day) or cell-free saline intravenously on 3 consecutive days. To investigate the duration of the transfusion effect, endothelial function of aortic segments was assessed 14 and 45 days after the last transfusion, respectively. Figure 3 shows that endothelium-dependent vasodilation was still almost completely normalized 14 days after MNC transfusion (maximal relaxation: 14-day MNCs, 87.1±3.3%; P<0.05 versus apoE−/− control). Endothelium-dependent vasodilation was still significantly improved 45 days after the last transfusion of MNCs, indicating a stable effect on endothelial function, although the effect was not as pronounced as 7 and 14 days after MNC transfusion (maximal relaxation: 74.2±2.9%; P<0.05 versus apoE−/− control and versus 14-day MNCs) (Figure 3). Endothelium-independent vasorelaxation was not impaired as compared with wild-type mice and was not affected by MNC treatment after 14 and 45 days, respectively (data not shown).
Effect of Transfusion of Spleen-Derived MNC Subgroups on Endothelial Function
To investigate which MNC subtype mediates the improvement of endothelial function in apoE−/− mice, spleen-derived MNCs from wild-type animals (2×107 cells per day) were subjected to further processing before transfusion.
First, to explore the role of vascular premature cells, spleen-derived MNCs from wild-type mice (2×107 cells per day) were differentiated in vitro into EPCs. Transfusion of this cell population resulted in significant improvement of endothelium-dependent vasodilation after 7 days but was not as effective as transfusion of complete MNCs before differentiation into EPCs (maximal relaxation: 72.0±2.8%; P<0.05 versus apoE−/− control and versus undifferentiated MNCs) (Figure 4A). Endothelium-independent vasorelaxation was not altered (data not shown).
Second, CD11b-positive (monocyte marker), Sca-1–positive (stem cell marker), and CD45R-positive (B-cell marker) MNCs were obtained by magnetic bead separation. Separate transfusion of these cell preparations significantly improved endothelium-dependent vasodilation after 7 days, although these treatments were not as effective as the transfusion of the complete MNC population (maximal relaxation: saline control, 40.1±4.7%; CD11b-positive MNCs, 73.7±1.8%; CD45R-positive MNCs, 65.7±2.2%; Sca-1–positive MNCs, 56.2±2.6%; P<0.05 versus apoE−/− control and versus complete MNCs) (Figure 4A). Endothelium-independent vasorelaxation was not altered (data not shown).
Third, spleen-derived MNCs were depleted of either CD11b-positive, Sca-1–positive, or CD45R-positive cells by magnetic bead separation. Separate transfusion of these depleted MNC preparations resulted in a significantly attenuated effect on endothelium-dependent vasodilation after 7 days as compared with transfusion of nondepleted MNCs; however, endothelial function was still significantly improved as compared with saline-treated apoE−/− mice (maximal relaxation: CD11b-negative MNCs, 64.0±3.9%; CD45R-negative MNCs, 71.9±3.0%; Sca-1–negative MNCs, 68.1±2.2%; P<0.05 versus apoE−/− control and versus nondepleted MNCs) (Figure 4B). Endothelium-independent vasorelaxation was not altered (data not shown).
Histological Analysis of Aortic Tissue
Treatment of apoE−/− mice with high-fat, high-cholesterol diet for 5 weeks resulted in atherosclerotic lesion formation in the aortic root and thoracic aorta, as demonstrated by oil red O staining (Figure 5A and 5B). Before intravenous transfusion, MNCs and EPCs were stained with the fluorescent dye PKH-26, allowing identification of these cells in the vessel wall. After transfusion, PKH-26–positive cells were located mainly in aortic atherosclerotic plaque area but not in the liver or lungs. Immunohistological analysis of PKH-26–positive cells attached to the luminal side of the plaque revealed that these cells expressed CD31 (PECAM-1), indicating the expression of an endothelial cell-specific marker in the transfused cells after homing within the endothelial cell layer. In addition, these cells showed typical histoanatomical features of endothelial cells in the microscopic high-magnification analyses of 7-μm-thin cryosections. PKH-26/CD31 double-positive cells were found in the endothelial cell layer after transfusion of EPCs (Figure 5C through 5F) as well as of MNCs (Figure 5G through 5J). In addition, PKH-26/CD31 double-positive cells were also identified in the endothelial cell layer after transfusion of CD11b-positive MNCs, but not after transfusion of CD45R-positive MNCs (data not shown).
Vascular Activity of NOS
Increased NO bioavailability attributable to enhanced endothelial cell-mediated NO production after progenitor cell transfusion may account for the improvement of endothelium-dependent vasodilation. Therefore, NO synthase (NOS) activity was assessed in aortic tissue from apoE−/− mice that received cell-free saline or transfusion of wild-type MNCs or EPCs. Figure 6A demonstrates that aortic NOS activity was significantly increased 7 and 14 days after treatment with wild-type MNCs (167.6±16.1% and 200.7±28.3% of saline-treated apoE−/−, respectively; both P<0.05 versus apoE−/−). Treatment with in vitro–differentiated EPCs also resulted in significant enhancement of NOS activity, although the effect was not as pronounced as compared with wild-type MNCs (153.3±24.6% of saline-treated apoE−/−, P<0.05 versus apoE−/−).
Vascular Production of ROS
Decreased vascular oxidative stress may contribute to increased NO bioavailability. Therefore, vascular ROS production was assessed in aortic segments of apoE−/− mice after treatment with cell-free saline or progenitor cells by L-012 chemiluminescence assays. Figure 6B shows that aortic ROS production was not significantly influenced by treatment with MNCs or EPCs.
Effect of Transfusion of Spleen-Derived MNCs on Atherosclerotic Plaque Formation in ApoE−/− Mice
After splenectomy and treatment with high-cholesterol diet for 5 weeks, apoE−/− mice received 3 transfusions of spleen-derived MNCs from wild-type mice (2×107 cells per day) or cell-free saline intravenously on 3 consecutive days. The effect of MNC treatment on atherosclerotic plaque formation was investigated in the aortic root of these animals 45 days after the last transfusion. Figure 7A and 7B demonstrate that atherosclerotic lesion formation in the aortic root was not significantly altered by cell treatment as compared with saline-treated apoE−/− mice (atherosclerotic plaque area: MNCs, 35.6±1.4%; saline control, 30.5±2.2%; P=NS).
We and others have recently shown that progenitor cells derived from spleen homogenates and in vitro–differentiated EPCs drive the process of reendothelialization after local vascular injury.17,18 These findings established the notion that, rather than local endothelial cells, circulating progenitor cells play an important role in regeneration processes of the endothelium and may therefore be an important target for therapeutic intervention. Interestingly, increasing the number of circulating EPCs by statin, estrogen, or granulocyte colony–stimulating factor (G-CSF) treatment, physical exercise, or cell transfusion not only accelerated reendothelialization but also profoundly inhibited neointima formation after vascular injury.17–19,25–27Studies in patients with in-stent restenosis after percutaneous coronary intervention demonstrating reduced numbers and impaired adhesion of circulating EPCs and novel approaches with EPC-capturing coronary stents, leading to enhanced reendothelialization and diminished restenosis point toward an important role of EPCs for the inhibition of restenosis in a clinical setting.28–30 To extend these observations, it is crucial to show that application of EPCs and related cell populations also interferes with the more disseminated vascular disease of atherosclerosis as opposed to the described effects after focal vascular injury. This is of relevance because neointima formation after vascular injury only in part resembles the pathology of atherosclerosis, and surgical injury models can only be crudely correlated to the situation in humans. Therefore, additional experiments in models that display typical characteristics of atherosclerosis, such as the apoE−/− mouse,20 are warranted. These mice develop endothelial dysfunction, as characterized by abnormal vasoreactivity, reflecting the extent of atherosclerosis.
A recently published study has shown that chronic treatment of young apoE−/− mice with bone marrow–derived progenitor cells from young nonatherosclerotic apoE−/− mice or wild-type mice prevented the development of atherosclerotic lesions.31 Treatment was started at an age of 3 weeks and repeated every 2 weeks in the presence of a cholesterol-rich diet. However, this does not resemble the situation in humans, who usually present in the clinic with established forms of atherosclerosis. In contrast to the aforementioned study, increased atherosclerotic plaque size and altered plaque composition in apoE−/− mice treated intravenously with EPCs or bone marrow cells was demonstrated in one study, whereas in another study no effect on atherosclerotic plaque size and structure was demonstrated in nonischemic apoE−/− mice treated intravenously with bone marrow MNCs, but an increased lesion size was seen in the apoE−/− group with hindlimb ischemia.32,33 In the present study, we did not observe a significant effect of MNC treatment on the extent of atherosclerotic lesion formation in animals with already established atherosclerosis. However, atherosclerotic plaque area in the aortic root was increased by approximately 17% after MNC treatment as compared with saline treatment. The relevance of this nonsignificant trend in this small group of investigated animals is not clear at present. These contradictory findings may possibly result from different methodological approaches in the apoE−/− mice, and the true nature of the contribution of EPCs and MNCs to atherosclerotic plaque development remains unclear in this animal model of atherosclerosis. However, a prospective clinical study demonstrated that low numbers and impaired function of circulating EPCs were associated with increased cardiovascular mortality in patients with coronary artery disease,34 supporting a protective role of EPCs in the atherosclerotic process in humans.
In the present study, we treated 17-week-old apoE−/− mice that had already developed endothelial dysfunction and atherosclerotic plaque formation attributable to the preceding cholesterol-rich diet. Intravenous application of MNCs as well as of EPC and MNC subpopulations improved endothelium-dependent vasodilation, suggesting that this cell-based treatment can limit abnormalities in vasoreactivity associated with atherosclerotic plaque development. Of note, the improvement of endothelial function sustained for at least 45 days after cell therapy. Cell treatment with MNCs and EPCs was associated with enhanced vascular NOS activity, which finally results in increased NO production. It is conceivable that this effect is an important mechanism underlying the observed improvement of vascular function, because increased NO concentrations together with unaltered vascular ROS production result in enhanced NO bioavailability, which leads to improvement of endothelium-dependent vasodilation.5 In addition, NO was shown to differentially regulate proliferation of EPCs and to promote EPC differentiation into mature endothelial cells, which may serve as an important autocrine/paracrine effect after EPC homing.35 The results of our study are in agreement with clinical studies that showed a positive correlation between EPC counts in vitro and endothelium-dependent vasodilation of the forearm36 and demonstrated improvement of endothelium-dependent vasorelaxation of the leg after bone marrow MNC implantation in patients with limb ischemia.37
The histological analyses after cell treatment revealed that the exogenously administered MNCs and EPCs incorporated into the vessel wall predominantly in aortic atherosclerotic plaque areas. Localization of the transfused cells within the endothelial cell monolayer, presence of histoanatomical characteristics of endothelial cells, and expression of the endothelial cell marker CD31 suggest that these initially premature cells turned into mature endothelial cells by differentiation or fusion processes and subsequently contribute to improvement of endothelium-dependent vasoreactivity by direct and/or paracrine effects. The fact that endothelium-dependent vasodilation was still markedly improved 45 days after cell application demonstrates that the transfusion of progenitor cells induced a prolonged effect on endothelial function and suggests a stable homing of the cells within the vessel wall. This is an important finding contrasting the results of a recent study that demonstrated only transient engraftment (<28 days) of bone marrow–derived hematopoietic stem cells in ischemic myocardium.38
Atherosclerosis in humans is often associated with abnormalities of both endothelium-dependent and -independent vasoreactivity. In contrast to endothelium-dependent vasodilation, we found no alteration of endothelium-independent vasorelaxation in our model of apoE−/− mice. Furthermore, we observed no effect of treatment with progenitor cells on the latter parameter. Data concerning endothelium-independent vasodilation at baseline and after experimental intervention are inconsistent in hypercholesterolemic mouse models, which may be caused by differences in the specific mouse models and strains, diet, duration of treatment, and model and protocol of assessment of vascular function. In addition, there are differences in human and rodent atherosclerosis. In agreement with animal data, impairment of endothelium-independent vasorelaxation is not found in all clinical studies assessing endothelial function in human atherosclerotic subjects. A trial investigating endothelial function in patients with limb ischemia demonstrated a selective improvement of endothelium-dependent vasodilation but not of endothelium-independent vasodilation after MNC treatment.37 In a study by Hill and colleagues, a weak correlation of EPC number and endothelium-independent vasoreactivity was found; however, only subjects with a high ratio of flow-mediated to nitroglycerin-induced brachial reactivity had higher EPC counts than did subjects with a low ratio.36 However, it cannot be excluded that treatment with premature cells improves endothelium-dependent vasodilation without affecting endothelium-independent abnormalities in vasoreactivity in atherosclerotic humans.
It remains incompletely understood from the data of our study which exact cell type of the spleen-derived MNCs is responsible for the observed beneficial effect on endothelium-dependent vasoreactivity. EPCs are good candidates because these cells obviously can differentiate into mature endothelial cells. The results of our study support an important role of this cell type, because in vitro–differentiated EPCs markedly improved endothelium-dependent vasodilation. Mouse EPCs are in part characterized by the expression of the stem cell marker Sca-1. Consistently, transfusion of Sca-1–positive cells led to an improvement of endothelium-dependent vasodilation in the present study. However, transfusion of MNCs that were depleted of Sca-1–positive cells resulted in an attenuation but not complete inhibition of the beneficial effect on endothelial function, indicating that this premature cell population is not the only cell population mediating the improvement of endothelium-dependent vasoreactivity. It was recently shown that endothelial (progenitor) cells could be differentiated from monocytic cell types, and in addition, it was reported that B cells could decrease atherosclerotic disease progression in apoE−/− mice.39–42 Therefore, we also explored the effect of MNC subpopulations that were positive or negative of the CD11b (monocyte marker) or CD45R (B-cell marker) surface antigens. CD11b-positive and CD45R-positive MNCs significantly improved endothelium-dependent vasodilation, respectively, but these treatments were not as effective as transfusion of total MNCs. Moreover, depletion of total MNCs of either CD11b-positive or CD45R-positive cells resulted in a significant attenuation of endothelium-dependent vasodilation as compared with nondepleted MNCs, but this vasoreactivity was still significantly improved as compared with saline-treated apoE−/− mice. These data suggest that both CD11b- and CD45R-positive MNCs are essential for the improvement of endothelium-dependent vasoreactivity but that neither CD11b- nor CD45R-positive subpopulations are exclusively important for the observed effect on endothelium-dependent vasodilation and that other cell types, as for example EPCs or others, are also involved in the restoration of vascular function. A recent study has shown that EPCs could be derived from both CD14-positive and -negative MNCs and that EPC phenotype after differentiation was important for the functional neovascularization capacity as compared with freshly isolated, nondifferentiated CD14-positive MNCs.43 In our study, both CD11b-positive and -negative MNC subpopulations had an effect on endothelium-dependent vasoreactivity. Interestingly, we found CD31-positive, exogenously applied, PKH-26–positive cells in the endothelial layer after transfusion of CD11b-positive MNCs but not of CD45R-positive MNCs. It may be speculated that the special composition of spleen-derived MNCs, including monocytes, B cells, EPCs, and possibly other (premature) cells, allows important interactions that could lead to a more efficient homing and differentiation of these cells via autocrine and paracrine mechanisms and finally to an improved function of mature endothelial cells. The results of our study suggest that more than 1 subpopulation of the MNC fraction is needed to exert the described beneficial effect on endothelial function. On the other hand, other cell types may differentiate into endothelial (progenitor) cells, and increased transdifferentiation rates as well as enhanced fusion may account for the pronounced effects on endothelium-dependent vasoreactivity. Further studies will have to clarify the cellular and molecular features which determine the successful homing and differentiation of premature cells, to maximize the therapeutic potential of this cell-based treatment.
Application of MNCs isolated from age-matched apoE−/− mice fed normal chow was less efficient in the enhancement of endothelium-dependent vasodilation as compared with the transfusion of wild-type MNCs. Therefore, it may be assumed that the inherited lipid disorder seems to be of importance for the beneficial effects of premature cells, although the underlying mechanisms remain obscure. It is improbable that the effect relates to a potential influence of wild-type cells on lipid metabolism, because lipid levels were not affected by cell treatment and the transfused cells were almost exclusively detected within vascular lesion sites and were not found in other organs such as the liver.
The presented data reveal that severe abnormalities in endothelium-dependent vasoreactivity can be limited by progenitor cell treatment in atherosclerotic mice. Our results establish another intriguing role of progenitor cells besides myocardial repair after infarction, angiogenesis, and vascular repair after local injury. Further studies are warranted to elucidate the detailed molecular and cellular mechanisms involved in this regenerative process. It remains to be determined whether the findings of the present study can be translated to human disease. In our study, a high number of premature cells was obtained from spleens and transfused intravenously on 3 consecutive days. This approach was used to gain direct mechanistic insight into the regenerative potential of progenitor cells in disseminated atherosclerotic disease in an animal model of human disease, which is, of course, not feasible in humans. However, the results of our model show that this premature cell-based approach has a profound regenerative potential on endothelial function in atherosclerotic disease and demonstrate a rather stable effect on endothelium-dependent vasoreactivity as long as 45 days. It is not clear how long lasting the treatment effect is in ongoing disseminated vascular injury during hypercholesterolemia, whether repeated treatments could maintain the beneficial effect, and whether the effect on endothelium-dependent vasodilation may be counterbalanced by the lack of effect on endothelium-independent vasoreactivity in atherosclerotic humans. The prospective Endothelial Progenitor Cells in Coronary Artery Disease study supports the protective role of high numbers of circulating progenitor cells in the atherosclerotic process in humans.34 As a perspective, enhanced endogenous mobilization, increased homing, and improved function of vascular progenitor cells after medical treatment may be options to possibly transfer progenitor cell-based therapeutic approaches in humans to improve vascular function in organs endangered by atherosclerosis.
The excellent technical assistance of Sybille Richter, Simone Jäger, Isabel Paez-Maletz, Bianca Klöckner, Susanne Schnell, Annika Bohner, and Kathrin Paul is greatly appreciated.
Sources of Funding
This work was supported by the Deutsche Forschungsgemeinschaft and a research grant from the University of the Saarland, Homburg/Saar, Germany.
Original received June 11, 2004; resubmission received July 13, 2006; revised resubmission September 1, 2006; accepted September 8, 2006.
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