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G-CSF administration after myocardial infarction in mice attenuates late ischemic cardiomyopathy by enhanced arteriogenesis Elisabeth Deindl,*,1 Marc-Michael Zaruba*,1 Stefan Brunner,* Bruno Huber,* Ursula Mehl,* Gerald Assmann,† Imo E. Hoefer,‡ Josef Mueller-Hoecker,† and Wolfgang-Michael Franz*,2 *Ludwig-Maximilians-University, Klinikum Grosshadern, Medical Department I, Munich, Germany; † Institute of Pathology, Ludwig Maximilians University, Munich, Germany; and ‡Department of Experimental Cardiology, UMC, Utrecht, The Netherlands To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4763fje SPECIFIC AIMS Granulocyte-colony stimulating factor (G-CSF) has been shown to improve cardiac function after myocardial infarction (MI) by bone marrow cell mobilization and protecting cardiomyocytes from apoptotic cell death. However, its role in adaptive collateral artery growth (arteriogenesis) has not yet been elucidated. Therefore, we investigated the effect of G-CSF administration [G-CSF: 100 ␮g/kg/day; bromodeoxyuridine (BrdU), 50 ␮g/kg/day, daily for 5 consecutive days after MI] on arteriolar growth, cardiac function, and histopathological changes 6 and 30 days after ligation of the LAD reflecting the complete time span of post-MI remodeling in mice. PRINCIPAL FINDINGS 1. Accumulation of bone marrow derived cells in the peripheral blood After treatment of mice with G-CSF for 5 consecutive days (200 ␮g/kg/day), we found a significant 4.5-fold increase of leukocytes in heparinized blood samples at day 6. Flow cytometry analyses on mononuclear cells evidenced a significant accumulation of different subpopulations of CD45⫹/CD34⫹ as well as of CD45⫹/ CD34⫺ leukocytes in the peripheral blood: CD45⫹/ CD34⫹, 13-fold; CD45⫹/CD34⫹/CD31⫹, 9-fold; CD45⫹/CD34⫹/Sca-1⫹, 6-fold; CD45⫹/CD34⫹/c-kit⫹, 31-fold; CD45⫹/CD34⫺, 1.9-fold; CD45⫹/CD34⫺/ CD31⫹, 1.6-fold; CD45⫹/CD34⫺/Sca-1⫹, 1.7-fold; and CD45⫹/CD34⫺/c-kit⫹, 3.3-fold. 2. Increased survival 4 wk after MI Four weeks after MI, G-CSF treated mice showed a significant increase in the survival rate compared to saline-treated animals (68.8 vs. 46.2%). Mortality 956

among untreated animals was very high within the first 8 days after MI, whereas mice surviving the first 3– 8 days showed a lower mortality in both groups. 3. Beneficial hemodynamical effects reflected by PV-loops in vivo Using conductance catheters, we measured pressurevolume relations at day 6 and day 30 after the surgical procedure in vivo. Pressure-volume relations (Fig. 1) on G-CSF and saline-treated animals, respectively, showed no significant differences at day 6 but revealed an improvement in all contractile and relaxation parameters in G-CSF treated mice compared to saline-treated animals at day 30: (MLVP: 81.0⫾3.6 vs. 69.0⫾2.9 mmHg, P⬍0.01), ejection fraction (EF: 32.5⫾2.5 vs. 17.2⫾1.2%, P⬍0.001), and contractility (4735⫾413 vs. 3229⫾200 mmHg/sec, P⬍0.01) were significantly improved, and the end diastolic and end systolic volume was reduced (EDV: 30.4⫾3.8 vs. 41.0⫾2.1 ␮l, P⬍0.05). Stroke work (738⫾119⫻ vs. 291⫾37 mmHg⫻␮l, P⬍0.01) and maximum power (4.3⫾0.8 vs. 2.3⫾0.2 mW, P⬍0.05) were improved. Diastolic relaxation was also restored (Tau Glantz: 10.5⫾1.3 vs. 14.8⫾2.0 ms, ⌬p/⌬tmin: ⫺4899⫾444 vs. 3270⫾295 mmHg/sec). 4. G-CSF treatment is associated with positive histopathological effects LV-infarct size was comparable in saline and G-CSF treated mice at 6 days (37.2⫾3.3 vs. 38.6⫾4.0% of total 1 Elisabeth Deindl and Marc-Michael Zaruba contributed equally to this work. 2 Correspondence: Ludwig-Maximilians-University, Klinikum Grosshadern, Medical Department I, Marchioninistr. 15, D-81377 Munich, Germany. E-mail address: wolfgang.franz@ med.uni-muenchen.de doi: 10.1096/fj.05-4763fje

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Figure 1. Histological findings and pressurevolume relations. Representative Masson Trichrom stainings of hearts (top) in relation to in vivo measured pressure-volume relations (bottom) of same mice 30 days after MI. Sham (A), infarcted and G-CSF-treated (B), and infarcted and saline-treated mice (C). Sham-operated mice revealed normal pressure-volume relations with low end systolic and end diastolic volumes (A), whereas infarcted animals revealed low LV pressures with high filling volumes resulting in a lower ejection fraction (C), which was partially restored in G-CSF treated animals resulting in a shift to the left of the PV loops (B).

LV area, P⫽ns), however, the cellular pattern of the infarcted area (consisting of granulation- and necrotic tissue) was clearly different: G-CSF treatment was associated with less granulation tissue (67.8⫾5.8 vs. 84.4⫾3.3% of total infarct area, P⬍0.001), less prominent collagen deposition, and decreased cellular density of granulation tissue (3271⫾190 vs. 4645⫾325/ mm2, P⬍0.01). The anterior wall thickness declined over time in both groups, however, to a smaller extent in G-CSF treated mice (day 6: 0.67⫾0.9 vs. 0.42⫾0.07 mm; day 30: 0.28⫾0.05 vs. 0.13⫾0.01 mm, P⬍0.05). At day 30 G-CSF treated mice showed less prominent scar extension and lower frequency of LV-aneurisms than saline-treated mice (23.1⫾3.0 vs. 30.8⫾2.7%, P⬍0.05). Moreover, cytokine treatment revealed a preservation of myocardial thickness in the remote area. 5. G-CSF administration results in enhanced arteriogenesis At day 6 after MI the granulation tissue of G-CSF treated and saline-treated animals revealed a strong infiltration of CD45⫹ cells, mostly monocytes and granulocytes. The number of Ki67 and BrdU positive cells was not significantly different between G-CSF and saline-treated animals, either within the granulation tissue (Ki67: 56.5⫾2.0 vs. 53.5⫾10.4%, P⫽ns; BrdU: 73.7⫾1.3 vs. 64.8⫾6.8%, P⫽ns) or within the remote area (⬍1%). However, we found a significant increase of Ki67 positive endothelial cells (ECs) and smooth muscle cells SMCs) in arterioles located at the boarder zone of the MI in G-CSF treated animals compared to saline-treated mice (23.4⫾4.5 vs. 4.7⫾2.6%, P⬍0.001). Investigations on Ki67 positive arterioles revealed an increased expression of ICAM-1 that was associated with a pronounced infiltration of CD45⫹ cells. Comparable numbers of BrdU positive arteriolar ECs and SMCs were found at day 6 and day 30.

CONCLUSIONS In the present study, we examined the effect of G-CSF administration on collateral artery growth, histopathological effects, and cardiac function in a long-term follow up in mice. Recent studies have demonstrated that G-CSF prevents cardiac remodeling by protecting cardiomyocytes and ECs from apoptotic cell death. Furthermore, increased sprouting of capillaries after G-CSF treatment was found. However, even a dense network of capillaries is not able to restore the blood flow diminished by the occlusion of an artery and ischemic cardiac tissue will finally suffer necrotic cell death. Since arteriogenesis is the only way to compensate for the loss of an artery, we investigated whether G-CSF has the potential to promote collateral artery growth accounting for a beneficial long-term effect. G-CSF administration is associated with enhanced arteriogenesis after MI Arteriogenesis is mediated by growth factors and cytokines supplied by leukocytes and is strongly dependent on the concentration of leukocytes in the peripheral blood, on their infiltration in growing arteries, and on the availability of ICAM-1 mediating leukocyte adhesion. Our study showed for the first time that G-CSF increased the expression of ICAM-1 in arterioles located at the border zone of the MI in vivo. This process was associated with a pronounced accumulation of growth promoting leukocytes and an augmented proliferation of arteriolar ECs and SMCs as shown by Ki67 staining at day 6 after MI. At day 30, we found comparable numbers of BrdU positive ECs and SMCs as on day 6, indicating that the growing arterioles were true collateral arteries and did not represent vessels functioning in removal of necrotic tissue since the latter regress with scar formation. Our finding that G-CSF

ADMINISTRATION OF G-CSF AFTER MI PROMOTES ARTERIOGENESIS

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Figure 2. Proposed mechanisms of G-CSF action Upon occlusion of an artery, blood flow is redirected to preexisting arteriolar connections, thereby increasing shear stress and inducing collateral artery growth. During uninfluenced arteriogenesis, this process is not effective enough to save myocardial tissue from extensive damage. Application of G-CSF, however, promotes arteriogenesis by mobilizing leukocytes from bone marrow and by up-regulation of ICAM-1 in arteriolar connections. Upon binding to ICAM-1 and extravasation leukocytes supply growth factors and cytokines to collateral arteries entailing an augmented proliferation of arteriolar cells and therefore collateral artery growth. This effect is associated with an improved blood supply in ischemic region showing beneficial effects on development of infarct size and consequently myocardial function.

promoted arteriogenesis is corroborated by several aspects: 1) own results on a hind-limb model of arteriogenesis showed enhanced arteriogenesis after G-CSF treatment. 2) A previous study on baboons showed an improved perfusion of the periinfarct region after G-CSF treatment. All previous studies on G-CSF administration after MI in C57BL/6 mice showed beneficial effects, whereas a study on Balb/C mice did not. However, in contrast to C57BL/6 mice Balb/C mice show only a minor arteriogenic response on artery ligation. 3) G-CSF has been shown to repair injured arteries, indicating that G-CSF has the capacity to remodel arteries in a positive manner. 4) Another closely related cytokine, GM-CSF, has been reported to stimulate arteriogenesis in experimental and in clinical studies. G-CSF treatment after MI improves long-term survival and partially restores myocardial function Our results showed beneficial effects of G-CSF treatment after MI on survival and cardiac function in a follow up of 4 wk extending previous data. The beneficial outcome on survival and cardiac function was related to 1) a reduced decline of LV wall at day 6 and

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day 30, 2) a reduced infarct size at day 30, and 3) a reduced number of animals developing ischemic related ventricular wall expansion. According to the modified law of Laplace, the reduced decline of LV wall thickness prevents high LV wall tension attenuating ventricular expansion and restoring ejection fraction. The mechanistic background of our findings are 1) a better perfusion of the peri-infarct region mediated by an improved growth of collateral vessels; 2) a reduced number of apoptotic ECs, and cardiomyocytes in the infarct and peri-infarct area as described previously by others; and 3) a moderately mediated remodeling process reflected by a lower degree of granulation tissue and fibrosis. It has previously been shown that granulocytes and monocytes release metalloproteinase on G-CSF stimulation. Furthermore, it increased the activity of MMP-1 and ⫺9, which was associated with a reduced area of fibrotic tissue and collagen. Theses results are likely to apply also for our study. In summary, our results show that G-CSF administration after MI enhances arteriogenesis by increasing the availability of ICAM-1 mediating leukocyte adhesion (Fig. 2). Furthermore, it improves myocardial function and reduces mortality after MI in mice.

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G-CSF administration after myocardial infarction in mice attenuates late ischemic cardiomyopathy by enhanced arteriogenesis Elisabeth Deindl,*,1 Marc-Michael Zaruba,*,1 Stefan Brunner,* Bruno Huber,* Ursula Mehl,* Gerald Assmann,† Imo E. Hoefer,‡ Josef Mueller-Hoecker,† and Wolfgang-Michael Franz,*,2 *Ludwig Maximilians University, Klinikum Grosshadern, Medical Department I, Munich, Germany; † Institute of Pathology, Ludwig Maximilians University, Munich, Germany; and ‡Department of Experimental Cardiology, UMC, Utrecht, The Netherlands Granulocyte-colony stimulating factor (G-CSF) has been shown to improve cardiac function after myocardial infarction (MI) by bone marrow cell mobilization and by protecting cardiomyocytes from apoptotic cell death. However, its role in collateral artery growth (arteriogenesis) has not been elucidated. Here, we investigated the effect of G-CSF on arteriolar growth and cardiac function in a murine MI model. Mice were treated with G-CSF (100 ␮g/kg/day) directly after MI for 5 consecutive days. G-CSF application resulted in a significant increase of circulating mononuclear cells expressing stem cell markers. Arterioles in the border zone of infarcted myocardium showed an increased expression of ICAM-1 accompanied by an accumulation of bone marrow derived cells and a pronounced proliferation of endothelial and smooth muscle cells. Histology of G-CSF treated mice revealed a lower amount of granulation tissue (67.8 vs. 84.4%) associated with a subsequent reduction in free LV wall thinning and scar extension (23.1 vs. 30.8% of LV). Furthermore, G-CSF treated animals showed a significant improvement of post-MI survival (68.8 vs. 46.2%). Pressure-volume relations revealed a partially restored myocardial function at day 30 (EF: 32.5 vs. 17.2%). Our results demonstrate that G-CSF administration after MI stimulates arteriogenesis and attenuates ischemic cardiomyopathy after MI.—Deindl, E., Zaruba, M.-M., Brunner, S., Huber, B., Mehl, U., Assmann, G., Hoefer, I. E., Mueller-Hoecker, J., Franz, W.-M. G-CSF administration after myocardial infarction (MI) in mice attenuates late ischemic cardiomyopathy by enhanced arteriogenesis. FASEB J. 20, E27–E36 (2006)

ABSTRACT

Key Words: granulocyte-colony stimulating factor 䡠 bone marrow derived cells 䡠 intercellular adhesion molecule-1

regional systolic and diastolic dysfunction (1). Although new medical therapies achieved a significant reduction in mortality and progress to chronic heart failure, once lost, functional cardiomyocytes cannot be replaced (2, 3). Remodeling caused by MI is a common cause of ventricular dilation and heart failure (4). In the course of remodeling, necrotic cardiomyocytes are lost and replaced by fibrous tissue. Simultaneously, neovascularization in the border zone of the infarcted area takes place. The latter is required for the survival of surrounding cardiomyocytes and is meant to prevent further loss of cardiomyocytes caused by apoptosis (5). Finally, fibrous scar tissue that is noncontractile and may expand causes further cardiac impairment and heart failure as well as electrophysiological instability (4). Angioplasty and thrombolytic agents can relieve the cause of infarction, but the time from onset of occlusion to reperfusion determines the degree of irreversible myocardial loss (6). Recently, promising data on animal models as well as clinical studies have shown that transplantation of bone marrow derived cells show positive effects on cardiac function after MI (6 –10). However, direct administration of stem cells to the infarcted myocardium is hampered by the invasive approach of administration including general anesthesia needed to obtain bone marrow cells, as well as by the limited number of cells that can be applied during single catheter-based delivery. Cytokine-induced mobilization of stem cells is an elegant alternative. Previous studies have evidenced that administration of G-CSF and stem cell factor (SCF) after MI reduces myocardial damage and mortality (6, 11, 12). Furthermore, first clinical studies have shown a 1

These authors contributed equally to this work. Correspondence: Ludwig-Maximilians University, Klinikum Grosshadern, Medical Department I, Marchioninistr. 15, D-81377 Munich, Germany, Tel.: ⫹⫹49 – 89-7095–3094, Fax: ⫹⫹49 – 89-7095– 6094, E-mail address: wolfgang.franz@ med.uni-muenchen.de doi: 10.1096/fj.05-4763fje 2

Myocardial infarction (MI) leads to a decrease in cardiomyocyte metabolism within a few minutes after occlusion of a main coronary artery, resulting in an irreversible injury of functional myocardial tissue with 0892-6638/06/0020-0027 © FASEB

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beneficial role of G-CSF treatment on post-MI function (13, 14). Therefore, we investigated the role of G-CSF application after MI in mice. We aimed to define the impact of G-CSF on vessel growth and post-MI survival as well as functional parameters of infarcted myocardium 6 and 30 days after the surgical procedure, time points that reflect the early and late phase of post-MI remodeling in mice (15). Myocardial function was assessed by in vivo pressure-volume relation measurements (16, 17).

MATERIALS AND METHODS Animal model MI was induced in male C57BL/6 mice 8 –12 wk of age by surgical occlusion of the left anterior descending artery (LAD) through a left anterolateral approach. Mice were anesthetized by intraperitoneal (ip) injection of a mixture of 100 mg/kg ketamine (Sigma Chemical Co., St. Louis, MO) and 5 mg/kg Xylazine (Sigma), intubated, and artificially ventilated by a mouse ventilator (HUGO SACHS, March, Germany) with 200 strokes/min and 200 ␮l/stroke. Animal care and all experimental procedures were performed in strict accordance to the German and National Institutes of Health animal legislation guidelines and were approved by the local animal care and use committees. Administration of G-CSF and bromodeoxyuridine After induction of MI, mice were divided into the following groups: 1) subcutaneous (sc) administration of saline daily for 5 days, killed on day 6 (n⫽10) and day 30 (n⫽26); 2) administration of G-CSF daily for 5 consecutive days (100 ␮g/kg/day sc, Amgen Biologicals) and killed on day 6 (n⫽10) and day 30 (n⫽30); and 3) sham-operated animals killed on day 6 (n⫽3) and day 30 (n⫽5) and not operated control animals receiving saline (n⫽6) killed on day 6 and at day 30. All animals received bromodeoxyuridine (BrdU; 50 ␮g/kg/day for 5 consecutive days). BrdU and cytokine treatment was started 30 min after ligation of the LAD. Flow cytometry analyses Eight- to twelve-week-old C57BL/6 mice (n⫽5) were either treated with G-CSF (200 ␮g/kg/day) or saline daily for 5 consecutive days. At day 6, 1 ml of peripheral blood was harvested from each mouse by aspirating the carotid artery. To define the number of leukocytes, heparinized blood samples were analyzed using a conventional hematological cell analyzer (Sysmex XE 2100). Mononuclear cells were separated by density-gradient centrifugation using 1.077 g/ml Histopaque solution (Sigma Chemicals), purified, and resuspended in PBS containing 1% BSA. Cells were incubated for 40 min in the dark at 4°C with the following fluoresceinisothiocyanate (FITC), phycoerythrin (PE), and peridininchlorophyll-protein (PerCP) conjugated monoclonal antibodies: CD45-PerCP, CD34-FITC, CD31-PE, Sca-1-PE, and c-kit-PE (all from BD Pharmingen). Matching isotype antibodies (BD Pharmingen) served as controls. Cells were analyzed by three-color flow cytometry using a Coulter® Epics® XL-MCLTM flow cytometer (Beckman Coulter). Each analysis included 20000 events. E28

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Histology and immunohistochemical analyses At day 6 (n⫽6; for sham-operated animals: n⫽3) and day 30 (n⫽10), hearts were excised. After fixation in 4% phosphate buffered formalin, the hearts were cut transversally into 2 mm thick slices, processed, and embedded in paraffin by standard methods; 4 ␮m thick sections were cut and mounted on positively charged glass slides. Standard histological procedures (hematoxylin/eosin and Masson trichrome) and immunostaining (see below) were performed. Infarct size was determined as area of infarction (AI) correlated to the area of the left ventricle (including LVseptum) in four different slices from the base to the apex of a heart. Total infarct size was calculated by multiplication of the mean percent value of the circular infarct area with the quotient: vertical extension of the infarct area/total ventricular extension. Wall thickness was measured by taking the average length of five segments along radii from the center of the left ventricle through the thinnest points of the free LV wall and the septal wall. For immunostaining, mounted tissue sections were deparaffinized by rinsing 3 ⫻ 5 min in xylene followed by 2 ⫻ 5 min 100%, 2 ⫻ 5 min 96%, and 2 ⫻ 5 min 70% ethanol rinses. Endogenous peroxidases were quenched in 7.5% H202 in distilled water for 10 min. After being rinsed in distilled water for 10 min and 2 ⫻ 5 min in TRIS-buffer, pH 7.5, the slides were incubated at room temperature for 60 min with the following primary antibodies: CD45 (rat antimouse, BD Pharmingen), ICAM-1 (goat anti-mouse, R&D), CD34 (rat anti-mouse, Linaris), Ki67 (goat anti-mouse, Santa Cruz), or BrdU (mouse monoclonal anti-BrdU, BD Pharmingen). Pretreatment was performed for 30 min (microwave 750 W) using TRS 6 (Dako) for CD45, Glykol (biologo) for CD34, Retrievagen A (BD Pharmingen) for BrdU, and citrate buffer (10 mM, pH 6.0) for Ki67. For detection of the immunoreaction avidin-biotinylated enzyme complex-Rat IgG, avidin-biotinylated enzyme complex-goat IgG (both from Vector), or rabbit anti-goat IgG (DAKO) was used. Aminoethylcarbazol was used as chromogen (incubation 10 min). Thereafter, the slides were rinsed in running water and counterstained with hematoxylin Gill’s sedimentary coeffcient formula (Vector). Cover slides were mounted with Kaiser sedimentary coefficient glycerol gelatin. Double staining was performed for CD31 and Ki67 using an avidin-biotinylated enzyme complex-goat IgG detection system and diaminobenzidine as chromogen and the APAAP-Rat system and chromogen red (all from Dako), respectively. Quantitative assessments were as follows: 1) granulation tissue: the number of BrdU and Ki76 positive nuclei was related to the total number of nuclei quantified in the granulation tissue; 2) arterioles: only arterioles with high proliferative activity were enclosed. Functional parameters For evaluation of pressure-volume relationships in vivo, surviving mice of the groups 1) MI day 6 (n⫽4) and day 30 (n⫽10); 2) MI⫹GCSF day 6 (n⫽4) and day 30 (n⫽10); and 3) sham day 30 (n⫽4), and control (n⫽6) animals were anesthetized with thiopental (100 mg/kg ip), intubated, and artificially ventilated by a mouse ventilator (HUGO SACHS). The left ventricle was catheterized via the right carotid artery using an impedance-micromanometer catheter (Millar Instruments, Houston, TX). In brief, the method is based on measuring the time-varying electrical conductance signal of two segments of blood in the left ventricle from which total volume is calculated. Raw conductance volumes were corrected for parallel conductance by the hypertonic saline

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TABLE 1. Mobilization of bone marrow derived cells

CD45⫹/CD34⫹ (%) CD45⫹/CD34⫹/CD31⫹ (%) CD45⫹/CD34⫹/Sca-1⫹ (%) CD45⫹/CD34⫹/c-kit⫹ (%) CD45⫹/CD34⫺/(%) CD45⫹/CD34⫺/CD31⫹ (%) CD45⫹/CD34⫺/Sca-1⫹ (%) CD45⫹/CD34⫺/c-kit⫹ (%)

Saline

G-CSF

Fold Increase

0.041 ⫾ 0.015 0.023 ⫾ 0.008 0.024 ⫾ 0.017 0.005 ⫾ 0.003 40.6 ⫾ 5.9 36.5 ⫾ 5.5 27.8 ⫾ 4.0 1.1 ⫾ 0.2

0.538 ⫾ 0.108** 0.211 ⫾ 0.051* 0.143 ⫾ 0.021** 0.169 ⫾ 0.018*** 77.5 ⫾ 5.4** 58.6 ⫾ 3.3* 46.4 ⫾ 2.8* 3.6 ⫾ 0.5***

13-fold 9-fold 6-fold 31-fold 1.9-fold 1.6-fold 1.7-fold 3.3-fold

Flow cytometry results on mononuclear cells in the peripheral blood after 5 days of G-CSF administration or saline treatment. *P ⬍ 0.15, **P ⬍ 0.005, ***P ⬍ 0.001. Values are mean ⫾ se.

dilution method. For absolute volume measurements, the catheter was calibrated with known volumes of heparin treated mouse blood. Pressure-volume signals were recorded at steady state and during transient preload reduction achieved by vena cava occlusion to obtain values independent of cardiac afterload (17). Data analyses were performed as described previously (16) using PVAN analysis software (HUGO SACHS). Statistical analyses Results are mean ⫾ sem. For statistical analyses, the unpaired Student’s t test was used. Mortality was analyzed by means of the Kaplan-Meier-method. Animals dying within the first 24 h after surgery were not included in the statistical analyses to exclude the influence of perioperative traumas. Data were considered statistically significant at a value of P ⱕ 0.05.

RESULTS Mobilization of bone marrow derived cells by G-CSF treatment Heparinized blood samples were analyzed for the number of leukocytes (CD45 positive cells) at day 6. Results showed a 4.5-fold increase in G-CSF treated animals compared to the saline-treated group (16.1⫾1.9⫻ 103/␮l vs. 3.7⫾0.4⫻103/␮l, P⬍0.001). Investigating CD45 positive cells via flow cytometry, we found a significant increase of different subtypes of CD34 positive as well as of CD34 negative bone marrow derived cells after G-CSF treatment: CD45⫹/CD34⫹, 13-fold; CD45⫹/CD34⫹/CD31⫹, 9-fold; CD45⫹/CD34⫹/Sca-1⫹, 6-fold; CD45⫹/CD34⫹/c-kit⫹, 31-fold; CD45⫹/CD34⫺, 1.9-fold; CD45⫹/CD34⫺/CD31⫹, 1.6-fold; CD45⫹/ CD34⫺/Sca-1⫹, 1.7-fold; and CD45⫹/CD34⫺/c-kit⫹, 3.3fold (Table 1).

Systolic and diastolic function measured in a longterm follow up Using conductance catheters, we measured pressurevolume relations from baseline, G-CSF treated as well as saline-treated mice at day 6 and day 30 after the surgical procedure in vivo (Table 2, Fig. 2). Compared to baseline, saline-treated mice as well as G-CSF treated animals bearing MI showed a significantly decreased systolic and diastolic function at day 6. There were no statistical significant differences between the G-CSF and the saline-treated group. However, compared to day 6, at day 30 pressurevolume relations revealed a partially restored systolic and diastolic cardiac function in G-CSF treated animals but not in the saline-treated mice. Moreover, our results showed an improvement in all contractile and relaxation parameters in G-CSF treated mice compared to saline-treated animals at day 30: LV pressures (MLVP: 81.0⫾3.6 vs. 69.0⫾2.9 mmHg, P⬍0.01), ejection fraction (EF: 32.5⫾2.5 vs. 17.2⫾1.2%, P⬍0.001), stroke work (738⫾119 mmHg⫻␮l vs. 291⫾37 mmHg⫻ ␮l, P⬍0.01) and contractility (4735⫾413 mmHg/sec vs. 3229⫾200 mmHg/sec, P⬍0.01) were significantly improved. Moreover, diastolic relaxation was also restored

Survival 4 wk after MI Four weeks after MI, G-CSF treated mice showed a significant increase in the survival rate compared to salinetreated animals (68.8 vs. 46.2%). Mortality among untreated animals was very high within the first 8 days after MI, whereas mice surviving the first 3– 8 day after MI showed a lower mortality in both groups (Fig. 1). G-CSF APPLICATION AFTER MI PROMOTES ARTERIOGENESIS

Figure 1. Cumulative survival. Cumulative survival of mice after MI either treated with G-CSF (n⫽32) or saline-treated (n⫽26) was calculated using the Kaplan-Meier method. E29

TABLE 2. Hemodynamical data Parameters

Baseline

HR (bpm) Pmax (mmHg) EDV (␮l) Contractile parameters EF (%) SV (␮l) dP/dt max (mmHg/sec) SW (mmHg x ␮l) Relaxation parameters Tau Glantz (msec) dP/dt min (mmHg/sec) Maximal power (mW)

MI ⫹ Saline, d6

MI ⫹ G-CSF, d6

MI ⫹ Saline, d30

MI ⫹ G-CSF, d30

(n⫽12) 477 ⫾ 13 90.0 ⫾ 3.4 23.0 ⫾ 2.5

(n⫽4) 425 ⫾ 10 58.0 ⫾ 3.5 57.8 ⫾ 8.9

(n⫽4) 405 ⫾ 24 51.0 ⫾ 1.6 37.5 ⫾ 7.5

(n⫽10) 430 ⫾ 20 69.0 ⫾ 2.9* 41.0 ⫾ 2.1

(n ⫽ 10) 440 ⫾ 20 81.0 ⫾ 3.6†* 30.4 ⫾ 3.8†

62.0 ⫾ 2.5 16.7 ⫾ 2.3 6536 ⫾ 376 1100 ⫾ 194

15.4 ⫾ 1.4 8.8 ⫾ 1.4 2953 ⫾ 291 277 ⫾ 30

15.3 ⫾ 6.5 6.4 ⫾ 3.2 1818 ⫾ 419 235 ⫾ 128

17.2 ⫾ 1.2 6.7 ⫾ 0.6 3229 ⫾ 200 291 ⫾ 37

32.5 ⫾ 2.5†* 11.8 ⫾ 1.4†* 4735 ⫾ 413†* 738 ⫾ 119†*

9.4 ⫾ 0.5 ⫺6513 ⫾ 299 7.0 ⫾ 1.2

12.0 ⫾ 0.3 ⫺2953 ⫾ 291 2.3 ⫾ 0.3

13.8 ⫾ 0.8 ⫺2534 ⫾ 253 1.6 ⫾ 0.4

14.8 ⫾ 2.0 ⫺3270 ⫾ 295 2.3 ⫾ 0.2

10.5 ⫾ 1.3†* ⫺4899 ⫾ 444†* 4.3 ⫾ 0.8†*

Pressure volume relations were gained from control and sham operated mice (baseline), from G-CSF treated as well as saline treated mice at day 6 and day 30 after MI in vivo using Millar-tip conductance catheters. HR: heart rate; Pmax: maximal pressure of LV; EDV: enddiastolic volume; EF: ejection fraction; SV: stroke volume; SW: stroke work (area enclosed by pressure loops PxV); Tau-Glantz: regression function of dp/dt vs. pressure; maximal power: maximal value of power during cardiac cycle (power⫽stroke work/time). M I ⫹ saline d6 vs. MI ⫹ saline d30 and MI ⫹ G-CSF d6 vs. MI ⫹ G-CSF d30; *P ⬍ 0.05. MI ⫹ saline d6 vs. MI ⫹ G-CSF d6 and MI ⫹ saline d30 vs. MI ⫹ G-CSF d30; †P ⬍ 0.05. Values are mean ⫾ sem.

reflected by an accelerated diastolic relaxation (Tau Glantz: 10.5⫾1.3 vs. 14.8⫾2.0 ms) and an improved contractility index (⌬p/⌬tmin: ⫺4899⫾444 vs. 3270⫾295 mmHg/sec). Furthermore, end diastolic volumes were significantly reduced in cytokine treated animals (Table 2). Histopathological effects Histopathological analyses of tissue samples at day 30 revealed a transmural MI with pronounced wall thinning and apical aneurysms in infarcted animals, whereas G-CSF treatment was associated with a lower frequency of large LV-aneurysms (Fig. 3). LV-infarct size was comparable in both groups at 6 days (37.2⫾3.3 vs. 38.6⫾4.0% of total LV area, P⫽ns); however, the

cellular pattern of the infarcted area (consisting of granulation and necrotic tissue) was clearly different: G-CSF treatment was associated with less granulation tissue (67.8⫾5.8 vs. 84.4⫾3.3% of total infarct area, P⬍0.001) and less prominent collagen deposition (Table 3 and Fig. 3). Moreover, cellular density of granulation tissue was significantly decreased (3271⫾190 vs. 4645⫾325/mm2, P⬍0.01) in G-CSF treated mice (Fig. 4). The anterior wall thickness declined over time in both groups, however, to a smaller extent in G-CSF treated mice (day 6: 0.67⫾0.9 mm vs. 0.42⫾0.07 mm; day 30: 0.28⫾0.05 mm vs. 0.13⫾0.01 mm, P⬍0.05; Table 3 and Fig. 3). At day 30, G-CSF treated mice showed a less prominent scar extension than salinetreated mice (23.1⫾3.0 vs. 30.8⫾2.7%, P⬍0.05). Furthermore, cytokine treatment resulted in a preservation of myocardial thickness in the remote area.

Figure 2. Histological findings and pressurevolume relations in mice. Representative Masson Trichrom stainings of infarcted or shamoperated hearts (top) in relation to in vivo measured pressure-volume relations (bottom) of same mice 30 days after MI. Sham-operated mice (A), infarcted and G-CSF treated mice (B), and infarcted and saline-treated mice (C). Sham-operated mice revealed normal pressurevolume relations with low end systolic and end diastolic volumes (A), whereas infarcted animals revealed low LV pressures with high filling volumes resulting in a lower ejection fraction (C), which was partially restored in G-CSF treated animals resulting in a shift to left of PV loops (B).

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Figure 3. Infarct size and anterior wall thickness Bar graphs representing: A) the decline of left anterior free wall; B) infarct size in salinetreated infarcted animals (striped bars) and G-CSF treated infarcted animals (white bars) at day 6 (n⫽10) and day 30 (n⫽10) after MI; C) Representative Massons Trichrome stainings of saline-treated (top) and G-CSF treated mice (bottom) at day 6 (left) and day 30 (right) after MI. *P ⬍ 0.05 saline vs. G-CSF; n.s. ⫽ not significant.

Immunohistochemical analyses At day 6 after MI, the granulation tissue of G-CSF treated as well as saline-treated animals revealed a strong infiltration of CD45 positive cells (Fig. 4), mostly monocytes and granulocytes. The number of Ki67 and BrdU positive cells was not significantly different between G-CSF and saline-treated animals, either within the granulation tissue (Ki67: 56.5⫾2.0 vs. 53.5⫾10.4%, P⫽ns; BrdU: 73.7⫾1.3 vs. 64.8⫾6.8%, P⫽ns; Fig. 4) or within the remote area (⬍1%). However, we found high numbers of Ki67 and BrdU positive endothelial and smooth muscle cells that were associated with CD31 positive arterioles in G-CSF treated animals compared to saline-treated animals (23.4⫾4.5 vs. 4.7⫾2.6%, P⬍0.001) as shown in Fig. 5 (data shown for Ki67/ CD31 double staining). These vessels were characterized by a layer of smooth muscle cells and were mainly located in the border zone of the infarct. Further investigations on Ki67 positive arterioles revealed an increased expression of ICAM-1, which was associated with a pronounced infiltration of CD45 positive cells (Fig. 5). CD34 staining was observed on endothelial cells of capillaries and veins and the adventitia of arteries and further stromal cells but not on endothelial cells of arterioles or infiltrating cells. Furthermore,

there was no obvious difference either in strength of staining or in cell types staining positive for CD34 between G-CSF treated and saline-treated mice (data not shown). The number of BrdU positive cells in arterioles of G-CSF treated animals at day 30 (22.6⫾4.2%) was similar as on day 6. Furthermore, at day 30 some of the longitudinal cut arterioles showed cork-screw formation typical for growing collateral arteries (Fig. 6).

DISCUSSION In this study, we examined the effect of a clinically based time set of G-CSF administration on vessel growth, survival, cardiac function, and histopathological changes at day 6 and day 30 after LAD ligation reflecting the complete time span of post-MI remodeling in mice. Our main findings were as follows: 1) a beneficial effect of G-CSF treatment after MI on arteriogenesis that was related to 2) an increased infiltration of CD45 positive blood cells mediated by an up-regulation of ICAM-1 in the luminal vessel wall of arterioles, 3) a reduced scar extension and an attenuation of late ischemic cardiomyopathy reflected by 4) an improved survival of mice in a long-term follow up of

TABLE 3. Histological analyses Parameter

Thickness anterior LV wall (mm) Thickness septal wall (mm) Infarct size (%LV) Granulation tissue (%infarct)

MI ⫹ Saline, d6

G-CSF ⫹ MI, d6

MI ⫹ Saline, d30

G-CSF ⫹ MI, d30

0.42 ⫾ 0.07 1.06 ⫾ 0.09 38.6 ⫾ 4.00 84.4 ⫾ 3.30

0.67 ⫾ 0.09* 0.80 ⫾ 0.09 37.2 ⫾ 3.30 67.8 ⫾ 5.80*

0.13 ⫾ 0.01† 1.03 ⫾ 0.20 30.8 ⫾ 2.70† —

0.28 ⫾ 0.05*† 1.16 ⫾ 0.12 23.1 ⫾ 3.00† —

Histological analysis of infarct size (n⫽10), LV anterior and septal wall thickness (n⫽10) as well as percentage of granulation tissue within the area of infarction (n⫽6) at 6 days and 30 days after MI. MI ⫹ saline d6 vs. MI ⫹ G-CSF d6 and MI ⫹ saline d30 vs. MI ⫹ G-CSF d30; *P ⬍ 0.05. MI ⫹ saline d6 vs. MI ⫹ saline d30 and MI ⫹ G-CSF d6 vs. MI ⫹ G-CSF d30; †P ⬍ 0.05. Values are mean ⫾ se.

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Figure 4. Cell proliferation and remodeling within the area of infarction. A) Bar graphs representing amount of BrdU and Ki67 and positive cells, respectively, within granulation tissue of myocardial infarctions of saline-treated animals (striped bars), of G-CSF treated animals (white bars), and of the remote areas (dotted bars) at day 6 after ligation of the LAD. B) Granulation tissue: representative nuclear BrdU staining (brown) of saline-treated (right) and G-CSF treated mice (left) at day 6 after MI. C) Representative immunohistochemical staining showing CD45 positive cells (brown) in the granulation tissue of a mouse at day 6 d MI. D) Bar graphs representing the number of cells within the granulation tissue of saline-treated (striped bars), and G-CSF treated mice (white bars) and the remote area (dotted bars), at day 6 after the surgical procedure. *P ⬍ 0.05 saline vs. G-CSF, n.s. ⫽ not significant.

Figure 5. Immunohistochemical analyses of arterioles at day 6. Left, A–C) Immunohistochemical analyses of arterioles of G-CSF treated mice 6 d after MI. Right, A–C): stainings of arteries of saline-treated mice 6 d after MI. A) Ki/67/ CD31 double staining: endothelial cells are stained in brown, the nuclei of Ki67 positive cells in violet. Arrows point to nuclei of proliferating endothelial- and smooth muscle cells in a growing arteriole; B) ICAM-1 positive cells; and C) CD45 positive cells are stained in brown. In all sections, nuclei (blue) were counterstained with hematoxylin.

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Figure 6. Immunohistochemical analyses of arterioles at day 30. BrdU staining of transversal (A) and longitudinal (B, C) cut arterioles of G-CSF treated mice at day 30 after LAD ligation. Nuclei of BrdU positive endothelial and smooth muscle cells are stained in brown. A longitudinally cut arteriole showing cork-screw formation is visible along red arrow (C). In all sections, nuclei (blue) were counterstained with hematoxylin.

30 day, and 5) an improvement of myocardial function confirmed by in vivo measured pressure-volume relations up to 30 days after MI. G-CSF is a powerful cytokine mobilizing stem cells from bone marrow to the peripheral blood (18) that has been used in several experimental as well as clinical studies to promote cardiac function after MI (11– 14,19 –22). No major adverse effects of G-CSF have been observed in patients with acute MI when treated after revascularization (13, 14, 21, 22). However, when treatment was started before revascularization, a high rate of instent restenosis was found (19), indicating that G-CSF APPLICATION AFTER MI PROMOTES ARTERIOGENESIS

G-CSF may be involved in vessel remodeling. In patients with therapy refractory coronary artery disease administration of G-CSF without medication of clopidogrel was described to be associated with an increased rate of MI and death (20). For GM-CSF, another cytokine used to treat patients with coronary artery disease, acute coronary syndromes that were supposed to be due to atherosclerotic plaque progression have been observed (23). It has previously been reported that G-CSF mobilized bone marrow derived stem cells induce neovascularization (CD34 and CD31 positive cells) (7) and differentiate into endothelial cells and cardiomyocytes (Sca-1 and c-kit positive cells) (6, 11) after MI. However, others were not able to confirm the transdifferentiation (24, 25). Therefore, the mechanism, by which G-CSF improves cardiac function after MI is still unclear. Recently, studies by the group of Komura et al. (12, 26) have demonstrated that G-CSF prevents cardiac remodeling by protecting cardiomyocytes and endothelial cells from apoptotic cell death through activation of the Akt/Stat pathway showing for the first time an intrinsic and not a stem cell dependent effect of G-CSF. Furthermore, they found an increased sprouting of capillaries after G-CSF treatment. The prevention of apoptosis and induction of angiogenesis, although having undoubtedly beneficial effects in a short-term manner, cannot account for long-term effects. Capillaries are designed to supply oxygen and metabolites locally but are unfit to restore blood flow diminished by the occlusion of a larger artery. Therefore, without adequate perfusion restoration, affected cardiomyocytes will finally suffer necrotic cell death. Since muscular arteries are the only type of vessels showing the capacity to transport sufficient blood from normoxic to ischemic regions, we hypothesized that G-CSF promotes collateral artery growth (arteriogenesis) and accounts for the beneficial long-term effect of the cytokine administration. To investigate the role of G-CSF for arteriogenesis with related effects on cardiac function and survival, we induced MI in mice via ligation of the LAD and applied human recombinant G-CSF 30 min after MI daily for 5 consecutive days. At days 6 and 30 after MI, we performed immunohistochemical as well as functional analyses. G-CSF administration is associated with enhanced arteriogenesis after MI In our study, bone marrow derived cell mobilization via application of G-CSF resulted in a massive accumulation of CD34 positive as well as CD34 negative cells expressing the common leukocyte antigen CD45 in the peripheral blood at day 6. Immunohistochemical analyses on infarcted myocardium at day 6 revealed a strong infiltration of CD45 positive cells in the granulation tissue. Most of these cells were identified as macrophages and granulocytes. At the same time, we found a pronounced accumulation of CD45 positive cells in arteries of G-CSF treated mice that was not observed in E33

untreated mice at a comparable level. These arteries were mainly located at the border zone of the MI and showed a significant proliferation of endothelial and smooth muscle cells as shown by Ki67 and BrdU staining. Furthermore, these arterioles showed an increased expression of ICAM-1. The latter findings are corroborated by in vitro data from Fuste et al. (27). Besides cardiomyocytes, endothelial cells are the only known cells of the heart expressing the G-CSF-receptor (26, 28). Fuste et al. (27) showed that stimulation of endothelial cells with G-CSF in vitro results in an increased expression of adhesion molecules like ICAM-1 through activation of p38 MAPK. Arteriogenesis is mediated by growth factors and cytokines supplied by leukocytes, in particular monocytes (29), and is strongly dependent on the concentration of leukocytes in the peripheral blood (30), on their infiltration in growing arteries (30 –32), as well as on the availability of ICAM-1 mediating leukocyte adhesion (31). Since stem cells do not incorporate in growing collaterals (33), our data do not only prove that G-CSF promotes arteriogenesis directly but demonstrate that G-CSF shows, besides its effect to reduce apoptosis (12, 26), a second intrinsic effect, namely increasing the expression of ICAM-1 in arteriolar vessels being associated with an accumulation of growth promoting leukocytes. Since most of the growing arterioles identified were located at the border zone of the MI, it is most likely that these vessels are growing collateral arteries playing a causative role for improved cardiac function as well as reduced mortality after G-CSF treatment (see below). Our assumption is corroborated by several aspects. First of all, our own observations on a pure model of arteriogenesis in the rabbit hind-limb showed enhanced collateral artery growth after G-CSF application (unpublished observations). Second, a study of Norol et al. (34) on baboons showed an improved perfusion of the peri-infarct region after stimulation with G-CSF. Third, all previous studies on G-CSF administration after MI in C57BL/6 mice showed beneficial effects (12, 26), whereas a study on Balb/C mice did not (35). However, in contrast to C57BL/6 mice the Balb/C mouse strain shows only a minor arteriogenic response on occlusion of an artery (36). Fourth, in another experimental set up, G-CSF has been shown to repair injured arteries indicating that G-CSF has the capacity to remodel arteries in a positive manner (37). Finally, another closely related colony stimulating factor, i.e., GM-CSF, has been reported to stimulate arteriogenesis in experimental settings (38 – 41) as well as in clinical studies (42). One could however hypothesize that the observed growing arterioles do not represent collateral arteries but function in removal of necrotic tissue, in particular since no perfusion measurements have been performed. In contrast to collateral arteries, these vessels regress during the process of scar formation. However, our immunohistochemical results revealed BrdU positive endothelial and smooth muscle cells at day 30 in E34

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similar numbers as on day 6 after ligation of the LAD. Since BrdU was administered only during the first 5 days after the surgical procedure, these results clearly demonstrate that the grown vessels did not regress but present true collateral arteries. Furthermore, at day 30 some of the longitudinal cut arterioles showed corkscrew formation, a typical sign of growing collateral arteries. G-CSF treatment after MI improves long-term survival and partially restores myocardial function Our results showed that G-CSF treatment after MI led to beneficial effects on survival and global systolic and diastolic function over a time period of 4 wk. Previously, it was shown that G-CSF treatment in combination with SCF given for 3 days before MI improved survival and function (11). Furthermore, it was shown that high doses (100 ␮g/kg/day) of G-CSF application improved short-term survival and functional outcome 14 days after MI (12, 26). Our data extend these previous findings indicating that G-CSF application at a dose of 100 ␮g/kg/day given post-MI without addition of a second cytokine like SCF is sufficient to improve longterm survival and cardiac function. The beneficial outcome on survival and cardiac function in our study was morphologically related to 1) a reduced decline of the LV-wall at day 6 and day 30 after MI, 2) a reduced scar extension at day 30, and 3) a reduced number of animals developing ischemic related ventricular wall expansion. According to the modified law of Laplace, ventricular wall tension is negatively related to the wall thickness. Accordingly, the reduced decline of LV wall thickness found at day 6 after G-CSF treatment prevented high LV wall tension attenuating ventricular expansion and restoring ejection fraction. High end diastolic volumes and a worse ejection fraction are known determinants of post-MI mortality. However, our results evidenced that G-CSF treated animals showed relatively low end diastolic volumes and an improved ejection fraction explaining their better rate of post-MI survival. Our follow up study of 4 wk extends previous findings of other groups reporting a decreased ventricular expansion (43) and a reduced decline of the LV-wall in a short-term period of 2 wk after MI (12). Moreover, a recent study of Harada et al. (26) showed a dose-dependent effect of G-CSF treatment on ejection fraction. In accordance with our data, a dose of 100 ␮g/kg/day started directly after MI was associated with most beneficial effects on cardiac function. In summary, the mechanistic background of our findings are 1) a better perfusion of the peri-infarct region mediated by an enhanced growth of collateral vessels, 2) a reduced number of apoptotic endothelial cells and cardiomyocytes in the infarct and peri-infarct area as described previously by Ohtsuka et al. (26) and Harada et al. (12), and 3) a moderately mediated remodeling process being reflected by a lower degree of granulation tissue and fibrosis. It is well known that the content of reactive granulation and scar tissues

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after burn injury, surgery, etc., frequently becomes excessive (44, 45). This phenomenon is likely to be detrimental by itself by impairing cardiac contractility and vascular supply to the viable myocardium. CD45 positive cells such as granulocytes and monocytes carry G-CSF receptors and activation of these receptors via G-CSF stimulation results in a release of metalloproteinases (46). Since we found a decrease in the content of granulation tissue and collagen at 6 days after MI, our findings are likely to be related to investigations of others who found an increased expression and activity of proteases like MMP-1 and ⫺9 after G-CSF treatment, which was associated with a reduced area of fibrotic tissue and collagen (47). In summary, our results show that G-CSF administration after MI enhances arteriogenesis by increasing the availability of ICAM-1 mediating leukocyte adhesion. Furthermore, it improves myocardial function and reduces mortality after MI in mice. We want to thank Saskia Bangert, Andrea Sendlhofert, Anja Heier, and Sabine Schaefer for excellent technical assistance. The positions of E.D., Saskia Bangert, and U.M. were funded by the Fritz-Bender-Stiftung, and the Award Program for Research and Teaching of the Medical School (Fo¨FoLe program) of the Ludwig Maximilians University Munich, respectively. Additional financial support for flow cytometry was provided by the Dr. Helmut Legerlotz-Stiftung.

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The FASEB Journal

Received for publication September 27, 2005. Accepted for publication December 20, 2005.

DEINDL ET AL.