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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

²Correspondence: Ludwig-Maximilians-University, Klinikum Grosshadern, Medical Department I, Marchioninistr. 15, D-81377 Munich, Germany. E-mail address: wolfgang.franz{at}med.uni-muenchen.de

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 µg/kg/day; bromodeoxyuridine (BrdU), 50 µg/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 µg/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 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 pressure-volume 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 µl, P<0.05). Stroke work (738±119x vs. 291±37 mmHgxµl, 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/t_min: –4899±444 vs. 3270±295 mmHg/sec).

View larger version (61K): [in this window] [in a new window]   Figure 1. Histological findings and pressure-volume 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).

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 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 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 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.

View larger version (32K): [in this window] [in a new window]   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.

FOOTNOTES

¹ Elisabeth Deindl and Marc-Michael Zaruba contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4763fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-4763fjev1 20/7/956    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Deindl, E. Articles by Franz, W.-M. Search for Related Content PubMed PubMed Citation Articles by Deindl, E. Articles by Franz, W.-M.