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Intracardiac injection of erythropoietin induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model.

Klopsch C, Furlani D, Gäbel R, Li W, Pittermann E, Ugurlucan M, Kundt G, Zingler C, Titze U, Wang W, Ong LL, Wagner K, Li RK, Ma N, Steinhoff G - J. Cell. Mol. Med. (2009)

Bottom Line: MI-EPO hearts exhibited smaller infarction size (20.1 +/- 1.1% versus 27.8 +/- 1.2%; n = 6-8, P < 0.001) and greater capillary density (338.5 +/- 14.7 versus 259.8 +/- 9.2 vessels per mm2; n = 6-8, P < 0.001) than MIC hearts.The chemoattractant SDF-1 was up-regulated significantly assessed by quantitative realtime PCR and immunohistology. c-Kit(+) and CD34(+) stem cells were significantly more numerous in MI-EPO than in MIC at 24 hrs in peripheral blood (n = 7, P < 0.05) and 48 hrs in the infarcted hearts (n = 6, P < 0.001).Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in MI-EPO hearts (n = 7, P < 0.05).

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiac Surgery, University of Rostock, Germany.

ABSTRACT
Erythropoietin (EPO) protects the myocardium from ischaemic injury and promotes beneficial remodelling. We assessed the therapeutic efficacy of intracardiac EPO injection and EPO-mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, EPO (3000 U/kg) or saline was delivered by intracardiac injection. Compared to myocardial infarction control group (MIC), EPO significantly improved left ventricular function (n =11-14, P < 0.05) and decreased right ventricular wall stress (n = 8, P < 0.05) assessed by pressure-volume loops after 6 weeks. MI-EPO hearts exhibited smaller infarction size (20.1 +/- 1.1% versus 27.8 +/- 1.2%; n = 6-8, P < 0.001) and greater capillary density (338.5 +/- 14.7 versus 259.8 +/- 9.2 vessels per mm2; n = 6-8, P < 0.001) than MIC hearts. Direct EPO injection reduced post-MI myocardial apoptosis by approximately 41% (0.27 +/- 0.03% versus 0.42 +/- 0.03%; n = 6, P= 0.005). The chemoattractant SDF-1 was up-regulated significantly assessed by quantitative realtime PCR and immunohistology. c-Kit(+) and CD34(+) stem cells were significantly more numerous in MI-EPO than in MIC at 24 hrs in peripheral blood (n = 7, P < 0.05) and 48 hrs in the infarcted hearts (n = 6, P < 0.001). Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in MI-EPO hearts (n = 7, P < 0.05). Intracardiac EPO injection restores myocardial functions following MI, which may attribute to the improved early recruitment of c-Kit(+) and CD34(+) stem cells via the enhanced expression of chemoattractant SDF-1.

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Effects of direct EPO injection on cardiac remodelling 6 weeks after MI. (A, B) Representative left ventricular cross-sections stained with Sirius Red (red, fibrosis) and Fast Green FCF (green, myocytes) from rats without (left) and with (right) EPO treatment. (C) Ratio of infarction area to entire LV is decreased in MI-EPO (n= 6) compared with MIC (n= 8). (D) Wall thickness of RA is significantly reduced in MI-EPO (n= 6) compared with MIC (n= 8). (E, F) EPO treatment results in a significantly decreased HW/BW ratio, and RVW/BW ratio (Sham n= 11, MIC n= 14, MI-EPO n= 11). (G, H) Collagen density (G) and cardiomyocyte size (H) in RA of MI-EPO hearts (n= 6) were reduced compared with MIC (n= 6). (I-K) Representative staining for Sirius Red (red, fibrosis) and Fast Green FCF (green, myocytes) in the RA of Sham (I, upper panel), MIC (J, middle panel) and MI-EPO (K, lower panel). Scale bars = 150 μm. (L, M) Representative immunostaining for TUNEL (green, square) and cardiac myosin (red) in RA (L, upper panel) and the border zone (M, lower panel) of MIC hearts. Scale bars = 25 μm (left panel). Scale bars = 5 μm (right panel). Blue, TOPRO3 in nuclei. (N) Cardiomyocyte apoptosis was reduced in both the border zone (left) and the RA (right) in MI-EPO (n= 6) compared with MIC (n= 6). (0) Quantitative real-time PCR analysis for Bcl2 gene in the IZ (left) and NIZ (right) of MI-EPO (n= 6), MIC (n= 6) and Sham (n= 6) hearts. The average mRNA expression level in the sham hearts was arbitrarily given a value of 1 (2°). *P< 0.05, **P< 0.01.
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fig02: Effects of direct EPO injection on cardiac remodelling 6 weeks after MI. (A, B) Representative left ventricular cross-sections stained with Sirius Red (red, fibrosis) and Fast Green FCF (green, myocytes) from rats without (left) and with (right) EPO treatment. (C) Ratio of infarction area to entire LV is decreased in MI-EPO (n= 6) compared with MIC (n= 8). (D) Wall thickness of RA is significantly reduced in MI-EPO (n= 6) compared with MIC (n= 8). (E, F) EPO treatment results in a significantly decreased HW/BW ratio, and RVW/BW ratio (Sham n= 11, MIC n= 14, MI-EPO n= 11). (G, H) Collagen density (G) and cardiomyocyte size (H) in RA of MI-EPO hearts (n= 6) were reduced compared with MIC (n= 6). (I-K) Representative staining for Sirius Red (red, fibrosis) and Fast Green FCF (green, myocytes) in the RA of Sham (I, upper panel), MIC (J, middle panel) and MI-EPO (K, lower panel). Scale bars = 150 μm. (L, M) Representative immunostaining for TUNEL (green, square) and cardiac myosin (red) in RA (L, upper panel) and the border zone (M, lower panel) of MIC hearts. Scale bars = 25 μm (left panel). Scale bars = 5 μm (right panel). Blue, TOPRO3 in nuclei. (N) Cardiomyocyte apoptosis was reduced in both the border zone (left) and the RA (right) in MI-EPO (n= 6) compared with MIC (n= 6). (0) Quantitative real-time PCR analysis for Bcl2 gene in the IZ (left) and NIZ (right) of MI-EPO (n= 6), MIC (n= 6) and Sham (n= 6) hearts. The average mRNA expression level in the sham hearts was arbitrarily given a value of 1 (2°). *P< 0.05, **P< 0.01.

Mentions: Computerized planimetry of the heart cross-sections revealed that scar area was remarkably lower in MI-EPO than in MIC (Fig. 2A and B). The average infarction size decreased significantly from 27.76 ± 1.20% in MIC to 20.11 ± 1.13% in MI-EPO (P< 0.001, Fig. 2C). Moreover, the interventricular septum thickness of EPO-treated rats in the RA was decreased compared with MIC rats (P= 0.014, Fig. 2D). Wall thickness in the infarcted area did not differ among experimental groups (P= 0.109). Furthermore, EPO-treated rats showed a 12% reduction in heart weight-to-body weight (HW/BW) ratio (P= 0.003, Fig. 2E), a commonly used index of cardiac hypertrophy. Therefore, these results suggest that EPO limits the development of post-infarction hypertrophy. More specifically, we found a 26% reduction in right ventricular free wall weight-to-body weight (RVW/BW) ratio (P= 0.041, Fig. 2F) in MI-EPO, another highly reliable indicator of reduced left ventri cular decompensation. There was no significant reduction in left ventricular weight-to-body weight (LVW/BW) ratio in EPO-treated rats (P= 0.061). Body weight did not differ among experimental groups. Collagen density in RA decreased significantly from 12.36 ± 0.20% in MIC to 9.53 ± 0.13% in MI-EPO (P< 0.001, Fig. 2G, I–K). Average cardiomyocyte size was significantly smaller in MI-EPO (765.21 ± 13.74 μm2) when compared to MIC (882.98 ± 36.91 μm2) (P= 0.015, Fig. 2H, I–K). Further, percentage of apoptotic cardiomyocyte reduced from 0.19 ± 0.02% (MIC group) to 0.12 ± 0.01% (MI-EPO group, P= 0.023) in RA and from 0.42 ± 0.03% (MIC group) to 0.27 ± 0.03% (MI-EPO group, P= 0.005) in the border zone (Fig. 2L-N). Quantitative real-time PCR revealed a 3-fold increase in mRNA expression of pro-survival Bcl2 gene after 6 weeks in the non-infarcted myocardium of MI-EPO hearts compared with MIC hearts (P= 0.001, Fig. 2O).


Intracardiac injection of erythropoietin induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model.

Klopsch C, Furlani D, Gäbel R, Li W, Pittermann E, Ugurlucan M, Kundt G, Zingler C, Titze U, Wang W, Ong LL, Wagner K, Li RK, Ma N, Steinhoff G - J. Cell. Mol. Med. (2009)

Effects of direct EPO injection on cardiac remodelling 6 weeks after MI. (A, B) Representative left ventricular cross-sections stained with Sirius Red (red, fibrosis) and Fast Green FCF (green, myocytes) from rats without (left) and with (right) EPO treatment. (C) Ratio of infarction area to entire LV is decreased in MI-EPO (n= 6) compared with MIC (n= 8). (D) Wall thickness of RA is significantly reduced in MI-EPO (n= 6) compared with MIC (n= 8). (E, F) EPO treatment results in a significantly decreased HW/BW ratio, and RVW/BW ratio (Sham n= 11, MIC n= 14, MI-EPO n= 11). (G, H) Collagen density (G) and cardiomyocyte size (H) in RA of MI-EPO hearts (n= 6) were reduced compared with MIC (n= 6). (I-K) Representative staining for Sirius Red (red, fibrosis) and Fast Green FCF (green, myocytes) in the RA of Sham (I, upper panel), MIC (J, middle panel) and MI-EPO (K, lower panel). Scale bars = 150 μm. (L, M) Representative immunostaining for TUNEL (green, square) and cardiac myosin (red) in RA (L, upper panel) and the border zone (M, lower panel) of MIC hearts. Scale bars = 25 μm (left panel). Scale bars = 5 μm (right panel). Blue, TOPRO3 in nuclei. (N) Cardiomyocyte apoptosis was reduced in both the border zone (left) and the RA (right) in MI-EPO (n= 6) compared with MIC (n= 6). (0) Quantitative real-time PCR analysis for Bcl2 gene in the IZ (left) and NIZ (right) of MI-EPO (n= 6), MIC (n= 6) and Sham (n= 6) hearts. The average mRNA expression level in the sham hearts was arbitrarily given a value of 1 (2°). *P< 0.05, **P< 0.01.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822874&req=5

fig02: Effects of direct EPO injection on cardiac remodelling 6 weeks after MI. (A, B) Representative left ventricular cross-sections stained with Sirius Red (red, fibrosis) and Fast Green FCF (green, myocytes) from rats without (left) and with (right) EPO treatment. (C) Ratio of infarction area to entire LV is decreased in MI-EPO (n= 6) compared with MIC (n= 8). (D) Wall thickness of RA is significantly reduced in MI-EPO (n= 6) compared with MIC (n= 8). (E, F) EPO treatment results in a significantly decreased HW/BW ratio, and RVW/BW ratio (Sham n= 11, MIC n= 14, MI-EPO n= 11). (G, H) Collagen density (G) and cardiomyocyte size (H) in RA of MI-EPO hearts (n= 6) were reduced compared with MIC (n= 6). (I-K) Representative staining for Sirius Red (red, fibrosis) and Fast Green FCF (green, myocytes) in the RA of Sham (I, upper panel), MIC (J, middle panel) and MI-EPO (K, lower panel). Scale bars = 150 μm. (L, M) Representative immunostaining for TUNEL (green, square) and cardiac myosin (red) in RA (L, upper panel) and the border zone (M, lower panel) of MIC hearts. Scale bars = 25 μm (left panel). Scale bars = 5 μm (right panel). Blue, TOPRO3 in nuclei. (N) Cardiomyocyte apoptosis was reduced in both the border zone (left) and the RA (right) in MI-EPO (n= 6) compared with MIC (n= 6). (0) Quantitative real-time PCR analysis for Bcl2 gene in the IZ (left) and NIZ (right) of MI-EPO (n= 6), MIC (n= 6) and Sham (n= 6) hearts. The average mRNA expression level in the sham hearts was arbitrarily given a value of 1 (2°). *P< 0.05, **P< 0.01.
Mentions: Computerized planimetry of the heart cross-sections revealed that scar area was remarkably lower in MI-EPO than in MIC (Fig. 2A and B). The average infarction size decreased significantly from 27.76 ± 1.20% in MIC to 20.11 ± 1.13% in MI-EPO (P< 0.001, Fig. 2C). Moreover, the interventricular septum thickness of EPO-treated rats in the RA was decreased compared with MIC rats (P= 0.014, Fig. 2D). Wall thickness in the infarcted area did not differ among experimental groups (P= 0.109). Furthermore, EPO-treated rats showed a 12% reduction in heart weight-to-body weight (HW/BW) ratio (P= 0.003, Fig. 2E), a commonly used index of cardiac hypertrophy. Therefore, these results suggest that EPO limits the development of post-infarction hypertrophy. More specifically, we found a 26% reduction in right ventricular free wall weight-to-body weight (RVW/BW) ratio (P= 0.041, Fig. 2F) in MI-EPO, another highly reliable indicator of reduced left ventri cular decompensation. There was no significant reduction in left ventricular weight-to-body weight (LVW/BW) ratio in EPO-treated rats (P= 0.061). Body weight did not differ among experimental groups. Collagen density in RA decreased significantly from 12.36 ± 0.20% in MIC to 9.53 ± 0.13% in MI-EPO (P< 0.001, Fig. 2G, I–K). Average cardiomyocyte size was significantly smaller in MI-EPO (765.21 ± 13.74 μm2) when compared to MIC (882.98 ± 36.91 μm2) (P= 0.015, Fig. 2H, I–K). Further, percentage of apoptotic cardiomyocyte reduced from 0.19 ± 0.02% (MIC group) to 0.12 ± 0.01% (MI-EPO group, P= 0.023) in RA and from 0.42 ± 0.03% (MIC group) to 0.27 ± 0.03% (MI-EPO group, P= 0.005) in the border zone (Fig. 2L-N). Quantitative real-time PCR revealed a 3-fold increase in mRNA expression of pro-survival Bcl2 gene after 6 weeks in the non-infarcted myocardium of MI-EPO hearts compared with MIC hearts (P= 0.001, Fig. 2O).

Bottom Line: MI-EPO hearts exhibited smaller infarction size (20.1 +/- 1.1% versus 27.8 +/- 1.2%; n = 6-8, P < 0.001) and greater capillary density (338.5 +/- 14.7 versus 259.8 +/- 9.2 vessels per mm2; n = 6-8, P < 0.001) than MIC hearts.The chemoattractant SDF-1 was up-regulated significantly assessed by quantitative realtime PCR and immunohistology. c-Kit(+) and CD34(+) stem cells were significantly more numerous in MI-EPO than in MIC at 24 hrs in peripheral blood (n = 7, P < 0.05) and 48 hrs in the infarcted hearts (n = 6, P < 0.001).Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in MI-EPO hearts (n = 7, P < 0.05).

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiac Surgery, University of Rostock, Germany.

ABSTRACT
Erythropoietin (EPO) protects the myocardium from ischaemic injury and promotes beneficial remodelling. We assessed the therapeutic efficacy of intracardiac EPO injection and EPO-mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, EPO (3000 U/kg) or saline was delivered by intracardiac injection. Compared to myocardial infarction control group (MIC), EPO significantly improved left ventricular function (n =11-14, P < 0.05) and decreased right ventricular wall stress (n = 8, P < 0.05) assessed by pressure-volume loops after 6 weeks. MI-EPO hearts exhibited smaller infarction size (20.1 +/- 1.1% versus 27.8 +/- 1.2%; n = 6-8, P < 0.001) and greater capillary density (338.5 +/- 14.7 versus 259.8 +/- 9.2 vessels per mm2; n = 6-8, P < 0.001) than MIC hearts. Direct EPO injection reduced post-MI myocardial apoptosis by approximately 41% (0.27 +/- 0.03% versus 0.42 +/- 0.03%; n = 6, P= 0.005). The chemoattractant SDF-1 was up-regulated significantly assessed by quantitative realtime PCR and immunohistology. c-Kit(+) and CD34(+) stem cells were significantly more numerous in MI-EPO than in MIC at 24 hrs in peripheral blood (n = 7, P < 0.05) and 48 hrs in the infarcted hearts (n = 6, P < 0.001). Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in MI-EPO hearts (n = 7, P < 0.05). Intracardiac EPO injection restores myocardial functions following MI, which may attribute to the improved early recruitment of c-Kit(+) and CD34(+) stem cells via the enhanced expression of chemoattractant SDF-1.

Show MeSH
Related in: MedlinePlus