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Angiogenesis, cardiomyocyte proliferation and anti-fibrotic effects underlie structural preservation post-infarction by intramyocardially-injected cardiospheres.

Tseliou E, de Couto G, Terrovitis J, Sun B, Weixin L, Marbán L, Marbán E - PLoS ONE (2014)

Bottom Line: Collagen deposition was reduced, collagen degradation was enhanced, and MMPs were upregulated.The beneficial effects of CSp transplantation were not observed in the Small MI group, indicating that the effects are not solely due to CSp-induced cardioprotection.These synergistic effects underlie the attenuation of adverse remodeling by cardiospheres.

View Article: PubMed Central - PubMed

Affiliation: Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.

ABSTRACT

Objective: We sought to understand the cellular and tissue-level changes underlying the attenuation of adverse remodeling by cardiosphere transplantation in acute myocardial infarction (MI).

Background: Cardiospheres (CSps) are heart-derived multicellular clusters rich in stemness and capable of multilineage differentiation. Post-MI CSp transplantation improves left ventricular (LV) function and attenuates remodeling in both small and large animal studies. However, the mechanisms of benefit have not yet been fully elucidated.

Methods: Four groups were studied: 1) "Sham" (Wistar Kyoto rats with thoracotomy and ligature without infarction); 2) "MI" (proximal LAD ligation with peri-infarct injection of vehicle); 3) "MI+CSp" (MI with cardiospheres injected in the peri-infarct area); 4) "Small MI" (mid-LAD ligation only).

Results: In vivo 1 week after CSp transplantation, LV functional improvement was associated with an increase in cardiomyocyte proliferation. By 3 weeks, microvessel formation was enhanced, while cardiomyocyte hypertrophy and regional fibrosis were attenuated. Collagen deposition was reduced, collagen degradation was enhanced, and MMPs were upregulated. The beneficial effects of CSp transplantation were not observed in the Small MI group, indicating that the effects are not solely due to CSp-induced cardioprotection. In vitro, CSp-conditioned media reduced collagen production in coculture with fibroblasts and triggered neoangiogenesis in an ex vivo aortic ring assay.

Conclusion: Cardiospheres enhance cardiomyocyte proliferation and angiogenesis, and attenuate hypertrophy and fibrosis, in the ischemic myocardium. These synergistic effects underlie the attenuation of adverse remodeling by cardiospheres.

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Related in: MedlinePlus

Cardiac tissue structure.(A) Representative photomicrographs of immunohistochemical staining of smooth muscle actin, wheat germ agglutinin, and Dapi in myocardial tissue sections. Five different heart tissues per group were stained. Approximately 10–15 high-power fields per area from a minimum 3 slides per heart were analyzed to obtain the average regional cross sectional area and regional myocyte nuclear density. 100 cardiomyocytes per heart were evaluated. (B) Quantification of cross-sectional area in the peri-infarct and D. in the remote zone respectively. (C,E) Measurement of the total number of cell nuclei per field evaluated in both above-mentioned regions. (F) Representative photomicrographs of immunohistochemical staining of α-sarcomeric actin, Ki67 and Dapi, in the peri-infarct zone and (I) in the remote area. (G–H) Quantification of proliferating cardiomyocytes per total number of cardiomyocytes per field and of proliferating cardiomyocytes per total nuclei per field respectively. (J–K) Same quantification as in G–H but in the remote area. Arrows point to Ki67+/asa+ cells. (Scale bars 50 µm). Data are mean±SEM. ¶ p<0.05 control vs. MI+CSp, * p<0.05 vs. sham.
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pone-0088590-g002: Cardiac tissue structure.(A) Representative photomicrographs of immunohistochemical staining of smooth muscle actin, wheat germ agglutinin, and Dapi in myocardial tissue sections. Five different heart tissues per group were stained. Approximately 10–15 high-power fields per area from a minimum 3 slides per heart were analyzed to obtain the average regional cross sectional area and regional myocyte nuclear density. 100 cardiomyocytes per heart were evaluated. (B) Quantification of cross-sectional area in the peri-infarct and D. in the remote zone respectively. (C,E) Measurement of the total number of cell nuclei per field evaluated in both above-mentioned regions. (F) Representative photomicrographs of immunohistochemical staining of α-sarcomeric actin, Ki67 and Dapi, in the peri-infarct zone and (I) in the remote area. (G–H) Quantification of proliferating cardiomyocytes per total number of cardiomyocytes per field and of proliferating cardiomyocytes per total nuclei per field respectively. (J–K) Same quantification as in G–H but in the remote area. Arrows point to Ki67+/asa+ cells. (Scale bars 50 µm). Data are mean±SEM. ¶ p<0.05 control vs. MI+CSp, * p<0.05 vs. sham.

Mentions: A major compensatory mechanism post-MI is hypertrophy of surviving cardiomyocytes. We sought to determine whether the gross changes in viable mass reflect cardiomyocyte hypertrophy or increased myocyte number. Samples from 5 different hearts per group, 3 weeks post-MI were stained with alpha sarcomeric actin (a-sa), wheat germ agglutinin (wga, a stain for the surface cell membrane) and Ki67+ (a marker of active proliferation). The absolute number of cardiomyocytes was reduced in both the peri-infarct and remote zones of the MI group, and cross-sectional area was increased, indicating compensatory myocyte hypertrophy (Figures 2A–E). However, in the CSp-treated myocardium we found a ∼10% higher myocyte nuclear density and attenuated hypertrophy in both regions (Figures 2B–E). In addition, the number of α-sa+/Ki-67+ cells was markedly higher than in the placebo groups (Figures 2F–K). Thus, we conclude that the augmentation of viable mass in CSp-treated hearts (Figure 1C) reflects myocyte hyperplasia and/or myocyte preservation rather than hypertrophy.


Angiogenesis, cardiomyocyte proliferation and anti-fibrotic effects underlie structural preservation post-infarction by intramyocardially-injected cardiospheres.

Tseliou E, de Couto G, Terrovitis J, Sun B, Weixin L, Marbán L, Marbán E - PLoS ONE (2014)

Cardiac tissue structure.(A) Representative photomicrographs of immunohistochemical staining of smooth muscle actin, wheat germ agglutinin, and Dapi in myocardial tissue sections. Five different heart tissues per group were stained. Approximately 10–15 high-power fields per area from a minimum 3 slides per heart were analyzed to obtain the average regional cross sectional area and regional myocyte nuclear density. 100 cardiomyocytes per heart were evaluated. (B) Quantification of cross-sectional area in the peri-infarct and D. in the remote zone respectively. (C,E) Measurement of the total number of cell nuclei per field evaluated in both above-mentioned regions. (F) Representative photomicrographs of immunohistochemical staining of α-sarcomeric actin, Ki67 and Dapi, in the peri-infarct zone and (I) in the remote area. (G–H) Quantification of proliferating cardiomyocytes per total number of cardiomyocytes per field and of proliferating cardiomyocytes per total nuclei per field respectively. (J–K) Same quantification as in G–H but in the remote area. Arrows point to Ki67+/asa+ cells. (Scale bars 50 µm). Data are mean±SEM. ¶ p<0.05 control vs. MI+CSp, * p<0.05 vs. sham.
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pone-0088590-g002: Cardiac tissue structure.(A) Representative photomicrographs of immunohistochemical staining of smooth muscle actin, wheat germ agglutinin, and Dapi in myocardial tissue sections. Five different heart tissues per group were stained. Approximately 10–15 high-power fields per area from a minimum 3 slides per heart were analyzed to obtain the average regional cross sectional area and regional myocyte nuclear density. 100 cardiomyocytes per heart were evaluated. (B) Quantification of cross-sectional area in the peri-infarct and D. in the remote zone respectively. (C,E) Measurement of the total number of cell nuclei per field evaluated in both above-mentioned regions. (F) Representative photomicrographs of immunohistochemical staining of α-sarcomeric actin, Ki67 and Dapi, in the peri-infarct zone and (I) in the remote area. (G–H) Quantification of proliferating cardiomyocytes per total number of cardiomyocytes per field and of proliferating cardiomyocytes per total nuclei per field respectively. (J–K) Same quantification as in G–H but in the remote area. Arrows point to Ki67+/asa+ cells. (Scale bars 50 µm). Data are mean±SEM. ¶ p<0.05 control vs. MI+CSp, * p<0.05 vs. sham.
Mentions: A major compensatory mechanism post-MI is hypertrophy of surviving cardiomyocytes. We sought to determine whether the gross changes in viable mass reflect cardiomyocyte hypertrophy or increased myocyte number. Samples from 5 different hearts per group, 3 weeks post-MI were stained with alpha sarcomeric actin (a-sa), wheat germ agglutinin (wga, a stain for the surface cell membrane) and Ki67+ (a marker of active proliferation). The absolute number of cardiomyocytes was reduced in both the peri-infarct and remote zones of the MI group, and cross-sectional area was increased, indicating compensatory myocyte hypertrophy (Figures 2A–E). However, in the CSp-treated myocardium we found a ∼10% higher myocyte nuclear density and attenuated hypertrophy in both regions (Figures 2B–E). In addition, the number of α-sa+/Ki-67+ cells was markedly higher than in the placebo groups (Figures 2F–K). Thus, we conclude that the augmentation of viable mass in CSp-treated hearts (Figure 1C) reflects myocyte hyperplasia and/or myocyte preservation rather than hypertrophy.

Bottom Line: Collagen deposition was reduced, collagen degradation was enhanced, and MMPs were upregulated.The beneficial effects of CSp transplantation were not observed in the Small MI group, indicating that the effects are not solely due to CSp-induced cardioprotection.These synergistic effects underlie the attenuation of adverse remodeling by cardiospheres.

View Article: PubMed Central - PubMed

Affiliation: Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.

ABSTRACT

Objective: We sought to understand the cellular and tissue-level changes underlying the attenuation of adverse remodeling by cardiosphere transplantation in acute myocardial infarction (MI).

Background: Cardiospheres (CSps) are heart-derived multicellular clusters rich in stemness and capable of multilineage differentiation. Post-MI CSp transplantation improves left ventricular (LV) function and attenuates remodeling in both small and large animal studies. However, the mechanisms of benefit have not yet been fully elucidated.

Methods: Four groups were studied: 1) "Sham" (Wistar Kyoto rats with thoracotomy and ligature without infarction); 2) "MI" (proximal LAD ligation with peri-infarct injection of vehicle); 3) "MI+CSp" (MI with cardiospheres injected in the peri-infarct area); 4) "Small MI" (mid-LAD ligation only).

Results: In vivo 1 week after CSp transplantation, LV functional improvement was associated with an increase in cardiomyocyte proliferation. By 3 weeks, microvessel formation was enhanced, while cardiomyocyte hypertrophy and regional fibrosis were attenuated. Collagen deposition was reduced, collagen degradation was enhanced, and MMPs were upregulated. The beneficial effects of CSp transplantation were not observed in the Small MI group, indicating that the effects are not solely due to CSp-induced cardioprotection. In vitro, CSp-conditioned media reduced collagen production in coculture with fibroblasts and triggered neoangiogenesis in an ex vivo aortic ring assay.

Conclusion: Cardiospheres enhance cardiomyocyte proliferation and angiogenesis, and attenuate hypertrophy and fibrosis, in the ischemic myocardium. These synergistic effects underlie the attenuation of adverse remodeling by cardiospheres.

Show MeSH
Related in: MedlinePlus