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Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling.

Formigli L, Perna AM, Meacci E, Cinci L, Margheri M, Nistri S, Tani A, Silvertown J, Orlandini G, Porciani C, Zecchi-Orlandini S, Medin J, Bani D - J. Cell. Mol. Med. (2007 Sep-Oct)

Bottom Line: C2C12 myoblasts did not trans-differentiate towards a cardiac phenotype, but did induce extracellular matrix remodelling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of vascular endothelial growth factor (VEGF).By echocardiography, C2C12-engrafted swine showed improved heart contractility compared with the ungrafted controls, especially those producing RLX.We suggest that the beneficial effects of myoblast grafting on cardiac function are primarily dependent on the paracrine effects of transplanted cells on extracellular matrix remodelling and vascularization.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, University of Florence, Florence, Italy.

ABSTRACT
In the post-infarcted heart, grafting of precursor cells may partially restore heart function but the improvement is modest and the mechanisms involved remain to be elucidated. Here, we explored this issue by transplanting C2C12 myoblasts, genetically engineered to express enhanced green fluorescent protein (eGFP) or eGFP and the cardiotropic hormone relaxin (RLX) through coronary venous route to swine with experimental chronic myocardial infarction. The rationale was to deliver constant, biologically effective levels of RLX at the site of cell engraftment. One month after engraftment, histological analysis showed that C2C12 myoblasts selectively settled in the ischaemic scar and were located around blood vessels showing an activated endothelium (ICAM-1-,VCAM-positive). C2C12 myoblasts did not trans-differentiate towards a cardiac phenotype, but did induce extracellular matrix remodelling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of vascular endothelial growth factor (VEGF). Relaxin-producing C2C12 myoblasts displayed greater efficacy to engraft the post-ischaemic scar and to induce extracellular matrix re-modelling and angiogenesis as compared with the control cells. By echocardiography, C2C12-engrafted swine showed improved heart contractility compared with the ungrafted controls, especially those producing RLX. We suggest that the beneficial effects of myoblast grafting on cardiac function are primarily dependent on the paracrine effects of transplanted cells on extracellular matrix remodelling and vascularization. The combined treatment with myoblast transplantation and local RLX production may be helpful in preventing deleterious cardiac remodelling and may hold therapeutic possibility for post-infarcted patients.

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Upper and centre panels. Light micrographs of the microvascular network in control (A), C2C12/GFP- (B) and C2C12/RLX-transplanted hearts (C), and morphometric assessment of microvascular density. Both C2C12/ GFP- and C2C12/RLX-injected hearts exhibit higher microvascular density than the control hearts. Of note, C2C12/RLX-injected hearts show significantly higher microvascular density than C2C12/GFP-injected ones. (a) P<0.05 versus control;(b) P<0.01 versus control;(c) P<0.05 versus C2C12/ GFP. Lower panel. RT-PCR assay for porcine VEGF transcripts in scar tissue samples from controls (lanes 1, 2), C2C12/GFP- (lanes 3,4) and C2C12/ RLX-transplanted hearts (lanes 5, 6). Images are representative of five different animals per experimental groups. Porcine VEGF mRNA is absent in the control samples, is slightly expressed in one of the two samples of C2C12/GFP-injected hearts, and clearly detectable in both the samples from C2C12/RLX-injected hearts.
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fig05: Upper and centre panels. Light micrographs of the microvascular network in control (A), C2C12/GFP- (B) and C2C12/RLX-transplanted hearts (C), and morphometric assessment of microvascular density. Both C2C12/ GFP- and C2C12/RLX-injected hearts exhibit higher microvascular density than the control hearts. Of note, C2C12/RLX-injected hearts show significantly higher microvascular density than C2C12/GFP-injected ones. (a) P<0.05 versus control;(b) P<0.01 versus control;(c) P<0.05 versus C2C12/ GFP. Lower panel. RT-PCR assay for porcine VEGF transcripts in scar tissue samples from controls (lanes 1, 2), C2C12/GFP- (lanes 3,4) and C2C12/ RLX-transplanted hearts (lanes 5, 6). Images are representative of five different animals per experimental groups. Porcine VEGF mRNA is absent in the control samples, is slightly expressed in one of the two samples of C2C12/GFP-injected hearts, and clearly detectable in both the samples from C2C12/RLX-injected hearts.

Mentions: By Van Gieson staining, collagen fibres markedly accumulated in the post-infarcted myocardium of the control animals and were reduced in the transplanted groups, especially in C2C12/RLX-treated animals (Fig. 3A and C). Notably, ventricular tissue samples coming from the post-infarcted areas were very similar in histological appearance among the animals of each group. Morphometric analysis of tissue collagen content confirmed the visual observation (Fig. 3). By electron microscopy, fibroblasts from the control hearts featured activated cells, with cytoplasms filled with protein synthesizing organelles and surrounded by collagen bundles (Fig. 3D). Conversely, in the hearts receiving C2C12/RLX, fibroblasts were immersed in a loose extracellular matrix with few, thin collagen fibrils (Fig. 3E). We then analysed MMP/ collagenase activity in tissue homogenates from the post-infarction scar, as well as in primary cultures of swine cardiac cells incubated together with C2C12/GFP or C2C12/RLX. MMP-2 activity was very low in the control hearts, whereas it was significantly increased in the hearts containing grafted C2C12/GFP and C2C12/RLX cells (P<0.01).We also observed a tendency of C2C12/RLX-grafted hearts to possess higher MMP-2 activity compared to the C2C12/GFP ones, although the differences did not reach statistical significance. On the other hand, we found that both the co-cultures released collagen-degrading enzymes in the supernatants, with significantly higher levels in those containing C2C12/RLX cells (Fig. 4). Of interest, both resident cardiac cells and myoblasts contributed to MMP activity and this effect was significantly enhanced by exogenous or endogenous RLX (Fig. 4).Finally, we studied scar tissue vascularization. In C2C12/GFP-transplanted hearts, microvessel density was slightly but significantly increased compared to the control group, while it reached the highest values in C2C12/RLX transplanted hearts (Fig. 5).Interestingly, changes in microvascular density paralleled with porcine VEGF mRNA expression in the cardiac scar (Fig. 5).Indeed, VEGF mRNA was absent in control hearts, absent or only slightly expressed in C2C12/GFP-injected hearts and well detectable in the C2C12/GFP-injected hearts (Table 1). On the other hand, C2C12-derived mouse VEGF transcript was not detected in the specimens from transplanted animals (data not shown), suggesting that the engrafted myoblasts do not play a substantial contribution to local VEGF production.


Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling.

Formigli L, Perna AM, Meacci E, Cinci L, Margheri M, Nistri S, Tani A, Silvertown J, Orlandini G, Porciani C, Zecchi-Orlandini S, Medin J, Bani D - J. Cell. Mol. Med. (2007 Sep-Oct)

Upper and centre panels. Light micrographs of the microvascular network in control (A), C2C12/GFP- (B) and C2C12/RLX-transplanted hearts (C), and morphometric assessment of microvascular density. Both C2C12/ GFP- and C2C12/RLX-injected hearts exhibit higher microvascular density than the control hearts. Of note, C2C12/RLX-injected hearts show significantly higher microvascular density than C2C12/GFP-injected ones. (a) P<0.05 versus control;(b) P<0.01 versus control;(c) P<0.05 versus C2C12/ GFP. Lower panel. RT-PCR assay for porcine VEGF transcripts in scar tissue samples from controls (lanes 1, 2), C2C12/GFP- (lanes 3,4) and C2C12/ RLX-transplanted hearts (lanes 5, 6). Images are representative of five different animals per experimental groups. Porcine VEGF mRNA is absent in the control samples, is slightly expressed in one of the two samples of C2C12/GFP-injected hearts, and clearly detectable in both the samples from C2C12/RLX-injected hearts.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401276&req=5

fig05: Upper and centre panels. Light micrographs of the microvascular network in control (A), C2C12/GFP- (B) and C2C12/RLX-transplanted hearts (C), and morphometric assessment of microvascular density. Both C2C12/ GFP- and C2C12/RLX-injected hearts exhibit higher microvascular density than the control hearts. Of note, C2C12/RLX-injected hearts show significantly higher microvascular density than C2C12/GFP-injected ones. (a) P<0.05 versus control;(b) P<0.01 versus control;(c) P<0.05 versus C2C12/ GFP. Lower panel. RT-PCR assay for porcine VEGF transcripts in scar tissue samples from controls (lanes 1, 2), C2C12/GFP- (lanes 3,4) and C2C12/ RLX-transplanted hearts (lanes 5, 6). Images are representative of five different animals per experimental groups. Porcine VEGF mRNA is absent in the control samples, is slightly expressed in one of the two samples of C2C12/GFP-injected hearts, and clearly detectable in both the samples from C2C12/RLX-injected hearts.
Mentions: By Van Gieson staining, collagen fibres markedly accumulated in the post-infarcted myocardium of the control animals and were reduced in the transplanted groups, especially in C2C12/RLX-treated animals (Fig. 3A and C). Notably, ventricular tissue samples coming from the post-infarcted areas were very similar in histological appearance among the animals of each group. Morphometric analysis of tissue collagen content confirmed the visual observation (Fig. 3). By electron microscopy, fibroblasts from the control hearts featured activated cells, with cytoplasms filled with protein synthesizing organelles and surrounded by collagen bundles (Fig. 3D). Conversely, in the hearts receiving C2C12/RLX, fibroblasts were immersed in a loose extracellular matrix with few, thin collagen fibrils (Fig. 3E). We then analysed MMP/ collagenase activity in tissue homogenates from the post-infarction scar, as well as in primary cultures of swine cardiac cells incubated together with C2C12/GFP or C2C12/RLX. MMP-2 activity was very low in the control hearts, whereas it was significantly increased in the hearts containing grafted C2C12/GFP and C2C12/RLX cells (P<0.01).We also observed a tendency of C2C12/RLX-grafted hearts to possess higher MMP-2 activity compared to the C2C12/GFP ones, although the differences did not reach statistical significance. On the other hand, we found that both the co-cultures released collagen-degrading enzymes in the supernatants, with significantly higher levels in those containing C2C12/RLX cells (Fig. 4). Of interest, both resident cardiac cells and myoblasts contributed to MMP activity and this effect was significantly enhanced by exogenous or endogenous RLX (Fig. 4).Finally, we studied scar tissue vascularization. In C2C12/GFP-transplanted hearts, microvessel density was slightly but significantly increased compared to the control group, while it reached the highest values in C2C12/RLX transplanted hearts (Fig. 5).Interestingly, changes in microvascular density paralleled with porcine VEGF mRNA expression in the cardiac scar (Fig. 5).Indeed, VEGF mRNA was absent in control hearts, absent or only slightly expressed in C2C12/GFP-injected hearts and well detectable in the C2C12/GFP-injected hearts (Table 1). On the other hand, C2C12-derived mouse VEGF transcript was not detected in the specimens from transplanted animals (data not shown), suggesting that the engrafted myoblasts do not play a substantial contribution to local VEGF production.

Bottom Line: C2C12 myoblasts did not trans-differentiate towards a cardiac phenotype, but did induce extracellular matrix remodelling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of vascular endothelial growth factor (VEGF).By echocardiography, C2C12-engrafted swine showed improved heart contractility compared with the ungrafted controls, especially those producing RLX.We suggest that the beneficial effects of myoblast grafting on cardiac function are primarily dependent on the paracrine effects of transplanted cells on extracellular matrix remodelling and vascularization.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, University of Florence, Florence, Italy.

ABSTRACT
In the post-infarcted heart, grafting of precursor cells may partially restore heart function but the improvement is modest and the mechanisms involved remain to be elucidated. Here, we explored this issue by transplanting C2C12 myoblasts, genetically engineered to express enhanced green fluorescent protein (eGFP) or eGFP and the cardiotropic hormone relaxin (RLX) through coronary venous route to swine with experimental chronic myocardial infarction. The rationale was to deliver constant, biologically effective levels of RLX at the site of cell engraftment. One month after engraftment, histological analysis showed that C2C12 myoblasts selectively settled in the ischaemic scar and were located around blood vessels showing an activated endothelium (ICAM-1-,VCAM-positive). C2C12 myoblasts did not trans-differentiate towards a cardiac phenotype, but did induce extracellular matrix remodelling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of vascular endothelial growth factor (VEGF). Relaxin-producing C2C12 myoblasts displayed greater efficacy to engraft the post-ischaemic scar and to induce extracellular matrix re-modelling and angiogenesis as compared with the control cells. By echocardiography, C2C12-engrafted swine showed improved heart contractility compared with the ungrafted controls, especially those producing RLX. We suggest that the beneficial effects of myoblast grafting on cardiac function are primarily dependent on the paracrine effects of transplanted cells on extracellular matrix remodelling and vascularization. The combined treatment with myoblast transplantation and local RLX production may be helpful in preventing deleterious cardiac remodelling and may hold therapeutic possibility for post-infarcted patients.

Show MeSH
Related in: MedlinePlus