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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 panels. Light micro-graphs of sections from control (A), C2C12/GFP- (B) and C2C12/RLX-transplanted hearts (C), stained with Van Gieson for collagen. A marked reduction of the extent of fibrosis can be seen in the ischaemic scars of the myoblast-transplanted hearts; of note, fibrosis is particularly attenuated in the hearts receiving C2C12/RLX cells. Bar = 20 μm. Centre panel. Sclerosis index (optical x volume density of collagen fibres) in the post-infarcted myocardi-um, estimated by computer-aided mor-phometry; (a) P<0.05 versus controls; (b) P<0.001 versus controls; (c) P<0.001 versus C2C12/GFP. Lower panels. Electron micrographs of fibrob-lasts from control (D) C2C12/GFP- (E) and C2C12/RLX-transplanted hearts (F). In the control, collagen microfibrils form large bundles adjacent to the cell's plasma membrane (inset), as occurs during de novo fibre assembly; in the C2C12/GFP-transplanted hearts, and even more in the C2C12/RLX-transplanted ones, collagen microfibrils are scattered and loosely arranged. Bar = 0.5 μm.
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fig03: Upper panels. Light micro-graphs of sections from control (A), C2C12/GFP- (B) and C2C12/RLX-transplanted hearts (C), stained with Van Gieson for collagen. A marked reduction of the extent of fibrosis can be seen in the ischaemic scars of the myoblast-transplanted hearts; of note, fibrosis is particularly attenuated in the hearts receiving C2C12/RLX cells. Bar = 20 μm. Centre panel. Sclerosis index (optical x volume density of collagen fibres) in the post-infarcted myocardi-um, estimated by computer-aided mor-phometry; (a) P<0.05 versus controls; (b) P<0.001 versus controls; (c) P<0.001 versus C2C12/GFP. Lower panels. Electron micrographs of fibrob-lasts from control (D) C2C12/GFP- (E) and C2C12/RLX-transplanted hearts (F). In the control, collagen microfibrils form large bundles adjacent to the cell's plasma membrane (inset), as occurs during de novo fibre assembly; in the C2C12/GFP-transplanted hearts, and even more in the C2C12/RLX-transplanted ones, collagen microfibrils are scattered and loosely arranged. Bar = 0.5 μm.

Mentions: By confocal immunofluorescence, numerous GFP-immunoreactive cells, either C2C12/GFP or C2C12/RLX, were found in the scar tissue from the transplanted groups but not in the control group (Fig. 1 A–C). Their myoblastic nature was confirmed by positive staining for a sarcomeric actin (Fig. 1D). C2C12/GFP-injected hearts contained a lower number of immunoreactive cells compared with C2C12/RLX-injected hearts (40 ± 7 versus 83 ± 12 cells/microscopical field; Student's t-test: P<0.01). The engrafted cells were preferentially located around blood vessels or in their close proximity and were almost absent in the peri-infarcted, viable myocardium. Interestingly, a clear-cut endothelial expression of ICAM-1 and VCAM (Fig.1E and F) was detected in small-sized blood vessels within the post-infarcted zone, but not in the non-ischaemic myocardium (Fig.1G), suggesting a role for activated endothelium of the injured regions in myoblast extravasation and homing. Immunoelectron microscopy of hearts transplanted with C2C12/RLX revealed the presence of RLX-producing cells in the post-infarction area, containing RLX gold labelling (Fig. 2). As expected, RLX-immunoreactive cells were not found in the hearts transplanted with C2C12/GFP (Fig. 2).The engrafted myoblasts could be distinguished by the activated fibroblasts in the scar because they showed a discontinuous basement membrane and lack collagen microfibrils apposed to the plasma membrane typical of fibrob-lasts (compare with Fig. 3D). However, the injected myoblasts remained single elements with no tendency to fuse into multinucleated myotubes, which were never observed in the scar tissue (Figs.1 and 2). This finding suggests that C2C12 myoblasts are unable to regenerate contractile tissue at the grafting site, at least in the current experimental conditions.


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 panels. Light micro-graphs of sections from control (A), C2C12/GFP- (B) and C2C12/RLX-transplanted hearts (C), stained with Van Gieson for collagen. A marked reduction of the extent of fibrosis can be seen in the ischaemic scars of the myoblast-transplanted hearts; of note, fibrosis is particularly attenuated in the hearts receiving C2C12/RLX cells. Bar = 20 μm. Centre panel. Sclerosis index (optical x volume density of collagen fibres) in the post-infarcted myocardi-um, estimated by computer-aided mor-phometry; (a) P<0.05 versus controls; (b) P<0.001 versus controls; (c) P<0.001 versus C2C12/GFP. Lower panels. Electron micrographs of fibrob-lasts from control (D) C2C12/GFP- (E) and C2C12/RLX-transplanted hearts (F). In the control, collagen microfibrils form large bundles adjacent to the cell's plasma membrane (inset), as occurs during de novo fibre assembly; in the C2C12/GFP-transplanted hearts, and even more in the C2C12/RLX-transplanted ones, collagen microfibrils are scattered and loosely arranged. Bar = 0.5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401276&req=5

fig03: Upper panels. Light micro-graphs of sections from control (A), C2C12/GFP- (B) and C2C12/RLX-transplanted hearts (C), stained with Van Gieson for collagen. A marked reduction of the extent of fibrosis can be seen in the ischaemic scars of the myoblast-transplanted hearts; of note, fibrosis is particularly attenuated in the hearts receiving C2C12/RLX cells. Bar = 20 μm. Centre panel. Sclerosis index (optical x volume density of collagen fibres) in the post-infarcted myocardi-um, estimated by computer-aided mor-phometry; (a) P<0.05 versus controls; (b) P<0.001 versus controls; (c) P<0.001 versus C2C12/GFP. Lower panels. Electron micrographs of fibrob-lasts from control (D) C2C12/GFP- (E) and C2C12/RLX-transplanted hearts (F). In the control, collagen microfibrils form large bundles adjacent to the cell's plasma membrane (inset), as occurs during de novo fibre assembly; in the C2C12/GFP-transplanted hearts, and even more in the C2C12/RLX-transplanted ones, collagen microfibrils are scattered and loosely arranged. Bar = 0.5 μm.
Mentions: By confocal immunofluorescence, numerous GFP-immunoreactive cells, either C2C12/GFP or C2C12/RLX, were found in the scar tissue from the transplanted groups but not in the control group (Fig. 1 A–C). Their myoblastic nature was confirmed by positive staining for a sarcomeric actin (Fig. 1D). C2C12/GFP-injected hearts contained a lower number of immunoreactive cells compared with C2C12/RLX-injected hearts (40 ± 7 versus 83 ± 12 cells/microscopical field; Student's t-test: P<0.01). The engrafted cells were preferentially located around blood vessels or in their close proximity and were almost absent in the peri-infarcted, viable myocardium. Interestingly, a clear-cut endothelial expression of ICAM-1 and VCAM (Fig.1E and F) was detected in small-sized blood vessels within the post-infarcted zone, but not in the non-ischaemic myocardium (Fig.1G), suggesting a role for activated endothelium of the injured regions in myoblast extravasation and homing. Immunoelectron microscopy of hearts transplanted with C2C12/RLX revealed the presence of RLX-producing cells in the post-infarction area, containing RLX gold labelling (Fig. 2). As expected, RLX-immunoreactive cells were not found in the hearts transplanted with C2C12/GFP (Fig. 2).The engrafted myoblasts could be distinguished by the activated fibroblasts in the scar because they showed a discontinuous basement membrane and lack collagen microfibrils apposed to the plasma membrane typical of fibrob-lasts (compare with Fig. 3D). However, the injected myoblasts remained single elements with no tendency to fuse into multinucleated myotubes, which were never observed in the scar tissue (Figs.1 and 2). This finding suggests that C2C12 myoblasts are unable to regenerate contractile tissue at the grafting site, at least in the current experimental conditions.

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