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Published as doi: 10.1096/fj.07-8252com.
 (The FASEB Journal. 2008;22:1021-1031.)
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(The FASEB Journal. 2008;22:1021-1031.)
© 2008 FASEB

Up-regulation of nestin in the infarcted myocardium potentially indicates differentiation of resident cardiac stem cells into various lineages including cardiomyocytes

Sergiu Scobioala*,{ddagger}, Rainer Klocke*, Michael Kuhlmann*, Wen Tian*, Lekbira Hasib*,{ddagger}, Hendrik Milting§, Simone Koenig{ddagger}, Matthias Stelljes{dagger}, Aly El-Banayosy§, Gero Tenderich§, Guenter Michel*, Guenter Breithardt* and Sigrid Nikol*,{ddagger}

* Department of Cardiology and Angiology, and

{dagger} Department of Medicine/Hematology and Oncology, University Hospital of Muenster, Muenster, Germany;

{ddagger} Interdisciplinary Center for Clinical Research (IZKF), University of Muenster, Muenster, Germany; and

§ Heart and Diabetes Center NRW, Ruhr University Bochum, Erich und Hanna Klessman Institute for Cardiovascular Research and Development, Bad Oeynhausen, Germany

1Correspondence: Medizinische Klinik und Poliklinik C, (Cardiology and Angiology), University of Muenster, Albert-Schweitzer-Str. 33, Muenster 48149, Germany. E-mail: nikol{at}uni-muenster.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To identify proteins involved in cardiac regeneration, a proteomics approach was applied. A total of 26 proteins, which displayed aberrant expression in mouse hearts infarcted through ligation of the left anterior descending coronary artery, were identified. These included the intermediate filament protein nestin, which was up-regulated in the infarct border zone. Corresponding changes were observed for its mRNA. Nestin mRNA was also up-regulated in hearts from 17 of 19 patients with end-stage heart failure, including 4 with acute myocardial infarction in comparison with 8 donor hearts. Immunofluorescence confocal laser scanning microscopy revealed that nestin is expressed, on the one hand, in small proportions of cardiomyocytes, endothelial cells, smooth muscle cells, neuronal cells, and fibroblasts. On the other hand, it was found to be coexpressed with the stem cell markers c-kit, Sca-1, Mdr-1, and Abcg2 in small interstitial cells. In infarcted hearts from chimeric mice transplanted with bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice, less than 1% of nestin-positive cells coexpressed EGFP, although EGFP-positive cells were abundant in these. Consequently, enhanced expression of nestin in the injured myocardium might reflect spontaneous regenerative processes supposedly based on the differentiation of resident cardiac stem cells into diverse cardiac cell types.—Scobioala, S., Klocke, R., Kuhlmann, M., Tian, W., Hasib, L., Milting, H., Koenig, S., Stelljes, M., El-Banayosy, A., Tenderich, G., Michel, G., Breithardt, G., Nikol, S. Up-regulation of nestin in the infarcted myocardium potentially indicates differentiation of resident cardiac stem cells into various lineages including cardiomyocytes.


Key Words: cardiac regeneration • myocardial infarction • heart failure • progenitor cells • proteomics • intermediate filament


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FOR MANY YEARS, IT HAS BEEN assumed that the heart is an organ incapable of regeneration. Cardiomyocytes were considered to be exclusively formed during embryogenesis and fetal development, so that their loss by senescence or cardiac injury cannot be compensated. However, recently it has been discovered that the heart contains a pool of primitive cardiac stem cells (CSCs) that reside in so-called niches and are capable of both proliferation and differentiation into cardiac cells, including myocytes (1 , 2) . It is therefore believed today that the heart has a limited degree of regenerative capacity that, however, may not be sufficient to facilitate efficient repair after the occurrence of serious injury such as myocardial infarction (MI) (1 , 2) . Bone marrow (BM) -derived progenitor cells immigrating into the damaged heart might also contribute to this rudimental regenerative capacity through poorly characterized mechanisms (3 , 4) .

Cellular processes such as those associated with regeneration, e.g., proliferation and differentiation, are activated and controlled by proteins. Therefore, the identification of changes in protein expression should contribute to the understanding of the mechanisms underlying regenerative processes in the infarcted heart. This identification can be accomplished by proteomic analysis, i.e., the combined application of two-dimensional (2D) gel electrophoresis for protein separation and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) for protein assignment (5) .

Proteomic analysis of human tissue samples from patients is hampered by factors such as limited availability of biopsies, disease complexity and stage, tissue heterogeneity, multimorbidity and age of patients, and the patient’s medical therapy. Accordingly, the application of proteomics to appropriate animal models constitutes a suitable alternative (6 , 7) . In the present work we used a surgical mouse model of MI based on the permanent ligation of the left anterior descending coronary artery (LAD) to identify differentially expressed proteins in the infarcted and noninfarcted areas of mouse myocardium (8) .

One of the proteins identified by this approach was the intermediate filament protein nestin, which was increased in the infarcted area and its border zone. Nestin was previously demonstrated to be associated with the development and differentiation of neuronal (9) , muscular (10) , and other tissues such as developing tooth bud (11) and testis (12) . Nestin, which is expressed in proliferating immature cells, is down-regulated during differentiation. However, after injury to muscular or neuronal tissue its expression is transiently reinduced (13 , 14) . Although nestin is widely used now as a marker of proliferating immature cells, little is known about its function. Several studies indicated a participation of nestin in the regulation of the assembly and disassembly of intermediate filaments in mitotically active cells, thereby suggesting a principal role in tissue regeneration (13 , 14) . We investigated the expression of nestin in CSCs compared with differentiated cardiac cells to reveal its suitability as an indicator of regeneration in the infarcted heart based on the differentiation of CSCs into the most important types of adult cardiac cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human cardiac tissue samples
Myocardial tissue samples were collected during implantation of a total artificial heart (15) or from explanted failing hearts during orthotopic heart transplantation, respectively. Left ventricular (LV) tissue samples were obtained from 19 patients suffering from heart failure (1 female/18 males; mean age 59.63 yr, range 41–67 yr) with coronary heart disease and with a history of previous MI. Four of them had acute MI with need for replacement of the native heart by a total artificial heart. Control LV tissue samples were obtained from 8 donor hearts, which could not be used for heart transplantation for technical reasons (4 females/4 males; mean age 36 yr, range 23–64 yr). For clinical data, see Supplemental Table 1. A declaration of consent for the scientific use of the samples was obtained from all patients.

Mouse model of myocardial infarction (coronary artery ligation)
MI was induced in male CD1 mice (age 8–12 wk, obtained from Charles River Deutschland GmbH, Sulzfeld, Germany) by permanent ligation of the LAD as described (see Supplemental Data) (8) . All animal experiments performed in this work were in accordance with the German Law on the Care and Use of Laboratory Animals.

Generation of EGFP+ chimeric mice
As described in Kuhlmann et al. (8) , 11 male chimeric C57BL/6 mice with enhanced green fluorescent protein (EGFP) -transgenic BM were generated.

Proteomic analysis
For a detailed description, see Supplemental Data. Briefly, protein expression was analyzed in the infarcted area, including the border zone in comparison with the noninfarcted area of infarcted mouse hearts 3 wk after permanent LAD ligation using 2D gel electrophoresis.

Protein separation in 2D gels was performed according to the manufacturer’s protocol (Bio-Rad, Munich, Germany). Spot patterns were analyzed using PDQUEST 7.3 7.3 (Bio-Rad). Protein assignment was accomplished by MALDI-MS analysis using Voyager STR-DE (Applied Biosystems, Hamburg, Germany).

Western blotting
Total protein extract (50 µg) was loaded per lane of sodium dodecyl sulfate gel. The monoclonal mouse anti-rat nestin antibody MAB353 (Chemicon, Temecula, CA, USA) was used in a dilution of 1:250. Antibody-antigen reactions were performed using Low Cross buffer (Candor Bioscience GmbH, Weissensberg, Germany) and were detected by a horseradish peroxidase-coupled anti-mouse secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using the ECL chemiluminescence system (ECL plus, Amersham Bioscience) and X-ray film (Hyperfilm, Amersham Bioscience). Three-dimensional densitometry of the blots was performed, and the scans were analyzed using ImageQuant imaging software (Molecular Dynamics, Sunnyvale, CA, USA).

RNA isolation, reverse transcription, and real-time quantitative reverse transciptase PCR (qRT-PCR)
Isolation of RNA from cardiac tissue samples was performed using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. Reverse transcription of mRNA to cDNA was performed using the ImProm-IITM Reverse Transcription System (Promega, Mannheim, Germany) according to the manufacturer’s protocol.

Gene expression on the RNA level was analyzed in the infarcted and noninfarcted areas of infarcted mouse hearts at 24 h, 48 h, 1 wk, 3 wk, and 6 wk after LAD ligation (5 animals per time point) in comparison with myocardium from sham-operated animals (n=3 to 5) by qRT-PCR (for primer sequences see Supplemental Table 2). Nestin mRNA level was additionally analyzed in human heart samples. qRT-PCR was performed using SYBR Green Reaction Mix (Eurogentec, Seraing, Belgium) on an ABI PRISM 7900HT Detection System (Applied Biosystems). Each sample was run in triplicate.

The levels of the various mRNAs in the tissue samples were quantified relative to Cyclophilin A (mouse samples) and β-actin (human samples) mRNA levels according to Sequence Detector User Bulletin 2 (Applied Biosystems).

Immunofluorescent staining
Mouse hearts were fixed in 3.7% formaldehyde, followed by dehydration in 0.8 M sucrose solution, O.C.T. embedding, and cryoconservation. Cross sections (5 µm) were used for fluorescence detection of EGFP+ cells or immunofluorescence detection of various antigens according to an established protocol (8) . The primary antibodies used were directed against nestin, Nkx-2.5, troponin T, proliferating cell nuclear antigen (PCNA), discoidin domain receptor 2 (DDR2), smooth muscle (SM) {alpha}-actin, neuronal nuclear antigen (NeuN), von Willebrand factor (vWF), c-kit, MDR1, Sca-1, and Abcg2 (see Supplemental Data). The primary antibodies were labeled using appropriate biotinylated secondary antibodies and streptavidin conjugated fluorochromes (see Supplemental Data).

Sections were examined using a confocal laser scanning microscope (LSM 510 META; Carl Zeiss MicroImaging, Inc., New York, NY). Images were digitized and transferred to a personal computer. For a quantitative analysis of different cell populations in the infarcted heart, the number of EGFP+ cells and cells expressing the immunohistological markers mentioned above were counted in at least three randomly selected fields of view at the same magnification (x400) in the border zone surrounding the infarcted area and in the noninfarcted area of the infarcted left ventricle wall. Likewise, colocalization of nestin with EGFP+ autofluorescence or the differentiation markers and stem/progenitor cell markers listed above was quantitatively analyzed. Corresponding immunohistological sections from sham-operated mice were used as controls.

Statistical data analysis
One-way ANOVA or, in the case of nonparametric data, the equivalent nonparametric test (Kruskal-Wallis ANOVA on ranks) was used for comparison among groups, followed by pairwise multiple comparison procedures (Student-Newman-Keuls method or Dunn’s method). The statistic analysis was performed using SigmaStat 3.1.1 Software package (Systat Software GmbH, Erkrath, Germany). Probability values <0.05 were considered to be significant; <0.01 were considered to be highly significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Spatial and temporal patterns of nestin expression in the infarcted myocardium
To identify proteins potentially involved in spontaneous regenerative processes in the infarcted mouse heart, we compared 2D gel electrophoresis separation of proteins extracted from the noninfarcted area with the infarcted area plus border zone applied in combination with MALDI-MS. Of 26 unambiguously identified, differentially expressed proteins (see Supplemental Data), the intermediate filament protein nestin appeared to be the most interesting one with respect to potential relevance for cardiac regeneration. Also, nestin is known to be associated with neuronal and myocyte differentiation and it is expressed during heart development (9 , 10) .

Nestin expression was up-regulated predominantly in the infarcted area and its border zone, as indicated by 2D gel electrophoresis (Fig. 1 A) and Western blotting (Fig. 1B ). In 2D gels, nestin was detected as a protein with a molecular weight of ~50 kDa (Fig. 1A ). This result was confirmed by Western blotting, which, however, also revealed the existence of a 200 kDa nestin variant (Fig. 1B ; see also Supplemental Data).


Figure 1
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  		Figure 1. A) Top: Direct overlay of electronic images representing average 2D gels of proteins from infarcted and noninfarcted areas of the left ventricular wall of infarcted mouse hearts 3 wk after surgery. Each average gel image was created from electronic images from 5 separate, fluorescently stained gels (total 10 gels). Proteins displaying significant differences in staining intensity between infarcted region plus adjacent border zone and noninfarcted area are labeled with numbers and are listed in Supplemental Table 3. Bottom: Enlargements of fluorescently stained gels highlighting differences in the expression of nestin (spot 20) in the noninfarcted area (NI) and in the infarcted region plus border zone (I&BZ) of an infarcted mouse heart. B) Detection of nestin by Western blotting in I&BZ and in NI of infarcted mouse hearts 3 wk after LAD ligation in comparison with hearts from sham-operated animals (C=control). Left: Display of Western blotting. Two variants of nestin are identified with molecular weights of ~200 and 50 kDa with the anti-rat nestin antibody. Using the polyclonal goat anti-mouse nestin antibody G-20 or the polyclonal rabbit anti-human nestin antibody H-85 (both from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) the same pattern of bands was observed (data not shown). Right: Quantitative data (50 kDa variant) obtained by densitometry of ECL film. The OD of nestin bands of infarcted and noninfarcted areas of the infarcted hearts were normalized to the mean OD of the nestin signal obtained for control hearts. Asterisks indicate statistically significant difference. Data are representative for three independent experiments and presented as mean ± SE.

Nestin mRNA levels were additionally determined at various time points after LAD ligation (Fig. 2 A). At 24 h the level of the nestin transcript was slightly increased in the infarcted area, whereas at 48 h, we observed 5- and 3-fold increased levels compared to those in hearts from sham-operated animals in the infarcted area and the noninfarcted area, respectively. At later time points (1, 3, and 6 wk), nestin mRNA levels returned to lower levels (~1.5- to 2.5-fold of the control level) in the infarcted area and to normal levels in the noninfarcted area (Fig. 2A ). Corresponding changes for nestin were obtained by immunofluorescence detection at 48 h after LAD (Fig. 2B ).


Figure 2
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  		Figure 2. Up-regulation of nestin in the infarcted mouse myocardium. A) Determination of nestin mRNA levels in the noninfarcted and infarcted areas (the latter plus border zones) of infarcted hearts in comparison with levels in myocardial tissue of sham-operated animals (control). The data were obtained for various follow-up periods after infarct induction (24 h, 48 h, 1 wk, 3 wk, and 6 wk) from 5 animals each per time point. RNA values are given relative to the normalized mean values determined for control tissue. Cyclophillin A mRNA levels served as internal standard. All samples were run in triplicate. Statistically significant time course changes of mRNA levels were determined in the infarcted (P<0.001) as well as in the noninfarcted (P<0.001) area of the myocardium. Significant differences between groups at individual time points are indicated by asterisks above brackets. Data are presented as mean ± SE. B) Immunohistological analysis of the spatial pattern of nestin expression in the infarcted myocardium. Based on the data obtained for m-RNA levels, nestin immunofluorescence (red) was analyzed 48 h after induction of MI. Blue fluorescence: DAPI-stained nuclei. Diagram: Nestin expression measured as nestin+ area in relation to the whole area of myocardial tissue in the field of view. Analysis included a minimum of 10 images from the border zone and noninfarcted area of 9 infarcted hearts and 3 control individuals from the corresponding area of LV in hearts. Significant differences between groups are indicated by asterisk above brackets. Data are presented as mean ± SE.

Nestin expression in acutely infarcted and chronically failing human myocardium
Nestin mRNA levels were also determined in human hearts from patients suffering from chronic heart failure (n=15) with a history of previous MI and from patients with acute MI (n=4) in comparison with levels in unaffected donor hearts (n=8) (for clinical data see Supplemental Table 1). In tissue samples from 14 chronically failing hearts and in 3 samples from acutely infarcted hearts, the levels of nestin mRNA were increased compared to the levels in nonfailing donor hearts (Fig. 3 ).


Figure 3
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  		Figure 3. Expression of nestin in failing and infarcted human hearts. Nestin mRNA levels in hearts from patients with acute MI (n=4) and chronic heart failure (n=15) were compared with those in transplantation donor hearts (n=8). The RNA levels are given relative to the normalized mean level determined for donor hearts (control). β-actin mRNA levels served as an internal standard. All samples were run in triplicate. Significant differences between groups are indicated by asterisks. Data are presented as mean ± SE.

Nestin expression in primitive cardiac cells
To evaluate the suitability of nestin as a marker of stem cell-based regeneration in the infarcted heart, its expression in CSCs was investigated using coimmunofluorescence detection with the stem cell markers c-kit+ (16) , MDR1+ (17) , Sca-1+ (18) , and Abcg2+ (19 , 20) by confocal microscopy. Since the highest level of nestin expression was observed 48 h after LAD ligation (see above), hearts isolated 2 days after the induction of MI (n=9) were used. Cells expressing the four stem cell markers were typically small and round or spindle-shaped and were detected as groups localized in the so- called niches (Fig. 4 ). They had a very high nucleus/cytoplasm ratio and, therefore, the stem cell markers, which are cell surface antigens, exhibited as signals closely surrounding the nucleus (Fig. 4) . All tested stem cell antigens were found to colocalize with nestin (Fig. 4) . The percentages of nestin coexpressing cells were 6.8 ± 3% of c-kit+, 8 ± 2.2% of MDR1+, 10 ± 4.3% of Sca-1+, and 12 ± 3.7% of Abcg2+ cells (Fig. 6) . The stem cells coexpressing nestin were localized in the border zone surrounding the infarcted area, whereas none of these cells were found in the noninfarcted area and in hearts of sham-operated animals (n=3). Nestin coexpressing stem cells displayed less intensive signals of the stem cell antigens, as cells expressing these antigens only (Fig. 4) . This finding could reflect a gradual loss of stem cell antigen expression by such primitive cells. Together with the coexpression with nestin and a more spindle-shaped and bigger appearance, this finding might indicate the initiation of differentiation in such cells.


Figure 4
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  		Figure 4. Expression of nestin in primitive cardiac cells in sections of the border zone of infarcted myocardium 48 h after coronary artery occlusion. Left panels: immunofluorescence detection (green) of the indicated stem cell makers; middle: nestin immunofluorescence (red); right: merged images of both left and middle panels. Nuclei are stained by DAPI (blue). Cardiomyocytes are sometimes visible by a green autofluorescence.


Figure 6
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  		Figure 6. Quantitative determination of proportions of the indicated stem cell and differentiated cardiac cell types expressing nestin. Cells were counted in midwall slices of an infarcted LV in the border zone and noninfarcted areas from 9 infarcted mouse hearts in at least three randomly selected fields of view each (120 microscope slides). Proportions of nestin+ cells relative to a selected number (see below) of a given cell type are indicated as average percentage from 9 infarcted hearts. Data are presented as mean ± SE. Significant differences between noninfarcted area and border zone are indicated by asterisks. Stem cells expressing nestin were found only in the border zone, and their respective proportions were calculated from 20 c-kit+, 24 MDR1+, 30 Sca-1+, and 20 Abcg2+ cells per infarcted mouse heart. Nestin+ differentiated cells were calculated from 45 cardiomyocytes (Nkx-2.5+), 50 fibroblasts (DDR2+), 50 SMCs (SM {alpha}-actin+) and ECs (vWF+), and 30 neuronal cells (NeuN+) per heart. In the corresponding regions of LV of 3 control hearts (34 microscope slides), no expression of nestin was detectable in both the stem cells and differentiated cells.

Nestin expression in differentiated cardiac cells
Nestin is known as a differentiation marker for myocyte precursors in the developing heart (10 , 14) . Therefore, it was investigated whether nestin is expressed in cardiomyocytes in infarcted hearts (n=9) 2 days after LAD ligation by the detection of coimmunofluorescence with Nkx-2.5 (Csx), a transcription factor restricted to the initial phases of myocyte differentiation (21) . In the border zone surrounding the infarcted area, 55 ± 5% of the cells with a distinct cross-striated structure expressed Nkx-2.5. Nestin was expressed in 45 ± 4% of Nkx-2.5+ cells in the border zone and in 10 ± 6% of Nkx-2.5+ cells in the noninfarcted area (Fig. 5 and 6 ). In the hearts of sham-operated animals (n=3) no Nkx-2.5+ cells coexpressing nestin were found.


Figure 5
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  		Figure 5. Expression of nestin in differentiated cardiac cells in sections of the border zone of infarcted myocardium 48 h after coronary artery occlusion. Top panels (from left to right): first panel, immunofluorescence (green) of Nkx2.5 (cardiomyocyte marker); second, nestin immunofluorescence (red); third, merged images of first and second panels; fourth, cross-striation pattern of nestin immunofluorescence (red), potentially indicating its association with the contractile apparatus in an apparently fully differentiated cardiomyocyte (surrounding cardiomyocytes visible by green autofluorescence). Panels of the second, third, fourth, and fifth row: left panels, immunofluorescence detection (green) of the indicated cardiac cell markers; middle, nestin immunofluorescence (red); right, merged images of both left and middle panels. Nuclei are stained by DAPI (blue). Frames: vessel cross sections.

Furthermore, the expression of nestin in cardiac fibroblasts, smooth muscle cells (SMCs) and endothelial cells (ECs) in infarcted mouse hearts (n=9) were investigated. Nestin was expressed in cardiac fibroblasts as demonstrated by coimmunofluorescence detection with DDR2, a specific marker for fibroblasts (22) . About 45 ± 8% of the fibroblasts in the border zone were positive for nestin vs. only 8 ± 3% of fibroblasts in the noninfarcted area (Figs. 5 and 6) . Nestin+ cardiac fibroblasts were not detected in control hearts (n=3). A large proportion of the SMCs and ECs in the border zone—identified by SM {alpha}-actin or vWF staining, respectively—were found to be nestin positive (63±3 or 54±7%, respectively). In contrast, in the noninfarcted area, the proportion of such cells was significantly smaller (15±4 and 11±5%; Figs. 5 and 6 ) and in the hearts of sham-operated mice (n=3) nestin+ SMCs and ECs were missing.

To investigate nestin expression in neuronal cells in the infarcted mouse myocardium (n=5), its coexpression with NeuN—a specific marker for neuronal cells (23) —was examined. About 17 ± 4% of the neuronal cells in the border zone were positive for nestin vs. 5 ± 2% in the noninfarcted region (Figs. 5 and 6) . In the hearts of sham-operated mice (n=3) nestin+ neuronal cells were missing.

Nestin expression in proliferating cells
To reveal whether nestin was expressed in proliferating cells, coimmunofluorescence detection of nestin and PCNA in infarcted hearts (n=4) was performed. The vast majority of PCNA+ cells were localized in the border zone (Fig. 7 ). Of the PCNA+ cells in the border zone, 68 ± 3% were also nestin+ (Fig. 7) . In the noninfarcted area, a significantly smaller proportion of PCNA+ cells (3.7±0.4%) was also nestin+. In LV tissue from noninfarcted hearts (n=3), no PCNA+ cells were found.


Figure 7
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  		Figure 7. Expression of nestin in proliferating (PCNA+) cells. A) Sections of the border zone of infarcted myocardium 48 h after coronary artery ligation. Left panel: proliferating cells identified by the green PCNA immunofluorescence in nuclei; middle: nestin immunofluorescence; right: merged images of both PCNA and nestin immunofluorescence (white frames: cells expressing both markers). Nuclei are stained by the blue fluorescence of DAPI. B) Quantification of nestin+/PCNA+ cells in the border zone and in the noninfarcted area in at least three randomly selected fields of view from 4 infarcted mouse hearts (43 microscope slides). The same numbers of PCNA+ cells (n=40) were selected from the border zone as well as from the noninfarcted area. Proportions of nestin+/PCNA+ cells relative to all PCNA+ cells are given as average percentages from 4 infarcted hearts. Data are presented as mean ± SE. An asterisk indicates significant differences between groups. No PCNA+ cells were determined in the LV of control hearts (3 individuals, 22 images).

Origin of nestin-positive cells
Infarcted hearts of chimeric mice with EGFP-transgenic BM were analyzed by fluorescence microscopy for the presence of EGFP+ (BM-derived), nestin+, and nestin+/EGFP+ cells. Both nestin+ and EGFP+ cells were predominantly localized in the infarcted area and its border zone. Only a very small proportion of nestin+ cells (<1%) were found to coexpress EGFP, although EGFP+ cells were abundant in these hearts (Fig. 8 ).


Figure 8
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  		Figure 8. Expression of nestin in infarcted hearts (border zone) of chimeric mice with EGFP-transgenic BM. Left panel: EGFP fluorescence detection (dark green, autofluorescence of cardiomyocyte); left panel box: lower magnification reveals abundant EGFP fluorescence (green) in the border zone of the infracted myocardium (dark red, immunofluorescence of troponin T in cardiomyocytes); middle panel: nestin immunofluorescence detection; right panel: merged images. Seven nestin+/EGFP+ cells from 1523 nestin+ cells (counted in 135 microscope slides) were identified. Arrow: one of the 7 nestin+/EGFP+ cells. Blue: DAPI-stained nuclei.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Proteomic analysis of infarcted mouse hearts enabled the identification of 26 proteins aberrantly expressed in the infarcted area or its border zone. These proteins belong to various functional classes (see Supplemental Data). However, as this work aimed at the identification of proteins potentially involved in cardiac regeneration, the most interesting finding was the up-regulation of the intermediate filament protein nestin. Primarily, nestin was identified in proliferating neuronal progenitor cells in the central and peripheral nervous system (24) . It was found to be overexpressed in nerve tissue of patients suffering from various neurological diseases, e.g., Parkinson’s disease (25) , multiple sclerosis (26) , and pituitary tumors (27) , and also in malignant melanoma (28) . Nestin has also been shown to be expressed during the development of myogenic tissues (10) , the differentiation of myoblast cell cultures, and the regeneration of skeletal muscle (29 , 30) . Its significantly elevated expression, not only in the infarcted mouse myocardium but also in human acutely infarcted and chronically failing hearts, suggests that regenerative processes based on cell differentiation are spontaneously activated in the damaged heart, irrespective of whether it is acutely or chronically affected. This interpretation accords with recent findings by other groups who also presented evidence for cell differentiation in the damaged myocardium (31 32 33) .

However, regarding the origin of stem cells delivering progenitor cells that commit to differentiate into adult cardiac cells in the damaged heart, different but not mutually exclusive explanations are possible. Such cells may be of cardiac origin and/or they may be BM-derived. The mobilization of the latter by myocardial injury and their immigration into the injured heart have been independently demonstrated by several groups (8 , 31 , 34) . Nevertheless, whether these BM-derived cells can transdifferentiate into adult heart cells when exposed to the cardiac surrounding is still controversial (4 , 8 , 35) . However, several groups provided evidence for the existence of resident CSCs that can produce progenitors committed to differentiation into adult cardiac cells (31 , 36) . In the present work the investigation of infarcted hearts from chimeric mice transplanted with BM from EGFP transgenics revealed that the vast majority of nestin+ cells (more than 99%) were not BM-derived but had resident origin, although EGFP+, BM-derived cells were abundant in the damaged hearts. This result is in accordance with data published by Fukuhara et al. (37) , which demonstrated that, in the infarcted myocardium of cytokine (G-CSF)-treated mice, only 2% of nestin-positive cells were of BM origin.

To identify stem/progenitor cells potentially involved in spontaneous cardiac regeneration four stem cell markers, c-kit, Sca-1, MDR1, and Abcg2, were tested for coexpression with nestin. Proportions of 6.8 to 12% of the stem/progenitor cell subpopulations defined by these markers were found to coexpress nestin, suggesting that these cells were activated to differentiate in the infarcted hearts. Also, however, proportions (17 to 63%) of the cell populations defined by five markers of differentiated cardiac cells—Nkx-2.5 (cardiomyocytes), SM {alpha}-actin (vascular SMCs), vWF (ECs), DDR2 (fibroblasts), and NeuN (neuronal cells)—were found to coexpress nestin. Based on the assumption that coexpression of these markers with nestin indicates a more immature than fully differentiated state, these data suggest that new cardiomyocytes, ECs, SMCs, fibroblasts, and neuronal cells are formed in the infarcted heart by the differentiation of stem/progenitor cells (Fig. 9 ).


Figure 9
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  		Figure 9. Hypothetical role of nestin+ cells in the infarcted heart: coexpression of nestin with stem cell markers identifies resident CMCs delivering nestin+ progenitors and precursors, which are committed to differentiate into adult cardiac cells including cardiomyocytes and ECs. Immature variants of the latter are still nestin+.

The coexpression of nestin, with all tested stem/progenitor cell markers on the one hand and all lineage markers on the other, suggests that increased nestin expression indicates cell differentiation in the infarcted heart but does not significantly affect its specific direction. This finding is in accordance with the data of Beltrami et al. (31) , who showed that adult CSCs give rise to cardiomyocytes, SMCs, and ECs, and of Linke et al. (38) , who found that the relative proportion of myocardial cells generated by each class of CSC, as defined by the markers c-kit, MDR1, and Sca-1 in that work, is approximately identical, namely myocytes {approx}50%, SMCs {approx}40%, and ECs {approx}10%.

Lardon et al. (39) published that nestin is expressed by angiogenic ECs, which contributes to the formation of neocapillaries during pancreas regeneration. Correspondingly, the detection of nestin in cardiac ECs may reflect neoangiogenesis in the infarcted myocardium, which is known to be associated with postinfarction remodeling (40) . The differentiation of stem cells into vascular SMCs, as suggested by nestin expression in the latter, may reflect arteriogenesis, which is also known to be induced by ischemia and MI (41 , 42) .

Nestin was also detected in cardiac fibroblasts. Although this cell type significantly contributes to scar formation, which counteracts regeneration, fibroblasts are indispensable for the formation of an intact extracellular matrix in the heart due to their ability to produce several types of collagens and MMPs (43 , 44) . Expressing growth factors and being involved in cytokine signaling, cardiac fibroblasts can modulate many heart functions, such as growth and differentiation (45 , 46) .

The detection of nestin+ neuronal cells in the infarcted mouse heart accords with data obtained by Drapeau et al. (47) and El-Helou et al. (48) , who demonstrated the presence of nestin+ neuronal cells in the infarct and peri-infarct region of the infarcted rat heart. Apart from potentially contributing to innervation of the infarct and peri-infarct area (47 , 48) , neuronal progenitor cells in the infarcted heart might support the formation of vessels by paracrine mechanisms, as suggested by Raab and co-workers (49) .

Apart from differentiation, cell proliferation is a prerequisite for regeneration and may therefore be indicative for it (50) . In accordance with this notion and work from other groups (33 , 37) , many PCNA-positive cells were detected in the border zone of MI. As most of these were also nestin-positive, it can be assumed that the proliferating cells were progenitor or precursor cells derived from CSCs and committed to differentiate into adult cardiac cells.

In conclusion, the enhanced expression of nestin in hearts damaged by acute MI or chronic ischemia may indicate spontaneous activation of stem cells of resident cardiac origin, leading to the production of progenitor cells and their differentiation into various types of myocardial cells. Consequently, nestin could be used as a biomarker suitable to monitor stem cell-based activity underlying regenerative processes such as differentiation and proliferation in the infarcted or failing heart.


   ACKNOWLEDGMENTS
 
We thank the Interdisciplinary Center for Clinical Research IZKF Münster B15 (S.S., S.N.), the Hans und Gertie Fischer-Stiftung (S.S., R.K., M.K., S.N.) and Innovative Medical Research IMF Ni 510404 (S.S., S.N.) for financial support; Dr. Oliver Schmidt for technical advice; Sezen Maleki for expert technical assistance; and Dr. Jared Sterneckert for carefully reading the manuscript.

Received for publication February 27, 2007. Accepted for publication October 4, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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