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. Author manuscript; available in PMC: 2012 Feb 18.
Published in final edited form as: Circ Res. 2011 Jan 13;108(4):478–489. doi: 10.1161/CIRCRESAHA.110.229658

Myocardial Injection with GSK-3β-Overexpressing Bone Marrow-Derived Mesenchymal Stem Cells Attenuates Cardiac Dysfunction after Myocardial Infarction

Jaeyeaon Cho 1, Peiyong Zhai 1, Yasuhiro Maejima 1, Junichi Sadoshima 1
PMCID: PMC3109296  NIHMSID: NIHMS269243  PMID: 21233455

Abstract

Rationale

Glycogen synthase kinase-3β (GSK-3β) upregulates cardiac genes in bone marrow-derived mesenchymal stem cells (MSCs) in vitro. Ex vivo modification of signaling mechanisms in MSCs may improve the efficiency of cardiac cell based therapy (CRT).

Objective

To test the effect of GSK-3β upon the efficiency of CBT with MSCs after myocardial infarction (MI).

Methods and Results

MSCs overexpressing either GSK-3β (GSK-3β-MSCs) or LacZ (LacZ-MSCs) or saline was injected into the heart after coronary ligation. A significant improvement in the mortality and left ventricular (LV) function was observed at 12 weeks in GSK-3β-MSC-injected mice compared to in LacZ-MSC- or saline-injected mice. MI size and LV remodeling were reduced in GSK-3β-MSC-injected mice compared to in LacZ-MSC- or saline-injected ones. GSK-3β increased survival and increased cardiomyocyte (CM) differentiation of MSCs, as evidenced by activation of an Nkx2.5-LacZ reporter and upregulation of troponin T. Injection of GSK-3β-MSCs induced Ki67 positive myocytes and c-Kit positive cells, suggesting that GSK-3β-MSCs upregulate cardiac progenitor cells. GSK-3β-MSCs also increased capillary density and upregulated paracrine factors, including vascular endothelial growth factor A (Vegfa). Injection of GSK-3β-MSCs in which Vegfa had been knocked-down abolished the increase in survival and capillary density. However, the decrease in MI size and LV remodeling, and the improvement of LV function were still observed in MI mice injected with GSK-3β-MSCs without Vegfa.

Conclusions

GSK-3β significantly improves the efficiency of CBT with MSCs in the post-MI heart. GSK-3β not only increases survival of MSCs but also induces CM differentiation and angiogenesis through Vegfa-dependent and -independent mechanisms.

Keywords: mesenchymal stem cells, glycogen synthase kinase-3β, myocardial infarction, cell based therapy, vascular endothelial growth factor A

Introduction

The myocardium has been regarded as a terminally differentiated organ lacking regenerative potential. However, increasing lines of evidence suggest that the heart is an organ with a regenerative capacity 1. Cardiac progenitor cells localized in niches have the ability to differentiate into cardiomyocytes (CMs), thereby replenishing the CMs lost due to basal turnover 2, 3. However, the capacity for cardiac regeneration is insufficient to maintain cardiac function when a massive loss of CMs occurs in response to myocardial infarction (MI) and heart failure. Various adult stem cells, including bone marrow-derived cells 4, endothelial progenitor cells 5, 6, and mesenchymal stem cells (MSCs) 7-9, have been shown to be capable of differentiation into CMs, support survival of residential CMs, induce angiogenesis, or promote infarct healing when introduced into the heart after MI, clearly establishing the proof of concept that cell based therapy (CBT) has the potential to improve cardiac function in the post-MI heart. However, both basic studies with experimental animals and clinical studies have suggested that the improvement in cardiac function in the post-MI heart is modest and that a breakthrough is needed to improve survival and CM differentiation of the injected progenitor cell population 10.

MSCs are convenient as a source of CBT, since autologous MSCs can be isolated easily and manipulated ex vivo. MSCs have the ability to differentiate into various cell types in the heart, and secrete paracrine factors stimulating the survival of residential CMs, the tissue repair process, and angiogenesis, making MSCs an ideal source for cardiac CBT 11-13. However, the effectiveness of CBT with MSCs in the heart has not been remarkable thus far. CBT with MSCs in post-MI animal models caused only modest or transient functional improvement, possibly due to the rare survival and the very inefficient CM differentiation of MSCs in vivo 14. In order to circumvent these potential shortcomings of CBT with MSCs in the heart, ex vivo or in situ manipulations of MSCs have been reported. For example, ex vivo introduction of Akt increased the survival of MSCs and improved cardiac function of the post-MI heart in mice subjected to CBT 15. However, to our knowledge, the efficiency of in situ CM differentiation of MSCs is insufficient for the CBT to achieve significant functional improvement of the heart 16.

Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase which phosphorylates many intracellular substrates, including β-catenin, glycogen synthase, eIF2Bε, GATA4, myocardin, c-Jun, cyclin D1, and N-Myc, thereby regulating various intracellular functions 17. GSK-3 also regulates Wnt, Notch and hedgehog, major signaling proteins involved in cell growth/differentiation 18-20. We have shown recently that overexpression of GSK-3β induces expression of CM-specific genes and proteins, in part through downregulation of β-catenin, while it prevents expression of non-cardiac markers, such as neuronal markers, in MSCs in vitro 18. Since induction of CM differentiation in embryonic stem cells before injection improves the efficiency of CM differentiation and diminishes differentiation into other cell types 21, 22, we speculated that CM differentiation of MSCs with GSK-3β might also enhance the efficiency of CBT.

The goals of this study were 1) to evaluate whether myocardial injection of GSK-3β-overexpressing MSCs (GSK-3β-MSCs) improves survival of the animals and LV function, and attenuates cardiac remodeling in the post-MI heart compared to injection of control MSCs, and 2) to investigate the underlying mechanism through which injection of GSK-3β-MSCs improves the efficiency of CBT.

Materials and Methods

MSC Culture

MSCs were isolated from bone marrow aspirates from 2-3 week old C57BL/6 mice, Tet-off GSK-3β transgenic mice (Tg-Tet-GSK-3β-tTA) 17, transgenic mice harboring mouse Nkx2.5 (9.0 kb) promoter-driven LacZ 18, and green fluorescent protein (GFP) transgenic mice. MSCs were cultured in a 1:1 mixture of DMEM/F12 (Invitrogen) and mesenchymal basal medium (Stem Cell) supplemented with 10% fetal bovine serum (Atlanta Biologicals) and 1% L-glutamine (Invitrogen). MSCs passaged 3-5 times were transduced with adenoviruses 48 hours before myocardial injection. For induction of GSK-3β in MSCs prepared from Tg-Tet-GSK-3β-tTA mice, the mice were treated with doxycycline (Dox) as described 17 until MSC isolation and MSCs were then treated with Dox until 48 hours before myocardial injection.

MSC Injection into the Mouse Model of MI

The mouse model of chronic MI has been described previously 23. Three month old C57BL/6 mice were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg). The mice were ventilated via tracheal intubations connected to a rodent ventilator with 65% oxygen during the surgical procedure. The left anterior descending branch of the coronary artery (LAD) was ligated using an 8-0 nylon suture, and 30 μl of saline alone or MSCs suspended in saline (1.5×105 cells/30 μl) were injected into the MI border zone just after coronary artery ligation. Echocardiography was performed before the surgery and 2, 6 and 12 weeks after coronary ligation. Hemodynamic measurement was conducted at 12 weeks and the mice were then euthanized for histological and biochemical analysis. Mice which did not recover from the initial anesthesia were excluded from the analysis.

Statistical Analyses

All values are expressed as mean ± SEM. Statistical analyses were performed using ANOVA with Newman-Keuls multiple comparison test and unpaired t test with Welch's correction with a P<0.05 considered significant.

Results

Injection of GSK-3β-overexpressing MSCs prevents cardiac dysfunction after MI

The effect of GSK-3β expression upon the efficiency of CBT with MSCs was evaluated using a mouse model of MI. MSCs were transduced with either Ad-LacZ or Ad-GSK-3β ex vivo 48 h before surgery. MSCs were injected into the myocardium of the border zone immediately after permanent ligation of the coronary artery (Online Figure I). We prepared 4 groups of mice, namely sham and MI plus injection of saline, LacZ-overexpressing MSCs (LacZ-MSCs), or GSK-3β-overexpressing MSCs (GSK-3β-MSCs). All sham-operated mice survived, whereas only 70% of the saline-injected MI mice survived at 12 weeks. Although injection with LacZ-MSCs did not improve survival compared to injection with saline, all mice injected with GSK-3β-MSCs survived (Figure 1A). To evaluate changes in LV dimensions and contractility, echocardiographic analyses were conducted before the surgery and after 2, 6 and 12 weeks (Figure 1B-D). In the saline-injected MI mice, a significant increase in LV end-diastolic dimension (LVEDD) and LV end-systolic dimension (LEVSD), and a significant decrease in fractional shortening (%FS) were observed compared to in the sham operated mice at 2 weeks and thereafter, suggesting that the LV is dilated and the systolic function is compromised. In the LacZ-MSC-injected MI mice, both LVEDD and LVESD increased progressively, reaching significance (P<0.001) compared to those in sham operated mice at 12 weeks (Figure 1B-C), while %FS was significantly and progressively decreased from 2 weeks on (Figure 1D), suggesting that injection of LacZ-MSCs retards LV dilation but does not prevent LV dysfunction. In the GSK-3β-MSC-injected MI mice, neither LVEDD nor LVESD was significantly increased compared to those in the sham operated mice at 2, 6 and 12 weeks (Figure 1B-C). Although %FS in the GSK-3β-MSC-injected MI mice gradually decreased, it was significantly higher than in the saline- or LacZ-MSC-injected MI mice (Figure 1D). (-)dP/dt was significantly decreased in both the saline- and LacZ-MSC-injected MI mice compared to in the sham operated group, but was maintained in the GSK-3β-MSC-injected MI mice (Figure 1E and Online Table IA). LVEDP was significantly elevated in the saline-injected MI mice, but not in the LacZ-MSC- or GSK-3β-MSC-injected MI mice, compared to in the sham-operated group (Figure 1F and Table S2A). Since the saline- and LacZ-MSC-injected MI mice had a higher mortality rate than the sham operated or GSK-3β-MSC-injected MI mice, the actual chamber size could be even greater and LV function could be even lower in the former groups. These results suggest that injection of untreated MSCs alone does not have long-term therapeutic effects, but ex vivo introduction of GSK-3β significantly improves the therapeutic effect of CBT with MSCs after MI.

Figure 1. Injection of GSK-3β-MSCs prevents cardiac dysfunction after MI.

Figure 1

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(A) Survival of MI mice injected with LacZ-overexpressing MSCs (LacZ-MSCs), GSK-3β-overexpressing MSCs (GSK-3β-MSCs) or saline was followed up for 12 weeks. LacZ-MSCs or saline was injected as controls for GSK-3β-MSCs. Sham operated mice were prepared as a basal control. (B-D) Echocardiographic analyses were conducted at 0, 2, 6 and 12 weeks. Comparison of left ventricular end-diastolic dimension (LVEDD) (B), left ventricular end-systolic dimension (LVESD) (C) and fractional shortening (FS) (D). (E, F) Hemodynamic analyses at 12 weeks. Comparison of (-)dP/dt (E) and end-diastolic pressure (EDP) (F). Data are shown as mean ± SEM. * P<0.05, ** P<0.01 and *** P<0.001 vs. Sham mice; # P<0.05, ## P<0.01, ### P<0.001 vs. GSK-3β-MSC-injected MI mice.

GSK-3β-overexpressing MSCs prevent cardiac hypertrophic remodeling

Twelve weeks after MI, the heart weight/ body weight (HW/BW) and myocyte cross sectional area in the remote and adjacent areas were significantly greater in the saline- and LacZ-MSC-injected MI mice than in the sham-operated mice, indicating that these mice undergo cardiac remodeling (Figure 2A-D). Although HW/BW and myocyte cross sectional area in the adjacent area were significantly greater in the GSK-3β-MSC-injected mice than in the sham-operated mice, they were significantly smaller in the GSK-3β-MSC-injected mice than in saline- or LacZ-MSC-injected mice (Figure 2A-D). There was no significant difference in myocyte cross sectional area in the remote area between sham and GSK-3β-MSC-injected MI mice, and the latter had a significantly smaller myocyte cross sectional area in the remote area than either saline- or LacZ-MSC-injected MI mice (Figure 2A-D). Masson-Trichrome-stained horizontal sections showed a markedly enlarged LV in saline-injected MI mice and a slightly enlarged LV in LacZ-MSC-injected MI mice, but no significant LV dilation in GSK-3β-MSC-injected MI mice (Figure 2E). The area of infarction size/LV in GSK-3β-MSC-injected MI mice (19 ±3%) was significantly smaller than that in saline- and LacZ-MSC-injected MI mice (39±4% and 28±3%) (Figure 2F). The wall thickness of the MI area in the GSK-3β-MSC-injected MI mice was 2-fold greater than that of saline- and LacZ-MSC-injected MI mice (Figure 2G). These results suggest that injection with GSK-3β-MSCs promotes repair of MI and/or suppresses MI progression, thereby alleviating the cardiac remodeling seen with injection with saline or LacZ-MSCs.

Figure 2. Injection of GSK-3β-MSCs prevents MI progression and cardiac hypertrophy following MI.

Figure 2

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(A) Comparison of heart weight/body weight (HW/BW) at 12 weeks. (B-D) Cell size analyses at 12 weeks. (B) Relative cross-sectional area of myocytes in the remote area. (C) Relative cross-sectional area of myocytes in adjacent area. Cell size of sham operated mice was used as a control and set as 100%. (D) Representative pictures of wheat germ agglutinin (WGA) immunohistochemistry in remote and adjacent areas. (E-G) Massson's Trichrome staining. (E) Representative pictures of left ventricular sections. (F) Comparison of infarction size. (G) Comparison of the wall thickness in MI area. Data are shown as mean ± SEM. * P<0.05, ** P<0.01 and *** P<0.001 vs. Sham mice; # P<0.05, ## P<0.01, ### P<0.001 vs GSK-3β-MSC-injected MI mice.

Stable expression of GSK-3β in MSCs improves the efficiency of CBT for chronic myocardial infarction

In order to exclude non-specific effects of adenovirus transduction for the ex vivo introduction of GSK-3β upon the therapeutic effects of GSK-3β-MSCs, and to induce stable expression of GSK-3β overexpression in MSCs in situ, MSCs were isolated from tet-regulatable GSK-3β transgenic mice (Tet-GSK-3β-MSCs), in which expression of GSK-3β is induced in the absence of doxycycline (Dox) (Figure 3A). These MSCs were then injected into the mouse model of MI as described above. MSCs isolated from GFP transgenic mice (GFP-MSC) were injected as a control. All Tet-GSK-3β-MSC-injected MI mice survived, whereas GFP-MSC- and saline-injected MI mice exhibited significantly elevated mortality (Figure 3B). Echocardiographic analyses showed that %FS, an index of LV contraction, was significantly higher in Tet-GSK-3β-MSC-injected MI mice than in GFP-MSC-injected MI mice (Figure 3C). Hemodynamic measurement showed that LV end diastolic pressure (EDP) was significantly lower in Tet-GSK-3β-MSC-injected MI mice than in GFP-MSC-injected MI mice at 12 weeks (Figure 3D-E and Online Table IB). Furthermore, myocyte cross sectional area in the MI adjacent area was significantly greater in GFP-MSC-injected MI mice than in Tet-GSK-3β-MSC-injected MI mice (Figure 3F). These results suggest that stable overexpression of GSK-3β in MSCs is beneficial and inhibits cardiac remodeling after MI.

Figure 3. Stable overexpression of GSK-3β in MSCs improves the efficiency of CBT in a mouse model of MI.

Figure 3

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(A) Immunoblot assay of expression of GSK-3β, β-catenin and GAPDH in GFP-MSCs and Tet-GSK-3β-MSCs with or without doxycycline treatment. (B) Survival of MI mice injected with GFP-MSCs, Tet-GSK-3β-MSCs or saline was followed up for 12 weeks. Saline was injected as a vehicle control. (C) The raw echocardiographic data at 2 and 6 weeks, and comparison of fractional shortening (FS). (D, E) Hemodynamic analyses at 12 weeks. Comparison of end-diastolic pressure (EDP) (D) and (-)dP/dt (E). (F) Immunohistochemistry of wheat germ agglutinin (WGA) in adjacent areas and a quantitative analysis of cross-sectional area of myocytes. Data are shown as mean ± SEM. * P<0.05, ** P<0.01 vs. MI mice injected with GFP-MSCs.

GSK-3β overexpression in MSCs enhances survival and CM differentiation of MSCs in vivo

To examine CM differentiation of MSCs, MSCs isolated from GFP transgenic mice were injected after ex vivo GSK-3β transduction. Twelve weeks after MI, more GFP positive cells were found when GFP-MSCs transduced with GSK-3β were injected than when untreated or LacZ-transduced GFP-MSCs were injected (Figure 4AB and Online Figure II). In MI mice injected with GSK-3β-transduced GFP-MSCs, GFP positive cells were observed at a rate of 25.8 cells per 1.0×104 μm2 of MI area, and GFP and troponin T double positive cells were observed at a rate of 9 cells per 1.0×104 μm2 of MI area (Figure 4B). Thus, approximately 35% of the GFP positive cells were troponin T-positive, suggesting that upregulation of GSK-3β in MSCs facilitates CM differentiation in vivo. The histologically determined size of troponin T/GFP double positive cells was significantly smaller than that of troponin T positive/GFP negative cells (Figure 4C), suggesting that troponin T/GFP double positive cells could be newly generated CMs.

Figure 4. GSK-3β overexpression induces CM differentiation of MSCs in vivo.

Figure 4

Figure 4

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(A-C) MSCs isolated from GFP transgenic mice were injected with or without Ad-LacZ or Ad-GSK-3β transduction. (A) Immunohistochemistry of GFP, troponin T (TnT) and 4′,6-diamidino-2-phenylindole (DAPI). The rightmost panel indicates images from confocal microscopic analyses. (B) A quantitative analysis of GFP and TnT double positive cells at 12 weeks. (C) Comparisons of size of TnT positive myocytes and TnT and GFP double positive myocytes. (D-F) Nkx2.5-LacZ-MSCs (MSCs isolated from Nkx2.5 promotor-LacZ transgenic mice) were injected with or without GSK-3β transduction. (D, E) Immunohistochemistry of X-Gal (blue) and TnT (brown), and a quantitative analysis of X-Gal positive cell number per unit area. Slides were counterstained with Gill's formulation #2 hematoxylin. Data are shown as mean ± SEM. (F) Sections were subjected to X-Gal staining and immunofluorescent staining of TnT. An inset shows a large magnification of TnT staining in a X-gal positive cells.

In order to obtain genetic evidence of CM differentiation, MSCs were obtained from transgenic mice harboring LacZ driven by the cardiac-specific Nkx2.5 promoter. Nkx2.5-LacZ-MSCs were transduced with either control virus or GSK-3β virus ex vivo and then injected into the MI mice. X-Gal positive cells were observed only when Nkx2.5-LacZ-MSCs were transduced with GSK-3β. Approximately 34 cells per 1 mm2 were positive for X-Gal in the cytosol (Figure 4D-E and Online Figure III). Most of the troponin T- and X-Gal-double positive cells were clustered along the epicardium of the MI area. Co-expression of troponin T and LacZ was also confirmed by immunofluorescent staining for troponin T together with X-Gal staining (Figure 4F). Our results suggest that injection of GSK-3β-MSC into the MI mice induces CM differentiation more efficiently than that of control MSCs. It should be noted that a clear striation pattern of troponin T was not observed in the X-Gal positive cells, suggesting that, although the Nkx2.5 promoter is activated, these cells are not fully differentiated.

Injection of Tet-GSK-3β-MSCs or GSK-3β-MSCs in MI mice significantly increased clusters of c-Kit positive cells in both the adjacent and remote areas compared to saline injection (Figure 5A), suggesting that GSK-3β induces either expression of c-Kit in the existing cell population or recruitment of c-Kit positive cells in the MI heart. Furthermore, injection of Tet-GSK-3β-MSCs or GSK-3β-MSCs in MI mice also increased Ki67 positive troponin T positive cells (Figure 5B), suggesting that injection of GSK-3β-MSCs in the MI heart may stimulate proliferation of myocytes or myocyte progenitors.

Figure 5. Injection of GSK-3β-MSCs increases c-Kit and Ki67 positive cells.

Figure 5

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Saline, Tet-GSK-3β-MSCs (without doxycycline), LacZ-MSCs or GSK-3β-MSCs were injected into mouse hearts after permanent coronary ligation. (A) Immunohistochemistry of c-Kit and DAPI in remote and adjacent areas. (B) Immunohistochemistry of Ki67 (Green), troponin T (Red) and DAPI (Blue) staining in remote and adjacent areas. (C) MSCs were transduced with Ad-LacZ or Ad-GSK-3β in vitro and subjected to hypoxia for 72 hours. Expression of GSK-3β, caspase-3, cleaved caspase-3 and GAPDH was evaluated with immunoblot analyses. The result shown is representative of 3 experiments.

Since GSK-3β-MSCs appeared to survive better than LacZ-MSCs in the MI heart, we investigated the effect of hypoxia upon activation of cell death mechanisms in MSCs in vitro. Incubation of LacZ-MSCs and GSK-3β-MSCs under hypoxia induced significant increases in cleaved caspase-3 in LacZ-MSCs but not in GSK-3β-MSCs. Thus, suppression of apoptosis may contribute to the overall salutary effects of GSK-3β-MSCs in this study (Figure 5C).

The therapeutic benefits of GSK-3β-overexpressing MSCs is in part mediated by angiogenesis through Vegfa production in MSCs

Capillary density in the MI area, as evaluated by platelet endothelial cell adhesion molecule-1 (PECAM-1) and von Willebrand factor (vWF) staining, was significantly greater in GSK-3β-MSC-injected MI mice than in saline- or LacZ-MSC-injected MI mice (p<0.001) (Figure 6 and Online Figures IV and V). To identify paracrine factors involved in angiogenesis, mRNA was isolated from myocardium injected with GSK-3β-MSCs or LacZ-MSCs, and PCR array analysis was conducted. Among 84 genes screened, the initial PCR array screening and subsequent RT-PCR analyses showed that Vegfa, platelet factor 4 (Pf4), fibroblast growth factor 1/2 (FGF1/2), tumor necrosis factor (Tnf) and Hairy/enhancer-of-split related with YRPW motif protein 1/2 (Hey1/2) are upregulated in the myocardium injected with GSK-3β-MSCs (Figure 7A and Online Figure VI). GSK-3β directly upregulated Vegfa expression in MSCs in vitro, suggesting that the effect of GSK-3β is cell autonomous (Figure 7B). In order to evaluate the contribution of Vegfa to the beneficial effects of GSK-3β-MSCs, Vegfa was downregulated with adenovirus harboring shRNA-Vegfa in GSK-3β-MSCs ex vivo, and then GSK-3β-MSCs with or without downregulation of Vegfa or saline was injected into the myocardium of the MI mice. Although all mice injected with GSK-3β-MSCs without Vegfa downregulation survived after MI, only 6 out of 10 survived among mice injected with GSK-3β-MSCs in which Vegfa was downregulated, a survival rate which was not significantly different from that of the saline-injected MI mice (Figure 7C). These results suggest that increased production of Vegfa is critical in mediating the increased survival of GSK-3β-MSC-injected MI mice. The capillary density in the MI area was significantly greater in mice injected with GSK-3β-MSCs than in mice injected with GSK-3β-MSCs with Vegfa downregulation or saline, suggesting that Vegfa production in MSCs also plays an important role in mediating the increased angiogenesis in GSK-3β-MSC-injected MI mice (Figure 7D). Interestingly, injection of GSK-3β-MSCs with Vegfa downregulation was as effective as injection of GSK-3β-MSCs without Vegfa downregulation in reducing the size of MI (Figure 7E). Echocardiographic analyses showed that injection with the Vegfa-downregulated GSK-3β-MSCs and with GSK-3β-MSCs without Vegfa downregulation both prevented LV dilation (Online Figure VII) and increases in HW/BW (Online Figure VIII) equally well, suggesting that attenuation of LV remodeling by GSK-3β-MSCs may take place independently of Vefga expression. Injection with the Vegfa-downregulated GSK-3β-MSCs resulted in a significantly reduced %FS compared to that with GSK-3β-MSCs without Vegfa downregulation at 6 weeks, but not at 12 weeks (Online Figure IX), and a significantly lower LVEDP than injection with saline at 12 weeks (Online Table IC). These results suggest that upregulation of Vefga is partially involved in the improvement of LV function in the GSK-3β-MSC-injected MI mice, and that the therapeutic benefit of GSK-3β-MSCs injection is mediated by both Vegfa-dependent and -independent mechanisms.

Figure 6. Injection of GSK-3β-MSCs enhances angiogenesis.

Figure 6

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MSCs were transduced with Ad-LacZ, Ad-GSK-3β or Ad-sh(Vegfa) alone or in combination ex vivo and injected into mouse hearts after permanent coronary ligation. Immunostaining of remote and MI areas with anti-PECAM-1 and anti-troponin T (TnT) antibodies and DAPI staining is shown. A quantitative analysis of capillary density, as indicated by the number of PECAM-1 positive cells per unit area, is shown below.

Figure 7. Injection of GSK-3β-MSCs enhances Vegfa expression, which partially mediates their therapeutic benefits.

Figure 7

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(A) mRNA expression of Vegfa, Pf4, Fgf1, Fgf2, Tnf, Hey1, Hey2 and GAPDH in the injected area. Expression of GAPDH was evaluated as an internal control. (B) mRNA expression of Vegfa, GSK-3β and GAPDH in LacZ-MSCs or GSK-3β- MSCs and a quantitative analysis of mRNA expression of Vegfa. GAPDH was evaluated as an internal control. (C) Percent survival of MI mice after injection of GSK-3β-MSCs, GSK-3β-sh(Vegfa)-MSCs or saline. (D) Representative pictures of WGA staining in the MI adjacent area and a quantitative analysis of myocyte cross sectional area. (E) Masson's Trichrome staining and comparisons of infarction size and wall thickness in the MI area. Data are shown as mean ± SEM. # P<0.05, ## P<0.01, ### P<0.001 vs. GSK-3β-MSC-injected MI mice.

Discussion

Although MSCs are a promising source of CBT for post-MI hearts 24, MSCs survive poorly when injected into the MI heart, which limits their potential in CBT 15. Furthermore, the efficiency of CM differentiation from MSCs appears very low 9, 16, which may limit the functional improvement of the heart through CBT with MSCs. We have shown previously that stimulation of GSK-3β in MSCs induces CM differentiation in vitro 18. In this study, we show that ex vivo upregulation of GSK-3β in MSCs significantly increases both survival and CM differentiation of the MSCs after injection into the MI heart, thereby significantly enhancing the efficiency of the CBT.

Although injection of either LacZ-MSCs or GFP-MSCs into the MI hearts slightly but significantly prevented LV dilation and expansion of MI, it failed to improve LV systolic function, LV remodeling or survival of the animals. On the other hand, injection of GSK-3β-MSCs remarkably improved these parameters, indicating that GSK-3β enhances the efficiency of CBT with MSCs. Since both adenovirus-mediated transduction and stable expression of GSK-3β in MSCs improved the efficiency of CBT, the effect of GSK-3β is independent of adenovirus transduction. Furthermore, even episomal and, thus, transient expression of GSK-3β increases the efficiency of the CBT after MI. Thus, our results clearly suggest that ex vivo modification of signaling molecules, such as GSK-3β, can improve the therapeutic potential of MSCs.

One of the significant challenges in CBT is the fact that survival of the injected cells is very low, especially in the post-MI heart 15. In this study, continued survival of the injected MSCs in the MI heart at 12 weeks was seen only when the MSCs had been transduced with GSK-3β ex vivo, suggesting that GSK-3β may enhance the survival of MSCs. This finding is surprising in that ex vivo introduction of Akt into MSCs has been shown to increase cardiac function and enhance angiogenesis by enhancing the survival of MSCs after injection into the MI heart 15, 16. Inhibition of GSK-3β in the adult heart prevents remodeling and promotes cardiac regeneration 25. Since Akt inhibits GSK-3β and GSK-3β stimulates apoptosis in CM 17, the mechanism regulating survival and death of MSCs in the MI heart may be distinct from that of CMs. In addition, autocrine or paracrine factors whose production is modulated by GSK-3β may also be involved in the enhanced survival of GSK-3β-MSCs.

Another challenge in CBT with adult stem cells is the fact that the efficiency of CM differentiation is low 9, which would limit the improvement of cardiac contraction. In fact, no LacZ-MSCs survived 12 weeks after injection into the MI heart and, thus, there was no evidence that LacZ-MSCs differentiated into CMs in the heart in vivo. In contrast, approximately 60% of the GSK-3β-MSCs surviving at 12 weeks expressed either troponin T or the genetic reporter, suggesting that GSK-3β may stimulate CM differentiation in vivo. Since GSK-3β induces CM differentiation in MSCs in vitro, when GSK-3β is introduced ex vivo, MSCs may continue to differentiate into CMs even after injection into the MI heart. It has been shown that ex vivo priming of embryonic stem cells into the CM lineage not only induces efficient CM differentiation but also prevents differentiation into undesired phenotypes and teratomas 21. This concept may extend to MSCs as well.

It remains to be elucidated to what extent the CM differentiation contributes to the functional improvement of the MI heart after injection of GSK-3β-MSCs. The activity of the Nkx2.5 promoter is increased even at early stages of CM differentiation. GFP/troponin T double positive cells were significantly smaller than GFP negative residential heart cells, and troponin T was not clearly striated in these cells. Thus, even if GSK-3β-MSCs commit to the CM lineage, they may not be fully differentiated. Determining whether or not they contribute to the force generation in the post-MI heart may require analysis of isolated single myocyte contraction. In addition, following up to see whether or not CMs differentiated from GSK-3β-MSCs could mature with time would be of great interest. Cell fusion has been suggested as a mechanism by which adult stem cells might acquire CM properties 3. Since most of the CMs differentiated from GSK-3β-overexpressing MSCs exhibit a small cell size and a single nucleus, we believe that the possibility of cell fusion is remote. However, further investigation is required to address this issue.

CBT may improve cardiac regeneration in part through interactions with host precursor cells. The number of Ki67 positive myocytes increased significantly when GSK-3β-MSCs were injected into the post-MI hearts. Interestingly, CMs differentiated from MSCs were observed mostly in the MI area, but Ki67 positive proliferating myocytes were observed globally in the heart, suggesting that the proliferating myocytes may originate from the host CMs. Injection with GSK-3β-MSCs also increased c-Kit positive cells near the site of injection. These results suggest that GSK-3β-MSCs may produce factors to induce proliferation of host CMs or residential cardiac progenitor cells (CPCs) or recruit CPCs from the other part of the heart or the systemic circulation. Recent evidence suggests that MSCs stimulate cell-cell interactions with endogenous c-Kit positive cells and promote their differentiation 26. Whether or not GSK-3β expression in MSCs induces CM differentiation by stimulating direct cell-cell interaction remains to be tested.

Upregulation of paracrine factors plays an important role in mediating the beneficial effects of CBT with MSCs 11, 13. Using PCR array analyses, we have identified several growth factors/cytokines, including Vegfa, that are upregulated in the GSK-3β-MSCs-injected heart. Increases in angiogenesis and post-MI survival of the mice with injection of GSK-3β-MSCs were abolished when Vegfa was downregulated in GSK-3β-MSCs, suggesting that upregulation of Vegfa is an important mechanism by which GSK-3β-MSCs improve the efficiency of CBT.

Although the survival advantage to the animals conferred by injection of GSK-3β-MSCs was nullified when Vegfa in GSK-3β-MSCs was downregulated, decreases in MI size, suppression of cardiac remodeling and partial improvement of systolic function were still observed. This suggests that GSK-3β-MSCs mediate beneficial effects in the post-MI heart, including suppression of cardiac remodeling and improvement of LV function, largely through Vegfa- or angiogenesis-independent mechanisms. The fact that the lack of Vegfa diminishes the survival advantage of GSK-3β-MSCs suggests, however, that well-coordinated activation of multiple mechanisms is required for the improvement of the efficiency of CBT with MSCs. The specific mechanism by which the lack of Vegfa expression in MSCs abolishes the survival advantage of the MI animals remains to be elucidated.

Survival, expansion and differentiation of MSCs in the heart may also be regulated by complex signaling mechanisms which depend upon timing. For example, CM differentiation of progenitors may require biphasic activation of GSK-3β27. We show that MSCs isolated from Dox-regulatable GSK-3β transgenic mice can be used for CBT in the post-MI heart. Thus, transgene expression in MSCs can be regulated in situ by exogenously-given Dox. A future study includes defining the timing of GSK-3β in situ to further improve the efficiency of CBT with MSCs.

The effectiveness of CBT with MSCs has been demonstrated in large animals and humans after myocardial infarction. Although MSCs were injected immediately after permanent ligation in our model, they were injected 3 days after permanent coronary occlusion in the pig model 7. In addition, MSCs were infused intravenously in patients who have had a first myocardial infarction 1-10 days before the intervention 24. Whether or not treatment with GSK-3β-MSCs are effective in clinically more relevant settings remains to be tested.

In summary, ex vivo genetic reprogramming of MSCs with GSK-3β improves the therapeutic efficiency of MSCs in the post-MI heart. GSK-3β overexpression in MSCs achieves several therapeutic benefits, most likely by pre-directing MSCs to CM differentiation and inducing secretion of paracrine factors, including Vegfa. Future investigation of the downstream mechanism through which GSK-3β stimulates CM differentiation of MSCs in vivo, and the GSK-3β-induced secreted factors mediating the recruitment of CPCs may provide us with useful information to further improve the efficiency of CBT with MSCs in post-MI hearts.

Supplementary Material

1

Acknowledgments

We thank Daniela Zablocki for critical reading of the manuscript.

Sources of Funding: This work was supported in part by U.S. Public Health Service Grants HL59139, HL67724, HL69020, HL91469, HL102738, AG23039, and AG27211, and the Foundation of Leducq Transatlantic Network of Excellence.

Abbreviations

α-MHC

α-myosin heavy chain

Ad

adenovirus

BM

bone marrow

CBT

cell based therapy

CM

cardiomyocyte

Dox

doxycycline

GSK

glycogen synthase kinase

GSK-3β-MSCs

GSK-3β-overexpressing MSCs

LacZ-MSCs

LacZ-overexpressing MSCs

LAD

left anterior descending branch of the coronary artery

LV

left ventricle

MI

myocardial infarction

MSC

mesenchymal stem cell

shRNA

short hairpin RNA

Tg

transgenic mice

TnT

troponin T

Vegfa

vascular endothelial growth factor A

WGA

wheat germ agglutinin

Footnotes

Disclosures: None

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Supplementary Materials

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NIHMS269243-supplement-1.pdf (1.7MB, pdf)

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