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Mechanisms of the protective effects of BMSCs promoted by TMDR with heparinized bFGF-incorporated stent in pig model of acute myocardial ischemia

Abstract This study investigates the mechanism by which transmyocardial drilling revascularization combined with heparinized basic fibroblast growth factor incorporated degradable stent implantation (TMDRSI) enhanced effects of bone marrow mesenchymal stem cells (BMSCs) transplantation against acute ischemic myocardial injury. After the mid-third of left anterior descending artery was ligated, miniswine were divided into none-treatment group (control, n = 6), BMSCs implantation group (C, n = 6), TMDRSI group (TS, n = 6) and TMDRSI and BMSCs implantation group (TSC, n = 6). Two channels of 3.5 mm diameter were established by a self-made drill in the ischemic region, into which a stent was implanted for the TS and TSC groups. Autologous BMSCs were transplanted into the ischemic region in C group or around the channels in TSC group. Expression of von Willebrand factor, vascular endothelial growth factor, interleukin-1β, transforming growth factor-β3, cell proliferation and apoptosis, histological and morphological analyses, myocardial remodelling and cardiac function were evaluated at different time-points. Six weeks after the operation, the above indices were significantly improved in TSC group compared with others (P < 0.05), though C and TS groups also showed better results than the control group (P < 0.05). The new method was shown to have activated paracrine pathway of transplanted BMSCs, increased survival and differentiation of such cells, and enhanced effects of BMSCs transplantation on myocardial remodelling, which may provide a new strategy for cell therapies against acute ischemic myocardial injury.


Introduction
Although bone marrow mesenchymal stem cells (BMSCs) implantation has emerged as a promising treatment for ischemic heart disease for their potential effects to improve myocardial function and reverse ventricular remodelling [1][2][3][4], experimental and clinical studies also yielded some negative results [5][6][7]. It was speculated that the effect of cell transplantation may be largely impacted by the hostile microenvironment of the ischemic region [8,9].
In our previous report [10], we have described a novel method for myocardial revascularization, i.e. transmyocardial drilling revascularization (TMDR) combined with heparinized basic fibroblast growth factor (bFGF)-incorporated degradable tubular stent implantation (TMDRSI). Our preliminary results showed that, for a miniswine model of acute myocardial ischemia, this new method, combined with Flk1 ϩ CD34 -CD31 -BMSCs transplantation, enhanced angiogenesis and myocardial perfusion, and promoted cardiac function more effectively than single approaches [11]. To explore the underlying mechanism, the present study was set to assess the following parameters, which included differentiation and paracrine function of transplanted cells, cell proliferation and apoptosis, and myocardial remodelling.

Material and methods
Miniswine experiment was approved by Tianjin Administrative Committee for Laboratory Animals. Animal care and experimental procedures were carried out strictly in compliance with the Guide for the care and use of laboratory animals published by the National Institutes of Health in 1996.

Surgical procedure
Miniswine (25-30 kg) were anesthetized as described previously [10]. Briefly, the mid-third of left anterior descending artery was ligated for establishing the model of acute myocardial ischemia. Thereafter, the animals were randomly assigned to four groups (six each): Control group (no treatment), C group (cell transplantation only), TS group (TMDRSI only) and TSC group (TMDRSI ϩ cell implantation).
After the model was successfully established, two transmyocardial tunnels (pitch of 10 mm) were created at the centre of ischemic area of the left ventricle from epicardium with a self-made drill equipped with a hollow drill bit of 3.5 mm in diameter at 13,000 rpm (TS and TSC groups). Immediately after the channels were created, a stent was manually implanted. Epicardial purse-string stitches placed in advance were ligated to control bleeding and fix the stent. Subsequently, autologous BMSCs (2 ϫ 10 7 ) in 100 l saline were injected with a sterile microinjection at five sites within the ischemic region (C group) or around the channels (TSC group). The needle was advanced 5 mm into the myocardium, and the cells were injected 1.5 mm away from the centre of the channel. Antibiotics were administrated intramuscularly for 3 days after the operation.
Preparation of the stent has been described in our previous report [10]. In this study, each stent contained 15  Six weeks after the operation, the animals were killed with an overdose of potassium chloride. Tissues around the channels were harvested for RT-PCR and histological analysis.

Histological analysis
Thin myocardium sections (

MRI scan
All studies were performed on a 1. 5

Statistical analysis
All offline measurements were made by investigators blinded to the treatment. Results were presented as mean Ϯ SD. Statistical analysis was performed with a SPSS 13.0 software package (SPSS Inc, Chicago, IL, USA). Paired Student t-test was used for self-comparison. ANOVA with Bonferroni post hoc correction was used to compare experimental measurements in each group. A value of P Ͻ 0.05 was regarded as statistically significant.

Results
Of the 28 miniswine studied, 4 were excluded from further experiments due to intractable ventricular fibrillation after ligation of the left anterior descending artery, whereas 24 were ultimately determined for the subsequent procedures. No severe events of malignant arrhythmias, embolization, bleeding or haemodynamic abnormality had occurred during the stent or cell implantation procedures.

Morphology and immunophenotypes of BMSCs
After being cultured for 7 days, the cells had appeared as colonies of large flat cells or spindle-like cells. Fluorescence-activated cell sorting showed that such cells did not express haematopoietic markers CD34, CD45 or endothelial marker CD31, while the proportion of Flk1 ϩ cells was about 60%.

Enhanced expression of vWF, VEGF, IL-1␤ and TGF-␤ 3 in TSC group
Six weeks after treatment, semi-quantitative analysis of PCR product showed that the expression levels of vWF, VEGF, IL-1␤ and TGF-␤3 mRNA in TSC group were significantly increased compared with control, C and TS groups, though those in C and TS groups also up-regulated relative to the control group (P Ͻ 0.05). (Fig. 1)

Increased cell proliferation in TSC group
Representative photographs of Ki-67 stained sections of different groups are shown in Figure 2A to D. The count of Ki-67 ϩ cells revealed that cell proliferation has significantly increased in C group (15 Ϯ 2 cell/hpf) and TS group (23 Ϯ 2 cell/hpf) compared with the control group (9 Ϯ 1 cell/hpf, P Ͻ 0.001), which was more significant in TSC group (40 Ϯ 2 cell/hpf, P Ͻ 0.001) even at comparison with C and TS groups (Fig. 2E).

Inhibited cell apoptosis in TSC group
As shown in Figure 3, the amount of TUNEL-staining positive cells showed that cell apoptosis was significantly inhibited in C group (29 Ϯ 4 cell/hpf, P ϭ 0.021) and TS group (17 Ϯ 2 cell/hpf, P Ͻ 0.001) compared with the control (35 Ϯ 3 cell/hpf), which again was more significant in TSC group (10 Ϯ 3 cell/hpf, P Ͻ 0.01) even at comparison with C and TS groups (Fig. 3E).

Enhancement of myocardial viability in TSC group
Representative photographs of Masson trichrome stained section of different groups are shown in Figure 4. Qualitative analysis of MD demonstrated significant increases in myocardial viability in C group ( 0.001, Fig. 4E).

We tracked the engraftment of transplanted undifferentiated cells 6 weeks after the operation. Frozen sections within the core of the stent showed that DiI-labelled cells distributed in all regions and in particular around the stents in TSC group, whereas most trans-planted cells were found in the peri-infarct regions in C group.
Additionally, quantitative analysis of DiI revealed the enhanced survival of transplanted cells in TSC group (OD ϭ 10,297 Ϯ 658 pixels/hpf) compared with C group (OD ϭ 3762 Ϯ 213 pixels/hpf, P Ͻ 0.001, Fig. 5K). Immunofluorescence of cross-sections from TSC group showed that DiI-labelled cells not only expressed endothelial cell-specific marker vWF (Fig. 5A to C), but also smooth muscle cell-specific actin (Fig. 5D to F) and musclespecific marker cTnT (Fig. 5G to J). In C group, however, only differentiation into endothelial cells was found. In contrast,

Attenuated myocardial remodelling and improved left ventricular function in TSC group
No differences in EDV, ESV and local left ventricular wall thickness were found among the four groups prior to the treatment. Six weeks after treatment, no difference was found in local left ventricular wall thickness between C group (2.4 Ϯ 0.1 mm, P ϭ 1) and control group (2.3 Ϯ 0.1 mm). However, a significant increase was detected in TS group (3.3 Ϯ 0.2 mm, P Ͻ 0.001) when compared with C and control groups, and there was a further increase in TSC group (4.3 Ϯ 0.2 mm, P Ͻ 0.001) compared with TS groups (Fig. 6B) 0.006, Fig. 6C). Similar results were obtained for ESV. (Fig. 6D) 3%, P ϭ 0.006, Fig. 6E).

Discussion
Our study has demonstrated for the first time that TMDRSI can enhance the survival and differentiation of transplanted BMSCs, promoted paracrine function of such cells on repair of acute myocardial ischemia, and therefore can enable BMSCs transplantation to further attenuate myocardial remodelling at least for the subacute period.
Although BMSCs transplantation has provided a promising therapeutic option for patients with myocardial ischemia [9], no consensus was reached as to whether such therapy may bring protective effects at clinical trials. Some authors [1][2][3] reported   [6,7]. In present study, although we found that single BMSCs transplantation could slightly inhibit left ventricular remodelling after acute myocardial infarction, it was suggested that this minimal benefit should not be enough for repair of cardiac ischemic injury, which may limit its application for ischemic myocardial disease [8,9].

Fig. 5 Distribution and differentiation of transplanted cells. (A), (D) and (G) showed red fluorescence-positive cells distributing around the stent in the TSC group (100ϫ magnification). (B) is the photograph of vWF staining. (C) is the merged picture of (A) and (B), revealing that survived transplanted cells were factor vWF ϩ (arrow). (E) shows stained smooth muscle actin. (F) is the merged picture of (D) and (E), suggesting differentiation into smooth muscle cells from transplanted BMSCs (arrow). (H) and (I) are cTNT and DAPI stained picture. (J) is the merged picture of (G), (H) and (I), indicating differentiation from transplanted BMSCs into cardiomyocytes and a structural integration between resident and neonatal myocytes (arrow). (K) shows comparison of quantization of DiI between C and TSC groups, with an increased survival of transplanted cells in TSC
Recently, we developed a new method of TMDRSI for patients with end-stage coronary artery disease who are not eligible for surgical intervention and other conventional treatments [10]. Moreover, our experiments have demonstrated that TMDRSI combined with BMSCs transplantation could provide more effective protection than any single method for left ventricular function against myocardial ischemia [11]. In the present study, the mechanism of this protective effect was further delineated.
BMSCs may mediate cardiac repair following ischemic injury by direct and indirect ways. It has been shown that BMSCs can differentiate into cardiomyocytes, endothelial cells and smooth muscle cells [12][13][14]. Recently, Rota et al. [13] found junctional and adhesion complexes between transplanted BMSCs and between transplanted BMSCs and adjacent myocytes in mice with myocardial infarction, which confirmed structural integration between resident and neonatal myocytes from BMSCs, and also demonstrated that the regenerated myocardium from BMSCs were functionally competent both electrically and mechanically. In our experiment, it was detected that transplanted cells were tightly juxtaposed with host endothelial cells and myocytes. As demonstrated by immunofluorescence, differentiation from BMSCs to cardiomyocytes and endothelial cells was significantly enhanced by TMDRSI, although immunohistological co-staining as a means of defining cell type has some limitations as there could be potential false positives. Additionally, it has been demonstrated that BMSCs secreted angiogenic and anti-apoptotic factors such as vascular VEGF, IL-1␤ and TGF-␤3, which stimulate proliferation of endothelial cells and smooth muscle cells, and thereby can promote in vitro or in vivo arteriogenesis through paracrine pathways [9,[15][16][17]. Our results have shown an up-regulation of angiogenic and antiapoptotic factors 6 weeks after therapy in TSC group, indicating that the paracrine pathway of BMSCs was activated by TMDRSI. Although enhanced cell proliferation and inhibited cell apoptosis could not be identified for implanted BMSCs, it was demonstrated that they were related to the paracrine pathway of BMSCs.
The heparin-incorporated stent implanted in transmural channel had kept the channel patent, promoted vascular formation and improved myocardial perfusion for tissue around the channels more effectively than traditional transmyocardial revascularization [10,11]. The stent is degradable and can fulfil controlled release of bFGF, which may further promote angiogenesis and myocardial perfusion [18]. Hence, TMDRSI may provide adequate perfusion for the survival of BMSCs, allowing such cells to repair myocardial ischemic injury. As discovered, bFGF can promote proliferation and differentiation of BMSCs by activation of the ERK1/2 or the PKB signalling pathways [19,20].
Although TMDRSI was inclined to provide more benefits than BMSCs implantation only, the additive effects of combined therapy were rather clear, suggesting that activated BMSCs may play a key role against cardiac ischemic injury by differentiating into func-tional endothelial cells and cardiomyocytes or paracrine pathway. Therefore, it is indicated that TMDRSI or other assistant methods that can activate BMSCs are necessary for transplantation of BMSCs, which may become a new trend of stem cell therapy.
Despite encouraging results, there are still questions and considerations that need to be further addressed. For instance, it has not been directly documented whether there are functional gap junctional coupling and intercellular electrical coupling between cardiomyocytes from BMSCs and adjacent myocytes. In addition, our study was performed on animal models, and the differences between human and pig hearts has not been taken into account.
In summary, TMDRSI may improve protection of BMSCs transplantation against cardiac ischemic injury through activating the paracrine pathway of transplanted BMSCs and enhancing survival and differentiation of such cells. Our method therefore may provide a new strategy for cell therapy of acute myocardial ischemia.