*3.4. Improvement of Cardiac Function Through Cell Therapy Assessed by CMR*

In order to assess the value of 18F-FDG-based imaging of cellular inflammation post-MI as an early predictor of functional outcome following cardiac cell therapy, cine CMR was performed three weeks after LAD ligation in separate groups. LVEF was significantly reduced by LAD ligation as compared with healthy animals (24.2 ± 4.1% vs. 59.3 ± 3.7%; *p* < 0.001). Cell therapy led to a significant increase of LVEF (31.7 ± 3.5% vs. 24.2 ± 4.1%; *p* = 0.007) (Figure 10). From this we conclude, that the change in the 18F-FDG uptake pattern, as described above, is a valuable early predictor of therapeutic efficacy.

**Figure 10.** Improved cardiac function after transplantation of embryonic stem cells (ESC) derived CiC following acute myocardial infarction at 3 weeks following MI. (**A**) Magnetic resonance imaging analyses of infarcted animals receiving either CiC (MIC) or matrigel and healthy animals and (**B**) left ventricular ejection fraction was significantly higher in CiC animals vs. control animals (n = 6, *p*-value was calculated using the student *t*-test).

#### *3.5. Modulation of the Immune Response Through Cell Therapy Assessed by Flow Cytometry*

In order to identify the immune response within the myocardium, we analyzed the mononuclear cell (MNC) suspension isolated from the remote and infarct area of the hearts of 129Sv mice five days after MI or MIC (Figure 1).

We observed a significantly higher percentage of NK cells (CD45+/CD11b−/CD11c+/NK1.1+) in the infarct area (0.82 ± 0.04 vs. 0.56 ± 0.03; *p* = 0.01) as compared with the remote area in MI mice. Cell therapy influences the various immune subpopulations differently in infarct and remote areas of the heart. On the one hand, cell therapy led to a significantly higher percentage of CD11b<sup>+</sup> myeloid cells (Figure 11A) in the remote area (49.28 ± 3.92 vs. 31.17 ± 2.72; *p* = 0.01) of MIC hearts as compared with MI hearts. This agrees well with the increased 18F-FDG uptake observed in the remote area of MIC mice (Figure 9), which has been attributed mainly to CD11b<sup>+</sup> cells [1]. On the other hand, cell therapy led to a decrease in the percentage of NK cells (Figure 11A) in the infarct area (0.41 ± 0.05 vs. 0.82 ± 0.04; *p* = 0.007) when compared to MI hearts.

**Figure 11.** Effect of CiC transplantation on the percentage of various immune cell subpopulations in the heart based on flow cytometric analysis. Mice were subjected to MI/MI+ cells and the remote and infarct area of the hearts were dissected, digested, and the isolated mononuclear cells were stained using various antibodies. These immune cell populations were then characterized using the gating strategy described before and represented as three major groups. (**A**) CD11b<sup>+</sup> and NK cells, (**B**) populations based on their CCR2/MHC-II expression, and (**C**) contribution of the populations towards the M1 (Ly6Chi) and M2 (Ly6Clo) cells, n <sup>=</sup> 5. Values are represented as mean <sup>±</sup> SEM. Significance was calculated using the Mann–Whitney test.

Interestingly, we also observed various differences in various CD11b<sup>+</sup> subpopulations based on their relative CCR2 and MHC-II expression between the MI and MIC groups (Figure 11B,C).

In the remote area, cell therapy led to an increase in the percentage of fetal liver HSC-derived resident macrophages (4.68 ± 1.03 vs. 1.29 ± 0.32; *p* = 0.05) and an increase in the percentage of monocyte-derived macrophages (50.48 ± 3.24 vs. 24.35 ± 4.54; *p* = 0.007), and their relative contribution to the percentage of M1 (Ly6Chi) (58.48 <sup>±</sup> 5.41 vs. 28.11 <sup>±</sup> 7.72; *<sup>p</sup>* <sup>=</sup> 0.01) and M2 (Ly6Clo) (47.28 <sup>±</sup> 3.95 vs. 23.39 ± 3.38%; *p* = 0.007) cells along with a subsequent decrease in the percentage of monocytes (24.33 <sup>±</sup> 5.61 vs. 44.36 <sup>±</sup> 5.62; *<sup>p</sup>* <sup>=</sup> 0.05) and their relative contribution to the percentage of M1 (Ly6Chi) (33.76 <sup>±</sup> 6.9 vs. 67.85 <sup>±</sup> 7.22; *<sup>p</sup>* <sup>=</sup> 0.03) and M2 (Ly6Clo) (13.25 <sup>±</sup> 1.74 vs. 32.71 <sup>±</sup> 3.56; *<sup>p</sup>* <sup>=</sup> 0.007) cells as compared with MI.

In the infarct area, we observed an increase in the percentage of fetal liver HSC-derived resident macrophages (4.68 ± 1.03 vs. 1.29 ± 0.32; *p* = 0.05) as well as monocyte-derived macrophages (57.46 ± 4.37 vs. 38.89 <sup>±</sup> 8.13) and their relative contribution to the percentage of M2 (Ly6Clo) (51.44 <sup>±</sup> 2.47 vs. 33.25 ± 6.6%; *p* = 0.03) cells with a subsequent decrease in the percentage of monocytes (18.88 ± 5.99 vs. 32.25 <sup>±</sup> 6.88; *p* = 0.05) and their relative contribution to the percentage of M1 (Ly6Chi) (27.6 <sup>±</sup> 7.59 vs. 47.4 <sup>±</sup> 10.04; *<sup>p</sup>* <sup>=</sup> 0.05) and M2 (Ly6Clo) (8.39 <sup>±</sup> 1.17 vs. 25.85 <sup>±</sup> 6.15; *<sup>p</sup>* <sup>=</sup> 0.007) along with a decrease in the percentage of yolk sac-derived resident macrophages (20.16 ± 2.43 vs. 27.95 ± 2.15; *p* = 0.05) as compared with the MI group.

Therefore, CiC transplantation in the heart increases the CD11b<sup>+</sup> cells in the remote area of the heart while favoring an increase in the MHC-IIhi subset of CD11b<sup>+</sup> cells (monocyte derived macrophages and fetal liver HSC-derived resident macrophages) in both the infarct and remote areas.

#### **4. Discussion**

Translational cardiovascular research has evolved at an incredible pace within the last decade and resulted in significantly higher survival rates after acute myocardial infarction. In contrast to highly efficient approaches based on early revascularization and pharmacological prevention of adverse ventricular remodeling, replacement of irreversibly lost cardiomyocytes has not been achieved yet, despite huge efforts in the field of regenerative medicine.

Recently, cellular inflammation following ischemic myocardial injury has been identified as a key player in the process of myocardial healing. Thereby, the local distribution patterns of specific monocyte and macrophage subpopulations have been proposed to determine the quality of myocardial healing [15,16].The vast majority of data has been obtained from rodent studies because their hearts can easily be excised for in vitro experiments such as flow cytometric measurements of single cells suspensions. Most researchers focus on the invasion of two distinct macrophage subpopulations, usually referred to as M1 and M2 macrophages. The early M1 macrophage subset is attracted to the site of myocardial injury via CCL2, expressing pro-inflammatory mediators and proteases for degradation of infarcted tissue. Subsequently, M2 macrophages are recruited via CX3CL1 for mediating synthesis of extracellular matrix and angiogenesis [3]. However, the M1/M2 classification does not adequately explain the complete spectrum of macrophage phenotypes. Recently, macrophages which do not express CCR2 in the neonatal heart have been shown to regenerate the infarcted tissue [15] while the adult heart involves CCR2<sup>+</sup> monocyte-derived macrophages also taking part in the remodeling process [17].

Preclinical studies have examined the effect of therapeutic applications, such as stem cell injections, to modify the qualitative and quantitative composition of the post infarct cellular immune response [18–20]. This modulation of the innate cellular immune response resulted in improved cardiac pump function, reduction of scar size, and adverse remodeling [18]. Findings from a meta-analysis by our group have ascribed high therapeutic potential to cardiovascular cell preparations [21]. Therefore, we sought to transplant ESC-derived cardiac induced cells to improve myocardial healing and also investigate whether they influence the ischemic cellular immune response.

The above-mentioned experiments are based on post-mortem analyses such as flow cytometry and immunohistochemistry of excised organs, and thus have been restricted to preclinical research. However, clinical translation requires methods that allow in-vivo visualization and quantification of inflammatory cells. Lee et al. established 18F-FDG PET for imaging the inflammatory cell activity in the heart, based on suppressing glucose metabolism in myocytes [1]. Interestingly, anaesthesia with ketamine/xylazine is both sufficient and highly effective in reducing glucose uptake in cardiomyocytes, hence, enabling visualization of inflammatory activity in the heart [1]. According to Lee et al.,

18F-FDG uptake at the site of myocardial inflammation related to the content of local CD11b<sup>+</sup> monocyte/macrophage concentration in C57BL6 mice [1], at day 5 after MI induction.

On the basis of these findings by other groups, we hypothesized the following:


We were able to replicate the glucose uptake suppression protocol based on anaesthesia with KX [5] in 129sv mice and achieved an almost 88% reduction in the glucose uptake in the whole myocardium as compared with isoflurane anaesthesia. We did not find any significant difference in the 18F-FDG uptake levels between the remote and infarcted myocardium using KX anaesthesia, which is in line with findings from Thackeray et al. [4,5]. Furthermore, intense focal tracer uptake was localized in the border zone. Similarly, Lee et al. also reported accentuated FDG uptake in the border zone corresponding to high numbers of infiltrating CD11b<sup>+</sup> cells [1]. Despite visual assessment of this high focal tracer accumulation, quantitative analysis in this region was only performed by Thackeray et al. However, their VOI positioning strategy remains unclear from the manuscript. This might be due to a certain overlap of the border zone to the adjacent regions which makes demarcation of the border zone difficult. In order to produce comparable data in this aspect, VOI positioning strategies should be provided in future studies.

Interestingly, transplantation of 10<sup>6</sup> cardiac induced cells after MI significantly increased FDG uptake in both the whole heart and the remote myocardium, but not in the infarct region. Moreover, CiC transplantation led to a 7% improvement in LVEF three weeks after MI. The magnitude of LVEF improvement is well in line with previous reports [21]. Therefore, we conclude that the change in the myocardial 18F-FDG uptake pattern represents a valid tool for early PET-based in vivo prediction of myocardial healing post MI.

A deeper investigation of the immune response in the heart five days after MI using flow cytometry revealed an increase in the percentage of CD11b<sup>+</sup> cells and a shift towards increased monocyte-derived macrophages in the remote area of cell-treated mice. We also observed a reduction in the yolk sac-derived resident macrophages in the infarct area of cell-treated mice. We did not however observe a shift in the M1/M2 polarization phenotype, as observed in previous studies involving MSCs [18]. It is interesting to note that increased monocyte-derived macrophages and reduced yolk sac-derived macrophages in the heart have been previously attributed to adverse cardiac remodeling and scar formation [17]. However, these studies did not involve the transplantation of cells into the infarcted heart and it is well known that the gene expression profile and the characteristics of the immune cells are affected based on their location in the myocardium and the cells they interact with [22].

However, unbiased expression profiling of these cells over various time points and under the influence of CiC has yet to be carried out. The influence of CiC transplantation on the immune response in the heart has not been studied until now and our work suggests possible new mechanisms and targets for improving the efficiency of CiC.

The 18F-FDG-based imaging strategies for myocardial inflammation are highly attractive and have been applied in a clinical setting despite some limitations, which have yet to be overcome [3]. Both cardiomyocytes [4] and infiltrating inflammatory cells [23] in the setting of acute myocardial injury possess high levels of glucose metabolism. Metabolism of healthy cardiomyocytes is mainly based on fatty acid oxidation, whereas ischemia triggers increased anaerobic glycolysis, which requires much higher rates of glucose [24]. This might hamper direct correlation of focal 18F-FDG uptake and high concentration of CD11b<sup>+</sup> cells in the infarcted region. The use of KX for anaesthesia has been shown to reduce serum insulin levels in rodents, thus, preventing translocation of GLUT4 to

membranes of cardiomyocytes and reducing 18F-FDG uptake [25]. In contrast, leucocyte glucose influx in a setting of acute inflammation depends more on GLUT1 and GLUT3, which are expressed and translocated independently of insulin [26]. Whereas, this KX protocol is well suited for preclinical research, different strategies are used for suppression of myocardial glucose uptake in patients including prolonged fasting, dietary modifications, and heparin loading before imaging [27]. This might hamper straightforward translation of imaging protocols established in rodents to clinical application.

Furthermore, the transplantation of CiC adds more complexity to its use in imaging the myocardium. It becomes difficult to attribute the observed 18F-FDG uptake pattern to the inflammatory cells alone since the ability of CiC to alter the cardiac metabolism is poorly understood. It is, therefore, difficult to delineate the relative contribution of 18F-FDG uptake between the host cardiomyocytes, immune cells, and the CiC. The heterogeneity of the inflammatory response and the relative 18F-FDG uptake between the different immune cell populations is also clearly not understood. Further research is necessary to understand metabolic changes in the different cells involved in healing myocardium.

Nevertheless, this is the first study to investigate 18F-FDG-based PET imaging of inflammation as an early indicator for assessing long term therapeutic efficiency in a rodent model of acute myocardial infarction. Furthermore, the current work illustrates the benefit of CiC transplantation to improved cardiac function after MI possibly through its beneficial modulation of the innate inflammatory response. Using such non-invasive techniques in the field of translational research will foster a better understanding of the inflammatory response and how its modulation could lead to altered cardiac function. It is also a valuable tool for monitoring various immune modulation and cell therapies and broadens the horizon for understanding the mechanisms of these therapeutic strategies.

**Author Contributions:** Conceptualization, C.I.L. and P.V.; methodology, T.L., J.S., and J.K.; formal analysis, C.I.L., P.V., and P.D.; investigation, C.I.L., P.V., R.G., P.M., and H.L.; writing—original draft preparation, P.V. and C.I.L.; writing—review and editing, J.K., R.D, G.S., and H.I.; visualization, P.V. and C.I.L; supervision, C.I.L. and R.D.; funding acquisition, C.I.L., R.D, B.J.K., and B.V.

**Funding:** This work was supported by the Federal Ministry of Education and Research Germany (FKZ 0312138A and FKZ 316159), the State Mecklenburg-Western Pomerania with EU Structural Funds (ESF/IVWM-B34-0030/10 and ESF/IVBM-B35-0010/12) and the DFG (DA1296/6-1), the German Heart Foundation (F/01/12) and the German Heart Research Foundation (F/34/15). In addition, R.D. is supported by the EU Structural Fund (ESF/14-BM-A55-0024/18), the FORUN Program of Rostock University Medical Centre (889001 and 889003), the Josef and Käthe Klinz Foundation (T319/29737/2017), the DAMP Foundation, and the BMBF (VIP+ 00240). The small animal PET/CT was funded by the Deutsche Forschungsgemeinschaft (INST 2268/6-1 FUGG). The small animal MRI system was funded by the European Regional Development Fund (EFRE UHROM 16). C.I.L. was supported by the Clinician Scientist Program of the Rostock University Medical Center.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

