Total AGEs’ levels were measured in heart tissues from SHAM, MI, MI + CASCs, and MI + CASCs + PM, and representative images are provided in Figure 1A. AGEs’ content was significantly increased in MI animals compared with SHAM (Figure 1B; 15% ± 0.6 in MI vs. 8.2% ± 0.7 in SHAM) and PM significantly decreased the AGEs’ content compared with MI (Figure 1B; 9.7% ± 1.8 in MI + CASCs + PM).

In vivo cardiac function was assessed by echocardiographic and hemodynamic measurements. Representative examples of echocardiographic images of SHAM, MI, MI + CASCs, and MI + CASCs + PM 4 weeks post-operative are shown in Figure S1. Echocardiographic parameters of the different groups, SHAM, MI, MI + CASCs, and MI + CASCs + PM, 4 weeks post-operative are summarized in Table 1. Additional echocardiographic parameters are summarized in Table S1. MI animals undergoing CASCs transplantation with or without additional PM treatment displayed a significantly increased ejection fraction (EF) compared with MI (Table 1; 59% ± 4 in MI vs. 79% ± 3 in MI + CASCs; vs. 72% ± 3 in MI + CASCs + PM, p = 0.051).

Hemodynamic measurements of SHAM, MI, MI + CASCs, and MI + CASCs + PM were performed 4 weeks after surgery. Compared with MI, additional PM treatment significantly reduced the time constant for isovolumetric relaxation (Table 2; Tau; 0.0499 s ± 0.017 in MI vs. 0.0117 s ± 0.001 in MI + CASCs + PM).

Figure 2A demonstrates representative examples of Sirius Red/Fast Green stained cryosections from SHAM, MI, MI + CASCs, and MI + CASCs + PM 4 weeks after surgery. Infarct size tended to decrease in animals undergoing CASCs transplantation (Figure 2B; 19% ± 2 in MI vs. 12% ± 2 in MI + CASCs, p = 0.07). Additional PM treatment did not further reduce infarct size (Figure 2B; 12% ± 3 in MI + CASCs + PM, p = 0.16).

Representative images of interstitial collagen obtained with Sirius Red/Fast Green staining in LV sections from the groups are provided in Figure 3A. Interstitial collagen deposition was significantly lower in CASCs transplanted animals compared with MI (Figure 3B; 14% ± 3 in MI vs. 6% ± 0.4 in MI + CASCs). Fibrosis tended to be lower in PM animals (Figure 3B; 7% ± 1 in MI + CASCs + PM).

Unloaded cell shortening was measured in freshly isolated cardiomyocytes isolated from SHAM, MI, MI + CASCs, and MI + CASCs + PM animals 4 weeks post-surgery. As shown in Figure 4A, cells isolated from the border zone of infarcts displayed altered functional properties, namely reduced and slower unloaded cell shortening. CASCs transplantation, with or without PM treatment, could prevent the deterioration of the cardiomyocyte contractile properties (Figure 4A; L/L₀, 5% ± 0.3 in MI vs. 7% ± 0.5 in MI + CASCs; vs. 7% ± 0.4 in MI + CASCs + PM). The kinetics of cell contraction and cell relaxation, i.e., TTP and RT₅₀, were significantly better in MI + CASCs and tended to be improved in MI + CASCs + PM animals (Figure 4B TTP; 120 ms ± 1 in MI vs. 112 ms ± 2 in MI + CASCs; vs. 118 ms ± 3 in MI + CASCs + PM; Figure 4C RT₅₀; 197 ms ± 3 in MI vs. 184 ms ± 4 in MI + CASCs; vs. 190 ms ± 4 in MI + CASCs + PM).

Gene expressions of pro-inflammatory cytokines (IFN-γ and IL-6) were evaluated in the four groups of animals, 4 weeks post-surgery. As shown in Figure 5, the expression of inflammatory cytokines tended to be lower in MI + CASCs + PM animals compared with MI and MI + CASCs animals (Figure 5A IFN-γ; 1.60 ± 0.27 in MI vs. 1.83 ± 0.30 in MI + CASCs; vs. 1.06 ± 0.14 in MI + CASCs + PM; Figure 5B IL-6; 3.28 ± 1.06 in MI vs. 3.56 ± 0.81 in MI + CASCs; vs. 2.77 ± 0.48 in MI + CASCs + PM).

Oxidative stress is one of the main factors inducing AGEs’ synthesis, by formation of reactive carbonyl compounds and glycoxidation of Amadori products in the Maillard reaction. In patients with MI, AGEs’ levels are significantly increased ^[8][17][25,34] and have potential deleterious effects on cardiac function ^[18][19][35,36]. Moreover, in our study, AGEs are significantly increased in MI animals. In addition to increased oxidative stress, the inflammatory process induced after MI activates neutrophils and macrophages. These immune cells are known to further secrete AGEs and are reported to be key inducers of AGEs’ formation in MI ^[9][10][26,27].

Even if they provide new insights into tissue regeneration, stem cells are transplanted in the border zone of MI with increased oxidative stress, inflammation, and AGEs’ levels. Previous studies have shown that increased levels of AGEs affect stem cells’ properties, i.e., by reducing their proliferation and migration properties ^[11][28]. Recently, we have demonstrated that the same adverse effects of AGEs apply to CASCs’ properties in vitro ^[12][29]. In that context, reducing AGEs’ formation could potentially enhance CASCs’ regenerative properties upon in vivo transplantation. To find out whether such an approach would offer new therapeutic insights, was the goal of our study. In the context of improving stem cell therapy by targeting AGEs, we evaluated the effect of CASCs transplantation in combination with PM in a rat model of MI. This vitamin B derivate is an effective and safe AGEs-lowering therapy ^[20][37], which has different mechanisms of action ^[21][22][38,39]. First, PM can bind with catalytic redox metal ions, which are needed for glycoxidation of Amadori products. As such and related to its iron-chelator properties, Amadori-to-AGEs conversion is blocked. Secondly, PM can scavenge reactive carbonyl compounds, the latter being major AGEs’ precursors. In addition, studies have demonstrated that PM is a co-enzyme associated with multiple inflammatory pathways, thereby potentially inhibiting inflammation ^[15][32]. Finally, by inhibiting ROS formation or scavenging oxygen radicals, PM has been shown to be a potent antioxidant. Previous studies have shown that, even independent of stem cell transplantation, PM alone improves survival and reduces extracellular remodeling after MI, by reducing AGEs’ levels ^[23][40]. Moreover, in clinical trials, PM has been demonstrated as a safe and effective drug in diabetic patients ^[21][38]. However, owing to financial issues, a clinical trial of NephroGenex in 2014, testing PM as an anti-diabetic treatment, was stopped ^[24][41]. No other clinical trials are currently investigating PM as a therapy.

In our study, PM succeeded in reducing total AGEs’ tissue levels. However, PM did not further improve cardiac function obtained with CASCs transplantation alone, which was, at first sight, not expected in our study. This could be partially explained by the recent discovery of Vagnozzi et al. ^[25][42]. In their article, the authors show that cellular therapy itself induces an inflammatory response after MI that could be the primary beneficial effect underlying stem cell treatment. Pro-inflammatory macrophages, mobilized and activated by transplanted stem cells, could indeed rejuvenate the mechanical properties of the injured cardiac area. By affecting fibroblast activity, the ECM content and area occupied by scar tissue could be reduced. The precise underlying mechanisms responsible for the repair response of these immune cells are unclear and require further investigation. In our study, it is thus likely that, by reducing local inflammation, PM could not further enhance the repair process, when combined with CASCs transplantation. Inflammatory cytokines such as IFN-γ and IL-6 tended to be reduced by PM treatment in our study. As an immune reaction is thus needed as a base for would healing with stem cells, PM could counteract the positive effects of CASCs therapy by reducing local tissue inflammation. This could explain the lack of additive value of PM in the context of MI and stem cell transplantation.

Even if our data do not demonstrate an additional effect of PM to CASCs transplantation, the potential of other anti-AGEs therapies still needs to be investigated. It has been shown in Alzheimer’s, Parkinson’s, and rheumatoid arthritis disease animal models that stem cell survival was prolonged, migration capacity was enhanced, and the MSCs were better protected against apoptosis, when sRAGE-secreting MSCs were transplanted. By scavenging AGEs with sRAGE, the effectiveness of MSCs transplantation was improved, thus suggesting a role of AGEs in regenerative approaches with stem cells ^[26][27][28][43,44,45]. However, using genetically modified stem cells is still highly experimental and needs to be investigated in vivo before any possible translation into the clinical setting is possible. Therefore, one cannot exclude that other anti-AGEs therapy approaches such as RAGE inhibitors, sRAGE, or ALT-711 could potentially succeed in further lowering AGEs’ concentrations in MI and potentially have an additive effect on cardiac outcome. These therapeutic options need to be investigated in both pre-clinical and clinical studies in combination with CASCs therapy.

Independent of PM treatment, our data confirm that transplantation of CASCs can prevent worsening of cardiac function after an ischemic injury, as shown by Fanton et al. in the minipig model ^[2][19]. Indeed, we have shown that EF significantly increased up to 20% after CASCs transplantation compared with non-treated animals. Meta-analysis of other CSCs therapies for the treatment of MI in mice showed an overall increase in EF of 9.9% ^[29][46]. Therefore, CASCs do have more effective regenerative effects compared with other CSCs and are remarkable candidates for cellular therapy. Other parameters of global cardiac function, such as dP/dt_max, SV, CO, and ESV, even if not significantly affected, followed the same trend, indicating an overall improvement of systolic function upon CASCs transplantation. In addition, infarct size tended to decrease after CASCs transplantation. Furthermore, as shown by the prevention of adverse remodeling at the cardiomyocyte level, our data suggest that mechanical load subjected to the resident myocytes of the ischemic area was reduced with CASCs transplantation. The prevention of collagen deposition seen in our study is also in line with a potentially reduced mechanical load with CASCs transplantation. Indeed, an important pathway in post-MI remodeling and scar formation is the TGF-β1 signaling pathway. In this study, we did not evaluate the underlying mechanisms resulting in reduced fibrosis with CASCs. However, TGF-β1 could be an essential contributor. Indeed, increased TGF-β1 is detected after MI and is known to decrease the expression and function of enzymes responsible for matrix degradation and increase the inhibitors of proteases ^[30][31][47,48]. Whether CASCs transplantation results in a reduced lysyl oxidase expression and/or PI3K/Akt, Smad3, and MAPK signaling pathway as a consequence of increased TGF-β1 activation ^[32][49], remains to be confirmed. However, other studies have shown that MSCs transplantation is able to ameliorate cardiac fibrosis by decreasing TGF-β1 levels ^[33][50]. Therefore, it seems likely that this TGF-β1 pathway is also involved in our study as it is a common pathway found in many diseases, but this has to be confirmed. In addition, we have demonstrated that CASCs transplantation can prevent adverse cellular remodeling of resident cardiomyocytes, isolated from the border zone of the infarct. Indeed, we show that, compared with MI animals, the amplitude and kinetics of cardiomyocyte shortening isolated from the border zone of MI in transplanted animals are improved. In that context, it has been described that the extent of mechanical load determines the extent of remodeling in both peri-infarct and remote regions ^[34][51]. It is then very likely that even a small decrease in infarct size, which we observe upon CASCs transplantation, will reduce adverse cellular remodeling in the resident cardiomyocytes.

However, whether the beneficial cardiac outcome is solely attributed to new cardiomyocytes differentiated from CASCs or to the paracrine factors secreted by these stem cells remains to be investigated. This then raises the question of whether CASCs were still present 4 weeks after transplantation. Indeed, pre-clinical studies have shown that most of the stem cells injected at the site of injury are cleared out within seconds, resulting in only 1–3% of the injected cells persisting at the site of injury ^[35][52]. Yet, previously, the presence of differentiated CASCs 8 weeks post-MI was demonstrated by immunostainings ^[2][19]. In addition, engraftment of the CASCs after 8 weeks of acute MI has been shown to be 19%, a value higher than that previously described with other stem cells ^[2][19]. It is thus very likely that CASCs are still present in the cardiac tissue 4 weeks post-transplantation. In addition, previous studies have shown that stem cells are able to secrete paracrine factors to promote survival and proliferation or have immunomodulatory effects on resident cardiomyocytes ^[36][53]. The strong paracrine effects of stem cells have been well documented and are, for a part, related to the limited improvement of cardiac function seen in some studies, as those may compensate for the lack of cardiomyocyte differentiation ^[37][38][54,55]. Whether the differentiation of CASCs to new cardiomyocytes and/or the paracrine factors secreted by the CASCs are the reason for the improvements seen after MI, remains to be clarified.