In the present study, we investigated the role of Custodiol-supplemented with normoxic and hypoxic CM-BMSCs as a novel preservation solution to attenuate I/R injury-induced graft dysfunction in a rat heart transplantation model. The novelty of our work is that the application of Custodiol-supplemented with normoxic CM-BMSCs in the cold storage of donor hearts before heart transplantation is associated with shorter graft re-beating time, higher post-transplant cardiac contractility of donor hearts, a lower rate of cardiomyocytes apoptosis, and lower level of serum proinflammatory cytokines and indices for myocardial injury. Additionally, these beneficial cardioprotective effects are significantly enhanced after hypoxic preconditioning.
In heart transplantation, donor hearts inevitably suffer from cold ischemia and warm reperfusion injury, which can result in PGD for transplanted hearts. I/R injury is a critical pathological process influencing graft clinical outcomes(30) due to intracellular calcium overload, the generation of ROS, inflammatory cytokines and oxidative stress, and the absence of ATP(31). On the other hand, apoptosis and inflammation are regarded to be involved in the pathogenesis of I/R injury, leading to tissue damage of donor hearts. Therefore, the pre-conditioning with preservation solution has been demonstrated to be a promising method to protect donor hearts against I/R injury-induced graft dysfunction via the inhibition of inflammation and apoptosis. Recently, an increasing number of studies have demonstrated that BMSCs can secrete a vast array of paracrine factors, including cytokines, microRNA, growth factors, antioxidants, proteasomes, and exosomes(32), which are thought to be responsible for the observed cardioprotective effects for I/R injury-induced infarcted hearts in myocardial infarction(13, 33), brain-dead(23) and older(15) donor hearts in a rat heart transplantation model. In the present study, our antibody-based protein array analyses showed that the normoxic CM-BMSCs isolated by our group contained various kinds of cytokines, including Monocyte Chemoattractant Protein-1 (MCP-1), Tissue Inhibitor of Metalloproteinase (TIMP)-1, Interleukin-10 (IL-10), Decorin, Activin A, Galectin-1, HGF, Interleukin-6 (IL-6), VEGF, which may be involved in cardiac protection and functional improvement observed in the present study. It has been demonstrated that MCP-1 can attenuate LV dysfunction induced by global I/R injury via a reactive oxygen species-dependent but K(ATP) channel-independent pathway in the Langendorff-perfused hearts of wild-type mice(34). Additionally, the transplantation of TIMP-1 gels into the ischemic myocardium of rats has been shown to improve cardiac function and myocardial remodeling and suppress myocardial apoptosis(35). Furthermore, the administration of IL-10 has been suggested to improve myocardial function after acute global I/R injury and suppress inflammation via the STAT3 pathway(36). Li et al have shown that Decorin gene therapy attenuates cardiac remodeling and dysfunction via the inhibition of Smad2/3 activation(37). What’s more, Oshima et al reported that the treatment of recombinant Activin A mitigated hypoxia/reoxygenation-induced apoptosis via the upregulation of Bcl-2 protein expression(38). Besides, the treatment of mice with recombinant Galectin-1 attenuated cardiac damage by preventing cardiac inflammation in a mouse model of acute myocardial infarction(39). Moreover, Rong et al have demonstrated that adenovirus containing human HGF gene therapy attenuated ventricular remodeling in rat hearts after myocardial infarction through reducing myocardial inflammation(40). It also should be mentioned that IL-6 leads to a PI3K and NO-dependent protection of cardiomyocytes. This protection is associated with alterations in mitochondrial Ca2+ handling, inhibition of reperfusion-induced mitochondrial depolarisation, swelling and loss of structural integrity, and suppression of cytosolic Ca2+ transients(41). Furthermore, Yin et al revealed that injection of VEGF carrying plasmid into myocardium restored cardiac performance, reduced infarct size and cardiomyocyte apoptosis in a rat model of myocardial infarction(42). Last but not the least, CM derived from human multipotent MSCs has been reported to attenuate apoptosis of human endothelial cells submitted to hypoxia due to the existence of IL-6, VEGF, and MCP-1 contained in CM(43). Consistent with these results and previous studies(15, 23), our study demonstrated that the “cocktail therapy” effects of a vast array of cytokines identified in normoxic CM-BMSCs might contribute to donor heart preservation, thereby improving cardiac function, as reflected by increased LVEF, LVFS and decreased LVIDs in echocardiography, decreasing cardiomyocytes apoptosis of transplanted donor hearts, as evidenced by TUNEL staining, and reducing the level of serum proinflammatory cytokines (TNF-α, IL-1β, IL-6) in donor rats after heart transplantation. Therefore, our findings suggested Custodiol-supplemented with normoxic CM-BMSCs could be introduced as a novel preservation solution for donor hearts in the future, thereby providing a rational basis for cell-free therapy for donor heart preservation.
Recent studies have shown that hypoxic preconditioning can intensify the paracrine effects of BMSCs, thereby promoting BMSCs to secrete more nutritional bioactive factors, such as VEGF, HGF, and Activin A(17, 18). However, no studies have applied hypoxic CM-BMSCs into Custodiol as a preservation solution for cold storage of donor hearts. Therefore, we further compared the composition of the secretome, which was the cytokines secreted from BMSCs into the conditioned medium under either normoxic or hypoxic conditions, to determine differences that may underlie the improved post-transplant cardiac function.
In the present study, the cardioprotective effect of donor hearts from CM-BMSCs could be enhanced by hypoxic preconditioning. Compared with normoxic CM-BMSCs, the application of Custodiol-supplemented with hypoxic CM-BMSCs in the cold storage of donor hearts before heart transplantation was associated with shorter graft re-beating time, higher post-transplant cardiac contractility of donor hearts, as demonstrated by echocardiography, a lower rate of cardiomyocytes apoptosis, as reflected by TUNEL staining, and a lower level of serum proinflammatory cytokines (IL-6, TNF-α) and indices for myocardial injury (cTnI). In line with the above findings, recent studies have shown hypoxic CM-BMSCs can be used to treat retinal ischemia(20) and ischemic stroke(19) by exerting a more robust antiapoptosis effect. Furthermore, Xia et al have reported that hypoxia-preconditioned adipose-derived mesenchymal stem cells accelerated the repair of gastric mucosal injury through suppressing inflammation(44). Additionally, it has been found that hypoxic CM-BMSCs can attenuate oxygen-glucose deprivation/reoxygenation-induced injury and promote the anti-inflammatory polarization of microglia due to the beneficial effect of exosome(21).
The highly secreted soluble factors in hypoxic CM-BMSCs may account for improved post-transplant cardiac function through the inhibition of apoptosis and inflammation. The antibody array in the present study indicated that hypoxic preconditioning resulted in an increased expression of 9 cytokines. Among these identified cytokines, VEGF(42) and Activin A(38) have been demonstrated to have anti-apoptosis properties in I/R injury-induced myocardium. Additionally, Decorin secreted by human umbilical cord blood-derived mesenchymal stem cell was found to polarize inflammatory macrophages into anti-inflammatory macrophages to mitigate hyperoxic lung injury(45).
In the present study, GO term enrichment analysis showed that the biological process of increased proteins in hypoxic CM included the positive regulation of ERK1 and ERK2 cascade, positive regulation of vascular endothelial growth factor signaling pathway, and positive regulation of protein autophosphorylation. Interestingly, previous studies reported that the activation of ERK1 and ERK2 cascade(46) and vascular endothelial growth factor signaling pathway(47) were associated with the attenuation of I/R injury. These results showed that the application of Custodiol-supplemented with hypoxic CM-BMSCs for donor heart preservation protected graft against I/R injury via different signal pathways. Further studies can be carried out to investigate the contributions of these pathways involved in the cardioprotective effect of hypoxic CM-BMSCs. Additionally, our KEGG pathway enrichment analysis demonstrated that various signaling pathways were associated with increased proteins in hypoxic CM. Intriguingly, some of these signaling pathways are involved in the mitigation of myocardial I/R injury. Among these pathways, it should be mentioned that the PI3K/Akt pathway participates in the protective mechanism of ischemia preconditioning(48). PI3Ks are involved in the regulation of cell growth, proliferation, survival, and migration(49), whereas the phosphorylation of Akt, which is downstream of PI3K, can attenuate I/R injury(50). In our present study, the treatment of donor hearts with hypoxic CM-BMSCs was associated with the increased protein levels of PI3K and p‐Akt/Akt ratio compared with normoxic CM-BMSCs and vehicle groups. Therefore, hypoxic CM-BMSCs might exert a more robust cardioprotective effect for donor hearts after heart transplantation partly through activating PI3K/Akt pathway. However, no significant difference was observed for the protein levels of PI3K and p‐Akt/Akt ratio between the vehicle and the N-CM groups. This finding might indicate the PI3K/Akt pathway was not the primary mechanism underlying the beneficial effects of normoxic CM-BMSCs on donor heart preservation, which was also concluded from the recent study(16).
There are several limitations to our present studies. Firstly, although our rat model of heterotopic heart transplantation has been introduced as a suitable model to study global myocardial I/R injury, the LV unloading can lead to donor heart atrophy, thrombus formation in LV cavities, and faster myocardial recovery after I/R injury, which may not fully represent the real world of transplanted hearts. Secondly, although the concentration of CM-BMSCs (0.5 mg/ml) was selected according to previous studies, the dose-response relationship between both normoxic and hypoxic CM-BMSCs and beneficial effects for donor hearts preservation remain unclear. Thirdly, the present study was unable to identify precisely the specific cytokines or signaling pathways that account for the therapeutic effects of hypoxic CM-BMSCs for cold storage of donor hearts. To fully understand the precise mechanisms, future investigations can be performed by overexpressing or inhibiting some of the specific cytokines in hypoxic CM-BMSCs. Fourthly, the impact of longer cold ischemia time for donor hearts and the effect of CM-BMSCs on right ventricle structure and function were not evaluated.
Despite these limitations, the current study demonstrated promising potential for the hypoxic CM-BMSCs-based therapeutic strategy against myocardial I/R injury in heart transplantation. From a clinical perspective, preconditioning heart preservation solution to mitigate myocardial I/R injury during heart transplantation can reduce the incidence of PGD for donor hearts, thereby improving short- and long-term graft function and recipient’s survival. Thus, one of the most promising and novel alternatives may be the supplementation of heart preservation solution with secretome derived from BMSCs under either normoxic or hypoxic conditions. Compared with living BMSCs, CM-BMSCs can provide some advantages, including fewer complications for donor hearts (carcinogenic risk, immunological and ethical problems), convenience for storage and transportation, etc. However, before its successful clinical translation, some key issues should be solved, including the verification of optimal concentration and cardioprotective effect of hypoxic CM-BMSCs in a big animal model, the standardization of BMSC cultivation and hypoxic preconditioning BMSCs, isolation, storage, and transportation of CM-BMSCs in the clinical setting.