Donor Heart Preservation with Hypoxic Conditioned Medium Derived from Bone Marrow Mesenchymal Stem cell improves Cardiac Function in a Heart Transplantation Model

doi:10.21203/rs.3.rs-65531/v1

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Donor Heart Preservation with Hypoxic Conditioned Medium Derived from Bone Marrow Mesenchymal Stem cell improves Cardiac Function in a Heart Transplantation Model

https://doi.org/10.21203/rs.3.rs-65531/v1

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published 13 Jan, 2021

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Background: In heart transplantation, donor hearts inevitably suffer from ischemia/reperfusion (I/R) injury, which leads to primary graft dysfunction and affects patients’ survival rate. Bone marrow mesenchymal stem cells (BMSCs) have been reported to attenuate myocardial I/R injury via their paracrine effects, which can be enhanced by hypoxic preconditioning. We hypothesized that the donor heart preservation with hypoxic conditioned medium derived from BMSCs (CM-BMSCs) would improve post-transplant graft function.

Methods: Normoxic CM and hypoxic CM were isolated from rat BMSCs cultured under normoxic (20% O₂) or hypoxic (1% O₂) condition. Donor hearts were explanted, stored in cardioplegic solution supplemented with either a medium (Vehicle), normoxic CM (N-CM), or hypoxic CM (H-CM), and then heterotopically transplanted. Antibody arrays were performed to compare the differences between hypoxic CM and normoxic CM.

Results: After heart transplantation, the donor heart preservation with normoxic CM was associated with shorter re-beating time, histopathological scores, and left ventricular systolic diameter, higher ejection fraction, and fractional shortening of transplanted hearts. These protective effects may be associated with the inhibition of apoptosis and inflammation, as reflected by less TUNEL-positive cells and lower levels of serum proinflammatory cytokines (Interleukin-1β, Interleukin-6, tumor necrosis factor-α) and cardiac troponin I in the N-CM group compared with the vehicle group. These therapeutic effects can be further enhanced by hypoxic preconditioning. Antibody arrays revealed that nine proteins were significantly increased in hypoxic CM compared with normoxic CM. Furthermore, compared with vehicle and N-CM groups, the protein levels of PI3K and p‐Akt/Akt ratio in the transplanted hearts significantly increased in the H-CM group.

Conclusions: Our results indicate that cardioplegic solution-enriched with hypoxic CM-BMSCs can be a novel and promising preservation solution for donor hearts.

Stem Cell & Developmental Cell Biology

mesenchymal stem cells

hypoxic preconditioning

conditioned medium

secretome

ischemia/reperfusion injury

heterotopic heart transplantation

Heart transplantation remains a well-established therapy of choice for patients with refractory heart failure(1). Despite the increasing population of heart failure patients, the number of heart transplantation has not increased proportionally over the past 2 decades due to the shortage of donor hearts(2). Additionally, 5% to 10% of recipients have suffered from primary graft dysfunction (PGD) after heart transplantation(3).

Previous studies have shown that ischemia/reperfusion (I/R) injury acts as a significant role in PGD(4), contributing to adverse short- and long-term clinical outcomes in the recipients. The key mechanisms underlying myocardial I/R injury include increased intracellular calcium concentration, sudden generation of reactive oxygen species (ROS) and inflammatory cytokines, adenosine triphosphate (ATP) depletion, and development of metabolic acidosis. All of these factors are known to result in myocardial apoptosis(5) and the acceleration of allograft rejection or chronic allograft dysfunction. Therefore, attenuating myocardial I/R injury during the heart transplant procedure would have a favorable impact on improving short- and long-term graft function and recipient’s survival(6).

The administration of bone marrow-derived mesenchymal stem cells (BMSCs) is emerging as a promising approach for I/R injury-induced myocardial infarction due to its multiple differentiation potential(7, 8). However, recent reports have demonstrated paracrine activities are likely implicated in the therapeutic effects of BMSCs(9) due to the relatively low long-term survival rate of implanted BMSCs(10, 11). Increasing evidence has shown that BMSCs secrete a vast array of bioactive factors, such as chemokines/cytokines (12), anti-apoptotic, and growth factors (13, 14). In line with the paracrine hypothesis, hypothermic perfusion of donor hearts with conditioned medium (CM) from BMSCs (CM-BMSCs) could protect against myocardial I/R injury in old rats (15-month-old)(15). Additionally, the preservation of brain-dead donor hearts with cardioplegic solution-supplemented with CM-BMSCs improved graft contractility after transplantation(16). Notably, recent studies have demonstrated that hypoxic preconditioning BMSCs intensify their paracrine abilities by secreting more nutritional bioactive factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and Activin A(17, 18). It has been shown that the administration of hypoxic CM-BMSCs can reduce tissue damage and improve neurological recovery after ischemic stroke(19). Roth et al reported that the injection of hypoxic CM-BMSCs significantly restored retinal function and attenuated apoptosis after retinal ischemia(20). Additionally, hypoxic CM-BMSCs has been found to alleviate oxygen-glucose deprivation/reoxygenation-induced injury and promote the anti-inflammatory polarization of microglia due to the beneficial effect of exosome(21). However, no studies have applied hypoxic CM-BMSCs into Custodiol as a preservation solution for cold storage of donor hearts.

Therefore, in the current study, we hypothesized that CM-BMSCs under normoxic conditions (20% O₂ BMSCs) added to a preservation solution could improve in vivo left-ventricular (LV) graft dysfunction after heart transplantation and that these cardioprotective effects could be enhanced by hypoxic preconditioning (1% O₂ BMSCs) in our well-established heterotopic heart transplantation model(22-24).

Animals

Inbred male Lewis rats (8-12 week-old; Charles River Laboratories, Beijing, China) received care in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1996). All procedures and handling of animals during the investigations were reviewed and approved by the Ethical Committee of the Animal Experimental Center of Southern Medical University Nanfang Hospital. The animals were housed at constant ambient temperature (22±2 °C) in light-controlled rooms (12-12 hr light-dark cycles), were given food and water access ad libitum, and acclimatized for 1 week.

Experimental groups

Rats were randomly assigned to three groups. Donor hearts were arrested and stored for 1 hr in either cold Custodiol (Dr. Franz Köhler, Chemie GmbH, Bensheim, Germany) supplemented with a serum-free medium vehicle (Vehicle group, 6 to 8 rats), or Custodiol supplemented with normoxic CM-BMSCs (N-CM group, 6 to 8 rats), or Custodiol supplemented with hypoxic CM-BMSCs (H-CM group, 6 to 8 rats). Then, the hearts were heterotopically transplanted.

Isolation and culture of BMSCs

As previously reported(15), BMSCs were harvested from the bone marrow of 8-week-old male Lewis rats. Briefly, rats were euthanized with an overdose of pentobarbital sodium (100 mg/kg, intraperitoneally). Bone marrow was isolated by flushing femurs and tibias with phosphate-buffered saline (PBS, Life Technologies, Grand island, NY, USA). The cells were suspended in MEM Alpha basic(1X) (α-MEM, Life Technologies, Grand island, NY, USA) supplemented with 10% fetal bovine serum (Life Technologies, Grand island, NY, USA), 1% penicillin-streptomycin (Life Technologies, Grand island, NY, USA), and then incubated at 37°C with 5% CO₂on culture flasks. The primary culture was subcultured at a ratio of 1:2 when 80% confluency was reached. Only the third passage was used in subsequent experiments.

Preparation of hypoxic and normoxic CM

Normoxic CM and hypoxic CM were acquired as described previously with slight modifications(19). After BMSCs reached greater than 80% confluency at Passage 3, the medium was aspirated, and BMSCs were rinsed 3 times with PBS. Then, α-MEM was added to culture flasks with BMSCs, and the culture flasks were put into an incubator under the normoxic or hypoxic conditions for 24 hr. For the normoxic group, BMSCs were cultured at 37°C in a humidified atmosphere containing 5% CO₂and 20% O₂. The hypoxic group was cultured at 37°C in 1% O₂, 5% CO₂, and 94% N₂ in a hypoxic incubator (Galaxy 48, Eppendorf, Shanghai, China). The primary CM was collected after incubation for 24 hr, and the cell debris was removed using a syringe filter (Millex‐GP; Millipore, Burlington, MA, USA). Then the CM was added to dedicated ultrafiltration tubes (Amicon Ultra‐15 3K, Millipore, Burlington, MA, USA) and centrifuged (5,000g for 2 hr at 4°C) to yield concentrated CM according to the manufacturer’s protocol. Bradford protein assay was performed to quantify the protein concentration of CM to ensure that equal concentrations (0.5 mg/ml) of CM was applied in the preservation solution. α-MEM was regarded as a control (nonconditioned medium).

Screening of Secreted Proteins in CM

The cytokines/chemokines of isolated CM were qualitatively measured by using a G‐series rat cytokine array 67 (Raybiotech, Norcross, GA, USA) according to the manufacturer’s instructions. The Gene Ontology (GO) analysis with Blast2GO (https://www.blast2go.com/) and Kyoto encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to investigate the biological process categories and pathways of proteins that were differentially expressed between hypoxic CM and normoxic CM.

The rat model of heterotopic heart transplantation

Surgical technique of heart transplantation

As described elsewhere(24, 25), heterotopic heart transplantations were performed in an isogenic Lewis-to-Lewis rat strain model to exclude the effects of immune rejections and avoid the application of immunosuppressors such as cyclosporine. Cardiac arrest was induced by Custodiol cardioplegic solution. The degree of rat cardiac allografts injury correlates with increased cold ischemia time(26), therefore, to minimize the variability among experiments, the duration between harvest, cold storage, and reperfusion was standardized to 1.5 hr, as previously described (27). End-to-side anastomoses were performed between the aorta and the abdominal aorta and between the pulmonary artery and the inferior vena cava. After the completion of anastomoses, the hearts were heterotopically transplanted into recipients, and in-vivo reperfusion lasted for 1.5 hr. The re-beating time (time to restoration of heartbeat) after reperfusion was also recorded. Measurement of LV graft function and sample collection for molecular biological analysis were performed 1.5 hr after the beginning of reperfusion.

Functional measurement in the graft

As previously reported (28), at 1.5 hr after transplantation, Transabdominal echocardiographic imaging (ECG) was performed by a blinded observer using a dedicated Vevo® 2100 System (21 MHz broadband sector transducer, Visualsonics Inc., Toronto, Canada). Ejection fraction (EF), left ventricular internal diameter at end-diastole (LVIDd), and at end-systole (LVIDs) were measured between the anterior wall and the posterior wall of the left ventricle from the short-axis view at the level of papillary muscles. For each measurement, three consecutive cardiac cycles were traced and averaged. Fractional shortening (FS) was calculated as [(LVIDd - LVIDs)/ LVIDd] x 100 (%).

Histology and immunohistochemistry in the graft

The donor hearts were explanted after the functional measurements. Pieces of myocardial tissue were fixed immediately in paraformaldehyde solution (4%) and embedded in paraffin. Five-micron-thick slices of myocardium were stained with hematoxylin and eosin. The pathology changes of each heart slices were evaluated by scoring according to the grades 0-4(29), under a light microscope with four different visual fields from each sample in a blinded way.

Detection of DNA strand breaks in the graft

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to detect DNA-strand breaks was performed as previously described(23). The number of TUNEL-positive cells was counted under a fluorescence microscope and the final results were expressed as the ratio of 4′,6-diamidino-2-phenylindole (DAPI)-TUNEL double-labeled nuclei to the total number of nuclei stained with DAPI.

Proinflammatory cytokines and markers of myocardial injury

Blood samples from the recipient rats were obtained at 1.5 hr after reperfusion just before death. Blood samples were drawn from the abdominal aorta. Then, plasma samples were obtained after centrifugation (3000 rpm, 15 min, 4°C). The levels of proinflammatory cytokines (Tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, IL-6), and cardiac troponin I (cTnI) were measured via Rat ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA).

Western blotting

Myocardial protein expression was assessed by western blot as previously described(16). The ratio of phosphorylated-Akt to Akt (1:1000 dilution, Cell Signaling Technology (Shanghai) Biological Reagents Company Limited, Shanghai, China) was calculated. Additionally, western blot analysis for phosphatidylinositol 3-kinase (PI3K) was performed (1:1000 dilution, Cell Signaling Technology (Shanghai) Biological Reagents Company Limited, Shanghai, China).

Statistical Analysis

The results were expressed as mean ± standard error of the mean (SEM). GraphPad Prism 7.02 software (GraphPad Sofware, Inc., San Diego, CA, USA) was used to perform statistical analysis. Shapiro-Wilk test was performed to test the normality of data before statistical tests were applied. For data with normal distribution, a two-sample Student’s t-test was applied to analyze the difference between N-CM and H-CM groups. If the normality test failed, a nonparametric Mann-Whitney-test was used. One-way ANOVA followed by Tukey’s post-hoc-test was performed for multiple comparisons between three experimental groups. If the data failed the normality test, the nonparametric Kruskal-Wallis test followed by Dunn’s post-hoc-test was used. A value of p<0.05 was considered statistically significant.

Effect of normoxic and hypoxic CM on the transplanted heart

Re-beating time

Re-beating time of donor hearts after reperfusion was significantly decreased in the N-CM and H-CM groups compared to the vehicle group (Vehicle group 79.4 ± 12.6 s vs N-CM group 48.0 ± 4.7 s, p<0.05, Figure 1; Vehicle group 79.4 ± 12.6 s vs H-CM group 19.5 ± 2.1 s, p<0.05, Figure 1). In addition, hypoxic CM-BMSCs was associated with a significant shortening of graft re-beating time compared to the N-CM group (N-CM group 48.0 ± 4.7 s vs H-CM group 19.5 ± 2.1 s, p<0.05, Figure 1).

LV graft function

After heart transplantation, significantly increased LVEF and LVFS were observed in N-CM and H-CM groups compared to the vehicle group (LVEF: Vehicle group 42.6 ± 0.8 % vs N-CM group 60.0 ± 1.6 %, p<0.05, Vehicle group 42.6 ± 0.8 % vs H-CM group 81.4 ± 1.0 %, p<0.05, Figure 2A; LVFS: Vehicle group 20.7 ± 0.4 % vs N-CM group 31.6 ± 0.9 %, p<0.05, Vehicle group 20.7 ± 0.4 % vs H-CM group 50.2 ± 1.1 %, p<0.05, Figure 2B). Moreover, the preservation of grafts with Custodiol solution supplemented with hypoxic CM-BMSCs was associated with significantly increased LVEF and LVFS in donor hearts compared with normoxic CM-BMSCs (LVEF: N-CM group 60.0 ± 1.6 % vs H-CM group 81.4 ± 1.0 %, p<0.05, Figure 2A; LVFS: N-CM group 31.6 ± 0.9 % vs H-CM group 50.2 ± 1.1 %, p<0.05, Figure 2B).

A trend towards improved myocardial recovery in N-CM and H-CM groups compared with vehicle group was noted with regards to the increased LVIDs (Vehicle group 4.1 ± 0.1 mm vs N-CM group 3.4 ± 0.1 mm, p<0.05, Vehicle group 4.1 ± 0.1 mm vs H-CM group 2.3 ± 0.1 mm, p<0.05, Figure 2C). Furthermore, hypoxic CM-BMSCs exerted more robust effects than normoxic CM-BMSCs with respect to improved LVIDs (N-CM group 3.4 ± 0.1 mm vs H-CM group 2.3 ± 0.1 mm, p<0.05, Figure 2C). However, there was no significant difference in the LVIDd among the groups (Vehicle group 5.2 ± 0.2 mm vs N-CM group 4.9 ± 0.2 mm vs H-CM group 4.7 ± 0.2 mm, p>0.05, Figure 2D).

In general, the preservation of donor hearts with Custodiol solution supplemented with normoxic CM-BMSCs appeared to protect cardiac contractility in donor hearts subjected to acute I/R injury, and hypoxic CM-BMSCs added in the preservation solution could further enhance this cardioprotective effect for grafts (Figure 2E).

Histological and immunohistochemical evaluation of the graft

After heart transplantation, histological evaluation of hematoxylin and eosin-stained myocardial tissue demonstrated more inflammatory cell infiltration, irregularly arranged and disordered-structure myocardial fibers in the vehicle group when compared with N-CM and H-CM groups (Figures 3A and 3B). Hypoxic CM-BMSCs was associated with a significant lower histopathology score of myocardial tissue compared to the vehicle and the N-CM groups (Vehicle group 2.5 ± 0.2 vs N-CM group 1.9 ± 0.2, p<0.05, Vehicle group 2.5 ± 0.2 vs H-CM group 1.2 ± 0.1, p<0.05, Figure 3C). Additionally, the histopathology score showed a tendency to decrease (without reaching a significant difference) in the N-CM group compared to the vehicle group (Vehicle group 2.5 ± 0.2 vs N-CM group 1.9 ± 0.2, p>0.05, Figure 3C).

Apoptosis in the graft

After heart transplantation, a significantly increased number of TUNEL-positive nuclei was observed in the vehicle group compared with the N-CM and H-CM groups (Vehicle group 55.4 ± 2.6 % vs N-CM group 46.2 ± 2.3 %, p<0.05, Vehicle group 55.4 ± 2.6 % vs H-CM group 31.3 ± 1.3 %, p<0.05, Figure 4). Besides, treatment with hypoxic CM-BMSCs significantly decreased DNA strand breaks after heart transplantation compared with normoxic CM-BMSCs (N-CM group 46.2 ± 2.3 % vs H-CM group 31.3 ± 1.3 %, p<0.05, Figure 4).

Serum level of proinflammatory cytokines and indices for myocardial injury

The levels of proinflammatory cytokines plasma IL-1β, IL-6 and TNF-α measured at 1.5 hr after reperfusion were significantly lower in the N-CM and H-CM groups compared with the vehicle group (IL-1β: Vehicle group 86.2 ± 3.7 pg/ml vs N-CM group 66.0 ± 5.5 pg/ml, p<0.05, Vehicle group 86.2 ± 3.7 pg/ml vs H-CM group 64.3 ± 5.0 pg/ml, p<0.05, Figure 5A; IL-6: Vehicle group 258.3 ± 16.3 pg/ml vs N-CM group 153.1 ± 25.8 pg/ml, p<0.05, Vehicle group 258.3 ± 16.3 pg/ml vs H-CM group 68.5 ± 7.3 pg/ml, p<0.05, Figure 5B; TNF-α: Vehicle group 17.6 ± 2.0 pg/ml vs N-CM group 7.9 ± 0.4 pg/ml, p<0.05, Vehicle group 17.6 ± 2.0 pg/ml vs H-CM group 2.9 ± 0.1 pg/ml, p<0.05, Figure 5C). The treatment of donor hearts with hypoxic CM-BMSCs significantly decreased the level of plasma IL-6 and TNF-α compared with normoxic CM-BMSCs (IL-6: N-CM group 153.1 ± 25.8 pg/ml vs H-CM group 68.5 ± 7.3 pg/ml, p<0.05, Figure 5B; TNF-α: N-CM group 7.9 ± 0.4 pg/ml vs H-CM group 2.9 ± 0.1 pg/ml, p<0.05, Figure 5C). However, no significance was found for plasma IL-1β between in N-CM and H-CM groups (IL-1β: N-CM group 66.0 ± 5.5 pg/ml vs H-CM group 64.3 ± 5.0 pg/ml, p>0.05, Figure 5A). Additionally, circulating cTnI concentrations, indicating the degree of myocardial injury, were significantly lower in the H-CM group compared to the vehicle and N-CM groups (H-CM group 19.9 ± 2.2 pg/ml vs Vehicle group 272.3 ± 102.4 pg/ml, p<0.05, Figure 5D; H-CM group 19.9 ± 2.2 pg/ml vs N-CM group 179.6 ± 74.2 pg/ml, p<0.05, Figure 5D). Although not reaching a significant difference, the level of cTnI showed a tendency to decrease in the N-CM group compared to the vehicle group (Vehicle group 272.3 ± 102.4 pg/ml vs N-CM group 179.6 ± 74.2 pg/ml, p>0.05, Figure 5D).

Cytokine antibody assay

An antibody‐based protein array (67 rat cytokines) analysis was performed to investigate the component of BMSCs secretome (see the supplementary material). The screening standard for differentially expressed proteins were fold changes (hypoxic/normoxic) of ≥1.2/1 or ≤1/1.2. Compared with normoxic CM-BMSCs, there were significant increases in 9 cytokines in hypoxic CM-MSCs (Table 1, Figures 6A and 6B). Decorin (fold change = 1.90; p = 0.041), cytokine-induced neutrophil chemoattractant-2 (CINC-2) (fold change = 1.69; p=0.008), Platelet-derived growth factor-AA (PDGF-AA) (fold change= 1.67; p = 0.029), Neuropilin-2 (fold change=1.53; p=0.023), Activin A (fold change = 1.49; p = 0.021), and Beta-nerve growth factor (b-NGF) (fold change = 1.39; p = 0.013) showed the largest differences in expression.

Table 1

Differentially secreted cytokines levels between H-CM and N-CM measured by antibody array
Protein name	EntrezID	Hypoxic CM Mean ± SD	Normoxic CM mean ± SD	Fold change hypoxic/normoxic	p value
Decorin	29139	10,299.06±2,918.47	5,285.78±157.00	1.901	0.041
CINC-2	171551	96,123.26±9,191.66	57,240.66±10,395.19	1.692	0.008
PDGF-AA	25266	250,149.29±24,866.75	153,260.81±43,629.50	1.668	0.029
Neuropilin-2	81527	5,556.21±880.88	3,613.78±309.85	1.529	0.023
Activin A	29200	10,671.66±987.40	7,238.02±1,275.96	1.486	0.021
b-NGF	310738	23,323.71±2,472.75	16,720.57±1,002.78	1.391	0.013
IL-6	24498	661.95±74.93	497.30±33.82	1.327	0.026
VEGF	83785	246,817.39±15,669.76	198,745.19±16,099.43	1.243	0.021
Flt-3L	103691134	915.92±9.66	757.78±22.63	1.209	0.0004
IL-10	25325	1,824.10±59.43	2,406.64±264.84	0.761	0.021
IL-13	116553	818.78±205.59	1,273.62±57.60	0.630	0.021
CINC-1	81503	12,568.68±4,510.34	24,729.04±2,903.48	0.488	0.017

Significantly altered proteins listed first with a p-value for the fold change. N-CM and H-CM indicate normoxic and hypoxic conditioned medium, respectively. Data represent mean ± standard deviation (SD) (n = 3/group)

GO term enrichment analysis was performed to evaluate the biological value of increased proteins in secretome derived from hypoxic-preconditioning BMSCs. As for the biological process, the up-regulated proteins significantly enriched in positive regulation of ERK1 and ERK2 cascade, positive regulation of vascular endothelial growth factor signaling pathway, and positive regulation of protein autophosphorylation (Figure 7A). With regards to molecular function, the highly-expressed proteins significantly enriched in receptor ligand activity, receptor regulator activity, growth factor activity, cytokine activity, cytokine receptor binding, and growth factor receptor binding (Figure 7B). The KEGG pathway enrichment analysis demonstrated that various signaling pathways were associated with increased proteins in hypoxic CM-BMSCs compared to normoxic CM-BMSCs, such as PI3K/Akt signaling pathway (adjusted p-value = 0.00032) (Figure 8A).

Involvement of PI3K/Akt signaling in the cardioprotective effects of hypoxic CM

Our KEGG analysis demonstrated that PI3K/Akt signaling might participate in the protective effect of hypoxic CM against graft dysfunction associated with I/R injury in heart transplantation (Figures 8A). Therefore, the protein expressions of PI3K, Akt, and p‐Akt by western blotting were further investigated. The results showed that hypoxic CM-BMSCs added to the preservation solution significantly increased the protein levels of PI3K (Figure 8B) and p‐Akt/Akt ratio (Figure 8C) in the transplanted hearts compared with the vehicle group and the N-CM group. However, no significant difference was observed between the vehicle group and the N-CM group with regards to the protein levels of PI3K (Figure 8B) and the p‐Akt/Akt ratio (Figure 8C). Our results indicated that the beneficial effects of hypoxic CM-BMSCs on donor heart preservation might partly result from the activation of the PI3K/Akt signaling pathway.

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.

Taken together, the present in vivo study reveals that the preservation of donor hearts with cardioplegic solution supplemented with hypoxic CM-BMSCs significantly improves post-transplant cardiac graft contractility compared with normoxic CM-BMSCs in a rat model of heterotopic heart transplantation via the inhibition of apoptosis and inflammation. Furthermore, hypoxic-preconditioning BMSCs confers the ability to upregulate some cytokines which are associated with various potential molecular mechanisms underlying hypoxic CM-BMSCs‐mediated protection against myocardial I/R injury for donor hearts. In summary, our results indicate that cardioplegic solution-enriched with hypoxic CM-BMSCs can be a novel and promising preservation solution for donor hearts.

ATP: Adenosine triphosphate

BMSCs: Bone marrow mesenchymal stem cells

b-NGF: Beta-nerve growth factor

CM: Conditioned medium

CM-BMSCs: Conditioned medium derived from bone marrow mesenchymal stem cells

CINC-2: Cytokine-induced neutrophil chemoattractant-2

cTnI: Cardiac troponin I

DAPI: 4′,6-diamidino-2-phenylindole

ECG: Echocardiographic imaging

EF: Ejection fraction

FS: Fractional shortening

GO: Gene ontology

H-CM: Hypoxic conditioned medium

HGF: Hepatocyte growth factor

IL: Interleukin

IL-6: Interleukin-6

IL-10: Interleukin-10

I/R: Ischemia/reperfusion

KEGG: Kyoto encyclopedia of genes and genomes

LVIDd: Left ventricular internal diameter at end-diastole

LVIDs: Left ventricular internal diameter at end-systole

MCP-1: Monocyte chemoattractant protein-1

N-CM: Normoxic conditioned medium

PDGF-AA: Platelet-derived growth factor-AA

PGD: Primary graft dysfunction

PI3K: Phosphatidylinositol 3-kinase

ROS: Reactive oxygen species

SEM: Standard error of the mean

TIMP: Tissue inhibitor of metalloproteinase

TNF: Tumor necrosis factor

TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling

VEGF: Vascular endothelial growth factor

Ethics approval and consent to participate

All procedures and handling of animals during the investigations were reviewed and approved by the Ethical Committee of the Animal Experimental Center of Southern Medical University Nanfang Hospital.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was supported by grants from Guangzhou Science and Technology Planning Project (No. 201804010067), Guangdong Science and Technology Planning Project (No. 2017ZC0064), and President Foundation of Nanfang Hospital, Southern Medical University (No. 2019c030).

Authors’ contributions

P.Z., H.L., X.L., and X.L. performed the experiments, analyzed the data, and participated in the writing of the manuscript. Z.X. and P.Z. conducted the experiments and collected the data. P.Z., Y.Z., J.L., and S.Z. designed the experiments and revised the manuscript. The authors read and approved the final manuscript.

Acknowledgments

The technical assistance of Rongning Xu and Xiaoxia Huang is gratefully acknowledged.

Authors’ information

Pengyu Zhou, Hao Liu, Ximao Liu, and Xiao Ling contributed equally to this work.

Author details

¹ Department of Cardiovascular Surgery, Nanfang Hospital, Southern Medical University, 510000 Guangzhou, China

² Animal Experimental Center, Nanfang Hospital, Southern Medical University, 510000 Guangzhou, China

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Donor Heart Preservation with Hypoxic Conditioned Medium Derived from Bone Marrow Mesenchymal Stem cell improves Cardiac Function in a Heart Transplantation Model

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Journal Publication

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Abstract

Figures

Introduction

Materials And Methods

Animals

Experimental groups

Isolation and culture of BMSCs

Preparation of hypoxic and normoxic CM

Screening of Secreted Proteins in CM

The rat model of heterotopic heart transplantation

Surgical technique of heart transplantation

Functional measurement in the graft

Histology and immunohistochemistry in the graft

Detection of DNA strand breaks in the graft

Proinflammatory cytokines and markers of myocardial injury

Western blotting

Statistical Analysis

Results

Effect of normoxic and hypoxic CM on the transplanted heart

Re-beating time

LV graft function

Histological and immunohistochemical evaluation of the graft

Apoptosis in the graft

Serum level of proinflammatory cytokines and indices for myocardial injury

Cytokine antibody assay

Involvement of PI3K/Akt signaling in the cardioprotective effects of hypoxic CM

Discussion

Conclusions

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors’ contributions

Acknowledgments

Authors’ information

Author details

References

Supplementary Files

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