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Bone-derived Nestin-positive mesenchymal stem cells improve cardiac function via recruiting cardiac endothelial cells after myocardial infarction.

ABSTRACT:

Background

Bone-derived mesenchymal stem cell (BMSC) transplantation has been reported to be effective for the treatment of ischemic heart disease, but whether BMSCs are the optimal cell type remains under debate. Increasing numbers of studies have shown that Nestin, an intermediate filament protein, is a potential marker for MSCs, which raises the question of whether Nestin⁺ cells in BMSCs may play a more crucial role in myocardial repair.

Methods

Nestin⁺ cells were isolated using flow cytometry by gating for CD45^- Ter119^- CD31^- cells from the compact bone of Nestin-GFP transgenic mice, expressing GFP driven by the Nestin promoter. Colony-forming and proliferative curve assays were conducted to determine the proliferative capacity of these cells, while qRT-PCR was used to analyze the mRNA levels of relative chemokines and growth factors. Cardiac endothelial cell (CEC) recruitment was assessed via a transwell assay. Moreover, permanent ligation of the left anterior descending (LAD) coronary artery was performed to establish an acute myocardial infarction (AMI) mouse model. After cell transplantation, conventional echocardiography was conducted 1 and 4?weeks post-MI, and hearts were harvested for hematoxylin-and-eosin (HE) staining and immunofluorescence staining 1?week post-MI. Further evaluation of paracrine factor levels and administration of a neutralizing antibody (TIMP-1, TIMP-2, and CXCL12) or a CXCR4 antagonist (AMD3100) in MI hearts were performed to elucidate the mechanism involved in the chemotactic effect of Nestin⁺ BMSCs in vivo.

Results

Compared with Nestin^- BMSCs, a greater proliferative capacity of Nestin⁺ BMSCs was observed, which further exhibited moderately high expression of chemokines instead of growth factors. More CEC recruitment in the Nestin⁺ BMSC-cocultured group was observed in vitro, while this effect was obviously abolished after treatment with neutralizing antibodies against TIMP-1, TIMP-2, or CXCL12, and more importantly, blocking the CXCL12/CXCR4 axis with a AMD3100 significantly reduced the chemotactic effect of Nestin⁺ BMSCs. After transplantation into mice exposed to myocardial infarction (MI), Nestin⁺ BMSC-treated mice showed significantly improved survival and left ventricular function compared with Nestin^- BMSC-treated mice. Moreover, endogenous CECs were markedly increased, and chemokine levels were significantly higher, in the infarcted border zone with Nestin⁺ BMSC treatment. Meanwhile, neutralization of each TIMP-1, TIMP-2, or CXCL12 in vivo could reduce the left ventricular function at 1 and 4?weeks post-MI; importantly, the combined use of these three neutralizing antibodies could make a higher significance on cardiac function. Finally, blocking the CXCL12/CXCR4 axis with AMD3100 significantly reduced the left ventricular function and greatly inhibited Nestin⁺ BMSC-induced CEC chemotaxis in vivo.

Conclusions

These results suggest that Nestin⁺ BMSC transplantation can improve cardiac function in an AMI model by recruiting resident CECs to the infarcted border region via the CXCL12/CXCR4 chemokine pathway. And we demonstrated that Nestin⁺BMSC-secreted TIMP-1/2 enhances CXCL12(SDF1?)/CXCR4 axis-driven migration of endogenous Sca-1⁺ endothelial cells in ischemic heart post-AMI. Taken together, our results show that Nestin is a useful marker for the identification of functional BMSCs and indicate that Nestin⁺ BMSCs could be a better therapeutic candidate for cardiac repair.

SUBMITTER: Lu D

PROVIDER: S-EPMC6487029 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Project description:Acute myocardial infarction (AMI) is the most critical heart disease. Mesenchymal stem cells (MSCs) have been widely used as a therapy for AMI for several years. The human placenta has emerged as a valuable source of transplantable cells of mesenchymal origin that can be used for multiple cytotherapeutic purposes. However, the different abilities of first trimester placental chorion mesenchymal stem cells (FCMSCs) and third trimester placental chorion mesenchymal stem cells (TCMSCs) have not yet been explored. In this study, we aimed to compare the effectiveness of FCMSCs and TCMSCs on the treatment of AMI. FCMSCs and TCMSCs were isolated and characterized, and then they were subjected to in vitro endothelial cell (EC) differentiation induction and tube formation to evaluate angiogenic ability. Moreover, the in vivo effects of FCMSCs and TCMSCs on cardiac improvement were also evaluated in a rat MI model. Both FCSMCs and TCMSCs expressed a series of MSCs surface markers. After differentiation induction, FCMSCs-derived EC (FCMSCs-EC) exhibited morphology that was more similar to that of ECs and had higher CD31 and vWF levels than TCMSCs-EC. Furthermore, tube formation could be achieved by FCMSCs-EC that was significantly better than that of TCMSCs-EC. Especially, FCMSCs-EC expressed higher levels of pro-angiogenesis genes, PDGFD, VEGFA, and TNC, and lower levels of anti-angiogenesis genes, SPRY1 and ANGPTL1. In addition, cardiac improvement, indicated by left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVEDs), left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVSF), could be observed following treatment with FCMSCs, and it was superior to that of TCMSCs and Bone marrow MSCs (BMSCs). FCMSCs exhibited a superior ability to generate EC differentiation, as evidenced by in vitro morphology, angiogenic potential and in vivo cardiac function improvement; further, increased levels of expression of pro-angiogenesis genes may be the mechanism by which this effect occurred.

Project description:Background Cardiac-resident mesenchymal stem cells (cMSCs) can exhibit fibrotic, proinflammatory, and proangiogenic phenotype in response to myocardial ischemia (Isch). How their phenotypic fate decisions are determined remains poorly understood. Here, we demonstrate that the cooperation of Oct4 and c-Myc in cMSCs creates a preferable mesenchymal-to-endothelial transition (MEndoT) to promote angiogenesis and consequent myocardial repair. Methods We collected MSCs from cardiac and peripheral blood of rat with left ventricular Isch (LV Isch) 30 days after myocardial infarction (MI) or sham operation. After a comparison of characterization between cMSCs and peripheral blood MSCs (pbMSCs), we conducted transcriptome analysis and RNA sequencing of cMSCs. Using loss/gain-of-function approaches to understand the cooperation of c-Myc and Oct4 on MEndoT of cMSCs under hypoxic condition, we explored the mechanisms through transcriptome and functional experiment, and chromatin immunoprecipitation. Next, we transplanted male cMSCs with overexpression or inhibition of c-Myc/Oct4 into the infarcted myocardium of female rats and evaluated infarct size, cell retention, inflammation, remodeling, and function after 30 days. Results LV Isch switched cMSCs toward both inflammatory and proangiogenic phenotypes, with increased secretion of inflammatory cytokines as well as decreased expression of proangiogenic factors. The effect of LV Isch on pbMSCs was less remarkable. Gene expression heatmap showed imbalance in expression of Oct4 and c-Myc regulating genes associated with remodeling of cMSCs. We provided evidence that cMSCs-specific c-Myc- versus Oct4-overexpression showed divergent genomic signatures, and their corresponding target genes play an important role in regulating cMSCs phenotypic changes. In particular, Oct4 accelerated angiogenesis induced by c-Myc overexpression in cMSCs and inhibited their phenotypic transition into inflammatory cells and fibroblast. Mechanistically, exogenous Oct4 caused c-Myc to translocate from the nucleus to the cytoplasm and activated some of its target signalings including VEGF signaling. Although transplantation of cMSCs alone did not improve LV remodeling and function, cMSCs co-transfected with c-Myc and Oct4 promoted a more positive effect in their survival and reparative properties, increased animal survival, reduced infarct size, decreased scar thickness, inhibited LV remodeling, and improved heart function 30 days after MI. Significantly, Oct4 promoted MEndoT (“Rescue me” signal) of cMSCs after both c-Myc stimulation in vitro and transplantation into the infarcted heart. Conclusions Myocardial Isch drives resident cMSCs toward multiple phenotypes. Oct4 interacts with c-Myc to promote MEndoT capacity of cMSCs and improve their survival and reparative effects through upregulation of angiogenesis-related signaling pathways. These findings may identify novel targets for stem cell therapy. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-022-03120-7.