Bei Shi

Featured Article: Transplantation of mesenchymal stem cells carrying the human receptor activity-modifying protein 1 gene improves cardiac function and inhibits neointimal proliferation in the carotid angioplasty and myocardial infarction rabbit model

Although transplanting mesenchymal stem cells (MSCs) can improve cardiac function and contribute to endothelial recovery in a damaged artery, natural MSCs may induce neointimal hyperplasia by directly or indirectly acting on vascular smooth muscle cells (VSMCs). Receptor activity-modifying protein 1 (RAMP1) is the component and the determinant of ligand specificity of calcitonin gene-related peptide (CGRP). It is recently reported that CGRP and its receptor involve the proliferation and the apoptosis in vivo and in vitro, and the exogenous RAMP1 enhances the antiproliferation effect of CGRP in VSMCs. Here, we investigated the effects of MSCs overexpressing the human receptor activity-modifying protein 1 (hRAMP1) on heart function and artery repair in rabbit models of myocardial infarction (MI) reperfusion and carotid artery injury. MSCs transfected with a recombinant adenovirus containing the hRAMP1 gene (EGFP-hRAMP1-MSCs) were injected into the rabbit models via the ear vein at 24 h after carotid artery injury and MI 7 days post-EGFP-hRAMP1-MSC transplantation. The cells that expressed both enhance green fluorescent protein (EGFP) and CD31 were detected in the neointima of the damaged artery via immunofluorescence. EGFP-hRAMP1 expression was observed in the injured artery and infarcted myocardium by western blot analysis, confirming that the engineered MSCs targeted the injured artery and infarcted myocardium and expressed hRAMP1 protein. Compared with the EGFP-MSCs group, the EGFP-hRAMP1-MSCs group had a significantly smaller infarcted area and improved cardiac function by 28 days after cell transplantation, as detected by triphenyltetrazolium chloride staining and echocardiography. Additionally, arterial hematoxylin–eosin staining revealed that the area of the neointima and the area ratio of intima/media were significantly decreased in the EGFP-hRAMP1-MSCs group. An immunohistological study showed that the expression of α-smooth muscle antigen and proliferating cell nuclear antigen in the neointima cells of the carotid artery of the EGFP-hRAMP1-MSCs group was approximately 50% lower than that of the EGFP-MSCs group, suggesting that hRAMP1 expression may inhibit VSMCs proliferation within the neointima. Therefore, compared with natural MSCs, EGFP-hRAMP1-engineered MSCs improved infarcted heart function and endothelial recovery from artery injury more efficiently, which will provide valuable information for the development of MSC-based therapy.

miR-21 Reduces Hydrogen Peroxide-Induced Apoptosis in c-kit(+) Cardiac Stem Cells In Vitro through PTEN/PI3K/Akt Signaling.

The low survival rate of cardiac stem cells (CSCs) in the infarcted myocardium hampers cell therapy for ischemic cardiomyopathy. MicroRNA-21 (miR-21) and one of its target proteins, PTEN, contribute to the survival and proliferation of many cell types, but their prosurvival effects in c-kit+ CSC remain unclear. Thus, we hypothesized that miR-21 reduces hydrogen peroxide- (H2O2-) induced apoptosis in c-kit+ CSC and estimated the contribution of PTEN/PI3K/Akt signaling to this oxidative circumstance. miR-21 mimics efficiently reduced H2O2-induced apoptosis in c-kit+ CSC, as evidenced by the downregulation of the proapoptosis proteins caspase-3 and Bax and upregulation of the antiapoptotic Bcl-2. In addition, the gain of function of miR-21 in c-kit+ CSC downregulated the protein level of PTEN although its mRNA level changed slightly; in the meantime, miR-21 overexpression also increased phospho-Akt (p-Akt). The antiapoptotic effects of miR-21 were comparable with Phen (bpV), the selective inhibitor of PTEN, while miR-21 inhibitor or PI3Ks inhibitor LY294002 efficiently attenuated the antiapoptotic effect of miR-21. Taken together, these results indicate that the anti-H2O2-induced apoptosis effect of miR-21 in c-kit+ CSC is contributed by PTEN/PI3K/Akt signaling. miR-21 could be a potential molecule to facilitate the c-kit+ CSC therapy in ischemic myocardium.

Bone marrow mesenchymal stem cell-derived exosomal miR-21 protects C-kit+ cardiac stem cells from oxidative injury through the PTEN/PI3K/Akt axis

Stem cell (SC) therapy for ischemic cardiomyopathy is hampered by poor survival of the implanted cells. Recently, SC-derived exosomes have been shown to facilitate cell proliferation and survival by transporting various proteins and non-coding RNAs (such as microRNAs and lncRNAs). In this study, miR-21 was highly enriched in exosomes derived from bone marrow mesenchymal stem cells (MSCs). Interestingly, exosomes collected from hydrogen peroxide (H2O2)-treated MSCs (H-Exo) contained higher levels of miR-21 than exosomes released from MSCs under normal conditions (N-Exo). The pre-treatment of C-kit+ cardiac stem cells (CSCs) with H-Exos resulted in significantly increased levels of miR-21 and phosphor-Akt (pAkt) and decreased levels of PTEN, which is a known target of miR-21. AnnexinV-FITC/PI analysis further demonstrated that the degree of oxidative stress-induced apoptosis was markedly lower in H-Exo-treated C-kit+ CSCs than that in N-Exo-treated cells. These protective effects could be blocked by both a miR-21 inhibitor and the PI3K/Akt inhibitor LY294002. Therefore, exosomal miR-21 derived from H2O2-treated MSCs could be transported to C-kit+ cardiac stem cells to functionally inhibit PTEN expression, thereby activating PI3K/AKT signaling and leading to protection against oxidative stress-triggered cell death. Thus, exosomes derived from MSCs could be used as a new therapeutic vehicle to facilitate C-kit+ CSC therapies in the ischemic myocardium.

miR-21 increases c-kit+ cardiac stem cell proliferation in vitro through PTEN/PI3K/Akt signaling

The low survival rate of cardiac stem cells (CSCs) in the ischemic myocardium is one of the obstacles in ischemic cardiomyopathy cell therapy. The MicroRNA (miR)-21 and one of its target protein, the tensin homolog deleted on chromosome ten (PTEN), contributes to the proliferation of many kinds of tissues and cell types. It is reported that miR-21 promotes proliferation through PTEN/PI3K/Akt pathway, but its effects on c-kit+ CSC remain unclear. The authors hypothesized that miR-21 promotes the proliferation in c-kit+ CSC, and evaluated the involvement of PTEN/PI3K/Akt pathway in vitro. miR-21 up-regulation with miR-21 efficiently mimics accelerated cell viability and proliferation in c-kit+ CSC, which was evidenced by the CCK-8, EdU and cell cycle analyses. In addition, the over-expression of miR-21 in c-kit+ CSCs notably down-regulated the protein expression of PTEN although the mRNA level of PTEN showed little change. Gain-of-function of miR-21 also increased the phosphor-Akt (p-Akt) level. Phen, the selective inhibitor of PTEN, reproduced the pro-proliferation effects of miR-21, while PI3K inhibitor, LY294002, totally attenuated the pro-survival effect of miR-21. These results indicate that miR-21 is efficient in promoting proliferation in c-kit+ CSCs, which is contributed by the PTEN/PI3K/Akt pathway. miR-21 holds the potential to facilitate CSC therapy in ischemic myocardium.

e0073 Effect of bone marrow mesenchymal stem cells transplantation on expression of NFB and PCNA and vascular stenosis after carotid artery balloon injury of rabbit

Objective To investigate effect of bone marrow mesenchymal stem cells transplantation on expression of nuclear factor κB (NF-κB) and proliferating cell nuclear antigen (PCNA) and vascular stenosis after carotid artery balloon injury of rabbit. Methods 36 carotid artery atherosclerotic stenosis rabbits were randomly divided into the control group (balloon injury+PBS solution) and the MSCs transplantation group (balloon injury+MSCs transplantation). MSCs (5×107/ml) were pre-labelled by DAPI and then infused into MSCs transplantation group rabbits by the ear vein, and control group was infused with the same amount of PBS solution. 1 week after MSCs transplantation, DAPI labelled cells were detected under immunofluorescence microscope; The plasma tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were detected with ELISA on the 1st, 2nd and 4th week after MSCs transplantation. After 2 weeks and 4 weeks, the injured vessels were stained by HE and the immunohistochemical analysis of NF-κB and PCNA. Results The DAPI-labelled MSCs could be detected on impaired intimae 1 week after MSCs transplantation. NF-κB and PCNA expression was not seen in the normal blood vessels after 2 weeks, the expression of NF-κB and PCNA in MSCs transplantation group decreased significantly compared with control group. The plasma TNF-α and IL-6 levels in MSCs transplantation group were significantly lower than those in control group. The intimal area, the ratio of the intima/media area and the luminal stenosis ratio were significantly lower in MSCs transplantation group than control group at 4 weeks. Conclusions MSCs are capable of decreasing the inflammatory reaction of injured vessels and lighten the restenosis of injured vessels.

Exosomes Derived from miR-214-Enriched Bone Marrow-Derived Mesenchymal Stem Cells Regulate Oxidative Damage in Cardiac Stem Cells by Targeting CaMKII

Cardiac stem cells (CSCs) have emerged as one of the most promising stem cells for cardiac protection. Recently, exosomes from bone marrow-derived mesenchymal stem cells (BMSCs) have been found to facilitate cell proliferation and survival by transporting various bioactive molecules, including microRNAs (miRs). In this study, we found that BMSC-derived exosomes (BMSC-exos) significantly decreased apoptosis rates and reactive oxygen species (ROS) production in CSCs after oxidative stress injury. Moreover, a stronger effect was induced by exosomes collected from BMSCs cultured under hypoxic conditions (Hypoxic-exos) than those collected from BMSCs cultured under normal conditions (Nor-exos). We also observed greater miR-214 enrichment in Hypoxic-exos than in Nor-exos. In addition, a miR-214 inhibitor or mimics added to modulate miR-214 levels in BMSC-exos revealed that exosomes from miR-214-depleted BMSCs partially reversed the effects of hypoxia-induced exosomes on oxidative damage in CSCs. These data further confirmed that miR-214 is the main effector molecule in BMSC-exos that protects CSCs from oxidative damage. miR-214 mimic and inhibitor transfection assays verified that CaMKII is a target gene of miR-214 in CSCs, with exosome-pretreated CSCs exhibiting increased miR-214 levels but decreased CaMKII levels. Therefore, the miR-214/CaMKII axis regulates oxidative stress-related injury in CSCs, such as apoptosis, calcium homeostasis disequilibrium, and excessive ROS accumulation. Collectively, these findings suggest that BMSCs release miR-214-containing exosomes to suppress oxidative stress injury in CSCs through CaMKII silencing.

TCT-823 miR-21 reduces hydrogen peroxide-induced apoptosis in c-kit+ cardiac stem cells in vitro through PTEN/PI3K/Akt signaling

RESULTS In vitro cultured VSMCs, the expression of myocardin shows downregulation along with time expansion. The level of myocardin protein is higher at 48h and 72h than at baseline in the cultured VSMCs(p<0.05). However, the myocardin is lower at 48h and 72h than at baseline after the treatment of CGRP in the cultured VSMCs (p<0.05). Furthermore, at 48h in the cultured VSMCs, the myocardin decrease along with SM a-actin(p<0.05), but OPN increase (p<0.05)in the AngIIgroup than that in the control group; After the treatment of CGRP, the levels of myocardin and SM a-actin become higher(p<0.05)but OPN grow lower(p<0.05)in the CGRP group than the AngIIgroup. Also, CGRP8-37 abrogates CGRP-induced increase in myocardin and SM a-actin and decrease in OPN in CGRP8-37 group compared with the CGRP group.

TCT-822 Effect of calcitonin gene-related peptide on myocardin expression and phenotypic switch in the vascular smooth muscle cells

RESULTS In vitro cultured VSMCs, the expression of myocardin shows downregulation along with time expansion. The level of myocardin protein is higher at 48h and 72h than at baseline in the cultured VSMCs(p<0.05). However, the myocardin is lower at 48h and 72h than at baseline after the treatment of CGRP in the cultured VSMCs (p<0.05). Furthermore, at 48h in the cultured VSMCs, the myocardin decrease along with SM a-actin(p<0.05), but OPN increase (p<0.05)in the AngIIgroup than that in the control group; After the treatment of CGRP, the levels of myocardin and SM a-actin become higher(p<0.05)but OPN grow lower(p<0.05)in the CGRP group than the AngIIgroup. Also, CGRP8-37 abrogates CGRP-induced increase in myocardin and SM a-actin and decrease in OPN in CGRP8-37 group compared with the CGRP group.

TCT-556 Research and clinical applications of potical coherence tomography in 25 cases of the drug-eluting stents implantated patients with in-stent restenosis

Late in-stent restenosis (ISR) is an important clinical issue in the drug-eluting stent (DES) era. Optical coherence tomography (OCT) is the most advanced technique in detecting intravascular lesions. OCT not only can provide the detailed information about restenotic tissue, but also can identify

HRAMP1 MODIFIED MSCS IMPROVE CARDIAC FUNCTION AND INHIBIT NEOINTIMAL PROLIFERATION IN THE CAROTID ANGIOPLASTY AND MYOCARDIAL INFARCTION RABBIT MODEL

Objectives Although transplanting mesenchymal stem cells (MSCs) can improve cardiac function and contribute to endothelial recovery in a damaged artery, natural MSCs may induce neointimal hyperplasia by directly or indirectly acting on vascular smooth muscle cells (VSMCs). Here, we investigated the effects of MSCs overexpressing the human receptor activity-modifying protein 1 (hRAMP1) on heart function and artery repair in rabbit models of myocardial infarction reperfusion and carotid artery injury. Methods MSCs transfected with a recombinant adenovirus containing the hRAMP1 gene (EGFP-hRAMP1-MSCs) were injected into the rabbit models via the ear vein at 24 h after carotid artery injury and myocardial infarction. Results Seven days post EGFP-hRAMP1-MSC transplantation, the cells that expressed both EGFP and CD31 were detected in the neointima of the damaged artery via immunofluorescence. EGFP-hRAMP1 expression was observed in the injured artery and infarcted myocardium by western blot analysis, confirming that the engineered MSCs were targeted the injured artery and infarcted myocardium and expressed hRAMP1 protein. Compared with the EGFP-MSCs group, the EGFP-hRAMP1-MSCs group had a significantly smaller infarcted area and improved cardiac function by 28 day after cell transplantation, as detected by triphenyltetrazolium chloride (TTC) staining and echocardiography. Additionally, arterial HE staining revealed that the area of the neointima and the area ratio of intima-media were significantly decreased in the EGFP-hRAMP1-MSCs group. Conclusions Therefore, compared with natural MSCs, EGFP-hRAMP1-engineered MSCs improved infarcted heart function and endothelial recovery from artery injury more efficiently, which will provide valuable information for the development of MSCs-based therapy.