Dual-modal tracking of transplanted mesenchymal stem cells after myocardial infarction

Yefei Li*, Yuyu Yao*, Zulong Sheng, Yanxiaoxiao Yang, Genshan Ma,Department of Cardiology, Zhongda Hospital, Medical School of Southeast University, Nanjing, Jiangsu, China*These two authors contributed equally to this work.Purpose: Results for implantation efficiency and effective improvement of cardiac function in the field of mesenchymal stem cells (MSCs) are controversial. To attempt to clarify this debate, we utilized magnetic resonance imaging (MRI) and near-infrared optical imaging (OI) to explore the effects of different delivery modes of mesenchymal stem cells on cell retention time and cardiac function after myocardial infarction (MI).Methods: Rat MSCs were labeled with superparamagnetic iron oxide nanoparticles and 1, 1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) for noninvasive cell tracking in a rat MI model. Rats underwent coronary artery ligation and were randomized into three experimental groups: intravenous (IV), intramyocardial (IM), and a control group. The first two groups referred to the route of delivery of the transplanted dual-labeled MSCs; whereas the control group was given an IV injection of serum-free medium one day post-MI. Cellular engraftment was determined 1 day and 7 days post cell delivery by measuring the iron and optical signals in explanted organs. Prussian blue staining and fluorescent microscopy were performed on histological sections for iron and DiD, respectively. Cardiac function was measured by echocardiography on day 7.Results: The cardiac function of the IM group increased significantly compared to the IV and control groups at day 7. In the IM group, labeled cells were visualized in the infracted heart by serial MRI, and the intensity by OI was significantly higher on day 1. In the IV group, the heart signals were significantly attenuated by dual-modal tracking at two time points, but the lung signals in OI were significantly stronger than the IM group at both time points.Conclusion: IM injection of MSCs increased cell engraftment within infarcted hearts and improved cardiac function after MI. However, IV infusion has a low efficacy due to the cell trapping in the lung. Therefore, direct injection may provide an advantage over IV, with regard to retention of stem cells and protection of cardiac function.Keywords: stem cell tracking, superparamagnetic iron oxide, DiD, cardiac function, myocardial infarctio

Vasculoprotective effects of rosiglitazone through modulating renin-angiotensin system in vivo and vitro

<p>Abstract</p> <p>Background</p> <p>The peroxisome proliferator-activated receptor-γ (PPARγ) agonist rosiglitazone has been suggested to exert cardiovascular protection through the improvement of lipid metabolism, anti-inflammation, anti-proliferation etc. However, whether renin-angiotensin system (RAS) is involved in the vascular protective effects of PPARγ agonists is not fully understood. The present study aimed to investigate the effects of the renin-angiotensin system in vascular protection mediated by PPARγ agonists.</p> <p>Objective</p> <p>To investigate the actions of the renin-angiotensin system in vascular protection mediated by activation of PPARγ in vivo and in vitro.</p> <p>Methods</p> <p>Rats were fed a regular diet (n = 8), a cholesterol-rich diet plus methylthiouracil (80 mg/Kg/day, n = 10), a cholesterol-rich diet plus methylthiouracil and rosiglitazone (4 mg/kg/day, n = 10). The rosiglitazone treatment was started from one month after the start of cholesterol-rich diet plus methylthiouracil, and lasted five months. Cultured vascular smooth muscle cells (VSMCs) were pretreated with 1 μmol/L angiotensin II (ANG II) for 6 h and randomly divided into the control group; the ANG II group (1 μmol/L ANG II); the groups respectively treated with different concentration rosiglitazone (20, 30, 50) μmol/L for 12 h; the groups treated with 30 μmol/L rosiglitazone for (6, 12, 24) h. Morphology changes of the aortic tissues were observed by hematoxylin and eosin stain. The VSMC growth was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Angiotensin II and expression of angiotensin receptors were determined by radioimmunoassay, reverse transcription polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry.</p> <p>Results</p> <p>After 6 months, lipid deposition, VSMC proliferation and migration toward intima were observed in aortic tissues in the rats on a cholesterol-rich diet plus methylthiouracil, while these pathological changes induced by the cholesterol-rich diet were significantly suppressed by rosiglitazone. In addition, VSMC proliferation induced by ANG II was markedly inhibited by rosiglitazone. Rosiglitazone markedly down-regulated expression of angiotensin type 1 receptor (AT₁R) and up-regulated expression of angiotensin type 2 receptor (AT₂R) in the aortic tissues and ANG II-treated VSMCs.</p> <p>Conclusions</p> <p>The present study demonstrated that PPARγ agonist rosiglitazone suppressed ANG II-induced VSMC proliferation in vitro and early atherosclerotic formation evoked by cholesterol-rich diet in vivo. These vasculoprotective effects of rosiglitazone were mediated at least partially by reduction in local tissue ANG II concentration, down-regulation of AT₁R expression and up-regulation of AT₂R expression both at the mRNA and protein levels.</p

Hypoxic preconditioning improves survival of cardiac progenitor cells: role of stromal cell derived factor-1α-CXCR4 axis.

BACKGROUND: Cardiac progenitor cells (CPCs) have been shown to be suitable in stem cell therapy for resurrecting damaged myocardium, but poor retention of transplanted cells in the ischemic myocardium causes ineffective cell therapy. Hypoxic preconditioning of cells can increase the expression of CXCR4 and pro-survival genes to promote better cell survival; however, it is unknown whether hypoxia preconditioning will influence the survival and retention of CPCs via the SDF-1α/CXCR4 axis. METHODS AND RESULTS: CPCs were isolated from adult mouse hearts and purified by magnetic activated cell sorting using c-kit magnetic beads. These cells were cultured at various times in either normoxic or hypoxic conditions, and cell survival was analyzed using flow cytometry and the expression of hypoxia-inducible factor-1α (HIF-1α), CXCR4, phosphorylated Akt and Bcl-2 were measured by Western blot. Results showed that the expression of pro-survival genes significantly increased after hypoxia treatment, especially in cells cultured in hypoxic conditions for six hours. Upon completion of hypoxia preconditioning from c-kit+ CPCs for six hours, the anti-apoptosis, migration and cardiac repair potential were evaluated. Results showed a significant enhancement in anti-apoptosis and migration in vitro, and better survival and cardiac function after being transplanted into acute myocardial infarction (MI) mice in vivo. The beneficial effects induced by hypoxia preconditioning of c-kit+ CPCs could largely be blocked by the addition of CXCR4 selective antagonist AMD3100. CONCLUSIONS: Hypoxic preconditioning may improve the survival and retention of c-kit+ CPCs in the ischemic heart tissue through activating the SDF-1α/CXCR4 axis and the downstream anti-apoptosis pathway. Strategies targeting this aspect may enhance the effectiveness of cell-based cardiac regenerative therapy

Bradykinin Preconditioning Improves Therapeutic Potential of Human Endothelial Progenitor Cells in Infarcted Myocardium

<div><p>Objectives</p><p>Stem cell preconditioning (PC) is a powerful approach in reducing cell death after transplantation. We hypothesized that PC human endothelial progenitor cells (hEPCs) with bradykinin (BK) enhance cell survival, inhibit apoptosis and repair the infarcted myocardium.</p> <p>Methods</p><p>The hEPCs were preconditioned with or without BK. The hEPCs apoptosis induced by hypoxia along with serum deprivation was determined by annexin V-fluorescein isothiocyanate/ propidium iodide staining. Cleaved caspase-3, Akt and eNOS expressions were determined by Western blots. Caspase-3 activity and vascular endothelial growth factor (VEGF) levels were assessed in hEPCs. For <i>in</i><i>vivo</i> studies, the survival and cardiomyocytes apoptosis of transplanted hEPCs were assessed using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodi- carbocyanine,4-chlorobenzenesul-fonate salt labeled hEPCs and TUNEL staining. Infarct size and cardiac function were measured at 10 days after transplantation, and the survival of transplanted hEPCs were visualized using near-infrared optical imaging.</p> <p>Results</p><p><i>In</i><i>vitro</i> data showed a marked suppression in cell apoptosis following BK PC. The PC reduced caspase-3 activation, increased the Akt, eNOS phosphorylation and VEGF levels. <i>In</i><i>vivo</i> data in preconditioned group showed a robust cell anti-apoptosis, reduction in infarct size, and significant improvement in cardiac function. The effects of BK PC were abrogated by the B2 receptor antagonist HOE140, the Akt and eNOS antagonists LY294002 and L-NAME, respectively.</p> <p>Conclusions</p><p>The activation of B2 receptor-dependent PI3K/Akt/eNOS pathway by BK PC promotes VEGF secretion, hEPC survival and inhibits apoptosis, thereby improving cardiac function <i>in</i><i>vivo</i>. The BK PC hEPC transplantation for stem cell-based therapies is a novel approach that has potential for clinical used. </p> </div

Ex vivo optical imaging study.

<p>(A and B) Representative NIR fluorescent images of explanted organs at 2 days or 10 days following the implantation of DiD-labeled hEPCs into the ischemic myocardium of nude mice. Bars represent maximum radiance. (C) Quantitative analysis of NIR fluorescent signals in the explanted hearts of each group at two time points. (A: 2 days after cell delivery, B: 10 days after cell delivery). All values are expressed as mean ± SEM. n = 5 for each group, *<i>P</i> < 0.01 <i>vs</i>. other myocardial infarction groups. </p

Inhibitory effect of BK preconditioning on hypoxia along with serum deprivation-induced apoptosis of hEPCs.

<p>(A) hEPCs were exposed to hypoxia along with serum deprivation for 12 hours. Representative flow cytometry analysis of apoptotic cells after being labeled with annexin V and propidium iodide. (B) Quantitative analysis of the apoptotic cells (annexin V positive). Data were obtained from six independent experiments and are expressed as mean ± SEM. *<i>P</i> < 0.01 <i>vs</i>. other groups during cell apoptosis.</p