Xinxing Xie

Deep magnetic capture of magnetically loaded cells for spatially targeted therapeutics

Magnetic targeting has recently demonstrated potential in promoting magnetically loaded cell delivery to target lesion, but its application is limited by magnetic attenuation. For deep magnetic capture of cells for spatial targeting therapeutics, we designed a magnetic pole, in which the magnetic field density can be focused at a distance from the pole. As flowing through a tube served as a model of blood vessels, the magnetically loaded mesenchymal stem cells (MagMSCs) were highly enriched at the site distance from the magnetic pole. The cell capture efficiency was positively influenced by the magnetic flux density, and inversely influenced by the flow velocity, and well-fitted with the deductive value by theoretical considerations. It appeared to us that the spatially-focused property of the magnetic apparatus promises a new deep targeting strategy to promote homing and engraftment for cellular therapy.

High density lipoprotein protects mesenchymal stem cells from oxidative stress-induced apoptosis via activation of the PI3K/Akt pathway and suppression of reactive oxygen species.

The therapeutic effect of transplantation of mesenchymal stem cells (MSCs) in myocardial infarction (MI) appears to be limited by poor cell viability in the injured tissue, which is a consequence of oxidative stress and pro-apoptotic factors. High density lipoprotein (HDL) reverses cholesterol transport and has anti-oxidative and anti-apoptotic properties. We, therefore, investigated whether HDL could protect MSCs from oxidative stress-induced apoptosis. MSCs derived from the bone marrow of rats were pre-incubated with or without HDL, and then were exposed to hydrogen peroxide (H2O2) in vitro, or were transplanted into experimentally infarcted hearts of rats in vivo. Pre-incubation of MSCs with HDL increased cell viability, reduced apoptotic indices and resulted in parallel decreases in reactive oxygen species (ROS) in comparison with control MSCs. Each of the beneficial effects of HDL on MSCs was attenuated by inhibiting the PI3K/Akt pathway. Preconditioning with HDL resulted in higher MSC survival rates, improved cardiac remodeling and better myocardial function than in the MSC control group. Collectively, these results suggest that HDL may protect against H2O2-induced apoptosis in MSCs through activation of a PI3K/Akt pathway, and by suppressing the production of ROS.

High density lipoprotein cholesterol promotes the proliferation of bone-derived mesenchymal stem cells via binding scavenger receptor-B type I and activation of PI3K/Akt, MAPK/ERK1/2 pathways

High-density lipoprotein (HDL) possesses protective properties in cardiovascular diseases. However, the effect of HDL on the mesenchymal stem cells (MSCs), which could be mobilized to the damaged myocardial tissue, has not been well elucidated yet. In the current study, we investigated the effect of HDL on the proliferation of MSCs so as to reveal its molecular mechanisms. MSCs derived from rats were treated with HDL in different concentrations and for different periods. The proliferation of MSCs was measured with MTT and BrdU cell proliferation assay. The phosphorylation of Akt, ERK1/2 and the expression of p21 were evaluated by Western blotting. After the activity of respective pathways was down-regulated by the specific inhibitor and the gene of scavenger receptor-B type I (SR-BI) was knocked down by RNA interference, BrdU assay was performed to examine this effect of HDL on MSCs. We found that the proliferation of MSCs induced by HDL, in a time- and concentration-dependent manner, was the phosphorylation of Akt- and ERK1/2-dependent, which was significantly attenuated by the specific inhibitor to respective pathways. Moreover, MAPK/ERK1/2 pathway exerted a more dominating effect on this process. SR-BI contributed to HDL-induced proliferation of MSCs, which was effectively abolished by the silencing of SR-BI. The results suggested that HDL was capable of improving MSCs proliferation, in which MAPK/ERK1/2 and PI3K/Akt pathways involved and SR-BI played a critical role as well.

Magnetic resonance hypointensive signal primarily originates from extracellular iron particles in the long-term tracking of mesenchymal stem cells transplanted in the infarcted myocardium.

Purpose The long-lasting hypointensities in cardiac magnetic resonance (CMR) were believed to originate from superparamagnetic iron oxide (SPIO)-engulfed macrophages during long-term stem cell tracking. However, the iron clearance capacity of the ischemic heart was limited. Therefore, we speculated that the extracellular SPIO particles may also be involved in the generation of false-positive signals. Methods and results Male swine mesenchymal stem cells (MSCs) were incubated with SPIO for 24 hours, and SPIO labeling had no significant effects on either cell viability or differentiation. In vitro studies showed that magnetic resonance failed to distinguish SPIO from living SPIO-MSCs or dead SPIO-MSCs. Two hours after the establishment of the female swine acute myocardial infarction model, 2×107 male SPIO-labeled MSCs (n=5) or unlabeled MSCs (n=5) were transextracardially injected into the infarcted myocardium at ten distinct sites. In vivo CMR with T2 star weighted imaging-flash-2D sequence revealed a signal void corresponding to the initial SPIO-MSC injection sites. At 6 months after transplantation, CMR identified 32 (64%) of the 50 injection sites, where massive Prussian blue-positive iron deposits were detected by pathological examination. However, iron particles were predominantly distributed in the extracellular space, and a minority was distributed within CD68-positive macrophages and other CD68-negative cells. No sex-determining region Y DNA of donor MSCs was detected. Conclusion CMR hypointensive signal is primarily caused by extracellular iron particles in the long-term tracking of transplanted MSCs after myocardial infarction. Consideration should be given to both the false-positive signal and the potential cardiac toxicity of long-standing iron deposits in the heart.

Another possible cell source for cardiac regenerative medicine: Reprogramming adult fibroblasts to cardiomyocytes and endothelial progenitor cells

Ischemic heart disease (IHD) is a significant burden to healthcare systems in the world despite substantial advances in risk modification, pharmacological therapy and revascularization therapy. Stem cell therapy is emerging as a novel therapeutic paradigm for myocardial repair. Several cell types including embryonic stem cells, adult stem cells and induced pluripotent stem (iPS) cells have been used for the treatment of ischemic heart disease. But all these engrafted cells must be systematically or locally administered after being expanded in vitro, the rare differentiation into cardiomyocytes and low cellular survival of engrafted cells also limited the efficacy of stem cell therapy. Recent research indicated that it was feasible to reprogramme one mature cell type into another cell type directly by introducing several transcription factors, which was called transdifferentiation. We speculate that cell reprogramming might provide potential new cell sources for therapeutic cardiac regeneration. For these reasons, we hypothesize that converting cardiac fibroblasts to cardiomyocytes and endothelial progenitor cells (EPCs) either in vivo or in vitro might be a possible way for future therapeutic cardiac regeneration. Furthermore we also analyzed the possible difficulties we might face on way to realize this hypothesis.

Rosuvastatin Changes Cytokine Expressions in Ischemic Territory and Preserves Heart Function After Acute Myocardial Infarction in Rats

Aim: To investigate the mechanism of rosuvastatin in preserving cardiac function after acute myocardial infarction (AMI) in a rat model. Methods: Sprague-Dawley rats were randomized to receive either rosuvastatin (5 mg/kg every day) or placebo (0.5% CMC-Na), respectively, by daily gavage from 7 days before AMI. Acute myocardial infarction (AMI) model was induced by left anterior descending coronary artery ligation through a lateral thoracotomy in rats. The expressions of stromal-cell-derived factor 1 (SDF-1), chemokine motif CXC receptor 4 (CXCR-4), vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) in peri-infarction region and nonischemic region at different time points were determined by the Western blot analysis. Immunohistochemistry analysis was performed on the 28th day after AMI to investigate the accumulation of CD90+, CD133+, and c-kit+ progenitor cells in the peri-infarction region. Masson staining and echocardiograph were performed to evaluate the left ventricular remodeling and postinfarction cardiac function 4 weeks after AMI. Results: Western blot analysis showed that rosuvastatin could change the cytokine expressions in the peri-infarction region by upregulating the SDF-1 expression and downregulating the expressions of CXCR-4, ICAM-1, and VEGF in 4 to 14 days after AMI. Immunohistochemistry analysis showed that rosuvastatin treatment was associated with increased accumulation of CD90+, CD133+, and c-kit+ progenitor cells in the peri-infarction region. Masson staining and echocardiograph confirmed that rosuvastatin could attenuate left ventricular remodeling and improve postinfarction systolic function. Conclusion: The data suggest that rosuvastatin can protect the heart from ischemic injury and preserve the cardiac function in rats in vivo. The changing expressions of SDF-1, CXCR-4, ICAM-1, and VEGF, and the accumulation of progenitor cells were involved in this process.

A novel method to delivery stem cells to the injured heart: spatially focused magnetic targeting strategy.

Given the adult hearts minimal capacity for endogenous regeneration, cell therapy has emerged as a promising approach to the regeneration of damaged vascular and cardiac tissue after acute myocardial infarction and heart failure. However, systematic review suggests only mild improvement in global heart function, and high degree of heterogeneity among clinical trials [1]. The first prerequisite for cell therapy success is the engraftment and thus, homing of transplanted cells to the target area. Poor cell homing, retention and engraftment are major obstacles in achieving a significant functional benefit irrespective of the cell type or delivery route used. Data showed only 1–3% of the delivered cells were recruited at the infarct sites via intracoronary administration. The retention of cells in the heart is extremely low, even undetectable after a few weeks when administered by the intravenous route [2,3,4,5]. The predominant number of cells was found in non-targeting organs such as liver, spleen and lung. To induce migration and homing of transplanted cells to optimize the efficacy of cell-based therapies, much efforts have been made in identifying chemokine and its receptors (CXCR4/SDF-1 axis, et al.) in the last decades [6,7]. However, due to the extreme complicity of the ‘cell-extracellular matrix-cytokine’ network and the homing molecular mechanisms, the chemoattractant molecules-targeted method has been far away from being able to precisely and effectively regulate stem cell migrating to target tissue [8,9]. Magnetic targeting strategy, traditionally used in chemotherapy for tumour [10], had been introduced to localize magnetic nanoparticle-loaded cell delivery to target lesion in vivo in recent years [11,12,13,14,15,16,17]. The accumulation and retention of the magnetic responsive cells can be enhanced by using an external magnetic field produced by electromagnet, which is focused on the area of interest [18]. Cheng K. et al. [19 were the first to introduce magnetic targeting strategy to attract transplanted cells to the heart. Using a 1.3 Tesla magnet applied above the rat apex during the intramyocardial injection of magnetic responsive cardiosphere-derived cells, they found that cell retention and engraftment in the recipient hearts increased by approximately threefold compared to non-targeted cells. Chaudeurge A et al. 2011 adopted subcutaneous insertion of a magnet over the chest cavity during therapeutic intracavitary stem cell infusion, found that the average number of engrafted cells was significantly 10 times higher with than without magnetic targeting. This magnetically enhanced intracoronary cell delivery was confirmed by another study 2012. Thus, magnetic targeting is proved to enhance cell retention, engraftment and this novel method to improve cell therapy outcome offers the potential for clinical applications. However, the magnetic field has some inherent limitations as the magnetic flux density is maximal at the magnet pole face and cannot be focused at a distance from the magnet [10]. For conventional electromagnet therefore magnetically loaded cells are predominantly attracted to the surface of magnetic materials, and hard to be targeted to tissues localized deeper in the body. To promote the cell retention at targeting sites remote from the magnet surface, a greater magnetic force or invasive approaches (e.g. implant magnetized stent, magnetic particles or magnet at the target site) will be required. Magnetically loaded endothelial cells were homing to the magnetized stent deployed in rat arteries in the presence of a uniform magnetic field [12,13]. However, achievement of the cell engraftment necessary for therapeutic effects by using a ‘safe magnet force’ would be challenging. Moreover, it is not feasible that there could be the invasive implantation of a magnet or magnetized materials in parenchymatous organs such as heart. To overcome these limitations, it is of great significance to develope a magnetic field which can be focused at a distance from the magnet surface. Recently, we proposed that the spatially focused magnetic field is feasible in theoretical considerations [22]. Its deep capture property of this special magnetic field has been testified in our preliminary in vitro study [22,23]. The deep accumulation of magnetically loaded mesenchymal stem cells was observed while cells flowed through a tube served as a model of blood vessels in such a magnetic field. The cell capture efficiency was positively influenced by the magnetic flux density, and negatively influenced by the flow velocity. The capture efficiency reached 89.3% with 640 mT of the magnetic flux density, 38.4 T/m of the magnetic intensity gradient and 0.8 mm/sec. of flow velocity in our in vitro study [23].

Activation of MAPK/ERK1/2 pathways is an important mechanism of oxidative stress-induced apoptosis in mesenchymal stem cells of rats

Objective To investigate the role of MAPK/ERK1/2 pathways in oxidative stress-induced apoptosis of mesenchymal stem cells. Methods Hydrogen peroxide (H2O2) was used to induce apoptosis of mesenchymal stem cells (MSCs) of adult Sprague–Dawley rats to establish the oxidative stress damage model in vitro. MSCs were treated with H2O2 in different concentration (0, 0.2 mmol/l, 0.5 mmol/l, 1 mmol/l, 2 mmol/l) for 6 h, and were divided into five groups. The percentages of total MSCs apoptosis were analysed by flow cytometry; the expression of proteins related with MAPK/ERK1/2 pathways, such as phosphorylation of ERK1/2 and caspase-3 were compared by Western-Blot assay. With the use of U0126, an inhibitor of phosphorylation of ERK1/2, all above detections in moderately treated concentration group (0.5 mmol/l) were observed. Results H2O2 significantly induced accumulation of ROS in MSCs in a concentration-dependant manner (control group, (2.34±0.55)%; 0.2 mmol/l group, (6.25±1.23)%; 0.5 mmol/l group, (16.52±2.30)%; 1 mmol/l group, (25.8±4.51)%; and 2 mmol/l group, (56.87±6.25)%, p value all<0.05). Flow cytometry indicated that the percentages of total apoptosis of MSCs gradually increased as the concentration of H2O2 rose ((7.2±3.9)%, (11.4±5.8)%, (13.8±7.1)%, (20.5±6.3)%, (24.6±7.9)%, respectively, p value all<0.05). More interestingly, phosphorylation of ERK1/2 was up-regulated gradually as the concentration of H2O2 increased (11.31±0.25, 1.56±0.36, 2.81±1.03, 3.25±1.39 (folds of internal control), respectively; p value all<0.05). Similar effects occurred with the expression of caspase-3 (11.64±0.31, 1.87±0.41, 3.56±0.65, 5.45±1.30 (folds of internal control), respectively; p value all<0.05). In U0126 group, the percentages of total apoptotic MSCs dramatically decreased compared with 0.5 mmol/l group ((10.1±3.4)% vs (24.6±7.9)%, p<0.05); the expression of caspase-3 downregulated synchronously (1.12±0.57 vs 1.87±0.41 (folds of internal control), p<0.05) Conclusion The above results suggested that activation of MAPK/ERK1/2 pathways is an important mechanism of oxidative stress-induced apoptosis in mesenchymal stem cells.

High density lipoprotein induces rats mesenchymal stem cells proliferation through activating PI3K-Akt pathway

Objective To explore the effect of high density lipoprotein (HDL) on the proliferation of mesenchymal stem cells (MSCs), and to elucidate the role of PI3K-Akt pathway in the potential regulation of it. Methods MSCs were collected from the femora of Sprague–Dawley rats and were treated with HDL in different concentration (0, 20 ug/ml, 50 ug/ml, 100 ug/ml) for 24 h; and then were treated with HDL (50 ug/ml) for 24 h, 48 h and 72 h, respectively. The proliferation of MSCs in each group was compared by Cell Counting Kit-8 (CCK-8) and BrdU cell proliferation assay. The expression of phosphorylation of Akt was evaluated by Western Blotting. LY294002, an inhibitor of PI3K, was used to down-regulate the activity of PI3K-Akt pathway. Results The results showed that HDL induces markedly MSCs proliferation in time- and concentration-dependent manner. Akt phosphorylation was significantly increased by 2.35-, 4.52-, and 5.89-folds after simulation by 20 ug/ml, 50 ug/ml and 100 ug/ml HDL for 24 h (p value all<0.05). And when incubated with HLD (50 ug/ml), the phosphorylation of Akt was activated at 15 min, and peaked at 60 min With the use of LY294002, the proliferation of MSCs was attenuated by 32% (26∼40%, p value<0.05) when treated with HLD (50 ug/ml) for 24 h. Conclusion HDL improved the proliferation of MSCs in time- and concentration-dependant manner, and PI3K/Akt pathway was one of the underlying mechanisms involved in it.

Mesenchymal stem cells preconditioned with high density lipoprotein resist oxidative stress-induced apoptosis and improve cardiac function in a rat model of myocardial infarction

Objective To explore the effect of high density lipoprotein (HDL) on mesenchymal stem cells (MSCs) implanted in infarcted myocardium, and to unveil the role of MAPK/ERK1/2 pathways in the potential mechanism of it. Methods MSCs were collected from the femora of Sprague–Dawley rats and were treated with PBS (CON group), HDL (HDL group), H2O2 (H2O2 group), HDL and H2O2 (EXP group), respectively. The expressions of proteins related with apoptosis, such as phosphor-ERK1/2, Bcl-2 and Bax were examined by Western-Blot assay; cell apoptosis was detected by TUNEL staining. In vivo study, acute myocardial infarction model was developed in female SD rats which were given an intramyocardial injection of one of the following cells derived from male rats: MSCs or HDL-preconditioned MSCs. After 4 days, the survival rates of MSCs were compared by means of measuring sry gene with real-time PCR. After 4 weeks, the cardiac remodelling and the percentage of fibrosis of heart were measured by echocardiograph and Massons staining respectively. Results In comparison with H2O2 group, expression of phospho-ERK1/2 was significantly lower in EXP group, and the ratio of expression of Bcl-2 to that of Bax increased (3.4±0.7 vs 5.2±1.2, p<0.05). TUNEL assay indicated that the percentages of MSCs apoptosis reduced evidently in EXP group compared with in H2O2 group ((13.3±2.7)% vs (22.8±3.9)%, p<0.05). In vivo study, the expression of sry gene showed that preconditioning with HDL improved the survival rates of the transplanted MSCs compared to the untreated MSCs (6.5±1.7 folds vs 2.3±0.5 folds, p<0.05); the echocardiological analysis demonstrated that HDL-preconditioned MSCs improved left ventricular ejection fraction significantly ((50.97±10.4)% vs (26.54±7.2)%, p<0.05). Furthermore, the percentage of fibrosis in the left ventricle wall was lower in HDL-preconditioned MSCs group than that in MSCs group ((18.35±7.8)% vs (30.62±6.2)%, p<0.05). Conclusion HDL enhanced the viability of MSCs in an oxidative stress circumstance, leading to prevent myocardial remodelling and to improve cardiac function. MAPK/ERK1/2 pathways were probably one of the underlying mechanisms involved in it.