Detection of Vulnerable Atherosclerotic Plaques in Experimental Atherosclerosis with the USPIO-Enhanced MRI.

This study's goal was to assess the diagnostic value of the USPIO-(ultra-small superparamagnetic iron oxide) enhanced magnetic resonance imaging (MRI) in detection of vulnerable atherosclerotic plaques in abdominal aorta in experimental atherosclerosis. Thirty New Zealand rabbits were randomly divided into two groups, Group A and Group B. Each group comprised 15 animals which were fed with high cholesterol diet for 8 weeks and then subjected to balloon-induced endothelial injury of the abdominal aorta. After another 8 weeks, animals in Group B received adenovirus carrying p53 gene that was injected through a catheter into the aortic segments rich in plaques. Two weeks later, all rabbits were challenged with the injection of Chinese Russell's viper venom and histamine. Pre-contrast images and USPIO-enhanced MRI images were obtained after pharmacological triggering with injection of USPIO for 5 days. Blood specimens were taken for biochemical and serological tests at 0 and 18 weeks. Abdominal aorta was histologically studied. The levels of serum ICAM-1 and VCAM-1 were quantified by ELISA. Vulnerable plaques appeared as a local hypo-intense signal on the USPIO-enhanced MRI, especially on T2*-weighted sequences. The signal strength of plaques reached the peak at 96 h. Lipid levels were significantly (p < 0.05) higher in both Group A and B compared with the levels before the high cholesterol diet. The ICAM-1 and VCAM-1 levels were significantly (p < 0.05) higher in Group B compared with Group A. The USPIO-enhanced MRI efficiently identifies vulnerable plaques due to accumulation of USPIO within macrophages in abdominal aorta plaques.

Efficiently tracking of stem cells in vivo using different kinds of superparamagnetic iron oxide in swine with myocardial infarction.

BACKGROUND: Superparamagnetic iron oxide (SPIO) particles have shown much promise as a means to visualize labeled cells using molecular magnetic resonance imaging (MRI). Micrometer-sized superparamagnetic iron oxide (MPIO) particles and nanometer-sized ultrasmall superparamagnetic iron oxide (USPIO) are two kinds of SPIO widely used for monitoring stem cells migration. Here we compare the efficiency of two kinds of SPIO during the use of stem cells to treat acute myocardial infarction (AMI). METHODS: An AMI model in swine was created by 60 minutes of balloon occlusion of the left anterior descending coronary artery. Two kinds of SPIO particles were used to track after intracoronary delivered 10(7) magnetically labeled mesenchymal stem cells (MR-MSCs). The distribution and migration of the MR-MSCs were assessed with the use of 3.0T MR scanner and then the results were confirmed by histological examination. RESULTS: MR-MSCs appeared as a local hypointense signal on T2*-weighted MRI and there was a gradual loss of the signal intensity after intracoronary transplantation. All of the hypointense signals in the USPIO-labeled group were found on T2*-weighted MRI, contrast to noise ratio (CNR) decreased in the MPIO-labeled group (16.07 ± 5.85 vs. 10.96 ± 1.34) and USPIO-labeled group (11.72 ± 1.27 vs. 10.03 ± 0.96) from 4 to 8 weeks after transplantation. However, the hypointense signals were not detected in MPIO-labeled group in two animals. MRI and the results were verified by histological examination. CONCLUSIONS: We demonstrated that two kinds of SPIO particles in vitro have similar labeling efficiency and viability. USPIO is more suitable for labeling stem cells when they are transplanted via a coronary route.

Detection of quantitative trait loci affecting haematological traits in swine via genome scanning.

BACKGROUND: Haematological traits, which consist of mainly three components: leukocyte traits, erythrocyte traits and platelet traits, play extremely important role in animal immune function and disease resistance. But knowledge of the genetic background controlling variability of these traits is very limited, especially in swine. RESULTS: In the present study, 18 haematological traits (7 leukocyte traits, 7 erythrocyte traits and 4 platelet traits) were measured in a pig resource population consisting of 368 purebred piglets of three breeds (Landrace, Large White and Songliao Black Pig), after inoculation with the swine fever vaccine when the pigs were 21 days old. A whole-genome scan of QTL for these traits was performed using 206 microsatellite markers covering all 18 autosomes and the X chromosome. Using variance component analysis based on a linear mixed model and the false discovery rate (FDR) test, 35 QTL with FDR < 0.10 were identified: 3 for the leukocyte traits, 28 for the erythrocyte traits, and 4 for the platelet traits. Of the 35 QTL, 25 were significant at FDR < 0.05 level, including 9 significant at FDR < 0.01 level. CONCLUSIONS: Very few QTL were previously identified for hematological traits of pigs and never in purebred populations. Most of the QTL detected here, in particular the QTL for the platelet traits, have not been reported before. Our results lay important foundation for identifying the causal genes underlying the hematological trait variations in pigs.

[Effects of autologous mesenchymal stem cells transfected with heme oxygenase-1 gene transplantation on ischemic Swine hearts].

OBJECTIVE: To observe the effect of intracoronary transfer of autologous HO-1 overexpressed MSCs in porcine model of myocardial ischemia (1 h)/reperfusion. METHODS: Apoptosis was assayed and cytokine concentrations in supernatant were measured in cells exposed to hypoxia-reoxygen in vitro. In vivo, Chinese male mini-pigs were allocated to the following treatment groups: control group (saline), MSCs group (MSCs), MSCs transfected with pcDNA3.1-nHO-1 (HO-1-MSCs). 1 x 10(7) of autologous stem cells or identical volume of saline was injected intracoronary into porcine hearts 1 h after ischemia. MRI assay and postmortem analysis were assessed 3 months after stem cell transplantation. RESULTS: In vitro, cell apoptosis rate post hypoxia-reoxygen was significantly reduced in HO-1-MSCs group (30.30% +/- 7.64%) compared with that in MSCs group (56.93% +/- 4.68%, P < 0.001) and LacZ-MSCs group (55.88% +/- 4.38%, P < 0.001), VEGF was also significantly upregulated in HO-1-MSCs group [(768.44 +/- 78.38) pg/ml] compared with that in MSCs group [(555.27 +/- 67.67) pg/ml, P < 0.001] and LacZ-MSCs group [(522.97 +/- 71.45) pg/ml, P < 0.001]. In vivo, cardiac function was significantly improved in both MSCs transplantation groups compared to saline group (all P < 0.05 vs.saline) and the left ventricular ejection fraction was significantly higher in HO-1-MSCs group compared with that in MSCs group at 3 months after transplantation (53.50% +/- 2.09% vs. 49.54% +/- 2.74%, P = 0.017), capillary density in the peri-infarct area was also significantly higher in HO-1-MSC group than that in MSCs group [(14.59 +/- 2.39)/HPF vs. (11.78 +/- 2.48)/HPF, P = 0.033]. CONCLUSIONS: Efficacy of HO-1 overexpressed MSCs on improving cardiac function and promoting angiogenesis was greater than those by MSCs in this porcine ischemia/reperfusion model.

Identification and differentiation of magnetically labeled mesenchymal stem cells in vivo in swines with myocardial infarction.

We aim to track mesenchymal stem cells (MSCs) after magnetically labeling and test the ability of these cells differentiate into cardiomyocytes in vivo. Therefore, 20 swines were divided into four groups, sham-operated group (n=3); acute myocardial infarction (AMI) transplanted with PBS (n=3); labeled MSCs (n=7) and unlabeled MSCs (n=7) group. 10(7) labeled or unlabeled cells were intracoronary delivered after MI (4.8+/-1.3 days), and serial cardiac MR (3.0T) imaging studies were performed at 0, 4 and 8 weeks after transplantation, then the results were confirmed by histological and western blot analysis. We demonstrated that labeled MSCs can be reliably detected and tracked in vivo using MR imaging. In particular, we provided the evidence of regeneration of labeled MSCs in vivo by histological examination and western blot analysis.

[Therapeutic effects of magnetically labeled mononuclear and mesenchymal stem cells transplantation in a swine myocardial infarction model assessed by magnetic resonance imaging].

OBJECTIVE: To evaluate the therapeutic effects of magnetically labeled mononuclear stem cells (MR-MNC) and mesenchymal stem cells (MR-MSC) transplantation in a swine acute myocardial infarction (AMI) model by MR imaging. METHODS: AMI model was established in swines by balloon occlusion of the left anterior descending coronary artery, 10(7) autologous MR-MSC (n = 7), MR-MNC (n = 6) or PBS (n = 6) were delivered via intracoronary infusion within 1 week after AMI [(4.8 +/- 1.3) days]. Changes of infarct size and cardiac function were assessed with the use of 3.0T MR scanner before AMI, at 1 and 8 weeks post AMI. RESULTS: Magnetically labeled stem cells could be identified in the region of AMI by cardiac MR imaging. Eight weeks post transplantation, infarct size was significantly reduced in MR-MSC transplantation group (8.5% +/- 0.5% vs. 24.7% +/- 3.1%, P < 0.05) and in MR-MNC transplantation (12.3% +/- 1.5% vs. 26.1% +/- 1.5%, P < 0.05) while infarct size remained unchanged in PBS group (P > 0.05) compared to values at 1 week post AMI, left ventricular ejection fraction (LVEF) was also significantly higher in MR-MSC transplantation group (56.9% +/- 1.3% vs. 40.7% +/- 2.0%, P < 0.05) and MR-MNC transplantation group (52.8% +/- 1.4% vs. 41.9% +/- 3.3%, P < 0.05) compared to LVEF at 1 week post AMI. LVEF increase was more significant in swines received MR-MSC transplantation than MR-MNC transplantation (16.2% +/- 1.2% vs. 10.9% +/- 3.0%, P < 0.05). Prussian blue staining identified stem cells in corresponding myocardial regions with as by MRI. Western blot analysis demonstrated that cardiac expressions of myosin heavy chain (MHC) in MR-MSC group (100.3 +/- 5.5) and in MR-MNCs group (95.5 +/- 4.2) were significantly higher than that in PBS group (75.7 +/- 5.7, P < 0.05), myocardial troponin T (cTNT) expression in MR-MSC group (124.0 +/- 5.8) and MR-MNC group (118.4 +/- 4.4) were also significantly higher than in PBS group (93.3 +/- 3.9, P < 0.05) while MMP2/TIMP1 ratios in MR-MSC group (0.6 +/- 0.1) and MR-MNC group (0.6 +/- 0.1) were significantly lower than that in PBS group (4.2 +/- 0.2, P < 0.05). CONCLUSIONS: Magnetically labeled MR-MSC and MR-MNC homed to heart post myocardial infarction and reduced infarct size, improved cardiac function. MR-MSC is superior to MR-MNC on improving cardiac function.

Transplantation of magnetically labeled mesenchymal stem cells improves cardiac function in a swine myocardial infarction model.

BACKGROUND: Mesenchymal stem cells (MSCs) transplantation provides a new approach for myocardial repair. However, many important fundamental questions about MSCs transplantation remain unanswered. There is an urgent need to identify MSCs from the beating heart and analyze the efficacy of this new approach. This study aimed to localize the magnetically labeled MSCs (MR-MSCs) and monitor the restorative effects of MR-MSCs with magnetic resonance (MR) imaging. METHODS: Acute myocardial infarction (AMI) was created in swine by a balloon occlusion of the left anterior descending coronary artery. Cells were delivered via intracoronary infusion after myocardial infarction. Infarct size change and cardiac function were assessed with 3.0T MR scanner. The results were then confirmed by histological and western blot analysis. All statistical procedures were performed with Systat (SPSS version 12.01). RESULTS: A total of 26 swine were divided into four groups (sham-operated group, n=6; AMI group with PBS transplantation, n=6; labeled MSCs group, n=7; unlabeled MSCs group, n=7). MSCs, MR-MSCs (10(7) cells) or PBS were delivered by intracoronary injection after MI and serial cardiac MR imaging studies were performed at 0, 4 and 8 weeks after transplantation. MR imaging demonstrated MI size decreased after MSCs transplantation in labeled and unlabeled groups, however, increases were seen in the AMI group at 8 weeks after MI. The left ventricular ejection fraction (LVEF) was slightly increased in the AMI group ((41.87+/-2.45)% vs (39.04+/-2.80)%, P>0.05), but significantly improved in the MR-MSCs group ((56.85+/-1.29)% vs (40.67+/-2.00)%, P<0.05) and unlabeled group ((55.38+/-1.07)% vs (41.78+/-2.08)%, P<0.05) at 8 weeks after treatment. MR-MSCs were further confirmed by Prussian blue and immunofluorescent staining. Western blot analysis demonstrated that there was an increased expression of cardiomyocyte markers such as myosin heavy chain and troponin T in the MSCs treatment groups and the ratio of matrix metalloproteinase 2 to tissue inhibitor of metalloproteinase 1 decreased in the labeled group and unlabeled group compared with the AMI group and sham-operated group. CONCLUSION: Transplanted MR-MSCs can regenerate new myocardium and prevent remolding in an MI model at 2-month follow-up and represent a preferred method to better understand the mechanisms of stem cell therapy in future clinical studies.

[MR imaging of injected magnetically labeled stem cells in myocardial infarction: experiment with pigs].

OBJECTIVE: To investigate the efficacy of magnetic resonance imaging (MRI) in tracking bone marrow derived mononuclear cells (BM-MNCs) labeled with superparamagnetic iron oxide (SPIO) nanoparticles. METHODS: BM-MNCs were isolated from the bone marrow of 14 pigs. These 14 pigs underwent occlusion of the left anterior descending coronary artery (LAD) to establish myocardial infarction (MI) models and then randomly divided into 2 groups: experimental group (n = 9) to be injected with BM-MNCs labeled with SPIO intracoronarily under X-ray fluoroscopy, and control group (n = 5), to be injected with unlabelled BM-MNCs MRI was performed with a 1.5T MR scanner to demonstrate the location of the BM-MNCs once a week. T pigs were killed when no labeled BM-MSC was detected. The hearts were taken out to undergo HE staging and Prussian blue staining. Immunohistochemistry was used to detect the desmin and myosin. RESULTS: The cell labeling efficiency was almost 100%. Contrast-enhanced MRI demonstrated successful establishment of MI models. Effective MRI tracking findings were obtained in 8 pigs, 7 of the experimental group and 3 of the control group. In 3 pigs T2* weighted MRI showed the zone of labeled cell accumulation shows vague low-signal area around the infarction area and much better conspicuity of the zone of hypoenhancement was shown under contrast-enhanced MRI. The hypoenhancement zone disappeared 14 - 21 days after the injection of BM-MSCs. Histological analyses showed that most Prussian blue positive cells were well correlated with the area where a signal intensity loss was observed in MRI. CONCLUSION: 1.5T MR imaging can monitor the magnetically labeled BM-MNC in vivo in myocardial infarction provided the number of injected is nor less than 10(6).

[Expression of human HSF in E. coli and its effects on mobilization of hematopoietic stem cells in rhesus monkeys].

OBJECTIVE: To study the expression of hHSF in E. coli and its effect on the mobilization of hematopoietic stem/progenitor cells. METHODS: The hHSF gene was obtained by overlapping PCR and cloned into the vector pET30a to yield pET30a-hHSF, which was transformed into E. coli BL21(DE3) and expressed with IPTG induction. Subsequently, rhHSF was purified by gel filtration and cation exchange chromatography and subjected to refolding. Molecular weight of hHSF was measured by MALDI-TOF Mass Spectroscopy. The N terminal amino acid sequence rhHSF was determined by protein sequencing. rhHSF was profiled in rhesus monkey for mobilization of peripheral blood stem cells. Eight rhesus monkeys were equally divided into two groups. The first group was administered single subcutaneous injection of 500 microg/kg hHSF, while the other one was administered 10 microg.kg(-1).d(-1) G-CSF for 4 days followed by a single subcutaneous injection of 500 microg/kg rhHSF. RESULTS: The sequence coding hHSF was confirmed by sequencing and the induced-expression level was about 30% of total cell proteins. The purity of target protein was over 95%. The sequence of N terminal 10 amino acids and the amino acid composition were consistent with the theoretical parameters; molecular weight of rhHSF was 7540. The peripheral CD34(+) cells, CFU-GM yields, and neutrophils peaked at 3 h (16.3-folds increase compared with baseline), 1 h (1.9-folds increase) and 45 min (4.4-folds increase) respectively after the single injection of rhHSF. The addition of rhHSF after the last dose of G-CSF boosted these levels to 25.8-folds, 8.7-folds and 8.3-folds respectively. CONCLUSION: hHSF is highly expressed in E. coli and rapidly mobilizes the hematopoietic stem/progenitor cells and neutrophils in rhesus monkeys. hHSF shows distinct synergistic effect with G-CSF.