Effects And Mechanism Of Ly6C^high Monocytes’ Deployment On MSCs Trabsplanation After AMI

Part One: Exposure to supernatants of macrophages that phagocytized dead mesenchymal stem cells improves hypoxic cardiomyocytes survival.Objective:To observe the impact of supernatants from macrophages that phagocytized dead MSCs (pM<D) on the survival of hypoxic cardiomyocytes.Methods:MSCs were isolated from bone marrow of mice and dead MSCs were harvested after 6 hours hypoxia. Macrophages were obtained from thioglycolate-elicited peritoneal cavity. Macrophages and dead MSCs were co-cultured for 2 days in the presence or absence of LPS (1 μg/ml). Cardiomyocytes obtained from neonatal mice were exposed to various medium including supernatants from pMO. MTT cell proliferation assay and mitochondria membrane potential kit were used to evaluate the viability of cardiomyocytes. Cytokines and chemokines (TNF-a, IFN-y, IL-6,IL-12, PGE2, VEGF-a, Ang-1, KGF, IGF-1, PDGF-BB, and EPO) in culture medium of macrophages, MSCs and pMO were detected by ELISA and Real-Time-PCR. Phosphate buffer saline (PBS, Negative control group.), MSCs(Positive control group), pMO (group 1), Macrophage inflammatory protein-1α(MIP-1α)+pMΦ (group 2) were each injected into acute myocardial infarction(AMI) BALB/C mice. MRI and multi-spectrometer analyzer were used to identify the colonization intensity of the transplanted cells in the infarct area;Results:Phagocytic activity of macrophages to dMSC was significantly enhanced by LPS. PGE2, VEGF-a, Ang-1, KGF, IGF-1, PDGF-BB, and EPO levels were significantly increased in supernatants of pM$. Exposure to supernatants of pMΦ significantly improved viability and survival time of hypoxic cardiomyocytes. Surface marker of CD206 is significantly lower than that of MΦ (21%±7% vs.82%±16% P <0.001), and expression of CD86 is significantly higher that of MO (69%±13% vs. 28%±9% P<0.001). Compared to PBS group, the other three groups of cells can be planted in myocardial infarction effectively, the relative fluorescence intensity in group 1 is significantly higher than the positive control group (P<0.001), but lower than the group2 (P<0.001); Pairwise comparisons were statistically significant (P <0.001), Elastic fiber staining showed significantly reduced myocardial infarct size in positive control group, as well as group 1 and 2 compared to PBS group (P<0.001), the ratio for the infarction size in left ventricular area in group 1 is significantly lower than the positive control group (P<0.001), but higher than group 2(P<0.001);Conclusion:Exposure to supernatants of pMΦ significantly improved viability and survival time of hypoxic cardiomyocytes, which might be linked to increased cytokines and chemokines secretion by pMΦ. pMΦ can effectively colonize in the infarcted myocardium after being transplanted into acute myocardial infarction mice and can significantly improve cardiac function, the possible mechanisms may be associated with the promotion of elastic fibers proliferation and mobilization of more reparative macrophages.Part Two: Intermediate monocytes leads to enhanced myocardial remodeling in STEMI patients with diabetesObjectives:This study aimed to evaluate the potential associations of intermediate monocytes (CD14^CD16+), with myocardial remodeling in ST segment elevation myocardial infarction (STEMI) patients with diabetes.Methods:A total of 67 STEMI patients with diabetes were enrolled, and the other 65 STEMI patients without diabetes were allocated as controls. All patients received emergency medical services for reperfusion therapy in less than 12 hours after onset of AMI. Blinded to patient clinical characteristics, monocyte subset analysis was performed using flow cytometry immediately after admission. mRNA of Chemokine Decoy Receptor D6 in each subset of monocytes was validated by Q-PCR. Expression of CCL2 in the patients’plasma was determined by Elisa kit.Infarct size and left ventricular ejection fraction (LVEF) were measured using three-dimensional echocardiography 3 days and 6 months after AMI. The incidences of recurrent cardiovascular events and death in each group were measured using Kaplan-Meier estimator in follow-up during the next 24 months. Cox proportional-hazard models were further used to analyze the relationship of monocyte cell counts and event-free survival after adjusting for confounding factors.Results:The number of circulating intermediate monocytes were significantly correlated with LVEF% and infarct size (r=-0.32; p= 0.008; r= 0.57, p< 0.001) in STEMI patients with diabetes compared with those without diabetes 6 months after AMI. Chemokine Decoy Receptor D6 transcript levels were lower in intermediate monocytes of STEMI patients with diabetes compared to the subsets in STEMI patients without diabetes (p< 0.001). Higher levels of CCL2 (pg/ml) was shown in STEMI patients with diabetes compared to STEMI patients without diabetes (p<0.001).During a mean follow-up period of 24 ± 1 months, recurrent cardiovascular events or death occurred in 23 patients belonging to the STEMI with diabetes group and 10 belonging to the control group. Univariate Kaplan-Meier analysis revealed that counts of the intermediate monocytes according to median showed statistical significance in STEMI patients with diabetes (p= 0.010). After full adjustment for confounding factors, the cells were found to remain independently related to recurrent cardiovascular events or death in this group (p= 0.004,95% CI:1.62-12.49).Conclusion:Intermediate monocytes were associated with LV remodeling in STEMI patients with diabetes. The cells were predictive for recurrent cardiovascular events or death in these patients. Low level of D6 mRNA in the intermediate monocytes of STEMI patients with diabetes and high level of CCL2 in these patients may partially explain the causality.Part Three: Inhibiting the Mobilization of Ly6Chigh Monocytes after Acute Myocardial Infarction Enhances the Efficiency of Mesenchymal Stromal Cell Transplantation and Curbs Myocardial RemodelingBackground:Ischemia related inflammation is the most critical factor for the survival of transplanted mesenchymal stem cells (MSCs), and strategies for controlling excessive inflammation after acute myocardial infarction (AMI) are essential and necessary for cell transplantation therapy. Our present study tested the effect of decreased Ly6Chlgh monocytes on mouse MSCs transplantation after AMI.Methods:BALB/c AMI mice were treated systemically with a CCR2 antagonist (RS 504393,2 mg/kg, subcutaneously) or normal saline (control group). Next,1(P EdU-labeled MSCs were administered by intramyocardial injection to the mice in each group. TUNEL kits were used to identify the apoptotic cardiomyocytes in the infarct. The slides of the infarct border zone were stained with wheat germ agglutinin to measure the vessel density, and anti-myosin heavy chain eFluor 660 was used to measure the cardiac myosin-positive area. A transwell chamber was used to examine the interactions between Ly6Chigh monocytes and MSCs. The inflammatory cytokines expressed by Ly6Chigh monocytes and the SDF-1 expressed by MSCs were detected using ELISA kits. MSC viability was further examined by MTT and mitochondrial membrane potential assays by flow cytometry using JC-1 kits.Results:We first observed the increased survival of transplanted MSCs (11.2±3.4/mm2 vs.3.5±1.6/mm2, p<0.001), and the decreased apoptosis of cardiomyocytes (11.20%±3.55% vs.20.51%±8.17%, p<0.001) in the infarcts at 3 days in the CCR2 antagonist group. An increased number of capillaries and small arterioles (139.6±21.7/mm2 vs.95.4±17.6/mm2, p<0.001) and an increased cardiac myosin-positive area (17.9%±6.6% vs.11.8%±3.5%, p<0.001) were also observed in the infarct zone at 21 days post MSC infusion in the CCR2 antagonist group. In addition, a significantly increased LvEF%(50.17±10.06 vs.45.44±9.45, p<0.001) was detected at the same time compared to the control mice. We further demonstrated that both the mitochondrial membrane potential of the MSCs (0.45±0.11 vs.3.4±0.3, p<0.001) and stromal cell-derived factor-1 (SDF-1) secreted by the MSCs significantly decreased (80.77±39.02 pg/ml vs.435.5±77.41 pg/ml, p<0.001) when co-cultured with Ly6Chigh monocytes. This is possibly mediated by the over-expressed cytokines secreted by the Ly6Chigh monocytes compared to the Ly6Clow monocytes, including IL-1 (139.45±30.44 vs.80.05±19.33, p<0.001), IL-6 (187.82±40.43 vs. 135.5±22.09, p<0.001), TNF-a (121.77±31.65 vs.75.3±22.14, p<0.001) and IFN-y (142.46±27.55 vs.88.25±19.91, p<0.001).Conclusion:We conclude that the mobilization of Ly6Chigh monocytes after AMI negatively affect the local trophic effects of transplanted MSCs, which may occur because the over-expressed TNF-a, IL-1/6, and INF-y by the Ly6Chigh monocytes decreases the survival of MSCs and the subsequent secretion of SDF-1.Part Four: Photoluminescent Mesoporous Silicon Nanoparticles with siCCR2 Improve the Effects of Mesenchymal Stromal Cell Transplantation after Acute Myocardial InfarctionBackground:Despite the benefits of mesenchymal stromal cell (MSC) transplantation in cardiac tissue, detailed in vivo observations have shown that MSCs only survive for a brief period after transplantation due to harsh microenvironmental conditions, including ischemia, inflammation and anoikis, in the infarcted myocardium. Thus, new strategies are needed to enhance MSC survival and inhibit cardiac remodeling. Studies have now demonstrated that chemokine [C-C motif] ligand 2 (CCL2) and its cognate receptor C-C chemokine receptor 2 (CCR2) promote excessive Ly6Chlgh inflammatory monocyte infiltration at the infarct in response to ischemic myocardial injury. Therefore, decreasing the activities of these monocytes immediately after acute myocardial infarction (AMI) could be beneficial for AMI patients.Objectives:This study tested the hypothesis that therapeutic siRNA-loaded photoluminescent mesoporous silicon nanoparticles (PMSNs) targeting CCR2 expression in Ly6Chlgh inflammatory monocytes decrease the accumulation of these cells in the infarct, improve the efficacy of MSC transplantation and attenuate myocardial remodeling.Methods:PMSNs carrying therapeutic siCCR2 were first synthesized without the inclusion of fluorescent materials or dyes. After AMI BALB/c mice were established, 1055-ethynyl-2’-deoxyuridine (EdU)-labeled MSCs suspended in 100 μl of phosphate buffered saline (PBS) were injected into the border zone of the infarct of each mouse. PMSNs-siCCR2 (25 μg/g) were also intravenously injected via the tail vein immediately following AMI induction. Control mice were injected with an equal amount of PMSNs without siCCR2. PMSNs-siCCR2 were examined in vivo using near-infrared imaging technology. The therapeutic effects of PMSNs-siCCR2 for MSC transplantation were determined at the mRNA, protein and functional levels.Results:PMSNs-siCCR2 circulated freely in vivo and were cleared in a relatively short period of time (t1/2=37 min) with no evidence of toxicity. The therapeutic PMSNs-siCCR2 showed higher levels of cellular accumulation in Ly6Chigh monocytes in the spleen and more efficient degradation of CCR2 compared with the control (8.04%±2.17% vs.20.02%±4.55%, p<0.001). Subsequently, the PMSNs-siCCR2 decreased the accumulation of CD11b-positive monocytes at the infarct (49.3%±17.34% vs.61.32%±22.43%, p<0.001) on day 1. Increased survival of transplanted MSCs (13±3/mm2 vs.4±1/mm2, p<0.001) and significantly decreased TdT-mediated dUTP nick end labeling (TUNEL)+cardiac myocytes (17.44%±6.26% vs.39.49%±13.28%, p<0.001) were then identified in the infarct zone three days after AMI induction in the PMSNs-siCCR2 group. Three weeks after MSC injection, significant increases were observed in the vascular density (235.5±39.6/mm2 vs. 147.4±20.3/mm2, p<0.001) and the cardiac myosin-positive area (21.7%±8.4% vs. 13.2%±4.4%, p<0.001) of the infarct border zone. In addition, significant amelioration of left ventricular (LV) remodeling (thickness of the LV posterior walls) (0.84±0.11 mm vs.0.61±0.08 mm, p<0.001) was also observed at the same time compared with the control group.Conclusions:PMSNs-siCCR2-mediated CCR2 gene silencing in LyeChigh monocytes improved the effectiveness of MSC transplantation and selectively ameliorated myocardial remodeling after AMI. These results suggest that PMSNs-siCCR2 could potentially be used to develop an anti-inflammatory therapy for post-AMI MSC transplantation.