Lung-Homing of Endothelial Progenitor Cells and Airway Vascularization Is Only Partially Dependant on Eosinophils in a House Dust Mite-Exposed Mouse Model of Allergic Asthma

Background

Asthmatic responses involve a systemic component where activation of the bone marrow leads to mobilization and lung-homing of progenitor cells. This traffic may be driven by stromal cell derived factor-1 (SDF-1), a potent progenitor chemoattractant. We have previously shown that airway angiogenesis, an early remodeling event, can be inhibited by preventing the migration of endothelial progenitor cells (EPC) to the lungs. Given intranasally, AMD3100, a CXCR4 antagonist that inhibits SDF-1 mediated effects, attenuated allergen-induced lung-homing of EPC, vascularization of pulmonary tissue, airway eosinophilia and development of airway hyperresponsiveness. Since SDF-1 is also an eosinophil chemoattractant, we investigated, using a transgenic eosinophil deficient mouse strain (PHIL) whether EPC lung accumulation and lung vascularization in allergic airway responses is dependent on eosinophilic inflammation.

Methods

Wild-type (WT) BALB/c and eosinophil deficient (PHIL) mice were sensitized to house dust mite (HDM) using a chronic exposure protocol and treated with AMD3100 to modulate SDF-1 stimulated progenitor traffic. Following HDM challenge, lung-extracted EPCs were enumerated along with airway inflammation, microvessel density (MVD) and airway methacholine responsiveness (AHR).

Results

Following Ag sensitization, both WT and PHIL mice exhibited HDM-induced increase in airway inflammation, EPC lung-accumulation, lung angiogenesis and AHR. Treatment with AMD3100 significantly attenuated outcome measures in both groups of mice. Significantly lower levels of EPC and a trend for lower vascularization were detected in PHIL versus WT mice.

Conclusions

This study shows that while allergen-induced lung-homing of endothelial progenitor cells, increased tissue vascularization and development lung dysfunction can occur in the absence of eosinophils, the presence of these cells worsens the pathology of the allergic response.     

Central Administration of C-X-C Chemokine Receptor Type 4 Antagonist Alleviates the Development and Maintenance of Peripheral Neuropathic Pain in Mice

Aim

To explore the roles of C-X-C chemokine receptor type 4 (CXCR4) in spinal processing of neuropathic pain at the central nervous system (CNS).

Methods

Peripheral neuropathic pain (PNP) induced by partial sciatic nerve ligation (pSNL) model was assessed in mice. Effects of a single intrathecal (central) administration of AMD3100 (intrathecal AMD3100), a CXCR4 antagonist, on pain behavior and pain-related spinal pathways and molecules in the L3-L5 spinal cord segment was studied compare to saline treatment.

Results

Rotarod test showed that intrathecal AMD3100 did not impair mice motor function. In pSNL-induced mice, intrathecal AMD3100 delayed the development of mechanical allodynia and reversed the established mechanical allodynia in a dose-dependent way. Moreover, intrathecal AMD3100 downregulated the activation of JNK1 and p38 pathways and the protein expression of p65 as assessed by western blotting. Real-time PCR test also demonstrated that substance P mRNA was decreased, while adrenomedullin and intercellular adhesion molecule mRNA was increased following AMD3100 treatment.

Conclusion

Our results suggest that central (spinal) CXCR4 is involved in the development and maintenance of PNP and the regulation of multiple spinal molecular events under pain condition, implicating that CXCR4 would potentially be a therapeutic target for chronic neuropathic pain.     

Intramyocardial Delivery of Mesenchymal Stem Cell-Seeded Hydrogel Preserves Cardiac Function and Attenuates Ventricular Remodeling after Myocardial Infarction

Background

To improve the efficacy of bone marrow-derived mesenchymal stem cell (MSC) therapy targeted to infarcted myocardium, we investigated whether a self-setting silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel seeded with MSC (MSC+hydrogel) could preserve cardiac function and attenuate left ventricular (LV) remodeling during an 8-week follow-up study in a rat model of myocardial infarction (MI).

Methodology/Principal Finding

Si-HPMC hydrogel alone, MSC alone or MSC+hydrogel were injected into the myocardium immediately after coronary artery ligation in female Lewis rats. Animals in the MSC+hydrogel group showed an increase in cardiac function up to 28 days after MI and a mid-term prevention of cardiac function alteration at day 56. Histological analyses indicated that the injection of MSC+hydrogel induced a decrease in MI size and an increase in scar thickness and ultimately limited the transmural extent of MI. These findings show that intramyocardial injection of MSC+hydrogel induced short-term recovery of ventricular function and mid-term attenuation of remodeling after MI.

Conclusion/Significance

These beneficial effects may be related to the specific scaffolding properties of the Si-HPMC hydrogel that may provide the ability to support MSC injection and engraftment within myocardium.     

Comparison of Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes with Human Mesenchymal Stem Cells following Acute Myocardial Infarction

Introduction

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have recently been shown to express key cardiac proteins and improve in vivo cardiac function when administered following myocardial infarction. However, the efficacy of hiPSC-derived cell therapies, in direct comparison to current, well-established stem cell-based therapies, is yet to be elucidated. The goal of the current study was to compare the therapeutic efficacy of human mesenchymal stem cells (hMSCs) with hiPSC-CMs in mitigating myocardial infarction (MI).

Methods

Male athymic nude hyrats were subjected to permanent ligation of the left-anterior-descending (LAD) coronary artery to induce acute MI. Four experimental groups were studied: 1) control (non-MI), 2) MI, 3) hMSCs (MI+MSC), and 4) hiPSC-CMs (MI+hiPSC-derived cardiomyocytes). The hiPSC-CMs and hMSCs were labeled with superparamagnetic iron oxide (SPIO) in vitro to track the transplanted cells in the ischemic heart by high-field cardiac MRI. These cells were injected into the ischemic heart 30-min after LAD ligation. Four-weeks after MI, cardiac MRI was performed to track the transplanted cells in the infarct heart. Additionally, echocardiography (M-mode) was performed to evaluate the cardiac function. Immunohistological and western blot studies were performed to assess the cell tracking, engraftment and cardiac fibrosis in the infarct heart tissues.

Results

Echocardiography data showed a significantly improved cardiac function in the hiPSC-CMs and hMSCs groups, when compared to MI. Immunohistological studies showed expression of connexin-43, α-actinin and myosin heavy chain in engrafted hiPSC-CMs. Cardiac fibrosis was significantly decreased in hiPSC-CMs group when compared to hMSCs or MI groups. Overall, this study demonstrated improved cardiac function with decreased fibrosis with both hiPSC-CMs and hMSCs groups when compared with MI group.     

SDF-1α upregulation by atorvastatin in rats with acute myocardial infarction via nitric oxide production confers anti-inflammatory and anti-apoptotic effects

Background

The effects of atorvastatin on SDF-1α expression under acute myocardial infarction (AMI) are still unclear. Therefore, our present study is to investigate the roles and mechanisms of atorvastatin treatment on SDF-1α expression in rats with AMI.

Methods

Male Sprague–Dawley rats were underwent permanent coronary artery ligation and randomly assigned into four groups as follow: blank control (B), atorvastatin (A), atorvastatin plus L-NAME (A+L-NAME), and atorvastatin plus AMD3100 (A+AMD3100). Rats underwent similar procedure but without ligation were used as group sham operated (S). Atorvastatin (10mg/Kg/d body weight) was administrated by gavage to rats in three atorvastatin treated groups, and L-NAME (40mg/Kg/d body weight) or AMD3100 (5mg/Kg/d body weight) was given to group A+L-NAME or A+AMD3100, respectively.

Results

Comparing with group B, NO production, SDF-1α and CXCR4 expression were significantly up-regulated in three atorvastatin treated groups at the seventh day. However, the increments of SDF-1α and CXCR4 expression in group A+L-NAME were reduced when NO production was inhibited by L-NAME. Anti-inflammatory and anti-apoptotic effects of atorvastatin were offset either by decrease of SDF-1α and CXCR4 expression (by L-NAME) or blockage of SDF-1α coupling with CXCR4 (by AMD3100). Expression of STAT3, a cardioprotective factor mediating SDF-1α/CXCR4 axis induced cardiac protection, was up-regulated most significantly in group A. The effects of atorvastatin therapy on cardiac function were also abrogated either when SDF-1α and CXCR4 expression was diminished or the coupling of SDF-1α with CXCR4 was blocked.

Conclusion

SDF-1α upregulation by atorvastatin in rats with AMI was, at least partially, via the eNOS/NO dependent pathway, and SDF-1α upregulation and SDF-1α coupling with CXCR4 conferred anti-inflammatory and anti-apoptotic effects under AMI setting which we speculated that ultimately contributed to cardiac function improvement.     

Sca-1+ cardiac stem cells mediate acute cardioprotection via paracrine factor SDF-1 following myocardial ischemia/reperfusion

Background

Cardiac stem cells (CSCs) promote myocardial recovery following ischemia through their regenerative properties. However, little is known regarding the implication of paracrine action by CSCs in the setting of myocardial ischemia/reperfusion (I/R) injury although it is well documented that non-cardiac stem cells mediate cardioprotection via the production of paracrine protective factors. Here, we studied whether CSCs could initiate acute protection following global myocardial I/R via paracrine effect and what component from CSCs is critical to this protection.

Methodology/Principal Findings

A murine model of global myocardial I/R was utilized to investigate paracrine effect of Sca-1+ CSCs on cardiac function. Intracoronary delivery of CSCs or CSC conditioned medium (CSC CM) prior to ischemia significantly improved myocardial function following I/R. siRNA targeting of VEGF in CSCs did not affect CSC-preserved myocardial function in response to I/R injury. However, differentiation of CSCs to cardiomyocytes (DCSCs) abolished this protection. Through direct comparison of the protein expression profiles of CSCs and DCSCs, SDF-1 was identified as one of the dominant paracrine factors secreted by CSCs. Blockade of the SDF-1 receptor by AMD3100 or downregulated SDF-1 expression in CSCs by specific SDF-1 siRNA dramatically impaired CSC-induced improvement in cardiac function and increased myocardial damage following I/R. Of note, CSC treatment increased myocardial STAT3 activation after I/R, whereas downregulation of SDF-1 action by blockade of the SDF-1 receptor or SDF-1 siRNA transfection abolished CSC-induced STAT3 activation. In addition, inhibition of STAT3 activation attenuated CSC-mediated cardioprotection following I/R. Finally, post-ischemic infusion of CSC CM was shown to significantly protect I/R-caused myocardial dysfunction.

Conclusions/Significance

This study suggests that CSCs acutely improve post-ischemic myocardial function through paracrine factor SDF-1 and up-regulated myocardial STAT3 activation.     

Granulocyte Colony-Stimulating Factor Reduces Fibrosis in a Mouse Model of Chronic Pancreatitis

Background

Chronic pancreatitis (CP) is a necroinflammatory process resulting in extensive pancreatic fibrosis. Granulocyte colony-stimulating factor (G-CSF), a hematopoietic stem cell mobilizer, has been shown to exert an anti-fibrotic effect partly through the enrichment of bone marrow (BM) cells in fibrotic organ. We aimed to test the effect of G-CSF on fibrosis in a mouse model of CP.

Methods

CP was induced in C57Bl/6J mice by consecutive cerulein injection (50 µg/kg/day, 2 days a week) for 6 weeks. Mice were then treated with G-CSF (200 µg/kg/day, 5 day a week) or normal saline for 1 week, and sacrificed at week 7 or week 9 after first cerulein injection. Pancreatic histology, pancreatic matrix metallopeptidase 9 (MMP-9), MMP-13 and collagen expression were examined. Pancreatic myofibroblasts were isolated and cultured with G-CSF. Collagen, MMP-9 and MMP-13 expression by myofibroblasts was examined. The BM-mismatched mice model was used to examine the change of BM-derived myofibroblasts and non-myofibroblastic BM cells by G-CSF in the pancreas.

Results

The pancreatic collagen expression were significantly decreased in the G-CSF-treated group sacrificed at week 9. While collagen produced from myofibroblasts was not affected by G-CSF, the increase of MMP13 expression was observed in vitro. There were no effect of G-CSF in the number of myofibroblasts and BM-derived myofibroblasts. However, the number of non-myofibroblastic BM cells and macrophages were significantly increased in the pancreata of cerulein- and G-CSF-treated mice, suggesting a potential anti-fibrotic role of non-myofibroblastic BM cells and macrophages stimulated by G-CSF.

Conclusions

Our data indicated that G-CSF contributed to the regression of pancreatic fibrosis. The anti-fibrotic effects were possibly through the stimulation of MMP-13 from myofibroblasts, and the enhanced accumulation of non-myofibroblastic BM cells and macrophages in the pancreas.     

Mobilisation of hematopoietic CD34+ precursor cells in patients with acute stroke is safe--results of an open-labeled non randomized phase I/II trial

Background

Regenerative strategies in the treatment of acute stroke may have great potential. Hematopoietic growth factors mobilize hematopoietic stem cells and may convey neuroprotective effects. We examined the safety, potential functional and structural changes, and CD34⁺ cell–mobilization characteristics of G-CSF treatment in patients with acute ischemic stroke.

Methods and Results

Three cohorts of patients (8, 6, and 6 patients per cohort) were treated subcutaneously with 2.5, 5, or 10 µg/kg body weight rhG-CSF for 5 consecutive days within 12 hrs of onset of acute stroke. Standard treatment included IV thrombolysis. Safety monitoring consisted of obtaining standardized clinical assessment scores, monitoring of CD34⁺ stem cells, blood chemistry, serial neuroradiology, and neuropsychology. Voxel-guided morphometry (VGM) enabled an assessment of changes in the patients'' structural parenchyma. 20 patients (mean age 55 yrs) were enrolled in this study, 5 of whom received routine thrombolytic therapy with r-tPA. G-CSF treatment was discontinued in 4 patients because of unrelated adverse events. Mobilization of CD34⁺ cells was observed with no concomitant changes in blood chemistry, except for an increase in the leukocyte count up to 75,500/µl. Neuroradiological and neuropsychological follow-up studies did not disclose any specific G-CSF toxicity. VGM findings indicated substantial atrophy of related hemispheres, a substantial increase in the CSF space, and a localized increase in parenchyma within the ischemic area in 2 patients.

Conclusions

We demonstrate a good safety profile for daily administration of G-CSF when begun within 12 hours after onset of ischemic stroke and, in part in combination with routine IV thrombolysis. Additional analyses using VGM and a battery of neuropsychological tests indicated a positive functional and potentially structural effect of G-CSF treatment in some of our patients.

Trial Registration

German Clinical Trial Register DRKS 00000723     

G-CSF prevents the progression of structural disintegration of white matter tracts in amyotrophic lateral sclerosis: a pilot trial

Background

The hematopoietic protein Granulocyte-colony stimulating factor (G-CSF) has neuroprotective and -regenerative properties. The G-CSF receptor is expressed by motoneurons, and G-CSF protects cultured motoneuronal cells from apoptosis. It therefore appears as an attractive and feasible drug candidate for the treatment of amyotrophic lateral sclerosis (ALS). The current pilot study was performed to determine whether treatment with G-CSF in ALS patients is feasible.

Methods

Ten patients with definite ALS were entered into a double-blind, placebo-controlled, randomized trial. Patients received either 10 µg/kg BW G-CSF or placebo subcutaneously for the first 10 days and from day 20 to 25 of the study. Clinical outcome was assessed by changes in the ALS functional rating scale (ALSFRS), a comprehensive neuropsychological test battery, and by examining hand activities of daily living over the course of the study (100 days). The total number of adverse events (AE) and treatment-related AEs, discontinuation due to treatment-related AEs, laboratory parameters including leukocyte, erythrocyte, and platelet count, as well as vital signs were examined as safety endpoints.Furthermore, we explored potential effects of G-CSF on structural cerebral abnormalities on the basis of voxel-wise statistics of Diffusion Tensor Imaging (DTI), brain volumetry, and voxel-based morphometry.

Results

Treatment was well-tolerated. No significant differences were found between groups in clinical tests and brain volumetry from baseline to day 100. However, DTI analysis revealed significant reductions of fractional anisotropy (FA) encompassing diffuse areas of the brain when patients were compared to controls. On longitudinal analysis, the placebo group showed significant greater and more widespread decline in FA than the ALS patients treated with G-CSF.

Conclusions

Subcutaneous G-CSF treatment in ALS patients appears as feasible approach. Although exploratory analysis of clinical data showed no significant effect, DTI measurements suggest that the widespread and progressive microstructural neural damage in ALS can be modulated by G-CSF treatment. These findings may carry significant implications for further clinical trials on ALS using growth factors.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00298597","term_id":"NCT00298597"}}NCT00298597     

Regulation of gene expression with thyroid hormone in rats with myocardial infarction

Introduction

The expression of hundreds of genes is altered in response to left ventricular (LV) remodeling following large transmural myocardial infarction (MI). Thyroid hormone (TH) improves LV remodeling and cardiac performance after MI. However, the molecular basis is unknown.

Methods

MI was produced by ligation of the left anterior descending coronary artery in female SD rats. Rats were divided into the following groups: (1) Sham MI, (2) MI, and (3) MI+T4 treatment (T4 pellet 3.3 mg, 60 days release, implanted subcutaneously immediately following MI). Four weeks after surgery, total RNA was isolated from LV non-infarcted areas for microarray analysis using the Illumina RatRef-12 Expression BeadChip Platform.

Results

Signals were detected in 13,188 genes (out of 22,523), of which the expression of 154 genes were decreased and the expression of 200 genes were increased in MI rats compared with Sham MI rats (false discovery rate (FDR) <0.05). Compared to MI rats, T4 treatment decreased expression of 27 genes and increased expression of 28 genes. In particular, 6 genes down-regulated by MI and 12 genes up-regulated by MI were reversed by T4. Most of the 55 genes altered by T4 treatment are in the category of molecular function under binding (24) and biological processes which includes immune system process (9), multi-organism process (5) and biological regulation (19) nonexclusively.

Conclusions

These results suggest that altered expression of genes for molecular function and biological process may be involved in the beneficial effects of thyroid hormone treatment following MI in rats.     

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.     

Sustained-Release Delivery of Prostacyclin Analogue Enhances Bone Marrow-Cell Recruitment and Yields Functional Benefits for Acute Myocardial Infarction in Mice

Background

A prostacyclin analogue, ONO-1301, is reported to upregulate beneficial proteins, including stromal cell derived factor-1 (SDF-1). We hypothesized that the sustained-release delivery of ONO-1301 would enhance SDF-1 expression in the acute myocardial infarction (MI) heart and induce bone marrow cells (BMCs) to home to the myocardium, leading to improved cardiac function in mice.

Methods and Results

ONO-1301 significantly upregulated SDF-1 secretion by fibroblasts. BMC migration was greater to ONO-1301-stimulated than unstimulated conditioned medium. This increase was diminished by treating the BMCs with a CXCR4-neutralizing antibody or CXCR4 antagonist (AMD3100). Atelocollagen sheets containing a sustained-release form of ONO-1301 (n = 33) or ONO-1301-free vehicle (n = 48) were implanted on the left ventricular (LV) anterior wall immediately after permanent left-anterior descending artery occlusion in C57BL6/N mice (male, 8-weeks-old). The SDF-1 expression in the infarct border zone was significantly elevated for 1 month in the ONO-1301-treated group. BMC accumulation in the infarcted hearts, detected by in vivo imaging after intravenous injection of labeled BMCs, was enhanced in the ONO-1301-treated hearts. This increase was inhibited by AMD3100. The accumulated BMCs differentiated into capillary structures. The survival rates and cardiac function were significantly improved in the ONO-1301-treated group (fractional area change 23±1%; n = 22) compared to the vehicle group (19±1%; n = 20; P = 0.004). LV anterior wall thinning, expansion of infarction, and fibrosis were lower in the ONO-1301-treated group.

Conclusions

Sustained-release delivery of ONO-1301 promoted BMC recruitment to the acute MI heart via SDF-1/CXCR4 signaling and restored cardiac performance, suggesting a novel mechanism for ONO-1301-mediated acute-MI heart repair.     

Exogenous Administration of a Recombinant Variant of TWEAK Impairs Healing after Myocardial Infarction by Aggravation of Inflammation

Background

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are upregulated after myocardial infarction (MI) in both humans and mice. They modulate inflammation and the extracellular matrix, and could therefore be important for healing and remodeling after MI. However, the function of TWEAK after MI remains poorly defined.

Methods and results

Following ligation of the left coronary artery, mice were injected twice per week with a recombinant human serum albumin conjugated variant of TWEAK (HSA-Flag-TWEAK), mimicking the activity of soluble TWEAK. Treatment with HSA-Flag-TWEAK resulted in significantly increased mortality in comparison to the placebo group due to myocardial rupture. Infarct size, extracellular matrix remodeling, and apoptosis rates were not different after MI. However, HSA-Flag-TWEAK treatment increased infiltration of proinflammatory cells into the myocardium. Accordingly, depletion of neutrophils prevented cardiac ruptures without modulating all-cause mortality.

Conclusion

Treatment of mice with HSA-Flag-TWEAK induces myocardial healing defects after experimental MI. This is mediated by an exaggerated neutrophil infiltration into the myocardium.     

Epigenetic Regulation of Cardiac Progenitor Cells Marker c-kit by Stromal Cell Derived Factor-1α

Background

Cardiac progenitor cells (CPCs) have been proven suitable for stem cell therapy after myocardial infarction, especially c-kit(+)CPCs. CPCs marker c-kit and its ligand, the stem cell factor (SCF), are linked as c-kit/SCF axis, which is associated with the functions of proliferation and differentiation. In our previous study, we found that stromal cell-derived factor-1α (SDF-1α) could enhance the expression of c-kit. However, the mechanism is unknown.

Methods and Results

CPCs were isolated from adult mouse hearts, c-kit(+) and c-kit(−) CPCs were separated by magnetic beads. The cells were cultured with SDF-1α and CXCR4-selective antagonist AMD3100, and c-kit expression was measured by qPCR and Western blotting. Results showed that SDF-1α could enhance c-kit expression of c-kit(+)CPCs, made c-kit(−)CPCs expressing c-kit, and AMD3100 could inhibit the function of SDF-1α. After the intervention of SDF-1α and AMD3100, proliferation and migration of CPCs were measured by CCK-8 and transwell assay. Results showed that SDF-1α could enhance the proliferation and migration of both c-kit(+) and c-kit(−) CPCs, and AMD3100 could inhibit these functions. DNA methyltransferase (DNMT) mRNA were measured by qPCR, DNMT activity was measured using the DNMT activity assay kit, and DNA methylation was analyzed using Sequenom''s MassARRAY platform, after the CPCs were cultured with SDF-1α. The results showed that SDF-1α stimulation inhibited the expression of DNMT1 and DNMT3β, which are critical for the maintenance of regional DNA methylation. Global DNMT activity was also inhibited by SDF-1α. Lastly, SDF-1α treatment led to significant demethylation in both c-kit(+) and c-kit(−) CPCs.

Conclusions

SDF-1α combined with CXCR4 could up-regulate c-kit expression of c-kit(+)CPCs and make c-kit(−)CPCs expressing c-kit, which result in the CPCs proliferation and migration ability improvement, through the inhibition of DNMT1 and DNMT3β expression and global DNMT activity, as well as the subsequent demethylation of the c-kit gene.     

Altered Gene Expression Pattern in Peripheral Blood Mononuclear Cells in Patients with Acute Myocardial Infarction

Background

Despite a substantial progress in diagnosis and therapy, acute myocardial infarction (MI) is a major cause of mortality in the general population. A novel insight into the pathophysiology of myocardial infarction obtained by studying gene expression should help to discover novel biomarkers of MI and to suggest novel strategies of therapy. The aim of our study was to establish gene expression patterns in leukocytes from acute myocardial infarction patients.

Methods and Results

Twenty-eight patients with ST-segment elevation myocardial infarction (STEMI) were included. The blood was collected on the 1^st day of myocardial infarction, after 4–6 days, and after 6 months. Control group comprised 14 patients with stable coronary artery disease, without history of myocardial infarction. Gene expression analysis was performed with Affymetrix Human Gene 1.0 ST microarrays and GCS3000 TG system. Lists of genes showing altered expression levels (fold change >1.5, p<0.05) were submitted to Ingenuity Pathway Analysis. Gene lists from each group were examined for canonical pathways and molecular and cellular functions. Comparing acute phase of MI with the same patients after 6 months (stable phase) and with control group we found 24 genes with changed expression. In canonical analysis three pathways were highlighted: signaling of PPAR (peroxisome proliferator-activated receptor), IL-10 and IL-6 (interleukin 10 and 6).

Conclusions

In the acute phase of STEMI, dozens of genes from several pathways linked with lipid/glucose metabolism, platelet function and atherosclerotic plaque stability show altered expression. Up-regulation of SOCS3 and FAM20 genes in the first days of myocardial infarction is observed in the vast majority of patients.     

Ultrastructural Features of Ischemic Tissue following Application of a Bio-Membrane Based Progenitor Cardiomyocyte Patch for Myocardial Infarction Repair

Background and Objective

Implantation of cell-sheets into damaged regions of the heart after myocardial infarction (MI) has been shown to improve heart function. However, the tissue morphology following application of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) has not been studied in detail at the level afforded by electron microscopy. We hypothesized that increasing the number of CM derived from iPSC would increase the effectiveness of cell-sheets used to treat ischemic cardiomyopathy. We report here on the ultrastructural features after application of a bio-membrane ‘cell patch’.

Methods

iPSC-derived progenitor cells were transduced using lentivirus vectors with or without NCX1 promoter. iPSC-CM sheets were transplanted over the transmural MI region in a mouse model of regional ischemic cardiomyopathy. Mice were divided into four groups, 1) Sham; 2) MI; 3) MI + iPSC without NCX1 treated cells (MI + iPSC^Null) and 4) MI + iPSC receiving NCX1 promoter treated cells (MI + iPSC^NCX1). Echocardiography was performed 4 weeks after cell patch application, followed by histological and transmission electron microscopy (TEM) analysis.

Results

Large numbers of transplanted CM were observed with significant improvements in left ventricular performance and remodeling in group 4 as compared with group 3. No teratoma formation was detected in any of the treatment groups.

Conclusion

Manipulation of iPSC yields large numbers of iPSC-CM and favorable morphological and ultrastructural tissue changes. These changes have the potential to enhance current methods used for restoration of cardiac function after MI.     

Atorvastatin Treatment of Rats with Ischemia-Reperfusion Injury Improves Adipose-Derived Mesenchymal Stem Cell Migration and Survival via the SDF-1α/CXCR-4 Axis

Background

Adipose-derived mesenchymal stem cells (ASCs) transplantation is a promising approach for myocardium repair. Promotion of ASCs migration and survival is the key for improving ASCs efficiency. SDF-1α is a critical factor responsible for ASCs migration and survival. Atorvastatin (Ator) is capable of up-regulating SDF-1α. Therefore, we''re going to investigate whether ASCs migration and survival could be improved with atorvastatin.

Methods

In vitro study, cardiomyocytes were subjected to anoxia-reoxygenation injury and subsequently divided into different groups: group blank control, Ator, Ator plus L-NAME (A+L-NAME) and Ator plus AMD3100 (A+AMD3100).When migration analysis completed, cardiomyocytes were used for subsequent analyses. In vivo study, rats underwent ischemia-reperfusion injury were assigned into different groups corresponding to in vitro protocols. ASCs were transplanted on the seventh day of atorvastatin therapy. Seven days later, the rates of migration, differentiation and apoptosis were evaluated.

Results

Compared with other groups, ASCs migration in vitro was significantly improved in group Ator, which was dependent on SDF-1α/CXCR-4 coupling. Results of in vivo study were consistent with that of in vitro study, further supporting the notion that the efficacy of atorvastatin on ASCs migration improvement was related to SDF-1α/CXCR-4 axis. Higher vessel density in group Ator might be another mechanism responsible for migration improvement. Concomitantly, apoptosis was significantly reduced in group Ator, whereas no significant difference of differentiation was found.

Conclusion

Migration and survival of ASCs could be improved by atorvastatin under ischemia-reperfusion injury, which were ascribed to SDF-1α/CXCR-4 axis.     

Granulocyte colony-stimulating factor therapy for stem cell mobilization following anterior wall myocardial infarction: the CAPITAL STEM MI randomized trial

Background:

Small studies have yielded divergent results for administration of granulocyte colony-stimulating factor (G-CSF) after acute myocardial infarction. Adequately powered studies involving patients with at least moderate left ventricular dysfunction are lacking.

Methods:

Patients with left ventricular ejection fraction less than 45% after anterior-wall myocardial infarction were treated with G-CSF (10 μg/kg daily for 4 days) or placebo. After initial randomization of 86 patients, 41 in the placebo group and 39 in the G-CSF group completed 6-month follow-up and underwent measurement of left ventricular ejection fraction by radionuclide angiography.

Results:

Baseline and 6-week mean ejection fraction was similar for the G-CSF and placebo groups: 34.8% (95% confidence interval [CI] 32.6%–37.0%) v. 36.4% (95% CI 33.5%–39.2%) at baseline and 39.8% (95% CI 36.2%–43.4%) v. 43.1% (95% CI 39.2%–47.0%) at 6 weeks. However, G-CSF therapy was associated with a lower ejection fraction at 6 months relative to placebo (40.8% [95% CI 37.4%–44.2%] v. 46.0% [95% CI 42.7%–44.3%]). Both groups had improved left ventricular function, but change in left ventricular ejection fraction was lower in patients treated with G-CSF than in those who received placebo (5.7 [95% CI 3.4–8.1] percentage points v. 9.2 [95% CI 6.3–12.1] percentage points). One or more of a composite of several major adverse cardiac events occurred in 8 patients (19%) within each group, with similar rates of target-vessel revascularization.

Interpretation:

In patients with moderate left ventricular dysfunction following anterior-wall infarction, G-CSF therapy was associated with a lower 6-month left ventricular ejection fraction but no increased risk of major adverse cardiac events. Future studies of G-CSF in patients with left ventricular dysfunction should be monitored closely for safety. Trial registration: ClinicalTrials.gov, no. {"type":"clinical-trial","attrs":{"text":"NCT00394498","term_id":"NCT00394498"}}NCT00394498Rapid reperfusion therapy has become the standard treatment for ST-segment elevation myocardial infarction (STEMI), with congestive heart failure and left ventricular dysfunction continuing as the strongest predictors of higher long-term risk.¹ To date, no definitive therapies exist to regenerate myocardium following myocardial necrosis, and myocardial preservation is therefore the goal of STEMI care. Contemporary studies have suggested the possibility of myocardial regeneration by endogenous stem and progenitor cell populations, and preliminary clinical studies have hinted at potential benefit.^2,3 Studies investigating whether postinfarction myocardial function can be improved by enhancing stem cell–mediated repair are in progress ({"type":"clinical-trial","attrs":{"text":"NCT00936819","term_id":"NCT00936819"}}NCT00936819 and {"type":"clinical-trial","attrs":{"text":"NCT00984178","term_id":"NCT00984178"}}NCT00984178).Granulocyte colony-stimulating factor (G-CSF), an endogenously produced glycoprotein growth factor, when given in pharmacologic doses, stimulates mobilization of hematopoietic stem cells into the peripheral blood. Therapeutically, recombinant synthetic forms have been used to enhance recovery from neutropenia following chemotherapy and for mobilization of stem cells before hematopoietic stem cell transplant.⁴ Numerous small clinical studies have investigated the potential of G-CSF–induced mobilization of stem cells in the peri-infarction period to enhance left ventricular recovery, but they have yielded discordant results. However, meta-analyses have suggested benefit for left ventricular ejection fraction in subgroups who received G-CSF early after infarction or in patients whose left ventricular dysfunction was mild to moderate.^5,6 Larger trials are necessary because, in addition to mobilizing stem cells, G-CSF modulates intracellular signalling cascades within cardiomyocytes and can activate neutrophils, and several trials have been stopped early as a result of excessive in-stent restenosis and acute coronary syndromes in patients with coronary artery disease.^7–11 Animal data have similarly yielded discordant results, depending on the dose and timing of G-CSF.¹²To clarify the role of G-CSF in promoting left ventricular recovery after acute myocardial infarction, we performed an adequately powered randomized clinical trial in patients with moderate left ventricular dysfunction following anterior-wall STEMI.     

Attenuation of osteoarthritis via blockade of the SDF-1/CXCR4 signaling pathway

Introduction

This study was performed to evaluate the attenuation of osteoarthritic (OA) pathogenesis via disruption of the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signaling with AMD3100 in a guinea pig OA model.

Methods

OA chondrocytes and cartilage explants were incubated with SDF-1, siRNA CXCR4, or anti-CXCR4 antibody before treatment with SDF-1. Matrix metalloproteases (MMPs) mRNA and protein levels were measured with real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The 35 9-month-old male Hartley guinea pigs (0.88 kg ± 0.21 kg) were divided into three groups: AMD-treated group (n = 13); OA group (n = 11); and sham group (n = 11). At 3 months after treatment, knee joints, synovial fluid, and serum were collected for histologic and biochemical analysis. The severity of cartilage damage was assessed by using the modified Mankin score. The levels of SDF-1, glycosaminoglycans (GAGs), MMP-1, MMP-13, and interleukin-1 (IL-1β) were quantified with ELISA.

Results

SDF-1 infiltrated cartilage and decreased proteoglycan staining. Increased glycosaminoglycans and MMP-13 activity were found in the culture media in response to SDF-1 treatment. Disrupting the interaction between SDF-1 and CXCR4 with siRNA CXCR4 or CXCR4 antibody attenuated the effect of SDF-1. Safranin-O staining revealed less cartilage damage in the AMD3100-treated animals with the lowest Mankin score compared with the control animals. The levels of SDF-1, GAG, MMP1, MMP-13, and IL-1β were much lower in the synovial fluid of the AMD3100 group than in that of control group.

Conclusions

The binding of SDF-1 to CXCR4 induces OA cartilage degeneration. The catabolic processes can be disrupted by pharmacologic blockade of SDF-1/CXCR4 signaling. Together, these findings raise the possibility that disruption of the SDF-1/CXCR4 signaling can be used as a therapeutic approach to attenuate cartilage degeneration.     

A Plant-Derived Remedy for Repair of Infarcted Heart

Background

Myocardial infarction (MI) due to coronary artery disease remains one of the leading causes of premature death. Replacement of infarcted heart tissue with regenerating myocardium from endogenous progenitor pools or exogenously introduced stem cells remains a therapeutic ideal. Their impracticality mainly lies in their low efficiency in cardiogenic differentiation (CD). Our recent studies with an acute MI animal model have already demonstrated the therapeutic effect of the MeOH extract of Geum japonicum (EGJ), providing clear evidence of myocardial regeneration.

Methods and Findings

The present study further isolated the active component contained in EGJ using bioassay-guided isolation and investigated its efficacy in the treatment of infarcted heart in animal MI models. We demonstrated that substantial repair of infarcted heart in animal MI models by EGJ can be mimicked by the isolated candidate compound (cardiogenin) in MI animal models. Clear evidence of newly regenerated endogenous mesenchymal stem cells (MSCs) derived cardiomyocytes was observed throughout the infarct zone, accompanied by significantly improved functional performance of the heart. Transplantation of MSCs pretreated with EGJ or cardiogenin into a MI animal model also resulted in substantial regeneration of functional myocardium, implying that the activated MSCs carry all the necessary blueprints for myocardial regeneration. Signaling pathways specific to cell survival, CD identified in embryonic heart induction and angiogenesis were activated in both cardiogenin-treated MSCs and cardiogenin-induced regenerating myocardium.

Conclusions

This study has demonstrated the therapeutic effects of cardiogenin in infarcted heart repair, and identified the associated signalling pathways for effective cardiogenic differentiation of MSCs, cell survival and angiogenesis. These findings should enable new treatment strategies for MI to be developed immediately.