The Mechanisms By Which Apolipoprotein-J Induces CXCR4 Expression And Migration And Potential Protection Of Cardiac Stem Cells

BackgroundIn mature human body, cardiac myocytes belong to the final differentiated cells, once damaged can only be replaced by scar tissue. How to Repair or reversing myocardial necrosis has been a hot spot for domestic and international experts to study for many years. A myoblast is a type of progenitor cell that gives rise to myocytes, Derived from mesodermal, with potential of proliferation, can be integrated with each other to form terminal muscle fibers. Many studies have now been reported to confirm that myoblast transplantation can inhibit the apoptosis of myocardial cells, reduce infarction size,improve cardiac function, participate in the treatment of myocardial infarction, heart failure and other ischemic heart disease. Although there are still many problems need to be solved, but this treatment may become the new method for myocardial damage and heart failure.The application of cell-based therapy for the treatment of heart disease remains in its preliminary phase, because cell therapy encounters significant challenges in isolation techniques, scalability and ease of clinical application. An alternative to cell therapy is to identify and modulate the molecules that mediate homing and engraftment of stem cells to the ischemic myocardium and to develop molecular therapies based on these discoveries. One of these molecules is surface receptor on stem cells. The chemokine, stromal cell-derived factor-1 (SDF-1), and its unique receptor CXCR4, are essential for normal cardiovascular development and play a critical role in postnatal vasculogenesis. Importantly, cell mobilization and homing of hematopoietic and mesenchymal stem cells depend on SDF-1 and CXCR4. Moreover, both MAP kinase (MAPK) and, particularly, phosphatidyl-inositol 3-kinase (PI 3-kinase)/Akt play central roles in SDF-1/CXCR4-mediated cell migration. However, it is still a challenge to enhance stem cell migration through modulating CXCR4/SDF-1, since the expression level of CXCR4 is low on some stem cells.Apolipoprotein-J (ApoJ, also designated clusterin), found in high-density lipoproteins (HDL), is a stress-responding, chaperone-like protein. The cytoprotection of ApoJ has been suggested in a number of different cardiovascular-related pathophysiological conditions, such as atheroscelrosis and myocarditis, whereas the mechanism of protection of ApoJ requires further study. The potential protective mechanisms of ApoJ include protecting stem cells against oxidative injury, modulating vascular smooth muscle cell and endothelial cell migration and proliferation during vascular injury. HDL, which is associated with ApoJ, is shown to be inversely related to the incidence of coronary heart disease and, hence, could be used as a powerful predictor for future coronary events. Potential protective mechanisms of HDL include reverse cholesterol transport, endothelial protection, anti-oxidation, and anti-inflammation. More recently, it has been reported that HDL promotes the repair of injured endothelium by stimulating the recruitment of endothelial progenitor cells into the endothelial layer. Some effects of HDL are mediated through PI3 kinase and MAPK, including PI3 kinase-dependent endothelial cell migration. These findings suggest that modulating migration of some stem cells is a potentially important atheroprotective mechanism to be added to the other known properties of these lipoproteins. We have recently shown that expression of ApoJ by cDNA transfection markedly reduces apoptosis in fetal cardiac stem cells or myoblasts, restores the mitochondrial membrane potential, and prevents the release of cytochrome C from the mitochondria into the cytoplasm. However, the effects of ApoJ on tissue-specific stem cell migration and its potential mechanisms are unknown. In this study, we examined the effects of ApoJ on canine fetal cardiac stem cell proliferation and migration, and the relationship of ApoJ with the SDF-1/CXCR4 signaling pathway. Furthermore, we need to know whether ApoJ have a direct protective effect on damaged cardiac stem cells?ObjectiveTo determine whether ApoJ exerts a protective effect on cardiovascular cells against a toxic agent, such as anti-HIV drug nucleotide analogue reverse transcriptase inhibitors(NRTI), and explore the effect of ApoJ on CXCR4 expression and SDF-1/CXCR4 mediated migration by tissue-specific stem cell without altering the cell cycle, ApoJ expression was induced in canine fetal myoblasts by stable cDNA transfection.Methods1. Cardiac stem cell cultures were prepared from myoblasts isolated from fetal dog heart by collagenase digestion. The myoblast tissues were suspended in trypsin and placed in a shaking water bath. After dissociation, the cells were counted using a hemacytometer and plated in tissue culture flasks. The cells were cultured in an incubator for 10-14 days before use. Overexpression of Apo J in stem cells:Human Apo J cDNA corresponding to human ApoJ was isolated from pCMV-sport 6 plasmid by using Hind III and EcoRV. The resulting fragment, containing the full length human ApoJ cDNA, was ligated into the mammalian expression vector pcDNA3, and transfected into stem cells using Lipofectamine2000. Transfected cells were split 48 h later and maintained in medium containing G418. Evaluate the ApoJ expression in the transfected myoblasts with Western blotting analysis. Cell proliferation was assessed with a BrdU Proliferation Assay. the cells were exposed to anti-BrdU monoclonal antibody followed by peroxidase-conjugated goat anti-mouse substrate. The colored reaction product was quantified by measuring absorbance at 450 nm and 595 nm. Cell cycle analysis were analyzed by FACScan flow cytometry.2. CXCR4 expression and migration to SDF-1:Applying real-time PCR and Western blot analysis to determine the expression of CXCR4.Flow cytometry is for its positive percentage which method is:Myoblast cells were stained with PE-conjugated anti-CXCR4, then sorted with a triple-laser fluorescence-activated cell sorter. The data were collected using a FACSCalibur machine and analyzed by using CELLQUEST. 10,000 events.Myoblast cells were placed in the culture medium and 100 nM SDF-1 for measuring the migratory capacity of myoblast cells. After incubation the medium was removed, and the insert was transferred to a second plate containing Calcein-AM solution. Then, the fluorescence of migrated cells was read in a fluorescence plate reader with bottom reading capabilities at excitation/emission wavelengths of 485/530 nm. To assess cell motility, cells were seeded at a density of 5×105 cells and grown to 70% confluence. The medium was removed from the plates and a wound was generated in each plate using a sterile pipette tip. Movement across the wound was assessed by microscopic examination and multiple fields were photographed with microscope equipped with a SPOT digital camera. Cells were stained with DAPI.3. Evaluat the protection of ApoJ to damaged cells:NRTI drug stock solution were made with 5 different NRTI drug and DPBS. Interfere in the wide-type myoblasts and ApoJ transfected myoblasts with concentrations of 1:1,1:2,1:4, andl:8 Cmax.24 hours later,counting method was used for cell mortality; In vitro toxicological analysis suite and spectrophotometry of LDH which was released in the culture medium after cell damage were used to test cell viability. Absorbance was measured at 490 nm. In the phase contrast microscope, cells were observed by morphological changes.Results:1. Synchronization of cell cycle in canine wild type and ApoJ-transfected fetal myoblasts. Canine fetal cardiac stem cells or myoblasts were isolated and transfected stably with or without ApoJ cDNA. The stable expression of ApoJ in the transfected cells was confirmed by PCR and Western blot. Because cells at different phases of cell cycle show different capacities of migration, ApoJ-transfected myoblasts were synchronized with wild type control cells through serum starvation. Analysis of myoblasts with BrdU incorporation by flow cytometry revealed that wild type and ApoJ-transfected myoblasts had a similar cell cycle. There were no differences in the percentages of cells in Go/Gl, S and M phases between untransfected myoblasts and ApoJ-transfected. Flow cytometry showed that 73.6%±4.6% of ApoJ-transfected canine fetal myoblasts and 70.1%±5.1% of wild-type myoblasts were in the Gl phase of the cell cycle (Figure 1B). Similarly, we found no substantial differences between the two groups in the percentage of cells in S phase (13.8%±5.3% of ApoJ-transfected fetal myoblasts vs.15.4%±4.9% of wild-type myoblasts). Furthermore, ApoJ did not alter the proliferation of canine fetal myoblasts as determined by BrdU incorporation assay (1.81±0.03 in wild-type myoblasts vs.1.79±0.02 absorbance in ApoJ-transfected myoblasts).2. After synchronization of cell cycle, expression of the chemokine receptor CXCR4 was analyzed by use of real time quantitative PCR. Compared to untransfected control cells, ApoJ-transfected myoblasts showed markedly increased expression of CXCR4 mRNA. Also, agarose gel electrophoresis demonstrated a four-fold increase in the expression of CXCR4 cDNA in the RNA samples from ApoJ-positive cells. By contrast, no difference in the 18S rRNA bands was found between wild type and ApoJ-expressing cells.Flow cytometry study clearly illustrates an increased of cell surface expression of CXCR4 in myoblasts transfected with ApoJ. Furthermore, ApoJ-transfected myoblast showed a much higher positivity in CXCR4 immunostaining as compared to wild type cells (29.8%±3.4% in ApoJ cells vs.10.5±2.1% in wild type cells, p< 0.01).Western blot analysis was conducted using total protein extracts of ApoJ-expressing and wild type cells with the same antibodies for flow cytometry. A single clear CXCR4 protein band was detected in ApoJ-transfected myoblast while this protein band was not found in wild type cells.In ApoJ-transfected cells, we observed a marked increase in the migratory response of canine fetal myoblasts to SDF-1 (100 nM). The ApoJ-induced increase in canine fetal myoblast migration in response to SDF-1 was attenuated by the addition of the PI3 kinase inhibitor, but not by the mitogen-activated protein/ERK kinase inhibitor,. In addition, the increased migration of ApoJ-transfected canine fetal myoblasts was inhibited by pretreatment with blocking anti-CXCR4 antibody (10μg/ml). The wound healing was increased in ApoJ-transfected myoblasts compared to wild type myoblast3. After 24 hours of the intervention of five NRTIdrugs(didanosin, Epivir, Zerit, ZIaGen, Viread),the cell morphology:the cell membrane rupture, necrosis, cell body broken, debris fused into pieces. Counting estimate the concentration of dead cells (control group vs ApoJ group) were didanosine 75.25%±9.22% vs70.31%±3.92%, Epivir 53.54%±2.76% vs 58.02%±10.8%, Zerit 62.65%±4.63% vs 32.21%±8.34%, ZIaGen 65.39%±4.75% vs 60.7%±14.6%, Viread 50.65%±2.63% vs 27.12%±5.35%, which Zerit and Viread have significant differences between two group cells (P<0.05). In vitro toxicological analysis showed that the release of LDH by myoblasts in the control group and Apo J transfected group increased with an increase in the drug concentration. Most drugs did not cause LDH release in the two kinds of cultured cells except for Viread (P=0.045) and Zerit (P=0.029), which caused the release of LDH at a low concentration by Apo J transgenic myogenic cells (P<0.05).Conclusions:1. ApoJ cDNA stable transfection of canine fetal cardiac stem cells has the same cell cycle and proliferation with non-transfected myoblast can exclude the impact of them to migration.2. ApoJ induced expression of CXCR4 in cardiac myoblasts. We also have shown that ApoJ overexpression by stable transfection increases SDF-1-induced migration in canine fetal cardiac myoblasts. These data indicate that the ApoJ-induced increase in migration of canine fetal myoblasts is CXCR4 dependent. These data indicate that the ApoJ-induced increase in migration of canine fetal myoblasts is CXCR4 dependent.3. The NRTIs drugs in our study are widely used in clinical treatment of HIV infection, studies have shown that these drugs can cause myoblasts damaged and death in vitro, meanwhile apolipoprotein J can counter the destructive effects of certain drugs, such as Viread and Zerit. The protection mechanism of ApoJ are perhaps mediated, in part, through attenuating cell apoptosis induced by cytotoxic actions,oxidative stress and altered calcium homeostasis.This study provides the first experimental evidence to the AIDS treatment propects and clinical drug screening.