Zulong Sheng

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.

Dual-modal tracking of transplanted mesenchymal stem cells after myocardial infarction.

Purpose: Results for implantation efficiency and effective improvement of cardiac function in the field of mesenchymal stem cells (MSCs) are controversial. To attempt to clarify this debate, we utilized magnetic resonance imaging (MRI) and near-infrared optical imaging (OI) to explore the effects of different delivery modes of mesenchymal stem cells on cell retention time and cardiac function after myocardial infarction (MI). Methods: Rat MSCs were labeled with superparamagnetic iron oxide nanoparticles and 1, 1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) for noninvasive cell tracking in a rat MI model. Rats underwent coronary artery ligation and were randomized into three experimental groups: intravenous (IV), intramyocardial (IM), and a control group. The first two groups referred to the route of delivery of the transplanted dual-labeled MSCs; whereas the control group was given an IV injection of serum-free medium one day post-MI. Cellular engraftment was determined 1 day and 7 days post cell delivery by measuring the iron and optical signals in explanted organs. Prussian blue staining and fluorescent microscopy were performed on histological sections for iron and DiD, respectively. Cardiac function was measured by echocardiography on day 7. Results: The cardiac function of the IM group increased significantly compared to the IV and control groups at day 7. In the IM group, labeled cells were visualized in the infracted heart by serial MRI, and the intensity by OI was significantly higher on day 1. In the IV group, the heart signals were significantly attenuated by dual-modal tracking at two time points, but the lung signals in OI were significantly stronger than the IM group at both time points. Conclusion: IM injection of MSCs increased cell engraftment within infarcted hearts and improved cardiac function after MI. However, IV infusion has a low efficacy due to the cell trapping in the lung. Therefore, direct injection may provide an advantage over IV, with regard to retention of stem cells and protection of cardiac function.

Tissue Kallikrein Promotes Cardiac Neovascularization by Enhancing Endothelial Progenitor Cell Functional Capacity

Tissue kallikrein (TK) has been demonstrated to improve neovasculogenesis after myocardial infarction (MI). In the present study, we examined the role and underlying mechanisms of TK in peripheral endothelial progenitor cell (EPC) function. Peripheral blood-derived mononuclear cells containing EPCs were isolated from rat. The in vitro effects of TK on EPC differentiation, apoptosis, migration, and vascular tube formation capacity were studied in the presence or absence of TK, kinin B(2) receptor antagonist (icatibant), and phosphatidylinositol-3 kinase inhibitor (LY294002). Apoptosis was evaluated by flow-cytometry analysis using Annexin V-FITC/PI staining, as well as western-blot analysis of Akt phosphorylation and cleaved caspase-3. Using an MI mouse model, we then examined the in vivo effects of human TK gene adenoviral vector (Ad.hTK) administration on the number of CD34(+)Flk-1(+) progenitors in the peripheral circulation, heart tissue, extent of vasculogenesis, and heart function. Administration of TK significantly increased the number of Dil-LDL/UEA-lectin double-positive early EPCs, as well as their migration and tube formation properties in vitro. Transduction of TK in cultured EPCs attenuated apoptosis induced by hypoxia and led to an increase in Akt phosphorylation and a decrease in cleaved caspase-3 levels. The beneficial effects of TK were blocked by pretreatment with icatibant and LY294002. The expression of recombinant human TK in the ischemic mouse heart significantly improved cardiac contractility and reduced infarct size 7 days after gene delivery. Compared with the Ad.Null group, Ad.hTK reduced mortality and preserved left ventricular function by increasing the number of CD34(+)Flk-1(+) EPCs and promoting the growth of capillaries and arterioles in the peri-infarct myocardium. These data provide direct evidence that TK promotes vessel growth by increasing the number of EPCs and enhancing their functional properties through the kinin B(2) receptor-Akt signaling pathway.

Bradykinin Preconditioning Improves Therapeutic Potential of Human Endothelial Progenitor Cells in Infarcted Myocardium

Objectives Stem cell preconditioning (PC) is a powerful approach in reducing cell death after transplantation. We hypothesized that PC human endothelial progenitor cells (hEPCs) with bradykinin (BK) enhance cell survival, inhibit apoptosis and repair the infarcted myocardium. Methods The hEPCs were preconditioned with or without BK. The hEPCs apoptosis induced by hypoxia along with serum deprivation was determined by annexin V-fluorescein isothiocyanate/ propidium iodide staining. Cleaved caspase-3, Akt and eNOS expressions were determined by Western blots. Caspase-3 activity and vascular endothelial growth factor (VEGF) levels were assessed in hEPCs. For in vivo studies, the survival and cardiomyocytes apoptosis of transplanted hEPCs were assessed using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodi- carbocyanine,4-chlorobenzenesul-fonate salt labeled hEPCs and TUNEL staining. Infarct size and cardiac function were measured at 10 days after transplantation, and the survival of transplanted hEPCs were visualized using near-infrared optical imaging. Results In vitro data showed a marked suppression in cell apoptosis following BK PC. The PC reduced caspase-3 activation, increased the Akt, eNOS phosphorylation and VEGF levels. In vivo data in preconditioned group showed a robust cell anti-apoptosis, reduction in infarct size, and significant improvement in cardiac function. The effects of BK PC were abrogated by the B2 receptor antagonist HOE140, the Akt and eNOS antagonists LY294002 and L-NAME, respectively. Conclusions The activation of B2 receptor-dependent PI3K/Akt/eNOS pathway by BK PC promotes VEGF secretion, hEPC survival and inhibits apoptosis, thereby improving cardiac function in vivo. The BK PC hEPC transplantation for stem cell-based therapies is a novel approach that has potential for clinical used.

In Vivo Imaging of Macrophages during the Early-Stages of Abdominal Aortic Aneurysm Using High Resolution MRI in ApoE−/− Mice

Background Angiotensin II (ANG II) promotes vascular inflammation and induces abdominal aortic aneurysm (AAA) in hyperlipidemic apolipoprotein E knock-out (apoE−/−) mice. The aim of the present study was to detect macrophage activities in an ANG II-induced early-stage AAA model using superparamagnetic iron oxide (SPIO) as a marker. Methodology/Principal Findings Twenty-six male apoE−/− mice received saline or ANG II (1000 or 500 ng/kg/min) infusion for 14 days. All animals underwent MRI scanning following administration of SPIO with the exception of three mice in the 1000 ng ANG II group, which were scanned without SPIO administration. MR imaging was performed using black-blood T2 to proton density -weighted multi-spin multi-echo sequence. In vivo MRI measurement of SPIO uptake and abdominal aortic diameter were obtained. Prussian blue, CD68,α-SMC and MAC3 immunohistological stains were used for the detection of SPIO, macrophages and smooth muscle cells. ANG II infusion with 1000 ng/kg/min induced AAA in all of the apoE−/− mice. ANG II infusion exhibited significantly higher degrees of SPIO uptake, which was detected using MRI as a distinct loss of signal intensity. The contrast-to-noise ratio value decreased in proportion to an increase in the number of iron-laden macrophages in the aneurysm. The aneurysmal vessel wall in both groups of ANG II treated mice contained more iron-positive macrophages than saline-treated mice. However, the presence of cells capable of phagocytosing haemosiderin in mural thrombi also induced low-signal-intensities via MRI imaging. Conclusions/Significance SPIO is taken up by macrophages in the shoulder and the outer layer of AAA. This alters the MRI signaling properties and can be used in imaging inflammation associated with AAA. It is important to compare images of the aorta before and after SPIO injection.

Tissue kallikrein-modified human endothelial progenitor cell implantation improves cardiac function via enhanced activation of akt and increased angiogenesis

Endothelial progenitor cells (EPCs) have been shown to enhance angiogenesis not only by incorporating into the vasculature but also by secreting cytokines, thereby serving as an ideal vehicle for gene transfer. As tissue kallikrein (TK) has pleiotropic effects in inhibiting apoptosis and oxidative stress, and promoting angiogenesis, we evaluated the salutary potential of kallikrein-modified human EPCs (hEPCs; Ad.hTK-hEPCs) after acute myocardial infarction (MI). We genetically modified hEPCs with a TK gene and evaluated cell survival, engraftment, revascularization, and functional improvement in a nude mouse left anterior descending ligation model. hEPCs were manipulated to overexpress the TK gene. In vitro, the antiapoptotic and paracrine effects were assessed under oxidative stress. TK protects hEPCs from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and -9, induction of Akt phosphorylation, and secretion of vascular endothelial growth factor. In vivo, the Ad.hTK-hEPCs were transplanted after MI via intracardiac injection. The surviving cells were tracked after transplantation using near-infrared optical imaging. Left ventricular (LV) function was evaluated by transthoracic echocardiography. Capillary density was quantified using immunohistochemical staining. Engrafted Ad.hTK-hEPCs exhibited advanced protection against ischemia by increasing LV ejection fraction. Compared with Ad.Null-hEPCs, transplantation with Ad.hTK-hEPCs significantly decreased cardiomyocyte apoptosis in association with increased retention of transplanted EPCs in the myocardium. Capillary density and arteriolar density in the infarct border zone was significantly higher in Ad.hTK-hEPC-transplanted mice than in Ad.Null-hEPC-treated mice. Transplanted hEPCs were clearly incorporated into CD31+ capillaries. These results indicate that implantation of kallikrein-modified EPCs in the heart provides advanced benefits in protection against ischemia-induced MI by enhanced angiogenesis and reducing apoptosis.

Analysis of in situ and ex vivo αVβ3 integrin expression during experimental carotid atherogenesis.

Objective Mural inflammation has been shown to contribute to the development of plaque, with the αVβ3 integrin highly expressed in atherosclerotic plaques. We herein examined αVβ3 integrin expression as a function of carotid atherosclerosis formation in the apolipoprotein E-deficient (apoE−/−) mouse. Methods and results Constrictive collars were placed around the left common carotid arteries of apo E−/− mice maintained on a high-fat diet (n = 14). Before and 21 days following collar placement, in vivo serial magnetic resonance imaging (MRI) measurements of the carotid aortic diameter were performed using a 7T magnetic resonance (MR) scanner. Near- infrared fluorescence (NIRF) imaging was performed (n = 6) using an in vivo imaging system 0–24 hours following administration of 1.0 nmol c(RGDyK)-Cy5.5 via the tail vein. A competition experiment was performed by the co-injection of a saturating dose of bicyclic RGD peptide H-Glu[cyclo(Arg-Gly-Asp-D-Tyr-Lys)]2 (n = 3). Following image acquisition and sacrifice at 24 hours after injection, carotid arteries were harvested for histological analyses. Neointima formation and arterial remodeling in the carotid arteries of apoE−/− mice were induced by the placement of a constrictive collar. Significantly greater fluorescent signals were obtained from constrictive collar left common carotid arteries as compared to uninvolved aortic segments in constrictive collar mice. Binding to stenotic lesions was efficiently blocked in competition experiments. Immunostaining confirmed the presence of mural αVβ3 integrin expression in macrophages in the neointima. Signal intensity increased in a macrophage density-dependent fashion in the stenotic segments. Conclusion Mural αVβ3 integrin expression, as determined using RGD-Cy5.5 near-infrared optical imaging, was increased in carotid arteries with constrictive collars in experimental mice. This expression can estimate the macrophage-bound inflammatory activity of atherosclerotic lesions.

DNA methylation regulates α-smooth muscle actin expression during cardiac fibroblast differentiation: HE et al.

Cardiac fibroblast (CF) differentiation to myofibroblasts expressing α‐smooth muscle actin (α‐SMA) plays a key role in cardiac fibrosis. Therefore, a study of the mechanism regulating α‐SMA expression is a means to understanding the mechanism of fibroblast differentiation and cardiac fibrosis. Previous studies have shown that DNA methylation is associated with gene expression and is related to the development of tissue fibrosis. However, the mechanisms by which CF differentiation is regulated by DNA methylation remain unclear. Here, we explored the epigenetic regulation of α‐SMA expression and its relevance in CF differentiation. In this study, we demonstrated that α‐SMA was overexpressed and DNMT1 expression was downregulated in the infarct area after myocardial infarction. Treatment of CFs with transforming growth factor‐β1 (TGF‐β1) in vitro upregulated α‐SMA expression via epigenetic modifications. TGF‐β1 also inhibited DNMT1 expression and activity during CF differentiation. In addition, α‐SMA expression was regulated by DNMT1. Conversely, increasing DNMT1 expression levels rescued the TGF‐β1‐induced upregulation of α‐SMA expression. Finally, TGF‐β1 regulated α‐SMA expression by inhibiting the DNMT1‐mediated DNA methylation of the α‐SMA promoter. Taken together, our research showed that inhibition of the DNMT1‐mediated DNA methylation of the α‐SMA promoter plays an essential role in CF differentiation. In addition, DNMT1 may be a new target for the prevention and treatment of myocardial fibrosis.

Kallistatin Inhibits Atherosclerotic Inflammation by Regulating Macrophage Polarization

Kallistatin (KS) has been recognized as a plasma protein with anti-inflammatory functions. Macrophages are the primary inflammatory cells in atherosclerotic plaques. However, it is unknown whether KS plays a role in macrophage development and the pathogenesis of atherosclerosis. This study investigated the role of KS in macrophage development, a key pathological process in atherosclerosis. An atherosclerosis model was established in ApoE-/- mice via partial left carotid artery (PLCA) ligation. An adenovirus vector (Ad. HKS) containing the human KS gene was delivered via the tail vein before PLCA ligation. The mice were divided into two groups: the PLCA + Ad. HKS and PLCA + adenovirus vector (Ad. Null) groups and followed for 2 and 4 weeks. Human KS was expressed in the mice after KS gene delivery. In addition, KS significantly inhibited plaque formation and reduced inflammation in the plaques and liver 4 weeks after gene delivery. Moreover, KS gene delivery significantly increased the expression of interleukin-10 and Arginase 1, which are M2 macrophage markers, and reduced the expression of inducible nitric oxide synthase and monocyte chemotactic protein 1, which are M1 macrophage markers. Furthermore, in cultured RAW 264.7 macrophages, KS significantly stimulated M2 marker expression and differentiation and decreased M1 marker expression, as determined by flow cytometry and real-time polymerase chain reaction. These effects were blocked by Krüppel-like factor 4 small-interfering RNA oligonucleotides. These findings demonstrate that KS inhibits atherosclerotic plaque formation and regulates M1/M2 macrophage polarization via Krüppel-like factor 4 activation.

TWEAK promotes endothelial progenitor cell vasculogenesis to alleviate acute myocardial infarction via the Fn14‑NF‑κB signaling pathway

Acute myocardial infarction (AMI) remains one of the leading causes of mortality worldwide; however, endothelial progenitor cell (EPC) transplantation has been proposed as a promising treatment strategy for EPC. High levels of tumor necrosis factor-related weak inducer of apoptosis (TWEAK) have been reported in AMI, although its effect on EPCs has not been reported. In the present study, immunofluorescence and flow cytometry were performed to assess the effect of TWEAK in isolated mouse EPCs. Echocardiography was used to evaluate the cardiac function of murine hearts following EPC treatment in the AMI model, while collagen synthesis within the heart tissue was assessed using Massons trichrome staining. A tube formation assay and Transwell migration assay were performed to investigate the effects of TWEAK on vessel formation and EPC migration in vitro. Angiogenesis and arteriogenesis were assessed in vivo using immunohistochemistry and western blotting was performed to determine the effect of TWEAK-mediated nuclear factor (NF)-κB pathway activation in EPCs. The results revealed that TWEAK promotes EPC migration, tube formation and viability in vitro. Furthermore, TWEAK treatment resulted in improved cardiac function, decreased heart collagen and vasculogenesis in mice with AMI, which was mediated by the TWEAK- fibroblast growth factor-inducible 14 (Fn14)-NF-κB signaling pathway, as determined using Fn14 small interfering (si)RNA and Bay 11–7082 (an NF-κB inhibitor). In summary, the results of the present study suggest that activation of the TWEAK-Fn14-NF-κB signaling pathway exerts a beneficial effect on EPCs for the treatment of AMI.