Brain metastasis is a defining component of tumor pathophysiology, and the underlying mechanisms responsible for this phenomenon are not well understood. Current dogma is that tumor cells stimulate and activate astrocytes, and this mutual relationship is critical for tumor cell sustenance in the brain. Here, we provide evidence that primary rat neonatal and adult astrocytes secrete factors that proactively induced human lung and breast tumor cell invasion and metastasis capabilities. Among which, tumor invasion factors namely matrix metalloprotease-2 (MMP-2) and MMP-9 were partly responsible for the astrocyte media-induced tumor cell invasion. Inhibiting MMPs reduced the ability of tumor cell to migrate and invade in vitro. Further, injection of astrocyte media-conditioned breast cancer cells in mice showed increased invasive activity to the brain and other distant sites. More importantly, blocking the preconditioned tumor cells with broad spectrum MMP inhibitor decreased the invasion and metastasis of the tumor cells, in particular to the brain in vivo. Collectively, our data implicate astrocyte-derived MMP-2 and MMP-9 as critical players that facilitate tumor cell migration and invasion leading to brain metastasis.
microRNAs (miRs), a novel class of small non-coding RNAs, are involved in cell proliferation, differentiation, development, and death. In this study, we found that miR-221 translocation by microvesicles (MVs) plays an important role in cardioprotection mediated by GATA-4 overexpressed mesenchymal stem cells (MSC).
Methods and Results
Adult rat bone marrow MSC and neonatal rat ventricle cardiomyocytes (CM) were harvested as primary cultures. MSC were transduced with GATA-4 (MSCGATA-4) using the murine stem cell virus (pMSCV) retroviral expression system. Empty vector transfection was used as a control (MSCNull). The expression of miRs was assessed by real-time PCR and localized using in situ hybridization (ISH). MVs collected from MSC cultures were characterized by expression of CD9, CD63, and HSP70, and photographed with electron microscopy. Cardioprotection during hypoxia afforded by conditioned medium (CdM) from MSC cultures was evaluated by lactate dehydrogenase (LDH) release, MTS uptake by CM, and caspase 3/7 activity. Expression of miR-221/222 was significantly higher in MSC than in CM and miR-221 was upregulated in MSCGATA-4. MSC overexpression of miR-221 significantly enhanced cardioprotection by reducing the expression of p53 upregulated modulator of apoptosis (PUMA). Moreover, expression of PUMA was significantly decreased in CM co-cultured with MSC. MVs derived from MSC expressed high levels of miR-221, and were internalized quickly by CM as documented in images obtained from a Time-Lapse Imaging System.
Our results demonstrate that cardioprotection by MSCGATA-4 may be regulated in part by a transfer of anti-apoptotic miRs contained within MVs.
The purpose of this study was to assess the effect of collagen composition on engraftment of progenitor cells within infarcted myocardium.
We previously reported that intramyocardial penetration of stem/progenitor cells in epicardial patches was enhanced when collagen was reduced in hearts overexpressing adenylyl cyclase-6 (AC6). In this study we hypothesized an alternative strategy wherein overexpression of microRNA-29b (miR-29b), inhibiting mRNAs that encode cardiac fibroblast proteins involved in fibrosis, would similarly facilitate progenitor cell migration into infarcted rat myocardium.
In vitro: A tri-cell patch (Tri-P) consisting of cardiac sodium-calcium exchanger-1 (NCX1) positive iPSC (iPSCNCX1+), endothelial cells (EC), and mouse embryonic fibroblasts (MEF) was created, co-cultured, and seeded on isolated peritoneum. The expression of fibrosis-related genes was analyzed in cardiac fibroblasts (CFb) by qPCR and Western blot. In vivo: Nude rat hearts were administered mimic miRNA-29b (miR-29b), miRNA-29b inhibitor (Anti-29b), or negative mimic (Ctrl) before creation of an ischemically induced regional myocardial infarction (MI). The Tri-P was placed over the infarcted region 7 days later. Angiomyogenesis was analyzed by micro-CT imaging and immunofluorescent staining. Echocardiography was performed weekly.
The number of green fluorescent protein positive (GFP+) cells, capillary density, and heart function were significantly increased in hearts overexpressing miR-29b as compared with Ctrl and Anti-29b groups. Conversely, down-regulation of miR-29b with anti-29b in vitro and in vivo induced interstitial fibrosis and cardiac remodeling.
Overexpression of miR-29b significantly reduced scar formation after MI and facilitated iPSCNCX1+ penetration from the cell patch into the infarcted area, resulting in restoration of heart function after MI.
DNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification.
We modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF). This aptamer enables better separation by fluorescence-activated cell sorting (FACS) of c-kit+ hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types.
Here we describe a novel procedure for the efficient generation of DNA aptamers that bind to specific cell membrane proteins and can be used as high affinity reagents. We have named the procedure STACS (Specific TArget Cell-SELEX).
Oroxylin A, a natural flavonoid, is one of the main bioactive compounds that underlie the anti-inflammatory effect of the medicinal herb Scutellariabaicalensis Georgi widely used in southeastern Asia; however, the molecular mechanisms for the therapeutic benefits remain largely unclear. In this study, we found that Oroxylin A induces estrogen-responsive gene expression and promoter activity. In macrophages, Oroxylin A treatment significantly attenuates lipopolysaccharide (LPS)-induced but not basal inflammatory response, including nitric oxide (NO) production and the expression of inflammatory mediators (i.e., iNOS and COX-2) and cytokines (i.e., TNF-α, IL-1β, and IL-6), in an estrogen receptor (ER)-dependent manner. Oroxylin A treatment also dramatically decreases LPS-induced secretion of pro-inflammatory cytokines. Furthermore, the downregulation of all these inflammatory parameters by Oroxylin A was abolished when cells were pretreated with specific ER antagonist. Thus, Oroxylin A is a novel phytoestrogen and exhibits anti-inflammatory effects that are mediated by ER activity.
Inflammation plays a critical role in adverse cardiac remodeling and heart failure. Therefore, approaches geared towards inhibiting inflammation may provide therapeutic benefits. We tested the hypothesis that genetic deletion of interleukin-10 (IL10), a potent anti-inflammatory cytokine, exacerbates pressure-overload induced adverse cardiac remodeling and hypertrophy and that IL10 therapy inhibits this pathology.
Methods and Results
Cardiac hypertrophy was induced in Wild-type (WT) and IL10-knockout (KO) mice by isoproterenol (ISO) infusion. ISO-induced left ventricular (LV) dysfunction and hypertrophic remodeling, including fibrosis and fetal gene expression, were further exaggerated in KO mice compared to WT. Systemic recombinant mouse IL10 administration markedly improved LV function and not only inhibited but also reversed ISO-induced cardiac remodeling. Intriguingly, very similar cardio-protective response of IL10 was found in transverse aortic constriction (TAC)-induced hypertrophy and heart failure model. In neonatal rat ventricular myocytes (NRCM) and H9c2 myoblasts, ISO activated NFκB while it inhibited STAT3 phosphorylation. Interestingly, IL10 suppressed ISO-induced NFκB activation and attenuated STAT3 inhibition. Moreover, pharmacological and genetic inhibition of STAT3 reversed the protective effects of IL10 while ectopic expression of constitutively active STAT3 mimicked the IL10 responses on the ISO effects, confirming that IL10 mediated inhibition of NFκB is STAT3 dependent.
Taken together our studies suggest IL10 treatment as a potential therapeutic approach to limit the progression of pressure overload-induced adverse cardiac remodeling.
heart failure; hypertrophy; interleukins; myocardium; signal transduction
While Bone-marrow endothelial progenitor cell based therapies (BM-EPC) improve the symptoms in patients with ischemic heart disease their limited plasticity and decreased function in patients with existing heart disease limits the full benefit of EPC therapy for cardiac regenerative medicine.
We hypothesized that reprogramming mouse and/or human EPCs using small molecules targeting key epigenetic repressive marks would lead to a global increase in active gene transcription, induce their cardiomyogenic potential and enhance their inherent angiogenic potential.
Method and Results
Mouse Lin-Sca1+CD31+ EPCs and human CD34+ cells were treated with inhibitors of DNA methyltransferases (5-Azacytidine), histone deacetylases (valproic acid) and G9a histone di-methyltransferase. Forty eight hour treatment led to global increase in active transcriptome including the reactivation of pluripotency associated and CMC specific mRNA expression while EC specific genes were significantly up-regulated. When cultured under appropriate differentiation conditions, reprogrammed EPCs showed efficient differentiation into CMC and vascular smooth muscle cells. Treatment with epigenetic modifying agents show marked increase in histone acetylation on cardiomyocyte and pluripotent cell specific gene promoters. Intra-myocardial transplantation of reprogrammed mouse and human EPCs in an acute myocardial infarction mouse model showed significant improvement in ventricular functions, which was histologically supported by their de novo CMC differentiation and increased capillary density and reduced fibrosis. Importantly, cell transplantation was safe and did not form teratomas.
Taken together, our results suggest that epigenetically reprogrammed EPCs display a safe, more plastic phenotype and improve post-infarct cardiac repair by both neo-cardiomyogenesis and neovascularization.
Epigenetic modification; EPC; cardiomyogenesis; myocardial ischemia; cell therapy; histone acetylation; trans-differentiation
It is now recognized that transplantation of bone marrow cells (BMCs) into infarcted hearts has the capacity to improve the cardiac function through paracrine effects. However, detailed expression levels of paracrine factors in BMCs in infarcted hearts are poorly described. By use of laser capture microdissection combined with real-time PCR, we depicted the expression profiles of paracrine factors in infarcted hearts versus normal hearts. Consistent with the in vivo observation, a similar expression pattern was evidenced in cultured BMCs. Furthermore, BMCs displayed heterogeneity of paracrine effects in infarcted hearts as analyzed at the single cell level using single cell PCR. Interestingly, the CD45+ subpopulation showed higher expression levels of angiogenic factors compared to other subpopulations. Finally, most angiogenic factors were induced under the microenvironment of infarction. Our study demonstrated the heterogeneity of paracrine effects in BMCs at single cell level in infarcted hearts, highlighting preferential expression of angiogenic factors in the CD45+ subpopulation. These findings broaden our understanding of paracrine effects of BMCs in vivo, and offer new insights into BMCs therapy in myocardial infarction (MI).
Insufficient neovascularization, characterized by poor endothelial cell (EC) growth, contributes to the pathogenesis of ischemic heart disease and limits cardiac tissue preservation and regeneration. The E2F family of transcription factors are critical regulators of the genes responsible for cell-cycle progression and growth; however, the specific roles of individual E2Fs in ECs are not well understood. Here we investigated the roles of E2F2 and E2F3 in EC growth, angiogenesis, and their functional impact on myocardial infarction (MI). An endothelial-specific E2F3-deficient mouse strain VE-Cre; E2F3fl/fl was generated, and MI was surgically induced in VE-Cre; E2F3fl/fl and E2F2-null (E2F2 KO) mice and their wild-type (WT) littermates, VE-Cre; E2F3+/+ and E2F2 WT, respectively. The cardiac function, infarct size, and vascular density were significantly better in E2F2 KO mice and significantly worse in VE-Cre; E2F3fl/fl mice than in their WT littermates. The loss of E2F2 expression was associated with an increase in the proliferation of ECs both in vivo and in vitro, while the loss of E2F3 expression led to declines in EC proliferation. Thus, E2F3 promotes while E2F2 suppresses ischemic cardiac repair through corresponding changes in EC proliferation; and differential targeting of specific E2F members may provide a novel strategy for therapeutic angiogenesis of ischemic heart disease.
In a swine model of acute myocardial infarction (AMI), Statins can enhance the therapeutic efficacy of mesenchymal stem cell (MSCs) transplantation. However, the mechanisms remain unclear. This study aims at assessing whether atorvastatin (Ator) facilitates the effects of MSCs through activation of nitric oxide synthase (NOS), especially endothelial nitric oxide synthase (eNOS), which is known to protect against ischemic injury.
Methods and Results
42 miniswines were randomized into six groups (n = 7/group): Sham operation; AMI control; Ator only; MSC only, Ator+MSCs and Ator+MSCs+NG-nitrol-L-arginine (L-NNA), an inhibitor of NOS. In an open-heart surgery, swine coronary artery ligation and reperfusion model were established, and autologous bone-marrow MSCs were injected intramyocardium. Four weeks after transplantation, compared with the control group, Ator+MSCs animals exhibited decreased defect areas of both “perfusion” defined by Single-Photon Emission Computed Tomography (−6.2±1.8% vs. 2.0±5.1%, P = 0.0001) and “metabolism” defined by Positron Emission Tomography (−3.00±1.41% vs. 4.20±4.09%, P = 0.0004); Ejection fraction by Magnetic Resonance Imaging increased substantially (14.22±12.8% vs. 1.64±2.64%, P = 0.019). In addition, indices of inflammation, fibrosis, and apoptosis were reduced and survivals of MSCs or MSC-derived cells were increased in Ator+MSCs animals. In Ator or MSCs alone group, perfusion, metabolism, inflammation, fibrosis or apoptosis were reduced but there were no benefits in terms of heart function and cell survival. Furthermore, the above benefits of Ator+MSCs treatment could be partially blocked by L-NNA.
Atorvastatin facilitates survival of implanted MSCs, improves function and morphology of infarcted hearts, mediated by activation of eNOS and alleviated by NOS inhibitor. The data reveal the cellular and molecular mechanism for anti-AMI therapy with a combination of statin and stem cells.
Circulating endothelial progenitor cells (circEPCs) of bone marrow (BM) origin contribute to postnatal neovascularization and represent a potential therapeutic target for ischemic disease. Statins are beneficial for ischemia disease and have been implicated to increase neovascularization via mechanisms independent of lipid lowering. However, the effect of Statins on EPC function is not completely understood. Here we sought to investigate the effects of Rosuvastatin (Ros) on EPC mobilization and EPC-mediated neovascularization during ischemic injury. In a mouse model of surgically-induced hindlimb ischemia (HLI), treatment of mice with low dose (0.1 mg/kg) but not high dose (5 mg/kg) significantly increased capillary density and accelerated blood flow recovery, as compared to saline-treated group. When HLI was induced in mice that had received Tie2/LacZ BM transplantation, Ros treatment led a significantly larger amount of endothelial cells (ECs) of BM origin incorporated at ischemic sites than saline. After treatment of mice with a single low dose of Ros, circEPCs significantly increased from 2 h, peaked at 4 h, declined until 8 h. In a growth-factor reduced Matrigel plug-in assay, Ros treatment for 5 d induced endothelial lineage differentiation in vivo. Interestingly, the enhanced circEPCs and post-HLI neovascularization stimulated by Ros were blunted in mice deficient in endothelial nitric oxide synthase (eNOS), and Ros increased p-Akt/p-eNOS levels in EPCs in vitro, indicating these effects of Ros are dependent on eNOS activity. We conclude that Ros increases circEPCs and promotes their de novo differentiation through eNOS pathway.
The morphogen Sonic hedgehog (Shh) holds great promise for repair or regeneration of tissues suffering ischemic injury, however clinical translation is limited by its short half-life in the body. Here, we describe a coacervate delivery system which incorporates Shh, protects it from degradation, and sustains its release for at least 3 weeks. Shh released from the coacervate stimulates cardiac fibroblasts to upregulate the expression of multiple trophic factors including VEGF, SDF-1α, IGF-1, and Shh itself, for at least 48 hours. Shh coacervate also demonstrates cytoprotective effects for cardiomyocytes in a hydrogen peroxide-induced oxidative stress environment. In each of these studies the bioactivity of the Shh coacervate is enhanced compared to free Shh. These results warrant further investigation of the in vivo efficacy of Shh coacervate for cardiac repair.
MicroRNA-1 (miR-1) is a cardio-specific/enriched microRNA. Our recent studies have revealed that serum and urine miR-1 could be a novel sensitive biomarker for acute myocardial infarction. Open-heart surgeries with cardiopulmonary bypass (CPB) are often accompanied with surgery injury and CPB-associated injury on the hearts. However, the association of miR-1 and these intra-operative and post-operative cardiac injures is unknown. The objective of this study was to test the hypothesis that urine and serum miR-1 might be a novel biomarker for myocardial injuries in open-heart surgeries with CPB. Serum and urine miR-1 levels in 20 patients with elective mitral valve surgery were measured at pre-surgery, pre-CPB, 60 min post-CBP, and 24h post-CBP. Serum cardiac troponin-I (cTnI) was used as a positive control biomarker for cardiac injury. Compared with these in pre-operative and pre-CPB groups, the levels of miR-1 in serum and urine from patients after open-heart surgeries and CPB were significant increased at all observed time points. A similar pattern of serum cTnI levels and their strong positive correlation with miR-1 levels were identified in these patients. The results suggest that serum and urine miR-1 may be a novel sensitive biomarker for myocardial injury in open-heart surgeries with CPB.
Interactions between epithelium and mesenchyme during wound healing are not fully understood, but Fibroblast Growth Factors (FGFs) and their receptors FGFRs are recognized as key elements. FGFR2 gene encodes for two splicing transcript variants, FGFR2-IIIb or Keratinocyte Growth Factor Receptor (KGFR) and FGFR2-IIIc, which differ for tissue localization and ligand specificity. Proinflammatory cytokines play an essential role in the regulation of epithelial-mesenchymal interactions, and have been indicated to stimulate FGFs production. Here we demonstrated that upregulation of FGFR2 mRNA and protein expression is induced by the proinflammatory cytokines Tumor Necrosis Factor-α, Interleukin-1β and Interleukin 2. Furthermore, we found that TNFα determines FGFR2 transcriptional induction through activation of pRb, mediated by Raf and/or p38 pathways, and subsequent release of the transcription factor E2F1. Experiments based on FGFR2 promoter serial deletions and site-directed mutagenesis allowed us to identify a minimal responsive element that retains the capacity to be activated by E2F1. Computational analysis indicated that this element is a non-canonical E2F responsive motif. Thus far, the molecular mechanisms of FGFR2 upregulation during wound healing or in pathological events are not known. Our data suggest that FGFR2 expression can be modulated by local recruitment of inflammatory cytokines. Furthermore, since alterations in FGFR2 expression have been linked to the pathogenesis of certain human cancers, these findings could also provide elements for diagnosis and potential targets for novel therapeutic approaches.
Recent studies have demonstrated that transplantation of adipose-derived stem cell (ADSC) can improve cardiac function in animal models of myocardial infarction (MI). However, the mechanisms underlying the beneficial effect are not fully understood. In this study, we characterized the paracrine effect of transplanted ADSC and investigated its relative importance versus direct differentiation in ADSC transplantation mediated cardiac repair.
MI was experimentally induced in mice by ligation of the left anterior descending coronary artery. Either human ADSC, conditioned medium (CM) collected from the same amount of ADSC or control medium was injected into the peri-infarct region immediately after MI. Compared with the control group, both ADSC and ADSC-CM significantly reduced myocardial infarct size and improved cardiac function. The therapeutic efficacy of ADSC was moderately superior to ADSC-CM. ADSC-CM significantly reduced cardiomyocyte apoptosis in the infarct border zone, to a similar degree with ADSC treatment. ADSC enhanced angiogenesis in the infarct border zone, but to a stronger degree than that seen in the ADSC-CM treatment. ADSC was able to differentiate to endothelial cell and smooth muscle cell in post-MI heart; these ADSC-derived vascular cells amount to about 9% of the enhanced angiogenesis. No cardiomyocyte differentiated from ADSC was found.
ADSC-CM is sufficient to improve cardiac function of infarcted hearts. The therapeutic function of ADSC transplantation is mainly induced by paracrine-mediated cardioprotection and angiogenesis, while ADSC differentiation contributes a minor benefit by being involved in angiogenesis.
Highlights 1 ADSC-CM is sufficient to exert a therapeutic potential. 2. ADSC was able to differentiate to vascular cells but not cardiomyocyte. 3. ADSC derived vascular cells amount to about 9% of the enhanced angiogenesis. 4. Paracrine effect is the major mechanism of ADSC therapeutic function for MI.
Progenitor cell retention and release are largely governed by the binding of stromal-cell-derived factor 1 (SDF-1) to CXC chemokine receptor 4 (CXCR4) and by α4-integrin signaling. Both of these pathways are dependent on c-kit activity: the mobilization of progenitor cells in response to either CXCR4 antagonism or α4-integrin blockade is impaired by the loss of c-kit kinase activity; and c-kit–kinase inactivation blocks the retention of CXCR4-positive progenitor cells in the bone marrow. SDF-1/CXCR4 and α4-integrin signaling are also crucial for the retention of progenitor cells in the ischemic region, which may explain, at least in part, why clinical trials of progenitor cell therapy have failed to display the efficacy observed in preclinical investigations. The lack of effectiveness is often attributed to poor retention of the transplanted cells and, to date, most of the trial protocols have mobilized cells with injections of granulocyte colony-stimulating factor (G-CSF), which activates extracellular proteases that irreversibly cleave cell-surface adhesion molecules, including α4-integrin and CXCR4. Thus, the retention of G-CSF-mobilized cells in the ischemic region may be impaired, and the mobilization of agents that reversibly disrupt SDF-1/CXCR4 binding, such as AMD3100, may improve patient response. Efforts to supplement SDF-1 levels in the ischemic region may also improve progenitor cell recruitment and the effectiveness of stem cell therapy.
Perhaps one of the most significant achievements in modern science is the discovery of human induced pluripotent stem cells (hiPSCs), which have paved the way for regeneration therapy using patients’ own cells. Cardiomyocytes differentiated from hiPSCs (hiPSC-CMs) could be used for modelling patients with heart failure, for testing new drugs, and for cellular therapy in the future. However, the present cardiomyocyte differentiation protocols exhibit variable differentiation efficiency across different hiPSC lines, which inhibit the application of this technology significantly. Here, we demonstrate a novel myocyte differentiation protocol that can yield a significant, high percentage of cardiac myocyte differentiation (>85%) in 2 hiPSC lines, which makes the fabrication of a human cardiac muscle patch possible. The established hiPSCs cell lines being examined include the transgene integrated UCBiPS7 derived from cord blood cells and non-integrated PCBC16iPS from skin fibroblasts. The results indicate that hiPSC-CMs derived from established hiPSC lines respond to adrenergic or acetylcholine stimulation and beat regularly for greater than 60 days. This data also demonstrates that this novel differentiation protocol can efficiently generate hiPSC-CMs from iPSC lines that are derived not only from fibroblasts, but also from blood mononuclear cells.
Bone-marrow derived endothelial progenitor cells (EPCs) play an important role in tumor neovasculature. Due to their tumor homing property, EPCs are regarded as promising targeted vectors for delivering therapeutic agents in cancer treatment. Consequently, non-invasive confirmation of targeted delivery via imaging is urgently needed. This study shows the development and application of a novel dual-modality probe for in vivo non-invasively tracking of the migration, homing and differentiation of EPCs.
The paramagnetic/near-infrared fluorescence probe Conjugate 1 labeled EPCs were systemically transplanted into mice bearing human breast MDA-MB-231 tumor xenografts. Magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence optical imaging were performed at different stages of tumor development. The homing of EPCs and the tumor neovascularization were further evaluated by immunofluorescence.
Conjugate 1 labeled EPCs can be monitored in vivo by MRI and NIR fluorescence optical imaging without altering tumor growth for up to three weeks after the systemic transplantation. Histopathological examination confirmed that EPCs were recruited into the tumor bed and then incorporated into new vessels two weeks after the transplantation. Tumor size and microvessel density was not influenced by EPCs transplantation in the first three weeks.
This preclinical study shows the feasibility of using a MRI and NIR fluorescence optical imaging detectable probe to non-invasively monitor transplanted EPCs and also provides strong evidence that EPCs are involved in the development of endothelial cells during the tumor neovascularization.
Endothelial progenitor cell (EPC) survival and function in the injured myocardium is adversely influenced by hostile microenvironment like ischemia, hypoxia and inflammatory response, thereby compromising full benefits of EPC-mediated myocardial repair.
We hypothesized that interleukin-10 (IL-10) modulates EPC biology leading to enhanced survival and function following transplantation in the ischemic myocardium.
Methods and Results
Myocardial infarction (MI)-induced mobilization of bone marrow EPC (Sca-1+Flk1+ cells) into the circulation was significantly impaired in IL-10 KO-mice. Bone marrow transplantation (BMT) to replace IL-10 KO-marrow with WT-marrow attenuated these effects. Impaired mobilization was associated with lower SDF-1 expression levels in the myocardium of KO-mice. Interestingly, SDF-1 administration reversed mobilization defect in KO-mice. In vitro, hypoxia-mediated increases in CXCR4 expression and cell survival were lower in IL-10-deficient-EPCs. Furthermore, SDF-1-induced migration of WT-EPCs was inhibited by AMD3100, an inhibitor of CXCR4. To further study the effect of IL-10 on in vivo EPC survival and engraftment into vascular structures, GFP-labeled EPC were injected intramyocardially after induction of MI, and the mice were treated with either saline or recombinant IL-10. IL-10-treated group showed increased retention of transplanted EPCs in the myocardium and was associated with significantly reduced EPC apoptosis post-MI. Interestingly, increased EPC retention and their association with the vascular structures was observed in IL-10 treated mice. Increased EPC survival and angiogenesis in the myocardium of IL-10-treated mice corroborated with improved LV function, reduced infarct size and fibrosis in the myocardium. In vitro, IL-10-induced increase in VEGF expression in WT-EPC was abrogated by STAT3 inhibitor suggesting IL-10 signals via STAT3 activation.
Taken together, our studies demonstrate that MI-induced EPC mobilization was impaired in IL-10 KO-mice and that IL-10 increases EPC survival and function possibly via activation of STAT3/VEGF signaling cascades, leading to attenuation of MI-induced LV dysfunction and remodeling.
Endothelial progenitor cells; survival; myocardial infarction; IL-10; bone marrow transplantation; inflammation; angiogenesis; left ventricular remodeling
MicroRNAs (miRNAs) are encoded in the genome as individual miRNA genes or as gene clusters transcribed as polycistronic units. About 50% of all miRNAs are estimated to be co-expressed with neighboring miRNAs. Recent studies have begun to illuminate the importance of the clustering of miRNAs from an evolutionary, as well as a functional standpoint. Many miRNA clusters coordinately regulate multiple members of cellular signaling pathways or protein interaction networks. This cooperative method of targeting could produce effects on an overall process that are much more dramatic than the smaller effects often associated with regulation by an individual miRNA. In this study, we screened 366 human miRNA minigenes to determine their effects on the major signaling pathways culminating in AP-1, NF-κB, c-Myc, or p53 transcriptional activity. By stratifying these data into miRNA clusters, this systematic screen provides experimental evidence for the combined effects of clustered miRNAs on these signaling pathways. We also verify p53 as a direct target of miR-200a. This study is the first to provide a panoramic view of miRNA clusters' effects on cellular pathways.
Peripheral arterial disease (PAD) is a common disease accounting for about 12% of the adult population, and causes significant morbidity and mortality. Therapeutic angiogenesis using angiogenic factors has been considered to be a potential treatment option for PAD patients. In this study, we assessed the potential of a new angiogenic factor AGGF1 for therapeutic angiogenesis in a critical limb ischemia model in mice for PAD.
Methods and Results
We generated a unilateral hindlimb ischemia model in mice by ligation of the right common iliac artery and femoral artery. Ischemic mice with intrasmuscular administration of DNA for an expression plasmid for human AGGF1 (AGGF1 group) resulted in increased expression of both AGGF1 mRNA and protein after the administration compared with control mice with injection of the empty vector (control group). Color PW Doppler echocardiography showed that the blood flow in ischemic hindlimbs was significantly increased in the AGGF1 group compared to control mice at time points of 7, 14, and 28 days after DNA administration (n = 9/group, P = 0.049, 0.001, and 0.001, respectively). Increased blood flow in the AGGF1 group was correlated to increased density of CD31-positive vessels and decreased necrosis in muscle tissues injected with AGGF1 DNA compared with the control tissue injected with the empty vector. Ambulatory impairment was significantly reduced in the AGGF1 group compared to the control group (P = 0.004). The effect of AGGF1 was dose-dependent. At day 28 after gene transfer, AGGF1 was significantly better in increasing blood flow than FGF-2 (P = 0.034), although no difference was found for tissue necrosis and ambulatory impairment.
These data establish AGGF1 as a candidate therapeutic agent for therapeutic angiogenesis to treat PAD.
Transplantation of human CD34+ stem cells to ischemic tissues has been associated with reduced angina, improved exercise time and reduced amputation rates in phase 2 clinical trials and has been shown to induce neo-vascularization in pre-clinical models. Previous studies have suggested that paracrine factors secreted by these pro-angiogenic cells are responsible, at least in part, for the angiogenic effects induced by CD34+ cell transplantation.
Our objective was to investigate the mechanism of CD34+ stem cell induced pro-angiogenic paracrine effects and to examine if exosomes, a component of paracrine secretion, are involved.
Methods and Results
Exosomes collected from the conditioned media of mobilized human CD34+ cells had the characteristic size (40–90 nm; determined via dynamic light scattering), cup-shaped morphology (electron microscopy), expressed exosome-marker proteins CD63, phosphatidylserine (flow cytometry) and TSG101 (immunoblotting), besides expressing CD34+ cell lineage marker protein, CD34. In vitro, CD34+ exosomes replicated the angiogenic activity of CD34+ cells by increasing endothelial cell viability, proliferation and tube formation on Matrigel. In vivo, the CD34+ exosomes stimulated angiogenesis in Matrigel plug and corneal assays. Interestingly, exosomes from CD34+ cells, but not from CD34+ cell-depleted mononuclear cells had angiogenic activity.
Our data demonstrate that human CD34+ cells secrete exosomes that have independent angiogenic activity both in vitro and in vivo. CD34+ exosomes may represent a significant component of the paracrine effect of progenitor-cell transplantation for therapeutic angiogenesis.
CD34+ cells; paracrine factor; exosomes; angiogenesis
Most metazoan microRNA (miRNA) target sites have perfect pairing to the “seed” sequence, a highly conserved region centering on miRNA nucleotides 2–7. Thus, complementarity to this region is a necessary requirement for target prediction algorithms. However, also non-canonical miRNA binding can confer target regulation. Here, we identified a seedless target of miR-210, a master miRNA of the hypoxic response. We analyzed 20 genes that were inversely correlated to miR-210 expression and did not display any complementarity with miR-210 seed sequence. We validated ROD1 (Regulator of Differentiation 1, also named PTBP3, Polypyrimidine Tract Binding protein 3) as a miR-210 seedless transcript enriched in miR-210-containing RNA-induced silencing complexes. ROD1 was not indirectly targeted by a miR-210-induced miRNA. Conversely, we identified a “centered” miR-210 binding site in ROD1 involving 10 consecutive bases in the central portion of miR-210. Reporter assays showed that miR-210 inhibited ROD1 by the direct binding to this sequence, demonstrating that ROD1 is a bona fide seedless target of miR-210. As expected, both ROD1 mRNA and protein were down-modulated upon hypoxia in a miR-210 dependent manner. ROD1 targeting by miR-210 was biologically significant: the rescue of ROD1 inhibition significantly increased hypoxia-induced cell death. These data highlight the importance of ROD1 regulation by miR-210 for cell homeostasis.
Cardiac stem cells (CSCs) can home to the infarcted area and regenerate myocardium. Stromal cell-derived factor-1α/C-X-C chemokine receptor type 4 (SDF-1α/CXCR4) axis is pivotal in inducing CSCs migration. However, the mechanisms remain unclear. This study set out to detect if SDF-1α promotes migration and engraftment of CSCs through the CXCR4/PI3K (phosphatidylinositol 3-kinase) pathway. In the in vitro experiment, c-kit+ cells were isolated from neonatal mouse heart fragment culture by magnetic cell sorting. Fluorescence-activated cell sorting results demonstrated that a few c-kit+ cells expressed CD45 (4.54%) and Sca-1 (2.58%), the hematopoietic stem cell marker. Conditioned culture could induce c-kit+ cells multipotent differentiation, which was confirmed by cardiac troponin I (cTn-I), α-smooth muscle actin (α-SMA), and von Willebrand factor (vWF) staining. In vitro chemotaxis assays were performed using Transwell cell chambers to detect CSCs migration. The results showed that the cardiomyocytes infected with rAAV1-SDF-1α-eGFP significantly increased SDF-1α concentration, 5-fold more in supernatant than that in the control group, and subsequently attracted more CSCs migration. This effect was diminished by administration of AMD3100 (10 µg/ml, CXCR4 antagonist) or LY294002 (20 µmol/L, PI3K inhibitor). In myocardial infarction mice, overexpression of SDF-1α in the infarcted area by rAAV1-SDF-1α-eGFP infection resulted in more CSCs retention to the infarcted myocardium, a higher percentage of proliferation, and reduced infarcted area which was attenuated by AMD3100 or ly294002 pretreatment. These results indicated that overexpression of SDF-1α enhanced CSCs migration in vitro and engraftment of transplanted CSCs and reduced infarcted size via CXCR4/PI3K pathway.
The antagonism of CXC-chemokine receptor 4 (CXCR4) with AMD3100 improves cardiac performance after myocardial infarction by augmenting the recruitment of endothelial progenitor cells (EPCs) from the bone marrow to the regenerating vasculature. We investigated whether AMD3100 may accelerate diabetes-impaired wound healing through a similar mechanism. Skin wounds were made on the backs of leptin-receptor–deficient mice and treated with AMD3100 or saline. Fourteen days after treatment, wound closure was significantly more complete in AMD3100-treated mice (AMD3100: 87.0±2.6%, Saline: 33.1±1.8%; P<0.0001) and was accompanied by greater collagen-fiber formation, capillary density, smooth-muscle-containing vessel density, and monocyte/macrophage infiltration. On day 7 after treatment, AMD3100 was associated with higher circulating EPC and macrophage counts and with significantly upregulated mRNA levels of stromal-cell–derived factor 1 and platelet-derived growth-factor B in the wound bed. AMD3100 also promoted macrophage proliferation and phagocytosis and the migration and proliferation of diabetic mouse primary dermal fibroblasts and 3T3 fibroblasts, which express very little CXCR4. In conclusion, a single topical application of AMD3100 promoted wound healing in diabetic mice by increasing cytokine production, mobilizing bone-marrow EPCs, and enhancing the activity of fibroblasts and monocytes/macrophages, thereby increasing both angiogenesis and vasculogenesis. Not all of the AMD3100-mediated effects evolved through CXCR4 antagonism.
Diabetes; Angiogenesis; Wound healing