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author:("Qin, gangjin")
1.  Infrared Fluorescent Protein 1.4 Genetic Labeling Tracks Engrafted Cardiac Progenitor Cells in Mouse Ischemic Hearts 
PLoS ONE  2014;9(10):e107841.
Stem cell therapy has a potential for regenerating damaged myocardium. However, a key obstacle to cell therapy’s success is the loss of engrafted cells due to apoptosis or necrosis in the ischemic myocardium. While many strategies have been developed to improve engrafted cell survival, tools to evaluate cell efficacy within the body are limited. Traditional genetic labeling tools, such as GFP-like fluorescent proteins (eGFP, DsRed, mCherry), have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent these limitations, a near-infrared fluorescent mutant of the DrBphP bacteriophytochrome from Deinococcus radiodurans, IFP1.4, was developed for in vivo imaging, but it has yet to be used for in vivo stem/progenitor cell tracking. In this study, we incorporated IFP1.4 into mouse cardiac progenitor cells (CPCs) by a lentiviral vector. Live IFP1.4-labeled CPCs were imaged by their near-infrared fluorescence (NIRF) using an Odyssey scanner following overnight incubation with biliverdin. A significant linear correlation was observed between the amount of cells and NIRF signal intensity in in vitro studies. Lentiviral mediated IFP1.4 gene labeling is stable, and does not impact the apoptosis and cardiac differentiation of CPC. To assess efficacy of our model for engrafted cells in vivo, IFP1.4-labeled CPCs were intramyocardially injected into infarcted hearts. NIRF signals were collected at 1-day, 7-days, and 14-days post-injection using the Kodak in vivo multispectral imaging system. Strong NIRF signals from engrafted cells were imaged 1 day after injection. At 1 week after injection, 70% of the NIRF signal was lost when compared to the intensity of the day 1 signal. The data collected 2 weeks following transplantation showed an 88% decrease when compared to day 1. Our studies have shown that IFP1.4 gene labeling can be used to track the viability of transplanted cells in vivo.
doi:10.1371/journal.pone.0107841
PMCID: PMC4214633  PMID: 25357000
2.  Assessing in vitro stem-cell function and tracking engraftment of stem cells in ischaemic hearts by using novel iRFP gene labelling 
Near-infrared fluorescence (NIRF) imaging by using infrared fluorescent protein (iRFP) gene labelling is a novel technology with potential value for in vivo applications. In this study, we expressed iRFP in mouse cardiac progenitor cells (CPC) by lentiviral vector and demonstrated that the iRFP-labelled CPC (CPCiRFP) can be detected by flow cytometry and fluorescent microscopy. We observed a linear correlation in vitro between cell numbers and infrared signal intensity by using the multiSpectral imaging system. CPCiRFP injected into the non-ischaemic mouse hindlimb were also readily detected by whole-animal NIRF imaging. We then compared iRFP against green fluorescent protein (GFP) for tracking survival of engrafted CPC in mouse ischaemic heart tissue. GFP-labelled CPC (CPCGFP) or CPC labelled with both iRFP and GFP (CPCiRFP GFP) were injected intramyocardially into mouse hearts after infarction. Three days after cell transplantation, a strong NIRF signal was detected in hearts into which CPCiRFP GFP, but not CPCGFP, were transplanted. Furthermore, iRFP fluorescence from engrafted CPCiRFP GFP was detected in tissue sections by confocal microscopy. In conclusion, the iRFP-labelling system provides a valuable molecular imaging tool to track the fate of transplanted progenitor cells in vivo.
doi:10.1111/jcmm.12321
PMCID: PMC4162818  PMID: 24912616
stem cells; myocardial infarction; cell transplantation; iRFP; GFP
3.  miR-92a inhibits vascular smooth muscle cell apoptosis: role of the MKK4–JNK pathway 
Vascular smooth muscle cell (VSMC) apoptosis plays an important role in vascular remodeling and atherosclerotic plaque instability. Oxidative stress in diseased vessels promotes VSMC apoptosis in part by activating the c-Jun N-terminal kinase (JNK) pathway, which has been identified as a molecular target of miR-92a in macrophages. Here, we examined the expression and biological activity of miR-92a in VSMC. Quiescent VSMC exhibited a low basal expression of miR-92a, which was positively regulated by serum stimulation and negatively regulated by H2O2. Overexpression of miR-92a decreased H2O2-induced VSMC apoptosis as indicated by TUNEL assay and cleaved caspase-3 protein levels. Using 3′UTRreporter assay, we found that miR-92a overexpression led to suppression of both mitogen-activated protein kinase kinase 4 (MKK4)- and JNK1-dependent luciferase activity. We also found that 10 mer seed match between miRNA: mRNA pair is more efficient than 8 mer seed match for us to identify authentic miRNA target. Protein levels of active phospho-JNK and phospho-c-Jun, downstream targets of the MKK4–JNK1 pathway, were also decreased by overexpressing miR-92a in VSMC under oxidative stress. Consistent with these findings, overexpression of MKK4 reversed the anti-apoptotic effects of miR-92a in oxidatively stressed VSMC. In conclusion, miR-92a overexpression inhibits H2O2-induced VSMC apoptosis by directly targeting the MKK4–JNK1 pathway.
doi:10.1007/s10495-014-0987-y
PMCID: PMC4143895  PMID: 24705900
Vascular smooth muscle cells; miR-92a; JNK; Apoptosis; Oxidative stress
4.  Non-Selective Cation Channels Mediate Chloroquine-Induced Relaxation in Precontracted Mouse Airway Smooth Muscle 
PLoS ONE  2014;9(7):e101578.
Bitter tastants can induce relaxation in precontracted airway smooth muscle by activating big-conductance potassium channels (BKs) or by inactivating voltage-dependent L-type Ca2+ channels (VDLCCs). In this study, a new pathway for bitter tastant-induced relaxation was defined and investigated. We found nifedipine-insensitive and bitter tastant chloroquine-sensitive relaxation in epithelium-denuded mouse tracheal rings (TRs) precontracted with acetylcholine (ACH). In the presence of nifedipine (10 µM), ACH induced cytosolic Ca2+ elevation and cell shortening in single airway smooth muscle cells (ASMCs), and these changes were inhibited by chloroquine. In TRs, ACH triggered a transient contraction under Ca2+-free conditions, and, following a restoration of Ca2+, a strong contraction occurred, which was inhibited by chloroquine. Moreover, the ACH-activated whole-cell and single channel currents of non-selective cation channels (NSCCs) were blocked by chloroquine. Pyrazole 3 (Pyr3), an inhibitor of transient receptor potential C3 (TRPC3) channels, partially inhibited ACH-induced contraction, intracellular Ca2+ elevation, and NSCC currents. These results demonstrate that NSCCs play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.
doi:10.1371/journal.pone.0101578
PMCID: PMC4081631  PMID: 24992312
5.  Contrasting roles of E2F2 and E2F3 in endothelial cell growth and ischemic angiogenesis 
The growth of new blood vessels after ischemic injury requires endothelial cells (ECs) to divide and proliferate, and the E2F transcription factors are key regulators of the genes responsible for cell-cycle progression; however, the specific roles of individual E2Fs in ECs are largely unknown. To determine the roles of E2F2 and E2F3 in EC proliferation and the angiogenic response to ischemic injury, hind-limb ischemia was surgically induced in E2F2−/− mice, endothelial-specific E2F3-knockout (EndoE2F3Δ/Δ) mice, and their littermates with wild-type E2F2 and E2F3 expression. Two weeks later, laser-Doppler perfusion measurements, capillary density, and endothelial proliferation were significantly greater in E2F2−/− mice and significantly lower in EndoE2F3Δ/Δ mice than in their littermates, and EndoE2F3Δ/Δ mice also developed toe and limb necrosis. The loss of E2F2 expression was associated with increases in the proliferation and G1/S-phase gene expression of isolated ECs, while the loss of E2F3 expression led to declines in these parameters. Thus E2F2 impairs, and endothelial E2F3 promotes, the angiogenic response to peripheral ischemic injury through corresponding changes in EC cell-cycle progression.
doi:10.1016/j.yjmcc.2013.04.009
PMCID: PMC3684263  PMID: 23603666
E2F; Endothelial cells; Proliferation; Angiogenesis; Ischemia
6.  BREAKING THE “HARMONY” OF TNF-α SIGNALING FOR CANCER TREATMENT 
Oncogene  2011;31(37):4117-4127.
Tumor necrosis factor-alpha (TNF-α) binds to two distinct receptors, TNFR1/p55 and TNFR2/p75. TNF-α is implicated in the processes of tumor growth, survival, differentiation, invasion, metastases, secretion of cytokines and pro-angiogenic factors. We have shown that TNFR2/p75 signaling promotes ischemia-induced angiogenesis via modulation of several angiogenic growth factors. We hypothesized that TNFR2/p75 may promote tumor growth and angiogenesis. Growth of mouse Lewis lung carcinoma (LLC1) and/or mouse melanoma B16 cell was evaluated in wild type (WT), p75 knockout (KO) and double p55KO/p75KO mouse tumor xenograft models. Compared to WT and p55KO/p75KO mice, growth of tumors in p75KO mice was significantly decreased (two-fold) in both LLC and B16 tumors. Tumor growth inhibition was correlated with decreases in VEGF expression and capillary density, as well as bone marrow (BM)-derived endothelial progenitor cells (EPCs) incorporation into the functional capillary network, and an increase in apoptotic cells in LLC xenografts. Gene array analysis of tumor tissues showed a decrease in gene expression in pathways that promote tumor angiogenesis and cell survival. Blocking p75 by shRNA in cultured LLCs led to increases in TNF-mediated apoptosis, as well as decreases in the constitutive and TNF-mediated expression of angiogenic growth factors (VEGF, HGF, PLGF), and SDF-1α receptor CXCR4. In summary, p75 is essential for tumor angiogenesis and survival in highly vascularized murine lung tumor xenografts. Blocking p75 expression may lead to tumor regression. This may represent new and effective therapy against lung neoplasms and potentially tumors of other origin.
doi:10.1038/onc.2011.567
PMCID: PMC3962797  PMID: 22158049
TNF-α; TNFR2/p75; cancer; angiogenesis; EPC; microenvironment
7.  Cardiac progenitor-derived Exosomes protect ischemic myocardium from acute ischemia/reperfusion injury 
Background
Cardiac progenitors (CPC) mediate cardioprotection via paracrine effects. To date, most of studies focused on secreted paracrine proteins. Here we investigated the CPC-derived-exosomes on protecting myocardium from acute ischemia/reperfusion (MI/R) injury.
Methods and Results
CPC were isolated from mouse heart using two-step protocol. Exosomes were purified from conditional medium, and confirmed by electron micrograph and Western blot using CD63 as a marker. qRT-PCR shows that CPC- exosomes have high level expression of GATA4-responsive-miR-451. Exosomes were ex vivo labeled with PKH26, We observed exosomes can be uptaken by H9C2 cardiomyoblasts with high efficiency after 12 hours incubation. CPC-exosomes protect H9C2 from oxidative stress by inhibiting caspase 3/7 activation in vitro. In vivo delivery of CPC-exosomes in an acute mouse myocardial ischemia/reperfusion model inhibited cardiomyocyte apoptosis by about 53% in comparison with PBS control(p<0.05).
Conclusion
Our results suggest, for the first time, the CPC-exosomes can be used as a therapeutic vehicle for cardioprotection, and highlights a new perspective for using non-cell exosomes for cardiac disease.
doi:10.1016/j.bbrc.2013.01.015
PMCID: PMC3732190  PMID: 23318173
cardiac progenitors; MicroRNA; Exosomes; ischemia/reperfusion; apoptosis
8.  CXCR4 Antagonist AMD3100 Promotes Cardiac Functional Recovery After Ischemia-Reperfusion Injury via eNOS-Dependent Mechanism 
Circulation  2012;127(1):63-73.
Background
CXC-chemokine receptor 4 (CXCR4) regulates the retention of stem/progenitor cells in the bone marrow (BM), and the CXCR4 antagonist AMD3100 improves recovery from coronary-ligation injury by mobilizing stem/progenitor cells from the BM to the peripheral blood. Thus, we investigated whether AMD3100 also improves recovery from ischemia-reperfusion (IR) injury, which more closely mimics myocardial infarction in patients, because blood flow is only temporarily obstructed.
Methods and Results
Mice were treated with single subcutaneous injections of AMD3100 (5 mg/kg) or saline after IR injury. Three days later, histological measurements of the infarct-area/area-at-risk ratio were smaller in AMD3100-treated mice than in mice administered saline, and echocardiographic measurements of left-ventricular function were greater in the AMD3100-treated mice at week 4. CXCR4+ cells were mobilized for just 1 day in both groups, but the mobilization of sca1+/flk1+ cells endured for 7days in AMD3100-treated mice compared to just 1 day in the saline-treated mice. AMD3100 upregulated BM levels of endothelial nitric oxide synthase (eNOS) and two targets of eNOS signaling, matrix-metalloproteinase 9 and soluble Kit ligand. Furthermore, the loss of BM eNOS expression abolished the benefit of AMD3100 on sca1+/flk1+ cell mobilization without altering the mobilization of CXCR4+ cells, and the cardioprotective effects of AMD3100 were retained in eNOS-knockout mice that had been transplanted with BM from wild-type mice, but not in wild-type mice with eNOS-knockout BM.
Conclusions
AMD3100 prolongs BM progenitor mobilization and improves recovery from IR injury, and these benefits appear to occur through a previously unidentified link between AMD3100 and BM eNOS expression.
doi:10.1161/CIRCULATIONAHA.112.099242
PMCID: PMC3560409  PMID: 23204107
Drugs; Myocardium; Ischemia; Reperfusion; Nitric oxide synthase
9.  IL 10 Treatment Attenuates Pressure Overload-Induced Hypertrophic Remodeling and Improves Heart Function via STAT3 Dependent Inhibition of NFκB 
Circulation  2012;126(4):418-429.
Background
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.
Conclusions
Taken together our studies suggest IL10 treatment as a potential therapeutic approach to limit the progression of pressure overload-induced adverse cardiac remodeling.
doi:10.1161/CIRCULATIONAHA.112.112185
PMCID: PMC3422741  PMID: 22705886
heart failure; hypertrophy; interleukins; myocardium; signal transduction
10.  Enhanced Angiogenic and Cardiomyocyte Differentiation Capacity of Epigenetically Reprogrammed Mouse and Human Endothelial Progenitor Cells Augments their Efficacy for Ischemic Myocardial Repair 
Circulation Research  2012;111(2):180-190.
Rationale
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.
Objective
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.
Conclusions
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.
doi:10.1161/CIRCRESAHA.112.270462
PMCID: PMC3406600  PMID: 22589372
Epigenetic modification; EPC; cardiomyogenesis; myocardial ischemia; cell therapy; histone acetylation; trans-differentiation
11.  Contrasting Roles of E2F2 and E2F3 in Cardiac Neovascularization 
PLoS ONE  2013;8(6):e65755.
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.
doi:10.1371/journal.pone.0065755
PMCID: PMC3683051  PMID: 23799044
12.  Rosuvastatin Enhances Angiogenesis via eNOS-Dependent Mobilization of Endothelial Progenitor Cells 
PLoS ONE  2013;8(5):e63126.
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.
doi:10.1371/journal.pone.0063126
PMCID: PMC3660394  PMID: 23704894
13.  Progenitor Cell Mobilization and Recruitment: SDF-1, CXCR4, α4-integrin, and c-kit 
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.
doi:10.1016/B978-0-12-398459-3.00011-3
PMCID: PMC3556394  PMID: 22917234
14.  IL-10 deficiency impairs bone marrow-derived endothelial progenitor cell (EPC) survival and function in ischemic myocardium 
Circulation research  2011;109(11):1280-1289.
Rationale
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.
Objective
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.
Conclusions
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.
doi:10.1161/CIRCRESAHA.111.248369
PMCID: PMC3235675  PMID: 21959218
Endothelial progenitor cells; survival; myocardial infarction; IL-10; bone marrow transplantation; inflammation; angiogenesis; left ventricular remodeling
15.  Angiogenic Factor AGGF1 Promotes Therapeutic Angiogenesis in a Mouse Limb Ischemia Model 
PLoS ONE  2012;7(10):e46998.
Background
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.
Conclusions
These data establish AGGF1 as a candidate therapeutic agent for therapeutic angiogenesis to treat PAD.
doi:10.1371/journal.pone.0046998
PMCID: PMC3479102  PMID: 23110058
16.  Exosomes from Human CD34+ Stem Cells Mediate their Pro-angiogenic Paracrine Activity 
Circulation research  2011;109(7):724-728.
Rational
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.
Objective
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.
Conclusions
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.
doi:10.1161/CIRCRESAHA.111.253286
PMCID: PMC3201702  PMID: 21835908
CD34+ cells; paracrine factor; exosomes; angiogenesis
17.  CXCR4 Antagonist AMD3100 Accelerates Impaired Wound Healing in Diabetic Mice 
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.
doi:10.1038/jid.2011.356
PMCID: PMC3276738  PMID: 22048734
Diabetes; Angiogenesis; Wound healing
18.  Adipocyte dysfunction and hypertension 
Obesity is increasingly a public health problem due to its high risk of developing insulin resistance, diabetes, atherosclerosis, hypertension, chronic kidney disease, and increased cardiovascular morbidity and mortality. In particular, the association of obesity and hypertension is well recognized; however, the underlying mechanisms are not fully understood. This article reviews recent advancements of cellular and molecular mechanisms by which adipocyte dysfunction and obesity contribute to hypertension through endocrine and paracrine effects of the adipose tissue-derived adipokines on the function of vascular endothelial cells, smooth muscle cells and macrophages.
PMCID: PMC3371623  PMID: 22720204
Obesity; adipocyte; adipokine; vascular function; hypertension
19.  The Role of Notch 1 Activation in Cardiosphere Derived Cell Differentiation 
Stem Cells and Development  2012;21(12):2122-2129.
Cardiosphere derived cells (CDC) are present in the human heart and include heterogeneous cell populations of cardiac progenitor cells, multipotent progenitors that play critical roles in the physiological and pathological turnover of heart tissue. Little is known about the molecular pathways that control the differentiation of CDC. In this study, we examined the role of Notch 1/J kappa-recombining binding protein (RBPJ) signaling, a critical cell-fate decision pathway, in CDC differentiation. We isolated CDC from mouse cardiospheres and analyzed the differentiation of transduced cells expressing the Notch1 intracellular domain (N1-ICD), the active form of Notch1, using a terminal differentiation marker polymerase chain reaction (PCR) array. We found that Notch1 primarily supported the differentiation of CDC into smooth muscle cells (SMC), as demonstrated by the upreguation of key SMC proteins, including smooth muscle myosin heavy chain (Myh11) and SM22α (Tagln), in N1-ICD expressing CDC. Conversely, genetic ablation of RBPJ in CDC diminished the expression of SMC differentiation markers, confirming that SMC differentiation CDC is dependent on RBPJ. Finally, in vivo experiments demonstrate enhanced numbers of smooth muscle actin-expressing implanted cells after an injection of N1-ICD-expressing CDC into ischemic myocardium (44±8/high power field (hpf) vs. 11±4/high power field (hpf), n=7 sections, P<0.05). Taken together, these results provide strong evidence that Notch1 promotes SMC differentiation of CDC through an RBPJ-dependent signaling pathway in vitro, which may have important implications for progenitor cell-mediated angiogenesis.
doi:10.1089/scd.2011.0463
PMCID: PMC3413090  PMID: 22239539
20.  E2F and microRNA regulation of angiogenesis 
E2F family of transcription factors are best known for regulating genes involved in cell cycle control, cell proliferation, tumorigenesis, and apoptosis. Recent evidences have revealed their critical involvement in modulating cellular response to hypoxia and ischemia in a variety of physiological and pathological processes. Of particular interest are findings that E2Fs act as both regulators and targets of microRNAs that govern hypoxic/ischemic angiogenesis. This review focuses on the crosstalk between E2Fs and microRNAs that have been shown to participate in the regulation of angiogenesis, hypoxia response and ischemic disease.
PMCID: PMC3244022  PMID: 22200034
E2F; microRNA; angiogenesis; ischemia; hypoxia
21.  CXCR4 mediated bone marrow progenitor cell maintenance and mobilization are modulated by c-kit activity 
Circulation research  2010;107(9):1083-1093.
Rationale
The mobilization of bone-marrow (BM) progenitor cells (PCs) is largely governed by interactions between stromal–cell derived factor 1 (SDF-1) and CXC-chemokine receptor 4 (CXCR4). Ischemic injury disrupts the SDF-1–CXCR4 interaction and releases BM PCs into the peripheral circulation, where the mobilized cells are recruited to the injured tissue and contribute to vessel growth. BM PCs can also be mobilized by the pharmacological CXCR4 antagonist AMD3100, but the other components of the SDF-1–CXCR4 signaling pathway are largely unknown. c-kit, a membrane bound tyrosine-kinase and the receptor for stem cell factor, has also been shown to play a critical role in BM PC mobilization and ischemic tissue repair.
Objective
To investigate the functional interaction between SDF-1–CXCR4 signaling and c-kit activity in BM PC mobilization.
Methods and Results
AMD3100 administration failed to mobilize BM PCs in mice defective in c-kit kinase activity or in mice transplanted with BM cells that expressed a constitutively active c-kit mutant. Furthermore, BM levels of phosphorylated c-kit (phospho–c-kit) declined after AMD3100 administration and after CXCR4 deletion. In cells adhering to culture plates coated with vascular cell adhesion molecule 1 (VCAM-1), SDF-1 and SCF increased phospho–c-kit levels, and AMD3100 treatment suppressed SDF-1–induced, but not SCF-induced, c-kit phosphorylation. SDF-1–induced c-kit phosphorylation also required the activation of Src non-receptor tyrosine kinase: pre-treatment of cells with a selective Src inhibitor blocked both c-kit phosphorylation and the interaction between c-kit and phosphorylated Src.
Conclusions
These findings indicate that the regulation of BM PC trafficking by SDF-1 and CXCR4 is dependent on Src-mediated c-kit phosphorylation.
doi:10.1161/CIRCRESAHA.110.220970
PMCID: PMC2966940  PMID: 20847314
CXCR4; c-kit; Integrin; Stem cells; Bone marrow; Niche; Mobilization; Homing
22.  Sonic Hedgehog Induces Angiogenesis via Rho Kinase-dependent Signaling in Endothelial Cells 
The morphogen Sonic Hedgehog (Shh) promotes neovascularization in adults by inducing pro-angiogenic cytokine expression in fibroblasts; however, the direct effects of Shh on endothelial cell (EC) function during angiogenesis are unknown. Our findings indicate that Shh promotes capillary morphogenesis (tube length on Matrigel™ increased to 271±50% of the length in untreated cells, p=0.00003), induces EC migration (modified Boyden chamber assay, 191±35% of migration in untreated cells, p=0.00009), and increases EC expression of matrix metalloproteinase 9 (MMP-9) and osteopontin (OPN) mRNA (real-time RT-PCR), which are essential for Shh-induced angiogenesis both in vitro and in vivo. Shh activity in ECs is mediated by Rho, rather than through the “classic” Shh signaling pathway, which involves the Gli transcription factors. The Rho dependence of Shh-induced EC angiogenic activity was documented both in vitro, with dominant-negative RhoA and Rho kinase (ROCK) constructs, and in vivo, with the ROCK inhibitor Y27632 in the mouse corneal angiogenesis model. Finally, experiments performed in MMP-9- and OPN-knockout mice confirmed the roles of the ROCK downstream targets MMP-9 and OPN in Shh-induced angiogenesis. Collectively, our results identify a “non-classical” pathway by which Shh directly modulates EC phenotype and angiogenic activity.
doi:10.1016/j.yjmcc.2010.05.003
PMCID: PMC2917529  PMID: 20478312
angiogenesis; Sonic Hedgehog; endothelial cells; Gli transcription factors; ischemia
23.  E2F and microRNA regulation of angiogenesis 
E2F family of transcription factors are best known for regulating genes involved in cell cycle control, cell proliferation, tumorigenesis, and apoptosis. Recent evidences have revealed their critical involvement in modulating cellular response to hypoxia and ischemia in a variety of physiological and pathological processes. Of particular interest are findings that E2Fs act as both regulators and targets of microRNAs that govern hypoxic/ischemic angiogenesis. This review focuses on the crosstalk between E2Fs and microRNAs that have been shown to participate in the regulation of angiogenesis, hypoxia response and ischemic disease.
PMCID: PMC3244022  PMID: 22200034
E2F; microRNA; angiogenesis; ischemia; hypoxia
24.  Regulation of Vascular Contractility and Blood Pressure by the E2F2 Transcription Factor 
Circulation  2009;120(13):1213-1221.
Background
Recent studies have identified a polymorphism in the ECE-1b promoter (−338C/A) that is strongly associated with hypertension in women, and the polymorphism is located in a consensus binding sequence for the E2F family of transcription factors. E2F proteins are crucially involved in cell-cycle regulation, but their roles in cardiovascular function are poorly understood. Here, we investigated the potential role of E2F2 in blood pressure (BP) regulation.
Methods and Results
Tail-cuff measurements of systolic and diastolic BP were significantly higher in E2F2-null (E2F2−/−) mice than in their wild-type (WT) littermates, and in ex vivo ring assays, aortas from the E2F2−/− mice exhibited significantly greater contractility in response to big endothelin-1 (BigET-1). BigET-1 is activated by endothelin converting enzyme-1 (ECE-1), and mRNA levels of ECE-1b, the repressive ECE-1 isoform, were significantly lower in E2F2−/− mice than in WT mice. In endothelial cells, chromatin-immunoprecipitation (ChIP) assays confirmed that E2F2 binds the ECE-1b promoter, and promoter-reporter assays indicated that E2F2 activates ECE-1b transcription. Furthermore, loss or downregulation of E2F2 led to a decline in ECE-1b levels, to higher levels of the membranous ECE-1 isoforms (i.e., ECE-1a, -1c, and -1d), and to deregulated ECE-1 activity. Lastly, Sam68 co-immunopreciptated with E2F2, occupied the ECE-1b promoter (ChIP), and repressed E2F2-mediated ECE-1b promoter activity (promoter-reporter assays).
Conclusions
Our results identify a cell cycle-independent mechanism by which E2F2 regulates endothelial function, arterial contractility, and BP.
doi:10.1161/CIRCULATIONAHA.109.859207
PMCID: PMC2785027  PMID: 19752322
E2F; Sam68; Endothelium; Endothelin; Blood pressure
25.  Inhibition of Melanoma Angiogenesis by Telomere Homolog Oligonucleotides 
Journal of Oncology  2010;2010:928628.
Telomere homolog oligonucleotides (T-oligos) activate an innate telomere-based program that leads to multiple anticancer effects. T-oligos act at telomeres to initiate signaling through the Werner protein and ATM kinase. We wanted to determine if T-oligos have antiangiogenic effects. We found that T-oligo-treated human melanoma (MM-AN) cells had decreased expression of vascular endothelial growth factor (VEGF), VEGF receptor 2, angiopoeitin-1 and -2 and decreased VEGF secretion. T-oligos activated the transcription factor E2F1 and inhibited the activity of the angiogenic transcription factor, HIF-1α. T-oligos inhibited EC tubulogenesis and total tumor microvascular density matrix invasion by MM-AN cells and ECs in vitro. In melanoma SCID xenografts, two systemic T-oligo injections decreased by 60% (P < .004) total tumor microvascular density and the functional vessels density by 80% (P < .002). These findings suggest that restriction of tumor angiogenesis is among the host's innate telomere-based anticancer responses and provide further evidence that T-oligos may offer a powerful new approach for melanoma treatment.
doi:10.1155/2010/928628
PMCID: PMC2906154  PMID: 20652008

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