The E2F transcription factors are best characterized for their roles in cell-cycle regulation, cell growth, and cell death. Here we investigated the potential role of E2F1 in cardiac neovascularization.
We have demonstrated that GLP-1 improved myocardial functional recovery in acute myocardial ischemic injury. However, whether stimulation of the GLP-1 receptor (GLP-1R) with exendin-4, a selective GLP-1R agonist, could initiate a protective effect in the heart remains to be determined. Mouse myocardial infarction (MI) was created by ligation of the left descending artery. After 48 h of MI, animals were divided into the following groups (n = 5-7/group): 1) sham (animals that underwent thoracotomy without ligation), 2) MI [animals that underwent MI and received a daily dose of intraperitoneal injection (ip) of saline]; and 3) MI + exendin-4 [infarcted mice that received injections of exendin-4 (0.1 mg/kg ip)]. Two weeks later, cardiac function was assessed by echocardiography and an isovolumetrically perfused heart. Compared with control MI hearts, stimulation of GLP-1R improved cardiac function, which was associated with attenuation of myocardial hypertrophy, the mitigation of interstitial fibrosis, and an increase in survival rate in post-MI hearts. Furthermore, H9c2 cardiomyoblasts were preconditioned with exendin-4 at a dose of 100 nmol/l and then subjected to hydrogen peroxide exposure at concentrations of 50 and 100 ?mol/l. The exendin-4 treatment decreased lactate dehydrogenase leakage and increased cell survival. Notably, this event was also associated with the reduction of cleaved caspase-3 and caspase-9 and attenuation of reactive oxygen species production. Exendin-4 treatments improved mitochondrial respiration and suppressed the opening of mitochondrial permeability transition pore and protected mitochondria function. Our results indicate that GLP-1R serves as a novel approach to eliciting cardioprotection and mitigating oxidative stress-induced injury.
We have recently shown that in vivo inhibition of histone deacetylase (HDAC) stimulates endogenous myocardial regeneration in infarcted hearts (Zhang L et al. J Pharmacol Exp Ther 341: 285-293, 2012). Furthermore, our observation demonstrates that HDAC inhibition promotes cardiogenesis, which is associated with HDAC4 reduction. However, it remains unknown as to whether specific inhibition of HDAC4 modulates cardiac stem cells (CSCs) to facilitate myocardial repair and to preserve cardiac performance. c-kit(+) CSCs were isolated from adult mouse hearts and were transfected with HDAC4 siRNA to knockdown HDAC4 of c-kit(+) CSCs. The transfection of HDAC4 siRNA caused a marked reduction of HDAC4 mRNA and proteins in c-kit(+) CSCs. Mouse myocardial infarction (MI) was created to assess the effect of HDAC4 inhibition in c-kit(+) CSCs on myocardial regeneration in vivo when cells were introduced into MI hearts. Transplantation of HDAC4 siRNA-treated c-kit(+) CSCs into MI hearts improved ventricular function, attenuated ventricular remodeling, and promoted CSC-derived regeneration and neovascularization. Furthermore, Ki67 and BrdU positively proliferative myocytes increased in MI hearts receiving HDAC4 siRNA-treated c-kit(+) CSCs compared with MI hearts engrafted with control siRNA-treated c-kit(+) CSCs. In addition, compared with MI hearts engrafted with control adenoviral GFP-infected c-kit(+) CSCs, MI hearts receiving adenoviral HDAC4-infected c-kit(+) CSCs exhibited attenuated cardiac functional recovery, CSC-derived regeneration, and neovascularization, which was accompanied with adverse ventricular remodeling and decrease in Ki67 and BrdU positively proliferative myocytes. HDAC4 inhibition facilitated c-kit(+) CSCs into the differentiation into cardiac lineage commitments in vitro, while HDAC4 overexpression attenuated c-kit(+) CSC-derived cardiogenesis. Our results indicate that HDAC4 inhibition promotes CSC-derived cardiac regeneration and improves the restoration of cardiac function.
The translation of cell-based therapies for ischemic tissue repair remains limited by several factors, including poor cell survival and limited target site retention. Advances in nanotechnology enable the development of specifically designed delivery matrices to address these limitations and thereby improve the efficacy of cell-based therapies. Given the relevance of integrin signaling for cellular homeostasis, we developed an injectable, bioactive peptide-based nanofiber matrix that presents an integrin-binding epitope derived from fibronectin, and evaluated its feasibility as a supportive artificial matrix for bone marrow-derived pro-angiogenic cells (BMPACs) used as a therapy in ischemic tissue repair. Incubation of BMPACs with these peptide nanofibers in vitro significantly attenuated apoptosis while enhancing proliferation and adhesion. Pro-angiogenic function was enhanced, as cells readily formed tubes. These effects were, in part, mediated via p38, and p44/p42 MAP kinases, which are downstream pathways of focal adhesion kinase. In a murine model of hind limb ischemia, an intramuscular injection of BMPACs within this bioactive peptide nanofiber matrix resulted in greater retention of cells, enhanced capillary density, increased limb perfusion, reduced necrosis/amputation, and preserved function of the ischemic limb compared to treatment with cells alone. This self-assembling, bioactive peptide nanofiber matrix presenting an integrin-binding domain of fibronectin improves regenerative efficacy of cell-based strategies in ischemic tissue by enhancing cell survival, retention, and reparative functions.
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'UTR-reporter 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.
Oxidative stress contributes to endothelial cell (EC) dysfunction, which is prevalent in ageing and atherosclerosis. MicroRNAs (miRs) are small, non-coding RNAs that post-transcriptionally regulate gene expression and play a key role in fine-tuning EC functional responses, including apoptosis and angiogenesis. MiR-92a is highly expressed in young endothelial cells in comparison with senescent endothelial cells, which exhibit increased oxidative stress and apoptosis. However, the impact of miR-92a treatment on EC viability and angiogenesis under oxidative stress is unknown. Hydrogen peroxide (H2O2) was used to induce oxidative stress in human umbilical vein endothelial cells (HUVEC). Pre-miR-92a treatment decreased H2O2-induced apoptosis of HUVEC as determined by TUNEL assay. Pre-miR-92a treatment enhanced capillary tube formation by HUVEC under oxidative stress, which was blocked by LY294002, an inhibitor of Akt phosphorylation. Interestingly, we also observed that inhibition of miR-92a by anti-miR-92a antisense can also enhance angiogenesis in HUVEC with and without oxidative stress exposure. Our results show that perturbation of miR-92a levels outside of its narrow "homeostatic" range may trigger endothelial cell angiogenesis, suggesting that the role of miR-92a in regulating angiogenesis is controversial and may vary depending on the experimental model and method of regulating miR-92a.
The acetylation and deacetylation of histones plays an important role in the regulation of gene transcriptions. Histone acetylation is mediated by histone acetyltransferase; the resulting modification in the structure of chromatin leads to nucleosomal relaxation and altered transcriptional activation. The reverse reaction is mediated by histone deacetylase (HDAC), which induces deacetylation, chromatin condensation and transcriptional repression. HDACs are divided into three distinct classes: I, II, and III, on the basis of size and sequence homology, as well as formation of distinct complexes. Among class II HDACs, HDAC4 is implicated in controlling gene expression important for diverse cellular functions. Basic and clinical experimental evidence has established that HDAC4 performs a wide variety of functions. Understanding the biological significance of HDAC4 will not only provide new insight into the mechanisms of HDAC4 involved in mediating biological response, but also form a platform to develop a therapeutic strategy to achieve clinical implications.
It has been reported that bitter tastants decrease blood pressure and relax precontracted vascular smooth muscle. However, the underlying mechanisms remain unclear. The aim of the present study was to determine the mechanism underlying the vasorelaxant effect of the bitter tastants. Thoracic aortic rings were isolated from Wistar rats and contractions were measured using an isometric myograph. Intracellular Ca(2+) ([Ca(2+)]i) in single rat thoracic aortic smooth muscle cells was recorded by calcium imaging. Calcium currents in single cells were recorded using patch-clamp techniques. High K(+) (140 mmol/L) induced contractions in rat thoracic aortic rings that were inhibited by 3 mmol/L chloroquine, 3 mmol/L denatonium and 10 ?mol/L nifedipine. In single rat thoracic aortic smooth muscle cells, high K(+) increased [Ca(2+)]i and this effect was also blocked by 3 mmol/L chloroquine and 10 ?mol/L nifedipine. Under Ca(2+) -free conditions, high K(+) failed to induce contractions in rat thoracic aortic rings. On its own, chloroquine had no effect on the muscle tension of rat aortic rings and [Ca(2+) ]i. The vasorelaxant effects of chloroquine on precontracted rat thoracic aortic rings were not altered by either 1 ?g/mL pertussis toxin (PTX), an inhibitor of G?o/i-protein, or 1 mmol/L gallein, an inhibitor of G??-protein. The results of patch-clamp analysis in single cells indicate that 1 mmol/L chloroquine blocks voltage-dependent L-type Ca(2+) channel (VDLCC) currents from both extracellular and intracellular sides. Together, the results indicate that chloroquine can block VDLCC, independent of PTX- and gallein-sensitive G-proteins, resulting in relaxation of high K(+)-precontracted thoracic aortic smooth muscle.
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 (CPC(iRFP)) 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. CPC(iRFP) 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 (CPC(GFP)) or CPC labelled with both iRFP and GFP (CPC(iRFP) (GFP)) were injected intramyocardially into mouse hearts after infarction. Three days after cell transplantation, a strong NIRF signal was detected in hearts into which CPC(iRFP) (GFP), but not CPC(GFP), were transplanted. Furthermore, iRFP fluorescence from engrafted CPC(iRFP) (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.
Induced pluripotent stem cells (iPS) can differentiate into cardiomyocytes (CM) and represent a promising form of cellular therapy for heart regeneration. However, residual undifferentiated iPS derivates (iPSD), which are not fully eliminated by cell differentiation or purification protocols, may form tumors after transplantation, thus compromising therapeutic application. Inhibition of stearoyl-coA desaturase (SCD) has recently been reported to eliminate undifferentiated human embryonic stem cells, which share many features with iPSD. Here, we tested the effects of PluriSin#1, a small-molecule inhibitor of SCD, on iPS-derived CM. We found that plurisin#1 treatment significantly decreased the mRNA and protein level of Nanog, a marker for both cell pluripotency and tumor progression; importantly, we provide evidence that PluriSin#1 treatment at 20 µM for 1 day significantly induces the apoptosis of Nanog-positive iPSD. In addition, PluriSin#1 treatment at 20 µM for 4 days diminished Nanog-positive stem cells in cultured iPSD while not increasing apoptosis of iPS-derived CM. To investigate whether PluriSin#1 treatment prevents tumorigenicity of iPSD after cell transplantation, we intramyocardially injected PluriSin#1- or DMSO-treated iPSD in a mouse model of myocardial infarction (MI). DMSO-treated iPSD readily formed Nanog-expressing tumors 2 weeks after injection, which was prevented by treatment with PluriSin#1. Moreover, treatment with PluriSin#1 did not change the expression of cTnI, ?-MHC, or MLC-2v, markers of cardiac differentiation (P>0.05, n = 4). Importantly, pluriSin#1-treated iPS-derived CM exhibited the ability to engraft and survive in the infarcted myocardium. We conclude that inhibition of SCD holds the potential to enhance the safety of therapeutic application of iPS cells for heart regeneration.
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.
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.
A better understanding of the mechanism underlying skeletal muscle repair is required to develop therapies that promote tissue regeneration in adults. Hedgehog signaling has been shown previously to be involved in myogenesis and angiogenesis: 2 crucial processes for muscle development and regeneration.
We have shown previously that estrogen (estradiol, E2) supplementation enhances voluntary alcohol consumption in ovariectomized female rodents and that increased alcohol consumption impairs ischemic hind limb vascular repair. However, the effect of E2-induced alcohol consumption on post-infarct myocardial repair and on the phenotypic/functional properties of endothelial progenitor cells (EPCs) is not known. Additionally, the molecular signaling of alcohol-estrogen interactions remains to be elucidated. This study examined the effect of E2-induced increases in ethanol consumption on post-infarct myocardial function/repair. Ovariectomized female mice, implanted with 17?-E2 or placebo pellets were given access to alcohol for 6 weeks and subjected to acute myocardial infarction. Left ventricular functions were consistently depressed in mice consuming ethanol compared with those receiving only E2. Alcohol-consuming mice also displayed significantly increased infarct size and reduced capillary density. Ethanol consumption also reduced E2-induced mobilization and homing of EPCs to injured myocardium compared with the E2-alone group. In vitro, exposure of EPCs to ethanol suppressed E2-induced proliferation, survival, and migration and markedly altered E2-induced estrogen receptor-dependent cell survival signaling and gene expression. Furthermore, ethanol-mediated suppression of EPC biology was endothelial nitric oxide synthase-dependent because endothelial nitric oxide synthase-null mice displayed an exaggerated response to post-acute myocardial infarction left ventricular functions. These data suggest that E2 modulation of alcohol consumption, and the ensuing EPC dysfunction, may negatively compete with the beneficial effects of estrogen on post-infarct myocardial repair.
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 (EndoE2F3(?/?)) 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 EndoE2F3(?/?) mice than in their littermates, and EndoE2F3(?/?) 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.
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.
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; E2F3(fl/fl) was generated, and MI was surgically induced in VE-Cre; E2F3(fl/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; E2F3(fl/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.
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.
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.
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.
Endothelial progenitor cell (EPC) survival and function in the injured myocardium is adversely influenced by hostile microenvironment such as ischemia, hypoxia, and inflammatory response, thereby compromising full benefits of EPC-mediated myocardial repair.
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 neovascularization in preclinical models. Previous studies have suggested that paracrine factors secreted by these proangiogenic cells are responsible, at least in part, for the angiogenic effects induced by CD34(+) cell transplantation.
The mobilization of bone marrow (BM) progenitor cells (PCs) is largely governed by interactions between stromal cell-derived factor (SDF)-1 and CXC chemokine receptor (CXCR)4. 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.
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-1alpha. 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 hosts innate telomere-based anticancer responses and provide further evidence that T-oligos may offer a powerful new approach for melanoma treatment.
Prolonged inflammatory response is associated with left ventricular (LV) dysfunction and adverse remodeling following myocardial infarction (MI). IL-10 inhibits inflammation by suppressing HuR-mediated mRNA stabilization of proinflammatory cytokines. Here we report that following MI, IL-10(-/-) mice showed exaggerated LV dysfunction, fibrosis, and cardiomyocyte apoptosis. Short-hairpin RNA (shRNA)-mediated knockdown of HuR in the myocardium significantly reversed MI-induced LV dysfunctions and LV remodeling. HuR knockdown significantly reduced MI-induced cardiomyocyte apoptosis concomitant with reduced p53 expression. Moreover, HuR knockdown significantly reduced infarct size and fibrosis area, which in turn was associated with decreased TGF-beta expression. In vitro, stable knockdown of HuR in mouse macrophage cell line RAW 264.7 corroborated in vivo data and revealed reduced mRNA expression of TNF-alpha, TGF-beta, and p53 following LPS challenge, which was associated with a marked reduction in the mRNA stability of these genes. Taken together, our studies suggest that HuR is a direct target of IL-10, and HuR knockdown mimics anti-inflammatory effects of IL-10.
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 "nonclassical" pathway by which Shh directly modulates EC phenotype and angiogenic activity.
Recent studies have identified a polymorphism in the endothelin-converting enzyme (ECE)-1b promoter (-338C/A) that is strongly associated with hypertension in women. 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 regulation.
Myocardial infarction rapidly depletes the endogenous cardiac progenitor cell pool, and the inefficient recruitment of exogenously administered progenitor cells limits the effectiveness of cardiac cell therapy. Recent reports indicate that interactions between the CXC chemokine stromal cell-derived factor 1 and its receptor CXC chemokine receptor 4 (CXCR4) critically mediate the ischemia-induced recruitment of bone marrow-derived circulating stem/progenitor cells, but the expression of CXCR4 in cardiac progenitor cells is very low. Here, we studied the influence of hypoxia on CXCR4 expression in cardiac progenitor cells, on the recruitment of intravenously administered cells to ischemic heart tissue, and on the preservation of heart function in a murine myocardial infarction model. We found that hypoxic preconditioning increased CXCR4 expression in CLK (cardiosphere-derived, Lin(-)c-kit(+) progenitor) cells and markedly augmented CLK cell migration (in vitro) and recruitment (in vivo) to the ischemic myocardium. Four weeks after surgically induced myocardial infarction, infarct size and heart function were significantly better in mice administered hypoxia-preconditioned CLK cells than in mice treated with cells cultured under normoxic conditions. Furthermore, these effects were largely abolished by the addition of a CXCR4 inhibitor, indicating that the benefits of hypoxic preconditioning are mediated by the stromal cell-derived factor 1/CXCR4 axis, and that therapies targeting this axis may enhance cardiac-progenitor cell-based regenerative therapy.
Persistent inflammatory response has adverse effects on left ventricular (LV) function and remodeling following acute myocardial infarction. We hypothesized that suppression of inflammation with interleukin (IL)-10 treatment attenuates LV dysfunction and remodeling after acute myocardial infarction. After the induction of acute myocardial infarction, mice were treated with either saline or recombinant IL-10, and inflammatory response and LV functional and structural remodeling changes were evaluated. IL-10 significantly suppressed infiltration of inflammatory cells and expression of proinflammatory cytokines in the myocardium. These changes were associated with IL-10-mediated inhibition of p38 mitogen-activated protein kinase activation and repression of the cytokine mRNA-stabilizing protein HuR. IL-10 treatment significantly improved LV functions, reduced infarct size, and attenuated infarct wall thinning. Myocardial infarction-induced increase in matrix metalloproteinase (MMP)-9 expression and activity was associated with increased fibrosis, whereas IL-10 treatment reduced both MMP-9 activity and fibrosis. Small interfering RNA knockdown of HuR mimicked IL-10-mediated reduction in MMP-9 expression and activity in NIH3T3 cells. Moreover, IL-10 treatment significantly increased capillary density in the infarcted myocardium which was associated with enhanced STAT3 phosphorylation. Taken together, our studies demonstrate that IL-10 suppresses inflammatory response and contributes to improved LV function and remodeling by inhibiting fibrosis via suppression of HuR/MMP-9 and by enhancing capillary density through activation of STAT3.
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 injury, which more closely mimics myocardial infarction in patients, because blood flow is only temporarily obstructed.
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.
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 ?4-integrin signaling. Both of these pathways are dependent on c-kit activity: the mobilization of progenitor cells in response to either CXCR4 antagonism or ?4-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 ?4-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 ?4-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.
Nitric oxide (NO) and related nitrovasodilators regulate blood pressure by activation of soluble guanylate cyclase, elevation of cyclic guanosine monophosphate (cGMP), and activation of cGMP-dependent protein kinase (cGPK). Despite the progress of our understanding of the NO/cGMP mediated vasorelaxation, partly through conventional cGPK knock-out mice, the role of cGPK remains unclear. In particular, the downstream target(s) of the kinase are not well defined. We hypothesized that highly selective inhibitors of cGPK delivered in vivo in adult may elucidate the role of the kinase in vasorelaxation and regulation of blood pressure.
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.
Inflammation plays a critical role in adverse cardiac remodeling and heart failure. Therefore, approaches geared toward inhibiting inflammation may provide therapeutic benefits. We tested the hypotheses that genetic deletion of interleukin-10 (IL-10), a potent antiinflammatory cytokine, exacerbates pressure overload-induced adverse cardiac remodeling and hypertrophy and that IL-10 therapy inhibits this pathology.
Although bone marrow endothelial progenitor cell (EPC)-based therapies improve the symptoms in patients with ischemic heart disease, their limited plasticity and decreased function in patients with existing heart disease limit the full benefit of EPC therapy for cardiac regenerative medicine.
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 SM22? (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.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.