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1.
Vascul Pharmacol ; 127: 106651, 2020 04.
Article in English | MEDLINE | ID: mdl-32044414

ABSTRACT

Aspirin is a widely used drug with anti-coagulating and anti-inflammatory effects on atherosclerotic vascular disease. The goal of this study was to investigate expression of microRNA (miR) in association with changes in arachidonic acid (AA) metabolism in cardiac and surrounding fat mesenchymal stem cells (MSCs) treated with or without aspirin. Aspirin-targeted endogenous lipid metabolites that impact specific miRNA expression were examined by mass spectrometry. The pattern of miR expression was characterized using a microarray of 1100 miRs. There were a dozen miRs expressed differentially in MSCs from human myocardium and peri-myocardial fat tissue at baseline, including hsa-miR-1307-3p, 765, 4739, 3613-3p, 4281, 6816-5p, 2861, and 146b-5p. After exposure to aspirin, cardiac MSCs expressed an array of of miRs (eg, hsa-miR-4734, 10a-5p, 4267, 3197, and 3182), while generation of their endogenous AA metabolites was depressed. However, in the peri-cardiac adipose tissue-derived MSCs, treatment with the same doses of aspirin caused mild changes in the miR expression levels. In conclusion, MSCs from human myocardium and peri-myocardial fat tissue respond differentially to aspirin treatment by alterations in miR expression and AA metabolism. The study further raises an intriguing issue as to whether the copious amounts of aspirin taken worldwide by patients with cardiovascular disease may have direct impacts on their heart repair processes by regulation of stromal cell miR expression and AA metabolism.


Subject(s)
Adipose Tissue/cytology , Anticoagulants/pharmacology , Arachidonic Acid/metabolism , Aspirin/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Mesenchymal Stem Cells/drug effects , MicroRNAs/metabolism , Myocardium/cytology , Cells, Cultured , Humans , Mesenchymal Stem Cells/metabolism , MicroRNAs/genetics , Phenotype , Transcriptome
2.
Chin Med J (Engl) ; 131(5): 544-552, 2018 Mar 05.
Article in English | MEDLINE | ID: mdl-29483388

ABSTRACT

BACKGROUND: Our previous studies have shown that Tongxinluo (TXL), a compound Chinese medicine, can decrease myocardial ischemia-reperfusion injury, protect capillary endothelium function, and lessen cardiac ventricle reconstitution in animal models. The aim of this study was to illuminate whether TXL can improve hypercholesterolemia-impaired heart function by protecting artery endothelial function and increasing microvascular density (MVD) in heart. Furthermore, we will explore the underlying molecular mechanism of TXL cardiovascular protection. METHODS: After intragastric administration of TXL (0.1 ml/10 g body weight) to C57BL/6J wild-type mice (n = 8) and ApoE-/- mice (n = 8), total cholesterol, high-density lipoprotein-cholesterol, very-low-density lipoprotein (VLDL)-cholesterol, triglyceride, and blood glucose levels in serum were measured. The parameters of heart rate (HR), left ventricular diastolic end diameter, and left ventricular systolic end diameter were harvested by ultrasonic cardiogram. The left ventricular ejection fraction, stroke volume, cardiac output, and left ventricular fractional shortening were calculated. Meanwhile, aorta peak systolic flow velocity (PSV), end diastolic flow velocity, and mean flow velocity (MFV) were measured. The pulsatility index (PI) and resistant index were calculated in order to evaluate the vascular elasticity and resistance. The endothelium-dependent vasodilatation was evaluated by relaxation of aortic rings in response to acetylcholine. Western blotting and real-time quantitative reverse transcription polymerase chain reaction were performed for protein and gene analyses of vascular endothelial growth factor (VEGF). Immunohistochemical detection was performed for myocardial CD34 expression. Data in this study were compared by one-way analysis of variance between groups. A value of P < 0.05 was considered statistically significant. RESULTS: Although there was no significant decrease of cholesterol level (F = 2.300, P = 0.240), TXL inhibited the level of triglyceride and VLDL (F = 9.209, P = 0.024 and F = 9.786, P = 0.020, respectively) in ApoE-/- mice. TXL improved heart function of ApoE-/- mice owing to the elevations of LVEF, SV, CO, and LVFS (all P < 0.05). TXL enhanced aortic PSV and MFV (F = 10.774, P = 0.024 and F = 11.354, P = 0.020, respectively) and reduced PI of ApoE-/- mice (1.41 ± 0.17 vs. 1.60 ± 0.17; P = 0.037). After incubation with 10 µmol/L acetylcholine, the ApoE-/- mice treated with TXL aortic segment relaxed by 44% ± 3%, significantly higher than control group mice (F = 9.280, P = 0.040). TXL also restrain the angiogenesis of ApoE-/- mice aorta (F = 21.223, P = 0.010). Compared with C57BL/6J mice, the MVD was decreased in heart tissue of untreated ApoE-/- mice (54.0 ± 3.0/mm2 vs. 75.0 ± 2.0/mm2; F = 16.054, P = 0.010). However, TXL could significantly enhance MVD (65.0 ± 5.0/mm2 vs. 54.0 ± 3.0/mm2; F = 11.929, P = 0.020) in treated ApoE-/- mice. In addition, TXL obviously increased the expression of VEGF protein determined by Western blot (F = 20.247, P = 0.004). CONCLUSIONS: TXL obviously improves the ApoE-/- mouse heart function from different pathways, including reduces blood fat to lessen atherosclerosis; enhances aortic impulsivity, blood supply capacity, and vessel elasticity; improves endothelium-dependent vasodilatation; restraines angiogenesis of aorta-contained plaque; and enhances MVD of heart. The molecular mechanism of MVD enhancement maybe relate with increased VEGF expression.


Subject(s)
Apolipoproteins E/blood , Drugs, Chinese Herbal/therapeutic use , Animals , Atherosclerosis/blood , Atherosclerosis/drug therapy , Blotting, Western , Echocardiography , Hypercholesterolemia/blood , Hypercholesterolemia/drug therapy , Immunohistochemistry , Lipoproteins, VLDL/blood , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardium/metabolism , Real-Time Polymerase Chain Reaction , Stroke Volume/drug effects , Triglycerides/blood
3.
Stem Cell Reports ; 8(2): 290-304, 2017 02 14.
Article in English | MEDLINE | ID: mdl-28111280

ABSTRACT

Maternal nicotine exposure causes alteration of gene expression and cardiovascular programming. The discovery of nicotine-medicated regulation in cardiogenesis is of major importance for the study of cardiac defects. The present study investigated the effect of nicotine on cardiac gene expression and epigenetic regulation during myocardial differentiation. Persistent nicotine exposure selectively inhibited expression of two cardiac genes, Tbx5 and Gata4, by promoter DNA hypermethylation. The nicotine-induced suppression on cardiac differentiation was restored by general nicotinic acetylcholine receptor inhibition. Consistent results of Tbx5 and Gata4 gene suppression and cardiac function impairment with decreased left ventricular ejection fraction were obtained from in vivo studies in offspring. Our results present a direct repressive effect of nicotine on myocardial differentiation by regulating cardiac gene suppression via promoter DNA hypermethylation, contributing to the etiology of smoking-associated cardiac defects.


Subject(s)
Cell Differentiation/genetics , DNA Methylation/drug effects , GATA4 Transcription Factor/genetics , Gene Expression Regulation/drug effects , Muscle Cells/cytology , Muscle Cells/metabolism , Nicotine/pharmacology , T-Box Domain Proteins/genetics , Animals , Base Sequence , Cell Line , Cell Survival/genetics , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Embryoid Bodies , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Female , Male , Mice , Nicotinic Antagonists/pharmacology , Pregnancy , Promoter Regions, Genetic , Rats , Receptors, Nicotinic/metabolism
4.
Biochim Biophys Acta ; 1842(11): 2266-75, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25108283

ABSTRACT

BACKGROUND AND OBJECTIVE: Hyperglycemia leads to adaptive cell responses in part due to hyperosmolarity. In endothelial and epithelial cells, hyperosmolarity induces aquaporin-1 (AQP1) which plays a role in cytoskeletal remodeling, cell proliferation and migration. Whether such impairments also occur in human induced pluripotent stem cells (iPS) is not known. We therefore investigated whether high glucose-induced hyperosmolarity impacts proliferation, migration, expression of pluripotency markers and actin skeleton remodeling in iPS cells in an AQP1-dependent manner. METHODS AND RESULTS: Human iPS cells were generated from skin fibroblasts by lentiviral transduction of four reprogramming factors (Oct4, Sox2, Klf4, c-Myc). After reprogramming, iPS cells were characterized by their adaptive responses to high glucose-induced hyperosmolarity by incubation with 5.5mmol/L glucose, high glucose (HG) at 30.5mM, or with the hyperosmolar control mannitol (HM). Exposure to either HG or HM increased the expression of AQP1. AQP1 co-immunoprecipitated with ß-catenin. HG and HM induced the expression of ß-catenin. Under these conditions, iPS cells showed increased ratios of F-actin to G-actin and formed increased tubing networks. Inhibition of AQP1 with small interfering RNA (siRNA) reverted the inducing effects of HG and HM. CONCLUSIONS: High glucose enhances human iPS cell proliferation and cytoskeletal remodeling due to hyperosmolarity-induced upregulation of AQP1.

5.
Atherosclerosis ; 232(1): 171-9, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24401233

ABSTRACT

OBJECTIVE: MicroRNA-133a (miR-133a) and insulin-like growth factor-1 (IGF-1) are two different molecules known to regulate cardiovascular cell proliferation. This study tested whether miR-133a affects expression of IGF-1 receptor (IGF-1R) and proliferation of IGF-1-stimulated vascular smooth muscle cells (VSMC) in a murine model of atherosclerosis. METHODS AND RESULTS: Expression of IGF-1R was analyzed by immuno-fluorescence and immuno-blotting, and miR-133a by qRT-PCR in the aortas of wild-type C57BL/6J (WT) and apolipoprotein-E deficient (ApoE(-/-)) mice. Compared to those in WT aortas, the IGF-1R and miR-133a levels were lower in ApoE(-/-) aortas. ApoE(-/-) VSMC grew slower than WT cells in the cultures with IGF-1-containing medium. MiR-133a-specific inhibitor decreased miR-133a, IGF-1R expression, IGF-1-stimulated VSMC growth in lipoprotein deficient media. By contrast, miR-133a precursor increased IGF-1R levels and promoted IGF-1-induced VSMC proliferation. In the luciferase-IGF-1R 3'UTR reporter system, the reporter luciferase activity was not inhibited in VSMC with miR-133a overexpression. IGF-1R mRNA half-life in ApoE(-/-) VSMC was shorter than that in WT VSMC. MiR-133a inhibitor reduced but precursor increased the mRNA half-life, although the effects appeared less striking in ApoE(-/-) VSMC than in WT cells. CONCLUSION: MiR-133a serves as a stimulatory factor for IGF-1R expression through prolonging IGF-1R mRNA half-life. In atherosclerosis induced by ApoE deficiency, reduced miR-133a expression is associated with lower IGF-1R levels and suppressive VSMC growth. Administration of miR-133a precursor may potentiate IGF-1-stimulated VSMC survival and growth.


Subject(s)
Atherosclerosis/blood , Atherosclerosis/physiopathology , Gene Expression Regulation , MicroRNAs/metabolism , Myocytes, Smooth Muscle/cytology , Receptor, IGF Type 1/metabolism , 3' Untranslated Regions , Animals , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscle, Smooth, Vascular/cytology , RNA, Messenger/metabolism
7.
Cell Cycle ; 12(23): 3594-8, 2013 Dec 01.
Article in English | MEDLINE | ID: mdl-24189530

ABSTRACT

In 2006, Dr Shinya Yamanaka succeeded to reprogram somatic cells into pluripotent stem cells (iPSC) by delivering the genes encoding Oct4, Sox2, Klf4, and c-Myc. This achievement represents a fundamental breakthrough in stem cell biology and opens up a new era in regenerative medicine. However, the molecular processes by which somatic cells are reprogrammed into iPSC remain poorly understood. In 2009, Yamanaka proposed the elite and stochastic models for reprogramming mechanisms. To date, many investigators in the field of iPSC research support the concept of stochastic model, i.e., somatic cell reprogramming is an event of epigenetic transformation. A mathematical model, f (Cd, k), has also been proposed to predict the stochastic process. Here we wish to revisit the Yamanaka model and summarize the recent advances in this research field.


Subject(s)
Cellular Reprogramming , Induced Pluripotent Stem Cells/cytology , Acetylation , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Epigenesis, Genetic , Epithelial-Mesenchymal Transition , Histones/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4 , Methylation , MicroRNAs/metabolism , Models, Theoretical , Promoter Regions, Genetic , Stochastic Processes , Transcription Factors/genetics , Transcription Factors/metabolism
8.
Circ Res ; 113(7): 902-14, 2013 Sep 13.
Article in English | MEDLINE | ID: mdl-23780385

ABSTRACT

RATIONALE: The number and function of stem cells decline with aging, reducing the ability of stem cells to contribute to endogenous repair processes. The repair capacity of stem cells in older individuals may be improved by genetically reprogramming the stem cells to exhibit delayed senescence and enhanced regenerative properties. OBJECTIVE: We examined whether the overexpression of myocardin (MYOCD) and telomerase reverse transcriptase (TERT) enhanced the survival, growth, and myogenic differentiation of mesenchymal stromal cells (MSCs) isolated from adipose or bone marrow tissues of aged mice. We also examined the therapeutic efficacy of transplanted MSCs overexpressing MYOCD and TERT in a murine model of hindlimb ischemia. METHODS AND RESULTS: MSCs from adipose or bone marrow tissues of young (1 month old) and aged (12 months old) male C57BL/6 and apolipoprotein E-null mice were transiently transduced with lentiviral vectors encoding TERT, MYOCD, or both TERT and MYOCD. Flow cytometry and bromodeoxyuridine cell proliferation assays showed that transduction with TERT and, to a lesser extent, MYOCD, increased MSC viability and proliferation. In colony-forming assays, MSCs overexpressing TERT and MYOCD were more clonogenic than mock-transduced MSCs. Fas-induced apoptosis was inhibited in MSCs overexpressing MYOCD or TERT. When compared with aged mock-transduced MSCs, aged MSCs overexpressing TERT, MYOCD, or both TERT and MYOCD increased myogenic marker expression, blood flow, and arteriogenesis when transplanted into the ischemic hindlimbs of apolipoprotein E-null mice. CONCLUSIONS: The delivery of the TERT and MYOCD genes into MSCs may have therapeutic applications for restoring, or rejuvenating, aged MSCs from adipose and bone marrow tissues.


Subject(s)
Hindlimb/blood supply , Ischemia/therapy , Mesenchymal Stem Cell Transplantation , Neovascularization, Physiologic , Nuclear Proteins/metabolism , Telomerase/metabolism , Trans-Activators/metabolism , Adipose Tissue/cytology , Animals , Cell Differentiation , Male , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Nuclear Proteins/genetics , Telomerase/genetics , Trans-Activators/genetics
10.
Biochem Biophys Res Commun ; 426(4): 615-9, 2012 Oct 05.
Article in English | MEDLINE | ID: mdl-22982320

ABSTRACT

Oxidative stress contributes to tissue injury and cell death during the development of various diseases. The present study aims at investigating whether oxidative stress triggered by the exposure to hydrogen peroxide (H(2)O(2)) can induce apoptosis of induced pluripotent stem cells (iPS cells) in a mechanism mediated by insulin-like growth factor (IGF-1) and microRNA-1 (miR-1). iPS cells treated with H(2)O(2) showed increases in miR-1 expression, mitochondria dysfunction, cytochrome-c release and apoptosis, Addition of IGF-1 into the iPS cell cultures reduced the H(2)O(2) cytotoxicity. Prediction algorithms showed that 3'-untranslated regions of IGF-1 gene as a target of miR-1. Moreover, miR-1 mimic, but not miR-1 mimic negative control, diminished the protective effect of IGF-1 on H(2)O(2)-induced mitochondrial dysfunction, cytochrome-c release and apoptosis in iPS cells. In conclusion, IGF-1 inhibits H(2)O(2)-induced mitochondrial dysfunction, cytochrome-c release and apoptosis. IGF-1's effect is, at least partially, regulated by miR-1 in iPS cells.


Subject(s)
Apoptosis , Induced Pluripotent Stem Cells/physiology , Insulin-Like Growth Factor I/physiology , MicroRNAs/metabolism , Oxidative Stress , 3' Untranslated Regions/genetics , Cells, Cultured , Cytochromes c/metabolism , Humans , Hydrogen Peroxide/pharmacology , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Insulin-Like Growth Factor I/genetics , MicroRNAs/genetics , Mitochondria/metabolism , Mitochondrial Diseases/metabolism
11.
Tissue Eng Part A ; 18(21-22): 2376-85, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22712633

ABSTRACT

Embryonic stem (ES) cells are pluripotent cells that are capable of differentiating all the somatic cell lineages, including those in the liver tissue. We describe the generation of functional hepatic-like cells from mouse ES (mES) cells using a biodegradable polymer scaffold and a rotating bioreactor that allows simulated microgravity. Cells derived from ES cells cultured in the three-dimensional (3D) culture system with exogenous growth factors and hormones can differentiate into hepatic-like cells with morphologic characteristics of typical mature hepatocytes. Reverse-transcription polymerase chain-reaction testing, Western blot testing, immunostaining, and flow cytometric analysis show that these cells express hepatic-specific genes and proteins during differentiation. Differentiated cells on scaffolds further exhibit morphologic traits and biomarkers characteristic of liver cells, including albumin production, cytochrome P450 activity, and low-density lipoprotein uptake. When these stem cell-bearing scaffolds are transplanted into severe combined immunodeficient mice, the 3D constructs remained viable, undergoing further differentiation and maturation of hepatic-like cells in vivo. In conclusion, the growth and differentiation of ES cells in a biodegradable polymer scaffold and a rotating microgravity bioreactor can yield functional and organizational hepatocytes useful for research involving bioartificial liver and engineered liver tissue.


Subject(s)
Bioreactors , Cell Culture Techniques/instrumentation , Cell Differentiation , Embryonic Stem Cells/cytology , Lactic Acid/pharmacology , Liver/cytology , Polymers/pharmacology , Weightlessness , Albumins/metabolism , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Proliferation/drug effects , Cell Shape/drug effects , Cytochrome P-450 Enzyme System/metabolism , Embryoid Bodies/cytology , Embryoid Bodies/drug effects , Embryoid Bodies/transplantation , Embryoid Bodies/ultrastructure , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/ultrastructure , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression Regulation/drug effects , Lipoproteins, LDL/metabolism , Liver/metabolism , Mice , Mice, SCID , Organ Specificity/drug effects , Organ Specificity/genetics , Polyesters , Rotation , Tissue Scaffolds/chemistry
12.
Int J Cardiol ; 150(3): 253-9, 2011 Aug 04.
Article in English | MEDLINE | ID: mdl-20451268

ABSTRACT

INTRODUCTION: An emerging technology using human embryonic stem cells (hESCs) to regenerate infarcted heart tissue has been underdeveloped. However, because non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, are taken during the infarction, it becomes critical to know whether the NSAIDs have negative impacts on heart tissue regeneration when using hESCs. METHODS: Mass spectrometry (LC/MS/MS) and high performance liquid chromatography (HPLC) analyses were used to analyze the functional presence of the elaborate prostanoids' biosynthesis and signaling systems in hESCs. The detected endogenous arachidonic acid (AA) released in the hESC membranes reflects the activity of phospholipase which directly controls the biosyntheses of the prostanoids. RESULTS: The complete inhibition of the endogenous prostaglandin E(2) (PGE(2)) biosynthesis by the cyclooxygenase-2 (COX-2) inhibitor, NS398, confirmed that the major prostanoids synthesized in the hESCs are mediated by the COX-2 enzyme. We also found that PGE(2) and the prostacyclin (PGI(2)) metabolite, 6-keto-PGF(1α), are present in the undifferentiated hESCs. CONCLUSION: This indicated different cyclooxygenase (COX)-downstream synthases and metabolizing enzymes are involved in the AA products' signaling through the COX-1 and COX-2 pathways. The presence of many enzymes' and receptors' [(COX-1, COX-2, microsomal prostaglandin E synthase (mPGES), cytosolic prostaglandin E synthase (cPGES), prostaglandin I synthase (PGIS), the PGE(2) subtype receptors (EP(1), EP(2), and EP(4)) and the prostacyclin receptor (IP)] involvement in the prostanoid biosynthesis and activity was confirmed by western blot. The studies implied the negative effects of NSAIDs, such as aspirin and COX-2 inhibitors, which suppress prostanoid production during tissue regeneration for infarcted heart when using hESCs.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Arachidonic Acid/metabolism , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Heart/drug effects , Regeneration/drug effects , Arachidonic Acid/antagonists & inhibitors , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Drug Delivery Systems/methods , Feeder Cells/drug effects , Feeder Cells/metabolism , Growth Inhibitors/adverse effects , Heart/physiology , Humans , Prostaglandins/biosynthesis , Regeneration/physiology
13.
Exp Cell Res ; 316(20): 3435-42, 2010 Dec 10.
Article in English | MEDLINE | ID: mdl-20813109

ABSTRACT

Clusterin (CST) is a stress-responding protein with multiple biological functions, including the inhibition of apoptosis and inflammation and transport of lipids. It may also participate in cell traffic and migration. In the process of post-infarct cardiac tissue repair, stem cells migrate into the damaged myocardium under the influence of chemoattractive substances such as stromal cell-derived factor (SDF). This study aimed at testing whether CST enhances expression of stem cell homing receptor and migration of cardiac progenitor cells (CPCs). CPCs isolated from fetal canine hearts transduced by CST cDNA expressed high levels of CXCR4, a receptor for SDF-1. The transfected cells also showed an increased migratory response to SDF-1 stimulation. The SDF-1-mediated migration of the CST-expressing CPCs was attenuated by PI3 kinase inhibitor LY294002 but not by mitogen-activated protein/ERK kinase inhibitor PD98059. Analysis of cell cycle by flow cytometry revealed no significant difference in cell cycle between the transduced and control CPCs. Thus, CST expression may increase CPCs migration via increasing CXCR4 expression and SDF-1/chemokine receptor signaling in a PI3/Akt-dependent manner.


Subject(s)
Cell Movement , Clusterin/metabolism , Myocardium/cytology , Receptors, CXCR4/biosynthesis , Stem Cells/cytology , Stem Cells/metabolism , Animals , Antibodies/immunology , Antibodies/pharmacology , Cell Cycle/genetics , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation , Chemokine CXCL12/pharmacology , Clusterin/genetics , Dogs , Enzyme Inhibitors/pharmacology , Fetus/cytology , Gene Expression/genetics , Humans , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Receptors, CXCR4/genetics , Receptors, CXCR4/immunology , Receptors, CXCR4/metabolism , Signal Transduction/drug effects , Transfection
14.
Circulation ; 121(3): 436-44, 2010 Jan 26.
Article in English | MEDLINE | ID: mdl-20065159

ABSTRACT

BACKGROUND: Preeclampsia is a prevalent life-threatening hypertensive disorder of pregnancy. The circulating antiangiogenic factor, soluble endoglin (sEng), is elevated in the blood circulation of women with preeclampsia and contributes to disease pathology; however, the underlying mechanisms responsible for its induction in preeclampsia are unknown. METHODS AND RESULTS: Here, we discovered that a circulating autoantibody, the angiotensin receptor agonistic autoantibody (AT(1)-AA), stimulates sEng production via AT(1) angiotensin receptor activation in pregnant mice but not in nonpregnant mice. We subsequently demonstrated that the placenta is a major source contributing to sEng induction in vivo and that AT(1)-AA-injected pregnant mice display impaired placental angiogenesis. Using drug screening, we identified tumor necrosis factor-alpha as a circulating factor increased in the serum of autoantibody-injected pregnant mice contributing to AT(1)-AA-mediated sEng induction in human umbilical vascular endothelial cells. Subsequently, among all the drugs screened, we found that hemin, an inducer of heme oxygenase, functions as a break to control AT(1)-AA-mediated sEng induction by suppressing tumor necrosis factor-alpha signaling in human umbilical vascular endothelial cells. Finally, we demonstrated that the AT(1)-AA-mediated decreased angiogenesis seen in human placenta villous explants was attenuated by tumor necrosis factor-alpha-neutralizing antibodies, soluble tumor necrosis factor-alpha receptors, and hemin by abolishing both sEng and soluble fms-like tyrosine kinase-1 induction. CONCLUSIONS: Our findings demonstrate that AT(1)-AA-mediated tumor necrosis factor-alpha induction, by overcoming its negative regulator, heme oxygenase-1, is a key underlying mechanism responsible for impaired placental angiogenesis by inducing both sEng and soluble fms-like tyrosine kinase-1 secretion from human villous explants. Our results provide important new targets for diagnosis and therapeutic intervention in the management of preeclampsia.


Subject(s)
Autoantibodies/blood , Intracellular Signaling Peptides and Proteins/blood , Pre-Eclampsia/blood , Pre-Eclampsia/immunology , Receptor, Angiotensin, Type 1/immunology , Tumor Necrosis Factor-alpha/blood , Animals , Autoantibodies/pharmacology , Chorionic Villi/blood supply , Chorionic Villi/immunology , Chorionic Villi/metabolism , Endoglin , Endothelial Cells/metabolism , Female , Heme Oxygenase-1/metabolism , Humans , Immunoglobulin G/blood , Immunoglobulin G/pharmacology , Mice , Neovascularization, Pathologic/blood , Neovascularization, Pathologic/immunology , Placenta/blood supply , Placenta/immunology , Placenta/metabolism , Pregnancy , Receptor, Angiotensin, Type 1/metabolism , Signal Transduction/physiology , Solubility
15.
J Control Release ; 142(3): 326-31, 2010 Mar 19.
Article in English | MEDLINE | ID: mdl-19903503

ABSTRACT

Intrinsically echogenic liposomes (ELIP) can be adapted to encapsulate nitric oxide to facilitate ultrasound-enhanced delivery of therapeutic agents to atherosclerotic plaques. However, the NO loading of targeted ELIP caused a 93% decrease of antibody (Ab) immunoreactivity. The following hypothesis was tested: biotin/avidin-mediated coupling of NO-ELIP and Ab-conjugated ELIP will enable co-delivery of bioactive gases and ELIP that can encapsulate other agents without loss of targeting efficiency. Complex formation was initiated by addition of excess streptavidin to equal proportions of biotinylated Ab-ELIP and NO-ELIP. Fluorescence deconvolution microscopy, Coulter Multisizer 3 analysis and flow cytometry demonstrated that the ELIP coupling procedure formed mixed aggregates of >or=10 liposomes within 1 min. Intravascular ultrasound imaging and ELISA showed that echogenicity and targeting efficiency were completely and 69-99% retained, respectively. When complexed to NO-ELIP, ELIP bifunctionally targeted to both CD34 and ICAM-1 (BF-ELIP) increased human mononuclear cell migration through human coronary artery endothelial cell monolayers in transwell plates 4-fold relative to a nonspecific IgG-ELIP control and 2-fold relative to BF-ELIP alone. It was concluded that this novel multi-functional conjugation methodology provides a platform technology for site-specific co-delivery of bioactive gases and other agents.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Drug Compounding/methods , Drug Delivery Systems/methods , Nitric Oxide/administration & dosage , Antibodies, Monoclonal/therapeutic use , Antigens, CD34/immunology , Antigens, CD34/metabolism , Biotinylation , Cell Adhesion , Cell Movement , Cells, Cultured , Coculture Techniques , Coronary Vessels/cytology , Coronary Vessels/diagnostic imaging , Drug Therapy, Combination , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Intercellular Adhesion Molecule-1/immunology , Intercellular Adhesion Molecule-1/metabolism , Liposomes , Microscopy, Fluorescence , Nitric Oxide/therapeutic use , Stem Cells/cytology , Stem Cells/drug effects , Stem Cells/metabolism , Ultrasonography, Interventional
16.
Mol Pharm ; 7(1): 3-11, 2010 Feb 01.
Article in English | MEDLINE | ID: mdl-19719324

ABSTRACT

In atherosclerosis, the loss of vascular stem cells via apoptosis impairs the capacity of the vascular wall to repair or regenerate the tissue damaged by atherogenic factors. Recruitment of exogenous stem cells to the plaque tissue may repopulate vascular cells and help repair the arterial tissue. Ultrasound-enhanced liposomal targeting may provide a feasible method for stem cell delivery into atheroma. Bifunctional echogenic immunoliposomes (BF-ELIP) were generated by covalently coupling two antibodies to liposomes; the first one specific for CD34 antigens on the surface of stem cells and the second directed against the intercellular adhesion molecule-1 (ICAM-1) antigens on the inflammatory endothelium covering atheroma. CD34+ stem cells from adult bone marrow were incubated on the ICAM-1-expressing endothelium of the aorta of swine fed high cholesterol diets, which was preloaded with BF-ELIP. Significantly increased stem cell adherence and penetration were detected in particular in the aortic segments treated with 1 MHz low-amplitude continuous wave ultrasound. Fluorescence and scanning electron microscopy confirmed the presence of BF-ELIP-bound CD34+ cells in the intimal compartment of the atheromatous arterial wall. Ultrasound treatment increased the number of endothelial cell progenitors migrating into the intima. Thus, under ultrasound enhancement, BF-ELIP bound CD34+ stem cells selectively bind to the ICAM-1 expressing endothelium of atherosclerotic lesions.


Subject(s)
Arteries/cytology , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Animals , Antibodies/administration & dosage , Antigens, CD34/immunology , Antigens, CD34/metabolism , Arteries/diagnostic imaging , Atherosclerosis/metabolism , Atherosclerosis/pathology , Atherosclerosis/therapy , Cell Adhesion , Humans , In Vitro Techniques , Intercellular Adhesion Molecule-1/immunology , Intercellular Adhesion Molecule-1/metabolism , Liposomes , Male , Swine , Swine, Miniature , Ultrasonography
17.
Exp Cell Res ; 315(17): 2921-8, 2009 Oct 15.
Article in English | MEDLINE | ID: mdl-19631208

ABSTRACT

Cardiac stem cells are vulnerable to inflammation caused by infarction or ischemic injury. The growth factor, erythropoietin (Epo), ameliorates the inflammatory response of the myocardium to ischemic injury. This study was designed to assess the role of Epo in regulation of expression and activation of the cell death-associated intracellular signaling components in cardiac myoblasts stimulated with the proinflammatory cytokine tumor necrosis factor (TNF)-alpha. Cardiac myoblasts isolated from canine embryonic hearts characterized by expression of myocardin A, a promyogenic transcription factor for cardiovascular muscle development were pretreated with Epo and then exposed to TNF-alpha. Compared to untreated cells, the Epo-treated cardiac myoblasts exhibited better morphology and viability. Immunoblotting revealed lower levels of active caspase-3 and reductions in iNOS expression and NO production in Epo-treated cells. Furthermore, Epo pretreatment reduced nuclear translocation of NF-kappaB and inhibited phosphorylation of inhibitor of kappa B (IkappaB) in TNF-alpha-stimulated cardiac myoblasts. Thus, Epo protects cardiac myocyte progenitors or myoblasts against the cytotoxic effects of TNF-alpha by inhibiting NF-kappaB-mediated iNOS expression and NO production and by preventing caspase-3 activation.


Subject(s)
Cell Survival/drug effects , Erythropoietin/pharmacology , Heart/physiology , Stem Cells/cytology , Stem Cells/physiology , Tumor Necrosis Factor-alpha/toxicity , Animals , Apoptosis , Caspase 3/metabolism , Cell Culture Techniques , Cell Death , Cell Division , Dogs , Flow Cytometry , Heart/drug effects , Myoblasts/cytology , Myoblasts/drug effects , Myoblasts/physiology , Myocardium/cytology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/physiology , Nitrites/metabolism , Nuclear Proteins/analysis , Nuclear Proteins/genetics , Stem Cells/drug effects , Trans-Activators/analysis , Trans-Activators/genetics
18.
Biochem Biophys Res Commun ; 357(1): 157-61, 2007 May 25.
Article in English | MEDLINE | ID: mdl-17416353

ABSTRACT

Over-consumption of ethanol (EtOH) represents a major health problem. This study was to test the cytotoxicity of EtOH in cardiac stem cells or myoblasts, and the potential protective effect of apolipoprotein-J (ApoJ), a stress-responding, chaperone-like protein in high-density lipoprotein, on EtOH-injured cardiac myoblasts. In culture, EtOH-exposed canine fetal myoblasts underwent apoptosis in a concentration- and time-dependent manner. Expression ApoJ by cDNA transfection markedly reduced EtOH-induced apoptosis in the cells. ApoJ expression also restored partially the mitochondrial membrane potential and prevented the release of cytochrome-c from mitochondria into cytoplasma. Thus, ApoJ serves as a cytoprotective protein that protects cardiac stem cells against EtOH cytotoxicity.


Subject(s)
Apoptosis/drug effects , Clusterin/metabolism , Ethanol/administration & dosage , Heart/embryology , Myoblasts, Cardiac/cytology , Myoblasts, Cardiac/metabolism , Animals , Apoptosis/physiology , Cardiotonic Agents/metabolism , Cells, Cultured , Clusterin/genetics , Dogs , Dose-Response Relationship, Drug , Heart/drug effects , Myoblasts, Cardiac/drug effects
19.
Circulation ; 107(1): 98-105, 2003 Jan 07.
Article in English | MEDLINE | ID: mdl-12515750

ABSTRACT

BACKGROUND: Fortilin, a recently characterized nuclear antiapoptotic factor structurally distinct from inhibitor of apoptosis proteins (IAPs) and Bcl-2 family member proteins, has been suggested to be involved in cell survival and regulation of apoptosis within the cardiovascular system. In this continued investigation, we characterized the influence of adenovirus-mediated fortilin (Ad-fortilin) gene delivery on vascular remodeling after experimental angioplasty. METHODS AND RESULTS: Vessel wall expression of Ad-fortilin or adenoviral luciferase (Ad-luc) was demonstrated 72 hours and 14 days after rat carotid artery (CA) balloon angioplasty. Morphometric analyses 14 days after injury revealed significantly diminished neointima development in the Ad-fortilin-treated CAs compared with Ad-luc or PBS controls, with no changes in medial wall morphometry observed between the 3 groups. The Ad-fortilin-treated CAs demonstrated a 50% reduction in medial wall proliferating cell nuclear antigen (PCNA) labeling after 72 hours, with significantly reduced neointimal and medial wall PCNA labeling and cell counts after 14 days. Terminal dUTP nick-end labeling results and morphological changes characteristic of programmed cell death suggest a trend toward reduced apoptosis in the fortilin-transfected balloon-injured vessels compared with Ad-luc injured controls. Temporal analysis of human aorta smooth muscle cell (SMC) proliferation demonstrated a marked time-dependent inhibition in Ad-fortilin treated SMCs without the influence of elevated apoptosis. Thymidine incorporation was significantly inhibited in the Ad-fortilin-treated cells compared with Ad-luc controls. Ad-fortilin transfected SMCs also demonstrated significantly decreased migration compared with Ad-luc controls. CONCLUSIONS: These cumulative results suggest that the novel antiapoptotic protein fortilin may play important redundant pathophysiological roles in modulating the vascular response to experimental angioplasty through suppression of SMC proliferation and migration concomitant with reduction of vessel wall apoptosis.


Subject(s)
Adenoviridae/genetics , Arterial Occlusive Diseases/therapy , Genetic Therapy , Muscle, Smooth, Vascular/physiopathology , Neoplasm Proteins , Nuclear Proteins/genetics , Angioplasty, Balloon/adverse effects , Animals , Arterial Occlusive Diseases/pathology , Arterial Occlusive Diseases/physiopathology , Biomarkers, Tumor , Carotid Stenosis/pathology , Carotid Stenosis/physiopathology , Carotid Stenosis/therapy , Cell Division , Cell Movement , Cells, Cultured , Genetic Vectors , Kinetics , Male , Proliferating Cell Nuclear Antigen/analysis , Rats , Rats, Sprague-Dawley , Thymidine/biosynthesis , Transfection , Tumor Protein, Translationally-Controlled 1
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