Inhibition of Neointima Hyperplasia, Inflammation, and Reactive Oxygen Species in Balloon-Injured Arteries by HVJ Envelope Vector-Mediated Delivery of Superoxide Dismutase Gene.

Extracellular superoxide dismutase (EC-SOD) has been implicated in regulation of vascular function but its underlying molecular mechanism is largely unknown. These two-step experiments investigate whether hemagglutinating virus of Japan envelope (HVJ-E) vector-mediated EC-SOD gene delivery might protect against neointima formation, vascular inflammation, and reactive oxygen species (ROS) generation, and also explore cell growth signaling pathways. The first in-vitro experiment was performed to assess the transfection efficacy and safety of HVJ-E compared to lipofectamine®. Results revealed that HVJ-E has higher transfection efficiency and lower cytotoxicity than those of lipofectamine®. Another in-vivo study initially used balloon denudation to rat carotid artery, then delivered EC-SOD cDNA through the vector of HVJ-E. Arterial section with H&E staining from the animals 14 days after balloon injury showed a significant reduction of intima-to-media area ratio in EC-SOD transfected arteries when compared with control (empty vector-transfected arteries) (p < 0.05). Arterial tissue with EC-SOD gene delivery also exhibited lower levels of ROS, as assessed by fluorescent microphotography with dihydroethidium staining. Quantitative RT-PCR revealed that EC-SOD gene delivery significantly diminished mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß (p < 0.05 in all comparisons). An immunoblotting assay from vascular smooth muscle cell (VSMC) cultures showed that the EC-SOD transfected group attenuated the activation of MEK1/2, ERK1/2, and Akt signaling significantly. In conclusion, EC-SOD overexpression by HVJ-E vector inhibits neointima hyperplasia, inflammation, and ROS level triggered by balloon injury. The modulation of cell growth-signaling pathways by EC-SOD in VSMCs might play an important role in these inhibitory effects.

Inhibition of Gata4 and Tbx5 by Nicotine-Mediated DNA Methylation in Myocardial Differentiation.

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.

MicroRNA-133a regulates insulin-like growth factor-1 receptor expression and vascular smooth muscle cell proliferation in murine atherosclerosis.

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.

Myocardin-A enhances expression of promyogenic genes without depressing telomerase activity in adipose tissue-derived mesenchymal stem cells.

BACKGROUND: The stromal compartment of adult adipose tissue consists of mesenchymal stem cells (MSCs) characterized by co-expression of myocardin-A (McA) and telomerase reverse transcriptase (TERT). OBJECTIVE: This study aims at testing the growth and myogenic regenerative properties of adipose tissue-derived MSCs, and the outcome of McA and TERT overexpression or suppression. METHODS AND RESULTS: TERT and/or McA in murine MSCs derived from adult adipose tissue were transduced with cDNA and co-immunoprecipitated with specific antibodies. The MSCs with TERT or TERT plus McA overexpressed were highly proliferative. Bioluminescence resonance energy transfer assay evidenced close interactions between TERT and McA in MSCs co-transfected with TERT and McA, which expressed the stem cell oncogene Oct-4, and promyogenic genes GATA-4, Nkx2.5, MLC2v, Mef2c, DTEF and McA. The increased myogenic gene expression depended on TERT and McA expression as siRNAs for TERT and McA diminished the TERT activities. The co-transfected MSCs also developed a stronger activity of serum response factor, a key factor for expression of cardiomyogenic genes. McA overexpression did not interfere with TERT-mediated proliferative effects, and rather slightly increased proliferation of MSCs. CONCLUSIONS: McA and TERT may interplay in promoting promyogenic gene expression and maintaining growth capacity of MSCs.

Rotating microgravity-bioreactor cultivation enhances the hepatic differentiation of mouse embryonic stem cells on biodegradable polymer scaffolds.

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.

Clusterin induces CXCR4 expression and migration of cardiac progenitor cells.

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.

Liposomal modular complexes for simultaneous targeted delivery of bioactive gases and therapeutics.

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.

Delivery of stem cells to porcine arterial wall with echogenic liposomes conjugated to antibodies against CD34 and intercellular adhesion molecule-1.

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.

Characterization of a novel ubiquitin-conjugating enzyme that regulates beta1,4-galactosyltransferase-1 in embryonic stem cells.

In this study we identified a novel galactosyltransferase 1-associating protein (GTAP) by cDNA cloning from a murine embryonic cDNA library using the two-hybrid yeast system. GTAP is expressed in early embryonic tissues, as well as in adult tissues with active cell turnover, and belongs to the class III ubiquitin-conjugating (E2) enzyme family. Its COOH-terminal domain contains a consensus sequence for ubiquitin binding shared by all the ubiquitin-conjugating enzymes, whereas its NH(2)-terminal domain appears critical for the binding and internalization of cell surface galactosyltransferase 1 (GalT1) in embryonic stem cells through a monensin- and MG132-dependent pathway. We have found that GTAP regulates GalT1-associated, laminin-dependent embryonic cell adhesion and the formation of embryoid bodies. Thus, GTAP functions as an evolutionarily conserved E2 enzyme, which may participate in intercellular adhesion and embryonic development. Disclosure of potential conflicts of interest is found at the end of this article.

Apolipoprotein-J prevention of fetal cardiac myoblast apoptosis induced by ethanol.

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.

Identification and characterization of cystatin-related epididymal spermatogenic protein in human spermatozoa: localization in the equatorial segment.

Our earlier studies in mouse have shown that the cystatin-related epididymal spermatogenic (CRES) protein is highly expressed in elongating spermatids in the testis and is present in mouse sperm acrosomes, suggesting specific roles in sperm function, fertilization, or both. However, whether the human CRES gene is similar to that of the mouse and is expressed in germ cells has not yet been determined. Therefore, the present study was undertaken to characterize the human ortholog of mouse CRES: Northern blot and in situ hybridization experiments showed that CRES is highly expressed in the human testis, specifically within clusters of round spermatids. Furthermore, reverse transcription-polymerase chain reaction detected CRES mRNA in the epididymis. Western blot analysis of protein lysates prepared from human testis and ejaculated spermatozoa showed a predominant 19-kDa protein and a minor 14-kDa protein. However, in contrast to the acrosomal localization of CRES protein in mouse spermatozoa, indirect immunofluorescence of human spermatozoa treated with methanol/acetic acid using anti-human CRES antibodies revealed that CRES was strictly localized to the equatorial segment. Furthermore, the same staining was observed in both capacitated and acrosome-reacted spermatozoa. To determine whether CRES was associated with the plasma membrane, live spermatozoa were incubated with CRES antibody after capacitation and acrosome reaction. Only acrosome-reacted spermatozoa showed a weak but specific equatorial staining. Taken together, these studies show that CRES protein is present in the sperm equatorial segment and becomes accessible to the extracellular environment during fertilization.