ABSTRACT
Contact inhibition and its disruption of vascular smooth muscle cells (VSMCs) are important cellular events in vascular diseases. But the underlying molecular mechanisms are unclear. In this study we investigated the roles of microRNAs (miRNAs) in the contact inhibition and its disruption of VSMCs and the molecular mechanisms involved. Rat VSMCs were seeded at 30% or 90% confluence. MiRNA expression profiles in contact-inhibited conï¬uent VSMCs (90% confluence) and non-contact-inhibited low-density VSMCs (30% confluence) were determined. We found that multiple miRNAs were differentially expressed between the two groups. Among them, miR-145 was significantly increased in contact-inhibited VSMCs. Serum could disrupt the contact inhibition as shown by the elicited proliferation of conï¬uent VSMCs. The contact inhibition disruption accompanied with a down-regulation of miR-145. Serum-induced contact inhibition disruption of VSMCs was blocked by overexpression of miR-145. Moreover, downregulation of miR-145 was sufficient to disrupt the contact inhibition of VSMCs. The downregulation of miR-145 in serum-induced contact inhibition disruption was related to the activation PI3-kinase/Akt pathway, which was blocked by the PI3-kinase inhibitor LY294002. KLF5, a target gene of miR-145, was identified to be involved in miR-145-mediated effect on VSMC contact inhibition disruption, as it could be inhibited by knockdown of KLF5. In summary, our results show that multiple miRNAs are differentially expressed in contact-inhibited VSMCs and in non-contact-inhibited VSMCs. Among them, miR-145 is a critical gene in contact inhibition and its disruption of VSMCs. PI3-kinase/Akt/miR-145/KLF5 is a critical signaling pathway in serum-induced contact inhibition disruption. Targeting of miRNAs related to the contact inhibition of VSMCs may represent a novel therapeutic approach for vascular diseases.
Subject(s)
Contact Inhibition/physiology , MicroRNAs/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Animals , Cell Count , Cell Proliferation/physiology , Chromones/pharmacology , Down-Regulation , Kruppel-Like Transcription Factors/metabolism , Male , MicroRNAs/genetics , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Rats, Sprague-Dawley , Signal Transduction/physiologyABSTRACT
OBJECTIVE: To construct a lentivirus vector carrying wheat germ agglutinin (WGA) and evaluate its ability of tracing WGA in the brain of mice with ischemic brain injury. METHODS: WGA gene was inserted into the lentiviral vector Plvx IRES-ZsGreen1 using genetic engineering methods. 293T cells were transfected with the vector and 3 packaging plasmids (RPEV, PRRE, and VSVG) to obtain the recombinant lentivirus for infection of human adipose-derived stem cells (hADSCs). The infected hADSCs were injected into the damaged brain area by in situ injection in a mouse model of middle cerebral artery occlusion (MCAO) and the expression of GFP was traced. RESULTS: Immunofluorescence identification detected WGA protein expression in the infected hADSCs, which survived in the infarct area of mice with MCAO. CONCLUSION: Packaging WGA gene in lentivirus is a reliable approach to allow efficient neuroanatomical tracing of various cells.
Subject(s)
Adipose Tissue/cytology , Genetic Vectors , Stem Cells/cytology , Transfection , Wheat Germ Agglutinins/metabolism , Animals , HEK293 Cells , Humans , Lentivirus , Mice , Plasmids , Wheat Germ Agglutinins/geneticsABSTRACT
OBJECTIVES: To observe the expression of brain-derived neurotrophic factor (BDNF) in hypothalamic and adipose tissue in mice with monosodium glutamate (MSG)-induced obesity. METHODS: The effects of hypothalamic lesions, specifically arcuate nucleus (ARC) lesions, induced by MSG injection were studied in male ICR mice at the neonatal stage. The following parameters were compared: body weight, body length, Lee's index, food intake, body temperature, fat weight, and levels of total cholesterol (CHOL), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and blood glucose (GLU). The BDNF expression levels in hypothalamic and adipose tissue were measured using western blotting. Results Compared with the control group, the model group body had significantly higher weight, Lee's index, food intake, fat weight, CHOL, TG, LDL, HDL, and GLU levels. BDNF expression levels in hypothalamic and adipose tissue were markedly down-regulated in the model group. DISCUSSION: BDNF may be closely associated with MSG-induced hypothalamic obesity.