PURPOSE: MicroRNAs are small non-coding RNAs that play important roles in vascular smooth muscle cell (VSMC) function. This study investigated the role of miR-379 on proliferation, invasion, and migration of VSMCs and explored underlying mechanisms thereof. MATERIALS AND METHODS: MicroRNA, mRNA, and protein levels were determined by quantitative real-time PCR and western blot. The proliferative, invasive, and migratory abilities of VSMCs were measured by CCK-8, invasion, and wound healing assay, respectively. Luciferase reporter assay was used to confirm the target of miR-379. RESULTS: Platelet-derived growth factor-bb was found to promote cell proliferation and suppress miR-379 expression in VSMCs. Functional assays demonstrated that miR-379 inhibited cell proliferation, cell invasion, and migration. Flow cytometry results further showed that miR-379 induced apoptosis in VSMCs. TargetScan analysis and luciferase report assay confirmed that insulin-like growth factor-1 (IGF-1) 3'UTR is a direct target of miR-379, and mRNA and protein levels of miR-379 and IGF-1 were inversely correlated. Rescue experiments showed that enforced expression of IGF-1 sufficiently overcomes the inhibitory effect of miR-379 on cell proliferation, invasion, and migration in VSMCs. CONCLUSION: Our results suggest that miR-379 plays an important role in regulating VSMCs proliferation, invasion, and migration by targeting IGF-1.
Apoptosis ; Cell Movement/*physiology ; Cell Proliferation/*physiology ; Humans ; Insulin ; Insulin-Like Growth Factor I/*physiology ; MicroRNAs/*physiology ; Muscle, Smooth, Vascular/*cytology ; Proto-Oncogene Proteins c-sis/*physiology ; RNA, Messenger/metabolism ; Real-Time Polymerase Chain Reaction ; Sincalide/physiology ; Wound Healing/physiology

OBJECTIVETo study the role and possible mechanisms of gap junctional intercellular communication (GJIC) involved in mesangial cell (MC) proliferation which could be inhibited by bufalin.

METHODSRat mesangial cells were cultured in vitro. The effect of bufalin on platelet-derived growth factor-BB (PDGF-BB)-induced MC proliferation was evaluated by MTT assay. The function of GJIC was detected by Lucifer Yellow scrape loading and dye transfer (SLDT). mRNA levels of Cx43, Cx45 and Cx40 were measured by RT-PCR. Intracellular calcium concentrations ([Ca(2+)]i) were examined in laser scanning confocal microscopy after loading by Fura-3/AM.

RESULTSMTT indicated that bufalin could inhibited PDGF-BB-induced MC proliferation (P<0.01). Compared with the hormal control group, PDGF-BB inhibited GJIC function, increased the expression of Cx45 and Cx40 (P<0.01) without altering the Cx43 (P>0.05) in gene level and also increased [Ca(2+)]i. However, bufalin treatment enhanced GJIC function, decreased Cx45 mRNA and Cx40 mRNA expression (P<0.01), and reduced [Ca(2+)]i (P<0.01).

CONCLUSIONSBufalin inhibits PDGF-BB-induced MC proliferation, and its possible mechanisms may be related to regulation of Cx45 and Cx40 expression in the gene level, reduction of [Ca(2+)]i and enhancement of GJIC function.

Animals ; Bufanolides ; pharmacology ; Calcium ; metabolism ; Cell Communication ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Gap Junctions ; drug effects ; Mesangial Cells ; drug effects ; physiology ; ultrastructure ; Proto-Oncogene Proteins c-sis ; pharmacology ; Rats

BACKGROUNDAmong various treatments preventing vein graft restenosis, external stent is receiving more and more attention. This study aimed to investigate the effect of non-restrictive external stent on the prevention of vein graft restenosis and the potential mechanisms of platelet-derived growth factor (PDGF) in the process of restenosis.

METHODSThirty-six "New Zealand white rabbits" were randomly divided into two groups, stented group (group S) and control group (non-stented group, group NS). Each rabbit underwent a reversed autologous external jugular vein into common carotid artery bypass grafting. In group S, the vein grafts were surrounded by a non restrictive stent which was 6 mm in diameter (a kind of Dacron vascular prosthesis); and in group NS, there was no stent to support the vein grafts. The grafts were harvested at the first week (1W), second week (2W) and fourth week (4W) after surgery respectively. The dimensions (including the thickness and area of the intima and media, luminal area) were measured by computer-aided image analysis system, and the intimal hyperplasia ratio was defined as the percentage of the area enclosed by the internal elastic lamina occupied by the intima.

RESULTSAt 1W, the difference of the thickness and area of the intima between groups S and NS was not significant (P > 0.05); at 2W and 4W, the thickness and area of the intima and the intimal hyperplasia ratio in group S were less significant than those in group NS (P < 0.05); from 1W to 4W, the thickness and area of the media in group S were smaller than those in group NS (P < 0.05). Immunocytochemistry staining of PDGF-B showed that the percentage of positive cells of intima in both two groups was peaked at 2W, and a significantly smaller percentage was detected in group S compared with that in group NS at 2W and 4W (P < 0.05); the percentage of PDGF-B positive cells of media in both two groups was also peaked at 2W, and that in group S was smaller than that in group NS from 1W to 4W (P < 0.05); and the percentage of PDGF-B positive cells of adventitia in group S was peaked at 4W, whereas the percentage of adventitia in group NS peaked at 2W, and the percentage of adventitia in group S was greater than in group NS at 4W (P < 0.05).

CONCLUSIONSNon-restrictive external stenting inhibits the hyperplasia of the intima and media of the vein grafts and reduces the thickness and area of the intima and media; Non-restrictive external stenting inhibits the synthesis of PDGF and changes its distribution, and then inhibits the hyperplasia of the intima.

Animals ; Female ; Graft Occlusion, Vascular ; prevention & control ; Image Processing, Computer-Assisted ; Immunohistochemistry ; Jugular Veins ; transplantation ; Male ; Models, Animal ; Platelet-Derived Growth Factor ; physiology ; Proto-Oncogene Proteins c-sis ; Rabbits ; Stents

Angiopoietins ; metabolism ; physiology ; Animals ; Cell Proliferation ; Endothelial Cells ; cytology ; physiology ; Humans ; Neoplasms ; blood supply ; Neovascularization, Pathologic ; physiopathology ; Neovascularization, Physiologic ; physiology ; Pericytes ; cytology ; metabolism ; physiology ; Proto-Oncogene Proteins c-sis ; metabolism ; physiology ; Receptor, Platelet-Derived Growth Factor beta ; metabolism ; physiology ; Receptor, TIE-2 ; metabolism ; physiology ; Signal Transduction

OBJECTIVETo study the expression and significance of platelet derived growth factor (PDGF) and its receptor (PDGFR) in liver tissues of patients with chronic hepatitis fibrosis and liver cirrhosis.

METHODSThe expression, distribution, quantitation, and correlation of PDGF-A, PDGF-B, PDGFR-alpha, PDGFR-beta, and alpha-SMA in the liver tissues were analyzed by immunohistochemical techniques in 21 patients with chronic hepatitis and 42 patients with liver cirrhosis.

RESULTSIn the liver tissues of chronic hepatitis and liver cirrhosis, PDGF and its receptor and alpha-SMA mainly distributed in the fibrotic septa and the infiltration area of inflammation, particularly in branch spindle-shaped cells (activated HSC). The expression of PDGF-B and PDGFR-beta was stronger than that of PDGF-A and PDGFR-alpha with a significant difference between them (P<0.05 approximately 0.01). The expression and distribution of alpha-SMA was basically identical with the expression and distribution of PDGF-A, PDGF-B and PDGFR-alpha, PDGFR-beta and quantitative analysis showed a positive correlation (r=0.606, P<0.001).

CONCLUSIONSPDGF and PDGFR play a key role in liver fibrogenesis and development. The biologic effects of PDGF are elicited through activising HSC. Inhibiting PDGF and its receptor is a new approach to the treatment of liver fibrosis.

Actins ; biosynthesis ; physiology ; Adolescent ; Adult ; Aged ; Female ; Hepatocytes ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; Male ; Middle Aged ; Muscle, Smooth ; chemistry ; Platelet-Derived Growth Factor ; biosynthesis ; physiology ; Proto-Oncogene Proteins c-sis ; biosynthesis ; physiology ; Receptor, Platelet-Derived Growth Factor alpha ; biosynthesis ; physiology ; Receptor, Platelet-Derived Growth Factor beta ; biosynthesis ; physiology

BACKGROUNDAngiotensin II (AngII) and platelet-derived growth factor (PDGF)-BB can induce hypertrophy in the cultured rat cardiomyocytes through different signal transduction pathways. AngII stimulates growth through G protein coupled receptor (GPCR), while PDGF-BB acts via receptor tyrosine kinase (RTK). Although there has been much development on the individual AngII and PDGF-BB mediated signal pathways, little is known about the interactions between these two factors. Therefore, the crosstalk between AngII and PDGF-BB mediated signal pathways in the rat cardiomyocytes was investigated in this study.

METHODSPrimary culture of neonatal rat ventricular myocytes was prepared. The amount of tyrosine-phosphorylated and non-phosphorylated PDGF-beta receptor, G(alphaq/11), and phospholipase C (PLC) beta(3) were measured by immunoblotting analysis. The statistical analysis was done by one-way ANOVA.

RESULTSTyrosine-phosphorylated PDGF-beta receptor was increased by 120.60% at 1 minute and recovered to the control level at 10 minutes after AngII stimulation. Phosphorylation of PDGF-beta receptor triggered by AngII was blocked by losartan, a specific antagonist of AT1 receptor. PLC inhibitor U73122, protein kinase C (PKC) inhibitor staurosporine (STS) and mitogen-activated ERK activating kinase (MEK) inhibitor PD98059 also inhibited the AngII-induced phosphorylation of PDGF-beta receptor. PDGF-BB slightly increased the expression of G(alphaq/11) protein.

CONCLUSIONAngII transactivates PDGF-beta receptor via AT(1) receptor-G(alphaq/11)-PLC-PKC pathway in the rat cardiomyocytes. ERK also participates in the transactivation of PDGF-beta receptor triggered by AngII.

Angiotensin II ; pharmacology ; Animals ; Cells, Cultured ; GTP-Binding Protein alpha Subunits, Gq-G11 ; metabolism ; Myocytes, Cardiac ; metabolism ; Phosphorylation ; Platelet-Derived Growth Factor ; pharmacology ; Proto-Oncogene Proteins c-sis ; Rats ; Rats, Wistar ; Receptor, Platelet-Derived Growth Factor beta ; metabolism ; Signal Transduction ; physiology ; Type C Phospholipases ; physiology

Platelet-rich plasma (PRP) contains growth factors that promote tissue regeneration. Previously, we showed that heparin-conjugated fibrin (HCF) exerts the sustained release of growth factors with affinity for heparin. Here, we hypothesize that treatment of skin wound with a mixture of PRP and HCF exerts sustained release of several growth factors contained in PRP and promotes skin wound healing. The release of fibroblast growth factor 2, platelet-derived growth factor-BB, and vascular endothelial growth factor contained in PRP from HCF was sustained for a longer period than those from PRP, calcium-activated PRP (C-PRP), or a mixture of fibrin and PRP (F-PRP). Treatment of full-thickness skin wounds in mice with HCF-PRP resulted in much faster wound closure as well as dermal and epidermal regeneration at day 12 compared to treatment with either C-PRP or F-PRP. Enhanced skin regeneration observed in HCF-PRP group may have been at least partially due to enhanced angiogenesis in the wound beds. Therefore, this method could be useful for skin wound treatment.
Animals ; Blotting, Western ; *Cell Proliferation ; Dermis/cytology/metabolism ; Female ; Fibrin/*metabolism ; Fibroblast Growth Factor 2/genetics/metabolism ; Heparin/metabolism ; Immunoenzyme Techniques ; Intercellular Signaling Peptides and Proteins/*secretion ; Mice ; Mice, Inbred BALB C ; Platelet-Rich Plasma/*metabolism ; Proto-Oncogene Proteins c-sis/genetics/metabolism ; RNA, Messenger/genetics ; Real-Time Polymerase Chain Reaction ; Regeneration ; Skin/*cytology/*metabolism ; Vascular Endothelial Growth Factor A/genetics/metabolism ; Wound Healing/*physiology

OBJECTIVETo investigate the role of spleen tyrosine kinase (Syk) in rat pulmonary vascular smooth muscle cells (PVSMCs) proliferation induced by platelet-derived growth factor-BB (PDGF-BB).

METHODSPVSMCs from male Sprague-Dawley rats were cultured in vitro and the cells of passages 3-5 were used in the experiment. PVSMCs were stimulated by PDGF-BB and were treated with three different doses of piceatannol, a Syk selective inhibitor. Cell proliferation was assessed by methyl thiazolyl tetrazolium (MTT) assay. DNA synthesis was measured by ³H-thymidine incorporation (³H-TdR). Cellular cycle was observed by flow cytometry. Syk mRNA and protein expression were detected using real-time quantitative PCR and Western blot, respectively.

RESULTSThe expression of Syk protein of PVSMCs was significantly up-regulated following PDGF-BB stimulation. PDGF-BB stimulation dramatically increased PVSMCs proliferation. After piceatannol treatment, both Syk mRNA and protein expression decreased and the proliferation of PVSMCs was inhibited in a dose-dependent manner.

CONCLUSIONSSyk may promote PVSMCs proliferation induced by PDGF-BB.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Hypertension, Pulmonary ; pathology ; Intracellular Signaling Peptides and Proteins ; analysis ; genetics ; physiology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; Platelet-Derived Growth Factor ; pharmacology ; Protein-Tyrosine Kinases ; analysis ; genetics ; physiology ; Proto-Oncogene Proteins c-sis ; Pulmonary Artery ; cytology ; Rats ; Rats, Sprague-Dawley ; Stilbenes ; pharmacology ; Syk Kinase

OBJECTIVESThe aim of the present study was to determine the effects of angiotensin-converting enzyme inhibitor, perindopril, on the progression of rat hepatic fibrosis induced by CCl4.

METHODSMale wistar rats weighting about 250g were treated with perindopril (2mg/kg, daily gavage), except for model group and control group. After 4, 6 weeks, morphological examination was based on microscopy. RT-PCR was utilized to detect gene expression of angiotensin II type 1 receptor (AT1 receptor) in the liver. Meanwhile, the protein expressions of AT1 receptor, transforming growth factor beta 1 (TGF-beta1) and platelet-derived growth factor-BB (PDGF-BB) in liver tissue were examined by Western blot. The activity of matrix metalloproteinase-2 (MMP-2) was assessed by zymography. Serum laminin (LN) and hyaluronic acid (HA) were measured using radio-immunity technique.

RESULTSRT-PCR and Western blot revealed that there was a up-regulation in AT1 receptor expression in model group compared with control group. Perindopril treatment significantly reduced mean fibrosis score, messenger RNA and protein levels of AT1 receptor, protein levels of TGF-beta1 and PDGF-BB, Serum levels of HA and LN, and MMP-2 activity.

CONCLUSIONThese results suggest that angiotensin II may play an important role in fibrosis of liver. Perindopril may have a inhibiting effect on CCl4-induced hepatic fibrogenesis of rat.

Angiotensin II ; physiology ; Angiotensin II Type 1 Receptor Blockers ; Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Blotting, Western ; Carbon Tetrachloride ; toxicity ; Liver ; pathology ; Liver Cirrhosis, Experimental ; chemically induced ; prevention & control ; Male ; Perindopril ; pharmacology ; Platelet-Derived Growth Factor ; antagonists & inhibitors ; Proto-Oncogene Proteins c-sis ; Rats ; Rats, Wistar ; Transforming Growth Factor beta ; antagonists & inhibitors ; Transforming Growth Factor beta1

OBJECTIVEConstruction of a small interfering RNA (siRNA) eukaryotic expression vector specific to mouse myocardin gene and study on the role of myocardin-siRNA on differentiation of mouse bone mesenchymal stem cells (MSCs) into smooth muscle-like cells induced by PDGF-BB in vitro.

METHODSMouse MSCs were isolated from bone marrow and cultured with 50 mg/L PDGF-BB and fetal bovine serum (20%). Specific myocardin-siRNA sequence was cloned into a plasmid pGenesil-1.0 vector, which contained U6 promoter. The recombinant plasmid and control plasmid were transfected into MSCs which had been cultured with PDGF-BB for 6 days beforehand. The expression of myocardin mRNA was detected by RT-PCR 48 hours after the transfection. Immunohistochemistry was used to detect the SM-MHC and to identify the smooth muscle-like cells.

RESULTSThe recombinant plasmids carrying myocardin-siRNA sequences were constructed successfully and the myocardin mRNA was reduced 42.86% by pGen-myo-shRNA in comparing with that of the controls (P<0.01); and the expression of SM-MHC protein was down-regulated (P<0.01).

CONCLUSIONSubset of mouse MSCs have the potential to differentiate into smooth muscle-like cells, a possible cell source responsible for atherosclerotic plaque formation, and myocardin expression may play an important role during this process.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Down-Regulation ; Gene Silencing ; Genetic Vectors ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Myosin Heavy Chains ; metabolism ; Nuclear Proteins ; biosynthesis ; genetics ; physiology ; Plaque, Atherosclerotic ; pathology ; Plasmids ; Platelet-Derived Growth Factor ; pharmacology ; Proto-Oncogene Proteins c-sis ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Trans-Activators ; biosynthesis ; genetics ; physiology ; Transfection