Platelet-derived Growth factor-BB Group Research Articles

Role and mechanism of platelet-derived growth factor BB in thrombocytosis in Kawasaki disease

To study the role and mechanism of platelet-derived growth factor BB (PDGF-BB) on platelet production in Kawasaki disease (KD) mice and human megakaryocytic Dami cells through in vitro and invivo experiments. ELISA was used to measure the expression of PDGF in the serum of 40 children with KD and 40 healthy children. C57BL/6 mice were used to establish a model of KD and were then randomly divided into a normal group, a KD group, and an imatinib group (30 mice in each group). Routine blood test was performed for each group, and the expression of PDGF-BB, megakaryocyte colony forming unit (CFU-MK), and the megakaryocyte marker CD41 were measured. CCK-8, flow cytometry, quantitative real-time PCR, and Western blot were used to analyze the role and mechanism of PDGF-BB in platelet production in Dami cells. PDGF-BB was highly expressed in the serum of KD children (P<0.001). The KD group had a higher expression level of PDGF-BB in serum (P<0.05) and significant increases in the expression of CFU-MK and CD41 (P<0.001), and the imatinib group had significant reductions in the expression of CFU-MK and CD41 (P<0.001). In vitro experiments showed that PDGF-BB promoted Dami cell proliferation, platelet production, mRNA expression of PDGFR-β, and protein expression of p-Akt (P<0.05). Compared with the PDGF-BB group, the combination group (PDGF-BB 25 ng/mL + imatinib 20 μmol/L) had significantly lower levels of platelet production, mRNA expression of PDGFR-β, and protein expression of p-Akt (P<0.05). PDGF-BB may promote megakaryocyte proliferation, differentiation, and platelet production by binding to PDGFR-β and activating the PI3K/Akt pathway, and the PDGFR-β inhibitor imatinib can reduce platelet production, which provides a new strategy for the treatment of thrombocytosis in KD.

Effects of probenecid on the migration and proliferation ability of platelet derived growth factor-BB induced pulmonary artery smooth muscle cells in rats

To investigate whether probenecid (PROB) could improve the proliferation and migration ability of rats' pulmonary artery smooth muscle cells induced by platelet-derived growth factor-BB (PDGF-BB). Primary pulmonary artery smooth muscle cells (PASMCs) of SD rats were cultured in vitro, and were randomly divided into control group (CON group), PDGF-BB group (10 ng/ml PDGF-BB treatment for 24 h) and PDGF-BB+PROB group (10 ng/ml PDGF-BB and 200 μmol/L PROB treatment for 24 h, PROB is a specific blocker of pannexin-1). CCK-8 method was used to select the suitable intervention concentrations of PROB and PDGF-BB, and to detect the proliferation of PASMCs in each group. The migration ability of PASMCs was detected by TranswellTM assay and cell scratch test. Immunofluorescence cytochemistry and Western blot were used to detect the protein expressions and distribution of osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) in PASMCs. Compared with CON group, the migration and proliferation ability of PASMCs in PDGF-BB group were enhanced (P＜0.05). After treated with PROB, the migration and proliferation ability of PASMCs in PDGF-BB+PROB group were decreased significantly (P＜0.05). Compared with CON group, the expression and protein levels of OPN and PCNA in PDGF-BB group were increased significantly (P＜0.05), while the expression and protein levels of OPN and PCNA in PDGF-BB+PROB were decreased significantly (P＜0.05). Probenecid inhibits the migration and proliferation of PDGF-BB-induced PASMCs by blocking Pannexin-1.

Platelet-derived growth factor-BB regenerates functional periodontal ligament in the tooth replantation

Tooth ankylosis is a pathological condition of periodontal ligament (PDL) restoration after tooth replantation. Platelet-derived growth factor-BB (PDGF-BB) has been proposed as a promising factor for preventing tooth ankylosis. Using rat tooth replantation model, we investigated whether PDGF-BB accelerates the repair of PDL after tooth replantation without ankylosis, and its molecular mechanisms. In PDGF-BB pretreated replanted teeth (PDGF-BB group), ankylosis was markedly reduced and functionally organized PDL collagen fibers were restored; the mechanical strength of the healing PDL was restored to an average of 76% of that in non-replanted normal teeth at 21 days. The numbers of PDGF-Rβ- and BrdU-positive cells in the periodontal tissues of the PDGF-BB group were greater than those of atelocollagen pretreated replanted teeth (AC group). Moreover, in the PDGF-BB group, the periodontal tissues had fewer osteocalcin-positive cells and decreased number of nuclear β-catenin-positive cells compared to those in the AC group. In vitro analyses showed that PDGF-BB increased the proliferation and migration of human periodontal fibroblasts. PDGF-BB downregulated mRNA expressions of RUNX2 and ALP, and inhibited upregulatory effects of Wnt3a on β-catenin, AXIN2, RUNX2, COL1A1, and ALP mRNA expressions. These findings indicate that in tooth replantation, topical PDGF-BB treatment enhances cell proliferation and migration, and inhibits canonical Wnt signaling activation in bone-tooth ankylosis, leading to occlusal loading of the PDL tissues and subsequent functional restoration of the healing PDL. This suggests a possible clinical application of PDGF-BB to reduce ankylosis after tooth replantation and promote proper regeneration of PDL.

LncRNA H19 regulates smooth muscle cell functions and participates in the development of aortic dissection through sponging miR-193b-3p.

Background: Multiple studies showed that long-chain noncoding RNA H19 (LncRNA H19) is high-expressed in human and mouse abdominal aortic aneurysms (AAAs). We speculated that it plays an important role in arterial disease, and therefore studied the role and mechanism of H19 in aortic dissection (AD).Methods: The expressions of related genes in human aortic smooth muscle cells (HASMCs) induced by platelet-derived growth factor BB (PDGF-BB) or in the aortic tissue of AD patients/mice were identified by Western blot and quantitative real-time polymerase chain reaction. The targeting relationship between H19 and miR-193b-3p was predicted and verified by bioinformatics analysis, dual luciferase assay, RNA pull-down assay, RNA immunoprecipitation (RIP), and Pearson correlation coefficient. The H19 and miR-193b-3p effects on the biological functions of tissues and cells were examined by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, thiazolyl blue tetrazolium bromide) assay, wound-healing assay, and Hematoxylin–Eosin (HE) staining.Results: LncRNA H19 was abnormally high-expressed in thoracic aorta tissues of AD patients, and it could competitively bind to and inhibit miR-193b-3p. In the PDGF-BB group, the expressions of H19, matrix metallopeptidase (MMP) 2 (MMP-2) and MMP-9 were up-regulated and the expressions of miR-193b-3p, α-SMA, and SM22α were down-regulated; moreover, the proliferation and migration rate of HASMCs were increased. However, H19 silencing reversed the regulation of PDGF-BB on HASMCs. More interestingly, miR-193b-3p inhibitor could partially reverse the effect of H19 silencing. In addition, the above results were verified by animal experiments, showing that shH19 and up-regulated miR-193b-3p could significantly reduce the thoracic aorta pathological damage in AD mice.Conclusion: LncRNA H19 regulated smooth muscle cell function by sponging miR-193b-3p and it participated in the development of AD.

Induction of tenogenic differentiation of equine adipose-derived mesenchymal stem cells by platelet-derived growth factor-BB and growth differentiation factor-6.

Managingtendon healing process is complicated mainly due tothe limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and have been considered for tendon repair and regeneration. This study aimed to evaluate the capacity of equine adipose tissue-derived cells (eASCs) to differentiate into tenocytes in response to platelet-derived growth factor-BB (PDGF-BB) and growth differentiation factor-6 (GDF-6) in vitro. Frozen characterized eASCS of 3 mares were thawed and the cells were expanded in basic culture medium (DMEM supplementedwith 10% FBS). The cells at passage 5 were treated for 14 days in different conditions including: (1) control group in basic culture medium (CM), (2) induction medium as IM (CM containing L-prolin, and ascorbic acid (AA)) supplemented with PDGF-BB (20ng/ml), (3) IM supplemented with GDF-6 (20ng/ml), and (4) IM supplemented with PDGF-BB and GDF-6. At the end of culture period (14th day), tenogenic differentiation was evaluated. Sirius Red staining was used to assess collagen production, and H&E was used for assessing cell morphology. mRNA levels of collagen type 1 (colI), scleraxis (SCX), and Mohawk (MKX), as tenogenic markers, were analyzed using real-time reverse-transcription polymerase chain reaction (qPCR). H&E staining showed a stretching and spindle shape (tenocyte-like) cells in all treated groups compared to unchanged from of cells in control groups. Also, Sirius red staining data showed a significant increase in collagen production in all treated groups compared with the control group. MKX expression was significantly increased in PDGF-BB and mixed groups and COLI expression was significantly increased only in PDGF-BB group. In conclusion, our results showed that PDGF-BB and GDF-6 combinationcould induce tenogenic differentiation in eASCs. These in vitro findings could be useful for cell therapy in equine regenerative medicine.

Platelet derived growth factor-BB regulates phenotype transformation of pulmonary artery smooth muscle cells via SIRT3 affecting glycolytic pathway

Objective: To investigate whether platelet-derived growth factor-BB (PDGF-BB) can regulate phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs) via SIRT3 affecting glycolytic pathway. Methods: The PASMCs were isolated from Sprague Dawley rats. PASMCs were divided into 3 groups by using 2-deoxyglucose (2-DG), an inhibitor of the glycolytic pathway: normal control group, PDGF-BB group(30 ng/ml) and PDGF-BB (30 ng/ml)+2-DG (10 mmol/L) group. In lentivirus-mediated overexpression assay, cells were divided into control group, PDGF-BB group(30 ng/ml), PDGF-BB+deacetylase sirtuin-3 (SIRT3) overexpression group and PDGF-BB+empty vector group. The expression levels of phenotype related index such as α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), calponin, vimentin were detected by qRT-PCR and Western blot. Meanwhile, the expression of α-SMA was detected by cellular immunofluorescence staining. EDU staining was used to detect the proliferation of PASMCs. The expression of SIRT3 was detected by Western blot. The expressions of glucose transporter 1 and aerobic glycolytic enzymes were detected by qRT-PCR and Western blot in lentivirus-mediated overexpression assay. Results: (1) PDGF-BB affects PASMCs phenotypic transformation through glycolytic pathway: compared with normal control group, PDGF-BB significantly decreased the expressions of contractile phenotype markers such as α-SMA, SM-MHC, calponin mRNA and protein (all P<0.05), but it increased the expressions of the synthetic phenotype marker vimentin mRNA and protein (both P<0.05). Cellular immunofluorescence assay showed that PDGF-BB significantly decreased the number of α-SMA positive cells, while 2-DG reversed the process. (2) PDGF-BB promoted cell proliferation through glycolytic pathway: the proliferation of PASMCs was significantly higher in PDGF-BB group than in control group (P<0.05), and which could be significantly reduced by 2-DG (P<0.05). (3) PDGF-BB inhibited the expression of SIRT3 protein in PASMCs: the expression of SIRT3 protein in PDGF-BB group was lower than that in control group (P<0.05). (4) PDGF-BB affected glycolytic pathway through SIRT3:compared with the control group, PDGF-BB significantly increased the expression levels of glucose transporter 1 (Glut1), hexokinase 2 (HK2) and 6-phosphfructo-2-kinase 3 (PFKFB3) mRNA (all P<0.05), which was reserved by over-expression of SIRT3. There were no significant difference in mRNA expression levels between PDGF-BB group and PDGF-BB+empty vector group (P>0.05).Compared with the control group, PDGF-BB significantly increased the expression levels of Glut1, HK2 and PFKFB3 protein(all P<0.05), which was reserved by over-expression of SIRT3. There were no significant differences in protein expression levels between PDGF-BB group and PDGF-BB+empty vector group (all P>0.05). Conclusion: PDGF-BB regulates phenotypic transformation of PASMCs via SIRT3 affecting glycolytic pathway.

Endostatin attenuates PDGF-BB- or TGF-β1-induced HSCs activation via suppressing RhoA/ROCK1 signal pathways.

AimTo testify the hypothesis that endostatin exerts antifibrotic effects in hepatic stellate cells (HSCs) by modulating RhoA (ras homolog gene family, member A)/ROCK 1 (Rho-associated protein kinase 1) signal pathways.Materials and methodsHSCs-T6 of passages 3–5 were cultured in DMEM and serum starved for 48 hours. HSCs were grouped as follows: control group, TGF-β1 (transforming growth factor β1) group, endostatin+TGF-β1 group, PDGF-BB (platelet-derived growth factor-BB) group, and endostatin+PDGF-BB group. In the PDGF-BB group, HSCs were treated with PDGF-BB (200 ng/mL) for 72 hours; in the TGF-β1 group, they were treated with TGF-β1 (10 ng/mL) for 72 hours. In the Endostatin+TGF-β1 group or Endostatin+PDGF-BB group, HSCs were treated with TGF-β1 (10 ng/mL) or PDGF-BB (200 ng/mL) for 72 hours after pretreatment with endostatin (5 µg/mL) for 1 hour. In the control group, HSCs were only treated with serum-free DMEM for 72 hours. Collagen I was analyzed with ELISA. F-actin was detected with immunofluorescent staining. The mRNAs and proteins of α-smooth muscle actin, RhoA, and ROCK1 were analyzed by using real-time PCR and Western blot, respectively.ResultsTGF-β1 and PDGF-BB promote the proliferation of HSCs significantly at 48 and 72 hours. Endostatin inhibits the proliferation effect induced by TGF-β1 or PDGF-BB significantly (P<0.01). The expression of collagen I and F-actin was significantly upregulated in both TGF-β1 and PDGF-BB groups than in the control group (P<0.01). Both the collagen I and F-actin expression were downregulated significantly in the endostatin-treated groups (P<0.05). Endostatin significantly inhibited the upregulated expression of α-smooth muscle actin, RhoA, and ROCK1 induced by TGF-β1 or PDGF-BB (P<0.01).ConclusionThese results suggested that endostatin inhibited TGF-β1- or PDGF-BB-induced fibrosis in HSCs by modulating RhoA/ROCK signal pathways.

Mesenchymal Stem Cells Stimulated by Growth Factors Release Exosomes with Potent Proangiogenic Activity

To explore the proangiogenic activity of exsomes released by human umbilical cord mesenchymal stem cells (MSCs) stimulated by erythropoietin and platelet-derived growth factor BB (PDGF-BB). Human umbilical cord-derived MSCs were seeded and maintained in culture overnight. The media were then replaced by alpha-MEM containing EPO (1 U/ml) and/or PDGF-BB (50 ng/ml), and the culture was maintained for 72 hours. The exosomes from the culture supernatants were isolated with a routine ultra-catrifagation method. Flow cytometric analysis was performed to identify the origin of the exosomes, and their morphological features were observed by using a transmission electron microscopy. The exosomes were added at a concentration of 10 µg/ml into the culture system of human umbilical cord vein endothelial cells. MTT assay was used to evaluate the proliferative status. The Matrigel assay was used to observe the formation of net-work structures which were calculated after culture for 12 hours. Flow cytometric analysis showed that microparticles released by human umbilical cord MSCs expressed CD9, CD63 and CD81, which was in accordance with the surface molecular features of exosomes. Under an electron microscope, the exosomes took the featured cystic shape. The protein contents of exosomes released by untreated, EPO-stimulated, PDGF-BB-stimulated and EPO plus PDGF-BB stimulated MSCs (108 cells) were 256±124 µg, 1021±392 µg, 830±265 µg and 2207±733 µg, respectively. The results revealed that MSCs treated by EPO and PDGF-BB released significantly higher amounts of exosomes (P<0.01). MTT assay proved that the exosomes from EPO and PDGF-BB treated MSCs had more potent proliferation-promoting activity on human umbilical cord vein endothelial cells than those from untreated MSCs. The Matrigel assay showed that the numbers of capillary-like structures in untreated, EPO-, PDGF-BB and EPO plus PDGF-BB-treated groups were 2.6±0.84, 4.6±1.57, 4.2±0.78 and 6.3±1.34 per high power objective. Treatment with EPO or PDGF-BB dramatically enhanced the numbers of capillary liue structure, compared with that of untreated group (P<0.01) and those in EPO and PDGF-BB combination group was significantly greater than those of EPO or PDGF-BB group (P<0.01). EPO and PDGF-BB can stimulate MSCs to release exosomes with more potent proangiogenic activity.

Oxymatrine suppresses MOVAS proliferation induced by platelet-derived growth factor via G0/G1 arrest

Objective: The traditional Chinese medicine Oxymatrine (OMT) has numerous biological effects, such as anti-inflammatory, anti-fibrosis, anti-proliferation and anti-tumor. However, the underlying effect of OMT on vascular smooth muscle cells remains unclear. Herein, the aim of this study is to investigate the role and mechanism of OMT on MOVAS (a mouse vascular aortic smooth muscle cell line) proliferation induced by Platelet-Derived Growth Factor-BB (PDGF-BB). Methods: Firstly, we assessed proliferation in MOVAS subjected to PDGF-BB induction or controlled intervention with/without OMT treatment. And then we detected cell cycle, cell apoptosis and the expression levels of cyclins, Cyclin-Dependent Kinases (CDKs) and p21 of each group. Results: In the present study, we found that OMT remarkably restrained the proliferation induced by PDGF-BB stimulus. Additionally, it was demonstrated that cell population of MOVAS treated with OMT in the S phase was reduced, but that of MOVAS in the G0/G1 phase was increased when compared to the PDGF-BB group. However, OMT treatment showed no effect on MOVAS apoptosis. Mechanistically, the expression of cyclinD1-CDK4/6 and cyclinE2-CDK2 were inhibited, while the expression of p21 was increased in MOVAS treated with OMT. Conclusion: These results demonstrated that the inhibitory effect of OMT on proliferation of MOVAS were in part due to G0/G1 arrest by inhibiting cyclinD1-CDK4/6 and cyclinE2-CDK2, and promoting p21 expression. Therefore, OMT might become a new strategy for treating excessive proliferation of vascular smooth muscle cells in the future.

Effects of endostatin pretreatment on fibrosis of human skin fibroblasts and the mechanisms

Objective: To explore the effects of endostatin pretreatment on fibrosis of human skin fibroblasts and the mechanisms. Methods: Human skin fibroblasts were routinely cultured in vitro, and then the cells of passage 3 to 5 were used in the following experiments. The cells were divided into blank control, endostatin, platelet-derived growth factor-BB (PDGF-BB), endostatin+ PDGF-BB, transforming growth factor-β(1) (TGF-β(1)), and endostatin+ TGF-β(1) groups according to the random number table, with 3 wells in each group. Cells in blank control group were cultured with DMEM medium for 24 h. Cells in endostatin group were cultured with DMEM medium containing 5 μg/mL endostatin for 24 h. Cells in PDGF-BB group and TGF-β(1) group were cultured with DMEM medium containing 200 ng/mL PDGF-BB and 10 ng/mL TGF-β(1) for 24 h, respectively. Cells in endostatin+ PDGF-BB group were pretreated with DMEM medium containing 5 μg/mL endostatin for 48 h and then cultured with DMEM medium containing 200 ng/mL PDGF-BB for 24 h. Cells in endostatin+ TGF-β(1) group were pretreated with DMEM medium containing 5 μg/mL endostatin for 48 h and then cultured with DMEM medium containing 10 ng/mL TGF-β(1) for 24 h. The content of type Ⅰ collagen in the cell culture supernatant of three wells in each group was determined by enzyme-linked immunosorbent assay. The protein expression levels of α-smooth muscle actin (α-SMA), PDGF receptor β (PDGFRβ), phosphorylated PDGFRβ (p-PDGFRβ), and phosphorylated extracellular signal-regulated protein kinases 1/2 (p-ERK1/2) of three wells in each group were detected by Western blotting. Data were processed with one-way analysis of variance and SNK test. Results: (1) Compared with (5.05±0.29) pg/mL in blank control group, content of type Ⅰ collagen in the cell culture supernatant of endostatin group [(4.72±0.37) pg/mL] was close to it (P>0.05), content of type Ⅰ collagen in the cell culture supernatant of PDGF-BB group and TGF-β(1) group [(8.60±0.57) and (9.20±0.64) pg/mL, respectively] was higher (with P values below 0.05). Content of type Ⅰ collagen in the cell culture supernatant of endostatin+ PDGF-BB group [(5.32±0.17) pg/mL] was lower than that of PDGF-BB group (P<0.05), and content of type Ⅰ collagen in the cell culture supernatant of endostatin+ TGF-β(1) group [(5.41±0.20) pg/mL] was lower than that of TGF-β(1) group (P<0.05). (2) Compared with those in blank control group, protein expression levels of α-SMA, PDGFRβ, p-PDGFRβ, and p-ERK1/2 of cells in endostatin group showed no obvious differences (with P values above 0.05), while those in PDGF-BB and TGF-β(1) group were significantly higher (with P values below 0.01). Protein expression levels of α-SMA, PDGFRβ, p-PDGFRβ, and p-ERK1/2 of cells in endostatin+ PDGF-BB group and endostatin+ TGF-β(1) group were significantly lower than those in PDGF-BB group and TGF-β(1) group, respectively (with P values below 0.05). Conclusions: Pretreatment of endostatin can inhibit the fibrosis of human skin fibroblast and its transformation into myofibroblast, which may be related to the down-regulation of protein expression of p-PDGFRβ, PDGFRβ, and p-ERK.

The effects of miRNA-145 on the phenotypic modulation of rat corpus cavernosum smooth muscle cells.

To investigate the effect of miR-145 on the phenotypic modulation in rat corpus cavernosum smooth muscle cells. Corpus cavernosum smooth muscle cells were treated with either miR-145 mimics or miR-145 negative control. Cell proliferation were analyzed by the MTS assay and colony formation assay. Wound healing assay were performed to detect the effect of miR-145 on cell migration. The mRNA and protein levels of phenotype marker proteins were assessed by quantitative real-time polymerase chain reaction and western blotting. The intracavernosal pressure and mean arterial pressure were measured to assess erectile function at one month after the injection of platelet-derived growth factor-BB and miR-145. Our results showed that miR-145 inhibited the proliferation and migration of cavernosal smooth muscle cells. Smooth muscle cell phenotypic markers were also affected by overexpression of miR-145, as indicated by the increase in α-smooth muscle actin, calponin and smooth muscle myosin heavy chain expression. Moreover, significantly attenuated erectile function was observed in the platelet-derived growth factor-BB group as compared with the platelet-derived growth factor-BB+miR-145 group. These findings indicated that miR-145 regulate phenotypic modulation of corpus cavernosum smooth muscle cells.

The Role of Platelet-Derived Growth Factor Receptor in Early Brain Injury Following Subarachnoid Hemorrhage

This study aims to observe encephaledema and cell apoptosis in rats following subarachnoid hemorrhage (SAH) and to explore the mechanism of platelet-derived growth factor receptor (PDGFR) in the development of early brain injury (EBI). Adult and male Sprague-Dawley rats were randomly divided into 4 groups: sham operation, SAH, SAH + imatinib, and SAH + platelet-derived growth factor-BB (PDGF-BB). SAH model was established using intravascular silk puncture of the internal carotid artery crotch. The SAH + imatinib group was treated with intraperitoneal injection of imatinib 1 hour before establishing the model. The SAH + PDGF-BB group was administered with intracerebroventricular injection of PDGF-BB 1 hour before establishing the model. The mortality, encephaledema, and nerve functional scoring were observed after 24 hours in all groups. The expression of caspase-3 in hippocampus was tested by reverse transcription polymerase chain reaction and Western blotting. Mortality and encephaledema were the highest in the SAH + PDGF-BB group, which were alleviated when the rats were injected with imatinib (P < .01). PDGFR may participate in the pathogenesis of EBI following SAH. The antagonist of PDGFR, imatinib, can reduce brain damage to some degree.

Effect of astaxanthin on vascular smooth muscle cells proliferation induced by platelet derived growth factor-BB

To investigate the effect of astaxanthin (AST) on vascular smooth muscle cells (VSMCs) proliferation in vitro induced by platelet derived growth factor-BB (PDGF-BB), and to explore its possible mechanism. There were 4 groups in this experiment: blank control group, PDGF-BB group, PDGF-BB+ AST group, AST group. After the cells received different intervention for the indicated time, the cell growth was determined by Trypan blue staining; cell proliferation was demonstrated using CCK-8 kit; the cell cycle progression was analyzed by flow cytometry, and the mRNA expression of cyclin D1, cyclin E, CDK6, CDK4, cyclin kinase inhibitor protein P21 was determined by real-time PCR; reactive oxygen species (ROS) generation was detected using a Microplate reader; the total and phosphorylated forms of ERK1/2, p38 MAPK, JNK was observed in AST pretreated VSMCs in 5, 10 and 15 min after PDGF-BB treatment by Western blot analysis. (1) Cell viability: AST and/or PDGF-BB did not induce VSMCs necrosis with the different concentration compared with untreated cells (P>0.05). (2) Cell proliferation: PDGF-BB induced VSMCs proliferation (2.5±0.3 vs 1, P<0.01), while AST reversed the effect in a concentration-dependent manner when co-treated with PDGF-BB (all P<0.01); Cell cycle: Flow cytometry analysis showed that AST at a dose of 25 μmol/L reduced the percentages of cells in S phase and increased the G0/G1 populations in PDGF-BB-stimulated VSMCs; mRNA expression of the check-point proteins: Real Time PCR results demonstrated that, compared with the control group, the mRNA expression of CDK6, CDK4, cyclin D1, cyclin E in the PDGF-BB group was higher (4.20±0.30, 2.90±0.18, 3.50±0.30, 2.70±0.11 vs 1, all P<0.01), while p21 mRNA expression was lower (0.52±0.03 vs 1, P<0.01), while AST reversed these effects when co-treated with PDGF-BB. (3) ROS expression: compared with the control group, ROS level was significantly higher in the PDGF-BB group (2.10±0.09 vs 1, P<0.01), while AST reversed the effect in a concentration-dependent manner when co-treated with PDGF-BB (all P<0.01). (4) Signaling pathway: AST blocking the proliferation of VSMCs induced by PDGF-BB was related to suppress ERK1/2, p-p38 MAPK signaling pathway, but little effect to JNK. Conclutions: These results demonstrate that AST can block the proliferation and migration of VSMCs through G0/G1 to S phase of the cell cycle arrest. Further study indicates that AST suppress PDGF-BB-induced VSMCs proliferation is associated with an inhibition of ROS generation and ERK1/2, p-p38 MAPK signal pathways.

Serum PDGF-BB, sFlt-1 and U-II in patients with preeclampsia

Objective To explore the clinical diagnostic value of joint detection of serum platelet-derived growth factor BB (PDGF-BB), soluble vascular endothelial growth factor receptor -1 (sFlt-1) and urotensinⅡ(U-Ⅱ) in preeclampsia disease. Methods The cases of obstetric patients suffering from preeclampsia in the Third People's Hospital of Liaocheng, Shandong Province between October 2012 and April 2014 were enrolled, including 96 cases of mild preeclampsia and 81 cases of severe preeclampsia.Totally 68 cases of normal pregnant women with similar age and gestational age were selected as control group. A case-control study was applied for the following investigations. The concentrations of serum PDGF-BB, sFlt-1 and U-Ⅱwere measured using ELISA. The diagnostic value of PDGF-BB, sFlt-1 and U-Ⅱalone or in combination for preeclampsia was analyzed and evaluated with receiver operating characteristic curve (ROC) and Logistic regression analysis. Results Serum concentrations of PDGF-BB, sFlt-1 and U-Ⅱin mild preeclampsia group were(80.45±21.87)ng/L, (23.03±6.67)μg/L and(4.54±1.02)ng/L, and those in severe preeclampsia group were(124.91±47.54)ng/L, (35.65±12.45)μg/L and(6.29±2.31)ng/L, while those in control group were(60.89±19.38)ng/L, (17.19±7.867)μg/L and(3.81±1.01)ng/L, respectively. The three parameters in mild preeclampsia group and severe preeclampsia group were significantly higher than those in control group (P<0.01; F value was 79.43, 79.28 and 50.72 respectively). In the same situation, these three indicators in severe preeclampsia group were significantly higher than those in mild group (P<0.01). Moreover, the AUC of serum PDGF-BB, sFlt-1 and U-Ⅱalone or in combination were 0.821, 0.786, 0.772 and 0.933, respectively. The differences between joint detection and three individual detections were statistically significant (P<0.05), as combined detection having a sensitivity of 95.7% and a specificity of 86.6%. Conclusion The combined detection of Serum PDGF-BB, sFlt-1 and U-Ⅱ had an important value in early assessment and treatment of preeclampsia.(Chin J Lab Med, 2015, 38: 528-531) Key words: Pre-eclampsia; Vascular endothelial growth factor receptor-1; Urotensins