Platelet-derived Growth Factor Group Research Articles

Immediate implant placement with platelet rich fibrin as space filling material versus deproteinized bovine bone in maxillary premolars: A randomized clinical trial.

Several biomaterials have been suggested to augment the jumping gap during immediate implant placement. The aim of this study was to compare the effect of xenograft or platelet derived growth factor (PRF) to graft the jumping gap in immediate implant placement in the maxillary premolar region. Twenty patients underwent atraumatic extraction followed by immediate placement. The patients were equally divided into two groups. The first group received xenograft as a jumping gap filling material. The second group received PRF to graft the jumping gap. All patients received preoperative, immediate postoperative, and 6months postoperative cone beam CT scan (CBCT). Implant stability quotient ISQ values were taken for the installed implants immediate postoperative and at 6months. Implants receiving PRF as a jumping gap graft material demonstrated a significantly greater amount of crestal bone loss 1.85 ± 0.89 mm as compared to xenograft group 0.77 ± 0.32 mm (t=3.52, p=0.005). PRF group showed significantly greater reduction in buccopalatal direction 1.63 mm as compared to xenograft group 0.59 mm (t=4014, p <0.001). ISQ values were similar immediately postoperative (t=0.070, p=0.945) while the ISQ values were significantly lower in PRF group as compared to xenograft graft at the six-month interval (t=0.248, p=0.023). The use of xenograft material as a jumping gap filling material resulted in superior results compared to PRF with regards to crestal bone loss, buccolingual socket reduction, and ISQ values.

A bioactive synthetic membrane improves bone healing in a preclinical nonunion model

ObjectivesHigh energy long bone fractures with critical bone loss are at risk for nonunion without strategic intervention. We hypothesize that a synthetic membrane implanted at a single stage improves bone healing in a preclinical nonunion model. MethodsUsing standard laboratory techniques, microspheres encapsulating bone morphogenic protein-2 (BMP2) or platelet derived growth factor (PDGF) were designed and coupled to a type 1 collagen sheet. Critical femoral defects were created in rats and stabilized by locked retrograde intramedullary nailing. The negative control group had an empty defect. The induced membrane group (positive control) had a polymethylmethacrylate spacer inserted into the defect for four weeks and replaced with a bare polycaprolactone/beta-tricalcium phosphate (PCL/β-TCP) scaffold at a second stage. For the experimental groups, a bioactive synthetic membrane embedded with BMP2, PDGF or both enveloped a PCL/β-TCP scaffold was implanted in a single stage. Serial radiographs were taken at 1, 4, 8, and 12 weeks postoperatively from the definitive procedure and evaluated by two blinded observers using a previously described scoring system to judge union as primary outcome. ResultsAll experimental groups demonstrated better union than the negative control (p = 0.01). The groups with BMP2 incorporated into the membrane demonstrated higher average union scores than the other groups (p = 0.01). The induced membrane group performed similarly to the PDGF group. Complete union was only demonstrated in groups with BMP2-eluting membranes. ConclusionsA synthetic membrane comprised of type 1 collagen embedded with controlled release BMP2 improved union of critical bone defects in a preclinical nonunion model.

Platelet-Derived Growth Factor-Modulated Guided Tissue Regeneration with a Bioresorbable Membrane in Class III Furcation Defects: A Histometric Study in the Monkey.

It was the aim of this study to histometrically evaluate guided tissue regeneration (bioresorbable membrane plus bone mineral) (GTR) with or without platelet-derived growth factor (PDGF) in two different types of class III furcation defects (small keyhole defects and horizonal defects) in monkeys. In six cynomolgus monkeys, two types of class III furcation defects were created and allowed to chronify for 5 months in mandibular first and second molars. After a hygiene program the molars were assigned to GTR group (collagen membrane plus bovine bone mineral), PDGF group (collagen membrane plus bovine bone mineral plus PDGF), or negative control group (flap reposition only). Histologic sections were made after 7 months of healing and descriptive statistics were provided from the histometric parameters. Postoperative healing was uneventful despite marginal membrane exposures in the GTR and PDGF group. Bone regeneration of 23–35% of the original defect area was found in the two treatment groups. In none of the evaluated key parameters (formation of bone, root cementum, connective tissue, or epithelium) differences were detected between GTR and PDGF groups. However, the negative control teeth exhibited better bone regeneration than the treatment groups. The type of class III defect did not influence the regenerative outcome. Within the limits of this study PDGF was not able to enhance the histologic regeneration of class III furcation areas in monkeys compared to bone mineral enhanced GTR treatment regardless of the defect configuration. Membrane exposure during early healing might have influenced these outcomes.

Effects of a novel biodegredable implant system on a rat tibia fracture model.

This study aimed to determine the effects of a novel biodegradable implant releasing platelet-derived growth factor (PDGF) at the fracture site on fracture healing in a rat tibia fracture model. In this study, 35 male Sprague-Dawley rats weighing between 300 and 350 g were used. The rats were divided into four groups: Group A (control group without any treatment, n=10), Group B (spacer without PDGF Group, n=10), Group C (spacer with PDGF group, n=10), and Group D (healthy rat Group, n=5). Standardized fractures were created in the right tibias of rats, and then biodegradable implants made of poly-β-hydroxybutyrate-co-3-hydroxy valerate were implanted at the fracture sites in Groups B and C. In Group C, implants were loaded with 600 ng of PDGF. Animals were sacrificed 30 days after the operation, and fracture healing in each group was assessed radiologically based on the Goldberg score. Furthermore, the anteroposterior (AP) and mediolateral (ML) callus diameters were measured macroscopically, and fracture sites were mechanically tested. In the radiological assessment, Group C showed higher fracture healing rate than Groups A and B (p=0.001), whereas no significant difference was found between group C and Group D (p>0.05). In the macroscopic assessment, while Group C exhibited the thickest AP callus diameter (p=0.02), no significant differences in ML callus diameters existed among the groups (p>0.05). Mechanical testing revealed that Group C had higher torsional strength (p=0.001) and stiffness than Groups A and B (p=0.001) while there was no significant difference between Groups C and D (p>0.05). Biodegradable implant releasing PDGF may have positive effects on fracture healing.

The Efficacy of Recombinant Platelet-Derived Growth Factor on Beta-Tricalcium Phosphate to Regenerate Femoral Critical Sized Segmental Defects: Longitudinal In Vivo Micro-CT Study in a Rat Model

Background and Objectives: Beta-tricalcium phosphate (beta-TCP) has been used for bone regeneration. The objective of this study was to assess longitudinally, the regeneration of critical sized segmental defects (CSSD) in rat femur using beta-TCP with or without recombinant platelet-derived growth factor (PDGF) through in vivo micro-computed tomography (micro-CT). Materials and Methods: Following ethical approval unilateral femoral CSSD measuring 5 mm was surgically created, under general anesthesia, in 30 male Wistar-Albino rats (aged 12–18 months; weighing 450–500 g). CSSD was stabilized using titanium mini-plate (4 holes, 1.0 mm thick with 8 mm bar). Depending upon biomaterial used for regeneration, the animals were randomly divided into: Control group (N = 10): CSSD covered with resorbable collagen membrane (RCM) only; Beta-TCP group (N = 10): CSSD filled with beta-TCP and covered by RCM; Beta-TCP + PDGF group (N = 10): CSSD filled with beta-TCP soaked in recombinant PDGF and covered by RCM. Longitudinal in vivo micro-CT analysis of the CSSD was done postoperatively at baseline, 2nd, 4th, 6th, and 8th weeks to assess volume and mineral density of newly formed bone (NFB) and beta-TCP. Results: Significant increase in NFB volume (NFBV) and mineral density (NFBMD) were observed from baseline to 8-weeks in all groups. Based on longitudinal in vivo micro-CT at 8-weeks, beta-TCP + PDGF group had significantly higher (p < 0.01) NFBV (38.98 ± 7.36 mm3) and NFBMD (3.72 ± 0.32 g/mm3) than the beta-TCP (NFBV—31.15 ± 6.68 mm3; NFBMD—2.28 ± 0.86g/mm3) and control (NFBV: 5.60 ± 1.06 mm3; NFBMD: 0.27 ± 0.02 g/mm3) groups. Significantly, higher reduction in beta-TCP volume (TCPV) and mineral density (TCPMD) were 1 observed in the beta-TCP + PDGF group when compared to the beta-TCP group. Conclusion: Addition of recombinant PDGF to beta-TCP enhanced bone regeneration within rat femoral CSSD and increased resorption rates of beta-TCP particles.

Human antigen R regulates TGF-β1 expression in airway smooth muscle cells under the stimulation of platelet-derived growth factor

Objective: To study the role of human antigen R (HuR) regulated transforming growth factor β1 (TGF-β1) expression in airway smooth muscle cells under the stimulation of platelet-derived growth factor (PDGF). Methods: Airway smooth muscle (ASM) cells were cultured at 37 ℃ and 5% CO2 in dulbecco's modified eagle medium (DMEM) cell medium. Cells at passages between 4 and 11 were divided into different groups according to the different compounds added. For control group, no compounds were administrated. For PDGF group, cells were stimulated with PDGF (20 μg/L) and cultured for an additional time. Cells were harvested and real-time PCR was used to measure mRNA level and Western blotting to detect protein level of HuR and TGF-β1 in ASM cells for different groups. Cells were divided into HuR siRNA group and control group. RNA-interference was used to determine whether lowering HuR expression could decrease PDGF-induced TGF-β1 expression in HuR siRNA group and control group after the stimulation of PDGF for indicated times. Western blotting analysis was used to test the expression of TGF-β1 after interrupting HuR expression. The concentration of TGF-β1 in the cultured serum of HuR siRNA group and control group for 0, 6, 12 h under the stimulation of PDGF was measured by enzyme-link immunosorbent assay (ELISA). Cells were divided into control group, control+ PDGF 6 h group, HuR siRNA group and HuR siRNA+ PDGF 6 h group, then the half-life of TGF-β1 mRNA in different groups was determined by treating ASM cells with the transcriptional inhibitor actinomycin D (10 mg/L) for 0, 4, 8 and 12 h. Results: PDGF treatment for 0, 6, 12 and 24 h significantly promoted HuR mRNA and protein expression and the relative levels were 1.00±0.00, 1.35±0.14, 1.73±0.17, 2.07±0.10; 0.51±0.10, 0.67±0.05, 0.83±0.07, 0.95±0.02 (all P<0.05). Similar alterations could also be demonstrated at TGF-β1 mRNA and protein. The relative expression was 1.00±0.00, 1.27±0.06, 1.60±0.10, 1.87±0.10; 0.72±0.09, 0.87±0.07, 1.13±0.12, 1.33±0.05 (6 h versus 12 h and 12 h versus 24 h, P<0.05). HuR expression decreased 21.9% in HuR siRNA group compared with control group under the stimulation of PDGF for 12 h. HuR silencing also decreased PDGF-induced TGF-β1 over-expression in ASM cells. In the control group, the relative protein levels of PDGF treatment for 0, 6 and 12 h were 0.70±0.05, 0.89±0.06, 1.06±0.05 and the protein levels in HuR siRNA group were 0.67±0.09, 0.77±0.03, 0.89±0.05 (all P<0.05). The concentration of TGF-β1 in the cultured serum was measured by ELISA and the outcomes were (773.33±16.32, 877.97±16.03, 3 060.34±82.53) ng/L in control group for 0, 6 and 12 h. Under the same condition, the outcomes of HuR siRNA group were (277.33±9.93, 407.77±7.14, 828.05±11.67) ng/L (both P<0.05). Actinomycin D disturbed the process of transcription and the half-life of TGF-β1 mRNA in HuR siRNA group showed no significant change compared with the HuR siRNA+ PDGF 6 h group (P>0.05). However, compared with control group and control + PDGF 6 h group, the half-life of TGF-β1 mRNA in HuR siRNA group showed significant change (P<0.05). Conclusions: PDGF can elevate TGF-β1 expression in ASM cells. HuR regulates TGF-β1 expression by promoting its mRNA stability.

Effects of ursolic acid on the signal pathway in activated hepatic stellate cells

Objective To observe the effects of ursolic acid (UA) on the activation of nicotinamide adenine dinucleotide phosphate-oxidase (NOX) and the downstream signaling pathways in platelet derived growth factor (PDGF) activated rat hepatic stellate cell (HSC-T6). Methods Rat HSC-T6 cells were divided into blank control group (no treatment), UA control group (50 μmol/L UA), PDGF group (10 μg/L PDGF), UA intervention group (50 μmol/L UA+ 10 μg/L PDGF), diphenyleneiodonium intervention (DPI) group (20 μmol/L DPI+ 10 μg/L PDGF), SB203580 (p38 mitogen-activated protein kirase (p38MAPK) inhibitor) intervention group (10 μmol/L SB203580+ 10 μg/LPDGF), LY294002 (phosphatidylinositop 3 kinase (PI3K) inhibitor) intervention group (10 μmol/L LY294002+ 10 μg/L PDGF) and rosup positive control group (5 μg/mL rosup). Except rosup positive control group, the expression of typeⅠcollagen at mRNA level of each group was detected by fluorescence quantitave-polymerase chain reaction (RT-PCR). The expression of membrane protein p47phox (except rosup positive control group), PI3K (except rosup positive control group and SB203580 intervention group), p-protein kinase B (p-AKT, except rosup positive control group and SB203580 intervention group) and phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK, except rosup positive control group and LY294002 intervention group) were tested by Western blot. Except SB203580 intervention group and LY294002 intervention group, the fluorescence intensity in the cells of each group was analyzed with active oxygen detection kit and fluorescence microplate reader. Single factor analysis of variance and LSD test were performed for comparison between groups. Results Type I collagen at the mRNA level of PDGF group (3.74±0.32) was higher than that of blank control group (1.00±0.00); Type Ⅰ collagen at the mRNA level of UA group (0.21±0.02) was lower than that of blank control group, UA intervention group (1.02±0.12), DPI intervention group (1.09±0.21), SB203580 intervention group (1.18±0.27), and LY294002 intervention group (1.15±0.26) were all lower than PDGF group, and the differences were statistically significant (t=15.667, -4.501, -15.553, -15.154, -14.642 and -14.813, all P<0.05). p47phox at the protein expression level of PDGF group (1.98±0.53) was higher than that of blank control group (1.00±0.00); that of UA group (0.48±0.10) was lower than blank control group; those of UA intervention group (0.95±0.26), DPI intervention group (0.99±0.28), SB203580 intervention group (0.93±0.31), and LY294002 intervention group (1.07±0.19) were all lower than PDGF group (t=4.209, -2.234, -4.424, -4.252, -4.510 and -3.909, all P<0.05). The protein expression level of PI3K of PDGF group (2.27±0.46) was higher than that of blank control group (1.00±0.00); that of UA intervention group (0.14±0.07) was lower than PDGF group and blank control group; that of UA group (0.14±0.07) was lower than blank control group; those of DPI intervention group (0.53±0.25) and LY294002 intervention group (0.35±0.14) were all lower than PDGF group (t=6.205, -8.208, -2.003, -4.202, -8.502 and -9.831, all P<0.05). The protein expression level of p-Akt of PDGF group (2.54±0.49) was higher than that of blank control group (1.00±0.00); those of UA intervention group (0.74±0.20), DPI intervention group (0.94±0.37) and LY294002 intervention group (1.17±0.41) were all lower than PDGF group; that of UA group (0.59±0.15) was lower than blank control group (t=5.927, -6.928, -6.158, -5.273 and -1.578, all P<0.05). The protein expression level of p-p38MAPK of PDGF group (1.98±0.35) was higher than that of blank control group (1.00±0.00); those of UA intervention group (0.68±0.28), DPI intervention group (0.63±0.27) and SB203580 intervention group (0.67±0.29) was all lower than PDGF group; that of UA group (0.28±0.13) was lower than blank control group (t=4.897, -6.479, -6.727, -6.529 and -3.561, all P<0.05). The level of active oxygen of PDGF group (105.57±7.51) was higher than that of blank control group (69.60±8.63); those of UA intervention group (64.56±9.11), DPI intervention group (65.75±6.62) was lower than PDGF group, UA group (29.84±3.19) was lower than blank control group (t=6.368, -7.288, -7.071 and -7.255, all P<0.05). Conclusion UA could inhibit membrane displacement of NOX subunit p47phox and reduce active oxygen production in PDGF induced rat HSC-T6 cells, and then block phosphorylation of PI3K-Akt, p 38MAPK signal pathways and inhibited the expression of type Ⅰ collagen at mRNA level. Key words: Hepatic stellate cell; Ursolic acid; Platelet-derived growth factor; NADPH oxidase

Effect of recombinant hPTEN gene expression on PDGF induced VSMC proliferation.

Human phosphatase and tensin homolog (hPTEN) gene was expressed in vascular smooth muscle cells (VSMCs) to study its effect on VSMC proliferation induced in platelet-derived growth factor (PDGF) conditioned medium. After G418 selection, MTT assay was conducted to examine transfected VSMC proliferation induced in human PDGF conditioned medium. We successfully constructed eukaryotic expression vector pcDNA4/myc-His-PTEN and transferred into VSMC cells. We report that in vitro proliferation of VSMC was inhibited in PTEN transfected VSMCs induced in PDGF conditioned medium. RT-PCR and Western blot results indicated significantly high levels of protein kinase B-PKB and nuclear factor kappa B mRNA and protein, respectively, in PDGF group as compared with the control group.

Modulation of PDGF on the expression of MMP-2,MMP-9 and TIMP-1 in human RPE cells

Background Researches showed that platelet-derived growth factor (PDGF) modulate the expression of matrix metalloproteinase/tissue inhibitor of metalloproteinase (MMP/TIMP) in cells,but the association of expression of MMP/TIMP in retinal pigment epithelial (RPE) cells and the dose and active time of PDGF is unclear.Objective This study was to observe the effects of PDGF on the expressions of MMP-2,MMP-9 and TIMP-1 in cultured RPE cells in vitro.Methods RPE cell line,ARPE-19,was calculated in vitro,and the cells were divided into 5 groups when they reached 70％-80％ confluence.Different concentrations (0,0.1,1,10,50 mg/L) of PDGF was added into the medium respectively for 36 hours,and the expressing levels of mRNA and protein of MMP-2,MMP-9 and TIMP-1 were detected by reverse transcription PCR (RT-PCR) and Western blot assay.In addition,RPE cells in PDGF group were treated with 10 mg/L PDGF for 24,36,48 hours respectively to detect the expressions of mRNA and protein of MMP-2,MMP-9 and TIMP-1 in the cells and to compare with the control group without PDGF.Results PDGF stimulated proliferation of RPE cells in a dose-and time-dependent manner.As the increase of the PDGF concentrations,the expression values of MMP-2 mRNA and MMP-9 mRNA in RPE cells were gradually elevated,with a statistically significant difference among various groups (MMP-2 mRNA:F=79.304,P=0.000;MMP-9 mRNA:F =8.465,P=0.003),and the expressions of MMP-2 mRNA and MMP-9 mRNA were significantly higher in the 1,10,50 mg/L PDGF groups compared with 0 mg/L PDGF normal control group (all at P＜0.05).Also,the expression values of MMP-2 and MMP-9 proteins in RPE cells were gradually elevated with the increase of PDGF concentrations,showing statistically differences among the groups (MMP-2:F=26.550,P=0.000;MMP-9:F=80.993,P=0.000).Compared with the 0 mg/L PDGF group,MMP-2 and MMP-9 expression levels in the 1,10,50 mg/L PDGF groups were significantly up-regulated (all at P＜ 0.05).However,the expression levels of TIMP-1 mRNA and protein group in the cells were not significantly different among various groups (mRNA:F =0.143,P =0.962 ; protein:F =1.955,P =0.178).The expression levels of M MP-2 mRNA,M MP-9 mRNA in the cells were increased in the PDGF group compared with the control group at different time points (MMP-2 mRNA:Ftime =83.250,P=0.002 ; MMP-9 mRNA:Ftime =6.785,P =0.019).Also,the expression values of MMP-2 and MMP-9proteins in RPE cells were increased in the PDGF group compared with the control group at different time points (MMP-2:Ftime =1 l.185,P =0.041 ; MMP-9:Ftime =968.413,P =0.000).The expression levels of MMP-2 and MMP-9 mRNAs and proteins were significant between the two groups at different time points (all at Pgroup =0.000;all at Ptime＜0.05).While the expression changes of TIMP-1 werc not significant between the two groups and among various time points (all at P＞0.05) Conclusions PDGF up-regulates MMP-2 and MMP-9 expressions in RPE cells in a dose-and time-dependent manner.But,PDGF dose not alter the expression of TIMP-1.These results indicate that PDGF disrupt the balance of MMP/TIMP,which may damage the extracellular matrix and therefore facilitate the migration of RPE cells in the pathogenesis of proliferative vitreoretinopathy. Key words: Platelet-derived growth factor; Extracellular matrix; Matrix metalloproteinase; Tissue inhibitor of metalloproteinase; Retina; Pigment epithelium of eye/cytology; Proliferative vitreoretinopathy

Effect of a chitosan sponge impregnated with platelet-derived growth factor on bone augmentation beyond the skeletal envelope in rat calvaria

We evaluated the ability of platelet-derived growth factor (PDGF) to promote bone augmentation beyond the skeletal envelope in rat calvaria. The calvariae of 14 rats were exposed, and two plastic caps-one with 0.03% PDGF and a chitosan sponge and one with a chitosan sponge alone-were placed. Microcomputed tomography and histologic sections were used to determine the amount of bone augmentation within the plastic caps. Bone volume was calculated using measurement software. Bone volume and amount of bone augmentation were significantly greater in the PDGF group than in the control group. In conclusion, a chitosan sponge containing 0.03% PDGF enhanced bone formation beyond the skeletal envelope in rat calvaria.

Role of phosphatidyl inositol 3-kinase pathway in regulating dedifferentiation and proliferation of rat pulmonary arterial vascular smooth muscle cells

Objective To investigate the role of phosphatidyl inositol 3-kinase(PI3K) in rat pulmonary arte-rial vascular smooth muscle cell(PASMC) dedifferentiation and proliferation. Methods PASMC were isolated from Sprague-Dawley(SD) rat arteriae pulmonalis and subcultured to the third generation for experiment.PASMC were divided into blank group, platelet derived growth factor(PDGF) group, dimethylsul foxide(DMSO) group, LY294002 group, small interfening RNA(siRNA) group, control-siRNA group, empty-virus group, recombinant-adenovirus group.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was performed to detect the PIK3CA mRNA level in PASMC transfected with pAdxsi-GFP-PIK3CA recombinant adenovirus and PIK3CA-siRNA.Western blot analysis was used to measure the expression level of alpha smooth muscle actin(α-SM actin). The mRNA level of α-SM actin was detected by RT-qPCR.The methyl thiazolyl tetrazolium(MTT) assay and thiazole cell counting kit-8(CCK-8) method were used to assess proliferation activity of PASMC. Results The PIK3CA mRNA level was significantly increased in recombinant-adenovirus group compared with blank group(P<0.05). The mRNA level of PIK3CA in siRNA group was lower than that in blank group(P<0.05). Compared with blank group, significantly lower α-SM actin mRNA and protein levels and a significantly higher proliferation activity were detected in PDGF group(all P<0.05). PI3K inhibitor LY294002 blocked this down-regulation of α-SM actin gene expression and up-regulation of proliferation activity induced by PDGF-BB(P<0.05). PIK3CA-siRNA also attenuated these biological effects.The α-SM actin mRNA level and protein expression level in recombinant-adenovirus group were lower than those in blank group(P<0.05), while the proliferation activity in the former group was greater(P<0.05). Conclusions PI3K pathway plays a vital role in vascular remodeling through a positive regulation of dedifferentiation and proliferation activity in PASMC. Key words: Phosphatidyl inositol 3-kinase; Pulmonary arterial vascular smooth muscle cell; Dedifferentiation; Proliferation

Effect of neferine on hepatic stellate cells in Collagen-I, TIMP-1 and MMP-2

To observe the effect of neferine on Collagen-I, TIMP-1 and MMP-2 expressions and protein secretion of hepatic stellate cells. The hepatic stellate cell line HSC-T6 was cultured in vitro, and then randomly divided into 5 groups: the control group, the platelet-derived growth factor (PDGF) group and PDGF + neferine (2, 6, 10 micromol x L(-1)) groups. All of the groups were cultured for 48 h, and their cells were collected to extract mRNA and detect Collagen-I, TIMP-1 and MMP-2 expressions with RT-PCR. Their cell supernatants were also collected to determine the protein content of three factors with ELISA. Compared with the control group, PDGF could remarkably increase the Collagen-I, TIMP-1 and MMP-2 expressions and protein secretion of hepatic stellate cells. Compared with the PDGF group, PDGF + neferine (6, 10 micromol x L(-1)) groups showed a notable decrease in the Collagen-I and mRNA expression and protein secretion along with the increase in the concentration, whereas the PDGF + neferine (2 micromol x L(-1)) group showed no significant change in the Collagen-I and mRNA expression and protein secretion. Compared with the PDGF group, three PDGF + neferine groups showed no notable change in MMP-2 expression and protein secretion. Neferine can inhibit the Collagen-I, TIMP-1 and mRNA protein expression and protein secretion of PDGF-induced HSCs along with the increase in the concentration, but with not remarkable effect on the MMP-2 expression and secretion.

Role of extracellular signal-regulated kinase 1/2 on inhibition of N-acetyl-seryl-aspartyl-lysyl-proline on proliferation and collagen synthesis of cultured rat pulmonary fibroblasts induced by platelet-derived growth factor

To investigate the role of extracellular signal-regulated kinase 1/2 on the inhibition of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on the proliferation and collagen synthesis of cultured rat pulmonary fibroblasts induced by platelet-derived growth factor (PDGF). Pulmonary fibroblasts were prepared from lungs of neonatal Wistar rats as described previously. Cells were divided into 4 groups: (1) control group (0.4% FBS group); (2) PDGF (10 ng/ml) stimulated group; (3) PD98059+PDGF group (25 micromol/L PD98059+10 ng/ml PDGF); (4) AcSDKP+PDGF group (10(-8) mol/L AcSDKP+10 ng/ml PDGF). All experiments were performed in the fourth passages. Metabolic activity of fibroblasts was observed by MTT, and expressions of type I and type III collagen were measured by immunocytochemistry and western blot. Expressions of phospho-ERK1/2 and ERK1/2 were detected by western blot. Compared with control group, exposure of pulmonary fibroblasts to 10 ng/ml PDGF increased cell metabolic activity, expression of type I and type III collagen and phosphorylation of ERK1/2. 25 micromol/L PD98059 and AcSDKP both could inhibit the metabolic activity of pulmonary fibroblasts, type I and type III collagen synthesis and phosphorylation of ERK1/ 2 induced by PDGF, with significant differences (P < 0.05). AcSDKP+PDGF group compared with PDGF stimulated group, metabolic activity of pulmonary fibroblasts decreased to 77.4%. Immunocytochemistry result showed that in AcSDKP+PDGF group, expressions of type I and type III collagen decreased to 69.3% and 67.2% compared with those of PDGF stimulated group. Western blot result showed that in AcSDKP+PDGF group, expressions of type I and type III collagen decreased to 92.4% and 78.0%, phospho-ERK1/2 decreased to 83.5% compared with those of PDGF stimulated group, with significant differences (P < 0.05). ERK1/2 plays an important role in the inhibition of AcSDKP on the proliferation and collagen synthesis of cultured rat pulmonary fibroblasts induced by PDGF.

Lentiviral Transfection with the PDGF-B Gene Improves Diabetic Wound Healing

The treatment of diabetic wounds remains a difficult challenge. The present study investigates whether platelet-derived growth factor (PDGF) lentiviral gene therapy can improve diabetic wound healing in the diabetic db/dbmouse. PDGF cDNA was cloned and lentiviral vectors were constructed with either the PDGF-B or green fluorescence protein (GFP) gene. A 2 x 2-cm full-thickness dermal wound was made on each db/db mouse. Animals were divided into three groups, with eight animals in each group as follows: group I, empty wound; group II, lentiviral PDGF; and group III, lentiviral GFP. Lentiviral vectors were injected into the wounds and healing was assessed at 21 days. Harvested wounds were assessed for residual epithelial gap, granulation tissue area, PDGF expression, collagen formation (picrosirius red), and angiogenesis (CD31 staining). Lentiviral vectors were constructed and transfected dermal fibroblasts demonstrated in vitro production of PDGF mRNA as measured by reverse-transcriptase polymerase chain reaction. Immunohistochemistry for PDGF confirmed successful in vivo transfection of the PDGF gene. At 21 days, reepithelialization and granulation tissue area were similar in all groups. However, there was a statistically significant increase in angiogenesis and substantially thicker, more coherently aligned collagen fibers in the PDGF group compared with controls. PDGF lentiviral vectors were successfully transfected into the regenerated dermis in diabetic wounds. Although reepithelialization was similar among the groups, there was enhanced angiogenesis and collagen deposition in the lentiviral PDGF group. These results demonstrate that lentiviral PDGF transfection of the diabetic wound enhances PDGF production, improves vascularization and collagen organization, and has potential clinical applications in diabetic wound treatment.

Effects of platelet-derived growth factor and interleukin-10 on Fas/Fas-ligand and Bcl-2/Bax mRNA expression in rat hepatic stellate cells in vitro.

To investigate the effects of platelet-derived growth factor(PDGF) and interleukin-10 (IL-10) on Fas/Fas-ligand and Bcl-2/Bax mRNA expressions in rat hepatic stellate cells. Rat hepatic stellate cells (HSCs) were isolated and purified from rat liver by in situ digestion of collagenase and pronase and single-step density Nycodenz gradient. After activated by culture in vitro, HSCs were divided into 4 groups and treated with nothing (group N), PDGF (group P), IL-10 (group I) and PDGF in combination with IL-10 (group C), respectively. Semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was employed to compare the mRNA expression levels of Fas/FasL and Bcl-2/Bax in HSCs of each group. The expression levels of Fas between the 4 groups had no significant differences (P>0.05). FasL mRNA level in normal culture-activated HSCs (group N) was very low. It increased obviously after HSCs were treated with IL-10 (group I) (0.091+/-0.007 vs 0.385+/-0.051, P<0.01), but remained the low level after treated with PDGF alone (group P) or PDGF in combination with IL-10 (group C). Contrast to the control group, after treated with PDGF and IL-10, either alone or in combination, Bcl-2 mRNA expression was down-regulated and Bax mRNA expression was up-regulated, both following the turn from group P, group I to group C. Expression of Bcl-2 mRNA in group C was significantly lower than that in group P (0.126+/-0.008 vs 0.210+/-0.024, P<0.01). But no significant difference was found between group C and group I, as well as between group I and group P (P>0.05). Similarly, the expression of Bax in group C was higher than that in group P (0.513+/-0.016 vs 0.400+/-0.022, P<0.01). No significant difference was found between group I and group P (P>0.05). But compared with group C, Bax expressions in group I tended to decrease (0.449+/-0.028 vs 0.513+/-0.016, P<0.05). PDGF may promote proliferation of HSCs but is neutral with respect to HSC apoptosis. IL-10 may promote the apoptosis of HSCs by up-regulating the expressions of FasL and Bax and down-regulating the expression of Bcl-2, which may be involved in its antifibrosis mechanism.

Quantitative assessment of cerebral neocapillary network and its remodeling in mice using intravital fluorescence videomicroscopy.

To assess the responses of different growth factors on cerebral neocapillary density (NCD), cerebral angiogenesis was induced in mice using growth factors, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) at a concentration of 6 ng/ml each. Intravital fluorescence videomicroscopy was used to quantitatively evaluate microhemodynamic parameters such as diameter and red cell velocity. The gel-nylon mesh-sandwich system was implanted over the exposed cortex. After incubation for different periods of time (days 7, 14 or 28), fluorescein isothiocyanate (FITC)-labeled red cells were injected through a carotid artery and the neocapillaries on the upper surface of the nylon mesh were observed under a fluorescence videomicroscope. Based on the recorded videoimages, we evaluated the density, diameter and red cell velocity of the neocapillaries. The NCD in the bFGF group on day 7 was significantly higher than that in the PDGF group on day 7 (P < 0.01). The NCD (index) reached 100% on day 14, while it reduced significantly in both the groups on day 28. The neocapillary diameter was greater than that of the pre-existing capillaries on day 7. On day 14, a clear difference appeared in the capillary density between large and small vessels. The red cell velocity increased with the number of days after incubation. The response of cerebral neocapillaries to acetylcholine was measured after 28 days of incubation with growth factor bFGF and with PDGF. The red cell velocity increased significantly from its basal value in the PDGF group. These results suggest that the neocapillaries in the PDGF group matured earlier than those in the bFGF group.

Cyclic strain activates the pro-survival Akt protein kinase in bovine aortic smooth muscle cells

Background. Pulsatile pressure induced by the beating heart causes cyclic strain on arterial endothelial cells and smooth muscle cells (SMCs). This study examined whether Akt, a serine/threonine protein kinase known to promote cell survival by inhibiting apoptosis, is activated by cyclic strain in bovine aortic SMCs. Methods. Bovine aortic SMCs were cultured on flexible-bottomed membranes and then serum-starved for 24 to 36 hours. The cells were then exposed to 150-mm Hg repetitive deformations, which created an average of 10% strain on the monolayer SMCs at a frequency of 60 cycles/minute for 0 (negative control) and 30 minutes. Platelet-derived growth factor (PDGF)–stimulated SMCs were used as positive controls. Phosphorylation of Akt was determined by means of Western blot analysis. An apoptosis assay (TUNEL) was also performed on SMCs exposed to cyclic strain. Results. Akt phosphorylation was significantly increased over that of the negative control after 30 minutes of cyclic strain and in the PDGF group. Cyclic strain did not increase the prevalence of apoptosis in SMCs over the control. Conclusions. Cyclic strain activated the pro-survival Akt kinase. The pro-survival function was supported by the fact that cyclic strain did not increase apoptosis in bovine aortic SMCs. This experiment suggests that cyclic strain may induce arterial wall thickening by tipping the balance toward arterial SMC proliferation through the inhibition of apoptosis. (Surgery 2001;130:378-81.)

Usefulness of platelet-derived growth factor as a prognostic factor in pulmonary adenocarcinoma.

Platelet-derived growth factor (PDGF) in the resected pulmonary adenocarcinoma tissue of 88 patients was detected immunohistochemically by the avidin-biotin-peroxidase technique to determine whether or not it is a prognostic parameter. The 88 patients were divided into PDGF (-) and PDGF (+) groups according to the stainability of the factor. The PDGF (-) group included 39 patients and the PDGF (+) group 49. The 5-year survival rate was 53% for the PDGF (-) group and 17% for the PDGF (+) group (P < 0.01). These findings indicate that the stainability of PDGF can be a prognostic parameter for pulmonary adenocarcinoma.