Platelet-derived Growth factor-B Gene Research Articles

Exploring the Molecular Characteristics and Role of PDGFB in Testis and Epididymis Development of Tibetan Sheep.

Platelet-derived growth factor B (PDGFB), as an important cellular growth factor, is widely involved in the regulation of cellular events such as cell growth, proliferation, and differentiation. Although important, the expression characteristics and biological functions in the mammalian reproductive system remain poorly understood. In this study, the PDGFB gene of Tibetan sheep was cloned by RT-PCR, and its molecular characteristics were analyzed. Subsequently, the expression of the PDGFB gene in the testes and epididymides (caput, corpus, and cauda) of Tibetan sheep at different developmental stages (3 months, 1 year, and 3 years) was examined by qRT-PCR and immunofluorescence staining. A bioinformatic analysis of the cloned sequences revealed that the CDS region of the Tibetan sheep PDGFB gene is 726 bp in length and encodes 241 amino acids with high homology to other mammals, particularly goats and antelopes. With the increase in age, PDGFB expression showed an overall trend of first decreasing and then increasing in the testis and epididymis tissues of Tibetan sheep, and the PDGFB mRNA expression at 3 months of age was extremely significantly higher than that at 1 and 3 years of age (p < 0.05). The PDGFB protein is mainly distributed in testicular red blood cells and Leydig cells in Tibetan sheep at all stages of development, as well as red blood cells in the blood vessel, principal cells, and the pseudostratified columnar ciliated epithelial cells of each epididymal duct epithelium. In addition, PDGFB protein expression was also detected in the spermatocytes of the 3-month-old group, spermatids of the 1-year-old group, spermatozoa and interstitial cells of the 3-year-old group, and loose connective tissue in the epididymal duct space in each developmental period. The above results suggest that the PDGFB gene, as an evolutionarily conserved gene, may play multiple roles in the development and functional maintenance of testicular cells (such as red blood cells, Leydig cells, and germ cells) and epididymal cells (such as red blood cells, principal cells, and ciliated epithelial cells) during testicular and epididymal development, which lays a foundation for the further exploration of the mechanisms by which the PDGFB gene influences spermatogenesis in Tibetan sheep.

Endothelial HIFα/PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension.

Mechanisms underlying maintenance of pathological vascular hypermuscularization are poorly delineated. Herein, we investigated retention of smooth muscle cells (SMCs) coating normally unmuscularized distal pulmonary arterioles in pulmonary hypertension (PH) mediated by chronic hypoxia with or without Sugen 5416, and reversal of this pathology. With hypoxia in mice or culture, lung endothelial cells (ECs) upregulated hypoxia-inducible factor 1α (HIF1-α) and HIF2-α, which induce platelet-derived growth factor B (PDGF-B), and these factors were reduced to normoxic levels with re-normoxia. Re-normoxia reversed hypoxia-induced pulmonary vascular remodeling, but with EC HIFα overexpression during re-normoxia, pathological changes persisted. Conversely, after establishment of distal muscularization and PH, EC-specific deletion of Hif1a, Hif2a, or Pdgfb induced reversal. In human idiopathic pulmonary artery hypertension, HIF1-α, HIF2-α, PDGF-B, and autophagy-mediating gene products, including Beclin1, were upregulated in pulmonary artery SMCs and/or lung lysates. Furthermore, in mice, hypoxia-induced EC-derived PDGF-B upregulated Beclin1 in distal arteriole SMCs, and after distal muscularization was established, re-normoxia, EC Pdgfb deletion, or treatment with STI571 (which inhibits PDGF receptors) downregulated SMC Beclin1 and other autophagy products. Finally, SMC-specific Becn1 deletion induced apoptosis, reversing distal muscularization and PH mediated by hypoxia with or without Sugen 5416. Thus, chronic hypoxia induction of the HIFα/PDGF-B axis in ECs is required for non-cell-autonomous Beclin1-mediated survival of pathological distal arteriole SMCs.

Intratumoral PDGFB gene predominantly expressed in endothelial cells is associated with angiogenesis and lymphangiogenesis, but not with metastasis in breast cancer.

Platelet-derived growth factor B (PDGFB) is known to play essential roles in angiogenesis and lymphangiogenesis during development, and tumor growth and vessel stabilization in experimental models. However, whether these findings could be translated to breast cancer patients remains unclear. We hypothesized that PDGFB gene expression is associated with angiogenesis, cell proliferation, and clinical outcomes in breast cancer patients. A total of 7635 primary breast cancer patients with full transcriptome and clinical data available from 13 independent cohorts were analyzed using in silico approach. The median value was used to divide each cohort into high and low PDGFB expression groups. High PDGFB gene expression was associated with increased expression of angiogenesis-related genes, higher amount of vascular cell infiltrations, and with enrichment of angiogenesis gene set, lymphangiogenesis-related gene expressions, lymphangiogenesis-related cell infiltrations, and enrichmentof lymphangiogenesis gene set in GSE96058 and validated by TCGA cohorts; however, not with lymphatic metastasis. PDGFB expression was neither associated with cell proliferation as assessed by Ki67 expression nor with Nottingham histological grade, or response to neoadjuvant chemotherapy. We found that PDGFB was most extensively expressed by endothelial and perivascular-like cells in the tumor microenvironment, and minimally by cancer cells consistently in two single-cell sequence cohorts. High PDGFB expression enriched TGFβ, epithelial-mesenchymal transition, hypoxia, and cancer stem cell-associated pathways. However, no association with distant metastasis was observed. Disease-specific and disease-free survival were worse in the high PDGFB expression group consistently in TCGA and METABRIC cohorts. PDGFB is predominantly expressed in endothelial cells and is associated with angiogenesis and lymphangiogenesis, but not with cellular proliferation or metastasis in breast cancer.

MicroRNA-432-5p regulates sprouting and intussusceptive angiogenesis in osteosarcoma microenvironment by targeting PDGFB

Osteosarcoma (OS) is a type of bone tumor conferred with high metastatic potential. Attainable growth of tumors necessitates functional vasculature mediated by sprouting angiogenesis (SA) and intussusceptive angiogenesis (IA). However, the regulation of IA and SA is still unclear in OS. To understand the mechanisms adopted by OS to induce angiogenesis, initially, we assessed the expression profile of a set of miRNAs' in both OS cells (SaOS2 and MG63) and normal bone cells. Amongst them, miR-432-5p was found to be highly downregulated in OS. The functional role of miR-432-5p in OS was further analyzed using miR-432-5p mimic/inhibitor. Platelet-derived growth factor-B (PDGFB) was found to be a putative target of miR-432-5p and it was further confirmed that the PDGFB 3'UTR is directly targeted by miR-432-5p using the luciferase reporter gene system. PDGFB was found to be secreted by OS to regulate angiogenesis by targeting the cells in its microenvironment. The conditioned medium obtained from miR-432-5p mimic transfected MG63 and SaOS2 cells decreased cell viability, proliferation, migration, and aorta ring formation in endothelial cells. The miRNA mimic/inhibitor transfected MG63 and SaOS2 cells were placed on SA (day 6) and IA (day 9) phase of CAM development to analyze SA and IA mechanisms. It was found that miR-432-5p mimic transfection in OS promotes the transition of SA to IA which was documented by the angiogenic parameters and SA and IA-associated gene expression. Interestingly, this outcome was also supported by the zebrafish tumor xenograft model. Corroborating these results, it is clear that miR-432-5p expression in OS cells regulates SA and IA by targeting PDGFB genes. We conclude that targeting miR-432-5p/PDGFB signaling can be a potential therapeutic strategy to treat OS along with other existing strategies.

Immunomodulatory effects of vitamin D3 on gene expression of MDGF, EGF and PDGFB in endometriosis

Endometriosis, an inflammatory disease, is assumed to be associated with an increased production of growth-related cytokines. Based on the emerging immunomodulatory role of vitamin D3 in different inflammatory conditions, this study aimed to examine its modulatory effect on the expression levels of the genes for platelet-derived growth factor-B (PDGFB), monocyte/macrophage-derived growth factor (MDGF, also known as PPBP) and epidermal growth factor (EGF) in peritoneal fluid mononuclear cells (PFMC) in women with and without endometriosis. PFMC from 10 women with endometriosis and 10 control participants were treated with vitamin D3.The gene expression levels of PDGFB, MDGF and EGF were measured 6, 24 and 48h following vitamin D3 administration using real-time PCR. Gene expression levels of EGF and PDGFB were higher in the PFMC of women with endometriosis than the control group (P = 0.006, P<0.001, respectively). Although MDGF expression showed an increase in the endometriosis group compared with non-endometriotic controls, no significant difference was found. Vitamin D3 significantly decreased EGF expression at 6, 24 and 48 h (P<0.001, P<0.001 and P = 0.007, respectively), MDGF at 24 and 48 h (P<0.001 and P = 0.009, respectively) and PDGFB at 6 h (P = 0.047) in the endometriosis group. Vitamin D3 treatment had no significant effect on expression of the genes in the PFMC of non-endometriotic women. The study concluded that PDGFB and EGF gene expression increases in endometriosis, and vitamin D3 could markedly decrease this expression, suggesting its therapeutic potential in endometriosis.

The effectiveness of chitosan-mediated silencing of PDGF-B and PDGFR-β in the mesangial proliferative glomerulonephritis therapy

Platelet-derived growth factor-B (PDGF-B) is a growth factor that plays an important role in the progression of mesangial proliferative glomerulonephritis (MsPGN). PDGF-B may contribute to mesangioproliferative changes and is overexpressed in MsPGN. Recently, small interfering RNAs (siRNAs) have been widely used for gene silencing effects in experimental models of renal diseases. Nanoparticle-based therapeutics are preferred for reasons such as increasing therapeutic efficacy and reducing toxic effects caused by high doses. The distribution of nanoparticles to the kidney is a significant advantage in siRNA delivery. The aim of this study was to investigate the efficacy of chitosan/siRNA nanoplexes in silencing of PDGF-B and PDGFR-β genes in kidney and to decrease mesangial cell proliferation and matrix accumulation in MsPGN model induced by anti-Thy-1.1 antibody. The therapeutic effects of chitosan/siPDGF-B + siPDGFR-β nanoplexes in glomerulonephritic rats were studied by molecular, biochemical, and histopathologic evaluations. Chitosan/siPDGF-B + siPDGFR-β nanoplexes markedly reduced PDGF-B and PDGFR-β mRNA and protein expressions in experimental MsPGN model. Histopathologic examination results showed that the silencing of PDGF-B and its receptor PDGFR-β led to reduction in mesangial cell proliferation and matrix accumulation. The use of chitosan/siPDGF-B + siPDGFR-β nanoplexes for silencing the PDGF-B pathway in MsPGN can be considered as a new effective therapeutic strategy.

Angiogenic factors polymorphisms in gall bladder cancer (GBC) progression and survival

Introduction: Gall bladder cancer (GBC) is the commonest biliary tract cancer and the most common gastro-intestinal cancer among women in north Indian population. Angiogenesis plays an important role in growth, progression, and metastasis of tumors. Vascular endothelial growth factor (VEGF), platelet derived growth factor-B (PDGFB) and human epidermal growth factor receptor-2 (HER2) are potent mitogens belonging to a family of receptor tyrosine kinases, that are thought to participate in signal transduction mediating tumor cell proliferation. In the present study, we evaluated the association of functional polymorphisms in VEGF, PDGFB and HER2 genes with GBC progression and survival. Methods: VEGF -1154 G>A, -2578 C>A, PDGFB +286A>G and +1135A>C polymorphisms were investigated by amplification refractory mutation system (ARMS) PCR. VEGF +936C>T and HER2 Ile655Val polymorphisms by restriction fragment length polymorphism (PCR-RFLP) and VEGF-2549 Ins/Del by PCR in DNA extracted from venous blood of 80 GBC patients who were operated before 2010 (to ensure at least 5 years follow up) and in whom follow up data was available. Results: +286GA genotype of PDGFB showed significant risk protective association with regional lymph node metastasis (OR=0.27, p value=0.03). In Kaplan-Meir survival analysis, genotypes VEGF-1154 GA (Chi-square=10.18 and P=0.0006), +936 CC (Chi-square=7.332 and P=0.026) and PDGFB+1135CC (Chi-square=7.424 and p value=0.024) showed significantly longer survival but there was no significant survival correlation with HER2 polymorphism. Conclusion: VEGF and PDGFB gene polymorphisms may play a role in progression (lymph node metastasis) and survival in GBC.

Association assessment of platelet derived growth factor B gene polymorphism and its expression status with susceptibility to coronary artery disease

Abstract Background Coronary artery disease (CAD) is the most frequent cause of morbidity and mortality in the world and it is known as a multifactorial disorder which is influenced by both genetic and environmental factors. Based on different assays, the platelet derived growth factor B (PDGF-B) gene is shown to be amongst the inflammation inducers involved in different pathological conditions such as atherosclerosis. Aim In this case-control study we have examined the association of the functional PDGF-B +286 and +1135 polymorphisms and its expression status with susceptibility to CAD. Subjects and methods Study groups included 369 patients with CAD and 413 healthy individuals. Genotypic and allelic frequencies of PDGF-B +286 and +1135 polymorphisms were determined by the SSP-PCR method. PDGF-B expression status was identified by quantitative real-time PCR. Results The analysis of genotyping results revealed CAD patients had an increased frequency of PDGF-B +286A>G A/A genotype in comparison with healthy individuals. Furthermore, we found that the PDGF-B expression level in CAD patients group is nearly 2-folds greater than its level in control group. Conclusion There is probably a relationship between variations in PDGF-B gene and CAD influence. The increase in PDGF-B gene expression may has a role in susceptibility to CAD.

The association of functional polymorphisms in genes encoding growth factors for endothelial cells and smooth muscle cells with the severity of coronary artery disease.

BackgroundDespite the important roles of vascular smooth muscle cells and endothelial cells in atherosclerotic lesion formation, data regarding the associations of functional polymorphisms in the genes encoding growth factors with the severity of coronary artery disease (CAD) are lacking. The aim of the present study is to analyze the relationships between functional polymorphisms in genes encoding basic fibroblast growth factor (bFGF, FGF2), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), platelet derived growth factor-B (PDGFB), transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor A (VEGF-A) and the severity of coronary atherosclerosis in patients with stable CAD undergoing their first coronary angiography.MethodsIn total, 319 patients with stable CAD who underwent their first coronary angiography at the Silesian Centre for Heart Diseases in Zabrze, Poland were included in the analysis. CAD burden was assessed using the Gensini score. The TaqMan method was used for genotyping of selected functional polymorphisms in the FGF2, PDGFB, TGFB1, IGF1 and VEGFA genes, while rs4444903 in the EGF gene was genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The associations between the selected polymorphisms and the Gensini were calculated both for the whole cohort and for a subgroup of patients without previous myocardial infarction (MI).ResultsThere were no differences in the distribution of the Gensini score between the genotypes of the analyzed polymorphisms in FGF2, EGF, IGF1, PDFGB, and TGFB1 in the whole cohort and in the subgroup of patients without previous MI. The Gensini score for VEGFA rs699947 single-nucleotide polymorphism (SNP) in patients without previous myocardial infarction, after correction for multiple testing, was highest in patients with the A/A genotype, lower in heterozygotes and lowest in patients with the C/C genotype, (p value for trend = 0.013, false discovery rate (FDR) = 0.02). After adjustment for clinical variables, and correction for multiple comparisons the association between the VEGFA genotype and Gensini score remained only nominally significant (p = 0.04, FDR = 0.19) under the dominant genetic model in patients without previous MI.ConclusionsWe were unable to find strong association between analyzed polymorphisms in growth factors and the severity of coronary artery disease, although there was a trend toward association between rs699947 and the severity of CAD in patients without previous MI.Electronic supplementary materialThe online version of this article (doi:10.1186/s12872-016-0402-4) contains supplementary material, which is available to authorized users.

PM445. A family of primary familial brain calcification due to mutation in platelet-derived growth factor-B gene

PM445. A family of primary familial brain calcification due to mutation in platelet-derived growth factor-B gene

Expression of PDGF-B and methylation levels of its gene promoter in human gliomas

Objective To investigate the role of platelet-derived growth factor B (PDGF-B) expression levels and the methylation levels of its gene promoter on proliferation of human gliomas.Methods The expressions of PDGF-B,p-Smad2 and Ki-67 were determined by immunohistochemistry in 54 human glioma specimens.And the methylation levels of PDGF-B gene promoter were determined using pyrosequencing.Results (1) The expression levels of PDGF-B increased with the pathologic grades elevated (P ＜0.05).(2) There was a positive and significant correlation between the expression levels of PDGF-Band Ki-67 as well as p-Smad2 (r =0.839,P ＜0.001) (r =0.802,P ＜0.001),respectively.And an inverse and significant correlation was found between the expression levels of PDGF-B and its gene promoter methylation (r =-0.817,P ＜0.001).(3) The methylation levels of PDGF-B gene promoter were significantly higher in low grade gliomas (WHO Ⅰ Ⅱ) than those in high grade gliomas (WHO Ⅲ Ⅳ) (P =0.001).Conclusions The expression levels of PDGF-B and its gene promoter methylation play an important role in predicting the malignancy and proliferation of human gliomas. Key words: Glioma; Gene Methylation; Pyrosequencing; Platelet-derived growth factov B

The methylation status of the platelet-derived growth factor-B gene promoter and its regulation of cellular proliferation following folate treatment in human glioma cells

Platelet-derived growth factor-B (PDGF-B) is a growth factor that regulates cell migration, proliferation, and differentiation, and is involved in several physical and pathological processes. The overexpression of PDGF-B in glioma surgical samples revealed its effect on tumorigenesis. In this study, we determined that the expression of PDGF-B in 54 glioma samples varied among different grades and was correlated with the cell proliferation marker, Ki-67. Using pyrosequencing, we quantitatively assessed PDGF-B gene methylation levels and determined that hypomethylation promotes increased expression of PDGF-B in higher grade gliomas. Furthermore, we treated two glioma cell lines with a demethylating agent (5-aza-2'-deoxycitidine, 5-aza-dC) or a remethylating agent (folate) to alter the methylation status of PDGF-B. The epigenetic regulation of the PDGF-B gene not only modulated the expression levels of PDGF-B but also affected the cellular proliferation induced by TGFβ-Smad activity and the PDGF-B peptide itself. Our work showed the importance of the methylation status of the PDGF-B gene promoter, and suggests that the epigenetic regulation of the PDGF-B gene may serve as a potential therapeutic target for the inhibition of glioma proliferation.

Regulatory effects of introduction of an exogenous FGF2 gene on other growth factor genes in a healing tendon

In this study of a tendon injury model, we investigated how injection of a vector incorporating one growth factor gene changes expression levels of multiple growth factor genes in the healing process. The flexor tendon of chicken toes was completely cut and repaired surgically. The tendons in the experimental arm were injected with an adeno-associated virus-2 vector incorporating basic fibroblast growth-factor gene, whereas the tendons in the control arm were not injected or injected with sham vectors. Using real-time polymerase chain reaction, we found that, within the tendon healing period, a set of growth factor genes-transforming growth factor-β1, vascular endothelial growth factor, and connective tissue growth factor-were significantly up-regulated. Expression of the platelet-derived growth factor-B gene was not changed, and the insulin-like growth factor was down-regulated. A tendon marker gene, scleraxis, was significantly up-regulated in the period. Our study revealed an intriguing finding that introduction of one growth factor gene in the healing tendon modulated expression of multiple growth factor genes. We believe this study may have significant implications in determining the approach of gene therapy, and the findings substantiate that gene therapy using a single growth factor could affect multiple growth factors.

Effect of recombinant human platelet-derived growth factor B on cat corneal endothelial cell viability mediated by adeno-associated virus.

To transduce recombinant human platelet-derived growth factor B(PDGF-B) gene adeno-associated virus(AAV) to in vitro cultured cat corneal endothelial cell (CEC) and observe the effect of the expressed PDGF-BB protein on the viability of cat CEC. Cat cornea endothelium was torn under microscope and rapidly cultivated in DMEM to form single layer CEC and the passage 2 endothelial cells were used in this study. The recombinant human PDGF-B gene AAV was constructed and transduced into cat CEC directly. Three groups were as following: blank control group, AAV control group and recombinant AAV group. At 24 hours, 48 hours, and 5 days after transduction, total RNA was extracted from the CEC by Trizol and the expression of PDGF-B gene was detected by fluorescence quantitative polymerase chain reaction. Viability of the transduced CEC was detected at 48 hours after transduction by MTT assay. Cell morphology was observed under inverted phase contrast microscope. With the torn endothelium culture technique, we rapidly got single layer cat CEC. At 24 hours, 48 hours and 5 days after transduction, fluorescence quantitative polymerase chain reaction showed there was no significant difference of the expressed PDGF-B gene mRNA between blank control group and AAV control group (P>0.05). In contrast, there were significant differences between two control groups and recombinant AAV group (P<0.05). MTT assay showed that in recombinant AAV group, the expressed PDGF-BB protein could promote the viability of cat CEC. Morphology observation showed at 48 hours after transduction, cells in CEC-AAV-PDGF-B group proliferated into bigger scales in regular triangle to hexagon shape with distinct boundary, while the number of cells was significantly less in the two control groups. The recombinant AAV-PDGF-B expresses biological active PDGF-BB protein in cat CECs, which promotes the viability and proliferation of cells.

The study of human PDGF-B gene transferred to cat corneal endothelial cells.

To demonstrate that human platelet-derived growth factor-B (PDGF-B) cDNA could be expressed in primary cultured cat corneal endothelia cells by using gene transfer techniques; to explore a useful tool for the further studies of the molecular mechanisms of corneal endothelium failure and provide a potential effective genetic therapy for the blind patients. Human PDGF-B cDNA was isolated from human placent by RT-PCR and inserted into pcDNA(4) vector to construct recombinant eukaryotic expression plasmid pcDNA(4)-PDGF-B. The full length was confirmed by the DNA sequencing analysis. By tearing endothelium technique we obtained pure single layer of cat corneal endothelial cells. The pcDNA(4)-PDGF-B eukaryotic expression vector was transferred into cat corneal endothelial cells by Effectene™ lipofectine. The transfection efficiency of Effectene™ lipofectine in pcDNA(4)-B was detected with pcDNA(4)-GFP. 5 days later, RT-PCR was used to check the PDGF-B expression. Cell viability was tested by modified tertrozalium salt (MTT) method. Cell morphology was observed under inverted phase contrast microscope. The human PDGF-B cDNA was isolated successfully from healthy parturien placent tissue and the sequence was confirmed by computer automatic sequence and PCR analysis. Pure single layer cat corneal endothelial cells were successfully cultured by tearing endothelium technique. Effectene™ lipofectine transfection technique could be effectively used to transfer pcDNA(4)-PDGF-B into cat corneal endothelial cells in vitro, the transfection efficiency was 30%. RT-PCR result showed that human PDGF-B gene was highly expressed in transfected cat corneal endothelial cells. The expressed PDGF-BB protein promoted the viability of cat corneal endothelial cells. Human platelet-derived growth factor-B (PDGF-B) cDNA could be highly expressed in cultured cat corneal endothelial cells by gene transfection techniques. Expressed PDGF-BB protein significantly promoted the viability of cat corneal endothelial cells, thus provided a potential effective method for corneal endothelium blindness genetic therapy.

Construction and identification of recombinant human platelet-derived growth factor-B adenoviral vector and transfection into periodontal ligament stem cells

To construct a recombinant human platelet-derived growth factor-B (PDGF-B) adenoviral vector and to transfect it into human periodontal ligament stem cells (PDLSC). The recombinant plasmid pAd-PDGF-B was constructed by homologous recombination and confirmed by restriction endonucleases digestion. Recombinant adenovirus was packaged in HEK293 cells. PDLSC were transfected with recombinant adenovirus and PDGF-B expression was confirmed. Expression of collagen type I gene was determined by quantitative analysis of the products of RT-PCR. The cell proliferation was determined with MTT colorimetric assay. The recombinant plasmid pAd-PDGF-B was confirmed by restriction endonucleases digestion. EGFP expression was observed on the third day after transfecting, and the expression of PDGF-B was detected. Immunohistochemical methods revealed that PDGF-B was expressed in PDLSC. Levels of expression of collagen type I gene were increased significantly by transfer of the exogenous PDGF-B gene to PDLSC. At the same time, findings indicated that Ad-PDGF-B stimulated PDLSC proliferation. MTT assay indicated the absorbance of PDLSC by stimulating with Ad-PDGF-B was (0.68 +/- 0.02), P < 0.01. Using the AdEasy system, the human PDGF-B recombinant adenovirus can be rapidly obtained. These results indicate that recombinant adenoviruses encoding PDGF-B transgenes could modulate proliferative activity of PDLSC, enhance the high expression of collagen type I and lay the foundation for periodontal tissue regeneration and dental implant gene therapy.

Mycophenolic acid inhibits the autocrine PDGF-B synthesis and PDGF-BB-induced mRNA expression of Egr-1 in rat mesangial cells

Uncontrolled mesangial cell (MC) proliferation within the context of glomerular disease contributes to the development of glomerulosclerosis. Mesangial autocrine growth factor stimulation has been described as a pathogenic factor. We investigated the effects of mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), on proliferation factors of cultured rat MCs. MPA was tested on the expression of platelet-derived growth factor-B (PDGF-B) and its receptor beta (PDGFR-beta), the immediate early gene (IEG) c-fos and the early growth response gene-1 (Egr-1), and AP-1 activation. Growth-arrested rat MCs were stimulated with 10% fetal calf serum (FCS) or 10-25 ng/ml platelet-derived growth factor-BB (PDGF-BB) in the presence or absence of MPA (0.019-10 microM) with or without guanosine (100 microM). MC proliferation was quantified by 5-bromo-2'-deoxyuridine (BrdU) incorporation and direct cell counting. Cytotoxicity of MPA was evaluated using the MTT and LDH tests. Protein expression of PDGF-B and its receptor PDGFR-beta was quantified by western blot analysis. The effect of MPA on gene expression of PDGF-B, Egr-1 and c-fos was determined by the reverse transcriptase-polymerase chain reaction (RT-PCR). AP-1 activation was analysed by an electrophoretic mobility shift assay (EMSA). Exposure of MCs to MPA caused a concentration-dependent inhibition of FCS-induced cell proliferation (cell number increase) with an IC50 of 0.44 +/- 0.03 microM and DNA synthesis with an IC50 of 0.52 +/- 0.02 microM without cell cytotoxicity in the therapeutic range. MPA decreased the PDGF-B protein expression and mRNA self-induction of PDGF-B but did not alter the protein expression of PDGFR-beta. MPA strongly inhibited the PDGF-BB-induced mRNA expression of Egr-1 decreasing to 7.6 +/- 2.5% after 30 min (P <or= 0.001) and to 4.7 +/- 3.1% after 1 h (P <or= 0.05), both being compared to the maximal expression induced by PDGF-BB. PDGF-BB-induced c-fos expression under MPA was unchanged after 30 min and decreased to 57 +/- 26% after 1 h (n.s.). MPA treatment did not affect PDGF-BB-induced AP-1 activity determined after 1 h and 2 h. The inhibitory MPA effect on PDGF-BB-induced PDGF-B expression was not significantly restored by guanosine (56 +/- 18% versus 32 +/- 17% after 2 h, n.s.), and MPA inhibition of PDGF-BB-induced Egr-1 expression was not reversed by exogenous guanosine. Treatment of cultured MCs with MPA inhibits MC proliferation correlating with a downregulation of the PDGF-B gene and protein expression and a suppression of Egr-1 mRNA expression. Since exogenous guanosine was not able to reverse the inhibitory MPA effect on PDGF-B and Egr-1 expression, we conclude that the antiproliferative effect of MPA on MCs may not solely depend on dGTP depletion but on a specific interference with the autocrine PDGF-B synthesis and Egr-1 expression of MCs.

062 Application of gene therapy to tendon healing: modification of tenocytes with exogeneous pdgf and vegf genes to promote collagen production

Purpose: Gene therapy provides a useful method to enhance wound healing, but it has not been attempted in healing of wounds in flexor tendons in the hands, which inherently lack sufficient intrinsic healing capacity. To explore the potential of application of gene therapy to tendon wound healing, we genetically modified tenocytes with the platelet-derived growth factor-B (PDGF-B) and the vascular endothelial growth factor (VEGF) gene and investigated the expression of the genes for collagen production in an in vitro model of the proliferating tenocytes. Method: Tenocytes were obtained from cultures of rat intrasynovial tendons and randomly distributed to 34 dishes. The tenocytes in dishes in two experimental groups (n = 9, each group) were treated for 12 hours with the plasmid containing the PDGF-B cDNA or the VEGF cDNA and were then cultured for 5 days; tenocytes in the other eight dished received sham vector and the tenocytes in the control dishes (n = 8) did not receive the exogenous gene. Efficiency of the gene transfer was evaluated by detection of the presence of the transgene in the tenocytes by reverse transcription polymerase chain reactions (RT-PCR). Levels of expression of type I and III collagen, and TGF-β genes were determined by quantitative analysis of the products of RT-PCR. Results: Expression of the type I collagen gene was significantly increased by transfer of the exogenous PFDGF gene to the tenocytes (p < 0.001). Transfer of the PDGF-B gene did not affect the expression of TGF-β and the type III collagen genes significantly. Expression of TGF-β gene increased significantly in the cells treated with exogenous VEGF cDNA (p = 0.039). Expression of type I and III collagen genes by tenocytes was minimally affected by transfer of the VEGF gene to the tenocytes and was significantly weaker than that stimulated by PDGF-B gene therapy (p = 0.001). Efficient gene transfer was confirmed by the presence of the PDGF-B gene or the VEGF gene in the tenocytes receiving the transferred genes. Conclusions: Transfer of exogenous PDGF-B gene significantly enhances expression of the type I collagen gene of the tenocytes. Transfer of exogenous VEGF gene has very limited effects on promotion of collagen production in the proliferating tenocytes. In contrast, transfer of the VEGF gene significantly increases expression of the TGF-β gene. This study suggests that transfer of the PDGF-B gene may offer a novel way of effectively promoting healing of intrasynovial flexor tendons. VEGF gene therapy is not as beneficial as PDGF-B gene therapy to tendon healing and may increase activities of TGF-β that are associated with adhesion formations.

A platelet-derived growth factor releasing chitosan/coral composite scaffold for periodontal tissue engineering

In recent years, functional biomaterial research has been directed towards the development of improved scaffolds and new drug delivery systems. The objective of this study was to develop growth-factor gene releasing coral composites as a regenerative material for periodontal regeneration. In this study, porous chitosan/coral composites combined with plasmid encoding platelet-derived growth factor B (PDGFB) gene were prepared through a freeze-drying process. These scaffolds were evaluated in vitro by analysis of microscopic structure and cytocompatibility. The expression of PDGFB and type-I collagen were detected with RT-PCR after human periodontal ligament cells (HPLCs) were seeded in this scaffold. Then these scaffolds were implanted subcutaneously into athymic mice. Results indicated that HPLCs showed much better proliferation properties on the gene-activated scaffolds than on the pure coral scaffolds, and the expression of PDGFB and type-I collagen up-regulated in gene-activated scaffold. After implanted in vivo, HPLCs not only proliferate but also increased the expression of PDGFB. This study demonstrated the potential of coral scaffold combined PDGFB gene as a good substrate candidate in periodontal tissue regeneration.

Lentiviral Gene Therapy With Platelet-Derived Growth Factor B Sustains Accelerated Healing of Diabetic Wounds Over Time

The treatment of diabetic wounds is a formidable clinical challenge. In this study, lentiviral vectors carrying the human platelet-derived growth factor B (PDGF-B) gene were used to treated diabetic mouse wounds. Full-thickness 2.0-cm x 2.0-cm excisional wounds were created on the dorsa of genetically diabetic C57BL/KsJ-m+/+Lepr(db) mice. Lentiviral vectors containing the PDGF-B gene were injected into the wound margins and base. Mice were killed at 14-, 21-, and 35-day intervals. Measurement of the residual epithelial gap showed a trend towards increased healing in lentiviral PDGF-treated wounds compared with untreated and saline-treated wounds at all time points. At 21 days, there was significantly increased healing in lentiviral PDGF-treated wounds (0.98+/-0.17 cm) compared with saline-treated wounds (1.22+/-0.30 cm; P<0.05). Immunohistochemistry for CD31 revealed significantly increased neovascularization in lentiviral PDGF-treated wounds compared with untreated and saline-treated wounds at 14 and 21 days (P<0.01). Picrosirius red staining demonstrated thicker and more highly organized collagen fibers in treated wounds compared with untreated and saline-treated wounds. Quantitative analysis of collagen content showed a 3.5-fold and 2.3-fold increase in lentiviral PDGF-treated wounds versus untreated and saline-treated wounds, respectively (P<0.01). Lentiviral gene therapy with PDGF-B can sustain diabetic wound healing over time and may possess promising potential in the clinical setting.