PDGF-BB Research Articles

Migration properties of adipose-tissue-derived mesenchymal stromal cells cocultured with activated monocytes in vitro

Mesenchymal stromal cells (MSCs) are a promising tool in regenerative medicine. MSC migration to damaged inflammatory sites (homing) is essential for tissue repair. We have studied the migration properties of adipose-tissue-derived MSCs (AT-MSC) after their cocultivation with activated monocytes from the THP-1 cell line. We observed the increased migration rate of AT-MSC in vitro with the lack of chemoattractant gradient and to the platelet-derived growth factor (PDGF BB), which is a well-known chemoattractant for cells of mesenchymal origin. Moreover, the rate of directional AT-MSC migration through fibronectin was also increased. We demonstrated that signaling via PDGFR-β activated through the binding of integrin receptors with an extracellular matrix is a possible mechanism for stimulation of cellular migration under simulated inflammatory conditions.

FOXF1 transcription factor is required for formation of embryonic vasculature by regulating VEGF signaling in endothelial cells.

Inactivating mutations in the Forkhead Box transcription factor F1 (FOXF1) gene locus are frequently found in patients with alveolar capillary dysplasia with misalignment of pulmonary veins, a lethal congenital disorder, which is characterized by severe abnormalities in the respiratory, cardiovascular, and gastrointestinal systems. In mice, haploinsufficiency of the Foxf1 gene causes alveolar capillary dysplasia and developmental defects in lung, intestinal, and gall bladder morphogenesis. Although FOXF1 is expressed in multiple mesenchyme-derived cell types, cellular origins and molecular mechanisms of developmental abnormalities in FOXF1-deficient mice and patients with alveolar capillary dysplasia with misalignment of pulmonary veins remain uncharacterized because of lack of mouse models with cell-restricted inactivation of the Foxf1 gene. In the present study, the role of FOXF1 in endothelial cells was examined using a conditional knockout approach. A novel mouse line harboring Foxf1-floxed alleles was generated by homologous recombination. Tie2-Cre and Pdgfb-CreER transgenes were used to delete Foxf1 from endothelial cells. FOXF1-deficient embryos exhibited embryonic lethality, growth retardation, polyhydramnios, cardiac ventricular hypoplasia, and vascular abnormalities in the lung, placenta, yolk sac, and retina. Deletion of FOXF1 from endothelial cells reduced endothelial proliferation, increased apoptosis, inhibited vascular endothelial growth factor signaling, and decreased expression of endothelial genes critical for vascular development, including vascular endothelial growth factor receptors Flt1 and Flk1, Pdgfb, Pecam1, CD34, integrin β3, ephrin B2, Tie2, and the noncoding RNA Fendrr. Chromatin immunoprecipitation assay demonstrated that Flt1, Flk1, Pdgfb, Pecam1, and Tie2 genes are direct transcriptional targets of FOXF1. FOXF1 is required for the formation of embryonic vasculature by regulating endothelial genes critical for vascular development and vascular endothelial growth factor signaling.

Effect of hypoxia and PDGF-BB siRNA transfected on the production of PDGF-BB and proliferation in cultured human RPE cells

Background Proliferative vitreoretinopathy (PVR):no blood vessels,fibrous membrane cell proliferation occurs retinal surface.The development of specific mechanisms has not been completely clarified,but the retinal pigment epithelium(RPE) and platelet-derived growth factor(PDGF) for the past few years are the research hotspot.Objective To explore the effects of hypoxia on the production of PDGF-BB and proliferation in cultured human RPE (hRPE) cells.Methods In the experimental group,10,15,20,30,40 μmol/L CoCl2 were used to mimic hypoxia environment of hRPE cells,the control group did not add CoCl2 on cultured hRPE cells.By using reverse transcriptive PCR (RT-PCR) and enzyme linked immunosorbent assay(ELISA),the expression of PDGF-BB mRNA and protein was detected,and detected proliferation of hRPE cells by MTT.The cells were divided into 6 groups,different siRNAs were transfected into PDGF-BB siRNA1 group,PDGF-BB siRNA2 group,PDGF-BB siRNA3 group (because PDGF-BB has three different siRNAs,one of which is a valid siRNA),β-actin siRNA group,unrelated sequence siRNA group,only Lip2000 was plused in the blank control group.After transfection of 4-6 hours,except for blank control group,the other five groups were added to PDGF-BB mRNA and protein and its effect on the proliferation of hRPE cells obviously CoCl2 concentrations (15 μmol/L) simulated hypoxia environment of 24 hours,the detection of PDGF-BB mRNA and protein and hRPE cell proliferation rate.Results Neither PDGF-BB mRNA or protein was found in the blank control group.The production of PDGF-BB mRNA and protein in hRPE cells cultured by different concentrations were different,with significant differences among them (F=43.737,P＜0.01;F=54.612,P＜0.05),and the expressions of PDGF-BB mRNA and protein of 15 μmol/L CoCl2 group were more than other groups,with significant differences between the two groups (all at P＜0.05).MTT showed that PDGF-BB protein and hRPE cell proliferation rate in different concentions of CoCl2 groups were different,with significant differences among them(F=95.379,P＜0.01 ; F =63.375,P＜0.05),the expression of PDGF-BB protein was more and hRPE cell proliferation rate was higher in 15 μmol/L CoCl2group than other groups,with significant differences between the two groups (all at P＜0.05),postive correlation was found between PDGF-BB protein and hRPE cell proliferation rate (r =0.994,P＜0.05).The production of PDGF-BB mRNA and protein in the different siRNA transfected groups were different,with significant differences among them (F =156.330,125.650,both at P＜0.01),and the expressions of PDGF-BB mRNA and protein of PDGF-BB siRNA2 group were more than other groups,with significant differences between them(all at P＜0.05).MTT showed that PDGF-BB protein and hRPE cell proliferation rate in different siRNA transfectedgroups were different,with significant differences among them (F =73.131,98.564,both at P＜ 0.01).The expression of PDGF-BB protein was less and hRPE cell proliferation rate was lower in PDGF-BB siRNA2 group than other groups,with significant differences between them (all at P ＜ 0.05),postive correlation was found between PDGF-BB protein and hRPE cell proliferation rate (r =0.996,P＜0.05).Conclusions Proper hypoxia can induce the expression of PDGF-BB,PDGF-BB expression can significantly promote the proliferation of hRPE cells.In the transfection targeting PDGF-BB siRNA,PDGF-BB expression is suppressed,can effectively reduce the value of hRPE cell hyperplasia. Key words: Platelet-derived growth factor-BB protein; Human retinal pigment epithelium; CoCl2; Proliferation ; siRNA

Transport and Golgi organisation protein 1 is a novel tumour progressive factor in oral squamous cell carcinoma

Transport and Golgi organisation protein 1 (TANGO), also known as MIA3, belongs to the melanoma inhibitory activity (MIA) gene family. Although MIA acts as an oncogene, MIA2 and TANGO have a tumour-suppressive function in several malignancies; accordingly, the role and function of the MIA gene family in tumours remain controversial. Here the roles of TANGO were investigated in oral squamous cell carcinoma (OSCC). We analysed expression and function of TANGO in human OSCC cell lines. TANGO expression was also examined in 171 cases of primary OSCC by immunohistochemistry and statistically assessed the correlation between TANGO positivity and the clinicopathological parameters including vessel density. By TANGO knockdown in OSCC cells, the growth and invasion were repressed and apoptosis was induced. Activities of platelet-derived growth factor beta polypeptide (PDGFB) and Neuropilin2 were inhibited by TANGO knockdown. TANGO immunoreactivity was detected in 35.1% (60/171) cases of OSCC. TANGO expression was strongly associated with tumour progression, nodal metastasis, clinical stage and number of blood or lymph vessels in OSCC. Patients showing TANGO-expression fared significantly worse disease-free survival than cases without TANGO expression. These findings suggest that TANGO might promote angiogenesis and lymphangiogenesis by upregulation of PDGFB and Neuropilin2 in OSCC.

Neoadjuvant Imatinib in Advanced Primary or Locally Recurrent Dermatofibrosarcoma Protuberans: A Multicenter Phase II DeCOG Trial with Long-term Follow-up

Dermatofibrosarcoma protuberans (DFSP) is a rare cutaneous tumor. COL1A1-PDGFB gene fusion is frequent in DFSP, rendering tumor cell proliferation and survival dependent on PDGFRβ (platelet-derived growth factor receptor β) signaling. This trial investigated imatinib as neoadjuvant treatment of DFSP, including long-term follow-up. The primary endpoint of this multicenter phase II trial was response; secondary endpoints were safety, tumor relapse, and response biomarkers. Patients with advanced primary or locally recurrent DFSP and measurable disease by RECIST (response evaluation criteria in solid tumors) were eligible and received imatinib 600 mg/d until definitive surgery with histopathologic proof of tumor-free margins. Sixteen patients received imatinib, and 14 patients were evaluable for all endpoints. Median treatment duration was 3.1 months; median tumor shrinkage was 31.5%. Best overall response was 7.1% complete response (CR), 50.0% partial response (PR), 35.7% stable disease, and 7.1% progressive disease (PD). Toxicity was moderate with 25.0% grade 3 and 4 events. During a median follow-up of 6.4 years, one patient developed secondary resistance to imatinib but responded to second-line sunitinib. This patient also presented local recurrence, distant metastasis, and death from DFSP. Exploratory analysis showed that response to imatinib was associated with decreased tumor cellularity and formation of strong hyalinic fibrosis. Weak PDGFRB phosphorylation and pigmented-type DFSP were associated with nonresponse. Additional to PDGFRB, the kinases EGFR and insulin receptor were found activated in a high percentage of DFSPs. The neoadjuvant use of imatinib 600 mg/d in DFSP is efficacious and well tolerated. Long-term follow-up results do not definitely support smaller surgical margins after successful imatinib pretreatment, and presume that secondary resistance to imatinib might promote accelerated disease progression.

Sorafenib inhibition of hepatic stellate cell activation in the microenvironment of hepatocellular carcinoma

Objective To investigate the effects and mechanisms of sorafenib on hepatic stellate cell viability and activation in the microenvironment of liver tumor.Methods The effects of LX2 cells on HepG2 cell proliferation were observed by coculture of LX2 and HepG2 cells.MTT assay was used to observe the effects of sorafenib on LX2 proliferation,and expression of α-smooth muscle actin (α-SMA) was measured immunocytochemically in LX2 cells treated with different concentrations of sorafenib.Changes in PDGF-BB and TGF-β1 concentrations were detected in LX2 supernatant using ELISA.Expression of ERK1,ERK2,and AKT signaling pathways were measured using Western blot.Furthermore,LX2 cells were cocultured with HepG2 cells for 24 hours to observe their effects on the invasive ability of HepG2 cells.Result After coculture of LX2 and HepG2 cells,HepG2 cells increased in the experimental group more than those of in the control group.After treatment with various concentrations of sorafenib for 12,24,36 or 48 hours,the viability of treated LX2 cells was lower than the controls in a concentration-and time-dependent manner.As sorafenib concentration and time of exposure increased,α-SMA expression became weaker in treated cells.PDGF-BB and TGF-β1 concentrations decreased with higher sorafenib concentrations and with longer exposure under the same concentration.ERK1,ERK2 and Akt expression was identical between treated and control groups,but their phosphorylated expression decreased with increased concentrations of sorafenib.The invasive ability of HepG2 cells induced by LX2 gradually decreased as sorafenib concentrations increased.Conclusions Sorafenib suppressed α-SMA expression,inhibited PDGF-dependent signaling pathways in HSCs,downregulated PDGF-BB and TGF β1 expression in the supernatant of HSCs,and restrained the viability and activation of HSCs.Sorafenib treatment therefore resulted in suppressed proliferation and invasion of HepG2 cells. Key words: Sorafenib; Hepatocellular carcinoma; Hepatic stellate cells; Tumor microenvironment

Integrated molecular portrait of non-small cell lung cancers

BackgroundNon-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The objectives of this study were to utilize integrated genomic data including copy-number alteration, mRNA, microRNA expression and candidate-gene full sequencing data to characterize the molecular distinctions between AC and SCC.MethodsComparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC.ResultsAt DNA, mRNA and miRNA levels we could identify molecular markers that discriminated significantly between the various histopathological entities of NSCLC. We identified 34 genomic clusters using aCGH data; several genes exhibited a different profile of aberrations between AC and SCC, including PIK3CA, SOX2, THPO, TP63, PDGFB genes. Gene expression profiling analysis identified SPP1, CTHRC1and GREM1 as potential biomarkers for early diagnosis of the cancer, and SPINK1 and BMP7 to distinguish between AC and SCC in small biopsies or in blood samples. Using integrated genomics approach we found in recurrently altered regions a list of three potential driver genes, MRPS22, NDRG1 and RNF7, which were consistently over-expressed in amplified regions, had wide-spread correlation with an average of ~800 genes throughout the genome and highly associated with histological types. Using a network enrichment analysis, the targets of these potential drivers were seen to be involved in DNA replication, cell cycle, mismatch repair, p53 signalling pathway and other lung cancer related signalling pathways, and many immunological pathways. Furthermore, we also identified one potential driver miRNA hsa-miR-944.ConclusionsIntegrated molecular characterization of AC and SCC helped identify clinically relevant markers and potential drivers, which are recurrent and stable changes at DNA level that have functional implications at RNA level and have strong association with histological subtypes.

Cortisol and proinflammatory cytokine profiles in type 1 (reversal) reactions of leprosy

PurposeCortisol levels in the circulation and at the sites of peripheral inflammation regulate type 1 (Reversal) reactions in leprosy akin to delayed type hypersensitivity reactions (DTH). In this study we determine the extent to which the differential mRNA expression of genes encoding cortisone–cortisol shuttle enzymes (11 β hydroxysteriod dehydrogenase I & II (11 β HSD I & II)), circulatory levels of proinflammatory cytokines (IL-6, IL-7, IP-10, IL-17F, IL-23, TNF-α, IL-1β, PDGF BB and CRP) and cortisol are associated with development of type 1 reactions in leprosy. MethodsUrine, blood and incisional skin biopsy samples from site of lesions were collected from 49 newly diagnosed untreated leprosy cases in T1R and 51 cases not in reaction (NR). mRNA expression levels of genes encoding 11 β HSD I & II in skin biopsy samples were determined by realtime PCR. Cortisol levels from the lesional skin biopsies, serum and urine samples and serum proinflammatory cytokine levels were measured using ELISA. ResultsThe mean expression ratios of 11 β HSD I & II are significantly lower in leprosy cases with T1R when compared to the NR leprosy cases. Cortisol levels in lesional skin biopsies and in urine are significantly lower (p=0.001) in leprosy cases with T1R. Serum cytokine levels of IP-10, IL-17F, IL-IL-6 and TNF-α are significantly higher (p<0.05) in leprosy cases with T1R when compared the NR leprosy cases. ConclusionOur study indicated an association of urinary and lesional skin cortisol levels with the manifestation of T1R in leprosy. IP-10, IL-17F, IL-6 and TNF-α can be potential prognostic serological markers and gene expression markers for early detection of type 1 reactions in leprosy.

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

A review of the clinical experience with recombinant human platelet-derived growth factor-BB (rhPDGF-BB) in orthopaedic bone repair and regeneration

Recombinant human PDGF BB homodimer (rhPDGF-BB) is a potent chemotactic and mitogenic factor for cells crucial to musculoskeletal tissue repair, including mesenchymal stem cells (MSCs), osteogenic cells, and tenocytes. Furthermore, rhPDGF-BB promotes angiogenesis by upregulation of vascular endothelial growth factor (VEGF). These properties allow rhPDGF-BB to initiate the tissue repair and regeneration cascade, a mechanism of action that has been demonstrated through preclinical and clinical studies. The established mechanism of action and preclinical safety profile of rhPDGF-BB support clinical investigation of this molecule in diverse conditions that require osseous repair. Furthermore, the safety and efficacy profiles of rhPDGF-BB observed in the healing of chronic foot ulcers in diabetic patients along with previously reported preclinical data in orthopaedic models provide additional rationale to assess the application of rhPDGF-BB containing scaffolds in bone applications, including periodontal disease and end-stage ankle arthritis. Additional combinations of rhPDGF-BB with tissue-specific matrices are being studied in additional clinical musculoskeletal indications, such as rotator cuff repair involving tendon and bone integration. This paper reviews the clinical experience with rhPDGF-BB in bone repair.

Platelet‐Derived Growth Factor Promotes Periodontal Regeneration in Localized Osseous Defects: 36‐Month Extension Results From a Randomized, Controlled, Double‐Masked Clinical Trial

Recombinant human platelet-derived growth factor (rhPDGF) is safe and effective for the treatment of periodontal defects in short-term studies up to 6 months in duration. We now provide results from a 36-month extension study of a multicenter, randomized, controlled clinical trial evaluating the effect and long-term stability of PDGF-BB treatment in patients with localized severe periodontal osseous defects. A total of 135 participants were enrolled from six clinical centers for an extension trial. Eighty-three individuals completed the study at 36 months and were included in the analysis. The study investigated the local application of β-tricalcium phosphate scaffold matrix with or without two different dose levels of PDGF (0.3 or 1.0 mg/mL PDGF-BB) in patients possessing one localized periodontal osseous defect. Composite analysis for clinical and radiographic evidence of treatment success was defined as percentage of cases with clinical attachment level (CAL) ≥2.7 mm and linear bone growth (LBG) ≥1.1 mm. The participants exceeding this composite outcome benchmark in the 0.3 mg/mL rhPDGF-BB group went from 62.2% at 12 months, 75.9% at 24 months, to 87.0% at 36 months compared with 39.5%, 48.3%, and 53.8%, respectively, in the scaffold control group at these same time points (P <0.05). Although there were no significant increases in CAL and LBG at 36 months among all groups, there were continued increases in CAL gain, LBG, and percentage bone fill over time, suggesting overall stability of the regenerative response. PDGF-BB in a synthetic scaffold matrix promotes long-term stable clinical and radiographic improvements as measured by composite outcomes for CAL gain and LBG for patients possessing localized periodontal defects ( ClinicalTrials.gov no. CT01530126).

The Role of Recombinant Human Platelet-derived Growth Factor-BB (rhPDGF-BB) in Orthopaedic Bone Repair and Regeneration

Recombinant human PDGF BB homodimer (rhPDGF-BB) is a potent recruiter of, and strong mitogenic factor for, cells crucial to musculoskeletal tissue repair, including mesenchymal stem cells (MSCs), osteogenic cells and tenocytes. rhPDGF-BB also upregulates angiogenesis. These properties allow rhPDGF-BB to trigger the cascade of bone and adjoining soft tissue repair and regeneration. This mechanism of action has been established in numerous preclinical and clinical studies. Demonstration of the safety and efficacy of rhPDGF-BB in the healing of chronic foot ulcers in diabetic patients and regeneration of alveolar (jaw) bone lost due to chronic infection from periodontal disease has resulted in two FDA-approved products based on this molecule. A third product is in late stages of clinical development, with pilot and pivotal clinical studies of rhPDGF-BB mixed with an osteoconductive bone matrix (Augment(®) Bone Graft) in foot and ankle fusions demonstrating that this product is at least as effective as bone autograft, and has an improved safety profile. Additional combinations of rhPDGF-BB with tissue-specific matrices are also being studied clinically in additional musculoskeletal indications.

The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells

We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-κB (NF-κB) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB) plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-κB through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells.

Visceral Adipose Tissue-derived Serine Proteinase Inhibitor Inhibits Apoptosis of Endothelial Cells as a Ligand for the Cell-Surface GRP78/Voltage-dependent Anion Channel Complex

Visceral adipose tissue-derived serine proteinase inhibitor (vaspin) is an adipokine identified from visceral adipose tissues of genetically obese rats. The role of vaspin in the diabetic vascular complications remains elusive, and we investigated the effects of vaspin on the vascular function under the diabetic milieu. Adenovirus carrying the full length of the vaspin gene (Vaspin-Ad) ameliorated intimal proliferation of balloon-injured carotid arteries in diabetic Wistar rats. The expression of Ccl2, Pdgfb, and Pdgfrb genes was significantly reduced by the treatment of Vaspin-Ad. In cuff-injured femoral arteries, the intimal proliferation was ameliorated in vaspin transgenic (Vaspin Tg) mice. The application of recombinant vaspin and Vaspin-Ad promoted the proliferation and inhibited the apoptosis of human aortic endothelial cells. Adenovirus expressing vaspin with calmodulin and streptavidin-binding peptides was applied to human aortic endothelial cells, subjected to tandem tag purification and liquid chromatography-tandem mass spectrometry, and we identified GRP78 (78-kDa glucose-regulated protein) as an interacting molecule. The complex formation of vaspin, GRP78, and voltage-dependent anion channel on the plasma membrane was confirmed by the immunoprecipitation studies using aortas of Vaspin Tg mice. The binding assay using (125)I-vaspin in human aortic endothelial cells revealed high-affinity binding (dissociation constant = 0.565×10(-9) m) by the treatment of 5 μM thapsigargin, which recruited GRP78 from the endoplasmic reticulum to plasma membrane by inducing endoplasmic reticulum stress. In human aortic endothelial cells, vaspin induced phosphorylation of Akt and inhibited the kringle 5-induced Ca(2+) influx and subsequent apoptosis. Vaspin is a novel ligand for the cell-surface GRP78/voltage-dependent anion channel complex in endothelial cells and promotes proliferation, inhibits apoptosis, and protects vascular injuries in diabetes mellitus.

Knockout of Density-Enhanced Phosphatase-1 Impairs Cerebrovascular Reserve Capacity in an Arteriogenesis Model in Mice

Collateral growth, arteriogenesis, represents a proliferative mechanism involving endothelial cells, smooth muscle cells, and monocytes/macrophages. Here we investigated the role of Density-Enhanced Phosphatase-1 (DEP-1) in arteriogenesis in vivo, a protein-tyrosine-phosphatase that has controversially been discussed with regard to vascular cell biology. Wild-type C57BL/6 mice subjected to permanent left common carotid artery occlusion (CCAO) developed a significant diameter increase in distinct arteries of the circle of Willis, especially in the anterior cerebral artery. Analyzing the impact of loss of DEP-1 function, induction of collateralization was quantified after CCAO and hindlimb femoral artery ligation comparing wild-type and DEP-1−/− mice. Both cerebral collateralization assessed by latex perfusion and peripheral vessel growth in the femoral artery determined by microsphere perfusion and micro-CT analysis were not altered in DEP-1−/− compared to wild-type mice. Cerebrovascular reserve capacity, however, was significantly impaired in DEP-1−/− mice. Cerebrovascular transcriptional analysis of proarteriogenic growth factors and receptors showed specifically reduced transcripts of PDGF-B. SiRNA knockdown of DEP-1 in endothelial cells in vitro also resulted in significant PDGF-B downregulation, providing further evidence for DEP-1 in PDGF-B gene regulation. In summary, our data support the notion of DEP-1 as positive functional regulator in vascular cerebral arteriogenesis, involving differential PDGF-B gene expression.

Nano‐Sized Calcium Phosphate Particles for Periodontal Gene Therapy

Growth factors such as platelet-derived growth factor (PDGF) have significantly enhanced periodontal therapy outcomes with a high degree of variability, mostly due to the lack of continual supply for a required period of time. One method to overcome this barrier is gene therapy. The aim of this in vitro study is to evaluate PDGF-B gene delivery in fibroblasts using nano-sized calcium phosphate particles (NCaPP) as vectors. NCaPP incorporating green fluorescent protein (NCaPP-GFP) and PDGF-B (NCaPP-PDGF-B) plasmids were synthesized using an established precipitation system and characterized using transmission electron microscopy and 1.2% agarose gel electrophoresis. Biocompatibility and transfection of the nanoplexes in fibroblasts were evaluated using cytotoxicity assay and florescence microscopy, respectively. Polymerase chain reaction and enzyme-linked immunosorbent assay were performed to evaluate PDGF-B transfection after different time points of treatments, and the functionality of PDGF-B transfection was evaluated using the cell proliferation assay. Synthesized NCaPP nanoplexes incorporating the genes of GFP and PDGF-B were spherical in shape and measured about 30 to 50 nm in diameter. Gel electrophoresis confirmed DNA incorporation and stability within the nanoplexes, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reagent assay demonstrated their biocompatibility in fibroblasts. In vitro transfection studies revealed a higher and longer lasting transfection after NCaPP-PDGF-B treatment, which lasted up to 96 hours. Significantly enhanced fibroblast proliferation observed in NCaPP-PDGF-B-treated cells confirmed the functionality of these nanoplexes. NCaPP demonstrated higher levels of biocompatibility and efficiently transfected PDGF plasmids into fibroblasts under described in vitro conditions.

Molecular and clinicopathological analysis of dermatofibrosarcoma protuberans

Dermatofibrosarcoma protuberans (DFSP) is a dermal and subcutaneous tumor of intermediate malignancy. The most remarkable cytogenetic feature of DFSP is the chromosomal translocation t(17;22)(q22;q13), causing a fusion of the platelet-derived growth factor beta chain (PDGFB) gene at 22q13, and the collagen type 1 alpha 1 (COL1A1) at 17q22. The aim of the study was to analyze the molecular characteristic of DFSP in conjunction with histopathological and clinical features.We performed fluorescence in situ hybridization (FISH) and multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to detect chromosomal translocations and fusion gene transcripts in 16 formalin-fixed, paraffin-embedded DFSP samples. In addition, the amplification of PDGFB was also evaluated in the 16 DFSP samples by real-time PCR.FISH analysis revealed that all the 16 samples exhibited COL1A1-PDGFB gene fusion. Eleven out of 11 informative cases (100%) showed fusion transcripts by multiplex RT-PCR analysis. Various exons of the COL1A1 gene were fused with the PDGFB gene. Among them, exon 25 was found to be more frequently involved. Real-time PCR showed that the PDGFB copy number increase in the DFSP samples was higher than in normal skin tissues (p=0.007). Values of FISH fusion signals and PDGFB DNA analysis were variable between samples, but suggested that increased values might be associated with parameters of tumor progression.Our results confirm that analysis of the COL1A1-PDGFB status by FISH and RT-PCR is a useful tool in the confirmation of a DFSP diagnosis. In addition, the analysis of PDGFB copy number status may become a useful diagnostic marker since the gene is a potential target for treatment of DFSP patients.

Platelet derived growth factor-CC isoform is associated with hemorrhagic transformation in ischemic stroke patients treated with tissue plasminogen activator

ObjectivePlatelet derived growth factor-CC (PDGF–CC) isoform is activated by tissue plasminogen activator (tPA) regulating blood brain barrier permeability after ischemia. We aimed to study the association of PDGF isoforms serum levels with hemorrhagic transformation (HT) and edema after thrombolytic treatment in ischemic stroke. MethodsWe studied 129 patients with ischemic stroke treated with tPA within the first 4.5 h (h) from stroke onset. CT was performed on admission and at 24–36 h. On the 2nd CT, HT was classified according to ECASS II criteria, and severe brain edema was diagnosed if extensive swelling causing any shifting of the structures of the midline was detected. PDGF-AA, PDGF-AB, PDGF–BB and PDGF–CC serum levels were analyzed by ELISA on admission (before tPA bolus), at 24 and 72 h. ResultsPatients who developed HT showed only higher levels of PDGF–CC isoform on admission and at 24 h (all p < 0.0001). In the multivariate analysis, PDGF–CC levels on admission (OR, 1.02; CI 95%, 1.00–1.04) and at 24 h (OR, 1.05; CI 95%, 1.02–1.08) were independently associated with HT after adjustment by confounding factors. On the other hand, patients with severe edema showed also higher levels of PDGF–CC on admission and at 24 h (p < 0.0001), but this statistical association was lost in the logistic regression analysis. PDGF–CC levels ≥ 175 ng/mL at 24 h predict the development of PH with a sensitivity of 90% and specificity of 88% (area under the curve 0.936; p < 0.0001). ConclusionIncreased PDGF–CC levels after tPA treatment is associated with HT.

Expression of adhesion-related cytokines in the uterine fluid after transcervical resection of adhesion

To study the concentration of adhesion-related cytokines, transforming growth factor (TGF) β(1), platelet-derived growth factor (PDGF) BB, b-fibroblast growth factor (bFGF) in intrauterine fluid and their roles in the intrauterine-adhesion reformation. From August 2009 to February 2010, 18 patients with moderate or severe intrauterine adhesions underwent transcervical resection of intrauterine adhesion as study group, in the mean time, 20 patients with incomplete septate uterus underwent transcervical resection of septum as control group. The charge of each patient's intrauterine fluid was collected at the time of 3, 6, 9, 12, 24, 48 and 72 hours, respectively, then the concentration of the three cytokines was measured by enzyme-linked immunosorbent assay (ELISA). The concentration of the expression of TGF-β(1) in the intrauterine fluid at 3, 6, 9, 12 hours after operation in study group was (3.6 ± 0.9), (10.4 ± 1.1), (7.6 ± 1.2), (7.2 ± 1.3) ng/ml, respectively; PDGF-BB was (2.6 ± 0.6), (3.5 ± 0.5), (5.4 ± 1.0), (5.7 ± 0.8) ng/ml, respectively; bFGF was (16.9 ± 1.3), (95.8 ± 17.8), (330.9 ± 70.5), (1303.3 ± 117.4) ng/ml, respectively. The expression of TGF-β(1) in control group was (3.0 ± 0.6), (7.5 ± 0.6), (5.4 ± 0.6), (4.6 ± 0.8) ng/ml, respectively; PDGF-BB was (2.5 ± 0.4), (2.6 ± 0.5), (4.7 ± 0.6), (4.4 ± 0.4) ng/ml, respectively; bFGF was (19.1 ± 2.4), (82.9 ± 21.8), (249.0 ± 54.2), (775.6 ± 178.8) ng/ml, respectively. The concentration of TGF-β(1), PDGF-BB and bFGF reached the peak during the time of 3 - 24 hours, 6 - 48 hours, 6 - 72 hours after surgery, respectively. The concentration of TGF-β(1), PDGF-BB and bFGF in study group were significantly higher than that in control group (P < 0.05). Concentration of TGF-β(1), PDGF-BB and bFGF was positively associated with area of transcervical resection of intrauterine adhesion, those cytokines might be involved in reformatin of intrauterine-adhesion.

THE STUDY OF BONE MARROW MESENCHYMAL STEM CELLS GENE MODIFIED BY RECOMBINANT ADENO-ASSOCIATED VIRUS-9 COMBINED WITH PDGF-B IN VITRO

ObjectivesTo explore the feasibility, stability and safety of using recombinant adeno-associated virus-9 which contained platelet-derived growth factor-B (rAAV9-PDGF-B) transduction to bone marrow mesenchymal stem cells in vitro.MethodsBone marrow mesenchymal stem...