PDGF AA Research Articles

Microglial neuropilin-1 promotes oligodendrocyte expansion during development and remyelination by trans-activating platelet-derived growth factor receptor

Nerve-glia (NG2) glia or oligodendrocyte precursor cells (OPCs) are distributed throughout the gray and white matter and generate myelinating cells. OPCs in white matter proliferate more than those in gray matter in response to platelet-derived growth factor AA (PDGF AA), despite similar levels of its alpha receptor (PDGFRα) on their surface. Here we show that the type 1 integral membrane protein neuropilin-1 (Nrp1) is expressed not on OPCs but on amoeboid and activated microglia in white but not gray matter in an age- and activity-dependent manner. Microglia-specific deletion of Nrp1 compromised developmental OPC proliferation in white matter as well as OPC expansion and subsequent myelin repair after acute demyelination. Exogenous Nrp1 increased PDGF AA-induced OPC proliferation and PDGFRα phosphorylation on dissociated OPCs, most prominently in the presence of suboptimum concentrations of PDGF AA. These findings uncover a mechanism of regulating oligodendrocyte lineage cell density that involves trans-activation of PDGFRα on OPCs via Nrp1 expressed by adjacent microglia.

Airway Remodeling Factors During Early-Life Rhinovirus Infection and the Effect of Premature Birth.

Background: Early rhinovirus (RV) infection is a strong risk factor for asthma development. Airway remodeling factors play a key role in the progression of the asthmatic condition. We hypothesized that RV infection in young children elicits the secretion of growth factors implicated in airway remodeling and asthma progression.Methods: We examined the nasal airway production of remodeling factors in children ( ≤ 2 years old) hospitalized due to PCR-confirmed RV infection. Airway remodeling proteins included: MMP-1, MMP-2, MMP-7, MMP-9, MMP-10, TIMP-1, TIMP-2, EGF, Angiopoietin-2, G-CSF, BMP-9, Endoglin, Endothelin-1, Leptin, FGF-1, Follistatin, HGF, HB-EGF, PLGF, VEGF-A, VEGF-C, VEGF-D, FGF-2, TGF-β1, TGF-β2, TGF-β3, PDGF AA, PDGF BB, SPARC, Periostin, OPN, and TGF-α.Results: A total of 43 young children comprising RV cases (n = 26) and uninfected controls (n = 17) were included. Early RV infection was linked to (1) enhanced production of several remodeling factors (e.g., HGF, TGFα), (2) lower MMP-9/TIMP-2 and MMP-2/TIMP-2 ratios, and (3) increased MMP-10/TIMP-1 ratios. We also found that relative to term infants, severely premature children had reduced MMP-9/TIMP-2 ratios at baseline.Conclusion: RV infection in young children elicits the airway secretion of growth factors implicated in angiogenesis, fibrosis, and extracellular matrix deposition. Our results highlight the potential of investigating virus-induced airway remodeling growth factors during early infancy to monitor and potentially prevent chronic progression of respiratory disorders in all ages.

Conditioned media from mesenchymal stromal cells and periodontal ligament fibroblasts under cyclic stretch stimulation promote bone healing in mouse calvarial defects

Background aimsWhen cells are exposed to stresses such as mechanical stimuli, they release growth factors and adapt to the surrounding environment H ere, we demonstrated that mechanical stimulation during culture affects the production of osteogenic and angiogenic factors. MethodsHuman bone marrow derived mesenchymal stromal cells (hMSCs) and human periodontal ligament fibroblasts (HPLFs ) were cultured under cyclic stretch stimulation for 24 h. Collected of the cells and conditioned media (CM), the gene and protein expression levels of osteogenic and angiogenic factors were evaluated. CM was also evaluated for angiogenic activity and calc ification ability. In in vivo study, CM was administered to a mouse calvarial defect model and histologically and radiologically evaluated. ResultsQuantitative real time polymerase chain reaction results showed that the expression of bone morphogenetic pro tein 2, 4 (BMP 2, 4), vascular endothelial growth factor A (VEGF A), and platelet derived growth factor AA (PDGF AA) was upregulated in the cyclic stretch stimulation group in comparison with the non stretch group in each cell type. Enzyme linked immunosor bent assay results revealed that the expression of BMP 2,4, VEGF A was upregulated in the cyclic stretch group in comparison with the non stretch group in each cell type. Only HPLFs showed significant difference in PDGF AA expression between the cyclic str etch and the non stretch group. Tube formation assay and Alizarin Red S staining results showed that angiogenic activity and calcification ability of CM was upregulated in the cyclic stretch stimulation group in comparison with the non stretch group in eac h cell type. CM was administered to the mouse calvarial defect model. Histological and radiological examination showed that the bone healing was promoted by CM from the cyclic stretch culture group. Immunohistological staining revealed that CM from cyclic stretch group have greater angiogenic effect than CM from the non stretch group. ConclusionsThese results indicate that osteogenesis was promoted by CM obtained under cyclic stretch stimulation through the increase of angiogenesis in the mouse calvarial defect model.

Combinatory Multifactor Treatment Effects on Primary Nanofiber Oligodendrocyte Cultures.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system. Neurological deficits are attributed to inflammatory demyelination, which compromises axonal function and survival. These are mitigated in experimental models by rapid and often complete remyelination of affected axons, but in MS this endogenous repair mechanism frequently fails, leaving axons increasingly vulnerable to the detrimental effects of inflammatory and metabolic stress. Understanding the molecular basis of remyelination and remyelination failure is essential to develop improved therapies for this devastating disease. However, recent studies suggest that this is not due to a single dominant mechanism, but rather represents the biological outcome of multiple changes in the lesion microenvironment that combine to disrupt oligodendrocyte differentiation. This identifies a pressing need to develop technical platforms to investigate combinatory and/or synergistic effects of factors differentially expressed in MS lesions on oligodendrocyte proliferation and differentiation. Here we describe protocols using primary oligodendrocyte cultures from Bl6 mice on 384-well nanofiber plates to model changes affecting oligodendrogenesis and differentiation in the complex signaling environment associated with multiple sclerosis lesions. Using platelet-derived growth factor (PDGF–AA), fibroblast growth factor 2 (FGF2), bone morphogenetic protein 2 (BMP2) and bone morphogenetic protein 4 (BMP4) as representative targets, we demonstrate that we can assess their combinatory effects across a wide range of concentrations in a single experiment. This in vitro model is ideal for assessing the combinatory effects of changes in availability of multiple factors, thus more closely modelling the situation in vivo and furthering high-throughput screening possibilities.

Characterizing The Biological Constituents Of Platelet-poor Plasma--do The Platelets Matter? A Prospective Cohort Study

Objectives: Platelet-rich plasma (PRP) is comprised of several biologically active factors that can stimulate musculoskeletal healing processes. The supernatant of PRP, known as PPP, is biologically active and may also stimulate tissue regeneration. In some instances, such as muscle injury, PPP may be preferred to PRP in order to stimulate muscle regrowth in a basic science study that was previously performed. Platelet poor plasma (PPP) is has several biologically active molecular factors that may be utilized to stimulate tissue healing. While platelet rich plasma has been previously studied and characterized, few studies have sought to quantify the biological constituents of PPP. The purpose of this study was to quantitate and assess growth factors, other chemokines, and cytokines in PPP derived from human peripheral blood that has been centrifuged. Study Design: Non-randomized, prospective cohort study; Level of evidence: 2. Methods: Peripheral blood was drawn to create PPP at three time points from sixteen healthy volunteers. Hematology analysis was conducted on the PPP to quantify the platelet fold-difference from baseline measurements. The PPP samples were immediately assayed and analyzed on the MagPix® following processing completion. Specific immunoassay kits used were human cytokine/chemokine magnetic bead panel, TGF-β magnetic bead panel, MMP magnetic bead panel 1, and MMP magnetic bead panel 2. Results: Among the biological factors tested, there was a significant positive association, defined by two factors being associated in that when one factor increases the other also increases, between BMI and the biological composition of PPP with PDGF AA, PDGF AB, MMP-1, MMP-9, MMP-13, and MMP-12 (p<0.05). Similarly, there was a significant positive association (p<0.05), between age and biological composition of PPP for MMP-9 and MMP-7. Conclusion: PPP has several biological factors, both anabolic and catabolic, that can potentially be utilized in musculoskeletal medicine to treat various conditions, such as muscle injury. PPP is biologically active and this study characterizes its anabolic and catabolic profile. These factors are influenced by certain demographic factors such as age and body mass index (BMI). Higher BMI significantly correlated to higher levels of PDGF AA, PDGF AB, MMP-1, MMP-9, MMP-13, and MMP-12 in PPP. This supernatant of the better-studied PRP is biological active and warrants further investigation for its therapeutic potential. Platelets could change the biological composition of plasma utilized for regenerative medicine applications, but this study demonstrates that the plasma alone has biological properties that may provide benefit in treating certain musculoskeletal conditions. This study will help clinicians better understand the biological nature of PPP and may aide in the more targeted use of PPP therapeutically.

Reduction in Serum Levels of Inflammatory Cytokines in Subjects Consuming the Fermented Soy Beverage Q-CAN® Plus (P19-010-19)

ObjectivesWe want to investigate the effects a fermented soy drink (Q-CAN® Plus) on the serum levels of inflammatory cytokines in lean and obese individuals. MethodsProspective study comprised of lean (10) and obese (10) individuals without a known diagnosis. 11 clinic visits over 14 weeks (visit 1/wk −2, visit 2/wk −1, visit 3/wk 0, visit 4/wk 1, visit 5/wk 2, visit 6/wk 3, visit 7/wk 4, visit 8/wk 6, visit 9/wk 8, visit 10/wk 10, visit 11/wk 12). Visits 1, 2 and 3 occurred prior to treatment. 237 ml of soy was dispensed at visit 3 and was consumed twice daily for 4 weeks until visit 7 (weeks 1–4). Visits 8, 9, 10 and 11 were post treatment. Blood was collected during visits 1, 3, 5, 7, 8, 9, 10,11, and serum was subjected to quantification of cytokine levels using Human Magnetic Luminex® Assays - Human XL cytokine Discovery Premixed kit (CCL-2,4,5,11,20, FLT-3 ligand, CXCL-2,3,10, PDGF AB/BB, AA, IL-1RA, IL-10, PDL-1, VEGF, GMCSF and TRAIL, expressed as pg/ml). Paired t-test and one-way ANOVA (significance of P < 0.05) for the three therapeutic phases of pretreatment, during treatment and post treatmen ResultsIn lean subjects during treatment PDGF AA and AB/BB was significantly lower as compared to pretreatment, and this was sustained post-treatment (PDGF AA mean, pre = 1355 pg/ml, on = 913 pg/ml & post = 746, P < 0.05; PDGF AB/BB mean, pre = 611, on = 403 & post = 313 pg/ml, P < 0.05). In obese subjects only PDGF AA levels showed significant reduction on Q-CAN® Plus treatment, and returned to baseline post-treatment (PDGF AA mean pre = 922 pg/ml & on = 694 pg/ml; PDGF AB/BB mean in obese pretreatment = 267 pg/ml). In obese subjects, the levels of IL-1RA & GMCSF were also significantly lowered upon treatment as compared to their respective pre-treatment phase levels (IL-1RA mean, pre = 474 pg/ml & on = 353 pg/ml, P < 0.05; GMCSF mean, pre = 14 pg/ml & on = 10 pg/ml; P < 0.05). Post-treatment IL-1RA and GMCSF levels in obese subjects trended back to base line ConclusionsConsumption of Q-CAN® Plus fermented soy drink by healthy subjects resulted in reduction of serum levels of key inflammatory cytokines and growth factors. This suggests that Q-CAN® Plus consumption may prove beneficial for the management of chronic inflammatory and metabolic disorders. Future studies are necessary to confirm the effects of Q-CAN plus in patients with other inflammatory conditions Funding SourcesBESO Biological Research, Inc.

Role of plasma PlGF, PDGF-AA, ANG-1, ANG-2, and the ANG-1/ANG-2 ratio as predictors of preeclampsia in a cohort of pregnant women

Preeclampsia affects 3-5% of pregnancies worldwide and is the primary cause of maternal-fetal and neonatal mortality. Previous studies show that alterations in maternal concentrations of angiogenic factors, such as PlGF, PDGF AA, ANG-1, and ANG-2, may play fundamental roles in the pathophysiology of the disease. Determine whether the PlGF, PDGF AA, ANG-1, and ANG-2 are predictors of preeclampsia occurrence in a prenatal cohort study. This is a case-control study associated with a prospective cohort of pregnant women, with gestational ages between 20 and 25 weeks, composed of 30 pregnant women with preeclampsia (PE) and 90 healthy pregnant women (HP). The plasma concentrations of the markers were determined using the ELISA method. The comparison between the case and control groups was performed using the t test on the SAS® 9.4 software. Also, ROC curves were constructed to evaluate the predictive potential of the biomarkers. Differences in the concentrations of PlGF, PDGF AA, ANG-1 and ANG-2, and the ANG-1/ANG-2 ratio were not observed between the PE and the HP groups. The predictive capacity of the biomarkers was assessed using ROC curves, in which the area under the curve for PlGF AUC = 0.55; PDGF AA AUC = 0.55; ANG-1 AUC = 0.47; ANG-2 AUC = 0.51, and the ANG-1/ANG-2 ratio AUC = 0.57. In pregnant women, with gestational ages between 20 and 25 weeks significant differences in biomarker concentrations between groups PE and HP were not observed. The ROC curves showed that the biomarkers were ineffective as preeclampsia predictors in the analyzed cohort.

39 Evaluation of plasma concentration of the platelet-derived growth factor (PDGF AA) in the prediction of preeclampsia

Introduction Preeclampsia (PE) is a disease with severe complications for the mother and neonate and amid all aspects related to the development of the disease, we highlight the angiogenic biomarkers. These markers are increasingly being studied to help in understanding the pathophysiology of PE, and of these markers is the PDGF AA, a protein directly involved in cell proliferation and migration of placental vasculogenesis. Objective This study aimed to determine the effectiveness of PDGF AA as a factor in PE prediction compared to serum biomarker levels in pregnant women who developed the disease and in healthy pregnant women. Methods This study is a case-control linked to a cohort, which included the participation of 1.417 pregnant women with gestational age between 20 and 25 weeks, who were interviewed and had blood collected for analysis before the development of the disease. Through interviews and medical record analysis (after delivery), we selected 30 women who had a confirmed diagnosis of preeclampsia and 90 healthy pregnant women. The dosage of PDGF AA was made to these participants and enabled us to analyze and compare the plasma concentration of this biomarker in healthy pregnant women and in women who developed preeclampsia. For the analysis of quantitative variables was used analysis of variance (ANOVA) and where this assumption was not observed, logarithmic transformations in the response variable were used. Results We found no significant difference in the age of pregnant women, BMI, gestational age and heart rate. The values of the comparison between the two groups showed a significant difference in systolic blood pressure (p = 0.001) and diastolic (p ± 167.7 and in healthy patients was 337.80 ± 305.1 (p = 0.48), no significant difference. Conclusion We conclude that serum levels of PDGF AA is not effective as a predictor of preeclampsia, since the difference observed between healthy pregnant women and pregnant women who developed the disease is not significant.

Platelet-rich plasma as treatment for persistent ocular epithelial defects

Platelet- rich plasma (PRP) exhibits regenerative proprieties in wound healing but the biochemical mechanisms are unclear. In this study, autologous PRP with a mean value of 338 × 103 platelets/µL was used to treat corneal lesions of different aetiology, while homologous PRP with 1 × 106 platelets/µL was used to treat cornel lesions induced by a graft versus host disease. The impact of platelet count on the levels of PDGF AA and BB, VEGF, and EGF in the two PRPs was evaluated after a cycle of freezing/thawing. Treated corneal lesions healed or improved. The levels of PDGF AA and BB, VEGF, and EGF in the autologous PRP raised from 296 ± 61; 201.8 ± 24; 53 ± 14 and 8.9 ± 2 to 1017 ± 253; 924.7 ± 222; 101 ± 46.5 and 174 ± 15.5 pg/mL, while in the homologous PRP were 3.4, 4.5, 3.2 and 2 folds higher, respectively. High level of platelet counts seems not required to treat corneal lesions.

Abstract 165: Platelet Derived Growth Factor (pdgf) is Associated With Progression of Symptomatic Intracranial Large Artery Atherosclerosis

Background and Purpose: Studies on the molecular pathways involved in the progression of intracranial large artery atherosclerosis are rare. We aimed to study the relationship between biomarkers and the risk of progression of symptomatic intracranial large artery atherosclerosis. Methods: Of 409 patients in Trial of cilostazol in symptomatic intracranial stenosis-2 (TOSS-2) study, 52 patients showed progression of symptomatic intracranial large artery atherosclerosis on MRA after 7 months. We selected 20 patients with progression and 40 age- and sex- matched control patients. We collected blood sample initially, one month and 7 month after infarction, and multiplex analysis of biomarkers including interleukin-1,2,6,8,10, soluble CD40ligand, TNF alpha, PDGF, soluble ICAM, E-selectin and VCAM , MMP-2,3,9, SOD1,2,3 and adipokines, were performed. Results: Demographic features such as age, sex, hypertension, diabetes and smoking history were not different between both groups. On univariate analysis, 7 month PDGF-AA (3053 ± 2896 pg/ml, vs 1444±1548 pg/ml), PDGF-AB/BB (15304.0±15634 pg/ml vs 5627±8656 pg/ml) level and MPO(25.7±33.9vs 9.9±8.7ng/ml) were higher in progression group. On multivariate analysis using logistic model, 7 month PDGF AA is independent prognostic factor for progression of intracranial large artery atherosclerosis (p=0.012). Conclusion: PDGF-AB/BB level is associated with progression of symptomatic intracranial large artery atherosclerosis.

NG2 Cells in White Matter But Not Gray Matter Proliferate in Response to PDGF

Glial cells that express the NG2 proteoglycan and the α receptor for PDGF (NG2 cells, polydendrocytes) make up the fifth major cell population that serves as oligodendrocyte progenitor cells in the postnatal CNS. Although recent studies have suggested differences in their proliferation and oligodendrocyte differentiation in gray and white matter, the mechanism underlying the observed differences has been unclear. Using organotypic slice cultures from the forebrain and cerebellum of early postnatal NG2creBAC:ZEG mice, we have compared basal and growth factor-induced proliferation of NG2 cells in gray and white matter. NG2 cells in white matter exhibited greater proliferative response to PDGF AA than those in gray matter. Heterotopic slice transplant and explant cultures suggested intrinsic mechanisms for the differential proliferative response of gray and white matter cells. Additionally, younger white matter NG2 cells showed a more robust proliferative response to PDGF. Basal and PDGF-induced proliferation of gray and white matter NG2 cells was largely dependent on Wnt/β-catenin and phosphatidylinositol 3-kinase acting through the mammalian target of rapamycin pathway and not through ERK. These data uncover a previously unrecognized divergence between gray and white matter NG2 cells in the developing brain in their proliferative response to PDGF.

PDGF receptor-β modulates metanephric mesenchyme chemotaxis induced by PDGF AA

PDGF B chain or PDGF receptor (PDGFR)-beta-deficient (-/-) mice lack mesangial cells. To study responses of alpha- and beta-receptor activation to PDGF ligands, metanephric mesenchymal cells (MMCs) were established from embryonic day E11.5 wild-type (+/+) and -/- mouse embryos. PDGF BB stimulated cell migration in +/+ cells, whereas PDGF AA did not. Conversely, PDGF AA was chemotactic for -/- MMCs. The mechanism by which PDGFR-beta inhibited AA-induced migration was investigated. PDGF BB, but not PDGF AA, increased intracellular Ca(2+) and the production of reactive oxygen species (ROS) in +/+ cells. Transfection of -/- MMCs with the wild-type beta-receptor restored cell migration and ROS generation in response to PDGF BB and inhibited AA-induced migration. Inhibition of Ca(2+) signaling facilitated PDGF AA-induced chemotaxis in the wild-type cells. The antioxidant N-acetyl-l-cysteine (NAC) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) abolished the BB-induced increase in intracellular Ca(2+) concentration, suggesting that ROS act as upstream mediators of Ca(2+) in suppressing PDGF AA-induced migration. These data indicate that ROS and Ca(2+) generated by active PDGFR-beta play an essential role in suppressing PDGF AA-induced migration in +/+ MMCs. During kidney development, PDGFR beta-mediated ROS generation and Ca(2+) influx suppress PDGF AA-induced chemotaxis in metanephric mesenchyme.

Bioconjugated Gold Nanodots and Nanoparticles for Protein Assays Based on Photoluminescence Quenching

This study describes the first instance of the use of two differently sized Au nanoparticles (Au NPs), acting separately as donor and acceptor, in homogeneous photoluminescence quenching assays developed for the analysis of proteins. Introduction of a breast cancer marker protein, platelet-derived growth factor AA (PDGF AA), to a solution of 11-mercaptoundecanoic acid-protected, 2.0-nm photoluminescent Au nanodots (L(AuND)) led to the preparation of PDGF AA-L(AuND) as the donor. Thiol-derivative PDGF binding aptamers (Apt) and 13-nm spherical Au NPs were used to synthesize the Apt-Q(AuNP) acceptor. The photoluminescence of PDGF AA-L(AuND) at 520 nm decreased when photoluminescence quenching occurred between Apt-Q(AuNP) and PDGF AA-L(AuND). We used the PDGF AA-L(AuND)/Apt-Q(AuNP)-based molecular light switching system to analyze PDGFs and PDGF alpha-receptor in separate homogeneous solutions. In the presence of PDGFs, the interaction between Apt-Q(AuNP) and PDGF AA-L(AuND) decreased as a result of competitive reactions between the PDGFs and Apt-Q(AuNP). Similarly, the interaction between Apt-Q(AuNP) and PDGF AA-L(AuND) reduced as a result of competitive reactions between PDGF alpha-receptor and PDGF AA-L(AuND). The limits of detection (LODs) for PDGF AA and PDGF alpha-receptor were 80 pM and 0.25 nM, respectively, resulting from a low background photoluminescence signal. When using the Apt-Q(AuNP) as selectors for (a) the enrichment of PDGF AA and (b) the removal of matrixes possessing intense background fluorescence from cell media and urine samples, the LOD for PDGF AA decreased to 10 pM. Unlike quantum dots, the L(AuND) provide the advantages of biocompatibility, ease of bioconjugation, and minimal toxicity.

A Novel PF4-Heparin Microbead Assay and Plasma Protein Biomarker Profiling for Improved Diagnosis of Heparin Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening, immune-mediated complication of heparin administration resulting in a hypercoagulable state. The diagnosis is made using clinical scoring systems with confirmatory laboratory testing. However, current diagnostic criteria may lack predictive value for HIT-associated thrombosis and need for alternative anticoagulation. Therefore, there is a need to develop diagnostic methods that will have a better predictive value for HIT diagnosis. The pathogenesis of HIT involves platelet-activating antibodies that recognize platelet factor 4 (PF4) -heparin complex, and may also involve dysregulation of inflammatory mediators and growth factors from platelets and endothelial cells. Luminex multiplex microbead assay allows for the simultaneous detection of multiple serum analytes. The aim of this study is to develop a multiplex method to detect anti-PF4-heparin antibodies and to generate profiles of cytokines, chemokines, growth factors and adhesion molecules with better predictive value for the diagnosis of HIT than current diagnostic methods. In combination with measurement of antibodies to (PF4)-heparin complex, such a panel is expected to enhance the accuracy of HIT diagnosis. To develop the anti-PF4-heparin IgG microbead assay, carboxylated Luminex microbeads were coated at various concentrations with the optimal molar ratio of the PF4/polyvinyl sulfonate (PF4/PVS) complex, the antigenic determinant from the GTI ELISA kit, (provided by GTI, Waukesha WI). The anti-PF4-heparin microbead assay was standardized using plasma samples from patients and healthy controls. Furthermore, we compared the anti-PF4-heparin microbead assay to traditional commercial HIT antibody ELISA kits from GTI, Diagnostica Stago and Aniara. To identify potential plasma biomarkers for HIT, measurements were made on plasma levels of 63 cytokines, chemokines, growth factors and adhesion molecules in patients with and without HIT (as defined by the Chong's score) and healthy controls using commercial multiplex detection panels (Bio-Rad, Upstate, Linco). Multiplex data were analyzed using multivariate statistical methods and compared with their respective clinical scores. Preliminary anti-PF4-heparin microbead assay results show an overall concordance rate of 80% with GTI (Polyclonal) ELISA; 91% with Diagnostica Stago (Polyclonal); 78% with Aniara Polyclonal and 76% with Aniara IgG ELISA. The anti-PF4-heparin microbead assay results also show good correlation with clinical determination of HIT disease status as determined by Chong's scoring (72% sensitivity, and 58% specificity). Preliminary biomarker profiling suggest that Il-2Ra, b-NGF, GRO-a, TNF-b, PDGF AB/BB, PDGF AA, and s-ICAM1 are potentially useful markers in further analysis for distinguishing HIT status. We have identified these candidate biomarkers to evaluate in a larger number of patient samples with suspected HIT. Our data show that our novel microbead assay for anti-PF4-heparin antibodies correlate well with the existing conventional ELISA results, and also correlate with HIT diagnosis as determined by the Chong's score. Furthermore, we have identified a panel of biomarkers that could be useful for improved diagnosis and prognosis of HIT, and will establish a basis for further understanding the pathophysiology of this coagulation disorder.

Induction of VEGF secretion in ovarian cancer by PDGF BB

5557 Background: We identified the PDGFR as a potential target in epithelial ovarian carcinoma (EOC). This led us to test whether inhibition of the receptor affects ovarian cancer cell proliferation and survival and regulates other processes critical to tumor growth and metastasis. We postulated that the PDGF-PDGFR axis regulates VEGF secretion in EOC. Methods: VEGF secretion in ovarian tumors, cancer cells, serum and ascites was measured by IHC, Western Blot and ELISA. The HOG Gyn03–62 protocol was a phase II protocol for patients with recurrent platinum resistant EOC. Patients were treated with imatinib and docetaxel. Serum and tumor samples from patients enrolled on this protocol were analyzed for VEGF. Results: VEGF expression was quantified by IHC in ovarian tumors. Of 21 PDGFR expressing ovarian tumors, seven specimens immunostained strongly for VEGF and six tumors demonstrated 2+ VEGF reactive extracellular (secreted) material. PDGF and VEGF secretion was measured in 17 specimens of malignant EOC ascites. The levels of PDGF BB and VEGF were strongly correlated (Pearson coefficient =0.728, p-value=0.001), suggesting that the two pathways interconnect. There was no correlation between PDGF AA and VEGF levels. VEGF levels were measured in 13 paired serum samples from patients enrolled in the clinical protocol HOG: Gyn03–62, before and after treatment. VEGF serum levels were stabilized or decreased in 9 of 13 EOC patients treated with imatinib. In conditioned media from primary cells, VEGF secretion was four fold higher for tumor derived cells than for cells derived from the normal ovarian epithelium. PDGF increased ten-fold VEGF secretion in PDGFR expressing immortalized ovarian cells (C272/hTert/E7 and C889/hTert), while imatinib reduced VEGF production to basal state. The effects of imatinib were mediated via inhibition of Akt and MAPK pathways, by stabilization of HIF1 alpha. In ovarian cancer cells overexpressing consitutively active Akt, imatinib inhibited only partially the secretion of VEGF compared to control cells, suggesting that the PI3K/Akt pathway is significantly implicated in PDGF-stimulated VEGF secretion. Conclusions: These results suggest that by blocking the PDGFR, imatinib inhibits VEGF production. This affects the tumor microenvironment favoring ovarian tumor growth and metastasis. No significant financial relationships to disclose.

Autocrine activation of PDGFRα promotes the progression of ovarian cancer

Platelet-derived growth factor receptor (PDGFR)α expression was found in ovarian cancer cells and tumors by microarray hybridization. This led us to test whether ovarian cancers also produce ligands for this receptor, as this would demonstrate that such malignancies support their own growth and spread through autocrine activation. We assayed the expression of ligands for the PDGFR in ovarian tumors, cell lines and peritoneal fluid using RT-PCR, immunohistochemistry (IHC) and ELISA. We detected strong mRNA expression for the PDGFRa ligands in most ovarian tumors. Receptor and ligand expressions (PDGFRα and PDGF AB) were also detected by IHC in, respectively, 34 and 32 of 47 ovarian tumors. The stainings for PDGFRa and PDGF AB were strongly correlated (P-value = 0.014), suggesting that an autocrine loop is functional in ovarian cancer. PDGF AA and BB were quantified in peritoneal fluid by ELISA. Both ligands are secreted at higher levels in ovarian cancer ascites specimens (n=54) than in fluid from nonmalignant disorders (n=8). PDGF was detected in media conditioned by ovarian cancer cells. Such conditioned media induced activation of the PDGFR, Akt and MAPK and stimulated cell proliferation. A neutralizing PDGF antibody blocked these effects. Specific PDGFR inhibition by siRNA or a neutralizing antibody to the receptor inhibited PDGF-stimulated receptor activation and cell proliferation, suggesting that receptor targeting has a role in ovarian cancer treatment.

TGF β1 and PDGF AA override Collagen type I inhibition of proliferation in human liver connective tissue cells

BackgroundA marked expansion of the connective tissue population and an abnormal deposition of extracellular matrix proteins are hallmarks of chronic and acute injuries to liver tissue. Liver connective tissue cells, also called stellate cells, derived from fibrotic liver have been thoroughly characterized and correspond phenotypically to myofibroblasts. They are thought to derive from fat-storing Ito cells in the perisinusoidal space and acquire a contractile phenotype when activated by tissue injury. In the last few years it has become evident that several peptide growth factors such as PDGF AA and TGF-β are involved in the development of fibrosis by modulating myofibroblast proliferation and collagen secretion. The fact that during the development of chronic fibrosis there is concomitant deposition of collagen, a known inhibitory factor, and sustained cell proliferation, raises the possibility that stellate cells from chronic liver fibrosis patients fail to respond to normal physiologic controls.MethodsIn this study we address whether cells from fibrotic liver patients respond to normal controls of proliferation. We compared cell proliferation of primary human liver connective tissue cells (LCTC) from patients with liver fibrosis and skin fibroblasts (SF) in the presence of collagens type I and IV; TGF-β, PDGF AA and combinations of collagen type I and TGF-β or PDGF AA.ResultsOur results indicate that despite displaying normal contact and collagen-induced inhibition of proliferation LCTC respond more vigorously to lower concentrations of PDGF AA. In addition, we show that collagen type I synergizes with growth factors to promote mitogenesis of LCTC but not SF.ConclusionsThe synergistic interaction of growth factors and extracellular matrix proteins may underlie the development of chronic liver fibrosis.

Platelet-derived growth factor and platelet profiles in childhood nephrotic syndrome

The aim of the study was to investigate (1) whether there are any changes in release of platelet-derived growth factor AA (PDGF AA) in children with nephrotic syndrome without clinical thromboembolic symptoms 2; (2) whether serum PDGF AA correlates with the platelet count (PLT) and platelet indices; (3) whether prednisone therapy affects the serum PDGF AA and the PLT; (4) whether PDGF AA is a useful predictor of disease activity. The study involved two groups of children: 33 with nephrotic syndrome (I) who were evaluated twice (A during relapse and B after 2 weeks of prednisone treatment) and 34 healthy children (II). The serum concentration of PDGF was measured by ELISA. In group I/A the PLT (P<0.01) and platelet distribution width (P<0.05) were elevated, the mean platelet volume (MPV) (P<0.05) was decreased and the plateletcrit (P>0.05) was normal. In group I/B, the PLT was decreased and MPV increased. The concentration of PDGF AA was still increased and correlated negatively with the albumin concentration. Hence in children with nephrotic syndrome an increase in PLT, a decrease in MPV, and a higher concentration of PDGF were observed. Treatment of nephrotic syndrome with prednisone for 2 weeks is not sufficient to normalize platelet parameters. Further studies are necessary to confirm the role of PDGF AA in the hypercoagulation state in children with nephrotic syndrome.

VEGF-null cells require PDGFR alpha signaling-mediated stromal fibroblast recruitment for tumorigenesis.

We generated VEGF-null fibrosarcomas from VEGF-loxP mouse embryonic fibroblasts to investigate the mechanisms of tumor escape after VEGF inactivation. These cells were found to be tumorigenic and angiogenic in vivo in spite of the absence of tumor-derived VEGF. However, VEGF derived from host stroma was readily detected in the tumor mass and treatment with a newly developed anti-VEGF monoclonal antibody substantially inhibited tumor growth. The functional significance of stroma-derived VEGF indicates that the recruitment of stromal cells is critical for the angiogenic and tumorigenic properties of these cells. Here we identified PDGF AA as the major stromal fibroblast chemotactic factor produced by tumor cells, and demonstrated that disrupting the paracrine PDGFR alpha signaling between tumor cells and stromal fibroblasts by soluble PDGFR alpha-IgG significantly reduced tumor growth. Thus, PDGFR alpha signaling is required for the recruitment of VEGF-producing stromal fibroblasts for tumor angiogenesis and growth. Our findings highlight a novel aspect of PDGFR alpha signaling in tumorigenesis.

Mechanisms of cell transformation in the Syrian hamster embryo (SHE) cell transformation system.

The Syrian hamster embryo (SHE) cell transformation system has been used for investigational studies of basic mechanisms of neoplastic transformation, as well as determining the carcinogenic potential of chemical, physical, and biological agents. Many of these investigations utilize an intermediate step in the SHE cell neoplastic transformation process, known as morphological transformation, as an indicator that the cells have acquired an increased potential to progress to malignancy. While the nature of the morphologically transformed phenotype is not completely understood, it is believed to result from a block in the cellular differentiation of stem cells present within the SHE cell population. In terms of determination of the transforming potential of biological/chemical/physical agents, more than 500 agents have been tested in the SHE cell transformation assay with an 80-90% correlation between MT and carcinogenic potential. As such, the SHE cell transformation assay has utility as a test to provide short-term information on the carcinogenic potential of chemicals. One class of agents of current interest with regard to SHE cell transformation assay utilization consists of growth and differentiation factors (GDFs). Analysis of the SHE cell transformation potential of the GDFs, epidermal growth factor (EGF), fibroblast growth factor 4 (FGF-4), platelet-derived growth factor AA (PDGF AA), PDGF AB, PDGF BB, and the antimitogenic GDF, transforming growth factor beta one (TGF-beta1), was performed. All GDFs, with the exception of TGF-beta1, induced SHE cell transformation. However, an interesting difference between the GDFs was observed--PDGF A/B and PDGF B/B, but not PDGF A/A, EGF, or FGF-4, induced transformation after both a transient 1-day exposure and a continuous 7-day exposure, while continuous 7-day exposure was required for transformation by PDGF A/A, EGF, and FGF-4. Interestingly, both transient 1-day and continuous 7-day TGF-beta1 exposure resulted in suppression of transformation induced by a variety of transforming agents including growth factors, Ames assay-positive carcinogens, Ames assay-negative carcinogens, and spontaneous transformation. Interestingly TGF-beta1 was not able to suppress transformation by the tumor promoter, TPA. Together, these data demonstrate the utility of the Syrian hamster embryo cell transformation system for analyzing the transforming potential of GDFs and for characterizing differences in transforming mechanisms between different GDFs.