PDGF-B Expression Research Articles

Angiogenic escape and tumour progression in two patients with metastatic breast cancer receiving bevacizumab treatment.

Abstract Abstract #1029 Background: Vascular endothelial growth factor A (VEGF-A) has an important role in tumour progression by promoting angiogenesis. VEGF-A inhibitors, such as bevacizumab (Bev) and VEGF-Trap are being introduced into the treatment of breast cancer in order to target angiogenesis by inhibiting VEGF-A.
 Material and Methods: Two patients with metastatic breast cancer are described having tumour progression while being treated with single agent Bev. Both patients participated in the AVADO clinical phase III study, where shown to have received Bev in combination with docetaxel (D) as first line treatment for metastatic breast cancer. The first patient (A) had received 6 cycles of D and Bev and was for 4 months on single agent Bev (15mg/kg/3wk) before progressing. Pt B had received 9 cycles of D and Bev and was on 7 months of single agent Bev (15mg/kg/3wk) before disease progression. Tumour biopsies of progressing lesions were obtained after informed consent. Routine histological assessment and a CD34/Ki67 double staining were performed on their primary tumour as well as on the newly developed metastasis (A+B). Chalkley counts (CC) and endothelial cell proliferation fractions (ECP) were assessed by two independent observers. RT-PCR Taqman low density arrays with a gene panel of 94 angiogenesis related genes were performed in triplicate on both metastasis and compared to 10 other primary breast tumours.
 Results: Both lesions showed a high CC, respectively 7.5±0.62 (A) and 4.8±0.2 (B). Both lesions had elevated ECP values of 14% (A) and 8% (B).
 Using the 2 (-__CT) method and 18S as an internal control, the VEGFR1 mRNA was highly overexpressed in both A (25.18±0.12 fold change) and B (38.60±0.07 fold change) compared to the mean of 10 unselected primary breast tumours serving as controls (p<10-7). Similarly, in metastasis B, VEGF-B, TGFB1 and PDGFRA were found to be overexpressed, i.e. out of range [min-max] of the 10 primary breast tumours. A had out of range overexpression of VEGF-C. The gene expression of VEGF-A, VEGF-D, VEGFR2, VEGFR3, PDGFB and PDGFRB in both A and B were found to be in the range of the 10 controls.
 Conclusion: We describe two patients with progressive disease while being treated with Bev after an initial response on the combination of D and Bev. These new sites of disease showed a highly angiogenic and apparently vascular dependent growth pattern, in spite of high dosed anti-VEGF-A regimen. This suggests the existence of an important VEGF-A independent alternative modality of the tumour to promote angiogenesis. VEGFR1 was remarkably overexpressed in both metastases compared to controls. The expression of placental growth factor, a VEGFR1 specific ligand is further being explored. Knowledge of the biology of Bev resistance is essential since it could be useful in designing well considered combinations of targeted therapies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1029.

Effects of Batroxobin on distal anastomotic intimal hyperplasia after expanded polytetrafluoroethylene bypass grafting in dog common carotid artery

To study the morphological behavior, the expression of MCP-1 and PDGF-B of distal anastomotic neointima after expanded polytetrafluoroethylene (ePTFE) bypass grafting. And to identify the effects of Batroxobin (BX) on distal anastomotic intimal hyperplasia. 12 adult mongrel dogs underwent end to side bypass grafting in left common carotid artery using ePTFE vascular graft (6mm in diameter, 5cm in length) were divided randomly into two groups. All 12 bypass grafts were removed 28 days after grafting and distal anastomotic parts were obtained. Using HE, Masson, IHC, RT-PCR and western blot, we analyzed the thickness, relative content of ECM, SMC PCNA index, expression of MCP-1 and PDGF-B in distal anastomotic neointima. And we compared the differences between the two groups. 28 day after grafting, the neointima was formed by SMC and ECM and was covered by epithelial cells. After using BX, the thickness of neointima was reduced [( 381.3 +/- 144.7) microm versus (213.8 +/- 29.0) microm, P < 0.05] and the relative content of ECM were significantly decreased (P = 0.006). The PCNA index (P = 0.109) and PDGF-B level (P = 0.055 by RT-PCR and P = 0.337 by WB) between the two groups had no significant difference. MCP-1 level was markedly decreased (P = 0.025 by RT-PCR and P = 0.016 by WB). Using BX after ePTFE grafting could effectively reduce the relative content of ECM, reduce the expression of MCP-1 and decrease the thickness of neointima in the early time.

Effects of interferon-alpha on the expression of Smad7 and PDGF-B in fibrotic pancreas in rats

Effects of interferon-alpha on the expression of Smad7 and PDGF-B in fibrotic pancreas in rats

GFAP promoter driven transgenic expression of PDGFB in the mouse brain leads to glioblastoma in a Trp53 null background

Glioblastomas are the most common and malignant astrocytic brain tumors in human adults. The tumor suppressor gene TP53 is commonly mutated and/or lost in astrocytic brain tumors and the TP53 alterations are often found in combination with excessive growth factor signaling via PDGF/PDGFRalpha. Here, we have generated transgenic mice over-expressing human PDGFB in brain, under control of the human GFAP promoter. These mice showed no phenotype, but on a Trp53 null background a majority of them developed brain tumors. This occurred at 2-6 months of age and tumors displayed human glioblastoma-like features with integrated development of Pdgfralpha+ tumor cells and Pdgfrbeta+/Nestin+ vasculature. The transgene was expressed in subependymal astrocytic cells, in glia limitans, and in astrocytes throughout the brain substance, and subsequently, microscopic tumor lesions were initiated equally in all these areas. With tumor size, there was an increase in Nestin positivity and variability in lineage markers. These results indicate an unexpected plasticity of all astrocytic cells in the adult brain, not only of SVZ cells. The results also indicate a contribution of widely distributed Pdgfralpha+ precursor cells in the tumorigenic process.

Aberrant PDGFRA Signalling Is Sustained by An Autocrine Loop in Peripheral T-Cell Lymphoma Not Otherwise Specified

Background. Peripheral T-cell lymphoma not otherwise specified (PTCL/NOS) is the most common T-cell lymphoma; however, it remains a complex entity showing great variety regarding morphology, immunophenotype and clinical behaviour. Recently, gene expression profiling (GEP) revealed PDGFRA as possible PTCL-associated molecule, by nominating it as potential therapeutic target.Aims and Methods. We investigated the possible determinants of PDGFRA activity in PTCL/NOS. We performed GEP of 28 PTCLs/NOS and 20 samples of normal T-cell subpopulations, by using the Affymetrix HG-U133 2.0 plus microarray. Moreover, we built tissue-microarrays (TMAs) including 145 PTCLs/NOS for protein analyses. The PDGFRA locus (4q1.1–4q1.3) was studied by FISH on those TMAs, while direct sequencing of all PDGFRA exon and introns as well as of the promoter region was performed in 90 cases. Immunohistochemistry (IHC) and ELISA were adopted in order to study the expression of PDGF-A, PDGF-B and PDGF-C on tissue sections and in supernatants from PTCL/NOS cell cultures, respectively. Finally, the expression of PDGFRA and its activated (phosphorilated) form, p-PDGFRA, was assessed by IHC on TMAs, and by flow-citometry in PTCL/NOS cultured cells before and after the exposure to an anti-PDGF ligand neutralizing antibody (R&B System).Results. First, GEP showed over-expression of PDGFRA in all tested PTCLs/NOS, while IHC confirmed the expression of PDGFRA and p-PDGFRA. FISH, SNPs analysis and direct sequencing showed preserved integrity of PDGRA locus. We then studied PDGF-A, PDGF-B and PDGF-C, which turned out to be expressed and secreted by PTCL/NOS cells. In order to verify the hypothesis of an autocrine loop, we tested whether the remotion of PDGF ligands from the supernatants of cultured PTCL/NOS cells determined PDGFRA de-phosphorilation. Notably, 48h after the exposure to 20 mg of anti-PDGF antibody, we already appreciated a significant effect with reduction of PDGFRA phosphorilation up to 75%.Conclusions. Taken together, our data demonstrate that PDGFRA activity is sustained by an autocrine loop in PTCL/NOS.

Role of Hypoxia Induced Factor (HIF-1 alpha) in Pulmonary Hypertension in Sickle Cell Disease

Background. Pulmonary hypertension is a leading cause of mortality in sickle cell disease (SCD), however, the mechanisms leading to the development of pulmonary hypertension remain poorly understood. The pathogenesis of SCD revolves around chronic hypoxia and hypoxic events that are mediated through the upregulation of hypoxia-inducible factor-1alpha (HIF-1alpha), a transcriptional factor, that is involved in regulation of several redox sensitive genes including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and ApoE. The objective of this study was to examine the role of HIF-1 and its target genes in SCD-associated pulmonary lung disease.Methods. In this study, 3 groups of age-matched homo and hemizygous sickle and control C57/Bl6 mice (Jackson Labs) were sacrificed and organs harvested for RNA isolation in Trizol. Using quantitative RT-PCR we assessed the RNA expression of HIF-1 and its target genes, VEGF and PDGF-B from the lungs of homozygous sickle mice and compared these with the C57/BL6 and the hemizygous sickle controls. HPRT was used as the housekeeping gene control.Results. Homozygous sickle mice demonstrated an upregulation of HIF-1 alpha RNA (almost 2.3 fold) compared with the hemizygous sickle and the C57/Bl6 controls. VEGF RNA expression was elevated ~8.3 to 10.9 fold in sickle mice compared to the two control groups. Interestingly there was a down regulation of PDGF-B expression (0.3 fold) in the sickle mice group compared to the control groups. In contrast, expression of all the HIF-related genes tested was down-regulated in the spleens and brains of sickle mice compared with the respective control tissues.Discussion. These findings support the possibility that complex molecular pathways, including dysregulation of HIF pathway, may contribute to the development of pulmonary hypertension in sickle cell disease and underscore role of oxidative stress pathways in the onset of disease pathogenesis.

Platelet-derived growth factor expression and function in idiopathic pulmonary arterial hypertension

Rationale Platelet-derived growth factor (PDGF) promotes the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), and may play a role in the progression of pulmonary arterial hypertension (PAH), a condition characterized by proliferation of PASMCs resulting in the obstruction of small pulmonary arteries. Objectives To analyze the expression and pathogenic role of PDGF in idiopathic PAH. Methods PDGF and PDGF receptor mRNA expression was studied by real-time reverse transcription-polymerase chain reaction performed on laser capture microdissected pulmonary arteries from patients undergoing lung transplantation for idiopathic PAH Immunohistochemistry was used to localize PDGF, PDGF receptors, and phosphorylated PDGFR-beta. The effects of imatinib on PDGF-B- induced proliferation and chemotaxis were tested on human PASMCs. Results PDGF-A, PDGF-B, PDGFR-alpha, and PDGFR-beta mRNA expression was increased in small pulmonary arteries from patients displaying idiopathic PAH, as compared with control subjects. Western blot analysis revealed a significant increase in protein expression of PDGFR-beta in PAH lungs, as compared with control lungs. In small remodeled pulmonary arteries, PDGF-A and PDGF-B mainly localized to PASMCs and endothelial cells (perivascular inflammatory infiltrates, when present, showed intensive staining), PDGFR-alpha and PDGFR-beta mainly stained PASMCs and to a lesser extent endothelial cells. Proliferating pulmonary vascular lesions stained phosphorylated PDGFR-beta. PDGF- BB-induced proliferation and migration of PASMCs were inhibited by imatinib. This effect was not due to PASMC apoptosis. Conclusions PDGF may play an important role in human PAH Novel therapeutic strategies targeting the PDGF pathway should be tested in clinical trials.

Activation of hepatic stellate cells is associated with cytokine expression in thioacetamide-induced hepatic fibrosis in mice

The pathophysiological mechanisms of thioacetamide (TAA)-induced hepatic fibrogenesis are not yet fully understood. In particular, the role of hepatic stellate cells (HSCs) remains unclear. We therefore examined proliferation and transdifferentiation of HSC as well as the underlying molecular mechanisms in TAA-induced fibrosis. Hepatic fibrogenesis was induced in mice by addition of TAA to drinking water. Liver damage was determined by assessment of alanine aminotransferase and aspartate aminotransferase levels, and measurement of collagen deposition. Additionally, expression patterns of α-smooth muscle actin, glial fibrillary acidic protein (GFAP, specific hepatic biomarker for HSC), cysteine- and glycine-rich protein 2 (CRP2, specific marker of HSC transdifferentiation), tissue inhibitor of metalloproteinases-1, matrix metalloproteinase-9 (MMP-9), interleukins (IL-1β, IL-6), platelet-derived growth factors (PDGF-B, PDGF-D) , tumor necrosis factor (TNF)-α, and (transforming growth factor (TGF)-β1 were assessed by real-time PCR. Transcription of GFAP and CRP2 were transiently upregulated during TAA-induced fibrogenesis (punctum maxima (p.m.) week 10 for GFAP and week 14 for CRP2). Similar transient expression patterns were demonstrated for IL-1β, IL-6, TGF-β1, and PDGF-B (p.m. week 12) whereas TNF-α and PDGF-D continuously increased with ongoing liver injury. In particular, not only neutrophil granulocytes, but also macrophages and leukocytes served as a major source for MMP-9 expression. GFAP and CRP2 expression patterns demonstrated transiently increased HSC-activation during TAA-induced hepatic fibrogenesis. The rate of increase of transcription of GFAP correlated best with PDGF-B, whereas CRP2 levels correlated with PDGF-B, PDGF-D, and IL-1β expression. This study demonstrates for the first time that transiently increased activation patterns of HSC are observed in toxically induced hepatic fibrosis. Thus, TAA in drinking water is an effective and elegant model to induce reproducible states of liver fibrosis without parenchymal damage in mice.

An Id-like molecule, HHM, is a synexpression group-restricted regulator of TGF-β signalling

Transforming growth factor (TGF)-β induces various cellular responses principally through Smad-dependent transcriptional regulation. Activated Smad complexes cooperate with transcription factors in regulating a group of target genes. The target genes controlled by the same Smad-cofactor complexes are denoted a synexpression group. We found that an Id-like helix-loop-helix protein, human homologue of Maid (HHM), is a synexpression group-restricted regulator of TGF-β signalling. HHM suppressed TGF-β-induced growth inhibition and cell migration but not epithelial–mesenchymal transition. In addition, HHM inhibited TGF-β-induced expression of plasminogen activator inhibitor-type 1 (PAI-1), PDGF-B, and p21WAF, but not Snail. We identified a basic-helix-loop-helix protein, Olig1, as one of the Smad-binding transcription factors affected by HHM. Olig1 interacted with Smad2/3 in response to TGF-β stimulation, and was involved in transcriptional activation of PAI-1 and PDGF-B. HHM, but not Id proteins, inhibited TGF-β signalling-dependent association of Olig1 with Smad2/3 through physical interaction with Olig1. HHM thus appears to regulate a subset of TGF-β target genes including the Olig1-Smad synexpression group. HHM is the first example of a cellular response-selective regulator of TGF-β signalling with clearly determined mechanisms.

Spinal Glioma: Platelet-Derived Growth Factor B–Mediated Oncogenesis in the Spinal Cord

Human platelet-derived growth factor B (hPDGFB) has been characterized in vitro and shown to mediate numerous cellular responses including glial proliferation and differentiation. Expression of PDGFB is thought to be important in the pathogenesis of glioma and several animal models of cerebral glioma based on PDGF expression have been described. To examine whether PDGF could contribute to the pathogenesis of spinal cord glioma, we developed transgenic mice that express hPDGFB under the control of a tetracycline-responsive element (TRE/hPDGFB). These TRE/hPDGFB mice were mated with transgenic mice expressing the tetracycline transcriptional activator (tet-off), tTA, regulated by the human glial fibrillary acidic protein (GFAP) promoter and exhibiting uniquely strong promoter activity in the spinal cord. These transgenic mice (GFAP/tTA:TRE/hPDGFB) expressed hPDGFB in GFAP-expressing glia in a manner responsive to doxycycline administration. Without doxycycline, almost all GFAP/tTA:TRE/hPDGFB mice developed spinal cord neoplasms resembling human mixed oligoastrocytoma. Tumorigenesis in these animals was suppressed by doxycycline. To further examine the importance of PDGFB in mouse primary intramedullary spinal cord tumors, we also created transgenic mice expressing hPDGFB under the control of the human GFAP promoter (GFAP/hPDGFB). These GFAP/hPDGFB mice also developed spinal oligoastrocytoma. PDGFB can mediate the development of mouse spinal tumors that are histologically and pathologically indistinguishable from primary intramedullary spinal tumors of humans and may provide opportunities for both novel insights into the pathogenesis of these tumors and the development of new therapeutics.

Platelet-derived growth factor isoform expression in carbon tetrachloride-induced chronic liver injury

Platelet-derived growth factor (PDGF) has an essential role in liver fibrogenesis, as PDGF-B and -D both act as potent mitogens on culture-activated hepatic stellate cells (HSCs). Induction of PDGF receptor type-β (PDGFRβ) in HSC is well documented in single-dose carbon tetrachloride (CCl4)-induced acute liver injury. Of the newly discovered isoforms PDGF-C and -D, only PDGF-D shows significant upregulation in bile duct ligation (BDL) models. We have now investigated the expression of PDGF isoforms and receptors in chronic liver injury in vivo after long-term CCl4 treatment and demonstrated that isolated hepatocytes have the requisite PDGF signaling pathways, both in the naive state and when isolated from CCl4-treated rats. In vivo, PDGF gene expression showed upregulation of all PDGF isoforms and receptors, with values peaking at 4 weeks and decreasing to near basal levels by 8 and 12 weeks. Interestingly, PDGF-C increased significantly when compared to BDL-models. PDGF-A, PDGF-C and PDGF receptor type-α (PDGFRα) correlated closely with inflammation and steatosis. Immunohistochemistry revealed expression of PDGF-B, -C and -D in areas corresponding to centrilobular necrosis, inflammation and fibrosis, whereas PDGF-A localized in regenerative hepatocytes. PDGFRβ was identified along the fibrotic septa, whereas PDGFRα showed positive staining in fibrotic septa and regenerative hepatocytes. Despite a significant decline of PDGF isoforms, hepatocyte regeneration peaked at 8 weeks. A marked difference in the degree of fibrosis was observed amongst the individual animals. In summary, PDGF expression in liver damage primarily parallels mesenchymal cell proliferation and extracellular matrix production, rather than hepatocyte regeneration. We conclude that PDGF levels in chronic liver injury peak at 4 weeks after onset of injury, and that the outcome of chronic toxic liver injury strongly depends on the individual capacity for tissue regeneration in the weeks following the peak of PDGF expression.

Prognostic Impact of Platelet-Derived Growth Factors in Non-small Cell Lung Cancer Tumor and Stromal Cells

In tumor angiogenesis there is a complex interplay between endothelial, stromal, and tumor cells (neoplastic epithelial cells). Platelet-derived growth factors (PDGFs) and receptors (PDGFRs) are pivotal in this interaction, and important targets in novel antiangiogenic therapies. This study investigates the prognostic impact of these molecular markers in tumor cells and tumor stroma of resected non-small cell lung cancer (NSCLC) tumors. Tumor tissue samples from 335 resected patients with stage I to IIIA NSCLC were obtained and tissue microarrays were constructed from duplicate cores of tumor cells and tumor-related stroma from each specimen. Immunohistochemistry was used to evaluate the expression of the molecular markers PDGF-A, -B, -C, and -D and PDGFR-alpha and -beta. In univariate analyses, high tumor cell expression of PDGF-B (p = 0.001), PDGF-C (p = 0.01), and PDGFR-alpha (p = 0.026) were negative prognostic indicators for disease-specific survival. In tumor stroma, high expression of PDGF-A (p = 0.009), PDGF-B (p = 0.04), PDGF-D (p = 0.019), and PDGFR-alpha (p = 0.019) correlated with good prognosis. In multivariate analyses, high tumor cell PDGF-B (p = 0.001) and PDGFR-alpha (p = 0.047) expression were independent negative prognostic factors for disease-specific survival, whereas in stromal cells high PDGF-A (p = 0.001) expression had an independent positive survival impact. Our results indicate PDGF-B and PDGFR-alpha inhibition as an interesting approach in NSCLC treatment, but also demonstrates the importance of understanding the cellular crosstalk between endothelial, stromal, and tumor cells when targeting PDGF markers.

Intraglomerular expressions of IL-1 alpha and platelet-derived growth factor (PDGF-B) mRNA in experimental immune complex-mediated glomerulonephritis.

Both PDGF and IL-1 play important roles as autocrine growth factors for cultured mesangial cells, and may be closely associated with the progression of glomerulonephritis. In this study we investigated intraglomerular expressions of PDGF-B and IL-1 alpha mRNA in mice with bovine serum albumin (BSA) nephritis, a model of immune complex-mediated glomerulonephritis, using the reverse transcription-polymerase chain reaction (RT-PCR) method. We also quantified intraglomerular PDGF-B mRNA by the competitive PCR and studied the correlation between the level of intraglomerular PDGF-B mRNA expression and the degree of observed glomerular injury. While expression of neither PDGF-B nor IL-1 alpha mRNA was detected in glomeruli from control mice, both were strongly expressed in glomeruli from mice with BSA nephritis. IL-1 alpha mRNA in glomeruli showed low accumulation in mice with mild glomerular injury, and was increased in mice with moderate glomerular injury. In contrast, high intraglomerular expression of PDGF-B mRNA occurred in all mice with mild glomerular injury and continued throughout the course of the disease. We observed no correlation between the level of PDGF-B mRNA expression and the histologic grade of renal damage. These results suggest that PDGF and IL-1 have different growth properties, and PDGF might play a role as a competence factor rather than a progression factor in the pathogenesis of immune complex-mediated glomerulonephritis.

Platelet‐derived growth factor‐B gene delivery sustains gingival fibroblast signal transduction

Platelet-derived growth factor-BB is a potent mediator of tooth-supporting periodontal tissue repair and regeneration. A limitation of the effects of topical platelet-derived growth factor-BB application is its short half-life in vivo. Gene therapy has shown strong promise for the long-term delivery of platelet-derived growth factor in both skin ulcer healing and periodontal tissue engineering. However, little is known regarding the extended effects of platelet-derived growth factor-B on cell signaling via gene delivery, especially at the level of phosphorylation of intracellular kinases. This study sought to evaluate the effect of gene transfer by Ad-PDGF-B on human gingival fibroblasts (HGFs) and the subsequent regulation of genes and cell-surface proteins associated with cellular signaling. HGFs from human subjects were treated by adenoviral PDGF-B, PDGF-1308 (a dominant negative mutant of PDGF) and recombinant human platelet-derived growth factor-BB, and then incubated in serum-free conditions for various time points and harvested at 1, 6, 12, 24, 48, 72 and 96 h. Exogenous PDGF-B was measured by RT-PCR and Western blot. Cell proliferation was evaluated by [methyl-3H]thymidine incorporation assay. We used proteomic arrays to explore phosphorylation patterns of 23 different intracellular kinases after PDGF-B gene transfer. The expression of alpha and beta PDGFR and Akt were measured by Western blot analysis. Sustained in vitro expression of PDGF-B in HGFs by Ad-PDGF-B transduction was seen at both the mRNA and protein levels. Compared to rhPDGF-BB and Ad-PDGF-1308, Ad-PDGF-B maintained cell growth in serum-free conditions, with robust increases in DNA synthesis. Gene delivery of PDGF-B also prolonged downregulation of the growth arrest specific gene (gas) PDGF alpha R. Of the 23 intracellular kinases that we tested in proteomic arrays, Akt revealed the most notable long-term cell signaling effect as a result of the over-expression of Ad-PDGF-B, compared with pulse recombinant human platelet-derived growth factor BB. Prolonged Akt phosphorylation was induced by treatment with Ad-PDGF-B, for at least up to 96 h. These findings further demonstrate that gene delivery of PDGF-B displays sustained signal transduction effects in human gingival fibroblasts that are higher than those conveyed by treatment with recombinant human platelet-derived growth factor-BB protein. These data on platelet-derived growth factor gene delivery contribute to an improved understanding of these pathways that are likely to play a role in the control of clinical outcomes of periodontal regenerative therapy.

Lymphangiogenesis promotes inflammation and neointimal hyperplasia after adventitia removal in the rat carotid artery

Lymphatic vessels exist in adventitia in the atherosclerotic coronary artery and play an important role in the inflammatory and immune response. After adventitia removal, the carotid wall of rat model showed significantly increased ratio of intimal to medial area (I/M ratio), the number of adventitial lymphatic vessels (Ad-LV) and microvessels (Ad-MV), and macrophage index and expression of VEGF-C, VEGFR-3, PDGF-B and PDGFR-β. The I/M ratio was significantly correlated with Ad-LV and macrophage index but not Ad-MV. These results suggest that adventitial lymphangiogenesis is stimulated by growth factors released by inflammatory cells in vasculature after adventitia removal, and these neogenetic lymph vessels in turn promote intimal inflammation and hyperplasia, probably via delivery and activation of inflammatory cells.

Abstract 724: Synectin is Required for Normal PDGF Secretion and Smooth Muscle Cell Basic Science Recruitment

Background: Synectin, a ubiquitously expressed scaffold and PDZ protein, has been shown to be a key regulator in the formation of arterial vasculature. Primary arterial endothelial cells isolated from synectin−/− mice exhibited defects in PDGF signaling that were not present in arterial synectin+/+ or either venous synectin+/+ or synectin−/− endothelial cells. Methods: To address the role of synectin in mural cell recruitment, retinas from synectin−/− (KO) and synectin+/+ (WT) mice were stained for isolectin and alpha-smooth muscle actin. To determine the mechanisms responsible for defective smooth muscle cells recruitment, primary arterial and venous endothelial (EC) and smooth muscle (SMC) cells were isolated from synectin+/+ and synectin−/− mice. Results: Examination of the retinal vasculature in synectin−/− mice demonstrated poor smooth muscle cell coverage of the forming arterial tree with a number of SMC cells sitting some distance away from the vessels, a defect reminiscent of retinal abnormalities observed in PDGF-B−/− mice. Western blot analysis of lysates from primary KO SMC demonstrated levels of PDGFR-β analogous to WT, although expression of PDGF-B was markedly reduced in lysates from arterial, but not venous, primary KO endothelial cells. Transduction of KO EC with an adenoviral synectin construct was sufficient to restore PDGF-B protein levels. Further, qPCR was used to determine whether defects in PDGF-B protein production were at the transcription or translation level. Interestingly, there was only a minor decrease in the amount of PDGF-B mRNA in KO compared to WT EC, suggesting that synectin deficiency has selectively affected post-transcriptional regulation of PDGF-B expression. Migration of WT and KO SMC in response to PDGF-B or conditioned media from WT and KO endothelial cells was also assessed to determine the cell type specificity of PDGF defects in KO mice. Conclusions: Synectin is required for post-transcriptional regulation of PDGF-B in endothelial cells. Deficits in PDGF-B secretion by synectin−/− endothelial cells result in inadequate smooth muscle recruitment and coverage of arterial vessels.

Breast Cancer Expressing the Activated HER2/neu Is Sensitive to Gefitinib In vitro and In vivo and Acquires Resistance through a Novel Point Mutation in the HER2/neu

The HER2/neu oncogene is an important diagnostic and prognostic factor and therapeutic target in breast and other cancers. We developed and characterized a breast cancer cell line (Bam1a) that overexpresses the activated HER2/neu and ErbB-3 and has a gene expression profile consistent with the ErbB-2 genetic signature. We evaluated the effects of the epidermal growth factor receptor (EGFR)/HER2 inhibitor, gefitinib, on this breast tumor line in vitro and in vivo. We characterized the effects of gefitinib on EGFR, HER2, and ErbB-3 phosphorylation by Western blot and determined the effects on downstream signaling through growth, survival, and stress pathways and the effect on proliferation, cell cycle, and apoptosis. Gefitinib treatment diminished phosphorylation of the ErbB-3 > EGFR > HER2/neu and signal transducers and activators of transcriptions in a dose-dependent fashion. Downstream mitogenic signaling through mitogen-activated protein (MAP)/extracellular signal regulated kinase kinase, p44/42 MAP kinase (MAPK) and stress signaling through c-Jun-NH(2)-kinase (JNK) 1 and c-Jun was impaired (1 micromol/L, 4-24 h), leading to cytostasis and cell cycle arrest within 24 h by decreased cyclin D1, cyclin B1, and p(Ser795)Rb and increased p27. Proliferation and colony formation were inhibited at 0.5 and 1 micromol/L, respectively, and correlated with altered gene expression profiles. Diminished survival signaling through Akt, induction of bim, loss of connexin43, and decreased production of vascular endothelial growth factor-D preceded caspase-3 and poly(ADP)ribose polymerase (PARP) cleavage and apoptosis (>50% 2 micromol/L, 48 h). Oral administration of gefitinib was able to prevent the outgrowth of Bam1a tumor cells from palpable lesions, shrink established tumors, eliminate HER2 and HER3 phosphorylation, and decrease MAPK and Akt signaling in vivo. A variant of the Bam1a cell line, IR-5, with acquired ability to grow in 5 micromol/L gefitinib was developed and characterized. IR-5 bears a novel point mutation in the HER2/neu that corresponds to a L726I in the ATP-binding pocket and correlates with a log decrease in sensitivity to gefitinib, increased heterodimerization with EGFR and HER3, and impaired down-regulation. Gene expression profiling of IR-5 showed increased expression of EMP-1, NOTCH-1, FLT-1, PDGFB, and several other genes that may contribute to the resistant phenotype and sustain signaling through MAPK and Akt. This model will be useful in understanding the differences between intrinsic drug sensitivity and acquired resistance in the context of therapeutic strategies that target oncogene addicted diseases.

Insulin-like growth factor binding protein 2 promotes glioma development and progression

Overexpression of insulin-like growth factor binding protein 2 (IGFBP2) is associated with progression in many types of human cancer. In this study we used a glial-specific transgenic mouse model to examine the active role of IGFBP2 in tumorigenesis and progression. Our studies show that IGFBP2 coexpression results in progression to a higher-grade glioma in platelet-derived growth factor beta (PDGFB)-driven tumors. These anaplastic oligodendrogliomas are characterized by increased cellularity, vascular proliferation, small regions of necrosis, increased mitotic activity, and increased activation of the Akt pathway. Combined expression of IGFBP2 or Akt with K-Ras was required to form astrocytomas, indicating that activation of two separate pathways is necessary for gliomagenesis. In ex vivo experiments, blockade of Akt by an inhibitor led to decreased viability of cells coexpressing IGFBP2 versus PDGFB expression alone. Thus, this study provides definitive evidence that IGFBP2 plays a key role in activation of the Akt pathway and collaborates with K-Ras or PDGFB in the development and progression of two major types of glioma.

Detection of COL1A1-PDGFB fusion transcripts and PDGFB/PDGFRB mRNA expression in dermatofibrosarcoma protuberans

Fusion of the collagen type I alpha 1 (COL1A1) gene with the platelet-derived growth factor beta chain (PDGFB) gene has been described in dermatofibrosarcoma protuberans. The abnormal fusion transcripts probably cause PDGFB and its receptor (platelet-derived growth factor receptor beta, PDGFRB) autocrine stimulation and cell proliferation, which are responsible for the development of dermatofibrosarcoma protuberans. A reverse transcription–polymerase chain reaction assay was performed to detect the COL1A1-PDGFB fusion transcripts in 57 samples. In addition, the PDGFB gene amplification and PDGFB/PDGFRB mRNA levels were quantified by a real-time PCR system for the samples in which the fusion transcripts had been successfully detected. The fusion transcripts were detected in 42 of 57 samples. Various exons of the COL1A1 gene were fused in frame with the PDGFB gene; exons 7 and 25 were found to be slightly more frequently involved than the other exons. The PDGFB gene amplification levels varied from 0.6 to 8.3 (mean 2.4) in 42 tumor samples and from 0.4 to 3.0 (mean 1.2) in 20 adjacent normal tissue samples. In the 20 paired samples, the PDGFB gene amplification in the tumor was significantly higher than that in the normal tissue. The presence of PDGFB and PDGFRB mRNAs was demonstrated in 26 and 21 of 26 cases, respectively. The PDGFB and PDGFRB mRNA expression levels showed a good correlation (r=0.76, P<0.0001). These results indicate that the fusion protein, which is processed by the COL1A1-PDGFB transcripts, can serve as a functional ligand for PDGFRB.

Gene profile analysis of colorectal cancer cell lines by cDNA macroarray

In order to identify high-risk groups of patients with cancer, understanding the mechanisms of invasion and metastasis from the viewpoint of gene expression is required; in particular, the changes in gene expression as tumours gain the ability to metastasise. Using a rat model of metastatic colorectal cancer (CRC), we determined the expression profiles of CRC cells that metastasised to the liver and the lung. In the hepatopulmonary metastasis model, metastasising ability increased with successive transplanted cell line generations. No significant differences in cellular proliferation ability were seen between the original cell line and succeeding metastatic cell lines. Analysis using cDNA macroarray showed that metastasising ability was associated with increased expression of integrin beta4, gamma-catenin, Smad 7, Bax, Bcl-2, c-fos and TGF-alpha, and a particularly marked increased expression of TGF-beta, PDGFb, Cdk4 and Rho B. Expression of both Rho GDIbeta and Gelsolin was reduced. These results suggest that high metastasising ability does not derive from a single gene, but rather through accumulated changes in the expression of several different genes.