Platelet-derived Growth Factor Beta Research Articles

Use of genetic variants in pericyte-driven tumor vessel maturation genes to predict treatment efficacy in mCRC patients treated with FOLFIRI/bevacizumab.

3566 Background: Angiogenesis is maintained by the presence of PDGFB (platelet derived growth factor beta) which binds to the receptor, PDGFRB on pericytes, signaling them to stabilize stalk cells adjacent to tip cells. PDGFB signaling by endothelial tip cells promotes proliferation, migration, and stabilization of pericytes which are closely associated with endothelial cells. VEGF driven angiogenesis in response to tumor hypoxia relies on several genes. Previously, studies showed RALBP1, RGS5, and CSPG4 greatly impact the structure of blood vessels during angiogenesis. Recognizing these genes are important in angiogenesis through pericytes, we investigated the correlation between polymorphisms in these genes and clinical outcomes in patients with mCRC treated with FOLFIRI/bevacizumab (BV). Methods: Genomic DNA was extracted from 455 patients’ blood or tissue treated with first-line BV + FOLFIRI and prospectively enrolled in a prospective pharmacogenomic translational study. Median follow up is 24 months and median PFS and OS were 10.5 and 29.9 months, respectively. Eight functionally significant SNPs; RGS5 (rs1056515, rs2661280), PDGFRB (rs2229562, rs2302271), CSPG4 (rs8023621, rs1127648), and RALBP1 (rs10989, rs329007) were analyzed by PCR-based direct sequencing. All candidate SNPs were analyzed for association with tumor response rate (RR), progression free survival (PFS), and overall survival (OS). Results: Four SNPs showed significant results in the univariate analysis of either OS or PFS. PDGFRB rs2302273 remained significant upon multivariate analysis. Univariate analysis of PDGFRB rs2302273 concluded patients with any T (CT/TT) allele were significantly associated with longer PFS compared to those with CC genotypes (median 10.2 vs 11.6 months, HR=0.78 [95%CI: 0.63-.98], p=0.031, log-rank test) which remained significant upon multivariate analysis (HR=0.73 [95% CI: 0.57-0.92], p=0.008, log-rank test). Conclusions: Our results show that the PDGFRB polymorphism rs2302273 may serve as a prognostic marker for the efficacy of FOLFIRI + BV treatment in patients with mCRC. Studies to confirm and update these preliminary findings are ongoing.

Platelet-derived growth factor alpha and beta receptors have overlapping functional activities towards fibroblasts.

BackgroundPlatelet-derived growth factor (PDGF) signalling is essential for many key cellular processes in mesenchymal cells. As there is redundancy in signalling between the five PDGF ligand isoforms and three PDGF receptor isoforms, and deletion of either of the receptors in vivo produces an embryonic lethal phenotype, it is not know which ligand and receptor combinations mediate specific cellular functions. Fibroblasts are key mediators in wound healing and tissues repair. Recent clinical trials using broad spectrum tyrosine kinase inhibitors in fibrotic diseases have highlighted the need to further examine the specific cellular roles each of the tyrosine kinases plays in fibrotic processes. In this study, we used PDGFR-specific neutralising antibodies to dissect out receptor-specific signalling events in fibroblasts in vitro, to further understand key cellular processes involved in wound healing and tissue repair.ResultsNeutralising antibodies against PDGFRs were shown to block signalling through PDGFRα and PDGFRβ receptors, reduce human PDGF-AA and PDGF-BB-induced collagen gel remodelling in dermal fibroblasts, and reduce migration stimulated by all PDGF ligands in human dermal and lung fibroblasts.ConclusionsPDGFRα and PDGFRβ neutralising antibodies can be a useful tool in studying PDGFR isoform-specific cellular events.

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.

An ICP-MS, ESI-MS and molecular modelling investigation of homogeneous gallium affinity tagging (HMAT) of phosphopeptides

Protein phosphorylation and de-phosphorylation, provide one of the most common signalling pathways within cells, being involved in regulating cellular processes, mediating enzyme inhibition, protein–protein recognition and protein degradation. Compared with normal proteomics, phosphoproteomics poses some additional challenges requiring more initial separation and additional sensitivity to detect and quantify potentially ultra-low abundance species. In this work, the selective detection of phosphopeptides is described based on the incorporation of a metal tag, gallium–N,N-biscarboxymethyl lysine (Ga-LysNTA), in solution before separation and detection by liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC–ICP-MS). Experimental and theoretical characterisation of the resulting Ga–phosphopeptide complex is presented based on linear ion trap electrospray ionisation mass spectrometry (ESI-MS), Fourier transform mass spectrometry (FT-MS) and molecular modelling data. Linear ion trap electrospray ionisation mass spectrometry (ESI-MS) was employed to study the interaction of the gallium tag with platelet derived growth factor beta receptor (ß-PDGF), a small phosphopeptide. In addition high resolution Fourier transform mass spectrometry (FT-MS) was used for accurate mass determination and multistage tandem mass spectrometry of the gallium–ß-PDGF complex identified the fragmentation pathway. Finally, molecular modelling was used to investigate the energetically favoured structures of both the Ga-LysNTA material and the ß-PDGF–Ga-LysNTA complex.

Localized and metastatic myxoid/round cell liposarcoma

Myxoid liposarcoma (MLPS), a disease especially of young adults with potential for local recurrence and metastasis, currently lacks solid prognostic factors and therapeutic targets. The authors of this report evaluated the natural history and outcome of patients with MLPS and commonly deregulated protein biomarkers. Medical records were retrospectively reviewed for patients who presented to the authors' institution with localized (n = 207) or metastatic (n = 61) MLPS (1990 to 2010). A tissue microarray of MLPS patient specimens (n = 169) was constructed for immunohistochemical analysis of molecular markers. The 5-year and 10-year disease-specific survival rates among patients with localized disease were 93% and 87%, respectively; male gender, age >45 years, and recurrent tumor predicted poor outcome. The local recurrence rate was 7.4%, and the risk of local recurrence was associated with recurrent tumors and nonextremity disease location. Male gender was the main risk factor for metastatic disease, which occurred in 13% of patients. Forty percent of patients who had localized disease received chemotherapy, mostly in the neoadjuvant setting. Immunohistochemical analysis revealed significantly higher expression of C-X-C chemokine receptor type 4 (CXCR4) and platelet-derived growth factor beta (PDGFR-β) in metastatic lesions versus localized lesions. Tumors with a round cell phenotype expressed increased levels of CXCR4, p53, adipophilin, PDGFR-α, PDGFR-β, and vascular endothelial growth factor relative to myxoid phenotype. Only the receptor tyrosine kinase encoded by the AXL gene (AXL) was identified as a prognosticator of disease-specific survival in univariate analysis. In this study, the authors identified clinical and molecular outcome prognosticators for patients with MLPS as well as several potential therapeutic targets.

Dermatofibrosarcoma protuberans: a comprehensive review and update on diagnosis and management

Dermatofibrosarcoma protuberans (DFSP) is a rare superficial tumor characterized by high rates of local recurrence and low risk of metastasis. DFSP occurs most commonly on the trunk and proximal extremities, affects all races, and often develops between the second and fifth decade of life. The tumor grows slowly, typically over years. Histologically, several variants of DFSP have been described and should be well characterized to avoid misdiagnosis with other tumors. These include pigmented (Bednar tumor), myxoid, myoid, granular cell, sclerotic, atrophic DFSP, giant cell fibroblastoma, and DFSP with fibrosarcomatous areas. Of all these variants, only the DFSP with fibrosarcomatous areas is high grade, with a higher rate of local recurrence and distant metastasis. DFSP is genetically characterized by the t(17;22)(q22;q13), resulting in the fusion of alpha chain type 1 of collagen gene and platelet-derived growth factor beta gene. This translocation is present in 90% of DFSP and represents a very useful tool in the differential diagnosis of DFSP with other tumors with similar histology. The standard treatment is wide local excision with at least a 2-cm margin. However, local recurrence after apparently adequate surgical excision is well recognized. Mohs micrographic surgery would be the treatment of choice with a better cure rate and maximal conservation of tissue. When surgery is insufficient, clinical evidence has suggested that imatinib mesylate is a safe and effective treatment in DFSP, especially in cases of local advanced or metastatic disease. This article presents an overview of the state of the art in the clinicopathological management of this disease.

A novel murine model to deplete hepatic stellate cells uncovers their role in amplifying liver damage in mice

We have developed a novel model for depleting mouse hepatic stellate cells (HSCs) that has allowed us to clarify their contributions to hepatic injury and fibrosis. Transgenic (Tg) mice expressing the herpes simplex virus thymidine kinase gene (HSV-Tk) driven by the mouse GFAP promoter were used to render proliferating HSCs susceptible to killing in response to ganciclovir (GCV). Effects of GCV were explored in primary HSCs and in vivo. Panlobular damage was provoked to maximize HSC depletion by combining CCl(4) (centrilobular injury) with allyl alcohol (AA) (periportal injury), as well as in a bile duct ligation (BDL) model. Cell depletion in situ was quantified using dual immunofluorescence (IF) for desmin and GFAP. In primary HSCs isolated from both untreated wild-type (WT) and Tg mice, GCV induced cell death in ≈ 50% of HSCs from Tg, but not WT, mice. In TG mice treated with CCl(4) +AA+GCV, there was a significant decrease in GFAP and desmin-positive cells, compared to WT mice (≈ 65% reduction; P < 0.01), which was accompanied by a decrease in the expression of HSC-activation markers (alpha smooth muscle actin, beta platelet-derived growth factor receptor, and collagen I). Similar results were observed after BDL. Associated with HSC depletion in both fibrosis models, there was marked attenuation of fibrosis and liver injury, as indicated by Sirius Red/Fast Green, hematoxylin and eosin quantification, and serum alanine/aspartate aminotransferase. Hepatic expression of interleukin-10 and interferon-gamma was increased after HSC depletion. No toxicity of GCV in either WT or Tg mice accounted for the differences in injury. Activated HSCs significantly amplify the response to liver injury, further expanding this cell type's repertoire in orchestrating hepatic injury and repair.

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.

Abstract 9566: LRP1 Deficiency Promotes Smooth Muscle Cell Apoptosis in Atherosclerotic Plaques via PDGF Receptor Mediated Downregulation of Bcl-2

Apoptosis of smooth muscle cells (SMCs) promotes instability of atherosclerotic plaques. Apoptosis is believed to be suppressed by growth factor dependent activation of PI 3-kinase (PI3K). We investigated the effect of LRP1 knockout driven elevated platelet-derived growth factor beta receptor (PDGFR) signaling on SMC apoptosis in atherosclerotic plaques. We quantified apoptotic TUNEL-positive SMCs in lesions from 12 months old LDLR-/-/smLRP1-/- double knockout mice. LDLR (low-density lipoprotein receptor) deficiency accelerates atherosclerosis in mice. LDLR-/-/smLRP1-/- increased the percentage of TUNEL-positive cells compared to LDLR-/-/smLRP1+/+ controls (3.6±0.7% vs. 0.7±0.3%,respectively, P &lt; 0.05, n = 6). Mutation of the PI3K binding site in the PDGFR in LDLR-/-/smLRP1-/--F2/F2 mice suppressed SMC apoptosis (2.4%±0.3%, P &lt; 0.05, n = 6) suggesting that the PDGFR exerts proapoptotic effects via PI3K. In vitro studies (Cell Death Detection ELISA) revealed that chronic stimulation of SMCs with PDGF-BB (96 h) led to 3.1±0.4-fold increased apoptosis (n=3, P&lt;0.05). To characterize proapoptotic PDGFR driven signaling pathways we analysed PDGFR mutants in which the binding sites for different signaling molecules were deleted. Lack of PI3K binding completely abolished the proapoptotic effect, whereas a mutant which only binds PI3K was able to induce apoptosis like the WT receptor. Further analysis of PDGFR mutants revealed that activation of PI3K led to induction of c-Myc and decreased expression of antiapoptotic Bcl-2 as assessed by RT-PCR and Westernblotting. siRNA mediated knockdown of c-Myc prevented both, Bcl-2 downregulation and PDGF induced apoptosis. Consistently, immunohistochemical analysis of atherosclerotic plaques from LDLR-/-/smLRP1-/- and LDLR-/-/smLRP1-/--F2/F2 mice revealed that PDGFR dependent activation of PI3K impairs the expression of antiapoptotic Bcl-2. In conclusion, our study identified a new PDGFR driven proapoptotic signaling pathway in SMCs. Chronically elevated PDGFR/PI3K signaling downregulates Bcl-2 via induction of c-Myc. This has direct implications in atherogenesis, as PDGF promotes cell death in atherosclerotic plaques, which can ultimately lead to plaque instability and rupture.

Enhancement of Megakaryocyte Interactions with the Osteoblastic Hematopoietic Stem Cell Niche Improves Engraftment Efficiency Following Hematopoietic Stem Cell Transplantation

Abstract 211Bone marrow (BM) radioablation produces structural changes in the endosteal osteoblastic stem cell niche, a critical site of hematopoietic stem cell (HSC) engraftment following HSC transplantation (HSCT). We have previously shown that total body irradiation (TBI) in wildtype (WT) mice induces migration of recipient megakaryocytes to the niche and an expansion of niche osteoblasts that supports HSC engraftment following transplantation. We have also demonstrated that c-MPL-deficient (mpl−/−) recipients have decreases in total megakaryocytes (35% of WT), the percentage of megakaryocytes migrating to the endosteum (<20% of WT), and niche osteoblast expansion (<50% of WT) following TBI, leading to profound deficits in long-term (LT)-HSC engraftment following HSCT. We now present data examining mechanisms by which megakaryocytes facilitate both niche osteoblast expansion post-TBI and donor HSC engraftment following HSCT, and a therapeutic strategy utilizing these mechanisms to enhance donor HSC engraftment.The decrease in total megakaryocytes and absent thrombopoietin (TPO) signaling in mpl−/− mice resulted in a 90% reduction in post-TBI mpl−/− versus WT BM levels of platelet-derived growth factor beta (PDGFβ), a known osteoblast growth factor. In vitro, megakaryocytes cultured together or across a transwell membrane markedly enhanced osteoblast growth (> 2.5 fold, p < 0.001), but PDGFβ signaling inhibition completely abrogated megakaryocyte-driven osteoblast growth. In vivo, inhibition of PDGF receptor signaling in WT mice via imatinib treatment resulted in near complete blockade of TBI-induced osteoblast expansion, and imatinib treatment of primary recipients resulted in diminished LT-HSC engraftment in secondary transplant assays.Blockade of CD41 integrin-mediated adhesion of megakaryocytes in WT recipient BM blocked TBI-induced megakaryocyte migration to the endosteal niche and severely abrogated LT-HSC engraftment efficiency. However, in contrast to c-MPL deficiency, CD41 blockade did not decrease PDGFβ expression or niche osteoblast expansion, suggesting that in addition to PDGFβ-dependent effects on niche expansion, the megakaryocyte migration to the niche itself is also required to efficiently engraft HSC. Mice with decreased GATA-1 expression (Gata-1tm2sho/J), have a large increase in total BM megakaryocytes a >2-fold (p < 0.001) increase in PDGFβ levels, and greatly increased expansion of osteoblast and other mesenchymal elements 48 hours post-TBI compared to WT mice. However, Gata-1tm2sho/J megakaryocytes have known defective terminal differentiation and function including decreased platelet production, and Gata-1tm2sho/J primary recipients did not engraft LT-HSC more efficiently than WT primary recipients, demonstrating the need for fully functional megakaryocytes, and not only increased PDGFβ-induced mesenchymal proliferation, to foster HSC engraftment.Finally, we have examined whether TPO administration prior to radioablation and HSCT can enhance host megakaryocyte effects on the niche and HSC engraftment. TPO administration for 5 days prior to radioablation, resulted in a significant increase in BM megakaryocytes and a 50% increase in niche osteoblast expansion. Furthermore, competitive secondary transplantation assays demonstrated that TPO- versus sham-treatment of primary recipients prior to TBI and BM transplant, resulted in increased initial engraftment at 24 hours post-primary transplant (40% increase, p < 0.05) increased short-term HSC and progenitor engraftment 3–6 weeks following secondary transplant (4–20 fold increase, p < 0.02), and sustained LT-HSC engraftment at 28 weeks post-transplant in 47% versus 7% (p < 0.05) of secondary recipients of TPO- versus sham-treated primary recipient BM, respectively.Taken together, our results demonstrate that host megakaryocytes facilitate efficient HSC engraftment following TBI and HSCT through PDGFβ-dependent enhancement of niche osteoblast expansion and through direct interactions of megakaryocytes with the niche. TPO-treatment of transplant recipients prior to radioablation and stem cell infusion enhances these megakaryocyte-dependent pathways and subsequent donor HSC engraftment efficiency, providing a clinically applicable strategy to enhance niche function and stem cell engraftment following clinical transplantation. Disclosures:No relevant conflicts of interest to declare.

Expression of platelet derived growth factor β receptor, its activation and downstream signals in bovine cutaneous fibropapillomas

Bovine cutaneous fibropapillomas are benign skin tumours formed by proliferation of epidermal keratinocytes and dermal fibroblasts caused by bovine papillomaviruses (BPVs). BPV E5 oncoprotein plays a key role in neoplastic cell transformation by specifically binding to the platelet derived growth factor beta receptor (PDGFβR) causing its phosphorylation and activation of proliferation and survival signal transduction pathways, among these phosphatidyl inositol-3-kinase (PI3K)/Akt and Ras-mitogen-activated-protein-kinase-Erk (Ras-MAPK-Erk) pathways. The aim of this study was to investigate the expression of PDGFβR, its phosphorylation status and expression of the downstream molecules phospho-Akt (pAkt) and phospho-Erk (pErk), in naturally occurring bovine cutaneous fibropapillomas. By immunohistochemistry on serial sections we showed cytoplasmic co-expression of the PDGFβR and E5 protein in neoplastic tissue. Western blot analysis revealed that PDGFβR was phosphorylated in higher amount in tumour samples compared to normal skin. pAkt, but not pErk, was also overexpressed in tumour samples. These findings may provide new insights into the aetiopathogenic mechanisms underlying naturally occurring bovine fibropapillomas and contribute to understanding the molecular scenario underlying BPV induced tumourigenesis.

Selective Targeting of Interferon γ to Stromal Fibroblasts and Pericytes as a Novel Therapeutic Approach to Inhibit Angiogenesis and Tumor Growth

New approaches to block the function of tumor stromal cells such as cancer-associated fibroblasts and pericytes is an emerging field in cancer therapeutics as these cells play a crucial role in promoting angiogenesis and tumor growth via paracrine signals. Because of immunomodulatory and other antitumor activities, IFNγ, a pleiotropic cytokine, has been used as an anticancer agent in clinical trials. Unfortunately only modest beneficial effects, but severe side effects, were seen. In this study, we delivered IFNγ to stromal fibroblasts and pericytes, considering its direct antifibrotic activity, using our platelet-derived growth factor-beta receptor (PDGFβR)-binding carrier (pPB-HSA), as these cells abundantly express PDGFβR. We chemically conjugated IFNγ to pPB-HSA using a heterobifunctional PEG linker. In vitro in NIH3T3 fibroblasts, pPB-HSA-IFNγ conjugate activated IFNγ-signaling (pSTAT1α) and inhibited their activation and migration. Furthermore, pPB-HSA-IFNγ inhibited fibroblasts-induced tube formation of H5V endothelial cells. In vivo in B16 tumor-bearing mice, pPB-HSA-IFNγ rapidly accumulated in tumor stroma and pericytes and significantly inhibited the tumor growth while untargeted IFNγ and pPB-HSA carrier were ineffective. These antitumor effects of pPB-HSA-IFNγ were attributed to the inhibition of tumor vascularization, as shown with α-SMA and CD-31 staining. Moreover, pPB-HSA-IFNγ induced MHC-II expression specifically in tumors compared with untargeted IFNγ, indicating the specificity of this approach. This study thus shows the impact of drug targeting to tumor stromal cells in cancer therapy as well as provides new opportunities to use cytokines for therapeutic application.

Abstract 512: Angiotensin-(1-7) Attenuates PDGF-dependent Migration of Vascular Smooth Muscle Cells

Over one million percutaneous coronary intervention (PCI) procedures are performed each year and restenosis, the narrowing of the vessel lumen due to vascular damage, occurs in more than 40% of these patients. Previous studies by our laboratory demonstrated that infusion of angiotensin-(1-7) [Ang-(1-7)], an anti-proliferative peptide hormone of the renin-angiotensin system, attenuated neointimal formation after vascular injury which was associated with reduced vascular smooth muscle cell (VSMC) proliferation. However, prevention of restenosis after PCI requires inhibition of both VSMC proliferation and migration. In this study, the effect of Ang-(1-7) on VSMC migration was investigated to determine whether a decrease in VSMC migration also represents a component of the heptapeptide-induced reduction in neointimal formation. Platelet-derived growth factor (PDGF), a potent stimulus for VSMC migration, increased chemotaxis in a modified Boyden’s chamber; PDGF stimulated chemotaxis 228% over random migration and pre-treatment with Ang-(1-7) reduced the PDGF-mediated migration to 132% over random movement, a reduction of 42.1% (n=6, p&lt;0.05). PDGF also stimulated directional migration in a wounding assay, by 600% over random migration; pre-treatment with Ang-(1-7) reduced directional migration in response to PDGF to 317% of random migration, a 40.4% decrease (n=5, p&gt;0.01). The reduction in PDGF-mediated migration by Ang-(1-7) was associated with a significant decrease in the phosphorylation of tyrosine 1009 on the PDGF beta receptor as well as the src homology 2 phosphatase-2 (SHP-2) and the tyrosine kinase Src, two enzymes which are positive mediators of PDGF-induced migration. Pre-treatment with Ang-(1-7) attenuated the PDGF-dependent increase in the phosphoY1009-PDGF receptor by 60%, phosphoY580-SHP-2 by 56% and phosphoY416-Src by 45% (n=5, p&lt;0.05). These data demonstrate that Ang-(1-7) inhibits VSMC migration to attenuate neointimal formation, which may result from a reduction in the phosphorylation of the PDGF receptor or attenuation of the cellular events resulting from receptor activation. Further, these results suggest that Ang-(1-7) may be administered as a targeted therapeutic to prevent vascular restenosis.

Recurrent Dermatofibrosarcoma Protuberans of the Shoulder with Rare Distant Abdominal Metastasis detected by Fluorodeoxyglucose-Positron Emission Tomography - Computed Tomography ( FDG-PET / CT )

Recurrent Dermatofibrosarcoma Protuberans of the Shoulder with Rare Distant Abdominal Metastasis detected by Fluorodeoxyglucose-Positron Emission Tomography - Computed Tomography ( FDG-PET / CT )

Dermatofibrosarcoma Protuberans in Children: an Update on the Diagnosis and Treatment

Dermatofibrosarcoma protuberans (DFSP) is a fibrohistiocytic tumor of low grade malignant potential. Although rare, pediatric cases pose a particular challenge in diagnosis and management. In children, the clinical appearance may be heterogeneous and a high index of suspicion is necessary to avoid delays in diagnosis which can lead to further morbidity. Histologic examination, often with the use of appropriate immunostains, is necessary for diagnosis. Advances in the understanding of the molecular genetics of DFSP have led to further diagnostic and therapeutic modalities. DFSP is thought to result from a translocation between platelet-derived growth factor beta (PDGFB, 22q13.1) and type 1 collagen (COL1A1, 17q21≈22) leading to a fusion protein (PDGFB) which stimulates the PDGF receptor. Detection of this translocation in tissue via PCR or fluorescence in situ hybridization (FISH) can be helpful in difficult cases. While surgery with wide local excision or Mohs micrographic surgery is the mainstay of treatment, the use of targeted therapy with imatanib mesylate shows promise in large or unresectable tumors. Knowledge of the clinical features, histology, genetics, and treatment options is important for successful management of these tumors.

Antifibrotic effects of Artemisia capillaris and Artemisia iwayomogi in a carbon tetrachloride-induced chronic hepatic fibrosis animal model

Ethnopharmacological relevanceArtemisia capillaris and Artemisia iwayomogi, both members of the Compositae family, have been indiscriminately used for various liver disorders as traditional hepatotherapeutic medicines in Korea for many years. Aim of the studyIn this study, the anti-hepatofibrotic effects of Artemisia capillaris and Artemisia iwayomogi were comparatively analyzed using a carbon tetrachloride (CCl4)-induced liver fibrosis rat model. Materials and methodsHepatic fibrosis was induced via a 10-week course of intraperitoneal CCl4 injections (50% dissolved in olive oil, 2mL/kg, twice per week). Water extract of Artemisia capillaris (AC) or Artemisia iwayomogi (AI) was orally administered six times per week from the 5th to the 10th week. ResultsAI (50mg/kg) significantly attenuated the CCl4-induced excessive release of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in serum (p<0.05), and hydroxyproline and malondialdehyde (MDA) contents in liver tissue (p<0.05). Further, AI markedly ameliorated the depletion of total antioxidant capacity (TAC), glutathione (GSH), and superoxide dismutase (SOD) in liver tissue (p<0.01). Unexpectedly, AC did not exert any effects on the above parameters. Histopathological and immunohistochemical analyses revealed that AI drastically reduced inflammation, necrosis, fatty infiltration, collagen accumulation, and activation of hepatic satellite cells in liver tissue. These changes were not observed with AC treatment. Several critical genes of fibrosis-related cytokines including transforming growth factor beta (TGF-β), platelet-derived growth factor beta (PDGF-β), and alpha smooth muscle actin (α-SMA) were more prominently downregulated by AI compared to AC treatment. ConclusionOur results show that AI exerts greater hepatoprotective and anti-fibrotic effects as compared with AC via enhancing antioxidant capacity and downregulating fibrogentic cytokines.

Diphenylhydantoin Promotes Proliferation in The Subventricular Zone and Dentate Gyrus

Diphenylhydantoin (phenytoin) is an antiepileptic drug that generates hyperplasia in some tissue by stimulating Epidermal Growth Factor (EGFR) and Platelet-Derived Growth Factor beta (PDGFR-β) receptors and by increasing serum levels of basic fibroblast growth factor (bFGF, FGF2 or FGF-β). Neural stem cells in the adult brain have been isolated from three regions: the Subventricular Zone (SVZ) lining the lateral wall of the lateral ventricles, the Subgranular Zone (SGZ) in the dentate gyrus at the hippocampus and the Subgranular Zone (SZC) lining between the hippocampus and the corpus callosum. Neural stem cells actively respond to bFGF, PDGFR-β or EGF by increasing their proliferation, survival and differentiation. The aim of this study was to evaluate the effect of phenytoin on proliferation and apoptosis in the three neurogenic niches in the adult brain. We orally administrated phenytoin with an oropharyngeal cannula for 30 days: 0 mg kg-1 (controls), 1, 5, 10, 50 and 100 mg kg-1. To label proliferative cells, three injections of 100 mg kg-1 of BrdU was administrated every 12 h. Immunohistochemistry against BrdU or Caspase-3 active were performed to determine the number of proliferative or apoptotic cells. Our results showed that phenytoin induces proliferation in the SVZ and the SGZ in a dose-dependent manner. No statistically significant effects on cell proliferation in the SCZ neither in the apoptosis rate at the SVZ, SGZ and SCZ were found. These data indicate that phenytoin promotes a dose-dependent proliferation in the SVZ and SGZ of the adult brain. The clinical relevance of these findings remain to be elucidated.

Plasma Concentrations of Angiogenesis-related Molecules in Patients with Pancreatic Cancer

Anti-angiogenic agents are now being clinically evaluated for the treatment of pancreatic cancer and a detailed investigation of the angiogenic profile of pancreatic cancer is needed. The aim of this study was to evaluate the plasma concentrations of angiogenesis-related molecules in patients with pancreatic cancer, compared with those with other diseases. Plasma samples obtained from 45 patients with pancreatic cancer were analyzed and compared with those from 9 patients with pancreatitis, 16 patients with benign hepatobiliary diseases and 58 patients with colorectal cancers. The plasma levels of angiogenesis-related molecules including angiopoietin-2, follistatin, granulocyte-colony stimulating factor, hepatocyte growth factor, interleukin-8, leptin, platelet-derived growth factor beta polypeptide, platelet endothelial cell adhesion molecule-1 and vascular endothelial growth factor were determined using an antibody suspension bead arrays system. The plasma levels of all the angiogenesis-related molecules were not increased in patients with pancreatic cancer, compared with those with pancreatitis and benign hepatobiliary diseases, whereas the levels of those with colorectal cancer were markedly increased. The plasma interleukin-8 concentration was significantly elevated in patients with distant metastases and was associated with a poor treatment outcome of chemotherapy in patients with pancreatic cancer. The plasma levels of angiogenesis-related molecules were not elevated in patients with pancreatic cancer, compared with those with benign diseases or colorectal cancer. The plasma interleukin-8 level may be a novel biomarker for the response to chemotherapy in patients with pancreatic cancer and warrants further prospective study.

2165 POSTER Targeting of Interferon Gamma to Stromal Fibroblasts Using a PDGF Receptor Recognizing Carrier Reduces Tumour Growth in Vivo

Background: Stromal fibroblasts are the key cell types in tumour stroma, that support angiogenesis, tumour cell proliferation and metastasis. Therefore, inhibition of stromal fibroblasts activity might inhibit tumour growth. Interferon gamma (IFNγ) is a potent cytokine and has been used for the treatment of experimental fibrosis. However, poor pharmacokinetics and severe side effects prevented its clinical application. In this study, we hypothesized that specific delivery of IFNγ to stromal fibroblasts may be beneficial to inhibit the tumour growth. Since Platelet-derived Growth Factor beta receptor (PDGFβR) is abundantly expressed on stromal fibroblasts, we developed a PDGFβR-specific drug carrier (PPB-HSA) by modifying albumin with a PDGFβR-recognizing cyclic peptide to deliver IFNγ. Materials and Methods: The IFNγ was conjugated to PPB-HSA carrier via a heterobifunctional PEG linker and characterized with Western blot analyses and nitric oxide release assay in RAW monocytes. In vitro, PPBHSA- IFNγ was examined for its effectivity in 3T3 fibroblasts using wound healing assay, immunocytochemistry and qRT-PCR. To simulate fibroblastsinduced angiogenesis process, tube formation assay was developed in which conditioned medium from 3T3 fibroblasts (incubated with TGFb and IFNγ or IFNγ constructs) was added to the endothelial cells (H5V) and tubes formed were counted. In vivo, the effects of the targeted PPB-HSAIFNγ on tumour growth were determined in subcutaneous B16 melanoma tumour model in mice. Treatments with vehicle, IFNγ, PPB-HSA-IFNγ, PPBHSA (n = 5 per group) at the equivalent doses (5 μg/dose/mouse) were administered intravenously. Results: PPB-HSA-IFNγ construct was successfully synthesized and the conjugated IFNγ retained its biological activity. The construct showed PDGFβR-specific binding in 3T3 cells which was blocked with anti-PDGFR antibody. The IFNγ construct significantly inhibited the proliferation and migration of 3T3 cells as determined with wound healing assay. Treatment with the targeted IFNγ drastically reduced TGFb-induced collagen-I, alpha smooth muscle actin and fibronectin expression in staining and gene expression. Furthermore, the PPB-HSA-IFNγ inhibited the 3T3 fibroblastsinduced angiogenesis as determined with the tube formation assay in H5V cells. In vivo, the targeted IFNγ construct attenuated the tumour growth by 60% (p < 0.01) compared to vehicle whereas untargeted IFNγ and PPBHSA carrier did not induce any reduction in the tumour growth. Conclusions: These data demonstrate that specific targeting of IFNγ to the stromal fibroblasts using PPB-HSA carrier is a potential therapeutic strategy to inhibit tumour growth.

Peptide-Modified Albumin Carrier Explored as a Novel Strategy for a Cell-Specific Delivery of Interferon Gamma To Treat Liver Fibrosis

Excessive accumulation of the extracellular matrix proteins primarily produced by activated hepatic stellate cells (HSC) leads to liver fibrosis. To date, no successful therapeutic intervention is available for the treatment of this disease. Platelet derived growth factor beta receptor (PDGFβR) is highly upregulated on disease-inducing activated HSC and thus can be used for delivery of antifibrotic drugs to increase therapeutic efficacy with reduced adverse effects. Interferon gamma (IFNγ) has been recognized as a potent antifibrotic cytokine; however, poor pharmacokinetics and side effects due to frequent administration have limited its clinical use. For HSC-specific delivery, a PDGFβR-specific drug delivery carrier (PPB-HSA) was developed by modifying albumin with PDGFβR-recognizing cyclic peptides. Subsequently, IFNγ was conjugated to PPB-HSA via bifunctional PEG linkers to synthesize PPB-HSA-PEG-IFNγ. In vitro, PPB-HSA-PEG-IFNγ retained complete biological activity similar to unmodified IFNγ and showed PDGFβR-specific binding to human HSC and primary culture-activated rat HSC. In TGFβ-stimulated mouse fibroblasts and human HSC, PPB-HSA-PEG-IFNγ induced significant reduction in crucial fibrotic parameters. In vivo, the conjugate rapidly accumulated into PDGFβR-expressing HSC in fibrotic livers and activated IFNγ-mediated pstat1α signaling pathway. Furthermore, in a CCl(4)-induced acute liver injury model in mice, treatment with HSC-targeted IFNγ strongly ameliorated hepatic fibrogenesis by inducing significant reduction (about 60%; p < 0.01) in collagen I and α-SMA expression as well as enhanced fibrolysis (increased MMP/TIMP ratio; p < 0.05) while free unmodified IFNγ was ineffective. Furthermore, in contrast to free native IFNγ, the conjugate did not induce macrophage infiltration and IL-1β expression in the liver. In conclusion, these data demonstrate the enhanced antifibrotic efficacy and reduced off-target effects of PPB-HSA-PEG-IFNγ conjugate showing the potential of cell-specific targeting of IFNγ for the treatment of liver fibrosis.