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There is a growing recognition that myogenic stem cells are influenced by their microenvironment during regeneration. Several interstitial cell types have been described as supportive for myoblasts. In this role, both the pericyte as a possible progenitor for mesenchymal stem cells, and interstitial cells in the endomysium have been discussed. We have applied immunohistochemistry on normal and pathological human skeletal muscle using markers for pericytes, or progenitor cells and found a cell type co-expressing CD10, CD34, CD271, and platelet-derived growth factor receptor α omnipresent in the endomysium. The marker profile of these cells changed dynamically in response to muscle damage and atrophy, and they proliferated in response to damage. The cytology and expression profile of the CD10+ cells indicated a capacity to participate in myogenesis. Both morphology and indicated function of these cells matched properties of several previously described interstitial cell types. Our study suggests a limited number of cell types that could embrace many of these described cell types. Our study indicate that the CD10+, CD34+, CD271+, and platelet-derived growth factor receptor α+ cells could have a supportive role in human muscle regeneration, and thus the mechanisms by which they exert their influence could be implemented in stem cell therapy.

Background: Crenolanib is a tyrosine kinase inhibitor targeting PDGFR-α, PDGFR-β and Fms related tyrosine kinase-3 (FLT3) that is currently evaluated in several clinical trials. Although platelet-derived growth factor receptor (PDGFR) signalling pathway is believed to play an important role in angiogenesis and maintenance of functional vasculature, we here demonstrate a direct angiostatic activity of crenolanib independently of PDGFR signalling., Methods: The activity of crenolanib on cell viability, migration, sprouting, apoptosis and mitosis was assessed in endothelial cells, tumour cells and fibroblasts. Alterations in cell morphology were determined by immunofluorescence experiments. Flow-cytometry analysis and mRNA expression profiles were used to investigate cell differentiation. In vivo efficacy was investigated in human ovarian carcinoma implanted on the chicken chorioallantoic membrane (CAM)., Results: Crenolanib was found to inhibit endothelial cell viability, migration and sprout length, and induced apoptosis independently of PDGFR expression. Treated cells  showed altered actin arrangement and nuclear aberrations. Mitosis was affected at several levels including mitosis entry and centrosome clustering. Crenolanib suppressed human ovarian carcinoma tumour growth and angiogenesis in the CAM model., Conclusions: The PDGFR/FLT3 inhibitor crenolanib targets angiogenesis and inhibits tumour growth in vivo unrelated to PDGFR expression. Based on our findings, we suggest a broad mechanism of action of crenolanib.

Clinical and biomedical research seeks single-cell quantification to better understand their roles in a complex, multicell environment. Recently, quantification of vascular endothelial growth factor receptors (VEGFRs) provided important insights into endothelial cell characteristics and response in tumor microenvironments. However, existing technologies for quantifying plasma membrane receptor tyrosine kinases (RTKs) lack multiplexing capabilities, limiting detailed characterization. Here, we use the unique spectral properties of quantum dots (Qdots) to optimize and dually quantify VEGFR1 and VEGFR2 on human umbilical vein endothelial cells (HUVECs). To enable this quantification, we reduce nonspecific binding between Qdot-conjugated antibodies and cells via buffer optimization. Second, we identify optimal labeling conditions by examining Qdot-conjugated antibody binding to five receptors: VEGFRs (VEGFR1 and VEGFR2), their coreceptor neuropilin1 (NRP1), and platelet-derived growth factor receptor (PDGFRα and PDGFRβ). We establish that 800-20 000 is the dynamic range where accurate Qdot-enabled quantification can be achieved. Through these optimizations, we demonstrate measurement of 1 100 VEGFR1 and 6 900 VEGFR2 per HUVEC. We induce ∼90% upregulation of VEGFR1 and ∼30% downregulation of VEGFR2 concentration via 24 h VEGF-A 165 treatment. We observe no change in VEGFR1 or VEGFR2 concentration with 24 h VEGF-B 167 treatment. We further apply Qdots to analyze HUVEC heterogeneity and observe that 24 h VEGF-A 165 treatment induces a ∼15% decrease in VEGFR2 heterogeneity, but little to no change in VEGFR1 heterogeneity. We observe that VEGF-B 167 induces little to no change in either VEGFR1 or VEGFR2 heterogeneity. Overall, we demonstrate experimental and analytical strategies for quantifying two or more RTKs at single-level using Qdots, which will help provide new insights into biological systems.

Renal fibrosis is the final common pathway of various renal injuries and it leads to chronic kidney disease. Recent studies reported that FOXD1-lineage pericyte plays a critical role in tubulointerstitial fibrosis (TIF). However the regulatory mechanisms remain unclear. Autophagy is a cellular process of degradation of damaged cytoplasmic components that regulates cell death and proliferation. To investigate the role of autophagy in FOXD1-lineage pericytes on renal TIF, we generated the FOXD1-lineage stromal cell-specific Atg7 deletion (Atg7 △FOXD1 ) mice. FOXD1-lineage stromal cell-specific Atg7 deletion enhanced renal TIF through Smad-dependent transforming growth factor (TGF)-β signaling after unilateral ureteral obstruction (UUO). FOXD1-lineage stromal cell-specific Atg7 deletion increased the accumulation of interstitial myofibroblasts and enhanced the differentiation of pericytes into myofibroblasts after UUO. Peritubular capillary rarefaction was accelerated in Atg7 △FOXD1 mice after UUO. Atg7 △FOXD1 mice increased the accumulation of SQSTM1/p62-positive aggregates in the obstructed kidney and resulted in increased expression of NLRP3 inflammasome, interleukin (IL) 1-β and caspase-1 signaling pathway, which enhanced apoptosis of interstitial cells after UUO. In summary, our data showed that autophagy in FOXD1-lineage stromal cells plays a protective role in renal TIF through regulating the Smad4 dependent TGF-β an NLRP3 inflammasome signaling pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Kaposi sarcoma (KS) is a locally progressive, intermediate-grade vascular neoplasm with no known cure, high recurrence rates, and potential for wide dissemination. Low efficacy and high toxicity limit current therapeutic options for advanced disease. Activation of mammalian target of rapamycin (mTOR), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and c-kit signaling pathways has been implicated in KS pathogenesis and may suggest a role for targeted inhibitors. KS cases were retrospectively retrieved (N=274), most (90%) associated with human immunodeficiency virus. Tissue microarray slides were stained with human herpes virus-8, Friend leukemia integration 1 transcription factor, CD117 (c-kit), phospho-S6 (pS6), PDGF receptor-β, VEGF, and phospho-mTOR. Both intensity and extent of staining were scored. Multiplying these scores for each core yielded total staining H-scores. Human herpes virus-8 was positive in 87% and Friend leukemia integration 1 transcription factor in 95.7% of cases. Most were also VEGF+ (97.6%), pS6+ (95.7%), CD117+ (92.5%), and PDGFRB+ (87.4%). Approximately half (55.6%) were phospho-mTOR+. There was no significant difference in staining among patients with low (<500 cells/mm 3 ) or preserved CD4 T-cell counts. Immunohistochemistry confirms upregulation of the mTOR, PDGF, VEGF, and c-kit pathways in a large cohort of KS samples. Of proteins tested, pS6, downstream of mTOR, demonstrated the highest proportion of strong positivity (67.1%). These results support the possibility of using targeted inhibitors in KS. Overexpression was independent of CD4 count, suggesting that even patients with low counts may be targeted therapy candidates., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Progress in cancer biology has led to an increasing discovery of oncogenic alterations of the platelet-derived growth factor receptors (PDGFRs) in cancers. In addition, their overexpression in numerous cancers invariably makes PDGFRs and platelet-derived growth factors (PDGFs) prognostic and treatment markers in some cancers. The oncologic alterations of the PDGFR/PDGF system affect the extracellular, transmembrane and tyrosine kinase domains as well as the juxtamembrane segment of the receptor. The receptor is also involved in fusions with intracellular proteins and receptor tyrosine kinase. These discoveries undoubtedly make the system an attractive oncologic therapeutic target. This review covers elementary biology of PDGFR/PDGF system and its role as a prognostic and treatment marker in cancers. In addition, the multifarious therapeutic targets of PDGFR/PDGF system are discussed. Great potential exists in the role of PDGFR/PDGF system as a prognostic and treatment marker and for further exploration of its multifarious therapeutic targets in safe and efficacious management of cancer treatments.

Aim: To determine if expression of colonic tryptophan hydroxylase-2 (TPH2), a surrogate marker of neuronal 5-hydroxytryptamine, is altered in Hirschsprung's-associated enterocolitis., Methods: Entire resected colonic specimens were collected at the time of pull-through operation in children with Hirschsprung's disease (HSCR, n = 12). Five of these patients had a history of pre-operative Hirschsprung's-associated enterocolitis (HAEC). Controls were collected at colostomy closure in children with anorectal malformation (n = 10). The distribution of expression of TPH2 was evaluated using immunofluorescence and confocal microscopy. Protein expression of TPH2 was quantified using western blot analysis in the deep smooth muscle layers., Results: TPH2 was co-expressed in nitrergic and cholinergic ganglia in the myenteric and submucosal plexuses in ganglionic colon in HSCR and healthy controls. Co-expression was also seen in submucosal interstitial cells of Cajal and PDGFRα(+) cells. The density of TPH2 immuno-positive fibers decreased incrementally from ganglionic bowel to transition zone bowel to aganglionic bowel in the myenteric plexus. Expression of TPH2 was reduced in ganglionic bowel in those affected by pre-operative HAEC compared to those without HAEC and healthy controls. However, expression of TPH2 was similar or high compared to controls in the colons of children who had undergone diverting colostomy for medically refractory HAEC., Conclusion: Altered TPH2 expression in colonic serotonergic nerves of patients with HSCR complicated by HAEC may contribute to intestinal secretory and motor disturbances, including recurrent HAEC.

This study evaluated the messenger RNA (mRNA) expression and immunolocalization of all members of the platelet-derived growth factor (PDGF) family in caprine ovaries by quantitative PCR and immunohistochemistry, respectively. Detectable levels of PDGF-A mRNA were not observed in primordial follicles. Higher levels of PDGF-B mRNA were observed in primary follicles than in primordial follicles (P < 0.05). PDGF-D mRNA levels were higher in secondary follicles than in the other preantral follicle categories (P < 0.05). PDGF-B mRNA expression was higher than PDGF-C mRNA expression in primary follicles (P < 0.05). In antral follicles, PDGF-A mRNA expression was higher in cumulus-oocyte complexes (COCs) from small antral follicles than in those from large antral follicles and their respective granulosa/theca (GT) cells (P < 0.05). Furthermore, in COCs from small and large antral follicles, PDGF-A mRNA expression was higher than that of the other PDGF isoforms (P < 0.05). The mRNA levels of PDGF-B and PDGF-D and PDGFR-α and PDGFR-β were higher in GT cells from large antral follicles than in GT cells from small antral follicles and in their respective COCs (P < 0.05). In COCs and GT cells from small antral follicles, the mRNA levels of PDGFR-α were higher than those of PDGFR-β (P < 0.05). All proteins were observed in the cytoplasm of oocytes from all follicular categories. In granulosa cells, all PDGFs and PDGFR-β were detected from starting at the secondary stage, and in theca cells, all proteins, except PDGF-C, were detected starting at the antral stage. In conclusion, PDGF and its receptors are differentially expressed in the oocytes and ovarian cells according to the stage of follicular development, suggesting their role in the regulation of folliculogenesis in goats., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Various kinds of molecular targeted drugs to inhibit receptor tyrosine kinases (RTKs) have been recently developed. The relationship between the expression status of major RTKs and prognosis in gastric cancer remains unclear. We conducted a multicenter study to evaluate the prognostic impact of the expression of epidermal growth factor receptor (EGFR), c-Met, platelet-derived growth factor receptor (PDGFR), and c-Kit in gastric cancer., Methods: This study included 153 gastric cancer patients who underwent gastrectomy at 9 institutions between 2000 and 2006. Expression status of EGFR, c-Met, PDGFR, and c-Kit were evaluated with immunohistochemistry (IHC) centrally. Overall survival based on RTK expression status was statistically compared. Cox multivariate analysis was conducted to adjust for potentially confounding factors., Results: The positive rates for EGFR, c-Met, PDGFR, and c-Kit were 14.4, 24.8, 41.2, and 11.1 %, respectively. Significant interactions with expression status were observed for pathological N stage with EGFR; HER2-status with c-Met; tumor location, histology, and pathological N stage with PDGFR; and no examined variables with c-Kit. Concomitant HER2 positivity was observed for 0.7 % of tumors positive for EGFR, 3.9 % for c-Met, 4.6 % for PDGFR, and 1.3 % for c-Kit. There were some differences in overall survival between patients with or without RTK expression, but only c-Kit expression showed a significant survival difference in Cox multivariate analysis (P = 0.046)., Conclusions: Our multicenter study indicated that IHC expression of 4 RTKs had some prognostic impact and that c-Kit-positive status may be a significant indicator of good prognosis in gastric cancer patients.

Platelet-derived growth factors (PDGFs) belong to a family of polypeptide growth factors that signal through cell surface tyrosine kinase receptors to stimulate growth, proliferation and differentiation. Platelet-derived growth factor receptors (PDGFRs) are also considered important targets for specific kinase inhibitors in the treatment of several human tumours. The aim of this study was to investigate the role of PDGF-A, PDGF-B, PDGFR-α and PDGFR-β in canine lymphoma by determining gene and protein expression in lymph nodes of dogs with diffuse large B-cell lymphoma (DLBCL), peripheral T-cell lymphoma (PTCL), T-lymphoblastic lymphoma (T-LBL) and in healthy control dogs. One lymph node was also studied at the end of therapy in a subset of dogs in remission for DLBCL. In controls, PDGF-A, PDGFR-α and PDGFR-β mRNA levels were significantly higher than in DLBCLs, PTCLs and T-LBLs. However, PDGFR-α and PDGFR-β were minimally expressed by lymphocytes and plasma cells in normal lymph nodes as determined by immunohistochemistry, while neoplastic B and T cells showed the highest score (P <0.05). This discordant result may be compatible with the constitutive expression of these molecules by endothelial cells and fibroblasts in normal lymph nodes, thereby influencing gene expression results. Furthermore, these cells were not included in the immunohistochemical analysis. Similarly, dogs with DLBCL that were in remission at the end of therapy showed significantly higher gene expression of PDGFs and receptors than at the time of diagnosis and with an opposite trend to the protein assay. PDGF-B protein and mRNA were overexpressed in PTCLs and T-LBLs when compared with DLBCLs and controls (P <0.05). Additionally, there was a correlation between protein expression of PDGF-B and both PDGFRs in PTCLs and T-LBLs, suggesting an autocrine or paracrine loop in the aetiology of aggressive canine T-cell lymphomas. These data provide a rationale for the use of PDGFR antagonists in the therapy of aggressive T-cell lymphomas, but not in DLBCLs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: We reported an extremely rare case of atypical laryngeal carcinoid, and examined the expression of several proteins for application of molecular targeted therapy., Method: Case report and review of the literature concerning atypical carcinoid arising from the larynx. The expressions of proteins were determined by immunohistochemical analysis., Results: We present here a case of atypical laryngeal carcinoid in a 79-year-old Japanese man, which was completely resected, and with no evidence of recurrence. On immunohistochemical analysis, neoplastic elements revealed, strong positivity for platelet-derived growth factor receptor α (PDGFRα), vascular endothelial growth factor receptor 2 (VEGFR2), and epidermal growth factor receptor (EGFR), and were mild positivity for KIT., Conclusion: Our findings suggest that atypical laryngeal carcinoid could be completely removed if it is located in the limited lesion. PDGFRα, VEGFR2, and EGFR expressions in this case provide the evidence that atypical laryngeal carcinoid is the candidate for molecular targeted therapy, although further investigations are necessary., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: Currently, there is no standard treatment for metastatic anaplastic thyroid cancer (ATC). DNA microarray analysis has shown platelet-dervived growth factor receptor (PDGFR) overexpression in ATC relative to well-differentiated thyroid cancer. In p53-mutated/deficient ATC cell lines, cABL is overexpressed, and selective inhibition of cABL results in a cytostatic effect. Imatinib inhibits tyrosine kinase activity of Bcr-ABL and PDGF. We hypothesize that patients with ATC that over-expresses PDGF receptors or cABL will respond to imatinib., Methods: Patients with histologically confirmed ATC who had measurable disease and whose disease expressed PDGF receptors by immunohistochemistry were eligible for study. Imatinib was administered at 400 mg orally twice daily without drug holiday. Response to treatment was assessed every 8 weeks. Patients with complete response, partial responses, or stable disease were treated until disease progression. The study was terminated early due to poor accrual., Results: From February 2004 to May 2007, 11 patients were enrolled and were started on imatinib. At baseline, 4/11 had locoregional disease, 5/11 had distant metastases, and 2/11 had both. Nine of 11 had prior chemoradiation, and 7/11 had thyroidectomy. Eight of 11 were evaluable for response; 4 were excluded for lack of follow-up with radiologic evaluation. The overall response rates at 8 weeks were complete response 0/8, partial response 2/8, and stable disease 4/8. The median time to follow-up was 26 months (ranges 23-30 months). The rate of 6-month progression-free survival was 36% (95% confidence interval, 9%-65%). The rate of 6-month overall survival was 45% (95% confidence interval, 16%-70%). The most common grade 3 toxicity was edema in 25%; other grade 3 toxicities included fatigue and hyponatremia (12.5% each). There were no grade 4 toxicities or treatment related deaths., Conclusions: Imatinib appears to have activity in advanced ATC and is well tolerated. Due to difficulty of accruing patients with a rare malignancy at a single institution, further investigation of imatinib in ATC may be warranted in a multi-institutional setting.

Background: Imatinib myselate is a molecularly targeted drug that inhibits Abl tyrosine kinase, as well as type III tyrosine kinase receptors such as platelet-derived growth factor receptor (PDGFR), KIT, colony-stimulating factor 1 receptor (CSF-1R), and discoidin domain receptor (DDR). Ph1 chromosome-positive chronic myeloid leukemias (CMLs), KIT-positive gastrointestinal stromal tumors (GISTs), and PDGFR-positive dermatofibrosarcoma protuberans (DFSP) have been reported to be responsive to imatinib treatment. We conducted a multicenter Phase II trial of imatinib in patients with relapsed or refractory KIT-positive (excluding GISTs) or PDGFR-positive sarcomas., Methods: Patient ages ranged from 12 and 75 years. Eligibility criteria included (1) metastatic sarcomas with a definitive diagnosis based on histopathology or that were completely unresectable and locally advanced; (2) relapsed or refractory cases that had completed standard treatment; and (3) a tumor confirmed by immunohistochemical staining to be KIT- or PDGFR-positive. A 600-mg dose of imatinib was administered to patients once a day, with each patient receiving six courses of the drug and each course lasting 4 weeks. In cases categorized as stable or progressive, the imatinib dose was increased to 800 mg/day administered twice daily., Results: A total of 25 patients who met the eligibility criteria were enrolled in the trial; 22 were evaluated for response. The response rate with a 600 mg/day dose of imatinib was 4.5% (0 complete response, 1 partial response). There were no other objective responses after increasing imatinib to 800 mg/day (0/10). We estimated 50% progression-free survival to be 61.0 days for an imatinib dose of 600 mg/day based on the Kaplan-Meier method. Side effects of imatinib were generally similar to those observed in previous clinical trials., Conclusions: Our results did not indicate effectiveness of imatinib monotherapy at a dose of 600 or 800 mg/day in patients with relapsed or refractory KIT-positive (excluding GISTs) or PDGFR-positive sarcomas. Our findings suggest the need to evaluate the synergistic effect of combination therapy with other anticancer drugs.

Irradiation by light emitting diode (LED) promotes fibroblast proliferation and wound healing. However, its mechanism is still unknown. The purpose of this study was to clarify the mechanism of fibroblast proliferation by LED irradiation. Cultured NIH3T3 fibroblasts from normal mice were irradiated by LED with a center wavelength of 627 nm. LED irradiation was performed with an energy density of 4 J/cm(2), at subculture and 24 h later. The expression of several growth factors and their receptors was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR): platelet-derived growth factor (PDGF)-A, PDGF-B, and PDGF-C, transforming growth factor-beta (TGF-beta), basic fibroblast growth factor (bFGF), PDGF-alpha receptor, and TGF-beta receptor. Then, the activation of the extracellular signal-regulated kinase (ERK) pathway was examined by Western blotting with and without the PDGF receptor inhibitor. LED irradiation induced cell growth of NIH3T3 fibroblasts. The expression of PDGF-C had significantly increased in the irradiated group (P < 0.01). Although strong activation of the ERK pathway was observed in the irradiated group, its activation was completely suppressed by the PDGF receptor inhibitor. We concluded that LED irradiation promotes fibroblast proliferation by increasing autocrine production of PDGF-C and activating the ERK pathway through phosphorylation of the PDGF receptor.

Congenital nephrotic syndrome of the Finnish type (NPHS1, CNF) is an autosomal recessive disease caused by mutations in a major podocyte protein, nephrin. NPHS1 is associated with heavy proteinuria and the development of glomerular scarring. We studied the cellular and molecular changes affecting the glomerular mesangium in NPHS1 kidneys. Marked hyperplasia of mesangial cells (MC) was mainly responsible for the early mesangial expansion in NPHS1 glomeruli. The levels of the proliferation marker, mindbomb homolog 1 and the major MC mitogen, platelet-derived growth factor, and its receptors, however, were quite normal. Only a small number of cells were positive for CD68 (marker for phagocytic cells) and CD34 (marker for mesenchymal precursor cells) in the NPHS1 mesangium. MCs strongly expressed alpha-smooth muscle actin, indicating myofibloblast transformation. The expression levels of the profibrotic mediators osteopontin and transforming growth factor beta were up-regulated in NPHS1 glomeruli by 3.2 and 1.6-fold, respectively, compared to the controls. The synthesis by MCs of the typical fibroblast products collagen I, fibronectin, and tenascin, however, was low, and the extracellular matrix increase was caused by the accumulation of a normal MC product, collagen IV. The results indicate that severe glomerular sclerosis can develop without major qualitative cellular or molecular changes in the mesangium.

Tunable aptamer capillary electrophoresis (CE) techniques were developed to enable the separation and detection of platelet derived growth factor (PDGF) isomers and their receptors. Using an aptamer that formed a stable complex with the B chain but not with the A chain of PDGF, we were able to tweak the electrophoretic mobilities of the PDGF isomers for their separation. PDGF-AB bound to a single aptamer molecule was well resolved from PDGF-BB bound to two aptamer molecules. Simultaneous determination of 50 pM of two isomers was accomplished in a single analysis. Furthermore, PDGF-AB was used as a connector to bring receptor alpha and fluorescent aptamer into a single complex molecule. As a result, the formation of a (receptor alpha)-(PDGF-AB)-(fluorescent aptamer) ternary complex enabled the detection of the receptor alpha by tunable aptamer CE. A competitive assay was developed to determine receptor beta, making use of the competition between the receptor beta and fluorescent aptamer in binding to PDGF-BB. Detection limits were 0.5 nM for PDGF receptor alpha and 3 nM for receptor beta. Determination of PDGF isomers and their receptors in diluted serum samples showed no interference from the sample matrix.

Our goal was to discover genes differentially expressed in the perichondrium (PC) of the mandibular condylar cartilage (MCC) that might enhance regenerative medicine or orthopaedic therapies directed at the tissues of the temporomandibular joint. We used targeted gene arrays (osteogenesis, stem cell) to identify genes preferentially expressed in the PC and the cartilaginous (C) portions of the MCC in 2-day-old mice. Genes with higher expression in the PC sample related to growth factor ligand-receptor interactions [FGF-13 (6.4x), FGF-18 (4x), NCAM (2x); PGDF receptors, transforming growth factor (TGF)-beta and IGF-1], the Notch isoforms (especially Notch 3 and 4) and their ligands or structural proteins/proteoglycans [collagen XIV (21x), collagen XVIII (4x), decorin (2.5x)]. Genes with higher expression in the C sample consisted mostly of known cartilage-specific genes [aggrecan (11x), procollagens X (33x), XI (14x), IX (4.5x), Sox 9 (4.4x) and Indian hedgehog (6.7x)]. However, the functional or structural roles of several genes that were expressed at higher levels in the PC sample are unclear [myogenic factor (Myf) 9 (9x), tooth-related genes such as tuftelin (2.5x) and dentin sialophosphoprotein (1.6x), VEGF-B (2x) and its receptors (3-4x) and sclerostin (1.7x)]. FGF, Notch and TGF-beta signalling may be important regulators of MCC proliferation and differentiation; the relatively high expression of genes such as Myf6 and VEGF-B and its receptors suggests a degree of unsuspected plasticity in PC cells.

Background: Imatinib mesylate is a potent inhibitor of the Bcr-Abl, c-Kit, and platelet-derived growth factor receptor (PDGFR) tyrosine kinases. On the basis of variable expression of c-Kit and PDGFR in breast cancer and of in vitro data supporting synergy between imatinib and capecitabine, the Southwest Oncology Group conducted a phase II trial of the combination in metastatic breast cancer., Patients and Methods: Eligible patients had progressive, measurable metastatic breast cancer and received

Transient myeloproliferative disorder (TMD) is experienced by approximately 10% of neonates with Down syndrome (DS). Most TMD is asymptomatic and the patients undergo spontaneous remission within a few months. However, some cases are fatal because of systemic organ dysfunctions including hepatic fibrosis. Some cytokines such as platelet-derived growth factor (PDGF) may be involved in the development of hepatic fibrosis in TMD. The report describes a fatal case of TMD accompanying DS. The patient presented with pulmonary hypertension and hepatic failure. An autopsy disclosed severe fibrosis in the lung, liver, kidney and pancreas. Immunohistochemical analysis revealed high expression of PDGF receptor beta in the severe fibrotic areas of the fibrotic tissues. A real-time polymerase chain reaction (PCR) analysis demonstrated the expression of PDGFalpha and PDGFbeta in the peripheral blood samples of the patient. The finding indicates that the PDGF pathway may play an important role in the fibrosis of several organs in patients with TMD.

Aims: In view of available targeted therapies, we investigated the presence of c-kit, PDGFR gene mutations and protein expression in cancer of unknown primary (CUP) in order to study their contribution in pathogenesis, their prognostic value and potential as therapeutic targets., Methods: Mutations in hot spots c-kit exon 11 and PDGFR exons 12 and 18 were studied in paraffin-embedded tumour samples from 50 patients with CUP by means of PCR-based single-strand conformational polymorphism and protein expression by means of streptavidin-biotin immunoperoxidase assays. Molecular markers were screened for possible correlations with patient outcome., Results: No shifted band was detected in any of the polyacrylamide gel electrophoreses, indicating absence of c-kit exon 11 and PDGFR exon 12, 18 mutations. Immunohistochemical analysis in 37 tumours revealed positive membranous CD117 expression in 30 samples (81%) of which five exhibited strong (+3), four moderate (+2) and 21 weak (+1) staining. PDGFRa protein staining was seen in 15 out of 30 (50%) cases, mostly weak (13) and rarely moderate (1) or strong (1). The expression of KIT or PDGFRa protein did not correlate with the clinical outcome of the patients in our cohort., Conclusions: In a moderate-sized CUP patient cohort, KIT or PDGFRa protein overexpression is rare, does not have gross prognostic significance for survival and is not associated with presence of activating mutations.

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.

Purpose: To explore the molecular bases of potential new pharmacologic targets in aggressive fibromatosis (desmoid tumor)., Experimental Design: Tumor specimens from 14 patients surgically treated for aggressive fibromatosis (6 familial adenomatous polyposis and 8 sporadic cases), analyzed for adenomatous polyposis coli (APC) and CTNNB1 (beta-catenin) mutations, were further investigated for beta-catenin, cyclooxygenase-2 (COX-2), platelet-derived growth factor (PDGF) receptor alpha (PDGFRA)/PDGF receptor beta (PDGFRB), their cognate ligands (PDGFA and PDGFB), and KIT using a comprehensive immunohistochemical, biochemical, molecular, and cytogenetic approach., Results: No CTNNB1 (beta-catenin) mutations were found in the familial adenomatous polyposis patients, but previously reported activating mutations were found in six of the eight sporadic patients. All of the cases carrying an altered WNT pathway showed nuclear and cytoplasmic immunoreactivity for beta-catenin, whereas beta-catenin expression was restricted to the cytoplasm in the sporadic patients lacking CTNNB1 mutations. COX-2 protein and mRNA overexpression was detected in all 14 cases, together with the expression and phosphorylation of PDGFRA and PDGFRB, which in turn paralleled the presence of their cognate ligands. No PDGFRB mutations were found. The results are consistent with PDGFRA and PDGFRB activation sustained by an autocrine/paracrine loop., Conclusions: Aggressive fibromatosis is characterized by WNT/oncogene pathway alterations triggering COX-2-mediated constitutive coactivation of PDGFRA and PDGFRB, and may therefore benefit from combined nonsteroidal anti-inflammatory drug + tyrosine kinase inhibitor treatment.

High levels of PDGFR expression in primary rhabdomyosarcoma (RMS) have been associated with disease progression. To date however, there are no reports on the activity of imatinib mesylate, a selective PDGFR inhibitor, in RMS preclinical models. A panel of 5 RMS cell lines was used to investigate the expression of PDGFRalpha and PDGFRbeta, c-Kit and the multidrug transporter ABCG2 (also inhibited by imatinib). In vitro and in vivo experiments were performed using RD (embryonal) and RH30 (alveolar) cell lines to determine the efficacy of imatinib as single agent and in combination with topotecan (TPT). PDGFRbeta was significantly expressed in all cell lines, with the highest levels in RD, while PDGFR alpha and ABCG2 were significantly expressed only in RH30 and RMZ-RC2. c-Kit was not detected. PDGFRbeta signaling was active in RD but not in RH30, whilst PDGFRalpha signaling was not active in either cell lines. Significant ABCG2-mediated extrusion of Hoechst 33342 was demonstrated in RH30 but not in RD, and was inhibited by imatinib and the specific ABCG2 inhibitor Ko143. In vitro, imatinib was not active as a single agent at therapeutic concentrations, but significantly potentiated TPT antitumor activity in both cell lines. In vivo experiments using tumor xenografts confirmed the synergistic interaction in both cell lines. These results suggest that at least 2 different mechanisms--inhibition of ABCG2 and/or PDGFRbeta--are involved in the synergistic interaction between imatinib and TPT, and support the use of this combination for the treatment of high-risk RMS patients., (Copyright 2006 Wiley-Liss, Inc.)

Objective: The objective of this study was to determine whether STI-571 (Gleevec; imatinib mesylate) could sensitize established head and neck squamous cell carcinoma (HNSCC) cell lines to the effects of cisplatin., Methods: Western blot analysis and immunofluorescence were used to examine the expression of the tyrosine kinases that are known targets of Gleevec, including c-kit, c-Abl, and platelet-derived growth factor receptor, on the cell lines, and immunohistochemistry was performed to determine the expression of these kinases in human HNSCC tissue. Once these targets were confirmed, clonogenic cell survival assays were performed to determine the effect STI-571 had on growth and proliferation when used in combination with cisplatin compared with STI-571 alone or cisplatin alone. Cells were incubated with a range of doses of STI-571 24 hours before the addition of cisplatin. Flow cytometry analysis was performed to determine cell-cycle distribution and to measure apoptosis caused by the various treatments. An annexin V assay was also used to further measure apoptosis., Results: Our results indicate that STI-571 potentiates the effect of cisplatin and leads to a significant decrease in cell proliferation and colony formation compared with cisplatin alone in a dose-dependent fashion. Surprisingly, there was a slight decrease in the level of apoptosis when Gleevec was used in combination with cisplatin compared with cisplatin alone. Gleevec alone resulted in a slight increase in G1 phase of the cell cycle, whereas cisplatin alone resulted in a G2 arrest. The addition of Gleevec to cisplatin resulted in an enhanced S/G2 phase accumulation. Although we did not demonstrate an increase in cisplatin-induced cell death, we postulate that the increased S/G2 arrest resulting from the DNA damage in the presence of Gleevec results in decreased proliferation of HNSCC, resulting in a net decrease in colony formation., Conclusions: The small molecule inhibitor Gleevec, which targets specific tyrosine kinases that are expressed in HNSCC, can significantly potentiate the antiproliferative effects of cisplatin. Because Gleevec alone has minimal side effects, treatment with the combination treatment of cisplatin and Gleevec may result in increased efficacy of cisplatin in treating this cancer. Additional studies are warranted, keeping in mind that drug combinations may result in unexpected toxicities that are not frequently seen with either drug alone.

The platelet-derived growth factor receptor (PDGFR) is a tyrosine kinase, implicated in the development and progression of different tumors, including gliomas. Chemoresistance is a common feature of malignant gliomas. Since receptor tyrosine kinases contribute to chemoresistance in tumors, we addressed whether PDGFR signaling might confer selective growth advantage to chemoresistant cells. The effects of the PDGFR inhibitor STI571 on proliferation and PDGFR signaling were compared in chemosensitive and cisplatin-selected, chemoresistant sublines derived from glioma and from two other PDGFR-expressing tumors (ovarian carcinoma and neuroblastoma). The chemoresistant glioma U87/Pt cells were twofold more sensitive to STI571 growth-inhibitory effects than the chemosensitive U87 cells, and two- to threefold more sensitive than five unrelated glioma cell lines. The other two paired cell lines were equally responsive. Sensitization of U87/Pt cells correlated with upregulation of the PDGF-B isoform and with PDGF-BB-induced Akt overactivation, which was prevented by STI571. STI571 specifically inhibited PDGF-BB-, but not PDGF-AA- or stem cell factor-mediated signaling. In serum-containing medium, STI571 decreased phospho-Akt in U87/Pt cells, but not in U87, while activating extracellular signal-regulated kinase (Erk) in both. STI571 antiproliferative effects were partially reverted by constitutively active Akt. Cotreatment with inhibitors of phosphatidylinositol 3'-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK) resulted in enhanced growth inhibition in glioma cells. Our results suggest that increased PDGF-BB signaling may sensitize chemoresistant glioma cells to STI571, suggesting a therapeutic potential for STI571 in patients with malignant gliomas refractory to chemotherapy. Simultaneous blockade of PDGFR and PI3K or Erk pathway may enhance therapeutic targeting in gliomas., (Copyright 2006 Wiley-Liss, Inc.)

Imatinib mesylate (IM), a small molecule that is a selective inhibitor of the ABL, platelet derived growth factor receptor (PDGFR-R) and stem cell ligand receptor (c-kit) tyrosine kinases (TK). IM was also found to inhibit the TK activity of BCR/ABL fusion protein produced in chronic myelogenous leukemia, with marked clinical activity against the disease. Since both PDGF-R and c-kit both having a putative role in tumorigenesis, we investigated the efficacy and safety of the use of IM in patients with endocrine tumors unresponsive to conventional therapies that expressed c-kit and/or PDGF-R (within the framework of a comprehensive phase II multi-center study of IM in patients with solid tumors). IM was initiated at a dose of 400 mg/day, with possible dose escalation within 1 week to 600 mg/day and an option to raise the dose to 800 mg/day in the event of progression and in the absence of safety concerns for a period of up to 12 months. Between September 2002 and July 2003, 15 adult patients with disseminated endocrine tumors were recruited as follows: medullary thyroid carcinoma (MTC, n = 6); adrenocortical carcinoma (ACC, n = 4); malignant pheochromocytoma (pheo, n = 2); carcinoid (non-secreting, n = 2), neuroendocrine tumor (NET, n = 1). No objective responses were observed. MTC--disease progression in 4 patients, and treatment discontinuation in 2 patients due to adverse events; ACC--disease progression in 3 patients, and treatment discontinuation in 1 patient due to severe psychiatric adverse event; Pheo--disease progression in 2 patients; Carcinoid--stable disease in 1 patient (6.5 months), and disease progression in 1 patient; NET--disease progression in 1 patient. IM does not appear to be useful for treatment of malignant endocrine tumors, also causing significant toxicity in this patient population.

Objective: To investigate the messenger RNA (mRNA) and protein expression of the recently discovered platelet-derived growth factor C (PDGF-C) and PDGF-D in the synovial membrane (SM) of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to assess the localization and cellular source of these proteins in the SM and their functional influence on synovial fibroblasts., Methods: Expression of mRNA for PDGFs A, B, C, and D as well as for PDGF receptor (PDGFR) alpha and beta chains in RA and OA SM samples was assessed by real-time reverse transcription-polymerase chain reaction. Protein levels of PDGF-C and PDGF-D were quantified by immunoblotting. Regional and cellular localization of PDGF-C and PDGF-D in the SM was investigated by double-staining immunohistochemistry. In addition, the influence of PDGF-D on the proliferation of synovial fibroblasts and their matrix metalloproteinase (MMP-1) mRNA expression were determined., Results: The expression of mRNA for PDGFs A, B, and C and for PDGFR alpha and beta chains was comparable in RA and OA SM samples; in contrast, the expression of mRNA for PDGF-D was significantly higher in OA SM. PDGF-C protein was not differentially expressed in OA and RA. The expression of PDGF-D protein was significantly higher in RA SM (full-length and activated form). PDGF-C and PDGF-D were expressed throughout the SM (lining layer, diffuse infiltrates, and stroma) by both synovial fibroblasts and macrophages. In addition, PDGF-D increased the proliferation of synovial fibroblasts and the expression of mRNA for MMP-1., Conclusion: PDGF-C and PDGF-D are expressed by synovial fibroblasts and macrophages in RA and OA SMs. The levels of PDGF-D protein were significantly higher in RA SM. In addition, PDGF-D stimulated synovial fibroblast proliferation and expression of MMP-1. These findings may have pathogenetic implications for cellular transformation and matrix remodeling in the RA SM.

We have developed a highly specific sensing system for platelet-derived growth factors (PDGFs) and platelet-derived growth factor receptors (PDGFR) that uses gold nanoparticles (GNPs). We synthesized GNPs modified with an aptamer (Apt-GNPs) that is specific to PDGFs and used them to detect PDGFs by monitoring the changes in the color and extinction of the Apt-GNPs that occur as a result of aggregation. The color of the Apt-GNPs changes from red to purple at low concentrations (<400 nM), but changes only slightly at higher concentrations (>400 nM). We found that the sensitivity of the Apt-GNPs for the three PDGFs is highly salt-dependent, with an optimum condition of 200 mM NaCl. We obtained biphasic curves when plotting of the ratios of the extinction coefficients of the Apt-GNPs at 650 and 530 nm against the concentrations of PDGF-AA at various concentrations of Apt-GNPs. The linear ranges of the increases and decreases in this extinction ratio are 2.5-10 and 10-20 nM, respectively, for 0.42 nM Apt-GNPs and 25-75 and 75-200 nM, respectively, for 8.4 nM Apt-GNPs. When using 8.4 nM Apt-GNPs, the corresponding linear ranges of the increases and decreases in this extinction ratio are 15-100 and 100-400 nM, respectively, for PDGF-AB and 35-150 and 150-400 nM, respectively, for PDGF-BB. In addition, we have developed a homogeneous assay to detect the PDGF receptor-beta (PDGFR-beta) at concentrations as low as 3.2 nM, on the basis of the competition between the Apt-GNPs and PDGFR-beta for PDGF-BB. The results we present in this paper imply that there are practical applications of Apt-GNPs in protein analysis and cancer diagnosis.

Objective: To determine the efficacy of low-dose imatinib mesylate (STI571) alone or combined with a taxane (paclitaxel) in inhibiting the growth of experimental extra-osseous hormone-refractory prostate cancer., Materials and Methods: Orthotopic PC3 prostate tumours were established in male severe combined-immunodeficient mice; on day 3 the mice were randomly assigned to one of four groups: paclitaxel 10 mg/kg intraperitoneally once a week; STI571 50 mg/kg once a day for 6/7 weekdays; combined paclitaxel and STI571; and vehicle-treated controls. On day 40, the primary prostate tumour and metastatic lymphadenopathy were removed and measured. Effects were correlated with tumour cell proliferation and microvessel density., Results: Paclitaxel reduced the mean tumour weight and volume by 21.3% (not significant) and 73.7% (P < 0.05), respectively, compared to controls, and reduced the number of lymph node metastases by 49.1% (P < 0.05) and mean lymph node size by 13.5% (not significant). Adding low-dose STI571 had a small additive effect on tumour weight and the incidence of lymph node metastases, but this was not significant compared to paclitaxel alone. STI571 alone did not inhibit tumour progression. Antitumour effects were associated with parallel changes in tumour cell proliferation with no significant changes in neo-angiogenesis., Conclusion: Combined low-dose STI571 and paclitaxel had little synergy in this experimental model. Low-dose STI571 monotherapy was not effective in extra-osseous disease, apparently due to a site-specific failure to up-regulate beta-platelet-derived growth factor receptor expression in prostate cancer cells and associated tumour stroma.

Objective: To elucidate the relation of platelet-derived growth factor receptor (PDGFR) and c-Fos protein expressions with human bladder transitional epithelial cell carcinoma (BTCC)., Methods: The expressions of PDGFR and c-Fos were investigated in 11 normal bladder tissue samples, 14 adjacent non-carcinoma tissues and 43 BTCC tissues by means of SP immunohistochemical technique., Results: The c-Fos expression was found in the cell nuclei and cytoplasm, and PDGFR in the nuclear membrane, cytoplasm, and cellular membrane. PDGFR and c-Fos were detected in 81.40% and 48.83% of the BTCC tissues respectively, at the rates both significantly higher than those in normal and adjacent non-carcinoma tissues (P<0.05). Correlation between the expression of c-Fos and the tumor grading was noted (P<0.05). The expressions of PDGFR and c-Fos in tumor blood vessels were significantly higher than those in normal vessels., Conclusions: The expressions of PDGFR and c-Fos might be involved in the development of BTCC, possibly related to the angiogenesis of the tumors. c-Fos expression can indicated the cell proliferative status of the BTCC.

Tuberous sclerosis (TSC) is a familial tumor syndrome due to mutations in TSC1 or TSC2, in which progression to malignancy is rare. Primary Tsc2(-/-) murine embryo fibroblast cultures display early senescence with overexpression of p21CIP1/WAF1 that is rescued by loss of TP53. Tsc2(-/-)TP53(-/-) cells, as well as tumors from Tsc2(+/-) mice, display an mTOR-activation signature with constitutive activation of S6K, which is reverted by treatment with rapamycin. Rapamycin also reverts a growth advantage of Tsc2(-/-)TP53(-/-) cells. Tsc1/Tsc2 does not bind directly to mTOR, however, nor does it directly influence mTOR kinase activity or cellular phosphatase activity. There is a marked reduction in Akt activation in Tsc2(-/-)TP53(-/-) and Tsc1(-/-) cells in response to serum and PDGF, along with a reduction in cell ruffling. PDGFRalpha and PDGFRbeta expression is markedly reduced in both the cell lines and Tsc mouse renal cystadenomas, and ectopic expression of PDGFRbeta in Tsc2-null cells restores Akt phosphorylation in response to serum, PDGF, EGF, and insulin. This activation of mTOR along with downregulation of PDGFR PI3K-Akt signaling in cells lacking Tsc1 or Tsc2 may explain why these genes are rarely involved in human cancer. This is in contrast to PTEN, which is a negative upstream regulator of this pathway.

Downregulation of survival signaling pathways contributes to the cytotoxicity of reactive oxygen species (ROS) and may underlie certain therapies for hyperproliferative diseases. We have investigated the role of singlet oxygen, an ROS formed by photosensitization, in the regulation of survival signaling via the epidermal growth factor receptor (EGFR). Exposure of human keratinocytes to singlet oxygen resulted in rapid loss of EGFR, which was not blocked by either inhibition of receptor internalization or by interrupting the major proteolytic pathways (proteasome, lysosome or calpain). However, pretreatment with a caspase-3 inhibitor, DEVD-FMK, inhibited EGFR degradation. Caspase-3 cleavage was detected as early as 5 min after singlet oxygen treatment, and recombinant active caspase-3 completely cleaved EGFR in a keratinocyte membrane fraction. The singlet oxygen-induced loss of EGFR was accompanied by dephosphorylation of EGFR as well as of Akt and extracellular signal-regulated kinase 1/2 (ERK)1/2. Singlet oxygen-induced protein dephosphorylation was not dependent on activation of caspase-3. In contrast, inhibition of protein phosphatases (PPs) with okadaic acid completely blocked dephosphorylation of EGFR, ERK1/2 and Akt as well as degradation of EGFR. These results indicate that the oxidative stress produced by singlet oxygen rapidly disrupts EGFR-mediated signaling by decreasing both the protein level and its phosphorylation. These responses depended on intertwined activation of caspase-3 and PPs.

Although pulmonary fibrosis is a frequent and serious consequence of radiotherapy for thoracic malignant diseases such as lung cancer, the pathogenesis of this radiation-induced lung disorder remains unclear. To clarify the mechanisms underlying radiation pneumonitis and pulmonary fibrosis, we investigated the expression of platelet-derived growth factor receptor (PDGFR) on fibroblasts obtained from irradiated rat lungs and on control fibroblasts. Whole lungs of male Wistar rats were irradiated with a single dose of 15 Gy, and lung fibroblasts were isolated at 4 weeks after the irradiation. The chemotactic response of irradiated lung fibroblasts to PDGF-BB was significantly higher than that of control lung fibroblasts, whereas there was no significant difference between irradiated lung fibroblasts and control lung fibroblasts in the response to PDGF-AA. Receptor binding assay showed more specific binding sites for PDGF-BB on irradiated lung fibroblasts than on control lung fibroblasts, and the displacement of (125)I-labeled PDGF binding to fibroblasts by unlabeled PDGF showed that (125)I-labeled PDGF-BB was displaced by PDGF-BB but not by PDGF-AA. These results suggest that the increased binding sites for PDGF-BB on irradiated lung fibroblasts correspond mainly to PDGFRB. Scatchard analysis of the saturation data demonstrated an approximately twofold increase both in the number of PDGF-BB binding sites and in the binding affinity in irradiated lung fibroblasts compared to that in control lung fibroblasts. Those results suggest that the increased chemotactic response of irradiated lung fibroblasts to PDGF-BB is related to the overexpression of PDGFRB, which may have an important role in the pathogenesis of radiation-induced pneumonitis and pulmonary fibrosis.

Objectives: To elucidate the role of the fibroblast growth factors 1 and 3 (FGFR-1, -3) and the platelet derived growth factor (PDGFR) in the growth of the mandibular condylar cartilage in the rat., Setting and Sample Population: Institute of Dentistry and Department of Biostatistics, University of Turku, Turku, Finland. The material consisted of 1- to 21-day-old Long-Evans/Turku rats (total of 24 animals, three in each age group)., Design: An immunohistological in vivo study combined with histomorphometry and biostatistical analysis., Experimental Variable: The animals were killed with an overdose of carbon dioxide and thereafter decapitated. Heads were fixed in 4% paraformaldehyde, decalcified in 12.5% ethylenediaminetetraacetic acid, cut sagittally into two halves and sectioned sagittally at 6 microns. In order to detect FGFR-1, -3 and PDGFR the sections were treated with H2O2/methanol (1:100), after which FGFR-1 and PDGFR monoclonal and FGFR-3 polyclonal antibodies were applied. The reaction products were visualized by using the Vectastain ABC Elite Kit using peroxidase substrate kit DAB as substrate. Negative and positive controls were also prepared. The sections were counterstained with hematoxylin., Outcome Measure: In order to measure the depth of the cell layer labeled with FGF-1, -3 and PDGF receptors, the condylar head was divided into four regions: anterior, superior, posterosuperior and posterior. The measurements were made perpendicular to the articular surface using a computerized image analysis system, the images being acquired by means of a microscope connected to a CCD camera. The mean of five equally distributed measurements of each region was used to indicate the depth of the cell layers secreting the receptors. Regression analysis was used to evaluate the association between the depth of the labeled cell layer in relation to total depth of the condylar head, as a function of age., Results: Our results show that the depth of the cell layer labeled for FGFR-1, -3 and PDGFR increase significantly as a function of age in the mandibular condylar head of rats., Conclusion: Increase in the cell layer labeled for FGFR-1, -3 and PDGFR occurs during the stage when the articular function of the mandibular condyle intensifies. FGFR-1, -3 and PDGFR evidently have an important role in the growth regulation of the condylar cartilage during the most rapid growth period in the rat.

The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immunoprecipitation assays., ((c) 2002 Elsevier Science (USA).)

Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors have been reported to signal via caveolin-containing membranes called caveolae. In contrast, others report that EGF and PDGF receptors are exclusively associated with caveolin-devoid membranes called rafts. Our subcellular fractionation and coimmunoprecipitation studies demonstrate that, in the absence of ligand, EGF and PDGF receptors are associated with rafts. However, in the presence of ligand, EGF and PDGF receptors transiently associate with caveolae. Surprisingly, pretreatment of cells with EGF prevents PDGF-dependent phosphorylation of PDGF receptors and extracellular signal-regulated kinase (ERK) 1/2 kinase activation. Furthermore, cells pretreated with PDGF prevent EGF-dependent phosphorylation of EGF receptors and ERK1/2 kinase activation. Radioligand binding studies demonstrate that incubation of cells with EGF or PDGF causes both EGF and PDGF receptors to be reversibly sequestered from the extracellular space. Experiments with methyl-beta-cyclodextrin, filipin, and antisense caveolin-1 demonstrate that sequestration of the receptors is dependent on cholesterol and caveolin-1. We conclude that ligand-induced stimulation of EGF or PDGF receptors can cause the heterologous desensitization of the other receptor by sequestration in cholesterol-rich, caveolin-containing membranes or caveolae.

Platelet-derived growth factors (PDGFs) are paracrine factors with roles in mesenchymal-epithelial interactions during normal and pathologic processes. Previously, PDGF and its receptor (PDGFR) have been shown to be present in perinatal, peripubertal, and adult rat testes. The role of PDGF in embryonic testicular cord formation is not known. The hypothesis tested is that PDGFs and PDGFRs are expressed during cord formation and that inhibition of their action influences normal cord formation during embryonic testis development. Embryonic Day (E) 13 gonadal organ cultures were used. Organs were cultured for 3 days and treated daily with vehicle or a PDGFR-specific tyrosine phosphorylation inhibitor (i.e., the tyrphostin AG1295 or AG1296). Vehicle-treated testes formed normal cords, whereas tyrphostin-treated testes formed "swollen cords," a phenomenon characterized by a significant decrease in the number of cords per testis area and increased cord diameter due to fusion of cords. Expression of PDGF and PDGFR in E13, E14, E16, Postnatal Day (P) 0, and P20 testes was examined. Messenger RNAs for PDGF-A and -B and PDGF alpha- and beta-receptors were expressed in isolated testes during all developmental periods examined. Immunoreactivity for PDGF was present throughout the testicular compartment at E14, restricted primarily to testicular cords at E16, and present in cells of the testicular cords with a stronger immunoreactivity in certain interstitial cell types of P0 testis. PDGFR beta-receptor immunoreactivity was primarily localized to the mesonephros of E14 organs and the testicular interstitium of E16 and P0 testes. Tyrphostins did not affect apoptotic cell number in the testis. PDGF had no effect on cell growth in P0 testis cultures. The results show that PDGFs and PDGFRs are expressed in embryonic testis during cord formation in a tissue-specific manner. Inhibition of PDGF actions does not inhibit cord formation but does alter normal cord development and morphology. The observations provide insight into the factors involved in male sex differentiation and embryonic testis development.

Vascular smooth muscle cells (VSMCs) in the intact vascular wall are differentiated for contraction, whereas the response to vascular injury involves transition towards a synthetic phenotype, with increased tendency for proliferation. Platelet-derived growth factor (PDGF) is thought to be important for this process. We investigated expression and functional coupling of PDGF receptors (PDGFRs) alpha and beta in rat tail arterial rings kept in organ culture, in order to capture early events in the phenotypic transition. In freshly dissected rings no PDGFR immunoreactivity was found in medial VSMCs, whereas PDGFR alpha was detected in nerve fibres. After organ culture for 1-4 days PDGFR alpha and beta as well as phospholipase Cgamma2 (PLCgamma2), known to couple to PDGFR, were expressed in VSMCs within 100 microm of the cut ends. Calponin, a marker for the contractile phenotype, was decreased near the injured area, suggesting that cells were in transition towards synthetic phenotype. In these cells, which showed functional Ca2+-release from the sarcoplasmic reticulum, PDGF-AB (100 ng x mL(-1)) had no effect on [Ca2+]i, whereas cultured VSMCs obtained from explants of rat tail arterial rings responded to PDGF-AB with an increase in [Ca2+]i. However, PDGFR within the cultured rings coupled to growth signalling pathways, as PDGF-AB caused a tyrphostin AG1295-sensitive activation of extracellular signal-regulated kinases 1 and 2 and of [3H]-thymidine incorporation. Thus, early expression of PDGFR in VSMC adjacent to sites of vascular injury coincides with signs of dedifferentiation. These receptors couple to growth signalling, but do not activate intracellular Ca2+ release.

Platelet-derived growth factor (PDGF) overactivity has been implicated in atherosclerosis and several fibrotic conditions including lung and kidney fibrosis, liver cirrhosis and myelofibrosis. Low oxygen tension (hypoxia) is a known stimulus for transcriptional induction of PDGF ligand and receptor genes in different tissues. We studied the expression and localization of PDGF-A, PDGF-B, and PDGF receptor (PDGFR)-alpha and -beta subunits in adult rat isolated corpus cavernosum (CC) under generalized transient hypoxia (pO(2) 10%) in comparison with normoxic conditions. Semi-quantitative RT-PCR analysis of mRNA extracted from rat penis showed higher amounts of PDGF-A, PDGF-B and PDGFR-beta mRNA transcripts in hypoxic versus normoxic animals. The immunohistochemical analysis showed that the localization of PDGF subunits and PDGFR-beta was confined to the cytoplasm of the perivascular smooth muscle cells, endothelium and trabecular fibroblasts. Our findings indicate that transient low oxygen tension induces PDGF overexpression in rat CC, which in the long term may lead to an increase of connective tissue production. We suggest that a local impairment of the PDGF/PDGFR system may contribute to CC fibrosis, which is an established cause of erectile dysfunction in man.

In the early atherosclerotic lesion, monocytes accumulate at sites of inflammation and endothelial injury. Platelet-derived growth factor (PDGF), produced for example by macrophages, is a chemoattractant for smooth muscle cells and possibly also for macrophages. During early differentiation into macrophages, human monocytes (early hMDM) showed lower expression of PDGF alpha-receptor (PDGF-Ralpha) than beta-receptor (PDGF-Rbeta) mRNA. Early hMDM showed increased random motility (chemokinesis) in the presence of PDGF of the long (BB(L)) but not short (BB(S)) B-chain homodimer. Neither PDGF-AA(S) nor PDGF-AA(L) affected early hMDM motility. Since increased cytokine levels accompany inflammation, the influence of interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) on PDGF-R expression and migratory response were studied. Only PDGF-Ralpha mRNA was highly upregulated by IFN-gamma. TGF-beta only had minor effects on receptor mRNAs. Upregulation of PDGF-Ralpha levels by IFN-gamma was accompanied by significantly increased migration (chemotaxis) towards PDGF-AA(L) only. Consequently, IFN-gamma modulates PDGF-Rs expression in early hMDM and, subsequently, the chemotactic activity of PDGF-AA(L) on IFN-gamma-stimulated early hMDM. This suggests that PDGF-AA(L) may be involved in attracting activated monocytes to sites of inflammation and injury.

In this study, we detected the expression of platelet-derived growth factor (PDGF) and its receptor in 61 human meningiomas by immunohistochemistry and in situ hybridisation. The results showed that almost all the 61 meningiomas expressed PDGFBB and PDGF beta receptor and the positive rate of PDGFAA was 49%. Only two meningiomas expressed PDGF alpha receptor. The positive rate and the immunostaining intensity of PDGFBB and PDGF beta receptor were higher in atypical meningiomas than in benign types. There was no significant difference between the different types of benign meningiomas. The expression of PDGFB chain mRNA was coincident with that of PDGFB chain protein. There was no correlation between the expression of PDGFAA and the types or grades of meningiomas. The correlation between overexpression of PDGFBB/R beta and tumour grade provides a useful parameter in evaluating the prognosis of patients with meningiomas. The proliferative activity of meningiomas was evaluated by the proliferating cell nuclear antigen labelling index (PCNA LI). In the 61 meningiomas, the average PCNA LI (%) was 1.8+/-1.3, 1.9+/-1.3, 1.7+/-0.8 and 11.6+/-5.3 (in fibrous, meningothelial, transitional and atypical meningiomas, respectively). Statistic analysis shows that the PCNA LI is higher in atypical meningiomas than that in benign types, and there was no significant difference between the different types of benign meningiomas. The expression of PDGFBB and PDGF beta receptor was significant enhanced in ascending order from low PCNA LI meningiomas to high ones. This result suggested that PDGFBB/R beta autocrine loop may stimulate the growth of meningiomas. In this study, we also detected the cell apoptosis of meningiomas by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) method. The average apoptosis labelling index (%) was 0.11+/-0.05, 0.08+/-0.04, 0.09+/-0.03 and 0.35+/-0.15 in fibrous, meningothelial, transitional and atypical meningiomas respectively. The apoptosis labelling index was higher in atypical meningiomas than that in benign types. There was a positive correlation between the apoptosis labelling index and PC NA LI of meningioma. When the positive rate of PDGFBB and/or PDGF beta receptor was higher in meningioma, the apoptotic cells was also increased. In conclusion, the overexpression of PDGFBB and its relevant receptor PDGFR beta in meningiomas was correlated with grade of meningiomas and the proliferative activity of meningiomas; PDGFBB/R beta autocrine loop may play an critical role in the pathogenesis of meningiomas., (Copyright 2001 Harcourt Publishers Ltd.)

We explored the origin and participation of atrophic tubules in the progression of interstitial fibrosis using a new microembolic rat model of chronic renal failure in which foci of atrophic tubules with cuff-like basement membrane thickening developed at 4 weeks. Atrophic tubules, immunoreactive for vimentin and platelet-derived growth factor, were surrounded by transformed interstitial cells expressing platelet-derived growth factor receptor beta and alpha-smooth muscle actin. Some tubules in the deep cortex and the outer stripe of the outer medulla had a mosaic appearance. Tall, intact proximal tubular cells with a brush border and positivity for Phaseolus vulgaris erythroagglutinin, adjoined typical atrophic tubule cells having no brush border and an immunostaining pattern characteristic for atrophic tubules. The transformed interstitial cells expressing alpha-smooth muscle actin were located near atrophic but not intact tubular epithelial cells. Type IV collagen accumulated between damaged tubular cells and transformed interstitial cells. Heat shock protein 47 showed immunoreactivity in damaged epithelial cells and in interstitial myofibroblasts. Staining with an anti-endothelial antibody suggested damage to peritubular capillaries near atrophic tubules. By disturbance of microcirculation following microsphere injection, proximal tubular cells expressed vimentin and platelet-derived growth factor; diffusion of the latter presumably stimulated transformation of interstitial cells to myofibroblasts. Injured tubular epithelial cells and interstitial myofibroblasts both were responsible for interstitial fibrosis.

Oligodendrocytes are the myelinating cells of the mammalian central nervous system. In the mouse spinal cord, oligodendrocytes are generated from strictly restricted regions of the ventral ventricular zone. To investigate how they originate from these specific regions, we used an explant culture system of the E12 mouse cervical spinal cord and hindbrain. In this culture system O4(+) cells were first detected along the ventral midline of the explant and were subsequently expanded to the dorsal region similar to in vivo. When we cultured the ventral and dorsal spinal cords separately, a robust increase in the number of O4(+) cells was observed in the ventral fragment. The number of both progenitor cells and mature cells also increased in the ventral fragment. This phenomenon suggests the presence of inhibitory factor for oligodendrocyte development from dorsal spinal cord. BMP4, a strong candidate for this factor that is secreted from the dorsal spinal cord, did not affect oligodendrocyte development. Previous studies demonstrated that signals from the notochord and ventral spinal cord, such as sonic hedgehog and neuregulin, promote the ventral region-specific development of oligodendrocytes. Our present study demonstrates that the dorsal spinal cord negatively regulates oligodendrocyte development., (Copyright 2000 Academic Press.)

Airway smooth muscle (ASM) mass is likely to be an important determinant of airway responsiveness. Highly inbred Fisher rats model innate hyperresponsiveness, and also have more ASM in vivo than control Lewis rats. Platelet derived growth factor (PDGF) is an important endogenous growth factor for ASM, and partially purified PDGF-AB causes enhanced growth of Fisher rat ASM cells, compared to Lewis cells. The aim of the present study was to determine the mitogenic effects of all three recombinant PDGF isoforms on ASM cells, and investigate the mechanisms of enhanced Fisher ASM growth responses. The potential mechanisms assessed include PDGF receptor expression and activation (tyrosine phosphorylation), and intracellular calcium (Ca2+) responses to PDGF isoforms. Fisher ASM cells had a greater mitogenic response to PDGF-AB and -AA, and a greater Ca2+ response to -BB than Lewis ASM cells. A Ca2+ response was not necessary for a mitogenic response, and the effects of PDGF isoforms on Ca2+ were not associated with their effects on growth. Therefore, we suggest that enhanced Fisher mitogenic response to PDGF-AA and -AB is not mediated by differences in Ca2+ signalling. Western analysis of the PDGF receptor indicated a similar expression of beta-PDGF receptor in ASM cells from the two rat strains, but a greater expression of alpha-PDGF receptor in Fisher cells; however, phosphorylation of the PDGF receptor following growth stimulation did not differ between strains. This suggests a role for post-receptor signals, in addition to enhanced receptor expression, in the enhanced growth response of Fisher ASM cells to PDGF-AA and -AB.

PDGF-related gene expression has been well characterized during fetal rat lung development and adult rat lung injury, but not during normal postnatal lung growth or injury. Lung expression of the mRNA for PDGF-A, -B, -alpha R, and -beta R and immunoreactive PDGF-AA, -BB, -alpha R, and -beta R were assessed in rat pups raised in air or 60% O(2) for up to 14 d after birth. Expression of mRNA and immunoreactive ligand did not correlate for pups raised in air. Immunoreactive PDGF-alpha R and -beta R, but not PDGF-AA and -BB, were evident throughout the lung at birth. Both PDGF-AA and -BB were evident in airway epithelium, PDGF-BB in alveolar epithelial cells and PDGF-AA was widely distributed in parenchymal tissue at 4 d. PDGF-alpha R was localized to airway epithelium, and PDGF-beta R to subendothelial perivascular regions and to airway and alveolar epithelium at 4 d. Immunoreactive PDGF ligands all declined after 4 d. Intraperitoneal injection of neutralizing antibodies or truncated soluble receptors to PDGF-BB reduced lung DNA synthesis in air. Exposure to 60% O(2) significantly increased mRNA for PDGF-B, -beta R, and -alpha R, but not PDGF-A, relative to air-exposed lung at various time points after birth. PDGF-A, -B, and -alpha R immunoreactivities in these lungs were reduced and delayed, consistent with a global inhibition of lung growth. Pups exposed to 60% O(2) had a similar distribution of PDGF-beta R to that seen in air, except that at 14 d PDGF-beta R was distributed throughout the lung parenchyma. We conclude that PDGF ligands and receptors are important for normal postnatal lung growth and that their expression is delayed by O(2) exposure.

Osteoblast differentiation and function can be studied in situ in the metaphysis of growing long bones. Proliferation and apoptosis dominate in the primary spongiosa subjacent to the growth plate, and differentiation and function dominate in the proximal metaphysis. Apoptosis of osteocytes dominates at the termination of the trabeculae in diaphyseal marrow. As parathyroid hormone regulates all phases of osteoblast development, we studied the in vivo regulation by human parathyroid hormone (1-34) (PTH) of apoptosis in bone cells of the distal metaphysis of young male rats. Rats were given PTH at 80 microg/kg per day, once daily, for 1-28 days. Bone cells were defined for flow cytometry as PTH1-receptor-positive (PTH1R(+)) and growth factor-receptor-positive (GFR(+)) cells. Apoptotic cells stained positive for either TdT-mediated dUTP-X nick end labeling (TUNEL) or annexin V (annV(+)) were detected by either flow cytometry or immunohistochemistry. Apoptosis was also assessed at the tissue level by RNAse protection and caspase enzyme activity assays. PTH increased apoptotic osteoblasts in the proliferating zone and apoptotic osteocytes in the terminal trabecular zone, by 40%-60% within 2-6 days of PTH treatment, but values became equivalent to controls after 21-28 days of treatment. This transient increase was confirmed in PTH1R(+), GFR(+) bone cells isolated by flow cytometry. There was no detectable change in the steady-state mRNA levels of selected apoptotic genes. Starting at 3 days, at the tissue level, PTH inhibited activity of caspases, which recognize the DEVD peptide substrate (caspases 2, 3, and/or 7), but not those caspases recognizing LEHD or YVAD peptide sequences. We speculate that the localized and tissue level effects of PTH on apoptosis can be explained on the basis of its anabolic effect on bone. The transient increase in apoptosis in the proliferating zone and terminal trabecular zone may be the result of the increased activation frequency and bone turnover seen with daily PTH treatment. As once-daily PTH increases the number of differentiated osteoblasts, and as these and hematopoietic marrow cells dominate metaphyseal tissue, inhibition of caspase activity may contribute to their prolonged survival, enabling extension of trabecular bone into the diaphyseal marrow to increase bone mass.

The rare benign extra-abdominal desmoid tumor is characterized by aggressive invasion of normal tissue. Treatment is complicated by its recurrence, invasiveness, and persistence. The etiology is unknown and the pathophysiology is obscure. Because of exuberant fibroblastic proliferation with collagenous tissue being the primary tissue component, this desmoid tumor has been compared with keloids arising from excessive scar formation in healing wounds. Numerous cytokines are associated with signaling for growth and maintenance of mesenchymal cells. Altered expression of these proteins is associated with many pathologic conditions. It has been proposed that the enhanced expression of platelet-derived growth factor and its receptor characterize desmoid tumors. We tested the hypothesis that the exuberant fibrosis of desmoid tumors may have resulted from the initiation of the cascade of molecular events producing increased expression of cytokines. We used immunohistochemical analysis of cytokines in desmoid tumors compared with keloids and skin to localize the expression of cytokines. The results showed localized increased expression of the cytokines epidermal growth factor, transforming growth factor-beta, tumor necrosis factor-alpha, vascular endothelial growth factor, interleukin-1beta, and interleukin-6 in the endothelial cells of blood vessels in the tumors. Production of tumor necrosis factor-alpha and interleukin-1beta in tumor tissue was increased, but we did not find increased expression of platelet-derived growth factor. We concluded that the increased expression of cytokines associated with angiogenesis usually found in wound healing and invasive tumors may contribute to the pathophysiology of the desmoid tumor.

Cell migration is a key event in many biological processes and depends on signals from both extracellular matrix and soluble motogenic factors. During atherosclerotic plaque development, vascular smooth muscle cells migrate from the tunica media to the intima through a basement membrane and interstitial collagenous matrix and proliferate to form a neointima. Matrix metalloproteinases have previously been implicated in neointimal formation and in this study smooth muscle cell adhesion and migration on degraded collagen have been evaluated. Vascular smooth muscle cells adhered to native intact collagen type I and to its first degradation by-product, 3/4 fragment (generated by collagenase-3 cleavage), unwound at 35 degrees C to mimic physiological conditions. PDGF-BB pre-treatment induced a fourfold stimulation of smooth muscle cell motility on the collagen 3/4 fragment whereas no increase in smooth muscle cell motility on collagen type I was observed. Cell migration on collagen type I was mediated by alpha2 integrin, whereas PDGF-BB-stimulated migration on the 3/4 collagen fragment was dependent on alphavbeta3 integrin. alphavbeta3 integrin was organised in clusters concentrated at the leading and trailing edges of the cells and was only expressed when cells were exposed to the 3/4 collagen fragment. Tyrphostin A9, an inhibitor of PDGF receptor-beta tyrosine kinase activity, resulted in complete abolition of migration of PDGF-BB treated cells on collagen type I and 3/4 fragment. These results strongly support the hypothesis that the cellular migratory response to soluble motogens can be regulated by proteolytic modification of the extracellular matrix.

Platelet-derived growth factor (PDGF)- A-deficient male mice were found to develop progressive reduction of testicular size, Leydig cells loss, and spermatogenic arrest. In normal mice, the PDGF-A and PDGF-Ralpha expression pattern showed positive cells in the seminiferous epithelium and in interstitial mesenchymal cells, respectively. The testicular defects seen in PDGF-A-/- mice, combined with the normal developmental expression of PDGF-A and PDGF-Ralpha, indicate that through an epithelial-mesenchymal signaling, the PDGF-A gene is essential for the development of the Leydig cell lineage. These findings suggest that PDGF-A may play a role in the cascade of genes involved in male gonad differentiation. The Leydig cell loss and the spermatogenic impairment in the mutant mice are reminiscent of cases of testicular failure in man.

The remarkable ability of the fetus to heal early gestation skin wounds without scarring remains poorly understood. Taking advantage of recent advances in signal transduction, the tyrosine phosphorylation patterns of fetal rat fibroblasts, representing the scarless cutaneous repair phenotype, and adult rat fibroblasts, representing scarforming phenotype, were examined whether there were inherent differences in cellular signaling. Specifically, correlation of the phosphorylation patterns with the expression levels of the signaling molecules that transmit information from the plasma membrane receptor to the nucleus was sought. By using three different cell lines of explanted fibroblasts from gestational day 13 fetal rat skin (n = 24) and 1-month-old postnatal adult rat skin (n = 3), immunoblotting was performed to compare tyrosine phosphorylation patterns. The results revealed five major protein bands of interest in fetal rat fibroblasts, but not in the adult rat fibroblasts. These phosphorylated protein bands are of interest because of their possible role in wound repair and may have the potential to regulate cellular responses to the extracellular matrix and their secondary signaling molecules. It was hypothesized that these bands represented receptor tyrosine kinases, epidermal growth factor receptor, and discoidin domain receptor 1, and their downstream adaptor protein Shc that binds receptor tyrosine kinases to transduce signals intracellularly. Furthermore, elevated expression of platelet-derived growth factor receptor-beta in adult compared with fetal fibroblasts was demonstrated, suggesting that decreased expression of certain growth factors may also be important for the scarless phenomenon to occur.