Pirfenidone (PD) is known for its antifibrotic effects in the bleomycin (BL) hamster model of lung fibrosis. We evaluated whether pretreatment of hamsters with PD could influence the effects of BL-induced overexpression of platelet-derived growth factor (PDGF)-A and PDGF-B genes and proteins in the same model of lung fibrosis. We demonstrate elevated levels of PDGF-A and PDGF-B mRNAs in bronchoalveolar lavage (BAL) cells from lungs of BL-treated compared with saline control hamsters by RT-PCR analysis. However, these levels were not altered in BAL cells obtained from BL-treated hamsters on diets containing 0.5% PD. Western blot analysis of BAL fluid for PDGF isoforms demonstrated that PD treatment inhibited the synthesis of both PDGF-A and PDGF-B isoforms. PD treatment also decreased the mitogenic activity in the BAL fluid from BL-treated hamster lungs. Taken together, these data provide evidence that the protective effects of PD against BL-induced lung fibrosis may be mediated by a reduction in PDGF isoforms produced by lung macrophages.

The SRY-box (Sox) transcription factors regulate oligodendrocyte differentiation, but their signaling targets are largely unknown. We have identified a major signal transduction pathway regulated by Sox containing gene 17 (Sox17) in the oligodendrocyte lineage. Microarray analysis in oligodendrocyte progenitor cells (OPCs) after Sox17 attenuation revealed upregulated genes associated with cell cycle control and activation of the Wingless and integration site (Wnt)/β-catenin pathway. Sox17 knockdown also increases the levels of cyclin D1, Axin2, and activated β-catenin. In OPCs, the expression pattern of Sox17, cyclin D1, and secreted Frizzled-related protein-1 in the presence of platelet-derived growth factor (PDGF) was coordinately accelerated by addition of thyroid hormone, indicating differentiation-induced regulation of Sox17 targets. In developing white matter, decreased total β-catenin, activated β-catenin, and cyclin D1 levels coincided with the peak of Sox17 expression, and immunoprecipitates showed a developmentally regulated interaction among Sox17, T-cell transcription factor 4, and β-catenin proteins. In OPCs, PDGF stimulated phosphorylation of glycogen synthase 3β and the Wnt coreceptor LRP6, and enhanced β-catenin-dependent gene expression. Sox17 overexpression inhibited PDGF-induced TOPFLASH and cyclin D1 promoter activity, and decreased endogenous cyclin D1, activated β-catenin, as well as total β-catenin levels. Recombinant Sox17 prevented Wnt3a from repressing myelin protein expression, and inhibition of Sox17-mediated proteasomal degradation of β-catenin blocked myelin protein induction. These results indicate that Sox17 suppresses cyclin D1 expression and cell proliferation by directly antagonizing β-catenin, whose activity in OPCs is stimulated not only by Wnt3a, but also by PDGF. Our identification of downstream targets of Sox17 thus defines signaling pathways and molecular mechanisms in OPCs that are regulated by Sox17 during cell cycle exit and the onset of differentiation in oligodendrocyte development.

OBJECTIVE

To investigate the progression of urodynamic changes, as well as histological and biochemical outcomes over a prolonged period of partial bladder outlet obstruction (pBOO) in an animal model with physiologically relevant pBOO.

METHODS

Healthy, adult, female Fischer rats underwent surgical creation of a pBOO for either 2, 4, 8, or 13 weeks and were compared with sham-operated rats. Urodynamic measurements were used to compare bladder volumes and pressure. Tissue was grossly analysed with light microscopy and bladder weights and thicknesses were compared. Reverse transcription-polymerase chain reaction for collagen, transforming growth factor β (TGF-β), connective tissue growth factor (CTGF), hypoxia inducible factor 1α (HIF-1α), and platelet-derived growth factor (PDGF-A) was performed on all samples, as well as immunohistochemistry (IHC) for α-smooth muscle actin (α-SMA). Finally, mass spectrometry was used to quantify the collagen content of the bladders as a measure of fibrosis.

RESULTS

After induction of pBOO, all rats remained healthy. Initial urodynamics showed an increase in capacity while maintaining normal pressures, but then deteriorated into small capacity, high-pressure bladders. Haematoxylin and eosin (H&E) staining showed an initial inflammatory response, and this was confirmed with significantly increased mRNA levels of TGF-β, CTGF, HIF-1α, and PDGF. The progression to smooth muscle hypertrophy was evident on H&E and confirmed with increased bladder mass and thickness. IHC for α-SMA showed a progressive increase associated with the elevated bladder pressures. Masson's trichrome and mass spectrometry showed a progressive increase in collagen to 13 weeks.

CONCLUSIONS

With this model, we have effectively replicated the clinical scenario, with significant pathophysiological changes occurring insidiously in otherwise healthy rats. We believe that our observed changes represent distinct phases of bladder decompensation; with an initial inflammatory response to the stress of the pBOO, smooth muscle hypertrophy to overcome the increased urethral resistance, and eventual decompensation to fibrosis. The time course of the inflammatory markers implies the need for early intervention to prevent this cascade. Novel strategies targeting these observed physiological responses could lead to improved preventative strategies, with respect to biochemical pathways and the time course of their initiation.

BACKGROUND

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The kinase inhibitor nintedanib specific for vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) significantly reduced the rate of decline of forced vital capacity versus placebo.

OBJECTIVE

To determine the in vitro effect of nintedanib on primary human lung fibroblasts.

METHODS

Fibroblasts were isolated from lungs of IPF patients and from non-fibrotic controls. We assessed the effect of VEGF, PDGF-BB and basic FGF (bFGF) ± nintedanib on: (i) expression/activation of VEGFR, PDGFR, and FGFR, (ii) cell proliferation, secretion of (iii) matrix metalloproteinases (MMP), (iv) tissue inhibitor of metalloproteinase (TIMP), and (v) collagen.

RESULTS

IPF fibroblasts expressed higher levels of PDGFR and FGFR than controls. PDGF-BB, bFGF, and VEGF caused a pro-proliferative effect which was prevented by nintedanib. Nintedanib enhanced the expression of pro-MMP-2, and inhibited the expression of TIMP-2. Transforming growth factor-beta-induced secretion of collagens was inhibited by nintedanib.

CONCLUSIONS

Our data demonstrate a significant anti-fibrotic effect of nintedanib in IPF fibroblasts. This effect consists of the drug's anti-proliferative capacity, and on its effect on the extracellular matrix, the degradation of which seems to be enhanced.

Growth factor-derived mitogenic signals from the cell surface are transmitted to the nucleus via receptor tyrosine kinases (RTKs), the adaptor proteins Shc and Grb2, and a Ras-dependent protein kinase cascade that activates the extracellular signal regulated kinase (ERK) subfamily of mitogen-activated protein kinases. ERKs also are activated by hormones that stimulate G protein-coupled receptors (GPCRs). We report here that, in agreement with previous data, the epidermal growth factor receptor (EGFR) is a signaling intermediate in ERK activation by GPCRs. Of import, we show that cross-talk between two classes of surface receptors, RTKs and GPCRs, is a general feature. Lysophosphatidic acid not only induces ligand-independent tyrosine autophosphorylation of EGFR but also of platelet-derived growth factor beta receptor (PDGF-beta-R) as shown by detection of tyrosine phosphorylation and by the use of specific inhibitors of RTKs. The cross-talk appears to be cell type-specific: In L cells that lack EGFR, lysophosphatidic acid-induced Shc and ERK activation is prevented completely by specific inhibition of PDGFR, whereas in COS-7 cells expressing only EGFR, the pathway via EGFR is chosen. In Rat-1 cells, however, that express both EGFR and PDGFR, the EGFR pathway dominates.

A key mechanism for mesenchymal stem cells/bone marrow stromal cells (MSCs) to promote tissue repair is by secretion of soluble growth factors (GFs). Therefore, clinical application could be optimized by a combination of cell and gene therapies, where MSCs are genetically modified to express higher levels of a specific factor. However, it remains unknown how this overexpression may alter the fate of the MSCs. Here, we show effects of overexpressing the growth factors, such as basic fibroblast growth factor (bFGF), platelet derived growth factor B (PDGF-BB), transforming growth factor β(1) (TGF-β(1) ), and vascular endothelial growth factor (VEGF), in human bone marrow-derived MSCs. Ectopic expression of bFGF or PDGF-B lead to highly proliferating MSCs and lead to a robust increase in osteogenesis. In contrast, adipogenesis was strongly inhibited in MSCs overexpressing PDGF-B and only mildly affected in MSCs overexpressing bFGF. Overexpression of TGF-β(1) blocked both osteogenic and adipogenic differentiation while inducing the formation of stress fibers and increasing the expression of the smooth muscle marker calponin-1 and the chondrogenic marker collagen type II. In contrast, MSCs overexpressing VEGF did not vary from control MSCs in any parameters, likely due to the lack of VEGF receptor expression on MSCs. MSCs engineered to overexpress VEGF strongly induced the migration of endothelial cells and enhanced blood flow restoration in a xenograft model of hind limb ischemia. These data support the rationale for genetically modifying MSCs to enhance their therapeutically relevant trophic signals, when safety and efficacy can be demonstrated, and when it can be shown that there are no unwanted effects on their proliferation and differentiation.

Platelet-derived growth factor (PDGF) isoforms lead to mitogenic, survival, and chemotactic responses in a variety of mesenchymal cell types during development and in the adult. We have studied the importance of phosphatidylinositol-3' kinase (PI3K) signaling in these responses by mutating the PI3K-binding sites in the PDGF-beta receptor by gene targeting in embryonic stem cells. Homozygous mutant mice developed normally; however, cells derived from the mutants were less chemotactic and had largely lost their ability to contract collagen gels in response to PDGF. Injection of a mast cell degranulating agent in mice led to a decrease in interstitial fluid pressure resulting in edema formation. In contrast to wild-type mice, mutant mice were unable to normalize the pressure after treatment with PDGF. Taken together, these observations suggest a function for PDGF signaling through PI3K in interstitial fluid homeostasis by modulating the tension between cells and extracellular matrix structures.

Glioblastomas represent the most aggressive glioma grade and are associated with a poor patient prognosis. The current standard of care, consisting of surgery, radiation and chemotherapy, only results in a median survival of 14 months, underscoring the importance of developing effective new therapeutic strategies. Among the challenges in treating glioblastomas are primary resistance and the rapid emergence of recurrent disease, which can result from tumor cell-intrinsic mechanisms in addition to tumor microenvironment (TME)-mediated extrinsic resistance. Using a PDGF-B-driven proneural glioma mouse model, we assessed a panel of tyrosine kinase inhibitors with different selectivity profiles. We found that PLX3397, an inhibitor of colony stimulating factor-1 receptor (CSF-1R), blocks glioma progression, markedly suppresses tumor cell proliferation and reduces tumor grade. By contrast, the multi-targeted tyrosine kinase inhibitors dovitinib and vatalanib, which directly target tumor cells, exert minimal anti-tumoral effects in vivo, despite killing glioma cells in vitro, suggesting a TME-mediated resistance mechanism may be involved. Interestingly, PLX3397 interferes with tumor-mediated education of macrophages and consequently restores the sensitivity of glioma cells to tyrosine kinase inhibitors in vivo in preclinical combination trials. Our findings thus demonstrate that microenvironmental alteration by CSF-1R blockade renders tumor cells more susceptible to receptor tyrosine kinase inhibition in a preclinical glioblastoma model, which may have important translational relevance.

Idiopathic pulmonary fibrosis (IPF) is characterized by accumulation of alveolar macrophages spontaneously releasing exaggerated amounts of the potent mesenchymal cell growth factor platelet-derived growth factor (PDGF). To evaluate the relative contribution of the two PDGF genes to this process, PDGF-A and -B gene transcription rates and mRNA levels were examined in normal and IPF alveolar macrophages. While normal alveolar macrophages constitutively transcribe both PDGF-A and PDGF-B genes, LPS stimulation increases the transcription of both genes more than threefold. Importantly, IPF alveolar macrophages spontaneously transcribe both genes at a rate similar to that observed for normal macrophages after in vitro stimulation. Consistent with the transcription data, normal macrophages contain mRNA for both PDGF-A and -B, but PDGF-B mRNA is 10-fold more abundant. Strikingly, in IPF, both PDGF-A and -B mRNA levels were markedly increased, with persistence of the 10-fold dominance of PDGF-B mRNA. Thus, the exaggerated release of PDGF by IPF alveolar macrophages is likely modulated by upregulated PDGF gene transcription rates and concomitantly increased mRNA levels and the persistent 10-fold excess of B greater than A PDGF mRNA suggests that the PDGF released by alveolar macrophages is likely mostly of the potent B-chain homodimeric form.

OBJECTIVE

Platelet-derived growth factor (PDGF) induces cellular proliferation and differentiation by activating intracellular signaling mechanisms via their cognate receptors. In previous studies, we demonstrated that human brain tumors such as meningiomas, astrocytomas, medulloblastomas, and ependymomas expressed the messenger ribonucleic acid for the PDGF subunits and their receptors. In the present study, we investigated the expression of the messenger ribonucleic acid PDGF A and B chains and the PDGF alpha and beta receptors in 17 cases of oligodendrogliomas.

METHODS

Measurements of messenger ribonucleic acid levels were obtained using radioactive complementary deoxyribonucleic acid probes. Protein expression was analyzed with specific antibodies.

RESULTS

Sixteen of 17 tumors expressed the PDGF A subunit and all the PDGF alpha receptors. Furthermore, all the tumors expressed PDGF B and PDGF beta receptor subunits.

CONCLUSIONS

The results of this study suggest that oligodendrogliomas may have an autocrine loop stimulated by the interaction of PDGF and its receptor simultaneously produced by these tumors.

Studies of platelet-derived growth factor (PDGF) receptor biosynthesis and degradation have been limited by the lack of anti-receptor antibodies. In this study, peptides based on the cDNA-predicted amino acid sequence of the PDGF receptor were used to produce antisera that specifically immunoprecipitated the receptor. PDGF receptor biosynthesis was examined by pulse-chase labeling of cultured fibroblasts with [35S]methionine followed by immunoprecipitation. In BALB/c 3T3 fibroblasts the receptor was synthesized as a 160-kDa precursor that was converted to a mature 180-kDa form within 30-45 min. Removal of high mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H treatment reduced the apparent molecular weight of the 160-kDa precursor but did not affect the migration of the 180-kDa mature receptor. When mannosidase II was inhibited by swainsonine, the 160-kDa precursor failed to mature; instead a 168-kDa form of the receptor was observed. Nevertheless, swainsonine-treated cells responded mitogenically to PDGF. The mature 180-kDa form of the receptor had a half-life of approximately 3 h in the absence of ligand. Addition of PDGF reduced the receptor half-life to 45 min. These studies define and characterize a PDGF receptor precursor, show that receptor degradation is enhanced by PDGF, and demonstrate the functional integrity of incompletely processed PDGF receptors.

BACKGROUND

Nanotechnology is a rapidly advancing industry with many new products already available to the public. Therefore, it is essential to gain an understanding of the possible health risks associated with exposure to nanomaterials and to identify biomarkers of exposure. In this study, we investigated the fibrogenic potential of SWCNT synthesized by chemical vapor deposition using cobalt (Co) and molybdenum (Mo) as catalysts. Following a single oropharyngeal aspiration of SWCNT in rats, we evaluated lung histopathology, cell proliferation, and growth factor mRNAs at 1 and 21 days post-exposure. Comparisons were made to vehicle alone (saline containing a biocompatible nonionic surfactant), inert carbon black (CB) nanoparticles, or vanadium pentoxide (V2O5) as a known inducer of fibrosis.

RESULTS

SWCNT or CB caused no overt inflammatory response at 1 or 21 days post-exposure as determined by histopathology and evaluation of cells (>95% macrophages) in bronchoalveolar lavage (BAL) fluid. However, SWCNT induced the formation of small, focal interstitial fibrotic lesions within the alveolar region of the lung at 21 days. A small fraction of alveolar macrophages harvested by BAL from the lungs of SWCNT-exposed rats at 21 days were bridged by unique intercellular carbon structures that extended into the cytoplasm of each macrophage. These "carbon bridge" structures between macrophages were also observed in situ in the lungs of SWCNT-exposed rats. No carbon bridges were observed in CB-exposed rats. SWCNT caused cell proliferation only at sites of fibrotic lesion formation as measured by bromodeoxyuridine uptake into alveolar cells. SWCNT increased platelet-derived growth factor (PDGF)-A, PDGF-B, and PDGF-C mRNA levels significantly at 1 day as measured by Taqman quantitative real-time RT-PCR. At 21 days, SWCNT did not increase any mRNAs evaluated, while V2O5 significantly increased mRNAs encoding PDGF-A, -B, and -C chains, PDGF-R alpha, osteopontin (OPN), connective tissue growth factor (CTGF), and transforming growth factor (TGF)-beta1.

CONCLUSIONS

Our findings indicate that SWCNT do not cause lung inflammation and yet induce the formation of small, focal interstitial fibrotic lesions in the alveolar region of the lungs of rats. Of greatest interest was the discovery of unique intercellular carbon structures composed of SWCNT that bridged lung macrophages. These "carbon bridges" offer a novel and easily identifiable biomarker of exposure.

Binding analyses using 125I-labeled platelet-derived growth factor (PDGF)-AA and PDGF-BB were used to identify a clonal human glioma cell line (U-343 MGa 31L) which expresses the A type but not the B type receptor for PDGF. The glioma cells were devoid of a B type receptor transcript, and immunoprecipitation with an antiserum raised against a B type receptor peptide rendered no signal. Similar analyses using human foreskin fibroblasts, which express both A and B type PDGF receptors, revealed a B type PDGF receptor-specific 5.5-kilobase pair mRNA in a Northern blot experiment, and 160,000 and 180,000 molecular weight components upon immunoprecipitation. A second antiserum, raised against purified porcine PDGF receptor preparations, was reactive with Mr 140,000 and 170,000 components in the U-343 MGa 31L cells, as well as in human fibroblasts. In addition, this antiserum precipitated the Mr 160,000 and 180,000 components from the fibroblasts. Exposure of cells to PDGF-AA, as well as to PDGF-BB, induced an increased rate of degradation of the Mr 170,000 component in the clonal glioma cells and in fibroblasts. The Mr 180,000 component in fibroblasts was degraded only when cells were exposed to PDGF-BB. This allowed the identification of the Mr 170,000 component as the cell surface expressed form of the A type receptor for PDGF. A structural relatedness between the A and B type PDGF receptors was furthermore indicated by similarities in peptide patterns, after limited proteolytic digestion.

The tyrosine kinase inhibitor STI571 inhibits BCR/ABL and induces hematologic remission in most patients with chronic myeloid leukemia. In addition to BCR/ABL, STI571 also inhibits v-Abl, TEL/ABL, the native platelet-derived growth factor (PDGF)beta receptor, and c-KIT, but it does not inhibit SRC family kinases, c-FMS, FLT3, the epidermal growth factor receptor, or multiple other tyrosine kinases. ARG is a widely expressed tyrosine kinase that shares substantial sequence identity with c-ABL in the kinase domain and cooperates with ABL to regulate neurulation in the developing mouse embryo. As described here, ARG has recently been implicated in the pathogenesis of leukemia as a fusion partner of TEL. A TEL/ARG fusion was constructed to determine whether ARG can be inhibited by STI571. When expressed in the factor-dependent murine hematopoietic cell line Ba/F3, the TEL/ARG protein was heavily phosphorylated on tyrosine, increased tyrosine phosphorylation of multiple cellular proteins, and induced factor-independent proliferation. The effects of STI571 on Ba/F3 cells transformed with BCR/ABL, TEL/ABL, TEL/PDGFbetaR, or TEL/ARG were then compared. STI571 inhibited tyrosine phosphorylation and cell growth of Ba/F3 cells expressing BCR/ABL, TEL/ABL, TEL/PDGFbetaR, and TEL/ARG with an IC(50) of approximately 0.5 microM in each case, but it had no effect on untransformed Ba/F3 cells growing in IL-3 or on Ba/F3 cells transformed by TEL/JAK2. Culture of TEL/ARG-transfected Ba/F3 cells with IL-3 completely prevented STI571-induced apoptosis in these cells, similar to what has been observed with BCR/ABL- or TEL/ABL-transformed cells. These results indicate that ARG is a target of the small molecule, tyrosine kinase inhibitor STI571.

The purpose of this review was to determine whether imatinib mesylate (STI571, Gleevec) has a role in the treatment of osteosarcoma. The expression of platelet-derived growth factor (PDGF) receptor and its ligand was examined in a panel of surgical specimens obtained from 54 osteosarcoma patients, and then the expression was compared with prognosis. The effects of imatinib mesylate on growth and molecular events in 10 patient-derived osteosarcoma cell cultures were investigated. Immunohistochemical studies demonstrated frequent expression of PDGF-AA (80.4%) and PDGF-alpha receptor (79.6%) and their correlation with inferior event-free survival (P < .05). PDGF-B-B and PDGF-beta-receptor expressions were also frequent (75.4% and 86%, respectively); however, statistically significant inferior event-free survival was not demonstrated (P = .15). In vitro studies demonstrated that imatinib mesylate had a variable cytotoxic effect on various osteosarcoma primary cultures, with an IC(50) of 5.6 microM to 9.5 microM, and blocked the PDGF-induced intracellular signal transduction as well as inhibition of downstream Akt phosphorylation. Mitogen-activated protein kinase (MAPK) was constitutively activated despite PDGF stimulation and imatinib mesylate treatment in 7 of 10 osteosarcoma cultures, perhaps explaining uncontrolled proliferation and relative unresponsiveness to imatinib. Imatinib mesylate could not be viewed as having a role as a single agent at current conventional doses for the treatment of osteosarcoma. These findings predicted activity in osteosarcoma clinical trials and suggested that in vitro model systems predict clinical behavior and that PDGF and its receptor expression could potentially be used for determining prognosis of osteosarcoma.

Blood platelets link the processes of haemostasis and inflammation. This study examined the immunomodulatory factors released by platelets after Toll-Like Receptor 4 (TLR4) engagement on their surfaces. Monoclonal anti-human FcgammaRII Ab (IV.3)-treated human platelets were cultured with TLR4 ligands in the presence or absence of blocking monoclonal antibody to human TLR4. The release of sCD62p, epidermal growth factor (EGF), transforming growth factor beta (TGFbeta), interleukin (IL)-8, platelet activating factor 4 (PAF4), platelet-derived growth factor, alpha, beta polypeptide (PDGF-AB), Angiogenin, RANTES (regulated upon activation, normal T-cell expressed, and presumably secreted) and sCD40L were measured by specific enzyme-linked immunosorbent assay. TLR4 ligand [Escherichia coli lipopolysaccharide (LPS)] bound platelet TLR4, which differentially modulates the release of cytokines by platelets. It was noted that (i) sCD62p, IL-8, EGF and TGFbeta release were each independent of platelet activation after TLR4 engagement; (ii) RANTES, Angiogenin and PDGF-AB concentration were weaker in platelet supernatant after TLR4 engagement; (iii) sCD40L and PAF4 are present in large concentration in the releaseate of platelets stimulated by TLR4 ligand. The effects of LPS from E. coli on the modulation of secretory factors were attenuated by preincubation of platelets with an anti-TLR4 monoclonal antibody, consistent with the immunomodulation being specifically mediated by the TLR4 receptor. We propose that platelets adapt the subsequent responses, with polarized cytokine secretion, after TLR4 involvement.

The expression of growth factors for mesenchymal cells was investigated in 10 different human mammary carcinoma cell lines. Expression of mRNA for platelet-derived growth factor (PDGF) A-chain, PDGF B-chain, transforming growth factor-alpha, and insulin-like growth factor II were found in eight, nine, five, and two of the cell lines, respectively. The production of PDGF-like growth factors by the mammary carcinoma cell lines was investigated in detail. PDGF receptor competing activity and mitogenic activity, which could be neutralized by PDGF antibodies, were found in the conditioned medium of almost all the cell lines. A PDGF-like growth factor was partially purified from the conditioned medium of one of the cell lines, MCF7. A Mr 31,000 component, which was reduced to Mr 17,000, was furthermore precipitated by a PDGF antiserum from the conditioned medium of metabolically labeled MCF7 cells. These results indicate that growth factors for mesenchymal cells are frequently expressed in human mammary carcinoma cell lines. This is of interest in relation to the fact that mammary carcinoma tumors often contain a high proportion of proliferating connective tissue cells.

Recent work has indicated that oxygen-sensing mechanism(s) resembling those controlling erythropoietin production operate in many non-erythropoietin-producing cells. To pursue the implication that such a system might control other genes, we studied oxygen-regulated expression of mRNAs for vascular endothelial growth factor, platelet-derived growth factor (PDGF) A and B chains, placental growth factor (PLGF), and transforming growth factor in four different cell lines and compared the characteristics with those of erythropoietin regulation. Oxygen-regulated expression was demonstrated for each gene in at least one cell type. However, the response to hypoxia (1% oxygen) varied markedly, ranging from a 13-fold increase (PDGF-B in Hep G2 cells) to a 2-fold decrease (PLGF in the trophoblastic line BeWo). For each gene/cell combination, both the magnitude and direction of the response to hypoxia were mimicked by exposure to cobaltous ions or two different iron-chelating agents, desferrioxamine and hydroxypyridinones. These similarities with established characteristics of erythropoietin regulation indicate that a similar mechanism of oxygen sensing is operating on a variety of vascular growth factors, and they suggest that chelatable iron is closely involved in the mechanism.

OBJECTIVE

We have previously shown the presence of an activated platelet-derived growth factor (PDGF) receptor (PDGFR) B and its ligand PDGFB in a limited number of patients with clinical and radiological responses to imatinib mesylate treatment. This article describes the results of comprehensive molecular/biochemical analyses of the three receptors targeted by the drug (PDGFRB, PDGFRA, and KIT) in a series of 31 chordoma patients.

METHODS

The presence and activation status of PDGFRB, PDGFRA, and KIT receptors were investigated by means of immunoprecipitation and Western blot analyses complemented by immunohistochemistry, their expression level was analyzed by means of real-time PCR, and the occurrence of activating point mutations was investigated by means of cDNA sequencing. The PDGFB, PDGFA, and stem cell factor cognate ligands were investigated by reverse transcription-PCR, and gene status was assessed by fluorescence in situ hybridization.

RESULTS

The results show that PDGFRB was highly expressed and phosphorylated, whereas PDGFRA and KIT were less expressed but phosphorylated and thus activated. These findings, together with the absence of gain-of-function mutations and the presence of the cognate ligands, strongly support the hypothesis that the activation mechanism is the autocrine/paracrine loop. No role seems to be played by gene amplification.

CONCLUSIONS

In the light of our findings, the clinical benefit observed in chordoma patients treated with imatinib seems to be attributable to the switching off of all three receptors.

OBJECTIVE

To compare the mRNA expression of growth factor receptors in cultured human trabecular meshwork (HTM) cells with ex vivo HTM tissues and to determine whether HTM cells generate a physiologic response after exposure to exogenous growth factors.

METHODS

The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of various growth factor receptor mRNAs using early passaged, cultured HTM cells from donors of several ages. RT-PCR on ex vivo HTM tissues from healthy donors and donors with glaucoma were also used to compare and contrast mRNA expression with cell culture results. After the exogenous administration of growth factors, cell proliferation and extracellular acidification rate studies were used to measure the functional responses of HTM cells to growth factors.

RESULTS

Amplification products of the expected size for 15 growth factor receptors were detected in cultured HTM cells and in ex vivo HTM tissues. The administration of exogenous growth factors showed that (a) hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulinlike growth factor (IGF)-1, tumor necrosis factor (TNF) alpha, platelet-derived growth factor (<em>PDGF</em>)-AA, <em>PDGF</em>-BB, <em>PDGF</em>-AB, and <em>b</em>asic fi<em>b</em>ro<em>b</em>last growth factor (FGF-2) stimulated cell proliferation, whereas FGF-1 (acidic), transforming growth factor (TGF) alpha, interleukin (IL)-1alpha, nerve growth factor (NGF), and FGF-7 (keratinocyte growth factor [KGF]) had no significant influence on cell proliferation; (<em>b</em>) TGF-<em>beta</em> isoforms significantly inhi<em>b</em>ited EGF-stimulated tra<em>b</em>ecular meshwork cell proliferation; and (c) FGF-1 (acidic), TGF-alpha, EGF, IL-1alpha, IL-1<em>beta</em>, HGF, TNF-alpha, <em>PDGF</em>-AA, and IGF-1 significantly stimulated extracellular acidification, whereas FGF-2 (<em>b</em>asic), FGF-7 (KGF), TGF-<em>beta</em>1-<em>beta</em>3 and NGF had no significant influence on extracellular acidification.

CONCLUSIONS

These studies show that mRNA for numerous growth factor receptors can be detected in cultured HTM cells and in ex vivo HTM tissues. They also show that many of the receptors are functional, because exogenous growth factor administration elicits a physiologic response. In vivo, these receptors may be activated by growth factors present within the aqueous humor (aquecrine/paracrine) or by growth factors synthesized and released locally by trabecular meshwork cells themselves (autocrine). Specific growth factors acting through high-affinity receptors may be involved in maintaining the normal microenvironment of the HTM and also may be involved in the pathogenesis of primary open-angle glaucoma.

Fluid shear stress has been shown to be an important regulator of vascular structure and function through its effect on the endothelial cell. We have explored the effect of shear stress on the expression of the heparin-binding growth factors platelet-derived growth factor B chain (PDGF-B) and basic fibroblast growth factor (bFGF) in bovine aortic endothelial cells using a purpose-built cone-plate viscometer. Using morphometric analysis, we have mimicked the endothelial cell shape changes encountered in vivo in response to shear stress and correlated these with changes in gene expression. Steady laminar shear stress of 15 and 36 dyn/cm2 both resulted in endothelial cell shape change, but the higher shear stress induced greater and more uniform alignment in the direction of flow and nuclear protrusion after 24 h. Steady laminar shear stress of both 15 and 36 dyn/cm2 induced a significant 3.9- and 4.2-fold decrease, respectively, in PDGF-B mRNA at 9 h. In contrast, steady laminar shear of 15 dyn/cm2 induced a mild and transient 1.5-fold increase in bFGF mRNA while shear of 36 dyn/cm2 induced a significant 4.8-fold increase at 6 h of shear which remained at 2.9-fold at 9 h. Pulsatile and turbulent shear stress showed the same effect as steady laminar shear stress (all at 15 dyn/cm2 time-average magnitude) on PDGF-B and bFGF mRNA content. Cyclic stretch (20% strain, 20/min) of cells grown on silicone substrate did not significantly affect either PDGF-B or bFGF mRNA levels. These results suggest that expression of each peptide growth factor gene is differentially regulated by fluid shear stress in the vascular endothelial cell. These results may have implications on vascular structure and function in response to hemodynamic forces and present a model for the study of transduction of mechanical stimuli into altered gene expression.

Endothelial Wnt/β-catenin signaling is necessary for angiogenesis of the central nervous system and blood-brain barrier (BBB) differentiation, but its relevance for glioma vascularization is unknown. In this study, we show that doxycycline-dependent Wnt1 expression in subcutaneous and intracranial mouse glioma models induced endothelial Wnt/β-catenin signaling and led to diminished tumor growth, reduced vascular density, and normalized vessels with increased mural cell attachment. These findings were corroborated in GL261 glioma cells intracranially transplanted in mice expressing dominant-active β-catenin specifically in the endothelium. Enforced endothelial β-catenin signaling restored BBB characteristics, whereas inhibition by Dkk1 (Dickkopf-1) had opposing effects. By overactivating the Wnt pathway, we induced the Wnt/β-catenin-Dll4/Notch signaling cascade in tumor endothelia, blocking an angiogenic and favoring a quiescent vascular phenotype, indicated by induction of stalk cell genes. We show that β-catenin transcriptional activity directly regulated endothelial expression of platelet-derived growth factor B (PDGF-B), leading to mural cell recruitment thereby contributing to vascular quiescence and barrier function. We propose that reinforced Wnt/β-catenin signaling leads to inhibition of angiogenesis with normalized and less permeable vessels, which might prove to be a valuable therapeutic target for antiangiogenic and edema glioma therapy.

The purpose of this study was to evaluate the vasoformative response of isolated vascular explants to a variety of growth factors that have been shown to stimulate angiogenesis. Rings of rat aorta were cultured in collagen gels under serum-free conditions in the presence or absence of vascular endothelial growth factor (VEGF), natural platelet-derived growth factor (PDGF), PDGF-AA, PDGF-BB, insulin-like growth factor-1 (IGF-1), transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta 1 (TGF-beta 1), epidermal growth factor (EGF), interleukin-1 alpha (IL-1 alpha), or hepatocyte growth factor (HGF). The angiogenic response of the rat aorta was stimulated by VEGF, PDGF, PDGF-AA, PDGF-BB, and IGF-1. Maximum stimulatory effects were obtained with VEGF and PDGF-BB. By contrast, TGF-beta 1 and IL-1 alpha had inhibitory activity. No significant effects were observed with TGF-alpha, EGF, or HGF. The vascular outgrowth of VEGF-stimulated cultures was primarily composed of microvessels, whereas that of PDGF- and IGF-1-stimulated cultures contained an increased number of fibroblast-like cells. The inability of TGF-alpha, TGF-beta 1, IL-1 alpha, EGF, and HGF to stimulate rat aortic angiogenesis in serum-free culture suggests that either these factors require the mediatory activity of accessory cells that are not present in the rat aorta model or that blood vessels are heterogeneous in their capacity to respond to different angiogenic factors.

Polypeptide growth factors are a class of potent natural biologic mediators which regulate many of the activities of wound healing including cell proliferation, migration, and metabolism. Platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I) have been shown to regulate DNA and protein synthesis in bone cells in vitro and to interact synergistically to enhance soft tissue wound healing in vivo. We have hypothesized that the combination of PDGF and IGF-I may, therefore, enhance regeneration of both the soft and hard tissue components of the periodontium. To test this hypothesis we performed conventional periodontal surgery on all 4 quadrants of the mouth of 13 beagle dogs with naturally occurring periodontal disease. Following flap reflection, degranulation, and root planing, all premolar teeth in 2 quadrants of each dog received a combination of 3 micrograms of recombinant PDGF-B and IGF-I in a methylcellulose gel, while the premolar teeth in the contralateral quadrants received the gel alone. Teeth in 4 additional animals also received 125I-PDGF or 125I-IGF-I in the treated sites. The clearance rate of the 125I-labeled protein, changes in local bone metabolism, and amount of new bone and cementum with inserting collagen fibers were measured. The clearance studies revealed that the half-life of the factors at the site of application was 3.0 hours for IGF-I and to 4.2 hours for PDGF-B. Greater than 96% of the radio-labeled proteins was cleared by 96 hours and no radioactivity was detected 2 weeks after application. There was a significant (P less than 0.01) 2-fold increase in uptake of the bone-seeking radiopharmaceutical Technetium 99-MDP at 2 and 4 weeks in growth factor treated sites compared to controls, indicating that there was increased metabolic activity within the bone at these sites. Computer-aided histologic analyses of biopsies obtained at 2 and 5 weeks post-operatively revealed a significant (P less than 0.01), 5 to 10 fold increase in new bone and cementum in PDGF-B/IGF-I treated sites at both time points compared to controls receiving the placebo gel. The height and total area of new bone continued to increase from 2 to 5 weeks. The new bone underwent a normal maturation process as judged by histologic appearance. A physiologic periodontal ligament space was also formed between the new bone and new cementum. There was no increase in ankylosis in the treated sites.(ABSTRACT TRUNCATED AT 400 WORDS)