Glioblastoma multiforme, the most common form of malignant brain tumor,is resistant to all forms of therapy and causes death within 9-12 months of diagnosis. Glioblastomas are known to contain numerous genetic and physiological alterations affecting cell survival and proliferation; one of the most common alterations being platelet-derived growth factor (PDGF) autocrine signaling characterized by coexpression of PDGF and its receptor. The PDGF family consists of four members, PDGF-A, -B, -C, and -D, that signal through the alpha and beta PDGF receptor (PDGFR) tyrosine kinases. Numerous studies have demonstrated expression of PDGF-A, PDGF-B, and the PDGFRs in gliomablastomas, but such studies have not been conducted for the newly identified PDGF-C and -D. Therefore, we examined the expression of all PDGF ligands and receptors in 11 glioma cell lines and 5 primary glioblastoma tumor tissues by quantitative reverse transcription-PCR. Expression of PDGF/PDGFR pairs that are known to functionally interact were identified in all of the samples. Interestingly, PDGF-C expression was ubiquitous in brain tumor cells and tissues but was very low or absent in normal adult and fetal brain. PDGF-D was expressed in 10 of 11 brain tumor cell lines and 3 of 5 primary brain tumor samples. As a strategy for blocking PDGFR signaling, CT52923, a potent selective small molecule piperazinyl quinazoline kinase inhibitor of the PDGFR, was identified. In model systems using NIH/3T3 cells, CT52923 blocked PDGF autocrine-mediated phosphorylation of PDGFR, Akt, and mitogen-activated protein kinase (MAPK), while having no effect on v-fms or V12-ras-mediated Akt or extracellular signal-regulated protein kinase (Erk) phosphorylation. More importantly, p.o. administration of CT52923 to nude mice caused a significant 61% reduction (P < 0.006) in tumor growth of NIH/3T3 cells transformed by PDGF, whereas tumor formation by cells expressing v-fms was unaffected. We next characterized PDGF autocrine signaling in five glioblastoma cell lines. In all of the cases, PDGF autocrine signaling was evident because treatment with 1-10 microM CT52923 inhibited PDGFR autophosphorylation when present at a detectable level and blocked downstream Akt and/or Erk phosphorylation. The functional significance of PDGF autocrine signaling in these cells was demonstrated by the fact that the CT52923 inhibited soft agar colony formation, and, when given p.o. to nude mice, it effectively reduced tumor formation by 44% (P < 0.0019) after s.c. injection of CPDGF-C and -D in brain tumor formation and confirms the existence of autocrine signaling by PDGF-A and -B. More importantly, treatment with the PDGFR antagonist CT52923 inhibited survival and/or mitogenic pathways in all of the glioblastoma cell lines tested and prevented glioma formation in a nude mouse xenograft model. Together these findings demonstrate the potential therapeutic utility of this class of compounds for the treatment of glioblastoma.

Members of the platelet-derived growth factor (PDGF) ligand family are known to play important roles in wound healing and fibrotic disease. We show that both transient and stable expression of PDGF-C results in the development of liver fibrosis consisting of the deposition of collagen in a pericellular and perivenular pattern that resembles human alcoholic and nonalcoholic fatty liver disease. Fibrosis in PDGF-C transgenic mice, as demonstrated by staining and hydroxyproline content, is preceded by activation and proliferation of hepatic stellate cells, as shown by collagen, alpha-smooth muscle actin and glial fibrillary acidic protein staining and between 8 and 12 months of age is followed by the development of liver adenomas and hepatocellular carcinomas. The hepatic expression of a number of known profibrotic genes, including type beta1 TGF, PDGF receptors alpha and beta, and tissue inhibitors of matrix metalloproteinases-1 and -2, increased by 4 weeks of age. Increased PDGF receptor alpha and beta protein levels were associated with activation of extracellular regulated kinase-1 and -2 and protein kinase B. At 9 months of age, PDGF-C transgenic mice had enlarged livers associated with increased fibrosis, steatosis, cell dysplasia, and hepatocellular carcinomas. These studies indicate that hepatic expression of PDGF-C induces a number of profibrotic pathways, suggesting that this growth factor may act as an initiator of fibrosis. Moreover, PDGF-C transgenic mice represent a unique model for the study of hepatic fibrosis progressing to tumorigenesis.

OBJECTIVE

Vascular endothelial growth factor (VEGF) is upregulated by hypoxia and is a major stimulatory factor for retinal neovascularization in ischemic retinopathies such as diabetic retinopathy. This study sought to determine if VEGF is a stimulatory factor in a murine model of choroidal neovascularization (CNV).

METHODS

Mice with laser-induced ruptures in Bruch's membrane were treated with vehicle alone; a drug that inhibits both VEGF and platelet-derived growth factor (PDGF) receptor kinases; a drug that inhibits PDGF, but not VEGF receptor kinase; or genistein, a nonspecific kinase inhibitor. After two weeks, CNV was quantified and compared.

RESULTS

Blockade of phosphorylation by VEGF and PDGF receptors caused dramatic, almost complete inhibition of CNV. Genistein also had an inhibitory effect, but less so than the VEGF/PDGF receptor blocker. Blockade of phosphorylation by PDGF receptors, but not VEGF receptors, had no significant effect on CNV.

CONCLUSIONS

These data and our previous study, which demonstrated that a kinase inhibitor that blocks VEGF and PDGF receptors and several isoforms of protein kinase C causing dramatic inhibition of CNV, suggest that VEGF signaling plays a critical role in the development of CNV in this model. If safety is established, the effect of inhibiting VEGF receptor kinase activity should be investigated in patients with CNV.

Treatment of quiescent cultures of mouse embryo-derived AKR-2B cells with transforming growth factor beta resulted in an early induction of c-sis mRNA. The increase in c-sis mRNA was followed by a corresponding increase in protein similar to platelet-derived growth factor (PDGF) in the culture medium. In addition, PDGF-regulated genes (c-fos and c-myc) were stimulated by transforming growth factor beta with delayed kinetics relative to that seen in other cell systems with direct PDGF stimulation. A model is proposed in which the monolayer mitogenicity of transforming growth factor beta is mediated by the induction of c-sis and PDGF and the subsequent autocrine stimulation of c-fos, c-myc, and other PDGF-inducible genes.

In Chinese hamster embryo fibroblasts (IIC9 cells), platelet-derived growth factor (PDGF) stimulated mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAP kinase/ERK) activity, but not that of c-jun N-terminal kinase (JNK), and induced G1 phase progression. ERK1 activation was biphasic and was sustained throughout the G1 phase of the cell cycle. PDGF induced cyclin D1 protein and mRNA levels in a time-dependent manner. Inhibition of PDGF-induced ERK1 activity by the addition of a selective inhibitor of MEK1 (MAP kinase kinase/ERK kinase 1) activation, PD98059, or transfection with a dominant-negative ERK1 (dnERK-) was correlated with growth arrest. In contrast, growth was unaffected by expression of dominant-negative JNK (dnJNK-). Interestingly, addition of PD98059 or dnERK-, but not dnJNK-, resulted in a dramatic decrease in cyclin D1 protein and mRNA levels, concomitant with a decrease in cyclin D1-cyclin-dependent kinase activity. To investigate the importance of sustained ERK1 activation, ERK1 activity was blocked by the addition of PD98059 throughout G1. Addition of PD98059 up to 4 h after PDGF treatment decreased ERK1 activity to the levels found in growth-arrested IIC9 cells. Loss of cyclin D1 mRNA and protein expression was observed within 1 h after inhibition of the second sustained phase of ERK1 activity. Disruption of sustained ERK1 activity also resulted in G1 growth arrest. These data provide evidence for a role for sustained ERK activity in controlling G1 progression through positive regulation of the continued expression of cyclin D1, a protein known to positively regulate G1 progression.

Capacitative Ca(2+) entry (CCE) through store-operated Ca(2+) (SOC) channels plays an important role in returning Ca(2+) to the sarcoplasmic reticulum (SR) and regulating cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)). A rise in [Ca(2+)](cyt) and sufficient Ca(2+) in the SR are required for pulmonary artery smooth muscle cell (PASMC) proliferation. We tested the hypothesis that platelet-derived growth factor (PDGF)-mediated PASMC growth involves upregulation of c-Jun and TRPC6, a transient receptor potential cation channel. In rat PASMC, PDGF (10 ng/ml for 0.5-48 h) phosphorylated signal transducer and activator of transcription (STAT3), increased mRNA and protein levels of c-Jun, and stimulated cell proliferation. PDGF treatment also upregulated TRPC6 expression and augmented CCE, elicited by passive depletion of Ca(2+) from the SR using cyclopiazonic acid. Furthermore, overexpression of c-Jun stimulated TRPC6 expression and CCE amplitude in PASMC. Downregulation of TRPC6 using an antisense oligonucleotide specifically for human TRPC6 decreased CCE and inhibited PDGF-mediated PASMC proliferation. These results suggest that PDGF-mediated PASMC proliferation is associated with c-Jun/STAT3-induced upregulation of TRPC6 expression. The resultant increase in CCE raises [Ca(2+)](cyt), facilitates return of Ca(2+) to the SR, and enhances PASMC growth.

Phospholipase C-gamma (PLC-gamma) and GTPase activating protein (GAP) are substrates of EGF, PDGF and other growth factor receptors. Since either PLC-gamma or GAP also bind to the activated receptors it was suggested that their SH2 domains are mediating this association. We attempted to delineate the specific region of the EGF receptor that is responsible for the binding, utilizing EGF receptor mutants, PLC-gamma, and a bacterially expressed TRP E fusion protein containing the SH2 domains of GAP. As previously shown, tyrosine autophosphorylation of the wild-type receptor wsa crucial in mediating the association and in agreement, a kinase negative EGF receptor could bind PLC-gamma or TRP E GAP SH2, but only when cross tyrosine phosphorylated by an active EGF receptor kinase. The importance of autophosphorylation for association was confirmed by demonstrating that a carboxy-terminal deletion of the EGFR missing four autophosphorylation sites bound these proteins poorly. To study the role of EGF receptor autophosphorylation further, a 203 amino acid EGF receptor fragment was generated with cyanogen bromide that contained all known tyrosine autophosphorylation sites. This fragment bound both TRP E GAP SH2 and PLC-gamma but only when tyrosine phosphorylated. This data localizes a major binding site for SH2 domain containing proteins to the carboxy-terminus of the EGF receptor and points to the importance of tyrosine phosphorylation in mediating this association.

The platelet-derived growth factor (PDGF) binds specifically to high-affinity receptors on the surface of bovine aortic smooth muscle cells and 3T3 cells. Conditioned medium from cultured bovine aortic endothelial cells (EC) prevents PDGF binding to these receptors in a dose-dependent manner at 4 degrees C. The (125)I-labeled PDGF that is displaced by the conditioned medium shows no increase in trichloroacetic acid solubility or decrease in binding capability to fresh cells. The competitor activity was identified as a protein by ammonium sulfate precipitability and sensitivity to trypsin. The competitor protein also is found in the serum-free conditioned media from porcine aortic EC and human umbilical vein EC but not in media from bovine aortic smooth muscle cells, human neonatal foreskin fibroblasts, or the interleukin-producing thyoma cell line EL-4. The competitor protein, like PDGF, has no effect on the specific 4 degrees C binding of either (125)I-labeled insulin to 3T3 cells or (125)I-labeled epidermal growth factor to human epidermoid A431 cells. Saturation curves of PDGF binding to smooth muscle cells that had been preincubated in the presence and absence of competitor indicate that the concentration for half-maximal binding of (125)I-labeled PDGF to its receptor ( approximately 30 pM) is unchanged by the competitor, whereas the apparent number of available receptor sites or maximal level of binding is greatly diminished. The competitor activity produced by cultured human umbilical vein EC is completely inhibited by antiserum against pure human PDGF, whereas the same PDGF antiserum only partially inhibits the mitogenic activity of the conditioned media. In addition, approximately 7-fold more crude endothelium-derived growth factor is required for half-maximal inhibition of (125)I-labeled PDGF binding as is required for half-maximal stimulation of DNA synthesis. These results suggest that EC secrete a PDGF-like protein that is biochemically distinct from the majority of EC-derived mitogenic activity.

Chemotaxis is an important component of wound healing, development, immunity and metastasis, yet the signalling pathways that mediate chemotaxis are poorly understood. Platelet-derived growth factor (PDGF) acts both as a mitogen and a chemoattractant. Upon stimulation, the tyrosine kinase PDGF receptor-beta (PDGFR-beta) autophosphorylates and forms a complex that includes SII2(Src homology 2)-domain-containing proteins such as the phosphatidylinositol-specific phospholipase C-gamma, Ras-GTPase-activating protein (GAP), and phosphatidylinositol-3-OH kinase. Specific tyrosine-to-phenylalanine substitutions in the PDGFR-beta can prevent binding of one SH2-domain-containing protein without affecting binding of other receptor-associated proteins. Here we use phospholipase C-gamma and PDGFR-beta mutants to map specific tyrosines involved in both positive and negative regulation of chemotaxis towards the PDGF-BB homodimer. Our results indicate that a delicate balance of migration-promoting (phospholipase C-gamma and phosphatidylinositol-3-OH kinase) and migration-suppressing (GAP) activities are recruited by the PDGFR-beta to drive chemotaxis towards PDGF-BB.

The classic pathway for agonist-induced generation of diacylglycerol is via activation of a phospholipase C-mediated hydrolysis of the "phosphoinositides." We now report findings from a variety of cell types, which indicate that tumor-promoting phorbol diesters, serum, and platelet-derived growth factor activate within seconds the hydrolysis of phosphatidylcholine, as detected by the formation of diacylglycerol and phosphocholine. It is known that phorbol diesters do not stimulate hydrolysis of the phosphoinositides. Yet, in cells prelabeled with either [14C]oleate or [32P]orthophosphate, addition of the tumor promoter phorbol dibutyrate (PBt2) resulted in the rapid generation of both diacylglycerol and phosphatidate in a time- and dose-dependent manner. The fatty acid composition of the phosphatidate most resembled the fatty acid profile of phosphatidylcholine from the same cell type. Taken together, these findings suggested a role for protein kinase C in the generation of diacylglycerol (and phosphatidate) from phosphatidylcholine. To define further the pathways involved, the metabolism of cellular phosphatidylcholine was studied. In cells prelabeled with [3H]choline, addition of PBt2, but not 4 alpha-phorbol, stimulated the formation of intracellular phosphocholine within 45 sec. Furthermore, addition of platelet-derived growth factor (PDGF) or serum to "serum-starved" cells prelabeled with [3H]choline resulted in increased levels of intracellular phosphocholine within 15-30 sec. Thus, the data suggest that agonists that stimulate protein kinase C either directly (e.g., PBt2) or indirectly via activation of phosphoinositide hydrolysis (e.g., PDGF and serum) may stimulate degradation of phosphatidylcholine by phospholipase C in intact cells. However, prior down-regulation of protein kinase C by prolonged pretreatment of cells with PBt2 almost totally abolished subsequent stimulation of phosphatidylcholine degradation by PBt2 but only partially attenuated subsequent stimulation by PDGF and serum. These observations suggest that PDGF and serum act, at least partially, through a protein kinase C-independent mechanism. Lastly, the size of the cellular choline and CDP-choline pools were shown to be small and relatively insensitive to agonist addition, as compared to the size and behavior of the phosphocholine pool. Thus, the rapidly increased levels of phosphocholine (and diacylglycerol) arising in response to agonist addition appear to be derived directly from phosphatidylcholine by a phospholipase C-mediated mechanism.

Platelet-derived growth factor (PDGF), as purified from fresh human platelets, is a protein of relative molecular mass (Mr) 30,000 composed of two disulphide-linked subunit chains of similar size, named A and B (ref. 1). The dimer structure of PDGRF seems to be important for its biological effects, as reduction irreversibly inactivates the factor; it is not known, however, whether PDGF exists as a heterodimer or as a mixture of homodimers. Amino-acid sequence analysis has revealed that the A- and B-chains of human PDGF are related to each other, and that the B-chain is almost identical to part of the v-sis gene product of simian sarcoma virus (SSV). There is experimental evidence that a PDGF-like protein is indeed operational in SSV-induced transformation and the biologically active v-sis product is probably structurally similar to a putative dimer of PDGF B-chains. PDGF-like growth factors and/or a 4.2-kilobase (kb) c-sis transcript are present in several transformed mammalian cell lines and in certain nontransformed cells; cloned c-sis complementary DNA from human T cells transformed with human T-lymphotropic virus (HTLV) or from human endothelial cells contains the coding sequence for a putative PDGF B-chain precursor, but apparently lacks PDGF A-chain sequences. We have previously partially purified and characterized a PDGF-like growth factor from U-2 OS cells (osteosarcoma-derived growth factor, ODGF) and shown that this factor has structural, functional and immunological characteristics in common with PDGF. We describe here a procedure for the preparation of homogeneous ODGF, and provide evidence that this factor, which binds to the PDGF receptor, has a structure similar to a homodimer of PDGF A-chains.

Formal proof for an involvement of autocrine stimulation in the disturbed growth of malignant cells has been difficult to obtain, in part due to lack of precise methods of assessing growth factor production and receptor occurrence. In this study we have analyzed the mRNA levels for two growth factors and the corresponding receptors in a number of established human malignant glioma cell lines. Twenty-one tested lines all contained transcripts for the platelet-derived growth factor (PDGF) A chain while 16-17 of 21 expressed the c-sis/PDGF B chain gene; these two genes were expressed independently of each other. PDGF receptor transcripts were present in 15-16 of the 21 lines. Transcripts for the epidermal growth factor receptor were found in all 15 tested lines, in 2 of them at high levels, and the corresponding ligand transforming growth factor-alpha was found in 11 of 15 lines. No amplification or structural rearrangements of the genes, as analyzed by Southern blot hybridization, could explain the varying expression of PDGF A and B chain transcripts or the elevated levels of epidermal growth factor receptor mRNA. A correlation was found between cell morphology and expression of growth factor and receptor mRNA in these lines. The highest amount of PDGF receptor transcripts was found in cells with fibroblast-like morphology, and c-sis/B chain transcripts were found in small cell types and in cells with astrocyte-like morphology, while no clear relationship was found between PDGF receptor and A chain transcript levels or between morphology and A chain transcripts. It is possible that the findings reflect a coordinated expression of these genes in the progenitor cells. In conclusion, the data imply the existence of two possible autocrine loops in human malignant glioma lines, affecting the PDGF and epidermal growth factor receptor pathways.

Swiss 3T3 cells incubated for 60 h with [3H]inositol incorporated radioactivity into phosphatidylinositol (PI) and the two polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). On stimulation with platelet-derived growth factor (PDGF) there were significant increases in the levels of inositol 1-phosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). The effect of PDGF and IP3 on Ca2+ mobilization was studied in both intact cells and in 'leaky' cells that had been permeabilized with saponin. In intact cells, PDGF stimulated the efflux of 45Ca2+, whereas IP3 had no effect. Conversely, IP3 stimulated 45Ca2+ efflux from 'leaky' cells, which were insensitive to PDGF. 'Leaky' cells, which accumulated 45Ca2+ to a steady state within 20 min, were found to release approx. 40% of the label within 1 min after addition of 10 microM-IP3. This stimulation of 45Ca2+ release by IP3 was reversible and was also dose-dependent, with a half-maximal effect at approx. 0.3 microM. It seems likely that an important action of PDGF on Swiss 3T3 cells is to stimulate the hydrolysis of PIP2 to form IP3 and diacylglycerol, both of which may function as second messengers. Our results indicate that IP3 mobilizes intracellular Ca2+, and we propose that diacylglycerol may act through C-kinase to activate the Na+/H+ antiport. By generating two second messengers, PDGF can simultaneously elevate the intracellular level of Ca2+ and alkalinize the cytoplasm by lowering the level of H+.

The presence and biological importance of DNA secondary structures in eukaryotic promoters are becoming increasingly recognized among chemists and biologists as bioinformatics in vitro and in vivo evidence for these structures in the c-Myc, c-Kit, KRAS, PDGF-A, hTERT, Rb, RET and Hif-1alpha promoters accumulates. Nevertheless, the evidence remains largely circumstantial. This minireview differs from previous ones in that here we examine the diversity of G-quadruplex and i-motif structures in promoter elements and attempt to categorize the different types of arrangements in which they are found. For the c-Myc G-quadruplex and Bcl-2 i-motif, we summarize recent biological and structural studies.

The relationship between platelet-derived growth factor (PDGF) and the proto-oncogene c-sis has been determined by amino acid sequence analysis of PDGF and nucleotide sequence analysis of c-sis genomic clones. The nucleotide sequences of five regions of the human c-sis gene which are homologous to sequences of the transforming region (v-sis) of simian sarcoma virus (SSV) were determined. By alignment of the c-sis and v-sis nucleotide sequences the predicted amino acid sequence of a polypeptide homologous to the putative transforming protein p28sis of SSV was deduced. Both predicted sequences use the same termination codon and additional coding sequences may lie 5' to the homologous regions. Amino acid sequence analysis of the PDGF B chain shows identity to the amino acid sequence predicted from the c-sis sequences over 109 amino acid residues. Polymorphism may exist at two amino acid residues. These results suggest that c-sis encodes a polypeptide precursor of the B chain. A partial amino acid sequence of the PDGF A chain is also described. This chain is 60% homologous to the B chain and cannot be encoded by that part of c-sis which has been sequenced but could be encoded by sequences which lie 5' to the five regions of v-sis homology in c-sis, or at a separate locus.

Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine-phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.

BIBF 1000 is a small molecule inhibitor targeting the receptor kinases of platelet-derived growth factor (PDGF), basic fibroblast growth factor and vascular endothelial growth factor, which have known roles in the pathogenesis of pulmonary fibrosis. The anti-fibrotic potential of BIBF 1000 was determined in a rat model of bleomycin-induced lung fibrosis and in an ex vivo fibroblast differentiation assay. Rats exposed to a single intra-tracheal injection of bleomycin were treated with BIBF 1000 starting 10 days after bleomycin administration. To gauge for anti-fibrotic activity, collagen deposition and pro-fibrotic growth factor gene expression was analysed in isolated lungs. Furthermore, the activity of BIBF 1000 was compared with imatinib mesylate (combined PDGF receptor, c-kit and c-abl kinase inhibitor) and SB-431542 (transforming growth factor (TGF)-beta receptor I kinase inhibitor) in an ex vivo TGF-beta-driven fibroblast to myofibroblast differentiation assay, performed in primary human bronchial fibroblasts. Treatment of rats with BIBF 1000 resulted in the attenuation of fibrosis as assessed by the reduction of collagen deposition and the inhibition of pro-fibrotic gene expression. In the cellular assay both SB-431542 and BIBF 1000 showed dose-dependent inhibition of TGF-beta-induced differentiation, whereas imatinib mesylate was inactive. BIBF 1000, or related small molecules with a similar kinase inhibition profile, may represent a novel therapeutic approach for the treatment of idiopathic pulmonary fibrosis.

OBJECTIVE

Platelet activation is accompanied by the release of microparticles. However, little is known about the role of platelet-derived microparticles (PMP) in the regulation of angiogenesis and related clinical situations. The aim of our study was to evaluate the effect of PMP on angiogenesis and to analyze its mechanisms.

METHODS

Both in vitro (rat aortic ring model, cell invasion test) and in vivo (agarose bead transplantation, artificial cardiac ischemia in Sabra rats) approaches were used in the study.

RESULTS

A dose-dependent pro-angiogenic effect of PMP was observed in the rat aortic ring model. This effect could be eliminated by inhibition of VEGF, bFGF, and PDGF, but not heparanase. PMP exerted their effect via PI 3-kinase, Src kinase, and ERK, whereas protein kinase C and p38 were not involved. Moreover, PMP induced invasion of endothelial cells through a layer of matrigel. This effect was mediated by VEGF, heparanase, and PDGF, but not bFGF. Furthermore, PMP induced angiogenesis in an in vivo model in which agarose beads containing PMP were transplanted subcutaneously into mice. In addition, the effect of PMP on angiogenesis was evaluated in the model of in vivo chronic myocardial ischemia in rats. Ischemia induced a decrease in the number of functioning capillaries (34+/-21.5 vs. 157+/-42.0 per view field), but their amount increased after injection of PMP into the myocarium (97+/-27.3; p<0.001 vs. ischemia without PMP).

CONCLUSIONS

PMP induce angiogenesis both in vitro and in vivo. Injection of PMP into the ischemic myocardium might improve the process of revascularization after chronic ischemia.

STAT proteins are a group of latent cytoplasmic transcription factors which function as signal transducers and activators of transcription. Stat1 and -2 were originally identified to function in interferon signaling, and Stat1 was also found to be activated by epidermal growth factor (EGF) and other cytokines. New members of the STAT gene family are identified. Among them, Stat3 has 52.5% amino acid sequence homology with Stat1 and is activated by platelet-derived growth factor (PDGF), colony-stimulating factor 1 (CSF-1), EGF, interleukin-6, and other cytokines. Treatment of cells with EGF activates Stat1 and Stat3, which become phosphorylated on tyrosine residues to form homo - or heterodimers and translocate into the nucleus, binding to the sis-inducible element (SIE) in the c-fos promoter. Somatic cell genetic analyses demonstrated that Jaks, a family of nontransmembrane protein tyrosine kinases, are required for the activation of Stat1 and Stat2 in interferon-treated cells. However, little is known about the activation of Stat3 by growth factors. Here we report that in all v-Src-transformed cell lines examined, Stat3 is constitutively activated to bind to DNA and the phosphorylation of tyrosine on Stat3 is enhanced by the induction of v-Src expression. We also report that Src is shown to be associated with Stat3 in vivo, as well as in vitro, and phosphorylates Stat3 in vitro. Stat3 is also activated by CSF-1, possibly through CSF-1 receptor-c Src association in NIH 3T3 cells overexpressing CSF-1 receptors. Together, the data suggest that Src is involved in activation of Stat3 in growth factor signal transduction.

This review of angiogenesis aims to describe (a) stimuli that either elicit or antagonize angiogenesis, (b) the response of the vasculature to angiogenic or anti-angiogenic stimuli, i.e., processes required for the formation of new vessels, (c) aspects of angiogenesis relating to tissue remodeling and disease, and (d) the potential of angiogenic or antiangiogenic therapeutic measures. Angiogenesis, the formation of new vessels from existing microvessels, is important in embryogenesis, wound healing, diabetic retinopathy, tumor growth, and other diseases. Hypoxia and other as yet ill-defined stimuli drive tumor, inflammatory, and connective tissue cells to generate angiogenic molecules such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), and others. Natural and synthetic angiogenesis inhibitors such as angiostatin and thalidomide can repress angiogenesis. Angiogenic and antiangiogenic molecules control the formation of new vessels via different mechanisms. VEGF and FGF elicit their effects mainly via direct action on relevant endothelial cells. TGF-beta and PDGF can attract inflammatory or connective tissue cells which in turn control angiogenesis. Additionally, PDGF may act differently on specific phenotypes of endothelial cells that are engaged in angiogenesis or that are of microvascular origin. Thus phenotypic traits of endothelial cells committed to angiogenesis may determine their cellular responses to given stimuli. Processes necessary for new vessel formation and regulated by angiogenic/antiangiogenic molecules include the migration and proliferation of endothelial cells from the microvasculature, the controlled expression of proteolytic enzymes, the breakdown and reassembly of extracellular matrix, and the morphogenic process of endothelial tube formation. In animal models some angiogenesis-dependent diseases can be controlled via induction or inhibition of new vessel formation. Life-threatening infantile hemangiomas are a first established indication for antiangiogenic therapy in humans. Treatment of other diseases by modulation of angiogenesis are currently tested in clinical trials. Thus the manipulation of new vessel formation in angiogenesis-dependent conditions such as wound healing, inflammatory diseases, ischemic heart and peripheral vascular disease, myocardial infarction, diabetic retinopathy, and cancer is likely to create new therapeutic options.

Current ideas about the mechanism of wound healing and the pathogenesis of atherosclerosis, pulmonary fibrosis and hepatic fibrosis suggest a central role for the mononuclear phagocyte in attracting and/or stimulating the proliferation of mesenchymal cells. We demonstrate here that activated human blood monocytes, but not resting monocytes, release a mediator that attracts smooth muscle cells and cooperates with other mediators to stimulate fibroblast proliferation. This mediator is very similar to platelet-derived growth factor (PDGF): its chromatographic properties and chemical stability are similar to those of PDGF, it competes with 125I-PDGF for binding to fibroblasts and it immunoprecipitates with anti-PDGF antibodies. In parallel, stimulated monocytes, but not resting monocytes, express the c-sis proto-oncogene, a gene coding for one of the PDGF chains, consistent with the concept that expression of the c-sis proto-oncogene may be involved in the ability of mononuclear phagocytes to modulate the accumulation of mesenchymal cells.

Fibrosis is a complex process involving an inflammatory reaction, fibroblast proliferation, and abnormal accumulation of interstitial collagens. Mononuclear cells are usually present in lung fibrosis. Activated monocytes and macrophages in culture have been shown to produce several growth factors including platelet-derived growth factor (PDGF). PDGF is a potent mitogen and chemoattractant for fibroblasts and smooth muscle cells and a stimulator of collagen synthesis. We have studied the expression of c-sis/PDGF-2 mRNA in lung tissues derived from five patients with idiopathic pulmonary fibrosis (IPF) and from four control individuals without IPF. Northern blot analysis of specimens obtained from four patients with IPF revealed the expression of the c-sis/PDGF-2 protooncogene. A control lung tissue without IPF did not express the c-sis protooncogene. In situ hybridization extended these studies demonstrating the expression of the c-sis mRNA in the five specimens with IPF but not in the four control specimens without IPF. The expression of c-sis mRNA was localized primarily in the epithelial cells. Invading alveolar macrophages also expressed c-sis mRNA. The expression of c-sis mRNA was accompanied by the expression of PDGF-like proteins in lung specimens with IPF but not in control lung specimens. These findings demonstrate the in vivo expression of the c-sis/PDGF-2 protooncogene and the production of PDGF-like proteins in the epithelial cells and macrophages of the fibrotic tissue. This localized and sustained production of PDGF-like mitogen may constitute an important contributing factor in the abnormal fibroblast proliferation and collagen production, events associated with pulmonary fibrosis.

A cellular receptor for platelet-derived growth factor (PDGF) was demonstrated by incubation of 125I-labeled PDGF with human foreskin fibroblast cultures followed by liberation of cell-bound radioactivity with Triton X-100. The cellular binding of labeled PDGF in the presence of increasing amounts of unlabeled PDGF showed saturation; Scatchard analysis of binding data indicated a single class of receptors having kd = 1 X 10(-9) M. The number of PDGF binding sites was approximately 3 X 10(5)/cell. Labeled PDGF binding reached an apparent equilibrium after 3 hr at 4 degrees C. At 37 degrees C, it passed a maximum after 30 min and then decreased with time due to degradation of the tracer. A large excess of unlabeled PDGF reduced labeled PDGF binding by more than 90% whereas similar doses of epidermal growth factor, fibroblast growth factor, or insulin had no effect. It was concluded that PDGF did not share receptors with these factors. PDGF receptors were found on skin fibroblasts, normal and malignant glial cells, smooth muscle cells, and 3T3 cells but not on epithelial-derived cells, neuroblastoma cells, endothelial cells, or peripheral lymphocytes.l As only the receptor-positive cells--i.e., the connective tissue- and glia-derived cells--are responsive to stimulation with PDGF, these findings imply a functional significance of the PDGF receptor.

Bone marrow mesenchymal stem cells (BMMSCs) are pluripotent cells capable of differentiating into several cell types and are thus an attractive cell source for connective tissue engineering. A challenge in such a use is expansion and directed seeding in vitro, requiring proliferation and survival, and directed migration, respectively, prior to functional differentiation. The epidermal growth factor (EGF) receptor (EGFR) is the prototypal growth factor receptor and elicits these responses from a wide variety of stromal, epithelial, and endothelial cells. Ligands for this receptor are appealing for use in tissue engineering because they are relatively resistant to biological extremes and amenable to high-volume production. Therefore, we determined whether an EGFR ligand, EGF, could be used for ex vivo expansion of BMMSCs. EGF stimulated motility in rat and immortalized human BMMSCs. EGF-induced proliferation was observed in immortalized human BMMSCs but was not apparent in rat BMMSCs under our experimental conditions. EGF did not, however, rescue either type of BMMSC from apoptosis due to lack of serum. During our examination of key signaling intermediaries, EGF caused robust phosphorylation of extracellular signal-regulated protein kinase (ERK) and protein kinase B/akt (AKT) but only minimal phosphorylation of EGFR and phospholipase C-gamma in rat BMMSCs, whereas in the human BMMSCs these intermediaries were all strongly activated. EGF also induced robust ERK activation in primary porcine mesenchymal stem cells. EGF pretreatment or cotreatment did not interfere with secondarily induced differentiation of either type of BMMSC into adipogenic or osteogenic lineages. Platelet-derived growth factor (PDGF) effects were similar to but not additive with those elicited by EGF, with some quantitative differences; however, PDGF did interfere with the differentiation of these BMMSCs. These findings suggest that EGFR ligands could be used for ex vivo expansion and direction of BMMSCs.