Class Ia phosphoinositide 3-kinases (PI3Ks) are heterodimers of p110 catalytic and p85 regulatory subunits that mediate a variety of cellular responses to growth and differentiation factors. Although embryonic development is not impaired in mice lacking all isoforms of the p85alpha gene (p85alpha-/- p55alpha-/- p50alpha-/-) or in mice lacking the p85beta gene (p85beta-/-) (D. A. Fruman, F. Mauvais-Jarvis, D. A. Pollard, C. M. Yballe, D. Brazil, R. T. Bronson, C. R. Kahn, and L. C. Cantley, Nat Genet. 26:379-382, 2000; K. Ueki, C. M. Yballe, S. M. Brachmann, D. Vicent, J. M. Watt, C. R. Kahn, and L. C. Cantley, Proc. Natl. Acad. Sci. USA 99:419-424, 2002), we show here that loss of both genes results in lethality at embryonic day 12.5 (E12.5). The phenotypes of these embryos, including subepidermal blebs flanking the neural tube at E8 and bleeding into the blebs during the turning process, are similar to defects observed in platelet-derived growth factor receptor alpha null (PDGFRalpha-/-) mice (P. Soriano, Development 124:2691-2700, 1997), suggesting that PI3K is an essential mediator of PDGFRalpha signaling at this developmental stage. p85alpha-/- p55alpha+/+ p50alpha+/+ p85beta-/- mice had similar but less severe defects, indicating that p85alpha and p85beta have a critical and redundant function in development. Mouse embryo fibroblasts deficient in all p85alpha and p85beta gene products (p85alpha-/- p55alpha-/- p50alpha-/- p85beta-/-) are defective in PDGF-induced membrane ruffling. Overexpression of the Rac-specific GDP-GTP exchange factor Vav2 or reintroduction of p85alpha or p85beta rescues the membrane ruffling defect. Surprisingly, reintroduction of p50alpha also restored PDGF-dependent membrane ruffling. These results indicate that class Ia PI3K is critical for PDGF-dependent actin rearrangement but that the SH3 domain and the Rho/Rac/Cdc42-interacting domain of p85, which lacks p50alpha, are not required for this response.

Regulated signal transduction in discrete microdomains of the cell surface is an attractive hypothesis for achieving spatial and temporal specificity in signaling. A procedure for purifying caveolae separately from other similarly buoyant microdomains including those rich in glycosylphosphatidylinositol-anchored proteins has been developed (Schnitzer, J. E., McIntosh, D. P., Dvorak, A. M., Liu, J., and Oh, P. (1995) Science 269, 1435-1439) and used here to show that caveolae contain many signaling molecules including select kinases (platelet-derived growth factor (PDGF) receptors, protein kinase C, phosphatidylinositol 3-kinase, and Src-like kinases), phospholipase C, sphingomyelin, and even phosphoinositides. More importantly, two different techniques reveal that caveolae function as signal transducing subcompartments of the plasma membrane. PDGF rapidly induces phosphorylation of endothelial cell plasmalemmal proteins residing in caveolae as detected by membrane subfractionation and confocal immunofluorescence microscopy. This PDGF signaling cascade is halted when the caveolar compartment is disassembled by filipin. Finally, in vitro kinase assays show that caveolae contain most of the intrinsic tyrosine kinase activity of the plasma membrane. As signal transducing organelles, caveolae organize a distinct set of signaling molecules to permit direct regionalized signal transduction within their boundaries.

Platelet-derived growth factor-D (PDGF-D) is a newly recognized growth factor known to regulate many cellular processes, including cell proliferation, transformation, invasion, and angiogenesis. Recent studies have shown that PDGF-D and its cognate receptor PDGFR-beta are expressed in prostate tumor tissues, suggesting that PDGF-D might play an important role in the development and progression of prostate cancer. However, the biological role of PDGF-D in tumorigenesis remains elusive. In this study, we found that PDGF-D-overexpressing PC3 cells (PC3 cells stably transfected with PDGF-D cDNA and referred to as PC3 PDGF-D) exhibited a rapid growth rate and enhanced cell invasion that was associated with the activation of mammalian target of rapamycin (mTOR) and reduced Akt activity. Rapamycin repressed mTOR activity and concomitantly resulted in the activation of Akt, which could attenuate the therapeutic effects of mTOR inhibitors. In contrast, B-DIM (BR-DIM from Bioresponse, Inc.; a chemopreventive agent) significantly inhibited both mTOR and Akt in PC3 PDGF-D cells, which were correlated with decreased cell proliferation and invasion. Moreover, conditioned medium from PC3 PDGF-D cells significantly increased the tube formation of human umbilical vein endothelial cells, which was inhibited by B-DIM treatment concomitant with reduced full-length and active form of PDGF-D. Our results suggest that B-DIM could serve as a novel and efficient chemopreventive and/or therapeutic agent by inactivation of both mTOR and Akt activity in PDGF-D-overexpressing prostate cancer.

Oligodendrocyte precursor cells (OPCs) persist in substantial numbers in the adult brain in a quiescent state suggesting that they may provide a source of new oligodendrocytes after injury. To determine whether adult OPCs have the capacity to divide rapidly, we have developed a method to highly purify OPCs from adult optic nerve and have directly compared their properties with their perinatal counterparts. When cultured in platelet-derived growth factor (PDGF), an astrocyte-derived mitogen, perinatal OPCs divided approximately once per day, whereas adult OPCs divided only once every 3 or 4 d. The proliferation rate of adult OPCs was not increased by addition of fibroblast growth factor (FGF) or of the neuregulin glial growth factor 2 (GGF2), two mitogens that are normally produced by retinal ganglion cells. cAMP elevation has been shown previously to be essential for Schwann cells to survive and divide in response to GGF2 and other mitogens. Similarly we found that when cAMP levels were elevated, GGF2 alone was sufficient to induce perinatal OPCs to divide slowly, approximately once every 4 d, but adult OPCs still did not divide. When PDGF was combined with GGF2 and cAMP elevation, however, the adult OPCs began to divide rapidly. These findings indicate that adult OPCs are intrinsically different than perinatal OPCs. They are not senescent cells, however, because they retain the capacity to divide rapidly. Thus, after demyelinating injuries, enhanced axonal release of GGF2 or a related neuregulin might collaborate with astrocyte-derived PDGF to induce rapid division of adult OPCs.

Diffuse intrinsic pontine glioma (DIPG) is an incurable tumor that arises in the brainstem of children. To date there is not a single approved drug to effectively treat these tumors and thus novel therapies are desperately needed. Recent studies suggest that a significant fraction of these tumors contain alterations in cell cycle regulatory genes including amplification of the D-type cyclins and CDK4/6, and less commonly, loss of Ink4a-ARF leading to aberrant cell proliferation. In this study, we evaluated the therapeutic approach of targeting the cyclin-CDK-Retinoblastoma (Rb) pathway in a genetically engineered PDGF-B-driven brainstem glioma (BSG) mouse model. We found that PD-0332991 (PD), a CDK4/6 inhibitor, induces cell-cycle arrest in our PDGF-B; Ink4a-ARF deficient model both in vitro and in vivo. By contrast, the PDGF-B; p53 deficient model was mostly resistant to treatment with PD. We noted that a 7-day treatment course with PD significantly prolonged survival by 12% in the PDGF-B; Ink4a-ARF deficient BSG model. Furthermore, a single dose of 10 Gy radiation therapy (RT) followed by 7 days of treatment with PD increased the survival by 19% in comparison to RT alone. These findings provide the rationale for evaluating PD in children with Ink4a-ARF deficient gliomas.

Platelet-derived growth factors (PDGFs) are important for normal tissue growth and maintenance. Overexpression of the classical PDGFs, PDGF-A and PDGF-B, has been linked to several diseases, including cancer, fibrotic disease and atherosclerosis. Recently, two novel PDGFs, PDGF-C and PDGF-D, were discovered. It has not yet been established whether PDGF-C and PDGF-D are linked to disease phenotypes like the classical PDGFs. PDGF-B, the cellular homologue of the viral simian sarcoma oncogene v-sis, is known to potently induce cellular transformation through activation of PDGF receptor (PDGFR)-beta. In this work, we have determined the transformation efficacy of PDGF-D in comparison with that of PDGF-C and PDGF-B. PDGF-D is a potent transforming growth factor for NIH/3T3 cells, and the transformed cells displayed stress fibre reorganization, increased proliferation rate, anchorage-independent growth in soft agar, ability to induce tumours in nude mice, and upregulation of vascular endothelial growth factor. Morphological analyses of the vasculatures from the PDGF-isoform-expressing tumours revealed marked differences suggesting differential signalling through the two PDGF receptors in tumour vessel development and remodelling. In summary, these results suggest that PDGF-D induce cellular transformation and promote tumour growth by accelerating the proliferation rate of the tumour cells, and by stimulation of tumour neovascularization.

OBJECTIVE

To explore the antitumor activity of imatinib in patients with advanced platelet-derived growth factor β (PDGFB)/PDGF receptor β (PDGFRB)-positive chordomas.

METHODS

In a collaborative Italian-Swiss, prospective, phase II clinical study conducted from November 2004 through April 2006, 56 patients with advanced PDGFB and/or PDGFRB chordoma received 800 mg/d of imatinib until progression. The primary end point was the overall tumor response rate (ORR), defined by RECIST. Secondary, exploratory end points included tissue response (ie, changes in tumor density or signal intensity/contrast enhancement, and/or [18F]-fluorodeoxyglucose positron emission tomography [PET] uptake), overall survival, progression-free survival (PFS), and pain score.

RESULTS

Among 50 patients evaluable by RECIST, the best response was one partial response (PR) obtained at 6 months (ORR, 2%). There were 35 patients with stable disease (SD, 70%) and a 64% clinical benefit rate (ie, RECIST complete response + PR + SD ≥ 6 months). A minor dimensional response (< 20%) was detected in nine patients. A maximum standard uptake value decrease ≥ 25% was observed in 10 (39%) of 26 patients evaluable for PET response at 3 months. Changes in the Brief Pain Inventory score were consistent with the response assessment. Median PFS (intention-to-treat population, 56 patients) was 9 months. No unexpected toxicities were observed.

CONCLUSIONS

This is the largest phase II study in chordoma to date. It confirms anecdotal evidence that imatinib has antitumor activity in this orphan disease, and therefore, it is worth further investigation.

Platelet-derived growth factor-D (PDGF-D) signaling plays critical roles in the pathogenesis and progression of human malignancies; however, the precise mechanism by which PDGF-D causes tumor cell invasion and angiogenesis remain unclear. Because Notch-1, nuclear factor-kappaB (NF-kappaB), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMP) are critically involved in the processes of tumor cell invasion and metastasis, we investigated whether PDGF-D down-regulation could be mechanistically associated with the down-regulation of Notch-1, NF-kappaB, VEGF, and MMP-9, resulting in the inhibition of tumor cell invasion and angiogenesis. Our data showed that down-regulation of PDGF-D leads to the inactivation of Notch-1 and NF-kappaB DNA-binding activity and, in turn, down regulates the expression of its target genes, such as VEGF and MMP-9. We also found that the down-regulation of PDGF-D by small interfering RNA (siRNA) decreased tumor cell invasion, whereas PDGF-D overexpression by cDNA transfection led to increased cell invasion. Consistent with these results, we also found that the down-regulation of PDGF-D not only decreased MMP-9 mRNA and its protein expression but also inhibited the processing of pro-MMP-9 protein to its active form. Moreover, conditioned medium from PDGF-D siRNA-transfected cells showed reduced levels of VEGF and, in turn, inhibited the tube formation of human umbilical vascular endothelial cells, suggesting that down-regulation of PDGF-D leads to the inhibition of angiogenesis. Taken together, we conclude that the down-regulation of PDGF-D by novel approaches could lead to the down-regulation of Notch-1 and, in turn, inactivate NF-kappaB and its target genes (i.e., MMP-9 and VEGF), resulting in the inhibition of invasion and angiogenesis.

The localized and temporally controlled delivery of growth factors is key to achieving optimal clinical efficacy. In sophisticated tissue engineering strategies, the biodegradable scaffold is preferred to serve as both a three-dimensional (3-D) substrate and a growth factor delivery vehicle to promote cellular activity and enhance tissue neogenesis. This study presents a novel approach to fabricate tissue engineering scaffolds capable of controlled growth factor delivery whereby growth factor containing microspheres were incorporated into 3-D scaffolds with good mechanical properties, well-interconnected macroporous and nano-fibrous structures. The microspheres were uniformly distributed throughout the nano-fibrous scaffold and their incorporation did not interfere the macro-, micro-, and nanostructures of the scaffold. The release kinetics of platelet-derived growth factor-BB (PDGF-BB) from microspheres and scaffolds was investigated using poly(lactic-co-glycolic acid) (PLGA50) microspheres with different molecular weights (6.5 and 64kDa, respectively) and microsphere-incorporated poly(l-lactic acid) (PLLA) nano-fibrous scaffolds. Incorporation of microspheres into scaffolds significantly reduced the initial burst release. Sustained release from several days to months was achieved through different microspheres in scaffolds. Released PDGF-BB was demonstrated to possess biological activity as evidenced by stimulation of human gingival fibroblast DNA synthesis in vitro. The successful generation of 3-D nano-fibrous scaffold incorporating controlled-release factors indicates significant potential for more complex tissue regeneration.

We investigated the role of the p38 mitogen-activated protein kinase (p38 MAPK) in the PDGF-BB-induced cytoskeleton remodeling that occurs during the migration of porcine aortic smooth muscle cells (SMC). We showed that p38 MAPK controlled the polymerization of actin that is required for PDGF-induced lamellipodia formation and migration. To investigate the mechanism of action of p38 MAPK, we explored its cellular localization and that of its indirect substrate, the heat shock protein Hsp27, during SMC spreading on fibronectin in the presence and absence of PDGF. Spreading of SMC on fibronectin activated p38 MAPK in a sustained manner only in the presence of PDGF. In these conditions, Hsp27 and p38 MAPK were localized all over the lamellipodia. A transiently phosphorylated form of p38 MAPK was observed at the leading edge, whereas p38 MAPK remained phosphorylated at the base of the lamellipodia. Phosphorylated Hsp27 was excluded from the leading edge and restricted to the base of the lamellipodia. These results were confirmed by Triton X-100 extraction of particulate membrane fraction. Displacement of Hsp27 from the leading edge by cytochalasin D treatment suggests that nonphosphorylated Hsp27 caps barbed ends in vivo. Our data indicate that nonphosphorylated Hsp27 might contribute to the formation of a short, branched actin network at the leading edge, whereas phosphorylated Hsp27 might stabilize the actin network at the base of lamellipodia, which is composed of long, unbranched actin filaments.

OBJECTIVE

We analyzed the expression of platelet-derived growth factor D (PDGF-D) in an experimental bile duct-ligated (BDL) rat model and assessed its biological function in cultured hepatic stellate cells (HSC) and myofibroblasts (MFB).

METHODS

The mRNA for PDGF-A, -B, -C, -D and for PDGF receptor-alpha and -beta chains (PDGFRalpha and PDGFRbeta) in normal and fibrotic rat livers was assessed quantitatively. Protein levels of PDGF-D were quantified by immunoblotting and immunohistochemistry.

RESULTS

The relative mRNA expression of all PDGF isoforms and receptors upregulated upon BDL and PDGF-A, -B and -D expression was significantly higher than that of PDGF-C. PDGF-D and PDGFRbeta protein also increased markedly. Immunostaining revealed that PDGF-D is localized along the fibrotic septa of the periportal- and perisinusoidal areas. Besides PDGF-B, PDGF-D is the second most potent PDGF isoform in PDGFRbeta signaling within HSC/MFB, evidenced by PDGFRbeta autophosphorylation and activation of the downstream signaling molecules ERK1/2-, JNK-, p38 MAPK, and PKB/Akt while PDGF-C effects were minimal. PDGF-D exerted mitogenic and fibrogenic effects in both cultured HSC and MFB comparable to PDGF-B but PDGF-A and -C showed only marginal fibrogenic effects.

CONCLUSIONS

PDGF-D possesses potential pathogenetic properties for HSC activation and matrix remodeling in liver fibrosis.

Adult rat arterial smooth muscle cells (SMC) in primary culture modulate from contractile to synthetic phenotype. This process includes partial loss of myofilaments and formation of an extensive rough endoplasmic reticulum and a large Golgi complex. It gives the cells the ability to initiate DNA synthesis and actively proliferate when stimulated with serum or isolated growth factors. After a few divisions, growth becomes partly independent of exogenous mitogens and does not cease until multiple cell layers have been formed. Here, it is demonstrated that serum-free conditioned medium from primary cultures of adult rat arterial SMC contains a factor that initiates DNA synthesis in growth-arrested secondary cultures of SMC. The mitogenic activity was neutralized by antibodies to platelet-derived growth factor (PDGF), and no mitogenic activity occurred in conditioned medium from cultures pretreated with actinomycin D, excluding release into the medium of PDGF adsorbed to the plastic vessels during the initial culture in serum-containing medium. Exposure of human fibroblasts to samples of the conditioned medium at 4 degrees C inhibited subsequent binding of 125I-labeled PDGF. It was further shown that the SMC of the primary cultures were able to initiate DNA synthesis in a chemically defined medium lacking PDGF and other growth factors. During the early, most active, and partly autonomous growth phase, the SMC had a low binding capacity for 125I-labeled PDGF and responded but little to stimulation with exogenous PDGF. Later on, with increasing cell density and decreasing growth rate, the ability to bind and respond to exogenous PDGF increased. Taken together, the observations suggest that modulation of SMC from contractile to synthetic phenotype is accompanied by production of a PDGF-like protein and autocrine or possibly by mitogen-independent initiation of DNA synthesis. Functionally, this may be important during wound healing and in the development of atherosclerotic lesions.

OBJECTIVE

Aberrant vascular smooth muscle cell (VSMC) proliferation and migration contribute significantly to the development of vascular pathologies, such as atherosclerosis and restenosis. MicroRNAs have recently emerged as critical modulators in cellular processes and the purpose of this study is to identify novel miRNA regulators implicated in human aortic VSMC proliferation and migration.

RESULTS

To identify miRNAs that are differentially expressed in human VSMCs, we performed miRNA microarray analysis in human aortic smooth muscle cells (SMCs) at different time points after platelet-derived growth factor (PDGF) stimulation. Here, we identified microRNA-638 (miR-638) as a transcript that was one of the most significantly down-regulated in human VSMCs after PDGF stimulation. Furthermore, we confirmed, by Quantitative RT-PCR, that miR-638 is highly expressed in human VSMCs, and its expression is markedly down-regulated in a dose- and time-dependent manner upon PDGF treatment. Consistent with a critical role in SMC proliferation, we found that miR-638 expression was significantly up-regulated in human VSMCs cultured in differentiation medium, a condition that inhibits SMC proliferation. Furthermore, we identified the orphan nuclear receptor NOR1 as a downstream target gene product of miR-638 and down-regulation of NOR1 is critical for miR-638-mediated inhibitory effects on PDGF-induced cyclin D1 expression, cell proliferation, and migration in human aortic SMCs.

CONCLUSIONS

These results indicate that miR-638 is a key molecule in regulating human VSMC proliferation and migration by targeting the NOR1/cyclin D pathway and suggest that specific modulation of miR-638 in human VSMCs may represent an attractive approach for the treatment of proliferative vascular diseases.

Increasing evidence indicates the significance of platelet-derived growth factor receptor-β (β-PDGFR) signaling in prostate cancer (PCa). Accordingly, preclinical studies suggest the potential of β-PDGFR as a therapeutic target in metastatic PCa. However, a ligand responsible for β-PDGFR activation in PCa was unknown, and recent clinical trials with imatinib mesylate showed limited success due to normal tissue toxicity. Similarly, in spite of mounting evidence indicating the significance of matriptase in PCa, little is known about its substrates or molecular actions during PCa progression. Here, we identified PDGF-D as a ligand for β-PDGFR in PCa and discovered matriptase as its regulator. Matriptase activates PDGF-D by proteolytic removal of the CUB domain in a 2-step process, creating a hemidimer, followed by growth factor domain dimer (GFD-D) generation. Matriptase can deactivate PDGF-D by further proteolytic cleavage within the GFD, revealing its biphasic regulation. Importantly, PDGF-D/matriptase colocalization is accompanied with β-PDGFR phosphorylation in human PCa tissues. This study unveiled a novel signaling axis of matriptase/PDGF-D/β-PDGFR in PCa, providing new insights into functional interplay between serine protease and growth factor signaling networks.

Activation of receptor tyrosine kinases, such as fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), and VEGF receptor (VEGFR), has been implicated in tumor progression and metastasis in human pancreatic cancer. In this study, we investigated the effects of TKI258, a tyrosine kinase inhibitor to FGFR, PDGFR, and VEGFR on pancreatic cancer cell lines (HPAF-II, BxPC-3, MiaPaCa2, and L3.6pl), endothelial cells, and vascular smooth muscle cells (VSMC). Results showed that treatment with TKI258 impaired activation of signaling intermediates in pancreatic cancer cells, endothelial cells, and VSMCs, even upon stimulation with FGF-1, FGF-2, VEGF-A, and PDGF-B. Furthermore, blockade of FGFR/PDGFR/VEGFR reduced survivin expression and improved activity of gemcitabine in MiaPaCa2 pancreatic cancer cells. In addition, motility of cancer cells, endothelial cells, and VSMCs was reduced upon treatment with TKI258. In vivo, therapy with TKI258 led to dose-dependent inhibition of subcutaneous (HPAF-II) and orthotopic (L3.6pl) tumor growth. Immunohistochemical analysis revealed effects on tumor cell proliferation [bromodeoxyuridine (BrdUrd)] and tumor vascularization (CD31). Moreover, lymph node metastases were significantly reduced in the orthotopic tumor model when treatment was initiated early with TKI258 (30 mg/kg/d). In established tumors, TKI258 (30 mg/kg/d) led to significant growth delay and improved survival in subcutaneous and orthotopic models, respectively. These data provide evidence that targeting FGFR/PDFGR/VEGFR with TKI258 may be effective in human pancreatic cancer and warrants further clinical evaluation.

The roles of polypeptide growth factors in promoting wound healing and in directing the specificity and sequence of responses of different tissues in wounds are little understood. We investigated the influence of four growth factors on the rates of healing of a novel full thickness dermal ulcer placed on an avascular base in the rabbit ear. The wound model precludes significant wound contraction and requires new granulation tissue and epithelial cells for healing to originate centripetally. 5 micrograms (7-31 pmol/mm2) of platelet-derived growth factor-B chain (PDGF-BB), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) applied locally at the time of wounding resulted in a twofold increase in complete reepithelialization of treated wounds (PDGF-BB, P = 0.02 chi square analysis; bFGF, P = 0.04; EGF, P = 0.05); transforming growth factor (TGF)-beta 1 significantly inhibited reepithelialization (P = 0.05). Both PDGF-BB and TGF-beta 1 uniquely increased the depth and area of new granulation tissue (P less than 0.005), the influx of fibroblasts, and the deposition of new matrix into wounds. Explants from 7-d old PDGF-BB-treated wounds remained metabolically far more active than controls, incorporating 473% more [3H]thymidine into DNA (P = 0.05) and significantly more [3H]leucine and [3H]proline into collagenase-sensitive protein (P = 0.04). The results establish that polypeptide growth factors have significant and selective positive influences on healing of full thickness ulcers in the rabbit.

Migration of eukaryotic cells toward a chemoattractant often relies on their ability to distinguish receptor-mediated signaling at different subcellular locations, a phenomenon known as spatial sensing. A prominent example that is seen during wound healing is fibroblast migration in platelet-derived growth factor (PDGF) gradients. As in the well-characterized chemotactic cells Dictyostelium discoideum and neutrophils, signaling to the cytoskeleton via the phosphoinositide 3-kinase pathway in fibroblasts is spatially polarized by a PDGF gradient; however, the sensitivity of this process and how it is regulated are unknown. Through a quantitative analysis of mathematical models and live cell total internal reflection fluorescence microscopy experiments, we demonstrate that PDGF detection is governed by mechanisms that are fundamentally different from those in D. discoideum and neutrophils. Robust PDGF sensing requires steeper gradients and a much narrower range of absolute chemoattractant concentration, which is consistent with a simpler system lacking the feedback loops that yield signal amplification and adaptation in amoeboid cells.

Lysophosphatidylcholine (lyso-PC) is a major phospholipid component of atherogenic lipoproteins (e.g., oxidized LDL and beta-VLDL) and also can be generated through the action of leukocyte-secreted phospholipase A2 at sites of inflammation. We have previously reported that lyso-PC can activate cultured endothelia, resulting in the selective upregulation of adhesion molecules, such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. In this study, we have found that lyso-PC increased steady state mRNA levels for two smooth muscle/fibroblast-directed growth factors, the A and B chains of PDGF and heparin-binding EGF-like protein (HB-EGF), in cultured human endothelial cells. Lyso-PC did not upregulate the expression of certain other inducible endothelial genes, including E-selectin, IL-8, or monocyte chemoattractant protein-1 in the same cells, in contrast to the coordinate pattern of activation typically observed with other stimuli, such as TNF alpha, bacterial endotoxin, or PMA. Nuclear runoff assays documented an increased transcriptional rate for the HB-EGF gene in lyso-PC-treated cells. Northern blot analyses, after actinomycin D treatment, further indicated that the increased amounts of mRNA for HB-EGF, PDGF A and B chains, and intercellular adhesion molecule-1 were not dependent upon message stabilization. We conclude that lyso-PC can induce growth factor gene expression in cultured endothelial cells and thus may contribute to the migration and proliferation of smooth muscle cells and fibroblasts in various response-to-injury settings in vivo.

OBJECTIVE

Investigations on the combination of radiotherapy with vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) antiangiogenic agents, which has the potential to improve the clinical outcome in cancer patients.

METHODS

Here, we analyze the combined VEGF (SU5416) and PDGF (SU6668) receptor tyrosine kinase inhibition with irradiation in human endothelium (HUVEC), prostate cancer (PC3), and glioblastoma (U87) in vitro and in vivo.

RESULTS

Combined inhibition of VEGF and PDGF signaling resulted in enhanced apoptosis, reduced cell proliferation, and clonogenic survival as well as reduced endothelial cell migration and tube formation compared with single pathway inhibition. These effects were further enhanced by additional irradiation. Likewise, in PC3 and U87 tumors growing s.c. on BALB/c nu/nu mice, dual inhibition of VEGF and PDGF signaling significantly increased tumor growth delay versus each monotherapy. Interestingly, radiation at approximately 20% of the dose necessary to induce local tumor control exerts similar tumor growth-inhibitory effects as the antiangiogenic drugs given at their maximum effective dose. Addition of radiotherapy to both mono- as well as dual-antiangiogenic treatment markedly increased tumor growth delay. With respect to tumor angiogenesis, radiation further decreased microvessel density (CD31 count) and tumor cell proliferation (Ki-67 index) in all drug-treated groups. Of note, the slowly growing PC3 tumor responded better to the antiangiogenic drug treatments than the faster-growing U87 tumor. In addition to the beneficial effect of abrogating VEGF survival signaling when combined with radiation, we identified here a novel mechanism for the tumor escape from radiation damage. We found that radiation induced up-regulation of all four isoforms of PDGF (A-D) in endothelial cells supporting adjacent smooth muscle cells resulting in a prosurvival effect of radiation. The addition of SU6668 attenuated this undesirable paracrine radiation effect, which may rationalize the combined application of radiation with PDGF signaling inhibition to increase antitumor effects.

CONCLUSIONS

A relative low radiation dose markedly enhances local antitumor effects of combined VEGF and PDGF signaling inhibition, suggesting a promising combination regimen for local tumor treatment with radiotherapy remaining an essential element.

OBJECTIVE

Airway remodelling in asthma is manifested, in part, as increased airway smooth muscle (ASM) mass, reflecting myocyte proliferation. We hypothesized that calcitriol, a secosteroidal vitamin D receptor (VDR) modulator, would inhibit growth factor-induced myocyte proliferation.

METHODS

Human ASM cell cultures were derived from bronchial samples taken during surgery. ASM cells were treated with platelet-derived growth factor (PDGF) (10 ng.mL(-1)) for 24 h in the presence of calcitriol, dexamethasone or a checkpoint kinase 1 (Chk1) inhibitor (SB218078). The effects of calcitriol on PDGF-mediated cell proliferation were assessed by thymidine incorporation assay, propidium iodide-based cell cycle analysis, caspase-3 assay and immunoblotting for specific cell cycle modulators.

RESULTS

Calcitriol, but not dexamethasone, inhibited PDGF-induced ASM DNA synthesis concentration dependently (IC(50)= 520 +/- 52 nM). These effects were associated with VDR-mediated expression of cytochrome CYP24A1 with no effects on ASM apoptosis. Calcitriol substantially inhibited (P < 0.01) PDGF-stimulated cell growth in ASM derived from both normal (59 +/- 8%) and asthmatic subjects (57 +/- 9%). Calcitriol inhibited PDGF-induced phosphorylation of retinoblastoma protein (Rb) and Chk1, with no effects on PDGF-mediated activation of extracellular signal-regulated kinases 1/2, PI3-kinase and S6 kinase, or expression of p21(Waf/Cip-1), p27(Kip1), cyclin D and E2F-1. Consistent with these observations, SB218078 also inhibited (IC(50)= 450 +/- 100 pM) PDGF-induced cell cycle progression.

CONCLUSIONS

Calcitriol decreased PDGF-induced ASM cell growth by inhibiting Rb and Chk1 phosphorylation.

Mesangial cell (MC) proliferation and extracellular matrix expansion are involved in the pathogenesis of glomerulosclerosis and renal failure. In vitro, PDGF and basic fibroblast growth factor (bFGF) regulate MC proliferation and/or matrix production. To elucidate the role of PDGF and bFGF in vivo, equimolar concentrations of recombinant PDGF-BB or bFGF or vehicle were infused intravenously into rats over a 7-d period. Rats were either nonmanipulated ("normals") or had received a subnephritogenic dose of anti-MC antibody ("anti-Thy 1.1 rats") before the infusion period. Glomerular cell proliferation (anti-proliferating cell nuclear antigen immunostaining) on days 2, 4, and 7 was unchanged in vehicle-infused normals or anti-Thy 1.1 rats. PDGF infusion increased glomerular cell proliferation 32-fold in anti-Thy 1.1 rats and an 11-fold in normals on day 2. bFGF increased glomerular cell proliferation fourfold in anti-Thy 1.1 rats but was ineffective in normals. Induction of cell proliferation in all kidneys was limited to the glomerulus. The majority of proliferating cells were identified as MC by double immunolabeling. No significant proteinuria, glomerular leukocyte, or platelet influx developed in any group. Glomerular matrix expansion with increased deposition of type IV collagen, laminin, and fibronectin, as well as upregulated laminin and collagen IV mRNA expression was confined to PDGF-infused anti-Thy 1.1 rats. These results show that PDGF and, to a lesser degree, bFGF are selective MC mitogens in vivo and that previous subclinical injury can enhance this MC response. The data thereby support a role of these cytokines in the pathogenesis of glomerulosclerosis.

Platelet-derived growth factor-D (PDGF-D) can regulate many cellular processes, including cell proliferation, apoptosis, transformation, migration, invasion, angiogenesis and metastasis. Therefore PDGF-D signaling has been considered to be important in human malignancies, and thus PDGF-D signaling may represent a novel therapeutic target, and as such suggests that the development of agents that will target PDGF-D signaling is likely to have a significant therapeutic impact on human cancers. This mini-review describes the mechanisms of signal transduction associated with PDGF-D signaling to support the role of PDGF-D in the carcinogenesis. Moreover, we summarize data on several PDGF-D inhibitors especially naturally occurring "chemopreventive agent" such an indole compound, which we believe could serve as a novel agent for the prevention of tumor progression and/or treatment of human malignancies by targeted inactivation of PDGF-D signaling.

Platelet-derived growth factor-D (PDGF-D) is a recently characterized member of the PDGF family with unknown in vivo functions. We investigated the effects of PDGF-D in transgenic mice by expressing it in basal epidermal cells and then analyzed skin histology, interstitial fluid pressure, and wound healing. When compared with control mice, PDGF-D transgenic mice displayed increased numbers of macrophages and elevated interstitial fluid pressure in the dermis. Wound healing in the transgenic mice was characterized by increased cell density and enhanced recruitment of macrophages. Macrophage recruitment was also the characteristic response when PDGF-D was expressed in skeletal muscle or ear by an adeno-associated virus vector. Combined expression of PDGF-D with vascular endothelial growth factor-E (VEGF-E) led to increased pericyte/smooth muscle cell coating of the VEGF-E-induced vessels and inhibition of the vascular leakiness that accompanies VEGF-E-induced angiogenesis. These results show that full-length PDGF-D is activated in tissues and is capable of increasing interstitial fluid pressure and macrophage recruitment and the maturation of blood vessels in angiogenic processes.

The recognition that periodontal regeneration can be achieved has resulted in increased efforts focused on understanding the mechanisms and factors required for restoring periodontal tissues so that clinical outcomes of such therapies are more predictable than those currently being used. In vitro models provide an excellent procedure for providing clues as to the mechanisms that may be required for regeneration of tissues. The investigations here were targeted at determining the ability of enamel matrix derivative (EMD) to influence specific properties of periodontal ligament cells in vitro. Properties of cells examined included migration, attachment, proliferation, biosynthetic activity and mineral nodule formation. Immunoassays were done to determine whether or not EMD retained known polypeptide factors. Results demonstrated that EMD under in vitro conditions formed protein aggregates, thereby providing a unique environment for cell-matrix interaction. Under these conditions, EMD: (a) enhanced proliferation of PDL cells, but not of epithelial cells; (b) increased total protein production by PDL cells; (c) promoted mineral nodule formation of PDL cells, as assayed by von Kossa staining; (d) had no significant effect on migration or attachment and spreading of cells within the limits of the assay systems used here. Next, EMD was screened for possible presence of specific molecules including: GM-CSF, calbindin D, EGF, fibronectin, bFGF, gamma-interferon, IL-1 beta, 2, 3, 6; IGF-1,2; NGF, PDGF, TNF, TGF beta. With immunoassays used, none of these molecules were identified in EMD. These in vitro studies support the concept that EMD can act as a positive matrix for cells at a regenerative site.