Hyperglycemia increases expression of platelet-derived growth factor (PDGF)-beta receptor and potentiates chemotaxis to PDGF-BB in human aortic vascular smooth muscle cells (VSMCs) via PI3K and ERK/MAPK signaling pathways. The purpose of this study was to determine whether increased activation of protein kinase C (PKC) isoforms had a modulatory effect on the PI3K and ERK/MAPK pathways, control of cell adhesiveness, and movement. All known PKC isoforms were assessed but only PKCalpha and PKCbetaII levels were increased in 25 mmol/L glucose. However, only PKCbetaII inhibition affected (decreased) PI3K pathway and MAPK pathway activities and inhibited PDGF-beta receptor upregulation in raised glucose, and specific MAPK inhibition was required to completely block the effect of glucose. In raised glucose conditions, activity of the ERK/MAPK pathway, PI3K pathway, and PKCbetaII were all sensitive to aldose reductase inhibition. Chemotaxis to PDGF-BB (360 pmol/L), absent in 5 mmol/L glucose, was present in raised glucose and could be blocked by PKCbetaII inhibition. Formation of lamellipodia was dependent on PI3K activation and filopodia on MAPK activation; both lamellipodia and filopodia were eliminated when PKCbetaII was inhibited. FAK phosphorylation and cell adhesion were reduced by PI3K inhibition, and although MAPK inhibition prevented chemotaxis, it did not affect FAK phosphorylation or cell adhesiveness. In conclusion, chemotaxis to PDGF-BB in 25 mmol/L glucose is PKCbetaII-dependent and requires activation of both the PI3K and MAPK pathways. Changes in cell adhesion and migration speed are mediated mainly through the PI3K pathway.

Platelet concentrates (PCs) constitute new biological mediators used in osseous reconstructive surgery. In this study, we assessed (i) the effects of various concentrations of calcium and thrombin on the kinetics of platelet-derived growth factor (PDGF-BB), transforming growth factor-betabeta 1), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) release by PCs and (ii) the contribution of PC supernatants to endothelial cell proliferation. Our results indicate that high concentrations of calcium (Ca) and thrombin (Thr) trigger an immediate and significant increase in bFGF, TGF-beta 1 and PDGF-BB concentrations. Thereafter, PDGF-BB, VEGF and TGF-beta 1 levels remained generally constant over a 6-day period while a decrease in bFGF concentrations was noted after 24h. Lower Ca and Thr concentrations tended to reduce and delay growth factors release from PCs. Endothelial cell proliferation was greatly enhanced with PC supernatants (mean: 20-fold increase). This was especially evident when endothelial cells were treated with supernatants harvested early after PC treatment with high concentrations of Ca and Thr or later after PC treatment with low Ca and Thr concentrations. Additional research aiming to measure the effects of Ca and Thr on bone formation in vivo is needed.

OBJECTIVE

It was the aim of our study to investigate the influence of a selective ET-A receptor antagonist LU 135252 alone and in combination with the ACE-inhibitor, trandolapril on podocyte number and morphology in streptozotocin diabetic rats.

METHODS

Male Sprague-Dawley rats were injected with 65 mg streptozotocin i.v. and subsequently developed diabetes. Animals were left untreated or received daily either trandolapril (0.3 mg/kg body weight), LU 135252 (50 mg/kg body weight) or a combination of both. After 6 months the experiment was terminated. Glomerular geometry and cellularity were assessed by stereological techniques. Protein expression of TGF-beta, ET-1, PDGF-AB, fibronectin, desmin and alpha-smooth muscle cell actin was investigated by immunohistochemistry.

RESULTS

The mean number of podocytes per glomerulus was lower (86+/-17 vs. 138+/-25; p<0.05) and mean podocyte volume was higher in untreated diabetic animals than in non-diabetic controls. Only ACE-i alone and in combination, but not ET(A)-RB alone prevented loss of podocytes and podocyte hypertrophy. In diabetic rats, increased numbers of PCNA positive and p27(kip1) positive cells (mainly podocytes) were reduced by all treatments, but only ACE-i decreased numbers of desmin positive podocytes and tubulointerstitial expression of TGF-beta. Albuminuria was increased in untreated diabetes and was prevented only by ACE-i and combination treatment.

CONCLUSIONS

Podocyte hypertrophy and degeneration is an early event in diabetic nephropathy leading to a loss of podocytes. Treatment with an ACE-i, but not with an ET(A)-RB, prevented the development of albuminuria as well as damage and loss of podocytes. The well known anti-proteinuric effect of ACE-i is presumably due at least in part to conservation of podocyte structure. Increased plasma endothelin-1 (ET-1) concentrations and urine excretion of ET-1 have been documented in patients with diabetes and proteinuria [1]. It has been shown that experimental diabetes mellitus increases renal ET-1 gene transcription [2]. To assess the relevance of the ET-system in the pathogenesis of renal structural changes in the model of the STZ-induced diabetic rat we compared the effect of an ET(A)-receptor specific antagonist with the well known beneficial effect of an ACE-i, especially on podocyte cell number and morphology.

We hypothesized that PDGF-B/PDGFR-beta-signaling is important in the cardiac contribution of epicardium-derived cells and cardiac neural crest, cell lineages crucial for heart development. We analyzed hearts of different embryonic stages of both Pdgf-b-/- and Pdgfr-beta-/- mouse embryos for structural aberrations with an established causal relation to defective contribution of these cell lineages. Immunohistochemical staining for alphaSMA, periostin, ephrinBBbryos. We observed that knockout embryos showed perimembranous and muscular ventricular septal defects, maldevelopment of the atrioventricular cushions and valves, impaired coronary arteriogenesis, and hypoplasia of the myocardium and cardiac nerves. The abnormalities correspond with models in which epicardial development is impaired and with neuronal neural crest-related innervation deficits. This implies a role for PDGF-B/PDGFR-beta-signaling specifically in the contribution of these cell lineages to cardiac development.

Platelet-derived growth factors (PDGFs) are important in embryonic development. To elucidate their role in avian heart and coronary development, we investigated protein expression patterns of PDGF-A, PDGF-B, and the receptors PDGFR-alpha and PDGFR-beta using immunohistochemistry on sections of pro-epicardial quail-chicken chimeras of Hamburger and Hamilton (HH) 28-HH35. PDGF-A and PDGFR-alpha were expressed in the atrial septum, sinus venosus, and throughout the myocardium, with PDGFR-alpha retreating to the trabeculae at later stages. Additionally, PDGF-A and PDGFR-alpha were present in outflow tract cushion mesenchyme and myocardium, respectively. Small cardiac nerves and (sub)epicardial cells expressed PDGF-B and PDGFR-beta. Furthermore, endothelial cells expressed PDGF-B, while vascular smooth muscle cells and interstitial epicardium-derived cells expressed PDGFR-beta, indicating a role in coronary maturation. PDGF-B is also present in ventricular septal development, in the absence of any PDGFR. Epicardium-derived cells in the atrioventricular cushions expressed PDGFR-beta. We conclude that all four proteins are involved in myocardial development, whereas PDGF-B and PDGFR-beta are specifically important in coronary maturation.

Macrophages play an integral role in the development of liver fibrosis by releasing mediators, such as platelet-derived growth factor-B (PDGF-B) and transforming growth factor-β1, which stimulate hepatic stellate cell proliferation, chemotaxis, and collagen production. However, the mechanism by which chronic liver injury stimulates macrophages to release these mediators is not completely understood. We tested the hypothesis that chronic liver injury activates hypoxia-inducible factor (HIF) transcription factors in macrophages that regulate the production of mediators that promote fibrosis. To test this hypothesis, Cre/lox technology was used to generate myeloid cell-specific HIF-1α or HIF-1β knockout mice. When these mice were subjected to bile duct ligation (BDL), levels of α-smooth muscle actin and type I collagen in the liver were reduced compared with those of mice with normal levels of HIFs. The deficiency of HIFs in macrophages did not affect liver injury or inflammation after BDL but reduced PDGF-B mRNA and protein, suggesting that HIF activation in macrophages may promote fibrosis by regulating the production of PDGF-B. Consistent with a role for HIFs in liver fibrosis in cholestatic liver disease, nuclear HIF-1α protein was present in macrophages, hepatocytes, and fibroblasts in the livers from patients with primary biliary cirrhosis and primary sclerosing cholangitis. These studies demonstrate that HIFs are important regulators of profibrotic mediator production by macrophages during the development of liver fibrosis and suggest that HIFs may be a novel therapeutic target for the treatment of chronic liver disease in patients.

The four platelet-derived growth factor (PDGF) ligands and PDGF receptors (PDGFRs), α and β (PDGFRA, PDGFRB), are essential proteins that are expressed during embryonic and mature nervous systems, i.e., in neural progenitors, neurons, astrocytes, oligodendrocytes, and vascular cells. PDGF exerts essential roles from the gastrulation period to adult neuronal maintenance by contributing to the regulation of development of preplacodal progenitors, placodal ectoderm, and neural crest cells to adult neural progenitors, in coordinating with other factors. In adulthood, PDGF plays critical roles for maintenance of many specific cell types in the nervous system together with vascular cells through controlling the blood brain barrier homeostasis. At injury or various stresses, PDGF modulates neuronal excitability through adjusting various ion channels, and affecting synaptic plasticity and function. Furthermore, PDGF stimulates survival signals, majorly PI3-K/Akt pathway but also other ways, rescuing cells from apoptosis. Studies imply an involvement of PDGF in dendrite spine morphology, being critical for memory in the developing brain. Recent studies suggest association of PDGF genes with neuropsychiatric disorders. In this review, we will describe the roles of PDGF in the nervous system, from the discovery to recent findings, in order to understand the broad spectrum of PDGF in the nervous system. Recent development of pharmacological and replacement therapies targeting the PDGF system is discussed.

OBJECTIVE

Epiretinal membranes (ERMs) arise from a variety of causes or, in some cases, for unknown reasons. Once established, ERMs tend to progress, becoming more extensive and exerting increasing traction along the inner surface of the retina. One possible cause for their progression is the production of growth factors by cells within ERMs that may provide autocrine or paracrine stimulation. Platelet derived growth factor (PDGF) and its receptors have been localised to cells of ERMs and may play such a role. In this study, comparative data were sought for several other growth factors that have been implicated in ERM formation.

METHODS

Immunohistochemical staining of ERMs was done for PDGF-A, PDGF-B, basic fibroblast growth factor (bFGF), three isoforms of transforming growth factor beta (TGF-beta), and vascular endothelial growth factor (VEGF) and its receptors, flt-1 and flk-1/KDR. Expression of flt-1 and flk-1/KDR was examined in cultured retinal pigmented epithelial (RPE) cells and retinal glia from postmortem eyes by immunohistochemistry and by reverse transcription coupled to polymerase chain reaction (RT-PCR).

RESULTS

Staining was most intense and most frequently observed for VEGF and PDGF-A, both in vascular and avascular ERMs. The majority of cells stained for VEGF in nine of 11 (81.8%) diabetic ERMs and in 14 of 24 (58.3%) proliferative vitreoretinopathy ERMs. The receptors for VEGF, flt-1, and flk-1/KDR were also identified on cells in ERMs and on cultured RPE cells. By RT-PCR, mRNA for flt-1 was identified in RPE cells and retinal glia, and mRNA for flk-1/KDR was identified in RPE cells.

CONCLUSIONS

These data show that VEGF and its receptors are localised to both vascular and avascular ERMs and suggest that VEGF, like PDGF-A, may be an autocrine and paracrine stimulator that may contribute to progression of vascular and avascular ERMs.

BACKGROUND

Saphenous vein grafts are widely used for aortocoronary bypass surgery as treatment for severe atherosclerosis and often are complicated by subsequent occlusion of the graft vessel.

RESULTS

We described a mouse model of venous bypass graft arteriosclerosis that can be effectively retarded by locally applied suramin, a growth factor receptor antagonist. Mouse isogeneic vessels of the vena cava veins pretreated with suramin were grafted end to end into the carotid arteries and enveloped with a mixture of suramin (1 mmol/L) and pluronic-127 gel. In the untreated group, vessel wall thickening was observed as early as 1 week after surgery and progressed to 4-fold and 10-fold the original thickness in grafted veins at 4 and 8 weeks, respectively. Pluronic-127 gel alone did not influence neointima formation. Suramin treatment reduced the neointima hyperplasia 50% to 70% compared with untreated controls. Immunohistochemical studies demonstrated that a significant proliferation of vascular smooth muscle cells (SMCs) constituted neointimal lesions between 4 and 8 weeks. The majority of SMCs expressed platelet-derived growth factor (PDGF) receptors-alpha and -beta, which were significantly reduced by suramin treatment. In vitro studies indicated that suramin completely blocked PDGF receptor activation or phosphorylation stimulated by PDGF-AB, inhibited activation of mitogen-activated protein kinase (ERK) kinases (MEK1/2) and ERK1/2, and abrogated transcription factor AP-1 DNA-binding activity.

CONCLUSIONS

Suramin inhibited SMC migration and proliferation in vivo and in vitro by blocking PDGF-initiated PDGF receptor and MAPK-AP-1 signaling. These findings indicate that locally applied suramin is effective in a mouse model of venous bypass graft arteriosclerosis.

Embryonic stem (ES) cells are exposed to fluid-mechanical forces, such as cyclic strain and shear stress, during the process of embryonic development but much remains to be elucidated concerning the role of fluid-mechanical forces in ES cell differentiation. Here, we show that cyclic strain induces vascular smooth muscle cell (VSMC) differentiation in murine ES cells. Flk-1-positive (Flk-1+) ES cells seeded on flexible silicone membranes were subjected to controlled levels of cyclic strain and examined for changes in cell proliferation and expression of various cell lineage markers. When exposed to cyclic strain (4-12% strain, 1 Hz, 24 h), the Flk-1+ ES cells significantly increased in cell number and became oriented perpendicular to the direction of strain. There were dose-dependent increases in the VSMC markers smooth muscle alpha-actin and smooth muscle-myosin heavy chain at both the protein and gene expression level in response to cyclic strain, whereas expression of the vascular endothelial cell marker Flk-1 decreased, and there were no changes in the other endothelial cell markers (Flt-1, VE-cadherin, and platelet endothelial cell adhesion molecule 1), the blood cell marker CD3, or the epithelial marker keratin. The PDGF receptor beta (PDGFR beta) kinase inhibitor AG-1296 completely blocked the cyclic strain-induced increase in cell number and VSMC marker expression. Cyclic strain immediately caused phosphorylation of PDGFR beta in a dose-dependent manner, but neutralizing antibody against PDGF-BB did not block the PDGFR beta phosphorylation. These results suggest that cyclic strain activates PDGFR beta in a ligand-independent manner and that the activation plays a critical role in VSMC differentiation from Flk-1+ ES cells.

Chronic pancreatitis (CP) is characterized by progressive fibrosis, pain and/or loss of exocrine and endocrine functions. Recent in vitro and in vivo experiments have proven objectively the role of activated pancreatic stellate cells (PSC) in fibrogenesis in CP. Molecular mediators shown to regulate the pathogenesis include transforming growth factor beta (TGF-beta), platelet-derived growth factor (PDGF), and pro-inflammatory cytokines such as IL-1, IL-6 and TNF-alpha. Furthermore, molecular pathways involving mitogen-activated protein kinases (MAPK), phosphatidyl inositol 3-kinase (PI3K), Ras superfamily G proteins, serine threonine protein kinase Raf-1 and peroxisome proliferator activated receptor gamma (PPAR-gamma) have been elucidated. Understanding of the pathogenesis has led to identification of novel molecular targets and development of potential newer therapeutic agents. Those found to retard the progression of experimental CP and fibrosis in animal models include interferon (IFN) beta and IFN-gamma; a Japanese herbal medicine called Saiko-keishi-to (TJ-10); curcumin; PPAR-gamma ligand (troglitazone); antioxidants (vitamin A, vitamin E, DA 9601 and epigallocatechin-3-gallate); a protease inhibitor (camostat mesilate) and hydroxymethylglutaryl-CoA inhibitor (lovastatin). This review summarizes the current literature addressing the role of different pharmacological agents aimed at reducing or preventing inflammation and the consequent fibrogenesis in CP.

During fibrosis the hepatic stellate cell (HSC) undergoes a complex activation process characterized by increased proliferation and extracellular matrix deposition. The 70-kDa ribosomal S6 kinase (p70S6K) is activated by mitogens, growth factors, and hormones in a phosphatidylinositol 3-kinase-dependent manner. p70S6K regulates protein synthesis, proliferation, and cell cycle control. Because these processes are involved in HSC activation, we investigated the role of p70S6K in HSC proliferation, cell cycle control, and type I collagen expression. Platelet-derived growth factor (PDGF) stimulated p70S6K phosphorylation, which was blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Rapamycin blocked phosphorylation of p70S6K but had no affect on PDGF-induced Akt phosphorylation, positioning p70S6K downstream of Akt. Transforming growth factor-beta, which inhibits HSC proliferation, did not affect PDGF-induced p70S6K phosphorylation. Rapamycin treatment did not affect alpha1(I) collagen mRNA but reduced type I collagen protein secretion. Expression of smooth muscle alpha-actin was not affected by rapamycin treatment, indicating that HSC activation was not altered. Rapamycin inhibited serum-induced DNA synthesis approximately 2-fold. Moreover, rapamycin decreased expression of cyclins D1, D3, and E but not cyclin D2, Rb-Ser780, and Rb-Ser795. Together, p70S6K plays a crucial role in HSC proliferation, collagen expression, and cell cycle control, thus representing a potential therapeutic target for liver fibrosis.

BACKGROUND

The elucidation of molecular mechanisms of vascular cell biology has markedly influenced our thinking on the pathophysiology of vascular disease. Antisense oligonucleotide gene therapy has helped identify proteins critical to cell-cycle progression and proliferation and possible therapeutic strategies to combat human disease. This approach, however, has not yet been used to examine the contribution of chemotactic proteins and/or their receptors. Platelet-derived growth factor-BB (PDGF-BB) released from activated platelets adherent to subendothelial connective tissue is a principal smooth muscle cell chemotactic factor.

RESULTS

A series of experiments was performed to assess the capacity of antisense oligonucleotides to reduce PDGF-beta receptor subunit (PDGFR-beta) expression and the contribution of PDGFR-beta in neointimal formation. Sustained, direct, and local perivascular administration of two different antisense oligonucleotide sequences complementary to PDGFR-beta mRNA almost completely abolished the expression of PDGFR-beta protein in the intima and media of injured carotid arteries and decreased neointimal formation by 80% and 60%, respectively. Furthermore, neointimal formation correlated precisely with PDGFR-beta expression in an exponential fashion.

CONCLUSIONS

Thus, myointimal proliferation depends on both PDGFR-beta overexpression and its activation by PDGF-BB. Removal of either of these two elements can suppress neointimal formation.

c-Kit encodes for the receptor tyrosine kinase (RTK) and belongs to type III receptor family. This includes platelet derived growth factor (PDGF) alpha and beta and macrophage colony stimulating factor (mCSF) apart from others. Their characteristic features are the presence of five immunologlobulin like domains in the extracellular region and 70-100 residues long kinase insert domain in the cytoplasmic region. The RTKs activate several signaling pathways within the cells leading to cell proliferation, differentiation, migration or metabolic changes. The Kit ligand-stem cell factor (SCF) induces a rapid and complete receptor dimerization resulting in activation by autophosphorylation of the catalytic tyrosine kinase and generation of signal transduction leading to regulation of cell growth. Various mutations in c-kit such as insertions and deletions (without affecting reading frame) and point mutations in the inhibitory juxtamembrane (JM) domain encoded by exon 11 have been reported in gastrointestinal stromal tumors (GISTs). Thus, c-kit signaling is believed to play a role in tumorigenesis. Efforts are being made to control and treat these tumors by blocking kit signaling using Imatinib with varying degrees of success. This review deals with the features of c-kit, its ligand and roles in gastrointestinal stromal tumors.

Although pathologic and hemodynamic changes in monocrotaline (MCT)-induced pulmonary hypertension have been studied extensively, relatively little is known about the inter- and intracellular signaling mechanisms underlying such alterations. As a first step to delineating signaling mechanisms governing adverse structural alterations in the hypertensive lungs, we examined changes in the steady-state levels of mRNAs encoding several growth factors including transforming growth factors (TGF), platelet-derived growth factors (PDGF), vascular endothelial cell growth factor (VEGF) and endothelin (ET) as a function of time in MCT-induced pulmonary hypertension in rats. These studies demonstrated a very diverse pattern of growth factor gene expression in response to MCT administration. In general, alterations in the steady-state levels of mRNAs encoding the growth factors preceded the onset of MCT-induced pulmonary hypertension. TGF-beta 1, -beta 2 and -beta 3 transcripts were seen to be elevated, whereas that of TGF-alpha and PDGF-A remained unchanged. Transcripts for PDGF-B and ET were increased in the early stages but declined to less than controls in the latter stages of MCT-induced hypertension. In contrast, levels of VEGF mRNA decreased to less than controls as the disease progressed. Viewed collectively, the diverse pattern of expression suggests that alterations in the levels of the growth factor transcripts may have a significant role in the development of pulmonary hypertensive disease and may be relevant to the pathological and structural changes in MCT-induced pulmonary hypertension.

OBJECTIVE

While natriuretic peptides can inhibit growth of vascular muscle cells (VSMC), controversy exists as to whether this effect is mediated via the guanylate cyclase-coupled receptors, NPR-A and NPR-B, or the clearance receptor, NPR-C. The original aim of this study was to examine the mechanism by which the NPR-C receptor regulates growth.

METHODS

Rat VSMC were characterized with regard to natriuretic peptide receptor expression by RT/PCR and radioligand binding studies. The effect on growth following addition of the peptides and the ligands for NPR-C was measured by [3H]thymidine incorporation. Cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay and mitogen activating protein kinase activity was based on the phosphorylation of myelin basic protein.

RESULTS

In rat VSMC, passages 4-12, both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) dose-dependently inhibited serum and PDGF-induced VSMC growth. In contrast, NPR-C specific ligands alone had no effect on cell growth but enhanced growth inhibition when co-administered with ANP and CNP. ANP and CNP also decreased PDGF-BB-stimulated MAP kinase activity. Once again, NPR-C specific ligands alone had no effect but enhanced the effects of ANP. Furthermore, a cGMP specific phosphodiesterase inhibitor dose-dependently inhibited VSMC growth and markedly enhanced natriuretic-peptide-induced inhibition at low peptide concentrations. To examine a potential mechanism for the controversy concerning the NPR-C, we investigated the autocrine expression of ANP and CNP by VSMC and found that mRNA encoding both peptides could be detected by RT/PCR.

CONCLUSIONS

Our findings indicate that the guanyl-cyclase-linked receptors mediate the antiproliferative actions of the natriuretic peptides on vascular smooth muscle cell growth. Moreover, we hypothesize that the apparent inhibition of growth by NPR-C specific ligands reported by others may be due to stabilization of natriuretic peptides produced by the cultured VSMC and subsequent action of these peptides at guanyl-cyclase-linked receptors.

The expression and localization of PDGF beta receptors and PDGF-AB/BB in human healing wounds was evaluated by immunohistochemical techniques and in situ hybridization. Expression of PDGF beta receptor protein and PDGF-AB/BB were analyzed in wound margin biopsies using the PDGFR-B2 and PDGF 007 antibodies. PDGF beta receptor expression was minor in normal skin. An increased expression of PDGF beta receptor protein was prominent in vessels in the proliferating tissue zone in wounds as early as 1 d after surgery and was apparent < or = 4 wk after surgery. There was also a concordant increase in PDGF beta receptor mRNA detected by in situ hybridization. PDGF-AB/BB was present in healing wounds as well as in normal skin. In normal skin, expression of PDGF-AB/BB was confined to peripheral nerve fibers and to solitary cells of the epidermis and of the superficial dermis. In wounds, infiltrating mononuclear cells also stained for PDGF-AB/BB. To identify cell types expressing PDGF AB/BB and PDGF beta receptors, respectively, we performed double immunofluorescence stainings. PDGF beta receptors were expressed by vascular smooth muscle cells and cells in capillary walls; the receptor protein could not be detected in neurofilament containing structures, T lymphocytes, or CD68 expressing macrophages. PDGF-AB/BB colocalized with neurofilaments, it was present in Langerhans cells of the epidermis and in HLA-DR positive cells located in the epidermal/dermal junction area. Of the macrophages infiltrating the wound, 43 +/- 18% stained positively for PDGF AB/BB. Since PDGF-AB/BB and PDGF beta receptors are expressed in the healing wound, two essential prerequisites for a role of PDGF in wound healing are fulfilled.

Platelet-derived growth factor (PDGF) plays a critical role in regulating the development and functional control of various tissues and has been implicated in the pathogenesis of serious diseases, including cancer and atherosclerosis. Given the emerging role of PDGF in the development and function of male gonads, we compared the expression profiles of the mRNAs of the PDGF A- and B-chains and of the PDGF receptor (PDGFR) alpha- and beta-subunits in fetal and adult human testis. The immunohistochemical localization of the corresponding proteins in fetal, adult, and diseased human testicular tissues was also analyzed. PDGFs and PDGFRs mRNAs were readily detected by both Northern analysis and RT-PCR. The transcript levels were higher during 16-20 wk gestation, significantly lower at 24-28 wk, and increased in the adult. An identical pattern of protein expression was confirmed by immunohistochemistry, although the cellular localization of the PDGF system changes during postnatal development, concomitantly with the progression of spermatogenesis. In the testicular samples from patients affected by either complete aplasia of germ cells or various grades of spermatogenic arrest, the immunohistochemical localization of PDGFs and PDGFRs was different from normal, confirming a close connection between germ cells and PDGF system distribution. These results indicate that PDGF, through complex interactions, could play a leading role in ontogenesis and testicular pathophysiology in humans. Finally, the expression of PDGF ligands and receptor proteins in Leydig cell tumors suggests a relationship of the PDGF system to tumorigenesis or tumor progression in this testicular neoplasm.

We have examined the histological and cytoskeletal changes in rat connective tissues induced by subcutaneous perfusion with cytokines. Granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), interleukin-1-alpha (IL-1-alpha), transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) produced a significant fibroblast accumulation, neovascular development and a weak to moderate leukocyte infiltration, while interleukin-2 (IL-2) and gamma-interferon (gamma-IFN) induced intense mononucleated leukocyte infiltration. Immunofluorescence staining showed that accumulated fibroblastic cells were positive for alpha-smooth muscle (SM) actin (but negative for the desmin and muscle myosin) only in GM-CSF-treated tissues. Electron microscopic examination established that a significant proportion of fibroblastic cell in GM-CSF-, IL-1-alpha- or TGF-beta-treated animals were typical myofibroblasts. Only in GM-CSF-treated animals did microfilament bundles of myofibroblasts contain alpha-SM actin, when examined by immuno electron microscopy. Our results suggest that locally applied cytokines induce the formation of distinct granulation tissues. In particular, GM-CSF stimulates alpha-SM actin synthesis in myofibroblasts, illustrating an unexpected extra-hematopoietic in vivo effect of this factor.

Treatment of Swiss 3T3 cells with a subsaturating concentration of recombinant Pasteurella multocida toxin (rPMT) markedly potentiated the production of inositol phosphates induced by bombesin, vasopressin, and endothelin but not by platelet-derived growth factor (PDGF) (AA and BB homodimers). Similarly, the neuropeptides but not PDGF caused a shift in the dose-dependent increase in inositol phosphates induced by rPMT. The rate of accumulation of inositol phosphates induced by bombesin was increased 2-fold by rPMT treatment while that of PDGF was unaffected. rPMT treatment also enhanced bombesin-induced inositol(1,4,5)trisphosphate, the direct product of phosphatidylinositol 4,5-bisphosphate hydrolysis. In contrast, treatment of cells with rPMT had no effect on the tyrosine phosphorylation of phospholipase C gamma. Depletion of protein kinase C increased rPMT-induced inositol phosphates in a manner similar to that observed for bombesin but not PDGF. Thus, rPMT selectively potentiates neuropeptide-mediated inositol phosphate production. The action of rPMT on phosphatidylinositol 4,5-bisphosphate hydrolysis persisted in streptolysin O-permeabilized cells. Addition of guanosine 5'-O-(beta-thiodiphosphate) to permeabilized cells markedly reduced rPMT-induced inositol phosphates in a time- and dose-dependent manner. rPMT also increased the sensitivity of phospholipase C for free calcium. Our results strongly suggest that the action of rPMT facilitates the coupling of G protein to phospholipase C.

Bone marrow stromal cells (BMSCs) are a heterogeneous population of cells derived from colony-forming units-fibroblastic (CFU-Fs). These cells reside in the bone marrow cavity and are capable of differentiating into several cell phenotypes including osteoblasts, chondroblasts, hematopoiesis-supporting stromal cells, and adipocytes. However, the factors that regulate the proliferation and differentiation of the BMSC population are for the most part unknown. Since many members of the receptor tyrosine kinase (RTK) family have been shown to participate in growth control of various mesenchymal cell populations, in this study we examined the expression and function of RTKs in the BMSC population. Degenerate oligonucleotides corresponding to two conserved catalytic domains of the RTK family and RT-PCR were used initially to determine which RTKs are expressed in the human BMSC (hBMSC) system. After subcloning the amplification product generated from mRNA of a multicolony-derived hBMSC strain, PDGF receptor (beta), EGF receptor, FGF receptor 1, and Axl were identified by DNA sequencing of 26 bacterial colonies. Furthermore, PDGF and EGF were found to enhance BMSC growth in a dose-dependent manner and to induce tyrosine phosphorylation of intracellular molecules, including the PDGF and EGF receptors themselves, demonstrating the functionality of these receptors. On the other hand, bFGF was found to have little effect on proliferation or tyrosine phosphorylation. Since single colony-derived hBMSC strains are known to vary from one colony to another in colony habit (growth rate and colony structure) and the ability to form bone in vivo, the expression levels of these RTKs were determined in 18 hBMSC clonal strains by semiquantitative RT-PCR and were found to vary from one clonal strain to another. While not absolutely predictive of the osteogenic capacity of individual clonal strains, on average, relatively high levels of PDGF-receptor were found in bone-forming strains, while on average, nonbone-forming strains had relatively high levels of EGF-receptor. Taken together, these results indicate that RTKs play a role in the control of hBMSC proliferation, and that the differential pattern of RTK expression may be useful in correlating the biochemical properties of individual clonal strains with their ability to produce bone in vivo.

The bovine papillomavirus E5 protein is a 44-aa transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor and induces constitutive tyrosine phosphorylation and activation of the receptor, resulting in cell transformation. The E5 protein does not resemble PDGF, but rather activates the receptor in a ligand-independent fashion, thus providing a unique system to examine activation of receptor tyrosine kinases. Here, we used a variety of approaches to explore the mechanism of receptor activation by the E5 protein. Chemical cross-linking experiments revealed that the E5 protein activated only a small fraction of the endogenous PDGF beta receptor in transformed fibroblasts and suggested that this fraction was constitutively dimerized. Coimmunoprecipitation experiments using extracts of cells engineered to coexpress full-length and truncated PDGF beta receptors confirmed that the E5 protein induced oligomerization of the receptor. Furthermore, in cells expressing the E5 protein, a kinase-active receptor was able to trans-phosphorylate a kinase-negative mutant receptor but was unable to catalyze intramolecular autophosphorylation. These results indicated that the E5 protein induced PDGF beta receptor activation by forming a stable complex with the receptor, resulting in receptor dimerization and trans-phosphorylation.

Connective tissue growth factor (CTGF) is a mitogenic and chemotactic factor for cultured fibroblasts that has been implicated in wound healing, fibrotic disorders and uterine function. Although the primary translational products of the mouse, human and pig CTGF (mCTGF, hCTGF, pCTGF) genes are predicted to be secreted and of approximate M(r) 38,000, 10 kDa biologically active forms of pCTGF have recently been described. In this report, we show that human foreskin fibroblasts (HFFs) and mouse connective tissue fibroblasts contained 2.4 kb CTGF transcripts, stained positively with an anti-CTGF[81-94] peptide antiserum, and produced a 38 kDa protein that was immunoprecipitated by an anti-CTGF[247-260] peptide antiserum. While 38 kDa CTGF was readily detected in cell lysates, it was non- or barely detectable in conditioned medium. 38 kDa CTGF remained cell-associated for at least 5 days after synthesis and was not releasable by treatment of the cells with trypsin, heparin, 1 M NaCl or low pH. Purification of CTGF from human or mouse fibroblast conditioned medium resulted in the isolation of 10-12 kDa CTGF proteins that were heparin-binding, bioactive, and reactive with anti-CTGF[247-260] on Western blots. Whereas 10 kDa CTGF stimulated DNA synthesis in 3T3 cells to the same extent as platelet-derived growth factor (PDGF)-AA, -AB, or -BB, it did not compete with 125I-PDGF-BB for binding to alpha alpha, alpha beta or beta beta PDGF receptors (PDGF-R), did not stimulate tyrosine phosphorylation of PDGF-alpha-R or -beta-R, and was not antagonized by a neutralizing PDGF-R-alpha antiserum. These data show that, in cultured fibroblasts, 38 kDa CTGF is principally cell-associated whereas low mass forms of CTGF are soluble and biologically active. They further demonstrate that, contrary to the previously proposed properties of 38 kDa CTGF, 10 kDa CTGF does not bind to PDGF-R and stimulates Balb/c 3T3 cell mitosis via a PDGF-R-independent mechanism.

Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.