Inhibition Of Platelet-derived Growth Factor Research Articles

Mesangial cell proliferation inhibitors for the treatment of proliferative glomerular disease.

Mesangial cells (MC) serve a number of functions in the renal glomerular capillary including structural support of the capillary tuft, modulation of glomerular hemodynamics, and a phagocytic function allowing removal of macromolecules and immune complexes. The proliferation of MC is a prominent feature of glomerular disease including IgA nephropathy, membranoproliferative glomerulonephritis, lupus nephritis, and diabetic nephropathy. In experimental animal models of nephritis, MC proliferation frequently precedes and is linked to the increase of extracellular matrix in the mesangium and glomerulosclerosis. Reduction of MC proliferation in glomerular disease models by treatment with heparin, low-protein diet, or antibodies to platelet-derived growth factor (PDGF), have been shown to reduce extracellular matrix expansion and glomerulosclerotic changes. Therefore, MC proliferation inhibitors may offer therapeutic opportunities for the treatment of proliferative glomerular disease. It is also known that the MC proliferation is inhibited by many kinds of pharmacological drugs, for example, angiotensin converting enzyme (ACE) inhibitors, leukotriene D(4) (LTD(4)) antagonists, PDGF inhibitors, matrix metalloproteinases (MMP) inhibitors, 3-hydroxy-3 methyl glutaryl-coenzymeA (HMG-CoA) inhibitors, cyclin-dependent kinases (CDK) inhibitors, and others. This review summarizes the recently reported MC proliferation inhibitors with their pharmacological properties on the basis of their chemical structures.

Effects of the prostanoids on the proliferation or hypertrophy of cultured murine aortic smooth muscle cells

Effects of the prostanoids on the growth of cultured aortic vascular smooth muscle cells (VSMCs) were examined using mice lacking prostanoid receptors. Proliferation of VSMCs was assessed by measuring [(3)H]-thymidine incorporation and the cell number, and their hypertrophy by [(14)C]-leucine incorporation and protein content. In VSMCs from wild-type mice, expressions of mRNAs for the EP(4) and TP were most abundant, followed by those for the IP, EP(3) and FP, when examined by competitive reverse transcriptase-PCR. Those for the EP(1), EP(2) and DP, however, could not be detected. AE1-329, an EP(4) agonist, and cicaprost, an IP agonist, inhibited platelet derived growth factor (PDGF)-induced proliferation of VSMCs from wild-type mice; these inhibitory effects disappeared completely in VSMCs from EP(4)(-/-) and IP(-/-) mice, respectively. In accordance with these effects, AE1-329 and cicaprost stimulated cAMP production in VSMCs from wild-type mice, which were absent in VSMCs from EP(4)(-/-) and IP(-/-) mice, respectively. Effects of PGE(2) on cell proliferation and adenylate cyclase were almost similar with those of AE1-329 in VSMCs from wild-type mice, which disappeared in VSMCs from EP(4)(-/-) mice. PGD(2) inhibited PDGF-induced proliferation of VSMCs from both wild-type and DP(-/-) mice to a similar extent. This action of PGD(2) was also observed in VSMCs from EP4(-/-) and IP(-/-) mice. In VSMCs from wild-type mice, I-BOP, a TP agonist, showed potentiation of PDGF-induced hypertrophy. I-BOP failed to show this action in VSMCs from TP(-/-) mice. The specific agonists for the EP(1), EP(2) or EP(3), and PGF(2)alpha showed little effect on the growth of VSMCs. These results show that PGE(2), PGI(2) and TXA(2) modulate PDGF-induced proliferation or hypertrophy of VSMCs via the EP(4), IP and TP, respectively, and that the inhibitory effect of PGD(2) on PDGF-induced proliferation is not mediated by the DP, EP(4) or IP.

Effect of Perilla frutescens on nitric oxide production and DNA synthesis in cultured murine vascular smooth muscle cells.

The effects of perilla (Perilla frutescens, Labiatae) on murine cultured vascular smooth muscle cells (VSMC) were investigated. The water extract of perilla leaves induced nitric oxide (NO) production of VSMC and this effect was synergistically augmented when combined with interferon (IFN)-gamma or tumour necrosis factor (TNF)-alpha, while the perilla extract significantly inhibited NO production induced by IFN-gamma combined with lipopolysaccharide (LPS). Northern blot analysis revealed that these effects of the perilla extract paralleled mRNA expression of inducible nitric oxide synthase. However, the perilla extract significantly inhibited platelet derived growth factor (PDGF) or TNF-alpha-induced VSMC proliferation measured as DNA synthesis. The inhibitory effect of the perilla extract on TNF-alpha-induced VSMC proliferation was significantly suppressed by N(G)-monomethyl-L-arginine, a non-specific nitric synthase inhibitor, suggesting that this effect was partially mediated by NO production as an autocrine/paracrine factor. The present findings suggest that perilla would be useful for the prevention of vascular diseases such as arteriosclerosis.

Dehydroepiandrosterone inhibits human vascular smooth muscle cell proliferation independent of ARs and ERs.

Dehyroepiandrosterone (DHEA), an adrenal-derived steroid, has been clinically implicated in protection against coronary artery disease and experimentally in inhibition of atherosclerosis and plaque progression. Because DHEA is enzymatically metabolized to androgens or estrogens, it is not clear whether DHEA exerts effects directly or after conversion to these hormones, both of which are associated with well-characterized pathways of action. We therefore examined the effects of DHEA on proliferation of human vascular smooth muscle cells (VSMCs) in culture in the presence or absence of the ER antagonist ICI 182,780 and the AR antagonist flutamide and compared them with the effects of 17beta-estradiol, androstenedione, and T. We also determined the affinity of DHEA for ERs and ARs in VSMC and its specific binding in intact cells. To explore a possible mechanism for DHEA action in these cells, we measured the phosphorylation of ERK-1, c-jun N-terminal protein kinase, and p38 (three members of the MAPK superfamily). Both DHEA and 17beta-estradiol significantly inhibited platelet derived growth factor (PDGF)-BB-induced increases in VSMC proliferation, whereas androstenedione and T increased proliferation. Although E2-induced inhibition of the PDGF effect was abolished by ICI 182,780 and T-induced stimulation was abolished by flutamide, neither receptor antagonist altered the inhibitory effect of DHEA. Binding studies confirmed the presence of both ERs and ARs; DHEA showed minimal affinity for either receptor but bound specifically and with high affinity to putative receptors in intact cells. Following 4-h incubation with DHEA (1-100 nM), ERK1 phosphorylation was significantly reduced in a dose-dependent manner, whereas neither c-jun N-terminal protein kinase nor p38 kinase activity was altered by either PDGF-BB or DHEA. DHEA inhibits human VSMC proliferation by a mechanism independent of either ARs or ERs, presumably via a DHEA-specific receptor that involves ERK1 signaling pathways.

Low density lipoprotein receptor-related protein mediates apolipoprotein E inhibition of smooth muscle cell migration.

This research was undertaken to identify the cell surface receptor responsible for mediating apolipoprotein E (apoE) inhibition of platelet-derived growth factor (PDGF)-directed smooth muscle cell migration. Initial studies revealed the expression of the low density lipoprotein receptor (LDLR), the LDL receptor-related protein (LRP), the very low density lipoprotein receptor (VLDL), and apoE receptor-2 in mouse aortic smooth muscle cells. Smooth muscle cells isolated from LDLR-null, VLDL-null, and apoE receptor-2-null mice were responsive to apoE inhibition of PDGF-directed smooth muscle cell migration, suggesting that these receptors were not involved. An antisense RNA expression knockdown strategy, utilizing morpholino antisense RNA against LRP, was used to reduce LRP expression in smooth muscle cells to assess the role of this receptor in apoE inhibition of cell migration. Results showed that apoE was unable to inhibit PDGF-directed migration of LRP-deficient smooth muscle cells. The role of LRP in mediating apoE inhibition of PDGF-directed smooth muscle cell migration was confirmed by experiments showing that antibodies against LRP effectively suppressed apoE inhibition of PDGF-directed smooth muscle cell migration. Taken together, these results document that apoE binding to LRP is required for its inhibition of PDGF-directed smooth muscle cell migration.

Application of LC/MS/MS in the quantitation of SU101 and SU0020 uptake by 3T3/PDGFr cells

SU101 or leflunomide, has been studied extensively because of its anti-cancer and immunomodulating properties. The parent isoxazole compound is converted in vitro and metabolized in vivo to an open ring isomeric form, SU0020. Several pharmacological activities have been reported for the parent and metabolite compounds including inhibition of platelet-derived growth factor (PDGF)-mediated signaling for the parent compound and inhibition of de novo pyrimidine biosynthesis for the metabolite. The inhibition of PDGF-mediated signaling and the anti-tumor properties have been ascribed to the parent compound. In spite of its short plasma half-life of the parent molecule, SU101 can be administered intermittently in animal tumor models and retain efficacy. Therefore, the relationship between plasma levels of SU101 and its efficacy in tumor-implanted immuno-compromised mice is not well established. This study was conducted to assess the concentration of SU101 in 3T3/PDGFr α and β cells (NIH3T3 mouse fibroblasts engineered to overexpress human PDGFr α or β) to better understand the cellular levels of SU101 and SU0020. Two strains of 3T3/PDGFr cells (α and β) were incubated with 1, 25, and 100 μM concentrations of SU101 for 1, 6, 24, and 48 hours. Quantitation of SU101 and SU0020 in these cell lines was achieved by a specific and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method. Interestingly, in both α and β cell lysates SU101 was much more concentrated than SU0020. The greater concentration of SU101 versus SU0020 that was observed may be due to the preferential partitioning of SU101 into the cells and this shows that significant levels of the parent drug can reach the pharmacological site of action for inhibition of PDGF receptors. The data suggest that the conversion of SU101 to SU0020 is much slower in these cells than in the incubation media.

PPARγ Expression and Function in Mesangial Cells: Implication for Renal Disease

7 Dominant negative mutations which inactivate the peroxisome proliferator-activated receptor gamma (PPARγ) nuclear receptor in man are associated with severe insulin resistance, diabetes and hypertension (Barroso I et al. Nature, 1999;402:880-883). Using RNase protection assay, Western blot analysis and immunostaining of cultured primary rat mesangial cells, we identified the presence of PPARγ message and protein. Electrophoretic mobility of a labeled PPARγ response element (PPRE) was retarded in the presence of mesangial cell nuclear extract, suggesting that PPARγ is functional in these cells. Addition of unlabelled PPRE efficiently competed away the PPARγ-PPRE protein complex, confirming specificity of binding of the PPARγ to the PPRE. PPARγ ligands, rosiglitazone (10 μM) and troglitazone (10 μM), both completely inhibited platelet derived growth factor (PDGF)-induced DNA synthesis measured as bromodeoxyuridine (BrdU) incorporation (p

PPARγ Expression and Function in Mesangial Cells: Implication for Renal Disease

7 Dominant negative mutations which inactivate the peroxisome proliferator-activated receptor gamma (PPARγ) nuclear receptor in man are associated with severe insulin resistance, diabetes and hypertension (Barroso I et al. Nature, 1999;402:880-883). Using RNase protection assay, Western blot analysis and immunostaining of cultured primary rat mesangial cells, we identified the presence of PPARγ message and protein. Electrophoretic mobility of a labeled PPARγ response element (PPRE) was retarded in the presence of mesangial cell nuclear extract, suggesting that PPARγ is functional in these cells. Addition of unlabelled PPRE efficiently competed away the PPARγ-PPRE protein complex, confirming specificity of binding of the PPARγ to the PPRE. PPARγ ligands, rosiglitazone (10 μM) and troglitazone (10 μM), both completely inhibited platelet derived growth factor (PDGF)-induced DNA synthesis measured as bromodeoxyuridine (BrdU) incorporation (p<0.05). When administered in anti-diabetic doses to streptozotocin-induced diabetic rats, troglitazone substantially normalized albumin excretion at 4 months (from 687.1 to 137.6 μg urinary albumin/mg creatinine, p<0.05), but did not affect hyperglycemia or hypertension in this model. These data suggest PPARγ activation may have direct effects to attenuate diabetic glomerular disease, possibly by inhibiting mesangial growth, which occurs early in the process of diabetic nephropathy. These results have therapeutic implications for diabetic glomerulosclerosis as well as proliferative mesangial diseases of the kidney.

Pyrrolo[3,4- c]-β-carboline-diones as a novel class of inhibitors of the platelet-derived growth factor receptor kinase

Members of the structurally diverse family of β-carbolines have previously been shown to exhibit a wide range of biological activities. A novel synthetic strategy for generation of β-carbolines was developed, allowing imido-β-carbolines to be created in three steps from known compounds. The compounds were screened for inhibition of platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation in Swiss 3T3 fibroblasts. A number of the newly synthesized β-carbolines with moderate to potent inhibitory activity were revealed. The most active derivative, 2,3-dihydro-8,9-dimethoxy-5-(2-methylphenyl)-1 H,6 H-pyrrolo[3,4- c]pyrido[3,4- b]indole-1,3-dione 2ee, inhibited purified PDGF receptor kinase and PDGF-receptor autophosphorylation in intact cells with IC 50 values of 0.4 and 2.6 μM, respectively. Dione 2ee also inhibited PDGF-stimulated DNA synthesis in Swiss 3T3 fibroblasts with an IC 50 of 3.2 μM. The compound had no effect on Src or epidermal growth factor (EGF) receptor kinase activity and a six–seven-fold higher IC 50 for inhibition of basic fibroblast growth factor (bFGF)-stimulated tyrosine phosphorylation or Kit/stem cell factor (SCF) receptor autophosphorylation, indicating a reasonable extent of kinase specificity. Thus, β-carbolines present a new lead of tyrosine kinase inhibitors with the capacity to selectively interfere with PDGF receptor signal transduction and PDGF-dependent cell growth.

Platelet-derived growth factor activates porcine thecal cell phosphatidylinositol-3-kinase-Akt/PKB and ras-extracellular signal-regulated kinase-1/2 kinase signaling pathways via the platelet-derived growth factor-beta receptor.

Platelet-derived growth factor (PDGF) is a potent mitogenic factor for ovarian thecal cells cultured in vitro. PDGF binds to and induces homo- or heterodimerization of PDGF receptor-a or -beta (PDGF-Ralpha or PDGF-Rbeta). Despite this, little information is available about which PDGF receptors are expressed in the ovary, what signaling cascades are activated by PDGF, and the effects of PDGF on thecal cell steroidogenesis. The present study demonstrates the expression of immunoreactive PDGF-Rbeta, but not PDGF-Ralpha, in the thecal and stromal compartments of intact porcine ovaries as well as in cultured porcine thecal cells. Treatment of porcine thecal cells in vitro with PDGF resulted in rapid and sustained tyrosine phosphorylation of PDGF-Rbeta, activation of Src tyrosine kinase and phosphatidylinositol-3-kinase (PI3-kinase), and serine 473 phosphorylation of Akt/protein kinase B. In addition, PDGF stimulated an increase in GTP-Ras (activated Ras) and extracellular signal-regulated kinase (ERK) phosphorylation. Both forms of PDGF, AB and BB, stimulated thecal cell growth approximately 3- to 4-fold over controls and inhibited LH-stimulated progesterone and androstenedione secretion. Blockade of PI3-kinase activation with wortmannin had no effect on PDGF-stimulated thecal cell growth or PDGF inhibition ofLH-stimulated steroid secretion, indicating that PI3-kinase activation is not necessary for PDGF-stimulated thecal cell growth or inhibition of LH-stimulated steroidogenesis. Conversely, blockade of the MEK-ERK pathway with PD98059 completely blocked PDGF-stimulated cell growth, indicating that activation of the MEK-ERK pathway is required for PDGF-stimulated thecal cell growth. Additionally, the MEK inhibitor PD98059 restored LH-stimulated steroid secretion, demonstrating that activation of the MEK-ERK pathway can lead to inhibition of LH-stimulated steroid secretion. The present study demonstrates that PDGF acts on ovarian thecal cells via activation of the PDGF beta-receptor and stimulates thecal cell growth via activation of a Rasmitogen-activated protein kinase-dependent, PI3-kinase-independent pathway. The strong expression of PDGF-Rbeta and the potent effects of PDGF on thecal cell growth and steroidogenesis suggest an important role for PDGF in thecal cell recruitment and growth during follicular development in vivo.

Small Mechanical Strains Selectively Suppress Matrix Metalloproteinase-1 Expression by Human Vascular Smooth Muscle Cells

Mechanical forces and biochemical stimuli may interact to regulate cellular responses. In this study, we tested the hypothesis that very small mechanical strains interact with growth factors in the regulation of matrix metalloproteinase (MMP)-1. Human vascular smooth muscle cells (VSMCs) were cultured on a precoated silicone membrane in a device that imposes a highly uniform biaxial strain. VSMCs cultured on fibronectin were treated with cyclic 1-Hz strains of 0, 1, or 4%, and MMPs were assayed by Western analysis or gelatin zymography. Small strains did not induce MMP-1 in VSMCs, but strain was a potent inhibitor of platelet-derived growth factor (PDGF)- or tumor necrosis factor-alpha-induced synthesis of MMP-1. In contrast, MMP-2 and TIMP-2 levels were not changed by PDGF and/or mechanical strain. VSMCs strained on the 120-kDa chymotryptic fragment of fibronectin or RGD peptides suppressed PDGF-induced expression of MMP-1, indicating that this effect is not mediated by the heparin-binding domain or connecting segment-1 of fibronectin. Northern analysis of ets-1, a transcriptional activator of MMP-1 expression, showed that strain down-regulated ets-1 expression, whereas c-fos expression was augmented. Thus, small deformations can selectively suppress MMP-1 synthesis by VSMCs, demonstrating the exquisite sensitivity of the cell to mechanical stimuli.

Effect of herbimycin A on tyrosine kinase receptors and platelet derived growth factor (PDGF)-induced signal transduction.

Herbimycin A is widely used as an inhibitor of Src family protein tyrosine kinases but is also reported to induce the downregulation of epidermal growth factor (EGF) receptor number in A431 cells without inhibiting its tyrosine kinase activity. To determine the specificity of the receptor downregulation, we examined its effect on a variety of cell lines which express different levels of EGF receptor and on other tyrosine kinase receptors. Long-term herbimycin A treatment decreased the amounts of all the tyrosine kinase receptors examined in a dose-dependent manner. It also reduced ligand-stimulated receptor autophosphorylation in accordance with the reduction in the receptor level. Herbimycin A inhibited platelet derived growth factor (PDGF)-induced tyrosine phosphorylation of cellular proteins and DNA synthesis in NIH3T3 cells but did not affect the serum-stimulated DNA synthesis. PDGF-induced tyrosine phosphorylation and activation of c-Src was inhibited but the protein level of c-Src was not reduced by herbimycin A. The reduced level of c-Src kinase activity correlated with the levels of both PDGF receptor and DNA synthesis. These results indicate that the herbimycin A treatment selectively downregulates receptor tyrosine kinases, independent of the number of receptors, and suggest that c-Src is to some degree involved in the selective inhibition of PDGF-induced mitogenesis by herbimycin A.

Identification of a cyclic peptide inhibitor of platelet-derived growth factor–BB receptor-binding and mitogen-induced DNA synthesis in human fibroblasts

Peptides corresponding to residues from Loops I and III of platelet-derived growth factor–BB (PDGF-BB) were examined for their potential to act as PDGF antagonists. We have identified two peptides which directly stimulated DNA synthesis in human dermal fibroblasts and a cyclic peptide which inhibited PDGF-induced DNA synthesis. The inhibitory action of cyclic PDGF-BB 73-81, on DNA synthesis was shown to be restricted to cells which express PDGF receptors. Also cyclic PDGF-BB 73-81 specifically competed for 125I-labelled PDGF-BB but not for 125I-labelled EGF binding to their respective cellular receptors. The cyclic peptide therefore provides a minimum structure to investigate PDGF/receptor interactions and our findings confirm the importance of the loop configuration of PDGF-BB 73-81 in the native molecule. The cyclic peptide may constitute a basis for developing more potent inhibitors of PDGF action.

Selective Inhibition of Platelet-Derived Growth Factor (PDGF) Receptor Autophosphorylation and PDGF-Mediated Cellular Events by a Quinoline Derivative

This report describes the biological effects of our original compound, Ki6783 ((3,4-dimethoxy)-4-phenoxy-6,7-dimethoxyquinoline), a potent and selective inhibitor of platelet-derived growth factor (PDGF) receptor autophosphorylation. This compound strongly inhibited autophosphorylation of the PDGF β-receptor in cultured rat glomerular mesangial cells (MC) bearing this receptor (IC500.1 μM), although it did not inhibit autophosphorylation of other growth factor receptors even at 100 μM.In a cell-free kinase experiment, it showed selective inhibition of PDGF β-receptor tyrosine kinase. A kinetic study of the compound to this tyrosine kinase revealed a competitive mode of action to ATP. [3H]Thymidine incorporation and cell proliferation of MC were inhibited by Ki6783 in a dose-dependent manner after Ki6783 and PDGF-BB were added to the culture medium. Furthermore, this compound normalized the fibrotic cell shape of v-sis-transformed NIH3T3 cells, which grow in an autocrine manner via the PDGF receptor. These effects could be explained by the inhibition of intracellular signal transduction triggered by PDGF receptor autophosphorylation, in which activation of mitogen-activated protein kinase occurs. These results suggest that Ki6783 is one of the more potent and selective inhibitors of PDGF receptor autophosphorylation and that it may be useful in ameliorating cell abnormalities due to excess action of PDGF and its receptor systems in several diseases.

Mesangial cell proliferation mediated by PDGF and bFGF is determined by levels of the cyclin kinase inhibitor p27Kip1

Mesangial cell proliferation in vitro is regulated by many cytokines. Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are potent mesangial cell mitogens, whereas transforming growth factor-beta1 (TGF-beta1) reduces their effects. We examined how these cytokines regulate rat mesangial cell proliferation at the level of the cell-cycle. Quiescent mesangial cells in vitro express the cyclin kinase inhibitor, p27Kip1 (p27), and PDGF- and bFGF-induced mesangial cell proliferation is associated with a substantial decrease in p27 levels. Consequently there is a marked increase in expression (Western blot analysis, immunostaining) of cyclin A and CDK2. The decline in p27 levels was prevented by TGF-beta1 during inhibition of PDGF- and bFGF-induced mesangial cell proliferation. To determine the functional role of p27 during cytokine-mediated mesangial cell proliferation, the expression of p27 was reduced with specific p27Kip1 antisense oligodeoxynucleotides. Reducing the levels of p27 resulted in an increased magnitude of mesangial cell proliferation (BrdU and 3H-thymidine incorporation) induced by PDGF and bFGF compared to non-transfected mesangial cells and mesangial cells transfected with control mismatch oligodeoxynucleotides. Furthermore, the onset of maximal proliferation occurred earlier in mesangial cells transfected with antisense compared to control. The reduction in proliferation by TGF-beta1 were not altered by decreased p27 expression. Reducing p27 expression in the absence of mitogens was not associated with entry into the cell-cycle. These results suggest cytokine mediated mesangial cell proliferation is associated with specific cell-cycle proteins, and that the levels of p27 may be important in determining the mesangial cell's proliferative response to PDGF and bFGF in vitro.

Role of platelets in progressive glomerular diseases.

There is increasing evidence that platelets are involved in the pathogenesis of glomerulonephritis. Intraglomerular platelets or their degradation products are observed in biopsies from patients with lupus nephritis, mesangioproliferative, membranous or IgA nephropathy. Moreover shortened platelet survival in patients with various glomerular diseases has also been described. In models of experimental glomerulonephritis, platelets may participate in glomerular injury, together with other mediators, by complex mechanisms. As extensively documented, platelets release within the glomerulus vasoactive, chemotactic and mitogenic substance that interact with a number of soluble mediators generated by renal resident or inflammatory cells and contribute to amplify glomerular injury. Thus platelet-activating factor and other platelet secretory products, polycationic macromolecules, platelet factor 4 and beta-thromboglobulin, alter glomerular permeability to proteins and enhance immune-mediated glomerular injury. Platelet-derived factors, like platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF beta) mediate renal disease progression in experimental and human glomerulonephritis via their chemotactic activity for infiltrating leucocytes and their effect of promoting extracellular matrix synthesis by resident renal cells. In these settings increased renal expression of PDGF and TGF beta has correlated with clinical features. Specific PDGF and TGF beta inhibitors ameliorated experimental glomerular disease. A wide variety of therapies to inhibit platelet function have been employed over the years, however the results of clinical studies are controversial and do not allow conclusions to be drawn about the efficacy of anti-platelet agents in progressive renal disease. Identification of specific platelet inhibitors or interventions specific for platelet secretory products and their target cells will be crucial for understanding the exact role of platelets and their products in glomerular disease.

Effect of SR 33805 on arterial smooth muscle cell proliferation and neointima formation following vascular injury

The possible activity of SR 33805 ([[ N-[dimethoxy-3,4-phenethyl]- N-methylamino-propoxyl]-4-benzenesulfonyl]-2-isopropyl-3-methyl-1-indole), a novel Ca 2+ channel blocker, in early atherogenesis was investigated. In vitro, SR 33805 strongly inhibited fetal calf serum-induced proliferation of cultured human aortic smooth muscle cells with an IC 50 value of 0.3 ± 0.1 μM ( n = 3). In this respect, SR 33805 was several fold more active than the reference compounds: diltiazem, verapamil, nifedipine and fantofarone. SR 33805 was also a potent inhibitor of platelet-derived growth factor- or basic fibroblast growth factor-induced proliferation of human smooth muscle cells. SR33805 inhibited serum-stimulated 45Ca 2+ uptake in these cells, with an IC 50 value of 47 ± 18 nM. The effect of SR 33805 on intimal smooth muscle hyperplasia in rabbit carotid arteries subjected to air-drying endothelial injury was then investigated. After a 16-day treatment, SR 33805 (6.0 mg/kg/day p.o.) inhibited the development of intimal thickening. Under the same experimental conditions, nifedipine, verapamil, diltiazem (2 × 6 mg/kg/day p.o. - 16 days) and fantofarone (12 mg/kg/day p.o. - 16 days) were inactive. These results show that SR 33805, a novel and potent Ca 2+ channel blocker, can reduce myointimal thickening following endothelial injury.

3BP-1, an SH3 domain binding protein, has GAP activity for Rac and inhibits growth factor-induced membrane ruffling in fibroblasts.

The SH3 binding protein, 3BP-1, was originally cloned as a partial cDNA from an expression library using the Abl SH3 domain as a probe. In addition to an SH3 binding domain, 3BP-1 displayed homology to a class of GTPase activating proteins (GAPs) active against Rac and Rho proteins. We report here a full length cDNA of 3BP-1 which extends the homology to GAP proteins previously noted. 3BP-1 functions in vitro as a GAP with a specificity for Rac-related G proteins. Microinjection of the 3BP-1 protein into serum-starved fibroblasts produces an inhibition of platelet-derived growth factor (PDGF)-induced membrane ruffling mediated by Rac. Co-injection of 3BP-1 with an activated Rac mutant that is unresponsive to GAPs, counter-acts this inhibition. 3BP-1 does not show in vitro activity towards Rho and, in agreement with this finding, microinjection of 3BP-1 into fibroblasts has no effect on lysophosphatidic acid (LPA)-induced stress fiber assembly mediated by Rho. Thus 3BP-1 is a new and specific Rac GAP that can act in cells to counter Rac-mediated membrane ruffling. How its SH3 binding site interacts with its GAP activity remains to be understood.

Inhibition of platelet-derived growth factor- and epidermal growth factor-mediated mitogenesis and signaling in 3T3 cells expressing delta Raf-1:ER, an estradiol-regulated form of Raf-1.

We have recently described the properties of delta Raf-1:ER, a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the human estrogen receptor. In this study, we demonstrate that activation of delta Raf-1:ER in quiescent 3T3 cells (C2 cells), while sufficient to promote morphological oncogenic transformation, was insufficient to promote the entry of cells into DNA synthesis. Indeed, activation of delta Raf-1:ER potently inhibited the mitogenic response of cells to platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) treatment. Addition of beta-estradiol to quiescent C2 cells led to rapid, sustained activation of delta Raf-1:ER and MEK but only two- to threefold activation of p42 mitogen-activating protein (MAP) kinase activity. Addition of PDGF or EGF to quiescent C2 cells in which delta Raf-1:ER was inactive led to rapid activation of Raf-1, MEK, and p42 MAP kinase activities, and entry of the cells into DNA synthesis. In contrast, when delta Raf-1:ER was activated in quiescent C2 cells prior to factor addition, there was a significant inhibition of certain aspects of the signaling response to subsequent treatment with PDGF or EGF. The expression and activation of PDGF receptors and the phosphorylation of p70S6K in response to PDGF treatment were unaffected by prior activation of delta Raf-1:ER. In contrast, PDGF-mediated activation of Raf-1 and p42 MAP kinases was significantly inhibited compared with that of controls. Interestingly, the mitogenic and signaling responses of quiescent C2 cells to stimulation with fetal bovine serum or phorbol myristate acetate were unaffected by prior activation of delta Raf-1:ER. It seems likely that at least two mechanisms contribute to the effects of delta Raf-1:ER in these cells. First, activation of delta Raf-1:ER appeared to uncouple the activation of Raf-1 from the activation of the PDGF receptor at the cell surface. This may be due to the fact that mSOS1 is constitutively phosphorylated as a consequence of the activation of delta Raf-1:ER. Second, quiescent C2 cells expressing activated delta Raf-1:ER appear to contain an inhibitor of the MAP kinase pathway that, because of its apparent sensitivity to sodium orthovanadate, may be a phosphotyrosine phosphatase. It is likely that the inhibitory effects of delta Raf-1:ER observed in these cells are a manifestation of the activation of some of the feedback inhibition pathways that normally modulate a cell's response to growth factors. 3T3 cells expressing delta Raf-1:ER will be a useful tool in unraveling the role of Raf-1 kinase activity in the regulation of such pathways.

Inhibition of platelet-derived growth factor- and epidermal growth factor-mediated mitogenesis and signaling in 3T3 cells expressing delta Raf-1:ER, an estradiol-regulated form of Raf-1.

We have recently described the properties of delta Raf-1:ER, a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the human estrogen receptor. In this study, we demonstrate that activation of delta Raf-1:ER in quiescent 3T3 cells (C2 cells), while sufficient to promote morphological oncogenic transformation, was insufficient to promote the entry of cells into DNA synthesis. Indeed, activation of delta Raf-1:ER potently inhibited the mitogenic response of cells to platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) treatment. Addition of beta-estradiol to quiescent C2 cells led to rapid, sustained activation of delta Raf-1:ER and MEK but only two- to threefold activation of p42 mitogen-activating protein (MAP) kinase activity. Addition of PDGF or EGF to quiescent C2 cells in which delta Raf-1:ER was inactive led to rapid activation of Raf-1, MEK, and p42 MAP kinase activities, and entry of the cells into DNA synthesis. In contrast, when delta Raf-1:ER was activated in quiescent C2 cells prior to factor addition, there was a significant inhibition of certain aspects of the signaling response to subsequent treatment with PDGF or EGF. The expression and activation of PDGF receptors and the phosphorylation of p70S6K in response to PDGF treatment were unaffected by prior activation of delta Raf-1:ER. In contrast, PDGF-mediated activation of Raf-1 and p42 MAP kinases was significantly inhibited compared with that of controls. Interestingly, the mitogenic and signaling responses of quiescent C2 cells to stimulation with fetal bovine serum or phorbol myristate acetate were unaffected by prior activation of delta Raf-1:ER. It seems likely that at least two mechanisms contribute to the effects of delta Raf-1:ER in these cells. First, activation of delta Raf-1:ER appeared to uncouple the activation of Raf-1 from the activation of the PDGF receptor at the cell surface. This may be due to the fact that mSOS1 is constitutively phosphorylated as a consequence of the activation of delta Raf-1:ER. Second, quiescent C2 cells expressing activated delta Raf-1:ER appear to contain an inhibitor of the MAP kinase pathway that, because of its apparent sensitivity to sodium orthovanadate, may be a phosphotyrosine phosphatase. It is likely that the inhibitory effects of delta Raf-1:ER observed in these cells are a manifestation of the activation of some of the feedback inhibition pathways that normally modulate a cell's response to growth factors. 3T3 cells expressing delta Raf-1:ER will be a useful tool in unraveling the role of Raf-1 kinase activity in the regulation of such pathways.