Platelet-derived Growth Factor Homodimer Research Articles

Wnt-GSK3β/β-Catenin Regulates the Differentiation of Dental Pulp Stem Cells into Bladder Smooth Muscle Cells.

Smooth muscle cell- (SMC-) based tissue engineering provides a promising therapeutic strategy for SMC-related disorders. It has been demonstrated that human dental pulp stem cells (DPSCs) possess the potential to differentiate into mature bladder SMCs by induction with condition medium (CM) from bladder SMC culture, in combination with the transforming growth factor-β1 (TGF-β1). However, the molecular mechanism of SMC differentiation from DPSCs has not been fully uncovered. The canonical Wnt signaling (also known as Wnt/β-catenin) pathway plays an essential role in stem cell fate decision. The aim of this study is to explore the regulation via GSK3β and associated downstream effectors for SMC differentiation from DPSCs. We characterized one of our DPSC clones with the best proliferation and differentiation abilities. This stem cell clone has shown the capacity to generate a smooth muscle layer-like phenotype after an extended differentiation duration using the SMC induction protocol we established before. We further found that Wnt-GSK3β/β-catenin signaling is involved in the process of SMC differentiation from DPSCs, as well as a serial of growth factors, including TGF-β1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor-homodimer polypeptide of B chain (BB) (PDGF-BB), and vascular endothelial growth factor (VEGF). Pharmacological inhibition on the canonical Wnt-GSK3β/β-catenin pathway significantly downregulated GSK3β phosphorylation and β-catenin activation, which in consequence reduced the augmented expression of the growth factors (including TGF-β1, HGF, PDGF-BB, and VEGF) as well as SMC markers (especially myosin) at a late stage of SMC differentiation. These results suggest that the canonical Wnt-GSK3β/β-catenin pathway contributes to DPSC differentiation into mature SMCs through the coordination of different growth factors.

Influence of Biochemical Cues in Human Corneal Stromal Cell Phenotype

ABSTRACTPurpose: To identify biochemical cues that could promote a keratocyte-like phenotype in human corneal stromal cells that had become fibroblastic when expanded in serum-supplemented media while also examining the effect on cell proliferation and migration.Methods: Proliferation was assessed by PrestoBlue™, morphology was monitored by phase contrast microscopy, phenotype was analyzed by real-time polymerase chain reaction (qPCR), immunochemistry and flow cytometry, and migration was studied with a scratch assay.Results: Ascorbic Acid (AA), Retinoic Acid (RA), Insulin-Transferrin-Selenium (ITS), Insulin-like Growth Factor 1 (IGF-1) and 3-isobutyl-1-methylxanthine (IBMX) promoted a dendritic morphology, increased the expression of keratocyte markers, such as keratocan, aldehyde dehydrogenase 3 family member A1 (ALDH3A1) and CD34, and prevented myofibroblast differentiation, while in some cases increasing proliferation. Transforming Growth Factor beta 1 (TGF-β1) and 3 (TGF-β3) promoted the differentiation toward myofibroblasts, with increased expression of α-SMA. Fibroblast Growth Factor 2 (FGF-2) supported a fibroblastic phenotype while Platelet-Derived Growth Factor Homodimer B (PDGF-BB) induced a pro-migratory fibroblastic phenotype. A combination of all the pro-keratocyte factors was also compared to the serum-free only, which significantly increased CD34 and keratocan expression.Conclusions: Partially recovery towards a quiescent keratocyte-like phenotype was achieved by the removal of serum and the addition of AA, IGF-1, RA, ITS and IBMX to a basal medium. These findings can be used to develop cell-based corneal therapies and to study corneal diseases in vitro.

Phloretin Inhibits Platelet-derived Growth Factor-BB-induced Rat Aortic Smooth Muscle Cell Proliferation, Migration, and Neointimal Formation After Carotid Injury.

Abnormal vascular smooth muscle cell proliferation and migration are key factors in many cardiovascular diseases. Here, we investigated the effects of phloretin on platelet-derived growth factor homodimer (PDGF-BB)-induced rat aortic smooth muscle cell (RASMC) proliferation, migration, and neointimal formation after carotid injury. Phloretin significantly inhibited the PDGF-BB-stimulated RASMC proliferation in a concentration-dependent manner (10-100 μM). Also, PDGF-BB-stimulated RASMC migration was inhibited by phloretin at 50 μM. Pretreating RASMC with phloretin dose-dependently inhibited PDGF-BB-induced Akt and p38 mitogen-activated protein kinases activation. Furthermore, phloretin increased p27 and decreased cyclin-dependent kinase 2, CDK4 expression, and p-Rb activation in PDGF-BB-stimulated RASMC in a concentration-dependent manner (10-50 μM). PDGF-BB-induced cell adhesion molecules and matrix metalloproteinase-9 expression were blocked by phloretin at 50 μM. Preincubation with phloretin dose-dependently reduced the intracellular reactive oxygen species production. In vivo study showed that phloretin (20 mg/kg) significantly reduced neointimal formation 14 days after carotid injury in rats. Thus, phloretin may have potential as a treatment against atherosclerosis and restenosis after vascular injury.

Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-β and VEGFR-2 expression

Despite intensive research studies, theories have yet to focus on the contribution of hypoxia to patency differences observed clinically between arterial vs. venous grafts. This study investigates the differential hypoxic response of smooth muscle cells (SMC) to hypoxia-derived endothelial cell (EC) growth factors. Initiation of SMC proliferation under hypoxia (<5% O(2)) occurred only after incubation with hypoxic endothelial cell-conditioned media (H-ECM). After the investigation of several possible growth factors in the H-ECM that may be responsible for SMC proliferation, the greatest difference was observed in vascular endothelial growth factor (VEGF-A) and platelet-derived growth factor homodimer B (PDGF-BB) expression. VEGF-A increased (2-fold) significantly (P < 0.05) in arterial-derived smooth muscle cells (ASMC) under hypoxia compared with venous-derived smooth muscle cells (VSMC), which showed no significant change. VSMC showed significant (P < 0.05) increase in VEGFR-2 expression under hypoxia compared with ASMC. Incubation with VEGFR-2-neutralizing antibody/PDGFR antagonist in VSMC before addition of H-ECM resulted in decreased proliferation. ASMC proliferation under hypoxia did not decrease during incubation with VEGFR-2-neutralizing antibody but did decrease upon PDGFR antagonist incubation. Current therapies focusing on treating intimal hyperplasia have negated the fact that combinational therapy might be required to combat induction of SMC proliferation. Clinically, therapy with PDGFR antagonists plus anti-VEGFR-2 may prove to be efficacious in managing SMC proliferation in venous-derived grafts.

Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Bladder Cells: Potential for Urological Tissue Engineering

Bone marrow mesenchymal stem cells (BMSCs) are capable of differentiating into multiple cell types, providing an alternative cell source for cell-based therapy and tissue engineering. Simultaneous differentiation of human BMSCs into smooth muscle cells (SMCs) and urothelium would be beneficial for clinical applications in bladder regeneration for patients with bladder exstrophy or cancer who need cystoplasty. We investigated the ability of human BMSCs to differentiate toward both SMCs and urothelium with cocultured or conditioned media and analyzed growth factors from a coculture system. After being cocultured with urothelium or cultured using urothelium-derived conditioned medium, human BMSCs expressed urothelium-specific genes and proteins: uroplakin-Ia, cytokeratin-7, and cytokeratin-13. When cocultured with SMCs or cultured in SMC-conditioned medium, human BMSCs expressed SMC-specific genes and proteins: desmin and myosin. Several growth factors (hepatocyte growth factor, platelet-derived growth factor-homodimer polypeptide of B chain (BB), transforming growth factor-beta1, and vascular endothelial growth factor) were detected in the SMC cocultured media and in the urothelium cocultured media (epidermal growth factor, platelet-derived growth factor-BB, transforming growth factor-beta1, and vascular endothelial growth factor). BMSC-scaffold constructs significantly improved cell contractility after myogenic differentiation. In vivo-grafted cells displayed significant matrix infiltration and expressed SMC-specific markers in the nanofibrous poly-l-lactic acid scaffolds. In conclusion, smooth muscle- and urothelium-like cells derived from human BMSCs provide an alternative cell source for potential use in bladder tissue engineering.

Interactive effects of growth factors and three‐dimensional scaffolds on multipotent mesenchymal stromal cells

The creation of tissue-engineered constructs with autologous cells is a central goal in regenerative medicine. With respect to ligament replacement, we have evaluated the influences of matrix and growth factors on hMSCs (human mesenchymal stromal cells). hMSCs were seeded in two different 3D (three-dimensional) systems consisting of either a collagen type I gel or a synthetic PLA [poly-(L-lactic acid)] scaffold. After cultivation for 14 days with rhTGFbeta1 (recombinant human transforming growth factor beta1), rhPDGF-BB (recombinant human platelet-derived growth factor homodimer of B-chain) or rhBMP13 (recombinant human bone morphogenetic protein 13), we assessed the proliferation potential, mRNA expression and protein expression of various matrix-interacting and matrix-degrading molecules by quantitative real-time RT (reverse transcriptase)-PCR, immunohistochemistry and gelatin zymography in comparison with unstimulated cells. Cellular reactions to the type of scaffold or soluble factors could be found in the expression of tenascin-C as well as integrin subunits alpha1, alpha3 and beta1. Collagen type X expression was induced by 3D culture and stimulated by rhTGFbeta1 on PLA. The expression of MMP-1 (matrix metalloproteinase 1) tended to increase, and MMP-13 was induced in the collagen culture system. The activation of MMP-2 was stimulated by the cultivation of MSCs within the collagenous matrix. These results demonstrated that various interactive effects of growth factors and scaffolds influence the cell-biological behaviour of MSCs. It is important to take these complex interactions, which partly differ from differentiated cells, into account in further tissue-engineering approaches.

Enhancement by PL 14736 of granulation and collagen organization in healing wounds and the potential role of egr-1 expression

Apart from becaplermin (recombinant human platelet-derived growth factor homodimer of B chains, PDGF-BB), for the treatment of lower extremity diabetic ulcers, few agents are available for pharmacological stimulation of wound healing. We have compared the mechanism of action of the potential wound healing agent, PL 14736 (G E P P P G K P A D D A G L V), with that of PDGF-BB on granulation tissue formation following sponge implantation in the normoglycemic rat and in healing full-thickness excisional wounds in db/db genetically diabetic mice. Expression of the immediate response gene, early growth response gene-1 ( egr-1) was studied in Caco-2 cells in vitro. While PDGF-BB and PL 14736 had similar selectivity for stimulation of granulation tissue in both sponge granuloma and in healing wounds in db/db mice, PL 14736 was more active in stimulating early collagen organization. It also stimulated expression of egr-1 and its repressor nerve growth factor 1-A binding protein-2 ( nab2) in non-differentiated Caco-2 cells more rapidly than PDGF-BB. EGR-1 induces cytokine and growth factor generation and early extracellular matrix (collagen) formation, offering an explanation for the beneficial effects of PL 14736 on wound healing.

Characterisation and application of antibodies specific for the long platelet-derived growth factor A and B chains

The platelet-derived growth factor (PDGF) family comprises important mitogens for mesenchymal cells. The active dimeric form of PDGF consists of four structurally related A, B, C, and D chains. All PDGF-variants bind to PDGF-receptors. The A and B chains occur with and without basic C-terminal amino acid extensions as long (A L and B L) and short (A S and B S) isoforms. PDGF-A and -B form homo- or heterodimers. The biological relevance of short and long isoforms is unknown, although it may relate to different affinities for glycosaminoglycans of the cell glycocalix and intercellular matrix. Commercially available anti-PDGF-A and anti-PDGF-B antibodies cannot discriminate between the short and the long isoforms. Thus, to investigate the function of the long and short isoforms, we raised antibodies specific for the long A and B chain isoforms. The antibodies were affinity-purified and their properties analysed by surface plasmon resonance. Inhibition studies with different PDGF homodimers and dot–blot studies proved their high specificity for the respective isoforms. Both antibodies recognised the target PDGF homodimers complexed to the glycocalix of human arterial smooth muscle cells and human monocyte-derived macrophages. By using these specific antibodies, we were able to confirm at the protein level the synthesis of PDGF-A and -B during differentiation of human monocyte-derived macrophages and to demonstrate the presence of the PDGF-A L and PDGF-B L isoforms in human arterial tissue.

Apolipoprotein J inhibits the migration, adhesion, and proliferation of vascular smooth muscle cells

Purpose: Apolipoprotein J (ApoJ) is expressed during tissue injury and remodeling and has been implicated in vascular smooth muscle cell (VSMC) differentiation. Recently, the gene for ApoJ was identified as upregulated in distal anastomotic intimal hyperplasia after prosthetic arterial grafting. In this study we investigate the effect of ApoJ on VSMC migration, adhesion, proliferation, and gene expression. Methods: To study the effect of ApoJ on cell migration, we used a microchemotaxis chamber with an intervening 8-μm pore semipermeable polycarbonate membrane. Assays were performed with ApoJ alone (1-50 μg/mL) or in combination with platelet-derived growth factor homodimer bb (PDGF-bb; 10 ng/mL) or 2% fetal bovine serum (FBS; n = 8) in the lower wells. The influence of extracellular matrix interactions on ApoJ and chemotaxis was studied in a similar way, except membranes were collagen coated. Furthermore, cells were exposed to ApoJ 15 minutes before or after seeding on the coated membrane (n = 10). The influence of ApoJ on cell adhesion to collagen was assessed with cell exposure to ApoJ 15 minutes before or after seeding on a collagen-coated membrane (n = 10). Migration and adhesion were quantified by counting the number of cells per three independent high-power fields with light microscopy. The effect of ApoJ in 0.4% FBS with or without PDGF-bb on VSMC proliferation (n = 12) was assessed by means of [Methyl-3H] thymidine incorporation. The transcriptional profile of VSMCs in 2% FBS exposed to ApoJ and a control for 24 hours was analyzed with an oligonucleotide microarray containing 12,560 genes. Results: ApoJ alone was not chemotactic for VSMCs. Without collagen, ApoJ decreased the migration of VSMCs toward 2% FBS by 96% or more starting at 10 μg/mL (P <.05) and toward PDGF-bb by 60.9% or more starting at 25 μg/mL (P <.05) compared with the control. When collagen was introduced, ApoJ (25 μg/mL) decreased migration toward 2% FBS by 64% (P <.01) and toward PDGF-bb by 67.5% (P <.01) and decreased adhesion by 26.8% (P <.01) only when VSMCs in solution were exposed to ApoJ before placement on collagen. ApoJ did not induce VSMC proliferation. ApoJ alone decreased VSMC thymidine incorporation by 41.1% at 25 μg/mL (P <.05). ApoJ decreased thymidine incorporation of PDGF-bb stimulated VSMCs by 42.8% at 50 μg/mL (P <.05). Interleukin-8 and endothelin-1 were demonstrated by means of the microarray to be differentially expressed more than twofold in VSMCs that were exposed to ApoJ. Conclusion: ApoJ is a potent inhibitor of VSMC migration, adhesion, and proliferation. Its genetic targets are linked to cell senescence and differentiation. Therefore, ApoJ may play a role, in part, in modulating the VSMC response to injury. (J Vasc Surg 2001;34:716-23.)

Rat Aortic Smooth Muscle Cell Density Affects Activation of MAP Kinase and Akt by Menadione and PDGF Homodimer BB

Mitogen-activated protein kinases (MAPK) and protein kinase B (PKB or Akt) are major signal transduction molecules regulating cell proliferation, differentiation, and apoptosis. We examined how cultured rat aortic vascular smooth muscle cells (VSMC) at different cell densities respond to selected stimuli and how this is reflected in the two distinct (MAPK and Akt) and yet cross-talking signaling pathways. VSMC were cultured to 100% confluence, reaching contact inhibition, and to 60–70% confluence, as sparse, proliferating cells. They were treated with menadione (an intracellular generator of O−2) and/or platelet-derived growth factor homodimer BB (PDGF). In sparse cells, menadione or PDGF alone activated ERK, and together the effect was synergistic, whereas in confluent cells menadione's and PDGF's activations of ERK were, at most, additive. Activation of the upstream ERK kinase (MEK-1) paralleled ERK activation except in sparse cells in which the synergistic effects of menadione and PDGF on ERK could not be fully accounted for by MEK-1 activation. Another member of the MAPK family, p38, did not show significant changes. Akt activation by PDGF alone was present under both cell culture conditions; Akt activation is blocked by menadione. Co-incubation with the reducing agent dithiothreitol or calcium chelators (EDTA/EGTA) inhibited partially or completely menadione's effects on MEK/ERK and Akt pathways, as well as menadione's effects on PDGF-induced ERK and Akt activations. These data suggest that in VSMC, the state of cell confluence determines how distinct pathways of MAPK activation cross talk. In addition while PDGF may function as a survival factor by inducing Akt activation, menadione could promote apoptosis by inhibiting PDGF-induced Akt activation independent of cell density. The effects of menadione, but not those of PDGF, are more dependent on the cellular redox status and extracellular calcium.

Overexpression of PDGF receptor β in dermal fibroblasts of lymphangitic post radiotherapy relapses of breast carcinoma

Abstract Lymphangitic relapses of breast cancer, initiating in the area irradiated for conservative treatment, are rare but not uncommon. Clinical and physiopathological observations such as intense fibrotic swelling ‘en cuirasse' of the lymphangitic area suggest cooperative stromal-epithelial interactions which promote and/or sustain the growth of tumour cells. In order to assess the role of paracrine interactions between irradiated stromal cells and tumour cells in this clinical type of relapse, we have performed a semi-quantitative immunohistochemical study of the PDGF Receptor β (PDGFRβ) on skin biopsies obtained from 36 patients and we have tested the sensitivity to its specific ligand, the platelet derived growth factor homodimer BB (PDGF BB) of dermal fibroblasts in culture from biopsies of six of these patients. We observed a consistently increased immunoreactivity of PDGFRβ in the dermal fibroblasts of irradiated lymphangitic skin, when compared to irradiated skin without lymphangitic recurrence ( P 3 H]thymidine incorporation and on cell growth in culture was significantly greater on fibroblasts from the irradiated field than on those from a non-irradiated field or from the skin of healthy donors. PDGF BB immunoreactivity was observed in the malignant epithelial mammary cells infiltrating the dermis. This altered phenotype of the stromal component may contribute to relapse of breast carcinoma within the irradiated field as an extensive inflammatory skin recurrence.

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

Induction of neutral proteinase and prostanoid production in bovine nasal chondrocytes by interleukin-1 and tumor necrosis factor α: Modulation of these cellular responses by interleukin-6 and platelet-derived growth factor

We have previously reported that recombinant human interleukin-1 (IL-1) stimulates matrix erosion in bovine nasal cartilage explants (R. J. Smith et al., Inflammation 13, 367–382, 1989). This action of IL-1 is believed to be caused by matrix-degrading neutral proteinases produced by activated chondrocytes. Accordingly, we investigated the effects of recombinant human interleukin-1α (IL-1α), recombinant human interleukin-1β (IL-1β), and recombinant human tumor necrosis factor α (TNFα) on bovine nasal chondrocyte (BNC) responsiveness. IL-1α and IL-1β stimulated a time (0–72 hr) and concentration-dependent (0.01–10 ng/ml) production of collagenase, gelatinase, caseinase, and prostaglandin E 2 (PGF 2) in BNC monolayer cultures. Neutral proteinase and PGE 2 production by BNC was also induced by TNFα (0.2–200 ng/ml) in a time-dependent (0–72 hr) manner. Recombinant human interleukin-6 (IL-6) caused a concentration-dependent (6–200 ng/ml) potentiation of IL-1-stimulated neutral proteinase and PGE 2 production by BNC. However, recombinant human platelet-derived growth factor homodimer BB suppressed BNC responsiveness to IL-1. A recombinant human IL-1 receptor antagonist protein inhibited BNC activation by IL-1 but not TNFα.

Platelet-derived growth factors and growth-related genes in rat lung. III. Immunolocalization during fetal development.

To further study the role of platelet-derived growth factor (PDGF) in fetal lung development, the distribution of the PDGF homodimers PDGF-AA and PDGF-BB was examined by immunohistochemistry in embryonic and fetal rat lung from d 12 to 22 of gestation (term = 22 d). PDGF-AA and PDGF-BB were localized to airway epithelial cells as early as d 12 of gestation, 2 d before their appearance in mesenchymal cells. Both PDGF homodimer immunoreactivities increased until the late pseudoglandular stage of lung development, followed by fluctuations in reactivity during the canalicular stage. Only weak immunoreactivity to either PDGF homodimer was evident during the saccular stage of lung development. Immunodetection by Western blotting revealed that PDGF-AA and PDGF-BB homodimer protein concentrations were high during the embryonic and pseudoglandular stage of lung development and decreased with advancing gestation. We conclude that the presence of PDGF in both developing airway epithelial cells and mesenchymal cells, as well as gestation-dependent changes of PDGF homodimers, is compatible with a role for this growth factor during fetal lung development.

Platelet‐derived growth factor potentiates cellular responses of articular chondrocytes to interleukin‐1

Recombinant human interleukin-1 alpha (IL-1 alpha) induced a time-dependent (0-72 hours) and concentration-dependent (0.01-10 ng/ml) production of metalloproteinases (collagenase, gelatinase, stromelysin) and prostaglandin E2 (PGE2) in rabbit articular chondrocytes (RAC). Exposure of RAC to recombinant human platelet-derived growth factor homodimer BB (PDGF-BB; 2-200 ng/ml) in the presence of stimulatory and substimulatory concentrations of IL-1 alpha resulted in a marked augmentation of metalloproteinase and PGE2 production. PDGF-BB exerted no agonist effects on RAC responsiveness. PDGF-BB up-regulated the number of IL-1 receptors per chondrocyte but had no effect on receptor affinity. Cycloheximide and actinomycin D caused a concentration-dependent suppression of the PDGF-BB-mediated potentiation of radiolabeled IL-1 alpha binding to RAC and cell responsiveness to IL-1 alpha. Similarly, IL-1 increased the number of PDGF receptors on RAC without changing receptor affinity. These data are discussed within the context of cytokine-growth factor interactions as components of the pathogenesis of arthritic diseases.

Cultured Kaposi's sarcoma-derived cells express functional PDGF A-type and B-type receptors

We have investigated the role of platelet-derived growth factor (PDGF) for the growth of cultured Kaposi's sarcoma (KS)-derived cells. They were found to bind 125I-PDGF-AA and 125I-PDGF-BB in a manner which is consistent with the presence of both PDGF A-type and PDGF B-type receptors. Furthermore, KS cells responded mitogenically to both PDGF homodimers. This shows that KS cells have functional A- and B-type PDGF receptors, suggesting an important role of PDGF for the growth of KS cells in vitro. In addition we found that KS cells produce PDGF-A chain mRNA in response to PDGF stimulation. This suggests that a PDGF-induced autocrine stimulation involving PDGF-AA which could amplify the response to PDGF occurs in these cells.

Early Signals in the Mitogenic Response of Swiss 3T3 Cells: A Comparative Study of Purified PDGF Homodimers

Platelet-derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB. Two distinct receptor subunits, alpha and beta, have been identified which bind either all three isoforms of PDGF (alpha) or PDGF-BB only (beta). Here, we have compared the effect of purified PDGF homodimers on the early intracellular signaling events and mitogenesis in Swiss 3T3 cells, which possess equivalent numbers of the alpha and beta subunits. Both PDGF-AA and PDGF-BB stimulated receptor phosphorylation, inositol phosphate formation, activation of protein kinase C, calcium mobilization, EGF receptor transmodulation, sodium uptake, arachidonic acid release, cyclic AMP accumulation, and c-fos induction in a comparable, dose-dependent manner (half-maximal values for all these response were in the 2-10 ng/ml range for both homodimers). At high concentrations of PDGF (greater than 10 ng/ml), the BB homodimer effect on early membrane and cytosolic signals was 20-30% greater than PDGF-AA, reflecting the greater number of available binding sites for PDGF-BB. DNA synthesis studies indicated that PDGF-AA and PDGF-BB were potent mitogens for Swiss 3T3 cells, displaying identical dose-response effects. Moreover, the mitogenic activities of both homodimers were equally potentiated in the presence of insulin. These results indicate that both PDGF-AA and PDGF-BB stimulate the full complement of molecular responses required for the synergistic interactions mediating long-term mitogenesis. We conclude that alpha and beta receptor subunits do not differ in their ability to transduce PDGF-mediated signals leading to DNA synthesis in Swiss 3T3 cells.

Ca2+-channel blockers inhibit the action of recombinant platelet-derived growth factor in vascular smooth muscle cells.

Human platelet-derived growth factor (PDGF) is mainly composed of two polypeptide chains (PDGF-AB). All three possible dimeric forms of PDGF--i.e., PDGF-AA, PDGF-BB and PDGF-AB--exist in nature. We have used two recombinant PDGF homodimers to determine the roles of each isoform in the activation of phosphatidylinositol turnover in vascular smooth muscle cells (VSMC) isolated from rat thoracic aorta, their mitogenic effect on VSMC, and their vasoconstrictor effect on intact strips of aortic vascular tissue. Three Ca2+-channel blockers, nifedipine, verapamil, and diltiazem, were used as antagonists for investigating the PDGF-dependent changes mediated by the homodimers. PDGF-BB had a greater efficacy than PDGF-AA on inositol 1,4,5-trisphosphate release, on the formation of diacylglycerol, and on Ca2+ mobilization, which was also associated with vasoconstrictor activity and effective mitogenicity. PDGF-AA, on the other hand, was more potent than PDGF-BB in stimulating protein kinase C. In all instances, the activation of the phosphatidylinositol turnover by the two homodimers was inhibited by the Ca2+-channel blockers.