Platelet-derived growth factor up-regulates Ca2+-sensing receptors in idiopathic pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease associated with remodeling of the pulmonary artery. We previously reported that the Ca2+-sensing receptor (CaSR) is up-regulated in pulmonary arterial smooth muscle cells (PASMCs) from patients with idiopathic PAH (IPAH) and contributes to enhanced Ca2+ responses and excessive cell proliferation. However, the mechanisms underlying the up-regulation of CaSR have not yet been elucidated. We herein examined involvement of platelet-derived growth factor (PDGF) on CaSR expression, Ca2+ responses, and proliferation in PASMCs. The expression of PDGF receptors was higher in PASMCs from patients with IPAH than in PASMCs from normal subjects. In addition, PDGF-induced activation of PDGF receptors and their downstream molecules [ERK1/2, p38, protein kinase B, and signal transducer and activator of transcription (STAT) 1/3] were sustained longer in PASMCs from patients with IPAH. The PDGF-induced CaSR up-regulation was attenuated by small interfering RNA knockdown of PDGF receptors and STAT1/3, and by the treatment with imatinib. In monocrotaline-induced pulmonary hypertensive rats, the up-regulation of CaSR was reduced by imatinib. The combination of NPS2143 and imatinib additively inhibited the development of pulmonary hypertension. These results suggest that enhanced PDGF signaling is involved in CaSR up-regulation, leading to excessive PASMC proliferation and vascular remodeling in patients with IPAH. The linkage between CaSR and PDGF signals is a novel pathophysiological mechanism contributing to the development of PAH.-Yamamura, A., Nayeem, M. J., Al Mamun, A., Takahashi, R., Hayashi, H., Sato, M. Platelet-derived growth factor up-regulates Ca2+-sensing receptors in idiopathic pulmonary arterial hypertension.

Transglutaminase 2 promotes PDGF-mediated activation of PDGFR/Akt1 and ß-catenin signaling in vascular smooth muscle cells and supports neointima formation.

BACKGROUND: Phenotypic switch of vascular smooth muscle cells (VSMCs) accompanies neointima formation and associates with vascular diseases. Platelet-derived growth factor (PDGF)-induced activation of PDGFR/Akt1 and ß-catenin signaling pathways in VSMCs has been implicated in vessel occlusion. Transglutaminase 2 (TG2) regulates these pathways and its levels are increased in the neointima. OBJECTIVE: The aim of this study was to evaluate the role of TG2 in PDGF/ß-catenin signaling cross-talk and assess its contribution to neointima. METHODS: Aortic VSMCs from wild-type and TG2 knockout mice were tested in vitro for levels of VSMC markers, proliferation, migration and PDGF-induced activation of PDGFR/Akt1 and ß-catenin pathways. Neointima in these mice was studied ex vivo in coronary vessels using a heart slice model and in vivo using a carotid artery ligation model. RESULTS: Genetic deletion of TG2 attenuated the PDGF-induced phenotypic switch of aortic VSMCs, reduced their proliferation and migration rates, and inhibited PDGF-induced activation of PDGFR/Akt1 and ß-catenin pathways in both ex vivo and in vivo neointima models. Importantly, genetic deletion of TG2 also markedly attenuated vessel occlusion. CONCLUSIONS: TG2 promotes neointima formation by mediating the PDGF-induced activation of the PDGFR/Akt1 and ß-catenin pathways in VSMCs. This study identifies TG2 as a potential therapeutic target for blocking neointima in blood vessels.

Piperlongumine inhibits atherosclerotic plaque formation and vascular smooth muscle cell proliferation by suppressing PDGF receptor signaling.

Piperlongumine (piplartine, PL) is an alkaloid found in the long pepper (Piper longum L.) and has well-documented anti-platelet aggregation, anti-inflammatory, and anti-cancer properties; however, the role of PL in prevention of atherosclerosis is unknown. We evaluated the anti-atherosclerotic potential of PL in an in vivo murine model of accelerated atherosclerosis and defined its mechanism of action in aortic vascular smooth muscle cells (VSMCs) in vitro. Local treatment with PL significantly reduced atherosclerotic plaque formation as well as proliferation and nuclear factor-kappa B (NF-κB) activation in an in vivo setting. PL treatment in VSMCs in vitro showed inhibition of migration and platelet-derived growth factor BB (PDGF-BB)-induced proliferation to the in vivo findings. We further identified that PL inhibited PDGF-BB-induced PDGF receptor beta activation and suppressed downstream signaling molecules such as phospholipase Cγ1, extracellular signal-regulated kinases 1 and 2 and Akt. Lastly, PL significantly attenuated activation of NF-κB-a downstream transcriptional regulator in PDGF receptor signaling, in response to PDGF-BB stimulation. In conclusion, our findings demonstrate a novel, therapeutic mechanism by which PL suppresses atherosclerosis plaque formation in vivo.

Mesothelioma cell proliferation through autocrine activation of PDGF-ßß receptor.

BACKGROUND/AIMS: Growth factors play a critical role in proliferation for a variety of cancer cells. The present study was conducted to understand the signaling cascades underlying PDGF-D/PDGF-ßß receptor-mediated proliferation of mesothelioma cells. METHODS: Cell growth and cell cycle were analyzed in human non-malignant Met5A cells and malignant mesothelioma cells such as MSTO-211H, NCI-H28, NCI-H2052, and NCI-H2452 cells. RESULTS: Growth of all the cells used here was not affected by PDGF-D, regardless of concentrations (1-30 ng/ml) or treatment time (48-72 h). Spontaneous growth of those cells was significantly inhibited by knocking-down PDGFD or PDGF-ßß receptor, without affecting cell cycling. The cell growth was significantly inhibited by the Akt inhibitor MK2206 and the ROCK inhibitor Y27632 for all the cell types, by the PDK1 inhibitor BX912 for NCI-H28 cells alone, and by the Rac1 inhibitor NSC23766 for NCI-H2052 cells alone, while the PI3 kinase inhibitor wortmannin had no effect. The cell growth, alternatively, was significantly attenuated by MAP kinase kinase inhibitor PD98059 or the ERK1/2 inhibitor FR180204 for all the cell types. CONCLUSION: The results of the present study show that PDGF-D promotes mesothelioma cell proliferation by targeting ROCK or MAP kinase through autocrine activation of PDGF-ßß receptor.

Salvianolic acid A inhibits PDGF-BB induced vascular smooth muscle cell migration and proliferation while does not constrain endothelial cell proliferation and nitric oxide biosynthesis.

Proliferation and migration of vascular smooth muscle cells (VSMCs) are critical events in the initiation and development of restenosis upon percutaneous transluminal coronary angioplasty (PTCA). Polyphenols have been suggested to ameliorate post-angioplasty restenosis. Salvianolic A (SalA) is one of the most abundant polyphenols extracted from salvia. In this study, we investigated the effect of salvianolic A (SalA) on the migration and proliferation of VSMCs. We found a preferential interaction of SalA with cellular systems that rely on the PDGF signal, but not on the EGF and bFGF signal. SalA inhibits PDGF-BB induced VSMC proliferation and migration in the concentration range from 0.01 to 0.1 µM. The inhibition of SalA on VSMC proliferation is associated with cell cycle arrest. We also found that SalA inhibits the PDGFRß-ERK1/2 signaling cascade activated by PDGF-BB in VSMCs. In addition, SalA does not influence the proliferation of endothelial cells, the synthesis of NO and eNOS protein expression. Our results suggest that SalA inhibits migration and proliferation of VSMCs induced by PDGF-BB via the inhibition of the PDGFRß-ERK1/2 cascade, but that it does not constrain endothelial cell proliferation and nitric oxide biosynthesis. Thus, the present study suggests a novel adjunct pharmacological strategy to prevent angioplasty-related restenosis.

Platelet-derived growth factor (PDGF)-BB produces NO-mediated relaxation and PDGF receptor ß-dependent tonic contraction in murine iliac lymph vessels.

We studied the effects of PDGF-BB on changes in the diameters of murine lymph vessels with or without intact endothelium. PDGF-BB induced dilation of the lymph vessels with endothelium. Pretreatment with l-NAME or removal of the endothelium caused a significant attenuation in the PDGF-BB-induced dilation. PDGF-BB also produced dose-related reduction of the diameters of the lymph vessels without endothelium. To evaluate intracellular signal transduction and Ca(2+) -dependence of the PDGF-BB-induced tonic contraction, we investigated the effects of imatinib, GW5074 (an inhibitor of Raf-1 kinase), U-73122 (an inhibitor of phospholipase C), and xestospongin C on the PDGF-BB-induced reduction responses. All of these inhibitors caused a significant attenuation in the PDGF-BB-induced reduction response that was significantly decreased by treatment with Ca(2+) -free Krebs-bicarbonate solution or nifedipine. Higher concentrations of PDGF-BB produced a marked reduction of lymph vessel diameter within both high K(+) Krebs-bicarbonate solution and Ca(2+) -free high K(+) Krebs solution containing 1mM EGTA. These findings suggest that PDGF-BB induced endothelium-dependent NO-mediated relaxation of lymphatic smooth muscles in murine lymph vessels. PDGF receptor ß-mediated tonic contraction of the muscles through increased Ca(2+) influx through the membrane and the release of membrane-bound and intracellular Ca(2+) .

Synergy in ERK activation by cytokine receptors and tyrosine kinase growth factor receptors.

Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) signal through EGF and PDGF receptors, which are important receptor tyrosine kinases (RTKs). Growth hormone (GH) and prolactin (PRL) are four helical bundle peptide hormones that signal via GHR and PRLR, members of the cytokine receptor superfamily. In this study, we examine crosstalk between signaling pathways emanating from these disparate receptor groups (RTKs and cytokine receptors). We find that GH and EGF specifically synergize for activation of ERK in murine preadipocytes. The locus of this synergy resides at the level of MEK activation, but not above this level (i.e., not at the level of EGFR, SHC, or Raf activation). Furthermore, dephosphorylation of the scaffold protein, KSR, at a critical serine residue is also synergistically promoted by GH and EGF, suggesting that GH sensitizes these cells to EGF-induced ERK activation by augmenting the actions of KSR in facilitating MEK-ERK activation. Similarly specific synergy in ERK activation is also detected in human T47D breast cancer cells by cotreatment with PRL and PDGF. This synergy also resides at the level of MEK activation. Consistent with this synergy, PRL and PDGF also synergized for c-fos-dependent transactivation of a luciferase reporter gene in T47D cells, indicating that events downstream of ERK activation reflect this signaling synergy. Important conceptual and physiological implications of these findings are discussed.

PDGF stimulation of Müller cell proliferation: Contributions of c-JNK and the PI3K/Akt pathway.

Platelet-derived growth factor (PDGF) has a critical role in proliferative vitreoretinopathy (PVR) as a chemoattractant and mitogen for retinal pigment epithelial cells and retinal glial cells. Here, we investigated the potential effects of PDGF on the proliferation of Müller cells and the intracellular signaling pathway mediating these changes. PDGF induced Müller cell proliferation and increased phosphorylation of the PDGF receptor (PDGFR), as shown by an MTT assay and immunoprecipitation analyses. Both effects were blocked by JNJ, a PDGFR-selective tyrosine kinase inhibitor. PDGF also stimulated phosphorylation of c-JNK and Akt. PDGF-induced Müller cell proliferation was significantly reduced by pre-treatment with SP600125 and LY294002, inhibitors of c-JNK and Akt phosphorylation, respectively. Our findings collectively indicate that PDGF-stimulated Müller cell proliferation occurs via activation of the c-JNK and PI3K/Akt signaling pathways. These data provide useful information in establishing the role of Müller cells in the development of proliferative vitreoretinopathy.

Targeted delivery of drugs for liver fibrosis.

Liver fibrosis and its end stage disease cirrhosis are a major cause of mortality and morbidity around the world. There is no effective pharmaceutical intervention for liver fibrosis at present. Many drugs that show potent antifibrotic activities in vitro often show only minor effects in vivo because of insufficient concentrations of drugs accumulating around the target cell and their adverse effects as a result of affecting other non-target cells. Hepatic stellate cells (HSC) play a critical role in the fibrogenesis of liver, so they are the target cells of antifibrotic therapy. Several kinds of targeted delivery system that could target the receptors expressed on HSC have been designed, and have shown an attractive targeted potential in vivo. After being carried by these delivery systems, many agents showed a powerful antifibrotic effect in animal models of liver fibrosis. These targeted delivery systems provide a new pathway for the therapy of liver fibrosis. The characteristics of theses targeted carriers are reviewed in this paper.

Stimulatory autoantibodies to the PDGF receptor: a link to fibrosis in scleroderma and a pathway for novel therapeutic targets.

Systemic sclerosis (scleroderma) is a complex disease characterized by excessive deposition of collagen and abnormalities of blood vessels. In addition, activation of the immune system is a central feature of scleroderma as shown by mononuclear cell infiltration of the skin, autoantibody production and release of inflammatory cytokines. The pathogenesis of the disease is poorly understood and the molecular events underlying the main clinical features are not known. The detection of agonistic autoantibodies targeting PDGF receptor in serum of patients with scleroderma may indicate a novel link between phenotypic features of the disease and a specific signalling pathway. Agonistic PDGF receptor antibodies induce in vitro the scleroderma phenotype in normal human fibroblasts and, thus, link autoimmunity to fibrosis. These findings pave the way to novel therapeutic strategies.

Discovery of a potent, highly selective, and orally efficacious small-molecule activator of the insulin receptor.

A series of 3,6-diaryl-2,5-dihydroxybenzoquinones were synthesized and evaluated for their abilities to selectively activate human insulin receptor tyrosine kinase (IRTK). 2, 5-Dihydroxy-6-(1-methylindol-3-yl)-3-phenyl-1,4-benzoquinone (2h) was identified as a potent, highly selective, and orally active small-molecule insulin receptor activator. It activated IRTK with an EC(50) of 300 nM and did not induce the activation of closely related receptors (IGFIR, EGFR, and PDGFR) at concentrations up to 30 000 nM. Oral administration of the compound to hyperglycemic db/db mice (0.1-10 mg/kg/day) elicited substantial to nearly complete correction of hyperglycemia in a dose-dependent manner. In ob/ob mice, the compound (10 mg/kg) caused significant reduction in hyperinsulinemia. A structurally related compound 2c, inactive in IRTK assay, failed to affect blood glucose level in db/db mice at equivalent exposure levels. Results from additional studies with compound 2h, aimed at evaluating classical quinone-related phenomena, provided sufficient grounds for optimism to allow more extensive toxicologic evaluation.

E5 oncoprotein mutants activate phosphoinositide 3-kinase independently of platelet-derived growth factor receptor activation.

The E5 oncoprotein of bovine papillomavirus type 1 is a Golgi-resident, 44-amino acid polypeptide that can transform fibroblast cell lines by activating endogenous platelet-derived growth factor receptor beta (PDGF-R). However, the recent discovery of E5 mutants that exhibit strong transforming activity but minimal PDGF-R tyrosine phosphorylation indicates that E5 can potentially use additional signal transduction pathway(s) to transform cells. We now show that two classes of E5 mutants, despite poorly activating the PDGF-R, induce tyrosine phosphorylation and activation of phosphoinositide 3-kinase (PI 3-K) and that this activation is resistant to a selective inhibitor of PDGF-R kinase activity, tyrphostin AG1296. Consistent with this independence from PDGF-R signaling, the E5 mutants fail to induce significant cell proliferation in the absence of PDGF, unlike wild-type E5 or the sis oncoprotein. Despite differences in growth factor requirements, however, both wild-type E5 and mutant E5 cell lines form colonies in agarose. Interestingly, activation of PI 3-K occurs without concomitant activation of the ras-dependent mitogen-activated protein kinase pathway. The known ability of constitutively activated PI 3-K to induce anchorage-independent cell proliferation suggests a mechanism by which the mutant E5 proteins transform cells.

Tyr1009 and Tyr1021 in the platelet-derived growth factor beta-receptor mediate agonist triggered calcium signalling.

Calcium signalling was studied in porcine aortic endothelial cells stably transfected with wild type or mutants of the human platelet-derived growth factor (PDGF) beta-receptor and fibroblast growth factor (FGF) receptor-1 (FGFR1). Phospholipase C-gamma (PLC-gamma) has a consensus binding site at phosphorylated Tyr1021 in the PDGF beta-receptor. The phosphorylated tyrosine at 1009 is a binding site for Syp/PTP1D, an adaptor molecule mediating Grb2/RAS signalling. Also, Tyr1009 has been shown to be a minor binding site for PLC-gamma; however previous data have indicated that it does not have any functional significance in PLC-gamma signalling. The concentration of cytoplasmic calcium ([Ca2+]i) was measured by microfluorometry and digital imaging. About 72% of the cells transfected with wild type PDGF beta-receptor responded to a challenge with PDGF-BB. Mutants in which both Tyr1009 and Tyr1021 in the PDGF beta-receptor were exchanged for phenylalanine totally lacked [Ca2+]i responses. However, in those with a single mutation at Tyr1009 or Tyr1021, 36% and 12% of the cells responded, respectively. In cells transfected with FGFR1 or FGFchim, with the kinase insert of FGFR1 replaced by the insert of the PDGF beta-receptor, a [Ca2+]i increases was observed in similar proportions of cells. The amplitudes of the growth factor-induced [Ca2+]i responses was comparable in the different transfectants. Thrombin, activating a G-protein coupled receptor, triggered [Ca2+]i peaks more rapidly, and in a higher proportion of cells compared to the growth factors. The present data indicate that both Tyr1009 and Tyr1021 alone and in cooperation mediate PDGF-BB triggered calcium signalling.

Association and direct activation of signal transducer and activator of transcription1alpha by platelet-derived growth factor receptor.

PDGF stimulates tyrosine phosphorylation of Janus kinase 1 (JAK1) and the signal transducer and activator of transcription 1 (STAT1alpha). However, it is not known whether JAKs are required for STAT1alpha phosphorylation or if the PDGF receptor itself can directly tyrosine phosphorylate and activate STAT1alpha. In vitro immunecomplex kinase assay of PDGF beta receptor (PDGFR) or STAT1alpha immunoprecipitates from lysates of mesangial cells treated with PDGF showed phosphorylation of a 91- and an 185-kD protein. Incubation of lysates prepared from quiescent mesangial cells with purified PDGFR resulted in STAT1alpha activation. Immunodepletion of Janus kinases from the cell lysate before incubation with the purified PDGFR showed no effect on STAT1alpha activation. Moreover, lysates from mesangial cells treated with JAK2 inhibitor, retained significant STAT1alpha activity. To confirm that STAT1alpha is a substrate for PDGFR, STAT1alpha protein was prepared by in vitro transcription and translation. The addition of purified PDGFR to the translated STAT1alpha resulted in its phosphorylation. This in vitro phosphorylated and activated protein also forms a specific protein-DNA complex. Dimerization of the translated STAT1alpha protein was also required for its DNA binding. Incubation of pure STAT1alpha with autophosphorylated PDGFR resulted in physical association of the two proteins. These data indicate that activated PDGFR may be sufficient to tyrosine phosphorylate and thus directly activate STAT1alpha.

PDGF-BB increases endothelial migration on cord movements during angiogenesis in vitro.

To explore direct effects of platelet-derived growth factor (PDGF) on endothelial cells during angiogenesis in vitro, we have used cloned bovine aortic endothelial cells that spontaneously form cord structures. Recently we have shown that cells forming these endothelial cords express PDGF beta-receptors and that PDGF-BB can contribute to cellular proliferation and cord formation. In this study we investigated whether PDGF-induced cellular migration might also contribute to endothelial repair and angiogenesis in vitro. Ten individual endothelial cells in cords were tracked at an early stage of cord formation by video-timelapse microscopy. PDGF-BB (100 ng/ml) induced an increase in endothelial cell movement of 67 +/- 15% as compared with diluent control. Interestingly, PDGF-BB also increased movements of entire cord structures, followed at branching points, by 53 +/- 12% over diluent control. Taken together, these video-timelapse experiments suggested that the apparent movements of single endothelial cord cells might also be due to the motion of entire underlying cord structures in response to PDGF. To analyze the response of single endothelial cord cells we therefore examined whether PDGF-induced migration contributes to endothelial repair. Abrasions were applied with a razor blade to confluent monolayers of endothelial cells at an intermediate stage of cord formation. PDGF-BB concentration-dependently increased the distance to which cord-forming endothelial cells migrated into the abrasion. An increased number of elongated, i.e., probably migrating, endothelial cells was found in the abrasion in response to PDGF-BB. However, there was no effect of PDGF-BB on the total number of endothelial cells found in the abrasion. PDGF-AA affected neither the distance to which the cells migrated nor the number of elongated cells. Actin and tubulin stainings revealed that these cytoskeletal structures were not appreciably altered by PDGF-BB. Furthermore, urokinase-type plasminogen activator transcripts were not modulated in response to PDGF-BB. We conclude that in this model of angiogenesis in vitro PDGF-BB can elicit the movement of entire cord structures, possibly via u-PA-independent mechanisms. PDGF-BB also controls the migration of single cord-forming endothelial cells. Thus, PDGF-BB possibly contributes to endothelial repair and angiogenesis by direct effects on proliferation and composite movements of PDGF beta-receptor-expressing endothelial cells and cords.