Human immunodeficiency virus (HIV)-associated pulmonary arterial hypertension (PAH) is a devastating, noninfectious complication of acquired immune deficiency syndrome, and the majority of HIV-PAH cases occur in individuals with a history of intravenous drug use (IVDU). However, although HIV-1 and IVDU have been associated with PAH independently or in combination, the pathogenesis of the disproportionate presence of HIV-PAH in association with IVDU has yet to be characterized. The objective of this study was to obtain a better understanding of the interactions between HIV-1 and cocaine to help uncover the mechanism(s) of the development of HIV-PAH. We observed that exposure of HIV-infected macrophages or HIV-Trans-Activator of Transcription (Tat)-treated pulmonary endothelial cells to cocaine enhanced the expression of platelet-derived growth factor (PDGF)-BB. Simultaneous treatment with Tat and cocaine, on the other hand, exacerbated both the disruption of tight junction proteins (TJPs), with enhanced permeability in pulmonary endothelial cells, and the proliferation of pulmonary smooth muscle cells (pSMCs) compared with either treatment alone. Histological examination of HIV plus IVDU human lung sections showed signs of early pulmonary arteriopathy, severe down-modulation of TJPs, and increased expression of PDGF-BB compared with the lung sections from individuals who are infected with HIV and without history of IVDU. Interestingly, blocking of PDGF receptor signaling with the receptor antagonist or small interfering RNA has been shown to inhibit the increase in proliferation of pSMCs on Tat and cocaine exposure. Our results, therefore, support an additive effect of cocaine to HIV infection in the development of pulmonary arteriopathy through enhancement of endothelial dysfunction and proliferation of pSMCs, while also suggesting PDGF-PDGF receptor axis as a potential target for use in clinical intervention.

Securin overexpression correlates with poor prognosis in various tumours. We have previously shown that securin depletion promotes radiation-induced senescence and enhances radiosensitivity in human cancer cells. However, the underlying molecular mechanisms and the paracrine effects remain unknown. In this study, we showed that radiation induced senescence in securin-deficient human breast cancer cells involving the ATM/Chk2 and p38 pathways. Conditioned medium (CM) from senescent cells promoted the invasion and migration of non-irradiated cancer and endothelial cells. Cytokine assay analysis showed the up-regulation of various senescence-associated secretory phenotypes (SASPs). The IL-6/STAT3 signalling loop and platelet-derived growth factor-BB (PDGF-BB)/PDGF receptor (PDGFR) pathway were important for CM-induced cell migration and invasion. Furthermore, CM promoted angiogenesis in the chicken chorioallantoic membrane though the induction of IL-6/STAT3- and PDGF-BB/PDGFR-dependent endothelial cell invasion. Taken together, our results provide the molecular mechanisms for radiation-induced senescence in securin-deficient human breast cancer cells and for the SASP responses.

Abnormal expression of platelet-derived growth factor (PDGF) receptors has been observed in malignant glioma and other tumors such as osteosarcomas and malignant melanomas. However, their role in the development and maintenance of the tumors is not understood. Signaling through the PDGF receptors is activated by ligand-induced dimerization. Thus, introduction of mutant receptors that are kinase deficient but still dimerization competent is one strategy to study the importance of PDGF receptors in glioma cell growth. A truncated PDGF-beta receptor was introduced into C6 rat glioma cells and the PDGF-mediated signaling and subsequent cell growth studied. In clones expressing the mutant receptor, PDGF-BB-induced tyrosine phosphorylation of the endogenous receptor was significantly reduced. In addition, these cells grew to lower density in culture and formed smaller colonies in soft agar than the C6 parental cells. Furthermore, the ability of cells expressing the truncated receptor to grow as xenografts in nude mice was significantly impaired. These results support the important role for the PDGF-beta receptor in C6 glioma cell growth. They also demonstrate the usefulness of dominant-negative mutants of the PDGF receptor for the evaluation of the role of the receptor in tumorigenesis.

Adenosine inhibits growth of vascular smooth muscle cells. The goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat aortic vascular smooth muscle cells, platelet-derived growth factor-BB (PDGF-BB) (25 ng/mL) stimulated DNA synthesis ([(3)H]thymidine incorporation), cellular proliferation (cell number), collagen synthesis ([(3)H]proline incorporation), total protein synthesis ([(3)H]leucine incorporation), and mitogen-activated protein (MAP) kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, but not N(6)-cyclopentyladenosine or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A(2B) receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1, 3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A(2) receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A(2B) receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and MAP kinase activity. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5'-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A(2B) receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of vascular smooth muscle cell growth by activating A(2B) receptors coupled to inhibition of MAP kinase activity. Pharmacological or molecular biological activation of A(2B) receptors may prevent vascular remodeling associated with hypertension, atherosclerosis, and restenosis following balloon angioplasty.

Human glioblastoma cells (A172) were found to concomitantly express PDGF-BB and PDGF beta-receptors. The receptors were constitutively autophosphorylated in the absence of exogenous ligand, suggesting the presence of an autocrine PDGF pathway. Neutralizing PDGF antibodies as well as suramin inhibited the autonomous PDGF receptor tyrosine kinase activity and resulted in up-regulation of receptor protein. The interruption of the autocrine loop by the PDGF antibodies reversed the transformed phenotype of the glioblastoma cell, as determined by (1) diminished DNA synthesis, (2) inhibition of tumor colony growth, and (3) reversion of the transformed morphology of the tumor cells. The PDGF antibodies showed no effect on the DNA synthesis of another glioblastoma cells line (U-343MGa 31L) or on Ki-ras-transformed fibroblasts. The present study demonstrates an endogenously activated PDGF pathway in a spontaneous human glioblastoma cell line. Furthermore, we provide evidence that the autocrine PDGF pathway drives the transformed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists.

The murine myeloid progenitor cell line 32D was recently shown to undergo monocytic differentiation when protein kinase C-delta (PKC-delta) was overexpressed and activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) (H. Mischak, J.H. Pierce, J. Goodnight, M.G. Kazanietz, P.M. Blumberg, and J.F. Mushinski, J. Biol. Chem. 268:20110-20115, 1993). Tyrosine phosphorylation of PKC-delta occurred when PKC-delta-transfected 32D cells were stimulated by TPA (W. Li, H. Mischak, J.-C. Yu, L.-M. Wang, J.F. Mushinski, M.A. Heidaran, and J.H. Pierce, J. Biol. Chem. 269:2349-2352, 1994). In order to elucidate the role played by PKC-delta in response to activation of a receptor tyrosine kinase, we transfected platelet-derived growth factor beta receptor (PDGF-beta R) alone (32D/PDGF-beta R) or together with PKC-delta (32D/PDGF-beta R/PKC-delta) into 32D cells. NIH 3T3 cells which endogenously express both PDGF-alpha R and PDGF-beta R were also transfected with PKC-delta (NIH 3T3/PKC-delta). Like TPA treatment, PDGF-BB stimulation caused striking phosphorylation of PKC-delta in vivo and translocation of some PKC-delta from the cytosol fraction to the membrane fraction in both cell systems. Some of the phosphorylation induced by PDGF-BB treatment was found to be on a tyrosine residue(s). Tyrosine-phosphorylated PKC-delta was observed only for the membrane fraction after stimulation with PDGF-BB or TPA. The enzymatic activity of PKC-delta in the membrane fraction also increased after stimulation with TPA or PDGF, providing a positive correlation between PKC-delta tyrosine phosphorylation and its activation. Overnight treatment of 32D/PDGF-beta R/PKC-delta cells with PDGF-BB induced monocytic differentiation as judged by an increase in expression of cell surface macrophage differentiation markers. PDGF-BB had much weaker effects on 32D/PDGF-beta R cell differentiation, suggesting that increased PKC-delta expression enhanced monocytic differentiation. These results indicate that PKC-delta is a downstream molecule in the PDGFR signaling pathway and may play a pivotal role in PDGF-beta R-mediated cell differentiation.

OBJECTIVE

We have previously reported that vascular injury or treatment of cultured vascular smooth muscle cells with platelet-derived growth factor-BB (PDGF-BB) or fibroblast growth factor-2 (FGF2) increases the levels of protein tyrosine phosphatase (PTP)1B. The current study was designed to test the hypothesis that PTP1B attenuates PDGF- or FGF-induced motility and proliferation of cultured cells, as well as neointima formation in injured rat carotid arteries.

RESULTS

Treatment of cultured cells with adenovirus expressing PTP1B decreased PDGF-BB- or FGF2-induced cell motility and blocked PDGF-BB- or FGF2-induced proliferation, whereas expression of dominant negative PTP1B (C215S-PTP1B) uncovered the motogenic effect of subthreshold levels of PDGF-BB or FGF2, increased neointimal and medial cell proliferation, and induced neointimal enlargement after balloon injury. The inhibitory effect of PTP1B directed against PDGF in cultured cells was associated with dephosphorylation of the PDGFbeta receptor.

CONCLUSIONS

PTP1B suppresses cell proliferation and motility in cultured smooth muscle cells treated with PDGF-BB or FGF2, and the phosphatase plays a counter-regulatory role in vascular injury-induced cell proliferation and neointima formation. Taken together with previous studies indicating increased PTP1B levels in cells treated with growth factors, the current findings are the first to report the existence of an inhibitory feedback loop involving PDGF or FGF, and PTP1B in blood vessels.

Humoral factors and extracellular matrix are critical co-regulators of smooth muscle cell (SMC) migration and proliferation. We reported previously that focal adhesion kinase (FAK)-related non-kinase (FRNK) is expressed selectively in SMC and can inhibit platelet-derived growth factor BB homodimer (PDGF-BB)-induced proliferation and migration of SMC by attenuating FAK activity. The goal of the current studies was to identify the mechanism by which FAK/FRNK regulates SMC growth and migration in response to diverse mitogenic signals. Transient overexpression of FRNK in SMC attenuated autophosphorylation of FAK at Tyr-397, reduced Src family-dependent tyrosine phosphorylation of FAK at Tyr-576, Tyr-577, and Tyr-881, and reduced phosphorylation of the FAK/Src substrates Cas and paxillin. However, FRNK expression did not alter the magnitude or dynamics of ERK activation induced by PDGF-BB or angiotensin II. Instead, FRNK expression markedly attenuated PDGF-BB-, angiotensin II-, and integrin-stimulated Rac1 activity and attenuates downstream signaling to JNK. Importantly, constitutively active Rac1 rescued the proliferation defects in FRNK expressing cells. Based on these observations, we hypothesize that FAK activation is required to integrate integrin signals with those from receptor tyrosine kinases and G protein-coupled receptors through downstream activation of Rac1 and that in SMC, FRNK may control proliferation and migration by buffering FAK-dependent Rac1 activation.

Ets-1 is a transcription factor that activates expression of matrix-degrading proteinases such as collagenase and stromelysin. To study the control of ets-1 gene expression in rat vascular smooth muscle cells (VSMC), cells were exposed to factors known to regulate VSMC migration and proliferation. Platelet-derived growth factor-BB (PDGF-BB), endothelin-1 (ET-1), and phorbol 12-myristate 13-acetate (PMA) induced a dose-dependent expression of ets-1 mRNA. These effects were abrogated by inhibition of protein kinase C (PKC) by H-7 or chronic PMA treatment. Ets-1 mRNA was superinduced by PDGF-BB and ET-1 in the presence of cycloheximide. The chelation of intracellular Ca2+ by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester and the depletion of endoplasmic reticulum intracellular Ca2+ concentration ([Ca2+]i) by thapsigargin inhibited PDGF-BB- and ET-1-induced ets-1 mRNA, whereas ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid had no effect. However, [Ca2+]i release alone was not sufficient to increase ets-1 mRNA. Forskolin blocked ET-1-, PDGF-BB-, and PMA-induced ets-1 mRNA, as well as inositol phosphate formation, consistent with an effect through impairment of PKC activation. Inhibitors of ets-1 gene expression, such as H-7 and herbimycin A, inhibited the ET-1 induction of collagenase I mRNA. We propose that ets-1 may be an important element in the orchestration of matrix proteinase expression and of vascular remodeling after arterial injury.

The lipoxygenase-derived leukotrienes (LTs) are important proinflammatory lipid mediators. Lipoxins (LXs), more recently described lipoxygenase products, modulate many proinflammatory actions of LTs and have impressive proresolution properties. Mesangial cell (MC) proliferation is a central event in the pathogenesis of glomerulonephritis. LTD4-induced proliferation of mesangial cells is modulated by LXA4. Here, we demonstrate that LXA4 inhibits PDGF- and LTD4-stimulated proliferation through modulation of platelet-derived growth factor receptor beta (PDGFRbeta) activation. Specifically, we demonstrate that LTD4 transactivates the PDGFRbeta, a process associated with c-src recruitment and ras activation. We demonstrate expression of cysLT1 and cysLT2 receptors in MCs. LTD4-induced c-src activation was insensitive to pertussis toxin and the cysLT1 receptor antagonist Zafirlukast but was blocked by the nonselective antagonist Pobilukast. We show that LXA4 inhibits LTD4-stimulated activation of the PDGFRbeta and that LXA4 modulates PDGF-BB-stimulated tyrosine phosphorylation of the PDGFRb and subsequent mitogenic events. Furthermore, expression of recombinant LXA4 receptor (ALXR) in CHOK1 cells was associated with an attenuation of serum-stimulated proliferation. These data demonstrate that LXA4 receptor (ALXR) activation is accompanied by antimitogenic effects coupled with inactivation of growth factor receptors, highlighting the complex cross-talk between G protein-coupled receptors and receptor tyrosine kinases in an inflammatory milieu. These data elaborate on the profile of cell signaling events that underpin the anti-inflammatory and proresolution bioactions of LX.

The inhibitory reversible oxidation of protein tyrosine phosphatases (PTPs) is an important regulatory mechanism in growth factor signaling. Studies on PTP oxidation have focused on pathways that increase or decrease reactive oxygen species levels and thereby affect PTP oxidation. The processes involved in reactivation of oxidized PTPs remain largely unknown. Here the role of the thioredoxin (Trx) system in reactivation of oxidized PTPs was analyzed using a combination of in vitro and cell-based assays. Cells lacking the major Trx reductase TrxR1 (Txnrd1(-/-)) displayed increased oxidation of PTP1B, whereas SHP2 oxidation was unchanged. Furthermore, in vivo-oxidized PTP1B was reduced by exogenously added Trx system components, whereas SHP2 oxidation remained unchanged. Trx1 reduced oxidized PTP1B in vitro but failed to reactivate oxidized SHP2. Interestingly, the alternative TrxR1 substrate TRP14 also reactivated oxidized PTP1B, but not SHP2. Txnrd1-depleted cells displayed increased phosphorylation of PDGF-β receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-β receptor phosphorylation. This effect was not observed in cells specifically lacking PTP1B. Together these results demonstrate that the Trx system, including both Trx1 and TRP14, impacts differentially on the oxidation of individual PTPs, with a preference of PTP1B over SHP2 activation. The studies demonstrate a previously unrecognized pathway for selective redox-regulated control of receptor tyrosine kinase signaling.

The antipsoriatic dimethylfumarate (DMF) has been anecdotically reported to reduce asthma symptoms and to improve quality of life of asthma patients. DMF decreases the expression of proinflammatory mediators by inhibiting the transcription factor NF-kappaB and might therefore be of interest for the therapy of inflammatory lung diseases. In this study, we determined the effect of DMF on platelet-derived growth factor (PDGF)-BB- and TNFalpha-induced asthma-relevant cytokines and NF-kappaB activation by primary human asthmatic and nonasthmatic airway smooth muscle cells (ASMC). Confluent nonasthmatic and asthmatic ASMC were incubated with DMF (0.1-100 microM) and/or dexamethasone (0.0001-0.1 microM), NF-kappaB p65 siRNA (100 nM), the NF-kappaB inhibitor helenalin (1 microM) before stimulation with PDGF-BB or TNFalpha (10 ng/ml). Cytokine release was measured by ELISA. NF-kappaB, mitogen and stress-activated kinase (MSK-1), and CREB activation was determined by immunoblotting and EMSA. TNFalpha-induced eotaxin, RANTES, and IL-6 as well as PDGF-BB-induced IL-6 expression was inhibited by DMF and by dexamethasone from asthmatic and nonasthmatic ASMC, but the combination of both drugs showed no glucocorticoid sparing effect in either of the two groups. NF-kappaB p65 siRNA and/or the NF-kappaB inhibitor helenalin reduced PDGF-BB- and TNFalpha-induced cytokine expression, suggesting the involvement of NF-kappaB signaling. DMF inhibited TNFalpha-induced NF-kappaB p65 phosphorylation, NF-kappaB nuclear entry, and NF-kappaB-DNA complex formation, whereas PDGF-BB appeared not to activate NF-kappaB within 60 min. Both stimuli induced the phosphorylation of MSK-1, NF-kappaB p65 at Ser276, and CREB, and all were inhibited by DMF. These data suggest that DMF downregulates cytokine secretion not only by inhibiting NF-kappaB but a wider range of NF-kappaB-linked signaling proteins, which may explain its potential beneficial effect in asthma.

De novo tissue generation stimulated by three angiogenic growth factors administered in a factorial design was studied in an in vivo murine tissue engineering chamber. A silicone chamber was implanted around the epigastric pedicle and filled with Matrigel with 100 ng/ml of recombinant mouse vascular endothelial growth factor-120 (VEGF120), recombinant human basic fibroblastic growth factor (FGF-2), or recombinant rat platelet-derived growth factor-BB (PDGF-BB) added as single, double, or triple combinations. Angiogenesis, supporting tissue ingrowth, and adipogenesis were assessed at 2 and 6 weeks by immunohistochemistry and morphometry. At 2 weeks angiogenesis was synergistically enhanced by VEGF120 + FGF-2 (P = 0.019). FGF-2 (P = 0.008) and PDGF-BB (P = 0.01) significantly increased connective tissue/inflammatory cell infiltrate (macrophages, pericytes, and preadipocytes) in double and triple combinations compared with control. At 6 weeks sequential addition of growth factors increased the percent volume of adipose tissue (P < 0.0005, each main effect), with a synergistic increase in adipose tissue in combination treatments (P < 0.0005). Groups containing 300 ng/ml of single growth factors produced significantly less adipose tissue than the triple growth factor combination (P < 0.0005, VEGF120 and PDGF-BB; P < 0.001, FGF-2). In conclusion, angiogenic growth factor combinations increased early angiogenesis and cell infiltration resulting in synergistically increased adipose tissue growth at 6 weeks. Two way and higher level synergies are likely to be important in therapeutic applications of angiogenic growth factors.

The platelet-derived growth factor (PDGF) family, which regulates many physiological and pathophysiological processes has recently been enlarged by two new members, the isoforms PDGF-C and -D. Little is known about the expression levels of these new members in hepatic fibrosis. We therefore investigated by quantitative real time PCR (Taqman) the mRNA expression profiles of all four PDGF isoforms in transdifferentiating primary cultured hepatic stellate cells (HSC), an in vitro model system of hepatic fibrogenesis, either with or without stimulation of the cells with PDGF-BB or TGF-beta1. All four isoforms were expressed in HSC transdifferentiating to myofibroblast-like cells (MFB) albeit with different profiles: while PDGF-A mRNA exhibited minor fluctuations only, PDGF-B was rapidly down-regulated. In contrast, both PDGF-C and -D mRNA were strongly induced: PDGF-C up to 5 fold from day 2 to day 8 and PDGF-D up to 8 fold from day 2 to day 5 of culture. Presence of PDGF-DD in activated HSC was confirmed at the protein level by immunocytochemistry. Stimulation of HSC and MFB with PDGF-BB led to down-regulation of the new isoforms, whereas TGF-beta1 upregulated PDGF-A only. We further show that PDGF receptor-beta (PDGFR-beta) mRNA was rapidly upregulated within the first day of culture and was constantly expressed from day 2 on while the expression profile of PDGFR-alpha mRNA was very similar to that of PDGF-A during transdifferentiation. Given the dramatic changes in PDGF-C and -D expression, which may compensate for down-regulation of PDGF-B, we hypothesize that the new PDGF isoforms may fulfil specific functions in hepatic fibrogenesis.

Platelet-derived growth factor-BB (PDGF-BB) is a potent motogen for vascular smooth muscle cells (VSMCs). To understand its motogenic signaling events, we have studied the role of the Janus-activated kinase/signal transducers and activators of transcription (Jak/STAT) pathway and cytosolic phospholipase A(2) (cPLA(2)). PDGF-BB stimulated tyrosine phosphorylation of Jak-2 and STAT-3 in a time-dependent manner in VSMCs. In addition, AG490 and Jak-2KEpRK5, a selective pharmacological inhibitor and a dominant negative mutant, respectively, of Jak-2, attenuated PDGF-BB-induced STAT-3 tyrosine phosphorylation and its DNA binding and reporter gene activities. PDGF-BB induced VSMC motility in a dose-dependent manner with a maximum effect at 10 ng/ml. Dominant negative mutant-dependent suppression of Jak-2 and STAT-3 blocked PDGF-BB-induced VSMC motility. PDGF-BB induced the expression of cPLA(2) in a Jak-2/STAT-3-dependent manner, and pharmacological inhibitors of cPLA(2) prevented PDGFBB-induced VSMC motility. Furthermore, either exogenous addition of arachidonic acid or forced expression of cPLA(2) rescued PDGF-BB-induced VSMC motility from inhibition by blockade of Jak-2 and STAT-3 activation. Together, these results for the first time show that PDGF-BB-induced VSMC motility requires activation of the Jak-2/STAT-3/cPLA(2) signaling axis.

BACKGROUND

Pulmonary hypertension and left ventricular diastolic dysfunction are complications of sickle cell disease. Pulmonary hypertension is associated with hemolysis and hypoxia, but other unidentified factors are likely involved in pathogenesis as well.

METHODS

Plasma concentrations of three angiogenic markers (fibroblast growth factor, platelet derived growth factor-BB [PDGF-BB], vascular endothelial growth factor [VEGF]) and seven inflammatory markers implicated in pulmonary hypertension in other settings were determined by Bio-Plex suspension array in 237 children and adolescents with sickle cell disease at steady state and 43 controls. Tricuspid regurgitation velocity (which reflects systolic pulmonary artery pressure), mitral valve E/Edti ratio (which reflects left ventricular diastolic dysfunction), and a hemolytic component derived from four markers of hemolysis and hemoglobin oxygen saturation were also determined.

RESULTS

Plasma concentrations of interleukin-8, interleukin-10 and VEGF were elevated in the patients with sickle cell disease compared to controls (P<or=0.003). By logistic regression, greater values for <em>PDGF</em>-<em>BB</em> (P = 0.009), interleukin-6 (P = 0.019) and the hemolytic component (P = 0.026) were independently associated with increased odds of elevated tricuspid regurgitation velocity while higher VEGF concentrations were associated with decreased odds (P = 0.005) among the patients with sickle cell disease. These findings, which are consistent with reports that <em>PDGF</em>-<em>BB</em> stimulates and VEGF inhibits vascular smooth muscle cell proliferation, did not apply to E/Etdi.

CONCLUSIONS

Circulating concentrations of angiogenic and pro-Inflammatory markers are altered in sickle cell disease children and adolescents with elevated tricuspid regurgitation velocity, a subgroup that may be at risk for developing worsening pulmonary hypertension. Further studies to understand the molecular changes in these children are indicated.

TRPM7, a non-selective cation channel of the TRP channel superfamily, is implicated in diverse physiological and pathological processes including cell proliferation. Recently, TRPM7 has been reported in hepatic stellate cells (HSCs). Here, we investigated the contribution role of TRPM7 in activated HSC-T6 cell (a rat hepatic stellate cell line) proliferation. TRPM7 mRNA and protein were measured by RT-PCR and Western blot in rat model of liver fibrosis in vivo and PDGF-BB-activated HSC-T6 cells in vitro. Both mRNA and protein of TRPM7 were dramatically increased in CCl4-treated rat livers. Stimulation of HSC-T6 cells with PDGF-BB resulted in a time-dependent increase of TRPM7 mRNA and protein. However, PDGF-BB-induced HSC-T6 cell proliferation was inhibited by non-specific TRPM7 blocker 2-aminoethoxydiphenyl borate (2-APB) or synthetic siRNA targeting TRPM7, and this was accompanied by downregulation of cell cycle proteins, cyclin D1, PCNA and CDK4. Blockade of TRPM7 channels also attenuated PDGF-BB induced expression of myofibroblast markers as measured by the induction of α-SMA and Col1α1. Furthermore, the phosphorylation of ERK and AKT, associated with cell proliferation, decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TRPM7 channels contribute to perpetuated fibroblast activation and proliferation of PDGF-BB induced HSC-T6 cells via the activation of ERK and PI3K pathways. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis.

Biomolecular environments encountered in vivo are complex and dynamic, with combinations of biomolecules presented in both freely diffusible (liquid-phase) and sequestered (bound to the extracellular matrix) states. Strategies for integrating multiple biomolecular signals into a biomimetic scaffold provide a platform to simultaneously control multiple cell activities, such as motility, proliferation, phenotype, and regenerative potential. Here we describe an investigation elucidating the influence of the dose and mode of presentation (soluble, sequestered) of five biomolecules (stromal cell-derived factor 1α [SDF-1α], platelet-derived growth factor BB [PDGF-BB], insulin-like growth factor 1 [IGF-1], basic fibroblast growth factor [bFGF], and growth/differentiation factor 5 [GDF-5]) on the recruitment, proliferation, collagen synthesis, and genomic stability of equine tenocytes within an anisotropic collagen-GAG scaffold for tendon regeneration applications. Critically, we found that single factors led to a dose-dependent trade-off between driving tenocyte proliferation (PDGF-BB, IGF-1) versus maintenance of a tenocyte phenotype (GDF-5, bFGF). We identified supplementation schemes using factor pairs (IGF-1, GDF-5) to rescue the tenocyte phenotype and gene expression profiles while simultaneously driving proliferation. These results suggest coincident application of multi-biomolecule cocktails has a significant value in regenerative medicine applications where control of cell proliferation and phenotype are required. Finally, we demonstrated an immobilization strategy that allows efficient sequestration of bioactive levels of these factors within the scaffold network. We showed sequestration can lead to a greater sustained bioactivity than soluble supplementation, making this approach particularly amenable to in vivo translation where diffusive loss is a concern and continuous biomolecule supplementation is not feasible.

Activated pancreatic stellate cells (PSCs) play a pivotal role in the pathogenesis of pancreatic fibrosis and inflammation. Inhibition of activation and cell functions of PSCs is a potential target for the treatment of pancreatic fibrosis and inflammation. The polyphenol compound curcumin is the yellow pigment in curry, and has anti-inflammatory and anti-fibrotic properties. We here evaluated the effects of curcumin on the activation and cell functions of PSCs. PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. The effects of curcumin on proliferation, alpha-smooth muscle actin gene expression, monocyte chemoattractant protein (MCP)-1 production, and collagen expression were examined. The effect of curcumin on the activation of freshly isolated cells in culture was also assessed. Curcumin inhibited platelet-derived growth factor (PDGF)-induced proliferation, alpha-smooth muscle actin gene expression, interleukin-1beta- and tumor necrosis factor (TNF)-alpha-induced MCP-1 production, type I collagen production, and expression of type I and type III collagen genes. Curcumin inhibited PDGF-BB-induced cyclin D1 expression and activation of extracellular signal-regulated kinase (ERK). Curcumin inhibited interleukin-1beta- and TNF-alpha-induced activation of activator protein-1 (AP-1) and mitogen-activated protein (MAP) kinases (ERK, c-Jun N-terminal kinase (JNK), and p38 MAP kinase), but not of nuclear factor-kappaB (NF-kappaB). In addition, curcumin inhibited transformation of freshly isolated cells to myofibroblast-like phenotype. In conclusion, curcumin inhibited key cell functions and activation of PSCs.

Burns, especially those involving large surface areas, represent a complex wound healing problem. Platelet-derived growth factor (PDGF) is released by activated platelets to recruit inflammatory cells toward the wound bed. It has effects on promoting angiogenesis and granulation tissue formation. However, the effectiveness of topical PDGF on wound closure is variable, ranging from little improvement observed in pig models to dramatic improvement reported in a diabetic mouse model. Here, we sought to determine the effectiveness of commercially sold PDGF-BB (Regranex) on wound closure in genetically diabetic mice. C57BL/KsJ db+/db+ mice and its host strain bearing dorsal 1.5-cm wounds were divided into groups (n = 8 in each group) receiving topical application of either Regranex (10 microg/wound) or vehicle for 5 consecutive days after wounding. The rate of wound closure was analyzed using computerized planimetry. The amount of granulation tissue was determined histologically. Our data indicate that diabetic mice exhibit a significant delay in wound closure when compared with their host strain. Topical application of Regranex did not improve the time to wound closure but did significantly increase the amount of granulation tissue. Our current study using commercially available Regranex failed to reproduce the previously reported finding that PDGF improved wound closure in healing impaired genetically diabetic mice.

OBJECTIVE

To evaluate the usefulness of a platelet-derived growth factor (PDGF)-B specific monoclonal antibody (mAb) as a therapeutic agent to treat chronic liver fibrosis.

METHODS

Liver fibrosis was induced in ICR mice by bile duct ligation (BDL) or BALB/c mice by weekly injection of concanavalin A (ConA) for 4 or 8 weeks. A mAb specific for mouse and human PDGF-B chain, AbyD3263, was generated, tested in vitro and administered twice a week throughout the experimental period.

RESULTS

AbyD3263 showed neutralizing activity against mouse and human PDGF-B chain in cell-based assays, as measured in vitro by inhibition of phosphorylation of PDGF receptor β and proliferation of hepatic stellate cells induced by PDGF-BB. The half life of AbyD3263 in mice exceeded 7 days and dosing of animals twice a week resulted in constant plasma levels of the mAb. Induction of liver fibrosis by BDL and ConA resulted in elevated levels of alanine aminotransferase (ALT) in plasma and hydroxyproline in the liver. Treatment with AbyD3263 did not modify ALT levels, but significantly reduced hydroxyproline content in the liver with a maximum reduction of 39% and 54% in the BDL and ConA models, respectively, compared to controls. Conclusios: Consistent with the notion that PDGF-BB plays an important role in the progression of liver fibrosis, AbyD3263 exhibits efficacy in pre-clinical disease models suggesting that pharmacological inhibition of PDGF-B chain may be a therapeutic approach to treat liver fibrosis.

alpha11beta1 constitutes the most recent addition to the integrin family and has been shown to display a binding preference for interstitial collagens found in mesenchymal tissues. We have previously observed that when alpha11beta1 integrin is expressed in cells lacking endogenous collagen receptors, it can mediate PDGF-BB-dependent chemotaxis on collagen I in vitro. To determine in which cells PDGF and alpha11beta1 might cooperate in regulating cell migration in vivo, we studied in detail the expression and distribution of alpha11 integrin chain in mouse embryos and tested the ability of PDGF isoforms to stimulate the alpha11beta1-mediated cell migration of embryonic fibroblasts. Full-length mouse alpha11 cDNA was sequenced and antibodies were raised to deduced alpha11 integrin amino acid sequence. In the embryonic mouse head, alpha11 protein and RNA were localized to ectomesenchymally derived cells. In the periodontal ligament, alpha11beta1 was expressed as the only detectable collagen-binding integrin, and alpha11beta1 is thus a major receptor for cell migration and matrix organization in this cell population. In the remainder of the embryo, the alpha11 chain was expressed in a subset of mesenchymal cells including tendon/ligament fibroblasts, perichondrial cells, and intestinal villi fibroblasts. Most of the alpha11-expressing cells also expressed the alpha2 integrin chain, but no detectable overlap was found with the alpha1 integrin chain. In cells expressing multiple collagen receptors, these might function to promote a more stable cell adhesion and render the cells more resistant to chemotactic stimuli. Wild-type embryonic fibroblasts activated mainly the PDGF beta receptor in response to PDGF-BB and migrated on collagens I, II, III, IV, V, and XI in response to PDGF-BB in vitro, whereas mutant fibroblasts that lacked alpha11beta1 in their collagen receptor repertoire showed a stronger chemotactic response on collagens when stimulated with PDGF-BB. In the cellular context of embryonic fibroblasts, alpha11beta1 is thus anti-migratory. We speculate that the PDGF BB-dependent cell migration of mesenchymal cells is tightly regulated by the collagen receptor repertoire, and disturbances of this repertoire might lead to unregulated cell migration that could affect normal embryonic development and tissue structure.

Platelet-derived growth factor BB (PDGF-BB) was an important inductive factor during wound healing, but the lack of efficient delivery system limited its clinical application. Here, a peptide of seven amino acids was firstly utilized to engineer PDGF-BB to construct a collagen-targeting system. This peptide functioned as collagen-binding domain (CBD) to specially target the PDGF-BB to the collagen and restrict its diffusion. In our study, in vitro activity assay showed that the CBD-fused PDGF (CBD-PDGF) and native PDGF (NAT-PDGF) possessed similar activity to stimulate the human fibroblast proliferation. As expected, this peptide promoted the binding of PDGF to collagen scaffolds, and CBD-PDGF caused more cells to proliferate on the collagen gel than NAT-PDGF when the same amounts of PDGF were added. In the in vivo experiment, CBD-PDGF-loaded collagen scaffolds were uniformly cellularized and vascularized compared to that of NAT-PDGF-loaded scaffolds. Our study demonstrated that the CBD targeted PDGF to the collagen scaffold to exert its functions, and it suggested this could be an effective system for targeting tissue regeneration and wound repair.

OBJECTIVE

To investigate possible correlations between vitreous and/or serum levels of platelet-derived growth factor isoforms (PDGF-AA, -AB and -BB) with parameters associated with non-proliferative diabetic retinopathy (NPDR) and clinically significant macula oedema (CSMO); to compare the results to relevant results regarding vascular endothelial growth factor (VEGF), which is an established growth factor affecting NPDR.

METHODS

Fifteen patients with NPDR, 31 patients with proliferative diabetic retinopathy (PDR) and 15 non-diabetic patients were included in the study. Vitreous and serum samples were obtained during vitrectomy. PDGF-AA, -AB and -BB, as well as VEGF, were measured by enzyme-linked immunosorbent assay (ELISA).

RESULTS

PDGF-AA, -AB and -BB and VEGF were all expressed in the serum and vitreous of controls and patients with NPDR. The levels of PDGF-AA, PDGF-AB and VEGF in vitreous were significantly increased in the NPDR group compared to controls, while PDGF-BB levels were significantly decreased in the NPDR group compared to controls. The levels of all PDGF isoforms and VEGF in vitreous were significantly increased in the PDR group compared to the NPDR group. No such differences were evident in serum. PDGF-AA and VEGF correlated significantly to the severity of NPDR. PDGF or VEGF in vitreous of NPDR patients did not correlate with retinal photocoagulation (RP) or the serum levels of haemoglobin A1c (HbA1c). There was no correlation between the vitreous and serum levels of VEGF or PDGF in patients with PDR. Only PDGF-AB vitreous levels correlated significantly with PDGF-BB vitreous levels in the NPDR group.

CONCLUSIONS

It appears that in addition to VEGF, almost all PDGF isoforms in the vitreous are also correlated with NPDR and CSMO.