Platelet-derived Growth Factor Induction Research Articles

Autophagy-Related Activation of Hepatic Stellate Cells Reduces Cellular miR-29a by Promoting Its Vesicular Secretion.

Background & AimsLiver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis.MethodsIntracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor β (TGFβ) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl4 induced liver fibrosis model.ResultsIn our study, we show that TGFβ and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets’ expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl4-treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease.ConclusionsWe provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion.

Hepatic microenvironment underlies fibrosis in chronic hepatitis B patients.

BACKGROUNDChronic hepatitis B virus (HBV) infection is a leading cause of liver morbidity and mortality worldwide. Liver fibrosis resulting from viral infection-associated inflammation and direct liver damage plays an important role in disease management and prognostication. The mechanisms underlying the contribution of the liver microenvironment to fibrosis in HBV patients are not fully understood. There is an absence of effective clinical treatments for liver fibrosis progression; thus, establishing a suitable in vitro microenvironment in order to design novel therapeutics and identify molecular biomarkers to stratify patients is urgently required.AIMTo examine a subset of pre-selected microenvironment factors of chronic HBV patients that may underlie fibrosis, with a focus on fibroblast activation.METHODSWe examined the gene expression of key microenvironment factors in liver samples from patients with more advanced fibrosis compared with those with less severe fibrosis. We also used the human stellate cell line LX-2 in the in vitro study. Using different recombinant cytokines and growth factors or their combination, we studied how these factors interacted with LX-2 cells and pinpointed the cross-talk between the aforementioned factors and screened the most important factors.RESULTSOf the secreted factors examined, transforming growth factor (TGF)-β1, interleukin (IL)-1β and tumor necrosis factor (TNF)-α were increased in patients with advanced fibrosis. We found that besides TGF-β1, IL-1β can also induce a profibrotic cascade by stimulating the expression of connective tissue growth factor and platelet-derived growth factor (PDGF) in LX-2 cells. Furthermore, the proinflammatory response can be elicited in LX-2 cells following treatment with IL-1β and TNF-α, suggesting that stellate cells can respond to proinflammatory stimuli. By combining IL-1β and TGF-β1, we observed not only fibroblast activation as shown by αlpha-smooth muscle actin and PDGF induction, but also the inflammatory response as shown by increased expression of IL-1β.CONCLUSIONCollectively, our data from HBV patients and in vitro studies demonstrate that the hepatic microenvironment plays an important role in mediating the crosstalk between profibrotic and proinflammatory responses and modulating fibrosis in chronic HBV patients. For the establishment of a suitable in vitro microenvironment for HBV-induced liver fibrosis, not only TGF-β1 but also IL-1β should be considered as a necessary environmental factor.

Enhanced Rb/E2F and TSC/mTOR Pathways Induce Synergistic Inhibition in PDGF-Induced Proliferation in Vascular Smooth Muscle Cells.

Platelet-derived growth factor (PDGF) plays an essential role in proliferation of vascular smooth muscle cells (VSMCs). The Rb/E2F and TSC/mTOR pathways contribute to the proliferation of VSMCs, but its exact roles in PDGF-induced proliferation are unclear. In this study, we demonstrated the roles of Rb/E2F and TSC/mTOR pathways in PDGF-induced proliferation in VSMCs. We found that PDGF stimulates the activity of E2F and mTOR pathways, and knockdown of either Rb or TSC2 increases PDGF-induced proliferation in VSMCs. More interestingly, we revealed that enhancing both E2F and mTOR activity leads to synergistic inhibition of PDGF-induced proliferation in VSMCs. We further identified that the synergistic inhibition effect is caused by the induced oxidative stress. Summarily, these data suggest the important regulations of Rb/E2F and TSC/mTOR pathways in PDGF-induced proliferation in VSMCs, and also present a promising way to limit deregulated proliferation by PDGF induction in VSMCs.

Dependence of fibroblast infiltration in tumor stroma on type IV collagen-initiated integrin signal through induction of platelet-derived growth factor

Cancer-associated fibroblasts play a crucial role in accelerating tumor progression, but there is a knowledge gap regarding the chemotactic signal activated in a tumor microenvironment. In this study, the expression of type IV collagen was knocked down using a lentiviral-mediated short hairpin RNA strategy. Although there was no obvious effect on cell growth in vitro, silencing the Col4-α1 gene decreased the tumorigenicity of B16F10 in C57BL/6 mice, which was accompanied by a reduction in the infiltration of alpha-smooth muscle actin-positive (α-SMA+) fibroblasts. Silencing the Col4-α1 gene or disrupting integrin engagement by blocking the antibody reduced the expression of platelet-derived growth factor A (PDGF-A), a potent chemotactic factor for fibroblasts. Furthermore, ectopic expression of the autoclustering integrin mutant significantly stimulated PDGF-A expression in murine B16F10 and human U118MG and Huh7 cells. PDGF-A-specific sh-RNA and neutralizing anti-PDGF-A antibody effectively inhibited the transwell migration of fibroblasts. Adding recombinant PDGF-A back to shCol cell-conditioned media restored the fibroblast-attraction ability indicating that PDGF-A is a major chemotactic factor for fibroblasts in the current study model. The integrin-associated PDGF-A production correlated with the activation of Src and ERK. High type IV collagen staining intensity colocalized with elevated PDGF-A expression was observed in tumor tissues obtained from hepatoma and glioma patients. The integrin signal pathway was activated by collagen engagement through Src and ERK, leading to enhanced PDGF-A production, which serves as a key regulator of fibroblast recruitment.

Different effects of tacrolimus and cyclosporine on PDGF induction and chronic allograft injury: Evidence for improved kidney graft outcome

IntroductionChronic allograft injury is the most frequent cause of long-term kidney allograft loss. Cyclosporine (CsA) nephrotoxicity is an important factor contributing to graft loss. Increased fibrosis has been reported in renal transplants under CsA as compared with tacrolimus (Tac). Platelet-derived growth factor (PDGF) is a well-characterized fibrogenic growth factor in renal disease. ObjectiveHere we investigated the effect of Tac on kidney grafts and PDGF expression both early after transplantation as well as during long-term follow-up of rat renal allografts, and compared it to CsA. Tac and CsA were also studied on PDGF secretion in macrophages in vitro. Materials and methodsKidney allografts were immunosuppressed with Tac or CsA. Grafts were analyzed by histology and immunohistochemistry. ELISA was used to measure PDGF-levels in Tac and CsA-treated macrophage cultures. ResultsTac ameliorated markedly both acute and chronic changes, whereas moderate to intense histological changes were seen in CsA-treated allografts. Tac also significantly inhibited PDGF expression compared to CsA. At clinically adjusted concentration CsA induced PDGF in macrophages whereas Tac did not. DiscussionTac may be less fibrogenic than CsA by decreasing PDGF expression in kidney grafts as well as in macrophages. Data presented here suggests that decreased PDGF induction by Tac may be beneficial for later outcome of the kidney graft.

Increased growth factors play a role in wound healing promoted by noninvasive oxygen-ozone therapy in diabetic patients with foot ulcers.

Management of diabetic foot ulcers (DFUs) is a great challenge for clinicians. Although the oxygen-ozone treatment improves the diabetic outcome, there are few clinical trials to verify the efficacy and illuminate the underlying mechanisms of oxygen-ozone treatment on DFUs. In the present study, a total of 50 type 2 diabetic patients complicated with DFUs, Wagner stage 2~4, were randomized into control group treated by standard therapy only and ozone group treated by standard therapy plus oxygen-ozone treatment. The therapeutic effects were graded into 4 levels from grade 0 (no change) to grade 3 (wound healing). The wound sizes were measured at baseline and day 20, respectively. Tissue biopsies were performed at baseline and day 11. The expressions of vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet-derived growth factor (PDGF) proteins in the pathologic specimens were determined by immunohistochemical examinations. The effective rate of ozone group was significantly higher than that of control group (92% versus 64%, P < 0.05). The wound size reduction was significantly more in ozone group than in control group (P < 0.001). After treatment, the expressions of VEGF, TGF-β, and PDGF proteins at day 11 were significantly higher in ozone group than in control group. Ozone therapy promotes the wound healing of DFUs via potential induction of VEGF, TGF-β, and PDGF at early stage of the treatment. (Clinical trial registry number is ChiCTR-TRC-14004415).

Platelet-Derived Growth Factor Induced Alpha-Smooth Muscle Actin Expression by Human Retinal Pigment Epithelium Cell

(1) To evaluate the association between expression of α-smooth muscle actin (α-SMA) in proliferative vitreoretinopathy (PVR) and the pathological grading of PVR, and the effect of platelet-derived growth factor (PDGF) on the expression of α-SMA by human retinal pigment epithelial (RPE) cells. (2) To investigate the potential induction of PDGF on the proliferation and migration of human RPE cells as well as the signaling pathways responsible. We immunohistochemically investigated the expression of α-SMA in PVR. To further investigate the effect of PDGF and the downstream signaling, exogenous PDGF-BB and signaling inhibitors were added to cultured human RPE cells. The MTT method was performed to detected cell proliferation, while cell migration was also measured. α-SMA expression was positively correlated with the pathological grading of PVR. PDGF-BB could stimulate the proliferation and migration of cultured RPE cells through the participation of mitogen-activated protein kinase. In addition, PDGF induced α-SMA expression. The promotion of proliferate/migration and α-SMA expression by PDGF-BB was enhanced by the presence of serum. PDGF and α-SMA are 2 potential therapeutic targets for the treatment of PVR.

Reactive Oxygen Species Regulate the Levels of Dual Oxidase (Duox1-2) in Human Neuroblastoma Cells

Dual Oxidases (DUOX) 1 and 2 are efficiently expressed in thyroid, gut, lung and immune system. The function and the regulation of these enzymes in mammals are still largely unknown. We report here that DUOX 1 and 2 are expressed in human neuroblastoma SK-N-BE cells as well as in a human oligodendrocyte cell line (MO3-13) and in rat brain and they are induced by platelet derived growth factor (PDGF). The levels of DUOX 1 and 2 proteins and mRNAs are induced by reactive oxygen species (ROS) produced by the membrane NADPH oxidase. As to the mechanism, we find that PDGF stimulates membrane NADPH oxidase to produce ROS, which stabilize DUOX1 and 2 mRNAs and increases the levels of the proteins. Silencing of gp91phox (NOX2), or of the other membrane subunit of NADPH oxidase, p22phox, blocks PDGF induction of DUOX1 and 2. These data unravel a novel mechanism of regulation of DUOX enzymes by ROS and identify a circuitry linking NADPH oxidase activity to DUOX1 and 2 levels in neuroblastoma cells.

PDGF upregulates Mcl-1 through activation of β-catenin and HIF-1α-dependent signaling in human prostate cancer cells.

BackgroundAberrant platelet derived growth factor (PDGF) signaling has been associated with prostate cancer (PCa) progression. However, its role in the regulation of PCa cell growth and survival has not been well characterized.Methodology/Principal FindingsUsing experimental models that closely mimic clinical pathophysiology of PCa progression, we demonstrated that PDGF is a survival factor in PCa cells through upregulation of myeloid cell leukemia-1 (Mcl-1). PDGF treatment induced rapid nuclear translocation of β-catenin, presumably mediated by c-Abl and p68 signaling. Intriguingly, PDGF promoted formation of a nuclear transcriptional complex consisting of β-catenin and hypoxia-inducible factor (HIF)-1α, and its binding to Mcl-1 promoter. Deletion of a putative hypoxia response element (HRE) within the Mcl-1 promoter attenuated PDGF effects on Mcl-1 expression. Blockade of PDGF receptor (PDGFR) signaling with a pharmacological inhibitor AG-17 abrogated PDGF induction of Mcl-1, and induced apoptosis in metastatic PCa cells.Conclusions/SignificanceOur study elucidated a crucial survival mechanism in PCa cells, indicating that interruption of the PDGF-Mcl-1 survival signal may provide a novel strategy for treating PCa metastasis.

Morphine Induces Expression of Platelet-Derived Growth Factor in Human Brain Microvascular Endothelial Cells: Implication for Vascular Permeability

Despite the advent of antiretroviral therapy, complications of HIV-1 infection with concurrent drug abuse are an emerging problem. Morphine, often abused by HIV-infected patients, is known to accelerate neuroinflammation associated with HIV-1 infection. Detailed molecular mechanisms of morphine action however, remain poorly understood. Platelet-derived growth factor (PDGF) has been implicated in a number of pathological conditions, primarily due to its potent mitogenic and permeability effects. Whether morphine exposure results in enhanced vascular permeability in brain endothelial cells, likely via induction of PDGF, remains to be established. In the present study, we demonstrated morphine-mediated induction of PDGF-BB in human brain microvascular endothelial cells, an effect that was abrogated by the opioid receptor antagonist-naltrexone. Pharmacological blockade (cell signaling) and loss-of-function (Egr-1) approaches demonstrated the role of mitogen-activated protein kinases (MAPKs), PI3K/Akt and the downstream transcription factor Egr-1 respectively, in morphine-mediated induction of PDGF-BB. Functional significance of increased PDGF-BB manifested as increased breach of the endothelial barrier as evidenced by decreased expression of the tight junction protein ZO-1 in an in vitro model system. Understanding the regulation of PDGF expression may provide insights into the development of potential therapeutic targets for intervention of morphine-mediated neuroinflammation.

HIV-1 Tat-Mediated Induction of Platelet-Derived Growth Factor in Astrocytes: Role of Early Growth Response Gene 1

HIV-associated neurologic disorders (HAND) are estimated to affect almost 60% of HIV-infected individuals. HIV encephalitis, the pathologic correlate of the most severe form of HAND, is often characterized by glial activation, cytokine-chemokine dysregulation, and neuronal damage and loss. However, the severity of HIV encephalitis correlates better with glial activation rather than viral load. Using the macaque model, it has been demonstrated that SIV encephalitis correlates with increased expression of the mitogen platelet-derived growth factor (PDGF) B chain in the brain. The goal of this study was to explore the role of PDGF-B chain in HIV-associated activation and proliferation of astrocytes. Specifically, the data demonstrate that exposure of rat and human astrocytes to the HIV-1 protein Tat resulted in the induction of PDGF at both the mRNA and protein levels. Furthermore, PDGF-BB induction was regulated by activation of ERK1/2 and JNK signaling pathways and the downstream transcription factor early growth response 1. Chromatin immunoprecipitation assays demonstrated binding of Egr-1 to the PDGF-B promoter. Exposure of astrocytes to PDGF-BB in turn led to increased proliferation and the release of proinflammatory cytokines MCP-1 and IL-1β. Because astrogliosis is linked to disease severity, understanding its regulation by PDGF-BB could aid in the development of therapeutic intervention strategies for HAND.

Cocaine-mediated induction of platelet-derived growth factor: implication for increased vascular permeability

AbstractNeuroinflammation associated with advanced HIV-1 infection is often exacerbated in cocaine-abusing, HIV-infected patients. The underlying mechanisms could, in part, be attributed to the increased impairment of blood brain barrier integrity in the presence of cocaine. Platelet-derived growth factor (PDGF) has been implicated in several pathologic conditions, specifically attributable to its potent mitogenic effects. Its modulation by drug abuse, however, has received very little attention. In the present study, we demonstrated cocaine-mediated induction of PDGF-BB in human brain microvascular endothelial cells through the binding to its cognate σ receptor. Furthermore, this effect was mediated, with subsequent activation of mitogen-activated protein kinases and Egr-1 pathways, culminating ultimately into increased expression of PDGF-BB. Cocaine exposure resulted in increased permeability of the endothelial barrier, and this effect was abrogated in mice exposed to PDGF-BB neutralizing antibody, thus underscoring its role as a vascular permeant. In vivo relevance of these findings was further corroborated in cocaine-treated mice that were administered neutralizing antibody specific for PDGF-BB as well as in Egr-1−/− mice. Understanding the regulation of PDGF-BB expression may provide insights into the development of potential therapeutic targets for neuroinflammation associated with HIV infection and drug abuse.

Early short-term imatinib treatment is sufficient to prevent the development of chronic allograft nephropathy

Chronic allograft nephropathy (CAN), now defined as interstitial fibrosis and tubular atrophy not otherwise specified, is a near universal finding in kidney grafts by the end of the first decade posttransplantation. Platelet-derived growth factor (PDGF) is a major mitogen mediating mesenchymal cell proliferation in CAN. Here, we investigated whether early short-term PDGF inhibition with imatinib could prevent CAN. Kidney transplantations were performed from Dark-Agouti (DA) to Wistar-Furth (WF) rats and syngenic controls were done between DA rats. Allografts were immunosuppressed with cyclosporine. One group was also treated with imatinib for the first 30 days after transplantation. Serum creatinine levels were measured once a week. Grafts were harvested 90 days after transplantation. In control allografts, moderate to intense chronic changes were seen, whereas in syngenic grafts, no changes were seen. The early imatinib treatment prevented the development of CAN significantly compared to control allografts. Only few histological changes were seen. Fibrogenic growth factor ligand and receptor induction as well as inflammatory cell response was significantly inhibited by imatinib. Creatinine values of imatinib-treated allografts were also significantly lower compared to controls. We show that short-term imatinib treatment is sufficient to prevent CAN significantly, indicating that early PDGF induction has an important role in the pathogenesis of CAN. Here, we provide preclinical work that will need to be confirmed in patients with CAN.

Tyrosine Phosphorylation of Munc18c Regulates Platelet-Derived Growth Factor-Stimulated Glucose Transporter 4 Translocation in 3T3L1 Adipocytes

Platelet-derived growth factor (PDGF) stimulation of skeletal muscle, cultured myotubes, and 3T3L1 adipocytes results in glucose transporter 4 (Glut4) translocation, albeit to a reduced level compared with insulin. To address the mechanism of PDGF action, we have determined that the Syntaxin 4 negative regulatory protein, Munc18c, undergoes PDGF-stimulated phosphorylation on tyrosine residue 521. The tyrosine phosphorylation of Munc18c on Y521 occurred concomitant with the dissociation of the Munc18c protein from Syntaxin 4 in a time frame consistent with Glut4 translocation. Moreover, expression of the wild-type Munc18c protein did not inhibit PDGF-induced Glut4 translocation, whereas expression of Y521A-Munc18c mutant was inhibitory and failed to dissociate from Syntaxin 4. In contrast, expression of either wild-type Munc18c or the Y521A-Munc18c mutant both resulted in a marked inhibition of insulin-stimulated Glut4 translocation. Together, these data demonstrate that one mechanism accounting for the PDGF induction of Glut4 translocation is the suppression of the Munc18c negative regulation of Syntaxin 4 function.

Suppressor of cytokine signaling 3 negative regulation of signal transducer and activator of transcription 3 in platelet‐derived growth factor‐induced fibroblast migration

Platelet-derived growth factor (PDGF) is involved in wound healing, but PDGF-induced fibroblast migration and the intracellular signaling mechanisms of fibroblast migration are poorly understood. Signal transducer and activator of transcription 3 (STAT3) is involved in migration and is negatively regulated by the suppressor of cytokine signaling 3 (SOCS3). We studied the PDGF induction of fibroblast migration in vitro and the involvement of STAT3 and SOCS3. We found that PDGF activated STAT3 and strongly induced fibroblast migration. Transfection with a dominant-negative mutant of STAT3 almost completely abolished PDGF-induced fibroblast migration and STAT3 phosphorylation. Next, we studied the mechanisms that regulate fibroblast migration. PDGF enhanced the expression of SOCS3 by 2.8-fold at 1 h. Transfection with SOCS3 almost completely abolished PDGF-induced STAT3 phosphorylation and reduced fibroblast migration to 47% of control, indicating that SOCS3 acts as a negative regulator of PDGF-induced fibroblast migration. In conclusion, PDGF induces fibroblast migration under the control of STAT3-SOCS3.

Gene Expression Profiles During Hepatic Stellate Cell Activation in Culture and In Vivo

Following hepatic injury, hepatic stellate cells (HSCs) transdifferentiate to become extracellular matrix-producing myofibroblasts and to promote hepatic fibrogenesis. In this study, we determine gene expression changes in 3 different models of HSC activation and investigate whether HSC culture activation reproduces gene expression changes of HSC in vivo activation. HSCs were isolated by density centrifugation and magnetic antibody cell sorting from normal mice, CCl(4)-treated mice, and mice that underwent bile duct ligation (BDL). Gene expression was analyzed by microarray and confirmed by polymerase chain reaction and Western blot analysis. Two thousand seventy-three probe sets were differentially expressed in at least 1 of 3 models of HSC activation, including novel genes that encode proinflammatory and antiapoptotic mediators; transcription factors; cell surface receptors; and cytoskeleton components such as CXCL14, survivin, septin 4, osteopontin, PRX1, LMCD1, GPR91, leiomodin, and anillin. BDL- and CCl(4)-activated HSCs showed highly correlated gene expression patterns, whereas culture activation only partially reproduced the gene expression changes observed during BDL- and CCl(4)-induced activation. Coculture with Kupffer cells or lipopolysaccharide treatment during culture activation shifted the expression of most examined genes toward the pattern observed during in vivo activation, suggesting a role for these factors in the microenvironment that drives HSC activation. The almost identical HSC gene expression patterns after BDL or CCl(4) treatment indicate that HSCs exert similar functions in different types of liver injury. Because culture activation does not properly regulate gene expression in HSCs, in vivo activation should be considered the gold standard for the study of HSC biology.

PKA promotes MEF2/HDAC repression of the c‐Jun promoter in vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) do not terminally differentiate, but can modulate their phenotype in response to extracellular stimuli. Although proliferative VSMCs are required during vascular repair, the activated phenotype also plays a role in vascular disease. To investigate the role of myocyte enhancer factor 2 (MEF2) in the induction of the growth-responsive gene, c-Jun, we utilized the A10 line. Mitogenic stimulation by platelet derived growth factor (PDGF) resulted in marked induction of c-Jun protein and promoter activity. This induction was attenuated by rottlerin and KN-62. Given that these signaling pathways have been shown to relieve the repressive effects of histone deacetylases (HDACs) on MEF2 proteins, we overexpressed HDAC4, which repressed the c-Jun promoter. Mutation of the MEF2 binding site in the c-Jun promoter resulted in activation during quiescent conditions, while treatment with trichostatin A, increased c-Jun protein. Interestingly, activation of protein kinase A (PKA) prevented PDGF induction of c-Jun, and repressed a MEF2-dependent reporter gene. PKA also caused nuclear accumulation of HDAC4 and stabilized the interaction of HDAC4 with MEF2D. Thus, it appears that MEF2 and HDAC4 act to repress c-Jun expression in quiescent VSMCs, PKA enhances this repression, and PDGF derepresses through CaMKs and novel PKCs. Supported by CIHR and the Heart and Stroke Foundation of Canada.

Early Short-Term Platelet-Derived Growth Factor Inhibition Prevents the Development of Chronic Allograft Nephropathy in Experimental Rat Kidney Transplantation

Chronic allograft nephropathy (CAN) remains the primary reason for late allograft loss in kidney transplantation. Platelet-derived growth factor (PDGF) is a major mitogen mediating mesenchymal cell proliferation in CAN. When administered continuously the PDGF receptor tyrosine kinase inhibitor imatinib prevents the development of CAN and restores kidney function in experimental kidney transplantation. Herein we investigated whether early short-term imatinib treatment prevented CAN. Kidney transplantations were performed from DA to WF rats and syngenic controls were done between DA rats. Allograft recipients were immunosuppressed with cyclosporine (CsA; 1.5 mg/kg/d sc). One group of allografts was also treated with imatinib (10 mg/kg/d po). Serum creatinine levels were measured once a week. Grafts were harvested 90 days after transplantation for histology and immunohistochemistry (PDGF-AA, -BB, PDGFR-α, -β). Histological changes were scored according to the Chronic Allograft Damage Index (CADI). Among syngenic grafts, no signs of CAN were observed, namely, CADI 0.3 ± 0.2 (mean ± SEM). Control allografts showed moderate to intense chronic changes, CADI 6.5 ± 1.3. Early short-term imatinib treatment significantly prevented the development of CAN compared with control allografts. Only a few histological changes were seen, namely, CADI 3.3 ± 1.4. Compared with control allografts PDGF ligand and receptor induction was significantly inhibited by imatinib to nearly the same level as in syngenic grafts. Creatinine values of imatinib-treated allografts were also lower than control allografts. Our results demonstrated that early short-term imatinib treatment significantly prevented CAN. This indicated that early PDGF induction has an important role in the pathogenesis of CAN.

TGF- type I receptor kinase inhibitor down-regulates rheumatoid synoviocytes and prevents the arthritis induced by type II collagen antibody

Rheumatoid arthritis (RA) is characterized by hypertrophic synovial tissues comprising excessively proliferating synovial fibroblasts and infiltrating inflammatory cells. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that regulates cell growth, inflammation and angiogenesis by acting on various cell types. In RA synovial tissues, TGF-beta is expressed at high levels. However, the precise role of TGF-beta in RA remains unclear. We herein demonstrated a causal link between the TGF-beta-induced RA synovial cell proliferation and induction of platelet-derived growth factor (PDGF)-AA. In addition, TGF-beta induced IL-6 and vascular endothelial growth factor (VEGF) production by RA synovial fibroblasts associated with nuclear factor-kappa B activation. These effects of TGF-beta on RA synovial fibroblasts were suppressed by TGF-beta type I receptor kinase inhibitor HTS466284. Furthermore, HTS466284 significantly prevented anti-collagen type II antibody-induced arthritis in mice according to the clinical manifestations, histology, tumor necrosis factor-alpha, PDGF and VEGF expression and 5-bromo-2'-deoxyuridine incorporation. These in vitro and in vivo results suggest that TGF-beta plays a role in the development of synovial hyperplasia consisting of synovial cell proliferation, inflammation and angiogenesis. The blockade of TGF-beta signaling may thus become an additional strategy for the treatment of RA.

Contrasting role of phospholipase C-γ1 in the expression of immediate early genes induced by epidermal or platelet-derived growth factors

While significant progress has been achieved in identifying the signal transduction elements that operate downstream of activated receptor tyrosine kinases, it remains unclear how different receptors utilize these signaling elements to achieve a common response. This study compares the capacity of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) to elicit the induction of immediate early gene (IEG) mRNAs in the presence or absence of phospholipase C-γ1 (PLC-γ1). The results show that while PDGF induction of nearly all IEG mRNAs is abrogated in plcg1 null cells, EGF induction of the same genes is variable in the null cells and exhibits three distinct responses. Five IEG mRNAs (Nup475, Cyr61, TF, Gly, TS7) are completely inducible by EGF in the presence or absence of PLC-γ1, while three others (JE, KC, FIC) exhibit a stringent requirement for the presence of PLC-γ1. The third type of response is exhibited by c-fos and COX-2. While these mRNAs are completely induced by EGF in the absence of PLC-γ1, the time course of their accumulation is significantly delayed. No IEG was identified as completely inducible by EGF and PDGF in the absence of PLC-γ1. Electrophoretic mobility shift assays (EMSA) demonstrate that PLC-γ1 is necessary for nuclear extracts from PDGF-treated cells, but not EGF-treated cells, to interact with probes for AP-1 or NF-κB.