Platelet-derived Growth factor-B mRNA Research Articles

Glucose degradation products in peritoneal dialysis solution impair angiogenesis by dysregulating angiogenetic factors in endothelial and vascular smooth muscle cells.

The accumulation of glucose degradation products (GDPs) during peritoneal dialysis (PD) can lead to immature angiogenesis in the peritoneum. However, the effect of GDPs on angiogenesis, at concentrations observed in dialysate effluent, has not been widely investigated. We do not know how the inflammation observed in PD-related peritonitis affects angiogenesis of the peritoneum. Human umbilical vessel endothelial cells (HUVEC) and human umbilical aortic smooth muscle cells (HUASMC) were used to examine the response to the three main GDPs found in peritoneal dialysate (methylglyoxal (MGO), 3-deoxyglucosone (3-DG), and 5-hydroxymethylfurfural (5-HMF). Supernatant from lipopolysaccharide (LPS)-activated murine macrophage cell lines (RAW 264.7 cells) were used to stimulate angiogenesis in the peritoneum. Changes in the expression of vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor B (PDGFB) in HUVEC, and PDGF-receptor beta (PDGF-Rβ) in HUASMC, were examined by real-time PCR, Western blot, and ELISA. In HUVECs, the expression of PDGFB mRNA and protein were decreased by exposure to MGO, 3-DG, and 5-HMF at concentrations observed in dialysate effluent. A subsequent decrease in secreted PDGF-BB was observed. In HUASMCs, MGO and 5-HMF increased the expression of VEGF-A mRNA and protein, while 5-HMF decreased the expression of PDGF-Rβ. VEGF-A is upregulated, and PDGF-Rβ is downregulated, by conditioned medium of LPS-stimulated macrophages in HUASMCs. The GDPs of PD effluent cause an imbalance of angiogenic factors in endothelial cells and vascular smooth muscle cells that may lead to immature angiogenesis in the peritoneum.

The effectiveness of chitosan-mediated silencing of PDGF-B and PDGFR-β in the mesangial proliferative glomerulonephritis therapy

Platelet-derived growth factor-B (PDGF-B) is a growth factor that plays an important role in the progression of mesangial proliferative glomerulonephritis (MsPGN). PDGF-B may contribute to mesangioproliferative changes and is overexpressed in MsPGN. Recently, small interfering RNAs (siRNAs) have been widely used for gene silencing effects in experimental models of renal diseases. Nanoparticle-based therapeutics are preferred for reasons such as increasing therapeutic efficacy and reducing toxic effects caused by high doses. The distribution of nanoparticles to the kidney is a significant advantage in siRNA delivery. The aim of this study was to investigate the efficacy of chitosan/siRNA nanoplexes in silencing of PDGF-B and PDGFR-β genes in kidney and to decrease mesangial cell proliferation and matrix accumulation in MsPGN model induced by anti-Thy-1.1 antibody. The therapeutic effects of chitosan/siPDGF-B + siPDGFR-β nanoplexes in glomerulonephritic rats were studied by molecular, biochemical, and histopathologic evaluations. Chitosan/siPDGF-B + siPDGFR-β nanoplexes markedly reduced PDGF-B and PDGFR-β mRNA and protein expressions in experimental MsPGN model. Histopathologic examination results showed that the silencing of PDGF-B and its receptor PDGFR-β led to reduction in mesangial cell proliferation and matrix accumulation. The use of chitosan/siPDGF-B + siPDGFR-β nanoplexes for silencing the PDGF-B pathway in MsPGN can be considered as a new effective therapeutic strategy.

Effect of 15-Deoxy-Δ12,14-prostaglandin J2Nanocapsules on Inflammation and Bone Regeneration in a Rat Bone Defect Model.

Background:15-Deoxy-∆12,14-prostaglandin J2 (15d-PGJ2), one of the major metabolites from prostaglandin D2 in arachidonic acid metabolic pathway, has potential anti-inflammatory properties. The objective of this study was to explore the effects of 15d-PGJ2-loaded poly(D,L-lactide-co-glycolide) nanocapsules (15d-PGJ2-NC) on inflammatory responses and bone regeneration in local bone defect.Methods:The study was conducted on 96 Wistar rats from June 2014 to March 2016. Saline, unloaded nanoparticles, free 15d-PGJ2 or 15d-PGJ2-NC, were delivered through a collagen vehicle inside surgically created transcortical defects in rat femurs. Interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) levels in the surrounding soft tissue were analyzed by Western blot and in the defect by quantitative real-time polymerase chain reaction over 14 days. Simultaneously, bone morphogenetic protein-6 (BMP-6) and platelet-derived growth factor-B (PDGF-B) messenger RNA (mRNA) in the defect were examined. New bone formation and EphrinB2 and osteoprotegerin (OPG) protein expression in the cortical defect were observed by Masson's Trichrome staining and immunohistochemistry over 28 days. Data were analyzed by one-way analysis of variance. Least-significant difference and Dunnett's T3 methods were used with a bilateral P < 0.05.Results:Application of l5d-PGJ2-NC (100 μg/ml) in the local bone defect significantly decreased IL-6, IL-1β, and TNF-α mRNA and protein, compared with saline-treated controls (P < 0.05). l5d-PGJ2-NC upregulated BMP-6 and PDGF-B mRNA (P < 0.05). New bone formation was observed in the cortical defect in l5d-PGJ2-NC-treated animals from 7th day onward (P < 0.001). Expression of EphrinB2 and OPG presented early on day 3 and persisted through day 28 in 15d-PGJ2-NC group (P < 0.05).Conclusion:Stable l5d-PGJ2-NC complexes were prepared that could attenuate IL-6, IL-1β, and TNF-α expression, while increasing new bone formation and growth factors related to bone regeneration.

Liver fibrosis with hypereosinophilia causing transient abnormal myelopoiesis

Transient abnormal myelopoesis is mostly self-resolving and has a good prognosis, but some patients subsequently die of liver fibrosis. We report the case of an infant with Down syndrome who developed life-threatening liver fibrosis at the same time as the blasts were about to disappear. This patient also had a marked increase in eosinophils, which were possibly harboring a GATA1 mutation and were expressing a high level of platelet-derived growth factor-B mRNA; these may have been involved in the development of liver fibrosis. Low-dose cytosine arabinoside therapy effectively treated both hypereosinophilia and liver fibrosis.

Chitosan nanoparticle carrying small interfering RNA to platelet-derived growth factor B mRNA inhibits proliferation of smooth muscle cells in rabbit injured arteries

The purpose of this study was to elucidate the transfection of chitosan nanoparticle carrying small interfering RNA against platelet-derived growth factor B (PDGF-B) to inhibit the expression of PDGF-B mRNA and proliferation of smooth muscle cells. A rabbit iliac artery injury model was constructed. A small interfering RNA (siRNA) against PDGF-B mRNA expression vector was constructed and packaged by chitosan nanoparticle to transfect into the vascular smooth muscle cells (vSMCs) of balloon catheter-injured rabbit iliac artery wall, using a therapeutic ultrasound for the gene delivery. The experiment was divided into two groups: experimental group, denudation and nano-PDGF-B siRNA treated, and only single denudation as control. Effects of the siRNA on the expressions of proliferating cell nuclear antigen (PCNA) and PDGF-B mRNA by vSMCs and the proliferation of vSMCs were observed with the methods of routine pathological, immunohistochemical staining, in situ hybridization and morphometry. The nano siRNA against PDGF-B was successfully transfected. The nano siRNA significantly inhibited the expressions of PCNA and PDGF-B mRNA in intimal vSMCs. The local intimal thickness and area were also reduced remarkably. In conclusion, transfection of chitosan nanoparticle carrying siRNA against PDGF-B mRNA could inhibit proliferation of vSMCs in the rabbit iliac artery injury model.

Hypoxia-Inducible Factor Activation in Myeloid Cells Contributes to the Development of Liver Fibrosis in Cholestatic Mice

Macrophages play an integral role in the development of liver fibrosis by releasing mediators, such as platelet-derived growth factor-B (PDGF-B) and transforming growth factor-β1, which stimulate hepatic stellate cell proliferation, chemotaxis, and collagen production. However, the mechanism by which chronic liver injury stimulates macrophages to release these mediators is not completely understood. We tested the hypothesis that chronic liver injury activates hypoxia-inducible factor (HIF) transcription factors in macrophages that regulate the production of mediators that promote fibrosis. To test this hypothesis, Cre/lox technology was used to generate myeloid cell-specific HIF-1α or HIF-1β knockout mice. When these mice were subjected to bile duct ligation (BDL), levels of α-smooth muscle actin and type I collagen in the liver were reduced compared with those of mice with normal levels of HIFs. The deficiency of HIFs in macrophages did not affect liver injury or inflammation after BDL but reduced PDGF-B mRNA and protein, suggesting that HIF activation in macrophages may promote fibrosis by regulating the production of PDGF-B. Consistent with a role for HIFs in liver fibrosis in cholestatic liver disease, nuclear HIF-1α protein was present in macrophages, hepatocytes, and fibroblasts in the livers from patients with primary biliary cirrhosis and primary sclerosing cholangitis. These studies demonstrate that HIFs are important regulators of profibrotic mediator production by macrophages during the development of liver fibrosis and suggest that HIFs may be a novel therapeutic target for the treatment of chronic liver disease in patients.

Lipopolysaccharide enhances TGFβ1 induction of PDGF‐B in bile duct epithelial cells: role of NF‐κB

Platelet‐derived growth factor‐B (PDGF‐B) is a potent fibrogenic mediator in the liver. The PDGF‐BB homodimer is expressed by bile duct epithelial cells (BDECs) and contributes to liver fibrosis after bile duct ligation. The mechanism of PDGF‐B induction in BDECs is not known. Transforming growth factor β (TGFβ) and lipopolysaccharide (LPS) also contribute to the fibrogenic response after bile duct ligation. We tested the hypothesis that LPS enhances TGFβ1 induction of PDGF‐B in BDECs. Stimulation of MMNK‐1 cells or primary rat BDECs with LPS did not induce PDGF‐B mRNA expression, but LPS stimulation enhanced TGFβ1 induction of PDGF‐B mRNA. LPS did not affect TGFβ1 activation of a SMAD‐responsive luciferase construct. Rather, stimulation of MMNK‐1 cells with LPS, but not TGFβ1, caused IκBα degradation and activation of a NF‐κB‐responsive luciferase construct. Expression of an IκBα super‐repressor or dominant negative IKK2 inhibited activation of the NF‐κB‐responsive luciferase construct and attenuated the LPS potentiation of TGFβ‐induced PDGF‐B mRNA. The results indicate that LPS activation of a NF‐κB pathway enhances TGFβ1‐induced PDGF‐B expression in BDECs. Financial support: NIH R01 ES017537.

Investigating structural and biochemical correlates of ganglion cell dysfunction in streptozotocin-induced diabetic rats

The aim of this study was to determine whether inner retinal dysfunction in diabetic rats is correlated with structural and/or biochemical changes in the retina and optic nerve. Using the electroretinogram (ERG; −5.83 to 1.28log cd.s.m−2) retinal function (photoreceptor, bipolar, amacrine and ganglion cell components) was measured in control (n=13; citrate buffer) and diabetic (n=13; streptozotocin, STZ, 50mg kg−1) rats, 12weeks following treatment. Retinae and optic nerves were analyzed for structural changes and retinae were assessed for alterations in growth factor/cytokine expression using quantitative real-time PCR. We found that phototransduction efficiency was reduced 12weeks after STZ-induced diabetes (−30%), leading to reduced amplitude of ON-bipolar (−18%) and amacrine cell (−29%) dominated responses; ganglion cell dysfunction (−84%) was more profound. In the optic nerve, nerve fascicle area and myelin sheath thickness were reduced (p<0.05), whereas the ratio of blood vessels and connective tissue to total nerve cross-sectional area was increased (p<0.05) in diabetic compared to control rats. In the retina, connective tissue growth factor (CTGF), transforming growth factor beta, type 2 receptor (TGFβ-r2) mRNA and platelet-derived growth factor B (PDGF-B) mRNA were increased (p<0.035). Reduced ganglion cell function was correlated with increased CTGF and TGFβ-r2, but not PDGF-B mRNA. In summary, the ganglion cell component exhibited the greatest level of dysfunction within the ERG components examined after 12weeks of STZ-induced diabetes; the level correlated with increased CTGF and TGFβ-r2 mRNA, but not with gross morphological changes in the retina or optic nerve.

Mycophenolic acid inhibits the autocrine PDGF-B synthesis and PDGF-BB-induced mRNA expression of Egr-1 in rat mesangial cells

Uncontrolled mesangial cell (MC) proliferation within the context of glomerular disease contributes to the development of glomerulosclerosis. Mesangial autocrine growth factor stimulation has been described as a pathogenic factor. We investigated the effects of mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), on proliferation factors of cultured rat MCs. MPA was tested on the expression of platelet-derived growth factor-B (PDGF-B) and its receptor beta (PDGFR-beta), the immediate early gene (IEG) c-fos and the early growth response gene-1 (Egr-1), and AP-1 activation. Growth-arrested rat MCs were stimulated with 10% fetal calf serum (FCS) or 10-25 ng/ml platelet-derived growth factor-BB (PDGF-BB) in the presence or absence of MPA (0.019-10 microM) with or without guanosine (100 microM). MC proliferation was quantified by 5-bromo-2'-deoxyuridine (BrdU) incorporation and direct cell counting. Cytotoxicity of MPA was evaluated using the MTT and LDH tests. Protein expression of PDGF-B and its receptor PDGFR-beta was quantified by western blot analysis. The effect of MPA on gene expression of PDGF-B, Egr-1 and c-fos was determined by the reverse transcriptase-polymerase chain reaction (RT-PCR). AP-1 activation was analysed by an electrophoretic mobility shift assay (EMSA). Exposure of MCs to MPA caused a concentration-dependent inhibition of FCS-induced cell proliferation (cell number increase) with an IC50 of 0.44 +/- 0.03 microM and DNA synthesis with an IC50 of 0.52 +/- 0.02 microM without cell cytotoxicity in the therapeutic range. MPA decreased the PDGF-B protein expression and mRNA self-induction of PDGF-B but did not alter the protein expression of PDGFR-beta. MPA strongly inhibited the PDGF-BB-induced mRNA expression of Egr-1 decreasing to 7.6 +/- 2.5% after 30 min (P <or= 0.001) and to 4.7 +/- 3.1% after 1 h (P <or= 0.05), both being compared to the maximal expression induced by PDGF-BB. PDGF-BB-induced c-fos expression under MPA was unchanged after 30 min and decreased to 57 +/- 26% after 1 h (n.s.). MPA treatment did not affect PDGF-BB-induced AP-1 activity determined after 1 h and 2 h. The inhibitory MPA effect on PDGF-BB-induced PDGF-B expression was not significantly restored by guanosine (56 +/- 18% versus 32 +/- 17% after 2 h, n.s.), and MPA inhibition of PDGF-BB-induced Egr-1 expression was not reversed by exogenous guanosine. Treatment of cultured MCs with MPA inhibits MC proliferation correlating with a downregulation of the PDGF-B gene and protein expression and a suppression of Egr-1 mRNA expression. Since exogenous guanosine was not able to reverse the inhibitory MPA effect on PDGF-B and Egr-1 expression, we conclude that the antiproliferative effect of MPA on MCs may not solely depend on dGTP depletion but on a specific interference with the autocrine PDGF-B synthesis and Egr-1 expression of MCs.

Effects of Positive End-Expiratory Pressure, Inhaled Nitric Oxide and Surfactant on Expression of Proinflammatory Cytokines and Growth Factors in Preterm Piglet Lungs

We hypothesized that imbalance of proinflammatory cytokines and growth factors (GFs) in immature lungs of early postnatal life may be affected by protective ventilation strategy, and evaluated correlations of these aspects. Preterm neonate piglets were mechanically ventilated with low tidal volume and 5-6 or 10-12 cm H2O positive end-expiratory pressure (PEEP) with or without surfactant and inhaled nitric oxide (iNO) for 6 h, followed by biochemical, biophysical, and histopathological assessment of lung injury severity. Compared with surfactant and the control, iNO combined with lower PEEP exerted better oxygenation, lower activity of myeloperoxidase, lower expression of mRNA of interleukin (IL)-1beta, IL-6, IL-8, and platelet derived growth factor-B (PDGF-B), but higher expression of insulin-like growth factor-I (IGF-I), whereas that of tumor necrosis factor-alpha, keratinocyte GF, hepatocyte GF, vascular endothelial growth factor, and TGF-beta1 had no or modest changes. IL-1beta, IL-6 mRNA were closely correlated to PDGF-B mRNA and myeloperoxidase, but inversely to IGF-I mRNA, Pao2/FiO2 and dynamic lung compliance at 6 h. These results indicate that the association of lower PEEP and iNO may be more protective than surfactant on preventing lung injury and facilitating reparation by affecting the expression of proinflammatory cytokines and GFs.

Up-regulation of alveolar macrophage platelet-derived growth factor-B (PDGF-B) mRNA by interferon-gamma fromMycobacterium tuberculosisantigen (PPD)-stimulated lymphocytes

Macrophage production of PDGF-B is believed to be important in the pathogenesis of diseases where chronic lung inflammation develops into fibrosis. Since tuberculosis is characterized by chronic inflammation and tissue fibrosis, we asked if lymphokines from lymphocytes stimulated by the Mycobacterium tuberculosis antigen PPD, contained factors capable of increasing human alveolar macrophage PDGF-B mRNA. Supernatants from both phytohaemagglutinin (PHA)- and purified protein derivative (PPD)-stimulated lymphocytes, when added to macrophages, induced an increase in the mRNA of PDGF-B, but not transforming growth factor-beta (TGF-beta). When lymphocytes from contacts of patients with tuberculosis, patients with tuberculosis, and normal subjects were compared following PPD stimulation, the lymphocytes from the contacts had the greatest proliferation response, the greatest production of interferon-gamma (IFN-gamma), and their lymphokines induced the greatest increase in PDGF-B mRNA in macrophages. Recombinant human IFN-gamma reproduced this ability of lymphokines to increase macrophage PDGF-B mRNA. Finally, the increase in macrophage PDGF-B mRNA following incubation with supernatants from PPD-stimulated lymphocytes was shown to be due to IFN-gamma, when the increase in macrophage PDGF-B mRNA was prevented by addition of anti-human IFN-gamma antibody to the lymphocyte supernatant. This study indicated that antigen-stimulated lymphocytes released IFN-gamma, which in turn resulted in an increase in PDGF-B mRNA in alveolar macrophages. Such a mechanism provides a link between the DTH response and the first stages of a fibrotic reaction, and may offer an explanation for the progression of chronic inflammation to fibrosis, as occurs in the lungs of patients with untreated pulmonary tuberculosis.

Age‐dependent impairment of HIF‐1α expression in diabetic mice: Correction with electroporation‐facilitated gene therapy increases wound healing, angiogenesis, and circulating angiogenic cells

Wound healing is impaired in elderly patients with diabetes mellitus. We hypothesized that age-dependent impairment of cutaneous wound healing in db/db diabetic mice: (a) would correlate with reduced expression of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha) as well as its downstream target genes; and (b) could be overcome by HIF-1alpha replacement therapy. Wound closure, angiogenesis, and mRNA expression in excisional skin wounds were analyzed and circulating angiogenic cells (CACs) were quantified in db/db mice that were untreated or received electroporation-facilitated HIF-1alpha gene therapy. HIF-1alpha mRNA levels in wound tissue were significantly reduced in older (4-6 months) as compared to younger (1.5-2 months) db/db mice. Expression of mRNAs encoding the angiogenic cytokines vascular endothelial growth factor (VEGF), angiopoietin 1 (ANGPT1), ANGPT2, platelet-derived growth factor B (PDGF-B), and placental growth factor (PLGF) was also impaired in wounds of older db/db mice. Intradermal injection of plasmid gWIZ-CA5, which encodes a constitutively active form of HIF-1alpha, followed by electroporation, induced increased levels of HIF-1alpha mRNA at the injection site on day 3 and increased levels of VEGF, PLGF, PDGF-B, and ANGPT2 mRNA on day 7. CACs in peripheral blood increased 10-fold in mice treated with gWIZ-CA5. Wound closure was significantly accelerated in db/db mice treated with gWIZ-CA5 as compared to mice treated with empty vector. Thus, HIF-1alpha gene therapy corrects the age-dependent impairment of HIF-1alpha expression, angiogenic cytokine expression, and CACs that contribute to the age-dependent impairment of wound healing in db/db mice.

Effect of non-restrictive external stent on the expression of platelet-derived growth factor in vein grafts of rabbits

To investigate the potential mechanism of non-restrictive external stent in preventing re-stenosis of vein grafts. Thirty-six rabbits underwent reversed bypass grafting of autologous external jugular vein into common carotid artery and then were randomly divided into two equal groups, stenting group (Group S), with an external stent 6 mm in diameter (Dacron vascular prosthesis) surrounding the vein graft, and control group (Group NS) , without stenting. The vein grafts were harvested 7, 14, and 28 days after the operation respectively. Immunocytochemistry was used to detect the platelet-derived growth factor B (PDGF-B) positive cells. The mRNA expression of PDGF-B was detected with RT-PCR. The PDGF-B positive cell percentages in the intima of Group S 7 and 14 days later were both significantly lower than those of Group NS (15.2% +/- 3.6% vs 21.6% +/- 4.6%, and 6.5% +/- 2.6% vs 12.5% +/- 4.4%, both P < 0.05) . The PDGF-B positive cell percentages in media 7, 14 and 28 days later of Group S were all significantly lower than those of Group NS (13.8% +/- 4.6% vs 25.4% +/- 6.2%, 21.3% +/- 4.4% vs 35.7% +/- 7.3%, and 7.2% +/- 3.2% vs 19.2% +/- 5.4%, all P < 0.01). The PDGF-B positive cell percentage in adventitia peaked 28 days later in Group S and 14 days later in Group NS, and the PDGF-B positive cell percentage in adventitia 28 days later of Group S was significantly higher than that of Group NS (42.5% +/- 8.6% vs 31.9% +/- 4.6%, P < 0.01). RT-PCR analysis revealed that PDGF-B amplification products (457 bp) appeared in both group S and NS from 7 to 28 days, and the PDGF-B mRNA expression levels 7, 14, and 28 days later of Group S were all significantly lower than those of Group NS (31.2% +/- 6.5% vs 45.4% +/- 8.4%, P < 0.05; 42.3% +/- 6.2% vs 65.2% +/- 11.5%, P < 0.01; and 21.3% +/- 5.6% vs 36.2% +/- 9.4%, P < 0.01). Non-restrictive external stenting can inhibit the synthesis of PDGF and change its distribution, which may be one of the mechanisms of external stent in preventing re-stenosis of vein grafts.

Cyclooxygenase‐2 inhibition selectively attenuates bone morphogenetic protein‐6 synthesis and bone formation during guided tissue regeneration in a rat model

Bone formation during guided tissue regeneration is a tightly regulated process involving cells, extracellular matrix and growth factors. The aims of this study were (i) to examine the expression of cyclooxygenase-2 (COX-2) during bone regeneration and (ii) the effects of selective COX-2 inhibition on osseous regeneration and growth factor expression in the rodent femur model. A standardized transcortical defect of 5 x 1.5 mm was prepared in the femur of 12 male rats and a closed half-cylindrical titanium chamber was placed over the defect. The expression of COX-2 and of platelet-derived growth factor-B (PDGF-B), bone morphogenetic protein-6 (BMP-6) and insulin-like growth factor-I/II (IGF-I/II) was analyzed at Days 3, 7, 21 and 28 semiquantitatively by reverse transcriptase-polymerase chain reaction and immunohistochemistry. The effects of COX-2 inhibition by intraperitoneal injection of NS-398 (3 mg/kg/day) were analyzed in five additional animals sacrificed at Day 14. Histomorphometry revealed that new bone formation occurred in the cortical defect area as well as in the supracortical region, i.e. region within the chamber by Day 7 and increased through Day 28. Immunohistochemical evidence of COX-2 and PDGF-B levels were observed early (i.e. Day 3) and decreased rapidly by Day 7. BMP-6 expression was maximal at Day 3 and slowly declined by Day 28. In contrast, IGF-I/II expression gradually increased during the 28-day period. Systemic administration NS-398 caused a statistically significant reduction (P<0.05) in new bone formation (25-30%) and was associated with a statistically significant reduction in BMP-6 protein and mRNA expression (50% and 65% at P<0.05 and P<0.01, respectively). PDGF-B mRNA or protein expression was not affected by NS-398 treatment. COX-2 inhibition resulted in reduced BMP-6 expression and impaired osseous regeneration suggesting an important role for COX-2-induced signaling in BMP synthesis and new bone formation.

Hypoxia Inducible Factor 1-α Regulates of Platelet Derived Growth Factor-B in Human Glioblastoma Cells

Hypoxia inducible factors (HIF) are transcription factors regulating expression of several genes related to oxygen homeostasis in response to hypoxic stress. Although HIF1-alpha and platelet derived growth factor-B (PDGF-B) are expressed in glioma tissue and closely related to tumor angiogenesis mediating vascular endothelial growth factor (VEGF) activity, their direct relationship has not yet been clarified. The aim of this study is to investigate whether HIF1-alpha regulates PDGF-B expression. The human glioblastoma cell lines, U87MG, U251MG, and A172, were exposed to 1-21% oxygen for 24 h. PDGF-B mRNA expression were quantitatively analyzed by real time RT-PCR, their intracellular protein levels were determined by computerized image analysis supported by flow cytometry to detect intracellular PDGF-B, and the concentration of secreted PDGF-B protein was assayed by ELIA. We also assayed following transfection of the cells with short interference RNA (siRNA) targeting HIF1-alpha mRNA. Relative PDGF-B mRNA and secretion of PDGF-B protein were significantly elevated at 1% oxygen. Following transfection of HIF1-alpha siRNA at 1% oxygen, PDGF-B expression was significantly suppressed at mRNA level. Our findings indicated that HIF1-alpha up-regulated expression of PDGF-B in human glioblastoma cells and showed the feasibility of siRNA technology in glioblastoma cell lines.

Tight Control of Platelet-derived Growth Factor B/c-sis Expression by Interplay between the 5′-Untranslated Region Sequence and the Major Upstream Promoter

The long and GC-rich 5'-untranslated region (5'-UTR) of the known 3.8-kb platelet-derived growth factor B (PDGF-B)/c-sis mRNA is highly conserved and inhibits its own translation. It has been thought that this 5'-UTR functions by regulating translation possibly using an internal ribosome entry site (IRES)-mediated mechanism. However, in the present study we found no evidence that the 5'-UTR sequence of PDGF-B mRNA contains any IRES activity. Instead, we found that the 5'-UTR sequence of PDGF-B functions as a promoter both constitutively and upon induction in a variety of cell lines. The 5'-UTR sequence contains two promoters (termed P1 and P2) when only the 5'-UTR sequence is analyzed. In the presence of the upstream TATA-box-containing promoter (P0), P1 and P0 promoters are integrated into one promoter, whereas the P2 promoter still functions. The full promoter with combined P0, P1, and P2 produced two transcripts, with the major one having the full-length 5'-UTR and the minor one the short 5'-UTR. The integrated P0/P1 promoter and P2 promoter are likely responsible for producing the endogenous 3.8- and 2.8-kb PDGF-B mRNAs that are detected in cultured human renal microvascular endothelial cells, a few tumor cells, and rat brain tissues. Furthermore, we detected the 2.8-kb PDGF-B mRNA in erythroleukemia K562 cells upon 12-O-tetradecanoylphorbol-13-acetate-induced differentiation. Considering that the 5'-UTR in the 3.8-kb mRNA contains no IRES activity and inhibits cap-dependent translation, we believe that the endogenous 2.8-kb mRNA produced from the 5'-UTR promoter is likely the major template responsible for protein production both constitutively and upon induction. We also found that the transcription from the 5'-UTR P2 promoter might be coordinated by the major upstream P0 promoter upon stimulation. Based on these observations, we propose that the TATA-containing P0 promoter and the 5'-UTR promoter work together to tightly control the expression of PDGF-B.

Accelerated diabetic wound healing using cultured dermal fibroblasts retrovirally transduced with the platelet-derived growth factor B gene.

The treatment of diabetic wounds is a considerable clinical challenge. In this study, mouse dermal fibroblasts retrovirally transduced with the human platelet-derived growth factor B (PDGF-B) gene were used to treat diabetic mouse wounds. The PDGF-B gene was obtained from human umbilical vein endothelial cells, cloned into retroviral vectors, and introduced into diabetic mouse C57B1/ks-db/db dermal fibroblasts. In vitro results demonstrated production of PDGF-B protein by these transduced cells at steady-state levels of 1000 ng PDGF-B/10(6) cells/24 hours, and expression of PDGF-B mRNA. These cells were seeded onto polyglycolic acid scaffold matrices and used to treat diabetic mouse 20-mm x 20-mm full-thickness excisional dorsal skin wounds. Measurement of the residual epithelial gap at 21 days showed significantly accelerated healing (P < 0.05) of wounds treated with PDGF-transduced cells (epithelial gap 10.46 +/- 1.20 mm) compared with untreated wounds (14.66 +/- 0.591 mm), wounds treated with polyglycolic acid alone (14.80 +/- 0.575 mm), or wounds treated with negative control LNCX-transduced cells (13.76 +/- 0.831 mm). Immunohistochemical staining showed intense staining for PDGF in wounds treated with PDGF-B-transduced cells. This study demonstrates the promising potential for gene therapy in diabetic wound healing.

Advanced Glycation Endproducts–Receptor Interactions Stimulate the Growth of Human Pancreatic Cancer Cells through the Induction of Platelet-Derived Growth Factor-B

The molecular basis for the clinical association betweendiabetes mellitusand pancreatic cancer was investigated, using Mia PaCa-2 human pancreatic cancer cells in culture. Advanced glycation endproducts (AGE) prepared with bovine serum albumin and glucose were found to stimulate Mia PaCa-2 cell synthesis of DNA in a dose-dependent manner and also to significantly increase the number of viable cells. Evidence that platelet-derived growth factor-B (PDGF-B) mediates this growth promotion was obtained; AGE upregulated the level of PDGF-B mRNA, and antibodies against PDGF-BB completely neutralized the AGE-induced DNA synthesis. Antisense oligodeoxyribonucleotides complementary to mRNA encoding a receptor for AGE were found to reverse both the PDGF-B upregulation and the AGE-induced DNA synthesis. These results thus indicate that AGE ligand–receptor interactions could play an active part in the progression of pancreatic cancer through the induction of autocrine PDGF-B.

Cyclosporine A Upregulates Platelet‐Derived Growth Factor B Chain in Hyperplastic Human Gingiva

Cyclosporine A (CSA) is a widely used immunosuppressant for transplant patients and is also used for the treatment of a wide variety of systemic diseases with immunologic components. A prominent side effect of CSA administration is gingival overgrowth (hyperplasia). It has been postulated that CSA alters fibroblast activity through effects on various growth factors/cytokines. However, as yet, data concerning the molecular mechanisms involved in pathologic connective tissue proliferation are preliminary in nature. Our previous investigations concerning phenytoin-induced effects on platelet-derived growth factor B (PDGF-B) gene expression have demonstrated that other drugs which cause gingival overgrowth can upregulate macrophage PDGF-B gene expression in vitro and in vivo. The purpose of the present study was to evaluate PDGF-B gene expression in gingival tissues of patients receiving CSA therapy and exhibiting gingival overgrowth to determine if similar PDGF-B upregulation occurs in response to CSA and to identify PDGF-B producing cells in these tissues. Quantitative competitive reverse transcription polymerase chain reaction (QC-RTPCR) techniques were utilized to measure PDGF-B mRNA levels in CSA overgrowth patients and normal controls (N = 6/group). Results were expressed as mean +/- mRNA copy number and tested for significance using unpaired t-tests. Gingival samples were harvested (standardized for local inflammation at the sample site), total RNA was extracted, and QC-RTPCR was performed using specific PDGF-B primers and a corresponding competitive internal standard. CSA-treated patients exhibiting gingival overgrowth demonstrated approximately 48-fold increase in PDGF-B mRNA (7667.1 +/- 477.4 copies for CSA patients vs. 158.2 +/- 37.1 copies for controls; P < 0.001). Additionally, dual fluorescence immunohistochemistry for mature macrophage marker antigen (CD51) and intracellular PDGF-B was utilized to identify and localize PDGF-B producing cells were demonstrated to be macrophages distributed in a non-uniform manner throughout the papillary connective tissue. These results further support the hypothesis that the molecular mechanisms responsible for drug-induced gingival overgrowth may involve upregulation of PDGF-B macrophage gene expression. We continue to investigated specific CSA-induced alterations of macrophage PDGF-B gene expression in vitro and in vivo.

Endothelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide.

CO is produced in vascular smooth muscle cells (VSMC) by heme oxygenase-1 (HO-1). CO increases cGMP levels in VSMC; however, its possible additional roles in the vasculature have not been examined. We report that a product of HO, released from VSMC and inhibited by hemoglobin, has paracrine effects on endothelial cells: it increases endothelial cGMP content and decreases the expression of the mitogens, endothelin-1 (ET-1) and platelet-derived growth factor-B (PDGF-B). This product has the characteristics of CO, and its production is increased sevenfold under hypoxia. The VSMC-derived CO caused a fourfold rise in endothelial cell cGMP. In addition, it inhibited the hypoxia-induced increases in mRNA levels of the ET-1 and PDGF-B genes. Inhibitors of HO, and hemoglobin, a scavenger of CO, prevented the rise in cGMP and also restored the hypoxic response of these genes. The inhibition of ET-1 and PDGF-B mRNA by CO resulted in decreased production of these endothelial-derived mitogens, and in turn, inhibition of VSMC proliferation. These findings suggest an important physiologic role for VSMC-derived CO in modulating cell-cell interaction and cell proliferation in the vessel wall during hypoxia.