Addition Of Platelet-derived Growth Factor Research Articles

In vitro modulation of Schwann cell behavior by VEGF and PDGF in an inflammatory environment

Peripheral glial cell transplantation with Schwann cells (SCs) is a promising approach for treating spinal cord injury (SCI). However, improvements are needed and one avenue to enhance regenerative functional outcomes is to combine growth factors with cell transplantation. Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) are neuroprotective, and a combination of these factors has improved outcomes in rat SCI models. Thus, transplantation of SCs combined with VEGF and PDGF may further improve regenerative outcomes. First, however, we must understand how the two factors modulate SCs. In this in vitro study, we show that an inflammatory environment decreased the rate of SC-mediated phagocytosis of myelin debris but the addition of VEGF and PDGF (alone and combined) improved phagocytosis. Cytokine expression by SCs in the inflammatory environment revealed that addition of PDGF led to significantly lower level of pro-inflammatory cytokine, TNF-α, but IL-6 and anti-inflammatory cytokines (TGF-β and IL-10), remained unaltered. Further, PDGF was able to decrease the expression of myelination associated gene Oct6 in the presence of inflammatory environment. Overall, these results suggest that the use of VEGF and/or PDGF combined with SC transplantation may be beneficial in SCI therapy.

Guided bone regeneration with osteoconductive grafts and PDGF: A tissue engineering option for segmental bone defect reconstruction.

Regeneration and reconstruction of segmental bone defects (SBD) is a clinical challenge in maxillofacial surgery and orthopedics. The present study evaluated efficacy of guided bone-regeneration (GBR) of rat femoral SBD using osteoconductive equine-bone (EB) and beta-tricalcium phosphate (beta-TCP) grafts, either with or without platelet-derived growth-factor (PDGF). Following ethical-approval, 50 male Wistar-Albino rats (aged ~12-15 months and weighing ~450-500 g) were included. A 5 mm femoral critical-size SBD was created and animals were divided into five groups depending on the graft material used for GBR (EB, EB + PDGF, Autograft, beta-TCP, beta-TCP + PDGF; n = 10/group). Following 12-weeks of healing, animals were sacrificed and femur specimens were analyzed through qualitative histology and quantitative histomorphometry. There was new bone bridging femoral SBD in all groups and qualitatively, better bone formation was seen in autograft and EB + PDGF groups. Histomorphometric bone-area (BA %) was significantly high in autograft group, followed by EB + PDGF, beta-TCP + PDGF, EB, and beta-TCP groups. Addition of PDGF to EB and beta-TCP during GBR resulted in significantly higher BA%. After 12-weeks of healing, EB + PDGF for GBR of rat femoral segmental defects resulted in new bone formation similar to that of autograft. Based on this study, GBR with EB and adjunct PDGF could be a potential clinical alternative for reconstruction and regeneration of segmental bone defects.

3D in vitro Model of Vascular Medial Thickening in Pulmonary Arterial Hypertension.

In pulmonary arterial hypertension (PAH), excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) causes vascular medial thickening. Medial thickening is a histopathological hallmark of pulmonary vascular remodeling, the central disease process driving PAH progression. Pulmonary vascular remodeling causes stenosis and/or obstruction of small pulmonary arteries. This leads to increased pulmonary vascular resistance, elevated pulmonary arterial pressure, and ultimately right heart failure. To improve the survival of PAH patients, which remains at approximately 60% at 3 years after diagnosis, the development of novel PAH-targeted drugs is desired. To this end, a detailed understanding of the mechanisms underlying excessive PASMC proliferation and the medial thickening that ensues is necessary. However, a lack of in vitro models that recapitulate medial thickening impedes our deeper understanding of the pathogenetic mechanisms involved. In the present study, we applied 3-dimensional (3D) cell culture technology to develop a novel in vitro model of the pulmonary artery medial layer using human PAH patient-derived PASMCs. The addition of platelet-derived growth factor (PDGF)-BB, a mitogen known to promote excessive PASMC proliferation in PAH, resulted in increased thickness of the 3D-PAH media tissues. Conversely, administration of the PDGF receptor inhibitor imatinib or other clinical PAH drugs inhibited this medial thickening-inducing effect of PDGF-BB. Altogether, by using 3D cell culture technology, we report the generation of an in vitro model of medial thickening in PAH, which had hitherto not been successfully modeled in vitro. This model is potentially useful for assessing the ability of candidate PAH drugs to suppress medial thickening.

Platelet-derived growth factor stabilises vascularisation in collagen-glycosaminoglycan scaffolds in vitro.

Collagen-glycosaminoglycan (CG) scaffolds have been widely developed for a range of regenerative medicine applications. To enhance their efficacy, CG scaffolds have previously been prevascularised in vitro using human umbilical vein endothelial cells and human mesenchymal stromal cells (hMSCs); however, at later timepoints, a regression of vascularisation is observed. This is undesirable for longer preculture periods (e.g., for partial/full organ regeneration) and for in vitro vascularised tissue model systems (e.g., for drug testing/modelling). We hypothesised that delayed platelet-derived growth factor (PDGF)-BB addition could stabilise vessels, preventing their regression. In 2D, we identified 25ng/ml as a suitable dose that enhanced hMSC metabolic activity and proliferation, without affecting endothelial cells, or migration in either cell type. In our 3D model of CG scaffold vascularisation, early addition of PDGF (Day 3) behaved similarly to no PDGF controls. However, PDGF addition at later timepoints (i.e., Days 4 and 5), with a second addition on Day 10, prevented vascular regression. In quantifying our observations, we identified a need for a tool to measure in vitro vascularisation in porous scaffolds. This was a second key objective of this work. A novel ImageJ macro was developed, which allowed us to analyse vessel-like structures, evaluating their number and morphology, and confirmed our qualitative observations. Finally, upregulation of angiogenic genes (ANG1, KDR, and TEK2) involved in vessel maturation illustrated how PDGF addition contributed to vascular stability. Taken together, the results suggest that addition of PDGF at specific timepoints can be used to stabilise vasculature in CG scaffolds.

The Efficacy of Recombinant Platelet-Derived Growth Factor on Beta-Tricalcium Phosphate to Regenerate Femoral Critical Sized Segmental Defects: Longitudinal In Vivo Micro-CT Study in a Rat Model

Background and Objectives: Beta-tricalcium phosphate (beta-TCP) has been used for bone regeneration. The objective of this study was to assess longitudinally, the regeneration of critical sized segmental defects (CSSD) in rat femur using beta-TCP with or without recombinant platelet-derived growth factor (PDGF) through in vivo micro-computed tomography (micro-CT). Materials and Methods: Following ethical approval unilateral femoral CSSD measuring 5 mm was surgically created, under general anesthesia, in 30 male Wistar-Albino rats (aged 12–18 months; weighing 450–500 g). CSSD was stabilized using titanium mini-plate (4 holes, 1.0 mm thick with 8 mm bar). Depending upon biomaterial used for regeneration, the animals were randomly divided into: Control group (N = 10): CSSD covered with resorbable collagen membrane (RCM) only; Beta-TCP group (N = 10): CSSD filled with beta-TCP and covered by RCM; Beta-TCP + PDGF group (N = 10): CSSD filled with beta-TCP soaked in recombinant PDGF and covered by RCM. Longitudinal in vivo micro-CT analysis of the CSSD was done postoperatively at baseline, 2nd, 4th, 6th, and 8th weeks to assess volume and mineral density of newly formed bone (NFB) and beta-TCP. Results: Significant increase in NFB volume (NFBV) and mineral density (NFBMD) were observed from baseline to 8-weeks in all groups. Based on longitudinal in vivo micro-CT at 8-weeks, beta-TCP + PDGF group had significantly higher (p < 0.01) NFBV (38.98 ± 7.36 mm3) and NFBMD (3.72 ± 0.32 g/mm3) than the beta-TCP (NFBV—31.15 ± 6.68 mm3; NFBMD—2.28 ± 0.86g/mm3) and control (NFBV: 5.60 ± 1.06 mm3; NFBMD: 0.27 ± 0.02 g/mm3) groups. Significantly, higher reduction in beta-TCP volume (TCPV) and mineral density (TCPMD) were 1 observed in the beta-TCP + PDGF group when compared to the beta-TCP group. Conclusion: Addition of recombinant PDGF to beta-TCP enhanced bone regeneration within rat femoral CSSD and increased resorption rates of beta-TCP particles.

Binding induced strand displacement amplification for homogeneous protein assay

An ultrasensitive and homogenous strategy for protein assay was established based upon binding-induced strand displacement amplification (BI-SDA). Binding-Induced DNA strand-displacement occurred between Apt-T•signal DNA and Apt-C, and release of signal DNA upon addition of platelet-derived growth factor (PDGF BB). The released signal DNA further hybridized with multifunctional hairpin DNA probe and induced the strand-displacement amplification in the presence of Klenow Fragment (exo-) and dNTPs. The BI-SDA product contain G-quaruplex DNA, which could be recognized and reported by the fluorescence of fluorochrome N-methyl porphyrin propionic acid IX (NMM). The fluorescence intensity was proportional to the concentration of PDGF-BB over the range of 1.0×10–11mol/L -2.0×10−9mol/L, with a detection limit of 3.6pmol/L. This proposed strategy showed good selectivity and practicality, and might be applied to other proteins in the future.

Platelet-derived growth factor (PDGF)-AA/AB in human serum are potential indicators of the proliferative capacity of human synovial mesenchymal stem cells.

IntroductionFor expansion of human mesenchymal stem cells (MSCs), autologous human serum is safer than fetal bovine serum in clinical situations. One of the problems with the use of autologous human serum is that its proliferative effect on MSCs varies widely between donors. The threefold goals of this study were: (1) to demonstrate an improved method for preparing human serum; (2) to identify growth factors predictive of proliferative potential; and (3) to identify a cytokine to promote MSC proliferation in human serum.MethodsFresh blood was collected using a closed bag system containing glass beads. The bag was shaken at 20 °C for 30 minutes for rapid preparation, or kept stationary at 4 °C for 24 hours for slow preparation. Passage 0 synovial MSCs derived from four donors were cultured with 10 % conventional rapid preparation serum or modified slow preparation serum from four different donors. To perform the colony-forming unit assay, synovial MSCs were cultured in these serums. The protein expression profile in serum was analyzed using cytokine array. The candidate proteins were speculated from the correlation between the colony-forming ability and protein expression. As an evaluation of the candidate proteins, proliferation ability, surface marker phenotype and differentiation capability of synovial MSCs were examined.ResultsCompared with rapid preparation serum, slow preparation serum resulted in a significantly higher total colony number and twofold higher expression levels of nine proteins (angiopoietin-1, BDNF, EGF, ENA-78, IGFBP-2, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, RANTES and TfR). Colony number was positively correlated with PDGF-AA/AB concentrations. Exogenous PDGF-AA significantly promoted proliferation of synovial MSCs, whereas PDGF receptor (PDGFR) inhibitor decreased it. Addition of PDGFs or PDGFR inhibitor did not affect surface epitopes of synovial MSCs. Pretreatment with PDGFs or PDGFR inhibitor did not affect chondrogenic, adipogenic, or calcification potentials of synovial MSCs.ConclusionSlow preparation serum contained higher concentrations of PDGF-AA/AB and increased the colony formation number of synovial MSCs. PDGF-AA/AB were indicators of the proliferative potential of human serum. Exogenous PDGF-AA increased proliferation of synovial MSCs without alteration of surface epitopes and differentiation potentials.

Combination of three angiogenic growth factors has synergistic effects on sprouting of endothelial cell/mesenchymal stem cell-based spheroids in a 3D matrix.

Combinations of angiogenic growth factors have been shown to have synergistic effects on angiogenesis and natural wound healing in various animal models. Each growth factor has unique roles during angiogenesis; vascular endothelial growth factor (VEGF) plays a key role during the initial step of angiogenesis, whereas PDGF functions in the maturation of blood vessels. We used a combination of three angiogenic growth factors to increase angiogenesis in vitro and in vivo. We chose VEGF as a basic factor and added platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) to induce angiogenesis in three in vitro and in vivo models: 3D angiogenesis assay, 3D co-culture, and matrigel plug implantation assay. Cell proliferation was significantly higher in co-cultured cells treated with PDGF + VEGF + FGF than in the control, single, or dual combination groups. mRNA expression of α-smooth muscle actin (α-SMA), von Willebrand factor (vWF), and CD105 was higher in the triple group (PDGF + VEGF + FGF) than in control, single, or dual combination groups. In the PDGF + VEGF + FGF group, the length and number of branches of spheroids was also significantly higher than in the control, single, or dual combination groups. Furthermore, in a nude mouse model, α-SMA expression was significantly higher in the PDGF + VEGF + FGF group than in other groups. In conclusion, the addition of PDGF and FGF to VEGF showed synergistic effects on angiogenesis in vitro and in vivo. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1535-1543, 2016.

Aptamer-based label-free detection of PDGF using ruthenium(II) complex as luminescent probe

We report a simple, cost-effective, and label-free detection method, consisting of a platelet-derived growth factor (PDGF) binding aptamer and hydrophobic Ru(II) complex as a sensor system for PDGF. The binding of PDGF with the aptamer results in the weakening of the aptamer-Ru(II) complex, monitored by luminescence signal. A substantial enhancement in the luminescence intensity of Ru(II) complex is observed in the presence of aptamer due to the hydrophobic interaction. Upon addition of PDGF, the luminescence intensity is decreased, due to the stronger interaction between the aptamer and PDGF resulting in the displacement of Ru(II) complex to the aqueous solution. Our assay can detect a target specifically in a complex medium such as the mixture of proteins, at a concentration of 0.8pM.

Porous calcium phosphate cement for alveolar bone regeneration

The present study aimed to provide information on material degradation and subsequent alveolar bone formation, using composites consisting of calcium phosphate cement (CPC) and poly(lactic-co-glycolic) acid (PLGA) with different microsphere morphology (hollow vs dense). In addition to the plain CPC-PLGA composites, loading the microspheres with the growth factors platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF) was investigated. A total of four different CPC composites were applied into one-wall mandible bone defects in beagle dogs in order to evaluate them as candidates for alveolar bone regeneration. These composites consisted of CPC and hollow or dense PLGA microspheres, with or without the addition of PDGF-IGF growth factor combination (CPC-hPLGA, CPC-dPLGA, CPC-hPLGAGF , CPC-dPLGAGF ). Histological evaluation revealed significantly more bone formation in CPC-dPLGA than in CPC-hPLGA composites. The combination PDGF-IGF enhanced bone formation in CPC-hPLGA materials, but significantly more bone formation occurred when CPC-dPLGA was used, with or without the addition of growth factors. The findings demonstrated that CPC-dPLGA composite was the biologically superior material for use as an off-the-shelf material, due to its good biocompatibility, enhanced degradability and superior bone formation.

Cooperative effects in differentiation and proliferation between PDGF-BB and matrix derived synthetic peptides in human osteoblasts

BackgroundEnhancing osteogenic capabilities of bone matrix for the treatment of fractures and segmental defects using growth factors is an active area of research. Recently, synthetic peptides like AC- 100, TP508 or p-15 corresponding to biologically active sequences of matrix proteins have been proven to stimulate bone formation. The platelet-derived growth factor (PDGF) BB has been identified as an important paracrine factor in early bone healing. We hypothesized that the combined use of PDGF-BB with synthetic peptides could result in an increase in proliferation and calcification of osteoblast-like cells.MethodsOsteoblast-like cell cultures were treated with PDGF and synthetic peptides, singly and as combinations, and compared to non-treated control cell cultures. The cultures were evaluated at days 2, 5, and 10 in terms of cell proliferation, calcification and gene expression of alkaline phosphate, collagen I and osteocalcin.ResultsExperimental findings revealed that the addition of PDGF, p-15 and TP508 and combinations of PDGF/AC-100, PDGF/p-15 and PDGF/TP508 resulted in an increase in proliferating osteoblasts, especially in the first 5 days of cultivation. Proliferation did not significantly differ between single factors and factor combinations (p > 0.05). The onset of calcification in osteoblasts occurred earlier and was more distinct compared to the corresponding control or PDGF stimulation alone. Significant difference was found for the combined use of PDGF/p-15 and PDGF/AC-100 (p < 0.05).ConclusionsOur findings indicate that PDGF exhibits cooperative effects with synthetic peptides in differentiation and proliferation. These cooperative effects cause a significant early calcification of osteoblast-like cells (p < 0.05). We suggest the combination of synthetic peptides and PDGF as a potential clinical approach for accelerating bone healing or coating osteosynthesis materials.

Gene Expression Profiling of Pulmonary Fibrosis Identifies Twist1 as an Antiapoptotic Molecular “Rectifier” of Growth Factor Signaling

Idiopathic pulmonary fibrosis (IPF) is a progressive and typically fatal lung disease. To gain insight into IPF pathogenesis, we performed gene expression profiling of IPF lungs. Twist1, a basic helix-loop-helix protein, was found among the most consistently and highly up-regulated genes and was expressed in nuclei of type II epithelial cells, macrophages, and fibroblasts in IPF lungs. We studied the function of Twist1 in fibroblasts further, because they are the major effector cells in this disease and persist despite an ambient proapoptotic environment. Twist1 was induced by the profibrotic growth factors (GFs) basic fibroblast growth factor, platelet-derived growth factor, and epidermal growth factor in primary rat lung fibroblasts (RLFs). Suppression of Twist1 expression resulted in decreased RLF accumulation due to increased apoptosis, whereas Twist1 overexpression protected RLFs against several apoptotic stimuli. Addition of platelet-derived growth factor in combination with other GFs led to an increase in proliferation. When Twist1 was depleted, GFs continued to act as mitogens but caused a marked increase in cell death. The increase in apoptosis under basal or growth factor-stimulated conditions was partly mediated by up-regulation of the proapoptotic Bcl-2 family members, Bim and PUMA. These findings indicate that Twist1 promotes survival and accumulation of fibroblasts by shaping their responsiveness to growth factor stimulation. We propose that Twist1 represents one of the factors that promotes pathogenic accumulation of fibroblasts in fibrotic lung disease.

Has Platelet-Rich Plasma Any Role in Anterior Cruciate Ligament Allograft Healing?

The aim of this study was to evaluate and compare the clinical and inflammatory parameters with the addition of platelet-derived growth factor (PDGF) in primary anterior cruciate ligament (ACL) reconstruction with bone-patellar tendon-bone allograft. We prospectively randomized 100 patients undergoing arthroscopic patellar tendon allograft ACL reconstruction to a group in whom platelet-enriched gel was used (n = 50) and a non-gel group (n = 50). The platelet concentration was 837 x 10(3)/mm(3), and the gel was introduced inside the graft and the tibial tunnel. Demographic data were comparable between groups. The mean follow-up was 24 months for both groups and included a history, clinical evaluation with the International Knee Documentation Committee score, radiographs, and magnetic resonance imaging. There were no differences in the number of associated injuries. The results did not show any statistically significant differences between the groups for inflammatory parameters (perimeters of the knee and C-reactive protein level), magnetic resonance imaging appearance of the graft, and clinical evaluation scores (visual analog scale, International Knee Documentation Committee, and KT-1000 arthrometer [MEDmetric, San Diego, CA]). At this time, the therapeutic role of PDGF in ACL reconstruction remains unclear. The use of PDGF, on the graft and inside the tibial tunnel, in patients treated with bone-patellar tendon-bone allografts has no discernable clinical or biomechanical effect at 2 years' follow-up. More clinical studies will be needed to show the efficacy and use of these factors in daily practice in ACL reconstruction. Level I, prospective, randomized, double-blind study.

Transactivation of RON receptor tyrosine kinase by interaction with PDGF receptor β during steady-state growth of human mesangial cells

Although it is well known that platelet-derived growth factor (PDGF) causes mesangial cell proliferation (presumably contributing to progression of glomerular disease), targeted inhibition of the PDGF receptor system has shown only limited efficacy against glomerular diseases. To examine whether this discrepancy is due to the involvement of other pathways, we used phosphorylated receptor tyrosine kinase arrays and found that RON (recepteur d'origine nantais) was phosphorylated while the PDGF receptor was dephosphorylated (thus inactive) in human mesangial cells (HMCs) at the time of cell cycle entry. Further, RON remained active during steady-state growth. Activation of RON was independent of its canonical ligand, macrophage-stimulating protein, but was mediated by transactivation from the PDGF-engaged PDGF receptor. Following stimulation with PDGF we found that the two receptors physically interacted. Knockdown of RON by siRNA increased the number of apoptotic cells without affecting the rate of DNA synthesis, suggesting that RON has anti-apoptotic functions. Immunohistochemical analysis found phosphorylated RON in glomerular lesions of patients with IgA nephropathy but not those with minimal change nephrotic syndrome, a disease not associated with mesangial proliferation. These results suggest that RON is involved in mesangial cell proliferation under both physiological and pathological conditions, and may be a relevant target for therapeutic intervention.

Keratinocyte-derived factors that stimulate fibroblast hyaluronan synthesis - the role of a low molecular weight factor

Hyaluronan-rich matrices are associated with cellular proliferation and migration, with high levels being expressed transiently during adult wound healing, and persistent high levels expressed during scar-free foetal wound healing. Cultured allogeneic keratinocytes and lysed keratinocyte extracts promote chronic wound healing, as does addition of exogenous hyaluronan. It is possible that the keratinocyte-stimulated effects on wound healing may be, at least in part mediated by the release of hyaluronan synthesis-stimulating factors. We have demonstrated that serum-free keratinocyte-conditioned medium (KCM), when added to confluent fibroblast cultures for 24 h stimulated up to an 8-fold increase in hyaluronan synthesis, as measured by 3H-glucosamine incorporation into cetylpyridinium chloride-precipitable glycosaminoglycans. This KCM can be fractionated by ultrafiltration into fractions > 30 kD and < 1 kD which are poorly active individually, but synergise strongly to stimulate fibroblast hyaluronan synthesis. We have examined the activity of both platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) to stimulate fibroblast hyaluronan synthesis in the presence and absence of the low molecular weight KCM fraction, and the effect of blocking antibodies to PDGF and bFGF on complete KCM. KCM stimulated a 7-fold increase in 3H-glucosamine incorporation into hyaluronan and a 8.3-fold increase into sulphated glycosaminoglycans. 35SO4 incorporation was increased only 1.5-fold, resulting in a significant decrease in the degree of polymer sulphation of the sulphated glycosaminoglycans. Blocking antibodies to PDGF (all isotypes) reduced the activity of the KCM by 27%, while blocking antibFGF reduced the activity by 58%. Addition of PDGF (20 ng/ml) to the < 1 kD KCM fraction restored full activity, while addition of bFGF had no significant effect. However, addition of heparin with the bFGF resulted in activity being restored. These results demonstrate that cultured human keratinocytes produce potent fibroblast glycosaminoglycan synthesis-stimulating factors. Both PDGF and bFGF appear to be involved, but require the presence of an as yet unidentified KCM low molecular weight factor. Identification of this factor may provide an important therapeutic aid in wound healing

Effect of local, long-term delivery of platelet-derived growth factor (PDGF) on injected fat graft survival in severe combined immunodeficient (SCID) mice

Autogenous fat injection is widely used for the correction of acquired and congenital soft tissue defects. However, the high absorption rate results in the need for over-correction of the defect and repeat procedures. We hypothesised that platelet-derived growth factor (PDGF), a potent mitogen and known stimulant for murine preadipocytes, would improve fat graft survival when concentrations were sustained with a gelatine microsphere delivery system. Abdominal fat was harvested from an otherwise healthy 43-year-old woman during a breast reconstruction. Prior to subdermal injection into severe combined immunodeficient (SCID) mice, the fat grafts were divided into 1-ml aliquots, mixed with microspheres bound to PDGF, free PDGF, or nothing depending on its experimental group, and weighed. The following experimental groups were thus created (minimum n=8 per group): (1) fat graft control, (2) fat graft with free PDGF, (3) fat graft with blank microspheres, and (4) fat graft with microspheres bound to PDGF. After 12 weeks, the fat xenografts were harvested for analysis of weight maintenance and histological and morphometric evaluation. The addition of PDGF bound to gelatine microspheres was effective in improving xenograft weight maintenance (P=0.018) and preservation of adipose tissue architecture (P<0.0005) compared to controls at 3 months. The microspheres were completely absorbed at 12 weeks. Sustained, local delivery of PDGF via a gelatine microsphere delivery system resulted in improved weight maintenance of the xenografts with greater preservation of adipose tissue architecture at 3 months compared to controls.

20-HETE inhibits the proliferation of vascular smooth muscle cells via transforming growth factor-β

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, has been shown to modulate the growth of vascular smooth muscle cells (VSMCs). We asked whether 20-HETE modulates the proliferation of R22D cells, a clonal VSMC from neonatal rats, by releasing transforming growth factor-beta (TGF-beta). Incubation of R22D cells with 20-HETE for 24 h attenuated [(3)H]thymidine incorporation in a concentration-dependent manner without causing the release of lactate dehydrogenase. 20-HETE also inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation in R22D cells and human VSMCs. At 5 muM, 20-HETE reduced [(3)H]thymidine incorporation by 34 +/- 6%; anti-TGF-beta neutralizing antibody, but not nonspecific IgG, completely reversed the attenuated [(3)H]thymidine incorporation induced by 20-HETE. In addition, 20-HETE attenuated fetal bovine serum- and PDGF-induced expression of cyclin D1, a downstream effector of TGF-beta(1), which was reversed by anti-TGF-beta antibody. Further studies demonstrated that 20-HETE may increase TGF-beta release to a level high enough to inhibit [(3)H]thymidine incorporation without altering the steady-state mRNA level of TGF-beta. Nevertheless, pretreatment of indomethacin (a cyclooxygenase inhibitor) or paxilline (a potassium channel inhibitor) did not affect the inhibitory effect on DNA synthesis induced by 20-HETE. These results demonstrate for the first time a growth-inhibitory effect induced by 20-HETE, which may be mediated by TGF-beta.

Adenosine monophosphate–activated protein kinase modulates the activated phenotype of hepatic stellate cells

Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate-activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time-dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet-derived growth factor (PDGF)-stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein-1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF-dependent activation of extracellular signal-regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short-term phosphorylation of ribosomal S6 kinase (p70(S6K)) and 4E binding protein-1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin-a (IL-1) to activate nuclear factor kappa B (NF-kappaB) was also reduced by AICAR. Activation of AMPK negatively modulates the activated phenotype of HSCs.

A fluorescence resonance energy transfer activation sensor for Arf6

The involvement of the small GTPase Arf6 in Rac activation, cell migration, and cancer invasiveness suggests that it is activated in a spatially and temporally regulated manner. Small GTPase activation has been imaged in cells using probes in which the GTPase and a fragment of a downstream effector protein are fused to fluorescent reporter proteins that constitute a fluorescence resonance energy transfer (FRET) donor/acceptor pair. Unlike other Ras family GTPases, the N terminus of Arf6 is critical for membrane targeting and, thus, cannot be modified by fusion to a fluorescent protein. We found that the previously described C-terminal green fluorescent protein (GFP) derivative also shows diminished membrane targeting. Therefore, we inserted a fluorescent protein into an inert loop within the Arf6 sequence. This fusion showed normal membrane targeting, nucleotide-dependent interaction with the downstream effector GGA3, and normal regulation by a GTPase-activating protein (GAP) and a guanine nucleotide exchange factor (GEF). Using the recently developed CyPET/YPET fluorescent proteins as a FRET pair, we found that Arf6–CyPET underwent efficient energy transfer when bound to YPET–GGA3 effector domain in intact cells. The addition of platelet-derived growth factor (PDGF) to fibroblasts triggered a rapid and transient increase in FRET, indicative of Arf6 activation. These reagents should be useful for investigations of Arf6 activation and function.

Use of freeze-dried bone allograft with platelet-derived growth factor for revision of a glenoid component

Glenoid replacement is technically challenging. Removal of a cemented glenoid component often results in a large osseous defect which makes the immediate introduction of a revision prosthesis almost impossible. We describe a two-stage revision procedure using a reversed shoulder prosthesis. Freeze-dried allograft with platelet-derived growth factor was used to fill the glenoid defect. Radiological incorporation of the allograft was seen and its consistency allowed the placement of a screwed glenoid component. There were no signs of new mature bone formation on histological examination. The addition of platelet-derived growth factor to the allograft seems to contribute to an increase in incorporation and hardness, but does not promote the growth of new bone.