Connective tissue growth factor (CTGF) is a 38-kd protein involved in several human fibrotic disorders including atherosclerosis and skin and renal fibrosis. Although it has been shown that human and experimental liver fibrosis is associated with CTGF expression through up-regulation of CTGF mRNA by hepatic stellate cells (HSC), the role of CTGF in the liver has not yet been determined. The aim of the present study was to assess the effects of CTGF on rat primary HSC and its regulation in a well-established model of in vitro liver fibrogenesis. Incubation of primary HSC with recombinant CTGF induced a significant migratory (2.3-fold, 50 ng/ml CTGF) and proliferative effect (1.8-fold, 100 ng/ml CTGF). Type I collagen mRNA expression, as assessed by a real-time RT-PCR procedure, was also increased when cells were incubated in the presence of CTGF (2-fold, 50 ng/ml). Transforming growth factor-beta1 (TGF-beta1) strongly stimulated CTGF mRNA expression, a direct mechanism observed in the absence of any intermediate protein synthesis. Furthermore, spontaneous activation of HSC plated on plastic and stimulation by vascular endothelial growth factor, lipid peroxidation products (HNE, MDA), acetaldehyde, and platelet-derived growth factor (PDGF)-BB significantly up-regulated CTGF mRNA expression in HSC. PDGF-induced CTGF stimulation might be related in part to TGF-beta1 secretion because CTGF mRNA up-regulation observed after PDGF-BB stimulation was abrogated in the presence of neutralizing TGF-beta1 antibody. In conclusion, this study extends the role of CTGF in HSC activation and suggests that CTGF up-regulation might be a central pathway during HSC activation.

MUC1 is a heterodimeric transmembrane glycoprotein that is overexpressed and aberrantly glycosylated in ductal adenocarcinomas. Differential phosphorylation of the MUC1 cytoplasmic tail (MUC1CT) has been associated with signaling events that influence the proliferation and metastasis of cancer cells. We identified a novel tyrosine phosphorylation site (HGRYVPP) in the MUC1CT by mass spectrometric analysis of MUC1 from human pancreatic adenocarcinoma cell lines. Analyses in vitro and in vivo showed that platelet-derived growth factor receptor beta (PDGFRbeta) catalyzed phosphorylation of this site and of tyrosine in the RDTYHPM site. Stimulation of S2-013.MUC1F cells with PDGF-BB increased nuclear colocalization of MUC1CT and beta-catenin. PDGF-BB stimulation had no significant effect on cell proliferation rate; however, it enhanced invasion in vitro through Matrigel and in vivo tumor growth and metastases. Invasive properties of the cells were significantly altered on expression of phosphorylation-abrogating or phosphorylation-mimicking mutations at these sites. We propose that interactions of MUC1 and PDGFRbeta induce signal transduction events that influence the metastatic properties of pancreatic adenocarcinoma.

There is clear evidence that the phenotypic modulation of smooth muscle cells (SMCs) contributes to the pathophysiology of vascular disease. Phenotypic modulation refers to the unique ability of SMCs to alter their phenotype in response to extracellular stimuli and is hallmarked by the loss of SMC marker gene expression. The transcription factor Krüppel-like factor 4 (KLF4) is a known powerful negative regulator of SMC marker gene expression that works, in part, by decreasing the expression of the serum response factor (SRF) myocardin. KLF4 is not expressed in healthy adult SMCs but is increased in SMCs in response to vascular injury in vivo or PDGF-BB treatment in vitro. The aim of the present study was to determine the molecular mechanisms that regulate the expression of KLF4 in phenotypically modulated SMCs. The results demonstrated that the transcription factor stimulating protein-1 (Sp1) regulated the expression of KLF4 in SMCs. The KLF4 promoter contains three consensus Sp1 binding sites. Using a series of truncated KLF4 promoters, we showed that only fragments containing these Sp1 sites could be activated by PDGF-BB. In addition, overexpression of Sp1 alone was sufficient to increase the activity of the KLF4 promoter. Moreover, inhibiting Sp1 expression with small-interfering RNA attenuated the effects of PDGF-BB on KLF4 expression. Mutation of the three Sp1 sites within the KLF4 promoter abolished both baseline and PDGF-BB-induced activity. Finally, the results demonstrated enhanced Sp1 binding to the KLF4 promoter in SMCs treated with PDGF-BB in vitro and following vascular injury in vivo. Taken together, the results suggest a novel role for Sp1 in increasing the expression of KLF4 in phenotypically modulated SMCs.

PDGF receptors have recently been found to be expressed in microvascular endothelium in vivo under circumstances of endothelial cell activation and angiogenesis suggesting that PDGF may have a direct effect on endothelial cells. We have tested the angiogenic activity of PDGF-AA and -BB homodimers in the chick chorioallantoic membrane in vivo. PDGF-BB was found to consistently induce an angiogenic response whereas PDGF-AA was less active. Morphological analyses revealed that there was little inflammation associated with this response but an increase in vessel density suggested a direct effect of PDGF on embryonic chorioallantoic endothelial cells. In vitro, PDGF-BB was found to be more potent than PDGF-AA in stimulating the chemotaxis of rat brain capillary endothelial cells. This is consistent with a direct effect of PDGF on endothelial cells. Thus, this novel angiogenic activity of PDGF has implications for several developmental and pathological events in which PDGF, particularly the B-chain, is expressed.

Selection of aptamers from nucleic acid libraries by in vitro evolution represents a powerful method of identifying high-affinity ligands for a broad range of molecular targets. Nevertheless, a sizeable fraction of proteins remain difficult targets due to inherently limited chemical diversity of nucleic acids. We have exploited synthetic nucleotide modifications that confer protein-like diversity on a nucleic acid scaffold, resulting in a new generation of binding reagents called SOMAmers (Slow Off-rate Modified Aptamers). Here we report a unique crystal structure of a SOMAmer bound to its target, platelet-derived growth factor B (PDGF-BB). The SOMAmer folds into a compact structure and exhibits a hydrophobic binding surface that mimics the interface between PDGF-BB and its receptor, contrasting sharply with mainly polar interactions seen in traditional protein-binding aptamers. The modified nucleotides circumvent the intrinsic diversity constraints of natural nucleic acids, thereby greatly expanding the structural vocabulary of nucleic acid ligands and considerably broadening the range of accessible protein targets.

Glioma cells in culture express platelet-derived growth factor (PDGF) A- and B-chains and secrete PDGF-like activity that is mainly PDGF-AA. In this work, we show that the PDGF alpha- and beta-receptors are independently expressed in human malignant glioma cells. We also define three different receptor phenotypes that are related to the morphology of glioma cells: cells with only alpha-receptors, only beta-receptors, or with both types of receptors. By the help of Northern blot analyses, 125I-PDGF-binding experiments, and immunoprecipitations the receptors are shown to be structurally normal PDGF receptors, except for minor variations in size that probably are due to differences in glycosylation. PDGF-BB induces DNA synthesis in cells of all three receptor phenotypes, whereas PDGF-AA or PDGF-AB has this effect only on cells with alpha- or with alpha- and beta-receptors. 125I-PDGF-AB binds with high affinity and down-regulates beta-receptors only in cells where alpha-receptors are present in addition to beta-receptors. Thus, the different functional capacities of PDGF isoforms on glioma cells fit with their known receptor-binding specificities and are compatible with the hypothesis that the isoforms act by inducing dimeric receptor complexes. When data on PDGF A- and B-chains, as well as alpha- and beta-receptor expression are compiled and the pattern of receptor binding specificity is taken into account, the majority of glioma cell lines are found to have a phenotype that makes autocrine stimulation possible.

The effects of i.c.v. infused platelet-derived growth factor and brain-derived neurotrophic factor on cell genesis, as assessed with bromodeoxyuridine (BrdU) incorporation, were studied in adult rats with unilateral 6-hydroxydopamine lesions. Both growth factors increased the numbers of newly formed cells in the striatum and substantia nigra to an equal extent following 10 days of treatment. At 3 weeks after termination of growth factor treatment, immunostaining of BrdU-labeled cells with the neuronal marker NeuN revealed a significant increase in newly generated neurons in the striatum. In correspondence, many doublecortin-labeled neuroblasts were also observed in the denervated striatum following growth factor treatment. Further evaluation suggested that a subset of these new neurons expresses the early marker for striatal neurons Pbx. However, no BrdU-positive cells were co-labeled with DARPP-32, a protein expressed by mature striatal projection neurons. Both in the striatum and in the substantia nigra there were no indications of any newly born cells differentiating into dopaminergic neurons following growth factor treatment, such that BrdU-labeled cells never co-expressed tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. In conclusion, our results suggest that administration of these growth factors is capable of recruiting new neurons into the striatum of hemiparkinsonian rats.

The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes, and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin (SOST) that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influenceaA osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or "osteokines" from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin (OCN) secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2 (LCN2) is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain. We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.

OBJECTIVE

We evaluated the expression of platelet-derived growth factor (PDGF) ligands and receptors in clinical specimens of human pancreatic adenocarcinomas and determined the therapeutic effect of STI571 (Gleevec), a protein tyrosine kinase inhibitor of PDGF receptor (PDGFR), on human pancreatic carcinoma cells growing in the pancreas and liver of nude mice.

METHODS

Immunohistochemical staining for PDGF-AA and -BB ligands, PDGFR-alpha and -beta, and phosphorylated PDGFR-alpha and -beta was performed on 31 specimens of human pancreatic cancer and L3.6pl human pancreatic adenocarcinoma cell line. To determine the in vivo effects of STI571, nude mice with L3.6pl cells injected into the pancreas were randomized 7 days later to receive one of the following treatments: sterile water p.o. (control), STI571, gemcitabine, or a combination of STI571 and gemcitabine.

RESULTS

In 29 of 31 clinical specimens of human pancreatic adenocarcinoma, both tumor cells and tumor-associated endothelial cells expressed phosphorylated PDGFR-alpha and -beta. L3.6pl cells growing in culture expressed moderate amounts of PDGF-AA and little to no PDGFR-alpha or -beta, whereas L3.6pl cells growing in the pancreas of nude mice expressed a high level of PDGF and receptors. Colocalization immunohistochemical analysis demonstrated expression of activated PDGFR-beta by tumor-associated endothelial cells in both the pancreas and in liver metastases. Tumors of mice treated for 4 weeks with STI571 (50 mg/kg or 100 mg/kg p.o. daily) were slightly smaller than controls. Tumors treated with gemcitabine and STI571 (50 mg/kg) were >70% smaller than tumors in control mice and 36% smaller than those in mice treated with gemcitabine only (P < 0.0002 and P < 0.04, respectively). Combination therapy also inhibited spontaneous metastasis to the liver. Tumors from mice treated with both STI571 and gemcitabine had decreased expression of activated (phosphorylated) PDGFR-alpha and -beta, decreased mean vessel density, decreased cell proliferation, and increased apoptosis of tumor cells.

CONCLUSIONS

Collectively, these data show that activated PDGFR on tumor cells and tumor-endothelial cells can be a novel target for therapy of pancreatic carcinoma.

The c-Abl tyrosine (Tyr) kinase is activated after platelet-derived-growth factor receptor (PDGFR) stimulation in a manner that is partially dependent on Src kinase activity. However, the activity of Src kinases alone is not sufficient for activation of c-Abl by PDGFR. Here we show that functional phospholipase C-gamma1 (PLC-gamma1) is required for c-Abl activation by PDGFR. Decreasing cellular levels of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) by PLC-gamma1-mediated hydrolysis or dephosphorylation by an inositol polyphosphate 5-phosphatase (Inp54) results in increased Abl kinase activity. c-Abl functions downstream of PLC-gamma1, as expression of kinase-inactive c-Abl blocks PLC-gamma1-induced chemotaxis towards PDGF-BB. PLC-gamma1 and c-Abl form a complex in cells that is enhanced by PDGF stimulation. After activation, c-Abl phosphorylates PLC-gamma1 and negatively modulates its function in vivo. These findings uncover a newly discovered functional interdependence between non-receptor Tyr kinase and lipid signalling pathways.

BACKGROUND

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral disease in China, caused by SFTS virus (SFTSV). Severe SFTS patients can quickly proceed to multiorgan dysfunction and death; however, underlying pathogenic mechanisms remain unclear.

METHODS

Serum samples from 15 fatal and 44 nonfatal SFTS cases were subjected to multiplex-microbead immunoassays to detect a broad spectrum of cytokines. The viral load and virus-specific IgG titers were also tested by real-time PCR and ELISA, respectively.

RESULTS

Cytokines IL-1RA, IL-6, IL-10, G-CSF, IP-10, and MCP-1 were elevated in SFTS patients and produced at robust levels in fatal cases. In contrast, cytokines PDGF-BB and RANTES decreased in SFTS patients. These cytokines reverted to normal ranges during the convalescent phase of SFTSV infection. Cytokines IL-1β, IL-8, MIP-1α, and MIP-1β showed a unique pattern of elevation in fatal cases but not in nonfatal cases. However, these cytokines increased in the convalescent phase of nonfatal SFTS cases. Our regression analysis revealed that the serum viral load correlated with these cytokines. Moreover, levels of these cytokines correlated with various clinical parameters and virus-specific IgG titers.

CONCLUSIONS

The study demonstrates that SFTSV infection induces a cytokine storm with abnormally expressed cytokine profiles, which are associated with the disease severity.

Retinal pigment epithelial (RPE) cells dedifferentiate and undergo epithelial-mesenchymal transition (EMT) following retinal detachment, playing a central role in formation of fibrous tissue on the detached retina and vitreous retraction (proliferative vitreoretinopathy (PVR)). We have developed a mouse model of subretinal fibrosis with implications for PVR in which retinal detachment is induced without direct damage to the RPE cells. Transforming growth factor-beta (TGF-beta) has long been implicated both in EMT of RPEs and the development of PVR. Using mice null for Smad3, a key signaling intermediate downstream of TGF-beta and activin receptors, we show that Smad3 is essential for EMT of RPE cells induced by retinal detachment. De novo accumulation of fibrous tissue derived from multilayered RPE cells was seen following experimental retinal detachment in eyes of wild type, but not Smad3-null mice. Expression of alpha-smooth muscle actin, a hallmark of EMT in this cell type, and extracellular matrix components, lumican and collagen VI, were also not observed in eyes of Smad3-null mice. Our data show that induction of PDGF-BB by Smad3-dependent TGF-beta signaling is likely an important secondary proliferative component of the disease process. The results suggest that blocking the Smad3 pathway might be beneficial in prevention/treatment of PVR.

Hypoxia enhances the proliferation and migration of adipose-derived stem cells (ASCs) via the generation of reactive oxygen species (ROS). Therefore, this study primarily investigated whether or not ROS generation could regulate microRNA-210 (miR-210) expression, and increase proliferation/migration of ASCs. In addition, we tried to identify the signaling pathways involved in miR-210 upregulation and the direct target genes of miR-210 that mediate these functions. Various sources of ROS generation such as hypoxia, antimycin, rotenone, and platelet-derived growth factor (PDGF)-BB upregulated miR-210 expression, and increased the proliferation/migration of ASCs. There is a positive feed-forward loop between ROS generation and miR-210, and miR-210 itself increases ROS generation by downregulation of iron-sulfur cluster scaffold homolog 2 (ISCU2). Although hypoxia-inducible factor-1α was not involved in miR-210 expression, pharmacological or small interfering RNA (siRNA)-driven inhibition of Akt and ERK1/2 molecules reduced miR-210 expression. Transfection of siRNAs of NF-κB and Elk1 also reduced miR-210 expression, indicating that these signaling pathways mediate miR-210 upregulation. Protein tyrosine phosphatase, non-receptor type 2 (PTPN2) was selected for miR-210 target gene, and it was downregulated by ROS generators or miR-210 mimic treatment. PTPN2 was first proven to be a direct miR-210 target in luciferase activity assay, and pharmacological inhibition or overexpression of PTPN2 regulated the proliferation and migration of ASC. In conclusion, ROS generation from diverse sources induces miR-210 expression in ASCs via PDGFR-β, Akt and ERK pathways. Transcription of miR-210 expression is regulated by NF-κB and Elk1, and miR-210 increases the proliferation and migration of ASCs via ISCU2 and PTPN2 downregulation.

BACKGROUND

The discrepancy between the functional improvements yielded experimentally by skeletal myoblasts (SM) transplanted in infarcted myocardium and the paucity of their long-term engraftment has raised the hypothesis of cell-mediated paracrine mechanisms.

RESULTS

We analyzed gene expression and growth factors released by undifferentiated human SM (CD56(+)), myotubes (SM cultured until confluence) and fibroblasts-like cells (CD56(-)). Gene expression revealed up-regulation of pro-angiogenic (PGF), anti-apoptotics (BAG-1, BCL-2), heart development (TNNT2, TNNC1) and extracellular matrix remodelling (MMP-2, MMP-7) genes in SM. In line with the gene expression profile, the analysis of culture supernatants of SM by ELISA identified the release of growth factors involved in angiogenesis (VEGF, PIGF, angiogenin, angiopoietin, HGF and PDGF-BB) as well as proteases involved in matrix remodelling (MMP2, MMP9 and MMP10) and their inhibitors (TIMPs). Culture of smooth muscle cells (SMC), cardiomyocytes (HL-1) and human umbilical vein endothelial cells (HUVECs) with SM-released conditioned media demonstrated an increased proliferation of HUVEC, SMC and cardiomyocytes (p<0.05) and a decrease in apoptosis of cardiomyocytes (p<0.05). Analysis of nude rats transplanted with human SM demonstrated expression of human-specific MMP-2, TNNI3, CNN3, PGF, TNNT2, PAX7, TGF-beta, and IGF-1 1 month after transplant.

CONCLUSIONS

Our data support the paracrine hypothesis whereby myoblast-secreted factors may contribute to the beneficial effects of myogenic cell transplantation in infarcted myocardium.

BACKGROUND

Platelet-derived growth factor (PDGF) and superoxide anion (O2.-) have been implicated in vascular diseases. We investigated whether PDGF stimulates the production of O2.- in human aortic smooth muscle cells (HSMCs) and whether O2.- leads in this way to the activation of nuclear factor-kappaB (NF-kappaB) and induction of monocyte chemoattractant protein 1 (MCP-1) in PDGF-stimulated HSMCs.

RESULTS

PDGF-AB concentration- and time-dependently stimulated O2.- generation from HSMCs. The stimulatory effect of PDGF-AB was mimicked by PDGF-BB but not by PDGF-AA. The generation of O2.- by PDGF-AB was attenuated by the NAD(P)H oxidase inhibitor iodonium diphenyl, the specific protein kinase C (PKC) inhibitor Ro 31-8220, and the phosphatidylinositol 3-kinase inhibitor wortmannin. Allopurinol and nifedipine had no effect on PDGF-AB-induced O2.- release, whereas indomethacin potentiated this response. Gel mobility shift assay revealed that PDGF-AB increased the binding activity of NF-kappaB, which contained predominantly the p50/p65 heterodimer in nuclear extracts from HSMCs. Superoxide dismutase as well as iodonium diphenyl, Ro 31-8220, and wortmannin attenuated PDGF-AB-induced activation of NF-kappaB and expression of MCP-1 mRNA. In contrast, superoxide dismutase did not inhibit the interleukin-1beta-induced NF-kappaB activation.

CONCLUSIONS

The results demonstrate that PDGF stimulates O2.- generation in HSMCs via PKC-dependent and wortmannin-sensitive pathways involving flavoenzyme(s). This PDGF-induced O2.- production may be involved in vascular lesion formation by mediating, at least in part, NF-kappaB activation and MCP-1 induction.

Platelet-derived growth factor, PDGF, purified from human platelets is a disulfide-bonded dimer consisting of two homologous polypeptide chains denoted A and B; it has not been known whether it is a heterodimer or a mixture of homodimers. We present here evidence that a major part of PDGF has a heterodimer structure. A highly homogeneous, 31-kDa PDGF was purified in the presence of protease inhibitors and shown to contain both chains by means of immunoprecipitations with peptide antisera specific for the A and B chains, respectively. The susceptibility of PDGF to mild acid treatment and its chromatographic behavior in reversed-phase high performance liquid chromatography and immobilized metal ion affinity chromatography, as compared to A and B chain homodimers, is consistent with a heterodimer structure. Analysis of PDGF purified according to our routine, large scale procedure revealed the major part to have a heterodimer structure. In addition, B chain homodimers were also found. With the demonstration that a major part of PDGF purified from human platelets occurs as a heterodimer, all three dimeric forms of PDGF have been identified. The following nomenclature to distinguish the various forms is suggested: PDGF-AA, a homodimer of A chains; PDGF-AB, a heterodimer; PDGF-BB, a homodimer of B chains; PDGF, any dimeric form of A or B chains.

The aim of the present study was to investigate which growth factors, receptors, and growth inhibiting factors are expressed in invasive breast cancer. Five (angiogenic) growth factors and their receptors: platelet-derived growth factor A chain (PDGF-AA) and PDGF receptor alpha (PDGF alpha R), PDGF-BB and PDGF beta receptor, transforming growth factor alpha (TGF alpha) and its receptor epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) and its receptors vascular endothelial growth factor receptor I (Flt-1) and vascular endothelial growth factor receptor II (Flk-1/KDR); two growth inhibiting factors: transforming growth factor-beta-1 (TGF beta 1) and (TGF beta 2) and their receptor couple transforming growth factor beta receptor I (TGF beta R-I) and TGF beta R-II; and basic fibroblast growth factor (bFGF) were stained by standard immunohistochemistry on frozen sections in 45 cases of invasive carcinoma of the breast. Staining was scored as negative or positive in tumour epithelium, stroma, and blood vessels. TGF beta 1 and TGF beta 2 were expressed in the tumour cells in 67 per cent and 76 per cent of cases, respectively, whereas PDG beta R and TGF beta R-II were expressed in 0 per cent and 2 per cent, respectively. The other factors showed variable expression in tumour cells. All factors were expressed in the stroma in most cases, except Flt-1, Flk-1/KDR, TGF beta 2, and TGF beta R-II, which showed variable expression, and EGFR, which showed no expression. The endothelium was in most cases positive for bFGF, PDGF-AA, PDGF-BB, VEGF, PDGF alpha R, PDGF beta R, and TGF beta 1 but TGF beta/ was negative in most cases and TGF alpha, EGFR, Flt-1, Flk-1/KDR, TGF beta R-I, and TGF beta R-II were variably expressed. The most interesting possible auto/paracrine loops, as demonstrated on serial sections and by fluorescence double staining, were the TGF alpha/EGFR, TGF beta s/TGF beta R, VEGF/Flt-1, and the VEGF/Flk-1 combinations. In conclusion, growth factors, growth inhibiting factors, and their receptors are frequently expressed in invasive breast cancer. Indications for some possible auto- and paracrine loops have been found, which should encourage further study on the role of these factors in breast cancer proliferation and angiogenesis.

OBJECTIVE

Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of osteoarthritis (OA). Growth factors (GFs) capable of antagonizing the catabolic actions of cytokines may have therapeutic potential in the treatment of OA. Herein, we investigated the potential synergistic effects of insulin-like growth factor (IGF-1) and platelet-derived growth factor (PDGF-bb) on different mechanisms participating in IL-1β-induced activation of nuclear transcription factor-κB (NF-κB) and apoptosis in chondrocytes.

METHODS

Primary chondrocytes were treated with IL-1β to induce dedifferentiation and co-treated with either IGF-1 or/and PDGF-bb and evaluated by immunoblotting and electron microscopy.

RESULTS

Pretreatment of chondrocytes with IGF-1 or/and PDGF-bb suppressed IL-1β-induced NF-κB activation via inhibition of IκB-α kinase. Inhibition of IκB-α kinase by GFs led to the suppression of IκB-α phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene products involved in inflammation and cartilage degradation (COX-2, MMPs) and apoptosis (caspase-3). GFs or BMS-345541 (specific inhibitor of the IKK) reversed the IL-1β-induced down-regulation of collagen type II, cartilage specific proteoglycans, β1-integrin, Shc, activated MAPKinase, Sox-9 and up-regulation of active caspase-3. Furthermore, the inhibitory effects of IGF-1 or/and PDGF-bb on IL-1β-induced NF-κB activation were sensitive to inhibitors of Src (PP1), PI-3K (wortmannin) and Akt (SH-5), suggesting that the pathway consisting of non-receptor tyrosine kinase (Src), phosphatidylinositol 3-kinase and protein kinase B must be involved in IL-1β signaling.

CONCLUSIONS

The results presented suggest that IGF-1 and PDGF-bb are potent inhibitors of IL-1β-mediated activation of NF-κB and apoptosis in chondrocytes, may be mediated in part through suppression of Src/PI-3K/AKT pathway, which may contribute to their anti-inflammatory effects.

This study describes the features of tissue-engineering scaffold capable of sequentially delivering three angiogenic factors. The scaffold consists of alginate-sulfate/alginate, wherein vascular endothelial growth factor (VEGF) platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1) are bound to alginate-sulfate with an affinity similar to that realized upon their binding to heparin. Factor release rate from the scaffold was correlated with the equilibrium binding constants of the factors to the matrix, thus enabling the sequential delivery of VEGF, PDGF-BB and TGF-beta1. In alginate scaffolds lacking alginate-sulfate, release of the adsorbed proteins was instantaneous. After subcutaneous implantation for 1 and 3 months in rats, the blood vessel density and percentage of mature vessels were 3-fold greater in the triple factor-bound scaffolds than in the factor-adsorbed or untreated scaffolds. Moreover, vascularization within the triple factor-bound scaffolds was superior to that found in scaffolds delivering only basic fibroblast growth factor. Application of this novel scaffold may be extended to the combined delivery of additional heparin-binding angiogenic factors or combinations of growth factors active in different tissue regeneration processes.

Growth factors may play an important role in tumour growth and angiogenesis by their influence on tumour cell proliferation or their effect on neovascularization. The aim of the present study was to determine which of the growth factors, growth-inhibiting factors, and their receptors investigated in a previous study are correlated with proliferation and angiogenesis in invasive breast cancer, with emphasis on the impact of possible autocrine and paracrine loops. Five growth factors and their receptors: platelet-derived growth factor A chain (PDGF-AA) and PDGF alpha receptor (PDGF alpha R), PDGF-BB and PDGF beta receptor (PDGF beta R), transforming growth factor alpha (TGF alpha) and its receptor epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) and its receptors (Flt-1 and Flk-1/KDR; two growth-inhibiting factors: transforming growth factor beta-1 (TGF beta 1) and TGF beta 2 and their receptor couple TGF beta R-I and TGF beta R-II; and basic fibroblast growth factor (bFGF) were stained in 45 cases of invasive breast cancer by standard immunohistochemistry on frozen sections. Staining in tumour cells, stromal cells, and endothelial cells was scored as negative or positive. Proliferation was determined by assessment of the mitotic activity index (MAI) and the degree of angiogenesis was measure by counting the number of microvessels (microvessel density: MVD) in the most vascularized area of the tumour. bFGF and EGFR showed positive correlations with the MAI, while TGF beta 2 showed a negative correlation. Expression of bFGF, TGF alpha, TGF beta 2, and EGFR correlated positively with the MVD. Co-expression of the TGF alpha/EGFR growth factor/receptor combination showed a stronger correlation with the MAI and the MVD than EGFR or TGF alpha alone, and the TGF beta 2/TGF beta R-I/TGE beta R-II combination showed a positive correlation with the MVD. In conclusion, the expression of several growth factors, growth factor receptors and growth-inhibiting factors showed correlations with the rate of proliferation and the degree of angiogenesis in invasive breast cancer. Some growth factor/receptor combinations showed stronger correlations with proliferation and angiogenesis than the growth factor or receptor alone, pointing to the importance of possible auto- and paracrine loops for stimulation of proliferation and angiogenesis by growth factors and their receptors.

Factors that regulate the growth and development of primitive bone marrow stromal cell precursors are not well defined. We have examined 25 purified recombinant growth factors for their ability to initiate and support clonogenic growth of fibroblast colony-forming cells (CFU-F) from adult human bone marrow. Assays were performed using bone marrow mononuclear cells (BMMNC) enriched in CFU-F by magnetic-activated cell sorting (MACS) using the monoclonal antibody (MoAb) STRO-1. A serum-deprived assay was developed to avoid components of fetal calf serum (FCS) that may mask or otherwise modify the response of CFU-F to exogenously added factors. L-ascorbate and the glucocorticoid dexamethasone were found to be essential for CFU-F colony development under serum-deprived conditions. Importantly, clonogenic growth of CFU-F in this culture system was absolutely dependent on an exogenous source of growth factor. Platelet-derived growth factor-BB (PDGF) and epidermal growth factor (EGF) demonstrated the greatest ability to support colony growth. Colony formation was dose-dependent, with half-maximal colony numbers at approximately 0.2 ng/mL for either factor and plateau numbers at concentrations in excess of 1.0 ng/mL. Simultaneous addition of PDGF and EGF had no effect on the number of colonies initiated but resulted in dose-dependent increases in mean colony diameter that were significant (P < or = .05) when compared with the effect of either factor alone or with the size of colonies elicited in control cultures by 20% FCS. Fluorescence-activated cell sorting (FACS) of BMMNC using MoAbs to the alpha chain of the PDGF receptor and to the EGF receptor in combination with the Moab STRO-1 demonstrated constitutive expression of both receptors by greater than 90% on CFU-F. Receptors for insulin-like growth factor-1 (IGF-1) and nerve growth factor (NGF) were also detected on STRO-1+ CFU-F, but in vitro both IGF-1 and NGF did not support colony growth. This report demonstrates the development of a simple, reproducible, and stringent culture system for the growth and assay of stromal precursors under serum-deprived conditions and represents an important prerequisite for future studies of the role of growth factors in the regulation of stromal cell proliferation, differentiation, and development.

The localized and temporally controlled delivery of growth factors is key to achieving optimal clinical efficacy. In sophisticated tissue engineering strategies, the biodegradable scaffold is preferred to serve as both a three-dimensional (3-D) substrate and a growth factor delivery vehicle to promote cellular activity and enhance tissue neogenesis. This study presents a novel approach to fabricate tissue engineering scaffolds capable of controlled growth factor delivery whereby growth factor containing microspheres were incorporated into 3-D scaffolds with good mechanical properties, well-interconnected macroporous and nano-fibrous structures. The microspheres were uniformly distributed throughout the nano-fibrous scaffold and their incorporation did not interfere the macro-, micro-, and nanostructures of the scaffold. The release kinetics of platelet-derived growth factor-BB (PDGF-BB) from microspheres and scaffolds was investigated using poly(lactic-co-glycolic acid) (PLGA50) microspheres with different molecular weights (6.5 and 64kDa, respectively) and microsphere-incorporated poly(l-lactic acid) (PLLA) nano-fibrous scaffolds. Incorporation of microspheres into scaffolds significantly reduced the initial burst release. Sustained release from several days to months was achieved through different microspheres in scaffolds. Released PDGF-BB was demonstrated to possess biological activity as evidenced by stimulation of human gingival fibroblast DNA synthesis in vitro. The successful generation of 3-D nano-fibrous scaffold incorporating controlled-release factors indicates significant potential for more complex tissue regeneration.

Activation of the PDGF receptor on human arterial smooth muscle cells (SMC) induces migration and proliferation via separable signal transduction pathways. Sphingosine-1-phosphate (Sph-1-P) can be formed following PDGF receptor activation and therefore may be implicated in PDGF-receptor signal transduction. Here we show that Sph-1-P does not significantly affect PDGF-induced DNA synthesis, proliferation, or activation of mitogenic signal transduction pathways, such as the mitogen-activated protein (MAP) kinase cascade and PI 3-kinase, in human arterial SMC. On the other hand, Sph-1-P strongly mimics PDGF receptor-induced chemotactic signal transduction favoring actin filament disassembly. Although Sph-1-P mimics PDGF, exogenously added Sph-1-P induces more prolonged and quantitatively greater PIP2 hydrolysis compared to PDGF-BB, a markedly stronger calcium mobilization and a subsequent increase in cyclic AMP levels and activation of cAMP-dependent protein kinase. This excessive and prolonged signaling favors actin filament disassembly by Sph-1-P, and results in inhibition of actin nucleation, actin filament assembly and formation of focal adhesion sites. Sph-1-P-induced interference with the dynamics of PDGF-stimulated actin filament disassembly and assembly results in a marked inhibition of cell spreading, of extension of the leading lamellae toward PDGF, and of chemotaxis toward PDGF. The results suggest that spatial and temporal changes in phosphatidylinositol turnover, calcium mobilization and actin filament disassembly may be critical to PDGF-induced chemotaxis and suggest a possible role for endogenous Sph-1-P in the regulation of PDGF receptor chemotactic signal transduction.

We investigated the role of the p38 mitogen-activated protein kinase (p38 MAPK) in the PDGF-BB-induced cytoskeleton remodeling that occurs during the migration of porcine aortic smooth muscle cells (SMC). We showed that p38 MAPK controlled the polymerization of actin that is required for PDGF-induced lamellipodia formation and migration. To investigate the mechanism of action of p38 MAPK, we explored its cellular localization and that of its indirect substrate, the heat shock protein Hsp27, during SMC spreading on fibronectin in the presence and absence of PDGF. Spreading of SMC on fibronectin activated p38 MAPK in a sustained manner only in the presence of PDGF. In these conditions, Hsp27 and p38 MAPK were localized all over the lamellipodia. A transiently phosphorylated form of p38 MAPK was observed at the leading edge, whereas p38 MAPK remained phosphorylated at the base of the lamellipodia. Phosphorylated Hsp27 was excluded from the leading edge and restricted to the base of the lamellipodia. These results were confirmed by Triton X-100 extraction of particulate membrane fraction. Displacement of Hsp27 from the leading edge by cytochalasin D treatment suggests that nonphosphorylated Hsp27 caps barbed ends in vivo. Our data indicate that nonphosphorylated Hsp27 might contribute to the formation of a short, branched actin network at the leading edge, whereas phosphorylated Hsp27 might stabilize the actin network at the base of lamellipodia, which is composed of long, unbranched actin filaments.