Fibroblastic response to treatment with different preparations rich in growth factors

Objectives:  Preparations rich in growth factors (PRGF) release them plus bioactive proteins at localized sites, with the aim of triggering healing and regenerative processes. The prevailing paradigm suggests that their influence on proliferation, angiogenesis and the extracellular matrix synthesis is minimal. However, variations in their composition and impact on different cell phenotypes have not been examined.
Materials and methods:  Sixteen fibroblast cultures obtained from three different anatomical sites (skin, synovium and tendon) of 16 donors were exposed to the molecular pool released from PRGF scaffolds, with increasing amounts of platelets. We evaluated cell proliferation, secretion of angiogenic growth factors (VEGF and HGF), synthesis of type I collagen and hyaluronic acid (HA), considering platelet dose and anatomical origin of the cells. Activity of transforming growth factor-beta (TGF-β) in type I procollagen and HA synthesis was examined by adding exogenous TGF-β to plasma preparations.
Results:  All plasma preparations induced a significant proliferative response compared to non-stimulated cells ( P  < 0.05). Maximum proliferation rate was obtained with PRGF with 2-fold or 4-fold platelet concentration. Exposure to PRGF stimulated VEGF synthesis exclusively in tendon cells ( P  < 0.05), which also exhibited a different pattern of HGF production ( P  < 0.05). PRGF enhanced HA synthesis ( P  < 0.05), but did not alter collagen I production. Platelet-secreted TGF-β may be involved in HA, but not in type I procollagen synthesis.
Conclusions:  Optimizing composition and use of platelet-rich products is crucial to enhancing the therapeutic potential of this technology. Our data show that the biological effects of PRGF may depend on concentration of platelets and on the anatomical source of the cells.     

Soy peptide-induced stem cell proliferation: involvement of ERK and TGF-β1

This study was conducted to investigate the proliferative effect of vegetable soy peptides on adult stem cells (ASCs) in the absence of serum and their possible mechanisms of action. The proliferation of human adipose tissue-derived mesenchymal stem cells (ADSCs) and cord blood-derived mesenchymal stem cells (CB-MSCs) treated with soy peptides was found to increase significantly upon 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Click-iT 5-ethynyl-2'-deoxyuridine flow cytometry assay. In addition, soy peptides led to stepwise phosphorylation of the p44/42 MAPK (ERK), mammalian target of rapamycin (mTOR), p70 S6 kinase, S6 ribosomal protein (S6RP) and eukaryotic initiation factor 4E (eIF4E) in ADSCs. Furthermore, quantitative analysis of the cytokines revealed that the production of transforming growth factor-beta1 (TGF-β1), vascular endothelial growth factor and interleukin-6 increased significantly in response to treatment with soy peptides in both ADSCs and CB-MSCs. Similarly, soy peptide-induced phosphorylation of the ERK/mTOR/S6RP/eIF4E pathway was blocked in response to pretreatment with PD98059, a specific ERK inhibitor. Moreover, inhibition of TGF-β1 through PD98059 pretreatment and a consecutive decrease in ADSC proliferation revealed that TGF-β1 induces the phosphorylation of mTOR/S6RP/eIF4E. Collectively, the results of this study indicate that ERK-dependent production of TGF-β1 plays a crucial role in the soy peptide-induced proliferation of ADSCs under serum-free conditions.     

Interleukin-6 receptor superantagonist Sant7 inhibits TGF-beta-induced proliferation of human lung fibroblasts

Abstract.  Objectives : Both interleukin-6 (IL-6) and transforming growth factor-β (TGF-β) are crucially involved in fibrotic events that characterize interstitial lung diseases (ILD). Therefore, the aim of this study was to investigate in primary cultures of normal and fibrotic human lung fibroblasts (HLF), exposed to either IL-6 or TGF-β1, the effects on phosphorylation of mitogen-activated protein kinases (MAPK) and cell growth of IL-6 signalling inhibition, performed by the IL-6 receptor superantagonist Sant7. Materials and methods : MAPK phosphorylation was detected by Western blotting, HLF viability and proliferation were evaluated using the trypan blue staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Results : Sant7, at a concentration of 1 µg/mL, was capable of significantly inhibiting HLF proliferation and MAPK phosphorylation induced by cell exposure to IL-6 (100 ng/mL) or TGF-β1 (10 ng/mL), whose actions were more evident in fibrotic cells. Conclusions : These findings suggest that, in HLFs derived from patients with ILDs, the proliferative mechanisms activated by TGF-β1 are at least in part mediated by an increased release of IL-6, leading to phosphorylation-dependent MAPK activation. Such preliminary findings may thus open new therapeutic perspectives for fibrogenic ILDs, based on inhibition of signal transduction pathways stimulated by the IL-6 receptor.     

Cytokine interactions in mesenchymal stem cells from cord blood

We used cytokine protein array to analyze the expression of cytokines from human cord blood-derived mesenchymal stem cells (CB-MSCs). Several cytokines, interleukins (IL), and growth factors, including ENA-78, GM-CSF, GRO, IL-1β, IL-6, IL-8, MCP-1, OSM, VEGF, FGF-4, FGF-7, FGF-9, GCP-2, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IP-10, LIF, MIF, MIP-3α, osteoprotegerin, PARC, PIGF, TGF-β2, TGF-β3, TIMP-1, as well as TIMP-2, were secreted by CB-MSCs, while IL-4, IL-5, IL-7, IL-13, TGF-β1, TNF-α, and TNF-β were not expressed under normal growth conditions. IL-6, IL-8, TIMP-1, and TIMP-2 were the most abundant interleukins expressed by CB-MSCs. A set of growth factors were selected to evaluate their stimulatory effects on the IL6 secretion for CB-MSCs. IL-1β was the most important factor inducing CB-MSC to secret IL-6. The mechanism by which IL-1β promoted IL-6 expression in CB-MSCs was studied. By using various inhibitors of signal transduction, we found that activation of p38 mitogen-activated protein kinases (MAPK) and MAPK kinase (MEK) is essential in the IL-1β stimulated signaling cascade which leads to the increase in IL-6 synthesis. Additionally, continuous supplement of IL-1β in the CB-MSCs culture will facilitate adipogenic maturation of CB-MSCs as evidenced by the presence of oil drops in the CB-MSCs and secretion of leptin, a molecule marker of adipocytes. These results strongly suggest that cytokine induction and signal transduction are important for the differentiation of CB-MSCs.     

Involvement of fibroblast growth factor 18 in dedifferentiation of cultured human chondrocytes

Objective:  Chondrocytes inevitably decrease production of cartilaginous matrices during long-term cultures with repeated passaging; this is termed dedifferentiation. To learn more concerning prevention of dedifferentiation, we have focused here on the fibroblast growth factor (FGF) family that influences chondrocyte proliferation or differentiation.
Materials and methods:  We have compared gene expression between differentiated cells in passage 3 (P3) and dedifferentiated ones in P8 of human cultured chondrocytes. We also performed ligand administration of the responsive factor or its gene silencing, using small interfering RNA (siRNA).
Results:  FGFs 1, 5, 10, 13 and 18 were higher at P8 compared to P3, while FGFs 9 and 14 were lower. Especially, FGF18 showed a 10-fold increase by P8. Ligand administration of FGF18 in the P3 cells, or its gene silencing using siRNA in the P8 cells, revealed dose-dependent increase and decrease respectively in type II collagen/type I collagen ratio. Exogenous FGF18 also upregulated expression of transforming growth factor beta (TGF-β), the anabolic factor of chondrocytes, in P3 chondrocytes, but P8 cells maintained a low level of TGF-β expression, suggesting a decrease in responsiveness of TGF-β to FGF18 stimulation in the dedifferentiated chondrocytes.
Conclusion:  FGF18 seems to play a role in maintenance of chondrocyte properties, although its expression was rather high in dedifferentiated chondrocytes. Upregulation of FGF18 in dedifferentiated chondrocytes implied that it may be a marker of dedifferentiation.     

Cytokine-Regulated Expression of Platelet-Derived Growth Factor Gene and Protein in Cultured Human Astrocytes

Abstract: To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-β₁ (TGF-β₁) or tumor necrosis factor-α (TNF-α); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1β (IL-1β) by itself had little or no effect but synergized with TGF-β₁ in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B-chain mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-β₁, TNF-α, TNF-α/TGF-β₁, or IL-1β/TGF-β₁, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-β₁, TNF-α, and IL-1β are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.     

Cellular Localization and Temporal Elevation of Tumor Necrosis Factor-α, Interleukin-1α, and Transforming Growth Factor-β1 mRNA in Hippocampal Injury Response Induced by Trimethyltin

Abstract: In certain pathologic states, cytokine production may become spatially and temporally dysregulated, leading to their inappropriate production and potentially detrimental consequences. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, and transforming growth factor-β (TGF-β) mediate a range of host responses affecting multiple cell types. To study the role of cytokines in the early stages of brain injury, we examined alterations in the 17-day-old mouse hippocampus during trimethyltin-induced neurodegeneration characterized by neuronal necrosis, microglia activation in the dentate, and astrocyte reactivity throughout the hippocampus. By 24 h after dosing, elevations in mRNA levels for TNF-α, IL-1α, IL-1β, and IL-6 mRNA were seen. TGF-β1 mRNA was elevated at 72 h. In situ hybridization showed that TNF-α and IL-1α were localized to the microglia, whereas TGF-β1 was expressed predominantly in hippocampal pyramidal cells. Intercellular adhesion molecule-1, EB-22, Mac-1, and glial fibrillary acidic protein mRNA levels were elevated within the first 3 days of exposure in the absence of increased inducible nitric oxide synthetase and interferon-γ mRNA. These data suggest that pro-inflammatory cytokines contribute to the progression and pattern of neuronal degeneration in the hippocampus.     

Chondrocyte proliferation in a new culture system

Objective:  This study has aimed to study different culture systems that might stimulate an increase in cell proliferation of normal and osteoarthritis chondrocytes from articular cartilage in rat model.
Material and Methods:  Three culture systems using chondrocytes embedded in alginate beads were tested: chondrocytes cultured in Dulbecco's modified Eagle's medium (DMEM) as control, a co-culture system consisting of a monolayer of de-differentiated chondrocytes as a source of mitotic factors, and an enriched medium containing culture medium obtained from a monolayer of chondrocytes and DMEM. Normal and osteoarthritis chondrocytes were stained with 5-carboxyfluorescein diacetate succinimidyl ester and were cultured in each of the three systems. After 5 days of culture cell, proliferation was detected by flow cytometry. Chondrocyte phenotype was confirmed by collagen type II and MMP-3 expression. To determine possible molecules released into the medium by the cultured chondrocyte monolayer and which would probably be involved in cell proliferation, a study of mRNA and expression of transforming growth factor-β1 (TGF-β1), fibroblastic growth factor-2 (FGF-2), epidermal growth factor (EGF), platelet derived growth factor-A (PDGF-A) and insulin-like growth factor-1 (IGF-1) proteins was conducted.
Results and Conclusions:  Chondrocytes in the co-culture system or in enriched medium showed an increase in proliferation; only when osteoarthritis chondrocytes were cultured in enriched medium would they display a statistically significant increase in their proliferation rate and in their viability. When chondrocytes from the monolayer were analysed, differential mRNA expression of TGF-β1 and IGF-1 was found during all passages, which suggests that these two growth factors might be involved in chondrocyte proliferation.     

TGFβ inhibits GM-CSF-induced phosphorylation of ERK and MEK in human myeloid leukaemia cell lines via inhibition of phosphatidylinositol 3-kinase (PI3-k)

Objectives:  Activation of SMAD-independent p44/42 MAPK (ERK1/2) signalling by TGFβ has been recently reported in various cell types. However, the mechanisms for the linkage between the SMAD-dependent and -independent pathways are poorly understood. In this study, we investigated whether TGF-β activates the ERK pathway and how TGFβ communicates with the MAP kinase signals induced by a mitogen, in human myeloid leukaemia cells.
Materials and methods and results:  TGFβ dramatically suppressed proliferation of MV4–11 and TF-1 cells without detectable phosphorylation of ERK1/2 and MEK1/2 for the duration of 48 h, as detected by MTT assay and Western blot analysis, respectively. In contrast, GM-CSF induced rapid and transient phosphorylation of MEK1/2 and ERK1/2 and up-regulated cell proliferation. Both GM-CSF-induced ERK1/2 activation and cell proliferation were significantly inhibited by TGFβ. GM-CSF also induced transient phosphorylation of the p85 subunit of PI3-kinase. Corresponding to this change, phosphorylated p85 was found to bind to the GM-CSF receptor-α subunit, as detected by immunoprecipitation and Western blot analysis. PD98059, a selective inhibitor of MEK, blocked GM-CSF-induced phosphorylation of MEK and ERK but not p85. However, TGFβ and LY294002, a potent inhibitor of PI3-kinase, significantly inhibited phosphorylation of both p85 and ERK1/2.
Conclusions:  These studies thus indicate that TGFβ does not activate the ERK pathway but turns off the GM-CSF-induced ERK signal via inhibition of the PI3-kinase-Akt pathway, in these human laeukemia cells.     

The core-aldehyde 9-oxononanoyl cholesterol increases the level of transforming growth factor β1-specific receptors on promonocytic U937 cell membranes

Among the broad variety of compounds generated via oxidative reactions in low-density lipoproteins (LDL) and subsequently found in the atherosclerotic plaque are aldehydes that are still esterified to the parent lipid, termed core aldehydes. The most represented cholesterol core aldehyde in LDL is 9-oxononanoyl cholesterol (9-ONC), an oxidation product of cholesteryl linoleate. 9-ONC, at a concentration detectable in biological material, markedly up-regulates mRNA expression and protein level of both the pro-fibrogenic and pro-apoptotic cytokine transforming growth factor β1 (TGF-β1) and the TGF-β receptor type I (TβRI) in human U937 promonocytic cells. We also observed increased membrane presentation of TGF-β receptor type II (TβRII). Experiments employing the TβRI inhibitor SB431542, or the TGFβ antagonist DANFc chimera, have shown that the effect on TβRI is directly induced by 9-ONC, while TβRII up-regulation seems stimulated by its specific ligand, i.e. TGFβ1, over-secreted meanwhile by treated cells. Increased levels of the cytokine and of its specific receptors in 9-ONC-treated cells clearly occurs through stimulation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), as demonstrated by ERK1/2 knockdown experiments using mitogen-activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MEK1 and MEK2) siRNAs, or PD98059, a selective MEK1/2 inhibitor. 9-ONC might thus sustain further vascular remodeling due to atherosclerosis, not simply by stimulating synthesis of the pro-fibrogenic cytokine TGF-β1 in vascular cells, but also and chiefly by enhancing the TGF-β1 autocrine loop, because of the marked up-regulation of the cytokine's specific receptors TβRI and TβRII.     

Adipose tissue-derived stem cells rescue Purkinje neurons and alleviate inflammatory responses in Niemann-Pick disease type C mice

Adult stem cells offer special therapeutic prospects because they can be isolated for autologous transplantation, expanded ex vivo, and differentiated into various cell types. We previously reported that bone marrow-derived mesenchymal stem cells improve neurological deficits in neurodegenerative disease animal models. However, the efficacy of adipose tissue-derived stem cells (ADSCs) transplantation in similar models remains unknown. Herein, we demonstrate that ADSCs, when transplanted into Niemann-Pick disease type C (NP-C) mouse cerebellum, elicit rescue of Purkinje neurons and restoration of motor coordination together with alleviation of inflammatory responses as verified by immunohistochemistry and real-time PCR using glial fibrillary acidic protein (GFAP), F4/80, IL-1β, IL-6, and TNF-α. Most importantly, ADSCs enhance electrically active Purkinje neurons with functional synaptic formation after transplantation in NP-C disease model mice. This report demonstrates for the first time that ADSCs can rescue imperiled Purkinje neurons and alleviate the inflammatory response in NP-C disease model mice, thereby signifying the therapeutic potential of ADSCs for neurodegenerative diseases.     

Neuroprotection of microglial conditioned medium on 6-hydroxydopamine-induced neuronal death: role of transforming growth factor beta-2

Microglia, the immune cells of the CNS, play essential roles in both physiological and pathological brain states. Here we have used an in vitro model to demonstrate neuroprotection of a 48 h-microglial conditioned medium (MCM) towards cerebellar granule neurons (CGNs) challenged with the neurotoxin 6-hydroxydopamine, which induces a Parkinson-like neurodegeneration, and to identify the protective factor(s). MCM nearly completely protects CGNs from 6-hydroxydopamine neurotoxicity and at least some of the protective factor(s) are peptidic in nature. While the fraction of the medium containing molecules < 30 kDa completely protects CGNs, fractions containing molecules < 10 kDa or > 10 kDa are not neuroprotective. We further demonstrate that microglia release high amounts of transforming growth factor-β2 (TGF-β2) and that its exogenous addition to the fraction of the medium not containing it (< 10 kDa) fully restores the neuroprotective action. Moreover, MCM neuroprotection is significantly counteracted by an inhibitor of TGF-β2 transduction pathway. Our results identify TGF-β2 as an essential neuroprotective factor released by microglia in its culture medium that requires to be fully effective the concomitant presence of other factor(s) of low molecular weight.     

Effect of Calcitriol in Combination with Corticosterone, Interleukin-1β, and Transforming Growth Factor-β1 on Nerve Growth Factor Secretion in an Astroglial Cell Line

Abstract: In astrocytes, nerve growth factor (NGF) synthesis has been described to be stimulated by the cytokines interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) and inhibited by corticosterone. As all three factors are present in the brain under certain conditions, we investigated the effect of their combined application on NGF secretion in the astroglial cell line RC7 and, in addition, studied the effect of calcitriol (1α,25-dihydroxyvitamin D₃). Calcitriol stimulated NGF secretion, whereas corticosterone reduced basal levels of NGF secretion as well as inhibited the NGF secretion induced by IL-1β, calcitriol, and TGF-β1. Calcitriol had an additive effect when applied together with IL-1β and a synergistic effect when applied with TGF-β1. Moreover, calcitriol not only counteracted the inhibitory effect of corticosterone on NGF secretion stimulated by TGF-β1 but even augmented it to a level more than threefold higher than that reached with TGF-β1 alone. Due to the trophic effect of NGF on basal forebrain cholinergic neurons, these findings might be of therapeutic relevance under conditions where cholinergic function is impaired and the endogenous levels of corticosterone, IL-1β, or TGF-β1 are elevated.     

Amyloid beta-derived neuroplasticity in bone marrow-derived mesenchymal stem cells is mediated by NPY and 5-HT2B receptors via ERK1/2 signalling pathways

Objective:  In Alzheimer's disease, toxic soluble and insoluble forms of amyloid beta (Aβ) cause synaptic dysfunction and neuronal loss. Given its potential role in producing a toxic host microenvironment for transplanted donor stem cells, we investigated the interaction between Aβ and proliferation, survival, and differentiation of bone marrow-derived mesenchymal stem cells (BM-MSC) in culture.
Materials and methods:  We used BM-MSC that had been isolated from mouse bone marrow and cultured, and we also assessed relevant reaction mechanisms using gene microarray, immunocytochemistry, and inhibitors of potential signalling molecules, such as mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)1/2 and tyrosine protein kinase.
Results and conclusions:  Interestingly, we found that treatment with aggregated (1–40 or 1–42) and oligomeric (1–42) Aβ promoted neuronal-like differentiation of BM-MSC without toxic effects. This was not dependent on soluble factors released from BM-MSC progeny nor solely on formation of Aβ fibrils. The effect of Aβ is mediated by G-protein coupled receptors, neuropeptide Y1 (NPY1R) and serotonin (5-hydroxytryptamine) receptor 2B, via phosphatidylinositol-3-OH kinase-dependent activation of the MAPK/ERK1/2. Our results lend support to the idea that reciprocal donor stem cell–host interactions may promote a regenerative response that can be exploited by epigenetic modulation of NPY/serotonergic gene expression, for stem cell therapy, in Alzheimer's disease.