Inflammation has an important role in the development of liver fibrosis in general and the activation of hepatic stellate cells (HSCs) in particular. It is known that HSCs are themselves able to produce cytokines and chemokines, and that this production may be a key event in the initiation of fibrogenesis. However, the direct involvement of cytokines and chemokines in HSC (self-)activation remains uncertain. In this study, the effects of pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-β1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8, and also the pro-fibrogenic mediator TGF-β1. Two drugs were also evaluated, the anti-TNF-α monoclonal antibody infliximab and the IL-1 receptor antagonist anakinra, regarding their inhibitory effects. In LX-2 human HSC, treatment with TGF-β1 are associated with downregulation of the metalloproteinase (MMP)-1 and MMP-3, with upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I α1, collagen type IV α1, α-SMA, endothelin-1 and PDGF-BB. Cytokines and chemokines expression were found to be downregulated, excepting IL-6. In contrast, we observed that LX-2 exposure to IL-1, TNF-α and IL-8 can reverse the phenotype of pro-fibrogenic activated cells. Indeed, MMP-1, MMP-3 and MMP-9 were found elevated, associated with downregulation of α-SMA and/or PDGF-BB, and a greater expression of IL-1β, IL-6, IL-8, CXCL1 and CCL2. Lastly, we found that infliximab and anakinra successfully inhibits effects of TNF-α and IL-1 respectively in LX-2 cells. Infliximab and anakinra may be of value in preclinical trials in chronic liver disease. Overall, our results suggest that (i) pro-inflammatory mediators exert complex effects in HSCs via an MMP/TIMP imbalance, and (ii) targeting IL-1 signaling may be a potentially valuable therapeutic strategy in chronic liver diseases.

Platelet-derived growth factors (PDGFs) play important roles in skeletal development and bone fracture healing, yet how PDGFs execute their functions remains incompletely understood. Here we show that PDGF-AA, but not -AB or -BB, could activate the BMP-Smad1/5/8 pathway in mesenchymal stem cells (MSCs), which requires BMPRIA as well as PDGFRα. PDGF-AA promotes MSC osteogenic differentiation through the BMP-Smad1/5/8-Runx2/Osx axis and MSC migration via the BMP-Smad1/5/8-Twist1/Atf4 axis. Mechanistic studies show that PDGF-AA activates BMP-Smad1/5/8 signaling by feedback down-regulating PDGFRα, which frees BMPRI and allows for BMPRI-BMPRII complex formation to activate smad1/5/8, using BMP molecules in the microenvironment. This study unravels a physical and functional interaction between PDGFRα and BMPRI, which plays an important role in MSC differentiation and migration, and establishes a link between PDGF-AA and BMPs pathways, two essential regulators of embryonic development and tissue homeostasis.

OBJECTIVE

Peptide growth factors are known accelerators of corneal wound healing, probably mediated through increased proliferation of the cells; however, information about their effect on keratocyte motility is lacking. The influence of peptide growth factors on keratocyte migratory activity was investigated, using the following growth factors: platelet derived growth factor (PDGF-BB), epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and transforming growth factor-beta-1 (TGF-beta 1).

METHODS

Keratocytes were seeded on gels of type 1 collagen, growth factor added, and the cells left to migrate for 72 hours. Subsequently, the number of keratocytes at the different levels in the collagen gel was evaluated by optically sectioning the gel at 20 microns, intervals, with an inverted phase contrast microscope.

RESULTS

PDGF, EGF and bFGF at 10 ng/ml, all increased the number of keratocytes at the different levels of the gel as compared to a non-stimulated control (p < 0.05 or p < 0.01, students t-test). TGF-beta proved to be a strong inhibitor of keratocyte migration, decreasing the number of keratocytes observed at every level in the gel (p < 0.05 and p < 0.01, students t-test), whereas no effect of IGF-I and aFGF was found. During the 72 hours of migration, no contraction of the collagen gels was observed. Autoradiography of histological sections of the gels showed that during the 72-hour period only TGF-beta and 10% fetal bovine serum induced an increase in keratocyte proliferation.

CONCLUSIONS

PDGF, EGF and bFGF increase keratocyte migration, independent of proliferation in a collagen gel invasion assay and might promote corneal wound healing, not only by increasing cell proliferation, but also through increased motility.

Purpose: Cancer metastasis can occur at the early stage of tumor development when a primary tumor is at the microscopic size. In particular, the interaction of malignant cells with other cell types including cancer-associated fibroblasts (CAF) in promoting metastasis at the early stage of tumor development remains largely unknown. Here, we investigated the role of CAFs in facilitating the initial events of cancer metastasis when primary tumors were at microscopic sizes.Experimental Design: Multicolor-coded cancer cells and CAFs were coimplanted into the transparent zebrafish body and metastasis at a single-cell level was monitored in living animals. Healthy fibroblasts, tumor factor-educated fibroblasts, and CAFs isolated from various tumors were tested for their ability to facilitate metastasis.Results: We showed that CAFs promoted cancer cell metastasis at the very early stage during primary tumor development. When a primary tumor was at the microscopic size consisting of a few hundred cells, CAFs were able to hijack cancer cells for dissemination from the primary site. Surprisingly, a majority of metastatic cancer cells remained in tight association with CAFs in the circulation. Furthermore, stimulation of non-metastasis-promoting normal fibroblasts with TGF-B, FGF-2, HGF, and PDGF-BB led to acquisition of their metastatic capacity.Conclusions: Cancer metastasis occurs at the very early stage of tumor formation consisting of only a few hundred cells. CAFs are the key cellular determinant for metastasis. Our findings provide novel mechanistic insights on CAFs in promoting cancer metastasis and targeting CAFs for cancer therapy should be aimed at the early stage during cancer development. Clin Cancer Res; 23(16); 4769-79. ©2017 AACR.

The purpose of this randomized controlled trial was to determine the effects of an 8-week (aerobic+strength) exercise training program (3 sessions/week) on the circulating cytokine levels of breast cancer survivors. We randomly allocated 16 female survivors of breast cancer (mean±SD age: 50±5 years) to an intervention or usual care (control) group (N=8 in each group). The intervention group followed an 8-week exercise program consisting of 3 sessions/week (session duration: 90 min). We measured the levels of the following cytokines before and after the intervention: beta-NGF, CTACK, eotaxin, FGF basic, G-CSF, gmCSFα, HGF, ICAM1, IFNα2, IFNγ, IL1α, IL1ß, IL1ra, IL2, IL2ra, IL3, IL4, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL16, IL17, IL18, IP10, LIF, MCS-F, MIP1α, MIP1β, MIF, MCP1, MCP3, MIG, PDGF bb, SCF, SCGFβ, SDF1α, TRAIL, TNFα, TNFβ, VCAM1, and VEGF. We only observed a significant interaction (group*time) effect for CTACK ( P=0.016), with mean values remaining stable in the intervention group but increasing over time in controls. The intervention program did not induce a significant decrease in the main breast cancer-related cytokines such as IL6 and IL8. A combined (aerobic+strength) 8-week exercise training intervention did not induce major changes in the basal cytokine levels of breast cancer survivors.

BACKGROUND

Aqueous flare as determined by laser flare photometry in the anterior chamber is a strong preoperative predictor for proliferative vitreoretinopathy (PVR) in patients with primary retinal detachment (RD). We analysed various cytokines in aqueous humour samples in relation to aqueous flare and postoperative PVR incidence in patients with RD.

METHODS

Preoperatively, the aqueous flare of patients with RD was measured quantitatively with a laser flare metre and aqueous humour samples were collected and analysed for interferon γ, tumour necrosis factor α, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, vascular endothelial growth factor (VEGF)-A, platelet derived growth factor (PDGF)-aa, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, fibroblast growth factor (FGF)-aa and FGF-bb by multiplex fluorescent bead-based immunoassays. Three months after RD surgery patients were examined for PVR development.

RESULTS

Of 67 consecutive patients, 10 developed at least PVR grade C. Patients with flare values >15 pc/ms (n=20) and the 10 patients with postoperative PVR all had significantly elevated levels of IL-6, IL-8, MCP-1 and TGF-β1 in aqueous humour (p≤0.05). Levels of VEGF-A, PDGF-aa and TGF-β2 were not significantly changed. Other cytokines were below the detection threshold. Eight of the 10 patients (80%) with PVR had elevated flare values of >15 pc/ms and 8 of the 20 patients (40%) with flare >15 pc/ms developed PVR. The OR for PVR with flare values >15 pc/ms was 30.7 (p=0.0001).

CONCLUSIONS

Laser flare photometry allows simple risk estimation for later PVR development. Elevated laser flare values correspond to an altered profibrotic intraocular cytokine milieu. These factors therefore constitute promising targets for a prophylactic intervention.

We have previously demonstrated that PDGF-BB enhances proliferation of C2 myoblasts. This has led us to examine whether the mitogenic influence of PDGF-BB in the C2 model correlates with modulation of specific steps associated with myogenic differentiation. C2 myoblasts transiting through these differentiation specific steps were monitored via immunocytochemistry. We show that the influence of PDGF on enhancing cell proliferation correlates with a delay in the emergence of cells positive for sarcomeric myosin. We further monitored the influence of PDGF-BB on differentiation steps preceding the emergence of myosin+ cells. We demonstrate that mononucleated C2 cells first express MyoD (MyoD+/myogenin- cells) and subsequently, myogenin. Cells negative for both MyoD and myogenin (the phenotype preceding the MyoD+ state) were present at all times in culture and comprised the majority, if not all, of the cells which responded mitogenically to PDGF. Additionally, the frequency of the MyoD+/myogenin+ cell phenotype was reduced in cultures receiving PDGF, suggesting that PDGF can modulate the transition of the cells into the myogenin+ state. We determined that many of the myogenin+ cells subsequently become MEF2A+ and this phenomenon is not influenced by PDGF-BB. FGF-2 also enhanced the proliferation of C2 myoblasts and suppressed the appearance of the myogenin+ cells, but did not influence the subsequent transition into the MEF2A+ state. The study raises the possibility that PDGF-BB and FGF-2 might delay the transition of the C2 cells into the MyoD+/myogenin+ state by depressing a paracrine signal that enhances differentiation.

We describe that stimulation of human fibroblasts with platelet-derived growth factor BB (PDGF-BB) induces a transient up-regulation of the PDGF alpha- and beta-receptor transcript and protein levels. The effect of PDGF-BB on the receptor transcript levels was more pronounced than those seen when other cytokines were used. Regulation of transcript levels by PDGF-BB was mediated through post-transcriptional mechanisms. No induction could be observed in a nuclear run-on analysis, but cycloheximide treatment attenuated the accumulation of both alpha- and beta-receptor transcripts induced by PDGF-BB. An increase in receptor protein levels was observed using two different experimental approaches. Increased amounts of receptor precursor forms could be immunoprecipitated from metabolically labeled cells, stimulated with PDGF-BB. In a second approach, cells were exposed to different concentrations of PDGF-BB, and, in a subsequent step, ligand binding analysis was performed. In this experiment, an initial down-regulation of receptors was followed by increased levels of the cell surface forms of the receptors. In conclusion, PDGF-BB, but not PDGF-AA, induces increased synthesis of both PDGF alpha- and beta-receptor protein; this constitutes a positive feed-back mechanism, which, for example, could serve to potentiate autocrine stimulation of growth.

Homeobox transcription factors specify body plan by regulating differentiation, proliferation, and migration at a cellular level. The homeobox transcription factor Gax is expressed in quiescent vascular smooth muscle cells (VSMCs), and its expression is downregulated by vascular injury or other conditions that lead to VSMC proliferation. Previous investigations demonstrate that Gax may regulate VSMC proliferation by upregulating the cyclin-dependent kinase (cdk) inhibitor p21. Here we examined whether Gax influences VSMC migration, a key feature in the development of stenotic lesions after balloon injury. Transduction of a Gax cDNA inhibited the migratory response of VSMCs toward PDGF-BB, basic fibroblast growth factor, or hepatocyte growth factor/scatter factor. Gax expression also inhibited migration of NIH.3T3 fibroblasts and embryonic fibroblasts lacking p53. Gax was unable to inhibit the migration of fibroblasts lacking p21, but this effect could be restored in these cells by providing exogenous p21 or by overexpressing another cdk inhibitor, p16. Flow cytometric analysis implicated a Gax-mediated downregulation of alpha(v)beta(3) and alpha(v)beta(5) integrin expression in VSMCs as a potential cause for reduced cell motility. Gax specifically downregulated beta(3) and beta(5) in VSMCs in culture and after acute vascular injury in vivo. Repression of integrin expression was also found in NIH 3T3 cells and p53 knockout fibroblasts, but not in p21-knockout fibroblasts, unless these cells express exogenous p21 or p16. These data suggest that cycle progression, integrin expression, and cell migration can be regulated in VSMCs by the homeobox gene product Gax.

We report a considerable number of cells in the ventricular and the subventricular zones (SVZ) of newborn mice to stain positive for the PDGF beta-receptor (PDGFRB). Many of them also stained for nestin and/or GFAP but less frequently for the neuroblast marker doublecortin and for the mitotic marker Ki-67. The SVZ of mice with nestin-Cre conditional deletion of PDGFRB expressed the receptor only on blood vessels and was devoid of any morphological abnormality. PDGFRB(-/-) neurospheres showed a higher rate of apoptosis without any significant decrease in proliferation. They demonstrated reduced capacities of migration and neuronal differentiation in response to not only PDGF-BB but also bFGF. Furthermore, the PDGFR kinase inhibitor STI571 blocked the effects of bFGF in control neurosphere cultures. bFGF increased the activity of the PDGFRB promoter as well as the expression and phosphorylation of PDGFRB. These results suggest the presence of the signaling convergence between PDGF and FGF. PDGFRB is needed for survival, and the effects of bFGF in migration and neural differentiation of the cells may be potentiated by induction of PDGFRB.

OBJECTIVE

Pericytes/pericyte precursors produce milk fat globule-associated protein with epidermal growth factor and factor VIII-like domains (MFG-E8) in vivo, and this α(v) integrin ligand enhances angiogenesis in tumors and in oxygen-induced retinopathy in mice. Inhibition of MFG-E8 production or function attenuates platelet-derived growth factor-BB (PDGF-BB)-induced migration of pericyte/pericyte precursor-like 10T1/2 cells in vitro. Herein, we describe mechanisms by which MFG-E8 modulates PDGF-BB:PDGF receptor β (PDGFRβ) signaling in 10T1/2 cells.

RESULTS

Small interfering RNA depletion of MFG-E8 from 10T1/2 cells or antibody inhibition of MFG-E8 action enhanced PDGF-BB-dependent degradation of PDGFRβ and attenuated signaling. Coimmunoprecipitation revealed transient association of MFG-E8 with PDGFRβ in PDGF-BB-treated 10T1/2 cells and reduced PDGFRβ-focal adhesion kinase association in MFG-E8-depleted cells. Confocal microscopy demonstrated that MFG-E8 binding to 10T1/2 cells was RGD motif and α(v) dependent but PDGF-BB treatment independent, whereas colocalization of MFG-E8 with PDGFRβ was enhanced by PDGF-BB. Ubiquitination of PDGFRβ was also increased in MFG-E8 small interfering RNA-transfected cells.

CONCLUSIONS

Integrin α(v)-bound MFG-E8 associates with PDGFRβ and focal adhesion kinase after PDGF-BB treatment, results in cell surface retention of PDGFRβ, delays receptor degradation, potentiates downstream signaling, and enhances migration of 10T1/2 cells. MFG-E8 may promote angiogenesis, in part, via cell autonomous actions on pericytes or pericyte precursors that result in enhanced PDGF-BB:PDGFRβ signaling mediated via integrin-growth factor receptor cross-talk.

Myelination by Schwann cells is likely to be regulated in vivo by positive and negative epigenetic factors. In vitro, the positive regulation of myelin differentiation, in particular expression of the major myelin protein Po, can be mimicked by cAMP elevating agents, while serum, transforming growth factor (TGF) beta s, and fibroblast growth factor (FGF)2 have been shown to exert a negative effect on this differentiation. Growth factors which promote Po induction have not, however, been identified previously. Using a forskolin concentration (0.4 microM) which alone produces little Po mRNA or protein induction, we show that insulin-like growth factor (IGF)-I, IGF-II and high concentrations of insulin promote high levels of Po induction, although in the absence of forskolin they have no effect. Another event related to Schwann cell differentiation, induction of galactocerebroside expression in response to cAMP analogues, is also potentiated by IGFs. In a different context, IGFs regulate Schwann cell DNA synthesis. We find that in defined medium forskolin plus FGF2, TGF beta or platelet-derived growth factor (PDGF) BB causes minimal DNA synthesis in the absence of IGFs and that IGFs act as potent mitogens under these conditions. IGFs also potentiate DNA synthesis induced by beta isoforms of neu-differentiation factors (NDFs), although in this case considerable DNA synthesis occurs even in the absence of IGF. These results show that IGFs can act as powerful stimulators of both proliferation and differentiation in Schwann cells, and that the total growth factor input determines which of these pathways IGFs will promote.

Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. The extent to which the endocytic trafficking routes can contribute to such RTK hyperactivation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor β-receptor (PDGFRβ) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRβ activation were synergistic in stimulating phosphatidylinositol (PI) 3-kinase activity, leading to receptor macropinocytosis. PDGFRβ macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of PI 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRβ activation in nontransformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor promoted macropinocytosis of both receptors and increased their activation in nontransformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis.

Gene therapy has yet to achieve reproducible clinical efficacy, due to inadequate gene delivery, inadequate gene expression, or dose-limiting toxicity. We have developed a gene therapy technology for tissue repair and regeneration that employs a structural matrix for DNA delivery. The matrix holds the DNA vector at the treatment site and provides a scaffolding for in-growth and accumulation of repair cells and efficient DNA transfection. We now report, for the first time, matrix-mediated delivery of targeted DNA vectors for soft tissue repair. A collagen matrix was used to deliver an adenoviral vector encoding platelet-derived growth factor-B (AdPDGF-B), resulting in efficient transgene expression in vitro and in vivo. Increases in the overall levels of expression and in the relative amounts of secreted PDGF-BB were achieved when AdPDGF-B was conjugated to fibroblast growth factor (FGF2) such that the virus was targeted for cellular uptake via FGF receptors. Matrix-mediated delivery of AdPDGF-B enhanced wound healing responses in vivo, and FGF2 targeting generated effects comparable to nontargeted vectors at significantly lower doses. Therefore, matrix-mediated delivery in combination with FGF2 targeting overcomes some of the safety and efficacy limitations of current gene therapy strategies and is an attractive therapeutic approach for tissue repair and regeneration.

In human malignant mesothelioma cell lines elevated expression of the platelet-derived growth factor (PDGF) beta-chain (c-sis) gene was previously reported, while normal mesothelial cells barely express this gene. Expression of the PDGF A-chain gene was only slightly elevated in these cell lines compared with normal mesothelial cells. For a putative autocrine function of the produced PDGF, in these cells expression of PDGF receptors is a prerequisite. In this paper we report on the expression of PDGF alpha- and beta-receptors in normal and malignant mesothelial cells. Cultured normal mesothelial cells expressed PDGF alpha-receptor mRNA and protein and had weak to undetectable levels of the PDGF beta-receptor mRNA and protein. In contrast, malignant mesothelioma cell lines were found to express PDGF beta-receptor mRNA and protein, while PDGF alpha-receptor expression was not detectable by Northern blotting and immunoprecipitation. Binding experiments with [125I]-PDGF-AA and [125I] PDGF-BB to normal and malignant mesothelial cell lines confirmed these observations. These results suggest that autocrine stimulation of growth may occur both in cultured normal mesothelial cells (PDGF-AA acting via the alpha-receptor) and in malignant mesothelioma cell lines (PDGF-BB acting via the beta-receptor).

We previously found that L6 myoblasts and skeletal muscle isolated from developing rats express the platelet-derived growth factor (PDGF) beta-receptor gene (Jin, P., Rahm, M., Claesson-Welsh, L., Heldin, C.-H., and Sejersen, T. (1990) J. Cell Biol. 110, 1665-1672). We now report that recombinant human PDGF-BB is a mitogen for L6 myoblasts and also a potent inhibitor of myogenic differentiation. Treatment of L6J1 myoblasts with PDGF-BB increased the rate of DNA synthesis and stimulated cell proliferation. In differentiation medium (Dulbecco's modified Eagle's medium/0.5% fetal calf serum or Dulbecco's modified Eagle's medium/insulin), PDGF-BB prevented fusion of confluent myoblasts and suppressed biochemical differentiation in L6J1 cells. Inhibition of myoblast differentiation was, however, reversible. Withdrawal of PDGF-BB from the medium allowed myoblast fusion to occur. Northern blot hybridization showed that the PDGF beta-receptor mRNA was down-regulated to an undetectable level when confluent cultures of L6J1 myoblasts in growth medium (Dulbecco's modified Eagle's medium/5% fetal calf serum) were shifted to differentiation medium. Receptor binding assays further indicated that binding of PDGF-BB to its receptors on L6J1 myoblasts declined rapidly before creatine kinase activity rose. Our results provide the first demonstration that PDGF-BB is a potent regulator of myogenesis of L6 rat myoblasts and suggest that it may regulate muscle differentiation in vivo.

This study examined the potential influence of platelet-derived growth factor-BB homodimers (PDGF-BB) on surgical incisions in irradiated animals with depressed wound healing. Rats were irradiated with either 800 rads total body or 2,500 rads surface irradiation. Parallel dorsal skin incisions were made 2 days later, and PDGF-BB was applied topically a single time to one of two incisions. In total body-irradiated rats, bone marrow-derived elements were severely depressed, wound macrophages were virtually eliminated, and PDGF-BB treatment was ineffective. However, in surface-irradiated rats, PDGF-BB treatment recruited macrophages into wounds and partially reversed impaired healing on day 7 (p less than 0.005) and day 12 (p less than 0.001). PDGF-BB-treated wounds were 50 percent stronger than the paired control wounds. The results suggest PDGF requires bone marrow-derived cells, likely wound macrophages, for activity and that it may be useful as a topical agent in postirradiation surgical incisions.

Glomerular crescents are a major determinant of progression in various renal diseases. Some types of growth factors are known to be involved in the evolution of crescents and the subsequent scar formation. Although glomerular parietal epithelial cells (PECs) are the major component of cellular crescents, the influence of growth factors on PECs is unknown. We performed immunohistochemical studies and in situ hybridization to examine alterations in connective tissue growth factor (CTGF) expression and to identify CTGF-synthesizing cells in crescents in the crescentic glomerulonephritis model of Wistar Kyoto rats. In addition, we examined the roles of fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)-BB, transforming growth factor (TGF)-beta, and CTGF in cell proliferation and matrix synthesis in an established rat PEC cell line (PEC line). In an acute phase of rat crescentic glomerulonephritis, a major component of the crescents were macrophages, which did not express CTGF mRNA. However, in the advanced phase, crescents strongly expressed CTGF mRNA and the epithelial marker pan-cadherin but did not express the macrophage marker ED1, suggesting that PECs synthesized the CTGF. In the PEC line, FGF-2 predominantly promoted [(3)H]thymidine incorporation compared with PDGF-BB. Both TGF-beta and PDGF-BB strongly stimulated extracellular matrix synthesis in association with up-regulation of endogenous CTGF, but TGF-beta showed a predominant role. FGF-2 had a minor effect on it. In addition, blockade of endogenous CTGF using an antisense oligodeoxynucleotide significantly attenuated both TGF-beta- and PDGF-BB-induced extracellular matrix synthesis. These results suggest that several growth factors promote cell proliferation and matrix production in PECs. CTGF-mediated matrix production via the TGF-beta or PDGF-BB pathway in PECs may, in part, play a role in the progression of scar formation in crescents.

Progressive contractile dysfunction of viable myocardium that surrounds a large infarct leads to heart failure following acute myocardial infarction (AMI). Experimental evidence indicates that cellular transplantation may improve the left ventricular (LV) contractile performance, even though the underlying mechanisms remain undefined. Here, we compared the effect of transplantation of murine multipotent adult progenitor cells (MAPCs), a population of adult bone marrow-derived cells that differentiate into cells of mesodermal, endodermal and ectodermal origin, with murine bone marrow cells (BMCs) or fibroblasts on post-infarct cardiac function by peri-infarct injection after coronary artery ligation in mice. We demonstrate that, in contrast to the other cell populations, transplantation of MAPCs significantly improved LV contractile function for at least 8 weeks post-transplantation and, although BMCs reduced infarct size, the decrease in scar size was substantially greater in MAPC-treated hearts. As neither MAPCs nor BMCs were present beyond 1 week, the beneficial effect was not due to differentiation and direct contribution of MAPCs to the vascular or cardiomyocyte compartment. Significantly more inflammatory cells were present in MAPC- than BMC-treated hearts at 1 week, which was accompanied by increased vascularity 8 weeks post-transplantation. We hypothesize that MAPCs indirectly contributed to these effects, by secreting inflammatory [monocyte chemoattractant protein-1 (MCP)-1], and vascular growth factors [vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-BB, and transforming growth factor (TGF)beta(1)), and others, resulting in increased angiogenensis and cardioprotection.

We have previously demonstrated specific insulin-like growth factor I (IGF I) messenger RNA (mRNA) transcripts in cultured rat aortic smooth muscle cells (RASM). To define the role of IGF I in the autocrine growth program of vascular smooth muscle cells, we quantitated IGF I mRNA levels in proliferating and quiescent (serum-deprived for 48 hours) RASM. IGF I mRNA levels were markedly decreased in quiescent cells, and this effect was reversible on reexposure to serum. Since platelet-derived growth factor (PDGF) acts synergistically with IGF I to stimulate vascular smooth muscle cell growth, we exposed quiescent RASM to PDGF AB or BB and quantitated IGF I transcript levels. Both PDGF dimers caused a marked, rapid increase in IGF I message levels. To determine whether induction of IGF I mRNA levels correlated with secretion of IGF I, we measured immunoreactive IGF I in RASM conditioned medium after separation of IGF I binding proteins by gel filtration chromatography. PDGF caused a significant increase in IGF I release at 24 hours. These findings indicate that IGF I mRNA levels in vitro are regulated by serum and by growth factors such as PDGF. Serum deprivation reversibly decreases IGF I transcript levels, and exposure of quiescent cells to PDGF increases IGF I mRNA levels and IGF I release. Regulation of IGF I expression by competence growth factors such as PDGF may play an important role in the control of vascular smooth muscle cell growth.

Platelet-derived growth factor (PDGF) is a critical regulator of cell proliferation. Because ethanol inhibits cell proliferation in vivo and in vitro, we hypothesize that ethanol-induced inhibition results from differential interference with signal transduction pathways activated by PDGF. Cultured cortical astrocytes were used to examine the effects of ethanol on PDGF-mediated signal transduction, on the expression of two PDGF monomers (A- and B-chains), and on the expression of two PDGF receptor subunits (PDGFalphar and PDGFbetar). PDGF-B chain homodimer (PDGF-BB), and to a lesser extent PDGF-A chain homodimer (PDGF-AA), stimulated the proliferation of astrocytes raised in a serum-free medium. Ethanol attenuated these actions in a concentration-dependent manner. Ethanol inhibited both PDGF-AA- and PDGF-BB-mediated phosphorylation of PDGFalphar, but it had little effect on PDGFbetar autophosphorylation. Likewise, ethanol abolished the association of PDGFalphar to Ras GTPase-activating protein (Ras-GAP), but it did not affect the binding of Ras-GAP to PDGFbetar. PDGF stimulated the activities of mitogen-activated protein kinase (MAPK) in protein kinase C (PKC) independent and dependent manners. Ethanol inhibited the PKC-independent, acute activation of MAPK; however, it stimulated the PKC-dependent, sustained activation of MAPK. The expression of neither ligand was altered by exposure to ethanol for 3 d. Moreover, such treatment specifically upregulated PDGFalphar expression in a concentration-dependent manner. It did not, however, affect the binding affinity of either receptor. Thus, the signal transduction pathways initiated by PDGF-AA and PDGF-BB were differentially affected by ethanol. This differential vulnerability resulted from the preferential effects of ethanol on PDGFalphar autophosphorylation. Hence, ethanol-induced alterations are transduced through specific receptors of mitogenic growth factors.

Platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), potent modulators of mesenchymal cell growth and differentiation, are often colocalizable in vivo. Previous in vitro studies in fibroblastic cell lines have shown variable, even antagonistic effects of TGF-beta on the mitogenic action of PDGF. This study demonstrates that in diploid human dermal fibroblasts, TGF-beta 1 is weakly mitogenic in the absence of serum or purified growth factors, and that TGF-beta 1 potentiates DNA synthesis in PDGF-stimulated fibroblasts with delayed kinetics when compared to stimulation with PDGF alone. TGF-beta 1 enhances mitogenic potency of all three PDGF isoforms and increases receptor binding of both 125I PDGF-AA and 125I PDGF-BB, consistent with the increased expression of the alpha type PDGF receptor. The induction of PDGF alpha receptor subunits by TGF-beta may play a role in enhancing the proliferative potential of human fibroblasts in certain physiologic and pathologic conditions.

Mesenchymal stem cells (MSCs) mobilize membrane type-1 matrix metalloproteinase (MT1-MMP) to traffic through both 3-dimensional (3D) collagen as well as basement membrane barriers, but factors capable of regulating the expression and activity of the protease remain unidentified. Herein, we report that the MT1-MMP-dependent invasive activities of rat MSCs are controlled by PDGF-BB. Furthermore, PDGF-BB also stimulates MSC proliferation in 3D type I collagen via an MT1-MMP-dependent process that is linked to pericellular collagen degradation. PDGF-BB stimulates MT1-MMP expression at both the mRNA and protein levels in concert with ERK1/2 and PI3K/AKT activation. Inhibition of ERK1/2 or PI3K/AKT activity potently suppresses both MT1-MMP-dependent invasive and proliferative activities. Basement membrane invasion is likewise stimulated by PDGF-BB in an MT1-MMP-dependent manner via ERK1/2 and PI3K/AKT signaling. Taken together, these data serve to identify PDGF-BB as an important MSC agonist that controls invasive and proliferative activities via MT1-MMP-dependent processes that are regulated by the ERK1/2 and PI3K/AKT signaling pathways.

Lovastatin inhibits 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase the rate limiting enzyme for synthesis of mevalonic acid, a precursor for cholesterol, farnesyl and geranylgeranyl pyrophosphate isoprenoids. Recent studies suggest it also has growth inhibitory properties. Posttranslational farnesyl or geranylgeranylation of low molecular weight GTP-binding proteins such as RAS and RHO are thought to be an essential step in activation of phosphorylation cascades such as the RAS-RAF-1-MEK-1-MAPK/ERK pathway which stimulate cell proliferation. In this study, we evaluated lovastatin effects on meningioma cell proliferation and activation of the MEK-1-MAPK/ERK pathway. The effect of lovastatin on cell proliferation was assessed in eight human meningioma cell cultures stimulated by platelet derived growth factor (PDGF)-BB, cerebrospinal fluid (CSF), and fetal bovine serum (FBS). Concomitant lovastatin effects on phosphorylation/activation of mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) kinase (MEK-1) and MAPK/ERK were assessed by Western blot. Whether lovastatin acts via a mevalonate-dependent mechanism was also evaluated. Coadministration of lovastatin completely blocked PDGF-BB, CSF, and FBS stimulation of [3H]-thymidine incorporation and cell proliferation. Lovastatin inhibited PDGF-BB's stimulatory effect in a dose dependent manner. Concomitant with its growth inhibitory effects, lovastatin reduced phosphorylation/activation of MEK-1/2 in five meningiomas and MAPK/ERK in seven. Coadministration of mevalonate with lovastatin partially restored PDGF's mitogenic effect. Lovastatin is a potent inhibitor of meningioma cell proliferation which may act in part by reducing activation of MEK-1-MAPK/ERK pathway. Additional studies are warranted to assess whether lovastatin and similar HMG-CoA reductase inhibitors represent a new adjunctive chemotherapy for recurrent meningiomas.