Platelet-derived growth factor (PDGF) has been implicated in promoting survival and proliferation of immature neurons, and even protecting neurons from gp120-induced cytotoxicity. However, the mechanisms involved in neuroprotection are not well understood. In the present study we demonstrate the role of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in PDGF-mediated neuroprotection. Pharmacological inhibition of PI3K greatly reduced the ability of PDGF-BB to block gp120 IIIB-mediated apoptosis and cell death in human neuroblastoma cells. The role of Akt in PDGF-mediated protection was further corroborated using a dominant-negative mutant of Akt, which was able to block the protective effect of PDGF. We next sequentially examined the signals downstream of Akt in PDGF-mediated protection in human neuroblastoma cells. In cells pretreated with PDGF prior to gp120 there was increased phosphorylation of both GSK-3beta and Bad, an effect that was inhibited by PI3-kinase inhibitor. Nuclear translocation of NF-kappaB, which lies downstream of GSK-3beta, however, remained unaffected in cells treated with PDGF. In addition to inducing phosphorylation of Bad, PDGF-mediated protection also involved down-regulation of the proapoptotic protein Bax. Furthermore, PDGF-mediated protection also involved the inhibition of gp120-induced release of mitochondrial cytochrome C. Our findings thus underscore the roles of both PI3K/Akt and Bcl family pathways in PDGF-mediated neuroprotection.

The redox state plays an important role in gene regulation. Thiols maintain the intracellular redox homeostasis. To understand the role of thiols in redox signaling, we have studied the effect of thiol alkylation on platelet-derived growth factor-BB (PDGF-BB)-induced cell survival events in vascular smooth muscle cells. PDGF-BB stimulated Akt phosphorylation predominantly at Ser-473. N-Ethylmaleimide (NEM), a thiol alkylating agent, blocked PDGF-BB-induced Akt phosphorylation without affecting its upstream phosphatidylinositol 3-kinase (PI3K). On the other hand, LY294002 and wortmannin, specific inhibitors of PI3K, prevented PDGF-BB-induced phosphorylation of Akt and its downstream effector molecules, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. NEM also abrogated the phosphorylation of p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E induced by PDGF-BB, suggesting that thiol alkylation interferes with the PI3K/Akt pathway at the level of Akt. In addition, NEM blocked PDGF-BB-induced phosphorylation of BAD and forkhead transcription factor FKHR-L1, and these events correlated with increased apoptosis. NEM alone and in concert with PDGF-BB increased reactive oxygen species (ROS) production and protein phosphatase 2A (PP2A) activity in VSMC. The inhibition of PDGF-BB-induced Akt phosphorylation by NEM was completely reversed by PP2A inhibitors fostriecin and okadaic acid, ceramide synthase inhibitor fumonisin B1, and ROS scavenger N-acetylcysteine (NAC). NAC also attenuated the apoptosis induced by NEM, alone or in combination with PDGF-BB. Together, these findings demonstrate for the first time that PP2A mediates thiol alkylation-dependent redox regulation of Akt and cell survival.

Glomerular mesangial cell proliferation constitutes a frequent pathological alteration in glomerulonephritis. In addition to platelet-derived growth factor (PDGF) inflammatory cytokines such as IL-1, IL-6 or tumour necrosis factor-alpha (TNF-alpha) have been proposed to have mitogenic activity for mesangial cells. A model was therefore established in which human mesangial cells (HMC) could be reversibly growth-arrested for prolonged times in serum-free medium without suffering irreversible functional or morphological changes. In this model 24 h stimulation with rhPDGF-BB induced an increase of the 3H-thymidine incorporation of 1190 +/- 280 (50 ng/ml) % +/- s.e.m. of medium control. Less growth induction was noted after stimulation with 50 ng/ml rhPDGF-AB (925 +/- 126%) or rhPDGF-AA (575 +/- 24%). Northern analysis confirmed the presence of both alpha- and beta-PDGF receptor subunit mRNA in growth-arrested HMCs. rhIL-1 alpha, rhIL-1 beta, rhTNF-alpha or rhIL-6 at various doses and times, despite increasing cellular PGE2-release, did not induce significant proliferation in HMCs. Inhibition of PGE2-release did not change the lack of mitogenicity of IL-1, TNF-alpha or IL-6. IL-6 did not alter the mitogenic response of the cells towards PDGF. In contrast, both IL-1 alpha and IL-1 beta (5 ng/ml) induced a delay but not augmentation of the PDGF growth response. This delay could be reversed by the concomitant addition of recombinant IL-6 or of anti-IL-1 antibody but not by inhibition of prostaglandin synthesis. High doses of TNF-alpha suppressed PDGF-induced proliferation. These data suggest that in growth-arrested HMCs inflammatory cytokines have a growth-modulating or -suppressive rather than (co-)mitogenic effect while PDGF-BB and -AB and to a lesser degree PDGF-AA are potent mitogens. The findings support the notion that the control of HMC proliferation in pathological situations depends on a complex network of interacting stimuli.

In addition to their ability to stimulate cell proliferation, polypeptide growth factors are able to maintain cell survival under conditions that otherwise lead to apoptotic death. Growth factors control cell viability through regulation of critical intracellular signal transduction pathways. We previously characterized C2 muscle cell lines that lacked endogenous expression of insulin-like growth factor II (IGF-II). These cells did not differentiate but underwent apoptotic death in low-serum differentiation medium. Death could be prevented by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we analyze the signaling pathways involved in growth factor-mediated myoblast survival. PDGF treatment caused sustained activation of extracellular-regulated kinases 1 and 2 (ERK1 and -2), while IGF-I only transiently induced these enzymes. Transient transfection of a constitutively active Mek1, a specific upstream activator of ERKs, maintained myoblast viability in the absence of growth factors, while inhibition of Mek1 by the drug UO126 blocked PDGF-mediated but not IGF-stimulated survival. Although both growth factors activated phosphatidylinositol 3-kinase (PI3-kinase) to similar extents, only IGF-I treatment led to sustained stimulation of its downstream kinase, Akt. Transient transfection of a constitutively active PI3-kinase or an inducible Akt promoted myoblast viability in the absence of growth factors, while inhibition of PI3-kinase activity by the drug LY294002 selectively blocked IGF- but not PDGF-mediated muscle cell survival. In aggregate, these observations demonstrate that distinct growth factor-regulated signaling pathways independently control myoblast survival. Since IGF action also stimulates muscle differentiation, these results suggest a means to regulate myogenesis through selective manipulation of different signal transduction pathways.

OBJECTIVE

The transmigratory capacity of bone marrow (BM) mesenchymal stromal cells (MSC) through the endothelial cell barrier into various tissues and their differentiation potential makes them ideal candidates for cell therapy. Nevertheless, the mechanisms and agents promoting their migration are not fully understood. We evaluated the effects of several inflammatory cytokines on the migration of BM MSC and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) production.

METHODS

The migratory potential of BM MSC was evaluated using a Boyden chamber coated with Matrigel in the presence and absence of stromal cell-derived (SDF)-1alpha, platelet-derived growth factor (PDGF)bb, insulin-like growth factor (IGF)-I and interleukin (IL)-6. The ability of inflammatory cytokines to induce MSC migration was tested in presence of their respective Ab or blocking peptide. We used immunofluorescence to check the expression of cytokine receptors, and MMP/TIMP production was analyzed at the protein (human cytokine array, enzyme-linked immunosorbent assay (ELISA), gelatine zymography and Western blot) and mRNA quantitative real-time polymerase chain reaction (qRT-PCR) levels.

RESULTS

We have demonstrated that inflammatory cytokines promote the migratory capacity of BM MSC according to the expression of their respective receptors. Higher migration through Matrigel was observed in response to IL-6 and PDGFbb. qRT-PCR and cytokine array revealed that migration was the result of the variable level of MMP/TIMP in response to inflammatory stimuli.

CONCLUSIONS

Our observations suggest that chemokines and cytokines involved in the regulation of the immunity or inflammatory process promote the migration of MSC into BM or damaged tissues. One of the mechanisms used by MSC to promote their migration though the extracellular matrix is modulation of the production of MMP-1, MMP-2, MMP-13, TIMP-1 and TIMP-2.

Genetic instability of tumor cells often leads to amplified expression of multiple growth factors that contribute to angiogenesis and tumor growth. Members of the platelet-derived growth factor (PDGF) family are frequently utilized growth factors by many tumors to support their growth. PDGFs have previously been found to induce tumor growth by directly stimulating cell growth of certain types of tumors. We have recently demonstrated that PDGFs are potent angiogenic factors. Particularly, the angiogenic activity of PDGFs can be potentiated in the presence of other angiogenic factors. In addition to stimulation of blood angiogenesis, we have recently found that PDGFs can directly stimulate lymphangiogenesis and lymphatic metastasis. In this review, multiple roles of PDGFs in control of tumor growth and metastasis are discussed.

The molecular mechanism involved in the stimulation of hyaluronan synthetase in normal human mesothelial cells was investigated. Exposure of mesothelial cells to platelet-derived growth factor (PDGF)-BB stimulated hyaluronan synthetase activity, measured in isolated membrane preparations, as well as hyaluronan secretion into the medium. The effect on hyaluronan synthetase was maximal after 6 h of treatment. In contrast, the stimulatory effect of transforming growth factor-beta 1 reached a maximum after 24 h. The stimulatory effect of PDGF-BB was inhibited by cycloheximide. The phosphotyrosine phosphatase inhibitor vanadate was found to stimulate hyaluronan synthetase activity, and to potentiate the effect of PDGF-BB. The protein kinase C (PKC) stimulator phorbol 12-myristate 13-acetate (PMA) also stimulated hyaluronan synthetase; furthermore, depletion of PKC by preincubation of the cells with PMA led to an inhibition of the PDGF-BB-induced stimulation of hyaluronan synthetase activity. Thus the PDGF-BB-induced stimulation of hyaluronan synthetase activity is dependent on protein synthesis and involves tyrosine phosphorylation and activation of PKC.

In this study, the effect of human erythropoietin Delta (Epo) on smooth muscle cell (SMC)-rich lesions was evaluated. Mice, of which the left carotid artery was ligated, were treated with suberythropoietic as well as erythropoietic doses of Epo and both doses of Epo enhanced SMC-rich lesion formation. No association was observed between hemoglobin levels and lesion size. Moreover, endothelial progenitor cell (EPC) numbers in the peripheral blood increased only in the erythropoietic dosing group, indicating that EPC numbers did not correlate with lesion size. Immunohistochemical analysis revealed that Epo-mediated enhancement of lesion formation correlates with increased signal transducer and activator of transcription 5 (Stat5) phosphorylation in the vessel wall. Experiments performed in cultured vascular cells demonstrated that Epo robustly induced phosphorylation of Stat5 in human umbilical vein endothelial cells (HUVECs), but only very weakly in SMCs. In tumor necrosis factor-alpha (TNFalpha)-activated HUVECS, Epo induced expression of platelet-derived growth factor B (PDGF-B), which was at least partially responsible for the induction of Stat5 phosphorylation in SMCs by HUVEC-conditioned medium. In conclusion, in mice Epo accelerates SMC-rich neointima formation, which correlates with increased Stat5 phosphorylation in the vessel wall but is independent of erythrocyte and EPC numbers.

The stem cell therapy for treating ischemic diseases is promising; however, the limited availability and compromised quality of progenitor cells in aged and diseased patients limit its therapeutic use. Here we report a nanofiber-based ex vivo stem cell expansion technology and proangiogenic growth factors overexpression of human umbilical cord blood (UCB)-derived progenitor cells to enhance angiogenic potential of therapeutic stem cells. The progenitor cells were expanded approximately 225-fold on nanofiber-based serum-free ex vivo expansion culture technique without inducing differentiation. The expanded cells express high levels of stem cell homing receptor, CXCR4, and adhesion molecule, LFA-1. The nanofiber-expanded stem cells uptake AcLDL effectively, and migrate efficiently in an in vitro transmigration assay. These expanded cells can also differentiate into endothelial and smooth muscle cells in vitro. In a NOD/SCID mouse hind limb vascular injury model, nanofiber-expanded cells were more effective in blood flow restoration and this effect was further augmented by VEGF(164) and PDGF-BB, growth factor overexpression. The data indicate that nanofiber-based ex vivo expansion technology can provide an essential number of therapeutic stem cells. Additionally, proangiogenic growth factors overexpression in progenitor cells can potentially improve autologous or allogeneic stem cell therapy for ischemic diseases.

The proliferation of vascular smooth muscle cells (VSMCs) is an integral part of the mechanism of vascular diseases such as restenosis. Post-translational modifications by histone deacetylase (HDAC) inhibitors play an important role in the regulation of gene expression by inducing cell cycle arrest. However, the role and mechanism of the HDAC inhibitor trichostatin A (TSA) on neointimal proliferation remain unknown. In this study, we investigated the effect and mechanism whereby TSA prevents the proliferation of VSMCs and neointimal hyperplasia induced by balloon injury in rat carotid artery. Local administration of TSA significantly prevented neointimal hyperplasia. TSA dramatically inhibited the proliferation and DNA synthesis of VSMCs in response to FBS or PDGF-BB. Overexpression of Krüppel like factor 4 (KLF4) blocked the cell proliferation and DNA synthesis, as determined by the MTT and [³H]thymidine incorporation assays, whereas knockdown of KLF4 resulted in an increase in VSMC proliferation. In VSMCs, TSA increased the mRNA level and protein expression of KLF4. Treatment with TSA or transfection of KLF4 increased the expression of both p21 and p27 and promoter activity. In addition, the anti-proliferative activity of TSA was recovered in KLF4-knockdown cells. These data demonstrate that TSA inhibits neointimal thickening and VSMC proliferation via activation of the KLF4/p21/p27 signaling pathway.

Fibrosarcomas are rare malignant mesenchymal tumors originating from fibroblasts. Importantly, fibrosarcoma cells were shown to have a high content and turnover of extracellular matrix (ECM) components including hyaluronan (HA), proteoglycans, collagens, fibronectin, and laminin. ECMs are complicated structures that surround and support cells within tissues. During cancer progression, significant changes can be observed in the structural and mechanical properties of the ECM components. Importantly, hyaluronan deposition is usually higher in malignant tumors as compared to benign tissues, predicting tumor progression in some tumor types. Furthermore, activated stromal cells are able to produce tissue structure rich in hyaluronan in order to promote tumor growth. Key biological roles of HA result from its interactions with its specific CD44 and RHAMM (receptor for HA-mediated motility) cell-surface receptors. HA-receptor downstream signaling pathways regulate in turn cellular processes implicated in tumorigenesis. Growth factors, including PDGF-BB, TGFβ2, and FGF-2, enhanced hyaluronan deposition to ECM and modulated HA-receptor expression in fibrosarcoma cells. Indeed, FGF-2 through upregulation of specific HAS isoforms and hyaluronan synthesis regulated secretion and net hyaluronan deposition to the fibrosarcoma pericellular matrix modulating these cells' migration capability. In this paper we discuss the involvement of hyaluronan/RHAMM/CD44 mediated signaling in the insidious pathways of fibrosarcoma progression.

OBJECTIVE

SSc is characterized by the overproduction of extracellular matrix (ECM) proteins, such as collagen and fibronectin, by activated fibroblasts, as well as oxidative stress. This study investigates the anti-fibrotic potential of the antioxidant epigallocatechin-3-gallate (EGCG) on activated dermal fibroblasts from SSc patients.

METHODS

Dermal fibroblasts from a cell line (AG), healthy individuals (CON) and SSc patients were treated with EGCG, TGF-β, PDGF-BB or other antioxidants [antioxidants superoxide dismutase (SOD), catalase, N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI)]. Collagen type I, fibronectin, connective tissue growth factor (CTGF), α-smooth muscle actin and mitogen-activated protein (MAP) kinases were measured by ELISA and western blot. Fibroblast contractile forces were measured by collagen gel contraction. Reactive oxygen species (ROS) were assessed by dichlorofluorescein assay and nuclear factor κ beta (NF-κB) activity by DNA binding assay.

RESULTS

EGCG (1-100 µM) dose-dependently decreased collagen type I secretion in culture medium after 24 h in AG fibroblasts. Collagen type I protein expression in cell lysates was also significantly reduced by 40% in EGCG-treated cells (40 µM). Furthermore, EGCG also down-regulated TGF-β-induced collagen type I, fibronectin and CTGF. Similarly, in CON fibroblasts EGCG decreased basal and stimulated collagen type I, fibronectin and CTGF after 24 h, while in SSc the effects of the antioxidant were apparent after 48 h. Fibroblast-mediated contraction of collagen gels was inhibited by EGCG as early as 1 h in AG fibroblasts, and in the CON and SSc fibroblasts. Additionally, EGCG also inhibited TGF-β-stimulated gel contraction similar to other antioxidants DPI and NAC, but not SOD or catalase. EGCG suppressed TGF-β-induced ROS production in all fibroblasts. Furthermore, EGCG inhibited TGF-β or PDGF-BB-induced phospho-extracellular signal-regulated kinase (ERK)1/2 MAP kinase and NF-κB activity in SSc fibroblasts.

CONCLUSIONS

The results suggest that the antioxidant, EGCG, can reduce ECM production, the fibrotic marker CTGF and inhibit contraction of dermal fibroblasts from SSc patients. Furthermore, EGCG was able to suppress intracellular ROS, ERK1/2 kinase signalling and NF-κB activity. Taken together, EGCG may be a possible candidate for therapeutic treatment aimed at reducing both oxidant stress and the fibrotic effects associated with SSc.

OBJECTIVE

Receptor tyrosine kinase (RTK) activation is critical for growth factor-mediated cell proliferation. The present study was designed to determine the effect of tyrphostin AG1295, a selective blocker of platelet-derived growth factor (PDGF) RTK, on proliferative vitreoretinopathy (PVR) development.

METHODS

Rabbit conjunctival fibroblasts cells (1 x 10(4)) were seeded into 96-well plates and maintained in Dulbecco's modified essentialmedium (DMEM) with 0.5% fetal bovine serum. The cells were exposed to 50 ng/mL PDGF-AAor PDGF-BBor phosphate-buffered saline with or without AG1295 (1 microM, 10 microM, and 100 microM). After 3 days, the viable cells in each well were measured by 3,(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Homologous rabbit conjunctival fibroblasts were injected intravitreally, followed by injection of 100 microM of AG1295. The development of tractional retinal detachment (TRD) was assessed to evaluate the effect of AG1295 in vivo. Electroretinography and histologic studies were performed after intravitreal injection of AG1295 into untreated eyes to evaluate toxicity.

RESULTS

Two concentrations of AG1295 (10 and 100 microM) significantly inhibited rabbit conjunctival fibroblast cell growth stimulated by PDGF-AA or PDGF-BB in vitro. Development of TRD was significantly attenuated (P <.01) with 100 microM of AG1295 until day 21. No significant histologic or retinal functional damage was found in the AG1295-treated group.

CONCLUSIONS

PDGF receptor specific inhibitor AG1295 attenuated PVR without significant side effects in rabbits. This reagent could be a useful treatment to prevent PVR.

We developed an effective regenerative therapy, referred to as platelet-derived growth factor-BB (PDGF-BB)-modulated guided tissue regenerative (GTR) therapy (P-GTR), capable of achieving periodontal regeneration of horizontal (Class III) furcation defects in the beagle dog. To determine its efficacy, repair and regeneration of horizontal furcation defects by P-GTR therapy and GTR therapy were compared. Chronically inflamed horizontal furcation defects were created around the second (P2) and fourth mandibular premolars (P4). After demineralization of the root surfaces with citric acid, the surfaces of left P2 and P4 were treated with PDGF-BB (P-GTR therapy) and those of contralateral teeth were treated with vehicle only (GTR therapy). Periodontal membranes were placed and retained 0.5 mm above the cemento-enamel junction for both groups. The mucoperiosteal flap was sutured in a coronal position and plaque control was achieved by daily irrigation with 2% chlorhexidine gluconate. At 5, 8, and 11 weeks, two animals each were sacrificed by perfusion with 2.5% glutaraldehyde through the carotid arteries, and the lesions were sliced mesio-distally, demineralized, dehydrated, and embedded. Periodontal healing and regeneration after GTR and P-GTR therapy were compared by histomorphometric as well as morphological analysis. Morphometric analysis for each time period was performed on the pooled samples of P2 and P4. Five weeks after both therapies, the lesions were filled primarily by tissue-free area, epithelium, inflamed tissue, and a small amount of newly formed fibrous connective tissue. At 8 and 11 weeks after P-GTR therapy, there was a statistically greater amount of bone and periodontal ligament formed in the lesions. The newly formed bone filled 80% of the lesion at 8 weeks and 87% at 11 weeks with P-GTR therapy, compared to 14% of the lesion at 8 weeks and 60% at 11 weeks with GTR therapy. Also, with P-GTR therapy there was less epithelium and tissue-free area, less inflamed tissue, and less connective tissue. Morphological analysis indicated that the defects around P2 revealed faster periodontal repair and regeneration than those around P4. While the lesions around P2 were effectively regenerated by 11 weeks even after GTR therapy, those around P4 failed to regenerate. On the other hand, P-GTR therapy further promoted periodontal repair and regeneration so that at 8 weeks the lesions around P2 and P4 demonstrated complete and nearly complete regeneration, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

OBJECTIVE

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is crucial for homeostasis of ocular extracellular matrices. To assess altered MMP activity as a determinant in the migration of human retinal pigment epithelial (RPE) cells, expression characteristics of several MMPs and TIMP-1 in RPE cell cultures were investigated.

METHODS

Expression studies were performed with RT-PCR, ELISA, and immunofluorescence analysis. Secretion of MMP-2 was demonstrated by zymography. Migration of cytokine-stimulated RPE cells was evaluated with microporous membranes of permeable chambers.

RESULTS

MMP-1, -2, -3, and -9; MT2-MMP; and TIMP-1 were expressed in cultured RPE cells. MMP-2 was detected on the cell surface and in secreted inactive and active forms. TGF-beta(2), IL-1beta, and TNF-alpha enhanced secretion of MMP-1, -2, and -3. TGF-beta(2) also stimulated MT2-MMP cell surface expression and release of TIMP-1. The mRNA levels of MMP-1, -2, and -3 and TIMP-1 were markedly increased by TNF-alpha and TGF-beta(2). MMP-2 mRNA levels were also upregulated by PDGF-BB. Migration of RPE cells stimulated by TGF-beta(2) or PDGF-BB was inhibited in presence of a synthetic MMP inhibitor.

CONCLUSIONS

Proinflammatory cytokines and TGF-beta(2) play an important role in the upregulation of expression of MMP-1, -2, and -3 in RPE cells and account for a directional shift in the balance between MMPs and TIMPs. Facilitation of RPE cell migration stimulated by cytokines (i.e., TGF-beta(2) or PDGF-BB) in ocular diseases may be due to increased release of MMPs, in the presence of comparatively lower levels of their inhibitors.

Tenascin C (TNC) is an extracellular matrix protein that is upregulated during development as well as tissue remodeling. TNC is comprised of multiple independent folding domains, including 15 fibronectin type III-like (TNCIII) domains. The fifth TNCIII domain (TNCIII5) has previously been shown to bind heparin. Our group has shown that the heparin-binding fibronectin type III domains of fibronectin (FNIII), specifically FNIII12-14, possess affinity towards a large number of growth factors. Here, we show that TNCIII5 binds growth factors promiscuously and with high affinity. We produced recombinant fragments of TNC representing the first five TNCIII repeats (TNCIII1-5), as well as subdomains, including TNCIII5, to study interactions with various growth factors. Multiple growth factors of the platelet-derived growth factor (PDGF) family, the fibroblast growth factor (FGF) family, the transforming growth factor beta (TGF-β) superfamily, the insulin-like growth factor binding proteins (IGF-BPs), and neurotrophins were found to bind with high affinity to this region of TNC, specifically to TNCIII5. Surface plasmon resonance was performed to analyze the kinetics of binding of TNCIII1-5 with TGF-β1, PDGF-BB, NT-3, and FGF-2. The promiscuous yet high affinity of TNC for a wide array of growth factors, mediated mainly by TNCIII5, may play a role in multiple physiological and pathological processes involving TNC.

In the present study, we analyzed the effect of conditioned media (CM) from bovine aortic endothelial cells exposed to laminar shear stress (SS) of 5 dyne/cm2 (SS5) or 15 dyne/cm2 (SS15) for 16 hours on smooth muscle cell (SMC) migration. In response to CM from bovine aortic endothelial cells exposed to SS5 (CMSS5) and SS15 (CMSS15), migration was 45 +/- 5.5 and 30 +/- 1.5 cells per field, respectively (P<0.05). Similar results were obtained with SS of 2 versus 20 dyne/cm2 and also when SS of 5 and 15 dyne/cm2 lasted 24 hours. Platelet-derived growth factor (PDGF)-AA levels in CMSS5 and CMSS15 were 9 +/- 7 and 18 +/- 5 ng/10(6) cells for 16 hours, respectively (P<0.05); PDGF-BB levels in CMSS5 and CMSS15 were 38 +/- 10 and 53 +/- 10 ng/10(6) cells for 16 hours, respectively (P<0.05). PDGF receptor alpha (PDGFRalpha) and PDGF receptor beta (PDGFRbeta) in SMCs were phosphorylated by CMSS15>CMSS5. In response to CMSS15, a neutralizing antibody against PDGF-AA enhanced SMC migration to a level comparable to that of CMSS5; in contrast, antibodies against PDGF-BB abolished SMC migration. Transfection of SMCs with a dominant-negative PDGFRalpha or PDGFRbeta increased or inhibited, respectively, SMC migration in response to CMSS15. Overexpression of wild-type PDGFRalpha inhibited SMC migration in response to CMSS5, CMSS15, or recombinant PDGF-BB (P<0.001). These results suggest that the ability of high SS to inhibit arterial wall thickening in vivo may be related to enhanced activation of PDGFRalpha in SMCs by PDGF isoforms secreted by the endothelium.

Platelet derived growth factor (PDGF) regulates gene transcription by binding to specific receptors. PDGF plays a critical role in oncogenesis in brain and other tumors, regulates angiogenesis, and remodels the stroma in physiologic conditions. Here, we show by using microRNA (miR) arrays that PDGFs regulate the expression and function of miRs in glioblastoma and ovarian cancer cells. The two PDGF ligands AA and BB affect expression of several miRs in ligand-specific manner; the most robust changes consisting of let-7d repression by PDGF-AA and miR-146b induction by PDGF-BB. Induction of miR-146b by PDGF-BB is modulated via MAPK-dependent induction of c-fos. We demonstrate that PDGF regulates expression of some of its known targets (e.g. cyclin D1) through miR alterations and identify the epidermal growth factor receptor (EGFR) as a new PDGF-BB target. We show that its expression and function are repressed by PDGF-induced miR-146b and that mir-146b and EGFR correlate inversely in human glioblastomas. We propose that PDGF-regulated gene transcription involves alterations in non-coding RNAs and provide evidence for a miR-dependent feedback mechanism balancing growth factor receptor signaling in cancer cells.

We investigated whether induction of transcription factor NF-kappa B is involved in the proliferation of cultured rat aortic smooth muscle cell using electrophoretic mobility shift assay and immunocytochemistry. NF-kappa B was induced in the nucleus in a dose-dependent manner when the smooth muscle cells were stimulated by various growth factors such as PDGF-BB, bFGF, EGF and IGF-1, but not growth inhibitors such as TGF-beta and IFN-gamma. Among growth factors, PDGF-BB and bFGF, more potent growth stimulators, induced higher kappa B binding activity than EGF or IGF-1. These evidences were also supported by the results obtained with immunocytochemistry. Immunocytochemistry also showed that the induced NF-kappa B contained p50 and p65. These results suggest that NF-kappa B induction may be involved in the proliferation of vascular smooth muscle cell.

OBJECTIVE

Ex vivo propagation of sparse populations of human mesenchymal stromal cells (hMSC) is critical for generating numbers sufficient for therapeutic applications. hMSC culture media have typically been supplemented with animal serum and, recently, human-sourced materials. However, these supplements are ill-defined and, thus, undesirable for clinical and research applications. Previously reported efforts to develop defined media for hMSC culture only resulted in slow or limited proliferation, and were unsuccessful in expanding these cells from primary cultures. Therefore a major step forward would be the identification of defined, serum-free culture conditions capable of supporting both the isolation and rapid expansion of hMSC.

METHODS

Using classical approaches of medium development, we were able to identify a set of growth and attachment factors that allowed the serum-free isolation and expansion of hMSC from bone marrow.

RESULTS

Heparin, selenium and platelet-derived growth factor (PDGF)-BB were found to be inhibitory for the growth of hMSC, whereas basic fibroblast growth factor (bFGF) was critical and worked synergistically with transforming growth factor (TGF)-beta1 to allow significant cell expansion. Ascorbic acid, hydrocortisone and fetuin were also found to be important growth and attachment factors that, in conjunction with substrate-coating proteins, allowed the isolation of hMSC from primary culture and their subsequent expansion.

CONCLUSIONS

We report a defined medium formulation (PPRF-msc6), consisting of key recombinant and serum-derived components, for the rapid isolation and expansion of hMSC in the absence of serum. This work represents an important step forward for achieving an ideal, completely defined synthetic medium composition for the safe use of hMSC in clinical settings.

OBJECTIVE

The tonicity-responsive transcription factor, nuclear factor of activated T cells 5 (NFAT5/tonicity enhancer binding protein [TonEBP]), has been well characterized in numerous cell types; however, NFAT5 function in vascular smooth muscle cells (SMCs) is unknown. Our main objective was to determine the role of NFAT5 regulation in SMCs.

RESULTS

We showed that NFAT5 is regulated by hypertonicity in SMCs and is upregulated in atherosclerosis and neointimal hyperplasia. RNAi knockdown of NFAT5 inhibited basal expression of several SMC differentiation marker genes, including smooth muscle α actin (SMαA). Bioinformatic analysis of SMαA revealed 7 putative NFAT5 binding sites in the first intron, and chromatin immunoprecipitation analysis showed NFAT5 enrichment of intronic DNA. Overexpression of NFAT5 increased SMαA promoter-intron activity, which requires an NFAT5 cis element at +1012, whereas dominant-negative NFAT5 decreased SMαA promoter-intron activity. Because it is unlikely that SMCs experience extreme changes in tonicity, we investigated other stimuli and uncovered 2 novel NFAT5-inducing factors: angiotensin II, a contractile agonist, and platelet-derived growth factor-BB (PDGF-BB), a potent mitogen in vascular injury. Angiotensin II stimulated NFAT5 translocation and activity, and NFAT5 knockdown inhibited an angiotensin II-mediated upregulation of SMαA mRNA. PDGF-BB increased NFAT5 protein, and loss of NFAT5 inhibited PDGF-BB-induced SMC migration.

CONCLUSIONS

We have identified NFAT5 as a novel regulator of SMC phenotypic modulation and have uncovered the role of NFAT5 in angiotensin II-induced SMαA expression and PDGF-BB-stimulated SMC migration.

Despite the advent of antiretroviral therapy, complications of HIV-1 infection with concurrent drug abuse are an emerging problem. Morphine, often abused by HIV-infected patients, is known to accelerate neuroinflammation associated with HIV-1 infection. Detailed molecular mechanisms of morphine action however, remain poorly understood. Platelet-derived growth factor (PDGF) has been implicated in a number of pathological conditions, primarily due to its potent mitogenic and permeability effects. Whether morphine exposure results in enhanced vascular permeability in brain endothelial cells, likely via induction of PDGF, remains to be established. In the present study, we demonstrated morphine-mediated induction of PDGF-BB in human brain microvascular endothelial cells, an effect that was abrogated by the opioid receptor antagonist-naltrexone. Pharmacological blockade (cell signaling) and loss-of-function (Egr-1) approaches demonstrated the role of mitogen-activated protein kinases (MAPKs), PI3K/Akt and the downstream transcription factor Egr-1 respectively, in morphine-mediated induction of PDGF-BB. Functional significance of increased PDGF-BB manifested as increased breach of the endothelial barrier as evidenced by decreased expression of the tight junction protein ZO-1 in an in vitro model system. Understanding the regulation of PDGF expression may provide insights into the development of potential therapeutic targets for intervention of morphine-mediated neuroinflammation.

Chemokine (C-C motif) ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1) is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND). The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS) via the disrupted blood-brain barrier (BBB). We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF)-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3K)/Akt pathways and the downstream transcription factor, nuclear factor κB (NFκB). Chromatin immunoprecipitation (ChIP) assays demonstrated increased binding of NFκB to the human MCP-1 promoter following PDGF-BB exposure. Conditioned media from PDGF-BB-treated astrocytes increased monocyte transmigration through human brain microvascular endothelial cells (HBMECs), an effect that was blocked by STI-571, a tyrosine kinase inhibitor (PDGF receptor (PDGF-R) blocker). PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays. Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte activation by PDGF-BB exaggerates monocyte recruitment into the brain via MCP-1 and underscores the critical role astrocytes play in HAND.

Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in targets cells. We recently reported that cultured cardiomyocytes are able to release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the transcriptional content of the released exosomes. Exosomes were isolated from media collected from cultured cardiomyocytes (HL-1) with or without growth factor treatment (TGF-β2 and PDGF-BB), with a series of differential centrifugations, including preparative ultracentrifugation and separation with a sucrose gradient. The exosomes were characterized with dynamic light scattering (DLS), electron microscopy (EM) and Western blot and analyzed with Illumina whole genome microarray gene expression. The exosomes were rounded in shape and had an average size of 50-90 nm in diameter with no difference between treatment groups. Analysis of the mRNA content in repeated experiments conclusively revealed 505 transcripts in the control group, 562 in the TGF-β2-treated group and 300 in the PDGF-BB-treated group. Common transcripts (217) were found in all 3 groups. We show that the mode of stimulation of parental cells affects the characteristics of exosomes released. Hence, there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated, or not stimulated, with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system.