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While flubendazole has been used as a macrofilaricide in humans and animals for some 40 years, work in vitro and in preclinical models over the last decade has suggested its potential use as an anticancer agent. This article reviews recent studies in a range of tumor types indicating novel functions for flubendazole in its control of processes associated with tumor growth, spread and renewal including ferroptosis, autophagy, cancer stem-like cell killing and suppression of intratumoral myeloid-derived suppressor cell accumulation and programmed cell death protein 1. Flubendazole’s potential use in clinical oncology will require further understanding of its mechanistic roles, range of inhibition of cancer types, capacity for adjunctive therapy and possible reformulation for enhanced solubility, bioavailability and potency.

The incidence of melanoma is increasing faster than any other cancer. In recent years, treatment of melanoma and a range of other deadly cancers has involved immunotherapy with programmed cell death protein-1 (PD-1)/PD-1 ligand (PD-L1) checkpoint blockade which has improved survival. However, many patients do not respond or have partial response, survival benefit is in the order of months and all available PD-1/PD-L1 strategies are antibodies requiring intravenous infusion. There are no clinically approved small molecule pharmacologic inhibitors of the PD-1/PD-L1 system. The benzimidazole derivative flubendazole is a widely used anthelmintic available over the counter in Europe. Here we demonstrate the ability of flubendazole to inhibit human melanoma growth and spread in mice. Flubendazole's ability to block tumor growth and spread was comparable to paclitaxel. Anti-tumor effects were observed when flubendazole was delivered systemically not locally. Flubendazole inhibited CD31/PECAM-1 staining indicating suppression of tumor angiogenesis. Most surprisingly, flubendazole inhibited PD-1 levels within the tumors, but not PD-L1. Western blotting and flow cytometry revealed that flubendazole inhibits PD-1 expression in cultured melanoma cells. Flubendazole also reduced myeloid-derived suppressor cell (MDSC) levels in tumor tissue. Further we found that flubendazole inhibited active (phospho-Tyr705) signal transducer and activator of transcription (STAT3), an upstream regulator of PD-1 expression. These findings uncover that flubendazole is a novel small molecule inhibitor of not only melanoma growth and spread but also of PD-1 and MDSC.

Egr-1, an immediate-early gene product and master regulator was originally described as a phosphoprotein following its discovery in the 1980s. However specific residue(s) phosphorylated in Egr-1 remain elusive. Here we phosphorylated recombinant Egr-1 in vitro with ERK1 prior to mass spectrometry, which identified phosphorylation of Ser12 and Ser26 with the latter ∼12 times more abundant than Ser12. Phosphorylation of wild-type recombinant Egr-1 (as compared with Ser26>Ala26 mutant Egr-1) revealed that Ser26 accounts for the majority of phosphorylation of Egr-1 by ERK1. N-FGSFPH(pS)PTMDNYC-C was used as an antigen to generate mouse monoclonal antibodies (pS26 MAb). pS26 MAb recognised ERK1-phosphorylated Egr-1 but not Egr-1 bearing a point mutation at Ser26. pS26 MAb recognised inducible ∼75 kDa and 100 kDa species in nuclear extracts of cells exposed to FGF-2. Peptide blocking revealed both inducible species were phosphosite-specific. Immunoprecipitation of nuclear extracts of cells exposed to FGF-2 with pS26 MAb followed by SDS-PAGE and mass spectrometry identified Egr-1 sequences corresponding to the ∼75 kDa species but not ∼100 kDa species. This study identifies a specific amino acid phosphorylated in endogenous Egr-1.

A thermostable drug suppresses a pERK-FosB/ΔFosB-VCAM-1 axis, endothelial activation, angiogenesis, and retinal permeability., Vascular permeability and angiogenesis underpin neovascular age-related macular degeneration and diabetic retinopathy. While anti-VEGF therapies are widely used clinically, many patients do not respond optimally, or at all, and small-molecule therapies are lacking. Here, we identified a dibenzoxazepinone BT2 that inhibits endothelial cell proliferation, migration, wound repair in vitro, network formation, and angiogenesis in mice bearing Matrigel plugs. BT2 interacts with MEK1 and inhibits ERK phosphorylation and the expression of FosB/ΔFosB, VCAM-1, and many genes involved in proliferation, migration, angiogenesis, and inflammation. BT2 reduced retinal vascular leakage following rat choroidal laser trauma and rabbit intravitreal VEGF-A165 administration. BT2 suppressed retinal CD31, pERK, VCAM-1, and VEGF-A165 expression. BT2 reduced retinal leakage in rats at least as effectively as aflibercept, a first-line therapy for nAMD/DR. BT2 withstands boiling or autoclaving and several months’ storage at 22°C. BT2 is a new small-molecule inhibitor of vascular permeability and angiogenesis.

Smooth muscle cells (SMC) in blood vessels are normally growth quiescent and transcriptionally inactive. Our objective was to understand promoter usage and dynamics in SMC acutely exposed to a prototypic growth factor or pro-inflammatory cytokine. Using cap analysis gene expression (FANTOM5 project) we report differences in promoter dynamics for immediate-early genes (IEG) and other genes when SMC are exposed to fibroblast growth factor-2 or interleukin-1β. Of the 1871 promoters responding to FGF2 or IL-1β considerably more responded to FGF2 (68.4%) than IL-1β (18.5%) and 13.2% responded to both. Expression clustering reveals sets of genes induced, repressed or unchanged. Among IEG responding rapidly to FGF2 or IL-1β were FOS, FOSB and EGR-1, which mediates human SMC migration. Motif activity response analysis (MARA) indicates most transcription factor binding motifs in response to FGF2 were associated with a sharp induction at 1 h, whereas in response to IL-1β, most motifs were associated with a biphasic change peaking generally later. MARA revealed motifs for FOS_FOS{B,L1}_JUN{B,D} and EGR-1..3 in the cluster peaking 1 h after FGF2 exposure whereas these motifs were in clusters peaking 1 h or later in response to IL-1β. Our findings interrogating CAGE data demonstrate important differences in promoter usage and dynamics in SMC exposed to FGF2 or IL-1β.

Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9–1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development., Histones, proteins that bind DNA, are toxic for pathogens outside cells but can also cause multi-organ damage as seen in sepsis. Here the authors develop small negatively charged molecules that can be used as histone antidotes, and show that they improve the phenotype in mouse models with histone-related pathologies.

The coronary collateral circulation is a rich anastomotic network of primitive vessels which have the ability to augment in size and function through the process of arteriogenesis. In this review, we evaluate the current understandings of the molecular and cellular mechanisms by which this process occurs, specifically focussing on elevated fluid shear stress (FSS), inflammation, the redox state and gene expression along with the integrative, parallel and simultaneous process by which this occurs. The initiating step of arteriogenesis occurs following occlusion of an epicardial coronary artery, with an increase in FSS detected by mechanoreceptors within the endothelium. This must occur within a 'redox window' where an equilibrium of oxidative and reductive factors are present. These factors initially result in an inflammatory milieu, mediated by neutrophils as well as lymphocytes, with resultant activation of a number of downstream molecular pathways resulting in increased expression of proteins involved in monocyte attraction and adherence; namely vascular cell adhesion molecule 1 (VCAM-1), monocyte chemoattractant protein 1 (MCP-1) and transforming growth factor beta (TGF-β). Once monocytes and other inflammatory cells adhere to the endothelium they enter the extracellular matrix and differentiate into macrophages in an effort to create a favourable environment for vessel growth and development. Activated macrophages secrete inflammatory cytokines such as tumour necrosis factor-α (TNF-α), growth factors such as fibroblast growth factor-2 (FGF-2) and matrix metalloproteinases. Finally, vascular smooth muscle cells proliferate and switch to a contractile phenotype, resulting in an increased diameter and functionality of the collateral vessel, thereby allowing improved perfusion of the distal myocardium subtended by the occluded vessel. This simultaneously reduces FSS within the collateral vessel, inhibiting further vessel growth.

Autophagy is a cellular process by which mammalian cells degrade and assist in recycling damaged organelles and proteins. This study aimed to ascertain the role of autophagy in remote ischemic preconditioning (RIPC)-induced cardioprotection. Sprague Dawley rats were subjected to RIPC at the hindlimb followed by a 30-min transient blockade of the left coronary artery to simulate ischemia reperfusion (I/R) injury. Hindlimb muscle and the heart were excised 24 h post reperfusion. RIPC prior to I/R upregulated autophagy in the rat heart at 24 h post reperfusion. In vitro, autophagy inhibition or stimulation prior to RIPC, respectively, either ameliorated or stimulated the cardioprotective effect, measured as improved cell viability to mimic the preconditioning effect. Recombinant interleukin-6 (IL-6) treatment prior to I/R increased in vitro autophagy in a dose-dependent manner, activating the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway without affecting the other kinase pathways, such as p38 mitogen-activated protein kinases (MAPK), and glycogen synthase kinase 3 Beta (GSK-3&beta, ) pathways. Prior to I/R, in vitro inhibition of the JAK-STAT pathway reduced autophagy upregulation despite recombinant IL-6 pre-treatment. Autophagy is an essential component of RIPC-induced cardioprotection that may upregulate autophagy through an IL-6/JAK-STAT-dependent mechanism, thus identifying a potentially new therapeutic option for the treatment of ischemic heart disease.

RNA-cleaving deoxyribozymes (DNAzymes) are synthetic single-stranded DNA-based catalytic molecules that can be engineered to bind to and cleave target mRNA at predetermined sites. These have been used as therapeutic agents in a range of preclinical cancer models and have entered clinical trials in Europe, China, and Australia. This review surveys regulatory insights into mechanisms of disease brought about by use of catalytic DNA in vitro and in vivo, including recent uses as nanosensors, nanoflowers, and nanosponges, and the emerging role of adaptive immunity underlying DNAzyme inhibition of cancer growth. DNAzymes represent a promising new class of nucleic acid–based therapeutics in cancer. This article discusses mechanistic and therapeutic insights brought about by DNAzyme use as nanotools and reagents in a range of basic science, experimental therapeutic and clinical applications. Current limitations and future perspectives are also discussed.

Percutaneous coronary intervention is widely used for the treatment of coronary artery disease; however, significant challenges such as restenosis remain. Key to solving these problems is to inhibit smooth muscle cell activation while enhancing re-endothelialization. Early growth response-1 (Egr-1) is a transcription factor that regulates vascular smooth muscle cell (SMC) proliferation and migration through its control of an array of downstream genes.A "cocktail" of vascular endothelial growth factor (VEGF)-A, VEGF-D and cyclic RGD was tested for its ability to inhibit neointima formation and accelerate re-endothelialization following balloon injury to carotid arteries of rats.In vitro, the cocktail stimulated endothelial cell growth yet inhibited smooth muscle cell growth. In vivo, cocktail-treated injured arteries exhibited reduced intimal thickening by50% (P0.05). It increased both re-endothelialization and endothelial nitric oxide synthase (NOS) expression. Cocktail reduced Egr-1 expression, an effect blocked by the NOS inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) that also prevented cocktail inhibition of neointima inhibition.This combination may potentially be useful for the treatment of restenosis with concomitant stimulation of revascularization.

The signaling pathways that regulate the synthesis and structure of proteoglycans secreted by vascular smooth muscle cells are potential therapeutic targets for preventing lipid deposition in the early stage of atherosclerosis. PDGF stimulates both core protein expression and elongation of glycosaminoglycan (GAG) chains on proteoglycans. In this study we investigated the effects of the tyrosine kinase inhibitor genistein on PDGF mediated receptor phosphorylation and proteoglycan synthesis in human vascular smooth muscle cells. We demonstrate that genistein does not block phosphorylation of the activation site of the PDGF receptor at Tyr(857) and two other downstream sites Tyr(751) and Tyr(1021). Genistein blocked PDGF-mediated proteoglycan core protein synthesis however it had no effect on GAG chain elongation. These results differ markedly to two other tyrosine kinase inhibitors, imatinib and Ki11502, that block PDGF receptor phosphorylation and PDGF mediated GAG elongation. We conclude that the action of genistein on core protein synthesis does not involve the PDGF receptor and that PDGF mediates GAG elongation via the PDGF receptor.

Wound healing is a complex dynamic process involving a variety of cell types, including fibroblasts that express and respond to cytokines and growth factors in the local microenvironment. The mechanisms controlling gene expression after injury at a transcriptional level are poorly understood. Here we show that decreased expression of a key receptor, PDGF-receptor (R)-α, after fibroblast injury is due to the release and paracrine activity of TNF-α. TNF-α inhibits PDGF-R-α expression and this involves formation of a c-Fos-Yin Yang 1 (YY1) complex and histone deacetylase (HDAC) activity. c-Fos, induced by TNF-α, negatively regulates PDGF-R-α transcription. Small interfering RNA (siRNA) targeting c-Fos or the zinc finger transcription factor YY1 inhibits TNF-α suppression of PDGF-R-α expression. Coimmunoprecipitation studies show that TNF-α stimulates the formation of a complex between c-Fos with YY1. Furthermore, chromatin immunoprecipitation (ChIP) analysis reveals the enrichment of c-Fos, YY1, and HDAC-1 at the PDGF-R-α promoter in cells exposed to TNF-α. With suberoylanilide hydroxamic acid (SAHA) and HDAC-1 siRNA, we demonstrate that HDAC mediates TNF-α repression of PDGF-R-α. These findings demonstrate that transcriptional repression of PDGF-R-α after fibroblast injury involves paracrine activity of endogenous TNF-α, the formation of a c-Fos-YY1 complex, and negative regulatory activity by HDAC.

Probucol inhibits the proliferation of vascular smooth muscle cells in vitro and in vivo, and the drug reduces intimal hyperplasia and atherosclerosis in animals via induction of heme oxygenase-1 (HO-1). Because the succinyl ester of probucol, succinobucol, recently failed as an antiatherogenic drug in humans, we investigated its effects on smooth muscle cell proliferation. Succinobucol and probucol induced HO-1 and decreased cell proliferation in rat aortic smooth muscle cells. However, whereas inhibition of HO-1 reversed the antiproliferative effects of probucol, this was not observed with succinobucol. Instead, succinobucol but not probucol induced caspase activity and apoptosis, and it increased mitochondrial oxidation of hydroethidine to ethidium, suggestive of the participation of H(2)O(2) and cytochrome c. Also, succinobucol but not probucol converted cytochrome c into a peroxidase in the presence of H(2)O(2), and succinobucol-induced apoptosis was decreased in cells that lacked cytochrome c or a functional mitochondrial complex II. In addition, succinobucol increased apoptosis of vascular smooth muscle cells in vivo after balloon angioplasty-mediated vascular injury. Our results suggest that succinobucol induces apoptosis via a pathway involving mitochondrial complex II, H(2)O(2), and cytochrome c. These unexpected results are discussed in light of the failure of succinobucol as an antiatherogenic drug in humans.

A three amino acid sequence, Ser/Thr-Pro-Ser/Thr, was recently identified and characterized as a novel nuclear localization signal (Chuderland et al., 2008). The immediate-early gene product, early growth response-1 is a three zinc finger containing transcription factor implicated in a wide variety of pathologies, and has a bipartite nuclear localization domain identified two decades ago. Efficient nuclear localization of Egr-1 is vital to its function as a transcription factor. Interestingly, Egr-1 also contains a C-terminal SPS domain (residues 482-484 in murine Egr-1). We hypothesized that (482)SPS(484) may also serve as a novel nuclear localization signal in Egr-1. We found that this sequence directs Egr-1 to the nucleus in transfected Chinese hamster ovary cells and show by co-immunoprecipitation analysis that Egr-1 forms a complex with importin-7. (482)SPS(484) is required for Egr-1's interaction with importin-7. Moreover, importin-7 knockdown with RNAi showed that Egr-1 nuclear translocation is importin-7-dependent. This study demonstrates that the nuclear translocation of Egr-1 is partially dependent on (482)SPS(484) and involves importin-7, and sheds light on the molecular mechanisms regulating the cellular localization of this pathophysiologically important transcription factor.

The initiation of atherosclerosis involves the subendothelial retention of lipoproteins by proteoglycans (PGs). Structural characteristics of glycosaminoglycan (GAG) chains on PGs influence lipoprotein binding and are altered adversely by platelet-derived growth factor (PDGF). The signaling pathway for PDGF-mediated GAG elongation via the PDGF receptor (PDGFR) was investigated. In human vascular smooth muscle cells, PDGF significantly increased 35S-sulfate incorporation into PGs and GAG chain size. PGs from PDGF-stimulated cells showed increased binding low-density lipoprotein (P < 0.001) in gel mobility shift assays. Knockdown of PDGFRβ using small interfering RNA demonstrated that PDGF mediated changes in PGs via PDGFRβ. GAG synthesis and hyperelongation was blocked by inhibition of receptor tyrosine kinase autophosphorylation site Tyr857 activity using Ki11502 or imatinib. Downstream signaling to GAG hyperelongation was mediated through ERK MAPK and not phosphatidylinositol-3 kinase or phospholipase Cγ. In high-fat-fed apolipoprotein E−/− mice, inhibition of PDGFRβ activity by imatinib reduced aortic total lipid staining area by 35% (P < 0.05). Inhibition of PDGFRβ tyrosine kinase activity leads to inhibition of GAG synthesis on vascular PGs and aortic lipid area in vivo. PDGFRβ and its signaling pathways are potential targets for novel therapeutic agents to prevent the earliest stages atherosclerosis.

Background Platelet-derived growth factor is involved in the regulation of hematopoiesis. Imatinib mesylate, a platelet-derived growth factor receptor inhibitor, induces thrombocytopenia in a significant proportion of patients with chronic myeloid leukemia. Although our previous studies showed that platelet-derived growth factor enhances megakaryocytopoiesis in vitro, the in vivo effect of platelet-derived growth factor in a model of radiation-induced thrombocytopenia has not been reported. Design and Methods In this study, we investigated the effect of platelet-derived growth factor on hematopoietic stem/progenitor cells and platelet production using an irradiated-mouse model. We also explored the potential molecular mechanisms of platelet-derived growth factor on thrombopoiesis in M-07e cells. Results Platelet-derived growth factor, like thrombopoietin, significantly promoted the recovery of platelets and the formation of bone marrow colony-forming unit-megakaryocyte in irradiated mice. Histology confirmed the protective effect of platelet-derived growth factor, as shown by an increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis. In a megakaryocytic apoptotic model, platelet-derived growth factor had a similar anti-apoptotic effect as thrombopoietin on megakaryocytes. We also demonstrated that platelet-derived growth factor activated the PI3-k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Conclusions Our findings show that platelet-derived growth factor enhances platelet recovery in mice with radiation-induced thrombocytopenia. This radioprotective effect is likely to be mediated via platelet-derived growth factor receptors with subsequent activation of the PI3-k/Akt pathway. We also provide a possible explanation that blockage of platelet-derived growth factor receptors may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia. © 2010 Ferrata Storti Foundation., link_to_OA_fulltext

Platelet-derived growth factors are a family of potent mitogens and chemoattractants for fibroblasts and other cells of mesenchymal origin. Platelet-derived growth factor (PDGF) dimeric ligands (composed of A-, B-, C-, and D-chains) exert their biological activity through high affinity interactions with cell surface receptor subunits (α and β). PDGF-receptor-α is widely implicated in the pathogenesis of hyperplastic fibrotic disease, yet the molecular mechanisms controlling its expression in response to injury are poorly understood. Here we show that PDGF-Rα expression is induced in fibroblasts by mechanical injury and interleukin (IL)-1β, which was abolished by neutralizing IL-1β antibodies in the culture supernatant or inhibitors of NF-κB. Chromatin immunoprecipitation and electrophoretic mobility shift assays revealed the existence of a new NF-κB binding site at −531/−521 bp in the PDGF-Rα promoter. We have recently shown that ATF-4 is also induced by injury (Malabanan, K. P., Kanellakis, P., Bobik, A., and Khachigian, L. M. (2008) Circ. Res. 103, 378–387), and we demonstrate here that ATF-4 binds a novel element −259/−254 and stimulates PDGF-Rα transcription. ATF-4 and NF-κB interact, occupy the PDGF-Rα promoter, and induce PDGF-Rα transcription in a cooperative manner. IL-1β facilitates the dissociation of histone deacetylase (HDAC)-1/2 from the PDGF-Rα promoter, whereas the HDAC inhibitors suberoylanilide hydroxamic acid and trichostatin A potentiate IL-1β induction of PDGF-Rα transcription. These findings, taken together, demonstrate that injury stimulates IL-1β secretion by fibroblasts, which activates NF-κB and ATF-4 and stimulates interaction with the PDGF-Rα promoter, triggering PDGF-Rα transcription. Physical and functional interactions between NF-κB and ATF-4 have not been reported in any gene. This is also the first report of HDAC regulation of PDGF-Rα transcription.

The oncogene c-Jun has been found to be up-regulated in a variety of cancers including osteosarcoma. DNA enzymes (DNAzymes) are oligonucleotides capable of specific catalysis of target mRNA. A c-Jun DNAzyme inhibited the growth and metastasis of osteosarcoma in an orthotopic spontaneously metastasizing model of the disease. c-Jun down-regulation–mediated apoptosis in osteosarcoma cells involved caspase-1, caspase-2, and caspase-8, but not the Fas/FasL pathway. Clinically, knockdown of c-Jun with DNAzymes may proffer an improved treatment outcome for these tumors originating in bone. (Mol Cancer Res 2008;6(8):1289–92)

The oncogene c-Jun has been found to be up-regulated in a variety of cancers, including osteosarcoma. Doxorubicin is a frontline chemotherapeutic against osteosarcoma, but is limited by toxicity. DNAzymes are oligonucleotides capable of specific catalysis of target mRNA. A biocompatible c-Jun DNAzyme nanoparticle formulated from chitosan regressed the growth and metastasis of pre-established tumors, especially in combination with doxorubicin. In vitro data confirmed that c-Jun knockdown chemosensitized these cells to doxorubicin treatment. c-Jun down-regulation–mediated tumor inhibition also led to concomitant decreased osteolysis. Clinically, knockdown of c-Jun with chitosan nanobiotechnology may proffer an improved treatment outcome for osteosarcoma. [Mol Cancer Ther 2008;7(7):1909–12]

DNAzymes are oligonucleotides capable of specific catalysis of target mRNA. To date, a delivery vehicle for DNAzymes has not been developed. Chitosan is a biomaterial obtained abundantly in nature. A biocompatible c-jun (an oncogene) DNAzyme nanoparticle formulated from chitosan was found to be more active against osteosarcoma (bone cancer) cells, inducing apoptotic cell death in these cells. The formulation was stable in serum for a week and at room temperature for a month. Clinically, knockdown of c-jun gene expression with chitosan nanobiotechnology may improve treatment outcome for tumours growing in bone.

Restenosis after balloon angioplasty and stenting is exacerbated by low flow. Flow-dependent restenosis after angioplasty but not stenting is prevented by the antioxidant pyrrolidine dithiocarbamate (PDTC). c-jun may play a role in these events as AP-1 activity is both flow and redox sensitive. Carotid arteries of cholesterol fed rabbits underwent stenting or balloon injury in the presence of low or normal flow. c-jun mRNA expression was enhanced by low flow and injury (stentballoon) and inhibited by the antioxidant PDTC irrespective of the injury type. The effect of locally delivered DZ13 (a DNAzyme specific for c-jun) or scrambled DZ13 (inactive DNAzyme) was assessed by histomorphometry at 28 days. Low flow significantly increased intimal hyperplasia in B and S relative to normal flow (P0.05). The active DNAzyme DZ13 markedly reduced intimal hyperplasia (P0.001) and increased lumen size (P0.05) in balloon-injured but not in stented segments, and abrogated the effect of low flow on restenosis after angioplasty, similar to the morphological effects of PDTC. We conclude that c-jun expression is enhanced by low flow and by injury (stentballoon) and markedly attenuated by PDTC, and that c-jun is an important mediator of flow-dependent restenosis in balloon-injured but not stented vessels.

Conventional anti-inflammatory strategies induce multiple side effects, highlighting the need for novel targeted therapies. Here we show that knockdown of the basic-region leucine zipper protein, c-Jun, by a catalytic DNA molecule, Dz13, suppresses vascular permeability and transendothelial emigration of leukocytes in murine models of vascular permeability, inflammation, acute inflammation and rheumatoid arthritis. Treatment with Dz13 reduced vascular permeability due to cutaneous anaphylactic challenge or VEGF administration in mice. Dz13 also abrogated monocyte-endothelial cell adhesion in vitro and abolished leukocyte rolling, adhesion and extravasation in a rat model of inflammation. Dz13 suppressed neutrophil infiltration in the lungs of mice challenged with endotoxin, a model of acute inflammation. Finally, Dz13 reduced joint swelling, inflammatory cell infiltration and bone erosion in a mouse model of rheumatoid arthritis. Mechanistic studies showed that Dz13 blocks cytokine-inducible endothelial c-Jun, E-selectin, ICAM-1, VCAM-1 and VE-cadherin expression but has no effect on JAM-1, PECAM-1, p-JNK-1 or c-Fos. These findings implicate c-Jun as a useful target for anti-inflammatory therapies.

The sulfated oligosaccharide PI-88 is a potent antiangiogenic, antitumor and anti-metastatic agent derived from yeast. It is primarily composed of sulfated phosphomannopentaose and phosphomannotetraose oligosaccharide units and is presently under evaluation in Phase II clinical trials for anticancer efficacy. PI-88 inhibits the heparan sulfate-degrading enzyme heparanase, exhibits antiangiogenic activity and has anticoagulant properties mediated by heparin cofactor II. It also inhibits vascular smooth muscle cell proliferation, kinase signalling and arterial intimal thickening following balloon injury. Many heparan sulfate-binding growth factors require heparan sulfate as a co-receptor in order to effectively deliver growth signals to cells. Thus, the antiangiogenic and antirestenotic activity of PI-88 may be at least partially due to this highly sulfated oligosaccharide competing with the interaction of growth factors, such as FGF-2 and VEGF, with cell surface heparan sulfate. This heparan sulfate mimetic has, therefore, multiple functions and therapeutic potential in a variety of vascular disorders.

This brief review discusses recent strategies targeting the zinc finger transcription factor and immediate-early gene product Egr-1 with catalytic DNA in efforts to inhibit postangioplasty restenosis.

Low flow (LF) promotes late lumen loss after angioplasty by exacerbating inward remodelling through redox-sensitive mechanisms. Stents eliminate inward remodelling and the effect of LF on in-stent restenosis is uncertain. We performed over-sized (1.3–1.5:1) stenting (S) and balloon injury (in the same vessel, B) to the carotid arteries of cholesterol-fed rabbits and compared 28-day late lumen loss with that in an uninjured segment in the same vessel (U). Vessels ( n = 5 animals per group) were subjected to high (H), normal (N) and low (L) flow in animals fed either vehicle (V) or the antioxidant pyrrolidine dithiocarbamate, PDTC (P). LF significantly increased in-stent neointima formation relative to normal and high flow (SLV 0.72 ± 0.07mm 2 versus SNV 0.43 ± 0.08mm 2 versus SHV 0.28 ± 0.04mm 2 , P 2 and SNV 5.32 ± 0.40mm 2 versus BLV 1.28 ± 0.33mm 2 and BNV 2.19 ± 0.28mm 2 ), by greater enhancement of inward remodelling. In addition, inward remodelling and lumen loss due to LF were greater in balloon-injured segments than in adjacent uninjured segments where shear homeostatic remodelling occurs (lumen BLV 1.28 ± 0.33mm 2 versus ULV 1.52 ± 0.22mm 2 ). Lastly, while PDTC effectively reduced intima formation and inward remodelling due to LF in balloon-injured vessels there was no effect on flow-dependent neointima formation in stented vessels. We conclude that LF accentuates in-stent neointima formation, but that flow-dependent lumen loss after stenting is less than that after balloon injury. When LF is present lumen loss can be minimised by antioxidants or stenting.

The platelet-derived growth factor (PDGF) family of ligands (composed of A-, B-, C-, and D-chains), potent mitogens, and chemoattractants for cells of mesenchymal origin has been implicated in numerous vascular pathologies involving smooth muscle cell (SMC) hyperplasia. Understanding the molecular mechanisms mediating PDGF transcription would provide new insights into strategies to control PDGF-dependent pathophysiologic processes. We demonstrated previously that PDGF-A expression is under the positive regulatory influence of Sp1, Sp3, and Egr-1 and is negatively controlled by GCF2, NF-1(X), and WT-1. In this article, we demonstrate that Ets-1 induces PDGF-A expression in primary rat aortic SMCs at the level of transcription and mRNA expression. Electrophoretic mobility shift, supershift, and mutational analyses revealed a functional role for the −555 TTCC −552 motif in the PDGF-A promoter that binds endogenous Ets-1. Chromatin immunoprecipitation analysis showed the interaction of endogenous and exogenous Ets-1 or glutathione S -transferase-tagged Ets-1, bearing only the DNA-binding domain with the authentic PDGF-A promoter. Conversely, dominant-negative mutant of Ets-1 blocked the promoter interaction of endogenous Ets-1. Overexpression of Ets-1 but not the mutant form of Ets-1 activates the PDGF-A promoter cooperatively with Sp1. Sp1, which interacts with Ets-1, failed to induce PDGF-A promoter-dependent expression if the promoter contained a site-specific mutation in this novel Ets-binding site. Small interfering RNA to Ets-1 and Sp1 blocked PDGF-BB- and serum-inducible PDGF-A expression. SMC growth was stimulated by Ets-1 and Sp1 separately and further increased by both factors together. Ets-1-inducible mitogenesis is blocked by antibodies neutralizing PDGF-A and involves activation of the PDGF α-receptor, which binds PDGF-A. These findings identify a functional cis -acting element for Ets-1 in the PDGF-A promoter and demonstrate that Sp1 and Ets-1 cooperatively activate PDGF-A transcription in vascular SMCs.

Reactive oxygen species (ROS) generated in and around vascular endothelium may play a role in normal cellular signaling mechanisms but may also be an important causative factor in endothelial dysfunction underlying the development of atherosclerosis, diabetes complications, and ischemia-reperfusion injury. ROS influence a variety of molecular and cellular activities, including changes in the cellular localization of regulatory factors, protein modification, and altered gene expression, which in turn influence cellular phenotype. One mechanism by which ROS exert their cellular effects involves their ability to modulate the expression and function of vascular genes, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), which play key atherogenic roles by their regulation of cell growth, differentiation, and fibroproliferative responsiveness. In this review the authors describe the changes induced by oxidative stress on the profile of growth factor gene expression in endothelial cells, and the impact these modifications have on endothelial phenotype as well as on the behavior of neighboring vascular smooth muscle cells and fibroblasts. The authors also discuss the involvement of redox-sensitive transcription factors in these regulatory processes.

Current understanding of key transcription factors regulating angiogenesis is limited. Here we show that RNA-cleaving phosphodiester-linked DNA-based enzymes (DNAzymes), targeting a specific motif in the 5' untranslated region of early growth response (Egr-1) mRNA, inhibit Egr-1 protein expression, microvascular endothelial cell replication and migration, and microtubule network formation on basement membrane matrices. Egr-1 DNAzymes blocked angiogenesis in subcutaneous Matrigel plugs in mice, an observation that was independently confirmed by plug analysis in Egr-1-deficient animals, and inhibited MCF-7 human breast carcinoma growth in nude mice. Egr-1 DNAzymes suppressed tumor growth without influencing body weight, wound healing, blood coagulation or other hematological parameters. These agents inhibited endothelial expression of fibroblast growth factor (FGF)-2, a proangiogenic factor downstream of Egr-1, but not that of vascular endothelial growth factor (VEGF). Egr-1 DNAzymes also repressed neovascularization of rat cornea. Thus, microvascular endothelial cell growth, neovascularization, tumor angiogenesis and tumor growth are processes that are critically dependent on Egr-1.

The cyclin-dependent kinase inhibitor (CKI) p21WAF1/Cip1 is regulated at the level of transcription by nuclear factors such as the co-activator p300. It is presently unknown whether the Ets family of transcription factors control p21WAF1/Cip1 gene expression. Ets-1 inhibits apoptosis in vascular smooth muscle cells as determined by both fluorescein isothiocyanate-linked annexin V/propidium iodide staining of cells and fluorescence-activated cell sorting analysis and quantitative cytoplasmic histone-associated internucleosomal DNA fragmentation. p21WAF1/Cip1 can play a mitogenic and anti-apoptotic role in smooth muscle cells. Using transient transfection and Western blot analysis, we determined that Ets-1 activates p21WAF1/Cip1 transcription and protein expression. Electrophoretic mobility shift assays revealed that Ets-1 interacts selectively with the -1350GGAA-1347 Ets element in the p21WAF1/Cip1 promoter. Mutation of this element reduced basal and Ets1-inducible p21WAF1/Cip1 promoter-dependent expression. In contrast, the -1577GGAT-1574 motif mediates basal but not Ets-1 activation of the p21WAF1/Cip1 promoter. Co-immunoprecipitation and co-transfection analysis showed that Ets-1 binds p300 and cooperatively activates p21WAF1/Cip1 transcription. The phenotypic importance of Ets-1 regulation of p21WAF1/Cip1 was demonstrated by the capacity of antisense p21WAF1/Cip1 strategies to block Ets-1-inhibition of apoptosis and inhibit Ets-1-induction of proliferation.

The capacity of DNA to bind RNA via Watson-Crick base-pairing is fundamental to antisense oligonucleotide strategies to inhibit gene expression, and is a property that has been exploited by bioengineers in the generation of catalytic molecules such as ribozymes, ribozyme subtypes, and more recently DNAzymes. This review describes the evolution of these gene-specific agents and summarizes recent efforts to inhibit smooth muscle cell growth with these molecules as candidate therapeutic tools in restenosis.

Platelet-derived growth factor (PDGF) is a broadly expressed mitogenic and chemotactic factor with diverse roles in a number of physiologic and pathologic settings. The zinc finger transcription factors Sp1, Sp3 and Egr-1 bind to overlapping elements in the proximal PDGF B-chain promoter and activate transcription of this gene. The anthracycline nogalamycin has previously been reported to inhibit the capacity of Egr-1 to bind DNA in vitro. Here we used electrophoretic mobility shift assays to show that nogalamycin added to cells in culture did not alter the interaction of Egr-1 with the PDGF-B promoter. Instead, it enhanced the capacity of Sp1 to bind DNA. Nogalamycin increased PDGF-B mRNA expression at the level of transcription, which was abrogated by mutation of the Sp1 binding site in the PDGF-B promoter or overexpression of mutant Sp1. Rather than increasing total levels of Sp1, nogalamycin altered the phosphorylation state of the transcription factor. Overexpression of dominant-negative PKC-ζ blocked nogalamycin-inducible Sp1 phosphorylation and PDGF-B promoter-dependent expression. Nogalamycin stimulated the phosphorylation of PKC-ζ (on residue Thr410). These findings demonstrate for the first time that PKC-ζ and Sp1 phosphorylation mediate the inducible expression of this growth factor.

Thrombopoietin (TPO), the specific cytokine that regulates platelet production, is expressed in human bone marrow (BM), kidney, and liver. There appears to be no regulation of TPO in the kidney and liver, but TPO messenger RNA (mRNA) expression can be modulated in the stromal cells of the BM. In this study, we used primary human BM stromal cells as a model to study the regulation of TPO mRNA expression in response to various platelet -granular proteins. We showed that platelet-derived growth factor (PDGF) BB and fibroblast growth factor (FGF) 2 stimulated TPO mRNA expression in both a dose-dependent and time-dependent manner. The addition of 50 ng/mL of PDGF and 20 ng/mL of FGF resulted in maximal induction of TPO mRNA expression in 4 hours. We also found that platelet factor 4 (PF4), thrombospondin (TSP), and transforming growth factor-beta (TGF-β) are negative modulators of megakaryocytopoiesis. We observed suppression in TPO mRNA expression with 1 μg/mL of both PF4 and TSP and 50 ng/mL of TGF-β, with maximal suppression occurring 4 hours after the addition of these proteins. Finally, the addition of whole-platelet lysate produced a dose-dependent inhibition of TPO expression. On the basis of these findings, we propose that the platelet -granular proteins studied may regulate TPO gene expression in BM stromal cells by means of a feedback mechanism.

Angiotensin II (ATII) and platelet-derived growth factor (PDGF) are two vasoconstrictors implicated in the maintenance of normal vascular homeostasis. PDGF A-chain levels increase in cultured vascular smooth muscle cells (SMCs) exposed to ATII. The molecular mechanisms underlying this induction are not known. We used transient transfection analysis to show that ATII can increase reporter gene activity driven by fragments of the PDGF-A promoter bearing recognition elements for the transcription factor, Egr-1. Nuclear run-off experiments indicate that ATII induces Egr-1 expression at the level of transcription. Gel shift and supershift studies show that Egr-1 protein accumulates in the nuclei of SMCs exposed to ATII and binds to the proximal region of the PDGF-A promoter in a specific, time-dependent manner. ATII induced extracellular-signal regulated kinase (p42/44 ERK) activity as did phorbol 12-myristate 13-acetate. The specific MEK1/2 inhibitor, PD98059, suppressed both PDGF-A and Egr-1 endogenous and promoter-dependent expression inducible by ATII. The ATII type 1 receptor (AT1) antagonist, Losartan, inhibited ATII-induction of p42/44 ERK, as well as Egr-1 and PDGF-A, whereas neither PD123319, an AT2 receptor antagonist, nor wortmannin, an inhibitor of phosphatidylinositol 3-kinase and c-Jun N-terminal kinase, had any effect. ATII-induction of Egr-1 and PDGF-A was blocked by SIN-1, a NO donor. In addition, this pathway was blocked by overexpression of NO synthase. Collectively, these findings demonstrate that ATII activation of the PDGF-A promoter is mediated via the MEK/ERK/Egr-1 pathway and AT1 receptor and that this process is antagonized by NO.

SummaryPlatelet-derived growth factor (PDGF) is a potent chemotactic and mitogenic factor implicated to play important roles in a variety of normal and pathophysiologic settings. We investigated PDGF receptor expression on human megakaryocytes and several megakaryocytic cell lines (CHRF, DAMI, Meg-01, M-07e) using enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunocytochemical staining. Both PDGF receptor subtypes were identified on CHRF, DAMI, and Meg-01 cells by ELISA; PDGF beta-receptor levels exceeded alpha-receptor levels. Flow cytometry revealed that beta-receptor levels on CHRF and DAMI cells exceeded those on Meg-01 cells, and that M-07e expressed neither receptor. Immunocytochemical staining confirmed these findings and determined that bone marrow megakaryocytes also expressed PDGF receptors. Exposure of megakaryocytes to PDGF-BB dramatically induced the expression of the immediate-early gene, c-fos, within 30 min. Moreover, PDGF-BB significantly stimulated CHRF proliferation and colony formation. The present study demonstrates the presence of functional PDGF receptors on human megakaryocytes and their ability to mediate a mitogenic response.

Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory protein first identified in T lymphocytes. We recently observed that GILZ is highly expressed in synovial endothelial cells in rheumatoid arthritis. However, the function of GILZ in endothelial cells is unknown. To investigate the actions of GILZ in this cell type, we induced GILZ expression in HUVECs via transient transfection. GILZ overexpression significantly reduced the capacity of TNF-stimulated HUVECs to support leukocyte rolling, adhesion, and transmigration. These effects were associated with decreased expression of E-selectin, ICAM-1, CCL2, CXCL8, and IL-6. Experiments in a human microvascular endothelial cell line demonstrated that TNF-inducible NF-κB activity was significantly inhibited by overexpression of GILZ. Exogenous GILZ inhibited TNF-induced NF-κB p65 DNA binding, although this occurred in the absence of an effect on p65 nuclear translocation, indicating that the mechanism of action of exogenous GILZ in endothelial cells differs from that reported in other cell types. GILZ overexpression also inhibited TNF-induced activation of p38, ERK, and JNK MAPKs, as well as increased expression of the MAPK inhibitory phosphatase, MKP-1. In contrast, silencing endogenous GILZ in glucocorticoid-treated HUVECs did not alter their capacity to support leukocyte interactions. These data demonstrate that exogenous GILZ exerts inhibitory effects on endothelial cell adhesive function via a novel pathway involving modulation of NF-κB p65 DNA binding and MAPK activity. Induction of GILZ expression in endothelial cells may represent a novel therapeutic modality with the potential to inhibit inflammatory leukocyte recruitment.

Platelet-derived growth factor (PDGF) is a chemotactic and mitogenic agent for fibroblasts and smooth muscle cells and plays a key role in the development of atherosclerotic lesions. PDGF is produced by a number of normal and transformed cell types and occurs as homo- or heterodimers of A and B polypeptide chains. Using Chinese hamster ovary (CHO) cells transfected with various forms of PDGF, we have previously shown that PDGF As (short splice version) is secreted, PDGF AL (long splice version) predominantly extracellular matrix-associated, and PDGF B divided between medium, cells, and matrix. In the present study we have demonstrated the mitogenic activity of matrix-localized PDGF in artificial and more physiologically relevant models by culturing Balb/c-3T3 cells (3T3), human foreskin fibroblasts (HFF), and rabbit aortic smooth muscle cells (SMC) on extracellular matrix (ECM) laid down by PDGF-expressing CHO cells and human umbilical vein endothelial cells (HUVEC). These cells responded to the local growth stimulus of PDGF-containing CHO ECM and HUVEC ECM. We showed that 3T3 cells required proteolytic activity to utilize matrix-localized PDGF, as aprotinin and η-ACA inhibited growth and 3T3 cells were shown to possess plasminogen activator activity. HFF and SMC did not appear to require proteolytic activity (including metalloproteinase and serine protease activity) as a prerequisite for mitogenesis but were able to access immobilized PDGF by contact with the matrix. An understanding of the mechanisms whereby the utilization of stored PDGF is controlled in situations of excessive cellular proliferation will aid in the development of therapy for these conditions. © 1996 Wiley-Liss, Inc.

Platelet-derived growth factor (PDGF) is a pleotropic mitogen involved in several normal processes and pathological settings which include embryonal development, wound healing, atherosclerosis and neoplasia. We have reviewed the literature on the structure, function and regulation of PDGF and its receptors, and the roles played by PDGF in these settings.

Conditions resulting in retinal angiogenesis and edema (exudative age-related macular degeneration, diabetic retinopathy, retinal vein occlusion and retinopathy of prematurity) are major causes of visual impairment, with significant impact on quality of life. There has been increasing clinical usage of anti-vascular endothelial growth factor (anti-VEGF) agents to stop retinal angiogenesis and resolve intraretinal fluid arising from these conditions. However, anti-VEGFs have not been completely successful in curing these conditions, and a range of emerging treatments aimed at supplementing or competing with anti-VEGF agents are being developed. We will discuss the proposed merits these emerging agents bring to the treatment arsenal and how they compare with anti-VEGFs with regards to therapeutic activity, potency, specificity and safety. This review will also highlight recent pre-clinical research findings and suggest where future research might be directed.

The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.

Rationale : TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) is well reported as an inducer of apoptosis in tumor models; however, its role and function in vivo in atherosclerosis and vascular injury has not been established. Objective : We sought to study the function of TRAIL in cardiovascular pathology and its regulation in vivo. Methods and Results : Here, we show that TRAIL was upregulated in medial vascular smooth muscle cells (VSMCs) 24 hours following perivascular cuff placement around femoral arteries of mice. We also show that TRAIL mRNA and promoter activity was induced in VSMCs following in vitro mechanical injury. Intimal thickening 15 days after cuff placement was reduced 2- to 3-fold in TRAIL −/− compared to wild-type mice and was reversible by administration of recombinant TRAIL. Additionally, reduced VSMC proliferation was observed in injured arteries of TRAIL −/− mice. Fibroblast growth factor (FGF)-2, a potent growth factor released following vascular injury, was also reduced in arteries of TRAIL −/− mice, and VSMCs isolated from these animals did not respond to FGF-2 in vitro. Injury and FGF-2 regulated TRAIL transcriptional activity via 2 specificity protein (Sp)1 elements in the proximal TRAIL promoter, a binding site also shared by nuclear factor (NF)κB. Mutational studies confirmed a role for Sp1 in injury- and FGF-2–inducible TRAIL transcription. Furthermore, increased NFκB expression after injury transactivated the TRAIL promoter. Interestingly, following mechanical injury, Sp1 phosphorylation (Thr453) and an increase in the physical interaction of p-Sp1(Thr453) with NFκB was observed. Conclusions : We conclude that TRAIL induction involves FGF-2, Sp1-phosphorylation and NFκB and that TRAIL promotes VSMC proliferation and neointima formation after arterial injury.

Objective: Restenosis after both angioplasty and stenting is flow dependent. The effects of flow are preventable with the antioxidant pyrrolidine dithiocarbamate (PDTC) after angioplasty but not after stenting. We examined to what extent these observations could be explained by the effect of PDTC on NF-κB and Egr-1, two transcription factors which are both flow- and redox-sensitive. Methods: In a flow-modified rabbit carotid model of angioplasty and stenting, we assessed the effects of altered flow, injury and PDTC on expression of Egr-1 and nuclear binding activity of NF-κB. We also examined the effects of local delivery of decoy oligodeoxynucleotides (ODN) specific for NF-κB and Egr-1 on morphology at 28 days in normal and low flow. Results: The activity of both transcription factors was enhanced by injury (stent > balloon alone) and was further augmented by low flow. PDTC markedly attenuated the activity of NF-κB but not Egr-1. Specific decoy ODN for Egr-1 attenuated intima formation in both stented and balloon injured vessels in both normal and low flow but had no effect on remodelling. In contrast while NF-κB decoy ODN caused a modest but significant reduction in intima formation, there was a striking effect on remodelling in low flow vessels only. Conclusions: We conclude that Egr-1 plays a pivotal role in intima formation under all flow conditions and that NF-κB plays a key role in flow-sensitive remodelling after angioplasty and that NF-κB inhibition likely accounts for a significant part of the morphological effects of PDTC after vessel injury.

Alternative splicing results in two distinct forms of the platelet-derived growth factor (PDGF) A-chain that differ by a hydrophilic carboxyl terminus consisting of 18 amino acids (A194-211). The functional significance of this region is unclear. Previous results indicate that a radioiodinated tyrosinated synthetic peptide corresponding to A194-211 binds specifically, saturably, and with low affinity to a large population of sites on Balb/c 3T3 and several other cell lines (Khachigian, L. M., Owensby, D. A., and Chesterman, C. N. (1992) J. Biol. Chem. 267, 1660-1666). In this paper, we report that (Y)A194-211 and A194-211 can modulate the cellular proliferative response to normal human serum and several individual polypeptide growth factors as a consequence. When these peptides were coincubated separately with whole serum, PDGF, epidermal growth factor, or fibroblast growth factor, DNA synthesis was inhibited in a dose-dependent fashion and maximally by 200 microM peptide. In addition, both peptides could attenuate the stimulation of cell division by serum and PDGF. Peptides with similar charge or length failed to modify the level of proliferation. These observations were not due to cytotoxicity. Synthetic peptides such as (Y)A194-211 and A194-211 that influence cellular proliferation may be useful in modulating the cellular response to selected growth factors.

Platelet-derived growth factor (PDGF) is a potent mitogen for cells of mesenchymal origin. Alternative exon splicing is responsible for two forms of the PDGF A-chain which differ at the carboxyl terminus by a highly basic region consisting of 18 amino acids. To clarify the function of the region, we synthesized an octadecapeptide corresponding to this extension (A194-211), incorporated a tyrosine residue at the amino terminus, and used the radioiodinated construct in binding studies with Balb/c3T3 cells and a variety of human cell lines. 125I-(Y)A194-211 bound specifically, reversibly, saturably, and with low affinity to a large population of binding sites on these cells. In addition, (Y)A194-211 markedly reduced the binding of its parent protein, 125I-PDGF-AAL, to its receptor. (Y)A194-211 also attenuated the binding of epidermal growth factor and several other isoforms of PDGF, but did not interfere with the binding of transferrin to its receptor. These observations were not due to competitive binding of peptide directly to known receptors for the respective growth factors, but was likely due to interaction of (Y)A194-211 with extracellular glycosaminoglycan. Thus, A194-211 may represent an additional heparin binding domain on mature PDGF-AAL, and as an isolate, is capable of modulating interactions between several potent growth factors and their respective receptors.

Activation transcription factor (ATF)-4 is a member of the ATF/CREB family of basic leucine zipper transcription factors that regulates cellular responses to a variety of stresses. The role of ATF-4 in smooth muscle cells of the vessel wall is completely unknown. Here, we show that ATF-4 expression is induced in smooth muscle cells in response to injury, both in vitro using a model of mechanical injury and in the media of balloon-injured rat carotid arteries. We demonstrate that ATF-4 is activated by fibroblast growth factor (FGF)-2, an injury-induced mitogen, through the phosphatidylinositol 3-kinase pathway. Injury also activates vascular endothelial growth factor (VEGF)-A, whose expression is stimulated by ATF-4 overexpression and exposure to FGF-2. FGF-2 induces ATF-4 binding to a recognition element located in the VEGF-A gene at +1767 bp and luciferase reporter gene expression dependent on this site. Moreover, ATF-4 knockdown with small interfering RNA or ATF-4 deficiency ameliorates FGF-2–inducible VEGF-A expression. Intraluminal delivery of ATF-4 small interfering RNA in rat carotid arteries blocks balloon injury–inducible ATF-4 and VEGF-A expression after 4 hours and intimal thickening after 14 days. These findings reveal, for the first time, the induction of ATF-4 by both vascular injury and FGF-2. ATF-4 serves as a conduit for the inducible expression of 1 growth factor by another during the process of intimal thickening.

Platelet-derived growth factors are a family of mitogens and chemoattractants comprising of four ligand genes (A-, B-, C-, D-chains) implicated in many physiologic and pathophysiologic processes, including atherosclerosis, fibrosis and tumorigenesis. Our understanding of the molecular mechanisms, which regulate PDGF-C transcription remains incomplete. Transient transfection analysis, conventional and quantitative real-time PCR revealed the induction of PDGF-C transcription and mRNA expression in smooth muscle cells (SMCs) exposed to the peptide hormone angiotensin (ATII), which induces Egr-1. Occupancy of a G + C-rich element in the proximal region of the PDGF-C promoter was unaffected by ATII. Instead we discovered, using both nuclear extracts and recombinant proteins with EMSA and ChIP analyses, the existence of a second Egr-1-binding element located 500 bp upstream. ATII induction of PDGF-C transcription is mediated by the angiotensin type 1 receptor (AT1R) and Egr-1 activation through this upstream element. DNAzyme ED5 targeting Egr-1 blocked ATII-inducible PDGF-C expression. Moreover, increased PDGF-C expression after exposure to ATII depends upon the differentiation state of the SMCs. This study demonstrates the existence of this novel ATII-AT1R-Egr-1-PDGF-C axis in SMCs of neonatal origin, but not in adult SMCs, where ATII induces Egr-1 but not PDGF-C.

Sp1, the first identified and cloned transcription factor, regulates gene expression via multiple mechanisms including direct protein–DNA interactions, protein–protein interactions, chromatin remodeling, and maintenance of methylation-free CpG islands. Sp1 is itself regulated at different levels, for example, by glycosylation, acetylation, and phosphorylation by kinases such as the atypical protein kinase C-ζ. Although Sp1 controls the basal and inducible regulation of many genes, the posttranslational processes regulating its function and their relevance to pathology are not well understood. Here we have used a variety of approaches to identify 3 amino acids (Thr668, Ser670, and Thr681) in the zinc finger domain of Sp1 that are modified by PKC-ζ and have generated novel anti-peptide antibodies recognizing the PKC-ζ–phosphorylated form of Sp1. Angiotensin II, which activates PKC-ζ phosphorylation (at Thr410) via the angiotensin II type 1 receptor, stimulates Sp1 phosphorylation and increases Sp1 binding to the platelet-derived growth factor-D promoter. All 3 residues in Sp1 (Thr668, Ser670, and Thr681) are required for Sp1-dependent platelet-derived growth factor-D activation in response to angiotensin II. Immunohistochemical analysis revealed that phosphorylated Sp1 is expressed in smooth muscle cells of human atherosclerotic plaques and is dynamically expressed together with platelet-derived growth factor-D in smooth muscle cells of the injured rat carotid artery wall. This study provides new insights into the regulatory mechanisms controlling the PKC-ζ–phospho-Sp1 axis and angiotensin II–inducible gene expression.