Netrin-1 and UNC5B Cooperate with Integrins to Mediate YAP-Driven Cytostasis.

Opposite expression and pro- or anti-cancer function of YAP and its paralog TAZ/WWTR1 stratify cancers into binary YAPon and YAPoff classes. These transcriptional coactivators are oncogenic in YAPon cancers. In contrast, YAP/TAZ are silenced epigenetically along with their integrin and extracellular matrix adhesion target genes in neural and neuroendocrine YAPoff cancers (e.g., small cell lung cancer, retinoblastoma). Forced YAP/TAZ expression induces these targets, causing cytostasis in part through Integrin-αV/ß5, independent of the integrin-binding RGD ligand. Other effectors of this anticancer YAP function are unknown. Here, using clustered regularly interspaced short palindromic repeats (CRISPR) screens, we link the Netrin receptor UNC5B to YAP-induced cytostasis in YAPoff cancers. Forced YAP expression induces UNC5B through TEAD DNA-binding partners, as either TEAD1/4-loss or a YAP mutation that disrupts TEAD-binding (S94A) blocks, whereas a TEAD-activator fusion (TEAD(DBD)-VP64) promotes UNC5B induction. Ectopic YAP expression also upregulates UNC5B relatives and their netrin ligands in YAPoff cancers. Netrins are considered protumorigenic, but knockout and peptide/decoy receptor blocking assays reveal that in YAPoff cancers, UNC5B and Netrin-1 can cooperate with integrin-αV/ß5 to mediate YAP-induced cytostasis. These data pinpoint an unsuspected Netrin-1/UNC5B/integrin-αV/ß5 axis as a critical effector of YAP tumor suppressor activity. SIGNIFICANCE: Netrins are widely perceived as procancer proteins; however, we uncover an anticancer function for Netrin-1 and its receptor UNC5B.

Cardiovascular Histopathology of a 11-Year Old with Mucopolysaccharidosis VII Demonstrates Fibrosis, Macrophage Infiltration, and Arterial Luminal Stenosis.

Mucopolysaccharidosis type VII (MPS VII) is caused by ß-glucuronidase deficiency, resulting in lysosomal accumulation of glycosaminoglycans (GAGs) and multisystemic disease. We present cardiovascular gross and histopathology findings from a 11-year-old MPS VII male, who expired after developing ventricular fibrillation following anesthesia induction. Gross anatomic observations were made at autopsy; postmortem formalin-fixed paraffin-embedded samples of the carotid artery, aorta, myocardium, and valves were sectioned and stained with hematoxylin-eosin, Verhoeff-Van Gieson, CD68, and trichrome stains. Gross heart findings include an enlarged, dilated heart, mitral valve prolapse with thick, shortened chordae tendinae, and thickened aortic valve cusps. The aorta contained raised intimal plaques mimicking conventional atherosclerosis. Cardiac myocytes included hypertrophic nuclei, subendocardial fibrosis, and increased interfascicular collagen. Coronary lumens were 40-70% stenosed by fibrointimal hyperplasia containing storage material-laden cells, CD68+ macrophages, and fragmented elastin laminae. Similar findings were visualized in aortic intimal plaques. We confirm that arterial plaques, elastin fragmentation, and activated CD68+ macrophage infiltration occur in human MPS VII, consistent with previously observed findings in murine and canine MPS VII. We also confirm ultrasonographically observed carotid intimal-medial thickening is an in vivo correlate of histopathologic vascular fibrointimal hyperplasia. MPS VII patients should be regularly monitored for cardiac disease, with methods such as Holter monitors and stress testing; MPS VII-directed treatments should effectively address cardiovascular disease.

Fractionated Radiation Exposure of Rat Spinal Cords Leads to Latent Neuro-Inflammation in Brain, Cognitive Deficits, and Alterations in Apurinic Endonuclease 1.

Ionizing radiation causes degeneration of myelin, the insulating sheaths of neuronal axons, leading to neurological impairment. As radiation research on the central nervous system has predominantly focused on neurons, with few studies addressing the role of glial cells, we have focused our present research on identifying the latent effects of single/ fractionated -low dose of low/ high energy radiation on the role of base excision repair protein Apurinic Endonuclease-1, in the rat spinal cords oligodendrocyte progenitor cells' differentiation. Apurinic endonuclease-1 is predominantly upregulated in response to oxidative stress by low- energy radiation, and previous studies show significant induction of Apurinic Endonuclease-1 in neurons and astrocytes. Our studies show for the first time, that fractionation of protons cause latent damage to spinal cord architecture while fractionation of HZE (28Si) induce increase in APE1 with single dose, which then decreased with fractionation. The oligodendrocyte progenitor cells differentiation was skewed with increase in immature oligodendrocytes and astrocytes, which likely cause the observed decrease in white matter, increased neuro-inflammation, together leading to the observed significant cognitive defects.

Graphene nanoribbons as a drug delivery agent for lucanthone mediated therapy of glioblastoma multiforme.

We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24h. However, their uptake was ~38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1.

Lucanthone and its derivative hycanthone inhibit apurinic endonuclease-1 (APE1) by direct protein binding.

Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC(50) values for inhibition of APE1 incision of depurinated plasmid DNA by lucanthone and hycanthone were 5 µM and 80 nM, respectively. The K(D) values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules like thioxanthenones can bind, and our modeling studies confirmed such docking. Circular dichroism spectra uncovered change in the helical structure of APE1 in the presence of lucanthone/hycanthone, and notably, this effect was decreased (Phe266Ala or Phe266Cys or Trp280Leu) or abolished (Phe266Ala/Trp280Ala) when hydrophobic site mutants were employed. Reduced inhibition by lucanthone of the diminished endonuclease activity of hydrophobic mutant proteins (as compared to wild type APE1) supports that binding of lucanthone to the hydrophobic pocket dictates APE1 inhibition. The DNA binding capacity of APE1 was marginally inhibited by lucanthone, and not at all by hycanthone, supporting our hypothesis that thioxanthenones inhibit APE1, predominantly, by direct interaction. Finally, lucanthone-induced degradation was drastically reduced in the presence of short and long lived free radical scavengers, e.g., TRIS and DMSO, suggesting that the mechanism of APE1 breakdown may involve free radical-induced peptide bond cleavage.

Radiation resistance in glioma cells determined by DNA damage repair activity of Ape1/Ref-1.

Since radiation therapy remains a primary treatment modality for gliomas, the radioresistance of glioma cells and targets to modify their radiation tolerance are of significant interest. Human apurinic endonuclease 1 (Ape1, Ref-1, APEX, HAP1, AP endo) is a multifunctional protein involved in base excision repair of DNA and a redox-dependent transcriptional co-activator. This study investigated whether there is a direct relationship between Ape1 and radioresistance in glioma cells, employing the human U87 and U251 cell lines. U87 is intrinsically more radioresistant than U251, which is partly attributable to more cycling U251 cells found in G2/M, the most radiosensitive cell stage, while more U87 cells are found in S and G1, the more radioresistant cell stages. But observed radioresistance is also related to Ape1 activity. U87 has higher levels of Ape1 than does U251, as assessed by Western blot and enzyme activity assays (approximately 1.5-2 fold higher in cycling cells, and approximately 10 fold higher at G2/M). A direct relationship was seen in cells transfected with CMV-Ape1 constructs; there was a dose-dependent relationship between increasing Ape1 overexpression and increasing radioresistance. Conversely, knock down by siRNA or by pharmacological down regulation of Ape1 resulted in decreased radioresistance. The inhibitors lucanthone and CRT004876 were employed, the former a thioxanthene previously under clinical evaluation as a radiosensitizer for brain tumors and the latter a more specific Ape1 inhibitor. These data suggest that Ape1 may be a useful target for modifying radiation tolerance.

Mitigation effect of an FGF-2 peptide on acute gastrointestinal syndrome after high-dose ionizing radiation.

PURPOSE: Acute gastrointestinal syndrome (AGS) resulting from ionizing radiation causes death within 7 days. Currently, no satisfactory agent exists for mitigation of AGS. A peptide derived from the receptor binding domain of fibroblast growth factor 2 (FGF-P) was synthesized and its mitigation effect on AGS was examined. METHODS AND MATERIALS: A subtotal body irradiation (sub-TBI) model was created to induce gastrointestinal (GI) death while avoiding bone marrow death. After 10.5 to 16 Gy sub-TBI, mice received an intramuscular injection of FGF-P (10 mg/kg/day) or saline (0.2 ml/day) for 5 days; survival (frequency and duration) was measured. Crypt cells and their proliferation were assessed by hematoxylin, eosin, and BrdU staining. In addition, GI hemoccult score, stool formation, and plasma levels of endotoxin, insulin, amylase, interleukin (IL)-6, keratinocyte-derived chemokine (KC) monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor (TNF)-alpha were evaluated. RESULTS: Treatment with FGF-P rescued a significant fraction of four strains of mice (33-50%) exposed to a lethal dose of sub-TBI. Use of FGF-P improved crypt survival and repopulation and partially preserved or restored GI function. Furthermore, whereas sub-TBI increased plasma endotoxin levels and several pro-inflammation cytokines (IL-6, KC, MCP-1, and TNF-alpha), FGF-P reduced these adverse responses. CONCLUSIONS: The study data support pursuing FGF-P as a mitigator for AGS.

Tissue-sparing effect of x-ray microplanar beams particularly in the CNS: is a bystander effect involved?

OBJECTIVE: Normal tissues, including the central nervous system, tolerate single exposures to narrow planes of synchrotron-generated x-rays (microplanar beams; microbeams) up to several hundred Gy. The repairs apparently involve the microvasculature and the glial system. We evaluate a hypothesis on the involvement of bystander effects in these repairs. METHODS: Confluent cultures of bovine aortic endothelial cells were irradiated with three parallel 27-microm microbeams at 24 Gy. Rats' spinal cords were transaxially irradiated with a single microplanar beam, 270 microm thick, at 750 Gy; the dose distribution in tissue was calculated. RESULTS: Within 6 hours following irradiation of the cell culture the hit cells died, apparently by apoptosis, were lost, and the confluency was maintained. The spinal cord study revealed a loss of oligodendrocytes, astrocytes, and myelin in 2 weeks, but by 3 months repopulation and remyelination was nearly complete. Monte Carlo simulations showed that the microbeam dose fell from the peak's 80% to 20% in 9 microm. CONCLUSIONS: In both studies the repair processes could have involved "beneficial" bystander effects leading to tissue restoration, most likely through the release of growth factors, such as cytokines, and the initiation of cell-signaling cascades. In cell culture these events could have promoted fast disappearance of the hit cells and fast structural response of the surviving neighboring cells, while in the spinal cord study similar events could have been promoting angiogenesis to replace damaged capillary blood vessels, and proliferation, migration, and differentiation of the progenitor glial cells to produce new, mature, and functional glial cells.

Fatal myocarditis related to Campylobacter jejuni infection: a case report.

Infectious myocarditis can be caused by a variety of agents, with enteroviruses being the most common. Myocarditis due to enteric bacteria is rare. We present pathological documentation of a rare case of a Campylobacter jejuni bowel infection leading to rapidly fatal myocarditis.

Augmentation of demineralized bone matrix (DBM) mineralization by a synthetic growth factor mimetic.

These studies evaluated whether F2A4-K-NS, a peptide mimetic of FGF-2, could augment ectopic bone production following the subcutaneous implant of human demineralized bone matrix (DBM). DBM was formulated into a gel with and without F2A4-K-NS, and injected subcutaneously into athymic rats. After 28 days the resultant tissue was excised and fixed. The tissue was examined with soft X-rays and microcomputerized tomography (micro-CT), and by histological methods. Inclusion of F2A4-K-NS with DBM resulted in an increased mineral deposition as determined by soft X-ray and micro-CT analysis and von Kossa staining. DBM-containing tissues showed extensive mineralization compared to the carrier alone, which was poorly mineralized. The mineralization was qualitatively and quantitatively the most extensive in the samples containing F2A4-K-NS plus DBM. Additionally, the highest amount of von Kossa staining for calcium was observed in tissues from animals that had received DBM plus F2A4-K-NS. In these studies, 100 ng of peptide per 0.2 mL of injectable DBM gel generated the most optimal results. The synthetic peptide F2A4-K-NS augmented DBM-induced ectopic mineralization in athymic animals.

Multidomain synthetic peptide B2A2 synergistically enhances BMP-2 in vitro.

UNLABELLED: A multidomain, synthetic peptide designated B2A2 synergizes the activity of BMP-2. B2A2 interacts with BMP receptor isoforms, potentiating the action of BMP-2 in activating alkaline phosphatase and triggering Smad and MAPK signaling. B2A2's design permits its delivery as a local surface coating as well as a soluble co-factor, thus broadening potential bioengineering applications. INTRODUCTION: BMP-2 induces osteogenic differentiation and accelerates bone repair. Although BMP-2 inhibitors have been discovered, no BMP-2 mimetics or enhancers that function in the physiological range have yet been found. Here we report that a synthetic peptide designated B2A2, consisting of (1) a BMP receptor-targeting sequence, (2) a hydrophobic spacer, and (3) a heparin-binding sequence, is a positive modulator of recombinant BMP-2. MATERIALS AND METHODS: Cultures of mesenchymal cell lines C2C12 and C3H10T1/2 were given B2A2, recombinant BMP-2, or both. Alkaline phosphatase (ALP) activity was assayed by conversion of paranitrophenol phosphate (PNPP). Signaling through Smad and MAP kinase pathways was monitored by Western blot. Receptor binding was assessed by incubating immobilized B2A2 with soluble recombinant receptor-Fc chimeras and detecting bound receptor by anti-Fc antibody ELISA. Surface coating of medical device materials was done by first dip-coating with silyl-heparin, followed by B2A2. RESULTS AND CONCLUSIONS: Treatment of cells with B2A2 alone marginally increased ALP activity. However, B2A2 plus BMP-2 resulted in 5- to 40-fold augmentation of ALP compared with BMP-2 alone in C3H10T1/2 or C2C12 cells, respectively. This synergistic enhancement was observed over a broad concentration range (4-1000 ng/ml BMP-2). B2A2 interacted directly with BMP receptor isoforms (preferentially to BMPR-Ib and ActivinR-II). In cells, B2A2 + BMP-2 led to a repression of MAP kinase and an increase of Smad activation, consistent with known activation pathways of BMP-2. B2A2 was ineffective when paired with other cytokine/growth factors (basic fibroblast growth factor [FGF-2], TGF-beta1, vascular endothelial growth factor [VEGF]). Simultaneous co-administration was not strictly required. Pulse-chase experiments revealed that temporal separations up to 1 h were still effective. B2A2 was also effective when delivered in a polystyrene- or stainless steel-coated surface through a heparin platform (silyl-heparin) while BMP-2 was added exogenously in solution. These results suggest that B2A2 might promote aggregation of receptor subunits, enabling BMP-2 to activate signaling pathways at effectively lower concentrations. Synthetic multidomain constructs like B2A2 may be useful to accelerate bone repair/deposition through augmentation of endogenous levels of BMP-2 or through local BMP-2 contained in artificial or engineered matrices.

Cell attachment and biocompatibility of polytetrafluoroethylene (PTFE) treated with glow-discharge plasma of mixed ammonia and oxygen.

The plasma generated from a gas mixture of NH3 plus O2 (NH3 + O2) has been used to impart unique chemical and biological characteristics to polytetrafluoroethylene (PTFE). PTFE treated with NH3 + O2 plasma was physiochemically distinct from surfaces treated with plasma of either NH3 or O2 alone, as determined by electron spectroscopy for chemical analysis (ESCA). The contact angle analysis revealed that the PTFE surfaces became less hydrophobic after plasma treatments. ESCA results indicate the presence of oxygen-containing groups and nitrogen-containing groups at the plasma-treated surfaces. PTFE treated with NH3 + O2 plasma resisted the attachment of platelets and leukocytes in a manner similar to untreated PTFE; however, the attachment of bovine aorta endothelial cells was substantially increased. Once attached, these cells grew to confluency. The increased endothelial cell attachment was higher than that observed following plasma treatment with each gas used separately, which could be attributed to the considerable amount of CF(OR)2-CF2 formed on the NH3 + O2 plasma-treated PTFE surface. At 14 days after subcutaneous implantation in rats, the PTFE wafers treated with NH3 + O2 plasma demonstrated less encapsulation and lower levels of inflammatory cells compared to controls. Collectively, the results suggest that NH3 + O2 plasma treatment imparts a unique character to PTFE and could be useful in certain in vivo applications.

NH3/O2 mixed gas plasmas alter the interaction of blood components with stainless steel.

Stainless steel treated with a mixed gas plasma of NH(3) plus O(2) had chemical and biologic characteristics distinct from untreated stainless steel or stainless steel treated with NH(3) or O(2) plasmas used separately. NH(3)/O(2) plasmas deposited nitrogen as both -CN (organic) and -NO (nitrate, nitrite)--materials not found on untreated stainless steel--and the contact angle changed from 44 degrees to 23 degrees. Treatment of stainless steel (and titanium) resulted in surfaces with enhanced resistance to platelet and leukocyte attachment. A gas plasma of N(2)O/O(2) also was found to reduce platelet and leukocyte attachment, suggesting that these properties may be common to surfaces coated with oxynitrites (nitrides). Upon subcutaneous implantation, no inflammation, hemolysis, or untoward thrombosis was noted in the tissue surrounding the wafers treated with the NH(3)/O(2) plasmas, although the cellular density was considerably reduced by 2 weeks after implant. Collectively, the results suggest that NH(3)/O(2) plasmas impart a unique character to stainless steel that may be useful in the construction of medical devices.

Local delivery of basic fibroblast growth factor (bFGF) using adsorbed silyl-heparin, benzyl-bis(dimethylsilylmethyl)oxycarbamoyl-heparin.

A growth factor delivery system was developed that is based on the use of silyl-heparin, a chemically modified analogue of heparin. The silyl-heparin was adsorbed onto surfaces by hydrophobic interaction via the prosthetic unit and can then be used as a solid-phase adsorbent for bFGF. All the coating steps were performed by adsorption, a process that allowed preparation of surfaces by immersion or "dip-coating". In this study a series of silyl-heparins were synthesized and each of the analogues found to function similar to unmodified heparin relative to their binding of antithrombin III and also the binding of bFGF. The silyl-heparins were found to be adsorbed onto a wide variety of substrates including polystyrene and lactide:glycolide copolymer. Enzyme-linked immumosorbant assay (ELISA) was used to establish that bFGF was readily bound to surface adsorbed silyl-heparin, and that the amount bound was directly related to amount offered for binding. Once adsorbed the silyl-heparin/FGF was able to induce capillary tube formation of endothelial cells and to increase the growth of endothelial cells. When coated onto suture material and implanted in muscle, the FGF/silyl-heparin coating caused an increased density of mesenchymal cells in the area of the implant. This coating method could prove to be useful in a number of tissue engineering applications for the local delivery of FGF and other growth factors.

Response of rat intracranial 9L gliosarcoma to microbeam radiation therapy.

Radiotherapeutic doses for malignant gliomas are generally palliative because greater, supposedly curative doses would impart clinically unacceptable damage to nearby vital CNS tissues. To improve radiation treatment for human gliomas, we evaluated microbeam radiation therapy, which utilizes an array of parallel, microscopically thin (<100 microm) planar beams (microbeams) of synchrotron-generated X rays. Rats with i.c. 9L gliosarcoma tumors were exposed laterally to a single microbeam, 27 pm wide and 3.8 mm high, stepwise, to produce irradiation arrays with 50, 75, or 100 microm of on-center beam spacings and 150, 250, 300, or 500 Gy of in-slice, skin-entrance, single-exposure doses. The resulting array size was 9 mm wide and 10.4 mm high (using three 3.8-mm vertical tiers); the beam's median energy was -70 keV. When all data were collated, the median survival was 70 days; no depletion of nerve cells was observed. However, when data from the highest skin-entrance dose and/or the smallest microbeam spacings were excluded, the median survival time of the subset of rats was 170 days, and no white matter necrosis was observed. Others have reported unilateral single-exposure broad-beam irradiation of i.c. 9L gliosarcomas at 22.5 Gy with a median survival of only -34 days and with severe depletion of neurons. These results suggest that the therapeutic index of unidirectional microbeams is larger than that of the broad beams and that an application for microbeam radiation therapy in treating certain malignant brain tumors may be found in the future.