Purpose: We sought a novel approach against glioblastomas (GBM) focused on targeting signaling molecules localized in the tumor extracellular matrix (ECM). We investigated fibulin-3, a glycoprotein that forms the ECM scaffold of GBMs and promotes tumor progression by driving Notch and NFkB signaling. Experimental Design:Weused deletion constructs to identify a key signaling motif of fibulin-3. An mAb (mAb428.2) was generated against this epitope and extensively validated for specific detection of human fibulin-3. mAb428.2 was tested in cultures to measure its inhibitory effect on fibulin-3 signaling. Nude mice carrying subcutaneous and intracranial GBM xenografts were treated with the maximum achievable dose of mAb428.2 to measure target engagement and antitumor efficacy. Results: We identified a critical 23-amino acid sequence of fibulin-3 that activates its signaling mechanisms. mAb428.2 binds to that epitope with nanomolar affinity and blocks the ability of fibulin-3 to activate ADAM17, Notch, and NFkB signaling inGBM cells. mAb428.2 treatment of subcutaneous GBM xenografts inhibited fibulin-3, increased tumor cell apoptosis, and enhanced the infiltration of inflammatory macrophages. The antibody reduced tumor growth and extended survival of mice carrying GBMs as well as other fibulin-3-expressing tumors. Locally infused mAb428.2 showed efficacy against intracranial GBMs, increasing tumor apoptosis and reducing tumor invasion and vascularization, which are enhanced by fibulin-3. Conclusions: To our knowledge, this is the first rationally developed, function-blocking antibody against an ECM target in GBM. Our results offer a proof of principle for using "anti-ECM" strategies toward more efficient targeted therapies for malignant glioma. Clin Cancer Res; 24(4); 821-33.

Macrophage accumulation is a critical step during development of chronic inflammation, initiating progression of many devastating diseases. Leukocyte-specific integrin αDβ2 (CD11d/CD18) is dramatically upregulated on macrophages at inflammatory sites. Previously we found that CD11d overexpression on cell surfaces inhibits in vitro cell migration due to excessive adhesion. In this study, we have investigated how inflammation-mediated CD11d upregulation contributes to macrophage retention at inflammatory sites during atherogenesis. Atherosclerosis was evaluated in CD11d-/-/ApoE2/2 mice after 16 wk on a Western diet. CD11d deficiency led to a marked reduction in lipid deposition in aortas and isolated macrophages. Macrophage numbers in aortic sinuses of CD11d-/- mice were reduced without affecting their apoptosis and proliferation. Adoptive transfer of fluorescently labeled wild-type and CD11d-/- monocytes into ApoE2/2 mice demonstrated similar recruitment from circulation, but reduced accumulation of CD11d-/- macrophages within the aortas. Furthermore, CD11d expression was significantly upregulated on macrophages in atherosclerotic lesions and M1 macrophages in vitro. Interestingly, expression of the related ligand-sharing integrin CD11b was not altered. This difference defines their distinct roles in the regulation of macrophage migration. CD11ddeficient M1 macrophages demonstrated improved migration in a three-dimensional fibrin matrix and during resolution of peritoneal inflammation, whereas migration of CD11b2/2 M1 macrophages was not affected. These results prove the contribution of high densities of CD11d to macrophage arrest during atherogenesis. Because high expression of CD11d was detected in several inflammation-dependent diseases, we suggest that CD11d/CD18 upregulation on proinflammatory macrophages may represent a common mechanism for macrophage retention at inflammatory sites, thereby promoting chronic inflammation and disease development.

The original version of this article unfortunately contained mistakes in the Author group and Abstract sections. Author D. P. Bezerra’s name was incorrectly presented as “D. P. Paiva”. This abstract version substitutes the previous one: Abstract PiT1 (SLC20A1) and PiT2 (SLC20A2) are members of the mammalian type-III inorganic phosphate transporters and recent studies linked SLC20A2 mutations with primary brain calcifications. MicroRNAs (miRNAs) are endogenous noncoding regulatory RNAs and MicroRNA-9 (miR-9) modulates neurogenesis but is also involved with different types of cancer. We evaluated possible interactions between miR-9 and the phosphate transporters (PiT1 and PiT2). SLC20A2, platelet-derived growth factor receptor beta (PDGFRB) and Fibrillin-2 (FBN2) showed binding sites with high affinity for mir-9, In silico. miR-9 mimic was transfected into HEK293 cells and expression was confirmed by RT-qPCR. Overexpression of miR-9 in these cells caused a significant reduction in PiT2 and FBN2. PDGFRB appeared to be decreased, but was not significantly down-regulated. PiT1 showed no significant difference relative to controls. The down-regulation of PiT2 protein by miR-9 was confirmed by western blotting. In conclusion, we showed that miR-9 can down-regulate PiT2, in HEK293 cells.

Nucleotide excision repair (NER) is a major repair pathway that recognizes and corrects various lesions in cellular DNA. We hypothesize that damage recognition is an initial step in NER that senses conformational anomalies in the DNA caused by lesions. We prepared three DNA duplexes containing the carcinogen adduct N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) at G1, G2 or G3 of NarI sequence (5′-CCG1G2CG3CC-3′). Our 19F-NMR/ICD results showed that FAAF at G1 and G3 prefer syn S-and W-conformers, whereas anti B-conformer was predominant for G2. We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G3 and-G1 duplexes incised more efficiently than the B-type G2 duplex (G3∼G1>G2). The melting and thermodynamic data indicate that the S-and W-conformers produce greater DNA distortion and thermodynamic destabilization. The N-deacetylated N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF) adducts in the same NarI sequence are repaired 2-to 3-fold less than FAAF: however, the incision efficiency was in order of G2∼G1>G 3, a reverse trend of the FAAF case. We have envisioned the so-called N-acetyl factor as it could raise conformational barriers of FAAF versus FAF. The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts.

We have shown that osteopontin (OPN), an extracellular matrix protein, plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Interleukin-1β (IL-1β), increased in the heart following MI, increases matrix metalloproteinase (MMP) activity in cardiac fibroblasts in vitro. Here, we show that OPN alone has no effect on MMP activity or expression. However, it reduces IL-1β-stimulated increases in MMP activity and expression in adult rat cardiac fibroblasts. Pretreatment with bovine serum albumin had no effect on MMP activity or protein content, whereas GRGDS (glycine-arginine-glycine-aspartic acid-serine)- pentapeptide (which interrupts binding of RGD-containing proteins to cell surface integrins) and monoclonal antibody m7E3 (a rat β3 integrins antagonist) inhibited the effects of OPN. Inhibition of PKC using chelerythrine inhibited the activities of both MMP-2 and MMP-9. Stimulation of cells using IL-1β increased phosphorylation and translocation of PKC to membrane fractions, which was inhibited by OPN. OPN inhibited IL-1β-stimulated increases in translocation of PKC-ζ from cytosolic to membrane fractions. Furthermore, the levels of phospho-PKC-ζ were lower in the cytosolic fractions of OPN knock-out mice hearts as compared with wild type 6 days post-MI. Inhibition of PKC-ζ using PKC-ζ pseudosubstrate inhibited IL-1β-stimulated increases in MMP-2 and MMP-9 activities. These observations suggest that OPN, acting via 3 integrins, inhibits IL-1β-stimulated increases in MMP-2 and MMP-9 activity, at least in part, via the involvement of PKC-ζ. Thus, OPN may play a key role in collagen deposition during myocardial remodeling following MI by modulating cytokine-stimulated MMP activity.

Elevated systemic levels of the acute phase C-reactive protein (CRP) are predictors of future cardiovascular events. There is evidence that CRP may also play a direct role in atherogenesis. Here we determined whether the proinflammatory interleukin (IL)-17 stimulates CRP expression in hepatocytes (Hep3B cell line and primary hepatocytes) and coronary artery smooth muscle cells (CASMC). Our results demonstrate that IL-17 potently induces CRP expression in Hep3B cells independent of IL-1β and IL-6. IL-17 induced CRP promoter-driven reporter gene activity that could be attenuated by dominant negative IκBα or C/EBPβ knockdown and stimulated both NF-κB and C/EBP DNA binding and reporter gene activities. Targeting NF-κB and C/EBPβ activation by pharmacological inhibitors, small interfering RNA interference and adenoviral transduction of dominant negative expression vectors blocked IL-17-mediated CRP induction. Overexpression of wild type p50, p65, and C/EBPβ stimulated CRP transcription. IL-17 stimulated p38 MAPK and ERK1/2 activation, and SB203580 and PD98059 blunted IL-17-mediated NF-κB and C/EBP activation and CRP transcription. These results, confirmed in primary human hepatocytes and CASMC, demonstrate for the first time that IL-17 is a potent inducer of CRP expression via p38 MAPK and ERK1/2-dependent NF-κB and C/EBPβ activation and suggest that IL-17 may mediate chronic inflammation, atherosclerosis, and thrombosis.

Ca2+ regulates a spectrum of cellular processes including many aspects of neuronal function. Ca2+-sensitive events such as neurite extension and axonal guidance are driven by Ca2+ signals that are precisely organized in both time and space. These complex cues result from both Ca2+ influx across the plasma membrane and the mobilization of intracellular Ca2+ stores. In the present study, using rat cortical neurons, we have examined the effects of the novel intracellular Ca 2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) on neurite length and cytosolic Ca2+ levels. We show that NAADP potentiates neurite extension in response to serum and nerve growth factor and stimulates increases in cytosolic Ca2+ from bafilomycin- sensitive Ca2+ stores. Simultaneous blockade of inositol trisphosphate and ryanodine receptors abolished the effects of NAADP on neurite length and reduced the magnitude of NAADP-mediated Ca2+ signals. This is the first report demonstrating functional NAADP receptors in a mammalian neuron. Interplay between NAADP receptors and more established intracellular Ca2+ channels may therefore play important signaling roles in the nervous system.

Matrix metalloproteinases (MMPs), a family of endoproteinases, are implicated in cardiac remodeling. Interleukin-1β (IL-1β), which is increased in the heart following myocardial infarction, increases expression and activity of MMP-2 (gelatinase A) and -9 (gelatinase B) in cardiac fibroblasts. Previously, we have shown that IL-1β activates ERK1/2, JNKs, and protein kinase C (PKC). However, signaling pathways involved in the regulation of MMP-2 and -9 expression and activity are not yet well understood. Using adult rat cardiac fibroblasts, we show that inhibition of ERK1/2 and JNKs inhibits IL-1β-stimulated increases in MMP-9, not MMP-2, expression and activity. Chelerythrine, an inhibitor of PKC, inhibited activation of ERK1/2 and JNKs and expression and activity of both MMPs. Selective inhibition of PKC-α/β1 using Gö6976 inhibited JNKs activation and the expression and activity of MMP-9, not MMP-2. Inhibition of PKC-θ and PKC-ζ using pseudosubstrates inhibited IL-1β-stimulated activation of ERK1/2 and JNKs and the expression and activity of MMP-2 and -9. Inhibition of PKC-ε had no effect. IL-1β activated NF-κB pathway as measured by increased phosphorylation of IKKα/β and Inhibition of ERK1/2, JNKs, and PKC-α/β1 had no effect on NF-κB activation, whereas inhibition of PKC-θ and PKC-ζ inhibited IL-1β-stimulated activation of NF-κB. SN50, NF-κB inhibitor peptide, inhibited IL-1β-stimulated increases in MMP-2 and -9 expression and activity. These observations suggest that 1) activation of ERK1/2 and JNKs plays a critical role in the regulation of MMP-9, not MMP-2, expression and activity; 2) PKC-α/β1 act upstream of JNKs, not ERK1/2; 3) PKC-ζ and -θ, not PKC-ε, act upstream of JNKs, ERK1/2, and NF-κB; and 4) activation of NF-κB stimulates expression and activity of MMP-2 and -9.

Cells of the vasculature, including macrophages, smooth muscle cells, and endothelial cells, exhibit apoptosis in culture upon treatment with oxidized low density lipoprotein, as do vascular cells of atherosclerotic plaque. Several lines of evidence support the hypothesis that the apoptotic component of oxidized low density lipoprotein is one or more oxysterols, which have been shown to induce apoptosis through the mitochondrial pathway. Activation of the mitochondrial pathway of apoptosis is regulated by members of the BCL family of proteins. In this study, we demonstrate that, in the murine macrophage-like cell line P388D1, oxysterols (25-hydroxycholesterol and 7-ketocholesterol) induced the degradation of the prosurvival protein kinase AKT (protein kinase B). This led, in turn, to the activation of the BCL-2 homology-3 domain-only proteins BIM and BAD and down-regulation of the anti-apoptotic multi-BCL homology domain protein BCL-xL. These responses would be expected to activate the pro-apoptotic multi-BCL homology domain proteins BAX and BAK, leading to the previously reported release of cytochrome c observed during oxysterol-induced apoptosis. Somewhat surprisingly, small interfering RNA knockdown of BAX resulted in a complete block of the induction of apoptosis by 25-hydroxycholesterol.

C-reactive protein (CRP) binds with high affinity to fibronectin (Fn), a major component of the extracellular matrix (ECM), but at physiological pH the binding is inhibited by calcium ions (Ca2+). Because CRP circulates in the blood in Ca2+-bound form, the occurrence of CRP-Fn interactions in vivo has been doubtful. To define the basis of inhibition of CRP-Fn interaction by Ca2+ at pH 7.0, we hypothesized that Fn-binding site on CRP consisted of amino acids co-ordinating Ca2+. Site-directed mutagenesis of amino acids co-ordinating Ca2+ drastically decreased the binding of CRP to Fn, indicating that the Ca 2+-binding site indeed formed the Fn-binding site. To determine the requirements for possible interaction between Ca2+-bound CRP and Fn, we investigated inhibition of CRP-Fn interaction by Ca2+ as a function of pH. Ca2+ did not inhibit binding of CRP to Fn at pH 6.5 and lower. The contrasting Fn binding properties of CRP at physiological and mildly acidic pH indicated that the interaction of Ca2+-bound CRP with Fn was controlled by pH. We conclude that the inhibition of binding of CRP to Fn by Ca2+ at pH 7.0 is a mechanism to prevent CRP-Fn interactions under normal conditions. CRP, in its Ca2+-bound state, is capable of binding Fn but only at the inflammatory sites and tumors with low pH. CRP, Fn, and the ECM all have been implicated in cancer. Taken together our data raise the possibility that CRP-Fn interactions may change the architecture of ECM to modify the development of tumors.