Temporary opening of the blood-brain barrier with the nitrone compound OKN-007

The blood-brain barrier (BBB) is usually impermeable to several drugs, which hampers treatment of various brain-related diseases/disorders. There have been several approaches to open the BBB, including intracarotid infusion of hyperosmotic concentrations of arabinose, mannitol, oleic or linoleic acids, or alkylglycerols, intravenous infusion of bradykinin B2, administration of a fragment of the ZO toxin from vibrio cholera, targeting specific components of the tight junctions (e.g. claudin-5) with siRNA or novel peptidomimetic drugs, or the use of ultrasound with microbubbles. We propose the use of a low molecular weight (MW), nitrone-type compound, OKN-007, which can temporarily open up the BBB for 1-2 hours. Gadolinium (Gd)-based compounds assessed ranged in MW from 546 (Gd-DTPA) to 465 kDa (beta-galactosidase Gd DOTA). We also included an albumin-based CA (albumin-Gd-DTPA-biotin) for assessment, as well as an antibody (Ab) against a neuron-specific biomarker conjugated to Gd-DOTA (anti-EphB2-Gd-DOTA). For the anti-EphB2 (goat Ab)-Gd-DOTA assessment, we utilized an anti goat Ab conjugated with horse radish peroxidase (HRP) for confirmation of the presence of the anti-EphB2-Gd-DOTA probe. In addition, a Cy5 labeled anti-EphB2 Ab was co-administered with the anti-EphB2-Gd-DOTA probe, and assessed ex vivo. This study demonstrates that OKN-007 may be able to temporarily open up the BBB to augment the delivery of various compounds ranging in MW from as small as similar to 550 to as large as similar to 470 kDa. This compound is an investigational new drugfor glioblastoma (GBM) therapy in clinical trials. The translational capability for human use to augment the delivery of non-BBB-permeable drugs is extremely high

The NuRD Chromatin-Remodeling Enzyme CHD4 Promotes Embryonic Vascular Integrity by Transcriptionally Regulating Extracellular Matrix Proteolysis

<div><p>The extracellular matrix (ECM) supports vascular integrity during embryonic development. Proteolytic degradation of ECM components is required for angiogenesis, but excessive ECM proteolysis causes blood vessel fragility and hemorrhage. Little is understood about how ECM proteolysis is transcriptionally regulated during embryonic vascular development. We now show that the NuRD ATP-dependent chromatin-remodeling complex promotes vascular integrity by preventing excessive ECM proteolysis <i>in vivo</i>. Mice lacking endothelial CHD4—a catalytic subunit of NuRD complexes—died at midgestation from vascular rupture. ECM components surrounding rupture-prone vessels in <i>Chd4</i> mutants were significantly downregulated prior to embryonic lethality. Using qPCR arrays, we found two critical mediators of ECM stability misregulated in mutant endothelial cells: the urokinase-type plasminogen activator receptor (uPAR or <i>Plaur</i>) was upregulated, and thrombospondin-1 (<i>Thbs1</i>) was downregulated. Chromatin immunoprecipitation assays showed that CHD4-containing NuRD complexes directly bound the promoters of these genes in endothelial cells. uPAR and THBS1 respectively promote and inhibit activation of the potent ECM protease plasmin, and we detected increased plasmin activity around rupture-prone vessels in <i>Chd4</i> mutants. We rescued ECM components and vascular rupture in <i>Chd4</i> mutants by genetically reducing urokinase (uPA or <i>Plau</i>), which cooperates with uPAR to activate plasmin. Our findings provide a novel mechanism by which a chromatin-remodeling enzyme regulates ECM stability to maintain vascular integrity during embryonic development.</p></div

Genetic reduction of urokinase restores ECM components and partially rescues vascular rupture in <i>Chd4^fl/fl;Tie2-Cre⁺</i> embryos.

<p>(A–C) Representative images of littermate control (A), <i>Chd4^fl/fl;Tie2-Cre⁺</i> (B), and <i>Chd4^fl/fl;Plau^+/−;Tie2-Cre⁺</i> (C) embryos at E12.5. All <i>Chd4^fl/fl;Tie2-Cre⁺</i> embryos examined (20/20) were pale and necrotic (B). 60% of <i>Chd4^fl/fl;Plau^+/−;Tie2-Cre⁺</i> embryos (9 out of 15) were comparable in size to littermate control embryos, although they displayed blood in the brain (arrow) and/or spinal cord (C). (D–L) Histological sections of dorsal aortae from E10.5 control, <i>Chd4^fl/fl;Tie2-Cre⁺</i>, and <i>Chd4^fl/fl;Plau^+/−;Tie2-Cre⁺</i> embryos were stained with antibodies against type IV collagen (Col-IV; D–F), fibronectin (G–I), or α-SMA (J–L). Scale bars: 1 mm (A–C); 50 µm (D–L). (M) Quantification of immunostained ECM components, such as those shown in panels D–L. Relative fluorescent intensity was measured and normalized to fluorescence in the control sections. Error bars represent SD of results from three independent experiments using three different sets of littermate embryos. Significant differences were calculated using a two-tailed Student's <i>t</i> test (*, <i>p</i><0.05).</p

<i>Chd4^fl/fl;Tie2-Cre⁺</i> embryos undergo vascular rupture by E11.25.

<p>(A) <i>Chd4^fl/+</i> females were mated with <i>Chd4^fl/+;Tie2-Cre⁺</i> males, and live progeny from 22 litters were genotyped and scored at weaning. No live <i>Chd4^fl/fl;Tie2-Cre⁺</i> mice were recovered [χ²(5_dof): <i>p</i><0.001]. (B–E) Gross images of E11.5 littermate control (B,D) and <i>Chd4^fl/fl;Tie2-Cre⁺</i> (C,E) embryos. Arrow in panel E indicates massive hemorrhage within the ventral trunk region of a <i>Chd4^fl/fl;Tie2-Cre⁺</i> embryo. (F) <i>Chd4^fl/fl</i> females were mated with <i>Chd4^fl/+;Tie2-Cre⁺</i> males, and dissections were performed on E10.5–12.5 embryos. Dead embryos were characterized by absence of heartbeat and onset of necrosis. No surviving <i>Chd4^fl/fl;Tie2-Cre⁺</i> embryos were found at E12.5 [χ²(3_dof): <i>p</i><0.01]. (G–L) Hematoxylin and eosin (H&E) staining of E10.5 (G,H) or E11.2±5 (I–L) littermate control (G,I,K) and <i>Chd4^fl/fl;Tie2-Cre⁺</i> (H,J,L) embryos. Boxed regions in panels I and J are shown at higher magnification in panels K and L respectively. Arrow in panel L reveals site of vascular rupture between the mutant dorsal aorta and cardinal vein; arrowhead indicates blood in extravascular tissues. DA, dorsal aorta; CV, cardinal vein. (M–P) Transmission electron micrographs from vessel walls of E10.5 littermate control (M,O) and <i>Chd4^fl/fl;Tie2-Cre⁺</i> (N,P) embryos. Arrowheads in panels M and N indicate smooth muscle cells adjacent to endothelial cells (ECs). Arrow in panel N points to a long, thin EC extension. (O,P) Endothelial cell junctions (*) are intact in both control and mutant sections. Scale bars: 1 mm (B–E); 100 µm (G–L); 2 µm (M,N); 500 nm (O,P).</p

CHD4 differentially regulates <i>Plaur</i> and <i>Thbs1</i> expression in endothelial cells.

<p>(A) qPCR with gene-specific primers for <i>Plau</i>, <i>Plaur</i>, and <i>Thbs1</i> was performed on endothelial cells isolated from E10.5 littermate control and <i>Chd4^fl/fl;Tie2-Cre⁺</i> embryos. Data were normalized to the relative expression of control samples. Error bars represent SD of results from three independent experiments. (B and C) C166 endothelial cells were transfected with nonspecific (NS) or CHD4-specific siRNA oligonucleotides for 24 h. (B) Western blot analysis was performed on cell lysates using antibodies that recognize CHD4 or GAPDH. A representative blot from 3 independent experiments is shown. (C) RNA was isolated, cDNA was synthesized, and qPCR was performed using <i>Plau</i>-, <i>Plaur</i>-, or <i>Thbs1</i>-specific primers. Data were normalized to the relative expression of NS siRNA-treated samples. Error bars represent SD of results from three to four independent experiments. (D) Chromatin immunoprecipitation (ChIP) assays were carried out in C166 endothelial cells using antibodies against normal mouse IgG (negative control), CHD4, or HDAC1. Immunoprecipitated DNA was analyzed by qPCR to examine CHD4 and HDAC1 enrichment at the <i>Plau</i>, <i>Plaur</i>, and <i>Thbs1</i> promoters. A transcriptionally inactive region approximately 5 kb upstream of the <i>Fzd5</i> transcription start site (<i>Fzd5UP</i>) was assessed as a negative control for CHD4 and HDAC1 binding. Data are represented as a percent of total input chromatin. Error bars represent SD of results from three independent experiments. Results for <i>Plau</i> and <i>Fzd5UP</i> ChIP experiments are magnified in the insets. For the <i>Plaur</i> and <i>Thbs1</i> ChIPs, CHD4 and HDAC1 binding were statistically compared against IgG binding at the respective loci or against CHD4 and HDAC1 binding at the <i>Fzd5UP</i> locus; both sets of comparisons revealed significant enrichment. (E) qPCR with gene-specific primers for <i>Chd4</i>, <i>Plaur</i>, and <i>Thbs1</i> was performed on C166 endothelial cells transfected with 0.02 ng of a CHD4 expression plasmid or the analogous empty vector backbone (control). Data were normalized to the relative expression of control samples. Error bars represent SD of results from three independent experiments. (F) Schematic of the region of the murine <i>Plaur</i> promoter that was cloned into a luciferase (LUC) reporter plasmid for use in G. The <i>Plaur</i> promoter fragment encompasses the region to which CHD4 and HDAC1 were shown to bind by ChIP in D. (G) Luciferase assays were performed in C166 cells co-transfected with 250 ng of the reporter schematized in F and 10 ng of a constitutive Renilla luciferase control plasmid. Cells were also transfected with either 10 pmol of non-specific (NS) siRNA or CHD4 siRNA oligonucleotides to knock down endogenous CHD4 or with the CHD4 expression plasmid or its relevant control (empty vector) described in E. All transfections were performed for 24 h. Ratios of relative luciferase∶renilla activity were normalized to results from the control samples. Error bars represent SD of results from four independent experiments (with triplicate samples) for the siRNA-transfected samples and from five independent experiments (with triplicate samples) for the CHD4/control plasmid-transfected samples. All statistical calculations for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004031#pgen-1004031-g003" target="_blank">Figure 3</a> were performed using a two-tailed Student's <i>t</i> test (*, <i>p</i><0.05).</p

Suppression of Tumor Growth in Mice by Rationally Designed Pseudopeptide Inhibitors of Fibroblast Activation Protein and Prolyl Oligopeptidase

Tumor microenvironments (TMEs) are composed of cancer cells, fibroblasts, extracellular matrix, microvessels, and endothelial cells. Two prolyl endopeptidases, fibroblast activation protein (FAP) and prolyl oligopeptidase (POP), are commonly overexpressed by epithelial-derived malignancies, with the specificity of FAP expression by cancer stromal fibroblasts suggesting FAP as a possible therapeutic target. Despite overexpression in most cancers and having a role in angiogenesis, inhibition of POP activity has received little attention as an approach to quench tumor growth. We developed two specific and highly effective pseudopeptide inhibitors, M83, which inhibits FAP and POP proteinase activities, and J94, which inhibits only POP. Both suppressed human colon cancer xenograft growth >90% in mice. By immunohistochemical stains, M83- and J94-treated tumors had fewer microvessels, and apoptotic areas were apparent in both. In response to M83, but not J94, disordered collagen accumulations were observed. Neither M83- nor J94-treated mice manifested changes in behavior, weight, or gastrointestinal function. Tumor growth suppression was more extensive than noted with recently reported efforts by others to inhibit FAP proteinase function or reduce FAP expression. Diminished angiogenesis and the accompanying profound reduction in tumor growth suggest that inhibition of either FAP or POP may offer new therapeutic approaches that directly target TMEs