fj.201701099(1).pdf

In the event of a radiologic catastrophe, endothelial cell and neutrophil dysfunction play important rolesin tissue injury.Clinically available therapeutics for radiation-induced vascular injury are largely supportive. PKCdwas identified as a critical regulator of the inflammatory response, and its inhibition was shown to protect criticalorgans during sepsis. We used a novel biomimetic microfluidic assay (bMFA) to interrogate the role of PKCd inradiation-induced neutrophil–endothelial cell interaction and endothelial cell function. HUVECs formed a completelumen in bMFA and were treated with 0.5, 2, or 5 Gy ionizing radiation (IR). At 24 h post-IR, the cells weretreatedwitha PKCd inhibitor for an additional 24h.Under physiologic shear flow, the role of PKCd on endotheliumfunction and neutrophil adherence/migration was determined. PKCd inhibition dramatically attenuated IRinducedendothelium permeability increase and significantly decreased neutrophil migration across IR-treatedendothelial cells. Moreover, neutrophil adhesion to irradiated endothelial cells was significantly decreased afterPKCd inhibition in a flow-dependentmanner. PKCd inhibition downregulated IR-induced P-selectin, intercellularadhesion molecule 1, and VCAM-1 but not E-selectin overexpression. PKCd is an important regulator ofneutrophil–endothelial cell interaction post-IR, and its inhibition can serve as a potential radiation medicalcountermeasure

Schematic illustration and images of neonatal blood-brain barrier on a chip (B³C).

<p>Schematic illustration of B³C showing the tissue compartment in the center of the device surrounded by two independent vascular channels with flow access openings. The dimensions of vascular channels are 200 μm x 100 μm x 2762 μm (width x height x length) and the dimensions of tissue compartment are 1575 μm x 100 μm (diameter x height). Vascular channels are in communication with the tissue compartment through a series of 3μm porous interface (pore dimensions are: 3μm x 3μm x 100 μm, width x height x length, spaced every 50 μm) along the length of the vascular channels (A). Schematic illustration of cell culture in B³C device showing one of two vascular channels (blue) with endothelial cells lining the channel walls, the tissue compartment (red) containing astrocytes, and the porous interface (white) separating the vascular channel and tissue compartment (B). The B³C device is assembled on a microscope glass slide with plastic tubes (dark blue) allowing access to individual vascular channels and the tissue compartment (C).</p

ACM enhances the barrier properties of neonatal RBEC more significantly in B³C as compared to transwell.

<p>Presence of ACM increases electrical resistance of neonatal RBEC in both B³C (A) and transwell (B), the electrical resistance measurements are from day 5. Presence of ACM increases resistance more significantly in B³C as compared to the transwell model (C). Please note that the units of electrical resistance for B³C and transwell are different as noted in the results section. [* indicates significant difference p<0.05, ** indicates significant difference p<0.01, *** indicates significant difference p<0.001, one-way ANOVA (panels A and B) or two-way ANOVA (panel C) with Tukey’s multiple comparison test; n = 3 experiments per group].</p

Tight junction formation by neonatal RBEC under static and flow conditions as indicated by immunofluorescence staining of ZO-1.

<p>RBEC cultured for 5 days under static conditions were stained with ZO-1 (red) and Hoechst 33342 (blue) (A). RBEC cultured for 5 days under flow of RBEC medium in B³C were stained with ZO-1 (red) and Hoechst 33342 (blue) (B). RBEC cultured under flow of ACM for 5 days in B³C were stained with ZO-1 (green) and Hoechst 33342 (blue) (C). Bright field image of B³C showing RBEC in the vascular channels after 5 days of co-culture with rat astrocytes in the tissue compartment of B³C (D). Immunofluorescence staining of RBEC in vascular channel for ZO-1 (green) and astrocytes in tissue compartment for GFAP (red) (E). Magnified view of interface with pores from panel E showing staining of cells inside the pores, ZO-1 (green), GFAP (red) and nuclei (blue) (F).</p

B³C exhibits significantly improved barrier function compared to the transwell model and closely approximates the permeability of neonatal <i>in vivo</i> BBB.

<p>B³C and Transwell BBB were constructed with neonatal RBEC in the presence of ACM. Permeability of 40 kDa dextran in B³C is significantly lower than transwell but not significantly different from that of <i>in vivo</i> BBB in neonatal rats. (** indicates significant difference p<0.01, *** indicates significant difference p<0.001, one-way ANOVA with Tukey’s multiple comparison test; n = 3–4 experiments per group).</p

Real-time analysis of passage of fluorescent dextran from the vascular to tissue compartment of B³C under shear flow.

<p>Compared to cell-free B³C (A) or RBEC alone (B), presence of ACM (C) and co-culture with rat astrocytes (D) improves barrier function of neonatal RBEC in B³C as detected by the passage of Texas Red 40 kDa dextran from the vascular channel to the tissue compartment of B³C under shear flow. The passage of Texas Red 40 kDa dextran was monitored and imaged by Fluorescent microscopy (Nikon TE200). Representative images acquired 60 min after the initiation of flow in the vascular channel (A-D). Quantification of permeability shows that RBEC cultured in the presence of ACM or RBEC co-cultured with astrocytes exhibit significant reduction in the permeability of 40 kDa dextran compared to RBEC alone (E). Coefficient of variance of measured intensities of fluorescent dextran at 12 regularly spaced ROIs in the tissue compartment immediately adjacent to the vascular channel at 60 min after the initiation of the flow was used as an index of permeability heterogeneity. Variation in permeability is lowest in cell-free B³C but increases as the microenvironment of B³C becomes more realistic (F). (*** indicates significant difference p<0.001, one-way ANOVA with Tukey’s multiple comparison test, n = 3–4 experiments per group).</p

Passage of fluorescent dextran from the vascular channel to tissue compartment of B³C under shear flow.

<p>Permeability of Texas Red 40 kDa dextran from vascular channel to the tissue compartment in a cell-free B³C after 5 min (A), 15 min (B), 30 min (C), 60 min (D) and 120 min (E) from the initiation of flow in vascular channel. Normalized tissue intensity in a cell-free B³C increases linearly with time in the tissue compartment (F).</p

Neonatal RBEC cultured under flow in the vascular channel of B³C form a complete lumen.

<p>Full view of B³C device showing RBEC cultured in the two vascular channels (A). Magnified view of inset from panel (A) showing a section of the vascular channel with cultured RBEC (B). 3D reconstruction of confocal images of neonatal RBEC cultured in B³C stained with f-actin (green) and Draq5 (red) after 5 days in culture maintained under flow of 0.01 μl/min in RBEC medium (C)—(F); images are shown with a Y-axis rotation of 0, 140, 210 and 330 degrees in (C), (D), (E) and (F) respectively. All images were acquired after 5 days of 0.01 μl/min of flow of RBEC medium over cultured RBEC in the vascular channel of B³C.</p

Neonatal RBEC exhibit distinct barrier structure and function compared to adult RBEC in B³C.

<p>Neonatal RBEC alone (A) and neonatal RBEC + ACM (B) exhibit distinctly weaker and less organized ZO-1 staining (arrows) compared to adult RBEC alone (C) and adult RBEC + ACM (D). In the presence of ACM, neonatal RBEC exhibit discontinuous bands of ZO-1 staining (B) compared to neonatal RBEC in the absence of ACM where ZO-1 staining is discontinuous and granular (A). Presence of ACM has a significantly larger impact on both permeability (E) and electrical resistance (F) in neonatal RBEC compared to adult RBEC. Inset panels show higher magnification of white squared regions. (** indicates significant difference p<0.01, student’s t-test; n = 3–4 experiments per group).</p

A Spiroligomer α-Helix Mimic That Binds HDM2, Penetrates Human Cells and Stabilizes HDM2 in Cell Culture

<div><p>We demonstrate functionalized spiroligomers that mimic the HDM2-bound conformation of the p53 activation domain. Spiroligomers are stereochemically defined, functionalized, spirocyclic monomers coupled through pairs of amide bonds to create spiro-ladder oligomers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045948#pone.0045948-Schafmeister1">[1]</a>. Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound <b>1</b>, that binds HDM2 with a Kd value of 400 nM. The spiroligomer <b>1</b> penetrates human liver cancer cells through passive diffusion and in a dose-dependent and time-dependent manner <em>increases</em> the levels of HDM2 more than 30-fold in Huh7 cells in which the p53/HDM2 negative feed-back loop is inoperative. This is a biological effect that is not seen with the HDM2 ligand nutlin-3a. We propose that compound <b>1</b> modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop.</p> </div