Expression of Lewis‐a glycans on polymorphonuclear leukocytes augments function by increasing transmigration

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141391/1/jlb0753.pd

Deposition of microparticles by neutrophils onto inflamed epithelium: a new mechanism to disrupt epithelial intercellular adhesions and promote transepithelial migration

Neutrophil [polymorphonuclear leukocyte (PMN)] transepithelial migration (TEM) is a hallmark of inflammatory mucosal disorders. PMN TEM is associated with epithelial injury; however, mechanisms involved in this process are not well defined. The current work describes a new mechanism whereby deposition of PMN membranederived microparticles (PMNâ MPs) onto intestinal epithelial cells (IECs) during TEM leads to loss of epithelial cadherins, thus promoting epithelial injury and increased PMN recruitment. PMNâ MPs secreted by activated PMNs during TEM displayed a high level of enzymatically activematrixmetalloproteinase 9 (MMPâ 9), and were capable of mediating potent effects on IEC integrity. Isolated PMNâ MPs efficiently bound to IEC monolayers and induced cleavage of desmogleinâ 2 (DSGâ 2) but not Eâ cadherin, leading to disruption of IEC intercellular adhesions. Furthermore, PMNâ MP binding to intestinal epithelium in vitro in transwell assays and in vivo in ligated intestinal loop preparations facilitated increases in PMN TEM. These effects were MMPâ 9 dependent and were reversed in the presence of specific pharmacological inhibitors. Finally, we demonstrated that IEC Dsgâ 2 serves as a barrier for migrating PMNs, and its removal by PMNâ MPâ associated MMPâ 9 facilitates PMNtrafficking across epithelial layers. Our findings thus implicate PMNâ MPs in PMNâ mediated inflammation and epithelial damage, as observed in inflammatory disorders ofmucosal surfaces.â Butinâ Israeli, V., Houser, M.C., Feng, M., Thorp, E. B., Nusrat, A., Parkos, C. A, Sumagin, R. Deposition of microparticles by neutrophils onto inflamed epithelium: anewmechanism to disrupt epithelial intercellular adhesions and promote transepithelialmigration. FASEB J. 30, 4007â 4020 (2016). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154234/1/fsb2fasebj30120734r.pd

Microvascular Endothelial Cells Exhibit Optimal Aspect Ratio for Minimizing Flow Resistance

A recent analytical solution of the three-dimensional Stokes flow through a bumpy tube predicts that for a given bump area, there exists an optimal circumferential wavenumber which minimizes flow resistance. This study uses measurements of microvessel endothelial cell morphology to test whether this prediction holds in the microvasculature. Endothelial cell (EC) morphology was measured in blood perfused in situ microvessels in anesthetized mice using confocal intravital microscopy. EC borders were identified by immunofluorescently labeling the EC surface molecule ICAM-1 which is expressed on the surface but not in the EC border regions. Comparison of this theory with extensive in situ measurements of microvascular EC geometry in mouse cremaster muscle using intravital microscopy reveals that the spacing of EC nuclei in venules ranging from 27 to 106 μm in diameter indeed lies quite close to this predicted optimal configuration. Interestingly, arteriolar ECs are configured to minimize flow resistance not in the resting state, but at the dilated vessel diameter. These results raise the question of whether less organized circulatory systems, such as that found in newly formed solid tumors or in the developing embryo, may deviate from the optimal bump spacing predicted to minimize flow resistance

Annexin A1–containing extracellular vesicles and polymeric nanoparticles promote epithelial wound repair

Epithelial restitution is an essential process that is required to repair barrier function at mucosal surfaces following injury. Prolonged breaches in epithelial barrier function result in inflammation and further damage; therefore, a better understanding of the epithelial restitution process has potential for improving the development of therapeutics. In this work, we demonstrate that endogenous annexin A1 (ANXA1) is released as a component of extracellular vesicles (EVs) derived from intestinal epithelial cells, and these ANXA1-containing EVs activate wound repair circuits. Compared with healthy controls, patients with active inflammatory bowel disease had elevated levels of secreted ANXA1-containing EVs in sera, indicating that ANXA1-containing EVs are systemically distributed in response to the inflammatory process and could potentially serve as a biomarker of intestinal mucosal inflammation. Local intestinal delivery of an exogenous ANXA1 mimetic peptide (Ac2-26) encapsulated within targeted polymeric nanoparticles (Ac2-26 Col IV NPs) accelerated healing of murine colonic wounds after biopsy-induced injury. Moreover, one-time systemic administration of Ac2-26 Col IV NPs accelerated recovery following experimentally induced colitis. Together, our results suggest that local delivery of proresolving peptides encapsulated within nanoparticles may represent a potential therapeutic strategy for clinical situations characterized by chronic mucosal injury, such as is seen in patients with IBD

Activation of PKCβII by PMA Facilitates Enhanced Epithelial Wound Repair through Increased Cell Spreading and Migration

<div><p>Rapid repair of epithelial wounds is essential for intestinal homeostasis, and involves cell proliferation and migration, which in turn are mediated by multiple cellular signaling events including PKC activation. PKC isoforms have been implicated in regulating cell proliferation and migration, however, the role of PKCs in intestinal epithelial cell (IEC) wound healing is still not completely understood. In the current work we used phorbol 12-myristate 13-acetate (PMA), a well recognized agonist of classical and non-conventional PKC subfamilies to investigate the effect of PKC activation on IEC wound healing. We found that PMA treatment of wounded IEC monolayers resulted in 5.8±0.7-fold increase in wound closure after 24 hours. The PMA effect was specifically mediated by PKCβII, as its inhibition significantly diminished the PMA-induced increase in wound closure. Furthermore, we show that the PKCβII-mediated increase in IEC wound closure after PMA stimulation was mediated by increased cell spreading/cell migration but not proliferation. Cell migration was mediated by PKCβII dependent actin cytoskeleton reorganization, enhanced formation of lamellipodial extrusions at the leading edge and increased activation of the focal adhesion protein, paxillin. These findings support a role for PKCβII in IEC wound repair and further demonstrate the ability of epithelial cells to migrate as a sheet thereby efficiently covering denuded surfaces to recover the intestinal epithelial barrier.</p> </div

PKCβII mediates PMA induced increase in wound closure.

<p>Confluent IEC monolayers were wounded by introduction of a single linear scratch wound, preincubated with the specified PKC inhibitors for 1 hour (Cheleretrine, pan PKC inhibitor (5 µM, pan); Calphostin C, Classical/non-conventional PKC inhibitor (10 µM, <i>Clas/Non-C</i>); KIC1-1, classical PKC inhibitor (5 µM, Clas); KIBI31-1, PKCβI inhibitor (1 µM, PKCβI); KIBII31-1 (5 µM, PKCβII); KIG31-1, PKCγ inhibitor (5 µM, PKCβγ) and stimulated with PMA (200nM). The area of the wound was measured at 12 and 24 hours. Inhibition of PKCβII, but not inhibition of other members of the classical PKCs significantly diminished PMA enhanced wound closure. N = 3 independent experiments. **significantly different from control (PMA alone, p<0.01).</p

PKC activation with PMA enhances epithelial wound healing.

<p>(A) Confluent T84 IEC monolayers were wounded by introduction of a single linear scratch wound. Wound closure was measured over 48 h in unstimulated (control) and PMA activated IEC monolayers as detailed in the methods section. (B) Representative images of unstimulated (control, left panels) and PMA activated (PMA, 200 nM right panels) IEC monolayers immediately after wounding (0 h, upper panels) and after 48 hours (48 h, bottom panels). PMA treatment induced a dramatic increase in wound closure as early as 12 and 24 hours. The bar is 50 µm. N = 4 independent experiments. **significantly different from each other (p<0.01).</p

PKCβII mediates extension of cell membrane protrusions in response to PMA stimulation.

<p>Scratch wounded cell monolayers were allowed to migrate for 4 hours in the absence (control) pr presence of PMA (PMA). (<b>A</b>) Representative images demonstrating the orientation of the Golgi apparatus in migrating cells at the wound edge with or without PMA activation. IECs were then fixed and stained for Golgi (red) and nuclei (blue). The bar is 20 µm. (<b>B</b>) Cell orientation in at least 8 randomly selected fields of N = 4 independent experiments were quantified as described in methods. No significant difference was observed in unstimulated versus PMA activated wounded monolayers. White arrows show correctly oriented migrated IECs. The bar is 20 µm. (<b>C</b>) IECs were fixed and stained for F-actin (green) and nuclei (blue). Representative images depict extended lamellipodium formation after PMA treatment, which was prevented by inhibition of PKCβII. (<b>D</b>) The area of the lamellipodium was defined as area past the nuclei of leading edge cells, as indicated by the white dashed line and quantified following the indicated treatment. At least 5 random fields per each condition were analyzed. N = 4 independent experiments. **significantly different (p<0.01).</p