Transendothelial migration (TEM), or diapedesis, is the step in which leukocytes squeeze between tightly apposed endothelial cells that line the post-capillary venules at sites of inflammation. Most of the good, the bad, and the ugly of inflammation occurs after leukocytes cross blood vessels. A thorough understanding of the molecules and mechanisms that regulate TEM should therefore enhance our ability to control the process therapeutically. Platelet/endothelial cell adhesion molecule-1 (PECAM) is a transmembrane glycoprotein expressed on the surfaces of leukocytes and platelets and concentrated at the borders between endothelial cells (EC). Homophilic interaction between PECAM on the leukocyte and PECAM at the endothelial cell border is responsible, in most cases, for initiating diapedesis. Other processes that promote efficient TEM include a transient increase in cyotosolic free calcium ion concentration (?[Ca2+]i ) and recruitment of the lateral border recycling compartment (LBRC) to the site of TEM. The LBRC is a network of membrane just below the junctional surface of the EC that serves as a reservoir of lateral border membrane. It is moved to the site of TEM along microtubules by kinesin-1 and a single splice variant of kinesin light chain 1 (KLC1c). We recently found that the scaffolding molecule IQGAP1 is physically associated with the LBRC and plays a role in targeted recycling of LBRC to the site of TEM. Blocking PECAM-PECAM interactions, ?[Ca+2]i , or targeted recycling blocks TEM. Leukocytes are arrested on the luminal surface of EC over the cell borders, actively probing but unable to transmigrate. Our overarching hypothesis is that PECAM initiates TEM by starting a set of signals that recruit the LBRC to the site of TEM. In this application we will uncover the mechanisms linking these three major drivers of TEM.
Aim I will determine how PECAM signaling activates TRPC6. Based on preliminary data, we hypothesize that the same mechanosensory machinery, involving PECAM, VE-cadherin, and VEGFR2, on the EC that responds to fluid shear also responds to the physical engagement of EC PECAM by leukocyte PECAM. We will also study calcium signaling in EC in real time in vivo by intravital microscopy using mice with a genetically encoded calcium sensor. We will study the effect of TRPC6 deficiency in EC in two models of ischemia/reperfusion (I/R) injury in vivo by intravital microscopy and in a model of myocardial infarction (MI).
Aim II will determine the role of IQGAP1 in targeted recycling of the LBRC and TEM. We will identify the domains involved and the critical binding partners of those domains. We will also determine whether mice with a lacking IQGAP1 in nonhematopoietic cells are protected from I/R injury and MI in vivo.
Aim III will determine whether KLC1c is really the light chain that binds LBRC cargo during TEM. We will carry out proof of principle experiments to test whether specifically targeting the LBRC with cell-permeable KLC1c peptide will block TEM in vivo in the same I/R injury models as in the other Aims.
Most diseases are due to inflammation that is maladaptive?damaging host tissues. The vast majority of the damage takes place after white blood cells cross blood vessels (transendothelial migration) to enter the inflamed tissues. We are studying how this process is regulated on the molecular level so that we can design better anti-inflammatory therapies to control it.
|Sullivan, David P; Bui, Triet; Muller, William A et al. (2018) In vivo imaging reveals unique neutrophil transendothelial migration patterns in inflamed intestines. Mucosal Immunol 11:1571-1581|
|Early, Merideth; Schroeder, William G; Unnithan, Ranajana et al. (2017) Differential effect of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) on leukocyte infiltration during contact hypersensitivity responses. PeerJ 5:e3555|
|Mei, Yang; Feng, Gong; Rahimi, Nina et al. (2017) Loss of mDia1 causes neutropenia via attenuated CD11b endocytosis and increased neutrophil adhesion to the endothelium. Blood Adv 1:1650-1656|
|Weber, Evan W; Muller, William A (2017) Roles of transient receptor potential channels in regulation of vascular and epithelial barriers. Tissue Barriers 5:e1331722|
|DeBerge, Matthew; Yeap, Xin Yi; Dehn, Shirley et al. (2017) MerTK Cleavage on Resident Cardiac Macrophages Compromises Repair After Myocardial Ischemia Reperfusion Injury. Circ Res 121:930-940|
|Muller, William A (2016) Transendothelial migration: unifying principles from the endothelial perspective. Immunol Rev 273:61-75|
|Cyrus, Bita F; Muller, William A (2016) A Unique Role for Endothelial Cell Kinesin Light Chain 1, Variant 1 in Leukocyte Transendothelial Migration. Am J Pathol 186:1375-86|
|Gonzalez, Annette M; Cyrus, Bita F; Muller, William A (2016) Targeted Recycling of the Lateral Border Recycling Compartment Precedes Adherens Junction Dissociation during Transendothelial Migration. Am J Pathol 186:1387-402|
|Muller, William A (2016) Localized signals that regulate transendothelial migration. Curr Opin Immunol 38:24-9|
|Muller, William A (2016) How monocytes guard the glomerulus. Proc Natl Acad Sci U S A 113:10453-5|
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