Potential Role of Axonal Chemorepellent Slit2 in Modulating Adventitial Inflammation in a Rat Carotid Artery Balloon Injury Model

Abstract

Leukocyte infiltration of adventitial and perivascular tissues is an early event in the development of vascular remodeling after injury. We investigated whether Slit/Robo—an axonal chemorepellent system in vertebrate and invertebrate development—is activated during the inflammatory phase that follows endothelial denudation. Using the rat carotid artery model of angioplasty, we conducted a time course analysis of mRNAs encoding Slit ligands (Slit2 and Slit3) and Robo receptors (Robo1, Robo2 and Robo4), as well as proinflammatory cell adhesion molecule (CAM) genes. Adventitial inflammatory cells were counted in immunostained arterial sections. E-selectin, vascular CAM-1 (VCAM-1), and intercellular CAM-1 (ICAM-1) were upregulated 2–3 hr after injury, followed by infiltration of neutrophils and monocytes as evidenced by real-time PCR, in situ hybridization, and immunohistochemistry. Slit2, Slit3, and Robo genes exhibited no expression changes at 3 hr; however, they were markedly upregulated 1 day after angioplasty. ICAM-1 expression was reduced by 50%, and the number of adventitial neutrophils decreased by >75% one day after angioplasty. Slit2 has been shown to be a potent chemorepelent of leukocytes, endothelial cells and smooth muscle cells. Thus, we decided to further investigate the localization of Slit2 in injured vessels. Immunohistochemical stainings revealed the presence of Slit2 within the vessel wall and in the perivascular vasa vasorum of naive and injured arteries. Double immunohistochemical analyses showed that infiltrating monocytes expressed Slit2 in the perivascular and adventitial tissues of injured arteries 1 and 3 days postangioplasty. In addition, recombinant full-length Slit2 and Slit2-N/1118, an N-terminal fragment of Slit2, inhibited stromal cell-derived factor 1 (SDF-1)-mediated migration of circulating rat peripheral blood mononuclear cells. In summary, adventitial activation of CAM genes and neutrophil infiltration preceded upregulation of Slit/Robo genes. Sli2 expression colocalized with infiltrating inflammatory cells in the adventitial layer. This temporospatial association suggests that leukocyte chemorepellent Slit2 may be involved in halting the adventitial accumulation of inflammatory cells in injured vessels.

Different Isoforms of the Neuronal Guidance Molecule Slit2 Directly Cause Chemoattraction or Chemorepulsion of Human Neutrophils

“…4A). Pre-incubation with Slit2 isoforms inhibit the ability of fMLP or TNFα to increase adhesion of neutrophils to activated endothelial cells (19, 42, 43). Both Slit2-N and Slit2-S significantly inhibited fMLP-induced adhesion to fibronectin, and Slit2-S but not Slit2-N inhibited TNFα-induced adhesion (Fig.…”

Guidance Molecules in Vascular Smooth Muscle

“…Slit2 is highly expressed in vascular smooth muscle cells following vascular injury. Since Slit2 is secreted, it may induce a paracrine effect on neighboring Robo receptors ( Liu D. et al, 2016 ). However, Slit2 inhibits smooth muscle migration through inhibition of Rac and Cdc42 activity ( Liu et al, 2006 ).…”

“…However, Slit2 inhibits smooth muscle migration through inhibition of Rac and Cdc42 activity ( Liu et al, 2006 ). Since balloon angioplasty additionally leads to local recruitment of inflammatory cells and generalized endothelial activation ( Liu D. et al, 2016 ), this upregulation of Slit2 may be beneficial because Slit2 maintains endothelial barrier function ( London et al, 2010 ) and minimizes leukocyte recruitment ( Kanellis et al, 2004 ). Therefore, Slit–Robo signaling may be a potential therapeutic target in vascular injury.…”

“…Together these lead to the incidence of restenosis, a common complication with vascular intervention. Since vascular smooth muscle upregulates Slit2 following vascular injury and Slit2 inhibits vascular smooth muscle migration ( Liu et al, 2006 ; Liu D. et al, 2016 ), Slit2 may serve as a useful therapeutic for restenosis.…”

“…Vertebrates express four different Robo receptors (Robo1-4), and three different Slit ligands (Slit1-3). While all isoforms are highly expressed in the central and peripheral nervous systems ( Carr et al, 2017 ), only Slit2, Slit3, Robo1, Robo2, and Robo4 are expressed in rat carotid arteries ( Liu D. et al, 2016 ). Classically, Slit–Robo interactions provide a repulsive cue to maintain midline deviation of neuronal axons in CNS development ( Dickinson and Duncan, 2010 ; Barak et al, 2014 ).…”