Abstract-Development of vascular restenosis is a multifaceted process characterized by migration and proliferation of vascular smooth muscle cells (VSMCs), resulting in loss of lumen diameter. Characterization of proteins that mediate this process is essential in our understanding of the pathogenesis of arterial injury. Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding protein that is expressed in VSMCs by allograft and balloon angioplasty injury. AIF-1 is not present in cultured human VSMCs but is induced by cytokines, and overexpression of AIF-1 results in increased VSMC growth and cell-cycle gene expression. To characterize AIF-1 modulatory effects in primary human VSMCs, AIF-1-interacting proteins were identified by an AIF-1/glutathione S transferase fusion protein affinity assay. MALDI-TOF mass spectrophotometric amino analysis identified actin as an AIF-1 interacting protein. This interaction was verified by coimmunoprecipitation. This is a functional interaction, because AIF-1 binds to and polymerizes F-actin in vitro. In unstimulated VSMCs, AIF-1 colocalizes with F-actin but translocates to lamellipodia on stimulation with platelet-derived growth factor. VSMCs stably transduced with AIF-1 retrovirus migrate 2.6-fold more rapidly (85.1Ϯ2.9 versus 225.5Ϯ16.6; PϽ0.001) in response to platelet-derived growth factor versus control cells. AIF-1 colocalizes with Rac1, and AIF-1-transduced VSMCs show a constitutive and enhanced activation of Rac1, providing a mechanism for the increased migration. These data indicate that AIF-1 binds and polymerizes F actin and also regulates Rac1 activity and VSMC migration. Considering the AIF-1 expression pattern in injured arteries, this suggests that AIF-1 may be involved in the cytoskeletal signaling network leading to vascular remodeling.

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Objective Interleukin-19 (IL-19) is putative Th2, anti-inflammatory interleukin. Its expression in, and potential role in atherogenesis is unknown. IL-19 is not detected in normal artery, and is expressed to a greater degree in plaque from symptomatic vs. asymptomatic patients, suggesting a compensatory-counter regulatory function. We tested if IL-19 could reduce atherosclerosis in susceptible mice, and identified plausible mechanisms. Approach and Results LDLR−/− mice fed an atherogenic diet and injected with either 1.0ng/g/day or 10.0ng/g/day rmIL-19 had significantly less plaque area in the aortic arch compared with controls (p<0.0001). Weight gain, cholesterol and triglyceride levels were not significantly different. Gene expression in splenocytes from IL-19 treated mice demonstrated immune cell Th2 polarization, with decreased expression of T-bet, IFNγ, IL-1β and IL-12β, and increased expression of GATA3 and FoxP3 mRNA. A greater percentage of lymphocytes were Th2 polarized in IL-19 treated mice. Cellular characterization of plaque by immunohistochemistry demonstrated IL-19 treated mice have significantly less macrophage infiltrate compared with controls (p<0.001). Intravital microscopy revealed significantly less leukocyte adhesion in wild-type mice injected with IL-19 and fed an atherogenic diet compared with controls. Treatment of cultured endothelial cells (EC), vascular smooth muscle cells (VSMC), and bone marrow-derived macrophages (BMDM) with IL-19 resulted in a significant decrease in chemokine mRNA, and in the mRNA-stability protein HuR. Conclusions These data suggest IL-19 is a potent inhibitor of experimental atherosclerosis, with diverse mechanisms including immune cell polarization, decrease in macrophage adhesion, and decrease in gene expression. This may identify IL-19 as a novel therapeutic to limit vascular inflammation.

Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding, inflammation-responsive scaffold protein. Several studies have reported increased AIF-1 expression in activated macrophages and have implicated AIF-1 as a marker of activated macrophages. However, the function of AIF-1 in macrophages and the mechanism whereby it participates in macrophage activation are unknown at this time. Immunohistochemical analysis colocalized AIF-1 expression with CD68-positive macrophages in atherosclerotic human coronary arteries. Subsequent experiments were designed to determine a role for AIF-1 in macrophage activation in response to atherogenic stimuli. Stimulation of human and murine macrophages with oxidized LDL significantly increased AIF-1 expression above basal levels. Stable transfection of AIF-1 small interfering RNA (siRNA) in macrophages reduced AIF-1 protein expression by 79% and reduced macrophage proliferation by 52% (P < 0.01). Inhibition of proliferation was not due to induction of apoptosis. Sequences that did not knock down AIF-1 expression had no effect on proliferation. AIF-1 siRNA expression reduced macrophage migration by 60% (P < 0.01). Both proliferation and migration of siRNA-expressing macrophages could be restored by adenoviral expression of AIF-1 (P < 0.001 and 0.005, respectively), suggesting a tight association between AIF-1 expression and macrophage activation. Phosphorylation of Akt, p44/42 MAPK, and p38 kinase were significantly reduced in siRNA macrophages challenged with oxidized LDL (P < 0.05). Phosphorylation of p38 kinase was significantly inhibited in siRNA macrophages stimulated with T lymphocyte conditioned medium (P < 0.05). These data indicate that AIF-1 mediates atherogenesis-initiated signaling and activation of macrophages.