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.

Objective-To characterize the expression and function of interleukin (IL) 19, a recently described T-helper 2 anti-inflammatory IL, on endothelial cell (EC) pathophysiological features.

Objective-To determine whether the ␤1 integrin/caveolin-1 signaling complex plays a role in shear stress regulation of RhoA activity. Methods and Results-Hemodynamic shear stress influences the phenotype of the endothelium. Integrins and RhoA are essential components in the process that allows endothelial cells to adapt to flow. However, the signaling mechanisms that relay from integrins to RhoA are not well defined. Bovine aortic endothelial cells were subjected to laminar shear stress (10 dyne/cm 2 ) for up to 6 hours. ␤1 integrin blockade inhibited Src family kinases and p190RhoGAP tyrosine phosphorylation observed after the immediate onset of shear stress. Depletion of caveolin-1 blocked the decline in p190RhoGAP tyrosine phosphorylation observed at later points by sustaining Src family kinase activity. The manipulation of ␤1 integrin and caveolin-1 also altered shear regulation of RhoA activity. More importantly, cells depleted of p190RhoGAP showed faulty temporal regulation of RhoA activity. Each of these treatments attenuated actin reorganization induced by flow. Similarly, stress fibers failed to form in endothelial cells exposed to enhanced blood flow in caveolin-1 knockout mice. Key Words: blood flow Ⅲ endothelium Ⅲ signal transduction Ⅲ vascular biology Ⅲ caveolae Ⅲ integrins Ⅲ mechanotransduction T he hemodynamic environment in which an endothelial cell resides strongly influences cell morphological features through regulation of cytoskeletal structures. 1 Several, now classic, studies [2][3][4][5] illustrate that in vitro and in vivo, actin stress fibers orient parallel to the direction of flow and are prominent in endothelial cells subjected to high shear velocities. These actin bundles reflect an adaptive response to shear stress that may aid endothelial cells in withstanding elevated hemodynamic stress, as in the case of hypertension. Conclusion-OurAlthough the influence of flow on regulating endothelial cell phenotype is widely recognized, the fundamental process by which these cells detect and transduce fluid mechanical forces into biochemical signals is not completely clear. Previous studies have described individual cellular elements with potential mechanosignaling properties, including ion channels, 6 integrins, 7 the glycocalyx, 8 cilia, 9 platelet endothelial cell adhesion molecule-1 (PECAM-1), 10 various receptors for humoral compounds, 11 the cytoskeleton, 12 and the plasma membrane, including lipid raft and caveolar subdomains. 13,14 Interestingly, many of these link to similar sets of second messenger signaling molecules and regulate the same flow responses, such as endothelial NO synthase regulation of NO production and events downstream of extracellular signal-regulated kinase activation. These observations suggest that primary mechanosignaling elements likely engage in a substantial degree of interaction and cross talk. Indeed, recent findings show that integrin/ vascular endothelial growth factor receptor 2 15 and PECAM-1/ vascular endothelial-cadherin 16 associations form importan...

Hypoxia in ischemic limbs typically initiates angiogenic and inflammatory factors to promote angiogenesis in attempt to restore perfusion. There is a gap in our knowledge concerning the role of anti-inflammatory interleukins in angiogenesis, macrophage polarization, and endothelial cell activation. Interleukin-19 is a uniquely anti-inflammatory Th2 cytokine that promotes angiogenic effects in cultured endothelial cells (EC); the purpose of this study was to characterize a role for IL-19 in restoration of blood flow in hind-limb ischemia, and define potential mechanisms. Hindlimb ischemia was induced by femoral artery ligation, and perfusion quantitated using Laser Doppler Perfusion Imaging (LDPI). Wild type mice which received i.p. injections of rIL-19 (10ng/g/day) showed significantly increased levels of perfusion compared to PBS controls. LDPI values were significantly decreased in IL-19−/− mice when compared to wild type mice. IL-19−/− mice injected with rIL-19 had significantly increased LDPI compared with PBS control mice. Significantly increased capillary density was quantitated in rIL-19 treated mice, and significantly less capillary density in IL-19−/−. Multiple cell types participate in IL-19 induced angiogenesis. IL-19 treatment of human microvascular EC induced expression of angiogenic cytokines. M2 macrophage marker and VEGF-A expression were significantly increased in macrophage and spleen from rIL-19 injected mice, and M1 marker expression was significantly increased in spleen from IL-19−/− compared with controls. Plasma VEGF-A levels are higher in rIL-19 injected mice. IL-19 decreased expression of anti-angiogenic IL-12 in spleen and macrophage. This study is the first to implicate IL-19 as a novel pro-angiogenic interleukin and suggests therapeutic potential for this cytokine.

Atherosclerosis regression is an important clinical goal, and treatments that can reverse atherosclerotic plaque formation are actively being sought. Our aim was to determine whether administration of exogenous IL-19, a Th2 cytokine, could attenuate progression of preformed atherosclerotic plaque and to identify molecular mechanisms. LDLR(-/-) mice were fed a Western diet for 12 weeks, then administered rIL-19 or phosphate-buffered saline concomitant with Western diet for an additional 8 weeks. Analysis of atherosclerosis burden showed that IL-19-treated mice were similar to baseline, in contrast to control mice which showed a 54% increase in plaque, suggesting that IL-19 halted the progression of atherosclerosis. Plaque characterization showed that IL-19-treated mice had key features of atherosclerosis regression, including a reduction in macrophage content and an enrichment in markers of M2 macrophages. Mechanistic studies revealed that IL-19 promotes the activation of key pathways leading to M2 macrophage polarization, including STAT3, STAT6, Kruppel-like factor 4, and peroxisome proliferator-activated receptor γ, and can reduce cytokine-induced inflammation in vivo. We identified a novel role for IL-19 in regulating macrophage lipid metabolism through peroxisome proliferator-activated receptor γ-dependent regulation of scavenger receptor-mediated cholesterol uptake and ABCA1-mediated cholesterol efflux. These data show that IL-19 can halt progression of preformed atherosclerotic plaques by regulating both macrophage inflammation and cholesterol homeostasis and implicate IL-19 as a link between inflammation and macrophage cholesterol metabolism.