BACKGROUND: Despite aggressive dietary modification, lipid lowering medications, and other medical therapy, vascular proliferative diseases continue to account for 50% of all mortality in the United States. It is a significant medical and socioeconomic problem contributing to the mortality of multiple diseases including myocardial infarction (MI), stroke, renal failure, and peripheral vascular disease. With a growing number of children becoming obese and an increase in the number of patients with co-morbidities such as metabolic syndrome and Type 2 diabetes mellitus, epidemiological studies project the morbidity and mortality of these diseases to increase. Among these vascular proliferative diseases are primary atherosclerosis, vascular restenosis, and allograft vasculopathy, all of which are the result of chronic inflammation believed to stem from initial endothelial injury. Once activated by any number of potential injurious agents, endothelial cells (ECs) secrete cytokines that act on multiple cell types. Stimulation of resident vascular smooth muscle cells (VSMCs) results in a phenotypic switch from a normally contractile state to a proliferative state. Following this phenotypic shift, VSMCs migrate from the media to the intima of the artery where they begin secretion of both pro- and anti-inflammatory cytokines. Vascular proliferative disease ensues as a result of the autocrine and paracrine signaling of these cytokines between many different cell types including ECs, VSMCs, macrophages, and T-cells. As a result of the integral role pro- and anti-inflammatory cytokines play in the development of vascular proliferative diseases, they have become the subject of intense study in the field of cardiovascular research. Interleukin-19 (IL-19) is a newly described member of the IL-10 sub-family of anti-inflammatory cytokines. Discovered in 2000, it was originally only thought to be basally expressed in monocytes and lymphocytes, however in 2005 our lab discovered that while uninjured arteries have no detectable IL-19, arteries of patients with vascular proliferative diseases have notable IL-19 expression. Since its discovery in multiple cell types of injured arteries, our lab has subsequently shown that IL-19 inhibits proliferation, migration, spreading, production of reactive oxygen species (ROSs), and expression of pro-inflammatory genes in VSMCs, while in ECs IL-19 has been shown to promote angiogenesis, proliferation, migration, and spreading. AIMS and HYPOTHESIS: The first aim of the current study is to show that IL-19 is expressed in atherosclerotic plaque, and to test that IL-19 can reduce experimental atherosclerosis in susceptible mice. The second aim of the study is to show that IL-19 can regulate development of intimal hyperplasia in a murine model of restenosis. For both aims, we sought to identify potential intracellular signaling mechanisms of IL-19 which produce the observed effect. These aims directed our overall hypothesis that the anti-inflammatory properties of IL-19 can attenuate the vascular response to injury in various animal models of vascular proliferative disease. METHODS and RESULTS: The first aim of this dissertation showed that 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 and serum lipid levels were not significantly different. IL-19 could halt, but not reverse expansion of existing plaque. Gene expression in splenocytes from IL-19 treated mice demonstrated immune cell Th2 polarization, with decreased expression of T-bet, IFNgamma, IL-1β and IL-12β, and increased expression of GATA3 messenger ribonucleic acid (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 EC, 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. In the second aim of this dissertation we showed that IL-19 attenuates vascular restenosis in response to carotid artery ligation. Carotid artery ligation of hyper-responsive friend leukemia virus B (FVB) wild-type mice injected with 10ng/g/day rIL-19 had significantly lower neointima/media ratio (I/M) compared with phosphate buffered saline (PBS) controls (p=0.006). Conversely, carotid artery of IL-19-/- mice demonstrated significantly higher I/M ratio compared with wild-type mice (p=0.04). Importantly, the increased I/M ratio in the knockout mice could be rescued by injection of 10ng/g/day IL-19 (p=0.04). VSMC explanted from IL-19-/- mice proliferated significantly more rapidly compared with wild-type (p=0.04). Surprisingly, in this model, IL-19 does not modulate adoptive immunity. Rather, addition of IL-19 to cultured wild-type VSMC did not significantly decrease VSMC proliferation, but could rescue proliferation in IL-19-/- VSMC to wild-type levels (p=0.02). IL-19-/- VSMC expressed significantly greater levels of inflammatory mRNA including IL-1β, TNFα, and MCP-1 in response to TNFα stimulation (p<0.01 for all). No polarization of adaptive immunity was noted in these mice. CONCLUSIONS: These data are the first to report that IL-19 is a potent inhibitor of experimental atherosclerosis via diverse mechanisms including immune cell polarization, decrease in macrophage adhesion, and decrease in gene expression. In addition, these data are also the first to show that IL-19 plays a previously unrecognized protective role in vascular restenosis. Together, these data suggest IL-19 is both anti-atherogenic and anti-restenotic and may identify IL-19 as a novel therapeutic to limit vascular inflammation. / Physiology

Objective: To test the hypothesis that loss of IL-19 exacerbates atherosclerosis. Approach and Results: Il19-/- mice were crossed into Ldlr-/- mice. Double knockout (dKO) mice had increased plaque burden in aortic arch and root compared to Ldlr-/- controls after 14 weeks of high fat diet (HFD). In a rescue study, dKO mice injected i.p. with 10ng/g/day of IL-19 had significantly less plaque burden compared to saline controls. Quantitative RT-PCR and western blot analysis revealed dKO mice had increased global and intraplaque polarization of T cells and macrophages to pro-inflammatory phenotypes, and also significantly increased TNFa expression in spleen and aortic arch compared to Ldlr-/- controls. Results from bone marrow transplantation experiments suggest immune cells participate in IL-19 mediated atheroprotection. Bone marrow derived macrophages (BMDMs) and vascular smooth muscle cells (VSMCs) isolated from dKO mice had significantly greater expression of TNFa mRNA and protein compared to controls. Importantly from a mechanistic standpoint, spleen and aortic arch from dKO mice had significantly increased expression of the mRNA stability protein Human antigen R (HuR). BMDMs and VSMCs isolated from dKO mice also had greater HuR abundance. HuR stabilizes pro-inflammatory transcripts by binding AU-rich elements (AREs) in the 3’ untranslated region (UTR). Cytokine and HuR mRNA stability were increased in dKO BMDMs and VSMCs compared to controls, which was rescued by addition of IL-19 to these cells. IL-19 induces expression of miR133a, which targets and reduces HuR abundance; miR133a levels were lower in dKO mice compared to controls. Conclusions: These data indicate that IL-19 is an atheroprotective cytokine that decreases abundance of HuR, leading to reduced inflammatory mRNA stability. / Biomedical Sciences

BACKGROUND: Atherosclerotic vascular disease is a significant medical and socioeconomic problem and contributes to mortality in multiple diseases including myocardial infarction (MI), stroke, renal failure, and peripheral vascular disease. Atherosclerosis, as well as other vascular diseases including post-intervention restenosis and allograft vasculopathy, is known to be driven by chronic inflammation and, consequently, pro- and anti-inflammatory cell signaling molecules have been an important target of cardiovascular research. Interleukin (IL)-19 is a recently discovered member of the IL-10 family of anti-inflammatory cytokines. IL-19 is expressed in injured vascular cells, including vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), where it exerts an anti-inflammatory effect. In VSMCs, IL-19 signaling results in inhibition of proliferation, migration, spreading, production of reactive oxygen species (ROSs), and expression of pro-inflammatory genes. In ECs, IL-19 signaling is pro-angiogenic and results in increased EC proliferation, migration, and spreading. AIMS and HYPOTHESIS: The goal of the present study was to explore the hypothesis that IL-19 mediates anti-inflammatory effects on vascular cells by inhibiting the expression of pro-inflammatory genes, such intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, IL-1β, IL-8, and monocyte chemotactic protein (MCP)-1, through modulation of the mRNA stability factor HuR by post- transcriptional (e.g., microRNA) and post-translational (e.g., serine phosphorylation) mechanisms. METHODS and RESULTS: We found that IL-19 can significantly inhibit tumor necrosis factor (TNF)-α-driven ICAM-1 and VCAM-1 mRNA and protein abundance in cultured human coronary artery ECs (p < 0.01). IL-19 treatment of ECs, but not monocytes, significantly inhibited monocyte adhesion to cultured EC monolayers (p < 0.01). In wild-type mice, systemic administration of IL-19 significantly reduced TNF-α-induced leukocyte rolling and adhesion as quantitated by intravital microscopy (p < 0.05). IL-19 failed to inhibit TNF-α-induced nuclear factor (NF)-κB activation in ECs. IL-19 inhibited nuclear-to-cytoplasmic translocation of HuR and significantly reduced mRNA stability of ICAM-1 and VCAM-1 (p < 0.01 ). IL-19 significantly inhibited serine-phosphorylation of HuR, which is required for its translocation, and significantly increased expression of the putative HuR regulator microRNA (miR)-133 in VSMCs. CONCLUSIONS: These data are the first to report that IL-19 can reduce leukocyte-EC interactions, and to propose reduction in HuR-mediated mRNA stability of ICAM-1 and VCAM-1 as a mechanism. We conclude that expression of IL-19 by ECs and VSMCs may represent an auto-regulatory mechanism to promote resolution of the vascular response to inflammation. These results suggest that IL-19 is anti-inflammatory in vascular cells and, therefore, may be of therapeutic value in atherosclerotic vascular disease. / Physiology

Angiogenesis is an important process in maintaining normal physiology as well as in the pathology of many diseases. Angiogenesis based therapies have the potential to have a phenomenal impact on a diseases affecting more than one billion people worldwide, including all cancers, cardiovascular disease, blindness, arthritis, complications of AIDS, diabetes, Alzheimer's disease, and more than 70 other major health conditions affecting children and adults, in developed and developing nations. In this study, we investigate the role of Interleukin-19 (IL-19) and Allograft inflammatory factor-1 (AIF-1) in endothelial cells (EC) proliferation, migration, activation and angiogenic potential. IL-19 is a recently described member of the IL-10 family of anti-inflammatory cytokines. Nothing has been reported on the expression or mechanism(s) of IL-19 effects in endothelial cells. We have found that IL-19 is expressed in aortic endothelium, and can be induced in cultured EC by serum and inflammatory cytokine challenge. IL-19 is chemotactic for EC, and promotes cell spreading, migration, and wound healing. IL-19 pretreatment also enhances the migration of EC to Vascular endothelial growth factor (VEGF). IL-19 activates the signaling proteins STAT3, p44/42 MAPK, and Rac1, and induces expression of Matrix Metalloproteinase-2, which is obligate for EC migration. IL-19 promotes tube -like structure formation on Matrigel by Human Umbilical Vein EC (HUVEC), and promotes microvessel formation in mouse aortic ring assay. Taken together, these data suggest that IL-19 is a chemotactic cytokine for, and promotes the proliferation, migration, activation and angiogenic potential of endothelial cells. AIF-1 is a cytoplasmic, calcium-binding, inflammation-responsive scaffold protein that has been implicated in the regulation of inflammation. The expression and function of AIF-1 in EC is uncharacterized. AIF-1 expression co-localized with CD31-positive endothelial cells in neointima of inflamed human arteries, but not normal arteries. AIF-1 is detected at low levels in unstimulated EC, but expression can be increased in response to serum and soluble factors. Stable transfection of AIF-1 siRNA in EC reduced AIF-1 protein expression by 73%, and significantly reduced EC proliferation and migration. Rescue of AIF-1 expression restored both proliferation and migration of siRNA expressing ECs, and AIF-1 over expression enhanced both of these activities, suggesting a strong association between AIF-1 expression and EC activation. Activation of MAPK p44/42 and PAK1 was significantly reduced in siRNA ECs challenged with inflammatory stimuli. Reduction of AIF-1 expression did not decrease EC tube-like structure or microvessel formation from aortic rings, but over-expression of AIF-1 did significantly increase the number and complexity of these structures. These data indicate that AIF-1 expression plays an important role in proliferation, migration, signal transduction, and may have a role in angiogenesis. / Microbiology and Immunology

Cardiovascular disease is the leading cause of mortality in the western world. The pro-inflammatory and pro-proliferative etiology of vascular proliferative diseases is well characterized, while much less is known about the mechanisms of anti-inflammatory and anti-proliferative processes. Interleukin-19 (IL-19) is a newly described member of the IL-10 family of anti-inflammatory interleukins, and our group was the first to discover IL-19 expression in activated, synthetic, but not quiescent, contractile human vascular smooth muscle cells (hVSMC). We also found that IL-19 is anti-inflammatory and anti-proliferative for hVSMC. IL-19 is able to reduce the abundance of COX-2, IL-1β, IL-8, and Cyclin D1 transcripts which contain AU-rich elements (ARE) in their 3'-untranslated regions (3'-UTR). IL-19 is able to reduce the abundance of HuR, a stabilizing RNA-binding protein, which we feel provides a mechanism for these effects. The overall goal of this study is to elucidate IL-19's anti-inflammatory and anti-proliferative mechanism(s) in hVSMC in the context of vascular proliferative diseases. This goal has directed our overall hypothesis: IL-19's anti-proliferative and anti-inflammatory effects in hVSMC are mediated, at least in part, by modulation of HuR abundance and translocation, resulting in decreased stability of mRNA transcripts. HuR functions through a translocation mechanism, and IL-19 is able to reduce HuR cytoplasmic abundance. IL-19 also reduces HuR phosphorylation, which is a pre-requisite for HuR translocation, possibly through a PKCα-dependent mechanism. The stability of ARE-containing transcripts is reduced with IL-19 treatment, and reducing HuR expression by siRNA has the same inhibitory effect. VSMC are important mediators in the initiation of atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) is able to induce IL-19 expression in these cells. VSMC are known to express scavenger receptors that take up ox-LDL. IL-19 is able to reduce the uptake of ox-LDL and the abundance of ox-LDL induced LOX-1 and CX-CL16 scavenger receptors. Interestingly, these scavenger receptors also have ARE in their 3'-UTR. IL-19 is able to reduce ox-LDL induced HuR cytoplasmic abundance. HuR knockdown by siRNA reduces the uptake of ox-LDL by hVSMC. These data suggest that IL-19 reduced scavenger receptor abundance may be due to decreased total and cytoplasmic HuR abundance. IL-19 reduces the abundance of ox-LDL induced COX-2 expression. Taken together, these results demonstrate that IL-19 down-regulates vital steps in vascular proliferative disease processes through an HuR-dependent mechanism. / Molecular and Cellular Physiology