Michael V. Autieri - US grants

DESCRIPTION (Applicant's abstract): Vascular restenosis induced by arterial trauma is one of the most critical factors which limits the success o solid organ transplantation and coronary interventional procedures. A popular hypothesis is that the cytokine-induced activation and proliferation of VSMC in the media, culminating in intimal hyperplasia, is the most critical cellular event in formation of both cardiac allograft vasculopathy (CAV) and balloon angioplastyinduced restenosis. Identification and functional characterization gene products involved in VSMC activation is a promising approach for the identification of targets to combat proliferative arteriopathy observed in vascular proliferative disorders. Our hypothesis is that allograft inflammatory factor-I (AIF-1) promotes development of vascular proliferative disease based on its ability to respond to inflammatory cytokines and participate in the growth stimulatory pathways leading to proliferation of VSMC. We have recently shown that modulation of AIF- 1 levels in human VSMC impacts the growth of these cells. The first aim of this project will determine the mechanism of AIF-l growth promoting effects in human VSMC through a combination of flow cytometric analysis and investigation of expression and turnover of cell cycle-associated proteins. We will also identify regions that mediate these effects by site-specific modification of the AIF- 1 protein. We have determined that AIF- 1 partners with several cytoplasmic proteins, including a newly described growth factor-activated lipid kinase termed LCBK5. The second aim of this proposal will determine the functional significance of the AIF- 1-LCBK5 interaction and characterize the other AIF-1-interacting peptides we have identified. Expression of AIF- 1 transcript is induced in mitogen-stimulated peripheral blood lymphocytes (PBL), and its expression in endomyocardial biopsies from transplanted hearts correlates with ISHLT rejection scores. A final aim of this proposal will correlate AIF-1 transcript levels in endomyocardial biopsies and PBL from heart transplant recipients with development of arteriopathy as determined by several clinical and imaging indices. It is anticipated that completion of these studies will implicate expression of this novel protein as a target of anti-restenotic therapy and a surrogate marker of transplant restenosis.

[unreadable] DESCRIPTION (provided by applicant): As part of the VSMC response to injury, VSMC migrate into the lumen of the vessel where they proliferate and synthesize cytokines which they respond to in an autocrine fashion, sustaining the loss of lumen diameter. In the course of HL63810, we have determined that expression of Allograft Inflammatory Factor-1 (AIF-1), a newly described cytoplasmic, calcium-binding protein, is predictive of development of clinical transplant vasculopathy. Expression of AIF-1 in injured carotid artery significantly exacerbates reduction of lumen diameter and recruits bone marrow cells to the adventitia. Knock-down of AIF-1 abrogates neointimal hyperplasia. Expression of AIF-1 in human VSMC increases migration, proliferation, induces expression of G-CSF, and activates the Pad GTPase. AIF-1 contains several signaling domains, binds to and polymerizes actin, and activates signal transduction proteins. Our data support our central hypothesis that AIF-1 is an inflammation-responsive scaffold protein that plays a key role in regulation of VSMC activation and development of neointimal hyperplasia. The overall goals of this application are to 1- determine a cause and effect relationship between AIF-1 expression and neointimal hyperplasia in vivo, and 2- characterize the cellular pathways and molecular mechanisms responsible for AIF-1 activity in VSMC. Three aims have been formulated to test the hypothesis that; 1- AIF-1 expression exacerbates neointimal hyperplasia, and that reduction of AIF-1 expression will reduce neointimal hyperplasia in angioplasty-injured rats and AIF-1 transgenic mice, 2- that AIF-1 directly activates Rac1 signaling pathways leading to G-CSF expression, and that AIF-1 directly activates G-CSF expression with a subsequent autocrine activation of the Rac signaling pathway, and 3- AIF-1 activates Rac1 by activation of GEFs, and induces G-CSF expression by activation of transcription factors. Characterization of AIF-1 function will clarify our understanding of inflammation-mediated signal transduction leading to VSMC pathobiology and vascular- immune cell cross talk. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The development of multiple vascular diseases ranging from atherosclerosis to transplant vasculopathy are inflammatory in nature. Although much is known about the deleterious effects of pro-inflammatory cytokines on vascular smooth muscle cells (VSMC) pathophysiology, we know very little about the direct protective effects of anti-inflammatory cytokines on VSMC. Our overall hypothesis is that IL -19 plays a protective role in the vascular response to injury by direct inhibitory effects on VSMC activation. IL-19 is a recently described member of the IL -10 family of anti-inflammatory cytokines. IL-19 expression is ascribed to be restricted to hematopoetic and inflammatory cells, where it has an anti-inflammatory effect. Nothing has been reported on the mechanism(s) of IL-19 effects, either in immune or vascular cells. We have found that; IL-19 is not expressed in quiescent VSMC or normal arteries, but is induced in VSMC by inflammatory cytokines and in arteries by injury; IL -19 is anti-proliferative for cultured, human coronary artery VSMC, induces activation of STAT-3; inhibits activation of signal transduction MAPK and expression of proliferative and inflammatory genes. IL -19 induces expression of the suppressor of cytokine signaling 5 (SOCS5), but inhibits expression and translocation of HuR, a stability factor which regulates decay of inflammatory and proliferative gene mRNA. IL-19 adenoviral gene transfer significantly reduces neointimal formation and VSMC proliferation in balloon angioplasty-injured rat carotid arteries. The overall goals of this application are designed to characterize the mechanism of IL -19 suppressive effects on VSMC and development of progression of intimal hyperplasia in response to vascular injury. We will test the hypothesis that STAT3 activation, SOCS5 expression, and HuR down-regulation are critical events in IL -19 mediated VSMC protection. We will test the hypothesis that IL-19 has protective effects on VSMC by decreasing expression of proliferative and inflammatory genes, and will define the mechanism(s) of these effects. We will test the hypothesis that IL -19 anti-restenotic effects in vivo are due at least in part by attenuation of inflammatory and proliferative gene expression, mediated by expression of SOCS5 and down regulation of HuR. PUBLIC HEALTH RELEVANCE: Cardiovascular disease is the number one cause of mortality in the United States and places an enormous medical and economic burden on our society. This application will address the novel concept of direct beneficial effects of anti- inflammatory cytokines on vascular pathophysiology.

DESCRIPTION (provided by applicant): The overall goals of this application are to demonstrate that Interleukin-19 (IL-19), a Th2 anti- inflammatory interleukin, can attenuate atherosclerosis, and identify the potential mechanisms of this inhibition. IL-19 is a newly described Th2, (T regulatory) anti-inflammatory interleukin which until our work, had been ascribed to be inflammatory cell-specific. We remain the only laboratory to investigate a role for this interleukin in vascular biology, particularly with respect to EC and VSMC pathophysiology, and to demonstrate molecular mechanisms for these effects. We previously reported that; 1- IL-19 is not detectible in normal artery, but is induced in EC and VSMC in human atherosclerotic lesions; 2- addition of IL-19 to VSMC reduces their migration, proliferation, and abundance of proliferative and inflammatory proteins; 3- IL-19 does NOT inhibit NF-kB, but does reduce the stability of inflammatory and proliferative mRNA transcripts in an HuR-dependent manner; 4- IL-19 induces expression of the vascular and cyto-protective protein Hemeoxygenase-1 (HO-1), and reduces apoptosis induced by vascular reactive oxygen species (ROS) in an HO-1 dependent manner. In this application we present preliminary data showing that addition of recombinant IL-19 to LDLR-/- mice fed an atherogenic diet significantly and dramatically decreases atherosclerotic plaque, and IL-19-/- mice have an exacerbated response to ligation injury. Based on published and preliminary data, we hypothesize that there are multiple, pleiotropic mechanisms for these protective effects, and Specific Aims are designed to test each of these mechanisms. In Aim 1, we will determine if absence of IL-19 exacerbates, and if over expression attenuates atherosclerosis. Aim 2 will test the hypothesis that one mechanism of IL-19 protection is primarily facilitated by adoptive immune system polarization to Th2. Aim 3 will test the hypothesis that IL-19 atheroprotection is mediated by reduction in leukocyte-endothelial cell interaction, and/or IL-19 induction of HO-1 expression. Aim 4 will determine the molecular mechanisms of how IL- 19 decreases inflammatory gene abundance. This application is potentially paradigm-changing as it will implicate a Th2 interleukin as an endogenous cytokine expressed by inflamed vascular cells with multiple autocrine and paracrine dampening effects. It will identify novel molecular mechanisms and targets of anti-inflammatory pathways in these cells.

DESCRIPTION (provided by applicant): The overall goal of this application is to demonstrate that Interleukin-19 (IL-19), an anti-inflammatory, Th2 interleukin, can drive angiogenesis and improve perfusion of ischemic tissue. IL-19 is a newly described Th2, (T regulatory) anti-inflammatory interleukin which until our work, had been ascribed to be inflammatory cell-specific. We remain the only laboratory to investigate a role for this Interleukin in vascular biology, particularly with respect to EC and VSMC pathophysiology. Both inflammatory and anti- inflammatory cytokines participate in wound healing and neo-vascularization, but the role of anti- inflammatory cytokines in angiogenesis and the cross-talk between these processes remain under characterized. In contrast to our previous work indicating that IL-19 suppresses vascular smooth muscle cells (VSMC) migration and proliferation, we have recently reported the surprising finding that IL-19 has potent pro-angiogenic effects on human endothelial cells (EC). IL-19 is not detected in normal EC but is expressed in EC in capillaries in human angiogenic tissue. IL-19 is mitogenic and chemotactic for EC, promotes cell spreading, and activates MAPK and Rac1. IL-19 promotes microvessel formation in aortic rings, and PECAM1-positive microvessels in vivo. IL-19 can induce angiogenic gene expression in EC. IL-19 effects are independent of VEGF and bFGF, as neither can induce IL-19 expression, and IL-19 cannot induce expression of either. Neutralization of bFGF and VEGF does not affect IL-19 activity, suggesting a novel, Th2-induced pathway to stimulate EC activation and angiogenesis. IL-19 can polarize human macrophage to the M2, wound healing phenotype, and induce angiogenic gene expression in macrophage. IL-19 expression and function is reciprocal to and regulates the pro- inflammatory anti-angiogenic cytokine IL-12. These preliminary and published data have driven the hypothesis that IL-19 is a novel vasculogenic cytokine with multiple effector cells. What needs to be determined is if IL-19 can restore blood flow in ischemic tissue, if the major effector cell is endothelial cells or the M2 macrophage, what soluble factors mediate IL-19 effects, and the molecular mechanisms and mediators of IL-19 differential effects in EC and VSMC. We will determine if absence of IL-19 attenuates, and if over expression of IL-19 promotes angiogenesis and restores blood flow in ischemic tissue, if IL-19 regulation of angiogenesis is facilitated by direct effects on vascular cells, or by macrophage M2 polarization, will identify and characterize IL-19 inducible factors necessary for IL-19- driven angiogenesis in vivo, and test the hypothesis that differential expression of IL-20 receptor subunits account for pleiotropic effects of IL-19 in VSMC compared with EC. This application is potentially paradigm-changing as it will implicate a Th2 interleukin as a novel anti-inflammatory, pro-vasculogenic cytokine expressed by inflamed resident vascular cells to promote neovascularization.

Our overarching hypothesis is that FXR1 activity may be a compensatory, anti-inflammatory activity in VSMC. Vascular smooth muscle cells (VSMC) play a critical role in the etiology and progression of many vascular diseases including atherosclerosis and restenosis. Reduction of inflammatory gene expression in VSMC is a rational approach to limit the severity of these diseases. Our laboratory has found that one anti-inflammatory interleukin, IL-19, can decrease vascular inflammation by reduction in mRNA stability of inflammatory transcripts by reduction of activity of HuR, an mRNA stability protein. HuR translocates from the nucleus to the cytoplasm where it recognizes elements present almost exclusively in the 3'UTR of pro-inflammatory genes. Proteins and pathways which limit HuR translocation may reduce inflammatory mRNA stability, but are currently understudied. Using LC-MS/MS to identify HuR-interacting proteins under different inflammatory conditions, we identified one protein termed Fragile X-related protein (FXR1), which interacts with HuR in inflammatory, but not basal conditions, a novel finding. Importantly, FXR1 mRNA expression is enhanced in muscle cells, and it's promoter contains multiple cholesterol-response elements. Nothing has been reported on expression of FXR1 in VSMC or function for FXR1 in vascular disease. We present preliminary data showing that FXR1 expression is increased in injured arteries and TNF? and oxLDL stimulated human VSMC, and this expression is increased by IL-19. We show that siRNA knock down of FXR1 in VSMC increases inflammatory mRNA stability, abundance of inflammatory proteins, and cholesterol uptake, while over-expression of FXR1 decreases mRNA stability and inflammatory protein abundance. Our hypothesis is that FXR1 expression is a compensatory, negative-regulatory mechanism and functions by decreasing HuR activity and vascular inflammation by decreasing the stability of pro-inflammatory transcripts by numerous mechanisms. The overall goal of this application is twofold: 1- to identify the molecular mechanisms of FXR1 function in regulation of HuR activity and mRNA stability of pro-inflammatory transcripts in VSMC, and; 2- determine if modulation of FXR1 activity can reduce severity of atherosclerosis and vascular restenosis.

The transformation of vascular smooth muscle cells (VSMC) into foam cells leading to increased plaque size and decreased stability is a key, yet understudied step in atherogenesis. Our work in the previous funding cycle (HL117724) demonstrated that Interleukin-19 (IL-19), a novel, anti-inflammatory cytokine, attenuates atherosclerosis by multiple anti-inflammatory effects on VSMC as well as by increasing lipid uptake and efflux in macrophage. In published and preliminary studies described here, we show that IL-19 reduces lipid accumulation in VSMC, an atheroprotective event, but without modifying expression of lipid receptors or transporters. IL-19 induces expression of miR133a, a muscle-specific miRNA previously ascribed to regulate VSMC phenotype. Although unrecognized and unreported, we have identified that miR133a can target and reduce expression of Low Density Lipoprotein Receptor Adaptor Protein 1, (LDLRAP1), an adaptor protein which functions to internalize the LDL receptor. Patients with mutations in LDLRAP1 have LDL receptor malfunction leading to hyperlipidemia and Autosomal Recessive Hypercholesterolemia (ARH) disorder. Nothing at all is known about a role for miR133a in regulation of lipid uptake and development of atherosclerosis. Similarly, nothing at all has been published about LDLRAP1 expression, function, and participation in VSMC foam cell formation. We have reported that that both miR133a and LDLRAP1 regulate oxLDL uptake in VSMC. LDLRAP1 is induced in VSMC by oxLDL, is not detectible in normal medial VSMC, but is expressed in plaque and neointimal VSMC of injured arteries. IL-19 can reduce LDLRAP1 expression and oxLDL uptake in VSMC. Both miR133a and LDLRAP1regulate VSMC proliferation, previously unrecognized functions for these molecules. Preliminary studies indicate that the LDLRAP1+/- mouse has increased atherosclerosis, but reduced restenosis. In this competitive renewal application, we hypothesize that LDLRAP1 can be selectively reduced in VSMC because miR133a is muscle specific, and that reduction of lipid uptake by VSMC is atheroprotective and could also attenuate vascular proliferative syndromes. The overall goals of this application are to determine causative roles for miR133a and LDLRAP1 in VSMC lipid uptake, proliferation, atherogenesis, cholesterol-induced phenotype modulation, and vascular restenosis.