A20: a regulator of IFN gamma triggered vascular remodeling

Interferon gamma (IFNg) is beneficial to the host defense system but could be detrimental to the vasculature. Strategies to reduce IFNg expression or signaling are of particular interest to reduce or prevent pathologic vascular remodeling, e.g. in transplant arteriosclerosis (TA). We have preliminary evidence that the anti-inflammatory and NF-kB inhibitory protein A20 decreases expression and activity of the key mediator of IFNg signaling, signal transducer and activator of transcription (STAT1), in smooth muscle cells (SMC). In preliminary work, we confirmed that overexpression of A20 in SMC, by means of recombinant adenovirus-mediated gene transfer, markedly decreased STAT1 mRNA, protein and phosphorylation levels, and consequently mRNA and protein levels of the IFNg-dependent genes indoleamine 2-3 dioxygenase (IDO) and interferon-induced protein 10 (IP10). Conversely, A20 knockdown in SMC, by means of siRNA transfection, significantly increased STAT1 and downstream IFNg target genes. These data agree with increased STAT1 mRNA levels in aortae of A20 knockout (KO), as compared to wild type mice. In vivo, overexpression of A20 in fully mismatched C57BL/6 to BALB/c mouse aortic to carotid vascular allografts reduced IFNg-dependent IDO expression in the media and protected against TA. In contrast, A20 knockdown, using A20 +/- allografts, increased IDO expression in the media and aggravated TA lesions. To our knowledge, these data are the first to show that A20 interrupts IFNg signaling in SMC through a STAT1-dependent mechanism, which likely contributes to containing TA. In this proposal, we aim to: 1) Elucidate in vitro the molecular basis for A20-dependent modulation of IFNg signaling, and in particular regulation of STAT1 expression and activity in SMC. 2) Evaluate in vivo the impact of A20 overexpression or knockdown (A20 +/-) upon STAT1 levels and IFNg signals in fully mismatched (a well-established model in our laboratory) and syngeneic mouse vascular allografts. In the syngeneic model, IFNg receptor (IFNGR) competent vessels are transplanted into IFNGR-null recipient and TA is strictly driven by IFNg infusion. The overarching goal of this proposal is to promote the development of novel A20-based therapeutic strategies to control pathologic interferon activity in vascular diseases such as TA, the hallmark of chronic rejection, but also extend these findings to other models of vascular disease, i.e. post-angioplasty restenosis, and atherosclerosis. With the synergism of the outstanding experience of Dr. Ferrans laboratory in inflammation, vascular biology, and immunology, and of Dr. Sexls laboratory in IFN/JAK-STAT signaling, we expect that this goal will be met and that both laboratories as well as the applicant will highly benefit from this collaborative effort.

Occlusive vascular diseases including atherosclerosis are the leading cause of death in the Western world. Blood flow restriction to the heart leads to heart failure, and often the only escape is heart transplantation. However, heart transplants are plagued by transplant associated vasculopathy (TAV), a certain type of coronary atherosclerosis, and therefore long term success is limited. Seeking for novel therapeutic approaches, we elucidated a unique pathway how the anti-inflammatory protein A20 protects blood vessels against vascular remodeling and narrowing of the vessel lumen. This athero-protective function of A20 supports its promise as a therapeutic tool and prognostic marker for atherosclerotic disease as well as for successful heart transplantation. In order to investigate the functions of A20 in the context of atherosclerotic disease, and in particular of TAV, we took advantage of an allograft model in mice, where we transplant a donor aorta into the carotid artery of a recipient mouse. In this model - by using MHC mismatched mice - aortic allografts develop severe lesions of TAV within 4 weeks in an identical process as seen in cardiac allografts. To specifically study the role of A20 we overexpressed this protein in aortic allografts immediately before transplantation. Vice versa, in our loss of function studies we were using aortas derived from A20 heterozygote animals, which lack one copy of the A20 gene and therefore express lower levels of the protein, similar to human individuals with a certain genetic make-up (i.e. single nuclear polymorphism in the A20/TNFAIP3 locus). As a backup for in vivo studies and to translate findings into the human system we studied human coronary artery endothelial and smooth muscle with A20 overexpressing or knockdown, respectively. Intriguingly, we identified A20 as a novel negative regulator of pathologic interferon gamma signaling, which is a well-defined pivotal culprit of atherosclerotic disease. Indeed, A20 significantly reduced levels of signal transducer and activator of transcription-1 (STAT1), the key interferon gamma signal mediator. Upstream of STAT1, A20 inhibits phosphorylation of tank binding kinase-1 (TBK-1), thereby inhibiting low basal interferon beta activity which is required to maintain adequate STAT1 levels in the cytosol. Most importantly, our data demonstrates that by blocking interferon signaling, overexpression of A20 protects aortic allografts against TAV, while partial knockdown of A20 aggravated lesions of TAV. Based on these results, we conclude that A20 plays an important physiologic role in the vascular remodeling of TA. We propose that tailored A20-based vascular therapies could decrease incidence and severity of TA, and synergize with or minimize use of immunosuppression in solid organ transplantation.