ADAR1 Non-Editing Function in Macrophage Activation and Abdominal Aortic Aneurysm.

BACKGROUND: Macrophage activation plays a critical role in abdominal aortic aneurysm (AAA) development. However, molecular mechanisms controlling macrophage activation and vascular inflammation in AAA remain largely unknown. The objective of the study was to identify novel mechanisms underlying adenosine deaminase acting on RNA (ADAR1) function in macrophage activation and AAA formation. METHODS: Aortic transplantation was conducted to determine the importance of nonvascular ADAR1 in AAA development/dissection. Ang II (Angiotensin II) infusion of ApoE-/- mouse model combined with macrophage-specific knockout of ADAR1 was used to study ADAR1 macrophage-specific role in AAA formation/dissection. The relevance of macrophage ADAR1 to human AAA was examined using human aneurysm specimens. Moreover, a novel humanized AAA model was established to test the role of human macrophages in aneurysm formation in human arteries. RESULTS: Allograft transplantation of wild-type abdominal aortas to ADAR1+/- recipient mice significantly attenuated AAA formation, suggesting that nonvascular ADAR1 is essential for AAA development. ADAR1 deficiency in hematopoietic cells decreased the prevalence and severity of AAA while inhibited macrophage infiltration and aorta wall inflammation. ADAR1 deletion blocked the classic macrophage activation, diminished NF-κB (nuclear factor kappa B) signaling, and enhanced the expression of a number of anti-inflammatory microRNAs. Mechanistically, ADAR1 interacted with Drosha to promote its degradation, which attenuated Drosha-DGCR8 (DiGeorge syndrome critical region 8) interaction, and consequently inhibited pri- to pre-microRNA processing of microRNAs targeting IKKß, resulting in an increased IKKß (inhibitor of nuclear factor kappa-B) expression and enhanced NF-κB signaling. Significantly, ADAR1 was induced in macrophages and interacted with Drosha in human AAA lesions. Reconstitution of ADAR1-deficient, but not the wild type, human monocytes to immunodeficient mice blocked the aneurysm formation in transplanted human arteries. CONCLUSIONS: Macrophage ADAR1 promotes aneurysm formation in both mouse and human arteries through a novel mechanism, that is, Drosha protein degradation, which inhibits the processing of microRNAs targeting NF-kB signaling and thus elicits macrophage-mediated vascular inflammation in AAA.

A Novel Mechanism Underlying Inflammatory Smooth Muscle Phenotype in Abdominal Aortic Aneurysm.

[Figure: see text].

Surfactant Protein A, a Novel Regulator for Smooth Muscle Phenotypic Modulation and Vascular Remodeling-Brief Report.

OBJECTIVE: The objective of this study is to determine the role of SPA (surfactant protein A) in vascular smooth muscle cell (SMC) phenotypic modulation and vascular remodeling. Approach and Results: PDGF-BB (Platelet-derived growth factor-BB) and serum induced SPA expression while downregulating SMC marker gene expression in SMCs. SPA deficiency increased the contractile protein expression. Mechanistically, SPA deficiency enhanced the expression of myocardin and TGF (transforming growth factor)-ß, the key regulators for contractile SMC phenotype. In vivo, SPA was induced in medial and neointimal SMCs following mechanical injury in both rat and mouse carotid arteries. SPA knockout in mice dramatically attenuated the wire injury-induced intimal hyperplasia while restoring SMC contractile protein expression in medial SMCs. These data indicate that SPA plays an important role in SMC phenotype modulation and vascular remodeling in vivo. CONCLUSIONS: SPA is a novel protein factor modulating SMC phenotype. Blocking the abnormal elevation of SPA may be a potential strategy to inhibit the development of proliferative vascular diseases.

Concomitant overexpression of triple antioxidant enzymes selectively increases circulating endothelial progenitor cells in mice with limb ischaemia.

Endothelial progenitor cells (EPCs) are a group of heterogeneous cells in bone marrow (BM) and blood. Ischaemia increases reactive oxygen species (ROS) production that regulates EPC number and function. The present study was conducted to determine if ischaemia-induced ROS differentially regulated individual EPC subpopulations using a mouse model concomitantly overexpressing superoxide dismutase (SOD)1, SOD3 and glutathione peroxidase. Limb ischaemia was induced by femoral artery ligation in male transgenic mice with their wild-type littermate as control. BM and blood cells were collected for EPCs analysis and mononuclear cell intracellular ROS production, apoptosis and proliferation at baseline, day 3 and day 21 after ischaemia. Cells positive for c-Kit+ /CD31+ or Sca-1+ /Flk-1+ or CD34+ /CD133+ or CD34+ /Flk-1+ were identified as EPCs. ischaemia significantly increased ROS production and cell apoptosis and decreased proliferation of circulating and BM mononuclear cells and increased BM and circulating EPCs levels. Overexpression of triple antioxidant enzymes effectively prevented ischaemia-induced ROS production with significantly decreased cell apoptosis and preserved proliferation and significantly increased circulating EPCs level without significant changes in BM EPC populations, associated with enhanced recovery of blood flow and function of the ischemic limb. These data suggested that ischaemia-induced ROS was differentially involved in the regulation of circulating EPC population.

Chronic cardiac allograft rejection in a rat model disparate for one single class I MHC molecule is associated with indirect recognition by CD4(+) T cells.

T-cell mediated immune responses play a critical role in chronic allograft dysfunction. The complex nature of allograft rejection, particularly with respect to the vast repertoire of alloantigens and their mode of recognition by T cells, presents a major challenge for the design of well-controlled studies into the immunobiology of chronic rejection. The purpose of this study was to develop a rat model with restricted antigenic specificity that develops chronic rejection without any immunologic manipulation to study the T-cell response. PVG.1U allogeneic hearts disparate for one single class I antigen, RT.1A(u), were transplanted into PVG.R8 rat recipients. Grafts from PVG.R8 were used as syngeneic controls. Chronic rejection was studied by histological analysis of the grafted hearts at various time points posttransplantation (20-100 days). Donor specific alloreactive response was studied in a mixed lymphocyte reaction assay. All allografts survived more than 90 days and showed extensive evidence of chronic rejection, which was characterized by interstitial fibrosis, vasculitis, and occlusive myointimal thickening. Chronic rejection was evident by day 20 and most extensive by day 100 posttransplantation. In marked contrast, syngeneic grafts remained free of chronic lesions. Lymphocytes harvested from graft recipients showed a more vigorous proliferative response to allogeneic splenocytes as compared with that of lymphocytes from nai;ve animals. The proliferative response was primarily mediated by CD4(+) T cells recognizing the RT1.A(a) molecule via the indirect pathway. A single class I disparity in this model generates chronic rejection associated with potent CD4(+) T-cell responses induced by the indirect recognition pathway. The use of this antigenically restricted model may facilitate the design of well-controlled studies for the characterization of immune mechanisms responsible for chronic rejection.

Blockade of indirect recognition mediated by CD4+ T cells leads to prolonged cardiac xenograft survival.

The T-cell response to xenografts is induced by direct and indirect recognition of xenoantigens. Although the importance of indirect recognition is well established in vitro, the contribution of this pathway to xenograft rejection in vivo remains to be fully elucidated. We herein investigated the direct contribution of indirect recognition to cardiac xenograft rejection in the rat-to-mouse (PVG.R8-to-C57BL/10) concordant model. Rat xenoantigens invoked a vigorous proliferative response in mouse T cells harvested from naïve or graft recipients at rejection. Indirect recognition predominated the response, as antibodies against mouse class II I-A(b), CD80, or CD86 molecules significantly (45 to 60%) blocked the proliferative response. Importantly, the blockade of indirect recognition in vivo by treating the graft recipients with a monoclonal antibody (mAb) against class II I-A(b) molecule on days 0, 1, and 3 post-transplantation resulted in significant (P < 0.009) prolongation of cardiac xenograft survival (Mean Survival Time (MST) >94 +/- 55 days vs. 7 +/- 0.8 days for controls). In contrast, treatment of recipients with a mAb against mouse class I H-2K(b)/D(b) molecules did not significantly affect graft rejection (MST = 8 +/- 1 days). These results demonstrate that indirect recognition mediated by CD4(+) T cells plays a critical role in the rejection of cardiac grafts in the rat-to-mouse xenogeneic model.

Predominant expression of the Th2 response in chronic cardiac allograft rejection.

Chronic rejection is the main cause of late allograft failure in patients. CD4+ T cells activated by indirect recognition of alloantigens are implicated in this rejection reaction. However, the type of T cell response (Th1 vs Th2) that contributes to chronic rejection has not been fully investigated. The purpose of this study is to examine whether chronic rejection is associated with a polarized T-cell response in a rat cardiac allograft model, where long-term graft survival is achieved by intrathymic immunomodulation with donor class I, RT1.Aa, allopeptides. All long-surviving allografts showed histological evidence of chronic rejection. Chronic rejection was associated with high levels of intragraft Th2 cytokines and the Th2-regulated alloantibodies. The Th2 response was systemic, since long-surviving allografts with chronic rejection had high levels of serum IL-10. The predominance of the Th2 cytokines demonstrates that the Th2 response was not sufficient for the prevention of chronic rejection in this model. The predominant expression of Th2 cytokines, together with the presence of Th2-regulated alloantibodies, suggests that the Th2 response may play a role in the development of chronic rejection.

Pattern of ischemia reperfusion injury in a mouse orthotopic liver transplant model.

BACKGROUND: The molecular pathways of ischemic injury after liver transplantation are complex and difficult to dissect because of the presence of many variables. Transgenic and genetically deficient strains of mice provide ideal models for the study of the contribution of a single gene product in biological processes in vivo. Although well described in rats, prolonged preservation has not been studied in a mouse model of orthotopic liver transplantation (mOLT). The aim of this study was to establish a model of cold ischemia and reperfusion injury in mOLT and describe the pattern of the regenerative response to various lengths of cold storage. MATERIALS AND METHODS: mOLT was performed using a syngeneic combination. Grafts were preserved at 4 degrees C in University of Wisconsin (Viaspan) solution for increasing periods of cold preservation. After cold storage, the liver grafts were transplanted and recipient survival was monitored. Hepatocellular injury was determined by histology, and the regenerative response was quantitated by interleukin 6 upregulation and DNA replication. RESULTS: Long-term survival was 100%, 100%, 88%, and 0% for cold preservation of 1, 4, 8, and 16 h, respectively. Grafts with short preservation times (1 and 4 h) demonstrated limited injury and a weak regenerative response, with slight IL-6 early upregulation and minimal cell division. Eight hours of cold ischemia resulted in prominent injury and an intense regenerative response accompanied by significant IL-6 upregulation and DNA synthesis. Sixteen hours of storage resulted in all recipients succumbing to liver failure, with histology showing extensive hepatic necrosis. CONCLUSIONS: This study demonstrated the feasibility of using the mOLT model for the study of molecular mechanisms associated with recovery from cold ischemia and reperfusion injury. Increasing lengths of cold ischemia correlate with progressive tissue damage whereas recovery is associated with a regenerative response that correlates with the severity of injury.

Interleukin-6 from intrahepatic cells of bone marrow origin is required for normal murine liver regeneration.

Interleukin-6 (IL-6) is required for normal liver regeneration, but the specific cellular source of this growth factor is unknown. We investigated whether this signal originates from the resident macrophage, the Kupffer cell. Using a murine model of bone marrow transplantation, we replaced recipient bone marrow-derived cells, including Kupffer cells, with cells of donor genetic phenotype. Recipients deficient in IL-6 (IL-6(-/-)) were lethally irradiated, then rescued with 10(7) donor bone marrow cells capable of expressing IL-6 (IL-6(+/+)). Conversely, IL-6(+/+) recipients received IL-6(-/-) marrow. Successful engraftment was measured by the presence of the Y chromosome SRY locus in the livers of female recipients receiving male marrow, in situ IL-6 expression by Kupffer cells, and up-regulation of IL-6 in splenocytes after activation with lipopolysaccharide (LPS). Kupffer cell isolation in IL-6(-/-) females receiving IL-6(+/+) male marrow clearly showed the presence of the SRY locus and IL-6 disrupted allele, whereas males receiving female marrow demonstrated no SRY or IL-6 signals, confirming the extent of replacement. Replacement of these cells in IL-6(-/-) mice with IL-6(+/+) bone marrow successfully restored the regenerative response after partial hepatectomy (PHx) as indicated by signal transduction and activator of transcription 3 (STAT3) activation and hepatocyte DNA replication. Alternatively, complete replacement of Kupffer cells in IL-6(+/+) mice by transplantation with IL-6(-/-) cells significantly inhibited liver regeneration and was partially restored by administration of IL-6. This investigation demonstrates a paracrine mechanism by which cells of bone marrow origin, most likely Kupffer cells, regulate the regenerative capacity of the hepatocyte through IL-6 expression.