Andy Lee

A20-mediated Modulation of Inflammatory and Immune Responses in Aortic Allografts and Development of Transplant Arteriosclerosis

Background. Transplant arteriosclerosis (TA) is the pathognomonic feature of chronic rejection, the primary cause of allograft failure. We have shown that the NF-&kgr;B inhibitory protein A20 exerts vasculoprotective effects in endothelial and smooth muscle cells (SMC), and hence is a candidate to prevent TA. We sought direct proof for this hypothesis. Methods. Fully mismatched, C57BL/6 (H2b) into BALB/c (H2d), aorta to carotid allografts were preperfused with saline, recombinant A20 adenovirus (rAd.A20) or rAd.&bgr;-galactosidase (&bgr;-gal), implanted, harvested 4 weeks after transplantation, and analyzed by histology, immunohistochemistry, and immunofluorescence staining. We measured indoleamine 2,3-dioxygenase, interleukin-6, and transforming growth factor-&bgr; mRNA and protein levels in nontransduced, and rAd.A20 or rAd.&bgr;-gal-transduced human SMC cultures after cytokine treatment. Results. Vascular overexpression of A20 significantly reduced TA lesions. This correlated with decreased graft inflammation and increased apoptosis of neointimal SMC. Paradoxically, T-cell infiltrates increased in A20-expressing allografts, including the immunoprivileged media, which related to A20 preventing indoleamine 2,3-dioxygenase upregulation in SMC. However, infiltrating T cells were predominantly T-regulatory cells (CD25+/Forkhead Box P3 [FoxP3+]). This agrees with A20 inhibiting interleukin-6 and promoting transforming growth factor-&bgr; production by medial SMC and in SMC cultures exposed to cytokines, which favors differentiation of regulatory over pathogenic T cells. Conclusions. In summary, A20 prevents immune-mediated remodeling of vascular allografts, therefore reduces TA lesions by affecting apoptotic and inflammatory signals and modifying the local cytokine milieu to promote an immunoregulatory response within the vessel wall. This highlights a novel function for A20 in local immunosurveillance, which added to its vasculoprotective effects, supports its therapeutic promise in TA.

A20 Regulates Atherogenic Interferon (IFN)-γ Signaling in Vascular Cells by Modulating Basal IFNβ Levels

Background: IFNγ signaling is a major culprit in occlusive vascular disease. Results: The anti-inflammatory protein A20 negatively regulates IFNγ signaling in vascular cells in vitro and in vivo. Conclusion: A20 impacts IFNγ signaling by modulating basal IFNβ and downstream STAT1 expression. Significance: This novel function of A20 supports its promise as a therapeutic target and prognostic marker for atherosclerotic disease. IFNγ signaling in endothelial (EC) and smooth muscle cells (SMC) is a key culprit of pathologic vascular remodeling. The impact of NF-κB inhibitory protein A20 on IFNγ signaling in vascular cells remains unknown. In gain- and loss-of-function studies, A20 inversely regulated expression of IFNγ-induced atherogenic genes in human EC and SMC by modulating STAT1 transcription. In vivo, inadequate A20 expression in A20 heterozygote mice aggravated intimal hyperplasia following partial carotid artery ligation. This outcome uniquely associated with increased levels of Stat1 and super-induction of Ifnγ-dependent genes. Transcriptome analysis of the aortic media from A20 heterozygote versus wild-type mice revealed increased basal Ifnβ signaling as the likely cause for higher Stat1 transcription. We confirmed higher basal IFNβ levels in A20-silenced human SMC and showed that neutralization or knockdown of IFNβ abrogates heightened STAT1 levels in these cells. Upstream of IFNβ, A20-silenced EC and SMC demonstrated higher levels of phosphorylated/activated TANK-binding kinase-1 (TBK1), a regulator of IFNβ transcription. This suggested that A20 knockdown increased STAT1 transcription by enhancing TBK1 activation and subsequently basal IFNβ levels. Altogether, these results uncover A20 as a key physiologic regulator of atherogenic IFNγ/STAT1 signaling. This novel function of A20 added to its ability to inhibit nuclear factor-κB (NF-κB) activation solidifies its promise as an ideal therapeutic candidate for treatment and prevention of vascular diseases. In light of recently discovered A20/TNFAIP3 (TNFα-induced protein 3) single nucleotide polymorphisms that impart lower A20 expression or function, these results also qualify A20 as a reliable clinical biomarker for vascular risk assessment.

A20 Haploinsufficiency Aggravates Transplant Arteriosclerosis in Mouse Vascular Allografts: Implications for Clinical Transplantation.

Background Inflammation is central to the pathogenesis of transplant arteriosclerosis (TA). We questioned whether physiologic levels of anti-inflammatory A20 influence TA severity. Methods We performed major histocompatibility complex mismatched aorta to carotid artery interposition grafts, using wild type (WT) or A20 heterozygote (HET) C57BL/6 (H-2b) donors and BALB/c (H-2d) recipients, and conversely BALB/c donors and WT/HET recipients. We analyzed aortic allografts by histology, immunohistochemistry, immunofluorescence, and gene profiling (quantitative real-time reverse-transcriptase polymerase chain reaction). We validated select in vivo A20 targets in human and mouse smooth muscle cell (SMC) cultures. Results We noted significantly greater intimal hyperplasia in HET versus WT allografts, indicating aggravated TA. Inadequate upregulation of A20 in HET allografts after transplantation was associated with excessive NF-кB activation, gauged by higher levels of IkB&agr;, p65, VCAM-1, ICAM-1, CXCL10, CCL2, TNF, and IL-6 (mostly localized to SMC). Correspondingly, cytokine-induced upregulation of TNF and IL-6 in human and mouse SMC cultures inversely correlated with A20 expression. Aggravated TA in HET versus WT allografts correlated with increased intimal SMC proliferation, and a higher number of infiltrating IFN&ggr;+ and Granzyme B+ CD4+ T cells and natural killer cells, and lower number of FoxP3+ regulatory T cells. A20 haploinsufficiency in allograft recipients did not influence TA. Conclusions A20 haploinsufficiency in vascular allografts aggravates lesions of TA by exacerbating inflammation, SMC proliferation, and infiltration of pathogenic T cells. A20 single nucleotide polymorphisms associating with lower A20 expression or function in donors of vascularized allografts may inform risk and severity of TA, highlighting the clinical implications of our findings.

Novel Use of Intravenous Immunoglobulin G in Complement Factor H Missense Mutation: A Case Report:

A white girl presented at 8 months of age with thrombotic microangiopathy, followed by recurrent episodes of renal dysfunction, hemolysis, and thrombocytopenia, compatible with atypical hemolytic uremic syndrome. The episodes of the syndrome were treated by a combination of infusions of fresh frozen plasma, plasmapheresis, and continuous venovenous hemodialysis. Interval resolution occurred between episodes. At 2 years of age, prophylactic infusions of fresh frozen plasma were started between relapses, but this proved to be poorly protective; however, introduction of prophylactic intravenous gamma globulin at age 3.5 years resulted in prolonged remission (42 months). Serum levels of the third and fourth components of complement, total hemolytic complement, and complement factor H were normal. Results of the third component functional assay were low before and normalized after the start of immunoglobulin G prophylaxis. A missense mutation of complement factor H was identified. At 6 years of age, the patient underwent bilateral native nephrectomy and started long-term peritoneal dialysis, followed by a combined liver-kidney transplant at age 8 years. Four and a half years after transplant, she has excellent renal and liver graft function without recurrence of atypical hemolytic uremic syndrome.

Abdominal and Thoracic Aortic Aneurysms

The management of aortic aneurysms has evolved over the last 15 years from open surgical repair as the standard therapy to endovascular stent-graft placement as the treatment of choice in anatomically suitable patients. This chapter introduces the basic pathophysiology, clinical symptoms, and indications for repair of both abdominal (AAA) and thoracic aortic aneurysms (TAA) and describes the conventional approaches of conservative medical management and open surgical repair for these lesions. The concept and rationale supporting endovascular AAA repair (EVAR) and TAA repair (TEVAR) are presented. The importance of pre-procedure planning is emphasized, and a detailed “How To” section explains the technical steps of the procedures. The most common risks and complications of EVAR and TEVAR are reviewed, and tips to avoid these adverse events are provided.