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-2) donors and BALB/c (H-2) 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 IkBα, 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 IFNγ 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.

The C-terminal domain of A1/Bfl-1 regulates its anti-inflammatory function in human endothelial cells.

A1/Bfl-1 is a NF-κB dependent, anti-apoptotic Bcl-2 family member that contains four Bcl-2 homology domains (BH) and an amphipathic C-terminal domain, and is expressed in endothelial cells (EC). Based on NF-κB reporter assays in bovine aortic EC, we have previously demonstrated that A1, like Bcl-2 and Bcl-xL, inhibits NF-κB activation. These results, however, do not fully translate when evaluating the cell's own NF-κB machinery in human EC overexpressing A1 by means of recombinant adenovirus (rAd.) mediated gene transfer. Indeed, overexpression of full-length A1 in human umbilical vein EC (HUVEC), and human dermal microvascular EC (HDMEC) failed to inhibit NF-κB activation. However, overexpression of a mutant lacking the C-terminal domain of A1 (A1ΔC) demonstrated a potent NF-κB inhibitory effect in these cells. Disparate effects of A1 and A1ΔC on NF-κB inhibition in human EC correlated with mitochondrial (A1) versus non-mitochondrial (A1ΔC) localization. In contrast, both full-length A1 and A1ΔC protected EC from staurosporine (STS)-induced cell death, indicating that mitochondrial localization was not necessary for A1's cytoprotective function in human EC. In conclusion, our data uncover a regulatory role for the C-terminal domain of A1 in human EC: anchoring A1 to the mitochondrion, which conserves but is not necessary for its cytoprotective function, or by its absence freeing A1 from the mitochondrion and uncovering an additional anti-inflammatory effect.

A20 promotes liver regeneration by decreasing SOCS3 expression to enhance IL-6/STAT3 proliferative signals.

UNLABELLED: Liver regeneration is of major clinical importance in the setting of liver injury, resection, and transplantation. A20, a potent antiinflammatory and nuclear factor kappa B (NF-κB) inhibitory protein, has established pro-proliferative properties in hepatocytes, in part through decreasing expression of the cyclin dependent kinase inhibitor, p21. Both C-terminal (7-zinc fingers; 7Zn) and N-terminal (Nter) domains of A20 were required to decrease p21 and inhibit NF-κB. However, both independently increased hepatocyte proliferation, suggesting that additional mechanisms contributed to the pro-proliferative function of A20 in hepatocytes. We ascribed one of A20's pro-proliferative mechanisms to increased and sustained interleukin (IL)-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation, as a result of decreased hepatocyte expression of the negative regulator of IL-6 signaling, suppressor of cytokine signaling 3 (SOCS3). This novel A20 function segregates with its 7Zn not Nter domain. Conversely, total and partial loss of A20 in hepatocytes increased SOCS3 expression, hampering IL-6-induced STAT3 phosphorylation. Following liver resection in mice pro-proliferative targets downstream of IL-6/STAT3 signaling were increased by A20 overexpression and decreased by A20 knockdown. In contrast, IL-6/STAT3 proinflammatory targets were increased in A20-deficient livers, and decreased or unchanged in A20 overexpressing livers. Upstream of SOCS3, levels of its microRNA regulator miR203 were significantly decreased in A20-deficient livers. CONCLUSION: A20 enhances IL-6/STAT3 pro-proliferative signals in hepatocytes by down-regulating SOCS3, likely through a miR203-dependent manner. This finding together with A20 reducing the levels of the potent cell cycle brake p21 establishes its pro-proliferative properties in hepatocytes and prompts the pursuit of A20-based therapies to promote liver regeneration and repair.

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-κB 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 (H2) into BALB/c (H2), aorta to carotid allografts were preperfused with saline, recombinant A20 adenovirus (rAd.A20) or rAd.ß-galactosidase (ß-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-ß mRNA and protein levels in nontransduced, and rAd.A20 or rAd.ß-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-ß 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.

Hepatocyte growth factor preferentially activates the anti-inflammatory arm of NF-κB signaling to induce A20 and protect renal proximal tubular epithelial cells from inflammation.

Inflammation induces the NF-κB dependent protein A20 in human renal proximal tubular epithelial cells (RPTEC), which secondarily contains inflammation by shutting down NF-κB activation. We surmised that inducing A20 without engaging the pro-inflammatory arm of NF-κB could improve outcomes in kidney disease. We showed that hepatocyte growth factor (HGF) increases A20 mRNA and protein levels in RPTEC without causing inflammation. Upregulation of A20 by HGF was NF-κB/RelA dependent as it was abolished by overexpressing IκBα or silencing p65/RelA. Unlike TNFα, HGF caused minimal IκBα and p65/RelA phosphorylation, with moderate IκBα degradation. Upstream, HGF led to robust and sustained AKT activation, which was required for p65 phosphorylation and A20 upregulation. While HGF treatment of RPTEC significantly increased A20 mRNA, it failed to induce NF-κB dependent, pro-inflammatory MCP-1, VCAM-1, and ICAM-1 mRNA. This indicates that HGF preferentially upregulates protective (A20) over pro-inflammatory NF-κB dependent genes. Upregulation of A20 supported the anti-inflammatory effects of HGF in RPTEC. HGF pretreatment significantly attenuated TNFα-mediated increase of ICAM-1, a finding partially reversed by silencing A20. In conclusion, this is the first demonstration that HGF activates an AKT-p65/RelA pathway to preferentially induce A20 but not inflammatory molecules. This could be highly desirable in acute and chronic renal injury where A20-based anti-inflammatory therapies are beneficial.

Volumetric effects of motor cortex injury on recovery of dexterous movements.

Due to the heterogeneous nature of most brain injuries, the contributions of gray and white matter involvement to motor deficits and recovery potential remain obscure. We tested the hypothesis that duration of hand motor impairment and recovery of skilled arm and hand motor function depends on the volume of gray and white matter damage of the frontal lobe. Lesions of the primary motor cortex (M1), M1 + lateral premotor cortex (LPMC), M1 + LPMC + supplementary motor cortex (M2) or multifocal lesions affecting motor areas and medial prefrontal cortex were evaluated in rhesus monkeys. Fine hand motor function was quantitatively assessed pre-lesion and for 3-12 months post-lesion using two motor tests. White and gray matter lesion volumes were determined using histological and quantitative methods. Regression analyses showed that duration of fine hand motor impairment was strongly correlated (R(2)>0.8) with the volume of gray and white matter lesions, with white matter lesion volume being the primary predictor of impairment duration. Level of recovery of fine hand motor skill was also well correlated (R(2)>0.5) with gray and white matter lesion volume. In some monkeys post-lesion skill exceeded pre-lesion skill in one or both motor tasks demonstrating that continued post-injury task practice can improve motor performance after localized loss of frontal motor cortex. These findings will assist in interpreting acute motor deficits, predicting the time course and expected level of functional recovery, and designing therapeutic strategies in patients with localized frontal lobe injury or neurosurgical resection.

Perception of hand motion direction uses a gravitational reference.

We studied possible frames of reference for kinesthetic perception of imposed hand motion direction in the frontal plane in ten young adult subjects with no history of neuromuscular disease. In one experiment, subjects were instructed to set unseen hand motion imposed by a motorized linear slide device parallel to the trunk-fixed longitudinal axis, seven visually specified axes and vertical (gravitational axis) while in a standard erect head/trunk posture and with head/trunk orientation varied. The visually specified axes were presented on a head-mounted display that also blocked vision of the external environment. In a second experiment using the same device, subjects set unseen hand motion parallel to vertical and to subjective oblique directions of 45 degrees clockwise (cw) and counter clockwise (ccw) from vertical in erect and varied head/trunk postures. Errors for setting hand motion to vertical and to verbally specified oblique axes (45 degrees cw and ccw from vertical) were lower than to the trunk longitudinal axis and visually specified axes. There were clear oblique effects in setting hand motion to visually specified axes and to subjective oblique (45 degrees cw and ccw) axes. When head and trunk orientation were varied, variable errors were higher for all axes, but remained lowest for vertical and subjective oblique axes. Moreover, errors for setting hand motion to all axes depended on head/trunk orientation. Overall, these results show that kinesthetic perception of imposed hand motion uses a subjective gravitational frame of reference that varies somewhat with head/trunk orientation.

Measurement of coordination of object manipulation in non-human primates.

We present a modification of the automated movement assessment panel [Gash DM, Zhang Z, Umberger G, Mahood K, Smith M, Smith C, et al. An automated movement assessment panel for upper limb motor functions in rhesus monkeys and humans. J Neurosci Methods 1999;89:111-7] that incorporates a three-dimensional load cell to record forces applied by monkeys while manipulating food targets. The absolute force-time integral (total absolute impulse) is used to characterize the total of the applied forces over time as the food (carrot chip with a hole punched through the center) is manipulated and lifted from a flat surface (easiest task) and threaded over a straight rod (medium difficulty) or curved rod (highest difficulty). The total impulse can be measured even on unsuccessful attempts to acquire the food. Thus, it can be used to evaluate changes in performance even before successful acquisition occurs as in learning or recovery following a nervous system insult. We show from tests in three rhesus monkeys that the total absolute impulse measure is sensitive to task complexity, learning and lesion of frontal lobe motor areas (in one case) and that there is good reliability in day-to-day performance (even with long periods between performances) after the monkey has learned the task. Importantly, the task requires minimal training as the monkeys can be successful on even the most difficult of these tasks with one or two training sessions, yet performance improvements continue to occur over several testing sessions. Furthermore, the three levels of task difficulty permit analysis of a progression of ability.