Haley Ramsey

A20, a modulator of smooth muscle cell proliferation and apoptosis, prevents and induces regression of neointimal hyperplasia

A20 is a NF‐κB‐dependent gene that has dual anti‐inflammatory and antiapoptotic functions in endothelial cells (EC). The function of A20 in smooth muscle cells (SMC) is unknown. We demonstrate that A20 is induced in SMC in response to inflammatory stimuli and serves an anti‐inflammatory function via blockade of NF‐κB and NF‐κB‐dependent proteins ICAM‐1 and MCP‐1. A20 inhibits SMC proliferation via increased expression of cyclin‐dependent kinase inhibitors p21waf1 and p27kip1. Surprisingly, A20 sensitizes SMC to cytokine‐ and Fas‐mediated apoptosis through a novel NO‐dependent mechanism. In vivo, adenoviral delivery of A20 to medial rat carotid artery SMC after balloon angioplasty prevents neointimal hyperplasia by blocking SMC proliferation and accelerating re‐endothelialization, without causing apoptosis. However, expression of A20 in established neointimal lesions leads to their regression through increased apoptosis. This is the first demonstration that A20 exerts two levels of control of vascular remodeling and healing. A20 prevents neointimal hyperplasia through combined anti‐inflammatory and antiproliferative functions in medial SMC. If SMC evade this first barrier and neointima is formed, A20 has a therapeutic potential by uniquely sensitizing neointimal SMC to apoptosis. A20‐based therapies hold promise for the prevention and treatment of neointimal disease.—Patel, V. I., Daniel, S., Longo, C. R., Shrikhande, G. V., Scali, S. T., Czismadia, E., Groft, C. M., Shukri, T., Motley‐Dore, C., Ramsey, H. E., Fisher, M. D., Grey, S. T., Arvelo, M. B., Ferran, C. A20, a modulator of smooth muscle cell proliferation and apoptosis, prevents and induces regression of neointimal hyperplasia. FASEB J. 20, 1418–1430 (2006)

A20 Protects Mice from Lethal Liver Ischemia Reperfusion Injury by Increasing Peroxisome Proliferator-Activated Receptor-α Expression

The nuclear factor‐κB inhibitory protein A20 demonstrates hepatoprotective abilities through combined antiapoptotic, anti‐inflammatory, and pro‐proliferative functions. Accordingly, overexpression of A20 in the liver protects mice from toxic hepatitis and lethal radical hepatectomy, whereas A20 knockout mice die prematurely from unfettered liver inflammation. The effect of A20 on oxidative liver damage, as seen in ischemia/reperfusion injury (IRI), is unknown. In this work, we evaluated the effects of A20 upon IRI using a mouse model of total hepatic ischemia. Hepatic overexpression of A20 was achieved by recombinant adenovirus (rAd.)‐mediated gene transfer. Although only 10%‐25% of control mice injected with saline or the control rAd.β galactosidase survived IRI, the survival rate reached 67% in mice treated with rAd.A20. This significant survival advantage in rAd.A20‐treated mice was associated with improved liver function, pathology, and repair potential. A20‐treated mice had significantly lower bilirubin and aminotransferase levels, decreased hemorrhagic necrosis and steatosis, and increased hepatocyte proliferation. A20 protected against liver IRI by increasing hepatic expression of peroxisome proliferator‐activated receptor alpha (PPARα), a regulator of lipid homeostasis and of oxidative damage. A20‐mediated protection of hepatocytes from hypoxia/reoxygenation and H2O2‐mediated necrosis was reverted by pretreatment with the PPARα inhibitor MK886. In conclusion, we demonstrate that PPARα is a novel target for A20 in hepatocytes, underscoring its novel protective effect against oxidative necrosis. By combining hepatocyte protection from necrosis and promotion of proliferation, A20‐based therapies are well‐poised to protect livers from IRI, especially in the context of small‐for‐size and steatotic liver grafts. Liver Transpl 15:1613–1621, 2009.

Expansion of the neonatal platelet mass is achieved via an extension of platelet lifespan.

The fetal/neonatal hematopoietic system must generate enough blood cells to meet the demands of rapid growth. This unique challenge might underlie the high incidence of thrombocytopenia among preterm neonates. In this study, neonatal platelet production and turnover were investigated in newborn mice. Based on a combination of blood volume expansion and increasing platelet counts, the platelet mass increased sevenfold during the first 2 weeks of murine life, a time during which thrombopoiesis shifted from liver to bone marrow. Studies applying in vivo biotinylation and mathematical modeling showed that newborn and adult mice had similar platelet production rates, but neonatal platelets survived 1 day longer in circulation. This prolonged lifespan fully accounted for the rise in platelet counts observed during the second week of murine postnatal life. A study of pro-apoptotic and anti-apoptotic Bcl-2 family proteins showed that neonatal platelets had higher levels of the anti-apoptotic protein Bcl-2 and were more resistant to apoptosis induced by the Bcl-2/Bcl-xL inhibitor ABT-737 than adult platelets. However, genetic ablation or pharmacologic inhibition of Bcl-2 alone did not shorten neonatal platelet survival or reduce platelet counts in newborn mice, indicating the existence of redundant or alternative mechanisms mediating the prolonged lifespan of neonatal platelets.

A20 Modulates Lipid Metabolism and Energy Production to Promote Liver Regeneration

Background Liver Regeneration is clinically of major importance in the setting of liver injury, resection or transplantation. We have demonstrated that the NF-κB inhibitory protein A20 significantly improves recovery of liver function and mass following extended liver resection (LR) in mice. In this study, we explored the Systems Biology modulated by A20 following extended LR in mice. Methodology and Principal Findings We performed transcriptional profiling using Affymetrix-Mouse 430.2 arrays on liver mRNA retrieved from recombinant adenovirus A20 (rAd.A20) and rAd.βgalactosidase treated livers, before and 24 hours after 78% LR. A20 overexpression impacted 1595 genes that were enriched for biological processes related to inflammatory and immune responses, cellular proliferation, energy production, oxidoreductase activity, and lipid and fatty acid metabolism. These pathways were modulated by A20 in a manner that favored decreased inflammation, heightened proliferation, and optimized metabolic control and energy production. Promoter analysis identified several transcriptional factors that implemented the effects of A20, including NF-κB, CEBPA, OCT-1, OCT-4 and EGR1. Interactive scale-free network analysis captured the key genes that delivered the specific functions of A20. Most of these genes were affected at basal level and after resection. We validated a number of A20s target genes by real-time PCR, including p21, the mitochondrial solute carriers SLC25a10 and SLC25a13, and the fatty acid metabolism regulator, peroxisome proliferator activated receptor alpha. This resulted in greater energy production in A20-expressing livers following LR, as demonstrated by increased enzymatic activity of cytochrome c oxidase, or mitochondrial complex IV. Conclusion This Systems Biology-based analysis unravels novel mechanisms supporting the pro-regenerative function of A20 in the liver, by optimizing energy production through improved lipid/fatty acid metabolism, and down-regulated inflammation. These findings support pursuit of A20-based therapies to improve patients’ outcomes in the context of extreme liver injury and extensive LR for tumor treatment or donation.

Rapamycin and CTLA4Ig Synergize to Induce Stable Mixed Chimerism Without the Need for CD40 Blockade

The mixed chimerism approach achieves donor‐specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti‐CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor‐specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC‐mismatched/minor antigen‐matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non‐MHC antigens cause graft rejection and split tolerance.

Anti-LFA-1 or rapamycin overcome costimulation blockade-resistant rejection in sensitized bone marrow recipients.

While costimulation blockade‐based mixed chimerism protocols work well for inducing tolerance in rodents, translation to preclinical large animal/nonhuman primate models has been less successful. One recognized cause for these difficulties is the high frequency of alloreactive memory T cells (Tmem) found in the (pre)clinical setting as opposed to laboratory mice. In the present study, we therefore developed a murine bone marrow transplantation (BMT) model employing recipients harboring polyclonal donor‐reactive Tmem without concomitant humoral sensitization. This model was then used to identify strategies to overcome this additional immune barrier. We found that B6 recipients that were enriched with 3 × 107 T cells isolated from B6 mice that had been previously grafted with Balb/c skin, rejected Balb/c BM despite costimulation blockade with anti‐CD40L and CTLA4Ig (while recipients not enriched developed chimerism). Adjunctive short‐term treatment of sensitized BMT recipients with rapamycin or anti‐LFA‐1 mAb was demonstrated to be effective in controlling Tmem in this model, leading to long‐term mixed chimerism and donor‐specific tolerance. Thus, rapamycin and anti‐LFA‐1 mAb are effective in overcoming the potent barrier that donor‐reactive Tmem pose to the induction of mixed chimerism and tolerance despite costimulation blockade.

IEX-1 deficiency induces browning of white adipose tissue and resists diet-induced obesity.

Chronic inflammation plays a crucial role in the pathogenesis of obesity and insulin resistance. However, the primary mediators that affect energy homeostasis remain ill defined. Here, we report an unexpected role for immediate early response gene X-1 (IEX-1), a downstream target of NF-κB, in energy metabolism. We found that IEX-1 expression was highly induced in white adipose tissue (WAT) in both epidydmal and subcutaneous depots but not in interscapular brown adipose tissue (BAT) in mice fed a high fat diet (HFD). Null mutation of IEX-1 protected mice against HFD-induced adipose and hepatic inflammation, hepatic steatosis, and insulin resistance. Unexpectedly, IEX-1 knockout (IEX-1−/−) mice gained markedly less weight on HFD for 20 weeks as compared to wild-type (WT) littermates (37 ± 3 versus 48 ± 2 gm) due to increased energy expenditure. Mechanistically, we showed that IEX-1 deficiency induced browning and activated thermogenic genes program in WAT but not in BAT by promoting alternative activation of adipose macrophages. Consequently, IEX-1−/− mice exhibited enhanced thermogenesis (24 ± 0.1 versus 22 ± 0.1 kcal/hour/kg in WT mice) explaining increased energy expenditure and lean phenotype in these mice. In conclusion, the present study suggests that IEX-1 is a novel physiological regulator of energy homeostasis via its action in WAT.

Stress-induced hematopoietic failure in the absence of immediate early response gene X-1 (IEX-1, IER3)

Expression of the immediate early response gene X-1 (IEX-1, IER3) is diminished significantly in hematopoietic stem cells in a subgroup of patients with early stage myelodysplastic syndromes, but it is not clear whether the deregulation contributes to the disease. The current study demonstrates increased apoptosis and a concomitant decrease in the number of hematopoietic stem cells lacking this early response gene. Null mutation of the gene also impeded platelet differentiation and shortened a lifespan of red blood cells. When bone marrow cells deficient in the gene were transplanted into wild-type mice, the deficient stem cells produced significantly fewer circulating platelets and red blood cells, despite their enhanced repopulation capability. Moreover, after exposure to a non-myeloablative dose of radiation, absence of the gene predisposed to thrombocytopenia, a significant decline in red blood cells, and dysplastic bone marrow morphology, typical characteristics of myelodysplastic syndromes. These findings highlight a previously unappreciated role for this early response gene in multiple differentiation steps within hematopoiesis, including thrombopoiesis, erythropoiesis and in the regulation of hematopoietic stem cell quiescence. The deficient mice offer a novel model for studying the initiation and progression of myelodysplastic syndromes as well as strategies to prevent this disorder.

Mitoquinone restores platelet production in irradiation-induced thrombocytopenia

Abstract Myelodysplastic syndromes (MDS) are hallmarked by cytopenia and dysplasia of hematopoietic cells, often accompanied by mitochondrial dysfunction and increases of reactive oxygen species (ROS) within affected cells. However, it is not known whether the increase in ROS production is an instigator or a byproduct of the disease. The present investigation shows that mice lacking immediate early responsive gene X-1 (IEX-1) exhibit lineage specific increases in ROS production and abnormal cytology upon radiation in blood cell types commonly identified in MDS. These affected cell lineages chiefly have the bone marrow as a primary site of differentiation and maturation, while cells with extramedullary differentiation and maturation like B- and T-cells remain unaffected. Increased ROS production is likely to contribute significantly to irradiation-induced thrombocytopenia in the absence of IEX-1 as demonstrated by effective reversal of the disorder after mitoquinone (MitoQ) treatment, a mitochondria-specific antioxidant. MitoQ reduced intracellular ROS production within megakaryocytes and platelets. It also normalized mitochondrial membrane potential and superoxide production in platelets in irradiated, IEX-1 deficient mice. The lineage-specific effects of mitochondrial ROS may help us understand the etiology of thrombocytopenia in association with MDS in a subgroup of the patients.

Dipeptidyl peptidase IV (DPPIV/CD26) inhibition does not improve engraftment of unfractionated syngeneic or allogeneic bone marrow after nonmyeloablative conditioning

In order to develop minimally toxic bone marrow transplantation (BMT) protocols suitable for use in a wider range of indications, it is important to identify ways to enhance BM engraftment at a given level of recipient conditioning. CXCL12/stromal cell-derived factor-1α plays a crucial physiological role in homing of hematopoietic stem cells to BM. It is regulated by the ectopeptidase dipeptidyl peptidase IV (DPPIV; DPP4) known as CD26, which cleaves dipeptides from the N-terminus of polypeptide chains. Blocking DPPIV enzymatic activity had a beneficial effect on hematopoietic stem cell engraftment in various but very specific experimental settings. Here we investigated whether inhibition of DPPIV enzymatic activity through Diprotin A or sitagliptin (Januvia) improves BM engraftment in nonmyeloablative murine models of syngeneic (i.e., CD45-congenic) and allogeneic (i.e., Balb/c to B6) BMT (1 Gy total body irradiation, 10–15 × 106 unseparated BM cells/mouse). Neither Diprotin A administered in vivo at the time of BMT and/or used for in vitro pretreatment of BM nor sitagliptin administered in vivo had a detectable effect on the level of multilineage chimerism (follow-up >20 weeks). Similarly, sitagliptin did not enhance chimerism after allogeneic BMT, even though DPPIV enzymatic activity measured in serum was profoundly inhibited (>98% inhibition at peak exposure). Our results provide evidence that DPPIV inhibition via Diprotin A or sitagliptin does not improve engraftment of unseparated BM in a nonmyeloablative BMT setting.