Fanconi anemia (FA) is a rare genomic instability disorder characterized by progressive bone marrow failure and predisposition to cancer. FA-associated gene products are involved in the repair of DNA interstrand crosslinks (ICLs). Fifteen FA-associated genes have been identified, but the genetic basis in some individuals still remains unresolved. Here, we used whole-exome and Sanger sequencing on DNA of unclassified FA individuals and discovered biallelic germline mutations in ERCC4 (XPF), a structure-specific nuclease-encoding gene previously connected to xeroderma pigmentosum and segmental XFE progeroid syndrome. Genetic reversion and wild-type ERCC4 cDNA complemented the phenotype of the FA cell lines, providing genetic evidence that mutations in ERCC4 cause this FA subtype. Further biochemical and functional analysis demonstrated that the identified FA-causing ERCC4 mutations strongly disrupt the function of XPF in DNA ICL repair without severely compromising nucleotide excision repair. Our data show that depending on the type of ERCC4 mutation and the resulting balance between both DNA repair activities, individuals present with one of the three clinically distinct disorders, highlighting the multifunctional nature of the XPF endonuclease in genome stability and human disease.

To date there is no effective therapy for Alzheimer disease (AD). High levels of circulating high density lipoprotein (HDL) and its main protein, apolipoprotein A-I (apoA-I), reduce the risk of cardiovascular disease. Clinical studies show that plasma HDL cholesterol and apoA-I levels are low in patients with AD. To investigate if increasing plasma apoA-I/HDL levels ameliorates AD-like memory deficits and amyloid-␤ (A␤) deposition, we generated a line of triple transgenic (Tg) mice overexpressing mutant forms of amyloid-␤ precursor protein (APP) and presenilin 1 (PS1) as well as human apoA-I (AI). Here we show that APP/PS1/AI triple Tg mice have a 2-fold increase of plasma HDL cholesterol levels. When tested in the Morris water maze for spatial orientation abilities, whereas APP/PS1 mice develop age-related learning and memory deficits, APP/PS1/AI mice continue to perform normally during aging. Interestingly, no significant differences were found in the total level and deposition of A␤ in the brains of APP/PS1 and APP/PS1/AI mice, but cerebral amyloid angiopathy was reduced in APP/PS1/AI mice. Also, consistent with the anti-inflammatory properties of apoA-I/HDL, glial activation was reduced in the brain of APP/ PS1/AI mice. In addition, A␤-induced production of proinflammatory chemokines/cytokines was decreased in mouse organotypic hippocampal slice cultures expressing human apoA-I. Therefore, we conclude that overexpression of human apoA-I in the circulation prevents learning and memory deficits in APP/ PS1 mice, partly by attenuating neuroinflammation and cerebral amyloid angiopathy. These findings suggest that elevating plasma apoA-I/HDL levels may be an effective approach to preserve cognitive function in patients with AD.

Objective-The majority of apoptotic cells in atherosclerotic lesions are macrophages. However, the pathogenic role of macrophage apoptosis in the development of atherosclerosis remains unclear. Elevated expression of Bax, one of the pivotal proapoptotic proteins of the Bcl-2 family, has been found in human atherosclerotic plaques. Activation of Bax also occurs in free cholesterol-loaded and oxysterol-treated mouse macrophages. In this study, we examined the effect of Bax deficiency in bone marrow-derived leukocytes on the development of atherosclerosis in low-density lipoprotein receptor-null (LDLRϪ/Ϫ) mice. Methods and Results-Fourteen 8-week-old male LDLRϪ/Ϫ mice were lethally irradiated and reconstituted with either wild-type (WT) C57BL6 or Bax-null (BaxϪ/Ϫ) bone marrow. Three weeks later, the mice were challenged with a Western diet for 10 weeks. No differences were found in the plasma cholesterol level between the WT and BaxϪ/Ϫ group. However, quantitation of cross sections from proximal aorta revealed a 49.2% increase (Pϭ0.0259) in the mean lesion area of the BaxϪ/Ϫ group compared with the WT group. A 53% decrease in apoptotic macrophages in the BaxϪ/Ϫ group was found by TUNEL staining (PϽ0.05). Conclusions-The reduction of apoptotic activity in macrophages stimulates atherosclerosis in LDLRϪ/Ϫ mice, which is consistent with the hypothesis that macrophage apoptosis suppresses the development of atherosclerosis.

Background: 8-OxoG is a major oxidative lesion in DNA and is associated with cancer. Results: Kinetic and mass spectrometric studies demonstrate that human polymerase bypasses 8-oxoG in a largely error-free manner. Conclusion: Arginine 61 from the finger domain plays a key role in error-free bypass at the insertion stage. Significance: In addition to photo-adducts and cisplatinated DNA, polymerase might also be involved in accurate bypass of 8-oxoG in vivo.

Background— Scavenger receptor class B type I (SR-BI) is expressed in macrophages, where it has been proposed to facilitate cholesterol efflux. However, direct evidence that the expression of macrophage SR-BI is protective against atherosclerosis is lacking. In this study, we examined the in vivo role of macrophage SR-BI in atherosclerotic lesion development in the apolipoprotein (apo) E–deficient mouse model. Methods and Results— ApoE-deficient mice with (n=16) or without (n=15) expression of macrophage SR-BI were created by transplanting lethally irradiated apoE-deficient mice with bone marrow cells collected from SR-BI −/− apoE −/− mice or SR-BI +/+ apoE −/− mice. The recipient mice were fed a chow diet for 12 weeks after transplantation for analysis of atherosclerosis. Quantification of macrophage SR-BI mRNA by real-time reverse transcription–polymerase chain reaction indicated successful engraftment of donor bone marrow and inactivation of macrophage SR-BI in recipient mice reconstituted with SR-BI −/− apoE −/− bone marrow. There were no significant differences in plasma lipid levels, lipoprotein distributions, and HDL subpopulations between the 2 groups. Analysis of the proximal aorta demonstrated an 86% increase in mean atherosclerotic lesion area in SR-BI −/− apoE −/− → apoE −/− mice compared with SR-BI +/+ apoE −/− → apoE −/− mice (109.50±18.08 versus 58.75±9.58×10 3 μm 2 ; mean±SEM, P =0.017). No difference in cholesterol efflux from SR-BI +/+ apoE −/− or SR-BI −/− apoE −/− macrophages to HDL or apoA-I discs was detected. Conclusions— Expression of macrophage SR-BI protects mice against atherosclerotic lesion development in apoE-deficient mice in vivo without influencing plasma lipids, HDL subpopulations, or cholesterol efflux. Thus, macrophage SR-BI plays an antiatherogenic role in vivo, providing a new therapeutic target for the design of strategies to prevent and treat atherosclerosis.

Background-Atherosclerosis is a disease marked by lipid accumulation and inflammation. Recently, atherosclerosis has gained recognition as an autoimmune-type syndrome characterized by increased activation of the innate and acquired immune systems. Natural killer T (NKT) cells have characteristics of both conventional T cells and NK cells and recognize glycolipid antigens presented in association with CD1d molecules on antigen-presenting cells. The capacity of NKT cells to respond to lipid antigens and modulate innate and acquired immunity suggests that they may play a role in atherogenesis. Methods and Results-We examined the role of NKT cells in atherogenesis and how the atherosclerotic environment affects the NKT cell population itself. The data show that CD1d-deficiency in male apolipoprotein E-deficient (apoE 0 ) mice results in reduction in atherosclerosis, and treatment of apoE 0 mice with ␣-galactosylceramide, a potent and specific NKT cell activator, results in a 2-fold increase in atherosclerosis. Interestingly, we demonstrate that ␣-galactosylceramide-induced interferon-␥ responses and numbers of NKT cells in apoE 0 mice show age-dependent qualitative and quantitative differences as compared with age-matched wild-type mice. Conclusions-Collectively, these findings reveal that hyperlipidemia and atherosclerosis have significant effects on NKT cell responses and that these cells are proatherogenic. ϩ or CD4 Ϫ CD8 Ϫ and express varying levels of CD161 (NK1.1 in mice). NKT cells are present in both humans and mice, are found in lymphoid organs and tissues, have a restricted T cell receptor (TCR) expression (V␣14-J␣18/V␤8 in mice and V␣24-J␣18/V␤11 in humans), and recognize glycolipid antigens presented in the context of the major histocompatibility complex (MHC) class I-like molecule CD1d. 1,2 Although a physiological ligand for NKT cells is still not known, these cells respond strongly to the marine sponge-derived glycolipid ␣-galactosylceramide (␣-GalCer), 3 which specifically binds to CD1d and selectively activates invariant NKT cells. Once activated, NKT cells rapidly produce large amounts of cytokines, including interleukin (IL)-4 and IL-10, which are associated with an antiinflammatory T helper 2 (Th2) response, and interferon (IFN)-␥ and tumor necrosis factor-␣, which are associated with a proinflammatory Th1 response. Recent studies have shown that activation of NKT cells by in vivo administration of ␣-GalCer, or its synthetic homolog KRN7000, has antimetastatic activities and suppresses inflammation in chronic autoimmune diseases such as type 1 diabetes and experimental autoimmune encephalomyelitis in mice. 3 Because atherosclerosis is a lipid-associated disease and has many characteristics in common with other autoimmune disorders, we hypothesized that NKT cells regulate immunity and progression of lesion growth in the artery wall. This hypothesis is consistent with the recent findings that CD1d is expressed in human atherosclerotic lesions and that numbers of CD4 ϩ NK1.1 ϩ cells producing IL-4 a...

Integrins control many cell functions, including generation of reactive oxygen species (ROS) and regulation of collagen synthesis. Mesangial cells, found in the glomerulus of the kidney, are able to produce large amounts of ROS via the NADPH oxidase. We previously demonstrated that integrin ␣1-null mice develop worse fibrosis than wild-type mice following glomerular injury and this is due, in part, to excessive ROS production by ␣1-null mesangial cells. In the present studies, we describe the mechanism whereby integrin ␣1-null mesangial cells produce excessive ROS. Integrin ␣1-null mesangial cells have constitutively increased basal levels of activated Rac1, which result in its increased translocation to the cell membrane, excessive ROS production, and consequent collagen IV deposition. Basal Rac1 activation is a direct consequence of ligand-independent increased epidermal growth factor receptor (EGFR) phosphorylation in ␣1-null mesangial cells. Thus, our study demonstrates that integrin ␣1␤1-EGFR cross talk is a key step in negatively regulating Rac1 activation, ROS production, and excessive collagen synthesis, which is a hallmark of diseases characterized by irreversible fibrosis.Integrin ␣1␤1, a major collagen binding receptor, is expressed in different cell types, including fibroblasts (45), endothelial cells (8), and mesangial cells in the glomerulus of the kidney (30, 53). Integrin ␣1␤1 confers the ability of cells to bind to collagenous substrata, including collagens I and IV (4, 45), and to proliferate on these substrata (45). Moreover, cells expressing integrin ␣1␤1 sense extracellular levels of collagen and downregulate its synthesis at both transcriptional and translational levels (4,14). Finally, we demonstrated that integrin ␣1␤1 also downregulates the production of reactive oxygen species (ROS) (4, 58).Following renal injury, mice lacking integrin ␣1␤1 develop more extensive glomerular fibrosis characterized by excessive accumulation of collagen type IV compared to wild-type (WT) mice (4, 58). Increased fibrosis is due to both a direct effect of the lack of integrin ␣1␤1-mediated downregulation of collagen IV synthesis and excessive ROS production by ␣1-null mesangial cells.Constitutive production of ROS by mesangial cells, a major cell type found in the glomerulus of the kidney, originates from an intrinsic NADPH oxidase (26, 48) that normally functions at a low level and increases in response to inflammatory stimuli, high glucose, or stress (25,34,35,37,55). The NADPH oxidase, highly characterized in phagocytes, is a multicomponent enzyme complex that consists of the membrane-bound cytochrome b 558 (p22 phox and gp91 phox ) and cytoplasmic proteins (p40 phox , p47 phox , p67 phox ) that translocate to the membrane following cellular stimulation to produce superoxide (3, 9, 47). A multicomponent phagocyte-like NADPH oxidase is also a major source of ROS in many nonphagocytic cells, including mesangial cells. In the phagocyte-like NADPH oxidase, the catalytic subunits are termed Nox proteins, with ...

Non-small cell lung cancer (NSCLC) not amenable to surgical resection has a high mortality rate, due to the ineffectiveness and toxicity of chemotherapy. Thus, there remains an urgent need of efficacious drugs that can combat this disease. In this study, we show that targeting the formation of pro-angiogenic epoxyeicosatrienoic acids (EETs) by the cytochrome P450 arachidonic acid epoxygenases (Cyp2c) represents a new and safe mechanism to treat NSCLC growth and progression. In the transgenic murine K-Ras model and human orthotopic models of NSCLC, we found that Cyp2c44 could be downregulated by activating the transcription factor PPARα with the ligands bezafibrate and Wyeth-14,643. Notably, both treatments reduced primary and metastatic NSCLC growth, tumor angiogenesis, endothelial Cyp2c44 expression and circulating EET levels. These beneficial effects were independent of the time of administration, whether before or after the onset of primary NSCLC, and they persisted after drug withdrawal, suggesting the benefits were durable. Our findings suggest that strategies to downregulate Cyp2c expression and/or its enzymatic activity may provide a safer and effective strategy to treat NSCLC. Moreover, as bezafibrate is a well-tolerated clinically approved drug used for managing lipidemia, our findings provide an immediate cue for clinical studies to evaluate the utility of PPARα ligands as safe agents for the treatment of lung cancer in humans.