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1.  Loss of fibroblast HIF-1α accelerates tumorigenesis 
Cancer research  2012;72(13):3187-3195.
Solid tumors consist of malignant cells and associated stromal components, including fibroblastic cells that contribute to tumor growth and progression. Although tumor fibrosis and aberrant vascularization contribute to the hypoxia often found in advanced tumors, the contribution of hypoxic signaling within tumor-associated fibroblasts to tumorigenesis remains unknown. In this study, we used a fibroblast-specific promoter to create mice in which key hypoxia regulatory genes, including VHL, HIF-1α, HIF-2α and VEGF-A, were knocked out specifically in tumor stromal fibroblasts. We found that loss of HIF-1α and its target gene VEGF-A accelerated tumor growth in murine model of mammary cancer. HIF-1α and VEGF-A loss also led to a reduction in vascular density and myeloid cell infiltration, which correlated with improved tumor perfusion. Together, our findings indicate that the fibroblast HIF-1α response is a critical component of tumor vascularization.
doi:10.1158/0008-5472.CAN-12-0534
PMCID: PMC4089958  PMID: 22556263
fibroblast; hypoxia; hypoxia-inducible factor-1
2.  Myeloid cell HIF-1α regulates asthma airway resistance and eosinophil function 
Hypoxia-inducible factor (HIF)-1α is a master regulator of inflammatory activities of myeloid cells, including neutrophils and macrophages. These studies examine the role of myeloid cell HIF-1α in regulating asthma induction and pathogenesis, and for the first time, evaluate the roles of HIF-1α and HIF-2α in the chemotactic properties of eosinophils, the myeloid cells most associated with asthma. Wild-type (WT) and myeloid cell-specific HIF-1α knockout (KO) C57BL/6 mice were studied in an ovalbumin (OVA) model of asthma. Administration of the pharmacological HIF-1α antagonist YC-1 was used to corroborate findings from the genetic model. WT, HIF-1α, and HIF-2α KO eosinophils underwent in vitro chemotaxis assays. We found that deletion of HIF-1α in myeloid cells and systemic treatment with YC-1 during asthma induction decreased airway hyperresponsiveness (AHR). Deletion of HIF-1α in myeloid cells in OVA-induced asthma also reduced eosinophil infiltration, goblet cell hyperplasia, and levels 34 of cytokines IL-4, IL-5, and IL-13 in the lung. HIF-1α inhibition with YC-1 during asthma induction decreased eosinophilia in bronchoalveolar lavage, lung parenchyma, and blood, as well as decreased total lung inflammation, IL-5, and serum OVA-specific IgE levels. Deletion of HIF-1α in eosinophils decreased their chemotaxis, while deletion of the isoform HIF-2α led to increased chemotaxis. This work demonstrates that HIF-1α in myeloid cells plays a role in asthma pathogenesis, particularly in AHR development. Additionally, treatment with HIF-1α inhibitors during asthma induction decreases AHR and eosinophilia. Finally, we show that HIF- 1α and HIF-2α regulate eosinophil migration in opposing ways.
doi:10.1007/s00109-012-0986-9
PMCID: PMC3646920  PMID: 23250618
Hypoxia inducible factor (HIF)-1α; Asthma; Allergic inflammation; Eosinophils; Chemotaxis; Airway hyperresponsiveness
3.  Hypoxia-inducible factors enhance the effector responses of CD8+ T cells to persistent antigen 
Nature immunology  2013;14(11):1173-1182.
Cytolytic activity by CD8+ cytotoxic T lymphocytes (CTLs) is a powerful strategy for the elimination of intracellular pathogens and tumor cells. The destructive capacity of CTLs is progressively dampened during chronic infection, yet the environmental cues and molecular pathways that influence immunological `exhaustion' remain unclear. Here we found that CTL immunity was regulated by the central transcriptional response to hypoxia, which is controlled in part by hypoxia-inducible factors (HIFs) and the von Hippel–Lindau tumor suppressor VHL. Loss of VHL, the main negative regulator of HIFs, led to lethal CTL-mediated immunopathology during chronic infection, and VHL-deficient CTLs displayed enhanced control of persistent viral infection and neoplastic growth. We found that HIFs and oxygen influenced the expression of pivotal transcription, effector and costimulatory-inhibitory molecules of CTLs, which was relevant to strategies that promote the clearance of viruses and tumors.
doi:10.1038/ni.2714
PMCID: PMC3977965  PMID: 24076634
4.  Activation of Hypoxia‐Inducible Factor‐2 in Adipocytes Results in Pathological Cardiac Hypertrophy 
Background
Obesity can cause structural and functional abnormalities of the heart via complex but largely undefined mechanisms. Emerging evidence has shown that obesity results in reduced oxygen concentrations, or hypoxia, in adipose tissue. We hypothesized that the adipocyte hypoxia‐signaling pathway plays an essential role in the development of obesity‐associated cardiomyopathy.
Methods and Results
Using a mouse model in which the hypoxia‐inducible factor (HIF) pathway is activated by deletion of the von Hippel–Lindau gene specifically in adipocytes, we found that mice with adipocyte–von Hippel–Lindau deletion developed lethal cardiac hypertrophy. HIF activation in adipocytes results in overexpression of key cardiomyopathy‐associated genes in adipose tissue, increased serum levels of several proinflammatory cytokines including interleukin‐1β and monocyte chemotactic protein‐1, and activation of nuclear factor–κB and nuclear factor of activated T cells in the heart. Interestingly, genetic deletion of Hif2a, but not Hif1a, was able to rescue cardiac hypertrophy and abrogate adipose inflammation.
Conclusion
We have discovered a previously uncharacterized mechanism underlying a critical and direct role of the adipocyte HIF‐2 transcription factor in the development of adipose inflammation and pathological cardiac hypertrophy.
doi:10.1161/JAHA.113.000548
PMCID: PMC3886757  PMID: 24326162
adipocytes; cardiomyopathy; hypoxia; inflammation; obesity
5.  Endothelial hypoxic metabolism in carcinogenesis and dissemination : HIF-A isoforms are a NO metastatic phenomenon 
Oncotarget  2013;4(12):2567-2576.
Tumor biology is a broad and encompassing field of research, particularly given recent demonstrations of the multicellular nature of solid tumors, which have led to studies of molecular and metabolic intercellular interactions that regulate cancer progression. Hypoxia is a broad stimulus that results in activation of hypoxia inducible factors (HIFs). Downstream HIF targets include angiogenic factors (e.g. vascular endothelial growth factor, VEGF) and highly reactive molecules (e.g. nitric oxide, NO) that act as cell-specific switches with unique spatial and temporal effects on cancer progression. The effect of cell-specific responses to hypoxia on tumour progression and spread, as well as potential therapeutic strategies to target metastatic disease, are currently under active investigation. Vascular endothelial remodelling events at tumour and metastatic sites are responsive to hypoxia, HIF activation, and NO signalling. Here, we describe the interactions between endothelial HIF and NO during tumor growth and spread, and outline the effects of endothelial HIF/NO signalling on cancer progression. In doing so, we attempt to identify areas of metastasis research that require attention, in order to ultimately facilitate the development of novel treatments that reduce or prevent tumour dissemination.
PMCID: PMC3926849  PMID: 24318195
Vascular endothelium; hypoxia; metastasis; HIF; HIF isoforms; nitric oxide; cell-specific responses
6.  Activating killer-cell immunoglobulin-like receptors (KIR) and their cognate HLA ligands are significantly increased in autism 
Brain, behavior, and immunity  2012;26(7):1122-1127.
Killer-cell immunoglobulin-like receptor (KIR) proteins are expressed on natural killer (NK) cells and appear important in innate and adaptive immunity. There are about 14 KIR genes on chromosome 19q13.4, composed of those that inhibit and those that activate NK cell killing. Haplotypes have different combinations of these genes meaning that not all genes are present in a subject. There are two main classes of cognate human leukocyte antigen (HLA) ligands (HLA-Bw4 and HLA-C1/C2) that bind to the inhibitory/activating receptors. As a general rule, the inhibitory state is maintained except when virally infected or tumor cells are encountered; however, both increased activation and inhibition states have been associated with susceptibility and protection against numerous disease states including cancer, arthritis, and psoriasis.
Utilizing DNA from 158 Caucasian subjects with autism and 176 KIR control subjects we show for the first time a highly significant increase in four activating KIR genes (2DS5, 3DS1, 2DS1 and 2DS4) as measured by chi square values and odds ratios. In addition, our data suggests a highly significant increase in the activating KIR gene 2DS1 and its cognate HLA-C2 ligand (2DS1+C2; p=0.00003 [Odds Ratio=2.87]). This information ties together two major immune gene complexes, the Human Leukocyte Complex and the Leukocyte Receptor Complex, and may partially explain immune abnormalities observed in many subjects with autism.
doi:10.1016/j.bbi.2012.07.014
PMCID: PMC3469320  PMID: 22884899
killer-cell immunoglobulin-like receptor; KIR genes; KIR haplotypes; human leukocyte antigen; HLA ligands; leukocyte receptor complex; autism; immune dysfunction; natural killer cells
7.  A New Pharmacological Agent (AKB-4924) Stabilizes Hypoxia Inducible Factor (HIF) and Increases Skin Innate Defenses Against Bacterial Infection 
Hypoxia inducible factor-1 (HIF-1) is a transcription factor that is a major regulator of energy homeostasis and cellular adaptation to low oxygen stress. HIF-1 is also activated in response to bacterial pathogens and supports the innate immune response of both phagocytes and keratinocytes. In this work, we show that a new pharmacological compound AKB-4924 (Akebia Therapeutics) increases HIF-1α levels and enhances the antibacterial activity of phagocytes and keratinocytes against both methicillin-sensitive and -resistant strains of Staphylococcus aureus in vitro. AKB-4924 is also effective in stimulating the killing capacity of keratinocytes against the important opportunistic skin pathogens Pseudomonas aeruginosa and Acinitobacter baumanii. The effect of AKB-4924 is mediated through the activity of host cells, as the compound exerts no direct antimicrobial activity. Administered locally as a single agent, AKB-4924 limits S. aureus proliferation and lesion formation in a mouse skin abscess model. This approach to pharmacologically boost the innate immune response via HIF-1 stabilization may serve as a useful adjunctive treatment for antibiotic-resistant bacterial infections.
doi:10.1007/s00109-012-0882-3
PMCID: PMC3606899  PMID: 22371073
Hypoxia inducible factor-1 (HIF-1); innate immunity; Staphylococcus aureus; bacterial infection; antibiotic-resistant bacteria
8.  Acute-Phase CD8 T Cell Responses That Select for Escape Variants Are Needed to Control Live Attenuated Simian Immunodeficiency Virus 
Journal of Virology  2013;87(16):9353-9364.
The overall CD8 T cell response to human/simian immunodeficiency virus (HIV/SIV) targets a collection of discrete epitope specificities. Some of these epitope-specific CD8 T cells emerge in the weeks and months following infection and rapidly select for sequence variants, whereas other CD8 T cell responses develop during the chronic infection phase and rarely select for sequence variants. In this study, we tested the hypothesis that acute-phase CD8 T cell responses that do not rapidly select for escape variants are unable to control viral replication in vivo as well as those that do rapidly select for escape variants. We created a derivative of live attenuated SIV (SIVmac239Δnef) in which we ablated five epitopes that elicit early CD8 T cell responses and rapidly accumulate sequence variants in SIVmac239-infected Mauritian cynomolgus macaques (MCMs) that are homozygous for the M3 major histocompatibility complex (MHC) haplotype. This live attenuated SIV variant was called m3KOΔnef. Viremia was significantly higher in M3 homozygous MCMs infected with m3KOΔnef than in either MHC-mismatched MCMs infected with m3KOΔnef or MCMs infected with SIVmac239Δnef. Three CD8 T cell responses, including two that do not rapidly select for escape variants, predominated during early m3KOΔnef infection in the M3 homozygous MCMs, but these animals were unable to control viral replication. These results provide evidence that acute-phase CD8 T cell responses that have the potential to rapidly select for escape variants in the early phase of infection are needed to establish viral control in vivo.
doi:10.1128/JVI.00909-13
PMCID: PMC3754066  PMID: 23785211
9.  APOBEC3B Deletion and Risk of HIV-1 Acquisition 
The Journal of infectious diseases  2009;200(7):1054-1058.
The human APOBEC3 family of cytidine deaminases provides intrinsic immunity to retroviral infection. A naturally occurring 29.5-kb deletion removes the entire APOBEC3B gene. We examined the impact of the APOBEC3B gene deletion in >4000 individuals from five HIV-1 natural history cohorts. The hemizygous genotype had no effect on either infection or progression. However, the homozygous deletion was significantly associated with unfavorable outcomes for HIV-1 acquisition (OR=7.37, P=0.024), progression to AIDS (RH = 4.01, P=0.03), and viral set-point (P=0.04). These findings suggest that the loss of APOBEC3B may increase host susceptibility to HIV-1/AIDS and warrant further study.
doi:10.1086/605644
PMCID: PMC3690486  PMID: 19698078
10.  Continuous Measurement of Endotracheal Tube Cuff Pressure 
AACN advanced critical care  2008;19(2):235-243.
Continuous monitoring and download of endotracheal tube cuff pressure for a 12-hour period were required to collect data for an ongoing program of research related to airway management of the critically ill patient. On the basis of reports from the anesthesia literature, continuous monitoring of cuff pressure via a traditional pressure transducer and monitor was identified as the best method to collect data. Although continuous pressure monitoring of many physiologic variables is routine in critical care settings, application of the technology to measurement of endotracheal tube cuff pressure has not been reported outside the operating room. The research team conducted bench testing and pilot testing in human subjects to establish feasibility, accuracy, and safety of continuous cuff pressure monitoring. Monitoring was feasible with stringent procedures applied to ensure safety. A bias of 0.5 cm H2O between continuous and intermittent measures was obtained in both in vitro and in vivo testing.
doi:10.1097/01.AACN.0000318126.79630.76
PMCID: PMC3506175  PMID: 18560292
endotracheal tube; monitoring; nursing; pressure; research
11.  HIF1α and HIF2α: sibling rivalry in hypoxic tumor growth and progression 
Nature reviews. Cancer  2011;12(1):9-22.
Preface
Hypoxia inducible factors (HIFs) are broadly expressed in human cancers, and HIF1α and HIF2α were previously suspected of promoting tumor progression through largely overlapping functions. However, this relatively simple model has now been challenged in light of recent data from genome-wide analyses of human tumors, genetically engineered mouse models of cancer, and systems biology approaches that reveal unique and sometimes opposing HIFa activities in both normal physiology and disease. These effects are mediated in part through regulation of unique target genes, as well as direct and indirect interactions with important oncoproteins and tumor suppressors, including MYC and p53. As HIF inhibitors are currently under clinical evaluation as cancer therapeutics, a more thorough understanding of unique roles performed by HIF1α and HIF2α in human neoplasia is warranted. This Review summarizes our rapidly changing understanding of shared and independent HIF1α and HIF2α activities in tumor growth and progression, and the implications for using selective HIF inhibitors as cancer therapeutics.
doi:10.1038/nrc3183
PMCID: PMC3401912  PMID: 22169972
12.  Genome-wide Association Study Implicates PARD3B-based AIDS Restriction 
The Journal of Infectious Diseases  2011;203(10):1491-1502.
Background. Host genetic variation influences human immunodeficiency virus (HIV) infection and progression to AIDS. Here we used clinically well-characterized subjects from 5 pretreatment HIV/AIDS cohorts for a genome-wide association study to identify gene associations with rate of AIDS progression.
Methods.  European American HIV seroconverters (n = 755) were interrogated for single-nucleotide polymorphisms (SNPs) (n = 700,022) associated with progression to AIDS 1987 (Cox proportional hazards regression analysis, co-dominant model).
Results.  Association with slower progression was observed for SNPs in the gene PARD3B. One of these, rs11884476, reached genome-wide significance (relative hazard = 0.3; P =3. 370 × 10−9) after statistical correction for 700,022 SNPs and contributes 4.52% of the overall variance in AIDS progression in this study. Nine of the top-ranked SNPs define a PARD3B haplotype that also displays significant association with progression to AIDS (hazard ratio, 0.3; P = 3.220 × 10−8). One of these SNPs, rs10185378, is a predicted exonic splicing enhancer; significant alteration in the expression profile of PARD3B splicing transcripts was observed in B cell lines with alternate rs10185378 genotypes. This SNP was typed in European cohorts of rapid progressors and was found to be protective for AIDS 1993 definition (odds ratio, 0.43, P = .025).
Conclusions. These observations suggest a potential unsuspected pathway of host genetic influence on the dynamics of AIDS progression.
doi:10.1093/infdis/jir046
PMCID: PMC3080910  PMID: 21502085
13.  Astrocyte pVHL and HIF-α isoforms are required for embryonic-to-adult vascular transition in the eye 
The Journal of Cell Biology  2011;195(4):689-701.
The von Hippel–Lindau tumor suppressor and the hypoxia-inducible factor-αs are essential for the transition from embryonic hyaloidal vascular system to adult retinal vasculature in the mouse eye.
Successful transition from embryonic to adult circulation is critical for survival of mammalian organisms. This shift occurs in the central cardiovascular circulation and in the eye as oxygen tension increases. However, its regulation is not well understood. We have used combinatorial gene deletion and overexpression assays to assess the effect of astrocyte-targeted deletion of von Hippel–Lindau tumor suppressor (Vhl), hypoxia-inducible factor-αs (Hif-αs), and Vegf on the normal regression of the hyaloidal vessels, the fetal ocular circulation system. Astrocytic Vhl deletion induced accelerated hyaloidal regression and subsequent massive secondary outgrowth. Combinatorial gene deletion involving Vhl, Hif-αs, and Vegf genes revealed that HIF-2α/vascular endothelial growth factor signaling induces secondary outgrowth in Vhl mutants. Conversely, HIF-1α regulated macrophage migration inhibitory factor and promoted macrophage infiltration that accelerates hyaloidal vessel regression. The phenotype observed in Vhl mutants strongly resembles human persistent hyperplastic primary vitreous cases and may provide insights into vascular remodeling mechanisms in other systems.
doi:10.1083/jcb.201107029
PMCID: PMC3257537  PMID: 22084310
14.  Endothelial Cell HIF-1α and HIF-2α Differentially Regulate Metastatic Success 
Cancer Cell  2012;21(1):52-65.
Summary
The hypoxia inducible transcription factors (HIFs) control many mediators of vascular response, including both angiogenic factors and small molecules such as nitric oxide (NO). In studying how endothelial HIF response itself affects metastasis, we found that loss of HIF-1α in endothelial cells reduces NO synthesis, retards tumor cell migration through endothelial layers, and restricts tumor cell metastasis, and that loss of HIF-2α has in each case the opposite effect. This results from differential regulation of NO homeostasis that in turn regulates vascular endothelial growth factor expression in an NO-dependent feedback loop. These opposing roles for the two HIF factors indicate that both they and endothelial cells regulate metastasis as malignancy progresses.
Highlights
► Endothelial cell hypoxic response is a critical determinant of metastatic success ► HIF isoforms differentially regulate NO homeostasis in endothelium ► iNOS is essential for hypoxic NO production in endothelial cells ► HIF isoforms in endothelium differentially promote and hinder metastasis
doi:10.1016/j.ccr.2011.11.017
PMCID: PMC3334270  PMID: 22264788
15.  von Hippel-Lindau protein regulates transition from fetal to adult circulatory system in retina 
Development (Cambridge, England)  2010;137(9):1563-1571.
SUMMARY
In the early neonatal stage, the fetal circulatory system undergoes dramatic transition to the adult circulatory system. Normally, embryonic connecting vessels such as ductus arteriosus and the foramen ovale close and regress. In the neonatal retina, hyaloid vessels maintaining blood flow in the embryonic retina regress, and retinal vessels take over to form adult-type circulatory system. This process is regulated by the programmed cell death switch mediated by macrophages via Wnt and Angiopoietin-2 pathways. In this study, we seek for the other mechanisms that regulate this process, and focus on the dramatic change in oxygen environment at the point of birth. The von Hippel–Lindau tumor suppressor protein (pVHL) is a substrate recognition component of an E3-ubiquitin ligase that rapidly destabilizes hypoxia-inducible factor-αs (HIF-αs) under normoxic conditions, but not hypoxic conditions. To examine the role of oxygen-sensing mechanisms in the retinal circulatory system transition, we generated retina-specific conditional knockout mice for VHL (VHLα-Cre KO mice). These mice exhibit arrested transition from fetal to adult circulatory system: persistence of hyaloid vessels and poorly formed retinal vessels. These defects are suppressed by intraocular injection of Flt1-Fc protein (vascular endothelial growth factor (VEGF) Receptor-1 (Flt1)/Fc chimeric protein that can bind VEGF and inhibit its activity), or by inactivating the HIF-1 α gene. Our results suggest that not only macrophages but also tissue oxygen-sensing mechanisms regulate the transition from fetal to adult circulatory system in retina.
doi:10.1242/dev.049015
PMCID: PMC3224975  PMID: 20388654
Angiogenesis; Circulatory system; Hypoxia-inducible factor 1
16.  Macrophage Expression of HIF-1α Suppresses T cell Function and Promotes Tumor Progression 
Cancer research  2010;70(19):7465-7475.
T cells can inhibit tumor growth, but their function in the tumor microenvironment is often suppressed. Many solid tumors exhibit abundant macrophage infiltration and low oxygen tension, yet how hypoxic conditions may affect innate immune cells and their impact on tumor progression is poorly understood. Targeted deletion of the hypoxia responsive transcription factor HIF-1α in macrophages in a progressive murine model of breast cancer resulted in reduced tumor growth, although VEGF-A and vascularization was unchanged. Tumor associated macrophages can suppress tumor infiltrating T cells by several mechanisms, and we found that hypoxia powerfully augmented macrophage-mediated T cell suppression in vitro in a manner dependent on macrophage expression of HIF-1α. Our findings link the innate immune hypoxic response to tumor progression through induction of T cell suppression in the tumor microenvironment.
doi:10.1158/0008-5472.CAN-10-1439
PMCID: PMC2948598  PMID: 20841473
17.  Astrocyte hypoxic response is essential for pathological but not developmental angiogenesis of the retina 
Glia  2010;58(10):1177-1185.
Vascular/parenchymal crosstalk is increasingly recognized as important in the development and maintenance of healthy vascularized tissues. The retina is an excellent model in which to study the role of cell type-specific contributions to the process of blood vessel and neuronal growth. During retinal vascular development, glial cells such as astrocytes provide the template over which endothelial cells migrate to form the retinal vascular network, and hypoxia-regulated vascular endothelial growth factor (VEGF) has been demonstrated to play a critical role in this process as well as pathological neovascularization. To investigate the nature of cell-specific contributions to this process, we deleted VEGF and its upstream regulators, the hypoxia-inducible transcription factors HIF-1α and HIF-2α, and the negative regulator of HIFα, von Hippel-Lindau protein (VHL), in astrocytes. We found that loss of hypoxic response and VEGF production in astrocytes does not impair normal development of retinal vasculature, indicating that astrocyte-derived VEGF is not essential for this process. In contrast, using a model of oxygen-induced ischemic retinopathy, we show that astrocyte-derived VEGF is essential for hypoxia-induced neovascularization. Thus, we demonstrate that astrocytes in the retina have highly divergent roles during developmental, physiological angiogenesis and ischemia-driven, pathological neovascularization.
doi:10.1002/glia.20997
PMCID: PMC2993327  PMID: 20544853
18.  O2 regulates stem cells through Wnt/β-catenin signalling 
Nature cell biology  2010;12(10):1007-1013.
Stem cells reside in specialized microenvironments or “niches” which regulate their function. In vitro studies employing hypoxic culture conditions (≤ 5% O2) have revealed strong regulatory links between O2 availability and stem/precursor cell functions1–6. Therefore, while some stem cells are perivascular, others may occupy hypoxic niches and be regulated by O2 gradients. However, the underlying mechanisms remain unclear. Here, we show that Hypoxia Inducible Factor-1α (HIF-1α), a principal mediator of hypoxic adaptations, modulates Wnt/β-catenin signalling in hypoxic embryonic stem (ES) cells by enhancing β-catenin activation and expression of downstream effectors LEF-1 and TCF-1. This regulation extends to primary cells, including isolated neural stem cells (NSCs), and is not observed in differentiated cells. In vivo, Wnt/β-catenin activity is closely associated with low O2 regions in the subgranular zone (SGZ) of the hippocampus, a key NSC niche7. Hif-1α deletion impairs hippocampal Wnt-dependent processes, including NSC proliferation, differentiation and neuronal maturation. This decline correlates with reduced Wnt/β-catenin signalling in the SGZ. Therefore, O2 availability may have a direct role in stem cell regulation via HIF-1α modulation of Wnt/β-catenin signalling.
doi:10.1038/ncb2102
PMCID: PMC3144143  PMID: 20852629
Embryonic stem cells; hypoxic niche; HIF-1α/ARNT; Wnt/β-catenin signalling; neurogenesis
19.  Differential Effects of MYH9 and APOL1 Risk Variants on FRMD3 Association with Diabetic ESRD in African Americans 
PLoS Genetics  2011;7(6):e1002150.
Single nucleotide polymorphisms (SNPs) in MYH9 and APOL1 on chromosome 22 (c22) are powerfully associated with non-diabetic end-stage renal disease (ESRD) in African Americans (AAs). Many AAs diagnosed with type 2 diabetic nephropathy (T2DN) have non-diabetic kidney disease, potentially masking detection of DN genes. Therefore, genome-wide association analyses were performed using the Affymetrix SNP Array 6.0 in 966 AA with T2DN and 1,032 non-diabetic, non-nephropathy (NDNN) controls, with and without adjustment for c22 nephropathy risk variants. No associations were seen between FRMD3 SNPs and T2DN before adjusting for c22 variants. However, logistic regression analysis revealed seven FRMD3 SNPs significantly interacting with MYH9—a finding replicated in 640 additional AA T2DN cases and 683 NDNN controls. Contrasting all 1,592 T2DN cases with all 1,671 NDNN controls, FRMD3 SNPs appeared to interact with the MYH9 E1 haplotype (e.g., rs942280 interaction p-value = 9.3E−7 additive; odds ratio [OR] 0.67). FRMD3 alleles were associated with increased risk of T2DN only in subjects lacking two MYH9 E1 risk haplotypes (rs942280 OR = 1.28), not in MYH9 E1 risk allele homozygotes (rs942280 OR = 0.80; homogeneity p-value = 4.3E−4). Effects were weaker stratifying on APOL1. FRMD3 SNPS were associated with T2DN, not type 2 diabetes per se, comparing AAs with T2DN to those with diabetes lacking nephropathy. T2DN-associated FRMD3 SNPs were detectable in AAs only after accounting for MYH9, with differential effects for APOL1. These analyses reveal a role for FRMD3 in AA T2DN susceptibility and accounting for c22 nephropathy risk variants can assist in detecting DN susceptibility genes.
Author Summary
African Americans have high rates of kidney disease attributed to type 2 diabetes mellitus. However, approximately 25% of patients are misclassified and have non-diabetic kidney disease on renal biopsy. The APOL1-MYH9 gene region on chromosome 22 is powerfully associated with non-diabetic kidney diseases in African Americans. Therefore, we tested for interactions between single nucleotide polymorphisms across the genome with APOL1 and MYH9 non-diabetic nephropathy risk variants in African Americans with presumed diabetic nephropathy. Markers in FRMD3, a gene associated with type 1 diabetic nephropathy in Caucasians, appeared to interact with MYH9; however, increased nephropathy risk was seen in diabetic cases lacking two MYH9 risk haplotypes, and protective effects were seen in those with two MYH9 risk haplotypes. Stratified analyses based on the chromosome 22 nephropathy risk haplotypes demonstrated that FRMD3 variants were associated with diabetic nephropathy risk in cases without two MYH9 (or APOL1) risk haplotypes. It appears that African Americans with diabetes and kidney disease who are not chromosome 22 nephropathy risk variant homozygotes are enriched for the presence of diabetic nephropathy and FRMD3 risk alleles. This genetic dissection ultimately allowed for detection of the FRMD3 diabetic nephropathy gene association in a subset of cases enriched for this disorder.
doi:10.1371/journal.pgen.1002150
PMCID: PMC3116917  PMID: 21698141
20.  Deletion of Vascular Endothelial Growth Factor in myeloid cells accelerates tumorigenesis 
Nature  2008;456(7223):814-818.
Angiogenesis and the development of a vascular network is required for tumor progression, and involves release of angiogenic factors, including vascular endothelial growth factor (VEGF), from both malignant and stromal cell types1. Infiltration by cells of the myeloid lineage is a hallmark of many tumors, and in many cases the macrophages in these infiltrates express VEGF2. Here we show that deletion of inflammatory cell-derived VEGF attenuates the formation of a typical high-density vessel network, thus blocking the angiogenic switch in solid tumors. Vasculature in tumors lacking myeloid cell-derived VEGF was less tortuous, with increased pericyte coverage and decreased vessel length, indicating vascular normalization. In addition, loss of myeloid-derived VEGF decreases VEGFR2 phosphorylation in tumors, even though overall VEGF levels in the tumors are unaffected. However, myeloid deletion of VEGF resulted in an accelerated tumor progression in multiple subcutaneous isograft models and an autochthonous transgenic model of mammary tumorigenesis, with less overall tumor cell death and decreased tumor hypoxia. Furthermore, loss of myeloid cell VEGF increased tumor susceptibility to chemotherapeutic cytotoxicity. This demonstrates that myeloid-derived VEGF is essential for tumorigenic alteration of vasculature and signaling to VEGFR2, and that these changes act to retard, not promote, tumor progression.
doi:10.1038/nature07445
PMCID: PMC3103772  PMID: 18997773
21.  The asparaginyl hydroxylase FIH (Factor Inhibiting HIF) is an essential regulator of metabolism 
Cell metabolism  2010;11(5):364-378.
SUMMARY
Factor Inhibiting HIF-1α (FIH) is an asparaginal hydroxylase. Hydroxylation of HIF-α proteins by FIH blocks association of HIFs with the transcriptional co-activators CBP/p300, thus inhibiting transcriptional activation. We have created mice with a null mutation in the FIH gene, and found that it has little or no discernable role in mice in altering classical aspects of HIF function, e.g., angiogenesis, erythropoiesis, or development. Rather, it is an essential regulator of metabolism: mice lacking FIH exhibit reduced body weight, elevated metabolic rate, hyperventilation, improved glucose and lipid homeostasis, and are resistant to high fat diet-induced weight gain and hepatic steatosis. Neuron-specific loss of FIH phenocopied some of the major metabolic phenotypes of the global null animals: those mice have reduced body weight, increased metabolic rate, enhanced insulin sensitivity, and are also protected against high fat diet-induced weight gain. These results demonstrate that FIH acts to a significant degree through the nervous system to regulate metabolism.
doi:10.1016/j.cmet.2010.03.001
PMCID: PMC2893150  PMID: 20399150
22.  Role of HIF-1α in skeletal development 
Angiogenesis and osteogenesis are tightly coupled during bone development and regeneration. Mesenchymal cells in the developing stroma elicit angiogenic signals to recruit new blood vessels into bone. Reciprocal signals, likely emanating from the incoming vascular endothelium, stimulate mesenchymal cell specification through additional interactions with cells within the vascular stem cell niche. The hypoxia-inducible factor-1 alpha (HIF-1) pathway has been identified as a key component in this process. We demonstrated that overexpression of HIF-1 in mature osteoblasts through disruption of the von Hippel-Lindau protein profoundly increases angiogenesis and osteogenesis; these processes appear to be coupled by cell nonautonomous mechanisms involving the action of vascular endothelial growth factor (VEGF) on the endothelial cells. The same occurred in the model of injury-mediated bone regeneration (distraction osteogenesis). Surprisingly, manipulation of HIF-1 does not influence angiogenesis of the skull bones, where earlier activation of HIF-1 in the condensing mesenchyme upregulates osterix during cranial bone formation.
doi:10.1111/j.1749-6632.2009.05238.x
PMCID: PMC3047468  PMID: 20392254
knockout mice; osteoblasts; hypoxia-inducible factor; angiogenesis
23.  Prolyl hydroxylase 3 (PHD3) is essential for hypoxic regulation of neutrophilic inflammation in humans and mice 
The Journal of Clinical Investigation  2011;121(3):1053-1063.
The regulation of neutrophil lifespan by induction of apoptosis is critical for maintaining an effective host response and preventing excessive inflammation. The hypoxia-inducible factor (HIF) oxygen-sensing pathway has a major effect on the susceptibility of neutrophils to apoptosis, with a marked delay in cell death observed under hypoxic conditions. HIF expression and transcriptional activity are regulated by the oxygen-sensitive prolyl hydroxylases (PHD1–3), but the role of PHDs in neutrophil survival is unclear. We examined PHD expression in human neutrophils and found that PHD3 was strongly induced in response to hypoxia and inflammatory stimuli in vitro and in vivo. Using neutrophils from mice deficient in Phd3, we demonstrated a unique role for Phd3 in prolonging neutrophil survival during hypoxia, distinct from other hypoxia-associated changes in neutrophil function and metabolic activity. Moreover, this selective defect in neutrophil survival occurred in the presence of preserved HIF transcriptional activity but was associated with upregulation of the proapoptotic mediator Siva1 and loss of its binding target Bcl-xL. In vivo, using an acute lung injury model, we observed increased levels of neutrophil apoptosis and clearance in Phd3-deficient mice compared with WT controls. We also observed reduced neutrophilic inflammation in an acute mouse model of colitis. These data support what we believe to be a novel function for PHD3 in regulating neutrophil survival in hypoxia and may enable the development of new therapeutics for inflammatory disease.
doi:10.1172/JCI43273
PMCID: PMC3049381  PMID: 21317538
24.  Accounting for multiple comparisons in a genome-wide association study (GWAS) 
BMC Genomics  2010;11:724.
Background
As we enter an era when testing millions of SNPs in a single gene association study will become the standard, consideration of multiple comparisons is an essential part of determining statistical significance. Bonferroni adjustments can be made but are conservative due to the preponderance of linkage disequilibrium (LD) between genetic markers, and permutation testing is not always a viable option. Three major classes of corrections have been proposed to correct the dependent nature of genetic data in Bonferroni adjustments: permutation testing and related alternatives, principal components analysis (PCA), and analysis of blocks of LD across the genome. We consider seven implementations of these commonly used methods using data from 1514 European American participants genotyped for 700,078 SNPs in a GWAS for AIDS.
Results
A Bonferroni correction using the number of LD blocks found by the three algorithms implemented by Haploview resulted in an insufficiently conservative threshold, corresponding to a genome-wide significance level of α = 0.15 - 0.20. We observed a moderate increase in power when using PRESTO, SLIDE, and simpleℳ when compared with traditional Bonferroni methods for population data genotyped on the Affymetrix 6.0 platform in European Americans (α = 0.05 thresholds between 1 × 10-7 and 7 × 10-8).
Conclusions
Correcting for the number of LD blocks resulted in an anti-conservative Bonferroni adjustment. SLIDE and simpleℳ are particularly useful when using a statistical test not handled in optimized permutation testing packages, and genome-wide corrected p-values using SLIDE, are much easier to interpret for consumers of GWAS studies.
doi:10.1186/1471-2164-11-724
PMCID: PMC3023815  PMID: 21176216
25.  MHC heterozygote advantage in simian immunodeficiency virus-infected Mauritian cynomolgus macaques 
Science translational medicine  2010;2(22):22ra18.
The importance of a broad CD8-T lymphocyte (CD8-TL) immune response to HIV is unknown. Ex vivo measurements of immunological activity directed at a limited number of defined epitopes provide an incomplete portrait of the actual immune response. Here we examined viral loads in SIV-infected MHC homozygous and heterozygous Mauritian cynomolgus macaques (MCM). Chronic viremia in MHC homozygous macaques was 80-fold greater than in MHC heterozygous macaques. Virus from MHC homozygous macaques accumulated 11 to 14 variants consistent with escape from CD8-TL responses after one year of SIV infection. The pattern of mutations detected in MHC heterozygous macaques suggests that their epitope-specific CD8-TL responses are a composite of those present in their MHC homozygous counterparts. These results provide the clearest example of MHC heterozygote advantage among individuals infected with the same immunodeficiency virus strain, suggesting that broad recognition of multiple CD8-TL epitopes should be a key feature of HIV vaccines.
doi:10.1126/scitranslmed.3000524
PMCID: PMC2865159  PMID: 20375000
MHC; heterozygosity; CD8; SIV

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