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1.  Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart 
The Journal of Physiology  2014;592(Pt 21):4715-4731.
Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l−1 NaCl (as control) or 0.7 mmol l−1 NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics.
PMCID: PMC4253472  PMID: 25172947
2.  HIF-1α Influences Myeloid Cell Antigen Presentation and Response to Subcutaneous OVA Vaccination 
Hypoxia-inducible factor (HIF)-1 is a transcription factor known to play an important role in regulating the innate immune response to infection. Under baseline conditions, cellular HIF-1 levels in leukocytes are scarce, but levels rise rapidly in response to hypoxia or molecular signals of infection or inflammation such as microbial surface molecules and host-derived cytokines. Innate immune cells such as macrophages, neutrophils and mast cells exhibit increased microbicidal activity when HIF-1 levels are increased, and mice lacking HIF-1 are more susceptible to invasive bacterial infection. In this study, we used genetic and pharmacologic means to determine whether HIF-1 also plays an important role in the adaptive immune response to infection. HIF-1α/Tie-2 Cre+ mice harboring a >90% knockdown of HIF-1 in myeloid cells were studied. We found antigen-presenting cells from these mice expressed lower levels of MHC-II and the costimulatory molecules CD80 and CD86, and were less able to induce T cell proliferation. These differences were present at baseline and persisted after activation. Increasing HIF-1 levels in WT cells by using the prolyl hydroxylase inhibitor drug AKB-4924 had the opposite effect, increasing MHC and co-stimulatory molecule expression and T cell proliferation. In experimental vaccination, HIF-1α/Tie-2 Cre+ mice exhibited a weaker T cell response and lower antibody levels in response to vaccination than WT mice, while WT mice treated with a drug to elevate HIF-1 responded more strongly to vaccination. Thus HIF-1 participates in bridging the innate and adaptive immune responses, and may merit further exploration as an adjuvant target.
PMCID: PMC3783576  PMID: 23686259
3.  Nonclinical pharmacokinetics and activity of etirinotecan pegol (NKTR-102), a long-acting topoisomerase 1 inhibitor, in multiple cancer models 
Cancer Chemotherapy and Pharmacology  2014;74(6):1125-1137.
The aim of the study was to demonstrate the activity of etirinotecan pegol, a polymer conjugate of irinotecan, in multiple human tumor models and to establish both the pharmacokinetic/pharmacodynamics (PK/PD) relationship and clinical relevance of the findings.
Experimental design
Anti-tumor activity was evaluated in mouse models of human lung, colorectal, breast, ovarian, and gastric cancers. Etirinotecan pegol was administered intravenously (once or every 3–7 days) to animals with established tumors. Activity was assessed by tumor growth delay (TGD) and regression. Mice bearing established colorectal and lung tumors were treated with etirinotecan pegol or irinotecan, and serial blood and tumor samples were collected at planned times between 0 and 60 days post-treatment for quantitation of etirinotecan pegol and SN38. For PK analysis, analyte concentration–time data were fit with compartmental models; PK/PD analysis was based on an inhibitory Emax response model.
Etirinotecan pegol was active in all tumor models. TGD was sustained for 2–10 weeks after last dose, while conventional irinotecan resulted in little suppression of tumor growth. Etirinotecan pegol was eliminated very slowly from the tumor (t1/2 = 17 days), achieving higher and more sustained tumor exposure when compared with conventional irinotecan. The increased tumor exposure following etirinotecan pegol correlated with strong and prolonged suppression of tumor growth. Sustained plasma exposure to active SN38 was consistently observed across nonclinical species (including mouse, rat, and dog) and translated to cancer patients.
Etirinotecan pegol is the first long-acting topoisomerase 1 inhibitor that provides sustained exposure, which results in prolonged anti-tumor activity in a wide variety of cancer models.
Electronic supplementary material
The online version of this article (doi:10.1007/s00280-014-2577-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4236622  PMID: 25228368
Topoisomerase 1 inhibition; Anticancer activity; Etirinotecan pegol; SN38; Pharmacokinetics
Wound closure requires a complex series of micro-environmentally influenced events. A key aspect of wound closure is the migration of keratinocytes across the open wound. It has been found previously that the response to hypoxia via the HIF-1α transcription factor is a key feature of wound closure. The need for hypoxic response is likely due to interrupted wound vasculature as well as infection, and in this work, we investigated the need for a highly related hypoxic response transcription factor, HIF-2α. This factor was deleted tissue-specifically in mice, and the resulting mice were found to have an accelerated rate of wound closure. This is correlated with a reduced bacterial load and inflammatory response in these mice. This indicates that manipulating or reducing the HIF-2α response in keratinocytes could be a useful means to accelerate wound healing and tissue repair.
PMCID: PMC3877686  PMID: 24037341
Wound closure requires a complex series of micro-environmentally influenced events. A key aspect of wound closure is the migration of keratinocytes across the open wound. It has been found previously that the response to hypoxia via the HIF-1α transcription factor is a key feature of wound closure. The need for hypoxic response is likely due to interrupted wound vasculature as well as infection, and in this work, we investigated the need for a highly related hypoxic response transcription factor, HIF-2α. This factor was deleted tissue-specifically in mice, and the resulting mice were found to have an accelerated rate of wound closure. This is correlated with a reduced bacterial load and inflammatory response in these mice. This indicates that manipulating or reducing the HIF-2α response in keratinocytes could be a useful means to accelerate wound healing and tissue repair.
PMCID: PMC3877686  PMID: 24037341
6.  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.
PMCID: PMC4089958  PMID: 22556263
fibroblast; hypoxia; hypoxia-inducible factor-1
7.  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.
PMCID: PMC3646920  PMID: 23250618
Hypoxia inducible factor (HIF)-1α; Asthma; Allergic inflammation; Eosinophils; Chemotaxis; Airway hyperresponsiveness
8.  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.
PMCID: PMC3977965  PMID: 24076634
9.  Activation of Hypoxia‐Inducible Factor‐2 in Adipocytes Results in Pathological Cardiac Hypertrophy 
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.
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.
PMCID: PMC3886757  PMID: 24326162
adipocytes; cardiomyopathy; hypoxia; inflammation; obesity
10.  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
11.  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.
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
12.  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.
PMCID: PMC3606899  PMID: 22371073
Hypoxia inducible factor-1 (HIF-1); innate immunity; Staphylococcus aureus; bacterial infection; antibiotic-resistant bacteria
13.  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.
PMCID: PMC3754066  PMID: 23785211
14.  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.
PMCID: PMC3690486  PMID: 19698078
15.  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.
PMCID: PMC3506175  PMID: 18560292
endotracheal tube; monitoring; nursing; pressure; research
16.  HIF1α and HIF2α: sibling rivalry in hypoxic tumor growth and progression 
Nature reviews. Cancer  2011;12(1):9-22.
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.
PMCID: PMC3401912  PMID: 22169972
17.  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.
PMCID: PMC3080910  PMID: 21502085
18.  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.
PMCID: PMC3257537  PMID: 22084310
19.  Endothelial Cell HIF-1α and HIF-2α Differentially Regulate Metastatic Success 
Cancer Cell  2012;21(1):52-65.
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.
► 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
PMCID: PMC3334270  PMID: 22264788
20.  von Hippel-Lindau protein regulates transition from fetal to adult circulatory system in retina 
Development (Cambridge, England)  2010;137(9):1563-1571.
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.
PMCID: PMC3224975  PMID: 20388654
Angiogenesis; Circulatory system; Hypoxia-inducible factor 1
21.  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.
PMCID: PMC2948598  PMID: 20841473
22.  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.
PMCID: PMC2993327  PMID: 20544853
23.  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.
PMCID: PMC3144143  PMID: 20852629
Embryonic stem cells; hypoxic niche; HIF-1α/ARNT; Wnt/β-catenin signalling; neurogenesis
24.  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.
PMCID: PMC3116917  PMID: 21698141
25.  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.
PMCID: PMC3103772  PMID: 18997773

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