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Hypoxia and Inflammation



Schematic Overview of the Link between Hypoxia and Inflammation in Cancer. In tumor cells, oncogenes, inflammatory signals (mediated in part through toll-like receptors [TLRs]), and hypoxia activate nuclear factor κB (NF-κB) and hypoxia-inducible factor (HIF) 1α (which activate each other). These factors induce a gene program that recruits and activates leukocytes (through release of chemokines and cytokines), stimulates angiogenesis and the formation of an abnormal vasculature and endothelium (through release of angiogenic signals), and increases tumor-cell invasion, metastasis, epithelial-to-mesenchymal transition (EMT), survival, proliferation, and metabolic reprogramming. In leukocytes, hypoxia also activates NF-κB and HIF-1α; endogenous ligands, released from necrotic cancer cells, activate TLRs upstream of NF-κB and HIF-1α, and HIF-1α up-regulates TLR expression. A resultant gene-expression profile leads to the production of cytokines and angiogenic signals and skews their polarization phenotype. Tumor vessels with two prolyl hydroxylase (PHD) domain 2 (PHD2) alleles have an abnormal endothelium, are hypoperfused, and cause tumor hypoxia, which fuels tumor-cell invasiveness and metastasis. In contrast, tumor vessels lacking one PHD2 allele have increased HIF-2α levels, which result in an up-regulation of factors that counteract the development of tumor endothelial abnormalities; this, in turn, results in improved tumor-vessel perfusion and oxygenation and, secondarily, reduced metastasis.
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Mechanisms of Disease
Robert S. Schwartz, M.D., Editor
Review article
n engl j med 364;7 february 17, 2011
Hypoxia and Inflammation
Holger K. Eltzschig, M.D., Ph.D., and Peter Carmeliet, M.D., Ph.D.
From the Department of Anesthesiology,
University of Colorado Denver, Aurora
(H.K.E.); the Department of Anesthesiol-
ogy and Intensive Care Medicine, Tübin-
gen University Hospital, Tübingen, Ger-
many (H.K.E.); and Vesalius Research
Center VIB, and Vesalius Research Cen-
ter, K.U. Leuven — both in Leuven, Bel-
gium (P.C.). Address reprint requests to
Dr. Eltzschig at the Department of Anes-
thesiology, University of Colorado Denver,
12700 E. 19th Ave., Mailstop B112, Re-
search Complex 2, Rm. 7124, Aurora, CO
80045, or at holger.eltzschig@ucdenver
N Engl J Med 2011;364:656-65.
Copyright © 2011 Massachusetts Medical Society.
ammals have oxygen-sensing mechanisms that help them
adapt quickly to hypoxia by increasing respiration, blood flow, and survival
responses. If an inadequate supply of oxygen persists, additional mecha-
nisms attempt to restore oxygenation or help the body adapt to hypoxia.
other mechanisms rely on oxygen-sensing prolyl hydroxylases (PHDs), which hydrox-
ylate prolines in the alpha subunit of the hypoxia-inducible transcription factor (HIF).
This transcription factor is a heterodimer with two subunits: HIF-or HIF-and
HIF-1β (or aryl hydrocarbon receptor nuclear translocator [ARNT] protein). HIF-
is ubiquitous, whereas HIF-is restricted to certain tissues.
In this review, we show the ways in which the PHD–HIF system affects inflam-
matory processes. We discuss the regulation of immune responses by hypoxia-
induced signaling, outline molecular aspects of the cross-talk between hypoxia and
inflammation, and illustrate the link between hypoxia and inflammation in in-
flammatory bowel disease, certain cancers, and infections.
Hy pox ia -Induced Infl amm at ion
The concept that hypoxia can induce inflammation has gained general acceptance
from studies of the hypoxia signaling pathway. In persons with mountain sickness,
for example, levels of circulating proinflammatory cytokines increase, and leakage
of fluid (“vascular leakage”) causes pulmonary or cerebral edema.
Increased se-
rum levels of interleukin-6, the interleukin-6 receptor, and C-reactive protein — all
markers of inflammation were increased in healthy volunteers who spent 3 nights
at an elevation higher than 3400 m.
At 8400 m, healthy climbers ascending Mount
Everest had severe hypoxemia (partial pressure of arterial oxygen [PaO
], 25 mm Hg).
Alveolar–arterial oxygen differences were elevated in these climbers, a finding that
is consistent with subclinical high-altitude pulmonary edema.
Moreover, vascular
leakage, accumulations of inflammatory cells in multiple organs, and elevated serum
levels of cytokines occur in mice after short-term exposure to low oxygen concen-
The development of inflammation in response to hypoxia is clinically relevant.
Ischemia in organ grafts increases the risk of inflammation and graft failure or
In patients undergoing kidney transplantation, the renal expression of
toll-like receptor (TLR) 4 — an extracellular receptor for bacterial lipopolysaccha-
ride — was shown to correlate with the degree of ischemic injury. In this study,
donor kidneys with a loss-of-function TLR4 allele, as compared with donor kidneys
that bore a functional allele of the TLR4 gene, had a higher rate of immediate graft
Moreover, increases in pulmonary cytokine levels and TLR expression
was shown to correlate with greater ischemic injury of transplanted lungs and loss
of graft function.
In the setting of obesity, an imbalance between the supply
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n engl j med 364;7 february 17, 2011
of and demand for oxygen in enlarged adipocytes
causes tissue hypoxia and an increase in inflam-
matory adipokines in fat. The resultant infiltration
by macrophages and chronic low-grade systemic
inflammation promote insulin resistance.
together, these clinical studies indicate that hy-
poxia promotes inflammation (Fig. 1).
Infl amm ation a nd T issu e
Hy poxi a
Just as hypoxia can induce inflammation, inflamed
lesions often become severely hypoxic. As a result
of the steep oxygen gradient between the anaero-
bic intestinal lumen and the metabolically active
lamina propria mucosae, intestinal epithelial cells
are normally hypoxic.
In inf lammatory bowel
disease, not only does the entire mucosa becomes
even more hypoxic,
but surgical specimens of
the inflamed intestine contain elevated levels of
HIF-1α and HIF-2α.
Contributors to tissue hypoxia during inflam-
mation include an increase in the metabolic
demands of cells and a reduction in metabolic
substrates caused by thrombosis, trauma, com-
pression (interstitial hypertension), or atelectasis
(airway plugging). Moreover, multiplication of in-
tracellular pathogens can deprive infected cells of
We stress that in the case of inflamed
tissue, hypoxia is not a bystander but instead can
influence the environment of the tissue, particu-
larly by regulating oxygen-dependent gene ex-
HIF a nd Ox ygen Sensor s
Cellular adaptations to hypoxia rely on the tran-
scription factor HIF, which is inactive when oxy-
gen is abundant but is activated in hypoxic condi-
tions (Fig. 2).
Oxygen-dependent hydroxylation
of prolyl residues in HIF-1α or HIF-2α in the HIF
heterodimer by PHDs creates a binding site for
the von Hippel–Lindau (VHL) gene product, which
is a component of the E3 ubiquitin ligase com-
plex; the binding of the VHL gene product to
HIF-(or HIF-2α) culminates in the destruction
of the α subunit in proteasomes.
In addition,
hydroxylation of asparagyl residues in HIF-1α (or
HIF-) by factor-inhibiting HIF an oxygen-
dependent asparagyl hydroxylase — reduces the
transcriptional activity of HIF.
The functions of
both hydroxylases (PHDs and factor-inhibiting
HIF) depend on oxygen.
Germline mutations
in the PHD2 gene have been found in association
with familial erythrocytosis and with a syndrome
of familial erythrocytosis with paraganglioma
inactivating mutations of both copies of the VHL
gene cause Von Hippel–Lindau disease (which is
characterized by hemangioblastomas, clear-cell re-
nal carcinomas, and pheochromocytomas).
HIF can be activated under normoxic condi-
tions, which allows the initiation of an inflam-
matory response before tissues become hypoxic.
Examples of this mechanism are the increase in
HIF-1α transcription by bacterial lipopolysaccha-
and the stabilization of HIF-1α when reac-
tive oxygen species and reduced cellular iron in-
hibit prolyl hydroxylase.
The phenotype of mice with HIF-1α deficiency
differs from that of mice with HIF-2α deficiency,
which implies that these components of the HIF
transcription-factor polypeptides have different
target genes.
The HIF2A gene in certain forms
of familial erythrocytosis has a gain-of-function
mutation, which probably causes normoxic stabi-
lization of the HIF-2α protein.
Hy pox ia Signa ling a nd NF -κB
Members of the nuclear factor κB (NF-κB) family
of transcription factors regulate inflammation and
orchestrate immune responses and tissue homeo-
Members of this family interact with
members of the PHD–HIF pathway in ways that
link inflammation to hypoxia (Fig. 2).
of a mouse model of inflammatory bowel disease
indicate that PHDs have a regulatory role in the
antiapoptotic effects of NF-κB in intestinal in-
The hypoxia of intestinal ischemia
reperfusion activates NF-κB in intestinal epithe-
lial cells, which in turn increases the production
of tumor necrosis factor α (TNF-α), a proinflam-
matory cytokine, but simultaneously attenuates
intestinal epithelial apoptosis.
Additional inter-
actions between hypoxia and inflammation are
seen in theB kinase complex, a regulatory com-
ponent of NF-κB (Fig. 2),
and in the regulation
of HIF-1α transcription by NF-κB before and
during inflammation.
Hypoxia amplifies the
NF-κB pathway by increasing the expression and
signaling of TLRs, which enhance the production
of antimicrobial factors and stimulate phagocy-
tosis, leukocyte recruitment, and adaptive im-
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Hy pox ia Signa ling a nd Inna te
Immunit y
The initial defense against pathogens relies on
the activation of neutrophils, macrophages, mast
cells, dendritic cells, and natural killer cells. These
cells of the innate immune system can rapidly
eradicate pathogens and transmit signals that
amplify the adaptive immune response. Myeloid
cells have HIF-dependent ways of functioning in
the oxygen-depleted conditions of hypoxic mi-
HIF-1α–null phagocytes can-
not efficiently eliminate bacterial loads but in-
stead form persistent ulcerative lesions.
HIF-1α regulates several functions of myeloid
cells (Fig. 3).
It allows myeloid cells to gener-
ate ATP in oxygen-deprived inflamed tissues,
thereby stimulating the aggregation, motility,
invasiveness, and bactericidal activity of myeloid
HIF-1α also prolongs the lifespan of
neutrophils in hypoxic conditions by inhibiting
In von Hippel–Lindau disease, neu-
trophils are characterized by reduced apoptosis
and enhanced phagocytosis of bacteria under
normoxic conditions, presumably owing to the
failure to degrade HIF-1α.
Hy pox ia a nd Ada pti ve I mmun it y
HIF-1α also influences adaptive immunity.
with HIF-1α-deficient lymphocytes have elevated
levels of anti–double-stranded DNA antibodies
and rheumatoid factor in serum, as well as pro-
teinuria and deposits of IgG and IgM in the kid-
Inflammation in Hypoxic Conditions Hypoxia in Inflammatory Conditions
Pulmonary edema Acute lung injury
Infections with pathogens
Organ transplantation
Adipose tissue
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Figure1.LinksbetweenHypoxiaandInf lammation.
Shown is an overview of clinical conditions characterized primarily by tissue hypoxia that causes inflammatory changes (left) and inflam-
matory diseases leading to tissue hypoxia (right).
mech anisms of disease
n engl j med 364;7 february 17, 2011
Increased production of HIF-in T cells
induces a shift from a type 1 helper T-cell (Th1)
phenotype, which enhances functions of macro-
phages and cytotoxic T cells, to a type 2 helper
T-cell (Th2) phenotype, which inhibits Th1-medi-
ated microbicidal actions of T cells by increasing
production of interleukin-10 and decreasing
interferon-γ levels.
HIF also influences regula-
tory T cells,
a specialized subgroup of inhibitory
T cells.
Hypoxia-induced signaling pathways
stimulate the differentiation and proliferation of
regulatory T cells
and increase extracellular lev-
els of adenosine,
which protects tissues by re-
straining effector functions of T cells.
Epithe li al R es ponses t o H yp ox ic
Infl am mat ion
Activation of the PHD–HIF pathway promotes the
resolution of mucosal inflammation in mice.
Hypoxia-induced changes in gene expression by
epithelial cells help to promote mucosal barrier
function (e.g., through activation of intestinal tre-
foil factor)
or to increase the production by the
High oxygen
p50 p65
p50 p65
p50 p65
Low oxygen
HIF Protein
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In hypoxic conditions (left), hypoxia-inducible factor (HIF) α and HIF-β subunits translocate to the nucleus, where they bind as heterodi-
mers to a hypoxia response promoter element (HRE), inducing transcription of numerous genes, including those of nuclear factor κB
(NF-κB) and toll-like receptors (TLRs). In normoxia, HIF-α is hydroxylated by prolyl hydroxylases (PHDs) and factor-inhibiting HIF (FIH)
and is thereby targeted for proteasomal degradation (in the case of PHDs) or rendered transcriptionally less active (in the case of FIH;
not shown here). In resting cells (right), NF-κB, a heterodimer consisting of p50 and p65 subunits, is inactive in the cytosol because it is
associated with nuclear factor of kappa light polypeptide gene enhancer in B cells alpha (IκBα), a regulatory component of NF-κB. At the
time of cellular activation, the beta subunit of the IκB kinase complex (IKKβ) phosphorylates the inhibitor IκBα, which thereby becomes
degraded and liberates NF-κB for translocation in the nucleus, where it can activate the transcription of inflammatory genes as well as
of HIF (genes involved in tissue protection and homeostasis are not shown). PHDs and FIH regulate NF-κB activation by controlling the
activity of IKKβ.
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epithelium of antiinflammatory signaling mole-
cules such as adenosine.
These adaptive re-
sponses to hypoxia are activated during muco-
sal inflammation and promote the resolution of
inflammatory bowel disease
or acute lung
In mice with targeted deletion of
HIF-1α in intestinal epithelia, as compared with
mice that have intact HIF-1α throughout, more
severe colitis develops after exposure to trinitro-
benzene sulfonic acid. In contrast, in mice with
inflammatory bowel disease and elevated HIF lev-
els due to deficiency of the VHL gene, as com-
pared with control animals, weight loss, disease
activity, and histologic signs of intestinal inflam-
mation are all reduced.
In mice with colitis that
is chemically induced by oral administration of
dextran sulfate sodium, treatment with pharma-
cologic compounds that enhance stabilization of
HIF reduces intestinal inflammation.
Several studies have shown that hypoxia en-
hances the enzymatic conversion of precursor nu-
cleotides such as ATP, adenosine diphosphate, or
AMP to adenosine,
thereby elevating extracel-
lular levels of adenosine, an antiinflammatory
signaling molecule involved in restraining innate
immune responses.
A single-nucleotide poly-
morphism in CD39, an enzyme required for ex-
tracellular generation of adenosine, is associated
with low levels of CD39
; in a case–control study,
this genetic variant was observed in patients with
High oxygenLow oxygen
and necrosis
Increase in
Increase in
Decrease in
Dendritic cell
Mast cell
Treg cell
CD4+ helper T cell
CD8+ T cell
Adaptive immune system
Innate immune system
Bacterial killing
Antimicrobial activity
Antigen presentation
Release of permeability factors and
Hypoxic survival
Endothelial adhesion
M2 macrophage polarization
Release of inflammatory cytokines
T-cell effector activity
Th1 polarization
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In the example shown in this schematic overview, the epithelium (left) is breached by invading pathogens, leading to tissue damage; as
a result, innate immune cells mount a host defense response, which is amplified by recruited adaptive immune cells. In general, hypoxia
amplifies the activity of innate immune cells while suppressing the response of the adaptive immune system, in part by promoting dif-
ferentiation of regulatory T cells and negatively regulating the function of CD4+ helper T (Th) cells and CD8+ cytotoxic T cells and the
polarization of type 1 Th (Th1) cells. By negatively regulating adaptive immunity, hypoxia prevents excessive activation of the immune
host defense, which might otherwise lead to collateral tissue damage.
mech anisms of disease
n engl j med 364;7 february 17, 2011
Crohn’s disease more frequently than it was seen
in healthy subjects.
HIF stimulates the production of extracellular
and suppresses both its uptake into
the intracellular compartment and its intracellu-
lar metabolism.
HIF also enhances adenosine-
receptor signaling by increasing the expression
on the cell surface of adenosine receptors
— an effect that attenuates immune responses,
vascular fluid leakage, and neutrophil accumula-
tion in the presence of myocardial, renal, he-
patic, or intestinal ischemia or acute lung inju-
HIF-dependent induction of the axon
guidance signal netrin-1 in epithelia interferes
with the entry of inflammatory cells into hypoxic
organs by enhancing extracellular adenosine sig-
naling events.
Other studies have shown that HIF
also attenuates epithelial inflammation through
induction of epithelial decay-accelerating factor
(which clears epithelia from neutrophils)
induction of barrier-protective genes in the case
of experimentally induced colitis or hypoxia.
Ca nce r
Concentrations of oxygen in solid tumors, as com-
pared with those in normal tissues, are frequently
Solid tumors contain increased levels
of HIF-1α and HIF-2α, and these elevated levels
correlate with cancer-related death.
Elevated lev-
els of HIF-1α and HIF-2α in biopsy specimens of
prostate tumors have been associated with an ad-
verse clinical course.
Hypoxia in a solid tumor
stabilizes HIF through hypoxia-dependent inhi-
bition of PHDs. Similarly, oncogenes, or the loss
of function of tumor-suppressor genes, result in
the stabilization of HIF, as happens in the case of
the VHL tumor-suppressor gene. In von Hippel–
Lindau disease, inactivating germline mutations of
the VHL tumor-suppressor gene increase the risk
of renal-cell carcinoma and other tumors.
poxia and inflammation meet at several points in
the setting of cancer (Fig. 4). Activation of HIF in
a hypoxic tumor or in stromal cells within the
tumor augments tumor vascularization.
increase in vascularization changes the morpho-
logic characteristics of tumor vessels and their
endothelial lining in ways that compromise oxy-
gen delivery.
Inflammatory cells also contrib-
ute to anomalies of vessels in tumors by releas-
ing vascular endothelial growth factor.
In mice, haplodeletion of PHD2 attenuates tu-
mor-vessel leakiness and vascular distortion while
improving tumor-vessel architecture (“vascular
normalization,as defined by more sharply demar-
cated boundaries and branching points of tumor
and tumor oxygenation.
This change
is associated with a reduction in tumor invasive-
ness and in the risk of metastasis.
This finding
suggests that endothelial cells use PHDs to sense
and correct imbalances in oxygen delivery. Anti-
PHD2 agents may offer a new approach to treat-
ing cancer, since they improve the architecture
and function of tumor vessels.
Experimental evidence indicates that inhibi-
tion of HIF within the inflamed tumor core at-
tenuates the growth and vascularization of tu-
mors and enhances the sensitivity of tumors to
In contrast, inhibition of PHD2 and
stabilization of HIF within the tumor vascula-
ture may play an important role in tumor thera-
py, if the means can be found to selectively di-
rect inhibitors of PHD to the tumor vasculature
and inhibitors of HIF to the hypoxic core.
Infec tions
Stabilization of HIF and induction of HIF-depen-
dent genes occur during infections with pathogens.
For example, infection with Bartonella henselae
the causative agent of bacillary angiomatosis —
is associated with stabilization of HIF-1α and the
transcription of genes that typically become tran-
scribed in hypoxic conditions.
In infected cells,
changes in oxygen consumption, as well as cel-
lular hypoxia and decreased ATP levels, correlate
with HIF stabilization and the release of angio-
genic factors during bacillary angiomatosis.
bilization of HIF during infections can also be
For example, under nor-
moxic conditions, iron uptake by bacteria attenu-
ates PHD activity, stabilizes HIF-1α, and induces
the expression of genes targeted by HIF.
bilization of HIF-has been found in liver-biopsy
specimens obtained from patients with chronic
hepatitis C
and in skin-biopsy specimens ob-
tained from patients with cutaneous infections
caused by Staphylococcus aureus, varicella–zoster vi-
rus, human herpesvirus 8, or Candida albicans.
Pathogens may highjack the host’s HIF path-
way for their own advantage. Pseudomonas aerugi-
nosa rapidly inactivates the adenosine that host
cells produce in an HIF-dependent manner, thus
depriving the host epithelium of the actions of
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extracellular adenosine signaling that promote in-
testinal barrier function during inflammation and
During infection with group A streptococcus
or P. aeruginosa, HIF-1α in immune cells induces
inflammation that helps to eliminate the patho-
Tumor cell
cancer cell
Decreased HIF-2α
Abnormal vessel,
hypoperfusion Oxygenation
Increased HIF-2α
Normal vessel,
better perfusion
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Dr. Eltzschig
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In tumor cells, oncogenes, inflammatory signals (mediated in part through toll-like receptors [TLRs]), and hypoxia activate nuclear factor κB
(NF-κB) and hypoxia-inducible factor (HIF) 1α (which activate each other). These factors induce a gene program that recruits and acti-
vates leukocytes (through release of chemokines and cytokines), stimulates angiogenesis and the formation of an abnormal vasculature
and endothelium (through release of angiogenic signals), and increases tumor-cell invasion, metastasis, epithelial-to-mesenchymal tran-
sition (EMT), survival, proliferation, and metabolic reprogramming. In leukocytes, hypoxia also activates NF-κB and HIF-1α; endogenous
ligands, released from necrotic cancer cells, activate TLRs upstream of NF-κB and HIF-1α, and HIF-1α up-regulates TLR expression.
A resultant gene-expression profile leads to the production of cytokines and angiogenic signals and skews their polarization phenotype.
Tumor vessels with two prolyl hydroxylase (PHD) domain 2 (PHD2) alleles have an abnormal endothelium, are hypoperfused, and cause
tumor hypoxia, which fuels tumor-cell invasiveness and metastasis. In contrast, tumor vessels lacking one PHD2 allele have increased
HIF-2α levels, which result in an up-regulation of factors that counteract the development of tumor endothelial abnormalities; this, in
turn, results in improved tumor-vessel perfusion and oxygenation and, secondarily, reduced metastasis.
mech anisms of disease
n engl j med 364;7 february 17, 2011
In mice lacking HIF-1α, bactericidal activ-
ity is decreased in myeloid cells, and the systemic
spread of infection cannot be contained.
Hypoxia and inflammation are intertwined at the
molecular, cellular, and clinical levels. Oxygen-
sensing mechanisms and hypoxia signaling are
potential therapeutic targets for the treatment of
inflammatory diseases. The value of such ap-
proaches could be tested in patients with acute
lung injury, myocardial ischemia, inflammatory
bowel disease, or cancer. Targeting hypoxia-depen-
dent signaling pathways could also help attenuate
organ failure due to ischemia in patients under-
going major surgery or alleviate hypoxia-driven
graft inflammation after solid-organ transplan-
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Copyright © 2011 Massachusetts Medical Society.
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... Hypoxia triggers nerve damage through inflammatory stimulation, eventually leading to neurological dysfunctions. 19,20 During neuroinflammation, the production of pro-inflammatory factors including interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) are increased. 21,22 Microglia are a type of glial cell, the brain equivalent of macrophages, that can clear the brain of damaged nerves, plaques, and infectious substances. ...
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Background: Central nervous system diseases are associated with hypoxia, which usually cause irreversible nerve damage, but the underlying mechanism is unclear and effective intervention strategies are lacking. This study was designed to explore the mechanism and treatment strategy of hypoxia-induced nerve injury. Methods: In this study, 13% O2 was used to treat mice for 0, 1, 3 7, and 14 days, Morris water maze and other animal behavior experiments were used to evaluate the neurological function of mice. TUNEL, BrdU, PCNA, DCX, and SOX2 staining were used to observe the apoptosis and proliferation of mouse neurons. RT-PCR and Iba1 staining were used to evaluate the release of inflammatory factors IL-1β, IL-6, and TNF-α and the activation of microglia. Results: Short-term hypoxia promotes neurogenesis, while long-term hypoxia inhibits neurogenesis. The changes in hypoxia-induced neurogenesis were positively correlated with neurological functions, but negatively correlated with apoptosis. Moreover, intermittent hypoxic conditioning restored long-term hypoxia-induced neurological dysfunction by promoting neural stem cell generation and inhibiting the release of inflammatory factors IL-1β, IL-6, and TNF-α and the activation of microglia. Conclusion: Hypoxia promoted neurogenesis in a time-dependent manner, and intermittent hypoxic conditioning exerted a neuroprotective effect through promoting neural stem cell generation and suppressing inflammation induced by long-term hypoxia stress, which provided a novel concept to develop a treatment for hypoxia-related brain injury.
... Previous studies have found that the development of PAH is closely related to the inflammatory response. 13,28,29 By downloading and re-analysing the gene expression data for patients with PAH from the public database (No. GSE703), 30 we observed increased expression of IL-1b in the blood of patients with PAH ( Figure 1G). ...
Aims Pulmonary arterial hypertension (PAH) is a pathophysiological syndrome associated with pulmonary/systemic inflammation. Melatonin relieves PAH, but the molecular mode of action remains unclear. Here, we investigated the role of melatonin in normalizing vascular homeostasis. Methods and results Light-time mean serum melatonin concentration was lower in patients with PAH than in normal controls [11.06 ± 3.44 (7.13–15.6) vs. 14.55 ± 1.28 (8.0–19.4) pg/mL], which was negatively correlated with increased serum levels of interleukin-1β (IL-1β) in patients with PAH. We showed that inflammasomes were activated in the PAH mice model and that melatonin attenuated IL-1β secretion. On one hand, melatonin reduced the number of macrophages in lung by inhibiting the endothelial chemokines and adhesion factors. Moreover, use of Il1r−/− mice, Caspase1/11−/− mice, and melatonin-treated mice revealed that melatonin reduced hypoxia-induced vascular endothelial leakage in the lung. On the other hand, we verified that melatonin reduced the formation of inflammasome multiprotein complexes by modulating calcium ions in macrophages using a live cell station, and melatonin decreased inositol triphosphate and increased cAMP. Furthermore, knockdown of melatonin membrane receptors blocked melatonin function, and a melatonin membrane receptors agonist inactivated inflammasomes in macrophages. Conclusion Melatonin attenuated inflammasome-associated vascular disorders by directly improving endothelial leakage and decreasing the formation of inflammasome multiprotein complexes in macrophages. Taken together, our data provide a theoretical basis for applying melatonin clinically, and inflammasomes may be a possible target of PAH treatment.
Increased pulmonary lactate production is correlated with severity of lung injury and outcome in acute respiratory distress syndrome (ARDS) patients. This study was conducted to investigate the relative contributions of inflammation and hypoxia to the lung's metabolic shift to glycolysis in an experimental animal model of ARDS using hyperpolarized (HP) ¹³C MRI. Fifty‐three intubated and mechanically ventilated male rats were imaged using HP ¹³C MRI before, and 1, 2.5 and 4 hours after saline (sham) or hydrochloric acid (HCl; 0.5 ml/kg) instillation in the trachea, followed by protective and nonprotective mechanical ventilation (HCl‐PEEP and HCl‐ZEEP) or the start of moderate or severe hypoxia (Hyp90 and Hyp75 groups). Pulmonary and cardiac HP lactate‐to‐pyruvate ratios were compared among groups for different time points. Postmortem histology and immunofluorescence were used to assess lung injury severity and quantify the expression of innate inflammatory markers and local tissue hypoxia. HP pulmonary lactate‐to‐pyruvate ratio progressively increased in rats with lung injury and moderate hypoxia (HCl‐ZEEP), with no significant change in pulmonary lactate‐to‐pyruvate ratio in noninjured but moderately hypoxic rats (Hyp90). Pulmonary lactate‐to‐pyruvate ratio was elevated in otherwise healthy lung tissue only in severe systemic hypoxia (Hyp75 group). ex vivo histological and immunopathological assessment further confirmed the link between elevated glycolysis and the recruitment into and presence of activated neutrophils in injured lungs. HP lactate‐to‐pyruvate ratio is elevated in injured lungs predominantly as a result of increased glycolysis in activated inflammatory cells, but can also increase due to severe inflammation‐induced hypoxia.
This project’s main objective was to develop and implement a molecular imaging tool based on hyperpolarized 13C magnetic resonance imaging (MRI) to quantitatively interrogate lung metabolism. The pulmonary system plays a major role in performing a variety of biochemical functions that maintain body homeostasis, but that undergo significant detrimental alteration in the setting of lung injury and/or inflammation. Such changes cause lactic acid to be released from the lungs and are associated with increased patient mortality. The ability to directly measure both changes in lung metabolism and its spatial heterogeneity can provide insight into the relationship between abnormal mechanics and cellularity in diseased lung tissue. This work primarily focuses on small and large mammalian species as a stepping stone toward translation to human subjects. The key deliverables of this project are acquisition and quantification tools for the regional assessment of hyperpolarized pyruvate’s conversion to lactate in lung tissue. To demonstrate the utility of our method, we used a two-hit animal model of acid aspiration and ventilator-induced lung injury that mimics a variety of inflammatory pulmonary diseases including acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). We measure the conversion of pyruvate to lactate using hyperpolarized lactate-to-pyruvate ratio, and show that this ratio is significantly correlated with inflammatory activity in the lung tissue as well as the degree of systemic hypoxemia. To further investigate hypoxia’s contribution to increased pulmonary lactate production, we assessed overall lung metabolism in non-injured hypoxic animals: while pulmonary pyruvate metabolism is resilient to moderate levels of hypoxemia, it changes significantly as a result of severe hypoxemia. Our data suggest that the increased lactate-to-pyruvate ratio in injured lungs is predominantly caused by inflammation. Next, we used our techniques to image both healthy and injured pigs on a clinical scanner in order to demonstrate the potential clinical translatability of hyperpolarized 13C imaging. Finally, we explored the possibility of using other imaging pulse sequences to achieve higher spatial and temporal resolution in both small and large animals, concluding that our method can serve as a future basis for rapid, high-resolution metabolic imaging of the lungs.
There is an overwhelming need for a simple, reliable tool that aids clinicians in diagnosing, assessing disease activity and treating rheumatic conditions. Identification of biomarkers in partially understood inflammatory disorders has long been sought after as the Holy Grail of Rheumatology. Given the complex nature of inflammatory conditions, it has been difficult to earmark the potential biomarkers. Metabolomics, however, is promising in providing new insights into inflammatory conditions and also identifying such biomarkers. Metabolomic studies have generally revealed increased energy requirements for by-products of a hypoxic environment, leading to a characteristic metabolic fingerprint. Here, we discuss the significance of such studies and their potential as a biomarker.
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Increasing evidence suggests the important role of metabolic reprogramming in the regulation of the innate inflammatory response, but the underlying mechanism remains unclear. Here we provide evidence to support a novel role for the pyruvate kinase M2 (PKM2)-mediated Warburg effect, namely aerobic glycolysis, in the regulation of high-mobility group box 1 (HMGB1) release. PKM2 interacts with hypoxia-inducible factor 1α (HIF1α) and activates the HIF-1α-dependent transcription of enzymes necessary for aerobic glycolysis in macrophages. Knockdown of PKM2, HIF1α and glycolysis-related genes uniformly decreases lactate production and HMGB1 release. Similarly, a potential PKM2 inhibitor, shikonin, reduces serum lactate and HMGB1 levels, and protects mice from lethal endotoxemia and sepsis. Collectively, these findings shed light on a novel mechanism for metabolic control of inflammation by regulating HMGB1 release and highlight the importance of targeting aerobic glycolysis in the treatment of sepsis and other inflammatory diseases.
Blood color of dialysis patients can be seen routinely. Darkened blood color is often observed in critically ill patients generally because of decreased oxygen saturation, but little is known about the other factors responsible for the color intensity. In addition, quantitative blood color examination has not been performed yet. Therefore, no one has evaluated the predictive power of blood color. The aim of this study was to evaluate if blood color darkness reflects some medical problems and is associated with survival disadvantage. Study design is a prospective cohort study. One hundred sixty-seven patients were enrolled in this study. Quantification of blood color was done using a reflected light colorimeter. Demographic and clinical data were collected to find out the factors that can be related to blood color. Follow-ups were performed for 2 years to analyze the risk factors for their survival. Regression analysis showed that C-reactive protein and white blood cell count were negatively correlated with blood color. In addition, blood color was positively correlated with mean corpuscular hemoglobin concentration and serum sodium concentration as well as blood oxygen saturation. During a follow-up, 34 (20.4%) patients died. Cox regression analysis revealed that darkened blood color was an independent significant risk factor of mortality in hemodialysis patients as well as low albumin and low Kt/V. These results suggest that inflammation independently affects blood color and quantification of blood color is useful to estimate prognosis in patients undergoing hemodialysis. It is possible that early detection of blood color worsening can improve patients' survival.
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The identification of biomarkers that distinguish between aggressive and indolent forms of prostate cancer (PCa) is crucial for diagnosis and treatment. In this study, we used cultured cells derived from prostate tissue from patients with PCa to define a molecular mechanism underlying the most aggressive form of PCa that involves the functional activation of eNOS and HIFs in association with estrogen receptor β (ERβ). Cells from patients with poor prognosis exhibited a constitutively hypoxic phenotype and increased NO production. Upon estrogen treatment, formation of ERβ/eNOS, ERβ/HIF-1α, or ERβ/HIF-2α combinatorial complexes led to chromatin remodeling and transcriptional induction of prognostic genes. Tissue microarray analysis, using an independent cohort of patients, established a hierarchical predictive power for these proteins, with expression of eNOS plus ERβ and nuclear eNOS plus HIF-2α being the most relevant indicators of adverse clinical outcome. Genetic or pharmacologic modulation of eNOS expression and activity resulted in reciprocal conversion of the transcriptional signature in cells from patients with bad or good outcome, respectively, highlighting the relevance of eNOS in PCa progression. Our work has considerable clinical relevance, since it may enable the earlier diagnosis of aggressive PCa through routine biopsy assessment of eNOS, ERβ, and HIF-2α expression. Furthermore, proposing eNOS as a therapeutic target fosters innovative therapies for PCa with NO inhibitors, which are employed in preclinical trials in non-oncological diseases. Occupancy of the hTERT promoter sites by ERβ or HIFs, on the other hand, was more strongly dependent upon the stimulus and the cell context. Upon treatment with only estrogen, we observed ERβ but not ERα occupancy (about a 4-fold increase over control) of the hTERT sites III and IV in C27IM (G1) cells and II and III in C38IM (G2) cells. Unexpectedly, estrogen treatment also induced recruitment of HIFs, primarily HIF-1α in C27IM cells and HIF-2α in C38IM cells (3- and 4-fold increase, respectively) with the same pattern as ERβ. Hypoxia caused enrichment of HIF-1α in C27IM cells and HIF-2α in C38IM cells, with maximum enrichment (up to a 6-fold increase) at site III in both cell lines. Notably, combined E2 and hypoxia markedly increased (to about a 12-fold increase) recruitment of both HIF-1α and ERβ at site III exclusively in C27IM cells. Moreover, while HIF-2α recruitment in C27IM cells was constant at site III (about a 3-fold increase) upon single or combined treatments, in C38IM cells, maximum recruitment (7-fold increase) occurred under hypoxia, and combined treatment reduced it to levels observed with E2 alone (4-fold increase). Occupancy by HIF-1α in C38IM cells or by ERβ or HIF-1α in both cell lines was absent or minimal under all conditions (Figure 7B and data not shown).
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Background: The level of environmental hypobaric hypoxia that affects climbers at the summit of Mount Everest (8848 m [29,029 ft]) is close to the limit of tolerance by humans. We performed direct field measurements of arterial blood gases in climbers breathing ambient air on Mount Everest. Methods: We obtained samples of arterial blood from 10 climbers during their ascent to and descent from the summit of Mount Everest. The partial pressures of arterial oxygen (PaO(2)) and carbon dioxide (PaCO(2)), pH, and hemoglobin and lactate concentrations were measured. The arterial oxygen saturation (SaO(2)), bicarbonate concentration, base excess, and alveolar-arterial oxygen difference were calculated. Results: PaO(2) fell with increasing altitude, whereas SaO(2) was relatively stable. The hemoglobin concentration increased such that the oxygen content of arterial blood was maintained at or above sea-level values until the climbers reached an elevation of 7100 m (23,294 ft). In four samples taken at 8400 m (27,559 ft)--at which altitude the barometric pressure was 272 mm Hg (36.3 kPa)--the mean PaO(2) in subjects breathing ambient air was 24.6 mm Hg (3.28 kPa), with a range of 19.1 to 29.5 mm Hg (2.55 to 3.93 kPa). The mean PaCO(2) was 13.3 mm Hg (1.77 kPa), with a range of 10.3 to 15.7 mm Hg (1.37 to 2.09 kPa). At 8400 m, the mean arterial oxygen content was 26% lower than it was at 7100 m (145.8 ml per liter as compared with 197.1 ml per liter). The mean calculated alveolar-arterial oxygen difference was 5.4 mm Hg (0.72 kPa). Conclusions: The elevated alveolar-arterial oxygen difference that is seen in subjects who are in conditions of extreme hypoxia may represent a degree of subclinical high-altitude pulmonary edema or a functional limitation in pulmonary diffusion.
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Hypoxia inducible factor (HIF)-1 is the key transcriptional factor involved in the adaptation process of cells and organisms to hypoxia. Recent findings suggest that HIF-1 plays also a crucial role in inflammatory and infectious diseases. Using patient skin biopsies, cell culture and murine infection models, HIF-1 activation was determined by immunohistochemistry, immunoblotting and reporter gene assays and was linked to cellular oxygen consumption. The course of a S. aureus peritonitis was determined upon pharmacological HIF-1 inhibition. Activation of HIF-1 was detectable (i) in all ex vivo in biopsies of patients suffering from skin infections, (ii) in vitro using cell culture infection models and (iii) in vivo using murine intravenous and peritoneal S. aureus infection models. HIF-1 activation by human pathogens was induced by oxygen-dependent mechanisms. Small colony variants (SCVs) of S. aureus known to cause chronic infections did not result in cellular hypoxia nor in HIF-1 activation. Pharmaceutical inhibition of HIF-1 activation resulted in increased survival rates of mice suffering from a S. aureus peritonitis. Activation of HIF-1 is a general phenomenon in infections with human pathogenic bacteria, viruses, fungi and protozoa. HIF-1-regulated pathways might be an attractive target to modulate the course of life-threatening infections.
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Hypoxia inducible factor (HIF) prolyl hydroxylase inhibitors are protective in mouse models of inflammatory bowel disease (IBD). Here, we investigated the therapeutic target(s) and mechanism(s) involved. The effect of genetic deletion of individual HIF-prolyl hydroxylase (PHD) enzymes on the development of dextran sulphate sodium (DSS)-induced colitis was examined in mice. PHD1(-/-), but not PHD2(+/-) or PHD3(-/-), mice were less susceptible to the development of colitis than wild-type controls as determined by weight loss, disease activity, colon histology, neutrophil infiltration, and cytokine expression. Reduced susceptibility of PHD1(-/-) mice to colitis was associated with increased density of colonic epithelial cells relative to wild-type controls, which was because of decreased levels of apoptosis that resulted in enhanced epithelial barrier function. Furthermore, with the use of cultured epithelial cells it was confirmed that hydroxylase inhibition reversed DSS-induced apoptosis and barrier dysfunction. Finally, PHD1 levels were increased with disease severity in intestinal tissue from patients with IBD and in colonic tissues from DSS-treated mice. These results imply a role for PHD1 as a positive regulator of intestinal epithelial cell apoptosis in the inflamed colon. Genetic loss of PHD1 is protective against colitis through decreased epithelial cell apoptosis and consequent enhancement of intestinal epithelial barrier function. Thus, targeted PHD1 inhibition may represent a new therapeutic approach in IBD.
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Sepsis and septic acute lung injury are among the leading causes for morbidity and mortality of critical illness. Extracellular adenosine is a signaling molecule implicated in the cellular adaptation to hypoxia, ischemia, or inflammation. Therefore, we pursued the role of the A2B adenosine receptor (AR) as potential therapeutic target in endotoxin-induced acute lung injury. We gained initial insight from in vitro studies of cultured endothelia or epithelia exposed to inflammatory mediators showing time-dependent induction of the A2BAR (up to 12.9 + or - 3.4-fold, p < 0.05). Similarly, murine studies of endotoxin-induced lung injury identified an almost 4.6-fold induction of A2BAR transcript and corresponding protein induction with LPS exposure. Studies utilizing A2BAR promoter constructs and RNA protection assays indicated that A2BAR induction involved mRNA stability. Functional studies of LPS-induced lung injury revealed that pharmacological inhibition or genetic deletion of the A2BAR was associated with dramatic increases in lung inflammation and histologic tissue injury. Studies of A2BAR bone marrow chimeric mice suggested pulmonary A2BAR signaling in lung protection. Finally, studies with a specific A2BAR agonist (BAY 60-6583) demonstrated attenuation of lung inflammation and pulmonary edema in wild-type but not in gene-targeted mice for the A2BAR. These studies suggest the A2BAR as potential therapeutic target in the treatment of endotoxin-induced forms of acute lung injury.
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CD39/ENTPD1 hydrolyzes proinflammatory nucleotides to generate adenosine. As purinergic mediators have been implicated in intestinal inflammation, we hypothesized that CD39 might protect against inflammatory bowel disease. We studied these possibilities in a mouse model of colitis using mice with global CD39 deletion. We then tested whether human genetic polymorphisms in the CD39 gene might influence susceptibility to Crohn's disease. We induced colitis in mice using Dextran Sodium Sulfate (DSS). Readouts included disease activity scores, histological evidence of injury, and markers of inflammatory activity. We used HapMap cell lines to find SNPs that tag for CD39 expression, and then compared the frequency of subjects with high vs. low CD39-expression genotypes in a case-control cohort for Crohn's disease. Mice null for CD39 were highly susceptible to DSS injury, with heterozygote mice showing an intermediate phenotype compared to wild type (WT). We identified a common SNP that tags CD39 mRNA expression levels in man. The SNP tagging low levels of CD39 expression was associated with increased susceptibility to Crohn's disease in a case-control cohort comprised of 1,748 Crohn's patients and 2,936 controls (P = 0.005-0.0006). Our data indicate that CD39 deficiency exacerbates murine colitis and suggest that CD39 polymorphisms are associated with inflammatory bowel disease in humans.
High-altitude pulmonary edema is a life-threatening condition occurring in predisposed but otherwise healthy individuals. It therefore permits the study of underlying mechanisms of pulmonary edema in the absence of confounding factors such as coexisting cardiovascular or pulmonary disease, and/or drug therapy. There is evidence that some degree of asymptomatic alveolar fluid accumulation may represent a normal phenomenon in healthy humans shortly after arrival at high altitude. Two fundamental mechanisms then determine whether this fluid accumulation is cleared or whether it progresses to HAPE: the quantity of liquid escaping from the pulmonary vasculature and the rate of its clearance by the alveolar respiratory epithelium. The former is directly related to the degree of hypoxia-induced pulmonary hypertension, whereas the latter is determined by the alveolar epithelial sodium transport. Here, we will review evidence that, in HAPE-prone subjects, impaired pulmonary endothelial and epithelial NO synthesis and/or bioavailability may represent a central underlying defect predisposing to exaggerated hypoxic pulmonary vasoconstriction and, in turn, capillary stress failure and alveolar fluid flooding. We will then demonstrate that exaggerated pulmonary hypertension, although possibly a conditio sine qua non, may not always be sufficient to induce HAPE and how defective alveolar fluid clearance may represent a second important pathogenic mechanism.
Intestinal epithelial cells that line the mucosal surface of the gastrointestinal tract are positioned between an anaerobic lumen and a highly metabolic lamina propria. As a result of this unique anatomy, intestinal epithelial cells function within a steep physiologic oxygen gradient relative to other cell types. Furthermore, during active inflammatory disease such as IBD, metabolic shifts towards hypoxia are severe. Studies in vitro and in vivo have shown that the activation of hypoxia-inducible factor (HIF) serves as an alarm signal to promote the resolution of inflammation in various mouse models of disease. Amelioration of disease occurs, at least in part, through transcriptional upregulation of nonclassic epithelial barrier genes. There is much interest in harnessing hypoxia-inducible pathways, including stabilizing HIF directly or via inhibition of prolyl hydroxylase enzymes, for therapy of IBD. In this Review, we discuss the signaling pathways involved in the regulation of hypoxia and discuss how hypoxia may serve as an endogenous alarm signal for the presence of mucosal inflammatory disease. We also discuss the pros and cons of targeting these pathways to treat patients with IBD.
Adaptation of cancer cells to their microenvironment is an important driving force in the clonal selection that leads to invasive and metastatic disease. O2 concentrations are markedly reduced in many human cancers compared with normal tissue, and a major mechanism mediating adaptive responses to reduced O2 availability (hypoxia) is the regulation of transcription by hypoxia-inducible factor 1 (HIF-1). This review summarizes the current state of knowledge regarding the molecular mechanisms by which HIF-1 contributes to cancer progression, focusing on (1) clinical data associating increased HIF-1 levels with patient mortality; (2) preclinical data linking HIF-1 activity with tumor growth; (3) molecular data linking specific HIF-1 target gene products to critical aspects of cancer biology and (4) pharmacological data showing anticancer effects of HIF-1 inhibitors in mouse models of human cancer.