Molecular imaging of peroxynitrite with HKGreen-4 in live cells and tissues

Article abstract of DOI:10.1021/ja504624q

Peroxynitrite (ONOO^-), the product of a radical combination reaction of nitric oxide and superoxide, is a potent biological oxidant involved in a broad spectrum of physiological and pathological processes. Herein we report the development, characterization, and biological applications of a new fluorescent probe, HKGreen-4, for peroxynitrite detection and imaging. HKGreen-4 utilizes a peroxynitrite-triggered oxidative N-dearylation reaction to achieve an exceptionally sensitive and selective fluorescence turn-on response toward peroxynitrite in chemical systems and biological samples. We have thoroughly evaluated the utility of HKGreen-4 for intracellular peroxynitrite imaging and, more importantly, demonstrated that HKGreen-4 can be efficiently employed to visualize endogenous peroxynitrite generated in Escherichia coli-challenged macrophages and in live tissues from a mouse model of atherosclerosis. This probe should serve as a powerful molecular imaging tool to explore peroxynitrite biology under a variety of physiological and pathological contexts.

Full text of DOI:10.1021/ja504624q

Article
pubs.acs.org/JACS
Molecular Imaging of Peroxynitrite with HKGreen‑4 in Live Cells and
Tissues
Tao Peng,†,‡ Nai-Kei Wong,†,‡ Xingmiao Chen,⊥ Yee-Kwan Chan,§ Derek Hoi-Hang Ho,†
Zhenning Sun,† Jun Jacob Hu,† Jiangang Shen,⊥ Hani El-Nezami,§ and Dan Yang*,†
⊥
§
†Morningside Laboratory for Chemical Biology and Department of Chemistry, School of Chinese Medicine, and School of
Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China
S
* Supporting Information
ABSTRACT: Peroxynitrite (ONOO−), the product of a radical
combination reaction of nitric oxide and superoxide, is a potent
biological oxidant involved in a broad spectrum of physiological
and pathological processes. Herein we report the development,
characterization, and biological applications of a new fluorescent
probe, HKGreen-4, for peroxynitrite detection and imaging.
HKGreen-4 utilizes a peroxynitrite-triggered oxidative N-
dearylation reaction to achieve an exceptionally sensitive and
selective fluorescence turn-on response toward peroxynitrite in chemical systems and biological samples. We have thoroughly
evaluated the utility of HKGreen-4 for intracellular peroxynitrite imaging and, more importantly, demonstrated that HKGreen-4
can be efficiently employed to visualize endogenous peroxynitrite generated in Escherichia coli-challenged macrophages and in live
tissues from a mouse model of atherosclerosis. This probe should serve as a powerful molecular imaging tool to explore
peroxynitrite biology under a variety of physiological and pathological contexts.
Historically, most of the studies about biological ONOO−
formation rely on its footprint reaction, i.e., nitration of tyrosine
INTRODUCTION
■
Peroxynitrite (ONOO−), the diffusion-controlled reaction
residues to form 3-nitrotyrosines, which, however, cannot be
product of nitric oxide (•NO) and superoxide (O2•−), has
used as unique evidence for ONOO− formation because of the
been considered a “stealthy” biological oxidant by virtue of its
existence of other nitrating species in vivo and for real-time
elusive nature and multiple in vivo reaction targets.1 Being a
ONOO− detection.8 To better define the physiological and
strong oxidant and good nucleophile, ONOO− can react with
pathological roles of ONOO− in vivo, there has been a strong
different biomolecules, including proteins, transition-metal-
containing enzyme centers, lipids, and nucleic acids, through
demand for reliable analytical methods with high spatiotempo-
ral resolution for ONOO− detection. Indeed, fluorescent
direct oxidation or decomposing into highly reactive secondary
probes, featuring high sensitivity and real-time spatial imaging
radicals such as hydroxyl radical (•OH), carbonate radical
capacity, have been accorded great importance in the detection
(CO3•−), and nitrogen dioxide (•NO2), eventually contributing
of reactive oxygen species (ROS), reactive nitrogen species
to cell death. Therefore, ONOO− is widely assumed to account
for most of the cytotoxicity previously ascribed to NO and
(RNS), and reactive sulfur species (RSS).9 During the past
several years, we and other groups have reported a number of
small-molecule and protein-based fluorescent probes for
ONOO− detection,10 some of which have found utility in
biological studies.11 Nevertheless, it remains challenging to
design probes that can be robustly employed in diverse
biological systems, especially in living mammalian tissues.
Moreover, the elusive nature of ONOO−, stemming from its
extremely short half-life (i.e., ∼10 ms), low steady-state
concentration (i.e., nM range),2b and complex chemistry amidst
various potentially interfering ROS and RNS in cellular milieu,
naturally calls for highly sensitive and selective probes for its
direct and unambiguous detection.
•
O2•− and has been implicated in a growing list of diseases, such
as cardiovascular and neurodegenerative disorders, metabolic
diseases, inflammation, pain, and cancer.2 Meanwhile, ONOO−
is also reported to display protective activities in vivo by
functioning as a cytotoxic effector against invading pathogens
and as a signaling molecule.1,3 For instance, ONOO− generated
from activated macrophages has been suggested to mediate
intracellular killing of invading Escherichia coli,4 Rhodococcus
equi,5 and Trypanosoma cruzi.6 Recent evidence indicates that
ONOO− also plays underappreciated roles in the redox
regulation of key signaling pathways that are dependent on
tyrosine phosphorylation via its ability to selectively nitrate
tyrosine residues.3 However, most of the beneficial effects of
ONOO− in vivo remain controversial or poorly characterized,7
probably due to a lack of reliable methods for unambiguously
monitoring ONOO− levels in vivo.2c
Herein we report the design and characterization of
HKGreen-4, a new small-molecule turn-on fluorescent probe
Received: May 8, 2014
Published: July 24, 2014
© 2014 American Chemical Society
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for exceptionally sensitive and selective detection of ONOO− in
aqueous solution, live cells, and tissues. HKGreen-4 exploits an
N-phenylrhodol-based fluorescence “off−on” switch and an
ONOO−-triggered oxidative N-dearylation reaction to achieve a
robust fluorescence turn-on response toward peroxynitrite over
a range of biologically relevant ROS/RNS. We demonstrate the
versatile utility of HKGreen-4 in biological contexts by showing
that this probe can be used for intracellular ONOO− imaging in
a variety of cell types by both single-photon and two-photon
excitation fluorescence microscopy. Moreover, we show that
HKGreen-4 can be employed to visualize the kinetics of
ONOO− generation in macrophages challenged with heat-
killed E. coli and prove that the ONOO− generation is
enzymatically regulated. Furthermore, the probe can also be
utilized to image the endogenous peroxynitrite generation in
live tissues from a mouse model of atherosclerosis.
detection. Therefore, we designed a new series of molecules,
1−5, bearing simple electron-donating substituents, such as
methoxyl, hydroxyl, and amino groups, on the phenyl ring of N-
phenyl-N-methylrhodol (Scheme 1). Upon reaction with
ONOO−, these molecules could readily undergo oxidative N-
dearylation to release strongly fluorescent N-methylrhodol.10f
Although previous work by Nagano’s group showed that an
analogous O-dearylation reaction can be used to develop
fluorescent probes, i.e., HPF and APF, for detection of highly
reactive oxygen species (hROS),14 those probes cannot
differentiate ONOO− from other interfering ROS/RNS. We
expect that the N-phenylrhodol scaffold would be uniquely
advantageous, as the rhodol fluorescence is highly efficiently
quenched by the N-phenyl group.13 More importantly, the
nitrogen atom of rhodol could further increase the electron
density of the N-phenyl ring, resulting in high reactivity toward
ONOO− over other ROS/RNS. Compounds 1−5 were
synthesized according to our previously developed synthetic
route to rhodol fluorophores (Schemes S2 and S3, Supporting
Information).13,15
Screening Probes for Peroxynitrite Detection. With
compounds 1−5 in hand, we first examined their reactivity
toward ONOO− and hypochlorous acid (HOCl), a potentially
interfering hROS in ONOO− detection, in aqueous solutions
buffered at physiological pH (0.1 M phosphate buffer, pH 7.4).
As expected, most of these compounds, except compound 1,
exhibited strong fluorescence increases upon treatment with
ONOO−, while displaying differential fluorescence responses
toward HOCl (Figures S1 and S2, Supporting Information).
Notably, compound 2 with a hydroxy substituent responded to
ONOO− much more dramatically than toward HOCl (Figures
S1 and S2). On the other hand, compounds 3−5 with various
amino substituents generally gave stronger fluorescence
responses toward HOCl than toward ONOO− when the
oxidants were present in excess amounts (Figure S2). We
propose that ONOO− oxidizes compounds 2−5 through direct
two-electron oxidation of the electron-rich phenyl rings to form
iminium ions,16 which are further hydrolyzed to give the N-
dearylation product (Schemes S6). By contrast, HOCl reacts
with compounds 2−5 through N-chlorination,17 which
ultimately leads to N-dearylation (Schemes S6). Due to steric
hindrance of the diarylamine moiety and competition with
chlorination at the phenol ortho positions (Schemes S6),18 N-
chlorination of compound 2 with HOCl is much slower and
less efficient than oxidation by ONOO−, thereby resulting in
efficient distinction of ONOO− from HOCl. Detailed
mechanistic studies on the N-dearylation reactions of those
compounds induced by ONOO− and HOCl are currently
underway.
RESULTS AND DISCUSSION
■
Design and Synthesis of Probes. The reaction-based
approach for developing selective fluorescent probes in
biological systems represents a new research focus that has
attracted increasing attention.9d Previously, our group discov-
ered that ONOO− can selectively react with a trifluoromethyl
ketone moiety through direct nucleophilic addition to form a
dioxirane intermediate, which can then oxidize a nearby anisole
ring to a dienone compound with concomitant C−O bond
cleavage of the anisole.10b,12 On the basis of this nucleophilic
reaction between ONOO− and trifluoromethyl ketone, we have
developed several reaction-based small-molecule fluorescent
probes for ONOO− detection.10b,c,f Notably, integration of this
ONOO−-selective reaction into an N-phenylrhodol fluores-
cence “off−on” switch (Scheme 1) afforded a rhodol-based
Scheme 1. Design of a New Series of Rhodol-Based
Compounds for Detecting Peroxynitrite
As the phenol compound 2, called HKGreen-4 hereafter
(Scheme 2), can differentiate ONOO− from HOCl and exhibits
high sensitivity toward ONOO−, we focused our subsequent
Scheme 2. N-Dearylation Reaction of HKGreen-4 Induced
by Peroxynitrite for Its Fluorescence Detection
fluorescent probe, HKGreen-3, for ONOO− detection.10f,13
Apart from its nucleophilicity, ONOO− also exhibits potent
oxidizing capability, allowing for direct oxidation of electron-
rich groups through one- or two-electron oxidation processes.2a
As highly electron-rich phenol and aniline derivatives could be
easily oxidized by ONOO−, we expect that incorporation of
simple phenol and anilines into the N-phenylrhodol scaffold
would afford novel reaction-based probes for ONOO−
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studies on this probe for detailed characterization in both
chemical and biological systems.
of the rhodol core fluorophore by the N-phenyl group.13
Addition of ONOO− to a solution of HKGreen-4 (1 μM in
phosphate buffer, pH 7.4) resulted in a dramatic and rapid
fluorescence turn-on response with maximal emission at 535
nm in a dose-dependent manner (Figure 1A). In less than 5 s
(Figure S4) after addition of 1 equiv of ONOO− into
HKGreen-4 solution, a 290-fold fluorescence increase was
observed, much higher than the responses of our previous-
generation fluorescent probes for ONOO−. A linear relation-
ship was observed between fluorescence intensity and ONOO−
concentration up to 1 equiv of ONOO− (Figure S5).
Fluorescence intensity then reached a plateau on further
addition of ONOO− up to 4 equiv (Figure S6). The detection
limit of HKGreen-4 for ONOO− was estimated to be as low as
10 nM, at which a 3-fold fluorescence increase could be
achieved. HPLC and LC-MS analysis confirmed that the
fluorescent product was indeed N-methylrhodol (Φ = 0.73;
Scheme 2 and Figure S10), resulting from the ONOO−-
induced N-dearylation reaction of HKGreen-4.
Reactivity and Selectivity of HKGreen-4 for Peroxyni-
trite. Spectroscopic properties of HKGreen-4 were evaluated in
aqueous solution buffered at physiological pH (0.1 M
phosphate buffer, pH 7.4). HKGreen-4 exhibited a strong
absorption peak at 517 nm in the UV spectrum (Figure S3),
suggesting that the probe exists predominately as the “open”
quinoid form in aqueous solution.19 As expected, HKGreen-4
was virtually non-fluorescent in aqueous solution (Φ = 0.001;
Figures 1A and S3), owing to effective fluorescence quenching
Reactivity of HKGreen-4 toward a panel of ROS and RNS
was tested to establish the selectivity of the probe for detecting
ONOO− (Figure 1B). While most of the biologically relevant
1
•
ROS and RNS, including H2O2, O2, NO, O2•−, and ROO•,
even when presented as 10 equiv, did not trigger any
fluorescence increases of the probe, potentially interfering
•
hROS, such as HOCl and OH, induced much weaker and
nearly negligible fluorescence increases, i.e., >18-fold lower
increases, compared to ONOO−. Together, these results
suggest that HKGreen-4 outperforms previously reported
probes for ONOO− detection in terms of lower working
concentration, greater fluorescence response, and better
•
selectivity over HOCl and OH.
To further evaluate HKGreen-4 for ONOO− detection in
chemical systems, we proceeded to perform additional
experiments. The probe exhibited efficient fluorescence turn-
on response to ONOO− within a biologically relevant pH range
from 6.5 to 9.5 (Figure S7). Moreover, HKGreen-4 still showed
comparable fluorescence increase toward ONOO− in the
presence of CO2 (Figure S8), which is ubiquitous in biological
systems and reacts with ONOO− through nucleophilic addition
at a high rate constant.1 Addition of DMSO (i.e., 0.1% v/v, ∼14
mM) as an effective OH scavenger into the probe solution
did not attenuate the fluorescence response of HKGreen-4 to
ONOO− (Figure S9), indicating that •OH, one of the
secondary decomposition radicals of ONOO−, does not
contribute to the fluorescence increase of HKGreen-4 in the
presence of ONOO−. By contrast, DMSO can completely
Figure 1. (A) Fluorescence response of 1 μM HKGreen-4 to different
amounts of ONOO−. (B) Relative fluorescence intensity (λem = 535
nm) of 1 μM HKGreen-4 toward various ROS and RNS. Data were
acquired at 25 °C in 0.1 M phosphate buffer at pH 7.4, with excitation
at 517 nm. ROS and RNS were slowly added to HKGreen-4 solution
with vigorously stirring. Reactions were then carried out for 30 min at
room temperature before the fluorescence intensity of the probe
solution was measured.
20
•
Figure 2. Validation of HKGreen-4 selectivity with exogenous ROS/RNS donors in RAW264.7 macrophages. Cells were coincubated with
HKGreen-4A (10 μM) and donors for •NO, O2•−, or ONOO− for 1 h, followed by confocal fluorescence imaging. NOC-18 (500 μM), MSB (100
μM), and SIN-1 (50 μM) were used for producing •NO, O2•−, and ONOO−, respectively. Urate (100 μM)/minocycline (100 μM) and FeTMPyP
(50 μM) are ONOO− scavengers and decomposition catalyst, respectively. Scale bar represents 20 μm.
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Figure 3. Confocal fluorescence imaging of endogenous ONOO− with HKGreen-4 in stimulated RAW264.7 macrophages. (A) Kinetics of ONOO−
generation upon stimulation with LPS (1 μg/mL) and IFN-γ (100 ng/mL), or heat-killed E. coli (multiplicity of infection (moi) = 100) for the
indicated time. ONOO− was detected by staining cells with HKGreen-4A (10 μM; 30 min) before confocal imaging. Minocycline (100 μM) was
added to scavenge ONOO−. (B) Cells were treated for 14 h with LPS/IFN-γ, or heat-killed E. coli, and then stained (30 min) with HKGreen-4A (10
μM), MitoTracker Red (100 nM), and Hoechst 33342 (75 ng/mL) before confocal imaging. Scale bars represent 10 μm.
suppress the fluorescence increases of HKGreen-4 and
compound 3 induced by HOCl (Figure S9), suggesting that
HOCl can readily oxidize DMSO21 even before it reacts with
the probes and that using DMSO as a co-solvent (i.e., 0.1% v/
v) for making HKGreen-4 solution can minimize the
interference from HOCl in ONOO− detection. These results
collectively indicate that HKGreen-4 can robustly detect
ONOO− in aqueous solution.
Salmonella typhimurium), pro-inflammatory cytokine interfer-
on-gamma (IFN-γ), and phorbol 12-myristate 13-acetate
(PMA).10c,f It has been suggested that ONOO− generated in
macrophages may contribute to the bactericidal activity of these
cells.4b,c,5 We therefore sought to use HKGreen-4 to examine
whether ONOO− is produced in macrophages when challenged
with heat-killed E. coli, a model bacterial species. First, a kinetic
analysis of ONOO− production in activated RAW264.7
macrophages was performed. Briefly, cells were challenged
with a combination of LPS and IFN-γ, or heat-killed E. coli for
different durations (t = 4, 15, 17, and 24 h), and incubated with
HKGreen-4A for 30 min before confocal imaging. Due to much
higher sensitivity of HKGreen-4 than our previously reported
ONOO− probes, a lower probe working concentration, i.e., 10
μM, of HKGreen-4A was needed to achieve optimal
fluorescence signals.10c,f In contrast to previously used
stimulation conditions of macrophages with LPS, IFN-γ, and
PMA,10c,f we omitted the usage of PMA throughout our studies,
as PMA is a plant toxin and irrelevant to the bacterial infection
model we are interested in. As shown in Figure 3A, upon LPS/
IFN-γ or E. coli stimulation, HKGreen-4 gave a strong
fluorescence increase in activated RAW264.7 cells in a time-
dependent manner, with detectable fluorescence signals at 4 h
and maximal signals being observed between 15 and 17 h post-
treatment, followed by a discernible decline, suggesting that
ONOO− production in macrophages is temporally controlled,
in a manner consistent with the temporally regulated
Evaluation of HKGreen-4 for Imaging Peroxynitrite in
Live Cells. We then examined the potential of HKGreen-4 to
visualize ONOO− in live cells by confocal fluorescence
microscopy. In order to improve the intracellular loading of
HKGreen-4, we prepared its acetate derivative, HKGreen-4A
(Scheme S5), which has superior cell membrane permeabili-
ty,10f,22 and used it in all subsequent cell imaging experiments.
Several exogenous ROS and RNS donors were employed to
validate the selectivity of HKGreen-4 in live cells. As shown in
Figure 2, mouse RAW264.7 macrophages loaded with
HKGreen-4 showed barely detectable background fluorescence.
A significant increase in intracellular fluorescence was observed
in cells treated with 3-morpholinosydnonimine hydrochloride
(SIN-1), an ONOO− generator, but not in cells treated with
3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18, an
•−
NO donor) or menadione sodium bisulfite (MSB, an O2
donor) (Figure 2). Fluorescence increase in SIN-1-treated cells
was significantly attenuated in the presence of ONOO−
scavengers, such as urate and minocycline,23 or FeTMPyP, an
ONOO− decomposition catalyst.1
24
•
generation of its precursor NO in stimulated macrophages.
We also established that HKGreen-4 can be robustly applied
in other cells types, such as C17.2 mouse neural progenitor
cells, CHO (Chinese hamster ovarian) cells, and BV-2 mouse
microglia, to imaging SIN-1 generated ONOO− (Figure S12).
Moreover, the cytotoxicity of HKGreen-4A was assessed by
MTT assay in RAW264.7 cells (Figure S11), which indicated
that this probe is virtually nontoxic to cells even at 80 μM after
24 h incubation. Taken together, these results confirmed that
HKGreen-4 is well-suited for selective intracellular imaging of
ONOO− in live biological samples.
Visualization of Endogenous Peroxynitrite Produc-
tion in Macrophages under Immune Stimulation. Next,
we focused on assessing the performance of HKGreen-4 in
imaging endogenous ONOO− in RAW264.7 mouse macro-
phages, which are known to produce ONOO− upon
stimulation with bacterial endotoxin lipopolysaccharide (LPS;
Having optimized the ONOO− induction time in macro-
phages, we proceeded to evaluate the intracellular localization
of HKGreen-4 in activated macrophages by confocal
microscopy. Co-staining experiments with MitoTracker Red
and HKGreen-4A in LPS/IFN-γ-stimulated macrophages
revealed broad spatial overlap of these two probes (Figure
3B), indicating that under low and moderate stimulation
conditions (i.e., LPS/IFN-γ stimulation) mitochondria might
be the major source for O2•−, and therefore for ONOO−. In
contrast, with strong stimulants like heat-killed E. coli,
macrophages exhibited maximum and broadly diffused green
fluorescence encompassing both nuclear and cytoplasmic
compartments, suggesting that HKGreen-4 and its fluorescent
product diffuse freely inside the cells and that under these
•−
conditions mitochondria might not be the only source of O2
and ONOO−.
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To expand the utility of HKGreen-4 for cellular imaging
studies, we also demonstrated that HKGreen-4 can be utilized
to visualize endogenous ONOO− production in primary mouse
macrophages that were differentiated with M-CSF from mouse
bone marrow progenitor cells and stimulated with LPS (E. coli
K12) for 18 h (Figure S13). Moreover, HKGreen-4 can be
applied to two-photon excitation microscopy (λex = 730 nm,
λem = 500−550 nm) for peroxynitrite imaging in stimulated
macrophages (Figure S14), further establishing the versatile
applicability of HKGreen-4 for molecular imaging studies.
Next, we used HKGreen-4 to validate whether ONOO−
formation in E. coli-challenged macrophages is enzymatically
regulated, which would provide important insights into the
biological functions of ONOO−. It has been controversial that
regulated ONOO− formation can be temporally achieved in
(AG), as a selective iNOS inhibitor and a nonselective NOS
inhibitor, respectively, both effectively abolished ONOO−
formation in E. coli-challenged macrophages (Figures 4 and
S15). Interestingly, 1400W also evidently reduced MitoSOX
Red fluorescence (Figure 4), consistent with recent observa-
tions that 1400W, in addition to inhibiting iNOS activity, also
•−
attenuates O2
production in vivo.26 Moreover, well-
documented NOX inhibitors, such as dibenziodolium chloride
(DPI) and apocynin, also greatly attenuated ONOO−
generation (Figures 4 and S15). Taken together, our cell
imaging experiments using HKGreen-4 provide the direct
visualization evidence that ONOO− is indeed generated in E.
coli-challenged macrophages, likely as an immune effector for
bacterial clearance, and suggest that ONOO− formation in E.
coli-challenged macrophages is enzymatically regulated and
•−
•−
stimulated macrophages, as the generations of O2 from
dependent on iNOS-generated NO and NOX-derived O2
.
•
NADPH oxidase (NOX) and NO from inducible nitric oxide
Imaging Peroxynitrite in Live Tissues from a Mouse
Model of Atherosclerosis. Finally, we sought to apply
HKGreen-4 to image ONOO− in live tissue samples. ONOO−
formation has long been implicated in the pathogenesis of
atherosclerosis on the basis of indirect detection, i.e.,
immunostaining of protein nitration, which, however, cannot
be used as unique evidence for ONOO− formation.8 To the
best of our knowledge, direct evidence about abnormal
ONOO− formation under atherosclerosis conditions is still
lacking.2a,27 To address this question, we employed an
apolipoprotein E knockout (ApoE−/−) mouse model that
readily develops atherosclerosis on a normal chow diet. Wild-
type C57BL/6 and ApoE−/− mice (n = 6 per group) were fed a
normal chow diet for 20 weeks before sacrifice. Under
anesthesia, living heart tissues were perfused with HKGreen-
4A (2 μM) in Hank’s balanced salt solution (HBSS) for 30 min,
followed by fixation and quenching with 4% PFA and glycine,
respectively. Mouse hearts were then excised and cryosectioned
at 10 μm intervals to reveal the aortic roots for confocal
imaging under two-photon excitation settings. As shown in
Figure 5, an obvious fluorescence increase, i.e., about 2-fold,
from HKGreen-4 was observed at the smooth muscle of the
aortic root from ApoE−/− mice compared to the samples from
wild-type mice (Figure S16). This result indicates that
HKGreen-4 is well-retained during histological sample
preparation and sensitive enough to visualize endogenous
ONOO− in live tissues. More importantly, our study also
provides the first direct evidence linking elevated ONOO−
formation with atherosclerotic conditions.
synthase (iNOS) seem temporally divergent.25 HKGreen-4
should serve as a unique tool to address this controversy. To
this end, imaging studies of E. coli-challenged macrophages
using HKGreen-4 were performed in the presence of well-
established inhibitors for NOX and iNOS to assess ONOO−
formation. MitoSOX Red was used in parallel for monitoring
•−
O2 throughout our studies. As shown in Figure 4, strong
Figure 4. Pharmacological validation of enzymatic pathways involved
in endogenous ONOO− formation in E. coli-challenged RAW264.7
macrophages. Cells were cotreated with heat-killed E. coli (moi = 100)
and various enzyme inhibitors for 14 h, and then stained with
HKGreen-4A (10 μM), MitoSOX (2.5 μM), and Hoechst 33342 (75
ng/mL) for 30 min before confocal imaging. ABAH (50 μM) is an
inhibitor of myeloperoxidase that produces HOCl. 1400W (100 nM)
and DPI (50 nM) are inhibitors for iNOS and NOX, respectively.
“Merge” represents overlay of all fluorescence channels including
Hoechst. Scale bar represents 10 μm.
CONCLUSION
■
In summary, we have presented the development, chemical
characterization, and molecular imaging applications of a new
fluorescent probe, HKGreen-4, for ONOO− detection in
aqueous solution, live cells, and tissues. HKGreen-4 contains
a rhodol core fluorophore and a peroxynitrite-triggered
fluorescence “off−on” switch that afford excellent sensitivity
and selectivity in fluorescence detection of ONOO− over other
biologically relevant ROS and RNS with visible excitation and
green emission profiles. We have thoroughly demonstrated that
HKGreen-4 can be utilized to image cellular ONOO− in
various cell types with minimal cytotoxicity by both single-
photon and two-photon excitation microscopy. Live cell
imaging with HKGreen-4 revealed that ONOO− formation in
activated macrophages is temporally and enzymatically
regulated, and may potentially be an important effector in
immune response. Moreover, HKGreen-4 can also be
intracellular fluorescence signals from HKGreen-4 and
MitoSOX Red were observed in E. coli-challenged macrophages
at 14 h post-treatment. Conventional scavengers for ONOO−,
O2•−, and •NO, i.e., urate, TEMPOL, and cPTIO, respectively,
can effectively suppress the green fluorescence signal of
HKGreen-4 (Figure S15). In addition, 4-aminobenzoic acid
hydrazide (ABAH), a potent inhibitor for myeloperoxidase
(MPO) that catalyzes the generation of HOCl, did not affect
fluorescence increase of HKGreen-4 (Figure 4). These results
further confirm the generation of ONOO− in E. coli-challenged
macrophages. On the other hand, 1400W and aminoguanidine
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Figure 5. Tissue staining of endogenous ONOO− with HKGreen-4 in
mouse heart smooth muscles. Living smooth muscles of the mouse
aortic root were stained with HKGreen-4A (2 μM) for 30 min by
perfusion, fixed, cryosectioned, and mounted for two-photon confocal
imaging. (A) Transverse section of wild-type smooth muscles. (B)
Transverse section of ApoE−/− smooth muscles. (C) Profile of
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were generated by merging 20 photosections from z-stack imaging and
are representative of HKGreen-4 staining of each group (n = 6 mice)
in two independent experiments. More representative images are
included in Figure S16 in Supporting Information.
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employed for live tissue staining, which confirmed that
ONOO− is generated at elevated levels in a mouse model of
atherosclerosis. Collectively, HKGreen-4 has been established
with practical utility and represents a robust molecular imaging
tool for unraveling the physiological and pathological
consequences of ONOO− formation under a variety of
biological contexts.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details for chemical synthesis of all compounds,
supplementary photophysical characterization of all probes, and
imaging methods and data. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
yangdan@hku.hk
■
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Author Contributions
‡T.P. and N.-K.W. contributed equally.
Notes
The authors declare the following competing financial
interest(s): D.Y. and T.P. hold patents on HKGreen-4 and
HKGreen-4A.
ACKNOWLEDGMENTS
■
This work was supported by The University of Hong Kong, the
University Development Fund, Hong Kong Research Grants
Council (HKU 706410, 776512M, and 777611M), and
Morningside Foundation. We thank Dr. Aimin Xu at
Department of Pharmacology and Pharmacy, The University
of Hong Kong, for kindly providing the ApoE−/− mouse model,
Dr. Jing Guo for technical assistance, and Faculty Core Facility
of the Li Ka Shing Faculty of Medicine at HKU for confocal
microscopy.
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