Visualizing Peroxynitrite Fluxes in Endothelial Cells Reveals the Dynamic Progression of Brain Vascular Injury

Article abstract of DOI:10.1021/jacs.5b06865

Accumulating evidence suggests that formation of peroxynitrite (ONOO^-) in the cerebral vasculature contributes to the progression of ischemic damage, while the underlying molecular mechanisms remain elusive. To fully understand ONOO^- biology, efficient tools that can realize the real-time tracing of endogenous ONOO^- fluxes are indispensable. While a few ONOO^- fluorescent probes have been reported, direct visualization of ONOO^- fluxes in the cerebral vasculature of live mice remains a challenge. Herein, we present a fluorescent switch-on probe (NP3) for ONOO^- imaging. NP3 exhibits good specificity, fast response, and high sensitivity toward ONOO^- both in vitro and in vivo. Moreover, NP3 is two-photon excitable and readily blood-brain barrier penetrable. These desired photophysical and pharmacokinetic properties endow NP3 with the capability to monitor brain vascular ONOO^- generation after injury with excellent temporal and spatial resolution. As a proof of concept, NP3 has enabled the direct visualization of neurovascular ONOO^- formation in ischemia progression in live mouse brain by use of two-photon laser scanning microscopy. Due to these favorable properties, NP3 holds great promise for visualizing endogenous peroxynitrite fluxes in a variety of pathophysiological progressions in vitro and in vivo.

Full text of DOI:10.1021/jacs.5b06865

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Article
Visualizing Peroxynitrite Fluxes in the Endothelial Cells
Reveals the Dynamic Progression of Brain Vascular Injury
Xin Li, Rong-Rong Tao, Ling-Juan Hong, Juan Cheng, Quan Jiang, Ying-Mei Lu, Mei-
Hua Liao, Wei-Feng Ye, Nan-Nan Lu, Feng Han, Yong-Zhou Hu, and You-Hong Hu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b06865 • Publication Date (Web): 09 Sep 2015
Downloaded from http://pubs.acs.org on September 15, 2015
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Visualizing Peroxynitrite Fluxes in the Endothelial Cells Reveals
the Dynamic Progression of Brain Vascular Injury
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†,
‡,
‡
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‡
§
∥
∥
Xin Li, RongꢀRong Tao, LingꢀJuan Hong, Juan Cheng, Quan Jiang, YingꢀMei Lu, MeiꢀHua
‡
‡
‡
,‡
,†
,†
Liao, WeiꢀFeng Ye, NanꢀNan Lu, Feng Han,* YongꢀZhou Hu,* YouꢀHong Hu*
†
ZJUꢀENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou
3
‡
10058, China
Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058,
China
§
School of Medicine, Zhejiang University City College, Hangzhou 310015, Zhejiang, China
ꢀ
ABSTRACT: Accumulating evidences suggest that the formation of peroxynitrite (ONOO ) in the cerebral vasculature contributes
ꢀ
to the progression of ischemic damage, while the underlying molecular mechanisms remain elusive. To fully understand ONOO
ꢀ
biology, efficient tools that can realize the realꢀtime tracing of endogenous ONOO fluxes are indispensable. Although there have
ꢀ
ꢀ
been a few ONOO fluorescent probes reported, a robust probe applicable for the direct visualization of ONOO fluxes in the cereꢀ
ꢀ
bral vasculature of live mice is still unavailable. While there are a few ONOO fluorescent probes reported, direct visualization of
ONOO fluxes in the cerebral vasculature of live mice remains a challenge. Herein, we present a fluorescent switchꢀon probe (NP3)
for ONOO imaging. NP3 exhibits good specificity, fast response and high sensitivity toward ONOO both in vitro and in vivo.
Moreover, NP3 is twoꢀphoton excitable and readily brainꢀblood barrier penetrable. These desired photophysical and pharmacokiꢀ
netic properties endow NP3 the capability to monitor brain vascular ONOO generation after injury with excellent temporal and
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
spatial resolution. As a proof of concept, NP3 has enabled the direct visualization of neurovascular ONOO formation in ischemia
progression in vivo in live mice brain using twoꢀphoton laser scanning microscopy. This is the first example of directly visualized
ꢀ
ONOO flux in the living mice brain. Due to these favorable properties, NP3 holds great promise for visualizing endogenous peroxꢀ
ynitrite fluxes in a variety of pathophysiological progressions in vitro and in vivo.
with high spatial resolution, which is pivotal for fully underꢀ
standing ONOO pathology in contexts of ischemia in vivo.
ꢀ
■ INTRODUCTION
ꢀ
Fluorescence imaging employing small molecular probes,
Peroxynitrite (ONOO ), a highly reactive nitrogen species
however, has emerged as a desirable and indispensable tool for
generated from the reaction between nitric oxide (NO) and
superoxide (O ) at a diffusionꢀlimited rate of 1.9×10 M S
under pathological conditions, attracts increasing attention
due to its "doubleꢀedged" character. ONOO may exert conꢀ
tributory effect by participating in nitrating tyrosine signaling.
Nevertheless, ONOO is more frequently regarded as deleteriꢀ
ous due to its nitrosative damage to lipids, proteins, and
DNA. ONOO has been implicated in various redoxꢀrelated
14,15
.
ꢀ
10
ꢀ1 ꢀ1
interrogating intact living samples.
Due to the obvious
2
1
technical and practical advantages of good membraneꢀ
permeability, high sensitivity and operational simplicity, fluoꢀ
rescence probes are attracting increasing attention in life sciꢀ
2,3
ꢀ
4
1
6ꢀ19
ꢀ
ence fields,
especially those twoꢀphoton excitable probes
because they are compatible with twoꢀphoton fluorescence
microscopy and can therefore realize the threeꢀdimensional
imaging of biological specimens with deeper tissue penetraꢀ
5
,6
ꢀ
5
,7,8
9
diseases,
including ischemiaꢀreperfusion injury. We have
2
0ꢀ22
ꢀ
tion and less photodamage.
Indeed, several fluorescent
been particularly interested in the roles of ONOO in the proꢀ
gression of brain ischemiaꢀinduced endothelial dysfunction
and neurovascular pathogenic cascades. Our preliminary reꢀ
sults have shown that ONOO is formed under conditions of
ischemia and that its nitrosative damage is implicated in neuꢀ
rovascular damage following cerebral ischemia.
previous work, ONOO was primarily detected by imꢀ
munostaining 3ꢀnitrotyrosine.
ONOO primarily relies on the immunostaining of 3ꢀ
ꢀ
probes are commercially available to detect ONOO , such as
aminophenyl fluorescein (APF) and hydroxyphenyl fluoresceꢀ
in (HPF). Unfortunately, these probes are limited by their
poor selectivity for ONOO against other highly reactive speꢀ
cies, such as •OH or ClO . To address this problem, several
23
ꢀ
ꢀ
ꢀ 24
1
0ꢀ12
In our
ꢀ
research groups have set out to develop new probes, and these
elegant work have resulted in the development of several seꢀ
1
0ꢀ12
Traditional biology assay for
ꢀ
25ꢀ37
ꢀ
lective probes suitable for imaging ONOO in live cells,
3
0
ꢀ
13
live mice, or even the redox cycles between ONOO and
30,32,38
nitrotyrosine. This method has the major limitation of being
incompatible with living systems and can therefore no longer
satisfy research needs for tracking native ONOO in real time
glutathione.
Facilitated by these probes, visualization of
ꢀ
25ꢀ
ꢀ
ONOO in macrophages during immune response,
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or in mouse hearts under atherosclerosis, has
which represents a great challenge in the development of brain
imaging agents, ascribing to the rigid requirements However,
study on the realꢀtime visualization of ONOO production in
been realized. However, none of these probes have realized
realꢀtime visualization of ONOO production in the brain of
live animals with ischemiaꢀinduced neurovascular damage,
ꢀ
ꢀ
the
brain
of
live
animals
with
Structures of HPF and APF
ESIPT effect of benzothiazole dyes
excitation
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O
O
O
S
N
S
NH
S
XH
N
H
-OOC
O
HO
O
HPF: X = O
APF: X = NH
emission
N
T
-
Design philosophy
Structures of ONOO probes
S
N
S
N
S
N
S
N
H
HO
H O
H C
N
3
ONOO-
H
S
N
S
N
HO
R2
NP1
R1
OH
H C
3
N
H N
CH3
NP2: R = R = H
1 2
NP3: R = H, R = OCH
1
2
3
NP4: R = R = OCH
1
2
3
OH
Figure 1. Structures of the BT series of ONOO probes and our design philosophy.
ꢀ
ischemiaꢀinduced neurovascular damage is still lacking, which
represents a great challenge ascribing to the rigid requirements
for brain imaging agents including high specificity, desired
photophysical properties and good bloodꢀbrain barrier (BBB)
penetrating infrared light, and possess desired pharmacokinetꢀ
ic profiles. Among the prevalent fluorescent markers for bioꢀ
imaging, 2ꢀ(2'ꢀhydroxyphenyl)benzothiazole (BT) provoked
our interest due to its drugꢀlike physicalꢀchemical performance
and good photophysical properties. First, the benzothiazole
skeleton may exhibit the desired pharmacokinetic properties,
especially the BBB penetration ability mandatory for brain
39,40
penetrability.
Herein, we report a twoꢀphoton fluorescent "switchꢀon"
ꢀ
11
probe for the detection of ONOO . The probe, judiciously deꢀ
imaging agents, as exemplified by [ C]PIB, an extensively
signed by combining the basic principles of fluorescent probe
design and drug design, is highly specific and sensitive toꢀ
studied positron emission tomography imaging probe for Aβ
41
plaques in humans. Second, the BT series of fluorophores
commonly exist in the normal form (N) with weak fluoresꢀ
cence but can automerize under excitation (T) via a process
called "excited state intramolecular proton transfer (ESIPT)"
ꢀ
wards ONOO , twoꢀphoton excitable, and most importantly,
readily BBB penetrable. It is fluorescenceꢀsilent in the absence
ꢀ
ꢀ
of ONOO , but can respond rapidly to ONOO with dramatic
emission enhancement (utmost 600ꢀfold). Its capability to
4
2
(
Figure 1), accompanied by both an enhancement and red
ꢀ
track in situ generation of ONOO in live cells and live mice
shift of their fluorescence. Blockage of this ESIPT effect with
a special chemical motif that can react selectively with intendꢀ
ed target in complex biological milieu to initiate the ESIPT
process enables the design of sensitive fluorescent probes.
Third, BT fluorophore is twoꢀphoton excitable and may be
with ischemiaꢀinduced neurovascular damage has been fully
characterized.
■
RESULTS AND DISCUSSION
43
compatible for live tissue imaging. With all these consideraꢀ
ꢀ
Probe Design and Synthesis. To image ONOO in live
animals, the fluorophore selected for probe construction
should be nonꢀtoxic, sufficiently bright, inert to other species
in the complex biological context, excitable with deepꢀ
tions, probe NP1 was designed by blocking the ESIPT process
with a saturated CꢀN bond (Figure 1). We envisioned that the
great tendency to be aromatic would render NP1 susceptible to
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ꢀ
oxidation by ONOO and therefore restore the ESIPT process.
various analytes. (b) NP3 responds to ONOO sufficiently fast to
ꢀ
ꢀ
NP1, as anticipated, could indeed respond to ONOO with a
reach its maximum intensity within seconds. ONOO (finally conꢀ
centration 10 ꢁM) was quickly injected into a solution of NP3
final concentration 5 ꢁM) and the fluorescent intensity at 470 nm
was plotted against time. (c) The fluorescent readout of the detecꢀ
tion reaction was linearly correlated with the concentrations of
ONOO . Fluorescence enhancement of NP3 (5 ꢁM) at 470 nm as
a function of ONOO (0ꢀ10 ꢁM) after 15 min of reaction. All the
data were acquired in PBS (10 mM, pH 7.4) with excitation 375
nm.
swift fluorescence intensity enhancement in PBS (10 mM, pH
7.4) (Figure S1 and S2). However, NP1 was found unstable
when exposed to air. This instability challenges its selectivity.
To develop probes with improved stability and specificity, we
next blocked the hydrogen donor of the ESIPT process and
designed NP2 by switching the hydroxyl group to Nꢀmethylꢀpꢀ
hydroxyl aniline, where the phenol group may be oxidized to
benzoquinone by ONOO , accompanied by the NꢀC (sp ) bond
cleavage, thereby furnishing a proton donor (Figure 1). NP3
and NP4 were also designed by installing electronꢀdonating
groups to the pꢀhydroxyl aniline ring in order to make the hyꢀ
droxyl group more susceptible to oxidants. All the probes were
facilely synthesized using Mills reaction (Scheme S1).
(
ꢀ
ꢀ
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ꢀ
2
26
Next, the sensing kinetics was studied, and the reaction beꢀ
tween NP3 and ONOO completed within seconds (Figure
2b, Figure S5), which is important given the extremely eluꢀ
sive nature of ONOO . Moreover, the fluorescence enhanceꢀ
ment of NP3 was linearly correlated with the concentrations
of ONOO ranging from 0ꢀ10 ꢁM, implying the great potenꢀ
tial of NP3 to quantify ONOO (Figure 2c, Figure S6). The
detection limit of NP3 was as low as 5.0 nmnM (S/N = 3)
ꢀ
ꢀ
ꢀ
Probe Evaluation. We first tested the fluorescence reꢀ
sponses of NP2-4 towards ONOO . These probes (5.0 ꢁM)
alone were nearly nonꢀemissive in PBS. In contrast, in the
presence of ONOO (10 ꢁM), all showed obvious fluorescence
enhancement, and NP3 was the most sensitive one with an
increase factor of 600ꢀfold, followed by NP2 and NP4 (Figure
S3). These results not only distinguish NP3 as a desirable canꢀ
didate for further study, but suggest that both electronic and
steric effects should be considered for probe design because
the steric effects of the methoxy groups adjacent to the hyꢀ
droxyl group in NP4 greatly outweigh their positive electronꢀ
donating effects.
ꢀ
ꢀ
ꢀ
ꢀ
(Figure S7). Notably, ONOO detection by NP3 was insusꢀ
ceptible to pH changes in the surroundings (Figure S8). Adꢀ
ditionally, sufficient photostability was observed for both
NP3 and the detection system (Figure S9), indicating the roꢀ
bustness of NP3.
ꢀ
We also evaluated the ability of NP3 to detect ONOO in a
twoꢀphoton excitation mode by measuring the fluorescence
spectra of the NP3ꢀONOO mixture with twoꢀphoton excitaꢀ
tion. As expected, sensitive signals retained. under twoꢀphoton
ꢀ
excitation. and the intensity at 470 nm (λ ) of the mixture
em
Characterization of the Spectroscopic Response of
NP3 towards ONOO . Spectroscopic Properties and Se-
lectivity. To profile the response of NP3 towards ONOO in
detail, we examined its specificity by recording its responses
towards various reactive oxygen species (ROS) and reactive
nitrogen species (RNS). Encouragingly, no analytes other than
ONOO could switch on the fluorescence of NP3. NP3 could
still recognize ONOO with a dramatic increase in fluoresꢀ
excited at 750 nm (or even 800 nm) was comparable to that
excited at 400 nm (Figure S13). The twoꢀphoton absorption
cross sections (σ) of the system at 760ꢀ820 nm were also deꢀ
termined with fluorescein in H O (pH = 13) as the standard,
and the σmax was observed at 820 nm with a value of 3.6 GM
(Figure S10). These results firmly support the feasibility of
-
ꢀ
44
2
ꢀ
ꢀ
NP3 as a highly specific and sensitive probe for ONOO in
vitro, and shows promise for in vivo imaging.
ꢀ
cence intensity even in the presence of other reactive species,
cations or amino acids commonly found in biological system,
implying the great specificity of NP3 towards ONOO (Figure
In agreement with the fluorescence switchꢀon response,
treating NP3 (10 ꢁM) with ONOO could also induce a draꢀ
matic change in its UV absorption profile. NP3 itself exhibited
a major absorption band centered at 300 nm (ε 16310), whereꢀ
as ONOO treatment resulted in the disappearance of this band
and the appearance of two new bands centered at 285 nm and
50 nm, both of which strengthened in a ONOO concentraꢀ
tionꢀdependent manner (Figure S11), indicating removal of the
phenol ring and generation of an intramolecular hydrogen
bond as shown in Figure 1. We also verified the detection
mechanism by isolating and characterizing the reaction prodꢀ
ꢀ
ꢀ
2
a, Figure S4).
ꢀ
ꢀ
3
1
uct using H NMR and HRMS spectra analysis (Figure S12,
45
S13).
Determination of Plasma and Brain NP3 Concentra-
tions. Pharmacokinetic study in C57 mice showed that NP3
readily crossed the brainꢀblood barrier (Figure S14). A 1.3ꢀ
fold brain/plasma ratio was achieved after 0.5 h of tail intraveꢀ
nous injection (I. V.) dosing at 10 mg/kg. The absolute brain
concentration reached a high level of 970 ng/mL 0.5 h after
NP3 administration and only 136 ng/mL remained after 2 h,
indicating the efficient brain penetration and fast brain clearꢀ
ance rate of NP3, which are deemed advantageous for brain
imaging agents.
Figure 2. Characterization of the fluorescent response of NP3
towards ONOO . (a) The fluorescent switchꢀon response of NP3
was preferentially towards ONOO , and the presence of other
analytes did not cause interference. Fluorescent responses of NP3
5 ꢁM) towards various analytes (10 ꢁM). Data shown represent
the fluorescent intensity at 470 nm 30 min after the addition of
ꢀ
ꢀ
(
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Fluorescent Response of NP3 to Dynamic Changes in
a timeꢀdependent manner until at least 50 min after treatment
(Figure 3a and b). In contrast, clearance of ONOO with uric
ꢀ
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Nitrosative Stress in Live Endothelial Cells. With the
photophysical profiles of NP3 fully characterized, we next
investigated its feasibility for dynamically tracking intracelluꢀ
acid (100 ꢁM) or FeTTPS (1 ꢁM) blunted the robustness of
ꢀ
NP3 fluorescence in ONOO ꢀtreated endothelial cells (Figure
ꢀ
lar ONOO generation. A timeꢀlapse series of single confocal
3c, Figure S15, S16). NP3 also worked well as an efficient
ꢀ
plane images were taken to observe the NP3 fluorescence
ONOO probe in other cell lines, as shown in human brain
ꢀ
response towards ONOO in live EA.hy926 endothelial cells
microvascular endothelial cells (HBMEC) in Figure S17. The
ꢀ
after incubation with or without 3ꢀmorpholinosydnonimine
ONOO (Figure S17a) or SINꢀ1 (Figure S17b) treatmentꢀ
ꢀ
(
SINꢀ1, 0.5 mM), an ONOO donor. As shown in Figure 3, no
induced NP3 fluorescence elevation was also observed in a
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detectable fluorescence signal was observed in endothelial
doseꢀdependent manner in HBMEC. These results indicate
ꢀ
ꢀ
cells loaded with NP3 in the absence of the ONOO donor
that NP3 is applicable for realꢀtime tracking of ONOO generꢀ
SINꢀ1 (Figure 3a and b). However, upon exposure to SINꢀ1,
intracellular fluorescence in endothelial cells gradually inꢀ
creased 10 min after SINꢀ1 stimulation and kept increasing in
ation in live cells and suggest its promise for serving as a moꢀ
ꢀ
lecular imaging tool to explore ONOO biology under pathoꢀ
logical conditions.
ꢀ
Figure 3. Characterization of ONOO formation by NP3 in endothelial cells upon nitrosative stress. (a) A timeꢀlapse series of single conꢀ
focal plane images taken from living EA.hy926 endothelial cells. The cells were seeded on glassꢀbottom 6ꢀwell plates overnight and then
preꢀincubated with NP3 (5.0 ꢁM) for 30 min, followed by stimulation with or without SINꢀ1 (0.5 mM). (b) Quantitative analysis of dyꢀ
namic changes of NP3 fluorescence after SINꢀ1 (0.5 mM) treatment in (a). Data are presented as a densitometry ratio change compared
ꢀ
with control. (c) Effects of ONOO scavengers uric acid (100 ꢁM) and FeTTPS (1 ꢁM) on changes in NP3 fluorescence in endothelial cells
ꢀ
ꢀ
ꢀ
in the presence of ONOO (60 ꢁM). The ONOO scavengers were preꢀincubated for 1 h prior to ONOO loading. PI counter staining indiꢀ
cates nuclear localization (red, λex 543 nm, λem 560ꢀ615 nm). NP3 fluorescence was collected at 420ꢀ480 nm with λex 405 nm.
46ꢀ48
-
Visualizing ONOO Fluxes in Endothelial Cells after
cells mediates cellular damage during cerebral ischemia.
Oxygen-Glucose Deprivation (OGD). Considerable eviꢀ
Thus, it would be interesting if NP3 could help to identify
ꢀ
ꢀ
dence has indicated that ONOO overproduction in endothelial
dynamic changes of ONOO formation during endothelial
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ischemic injury. As shown in Figure 4a, a timeꢀdependent
accumulation of NP3 fluorescence was observed in endotheliꢀ
al cells over 0.5 to 2 h following OGD exposure. A similar
pattern of fluorescence was observed in HBMEC (Figure 4b).
whether the OGDꢀinduced increase of NP3 fluorescence ocꢀ
2+
curs in a Ca /Calmodulinꢀdependent manner, calmodulin
2+
inhibitors (W7 and melatonin) and a Ca ꢀspecific aminopolyꢀ
carboxylic acid (1,2ꢀbis(oꢀaminophenoxy)ethaneꢀN,N,N',N'ꢀ
ꢀ
2+
To elaborate the specificity of NP3 towards ONOO during
tetraacetic acid, BAPTA) were used to bind Ca /Calmodulin
ꢀ
OGD treatment, FeTTPS (1 ꢁM), the ONOO decomposition
signaling. As shown in Figure 4c and Figure S19, the OGDꢀ
induced increase of NP3 fluorescence was reduced in the
presence of either the calmodulin inhibitor or BAPTA. Taken
together, these results suggest that NP3 is a highly selective
catalyst, blunted the elevation of NP3 fluorescence in OGDꢀ
treated endothelial cells (Figure 4c, Figure S18, S19), confirmꢀ
ꢀ
ing that NP3 is specific for ONOO during OGD insult.
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ꢀ
ꢀ
ONOO ꢀmediated stress can also be initiated by early intracelꢀ
and specific probe for monitoring ONOO fluxes during isꢀ
2+
49
lular Ca release and calmodulin activation. To discern
chemia.
ꢀ
Figure 4. Characterization of ONOO formation using NP3 in endothelial cells upon OGD. The representative confocal images show timeꢀ
dependent accumulation of NP3 fluorescence (green, λex 405 nm, λem 420ꢀ480 nm) in EA.hy926 endothelial cells over 0.5 to 2 h following
OGD exposure (a) as well as in human brain microvascular endothelial cells (HBMEC) in (b). PI counter staining indicated nuclear localiꢀ
ꢀ
zation (red, λ 543 nm, λem 560ꢀ615 nm). (c) The OGDꢀinitiated NP3 fluorescence response to ONOO was modulated by suppressing
ex
ꢀ
ONOO formation. EA.hy926 endothelial cells were pretreated with FeTTPS (1 ꢁM), W7 (1 ꢁM), melatonin (10 ꢁM) and BAPTA (1 ꢁM)
ꢀ
1
h prior to OGD treatment to suppress the ONOO signal.
Real-time Monitoring of Vascular Peroxynitrite
was primarily observed localizing in the same components
(Figure 5). Analysis of NP3 fluorescence (green) in mitochonꢀ
drial components of endothelial cells revealed a significant
elevation after 1 h of OGD, and continuous elevation could be
observed after 2 h until at least 6 h (Figure 5, Figure S20).
Fluxes with High Temporal and Spatial Resolution In
Vitro In Live Cells. Mitochondria constitutes a primary locus
ꢀ
50
for intracellular ONOO formation and target. For example,
ꢀ
modification of tyrosine residues by endogenous ONOO reꢀ
5
1
sults in the inhibition of mitochondrial complex I.ꢀ To further
visualize the subcellular distribution of ONOO labeled by
NP3 fluorescence, MitoRed (Invitrogen) was used to localize
mitochondrial components in endothelial cells. NP3 fluoresꢀ
cence in control endothelial cells was undetected (Figure 5,
Figure S20). Interestingly, consistent with MitoRed localizaꢀ
tion, elevated NP3 fluorescence in endothelial cells after OGD
Moreover, coinciding with the reports that lysosome response
11,52
is also associated with nitrosative stress,
our results
demonstrated that enhanced fluorescence partially located in
the lysosomal compartment (Figure S21), suggesting the dyꢀ
ꢀ
namic spatioꢀtemporal coordination between ONOO and lysoꢀ
somes. Similar changes in NP3 fluorescence were confirmed
in HBMEC after OGD (Figure S22, S23). The results indicatꢀ
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ed that NP3 can monitor ONOO flux during ischemic injury.
Accompanied by the increased NP3 fluorescence in mitoꢀ
chondria, as well as the spreading distribution in endothelial
(blue), in agreement with the results that ONOO could induce
apoptosis (Figure S24). Thus, these collective data demonꢀ
strated that NP3 efficiently visualized the progressive ONOO
fluxes in endothelial cells with excellent temporal and spatial
ability.
ꢀ
ꢀ
cell, ONOO accumulation challenged the endothelial cell and
contributed to apoptosis, as shown by the Annexin V staining
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Figure 5. The distribution of NP3 fluorescence was examined by counterstaining with the mitochondria indicator MitoRed after OGD. (a)
Representative confocal images show the temporal changes of ONOO ꢀdependent NP3 fluorescence (green, λ 405 nm, λem 420ꢀ480 nm)
and MitoRed (red, λex 543 nm, λem 560ꢀ615 nm), as well as the apoptosis maker Annexin V (blue, λex 488 nm, λem 505ꢀ550 nm), following
OGD treatment. (b) Orthogonal projections onto the x–z (upper) and yꢀz (right) planes are shown to confirm the colocalization of NP3 and
MitoRed throughout the endothelial cells shown in (a) after ischemia injury. All images were captured using a Zeiss LSM 510 confocal
microscope.
ꢀ
ex
The MTT assay showed that NP3 did not exhibit cytotoxiciꢀ
ty within 48 h except for such high concentrations as 150 and
200 ꢁM (Figure S25). Moreover, the pretty low cytotoxicity of
NP3 was confirmed by mitochondrial membrane potentialꢀ
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sensing dye JCꢀ1. Since the loss of mitochondrial transmemꢀ
brane potential (ΔΨm) signals is a hallmark of mitochondrial
ciently monitored over a recording period of 30 secs (Figure
6a, Movie S1). In contrast, no significant signal was observed
in negative control experiments in which mice were modeled
in the same way but not treated with NP3, indicating the caꢀ
5
3
dysfunction, cytotoxicity and apoptotic signaling, we used
JCꢀ1 to further investigate the potential cytotoxic effects of
NP3 on endothelial cells. No significant shift in JCꢀ1 fluoresꢀ
cence from red to green was observed following NP3 treatꢀ
ment up to 100 ꢁM in EA.hy926 endothelial cells compared
with control cells undergoing JCꢀ1 staining (Figure S26).
Thus, these results suggest that NP3 is nearly noncytotoxic up
to 100 ꢁM in endothelial cells, implying the excellent biocomꢀ
patibility for biological application, although further in vivo
testing is necessary prior to application for clinical diagnosis.
ꢀ
pacity of NP3 for the in vivo tracking of ONOO fluxes during
brain vascular damage (Figure S27).
Moreover, to provide another example of the in vivoꢀ
ꢀ
visualized ONOO flux enabled by NP3, realꢀtime imaging of
ꢀ
endogenous ONOO formation was performed in live mice
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suffering from laser irradiationꢀinduced cerebrovascular rupꢀ
ture at the twoꢀphoton wavelength of 800 nm. As shown in
ꢀ
Figure 6b, endogenous ONOO formation increased dramatiꢀ
cally upon laser irradiationꢀmediated microvessel injury at 10
s point and remained at its highest fluorescent intensity for at
least 30 secs (Figure 6b and c; Movie S2). Similar to our preꢀ
Real-time Imaging of Endogenous Peroxynitrite For-
mation After Brain Microvessel Injury in Live Mice.
Combined with in vivo twoꢀphoton laser scanning microscopy
ꢀ
viously reported results that ischemia induced ONOO producꢀ
(
TPLSM), NP3 enabled visualization of the dynamic changes
ꢀ
tion based on indirect evidence from immunoblot or imꢀ
of neurovascular ONOO formation upon ischemia in live
mice. The ischemia mice were modeled by RoseꢀBengalꢀ
induced vascular occlusion or laser irradiationꢀinduced miꢀ
10,12
munostaining,
we observed laser irradiationꢀinduced local
ꢀ
54
ONOO formation in injured cerebral microvessels in live
mice using the combination of in vivo TPLSM and probe NP3.
More importantly, we were able to visualize the dynamic
55
crovessel rupture. The time series images in Figure 6 are
individual frames from a continuous timeꢀlapse movie. The
ꢀ
ꢀ
changes of ONOO formation in a temporal and spatial manner
dynamic elevation of local ONOO formation (arrows) in the
with highꢀresolution images in live mice.
microvessel indicated by strong NP3 fluorescence was effiꢀ
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Figure 6. Realꢀtime visualization of endogenous peroxynitrite fluxes after brain microvessel injury with a combination of NP3 and in vivo
twoꢀphoton laser scanning microscopy. For twoꢀphoton imaging, cortical brain vessels 10~15 µm in diameter and 100 µm below the cortiꢀ
cal surface were selected for imaging. The timeꢀseries images in (a, b) are individual frames from a continuous timeꢀlapse movie and show
dynamic NP3 fluorescence elevation (arrows) following RoseꢀBengalꢀinduced vascular occlusion (a) and laser irradiationꢀinduced vascular
rapture (b) in live mice. (c) The mean values of NP3 fluorescence intensity from (b) were measured to quantify the progressive ONOO
formation in cerebrovessels after ischemia. Emission was collected at 420ꢀ480 nm for NP3 fluorescence and 575ꢀ630 nm for dextran
ꢀ
Texas or roseꢀBengal upon excitation at 800 nm.
■
CONCLUSION
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REFERENCES
To conclude, by considering basic principles of probe and
drug design, we developed a fluorescent probe with desired
pharmacokinetic and photophysical properties suitable for
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should be instructive for future probe design.
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SUPPORTING INFORMATION
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ABBREVIATIONS
ONOO , peroxynitrite; ROS, reactive oxygen species; RNS, reacꢀ
tive nitrogen species; ESIPT, excited state intramolecular proton
transfer; BBB, bloodꢀbrain barrier.
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Figure 2. Characterization of the fluorescent response of NP3 towards ONOO-. (a) Fluorescent responses of
NP3 (5 µM) to-wards various analytes (10 µM). Data shown represent the fluorescent intensity at 470 nm 30
min after the addition of various analytes. (b) ONOO- (finally concentration 10 µM) was quickly injected into
a solution of NP3 (final concentration 5 µM) and the fluorescent intensity at 470 nm was plotted against
time. (c) Fluorescence enhancement of NP3 (5 µM) at 470 nm as a function of ONOO- (0-10 µM) after 15
min of reaction. All the data were acquired in PBS (10 mM, pH 7.4) with excitation 375 nm.
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Figure 3. Characterization of ONOOꢀ formation by NP3 in endothelial cells upon nitrosative stress. (a) A
timeꢀlapse series of single confocal plane images taken from living EA.hy926 endothelial cells. The cells were
seeded on glassꢀbottom 6ꢀwell plates overnight and then preꢀincubated with NP3 (5.0 ꢁM) for 30 min,
followed by stimulation with or without SINꢀ1 (0.5 mM). (b) Quantitative analysis of dynamic changes of
NP3 fluorescence after SINꢀ1 (0.5 mM) treatment in (a). Data are presented as a densitometry ratio change
compared with control. (c) Effects of ONOOꢀ scavengers uric acid (100 ꢁM) and FeTTPS (1 ꢁM) on changes
in NP3 fluorescence in endothelial cells in the presence of ONOOꢀ (60 ꢁM). The ONOOꢀ scavengers were
preincubated for 1 h prior to ONOOꢀ loading. PI counter staining indicates nuclear localization (red, λex 543
nm, λem 560ꢀ615 nm). NP3 fluorescence was collected at 420ꢀ480 nm with λex 405 nm.
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Figure 4. Characterization of ONOOꢀ formation using NP3 in endothelial cells upon OGD. The representative
confocal images show timeꢀdependent accumulation of NP3 fluorescence (green, λex 405 nm, λem 420ꢀ480
nm) in EA.hy926 endothelial cells over 0.5 to 2 h following OGD exposure (a) as well as in human brain
microvascular endothelial cells (HBMEC) in (b). PI counter staining indicated nuclear localiꢀzation (red, λex
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43 nm, λem 560ꢀ615 nm). (c) The OGDꢀinitiated NP3 fluorescence response to ONOOꢀ was modulated by
suppressing ONOOꢀ formation. EA.hy926 endothelial cells were pretreated with FeTTPS (1 ꢁM), W7 (1 ꢁM),
melatonin (10 ꢁM) and BAPTA (1 ꢁM) 1 h prior to OGD treatment to suppress the ONOOꢀ signal.
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Figure 5. The distribution of NP3 fluorescence was examined by counterstaining with the mitochondria
indicator MitoRed after OGD. (a) Representative confocal images show the temporal changes of ONOOꢀꢀ
dependent NP3 fluorescence (green, λex 405 nm, λem 420ꢀ480 nm) and MitoRed (red, λex 543 nm, λem
560ꢀ615 nm), as well as the apoptosis maker Annexin V (blue, λex 488 nm, λem 505ꢀ550 nm), following
OGD treatment. (b) Orthogonal projections onto the x–z (upper) and yꢀz (right) planes are shown to confirm
the colocalization of NP3 and MitoRed throughout the endothelial cells shown in (a) after ischemia injury. All
images were captured using a Zeiss LSM 510 confocal microscope.
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Figure 6. Realꢀtime visualization of endogenous peroxynitrite fluxes after brain microvessel injury with a
combination of NP3 and in vivo twoꢀphoton laser scanning microscopy. For twoꢀphoton imaging, cortical
brain vessels 10~15 µm in diameter and 100 µm below the cortical surface were selected for imaging. The
timeꢀseries images in (a, b) are individual frames from a continuous timeꢀlapse movie and show dynamic
NP3 fluorescence elevation (arrows) following RoseꢀBengalꢀinduced vascular occlusion (a) and laser
irradiationꢀinduced vascular rapture (b) in live mice. (c) The mean values of NP3 fluorescence intensity from
(
b) were measured to quantify the progressive ONOOꢀ formation in cerebrovessels after ischemia. Emission
was collected at 420ꢀ480 nm for NP3 fluorescence and 575ꢀ630 nm for dextran Texas or roseꢀBengal upon
excitation at 800 nm.
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