Visualizing Peroxynitrite Fluxes in 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*,†
†ZJU-ENS Joint Laboratory of Medicinal Chemistry,College of Pharmaceutical Sciences,Zhejiang University,Hangzhou310058, China
‡Institute of Pharmacology and Toxicology,College of Pharmaceutical Sciences,Zhejiang University,Hangzhou310058,China
§School of Medicine,Zhejiang University City College,Hangzhou310015,China
*Supporting Information
pharmacokinetic properties endow NP3with the capability to monitor
temporal and spatial resolution.As a proof of concept,NP3has
风速歌formation in ischemia progression in live mou brain by u of two-
properties,NP3holds great promi for visualizing endogenous
progressions in vitro and in vivo.
INTRODUCTION
Peroxynitrite(ONOO−),a highly reactive nitrogen species
generated from the reaction between nitric oxide(NO)and
马诗二十三首其四superoxide(O2.‑)at a diffusion-limited rate of1.9×1010M−1·s−1under pathological conditions,1attracts increasing attention
due to its“double-edged”character.2,3ONOO−may exert a
contributory effect by participating in nitrating tyrosine
signaling.4Nevertheless,ONOO−is more frequently regarded
as deleterious due to its nitrosative damage to lipids,proteins,
and DNA.5,6ONOO−has been implicated in various redox-related dias,5,7,8including ischemia-reperfusion injury.9We have been particularly interested in the roles of ONOO−in the progression of brain ischemia-induced endothelial dysfunction and neurovascular pathogenic cascades.Our preliminary results have shown that ONOO−is formed under conditions of ischemia and that its nitrosative damage is implicated in neurovascular damage following cerebral ischemia.10−12Tradi-tional biological assay for ONOO−primarily relies on the immunostaining of3-nitrotyrosine.13This method has the major limitation of being incompatible with living systems and can therefore no longer satisfy rearch needs for tracking native ONOO−in real time with high spatial resolution,which is pivotal for fully understanding ONOO−pathology in
contexts of ischemia.
Fluorescence imaging employing small molecular probes, however,has emerged as a desirable and indispensable tool for
interrogating intact living samples.14,15Due to the obvious
technical and practical advantages of good membrane
permeability,high nsitivity,and operational simplicity,fluorescence probes are attracting increasing attention in life sciencefields,16−19especially two-photon excitable probes,
becau they are compatible with two-photonfluorescence
microscopy and can therefore realize three-dimensional
imaging of biological specimens with deeper tissue penetration and less photodamage.20−22Indeed,veralfluorescent probes are commercially available to detect ONOO−,such as aminophenylfluorescein(APF)and hydroxyphenylfluorescein (HPF).23Unfortunately,the probes are limited by their poor lectivity for ONOO−against other highly reactive species, such as•OH or ClO−.24To address this problem,veral rearch groups have t out to develop new probes,and this
Received:July2,2015
Published:September9,2015
elegant work has resulted in the development of veral lective probes suitable for imaging ONOO −in live cells,25−37live mice,30or even the redox cycles between ONOO −and glutathione.30,32,38Facilitated by the probes,visualization of O N O O −i n m a c r o p h a g e s d u r i n g i m m u n e r e -spon 25−27,29,30,32,33,35or in mou hearts under atheroscle-rosis 26has been realized.However,study on the real-time visualization of ONOO −production in the brain of live animals with ischemia-induced neurovascular damage is still lacking,which reprents a great challenge due to the rigid require-ments for brain imaging agents including high speci ficity,desired photophysical properties,and good blood −brain barrier (BBB)penetrability.39,40
Herein,we report a two-photon fluorescent “switch-on ”probe for the detection of ONOO −.The probe,judiciously designed by combining the basic principles of fluorescent probe design and drug design,is highly speci fic and nsitive toward ONOO −,two-photon excitable,and most importantly,readily BBB-penetrable.It is fluorescence-silent in the abnce of ONOO −but can respond rapidly to ONOO −with dramatic emission enhancement (utmost 600-fold).Its capability to track in situ generation of ONOO −in live cells and live mice with ischemia-induced neurovascular damage has been fully characterized.
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学简笔画
RESULTS AND DISCUSSION
Probe Design and Synthesis.To image ONOO −in live animals,the fluorophore lected for probe construction should be nontoxic,su fficiently bright,inert to other species in the complex biological context,excitable with deep-penetrating infrared light,and posss desired pharmacokinetic pro files.Among the prevalent fluorescent markers for bioimaging,2-(2′-hydroxyphenyl)benzothiazole (BT)provoked our interest due to its druglike physical −chemical performance and good photophysical properties.First,the benzothiazole skeleton may exhibit the desired pharmacokinetic properties,especially the BBB penetration ability mandatory for brain imaging
agents,as exempli fied by [11C]PIB,an extensively studied positron emission tomography imaging probe for A βplaques in humans.41Second,the BT ries of fluorophores commonly exist in the normal form (N)with weak fluorescence but can automerize under excitation (T)via a process called excited-state intramolecular proton transfer (ESIPT)(Figure 1),42accompanied by both enhancement and red shift of their fluorescence.Blockage of this ESIPT e ffect with a special chemical motif that can react lectively with intended target in complex biological milieu to initiate the ESIPT process enables the design of nsitive fluorescent probes.Third,BT fluorophore is two-photon excitable and may be compatible with live tissue imaging.43With all the considerations,probe NP1was designed
by blocking the ESIPT process with a saturated C −N bond (Figure 1).We envisioned that the great tendency to be aromatic would render NP1susceptible to oxidation by ONOO −and therefore restore the ESIPT process.NP1,as anticipated,could indeed respond to ONOO −with a swift fluorescence intensity enhancement in phosphate-bu ffered saline (PBS;10mM,pH 7.4)(Figures S1and S2).However,NP1was found to be unstable when expod to air.This instability challenges its lectivity.To develop probes with improved stability and speci ficity,we next blocked the hydrogen donor of the ESIPT process and designed NP2by switching the hydroxyl group to N -methyl-p -hydroxyaniline,where the phenol group may be oxidized to benzoquinone by ONOO −,accompanied by N −C (sp 2)bond cleavage,thereby furnishing a proton donor (Figure 1).26NP3and NP4were also designed by installing electron-donating groups to the p -hydroxyaniline ring in order to make the hydroxyl group more susceptible to oxidants.All the probes were facilely synthesized via Mills reaction (Scheme S1).
Probe Evaluation.We first tested the fluorescence respons of NP2−4toward ONOO −.The probes (5.0μM)alone were nearly nonemissive in PBS.In contrast,in the prence of ONOO −(10μM),all showed obvious fluorescence enhancement,and NP3was the most nsitive one with
an
Figure 1.Structures of BT ries of ONOO −probes and design philosophy.
革命战士increa factor of 600-fold,followed by NP2and NP4(Figure S3).The results not only distinguish NP3as a desirable candidate for further study but also suggest that both electronic and steric e ffects should be considered for probe design becau the steric e ffects of the methoxy groups adjacent to the hydroxyl group in NP4greatly outweigh their positive electron-donating e ffects.
Spectroscopic Properties and Selectivity.To pro file the respon of NP3toward ONOO −in detail,we examined its speci ficity by recording its respons toward various reactive oxygen species (ROS)and reactive nitrogen species (RNS).Encouragingly,no analytes other than ONOO −could switch on the fluorescence of NP3.NP3could still recognize ONOO −with a dramatic increa in fluorescence intensity even in the prence of other reactive species,cations,or amino acids commonly found in biological systems,implying great speci ficity of NP3toward ONOO −(Figure 2a and Figure S4).
Next,the nsing kinetics was studied,and the reaction between NP3and ONOO −was found to be completed within conds (Figure 2b and Figure S5),which is important given the extremely elusive
nature of ONOO −.Moreover,the fluorescence enhancement of NP3was linearly correlated with concentrations of ONOO −ranging from 0to 10μM,implying the great potential of NP3to quantify ONOO −(Figure 2c and Figure S6).The detection limit of NP3was as low as 5.0nM (S/N =3)(Figure S7).Notably,ONOO −detection by NP3was insusceptible to pH changes in the surroundings (Figure S8).Additionally,su fficient photostability was obrved for both NP3and the detection system (Figure S9),indicating the robustness of NP3.
We also evaluated the ability of NP3to detect ONOO −in two-photon excitation mode by measuring fluorescence spectra of the NP3−ONOO −mixture with two-photon excitation.As expected,nsitive signals remained.The two-photon absorp-tion cross ctions (σ)of the system at 760−820nm were also determined with fluorescein in H 2O (pH =13)as standard,44
and σmax was obrved at 820nm with a value of 3.6GM (Figure S10).The results firmly support the feasibility of NP3as a highly speci fic and nsitive probe for ONOO −in vitro that shows promi for in vivo imaging.
In agreement with the fluorescence switch-on respon,treating NP3(10μM)with ONOO −could also induce a dramatic change in its UV absorption pro file.NP3itlf exhibited a major absorption band c
entered at 300nm (ε16310),whereas ONOO −treatment resulted in the disappear-ance of this band and the appearance of two new bands centered at 285and 350nm,both of which strengthened in a ONOO −concentration-dependent manner (Figure S11),indicating removal of the phenol ring and generation of an intramolecular hydrogen bond as shown in Figure 1.We also veri fied the detection mechanism by isolating and characteriz-ing the reaction product by 1H NMR and HRMS spectral analysis (Figures S12and S13).45
Determination of Plasma and Brain NP3Concen-trations.Pharmacokinetic study in C57mice showed that NP3readily crosd the blood −brain barrier (Figure S14).A 1.3-fold brain/plasma ratio was achieved after 0.5h of tail intravenous (iv)injection dosing at 10mg/kg.The absolute brain concentration reached a high level of 970ng/mL at 0.5h after NP3administration and only 136ng/mL remained after 2h,indicating the e fficient brain penetration and fast brain clearance rate of NP3,which are deemed advantageous for brain imaging agents.
Fluorescent Respon of NP3to Dynamic Changes in Nitrosative Stress in Live Endothelial Cells.With the photophysical pro files of NP3fully characterized,we next investigated its feasibility for dynamically tracking intracellular ONOO −generation.A time-lap ries of single confocal plane images were taken to obrve the NP3fluorescence respon toward ONOO −in live EA.hy926endot
helial cells after incubation with or without 3-morpholinosydnonimine (SIN-1,0.5mM),an ONOO −donor.As shown in Figure 3,no detectable fluorescence signal was obrved in endothelial cells loaded with NP3in the abnce of the ONOO −donor SIN-1(Figure 3a,b).However,upon exposure to SIN-1,intracellular fluorescence in endothelial cells gradually incread 10min after SIN-1stimulation and kept increasing in a time-dependent manner until at least 50min after treatment (Figure 3a,b).In contrast,clearance of ONOO −with uric acid (100μM)or 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinatoiron(III)chloride (FeTTPS;1μM)blunted the robustness of NP3fluorescence in ONOO −-treated endothelial cells (Figure 3c and Figures S15and S16).NP3also worked well as an e fficient ONOO −probe in other cell lines,as shown in human brain microvascular endothelial cells (HBMEC)in Figure S17.The ONOO −(Figure S17a )or SIN-1(Figure S17b )treatment-induced NP3fluorescence elevation was also obrved in a do-dependent manner in HBMEC.The results indicate that NP3is applicable for real-time tracking of ONOO −generation in live cells and suggest its promi for rving as a molecular imaging tool to explore ONOO −biology under pathological conditions.胆小如鼠的意思是什么
Visualizing ONOO −Fluxes in Endothelial Cells after Oxygen −Gluco Deprivation.Considerable evidence has indicated that ONOO −overproduction in endothelial cells mediates cellular damage du
ring cerebral ischemia.46−48Thus,it would be interesting if NP3could help to identify dynamic changes of ONOO −formation during endothelial ischemic injury.As shown in Figure 4a,time-dependent accumulation
of
Figure 2.Characterization of fluorescent respon of NP3toward ONOO −.(a)Fluorescent respons of NP3(5μM)toward various analytes (10μM).Data shown reprent fluorescent intensity at 470nm,30min after addition of various analytes.(b)ONOO −(final concentration 10μM)was quickly injected into a solution of NP3(final concentration 5μM),and the fluorescent intensity at 470nm was plotted against time.(c)Fluorescence enhancement of NP3(5μM)at 470nm as a function of ONOO −(0−10μM)after 15min of reaction.All data were acquired in PBS (10mM,pH 7.4)with excitation at 375nm.
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NP3fluorescence was obrved in endothelial cells over 0.5−2h following oxygen −gluco deprivation (OGD)exposure.A similar pattern of fluorescence was obrved in HBMEC (Figure 4b).To elaborate the speci ficity of NP3toward ONOO −during OGD treatment,FeTTPS (1μM),the ONOO −decomposition catalyst,blunted the elevation of NP3fluorescence in OGD-treated endothelial cells (Figure 4c and Figures S18and S19),con firming that NP3is speci fic for ONOO −during OGD insult.ONOO −-mediated stress can also be initiated by early intracellular Ca 2+releas
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e and calmodulin activation.49To discern whether the OGD-induced increa of NP3fluorescence occurs in a Ca 2+/calmodulin-dependent manner,calmodulin inhibitors (W7and melatonin)and a Ca 2+-speci fic aminopoly(carboxylic acid)[1,2-bis(o -aminophenoxy)ethane-N ,N ,N ′,N ′-tetraacetic acid,BAPTA]were ud to bind Ca 2+/calmodulin signaling.As shown in Figure 4c and Figure S19,the OGD-induced increa of NP3fluorescence was reduced in the prence of either calmodulin inhibitor or BAPTA.Taken together,the results suggest that
NP3is a highly lective and speci fic probe for monitoring ONOO −fluxes during ischemia.
Real-Time Monitoring of Vascular Peroxynitrite Fluxes with High Temporal and Spatial Resolution in Live Cells.Mitochondria constitute a primary locus for intracellular ONOO −formation and targeting.50For example,modi fication of tyrosine residues by endogenous ONOO −results in inhibition of mitochondrial complex I.51To further visualize the subcellular distribution of ONOO −labeled by NP3fluorescence,MitoRed (Invitrogen)was ud to localize mitochondrial components in endothelial cells.NP3fluores-cence in control endothelial cells was undetected (Figure 5and Figure S20).Interestingly,consistent with MitoRed local-ization,elevated NP3fluorescence in endothelial cells after OGD was primarily obrved to localize in the same components (Figure 5).Analysis of NP3fluorescence (green)in mitochondrial components of endothelial cells revealed a signi ficant elev
孤独是常态ation after 1h of OGD,and continuous elevation could be obrved after 2h until
at
Figure 3.Characterization of ONOO −formation by NP3in endothelial cells upon nitrosative stress.(a)Time-lap ries of single confocal plane images taken from living EA.hy926endothelial cells.The cells were eded on glass-bottom 6-well plates overnight and then preincubated with NP3(5.0μM)for 30min,followed by stimulation with or without SIN-1(0.5mM).(b)Quantitative analysis of dynamic changes of NP3fluorescence after SIN-1(0.5mM)treatment in panel a.Data are prented as a densitometric ratio change compared with control.(c)E ffects of ONOO −scavengers uric acid (100μM)and FeTTPS (1μM)on changes in NP3fluorescence in endothelial cells in the prence of ONOO −(60μM).The ONOO −scavengers were preincubated for 1h prior to ONOO −loading.Propidium iodide (PI)counterstaining indicates nuclear localization (red;λex 543nm,λem 560−615nm).NP3fluorescence was collected at 420−480nm with λex 405nm.
least 6h (Figure 5and Figure S20).Moreover,coinciding with the reports that lysosome respon is also associated with nitrosative stress,11,52our results demonstrated that enhanced fluorescence is partially located in the lysosomal compartment (Figure S21),suggesting dynamic spatiotemporal coor
dination between ONOO −and lysosomes.Similar changes in NP3fluorescence were con firmed in HBMEC after OGD (Figures S22and S23).The results indicated that NP3can monitor ONOO −flux during ischemic injury.Accompanied by incread NP3fluorescence in mitochondria,as well as the spreading distribution in endothelial cells,ONOO −accumu-lation challenged the endothelial cell and contributed to apoptosis,as shown by annexin V staining (blue),in agreement with the results that ONOO −could induce apoptosis (Figure S24).Thus,the collective data demonstrated that NP3e fficiently visualized progressive ONOO −fluxes in endothelial cells with excellent temporal and spatial ability.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT)assay showed that NP3did not exhibit cytotoxicity within 48h except at such high concentrations as 150and 200μM (Figure S25).Moreover,the fairly low cytotoxicity of NP3was con firmed by mitochondrial membrane potential-nsing dye JC-1.Since the loss of mitochondrial transmembrane potential (ΔΨm )signals is a hallmark of mitochondrial dysfunction,cytotoxicity,and apoptotic signal-ing,53we ud JC-1to further investigate the potential cytotoxic e ffects of NP3on endothelial cells.No signi ficant shift in JC-1fluorescence from red to green was obrved following NP3treatment up to 100μM in EA.hy926endothelial cells,compared with control cells undergoing JC-1staining (Figure S26).Thus,th
e results suggest that NP3is nearly noncytotoxic up to 100μM in endothelial cells,implying excellent biocompatibility for biological application,although further in vivo testing is necessary prior to application for clinical diagnosis.
Real-Time Imaging of Endogenous Peroxynitrite Formation after Brain Microvesl Injury in Live Mice.Combined with in vivo two-photon lar scanning microscopy (TPLSM),NP3enabled visualization of dynamic changes of neurovascular ONOO −formation upon ischemia in live mice.The ischemia mice were modeled by ro bengal-induced vascular occlusion 54or lar irradiation-induced microvesl rupture.55The time ries images in Figure 6are individual frames from a continuous time-lap movie.The dynamic elevation of local ONOO −formation (arrows)in the microvesl indicated by strong NP3fluorescence was e fficiently monitored over a recording period of 30s (Figure 6a and Movie S1).In contrast,no signi ficant signal was obrved in negative control experiments in which mice were modeled in the same way but not treated with NP3,
indicating
Figure 4.Characterization of ONOO −formation by u of NP3in endothelial cells upon OGD.(a,b)Reprentative confocal images show time-dependent accumulation of NP3fluorescence (green;λex 405nm,λem 420−480nm)in (a)EA.hy926endothelial cells over 0.5−2h following OGD exposure as well as in (b)human brain microvascular endothelial cells (HBMEC).PI counterstaining indicated nuclear localization (red,λex 543nm,λem 560−615nm).(c)The OGD-initiated NP3fluorescence respon to ONOO −was modulated by suppressing ONOO −formation.EA.hy926endothelial cells were pretreated with FeTTPS (1μM),W7(1μM),melatonin (10μM),or BAPTA (1μM)1h prior to OGD treatment to suppress the ONOO −signal.
