MITOCHONDRIAL REDOX IMAGING FOR CANCER DIAGNOSTIC AND THERAPEUTIC STUDIES

Mitochondrial redox states provide important information about energy-linked biological processes and signaling events in tissues for various disease phenotypes including cancer.The redox scanning method developed at the Chance laboratory about 30 years ago has allowed 3D highresolution(∼50×50×10µm^(3))imaging of mitochondrial redox state in tissue on the basis of the fluorescence of NADH(reduced nicotinamide adenine dinucleotide)and Fp(oxidized flavoproteins including flavin adenine dinucleotide,i.e.,FAD).In this review,we illustrate its basic principles,recent technical developments,and biomedical applications to cancer diagnostic and therapeutic studies in small animal models.Recently developed calibration procedures for the redox imaging using reference standards allow quantification of nominal NADH and Fp concentrations,and the concentration-based redox ratios,e.g.,Fp/(Fp+NADH)and NADH/(Fp+NADH)in tissues.This calibration facilitates the comparison of redox imaging results acquired for different metabolic states at different times and/or with different instrumental settings.A redox imager using a CCD detector has been developed to acquire 3D images faster and with a higher in-plane resolution down to 10µm.Ex vivo imaging and in vivo imaging of tissue mitochondrial redox status have been demonstrated with the CCD imager.Applications of tissue redox imaging in small animal cancer models include metabolic imaging of glioma and myc-induced mouse mammary tumors,predicting the metastatic potentials of human melanoma and breast cancer mouse xenografts,differentiating precancerous and normal tissues,and monitoring the tumor treatment response to photodynamic therapy.Possible future directions for the development of redox imaging are also discussed.

Quantitative optical measurement of mitochondrial superoxide dynamics in pulmonary artery endothelial cells

Reactive oxygen species(ROS)play a vital role in cell signaling and redox regulation,but when present in excess,lead to numerous pathologies.Detailed quantitative characterization of mitochondrial superoxide anion(O^(·-)_(2))production in fetal pulmonary artery endothelia cells(PAECs)has never been reported.The aim of this study is to assess mitochondrial O^(·-)_(2)pro-duction in cultured PAECs over time using a novel quantitative optical approach.The rate,the sources,and the dynamics of O^(·-)_(2)production were assessed using targeted metabolic modulators of the mitochondrial electron transport chain(ETC)complexes,specifically an uncoupler and inhibitors of the various ETC complexes,and inhibitors of extra-mitochondrial sources of O^(·-)_(2).After stabilization,the cells were loaded with nanomolar mitochondrial-targeted hydroethidine(Mito-HE,MitoSOX)online during the experiment without washout of the residual dye.Time-lapse fuorescence microscopy was used to monitor the dynamic changes in O^(·-)_(2)fluorescence intensity over time in PAECs.The transient behaviors of the fuorescence time course showed exponential increases in the rate of O^(·-)_(2) production in the presence of the ETC uncoupler or inhibitors.The most dramatic and the fastest increase in O^(·-)_(2)production was observed when the cells were treated with the uncoupling agent,PCP.We also showed that only the complex IV inhibitor,KCN,attenuated the marked surge in O^(·-)_(2)production induced by PCP.The results showed that mitochondrial respiratory complexes I,III and IV are sources of O^(·-)_(2) production in PAECs,and a new observation that ROS production during uncoupling of mitochondrial res-piration is mediated in part via complex IV.This novel method can be applied in other studies that examine ROS production under stress condition and during ROS mediated injuries in vritro.

This research was supported by E.U.FP6 Integrated Project“Molecular Imaging”LSHG-CT-2003-503259 and E.U.FP7 Collaborative Project“FMT-XCT”.R.F.acknowledges support from the Marie Curie Program EST-MolecImag Early Stage Training MEST-CT-2004-007643.

We have imaged mitochondrial oxidation-reduction states by taking a ratio of mitochondrial fluorophores:NADH(reduced nicotinamide adenine dinucleotide)to Fp(oxidized flavoprotein).Although NADH has been investigated for tissue metabolic state in cancer and in oxygen deprived tissues,it alone is not an adequate measure of mitochondrial metabolic state since the NADH signal is altered by dependence on the number of mitochondria and by blood absorption.The redox ratio,NADH/(Fp+NADH),gives a more accurate measure of steady-state tissue metabolism since it is less dependent on mitochondrial number and it compensates effectively for hemodynamic changes.This ratio provides important diagnostic information in living tissues.In this study,the emitted fluorescence of mouse colon in situ is passed through an emission filter wheel and imaged on a CCD camera.Redox ratio images of the healthy and hypoxic mouse intestines clearly showed significant differences.Furthermore,the corrected redox ratio indicated an increase from an average value of 0.51±0.10 in the healthy state to 0.92±0.03 in dead tissue due to severe ischemia(N=5).We show that the CCD imaging system is capable of displaying the metabolic differences in normal and ischemic tissues as well as quantifying the redox ratio in vivo as a marker of these changes.

OPTICAL IMAGING OF LIPOPOLYSACCHARIDE-INDUCED OXIDATIVE STRESS IN ACUTE LUNG INJURY FROM HYPEROXIA AND SEPSIS

Reactive oxygen species(ROS)have been implicated in the pathogenesis of many acute and chronic pulmonary disorders such as acute lung injury(ALI)in adults and bronchopulmonary dysplasia(BPD)in premature infants.Bacterial infection and oxygen toxicity,which result in pulmonary vascular endot belial injury,contribute to impaired vascular growth and alveolar simplfcation soen in the lungs of premature infants with BPD.Hyperoxia induces ALI,reduces cell proliferation,cauuses DNA damage and promotes cell death by causi ng mitochondrial dys function.The objective of this study was to use an optical imaging technique to evaluate the variations in fluorescence intensities of the auto fuorescent mitochondrial metabolic coenzymes,NADH and FAD in four different groups of rats.The ratio of these fluorescence signals(NA DH/FAD),referred to as NADH redox ratio(NADH RR)has been used as an indicator of tisue metabolism in injuries.Here,we investigated whether the changes in metabolic state can be used as a marker of oxidative stress caused by hyperoxia and bacterial lipopolysaccharide(LPS)exposure in neonatal mat lungs.We examined the tissue redox states of lungs from four groups of rat pups:nomoxic(21%O_(2))pups,hyperoxic(90%O_(2))pups,pups treated with LPS(normoxic+LPS),and pups treated with LPS and hyperoxia(hyperoxic+LPS).Our results show that hyperoxia oxidized the respiratory chain as reflected by a~31%docrease in lung tssue NADH RR as compared to that for normoxic lungs.LPS treatment alone or with hyperoxia had no significant ffect on lung tssue NADH RR as compared to that for normoxic or hyperoxic lungs,respectively.Thus,NADH RR serves as a quantitative marker of oxidative stress level in lung injury caused by two clinically important conditions:hyperoxia and LPS exposure.

Near-infrared spectroscopy muscle oximetry of patients with postural orthostatic tachycardia syndrome

Postural orthostatic tachycardia syndrome(POTS)is a disabling condition characterized by orthostatic intolerance with tachycardia in the absence of drop-in blood pressure.A custom-built near-infrared spectroscopy device(NIRS)is applied to monitor the muscle oxygenation,noninvasively in patients undergoing incremental head-up tilt table(HUT).Subjects(6 POTS patients and 6 healthy controls)underwent 30 mins of 70
on a HUT.The results showed a signicant difference in deoxyhemoglobin(Hb),change-in-oxygenation(△Oxy)and blood volume(△BV)between patients and healthy controls.However,oxyhemoglobin(HbO_(2))showed a significantly faster rate of change in the healthy controls during the first 10 mins of the tilt and during the recovery.This NIRS muscle oximetry tool provides quantitative measurements of blood oxygenation monitoring in diseases such as POTS.