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Papers by Christian Stork

The Journal of Physiology, 2018

Circulating microparticles (MPs) are elevated in many cardiovascular diseases and have been consi... more Circulating microparticles (MPs) are elevated in many cardiovascular diseases and have been considered as biomarkers of disease prognosis; however, current knowledge of MP functions has been mainly derived from in vitro studies and their precise impact on vascular inflammation and disease progression remains obscure. r Using a diabetic rat model, we identified a >130-fold increase in MPs in plasma of diabetic rats compared to normal rats, the majority of which circulated as aggregates, expressing multiple cell markers and largely externalized phosphatidylserine; vascular images illustrate MP biogenesis and their manifestations in microvessels of diabetic rats. r Using combined single microvessel perfusion and systemic cross-transfusion approaches, we delineated how diabetic MPs propagate inflammation in the vasculature and transform normal microvessels into an inflammatory phenotype observed in the microvessels of diabetic rats. r Our observations derived from animal studies resembling conditions in diabetic patients, providing a mechanistic insight into MP-mediated pathogenesis of diabetes-associated multiorgan microvascular dysfunction.

International journal of physiology, pathophysiology and pharmacology, 2016

Zinc (Zn(2+)) is required for numerous cellular functions. As such, the homeostasis and distribut... more Zinc (Zn(2+)) is required for numerous cellular functions. As such, the homeostasis and distribution of intracellular zinc can influence cellular metabolism and signaling. However, the exact distribution of free zinc within live cells remains elusive. Previously we showed the release of zinc from thapsigargin/IP3-sensitive endoplasmic reticulum (ER) storage in cortical neurons. In the present study, we investigated if other cellular organelles also contain free chelatable zinc and function as organelle storage for zinc. To identify free zinc within the organelles, live cells were co-stained with Zinpyr-1, a zinc fluorescent dye, and organelle-specific fluorescent dyes (MitoFluor Red 589: mitochondria; ER Tracker Red: endoplasmic reticulum; BODIPY TR ceramide: Golgi apparatus; Syto Red 64: nucleus). We examined organelles that represent potential storing sites for intracellular zinc. We showed that zinc fluorescence staining was co-localized with MitoFluor Red 589, ER Tracker Red, an...

STORK, CHRISTIAN J., Ph.D., March 2011, Molecular and Cellular Biology The Role of Zinc in Neuron... more STORK, CHRISTIAN J., Ph.D., March 2011, Molecular and Cellular Biology The Role of Zinc in Neuronal Injury and Death in an Oxygen-Glucose Deprivation Model of Ischemic Stroke (183 pp.) Director of Dissertation: Yang V. Li Stroke injury is a devastating medical condition in which less than 3% of victims can even meet the qualifications to receive the one FDA-approved treatment, tissue plasminogen activator (tPA). The dominant theory to describe how cerebral ischemia damages the brain is centered on excessive Ca influx into ischemic neurons where a resultant “Ca-overload” triggers neuron death and overall injury. Directed by this theory, dozens of potential stroke therapeutics designed to prevent Ca-overload have been developed and tested in clinical trials, showing mixed results. Much of the evidence that has provided experimental support for the theory of Ca-overload has been made where Ca was identified on the basis of detection with fluorescent Ca indicators. Zn also accumulates in neurons under ischemic conditions. In the current work results are presented that demonstrated Zn accumulation in ischemic neurons comprised the majority of the signal from a popular fluorescent Ca indicator, showing how the Zn transient in ischemia could be readily interpreted as support for Ca-overload. Moreover, the Zn accumulation with ischemia was shown to correlate significantly with neuronal injury. And, prevention of the Zn accumulation prevented the development of damage from simulated ischemic injury. 4 The source of the ischemically-evolved Zn was determined to be of intracellular origin, and results of the investigation to localize sites of intracellular Zn storage are presented in the present work. Under basal conditions, primary cultured cells from the rat cerebral cortex showed labile Zn within mitochondria, the Golgi apparatus, and the endoplasmic reticulum. Zn localized in the endoplasmic reticulum was found to be released by thapsigargin and IP3. To further investigate ischemic Zn liberation and characterize the specific effects of this event independent from that of Ca-overload, the respective effects of Ca and Zn were tested to compare their relative toxicity, and the relative neuroprotection afforded by treatments targeting each ion in ischemia. As shown in this study, the induced elevation of Zn resulted in significantly greater neuronal injury compared to the induced elevation of Ca. Further, preventing Zn accumulation under ischemic conditions was highly protective for both 10 min and 30 min insult; whereas preventing Ca influx showed a protective effect for up to only 10 min of simulated ischemia. The collective work presented here shows how Zn can be misconstrued as Ca under ischemic conditions. It demonstrated that the Zn-mediated damage was of greater consequence than the Ca-mediated damage. Collectively, these data support Zn accumulation to be a more significant causal factor of the neuronal injury and death resultant from simulated ischemia. These findings have implications that are relevant to ischemic stroke and to the development of therapeutic interventions for ischemic stroke. 5 Approved: _____________________________________________________________ Yang V. Li Associate Professor of Neuroscience

Acta neurochirurgica. Supplement, 2016

Intracellular zinc release and the generation of reactive oxygen species (ROS) have been reported... more Intracellular zinc release and the generation of reactive oxygen species (ROS) have been reported to be common ingredients in numerous toxic signaling mechanisms in neurons. A key source for intracellular zinc release is its liberation from metallothionein-III (MT-III). MT-III binds and regulates intracellular zinc levels under physiological conditions, but the zinc-binding thiols readily react with certain ROS and reactive nitrogen species (RNS) to result in intracellular zinc liberation. Liberated zinc induces ROS and RNS generation by multiple mechanisms, including the induction of mitochondrial ROS production, and also promotes ROS formation outside the mitochondria by interaction with the enzymes NADPH oxidase and 12-lipoxygenase. Of particular relevance to neuronal injury in the context of ischemia and prolonged seizures, the positive feedback cycle between ROS/RNS generation and increasing zinc liberation will be examined.

Metal Ion in Stroke, 2012

Zinc is abundant in the brain, and dysregulation of zinc ion homeostasis has been implicated in A... more Zinc is abundant in the brain, and dysregulation of zinc ion homeostasis has been implicated in Alzheimer’s disease pathology and neuronal death after brain ischemia. Many studies have established that cytosolic free zinc ion concentrations are much lower than measured total average cellular concentrations, suggesting a large capacity for intracellular zinc buffering. It is generally believed that protein binding constitutes a major source of intracellular zinc ion buffering. In this study, we explored the subcellular distribution of zinc-binding sites in neurons using synchrotron radiation X-ray fluorescence microscopy. We observed that nearly all the intracellular zinc in resting neurons is bound and that zinc-binding sites were uniformly distributed in neurons resulting in similar average zinc concentrations (≈300 μM) throughout the cell body including the nucleus, peri-nuclear region, and processes. However, sites of elevated zinc concentration were observed in resting neurons, and were prevalent throughout the neuron (including the cell body and processes) when neurons were loaded with zinc. These sites of elevated zinc could represent either zinc bound to proteins or zinc sequestered in subcellular organelles. Additional studies are needed to completely describe the neuronal zinc metallome and the function of zinc-binding proteins in buffering zinc loads.

Methods in molecular biology (Clifton, N.J.), 2011

Zn(2+) ions are a critical component of cellular machinery. The ion is required for the function ... more Zn(2+) ions are a critical component of cellular machinery. The ion is required for the function of many cell components crucial to survival, such as transcription factors, protein synthetic machinery, metabolic enzymes, hormone packaging, among other roles. In stark contrast to the cells' necessity for a sufficient Zn(2+) supply, an excess of free Zn(2+) is a situation that results in acute toxicity. Under normal conditions, free Zn(2+) levels in the cell are extremely low; whereas estimates of free Zn(2+) are in the subpicomolar range. In this way, the detection of elevated intracellular Zn(2+) can be exploited as a highly sensitive and specific signal to indicate neuronal dysfunction. We have shown that the relationship between intracellular Zn(2+) accumulation and the development of cellular injury/death to be ubiquitous among each of five tissue types tested; demonstrating the broad application and utility of the present technique.

Journal of Neuroscience, 2006

Much of our current evidence concerning of the role of calcium (Ca 2ϩ) as a second messenger come... more Much of our current evidence concerning of the role of calcium (Ca 2ϩ) as a second messenger comes from its interaction with fluorescent probes; however, many Ca 2ϩ probes also have a higher affinity for another divalent cation: zinc (Zn 2ϩ). In this study, using a selective Zn 2ϩ probe (Newport Green), we investigated the accumulation of intracellular Zn 2ϩ transients in acute rat hippocampal slices during ischemia, simulated by oxygen and glucose deprivation (OGD). Subsequent reperfusion with glucose-containing oxygenated medium resulted in an additional increase in intracellular Zn 2ϩ. Such observations compelled us to investigate the contribution of Zn 2ϩ to the alleged intracellular Ca 2ϩ overload occurring in ischemia and reperfusion. Using confocal fluorescent microscopy of Calcium Green-1, a widely used Ca 2ϩ indicator, we detected increases in fluorescence intensity during OGD and reperfusion. However, application of a Zn 2ϩ chelator, at the peak of the fluorescence elevation (interpreted as Ca 2ϩ overload), resulted in a significant drop in intensity, suggesting that rising Zn 2ϩ is the primary source of the increasing Calcium Green-1 fluorescence. Finally, staining with the cell viability indicator propidium iodide revealed that Zn 2ϩ is responsible for the ischemic neuronal cell death, because Zn 2ϩ chelation prevented cells from sustaining ischemic damage. Current cellular models of ischemic injury center on Ca 2ϩ-mediated excitotoxicity. Our results indicate that Zn 2ϩ elevation contributes to conventionally recognized Ca 2ϩ overload and also suggest that the role of Ca 2ϩ in neurotoxicity described previously using Ca 2ϩ probes may need to be reexamined to determine whether effect previously attributed to Ca 2ϩ could, in part, be attributable to Zn 2ϩ .

Journal of Neuroscience Methods, 2006

Recent findings suggest that the accumulation of cytoplasmic zinc [Zn 2+ ] i is a ubiquitous comp... more Recent findings suggest that the accumulation of cytoplasmic zinc [Zn 2+ ] i is a ubiquitous component in the cell death cascade. Zn 2+ can be liberated from intracellular stores following oxidative stress and contribute to cell death processes. Here we show that the membrane/cell impermeable Zn 2+ fluorescent indicator Newport Green (NG), which is non-toxic and impermeable to the membranes of healthy cells, can label unhealthy cells in tissue slices in a manner comparable to the traditional viability indicator propidium iodide (PI). Using confocal microscopy, we detected PI labeled nuclei colocalized with NG fluorescence. Our results indicate that cells which absorbed PI into their nuclei also allowed cell-impermeable Zn 2+ dye to penetrate their plasma membranes, subsequently exhibiting cytosolic and nuclear fluorescence. As in PI staining, we observed marked increases in NG fluorescence in damaged/dead cells of tissue slices. Two other cell impermeable fluorescent Zn 2+ dyes, Fluozin-3 and Zinpyr-4, also stained cytosolic Zn 2+ in PI labeled cells. Our data indicates that the application of a Zn 2+ fluorescent indicator is a fast, simple, non-toxic and reliable method for visualizing cell viability within in vitro tissue preparations. Accordingly, we demonstrate that intracellular accumulation of Zn 2+ correlates with neuronal death.

Journal of Molecular Signaling, 2010

Background: Changes in ionic concentration have a fundamental effect on numerous physiological pr... more Background: Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP 3-gated thapsigargin sensitive intracellular calcium (Ca 2+) storage provides a source of the ion for many cellular signaling events. Less is known about the dynamics of other intracellular ions. The present study investigated the intracellular source of zinc (Zn 2+) that has been reported to play a role in cell signaling. Results: In primary cultured cortical cells (neurons) labeled with intracellular fluorescent Zn 2+ indicators, we showed that intracellular regions of Zn 2+ staining co-localized with the endoplasmic reticulum (ER). The latter was identified with ER-tracker Red, a marker for ER. The colocalization was abolished upon exposure to the Zn 2+ chelator TPEN, indicating that the local Zn 2+ fluorescence represented free Zn 2+ localized to the ER in the basal condition. Blockade of the ER Ca 2+ pump by thapsigargin produced a steady increase of intracellular Zn 2+. Furthermore, we determined that the thapsigargin-induced Zn 2+ increase was not dependent on extracellular Ca 2+ or extracellular Zn 2+ , suggesting that it was of intracellular origin. The applications of caged IP 3 or IP 3-3Kinase inhibitor (to increase available IP 3) produced a significant increase in intracellular Zn 2+. Conclusions: Taken together, these results suggest that Zn 2+ is sequestered into thapsigargin/IP 3-sensitive stores and is released upon agonist stimulation.

Journal of Cosmetic Dermatology, 2010

Zinc oxide (ZnO) is an active ingredient in sunscreen owing to its properties of broadly filterin... more Zinc oxide (ZnO) is an active ingredient in sunscreen owing to its properties of broadly filtering the ultraviolet (UV) light spectrum and it is used to protect against the carcinogenic and photodamaging effects of solar radiation on the skin. This study investigated the dissociation of zinc (Zn 2+) from ZnO in commercial sunscreens under ultraviolet type B light (UVB) irradiation and assessed the cytotoxicity of Zn 2+ accumulation in human epidermal keratinocytes (HEK). Using Zn 2+ fluorescent microscopy, we observed a significant increase in Zn 2+ when ZnO sunscreens were irradiated by UVB light. The amount of Zn 2+ increase was dependent on both the irradiation intensity as well as on the ZnO concentration. A reduction in cell viability as a function of ZnO concentration was observed with cytotoxic assays. In a real-time cytotoxicity assay using propidium iodide, the treatment of UVB-irradiated ZnO sunscreen caused a late-or delayed-type cytotoxicity in HEK. The addition of a Zn 2+ chelator provided a protective effect against cellular death in all assays. Furthermore, Zn 2+ was found to induce the production of reactive oxygen species (ROS) in HEK. Our data suggest that UVB irradiation produces an increase in Zn 2+ dissociation in ZnO sunscreen and, consequently, the accumulation of free or labile Zn 2+ from sunscreen causes cytotoxicity and oxidative stress.

Journal of Cerebral Blood Flow & Metabolism, 2009

There is a rising intracellular Zn2+transient during neuronal ischemic hypoxia (oxygen-glucose de... more There is a rising intracellular Zn2+transient during neuronal ischemic hypoxia (oxygen-glucose deprivation and reoxygenation, OGD/R). The results of our recent works suggest that the OGD/R-induced Zn2+transient can readily be mistaken for a Ca2+transient. The aim of this study was to examine the respective functions of Zn2+and Ca2+in OGD/R-induced neuronal injury. We showed that [Zn2+]iaccumulation was consistently met with the induction of OGD/R-induced cell injury. Ca2+accumulation induced with high [K+] (to open voltage-gated calcium channels) or ionomycin (a Ca2+ionophore) caused a moderate neuronal injury that was reduced significantly by the application of the Zn2+chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). In comparison, Zn2+accumulation, induced with the Zn2+ionophore pyrithione, resulted in significantly greater injury. The application of nimodipine and MK801 was shown to attenuate neuronal injury only from a mild (10 mins) OGD insult. Neuron...

Cell Calcium, 2007

Don't we want to know whether zinc accumulation contributes to the calcium transient measured wit... more Don't we want to know whether zinc accumulation contributes to the calcium transient measured with these 'calcium' fluorophores?

Cell Calcium, 2006

Investigations into the roles of Ca 2+ and Zn 2+ in cell biology have been facilitated by the dev... more Investigations into the roles of Ca 2+ and Zn 2+ in cell biology have been facilitated by the development of sensitive fluorometric probes that have enabled the measurement of Ca 2+ or Zn 2+ in both extracellular and intracellular environments. It is critical to be aware of the specificity and relative selectivity of a probe for the targeted ion. Here, we investigated metal-ion responses by screening nominally Zn 2+-or Ca 2+-selective fluorophores in solutions containing various concentrations of Ca 2+ , as a potential interferent for Zn 2+ , or Zn 2+ , as a potential interferent for Ca 2+. The results suggested that Zn 2+-sensitive dyes were more specific for their targeted ion than dyes that targeted Ca 2+. Ca 2+-sensitive dyes such as Calcium Green-1, Fura-2, and Fluo-3 showed a wide range of interaction with Zn 2+ , even responding to Zn 2+ in the presence of high concentrations of Ca 2+. We demonstrate that these Ca 2+ indicators can effectively measure dynamic changes of cytosolic Zn 2+. Our results appeal for a new generation of Ca 2+ fluorophores that are more specific for Ca 2+ over Zn 2+. One implication of these results is that data obtained using Ca 2+-sensitive dyes may need to be reexamined to determine if results previously attributed to Ca 2+ could, in part, be due to Zn 2+ .