Increased circulating microparticles in streptozotocin-induced diabetes propagate inflammation contributing to microvascular dysfunction.

KEY POINTS: 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. 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. 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. Our observations derived from animal studies resembling conditions in diabetic patients, providing a mechanistic insight into MP-mediated pathogenesis of diabetes-associated multi-organ microvascular dysfunction. ABSTRACT: In various cardiovascular diseases, microparticles (MPs), the membrane-derived vesicles released during cell activation, are markedly increased in the circulation. These MPs have been recognized to play diverse roles in the regulation of cellular functions. However, current knowledge of MP function has been largely derived from in vitro studies. The precise impact of disease-induced MPs on vascular inflammation and disease progression remains obscure. In this study we investigated the biogenesis, profile and functional roles of circulating MPs using a streptozotocin-induced diabetic rat model with well-characterized microvascular functions. Our study revealed a >130-fold increase in MPs in the plasma of diabetic rats compared to normal rats. The majority of these MPs originate from platelets, leukocytes and endothelial cells (ECs), and circulate as aggregates. Diabetic MPs show greater externalized phosphatidylserine (PS) than normal MPs. When diabetic plasma or isolated diabetic MPs were perfused into normal microvessels or systemically transfused into normal rats, MPs immediately adhered to endothelium and subsequently mediated leukocyte adhesion. These microvessels then exhibited augmented permeability responses to inflammatory mediators, replicating the microvascular manifestations observed in diabetic rats. These effects were abrogated when MPs were removed from diabetic plasma or when diabetic MPs were pre-coated with a lipid-binding protein, annexin V, suggesting externalized PS to be key in mediating MP interactions with endothelium and leukocytes. Our study demonstrated that the elevated MPs in diabetic plasma are actively involved in the propagation of vascular inflammation through their adhesive surfaces, providing mechanistic insight into the pathogenesis of multi-organ vascular dysfunction that commonly occurs in diabetic patients.

Elevated Cytoplasmic Free Zinc and Increased Reactive Oxygen Species Generation in the Context of Brain Injury.

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.

Intracellular zinc elevation measured with a "calcium-specific" indicator during ischemia and reperfusion in rat hippocampus: a question on calcium overload.

Much of our current evidence concerning of the role of calcium (Ca2+) as a second messenger comes from its interaction with fluorescent probes; however, many Ca2+ probes also have a higher affinity for another divalent cation: zinc (Zn2+). In this study, using a selective Zn2+ probe (Newport Green), we investigated the accumulation of intracellular Zn2+ 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 Zn2+. Such observations compelled us to investigate the contribution of Zn2+ to the alleged intracellular Ca2+ overload occurring in ischemia and reperfusion. Using confocal fluorescent microscopy of Calcium Green-1, a widely used Ca2+ indicator, we detected increases in fluorescence intensity during OGD and reperfusion. However, application of a Zn2+ chelator, at the peak of the fluorescence elevation (interpreted as Ca2+ overload), resulted in a significant drop in intensity, suggesting that rising Zn2+ is the primary source of the increasing Calcium Green-1 fluorescence. Finally, staining with the cell viability indicator propidium iodide revealed that Zn2+ is responsible for the ischemic neuronal cell death, because Zn2+ chelation prevented cells from sustaining ischemic damage. Current cellular models of ischemic injury center on Ca2+-mediated excitotoxicity. Our results indicate that Zn2+ elevation contributes to conventionally recognized Ca2+ overload and also suggest that the role of Ca2+ in neurotoxicity described previously using Ca2+ probes may need to be re-examined to determine whether effect previously attributed to Ca2+ could, in part, be attributable to Zn2+.

Determining zinc with commonly used calcium and zinc fluorescent indicators, a question on calcium signals.

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 re-examined to determine if results previously attributed to Ca(2+) could, in part, be due to Zn(2+).

Measuring cell viability with membrane impermeable zinc fluorescent indicator.

Recent findings suggest that the accumulation of cytoplasmic zinc [Zn2+]i is a ubiquitous component in the cell death cascade. Zn2+ can be liberated from intracellular stores following oxidative stress and contribute to cell death processes. Here we show that the membrane/cell impermeable Zn2+ 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 Zn2+ 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 Zn2+ dyes, Fluozin-3 and Zinpyr-4, also stained cytosolic Zn2+ in PI labeled cells. Our data indicates that the application of a Zn2+ 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 Zn2+ correlates with neuronal death.

Exploiting the liberation of Zn²+ to measure cell viability.

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.

Zinc release from thapsigargin/IP3-sensitive stores in cultured cortical neurons.

BACKGROUND: Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP3-gated thapsigargin sensitive intracellular calcium (Ca2+) 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 (Zn2+) that has been reported to play a role in cell signaling. RESULTS: In primary cultured cortical cells (neurons) labeled with intracellular fluorescent Zn2+ indicators, we showed that intracellular regions of Zn2+ 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 Zn2+ chelator TPEN, indicating that the local Zn2+ fluorescence represented free Zn2+ localized to the ER in the basal condition. Blockade of the ER Ca2+ pump by thapsigargin produced a steady increase of intracellular Zn2+. Furthermore, we determined that the thapsigargin-induced Zn2+ increase was not dependent on extracellular Ca2+ or extracellular Zn2+, suggesting that it was of intracellular origin. The applications of caged IP3 or IP3-3Kinase inhibitor (to increase available IP3) produced a significant increase in intracellular Zn2+. CONCLUSIONS: Taken together, these results suggest that Zn2+ is sequestered into thapsigargin/IP3-sensitive stores and is released upon agonist stimulation.

UVB radiation induces an increase in intracellular zinc in human epidermal keratinocytes.

Ultraviolet (UV) radiation is known to cause oxidative stress, inflammation, DNA damage and apoptotic cell death; however, many details of these malign mechanism have yet to be elucidated. In this study, the exposure of adult human epidermal keratinocytes (HEKa) with UVB (>100 mJ/cm(2)) resulted in the significant increase of intracellular zinc that was released from its storage and was detected by fluorescent zinc indicators. Toxicity testing revealed that UVB-induced zinc release in HEKa is associated with HEKa cell death. Cells that showed elevated intracellular zinc fluorescence upon UVB exposure were also stained by propidium iodide (PI), a traditional viability indicator whose fluorescent signal is as a result of its intercalating with DNA fragments and is unaffected by zinc concentration, showing significant colocalization [Pearson's correlation coefficients r=0.956 (n=6)]. The cytotoxicity of zinc was also determined by an MTT assay after applying the exogenous zinc (ZnCl2) along with its ionophore pyrithione (20 microM) into HEKa culture medium. A significant reduction in cell viability as a function of both zinc concentration and exposure time was observed. The treatments of 1, 10 and 100 microM ZnCl2 with pyrithione demonstrated 2.3, 60 and 84% cell deaths, respectively (control 0.5%) after 30 min. ZnCl2 (100 microM) was also found to induce complete HEKa death after 1 h. Thus, the present study demonstrates that UVB irradiation-induced increased zinc is detrimental to HEKa viability, and zinc may be a necessary step in UVB-induced cell death signaling pathways.

UV irradiation-induced zinc dissociation from commercial zinc oxide sunscreen and its action in human epidermal keratinocytes.

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.

Rising zinc: a significant cause of ischemic neuronal death in the CA1 region of rat hippocampus.

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+]i accumulation 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. Neuronal injury from more severe (30 mins) OGD was not mitigated by the ion channel antagonists, whereas treatment with the Zn2+ chelator TPEN did afford significant protection from cell injury. These results indicate Zn2+-mediated damage to be of greater consequence than Ca2+-mediated damage, and collectively support the suggestion that Zn2+ accumulation may be a more significant causal factor of OGD/R-induced neuronal injury.