Blockade of the mineralocorticoid receptor improves markers of human endothelial cell dysfunction and hematological indices in a mouse model of sickle cell disease.

Increased endothelin-1 (ET-1) levels in patients with sickle cell disease (SCD) and transgenic mouse models of SCD contribute to disordered hematological, vascular, and inflammatory responses. Mineralocorticoid receptor (MR) activation by aldosterone, a critical component of the Renin-Angiotensin-Aldosterone-System, modulates inflammation and vascular reactivity, partly through increased ET-1 expression. However, the role of MR in SCD remains unclear. We hypothesized that MR blockade in transgenic SCD mice would reduce ET-1 levels, improve hematological parameters, and reduce inflammation. Berkeley SCD (BERK) mice, a model of severe SCD, were randomized to either sickle standard chow or chow containing the MR antagonist (MRA), eplerenone (156 mg/Kg), for 14 days. We found that MRA treatment reduced ET-1 plasma levels (p = .04), improved red cell density gradient profile (D50 ; p < .002), and increased mean corpuscular volume in both erythrocytes (p < .02) and reticulocytes (p < .024). MRA treatment also reduced the activity of the erythroid intermediate-conductance Ca2+ -activated K+ channel - KCa 3.1 (Gardos channel, KCNN4), reduced cardiac levels of mRNAs encoding ET-1, Tumor Necrosis Factor Receptor-1, and protein disulfide isomerase (PDI) (p < .01), and decreased plasma PDI and myeloperoxidase activity. Aldosterone (10-8 M for 24 h in vitro) also increased PDI mRNA levels (p < .01) and activity (p < .003) in EA.hy926 human endothelial cells, in a manner blocked by pre-incubation with the MRA canrenoic acid (1 µM; p < .001). Our results suggest a novel role for MR activation in SCD that may exacerbate SCD pathophysiology and clinical complications.

Inhibitory effects of Syzygium jambos extract on biomarkers of endothelial cell activation.

BACKGROUND: Disordered endothelial cell activation plays an important role in the pathophysiology of atherosclerosis, cancer, sepsis, viral infections, and inflammatory responses. There is interest in developing novel therapeutics to regulate endothelial cell function in atherothrombotic, metabolic, vascular, and hematological diseases. Extracts from leaves of the Syzygium jambos (L.) Alston (S. jambos) trees have been proposed to treat cardiovascular diseases and diabetes through unclear mechanisms. We investigated the effects of the S. jambos extract on biomarkers of endothelial dysfunction and immune responses in the human endothelial cell line, EA.hy926. METHODS: Leaves of S. jambos were collected, concocted and lyophilized. To study the effects of S. jambos on endothelial cell activation, we used the human endothelial cell line. IL-6 levels were measured using qPCR and ELISA. PDI activity was measured using Insulin Turbidity and Di-E-GSSG assays. CM-H2DCFDA was used to study ROS levels. Migration assay was used to study S. jambos effect on ex vivo human polymorphonuclear and human mononuclear cells. RESULTS: Our results show that incubation of EA.hy926 cells with ET-1 led to a 6.5 ± 1.6 fold increase in IL-6 expression by qPCR, an event that was blocked by S. jambos. Also, we observed that ET-1 increased extracellular protein disulfide isomerase (PDI) activity that was likewise dose-dependently blocked by S. jambos (IC50 = 14 µg/mL). Consistent with these observations, ET-1 stimulated ex vivo human polymorphonuclear and mononuclear cell migration that also was dose-dependently blocked by S. jambos. In addition, ET-1 stimulation led to significant increases in ROS production that were sensitive to S. jambos. CONCLUSION: Our results suggest that the S. jambos extract represents a novel cardiovascular protective pharmacological approach to regulate endothelial cell activation, IL-6 expression, and immune-cell responses.

Aldosterone's rapid, nongenomic effects are mediated by striatin: a modulator of aldosterone's effect on estrogen action.

The cellular responses to steroids are mediated by 2 general mechanisms: genomic and rapid/nongenomic effects. Identification of the mechanisms underlying aldosterone (ALDO)'s rapid vs their genomic actions is difficult to study, and these mechanisms are not clearly understood. Recent data suggest that striatin is a mediator of nongenomic effects of estrogen. We explored the hypothesis that striatin is an intermediary of the rapid/nongenomic effects of ALDO and that striatin serves as a novel link between the actions of the mineralocorticoid and estrogen receptors. In human and mouse endothelial cells, ALDO promoted an increase in phosphorylated extracellular signal-regulated protein kinases 1/2 (pERK) that peaked at 15 minutes. In addition, we found that striatin is a critical intermediary in this process, because reducing striatin levels with small interfering RNA (siRNA) technology prevented the rise in pERK levels. In contrast, reducing striatin did not significantly affect 2 well-characterized genomic responses to ALDO. Down-regulation of striatin with siRNA produced similar effects on estrogen's actions, reducing nongenomic, but not some genomic, actions. ALDO, but not estrogen, increased striatin levels. When endothelial cells were pretreated with ALDO, the rapid/nongenomic response to estrogen on phosphorylated endothelial nitric oxide synthase (peNOS) was enhanced and accelerated significantly. Importantly, pretreatment with estrogen did not enhance ALDO's nongenomic response on pERK. In conclusion, our results indicate that striatin is a novel mediator for both ALDO's and estrogen's rapid and nongenomic mechanisms of action on pERK and phosphorylated eNOS, respectively, thereby suggesting a unique level of interactions between the mineralocorticoid receptor and the estrogen receptor in the cardiovascular system.

Laser microdissection sample preparation for RNA analyses.

Gene expression analysis provides an insight into the unique and defining biomolecular characteristics of a given cell type. However, heterogeneous cellular compositions hinder gene analysis studies from most tissue samples. The laser microdissection (LMD) technique allows for the unambiguous isolation of a desired cell population. However, preserving RNA integrity can be challenging because of the deliberately limited amount of starting material, sometimes as little as a single cell. General laboratory procedures for reducing ribonuclease (RNase) activity, both in reagents and in the laboratory environment, are required for successful downstream RNA isolation and quantitation. Quality RNA can be extracted from sections made from flash-frozen and paraffin-embedded tissue. The standard histological stains such as hematoxylin and eosin (H&E), or toluidine blue, can provide visualization of the cells of interest. Following LMD, validation of RNA integrity should precede downstream analysis.

Using the multifaceted Rasch model to improve the TAT/PSE measure of need for achievement.

We used the Rasch (1980) model to develop new pictures for the Thematic Apperception Test (C. D. Morgan & Murray, 1938; McClelland, Atkinson, Clark, & Lowell, 1953) or picture story exercise to measure need for achievement (nAch). In Experiments 1 and 2, we analyzed stories to assess the difficulty level of a total of 8 pictures using the multifaceted Rasch model with picture difficulty, story probe difficulty, and participant ability as facets with a partial credit model (FACETS; Linacre, 2005). A total of 6 pictures were retained and 4 new ones added for Experiment 3 in which 201 participants wrote 6 stories to a random set of the 10 pictures. FACETS analysis revealed improved person separation reliability. In Experiment 4, 206 participants wrote 1 story to the Studying picture either before or after filling out a battery of achievement-related questionnaires. The 2 experimental groups did not differ in the amount of nAch in their stories. The coder facet was demonstrated with 2 independent coders using the revised coding system for nAch.

Stem cell proliferative history in tissue revealed by temporal halogenated thymidine analog discrimination.

Detection of proliferating cells based on bromodeoxyuridine (BrdU) incorporation and determination of phenotype by immunofluorescence labeling are standard approaches for studying stem and progenitor cell populations in developing and adult tissue as well as in histopathology studies. We describe incorporation of different halogenated thymidine analogs for temporal discrimination of cell cycle in the rat. With equimolar delivery, these analogs are suitable for quantitative histological studies including assessment of the regulation of proliferation, clonal analysis and simultaneous profiling of cell phenotype relative to proliferative history.