Platelet Extracellular Regulated Protein Kinase 5 Is a Redox Switch and Triggers Maladaptive Platelet Responses and Myocardial Infarct Expansion.

BACKGROUND: Platelets have a pathophysiologic role in the ischemic microvascular environment of acute coronary syndromes. In comparison with platelet activation in normal healthy conditions, less attention is given to mechanisms of platelet activation in diseased states. Platelet function and mechanisms of activation in ischemic and reactive oxygen species-rich environments may not be the same as in normal healthy conditions. Extracellular regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase family member activated in hypoxic, reactive oxygen species-rich environments and in response to receptor-signaling mechanisms. Prior studies suggest a protective effect of ERK5 in endothelial and myocardial cells after ischemia. We present evidence that platelets express ERK5 and that platelet ERK5 has an adverse effect on platelet activation via selective receptor-dependent and receptor-independent reactive oxygen species-mediated mechanisms in ischemic myocardium. METHODS AND RESULTS: Using isolated human platelets and a mouse model of myocardial infarction (MI), we found that platelet ERK5 is activated post-MI and that platelet-specific ERK5(-/-) mice have less platelet activation, reduced MI size, and improved post-MI heart function. Furthermore, the expression of downstream ERK5-regulated proteins is reduced in ERK5(-/-) platelets post-MI. CONCLUSIONS: ERK5 functions as a platelet activator in ischemic conditions, and platelet ERK5 maintains the expression of some platelet proteins after MI, leading to infarct expansion. This demonstrates that platelet function in normal healthy conditions is different from platelet function in chronic ischemic and inflammatory conditions. Platelet ERK5 may be a target for acute therapeutic intervention in the thrombotic and inflammatory post-MI environment.

Ultraviolet light (UVB and UVA) induces the damage-responsive transcription factor CHOP/gadd153 in murine and human epidermis: evidence for a mechanism specific to intact skin.

C/EBP-homologous protein (CHOP)/gadd153 (or CHOP) is a transcription factor induced by endoplasmic reticulum (ER) stress. Forcible overexpression of CHOP causes apoptosis in keratinocytes in culture. Here, we asked whether CHOP might be increased in the skin after UVB (280-320 nm) exposure, thus implicating CHOP in sunburn cell (SBC) formation. SKH-1 hairless mice were exposed to a ultraviolet (UV) source (80 mJ per cm2; approximately 74% UVB, approximately 16% UVA), and skin biopsies examined by immunohistology and immunoprecipitation. Compared with non-irradiated epidermis, CHOP expression was significantly increased at 30 min, and reached maximal levels by 24 h. Similar increases in CHOP following UVB exposure were observed in human buttock skin. The time course of CHOP expression preceded SBC formation and another marker of apoptosis, caspase-3 cleavage. Intracellular CHOP accumulated mainly in cytoplasmic and perinuclear locations, with little remaining in the nucleus. To examine mechanisms, cultured keratinocytes were irradiated in vitro and examined by western blotting. Under conditions that eliminated ER stress because of cell handling, CHOP did not accumulate (and was in fact decreased) in the cells. Thus, induction of CHOP in keratinocytes requires factors present only in the native skin. Overall, the data suggest that CHOP participates in adaptive responses of the epidermis following UVB/UVA exposure in vivo.

Drug library screen to identify compounds that decrease secreted Abeta from a human cell line.

In order to discover compounds that may be useful in the prevention of Alzheimer disease, a drug library was screened for drugs that could decrease amyloid beta peptide (Abeta) accumulation secreted from a human CNS-derived cell line. Of the 2160 compounds subjected to a primary screen, 16, or 0.74%, reduced Abeta accumulation by at least 40%. Seven of these compounds were confirmed to be effective in a secondary screen using each compound at a dose of 5 microM. Three of these seven compounds were more effective than the others at reducing Abeta levels in a tertiary screen, and led to 68 to 85% reductions in total Abeta, at the 25 microM dosage. The effects of these three drugs on secreted Abeta40, Abeta42, and sAPPalpha were also determined. Thus, we have identified lead compounds that may be useful for subsequent studies to determine the mechanism of action of each drug, as well as for pre-clinical studies to determine whether each of these drugs is safe and effective in vivo.

Drug discovery: estrogen-related compounds in mouse models of Alzheimer's disease.

Despite promising epidemiological studies that showed a decreased incidence of Alzheimer disease (AD) in women who used hormone replacement therapy (HRT), the results of the recently released Women's Health Initiative Memory Study has dampened any enthusiasm for the use of HRT in women to prevent or delay the onset of AD. In this position paper, we review these data, along with our own--using estrogens in a transgenic mouse model of AD--and introduce our current working hypothesis and research.

Synthesis and biological evaluation of analogues of a novel inhibitor of beta-amyloid secretion.

A drug library of 17,200 compounds was screened to select small molecules that inhibit the secretion of amyloid beta peptide (Abeta), the major component of Alzheimer disease senile plaques, from a human neuronal cell line. Twenty-nine hits were validated that decreased Abeta secretion by >40% at 10 microM, for a 0.17% hit rate. A lead hit was selected for further study based on its activity and low cytotoxicity, and it was found to inhibit Abeta secretion through activation of the alpha-secretase pathway. Twenty-four commercially available and 53 synthesized analogues were analyzed for activity. Selected analogues were evaluated for biological stability by incubation with hepatoma cells and for transcellular permeability using Caco-2 cell monolayers. The analogue with the best permeability was evaluated in 2-month old amyloid precursor protein transgenic mice and found to acutely reduce cerebral Abeta levels by 40% after a single iv administration.

Sex specific gene regulation and expression QTLs in mouse macrophages from a strain intercross.

BACKGROUND: A powerful way to identify genes for complex traits it to combine genetic and genomic methods. Many trait quantitative trait loci (QTLs) for complex traits are sex specific, but the reason for this is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: RNA was prepared from bone marrow derived macrophages of 93 female and 114 male F(2) mice derived from a strain intercross between apoE-deficient mice on the AKR and DBA/2 genetic backgrounds, and was subjected to transcriptome profiling using microarrays. A high density genome scan was performed using a mouse SNP chip, and expression QTLs (eQTLs) were located for expressed transcripts. Using suggestive and significant LOD score cutoffs of 3.0 and 4.3, respectively, thousands of eQTLs in the female and male cohorts were identified. At the suggestive LOD threshold the majority of the eQTLs were trans eQTLs, mapping unlinked to the position of the gene. Cis eQTLs, which mapped to the location of the gene, had much higher LOD scores than trans eQTLs, indicating their more direct effect on gene expression. The majority of cis eQTLs were common to both males and females, but only approximately 1% of the trans eQTLs were shared by both sexes. At the significant LOD threshold, the majority of eQTLs were cis eQTLs, which were mostly sex-shared, while the trans eQTLs were overwhelmingly sex-specific. Pooling the male and female data, 31% of expressed transcripts were expressed at different levels in males vs. females after correction for multiple testing. CONCLUSIONS/SIGNIFICANCE: These studies demonstrate a large sex effect on gene expression and trans regulation, under conditions where male and female derived cells were cultured ex vivo and thus without the influence of endogenous sex steroids. These data suggest that eQTL data from male and female mice should be analyzed separately, as many effects, such as trans regulation are sex specific.

Transcriptome profile of macrophages from atherosclerosis-sensitive and atherosclerosis-resistant mice.

We performed a strain intercross between two apoE-deficient mouse strains with a large difference in lesion susceptibility and measured aortic root lesion area in 98 female F(2) progeny. Total RNA was prepared from bone marrow-derived macrophages, and RNA from the five mice with the smallest and largest lesions were used for microarray gene expression profiling. Remarkably, approximately 5% of the 12,288 expressed transcripts were differentially expressed in the atherosclerosis-susceptible and atherosclerosis-resistant bone marrow-derived macrophages (unadjusted p < 0.05), thus defining the transcriptome of macrophages associated with atherosclerosis susceptibility. Using more stringent criteria of twofold or greater change and p < 0.01, 116 and 70 transcripts were overexpressed in lesion-prone and lesion-resistant bone marrow-derived macrophages, respectively. Transcription factor binding site analysis identified two promoter elements that were found more often in the genes overexpressed in the large-lesion group, and one promoter element that was found more often in the small-lesion group. The combination of this expression profiling data with the genetic method of quantitative trait locus mapping should give powerful insights into the genes that affect atherosclerosis susceptibility in mice.