Laura F. Michael

Farnesoid X receptor agonist reduces serum asymmetric dimethylarginine levels through hepatic dimethylarginine dimethylaminohydrolase-1 gene regulation.

The farnesoid X receptor (FXR, NR1H4) is a bile acid-responsive nuclear receptor that plays critical roles in the transcriptional regulation genes involved in cholesterol, bile acid, triglyceride, and carbohydrate metabolism. By microarray analysis of hepatic genes from female Zucker diabetic fatty (ZDF) rats treated with the FXR agonist GW4064, we have identified dimethylarginine dimethylaminohydrolase-1 (DDAH1) as an FXR target gene. DDAH1 is a key catabolic enzyme of asymmetric dimethylarginine (ADMA), a major endogenous nitric-oxide synthase inhibitor. Sequence analysis of the DDAH1 gene reveals the presence of an FXR response element (FXRE) located 90 kb downstream of the transcription initiation site and within the first intron. Functional analysis of the putative FXRE demonstrated GW4064 dose-dependent transcriptional activation from the element, and we have demonstrated that the FXRE sequence binds the FXR-RXR heterodimer. In vivo administration of GW4064 to female ZDF rats promoted a dose-dependent and >6-fold increase in hepatic DDAH1 gene expression. The level of serum ADMA was reduced concomitantly. These findings provide a mechanism by which FXR may increase endothelium-derived nitric oxide levels through modulation of serum ADMA levels via direct regulation of hepatic DDAH1 gene expression. Thus, beneficial clinical outcomes of FXR agonist therapy may include prevention of atherosclerosis and improvement of the metabolic syndrome.

The pharmacology of LXR.

Liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. Two LXRs (LXRalpha and LXRbeta) were initially characterized as orphan members of this superfamily with disparate patterns of tissue expression. These two receptors later were recognized as sterol-responsive with the ability to directly bind several oxysterol metabolites. Many LXR target genes have been identified that implicate these receptors in a variety of physiological processes including cholesterol transport and metabolism, glucose metabolism, and inflammation. Synthetic LXR ligands have been designed with the potential to treat disorders such as atherosclerosis and diabetes. In this review, we describe the potential utility of LXR ligands in the treatment of disease.

Peroxisome Proliferator-Activated Receptor γ1 Expression Is Induced during Cyclic Adenosine Monophosphate-Stimulated Differentiation of Alveolar Type II Pneumonocytes1

The primary function of lung alveolar type II cells is to synthesize pulmonary surfactant, a lipoprotein enriched in dipalmitoylphosphatidylcholine. Because type II pneumonocytes are highly lipogenic, we considered the possible role of the adipogenic nuclear hormone receptor, peroxisome proliferator-activated receptorγ (PPARγ), in their differentiation from epithelial cell precursors. A degenerate PCR-screening strategy revealed that multiple PPARs, including PPARγ, are present in differentiated type II cells. A PCR-amplified PPARγ DNA-binding domain was used to isolate a full-length PPARγ1 complementary DNA clone from a rabbit type II cell complementary DNA library. Although another PPARγ isoform, PPARγ2, is known to be highly expressed in adipocytes, only PPARγ1 was detected in rabbit type II cells by use of RT-PCR and by library screening. Rabbit PPARγ1 has 90% nucleotide sequence identity and 95% amino acid identity to mouse PPARγ1. PPARγ1 messenger RNA was readily detected in total RNA isolated from ...

Concordant Changes of Plasma and Kidney MicroRNA in the Early Stages of Acute Kidney Injury: Time Course in a Mouse Model of Bilateral Renal Ischemia-Reperfusion

Background Acute kidney injury (AKI) is a syndrome characterized by the rapid loss of the kidney excretory function and is strongly associated with increased early and long-term patient morbidity and mortality. Early diagnosis of AKI is challenging; therefore we profiled plasma microRNA in an effort to identify potential diagnostic circulating markers of renal failure. The goal of the present study was to investigate the dynamic relationship of circulating and renal microRNA profiles within the first 24 hours after bilateral ischemia-reperfusion kidney injury in mice. Methodology/Principal Findings Bilateral renal ischemia was induced in C57Bl/6 mice (n = 10 per group) by clamping the renal pedicle for 27 min. Ischemia-reperfusion caused highly reproducible, progressive, concordant elevation of miR-714, miR-1188, miR-1897-3p, miR-877*, and miR-1224 in plasma and kidneys at 3, 6 and 24 hours after acute kidney injury compared to the sham-operated mice (n = 5). These dynamics correlated with histologic findings of kidney injury and with a conventional plasma marker of renal dysfunction (creatinine). Pathway analysis revealed close association between miR-1897-3p and Nucks1 gene expression, which putative downstream targets include genes linked to renal injury, inflammation and apoptosis. Conclusions/Significance Systematic profiling of renal and plasma microRNAs in the early stages of experimental AKI provides the first step in advancing circulating microRNAs to the level of promising novel biomarkers.

A 15-ketosterol is a liver X receptor ligand that suppresses sterol-responsive element binding protein-2 activity

Hypercholesterolemia is a major risk factor for coronary artery disease. Oxysterols are known to inhibit cholesterol biosynthesis and have been explored as potential antihypercholesterolemic agents. The ability of 3β-hydroxy-5α-cholest-8(14)-en-15-one (15-ketosterol) to lower non-HDL cholesterol has been demonstrated in rodent and primate models, but the mechanisms of action remain poorly understood. Here we show in a coactivator recruitment assay and cotransfection assays that the 15-ketosterol is a partial agonist for liver X receptor-α and -β (LXRα and LXRβ). The binding affinity for the LXRs was comparable to those of native oxysterols. In a macrophage cell line of human origin, the 15-ketosterol elevated ATP binding cassette transporter ABCA1 mRNA in a concentration-dependent fashion with a potency similar to those of other oxysterols. We further found that in human embryonic kidney HEK 293 cells, the 15-ketosterol suppressed sterol-responsive element binding protein processing activity and thus inhibited mRNA expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, LDL receptor, and PCSK9. Our data thus provide a molecular basis for the hypocholesterolemic activity of the 15-ketosterol and further suggest its potential antiatherosclerotic benefit as an LXR agonist.

Genome-wide screen for modulation of hepatic Apolipoprotein A-I (ApoA-I) secretion

Background: Increasing HDL-c through ApoA-I expression is hypothesized to reduce cardiovascular deaths significantly. Results: Genes that regulate hepatocyte ApoA-I secretion were identified using 21,789 siRNAs. Conclusion: Forty genes of interest were confirmed as regulators of ApoA-I production by hepatocytes. Significance: This study provides functional genomics-based data for exploring new mechanisms by which ApoA-I levels may be regulated. Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25–40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.

Molecular determinants of LXRα agonism

Liver X receptors (LXRs) are nuclear receptors that participate in the regulation of cholesterol, bile acid, and glucose metabolism. Despite the identification of the natural oxysterol and nonsteroidal ligands for LXRalpha, little is known about the structure of the LXRalpha ligand-binding domain (LBD). We constructed a three-dimensional (3D) homology model of the LBD of LXRalpha based on the crystal structure of the retinoic acid receptor gamma (RARgamma) and all-trans retinoic acid complex. We combined molecular modeling and classical structure-function techniques to define the interactions between the LBD and three structurally diverse ligands, 22(R)-hydroxycholesterol (22RHC), N-(2,2,2-trifluoro-ethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide (T0901317) and (3-[3-[(2-chloro-3-trifluoromethyl-benzyl)-(2,2-diphenyl-ethyl)-amino]-propoxy]-phenyl)-acetic acid (GW3965). Sixteen individual amino acid point mutations were made in the predicted ligand-binding cavity of the LBD, and each of these mutant receptors was assessed for their ability to be activated by these three ligands. The majority of individual mutations resulted in lack of activation by all three ligands. Two residues were identified that resulted in a significant increase in basal activity while retaining responsiveness to the ligands. Interestingly, a number of residues were identified that appear to be selective in their response to a particular ligand, indicating that these three ligands recognize distinct structural components within the ligand-binding cavity. These data, together with our docking study, enable us to identify the amino acids that coordinate the interaction of both steroidal and non-steroidal ligands in the ligand-binding pocket of LXRalpha.

Leukotriene B4 levels in human atherosclerotic plaques and abdominal aortic aneurysms.

Background Leukotriene B4 (LTB4) has been associated with the initiation and progression of atherosclerosis and abdominal aortic aneurysm (AAA) formation. However, associations of LTB4 levels with tissue characteristics and adverse clinical outcome of advanced atherosclerosis and AAA are scarcely studied. We hypothesized that LTB4 levels are associated with a vulnerable plaque phenotype and adverse clinical outcome. Furthermore, that LTB4 levels are associated with inflammatory AAA and adverse clinical outcome. Methods Atherosclerotic plaques and AAA specimens were selected from two independent databases for LTB4 measurements. Plaques were isolated during carotid endarterectomy from asymptomatic (n = 58) or symptomatic (n = 317) patients, classified prior to surgery. LTB4 levels were measured without prior lipid extraction and levels were corrected for protein content. LTB4 levels were related to plaque phenotype, baseline patient characteristics and clinical outcome within three years following surgery. Seven non-diseased mammary artery specimens served as controls. AAA specimens were isolated during open repair, classified as elective (n = 189), symptomatic (n = 29) or ruptured (n = 23). LTB4 levels were measured similar to the plaque measurements and were related to tissue characteristics, baseline patient characteristics and clinical outcome. Twenty-six non-diseased aortic specimens served as controls. Results LTB4 levels corrected for protein content were not significantly associated with histological characteristics specific for vulnerable plaques or inflammatory AAA as well as clinical presentation. Moreover, it could not predict secondary manifestations independently investigated in both databases. However, LTB4 levels were significantly lower in controls compared to plaque (p = 0.025) or AAA (p = 0.017). Conclusions LTB4 levels were not associated with a vulnerable plaque phenotype or inflammatory AAA or clinical presentation. This study does not provide supportive evidence for a role of LTB4 in atherosclerotic plaque destabilization or AAA expansion. However, these data should be interpreted with care, since LTB4 measurements were performed without prior lipid extractions.

Disentangling the genetic overlap between cholesterol and suicide risk

Suicide is major public health concern; one million individuals worldwide die by suicide each year of which there are many more attempts. Thus, it is imperative that robust and reliable indicators, or biomarkers, of suicide risk be identified so that individuals at risk can be identified and provided appropriate interventions as quickly as possible. Previous work has revealed a relationship between low levels of circulating cholesterol and suicide risk, implicating cholesterol level as one such potential biomarker, but the factors underlying this relationship remain unknown. In the present study, we applied a combination of bivariate polygenic and coefficient-of-relatedness analysis, followed by mediation analysis, in a large sample of Mexican-American individuals from extended pedigrees [N = 1897; 96 pedigrees (average size = 19.17 individuals, range = 2–189) 60% female; mean age = 42.58 years, range = 18–97 years, sd = 15.75 years] with no exclusion criteria for any given psychiatric disorder. We observed that total esterified cholesterol measured at the time of psychiatric assessment shared a significant genetic overlap with risk for suicide attempt (ρg = −0.64, p = 1.24 × 10−04). We also found that total unesterified cholesterol measured around 20 years prior to assessment varied as a function of genetic proximity to an affected individual (h2 = 0.21, se = 0.10, p = 8.73 × 10−04; βsuicide = −0.70, se = 0.25, p = 8.90 × 10−03). Finally, we found that the relationship between total unesterified cholesterol and suicide risk was significantly mediated by ABCA-1-specific cholesterol efflux capacity (βsuicide-efflux = −0.45, p = 0.039; βefflux-cholexterol = −0.34, p < 0.0001; βindirect = −0.15, p = 0.044). These findings suggest that the relatively well-delineated process of cholesterol metabolism and associated molecular pathways will be informative for understanding the neurobiological underpinnings of risk for suicide attempt.

Opportunities for Pharmacotherapy at the Intersection of Metabolic Syndrome and Hemostasis

Hemostatic balance is regulated by many factors that may become perturbed by cardio-metabolic abnormalities. Indeed, patients with multiple components of the metabolic syndrome have increased risk of atherosclerosis, hemostatic disorders and thrombotic events. This review focuses on the interrelationship between the metabolic syndrome components and thrombotic and thromboembolic events, the potential underlying mechanisms that lead to metabolic and hemostatic disorders in metabolic syndrome patients, the existing therapeutics aimed at reducing major cardiovascular events, and new therapeutic approaches to address pro-coagulant states.