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Objectives There has been limited success in achieving integration of patient-reported outcomes (PROs) in clinical trials. We describe how stakeholders envision a solution to this challenge. Methods Stakeholders from academia, industry, non-profits, insurers, clinicians, and the Food and Drug Administration convened at a Think Tank meeting funded by the Duke Clinical Research Institute to discuss the challenges of incorporating PROs into clinical trials and how to address those challenges. Using examples from cardiovascular trials, this article describes a potential path forward with a focus on applications in the United States. Results Think Tank members identified one key challenge: a common understanding of the level of evidence that is necessary to support patient-reported outcome measures (PROMs) in trials. Think Tank participants discussed the possibility of creating general evidentiary standards depending upon contextual factors, but such guidelines could not be feasibly developed because many contextual factors are at play. The attendees posited that a more informative approach to PROM evidentiary standards would be to develop validity arguments akin to courtroom briefs, which would emphasize a compelling rationale (interpretation/use argument) to support a PROM within a specific context. Participants envisioned a future in which validity arguments would be publicly available via a repository, which would be indexed by contextual factors, clinical populations, and types of claims. Conclusions A publicly available repository would help stakeholders better understand what a community believes constitutes compelling support for a specific PROM in a trial. Our proposed strategy is expected to facilitate the incorporation of PROMs into cardiovascular clinical trials and trials in general.

In this qualitative, experiential study, 300 members of the database of WomenHeart: The National Coalition for Women With Heart Disease completed an online survey about hypertension diagnosis and treatment, patient education, and perceptions of this and related conditions. Based on the findings from the survey, characteristics of the prototypical journey were identified. To the extent to which the surveyed WomenHeart members represent typical experiences, this survey provides insights into common hurdles women encounter in their journey throughout the hypertension diagnosis and treatment process. Results of this study suggest the need for a patient-centric approach to hypertension management and to implement programs with the intention of comprehensively assessing and meeting individual needs. Further studies would be of value to expand on patients' journeys in the management of hypertension and identify the types of products, services, and programming that most effectively support treatment adherence and achievement of optimal blood pressure control.

This study examined progression to type 2 diabetes and compared healthcare utilization and costs among patients with pre-diabetes, with or without comorbid hypertension.This study drew from a large national claims database (2003-2008). Patients were ≥18 years of age with a medical claim or lab value indicating the presence of pre-diabetes. The index date was the first pre-diabetes diagnosis (ICD-9 codes 790.21, 790.22, 790.29) or qualifying lab value of fasting plasma glucose or impaired glucose intolerance. All patients had ≥12-month data pre- and post- index date. Multivariate analysis was conducted to identify risk factors affecting progression to type 2 diabetes, and to estimate the impact of hypertension status and diabetes progression on healthcare utilization and cost.144,410 patients met study criteria, with an average follow-up of 802 (SD 344) days. Among participants, 30.7% progressed to diabetes, with a mean 288 (SD 340) days from pre-diabetes identification to diabetes diagnosis. Compared with patients who did not progress, the total adjusted medical costs for patients who developed diabetes increased by $1429 in 1 year, $2451 in 2 years, and $3621 in 3 years (p 0.001). Patients with concomitant hypertension were significantly more likely to progress to type 2 diabetes, and had higher total medical costs compared to patients without hypertension ($476 higher in 1 year, $949 in 2 years, $1378 in 3 years).Patients with pre-diabetes who progressed to type 2 diabetes had higher healthcare utilization and costs compared with patients who did not. The presence of hypertension substantially increased costs and was associated with higher likelihood of diabetes progression. Blood pressure, lifestyle intervention, body mass index, and other factors cannot be examined due to the limitations of the data. Results may not be generalizable to patients with insurance other than commercial or Medicare.

To evaluate the impact of angiotensin receptor blocker (ARBs)/dihydropyridine calcium channel blockers (CCBs) single-pill combination (SPC) on adherence to antihypertensive treatment in comparison to free combination of ARBs and CCBs.A retrospective data analysis was performed using pharmacy claims data from a national pharmacy benefit management company. The study included patients who were newly initiated on ARB/CCB treatment between 01/01/2007 and 08/31/2008, aged ≥ 18 years, and continuously enrolled in the same health plan for 12 months prior to and 13 months after starting ARB/CCB treatment. Outcome variables were persistence, defined as time to discontinuation of therapy, and adherence, defined as proportion of days covered (PDC) ≥ 0.80. Propensity score weighting was used to balance the characteristics of the two groups.The final sample contained 2312 patients in the free-combination group and 2213 patients in the SPC group. Patients in the SPC group and the free-combination group were different in age, gender, type of insurance, history of antihypertensive therapy and co-morbidities. These differences were largely normalized after propensity score adjustment. Multivariate logistic model regression showed that patients in the SPC group had a 90% greater odds of being adherent to index therapy compared to patients in the free-combination group (odds ratio [OR] 1.90, 95% confidence interval [CI] 1.75-2.08, p0.001). A Cox proportional hazards model showed that patients in the SPC group were less likely to discontinue ARB/CCB SPC therapy compared to patients in the free-combination group (hazard ratio [HR] 0.66, 95% CI 0.63-0.70, p0.001). In both models, higher copayment (copayment $50 and above) was associated with worse persistence and adherence in comparison to patients who had a lower copayment ($0-$5): HR = 1.23, p0.001 and OR = 0.67, p0.001.Patients using SPC ARB/CCB therapy were more likely to be persistent and adherent to treatment compared to patients taking free-combination therapy.

To explore the relative contributions of baseline systolic blood pressure (SBP) and diastolic blood pressure (DBP) and lipoproteins on the risk of recurrent stroke or first major cardiovascular event (MCVE) and their potential impact on the benefit of statin treatment.The SPARCL trial randomized 4731 patients with recent stroke or transient ischemic attack (TIA) and no known coronary heart disease and LDL-C between 100 and 190 mg/dL to either atorvastatin 80 mg/d or placebo. Baseline assessment included SBP, DBP and measurements of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), and triglyceride levels. After 4.9 years of follow-up, there were 575 primary end points (fatal and nonfatal stroke), including 491 ischemic strokes, and 740 MCVEs (stroke plus myocardial infarction and vascular death). Cox regression models analysis showed a trend (P0.05 and P0.10) for higher SBP but not DBP to be associated with an outcome stroke with only SBP associated with MCVE. Only baseline low HDL-C was associated with an outcome stroke. Baseline HDL-C, triglycerides, and LDL/HDL ratio were each associated with MCVEs. There were no interactions between any of these baseline variables and the effect of treatment on outcome strokes.In patients with recent stroke or TIA and no coronary heart disease, only lower baseline HDL-C predicted the risk of recurrent stroke with HDL-C, triglycerides, and LDL/HDL ratio associated with MCVE. Atorvastatin treatment was similarly effective regardless of baseline lipoprotein levels.

In clinical practice, persistence with statin therapy is poor. While little is known about relative persistence to specific statins, previous studies have observed greater persistence in patients who achieve greater degrees of lipid lowering. Identification of statin therapies which improve patient persistence has the potential to improve the quality of patient care and clinical outcomes. Therefore, we assessed patient persistence with atorvastatin and simvastatin in primary and secondary prevention patients enrolled in managed care.New statin users agedor =18 years, both with and without prior cardiovascular (CV) events within the 12 month pre-treatment period, were identified from a large national database of managed care patients. Patients initiated atorvastatin or simvastatin therapy from January 1, 2003 to September 30, 2005 and were continuously enrolled in a covered plan for at least 12 months before and after initiation of statin therapy. Subanalyses of patientsor =65 years were also conducted. Measures of interest included demographic and clinical characteristics of the study samples and persistence of statin utilization over the 1-year follow-up period. Persistence was defined as the number of days a patient remained on treatment in the first year following their index date, measured from the date of first fill to study end or the date of discontinuation.A total of 129 764 atorvastatin users and 45 558 simvastatin users without prior CV events were included in the study. For those patients with prior CV events, a total of 6888 atorvastatin users and 4443 simvastatin users were included in the study. Median persistence in patients without prior CV events was 50 days longer for patients initiating therapy with atorvastatin than simvastatin (207 vs. 157 days, p0.0001) and after adjusting for confounding factors, those treated with atorvastatin were 15% less likely to discontinue therapy during the first year than those treated with simvastatin (HR=0.85; 95% CI 0.84, 0.86; p0.001). In secondary prevention patients median persistence was 85 days longer in atorvastatin patients than simvastatin patients (266 vs. 181 days, p0.0001) and atorvastatin patients were 22% less likely to discontinue therapy (HR=0.78; 95% CI 0.75, 0.82; p0.001). Persistence was worse in the elderly patients, but the relative difference between atorvastatin and simvastatin was similar to the overall patient population.In patients with and without prior CV disease, persistence is generally poor, even worse in the elderly, but significantly better for atorvastatin patients than simvastatin patients (p0.001). Further studies are required to determine whether this is due to differences in cost, effectiveness, side-effects, or other attributes of the statins.Differences in persistence could be, in part, due to unmeasured confounders although all available variables were adjusted in multivariate analyses. Additionally, the claims database lacks some clinical data such as lipid levels, limiting assessments of statin efficacy, and does not include any reasons for discontinuation of therapy.

Mineralocorticoid receptor (MR) antagonism has proven to effectively attenuate the pathophysiological effects of aldosterone in clinical and experimental settings of hypertension and heart failure. MR activates transcription of target genes upon aldosterone binding, and eplerenone selectively binds to MR and blocks aldosterone- mediated activation. In this review, we summarize the preclinical and clinical evidence supporting the beneficial effects of eplerenone (INSPRATM), a selective aldosterone blocker, in the treatment of hypertension and heart failure. We also review the current status in understanding the molecular mechanisms of action of the MR and its ligand. In addition, we compare the effects of eplerenone and spironolactone, a nonselective aldosterone blocker, on the transcriptional activity of MR and provide a molecular explanation for the improved side-effect profile of eplerenone compared with spironolactone.

Background— In heart failure (HF), aldosterone has been implicated in the formation of reactive interstitial fibrosis, a maladaptation that contributes to left ventricular (LV) remodeling. Eplerenone is a novel selective aldosterone blocker. The present study examined the effects of long-term monotherapy with eplerenone on the progression of LV dysfunction and remodeling in dogs with chronic HF. Methods and Results— HF was produced in 14 dogs by intracoronary microembolizations that were discontinued when LV ejection fraction (EF) was between 30% and 40%. Two weeks after the last embolization, dogs were randomized to 3 months of oral therapy with eplerenone (10 mg/kg twice daily, n=7) or no therapy at all (control, n=7). Hemodynamic measurements were made just before randomization and were repeated at the end of 3 months of therapy. In control dogs, LV end-diastolic and end-systolic volume increased significantly (62±4 versus 68±4 mL, P P P Conclusions— Our results indicate that long-term therapy with eplerenone prevents progressive LV dysfunction and attenuates LV remodeling in dogs with chronic HF.

SummaryTo understand the role of the factor X (fX) activation peptide (AP), a deletion mutagenesis approach was employed. Two single-chain, variant enzymes were generated in which 41 residues were deleted from the AP: fXdes-137-183 and fXdes-137-183;N191A, which lacks a carbohydrate moiety at Asn191 due to an alanine substitution. Deletion of the fX AP did not impact fXa catalytic activity. Activation of the variant zymogens, however, was altered. Neither mutant enzyme was activated by the fX coagulant protein from Russell’s viper venom (RVV-X1). Activation by factor VIIa (fVIIa) and fVIIa in the presence of cofactor, lipidated tissue factor (TF), occurred at an accelerated rate for both variants as compared to wild-type fX (WTfX). Similar to fVII, the mutants auto-activated in a cofactor-independent manner, which was characterized by a lag period and accelerated dose-dependently by plasma fXa (kcat/Km, 0.046 ± 0.004 µM−1s−1). Both mutants were also found to be activated by fVIIa (0.31 ± 0.03 µM−1s−1), fIXa (0.30 ± 0.03 µM−1 s−1), and thrombin (0.00078 ± 0.00015 µM−1s−1). In all cases, the rate of activation was faster for fXdes-137-183;N191A as compared to fXdes-137-183. We propose that the fX AP and Asn191 carbohydrate serve primarily as negative autoregulation mechanisms to prevent spurious activation of fX and secondarily in cofactor dependence and activator specificity.The abbreviations used are: TF, human tissue factor; AP, activation peptide; HEPES, (N-[2-Hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid]); PEG, polyethylene glycol 8000; BSA, bovine serum albumin; Tris, (Tris[hydroxymethyl] aminomethane); EDTA, (Ethylenedinitrilo)-tetraacetic acid; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; RVV-X, X activating protein from Russell’s viper venom; gla, γ-carboxylated glutamic acid; WTfX, wild type factor X; PC:PS, phosphatidylcholine:phosphatidylserine; fXa, fIXa, f Va, fVIIa, and fVIIIa, activated coagulation factor X, IX, V, VII, and VIII; ATIII, antithrombin; TFPI, tissue factor pathway inhibitor.

Based on homology, amino acids 326–336 (143–154 in chymotrypsin numbering) of factor X (fX) comprise a flexible surface loop, which is susceptible to self-proteolysis and influences substrate catalysis. To investigate the role of this autolysis loop in fX function, a recombinant variant with a new site for asparagine-linked glycosylation has been produced by changing glutamine 333 to asparagine. Q333N fX is activated normally by factor VIIa and tissue factor, factors IXa and VIIIa, and Russell's viper venom. Proteolysis of the loop is prevented by the mutation. Reactivity of the free enzyme toward substrates and inhibitors is attenuated 4–20-fold; relative to wild type fXa, Spectrozyme XaTMhydrolysis is 25%, inhibition by antithrombin III and the tissue factor pathway inhibitor is ∼20%, and prothrombin activation in the absence of the cofactor Va is only 5%. Surprisingly, activities of the variant and wild type enzymes are equivalent when part of the prothrombinase complex. N-Glycanase cleaves the new oligosaccharide from Q333N fXa leaving aspartic acid. Q333D fXa is ∼1.6-fold more reactive with Spectrozyme XaTM, antithrombin III and tissue factor pathway inhibitor, and prothrombin than its glycosylated counterpart, Q333N fXa, but still quite abnormal relative to wild type fXa. Like Q333N fXa, Q333D fXa is fully functional as part of the prothrombinase complex. We conclude that Gln-333 is geographically close to a site of proteolytic degradation but not to activator, cofactor, or membrane binding sites. Mutation of Gln-333 impairs catalytic function, but given normal prothrombin activation by the complexed enzyme, the importance of Gln-333 for catalysis is not manifest in the prothrombinase assembly, suggesting a conformational change in complexed fXa.

Purified murine toxin (Ymt) of Yersinia pestis is highly toxic for mice and rats but less active in other animals such as guinea pigs, rabbits, dogs and monkeys. This suggested that Ymt contributes to the very low infectious dose of Y. pestis in mice. The gene encoding Ymt (ymt) is localised on the 100-kb plasmid pFra, which is unique for Y. pestis. Sequence analysis revealed that Ymt showed homology to proteins of the phospholipase D (PLD) superfamily of proteins. Y. pestis strains expressing Ymt possessed PLD activity whereas strains carrying deletions in the ymt gene showed no detectable PLD activity. Western blot analysis showed that Ymt was associated with bacteria under normal growth conditions, and immunogold EM revealed that Ymt was mainly localised in the bacterial cytoplasm. Ymt was purified to homogeneity, and the purified toxin showed a dose-dependent PLD activity. Substitution of amino acids in the PLD consensus motif of Ymt essentially abolished the enzymatic activity and these variants of the toxin were no longer toxic to mice. Interestingly, an in-frame deletion mutant of ymt in the Y pestis strain KIM was not significantly attenuated for mouse virulence. Together with the observation that expression of Ymt was higher at room temperature compared to 37 degrees C this prompted us to investigate the role of Ymt in the flea vector. Fleas were infected with isogenic ymt+ or ymt- mutant strains of Y. pestis. Preliminary results suggest that Ymt is important for survival of Y. pestis in the flea and thereby also for the flea-borne route of infection.

The bacterial pathogens of the genus Yersinia deliver several virulence factors into target cells using a type III secretion system. We demonstrate that Yersinia protein kinase A (YpkA), an essential bacterial virulence factor, is produced as an inactive serine/threonine kinase. The inactive kinase is activated within the host cell by a cytosolic eukaryotic activator. Using biochemical purification techniques, we demonstrate that actin is a cellular activator of YpkA. This stimulation of YpkA kinase activity by actin depends on the presence of the C-terminal twenty amino acids of YpkA, because deletion of these 20 aa not only obliterates YpkA activity, but it also destroys the interaction between YpkA and actin. Activated YpkA functions within cultured epithelial cells to disrupt the actin cytoskeleton. The disruption of the actin cytoskeleton by YpkA would be expected to inhibit macrophage function and phagocytosis of Yersinia .

The steroid aldosterone plays a major role in the maintenance of total body sodium homeostasis and also contributes to cardiovascular pathophysiology by mediating cardiac hypertrophy and fibrosis. In addition to classical adrenal production of aldosterone, endogenous tissue production of aldosterone has been observed in various organs; aldosterone biosynthesis in cardiac tissues, however, remains highly controversial. The current study provides a comprehensive evaluation of steroid hormone biosynthethic capabilities in multiple tissues from two distinct rat strains under unstimulated and stimulated conditions. Panels of tissues from Wistar and Sprague-Dawley rats were probed for 11 beta-hydroxylase (P45011beta) and aldosterone synthase (P450aldo) by reverse transcriptase-polymerase chain reaction (RT-PCR). Under unstimulated conditions, cardiac P45011beta and P450aldo were detected only in Wistar rats. Angiotensin II (100 microg/day) stimulated myocardial expression of both enzymes in both strains. Cerebral cortex and mesenteric artery message levels in both strains was reduced by angiotensin II. These data demonstrate the potential for local steroid synthesis in vascular, cardiac, renal, and neuronal tissues, and that biosynthesis of non-adrenal aldosterone may be differentially regulated between strains. This variability may thus resolve in part or whole the current controversy over the existence of non-adrenal steroidogenic systems.

Herein we describe a recombinant factor X (fX) with a single substitution at position 347 (fXR347N). Activated fXR347N had a reduced affinity for factor Va (fVa), although the catalytic impact of fVa binding remained intact. The mutation was selective as demonstrated by normal activation and inhibition, except in the presence of subsaturating heparin where the rate of inhibition by antithrombin III (ATIII) was 15% of normal. The reactivity of fXaR347N toward prothrombin was equivalent to wild-type fXa (fXaWT) in the absence of fVa and phospholipid. Addition (without phospholipid) of fVa dramatically increased the catalytic efficiency of fXaWT toward prothrombin but had a negligible effect on fXaR347N. On addition of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1) vesicles, fXaR347Ndisplayed an increased catalytic activity in response to fVa, but the apparent affinity for fVa on the phospholipid surface was 5-20-fold lower than that of fXaWT. On an activated platelet surface, however, fXaWT and fXaR347N activated prothrombin similarly. In a competitive binding assay that measures the displacement of radiolabeled fXa from fVa on a phospholipid surface, fXaR347N was approximately 10-fold less effective than fXaWT. Substitution of fXa at position 347 selectively attenuates the interaction between fXa and fVa without affecting its catalytic activity.

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X) 4 D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the k cat for hydrolysis by a factor of 10 5 , suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32 P-labeled protein could be trapped when the enzyme was incubated with 32 P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.

Blood coagulation factor X plays a pivotal role in the clotting cascade. When administered intravenously to mice, the majority of activated factor X (factor Xa) binds to α2-macroglobulin (α2M) and is rapidly cleared from the circulation into liver. We show here that the low-density lipoprotein receptor-related protein (LRP) is responsible for factor Xa catabolism in vivo. Mice overexpressing a 39-kD receptor-associated protein that binds to LRP and inhibits its ligand binding activity displayed dramatically prolonged plasma clearance of 125I-factor Xa. Preadministration of α2M-proteinase complexes (α2M*) also diminished the plasma clearance of125I-factor Xa in a dose-dependent fashion. The clearance of preformed complexes of 125I-factor Xa and α2M was similar to that of 125I-factor Xa alone and was also inhibited by mice overexpressing a 39-kD receptor-associated protein. These results thus suggest that, in vivo, factor Xa is metabolized via LRP after complex formation with α2M.

A molecular defect in factor X (fX) results from a point mutation that causes glycine substitution for γ-carboxylated glutamic acid at position 7. The variant (fXSt. Louis II) and wild type (fXWT) proteins were produced in a mammalian expression system and characterized. fXSt. Louis II has

Acquired inhibitors of factor V are rare causes of clinical bleeding, whose severity ranges from mild to life-threatening. Optimal treatment of patients with factor V inhibitors is uncertain. We report on our successful treatment approach in a patient with spontaneous, life-threatening intracranial bleeding caused by a factor V inhibitor. The patient deteriorated after initial treatment with fresh-frozen plasma and platelet transfusions. He was subsequently treated with a combination of plasma exchange and chemotherapy, which led to complete recovery. Our experience suggests that plasma exchange may be life-saving in cases of severe bleeding caused by factor V inhibitors. The use of plasmapheresis in conjunction with chemotherapy is an efficacious and well-tolerated treatment and should be considered in patients with factor V inhibitors.

Transmission by flea bite is a relatively recent adaptation that distinguishesYersinia pestis, the plague bacillus, from closely related enteric bacteria. Here, a plasmid-encoded phospholipase D (PLD), previously characterized as Yersinia murine toxin (Ymt), was shown to be required for survival ofY. pestisin the midgut of its principal vector, the rat fleaXenopsylla cheopis. Intracellular PLD activity appeared to protectY. pestisfrom a cytotoxic digestion product of blood plasma in the flea gut. By enabling colonization of the flea midgut, acquisition of this PLD may have precipitated the transition ofY. pestisto obligate arthropod-borne transmission.

Background and Purpose— The SPARCL trial showed that atorvastatin 80 mg/d reduces the risk of stroke and other cardiovascular events in patients with recent stroke or transient ischemic attack (TIA). We tested the hypothesis that the benefit of treatment varies according to index event stroke subtype. Methods— Subjects with stroke or TIA without known coronary heart disease were randomized to atorvastatin 80 mg/d or placebo. The SPARCL primary end point was fatal or nonfatal stroke. Secondary end points included major cardiovascular events (MCVE; stroke plus major coronary events). Cox regression models testing for an interaction with treatment assignment were used to explore potential differences in efficacy based on stroke subtype. Results— For subjects randomized to atorvastatin versus placebo, a primary end point occurred in 13.1% versus 18.6% of those classified as having large vessel disease (LVD, 15.8% of 4,731 participants), in 13.1% versus 15.5% of those with small vessel disease (SVD, 29.8%), in 11.2% versus 12.7% of those with ischemic stroke of unknown cause (21.5%), in 7.6% versus 8.8% of those with TIA (30.9%), and in 22.2% versus 8.3% of those with hemorrhagic stroke (HS, 2%) at baseline. There was no difference in the efficacy of treatment for either the primary end point (LVD hazard ratio [HR] 0.70, 95% confidence interval [CI] 0.49 to 1.02, TIA HR 0.81, CI 0.57 to 1.17, SVD HR 0.85, CI 0.64 to 1.12, unknown cause HR 0.87, CI 0.61 to 1.24, HS HR 3.24, CI 1.01 to 10.4; P for heterogeneity=0.421), or MCVEs ( P for heterogeneity=0.360) based on subtype of the index event. As compared to subjects with LVD strokes, those with SVD had similar MCVE rates (19.2% versus 18.5% over the course of the trial), and similar overall reductions in stroke and MCVEs. Conclusions— Atorvastatin 80 mg/d is similarly efficacious in preventing strokes and other cardiovascular events, irrespective of baseline ischemic stroke subtype.

While the results of randomized clinical trials have indicated that statins improve outcomes in patients without cardiovascular disease (CVD), it remains uncertain whether there are differences in efficacy between statins, particularly in clinical practice, where the public health implications could be substantial.We assessed cardiovascular (CV) outcomes among primary-prevention patients newly initiating therapy with atorvastatin or simvastatin.Using claims data from 92 US managed care plans, we identified new statin users without CVD who initiated atorvastatin (10 or 20 mg) or simvastatin (20 or 40 mg) from January 2003 to September 2005 and were continuously enrolled in a covered plan foror = 12 months before andor = 1 month after the initiation of statin therapy. The main outcome was the time to the first CV event (hospitalization related to myocardial infarction, angina, or coronary artery disease; stroke; amaurosis fugax; transient ischemic attack; peripheral or central nervous system vascular disease; or revascularization). Persistence with treatment was calculated in terms of the number of days during follow-up that a patient remained on statin treatment, starting from the date of the first prescription fill to the end of the study or the date at which therapy was discontinued.A total of 168,973 patients initiating atorvastatin (mean dose, 13.5 mg) and 50,658 patients initiating simvastatin (mean dose, 28.5 mg) were followed for a median of 1.5 years. Atorvastatin patients were significantly more persistent with treatment than simvastatin patients (median treatment duration, 158 vs 124 days, respectively; P0.001). After adjustment for age, sex, type of health plan, payer type, geographic region, calendar year of statin initiation, physician specialty, comorbidities, concomitant therapies, and total direct health care costs in the year before statin initiation, use of atorvastatin was associated with significantly fewer CV events compared with use of simvastatin (hazard ratio = 0.88; 95% CI, 0.83-0.93; P0.001).In these patients without CVD, atorvastatin 10 or 20 mg was associated with a significantly lower risk of CV events compared with simvastatin 20 or 40 mg. Further studies are required to determine whether differences in persistence, achieved low-density lipoprotein cholesterol levels, or other factors contribute to these differences in outcomes.

Background and Purpose— The intention-to-treat analysis of data from the placebo-controlled Stroke Prevention by Aggressive Reduction in Cholesterol Levels (SPARCL) trial found 80 mg atorvastatin per day reduced the risk of stroke and major coronary events in patients with recent stroke or transient ischemic attack. This benefit was present despite only a 78% net difference in adherence to randomized treatment over the course of the trial. In this exploratory analysis, our aim was to evaluate the benefit and risks associated with achieving a ≥50% low-density lipoprotein cholesterol (LDL-C) reduction from baseline. Methods— This post hoc analysis was based on 55 045 LDL-C measurements among the 4731 patients enrolled in SPARCL (average, 11.6 measurements per patient) during a mean follow-up of 4.9 years. At each postrandomization LDL-C assessment, percent change in LDL-C from baseline for each patient was classified as no change or increase from baseline (32.7% of measurements), Results— Compared with no change or an increase in LDL-C, analysis of time-varying LDL-C change showed that patients with ≥50% LDL-C reduction had a 31% reduction in stroke risk (hazard ratio, 0.69, 95% CI, 0.55 to 0.87, P =0.0016), a 33% reduction in ischemic stroke ( P =0.0018), no statistically significant increase in hemorrhagic stroke ( P =0.8864), and a 37% reduction in major coronary events ( P =0.0323). There was no increase in the incidence of myalgia or rhabdomyolysis. Persistent liver enzyme elevations were more frequent in the group with ≥50% LDL-C reduction. Conclusions— As compared with having no change or an increase in LDL-C, achieving a ≥50% lowering was associated with a greater reduction in the risk of stroke and major coronary events with no increase in brain hemorrhages.

Although multiple advancements have been made in the treatment of heart failure (HF), mortality rates remain alarmingly high. The accepted arsenal of therapeutics includes a diuretic, digitalis, a beta-blocking agent and an inhibitor of the renin-angiotensin-aldosterone system. Despite the employment of a vast array of agents, nearly 300,000 patients in the US die annually with HF as a primary or contributory cause of death. Additional molecular targets are being evaluated in preclinical and clinical settings including vasopeptidase inhibitors, endothelin-1 receptor antagonists, arginine vasopressin antagonists, selective aldosterone blockers, TNF-alpha blockers and matrix metalloproteinase inhibitors. Although these approaches hold promise as viable therapeutics, a thorough evaluation of clinical benefit from these agents requires additional trials. Future disease-modifying approaches will also undoubtedly include cell transplantation and gene therapy. It is likely that notable advances in HF treatment will come from agents that attenuate myocardial remodelling. Indeed, maintenance or improvement of cardiac structure can attenuate HF development and improve mortality.

Background Mitogen-activated protein kinases (MAPKs) have emerged as an important pathophysiologic regulator during the development of heart failure (HF). p38 MAPK activity is elevated in cardiac hypertrophy and HF. We used a mouse model of myocardial infarction (MI) to test the hypotheses that (1) inhibition of p38 MAPK activity may improve cardiac function and remodeling after myocardial infarction (MI) and (2) coadministration of a p38 inhibitor (p38i) and an angiotensin-converting enzyme inhibitor (ACEI) may provide only limited further cardioprotection in this model. Methods and results MI was induced in C57BL/6J mice by ligating the left anterior descending coronary artery and then either left untreated or treated with a p38i (SC-409, 30 mg/kg/day in chow), ACEI (enalapril, 20 mg/kg in drinking water), or p38i plus ACEI for 12 weeks. Echocardiography was performed and systolic blood pressure measured before MI and weekly thereafter. At the end of the study, interstitial collagen fraction (ICF) and myocyte cross-sectional area (MCSA) were examined histologically. We found that p38i significantly increased left ventricular ejection fraction and cardiac output and decreased left ventricular area at diastole, ICF, and MCSA. ACEi and p38i each had similar beneficial effects in this mouse model of HF produced by a large MI. Coadministration of p38i and ACEi did not provide any additional benefit. Conclusion Our data suggest that inhibition of p38 MAPK provides significant cardioprotection in mice with HF post-MI.

Background: The incidence of cardiovascular diseases increases rapidly with age, and the elderly suffer higher morbidity and mortality. Aldosterone blockers have shown. benefits in patients with left ventricular (LV) dysfunction and heart failure after myocardial infarction (MI). However, aldosterone blockade efficacy has not been explored in aged animals with MI. Methods and Results: Small-to-moderate MI was induced by coronary artery ligation in 16-month old rats, divided into 3 groups: sham-operated (control, n = 9), MI (n. = 9), and MI fed a diet containing eplerenone (120 mg/kg/day, MI+Eplerenone, n = 9). given 18 days postsurgery and up to sacrifice 3 months later. At. sacrifice, untreated MI rats did not show overt systolic dysfunctiom but they had (1) echocardiographic evidences of impaired relaxation (increase of E wave deceleration time and of isovolumic relaxation time, decrease of peak E wave velocity), (2) hemodynamically impaired LV relaxation (LV -dP/dt from 7413 +/- 720 to 4956 +/- 475 mm Hg/s, P < .05), and (3) significant increase of collagen content in LV interstitium (from 4.27 +/- 0.23 to 5.34 +/- 0.24%, P < .01), and in aorta. (from 19 +/- 1 to 24 +/- 2%, P < .05). Eplerenone normalized echocardiographic and hemodynamic evidences of diastolic dysfunction, as well as myocardial. interstitial collagen and aortic fibrosis. (all parameters statistically different from untreated MI). Conclusion: In aged rats with small to moderate MI, eplerenone normalized diastolic relaxation, possibly through a reduction of interstitial fibrosis.

The classical mineralocorticoid effect of aldosterone on unidirectional transepithelial sodium transport in the kidney was long thought to be the predominant effect of this hormone. However, there is convincing evidence for additional extrarenal actions of aldosterone that are mediated via activation of mineralocorticoid receptors (MRs) in the heart, vasculature and brain. It is now postulated that many of the detrimental effects of aldosterone are mediated through MR activation in these nonclassical target organs. The selective aldosterone blocker, eplerenone (Inspra), is under development for human therapeutic use for treatment of hypertension and heart failure post-myocardial infarction (MI). Clinical and preclinical studies have linked elevated aldosterone to hypertension, left ventricular and vascular remodeling, cardiac, renal, and cerebral vascular inflammation and injury, and increased risk of mortality in heart failure patients. Multiple studies in experimental models of hypertension and heart failure demonstrate that selective blockade of aldosterone by eplerenone effectively preserves cardiac function, attenuates maladaptive left ventricular remodeling and tissue and vascular injury in part by reducing vascular inflammation in aldosterone target organs.

Background Spironolactone, a nonselective aldosterone blocker, has a cardioprotective effect; however, significant endocrine side effects limit its use. Eplerenone is a new selective aldosterone blocker. We investigated whether eplerenone attenuates cardiac remodeling and improves function in a mouse model of heart failure and whether coadministration of eplerenone and an angiotensin-converting enzyme inhibitor (ACEi) provides better cardioprotection than either agent alone. Methods and results C57BL/6J mice were subjected to myocardial infarction (MI) by ligating the left anterior descending coronary artery. Two weeks later, the mice were either left untreated or treated with (1) eplerenone, (2) ACEi, or (3) eplerenone plus ACEi for 12 weeks. Systolic blood pressure (SBP) was measured and echocardiography performed before MI and weekly thereafter. At the end of the study, interstitial collagen fraction (ICF) and myocyte cross-sectional area (MCSA) were examined histologically. We found that (1) eplerenone significantly improved ejection fraction and cardiac output and decreased left ventricular (LV) systolic area, LV weight, ICF, and MCSA independently of changes in SBP compared with untreated animals; (2) ACEi had similar beneficial effects, accompanied by a significant reduction in SBP; and (3) combined treatment offered limited additional benefit beyond monotherapy. Conclusions In mice with MI, eplerenone attenuates progression of heart failure comparably to ACEi, and its effect is independent of BP lowering.

Aldosterone classically promotes unidirectional transepithelial sodium transport, thereby regulating blood volume and blood pressure. Recently, both clinical and experimental studies have suggested additional, direct roles for aldosterone in the cardiovascular system. To evaluate aldosterone activation of cardiomyocyte mineralocorticoid receptors, transgenic mice overexpressing 11β-hydroxysteroid dehydrogenase type 2 in cardiomyocytes were generated using the mouse α-myosin heavy chain promoter. This enzyme converts glucocorticoids to receptor-inactive metabolites, allowing aldosterone occupancy of cardiomyocyte mineralocorticoid receptors. Transgenic mice were normotensive but spontaneously developed cardiac hypertrophy, fibrosis, and heart failure and died prematurely on a normal salt diet. Eplerenone, a selective aldosterone blocker, ameliorated this phenotype. These studies confirm the deleterious consequences of inappropriate activation of cardiomyocyte mineralocorticoid receptors by aldosterone and reveal a tonic inhibitory role of glucocorticoids in preventing such outcomes under physiological conditions. In addition, these data support the hypothesis that aldosterone blockade may provide additional therapeutic benefit in the treatment of heart failure.

To understand the role of the factor X (fX) activation peptide (AP), a deletion mutagenesis approach was employed. Two single-chain, variant enzymes were generated in which 41 residues were deleted from the AP: fX (des-137-183) and fX(des-137-183;N191A), which lacks a carbohydrate moiety at Asn191 due to an alanine substitution. Deletion of the fX AP did not impact fXa catalytic activity. Activation of the variant zymogens, however, was altered. Neither mutant enzyme was activated by the fX coagulant protein from Russell's viper venom (RVV-X(1)). Activation by factor VIIa (fVIIa) and fVIIa in the presence of cofactor, lipidated tissue factor (TF), occurred at an accelerated rate for both variants as compared to wild-type fX (WTfX). Similar to fVII, the mutants auto-activated in a cofactor-independent manner, which was characterized by a lag period and accelerated dose-dependently by plasma fXa (kcat/Km, 0.046 +/- 0.004 micro M(-1) s(-1)). Both mutants were also found to be activated by fVIIa (0.31 +/- 0.03 micro M(-1) s(-1)), fIXa (0.30 +/- 0.03 micro M(-1) s(-1)), and thrombin (0.00078 +/- 0.00015 micro M(-1) s(-1)). In all cases, the rate of activation was faster for fX(des-137-183;N191A) as compared to fX(des-137-183). We propose that the fX AP and Asn191 carbohydrate serve primarily as negative autoregulation mechanisms to prevent spurious activation of fX and secondarily in cofactor dependence and activator specificity.

Heart failure is a complex multifactorial disease resulting in a myriad of progressive changes at the molecular, cellular, and physiological level. To better understand the mechanisms associated with the development of congestive heart failure, a comprehensive examination of the aging lean male spontaneously hypertensive, heart failure-prone rat (SHHF) was conducted. Myocardial function and structural integrity progressively diminished as evidenced by decreased ejection fraction and increased left ventricular volume measured using echocardiography. Functional and structural changes were accompanied by elevations in circulating inflammatory markers, including tumor necrosis factor-α (TNF-α), IL-6, and TNF receptors type 1 and 2. Increased systemic inflammatory marker levels were consistent with age-dependent changes in the expression pattern of genes that contribute to stress, inflammation, and the extracellular matrix in SHHF animals analyzed from age 4 to 18 mo. In summary, the SHHF rat shares many hallmark features of the human disease state and represents a key experimental model for the dissection of complex human heart failure pathophysiology.

Vascular inflammation was examined as a potential mechanism of aldosterone-mediated myocardial injury in uninephrectomized rats receiving 1% NaCl-0.3% KCl to drink for 1, 2, or 4 wk and 1) vehicle, 2) aldosterone infusion (0.75 μg/h), or 3) aldosterone infusion (0.75 μg/h) plus the selective aldosterone blocker eplerenone (100 mg · kg−1· day−1). Aldosterone induced severe hypertension at 4 wk [systolic blood pressure (SBP), 210 ± 3 mmHg vs. vehicle, 131 ± 2 mmHg, P < 0.001], which was partially attenuated by eplerenone (SBP, 180 ± 7 mmHg; P < 0.001 vs. aldosterone alone and vehicle). No significant increases in myocardial interstitial collagen fraction or hydroxyproline concentration were detected throughout the study. However, histopathological analysis of the heart revealed severe coronary inflammatory lesions, which were characterized by monocyte/macrophage infiltration and resulted in focal ischemic and necrotic changes. The histological evidence of coronary lesions was preceded by and associated with the elevation of cyclooxygenase-2 (up to ∼4-fold), macrophage chemoattractant protein-1 (up to ∼4-fold), and osteopontin (up to ∼13-fold) mRNA expression. Eplerenone attenuated proinflammatory molecule expression in the rat heart and subsequent vascular and myocardial damage. Thus aldosterone and salt treatment in uninephrectomized rats led to severe hypertension and the development of a vascular inflammatory phenotype in the heart, which may represent one mechanism by which aldosterone contributes to myocardial disease.

Myocardial infarction (MI) initiates adaptive tissue remodeling, which is essential for heart function (such as infarct healing) but is also important for maladaptive remodeling (for example, reactive fibrosis and left ventricular dilation). The effect of aldosterone receptor antagonism on these processes was evaluated in Sprague-Dawley rats using eplerenone, a selective aldosterone receptor antagonist. Infarct healing and left ventricular remodeling were evaluated at 3, 7, and 28 days after MI by determination of the diastolic pressure-volume relationship of the left ventricle, the infarct-thinning ratio, and the collagen-volume fraction. Eplerenone did not affect reparative collagen deposition as was evidenced by a similar collagen volume fraction in the infarcted myocardium between eplerenone and vehicle-treated groups at 7 and 28 days post-MI. In addition, the thinning ratio, which is an index of infarct expansion, was comparable between the eplerenone and vehicle-treated animals at 7 and 28 days post-MI. A protective effect of eplerenone was demonstrated at 28 days post-MI, where reactive fibrosis in the viable myocardium was reduced in eplerenone-treated animals compared with vehicle-treated animals. Thus aldosterone receptor antagonism does not retard infarct healing but rather protects against maladaptive responses after MI.

We reported previously that residue 347 in activated fX (fXa) contributes to binding of the cofactor, factor Va (fVa) (Rudolph, A. E., Porche-Sorbet, R. and Miletich, J. P. (2000) Biochemistry 39, 2861-2867). Four additional residues that participate in fVa binding have now been identified by mutagenesis. All five resulting fX species, fX(R306A), fX(E310N), fX(R347N), fX(K351A), and fX(K414A), are activated and inhibited normally. However, the rate of inhibition by antithrombin III in the presence of submaximal concentrations of heparin is reduced for all the enzymes. In the absence of fVa, all of the enzymes bind and activate prothrombin similarly except fXa(E310N), which has a reduced apparent affinity ( approximately 3-fold) for prothrombin compared with wild type fXa (fXa(WT)). In the absence of phospholipid, fVa enhances the catalytic activity of fXa(WT) significantly, but the response of the variant enzymes was greatly diminished. On addition of 100 nm PC:PS (3:1) vesicles, fVa enhanced fXa(WT), fXa(R306A), and fXa(E310N) similarly, whereas fXa(R347N), fXa(K351A), and fXa(K414A) demonstrated near-normal catalytic activity but reduced apparent affinity for fVa under these conditions. All enzymes function similarly to fXa(WT) on activated platelets, which provide saturating fVa on an ideal surface. Loss of binding affinity for fVa as a result of the substitutions in residues Arg-347, Lys-351, and Lys-414 was verified by a competition binding assay. Thus, Arg-347, Lys-351, and Lys-414 are likely part of a core fVa binding site, whereas Arg-306 and Glu-310 serve a less critical role.

The bacterial pathogen Yersinia uses a type III secretion system to inject several virulence factors into target cells. One of the Yersinia virulence factors, YopJ, was shown to bind directly to the superfamily of MAPK (mitogen-activated protein kinase) kinases (MKKs) blocking both phosphorylation and subsequent activation of the MKKs. These results explain the diverse activities of YopJ in inhibiting the extracellular signal–regulated kinase, c-Jun amino-terminal kinase, p38, and nuclear factor kappa B signaling pathways, preventing cytokine synthesis and promoting apoptosis. YopJ-related proteins that are found in a number of bacterial pathogens of animals and plants may function to block MKKs so that host signaling responses can be modulated upon infection.

A system is described for producing recombinant factor X with properties very similar to human plasma factor X. Optimization of the expression system for factor X resulted in the finding that human kidney cells (293 cells) are superior to the widely utilized baby hamster kidney cells (BHK cells) for the expression of functional factor X. It was also determined that production of factor X by 293 cells requires the substitution of the -2 residue (Thr-->Arg) which affords the removal of the factor X propeptide. Purification of recombinant and plasma factor X is accomplished using a calcium-dependent monoclonal antibody directed against the gla domain. The proteins are comparable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rate and extent of activation by the factor X coagulant protein from Russell's viper venom and by factors IXa and VIIIa are similar; activation of the recombinant protein by VIIa and tissue factor is mildly faster. The activated enzymes have the same activity toward a chromogenic substrate and the biologic substrate, prothrombin. Both enzymes have the same apparent affinity for the activated platelet surface as judged by their ability to activate prothrombin. Finally, inhibition by antithrombin, with or without heparin, and inhibition by the tissue factor pathway inhibitor are equivalent. Recombinant factor X produced by this method is therefore well suited for probing structure-function relationships by mutational analysis.

Herein, we demonstrate that nitric oxide is a potent (20% release) and highly selective inducer of [3H]arachidonic acid mobilization in the macrophage-like cell line RAW 264.7. Treatment of RAW 264.7 cells with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one resulted in the inhibition of the large majority (86%) of nitric oxide-induced [3H]arachidonic acid release into the medium (IC500.5 microM) and the concomitant inhibition of in vitro measurable calcium-independent phospholipase A2 activity (92% inhibition) without demonstrable effects on calcium-dependent phospholipase A2 activity. Since nitric oxide is a potent stimulator of glycolysis (and therefore glycolytically derived ATP) and since cytosolic calcium-independent phospholipase A2 exists as a catalytic complex comprised of ATP-modulated phosphofructokinase-like regulatory polypeptides and a catalytic subunit, we examined the role of glucose in facilitating nitric oxide-mediated arachidonic acid release. Nitric oxide-induced release of [3H]arachidonic acid possessed an obligatory requirement for glucose, was highly correlated with the concentration of glucose in the medium, and was dependent on the metabolism of glucose. Thus, [3H]arachidonic acid release is coupled to cellular glucose metabolism through alterations in the activity of calcium-independent phospholipase A2. Collectively, these results identify a unifying metabolic paradigm in which the generation of lipid second messengers is coordinately linked to the signalstimulated acceleration of glycolytic flux, thereby facilitating integrated metabolic responses to cellular stimuli.

Aldosterone/salt induces renal inflammation and fibrosis in hypertensive rats.BackgroundWe evaluated the role of aldosterone as a mediator of renal inflammation and fibrosis in a rat model of aldosterone/salt hypertension using the selective aldosterone blocker, eplerenone.MethodsUnnephrectomized, Sprague-Dawley rats were given 1% NaCl (salt) to drink and randomized to receive treatment for 28 days: vehicle infusion (control); 0.75 μg/hour aldosterone subcutaneous infusion; or aldosterone infusion + 100 mg/kg/day oral dose of eplerenone. Blood pressure and urinary albumin were measured and kidneys were evaluated histologically. Renal injury, inflammation, and fibrosis were assessed by immunohistochemistry, in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR).ResultsAldosterone/salt induced severe hypertension compared to controls (220 ± 4 mm Hg vs. 131 ± 4 mm Hg, P < 0.05), which was partially attenuated by eplerenone (179 ± 4 mm Hg, P < 0.05). In aldosterone/salt treated rats, renal histopathologic evaluation revealed severe vascular and glomerular sclerosis, fibrinoid necrosis and thrombosis, interstitial leukocyte infiltration, and tubular damage and regeneration. Aldosterone/salt increased circulating osteopontin (925.0 ± 80.2 ng/mL vs. 53.6 ± 6.3 ng/mL) and albuminuria (75.8 ± 10.9 mg/24 hours vs. 13.2 ± 3.0 mg/24 hours) compared to controls and increased expression of proinflammatory molecules. Treatment with eplerenone reduced systemic osteopontin (58.3 ± 4.2 ng/mL), albuminuria (41.5 ± 7.2 mg/24 hours), and proinflammatory gene expression: osteopontin (OPN), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and interleukin-1β (IL-1β).ConclusionThese findings indicate that aldosterone/salt–induced renal injury and fibrosis has inflammatory components involving macrophage infiltration and cytokine up-regulation. Attenuation of renal damage and inflammation by eplerenone supports the protective effects of aldosterone blockade in hypertensive renal disease.