David M. Kerins

Angiotensin induction of PAI-1 expression in endothelial cells is mediated by the hexapeptide angiotensin IV.

Recent studies from this laboratory have demonstrated that angiotensin II (Ang II) stimulates the expression of plasminogen activator inhibitor 1 (PAI-1) in cultured endothelial cells. This response does not appear to be mediated via an interaction with either the AT1 or the AT2 receptor subtype. Since a novel angiotensin receptor has been identified in a variety of tissues that specifically binds the hexapeptide Ang IV (Ang II, [3-8]), we therefore examined the effects of Ang IV on the expression of PAI-1 mRNA in bovine aortic endothelial cells. Ang IV stimulated dose- and time-dependent increases in the expression of PAI-1 mRNA. The effect of Ang IV (10 nM) was not inhibited by Dup 753 (1.0 microM), a highly specific antagonist of the AT1 receptor, or by PD123177 (1.0 microM), a highly specific antagonist of the AT2 receptor. In contrast, the AT4 receptor antagonist, WSU1291 (1.0 microM), effectively prevented PAI-1 expression. Although larger forms of angiotensin (i.e., Ang I, Ang II, and Ang III) are capable of inducing PAI-1 expression, this property is lost in the presence of converting enzyme or aminopeptidase inhibitors. These results indicate that the hexapeptide Ang IV is the form of angiotensin that stimulates endothelial expression of PAI-1. This effect appears to be mediated via the stimulation of an endothelial receptor that is specific for Ang IV.

Exercise and associated dietary extremes impact on gut microbial diversity

Objective The commensal microbiota, host immunity and metabolism participate in a signalling network, with diet influencing each component of this triad. In addition to diet, many elements of a modern lifestyle influence the gut microbiota but the degree to which exercise affects this population is unclear. Therefore, we explored exercise and diet for their impact on the gut microbiota. Design Since extremes of exercise often accompany extremes of diet, we addressed the issue by studying professional athletes from an international rugby union squad. Two groups were included to control for physical size, age and gender. Compositional analysis of the microbiota was explored by 16S rRNA amplicon sequencing. Each participant completed a detailed food frequency questionnaire. Results As expected, athletes and controls differed significantly with respect to plasma creatine kinase (a marker of extreme exercise), and inflammatory and metabolic markers. More importantly, athletes had a higher diversity of gut micro-organisms, representing 22 distinct phyla, which in turn positively correlated with protein consumption and creatine kinase. Conclusions The results provide evidence for a beneficial impact of exercise on gut microbiota diversity but also indicate that the relationship is complex and is related to accompanying dietary extremes.

The Urokinase-type Plasminogen Activator Receptor Mediates Tyrosine Phosphorylation of Focal Adhesion Proteins and Activation of Mitogen-activated Protein Kinase in Cultured Endothelial Cells

Urokinase-type plasminogen activator (uPA) binds to cells via a specific glycosylphosphatidylinositol-anchored receptor. Although occupancy of the uPA receptor (uPAR) has been shown to alter cellular function and to induce gene expression, the signaling mechanism has not been characterized. Urokinase induced an increase in the tyrosine phosphorylation of multiple proteins in bovine aortic endothelial cells. In contrast, low molecular weight uPA did not induce this response. Analysis by immunoblotting demonstrated tyrosine phosphorylation of focal adhesion kinase (FAK), the focal adhesion-associated proteins paxillin and p130 cas ,and mitogen-activated protein kinase (MAPK) following the occupancy of the uPAR by uPA. Treatment of cells with phosphatidylinositol-specific phospholipase C, which cleaves glycosylphosphatidylinositol-linked proteins from the cell surface, blocked the uPA-induced tyrosine phosphorylation of FAK, indicating the requirement of an intact uPAR on the cell surface. The uPA-induced activation of MAPK was completely inhibited by genistein, but not by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a specific inhibitor of Src family kinases. Thus, this study demonstrates a novel role for the uPAR in endothelial cell signal transduction that involves the activation of FAK and MAPK, which are mediated by the receptor-binding domain of uPA. This may have important implications for the mechanism through which uPA influences cell migration and differentiation.

Platelet and vascular function during coronary thrombolysis with tissue-type plasminogen activator.

Platelet activation may limit the response to tissue-type plasminogen activator (t-PA) during coronary thrombolysis in humans. As an index of platelet activation, we assessed thromboxane A2 biosynthesis during coronary thrombolysis with intravenous t-PA in patients with acute myocardial infarction. Urinary 2,3-dinor-thromboxane B2, a metabolite of thromboxane A2, was increased to a peak of 3,327 +/- 511 pg/mg creatinine (n = 12) following administration of intravenous t-PA and remained elevated for 48 hours. This increase was abolished by pretreatment with aspirin 325 mg orally (n = 6), indicating de novo biosynthesis of thromboxane A2 rather than washout of preformed metabolites during reperfusion. Prostacyclin (PGI2) biosynthesis, determined by excretion of 2,3-dinor-6-keto-PGF1 alpha, also increased after t-PA administration. However, this increase was less pronounced in patients who reperfused (28 +/- 3.3 ng.hr/mg creatinine) than in patients who failed to reperfuse (118 +/- 30 ng.hr/mg creatinine, p less than 0.05). These data provide evidence of platelet activation during coronary thrombolysis with t-PA. In patients who reperfuse, the reduction in PGI2 biosynthesis may be a marker of reperfusion injury to the vasculature and may further amplify platelet activation.

Liver regeneration is transiently impaired in urokinase-deficient mice

To test the hypothesis that urokinase-type plasminogen activator (uPA) plays an important role in liver regeneration in vivo, partial hepatectomy was performed on wild-type and uPA-deficient (uPA-/-) mice. Mice were studied at 24, 44, and 96 h and at 8 days and 4 wk post-partial hepatectomy for evidence of regeneration, as measured by mitotic indexes and [3H]thymidine incorporation. In wild-type mice, thymidine incorporation peaked at 44 h and this index was reduced by 47% in uPA-/- mice (P = 0.02). By 8 days, however, liver mass was comparable in both groups. Histological analysis revealed the presence of focal areas of fibrin deposition and cellular loss by 24 h that were more severe and prevalent in uPA-/- mice than in wild-type mice (62 and 23%, respectively; chi2 = 3.939, P = 0.047). In contrast, regeneration was not impaired in uPA receptor (uPAR)-deficient mice at 24 and 44 h. Taken together, these data indicate that uPA, independent of its interaction with the uPAR, plays an important role in liver regeneration in vivo.To test the hypothesis that urokinase-type plasminogen activator (uPA) plays an important role in liver regeneration in vivo, partial hepatectomy was performed on wild-type and uPA-deficient (uPA-/-) mice. Mice were studied at 24, 44, and 96 h and at 8 days and 4 wk post-partial hepatectomy for evidence of regeneration, as measured by mitotic indexes and [3H]thymidine incorporation. In wild-type mice, thymidine incorporation peaked at 44 h and this index was reduced by 47% in uPA-/- mice ( P= 0.02). By 8 days, however, liver mass was comparable in both groups. Histological analysis revealed the presence of focal areas of fibrin deposition and cellular loss by 24 h that were more severe and prevalent in uPA-/- mice than in wild-type mice (62 and 23%, respectively; χ2 = 3.939, P = 0.047). In contrast, regeneration was not impaired in uPA receptor (uPAR)-deficient mice at 24 and 44 h. Taken together, these data indicate that uPA, independent of its interaction with the uPAR, plays an important role in liver regeneration in vivo.

Effects of centrally acting ACE inhibitors on the rate of cognitive decline in dementia

Objectives There is growing evidence that antihypertensive agents, particularly centrally acting ACE inhibitors (CACE-Is), which cross the blood–brain barrier, are associated with a reduced rate of cognitive decline. Given this, we compared the rates of cognitive decline in clinic patients with dementia receiving CACE-Is (CACE-I) with those not currently treated with CACE-Is (NoCACE-I), and with those who started CACE-Is, during their first 6 months of treatment (NewCACE-I). Design Observational case–control study. Setting 2 university hospital memory clinics. Participants 817 patients diagnosed with Alzheimers disease, vascular or mixed dementia. Of these, 361 with valid cognitive scores were included for analysis, 85 CACE-I and 276 NoCACE-I. Measurements Patients were included if the baseline and end-point (standardised at 6 months apart) Standardised Mini-Mental State Examination (SMMSE) or Quick Mild Cognitive Impairment (Qmci) scores were available. Patients with comorbid depression or other dementia subtypes were excluded. The average 6-month rates of change in scores were compared between CACE-I, NoCACE-I and NewCACE-I patients. Results When the rate of decline was compared between groups, there was a significant difference in the median, 6-month rate of decline in Qmci scores between CACE-I (1.8 points) and NoCACE-I (2.1 points) patients (p=0.049), with similar, non-significant changes in SMMSE. Median SMMSE scores improved by 1.2 points in the first 6 months of CACE treatment (NewCACE-I), compared to a 0.8 point decline for the CACE-I (p=0.003) group and a 1 point decline for the NoCACE-I (p=0.001) group over the same period. Multivariate analysis, controlling for baseline characteristics, showed significant differences in the rates of decline, in SMMSE, between the three groups, p=0.002. Conclusions Cognitive scores may improve in the first 6 months after CACE-I treatment and use of CACE-Is is associated with a reduced rate of cognitive decline in patients with dementia.

Effects of centrally acting angiotensin converting enzyme inhibitors on functional decline in patients with Alzheimer's disease.

BACKGROUND Centrally acting angiotensin converting enzyme inhibitors (CACE-Is) are associated with reduced rates of cognitive decline in patients with dementia. CACE-Is may also improve exercise tolerance in functionally impaired older adults with normal cognition, suggesting that CACE-Is may positively influence activities of daily living (ADL) in dementia. OBJECTIVE To compare rates of decline in patients with mild to moderate Alzheimers disease (AD) receiving CACE-Is to those not currently treated with CACE-Is (NoCACE-I), included in the Doxycycline and Rifampicin for Alzheimers Disease study (n = 406). METHODS Patients were included if baseline and end-point (twelve months apart) scores were available for measures including the Standardized Alzheimers Disease Assessment Scale - Cognitive Subscale; Quick Mild Cognitive Impairment screen; Clinical Dementia Rating Scale (CDR-SB), and Lawton-Brody ADL Scale. RESULTS There was a significant, 25% difference (median one-point) in the 12-month rate of decline in ADL scores in patients taking CACE-Is (n = 91), compared to the NoCACE-I group (n = 274), p = 0.024. This remained significant after adjusting for age, gender, education, and blood pressure, p = 0.034. When individual CACE-Is were compared to the NoCACE-I group, a significant reduction in the rate of decline in ADLs (median one versus four points), were only observed for perindopril, p = 0.01. The CDR-SB was also reduced (median one-point) for the perindopril compared to the NoCACE-I group, p = 0.04. CONCLUSION This observational study suggests that CACE-Is, and potentially perindopril in particular, are associated with a reduced rate of functional decline in patients with AD, without an association with mood or behavior. This suggests that CACE-Is may slow disease progression in AD.

Heart rate variability during sleep in healthy term newborns in the early postnatal period

Normative time- and frequency-domain heart rate variability (HRV) measures were extracted during quiet sleep (QS) and active sleep (AS) periods in 30 healthy babies. All newborn infants studied were less than 12 h old and the sleep state was classified using multi-channel video EEG. Three bands were extracted from the heart rate (HR) spectrum: very low frequency (VLF), 0.01-0.04 Hz; low frequency (LF), 0.04-0.2 Hz, and high frequency (HF), >0.2 Hz. All metrics were averaged across all patients and per sleep state to produce a table of normative values. A noticeable peak corresponding to activity in the RSA band was found in 80% patients during QS and 0% of patients during AS, although some broadband activity was observed. The majority of HRV metrics showed a statistically significant separation between QS and AS. It can be concluded that (i) activity in the RSA band is present during QS in the healthy newborn, in the first 12 h of life, (ii) HRV measures are affected by sleep state and (iii) the averaged HRV metrics reported here could assist the interpretation of HRV data from newborns with neonatal illnesses.

Cardiovascular safety of anti-diabetic drugs

Cardiovascular disease is the leading cause of morbidity and mortality among patients with diabetes, underscoring the importance of choosing anti-diabetic drugs that do not increase cardiovascular risk but might reduce the risk of cardiovascular events. Most type 2 diabetic patients die from cardiovascular causes despite the beneficial effects of blood pressure (BP) and lipid-lowering medications. The prevalence of patients with cardiovascular disease and diabetes mellitus is growing exponentially. Approximately 40% of patients hospitalized with heart failure and reduced ejection fraction have diabetes mellitus. The recent trials conducted in patients with heart failure who had diabetes showed a different response to standard medication, with these patients being more prone to develop side effects than patients with the same degree of heart failure but without diabetes mellitus. Therefore, careful selection of drug therapy paying particular attention to cardiovascular safety is important in optimizing diabetic therapy. This review discusses the efficacy and safety of the most commonly prescribed anti-diabetic drugs in the context of cardiovascular impact.

Pharmacokinetics of tissue-type plasminogen activator during acute myocardial infarction in men. Effect of a prostacyclin analogue.

BackgroundCoronary reocclusion complicates the thrombolytic therapy of acute myocardial infarction despite the routine use of aspirin. This is consistent with experimental studies demonstrating that multiple agonists, in addition to thromboxane A2, mediate the platelet activation underlying reocclusion. Consequently, a more potent antiplatelet therapy with a broader spectrum of activity than aspirin may be required in this setting. Prostacyclin and its more stable analogue, iloprost, inhibit platelet aggregation to all known agonists and exert an additional effect over aspirin alone. Experiments in animal models have demonstrated, however, that iloprost increases the clearance of tissue-type plasminogen activator (t-PA) and impairs thrombolysis in vivo. This study examines whether a similar interaction occurs in humans. Methods and ResultsTwelve patients with acute myocardial infarction received t-PA intravenously, 60 mg in the first hour and a maintenance infusion of 13.3 mg/hr for 3 hours. Patients were assigned in a double-blind fashion to iloprost (2 ng/kg/min) or placebo following the initial 90 minutes of the maintenance infusion of t-PA. Iloprost decreased mean arterial blood pressure (−10±2.9 mm Hg, p<0.05) but did not alter heart rate. Steady-state plasma iloprost concentration was 591±64 pmol/l. At this concentration, iloprost markedly inhibited platelet aggregation in vitro, particularly in the presence of aspirin. Steady-state clearance of t-PA was unchanged by iloprost (454±65 versus 443±136 ml/min in controls, p=NS). Furthermore, neither elimination kinetics nor plasma protein binding of t-PA was altered by iloprost. ConclusionsAt plasma levels that exert a potent antiplatelet effect, iloprost did not alter the pharmacokinetics of t-PA in men. Prostacyclin analogues may prove useful as an adjunct to plasminogen activators, particularly in patients at high risk for thrombotic reocclusion.