Erkennungssequenzen und strukturelle Elemente als Determinanten der regulierten Spaltung von Membranproteinen

Eine funktionell und strukturell diverse Gruppe von Transmembranproteinen wie beispielsweise Mediatoren und deren Rezeptoren können proteolytisch gespalten werden. Dieser Prozess wird als Shedding bezeichnet. Kürzlich konnte die proteolytische Aktivität identifiziert werden, die für die Prozessierung von proTNFa verantwortlich ist. Sie wurde TACE (TNF Alpha Converting Enzyme) genannt. In Experimenten mit TACE-/- Fibroblasten konnte ich herausfinden, dass das durch PMA induzierte Shedding des IL-6Rs stark reduziert war. Eine basale hydroxamatsensitive Freisetzung des IL-6Rs konnte allerdings noch detektiert werden. Um Unterschiede im Shedding von IL-6R und proTNFa zu untersuchen, generierte ich chimäre Proteine aus diesen beiden Proteinen, bei denen die Spaltstellenregionen gegeneinander vertauscht worden waren. TNFa Chimären zeigten nur sehr geringes Shedding. Im Gegensatz dazu wurden IL-6R Chimären, die die proTNFa Spaltstelle enthielten spontan gespalten. Die PMA-Induzierbarkeit war verloren gegangen. Daraufhin wurden verschiedene Chimären des unspaltbaren Proteins gp130 und der Spaltstellenpeptide aus TNFa, TGFa und IL-6R generiert. Hierbei wurde ein kurzes membranproximales Peptid aus gp130 gegen die Spaltstellen ausgetauscht. Diese Peptide übertrugen sowohl spontane ale auch PMA-induzierte Spaltbarkeit auf gp130. Um die minimalen Bedingungen für Shedding zu untersuchen, setzte ich verkürzte IL-6R Spaltstellenpeptide in gp130 ein. Die resultierenden Chimären waren empfänglich für reguliertes Shedding. Überaschenderweise konnten auch spaltbare Chimären durch das Ersetzen der membrannahem Region von gp130 durch die entsprechende Region aus dem ebenfalls nicht spaltbaren LIFR generiert werden.

Evidence-based polytherapies and long-term mortality after acute myocardial infarction in very old subjects compared with elderly and adults: a nested case-control study

Background: Clinical trials have demonstrated that selected secondary prevention medications for patients after acute myocardial infarction (AMI) reduce mortality. Yet, these medications are generally underprescribed in daily practice, and older people are often absent from drug trials. Objectives: To examine the relationship between adherence to evidence-based (EB) drugs and post-AMI mortality, focusing on the effects of single therapy and polytherapy in very old patients (≥80 years) compared with elderly and adults (<80 years). Methods: Patients hospitalised for AMI between 01/01/2008 and 30/06/2011 and resident in the Local Health Authority of Bologna were followed up until 31/12/2011. Medication adherence was calculated as the proportion of days covered for filled prescriptions of angiotensin-converting enzyme inhibitors (ACEIs)/angiotensin receptor blockers (ARBs), β-blockers, antiplatelet drugs, and statins. We adopted a risk set sampling method, and the adjusted relationship between medication adherence (PDC≥75%) and mortality was investigated using conditional multiple logistic regression. Results: The study population comprised 4861 patients. During a median follow-up of 2.8 years, 1116 deaths (23.0%) were observed. Adherence to the 4 EB drugs was 7.1%, while nonadherence to any of the drugs was 19.7%. For both patients aged ≥80 years and those aged <80 years, rate ratios of death linearly decreased as the number of EB drugs taken increased. There was a significant inverse relationship between adherence to each of 4 medications and mortality, although its magnitude was higher for ACEIs/ARBs (adj. rate ratio=0.60, 95%CI=0.52–0.69) and statins (0.60, 0.50–0.72), and lower for β-blockers (0.75, 0.61–0.92) and antiplatelet drugs (0.73, 0.63–0.84). Conclusions: The beneficial effect of EB polytherapy on long-term mortality following AMI is evident also in nontrial older populations. Given that adherence to combination therapies is largely suboptimal, the implementation of strategies and initiatives to increase the use of post-AMI secondary preventive medications in old patients is crucial.

Angiotensinergic and noradrenergic neurons in the rat and human heart

Although the physiological and pharmacological evidences suggest a role for angiotensin II (Ang II) with the mammalian heart, the source and precise location of Ang II are unknown. To visualize and quantitate Ang II in atria, ventricular walls and interventricular septum of the rat and human heart and to explore the feasibility of local Ang II production and function, we investigated by different methods the expression of proteins involved in the generation and function of Ang II. We found mRNA of angiotensinogen (Ang-N), of angiotensin converting enzyme, of the angiotensin type receptors AT(1A) and AT(2) (AT(1B) not detected) as well as of cathepsin D in any part of the hearts. No renin mRNA was traceable. Ang-N mRNA was visualized by in situ hybridization in atrial ganglial neurons. Ang II and dopamine- -hydroxylase (D H) were either colocalized inside the same neuronal cell or the neurons were specialized for Ang II or D H. Within these neurons, the vesicular acetylcholine transporter (VAChT) was neither colocalized with Ang II nor D H, but VAChT-staining was found with synapses en passant encircle these neuronal cells. The fibers containing Ang II exhibited with blood vessels and with cardiomyocytes supposedly angiotensinergic synapses en passant. In rat heart, right atrial median Ang II concentration appeared higher than septal and ventricular Ang II. The distinct colocalization of neuronal Ang II with D H in the heart may indicate that Ang II participates together with norepinephrine in the regulation of cardiac functions: Produced as a cardiac neurotransmitter Ang II may have inotropic, chronotropic or dromotropic effects in atria and ventricles and contributes to blood pressure regulation.

Cardiorenal End Points in a Trial of Aliskiren for Type 2 Diabetes

BACKGROUND: This study was undertaken to determine whether use of the direct renin inhibitor aliskiren would reduce cardiovascular and renal events in patients with type 2 diabetes and chronic kidney disease, cardiovascular disease, or both. METHODS: In a double-blind fashion, we randomly assigned 8561 patients to aliskiren (300 mg daily) or placebo as an adjunct to an angiotensin-converting-enzyme inhibitor or an angiotensin-receptor blocker. The primary end point was a composite of the time to cardiovascular death or a first occurrence of cardiac arrest with resuscitation; nonfatal myocardial infarction; nonfatal stroke; unplanned hospitalization for heart failure; end-stage renal disease, death attributable to kidney failure, or the need for renal-replacement therapy with no dialysis or transplantation available or initiated; or doubling of the baseline serum creatinine level. RESULTS: The trial was stopped prematurely after the second interim efficacy analysis. After a median follow-up of 32.9 months, the primary end point had occurred in 783 patients (18.3%) assigned to aliskiren as compared with 732 (17.1%) assigned to placebo (hazard ratio, 1.08; 95% confidence interval [CI], 0.98 to 1.20; P=0.12). Effects on secondary renal end points were similar. Systolic and diastolic blood pressures were lower with aliskiren (between-group differences, 1.3 and 0.6 mm Hg, respectively) and the mean reduction in the urinary albumin-to-creatinine ratio was greater (between-group difference, 14 percentage points; 95% CI, 11 to 17). The proportion of patients with hyperkalemia (serum potassium level, ≥6 mmol per liter) was significantly higher in the aliskiren group than in the placebo group (11.2% vs. 7.2%), as was the proportion with reported hypotension (12.1% vs. 8.3%) (P<0.001 for both comparisons). CONCLUSIONS: The addition of aliskiren to standard therapy with renin-angiotensin system blockade in patients with type 2 diabetes who are at high risk for cardiovascular and renal events is not supported by these data and may even be harmful. (Funded by Novartis; ALTITUDE ClinicalTrials.gov number, NCT00549757.).

Arterial hypertension and proteinuria in pediatric chronic kidney disease

A variety of chronic kidney diseases tend to progress towards end-stage kidney disease. Progression is largely due to factors unrelated to the initial disease, including arterial hypertension and proteinuria. Intensive treatment of these two factors is potentially able to slow the progression of kidney disease. Blockers of the renin-angiotensin-aldosterone system, either converting enzyme inhibitors or angiotensin II receptor antagonists, reduce both blood pressure and proteinuria and appear superior to a conventional antihypertensive treatment regimen in preventing progression to end-stage kidney disease. The most recent recommendations state that in children with chronic kidney disease without proteinuria the blood pressure goal is the corresponding 75th centile for body length, age and gender; whereas the 50th centile should be aimed in children with chronic kidney disease and pathologically increased proteinuria.

[Microalbuminuria--a new cardiovascular risk factor?]

Microalbuminuria is an established risk factor for renal disease, especially in the diabetic population. Recent studies have shown that microalbuminuria has also a highly relevant predictive value for cardiovascular morbidity and mortality. From normal to overt proteinuria levels, albuminuria shows a continuous marked increase in cardiovascular risk. This association is independent of other "classical" cardiovascular risk factors such as hypertension, hyperlipidemia or smoking. Furthermore it has a predictive value not only for patients with diabetic or renal disease, but also for hypertensive individuals or the general population. Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers have been shown to display not only reno--but also cardioprotective effects. Their unique ability to lower albuminuria by 40% is related to a significant risk reduction in cardiovascular mortality. New clinical trials are needed to define "normal" albuminuria levels and how low we should go.

Are beta-blockers needed in patients receiving spironolactone for severe chronic heart failure? An analysis of the COPERNICUS study

BACKGROUND: The beneficial effects of beta-blockers and aldosterone receptor antagonists are now well established in patients with severe systolic chronic heart failure (CHF). However, it is unclear whether beta-blockers are able to provide additional benefit in patients already receiving aldosterone antagonists. We therefore examined this question in the COPERNICUS study of 2289 patients with severe CHF receiving the beta1-beta2/alpha1 blocker carvedilol compared with placebo. METHODS: Patients were divided post hoc into subgroups according to whether they were receiving spironolactone (n = 445) or not (n = 1844) at baseline. Consistency of the effect of carvedilol versus placebo was examined for these subgroups with respect to the predefined end points of all-cause mortality, death or CHF-related hospitalizations, death or cardiovascular hospitalizations, and death or all-cause hospitalizations. RESULTS: The beneficial effect of carvedilol was similar among patients who were or were not receiving spironolactone for each of the 4 efficacy measures. For all-cause mortality, the Cox model hazard ratio for carvedilol compared with placebo was 0.65 (95% CI 0.36-1.15) in patients receiving spironolactone and 0.65 (0.51-0.83) in patients not receiving spironolactone. Hazard ratios for death or all-cause hospitalization were 0.76 (0.55-1.05) versus 0.76 (0.66-0.88); for death or cardiovascular hospitalization, 0.61 (0.42-0.89) versus 0.75 (0.64-0.88); and for death or CHF hospitalization, 0.63 (0.43-0.94) versus 0.70 (0.59-0.84), in patients receiving and not receiving spironolactone, respectively. The safety and tolerability of treatment with carvedilol were also similar, regardless of background spironolactone. CONCLUSION: Carvedilol remained clinically efficacious in the COPERNICUS study of patients with severe CHF when added to background spironolactone in patients who were practically all receiving angiotensin-converting enzyme inhibitor (or angiotensin II antagonist) therapy. Therefore, the use of spironolactone in patients with severe CHF does not obviate the necessity of additional treatment that interferes with the adverse effects of sympathetic activation, specifically beta-blockade.

Effects of antihypertensive drugs on blood pressure and proteinuria in childhood

BACKGROUND: Historically, there have been few drug trials for antihypertensive treatment in childhood and recommendations have been extrapolated from data obtained in adulthood. During the last decade an increased awareness of the risks of childhood hypertension stimulated clinical trials of antihypertensive agents in children. OBJECTIVE: The aim of this article is to systematically review the studies published between 1995 and 2006 that deal with the effect of antihypertensive drugs on childhood hypertension or proteinuria. METHODS: Medline, Current Contents, personal files and reference lists were used as data sources. RESULTS: Fifty-two out of 79 initially found reports were excluded. Consequently 27 articles were retained for the final analysis. The blood pressure reduction was similar with angiotensin-converting enzyme inhibitors (10.7/8.1 mmHg), angiotensin II receptor antagonists (10.5/6.9 mmHg) and calcium-channel blockers (9.3/7.2 mmHg). In addition angiotensin-converting enzyme inhibitors (by 49%) and angiotensin II receptor antagonists (by 59%) significantly reduced pathological proteinuria. CONCLUSIONS: The blood pressure reduction of angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists and calcium-channel blockers is almost identical. In children with pathological proteinuria angiotensin-converting enzyme inhibitors or angiotensin II antagonists are superior to calcium-channel blockers.

Adjuvant TACE inhibitor treatment improves the outcome of TLR2-/- mice with experimental pneumococcal meningitis

BACKGROUND: Streptococcus (S.) pneumoniae meningitis has a high lethality despite antibiotic treatment. Inflammation is a major pathogenetic factor, which is unresponsive to antibiotics. Therefore adjunctive therapies with antiinflammatory compounds have been developed. TNF484 is a TNF-alpha converting enzyme (TACE) inhibitor and has been found efficacious in experimental meningitis. Toll-like receptor 2 (TLR2) contributes to host response in pneumococcal meningitis by enhancing bacterial clearing and downmodulating inflammation. In this study, TNF484 was applied in mice, which lacked TLR2 and exhibited a strong meningeal inflammation. METHODS: 103 CFU S. pneumoniae serotype 3 was inoculated subarachnoidally into C57BL/6 wild type (wt) mice or TLR2-/-, CD14-/- and CD14-/-/TLR2-/- mice. Severity of disease and survival was followed over 9 days. Response to antibiotics (80 mg/kg ceftriaxone i.p. for 5 days) and/or TACE inhibitor treatment (1 mg/kg s.c. twice daily for 4 days) was evaluated. Animals were sacrificed after 12, 24, and 48 h for analysis of bacterial load in cerebrospinal fluid (CSF) and brain and for TNF and leukocyte measurements in CSF. RESULTS: TLR2-/- mice were significantly sicker than the other mouse strains 24 h after infection. All knockout mice showed higher disease severity after 48 h and died earlier than wt mice. TNF release into CSF was significantly more elevated in TLR2-/- than in the other strains after 24 h. Brain bacterial numbers were significantly higher in all knockout than wt mice after 24 h. Modulation of outcome by antibiotic and TACE inhibitor treatment was evaluated. With antibiotic therapy all wt, CD14-/- and TLR2-/-/CD14-/- mice, but only 79% of TLR2-/- mice, were rescued. TACE inhibitor treatment alone did not rescue, but prolonged survival in wt mice, and in TLR2-/- and CD14-/- mice to the values observed in untreated wt mice. By combined antibiotic and TACE inhibitor treatment 95% of TLR2-/- mice were rescued. CONCLUSION: During pneumococcal meningitis strong inflammation in TLR2-deficiency was associated with incomplete responsiveness to antibiotics and complete response to combined antibiotic and TACE inhibitor treatment. TACE inhibitor treatment offers a promising adjuvant therapeutic strategy in pneumococcal meningitis.

Ramipril increases the protein level of skeletal muscle IRS-1 and alters protein tyrosine phosphatase activity in spontaneously hypertensive rats

To investigate mechanisms by which angiotensin converting enzyme (ACE)-inhibition increases insulin sensitivity, spontaneously hypertensive (SH) rats were treated with or without ramipril (1 mg/kg per day) for 12 weeks. Insulin binding and protein levels of insulin receptor substrate-1 (IRS-1), p85-subunit of phosphatidylinositol 3'-kinase (p85) and Src homology 2 domain-containing phosphatase-2 (SHP2) were then determined in hindlimb muscle and liver. Additionally, protein tyrosine phosphatase (PTPase) activities towards immobilized phosphorylated insulin receptor or phosphorylated IRS-1 of membrane (MF) and cytosolic fractions (CF) of these tissues were measured. Ramipril treatment increased IRS-1-protein content in muscle by 31+/-9% (P<0.05). No effects were observed on IRS-1 content in liver or on insulin binding or protein expression of p85 or SHP2 in both tissues. Ramipril treatment also increased dephosphorylation of insulin receptor by muscle CF (22.0+/-1.0%/60 min compared to 16.8+/-1.5%/60 min; P<0.05), and of IRS-1 by liver MF (37.2+/-1.7%/7.5 min compared to 33.8+/-1.7%/7.5 min; P<0.05) and CF (36.8+/-1.0%/7.5 min compared to 33.2+/-1.0%/7.5 min; P<0.05). We conclude that the observed effects of ACE-inhibition by ramipril on the protein expression of IRS-1 and on PTPase activity might contribute to its effect on insulin sensitivity.

Scleroderma Renal Crisis: A Rare but Severe Complication of Systemic Sclerosis

Scleroderma renal crisis (SRC) is a major complication in patients with systemic sclerosis (SSc). It is characterized by malignant hypertension and oligo/anuric acute renal failure. SRC occurs in 5% of patients with SSc, particularly in the first years of disease evolution and in the diffuse form. The occurrence of SRC is more common in patients treated with glucocorticoids, the risk increasing with increasing dose. Left ventricular insufficiency and hypertensive encephalopathy are typical clinical features. Thrombotic microangiopathy is detected in 43% of the cases. Anti-RNA-polymerase III antibodies are present in one third of patients who develop SRC. Renal biopsy is not necessary if SRC presents with classical features. However, it can help to define prognosis and guide treatment in atypical forms. The prognosis of SRC has dramatically improved with the introduction of angiotensin-converting enzyme inhibitors (ACEi). However, 5 years survival in SSc patients who develop the full picture of SRC remains low (65%). SRC is often triggered by nephrotoxic drugs and/or intravascular volume depletion. The treatment of SRC relies on aggressive control of blood pressure with ACEi, if needed in combination with other types of antihypertensive drugs. Dialysis is frequently indicated, but can be stopped in approximately half of patients, mainly in those for whom a perfect control of blood pressure is obtained. Patients who need dialysis for more than 2 years qualify for renal transplantation.

Proton-coupled oligopeptide transporter family SLC15: physiological, pharmacological and pathological implications

Mammalian members of the proton-coupled oligopeptide transporter family (SLC15) are integral membrane proteins that mediate the cellular uptake of di/tripeptides and peptide-like drugs. The driving force for uphill electrogenic symport is the chemical gradient and membrane potential which favors proton uptake into the cell along with the peptide/mimetic substrate. The peptide transporters are responsible for the absorption and conservation of dietary protein digestion products in the intestine and kidney, respectively, and in maintaining homeostasis of neuropeptides in the brain. They are also responsible for the absorption and disposition of a number of pharmacologically important compounds including some aminocephalosporins, angiotensin-converting enzyme inhibitors, antiviral prodrugs, and others. In this review, we provide updated information on the structure-function of PepT1 (SLC15A1), PepT2 (SLC15A2), PhT1 (SLC15A4) and PhT2 (SLC15A3), and their expression and localization in key tissues. Moreover, mammalian peptide transporters are discussed in regard to pharmacogenomic and regulatory implications on host pharmacology and disease, and as potential targets for drug delivery. Significant emphasis is placed on the evolving role of these peptide transporters as elucidated by studies using genetically modified animals. Whenever possible, the relevance of drug-drug interactions and regulatory mechanisms are evaluated using in vivo studies.

Quality of care after acute coronary syndromes in a prospective cohort with reasons for non-prescription of recommended medications

BACKGROUND Adherence to guidelines is associated with improved outcomes of patients with acute coronary syndrome (ACS). Clinical registries developed to assess quality of care at discharge often do not collect the reasons for non-prescription for proven efficacious preventive medication in Continental Europe. In a prospective cohort of patients hospitalized for an ACS, we aimed at measuring the rate of recommended treatment at discharge, using pre-specified quality indicators recommended in cardiologic guidelines and including systematic collection of reasons for non-prescription for preventive medications. METHODS In a prospective cohort with 1260 patients hospitalized for ACS, we measured the rate of recommended treatment at discharge in 4 academic centers in Switzerland. Performance measures for medication at discharge were pre-specified according to guidelines, systematically collected for all patients and included in a centralized database. RESULTS Six hundred and eighty eight patients(54.6%) were discharged with a main diagnosis of STEMI, 491(39%) of NSTEMI and 81(6.4%) of unstable angina. Mean age was 64 years and 21.3% were women. 94.6% were prescribed angiotensin converting enzyme inhibitors/angiotensin II receptor blockers at discharge when only considering raw prescription rates, but increased to 99.5% when including reasons non-prescription. For statins, rates increased from 98% to 98.6% when including reasons for non-prescription and for beta-blockers, from 82% to 93%. For aspirin, rates further increased from 99.4% to 100% and from to 99.8% to 100% for P2Y12 inhibitors. CONCLUSIONS We found a very high adherence to ACS guidelines for drug prescriptions at discharge when including reasons for non-prescription to drug therapy. For beta-blockers, prescription rates were suboptimal, even after taking into account reason for non-prescription. In an era of improving quality of care to achieve 100% prescription rates at discharge unless contra-indicated, pre-specification of reasons for non-prescription for cardiovascular preventive medication permits to identify remaining gaps in quality of care at discharge.

Lack of efficacy of low-dose spironolactone as adjunct treatment to conventional congestive heart failure treatment in dogs.

Aldosterone plays an important role in the pathophysiology of heart failure. Aldosterone receptor blockade has been shown to reduce morbidity and mortality in human patients with advanced congestive left ventricular heart failure. This study was designed to assess the efficacy and tolerance of long-term low-dose spironolactone when added to conventional heart failure treatment in dogs with advanced heart failure. Eighteen client-owned dogs with advanced congestive heart failure due to either degenerative valve disease (n=11) or dilated cardiomyopathy (n=7) were included in this prospective, placebo-controlled, double-blinded, randomized clinical study. After initial stabilization including furosemide, angiotensin-converting enzyme inhibitors, pimobendan and digoxin, spironolactone at a median dose of 0.52 mg/kg (range 0.49-0.8 mg/kg) once daily (n=9) or placebo (n=9) was added to the treatment, and the dogs were reassessed 3 and 6 months later. Clinical scoring, echocardiography, electrocardiogram, systolic blood pressure measurement, thoracic radiography, sodium, potassium, urea, creatinine, alanine aminotransferase, aldosterone and aminoterminal atrial natriuretic propeptide were assessed at baseline, 3 and 6 months. Survival times were not significantly different between the two treatment groups. Spironolactone was well tolerated when combined with conventional heart failure treatment.

The matrix metalloproteinase inhibitor RS-130830 attenuates brain injury in experimental pneumococcal meningitis.

BACKGROUND Pneumococcal meningitis (PM) is characterized by high mortality and morbidity including long-term neurofunctional deficits. Neuropathological correlates of these sequelae are apoptosis in the hippocampal dentate gyrus and necrosis in the cortex. Matrix metalloproteinases (MMPs) play a critical role in the pathophysiology of PM. RS-130830 (Ro-1130830, CTS-1027) is a potent partially selective inhibitor of MMPs of a second generation and has been evaluated in clinical trials as an anti-arthritis drug. It inhibits MMPs involved in acute inflammation but has low activity against MMP-1 (interstitial collagenase), MMP-7 (matrilysin) and tumour necrosis factor α converting enzyme (TACE). METHODS A well-established infant rat model of PM was used where live Streptococcus pneumoniae were injected intracisternally and antibiotic treatment with ceftriaxone was initiated 18 h post infection (hpi). Treatment with RS-130830 (75 mg/kg bis in die (bid) i.p., n = 40) was started at 3 hpi while control littermates received the vehicle (succinylated gelatine, n = 42). RESULTS Cortical necrosis was significantly attenuated in animals treated with RS-130830, while the extent of hippocampal apoptosis was not influenced. At 18 hpi, concentrations of interleukin (IL)-1β and IL-10 were significantly lower in the cerebrospinal fluid of treated animals compared to controls. RS-130830 significantly reduced weight loss and leukocyte counts in the cerebrospinal fluid of survivors of PM. CONCLUSION This study identifies MMP inhibition, specifically with RS-130830, as an efficient strategy to attenuate disease severity and cortical brain injury in PM.