Effect of cilobradine in cats with a first episode of congestive heart failure due to primary cardiomyopathy.

INTRODUCTION: Heart rate (HR) is often elevated in cats with cardiomyopathies (CMPs). Pharmacologic modulation of HR may reduce cardiac morbidity and mortality. OBJECTIVES: To investigate the effects of cilobradine vs. placebo, regarding time to cardiac mortality or morbidity in cats with first episode of congestive heart failure (CHF) due to primary CMP. ANIMALS: Three hundred and sixty-seven client-owned cats with primary CMP that had presented with a first episode of CHF at 50 centers in Europe. Per-protocol population comprised 193 cats (n = 89 cilobradine, n = 104 placebo). An interim analysis for futility was planned. METHODS: Prospective, randomized, placebo-controlled, double-blinded, multicenter clinical trial. Primary outcome variable was the time to a composite of cardiac mortality or cardiac morbidity. RESULTS: Median time to primary outcome was 84 days (95% confidence interval [CI]: 63-219 days) in the cilobradine group (CG) and 203 days in the placebo group (95% CI: 145-377 days) with observed hazard ratio of 1.44, indicating a higher hazard for the CG (P = 0.057). Mean HR was 28 beats per minute (bpm) lower at Day 7 (P < 0.0001) and remained 29 bpm lower at Day 360 (P = 0.026) in the CG than that in the placebo group. Although the number of adverse events did not differ, there were more serious adverse events in the CG. CONCLUSIONS: Heart rate reduction by cilobradine in cats with a first episode of CHF due to primary CMP did not reduce cardiac mortality and morbidity.

The Cause of Acute Respiratory Failure Predicts the Outcome of Noninvasive Ventilation in Immunocompromised Children.

BACKGROUND: Noninvasive ventilation (NIV) may be superior to conventional therapy in immunocompromised children with respiratory failure. METHODS: Mortality, success rate, prognostic factors and side effects of NIV for acute respiratory failure (ARF) were investigated retrospectively in 41 in children with primary immunodeficiency, after stem cell transplantation or chemotherapy for oncologic disease. RESULTS: In 11/41 (27%) children invasive ventilation was avoided and patients were discharged from ICU. In children with NIV failure ICU-mortality was 19/30 (63%). 8/11 (72%) children with NIV success had recurrence of ARF after 27 days. Only 4/11 (36%) children with first episode NIV success and 8/30 (27%) with NIV failure survived to hospital discharge. Lower FiO2, SpO2/FiO2 and blood culture positive bacterial sepsis were predictive for NIV success, while fungal sepsis or culture negative ARF were predictive for NIV failure. We observed catecholamine treatment in 14/41 (34%), pneumothorax in 2/41 (5%), mediastinal emphysema in 3/41 (7%), a life threatening nasopharyngeal hemorrhage and need for resuscitation during intubation in 5/41 (12%) NIV-episodes. CONCLUSIONS: The prognosis of ARF in immunocompromised children remains guarded independent of initial success or failure of NIV due to a high rate of recurrent ARF. Reversible causes like bacterial sepsis had a higher NIV response rate. Relevant side effects of NIV were observed.

[Prevalence of heart murmurs, aortic and pulmonic stenosis in boxers presented for pre-breeding exams in Switzerland]. / Prävalenz von Herzgeräuschen, Aorten- und Pulmonalstenosen bei Zuchtuntersuchungen von Boxern in der Schweiz.

Boxer are predisposed to subaortic (SAS) and pulmonic stenosis (PS). To decrease the prevalence, pre-breeding cardiologic exams were performed in the last years. In our study the results of 309 pre-breeding exams of boxers presented between 1999 and 2008 were evaluated retrospectively. The overall prevalence of heart murmurs was 26.5 %. A SAS was diagnosed in 25 (8.1 %) and a PS in 10 (3.3 %) dogs. A combination of both defects was found in 7 (2.3 %) Boxers. Animals with a heart murmur of at least grade 3/6 had a significantly higher peak aortic flow velocity (VmaxAo) than animals without or only soft heart murmurs. Over the study period both the frequency of heart murmurs and diagnosis of SAS and PS decreased.

ICBP90 belongs to a new family of proteins with an expression that is deregulated in cancer cells.

ICBP90 (Inverted CCAAT box Binding Protein of 90 kDa) is a recently identified nuclear protein that binds to one of the inverted CCAAT boxes of the topoisomerase IIalpha (TopoIIalpha) gene promoter. Here, we show that ICBP90 shares structural homology with several other proteins, including Np95, the human and mouse NIRF, suggesting the emergence of a new family of nuclear proteins. Towards elucidating the functions of this family, we analysed the expression of ICBP90 in various cancer or noncancer cell lines and in normal or breast carcinoma tissues. We found that cancer cell lines express higher levels of ICBP90 and TopoIIalpha than noncancer cell lines. By using cell-cycle phase-blocking drugs, we show that in primary cultured human lung fibroblasts, ICBP90 expression peaks at late G1 and during G2/M phases. In contrast, cancer cell lines such as HeLa, Jurkat and A549 show constant ICBP90 expression throughout the entire cell cycle. The effect of overexpression of E2F-1 is more efficient on ICBP90 and TopoIIalpha expression in noncancer cells (IMR90, WI38) than in cancer cells (U2OS, SaOs). Together, these results show that ICBP90 expression is altered in cancer cell lines and is upregulated by E2F-1 overexpression with an efficiency depending on the cancer status of the cell line.

Attenuated agonist evoked vasoconstrictor responses in the perfused mesenteric vascular bed of streptozotocin diabetic rats.

We compared agonist-evoked responses in the perfused mesenteric vascular bed (MVB) of streptozotocin (STZ) diabetic Sprague-Dawley rats 2 and 14 weeks after induction of diabetes. Endothelin-1 (ET-1)-, methoxamine (MTX)-, and KCl-evoked vasoconstrictor responses were unchanged in 2-week-old diabetic rats. In contrast, both the sensitivity (P < 0.01) and the maximal vasoconstrictor responses (P < 0.05) to ET-1 were attenuated in 14-week-old diabetic rats, whereas endothelin plasma levels were increased (P < 0.05). Although no differences were observed in responses to KCl in either the 2- or 14-week-old diabetic groups, MTX-evoked maximal responses were attenuated in the 14-week-old group (P < 0.01). Changes in agonist-evoked responses in the 14-week-old diabetic group were unaffected by the protein kinase C (PKC) inhibitor, staurosporine, the phospholipase C (PLC) inhibitor, U73122, the calcium channel blocker, nifedipine, the calcium pump inhibitor, cyclopiazonic acid (CPA), or by endothelial denudation. Sodium fluoride (NaF), an activator of guanosine triphosphate binding proteins (G proteins) normalized the responses in the 14-week-old diabetic group. These data suggest that advanced stages of STZ are associated with alterations in G protein receptor coupling and/or activity leading to the attenuation of responses to vasoconstrictor agonists.

Genomic structure and chromosomal mapping of the gene coding for ICBP90, a protein involved in the regulation of the topoisomerase IIalpha gene expression.

We have recently identified a novel CCAAT box binding protein (ICBP90) involved in the regulation of topoisomerase IIalpha gene expression. We have observed that it is expressed in non-tumoral proliferating human lung fibroblast cells whereas in HeLa cells, a tumoral cell line, ICBP90 was still present even when cells were at confluence. In the present study, we have determined the ICBP90 gene structure by screening of a human placenta genomic library and PCR analysis. We report that the ICBP90 gene spans about 35.8 kb and contains six coding exons named A to F. In the 5' upstream sequence of the region containing the coding exons, two additional exons (I and II) were found. Additionally, an internal splicing site was found in exon A. A promoter region, including three putative Sp1 binding sites between exons I and A, was identified by transient transfection. Northern blot analysis of several cancer cell lines revealed the existence of two ICBP90 mRNA species of 5.1 and 4.3 kb that are transcribed from the gene. The relative amounts of these mRNAs depended on the cell type. In MOLT-4 cells and Burkitt's lymphoma Raji cells, the 4.3 kb or the 5.1 kb transcripts were mainly observed, respectively. In other cell lines, such as HL-60 cells, chronic myelogenous leukaemia K-562, lung carcinoma A549, HeLa or colorectal SW480, both 4.3 and 5.1 kb forms of ICBP90 mRNA could be detected. Interestingly, western blot analysis showed several ICBP90 protein bands in HeLa but only a single band in MOLT-4 cell extracts. Taken together our results are consistent with the ICBP90 gene exhibiting alternative splicing and promoter usage in a cell-specific manner.

Altered paracrine effect of endothelin in blood vessels of the hyperinsulinemic, insulin resistant obese Zucker rat.

OBJECTIVE: Earlier, we reported that high insulin incubation in vitro leads to increased ETA receptor expression in cultured rat aortic smooth muscle cells (Diabetes 1998, 47: 934-944). Our later observation of enhanced endothelin-1 evoked vasoconstriction in aorta from the hyperinsulinemic obese Zucker rat indicated that this interaction might also be relevant in vivo. To further examine the relationship between insulinemia and endothelin, we characterized endothelin receptor expression and endothelin-1 peptide levels in vascular tissues and plasma from young and old obese Zucker rats. METHODS: 12 and 40-week-old Zucker obese and lean rats were used. Plasma endothelin-1 levels and endothelin-1 peptide content in the mesenteric artery and in the thoracic aorta were examined by radioimmunoassay. Messenger RNA levels of endothelin-1 peptide and ETA and ETB receptors were examined in the aortic and mesenteric vessels using RT-PCR. RESULTS: Obese rats from both age groups had significantly higher plasma levels of insulin (4-10 fold), total cholesterol (2-3 fold), triglycerides (10-fold), and glucose (approximately 1.5 fold) than their lean counterparts. There was a trend toward worsening lipoproteinemia and glycemia, but improved insulinemia with age in the obese rats. In association with these changes, obese rats exhibited attenuated endothelin-1 peptide and preproET-1 mRNA levels, but conversely elevated ETA and ETB receptor mRNA levels in both aortic and mesenteric vessels. CONCLUSION: These data suggest that vascular tissue from the metabolically dysregulated obese Zucker rat exhibits attenuated endothelin-1 peptide production and elevated endothelin receptor levels. Since elevated insulin levels have been linked to increased endothelin receptor expression, it is plausible that hyperinsulinemia upregulates endothelin receptors contributing to elevated vasoconstrictor responses to endothelin-1 in this model of obesity and hypertension.

ICBP90, a novel human CCAAT binding protein, involved in the regulation of topoisomerase IIalpha expression.

The one-hybrid system with an inverted CCAAT box as the DNA target sequence was used to identify proteins acting on key DNA sequences of the promoter of the topoisomerase IIalpha gene. Screening of cDNA libraries from the leukemia Jurkat cell line and from the adult human thymus resulted in the isolation of a novel protein of 793 amino acids (89,758 Da). This protein has in vitro CCAAT binding properties and has been called ICBP90. Adult thymus, fetal thymus, fetal liver, and bone marrow, known as active tissues in terms of cell proliferation, are the tissues richest in ICBP90 mRNA. In contrast, highly differentiated tissues and cells such as the central nervous system and peripheral leukocytes are free of ICBP90 mRNA. Western blotting experiments showed a simultaneous expression of topoisomerase IIalpha and ICBP90 in proliferating human lung fibroblasts. Simultaneous expression of both proteins has also been observed in HeLa cells, but in both proliferating and confluent cells. Overexpression of ICBP90 in COS-1-transfected cells induced an enhanced expression of endogenous topoisomerase IIalpha. Immunohistochemistry experiments showed that topoisomerase IIalpha and ICBP90 were coexpressed in proliferating areas of paraffin-embedded human appendix tissues and in high-grade breast carcinoma tissues. We have identified ICBP90, which is a novel CCAAT binding protein, and our results suggest that it may be involved in topoisomerase IIalpha expression. ICBP90 may also be useful as a new proliferation marker for cancer tissues.

Lanoteplase Genetics Institute.

Lanoteplase, a modified form of a tissue plasminogen activator (t-PA) lacking fibronectin finger-like and epidermal growth factor domains, was developed by the Genetics Institute for the potential treatment of thromboembolic disorders. Suntory, the Japanese licensee, has filed an NDA in Japan for the treatment of acute myocardial infarction (MI) [301222]. It was also licensed to Bristol-Myers Squibb (BMS) for worldwide development (excluding Japan, China, South Korea and Taiwan) [178225]. BMS conducted phase III trials for acute MI [272490] but discontinued development and returned the license to Genetics Institute during 1999 [359688]. In February 1999, Lehman Brothers predicted the drug had a 60% probability of reaching market, with an estimated first launch date in 2000. The analysts predicted peak sales would occur in 2006, with peak sales of $200 million in the US and US $100 million in the rest of the world at that time [319225].

Effect of sodium orthovanadate treatment on cardiovascular function in the hyperinsulinemic, insulin-resistant obese Zucker rat.

We recently demonstrated that oral vanadate treatment ameliorates exaggerated vasoconstriction in aortic tissue from the hyperinsulinemic/insulin resistant obese Zucker rat. It has been suggested that changes in large artery contractility might contribute to the development of hypertension in this strain. Thus we examined the effect of vanadate treatment (0.5 mg/ml, p.o.) on conductance and resistance vessel function as well as blood pressure (BP) in Zucker rats. Vasoconstrictor responses to endothelin-1 (ET-1) and methoxamine and vasodilator responses to acetylcholine in the aorta and perfused mesenteric vascular bed served as indices of conductance and resistance function, respectively. Separate groups were treated with insulin (12 mU/kg/min, s.c.) to determine its role in the actions of vanadate. Vanadate treatment reduced (2.5-fold; p < 0.05) elevated plasma insulin levels and abolished exaggerated aortic vasoconstriction in obese rats. Vasoconstrictor responses in the mesenteric bed, however, were similar between obese and lean rats, and were unaffected by vanadate. Vanadate did not affect elevated BP in obese rats and actually increased BP in the lean group. Insulin treatment per se failed to affect vasomotor function or BP in either strain, and acetylcholine-evoked relaxation was similar in all groups. We conclude that whereas vanadate overcomes exaggerated central artery contractility in obese Zucker rats, it fails to affect resistance vessel function or BP in this strain, and might conversely elevate BP in normotensive lean control rats. The vascular actions of vanadate in obese rats appear to occur independent of changes in plasma insulin or endothelial function.

Vasopressin-evoked [Ca2+]i responses in neonatal rat cardiomyocytes.

The presence of arginine vasopressin (AVP) V1 receptors on neonatal rat cardiomyocytes (NRCs) linked to processes capable of elevating intracellular free calcium ([Ca2+]i) is now firmly established. This study examined the sources and signaling involved in [Ca2+]i elevations evoked by AVP in NRCs. AVP promoted increases in both [Ca2+]i and 1,4,5-inositoltrisphosphate (IP3) levels in NRCs. The degree of [Ca2+]i elevation was less than that of angiotensin II, but greater than that of endothelin-1. Extracellular Mg2+ depletion led to diminution of the maximal [Ca2+]i response, with a rightward shift in the concentration-response curves to AVP. The phospholipase C inhibitors, D-609, NCDC, or U73122, and the IP3 receptor blocker, heparin, abolished the [Ca2+]i response to AVP. Neither cyclooxygenase inhibition with indomethacin nor PKC inhibition with staurosporine had any effect. Neither ryanodine nor caffeine, which deplete sarcoplasmic reticulum (SR) Ca2+ stores, nor ruthenium red, which inhibits both SR and mitochondrial Ca2+ stores, affected [Ca2+]i responses to AVP. The SR Ca2+ pump inhibitor, cyclopiazonic acid, abolished, and removal of extracellular Ca2+ attenuated, the response to AVP. These data indicate that activation of cardiac V1 receptors by AVP results in mobilization of Ca2+ from a distinct, non-SR, nonmitochondrial, intracellular Ca2+ pool that is Ca2+ pump replenished and IP3 sensitive. This process occurs secondary to phospholipase C (PLC)-mediated generation of IP3, requires the presence of Mg2+ and extracellular Ca2+, and occurs in a manner independent of PKC and cyclooxygenase activation. Such mechanisms of Ca2+ mobilization might indicate a distinct role for AVP in cardiac physiology and disease.

Phosphatidic acid increases inositol-1,4,5,-trisphosphate and [Ca2+]i levels in neonatal rat cardiomyocytes.

Phosphatidic acid (PA), which can be synthesized de novo, or as a product of phosphatidylcholine hydrolysis and/or phosphorylation of 1,2-diacylglycerol (DAG), mediates diverse cellular functions in various cell types, including cardiomyocytes. We set out to characterize the effect of PA on intracellular free calcium ([Ca2+]i) and inositol-1,4,5-trisphosphate (IP(3)) levels in primary cultures of neonatal rat cardiomyocytes. Addition of PA led to rapid, concentration and time dependent increases in both IP(3) and [Ca2+]i levels in adherent cells. There was strong correlation in the concentration-response relationships between IP(3) and [Ca2+]i increases evoked by PA. Incubation with the sarcoplasmic reticulum (SR) Ca2+ pump inhibitor, cyclopiazonic acid (CPA), significantly attenuated the PA evoked [Ca2+]i increase but had no significant effect on IP(3) accumulation. The phospholipase C (PLC) inhibitor, D-609, attenuated both IP(3) and [Ca2+]i elevations evoked by PA whereas staurosporine (STS), a potent and non-selective PKC inhibitor, had no significant effect on either. Another PLC inhibitor, U73122, but not its inactive analog, U73343, also inhibited PA evoked increases in [Ca2+]i. Depletion of extracellular calcium attenuated both basal and PA evoked increases in [Ca2+]i. The PLA(2) inhibitors, bromophenylacyl-bromide (BPB) and CDP-choline, had no effect on PA evoked [Ca2+]i responses. Neither the DAG analog, dioctanoylglycerol, nor the DAG kinase inhibitor, R59949, affected PA evoked changes in [Ca2+]i. Taken together, these data indicate that PA, in a manner independent of PKC, DAG, or PLA(2), may enhance Ca2+ release from IP(3) sensitive SR Ca(2+) stores via activation of PLC in neonatal rat cardiomyocytes.

Pneumadin-evoked intracellular free Ca2+ responses in rat aortic smooth muscle cells: effect of dexamethasone.

The direct vascular effect of pneumadin (PN) was determined by studying the changes in intracellular free calcium ([Ca2+]i) levels in cultured rat aortic smooth muscle cells maintained between the second and fifth passages. PN evoked a rapid, concentration-dependent, biphasic increase in [Ca2+]i. The [Ca2+]i level rose from a basal value of 108 nM to a maximum increase in peak value of 170 nM. Although the level of maximal [Ca2+]i response evoked by PN was less than with other vasoactive agonists, it was more potent (EC50 0.5 nM) than even endothelin-1 (EC50 3.1 nM). At concentrations > 100 nM, [Ca2+]i elevations induced by PN above basal levels were no longer observed. Pretreatment with dexamethasone (100 nM for 24 hr) resulted in a significant increase (P < 0.01) in the peak [Ca2+]i response (310 nM) to PN. However, the biphasic pattern in the peak [Ca2+]i responses encountered with increasing concentrations of PN remained unaffected. The exaggerated [Ca2+]i response to PN was abolished by preincubation of cells with either the glucocorticoid antagonist mifepristone (RU 486) or the protein synthesis inhibitor cycloheximide. Inclusion of either an AT1 antagonist (losartan), a V1 selective vasopressin antagonist (d(Ch2)5 Tyr (Me) AVP), or an alpha-adrenoceptor antagonist (phentolamine) failed to affect the increases in [Ca2+]i induced by PN. PN-evoked increases in inositol 1,4,5-trisphosphate levels paralleled the [Ca2+]i changes. These data suggest that PN increases Ca2+ mobilization in rat aortic smooth muscle cells via activation of phospholipase C coupled receptors. This effect is up-regulated by dexamethasone.

Vasopressin accelerates protein synthesis in neonatal rat cardiomyocytes.

Arginine vasopressin (AVP) has been shown to promote vascular smooth muscle cell hypertrophy and hyperplasia of fibroblasts. The present study examines the effect of AVP and endothelin-1 (ET-1) on protein, DNA, and RNA synthesis in primary cultures of serum deprived neonatal rat cardiomyocytes (RC) as assessed by changes in [3H] phenylalanine, [3H] thymidine, and [14C] uridine incorporation respectively. Both AVP and ET-1 evoked significant increases in protein synthesis in RC of 36 +/- 12% (p < 0.05) and 53 +/- 22% (p < 0.01) respectively. The stimulating action of AVP on [3H] phenylalanine incorporation was abolished by pretreatment with 2-nitro-4carboxyphenyl-N, N-diphenylcarbamate (NCDC), a phospholipase C (PLC) inhibitor. [14C] uridine incorporation was significantly higher in cells incubated with ET-1 (95 +/- 12%) but not AVP (9 +/- 11%). Neither AVP nor ET-1 significantly affected cell number or [3H]thymidine incorporation, suggesting a lack of a hyperplastic effect. AVP evoked an increase in [Ca2+]i levels (162 +/- 12 nmol/L from a basal value of 77 +/- 6 nmol/L) which was completely abolished by pretreatment with either NCDC or cyclopiazonic acid (sarcoplasmic reticulum (SR) Ca2+ pump inhibitor) but unaffected by ryanodine (ryanodine sensitive SR Ca2+ store depletor). Taken together, these data suggest that AVP, in a PLC dependent manner, both stimulates protein synthesis and augments [Ca2+]i release in RC from ryanodine insensitive (IP3 sensitive) Ca2+ stores. Thus, AVP may promote cardiac hypertrophy via direct effects on cardiomyocyte protein synthesis secondary to IP3 mediated [Ca2+]i release.

Plasma endothelin levels and vascular responses at different temporal stages of streptozotocin diabetes.

While alterations in the release and action of endothelial derived vasoactive factors such as endothelin- and endothelial derived relaxing factor (EDRF) occur in diabetes mellitus, the nature and direction of these changes is controversial. We examined the role of diabetes duration on endothelin- plasma levels and vasoconstrictor responses to endothelin-1 in aortic rings from streptozotocin-induced diabetic rats. Endothelin-1 plasma levels were attenuated at 2-weeks, but conversely elevated at 14-weeks, after diabetes induction. Similarly, maximal vasoconstriction to endothelin-1 in aorta was exaggerated in 2-week, but attenuated in 14-week diabetic rats. Also, sensitivity of aorta to endothelin-1 was enhanced in the 14-week group. Neither nitric oxide synthase inhibition with nitro-L-arginine-methyl-ester, nor endothelium removal affected alterations in maximal vasoconstriction, but both abolished changes in sensitivity to endothelin-1. Endothelium dependent (acetylcholine-evoked) vasorelaxation responses were attenuated in 14-week, but not 2-week, diabetic rats, while endothelium independent (sodium nitroprusside-evoked) responses remained unchanged. Together, these data indicate a deficiency in EDRF production in the 14-week group. Elevated plasma glucose and attenuated insulin levels were present in both groups, but plasma cholesterol and triglyceride levels were elevated only in 14-week rats. We conclude that differences in the pre-existing duration of diabetes differentially affect both plasma levels and action of endothelin-1. These changes might be linked to coincident diabetes duration dependent changes in EDRF production and plasma lipid levels. Such a shift in the production and action of endothelial derived relaxing and contracting factors might contribute to the characteristic early and late stage cardiovascular complications of diabetes mellitus.