Identification of two preclinical canine models of atrial fibrillation to facilitate drug discovery.

BACKGROUND: Atrial fibrillation (AF) is the most common arrhythmia occurring in humans, and new treatment strategies are critically needed. The lack of reliable preclinical animal models of AF is a major limitation to drug development of novel antiarrhythmic compounds. OBJECTIVE: The purpose of this study was to provide a comprehensive head-to-head assessment of 5 canine AF models. METHODS: Five canine models were evaluated for the efficacy of AF induction and AF duration. We tested 2 acute models: short-term atrial tachypacing (AT) for 6 hours with analysis of AF at hourly increments, and carbachol injection into a cardiac fat pad followed by short-term AT. We also tested 3 chronic models: pacemaker implantation followed by either 4 weeks of AT and subsequent atrial burst pacing or intermittent long-term AT for up to 4-5 months to generate AF ≥4.5 hours, and finally ventricular tachypacing to induce heart failure followed by atrial burst pacing to induce AF. RESULTS: Careful evaluation showed that acute AT, AT for 4 weeks, and the heart failure model all were unsuccessful in generating reproducible AF episodes of sufficient duration to study antiarrhythmic drugs. In contrast, intermittent long-term AT generated AF lasting ≥4.5 hours in ∼30% of animals. The acute model using carbachol and short-term AT resulted in AF induction of ≥15 minutes in ≥75% of animals, thus enabling testing of antiarrhythmic drugs. CONCLUSION: Intermittent long-term AT and the combination of local carbachol injection with successive short-term AT may contribute to future drug development efforts for AF treatment.

Spindle disturbances in human-hamster hybrid (A(L) ) cells induced by the electrical component of the mobile communication frequency range signal.

The production of spindle disturbances in a human-hamster hybrid (A(L) ) cell line by an electromagnetic field (EMF) with field strength of 90 V/m at a frequency of 900 MHz was studied in greater detail. The experimental setup presented allows investigating whether either the electrical (E) and/or the magnetic (H) field component of EMF can be associated with the effectiveness of the spindle-disturbing potential. Therefore, both field components of a transversal electromagnetic field (TEM) wave have been separated during exposure of the biological system. This procedure should give more insight on understanding the underlying mechanisms of non-thermal effects of EMF. A statistical comparison of the proportions of the fractions of ana- and telophases with spindle disturbances, obtained for five different exposure conditions with respect to unexposed controls (sham condition), showed that only cells exposed to the H-field component of the EMF were not different from the control. Therefore, the results of the present study indicate that an exposure of cells to EMF at E-field strengths of 45 and 90 V/m, as well as to the separated E component of the EMF, induces significant spindle disturbances in ana- and telophases of the cell cycle.

Design and synthesis of potent and selective azaindole-based Rho kinase (ROCK) inhibitors.

Rho kinase plays a pivotal role in several cellular processes such as vasoregulation, making it a suitable target for the treatment of hypertension and related disorders. We discovered a new compound class of Rho kinase (ROCK) inhibitors containing a 7-azaindole hinge-binding scaffold tethered to an aminopyrimidine core. Herein we describe the structure-activity relationships elucidated through biochemical and functional assays. The introduction of suitable substituents at the 3-position of the bicyclic moiety led to an increase in activity, which was required to design compounds with favorable pharmacokinetic profile. Azaindole 32 was identified as a highly selective and orally available ROCK inhibitor able to cause a sustained blood pressure reduction in vivo.

Spindle disturbances in human-hamster hybrid (AL) cells induced by mobile communication frequency range signals.

The production of spindle disturbances in FC2 cells, a human-hamster hybrid (A(L)) cell line, by non-ionizing radiation was studied using an electromagnetic field with a field strength of 90 V/m at a frequency of 835 MHz. Due to the given experimental conditions slide flask cultures were exposed at room temperature in a microTEM (transversal electromagnetic field) cell, which allows optimal experimental conditions for small samples of biological material. Numerical calculations suggest that specific absorption rates of up to 60 mW/kg are reached for maximum field exposure. All exposure field parameters--either measured or calculable--are precisely defined and, for the first time, traceable to the standards of the SI system of physical units. Compared with co-incident negative controls, the results of two independently performed experiments suggest that exposure periods of time from 0.5 to 2 h with an electric field strength of 90 V/m are spindle acting agents as predominately indicated by the appearance of spindle disturbances at the ana- and telophase stages (especially lagging and non-disjunction of single chromosomes) of cell divisions. The spindle disturbances do not change the fraction of mitotic cells with increasing exposure time up to 2 h. Due to the applied experimental conditions an influence of temperature as a confounder parameter for spindle disturbances can be excluded.

Beneficial cardiovascular effects of endothelin ET(A) receptor blockade in established long-term heart failure after myocardial infarction.

Although experimental prevention studies have suggested therapeutic potential of endothelin (ET) antagonists for the treatment of heart failure, the results of clinical trials using ET antagonists on top of standard heart failure medications have been largely disappointing. This experimental study investigated the effects of chronic ET(A) receptor blockade in long-term survivors of myocardial infarction who had developed stable chronic heart failure in the absence of other treatments. Systolic blood pressure, heart rate, organ weights of the right atrium and ventricle, and the lungs were determined, and tissue ET-1 peptide levels were measured in cardiac tissue, lung, and aorta. The results show that chronic blockade of ET(A) receptors stabilizes systolic blood pressure and reverses the heart failure-induced weight increases of right heart chambers and lung. The changes observed occurred independently of tissue ET-1 concentrations and heart rate, suggesting mechanisms independent of local cardiac or pulmonary ET-1 synthesis, which are yet to be identified.

Endothelin ETA receptor blockade with darusentan increases sodium and potassium excretion in aging rats.

This study investigated whether intrarenal endothelin-1(ET-1) contributes to sodium excretion in aged rats. Metabolic function studies were performed in male Wistar rats (3 and 24 months) treated with placebo or the orally active ET(A) receptor antagonist darusentan (20 mg/kg/d) for 4 weeks. Mean arterial pressure was measured using an intra-arterial catheter. Electrolytes, aldosterone levels, renin activity, and angiotensin converting enzyme activity were determined in plasma, and mRNA expression of epithelial sodium channel (ENaC) and Na(+), K(+)-ATPase subunits was measured in the renal cortex and medulla. Aging was associated with a marked decrease in urinary excretion of sodium, chloride, and potassium (all P < 0.001) as well as renin activity (P < 0.05), but had no significant effect on gene expression of ENaC or Na(+), K(+)-ATPase subunits. In aged rats, darusentan treatment increased ion excretion (P < 0.05), reduced cortical gene expression of alphaENaC and alpha(1)-Na(+), K(+)-ATPase (both P < 0.05), and increased plasma aldosterone levels (P < 0.01). These data demonstrate a decrease of sodium and potassium excretion in aged rats, changes that are partly sensitive to ETA receptor blockade. Treatment with darusentan also reduced cortical expression of alphaENaC and alpha(1)-Na(+), K(+)-ATPase and increased plasma aldosterone levels independently of blood pressure, electrolytes, renin activity, or angiotensin converting enzyme activity. These findings may provide new pathogenetic links between aging and sodium sensitivity.

Endothelin A antagonist LU-135252 and trandolapril in the treatment of the Cohen-Rosenthal diabetic hypertensive rat.

BACKGROUND: Hypertension and non-insulin-dependent diabetes mellitus (NIDDM) often occur simultaneously and the combination requires vigorous control of hypertension. This can generally be achieved by a combination of antihypertensive drugs. The present study examines the antihypertensive and possible hypoglycemic effects of combined therapy with endothelin A (ETA) receptor antagonist LU-135252 and angiotensin-converting enzyme (ACE) inhibitor trandolapril in male Cohen-Rosenthal Diabetic Hypertensive (CRDH) rats. METHODS: Rats were divided into four groups as follows: group I served as control; group II--LU-135252 30 mg/kg/day; group III--trandolapril 0.1 mg/kg/day and group IV--both LU-135252 30 mg/kg/day and trandolapril 0.1 mg/kg/day. Systolic blood pressure (SBP) and plasma glucose levels were evaluated at the beginning of the experiment and after 2, 4 and 6 weeks. RESULTS: SBP decreased significantly in all treated groups after 2, 4 and 6 weeks of treatment compared to baseline. Maximum decrease was in group IV (combination) from 174.8+/-3.7 to 136.1+/-2.4 mmHg (22%) (p<0.0001); in group III (trandolapril) from 165.8+/-2.7 to 137.5+/-2.9 mmHg (17%) (p=0.0002); and in group II (LU-135252) and from 169.1+/-3.1 to 147.8+/-2.5 mmHg (12%) (p=0.0004). Glucose levels in plasma decreased significantly after 6 weeks of treatment. Maximum decrease was in group IV: from 501.0+/-42.8 to 178.6+/-7.3 mg/dl (62%) (p<0.0001); in group III from 428.2+/-47.7 to 146.8+/-5.6 mg/dl (63%) (p<0.0001); and in group II from 491.2+/-39.3 to 272.2+/-28.3 mg/dl (42%) (p=0.0002). CONCLUSION: The SBP decrease was additive when both drugs were given together. Thus, combination of ETA antagonist and ACE inhibitor appears to offer a rational fixed-dose antihypertensive therapy, which is superior to that of either drug alone. The decrease in glucose level, which was the least impressive while on LU-135252 alone, was more prominent during combination after 2 weeks, although without further decrease.

Activation of pro-inflammatory and anti-inflammatory cytokines in host organs during chronic allograft rejection: role of endothelin receptor signaling.

This study investigated whether allograft rejection is associated with local inflammatory activation in host organs and whether endothelin ET(A) receptor signaling is involved. Expression of IL-1beta, IL-1ra, IL-6, IL-10 and TNF-alpha was investigated in host liver, lung and native heart in a rat model of chronic rejection 8 weeks after heterotopic cardiac transplantation in the absence of immunosuppression. In the presence of rejection, circulating levels of cytokines increased, while tissue level activation was dependent on the organ involved. Similarly, tissue-specific regulatory patterns were observed regarding transcriptional activation. Although chronic ET(A) receptor blockade did not reduce transplant vasculopathy or tissue protein expression, treatment had pronounced effects on plasma levels and transcriptional regulation of chemokines. These data provide evidence for distinct pro-inflammatory local activation in host organs during chronic rejection and suggest a role for ET(A) receptors contributing to regulation of cytokine plasma levels and transcriptional activity.

Anatomically distinct activation of endothelin-3 and the L-arginine/nitric oxide pathway in the kidney with advanced aging.

Aging is associated with spontaneous degenerative changes of renal function and structure. The aim of this study was to determine changes of the endothelin (ET) system and NO tissue bioactivity during the physiological aging process. Renal protein expression of ET-1 and ET-3, ETA, and ETB receptor mRNA expression, ET receptor binding and distribution, and tissue NO metabolite content were determined in adult, middle-aged, and senescent normotensive female Wistar rats. In senescent animals, medullary ET-3 content increased 3.4-fold (p<0.05 vs. adult), whereas aging did not affect ET-3 levels in the cortex. Local NO bioavailability, determined by NO metabolite tissue measurements, decreased in the cortex only. ET receptor binding capacity--predominantly due to ETB receptor binding--was lower in medulla than in cortex. Aging had no effect on ET-1 binding capacity or ET receptor distribution, whereas with advanced age gene expression of both receptors decreased. In conclusion, aging causes distinctive expressional changes of the renal endothelin system in otherwise healthy rats. The pronounced increase of endothelin-3 in the renal medulla is associated with preservation of local NO metabolite levels, changes not observed in the cortex. These findings could be important for pathologies and possibly therapy associated with renal aging.

Role of podocytes for reversal of glomerulosclerosis and proteinuria in the aging kidney after endothelin inhibition.

The cause of focal-segmental glomerulosclerosis as a consequence of physiological aging, which is believed to be inexorable, is unknown. This study investigated whether inhibition of endothelin-1, a growth-promoting peptide contributing to renal injury in hypertension and diabetes, affects established glomerulosclerosis and proteinuria in the aged kidney. We also determined the role of endothelin receptors for podocyte injury in vivo and in vitro. Aged Wistar rats, a model of spontaneous age-dependent glomerulosclerosis, were treated with the orally active endothelin subtype A (ET(A)) receptor antagonist darusentan, and evaluation of renal histology, renal function studies, and expression analyses were performed. In vitro experiments using puromycin aminonucleoside to induce podocyte injury investigated the role of ET(A) receptor signaling for apoptosis, cytoskeletal injury, and DNA synthesis. In aged Wistar rats, established glomerulosclerosis and proteinuria were reduced by >50% after 4 weeks of darusentan treatment, whereas blood pressure, glomerular filtration rate, or tubulo-interstitial renal injury remained unaffected. Improvement of structural injury in glomeruli and podocytes was accompanied by a reduction of the expression of matrix metalloproteinase-9 and p21Cip1/WAF1. In vitro experiments blocking ET(A) receptors using specific antagonists or RNA interference prevented apoptosis and structural damage to podocytes induced by puromycin aminonucleoside. In conclusion, these results support the hypothesis that endogenous endothelin contributes to glomerulosclerosis and proteinuria in the aging kidney. The results further suggest that age-dependent glomerulosclerosis is not merely a "degenerative" but a reversible process locally confined to the glomerulus involving recovery of podocytes from previous injury.

Renal damage in the SHR/N-cp type 2 diabetes model: comparison of an angiotensin-converting enzyme inhibitor and endothelin receptor blocker.

SUMMARY: The pathomechanisms that cause renal damage in diabetes have not been completely clarified. Treatment with angiotensin-converting enzyme inhibitors (ACE-i) is highly effective but fails to completely prevent end-stage renal disease. The effects of ET(A)-receptor blockers (ET(A)-RB) on renal damage are controversial and have rarely been investigated in type 2 diabetes. We compared the influence of the selective ET(A)-RB LU135252 and the ACE-i Trandolapril on renal structure in the SHR/N-cp rat model of type 2 diabetes. Three-month-old male SHR/N-cp rats were left untreated or received daily either Trandolapril or LU135252. The experiment was terminated after 6 months. The glomerulosclerosis index; tubulointerstitial damage index; and glomerular geometry, glomerular cell number, and capillary density were investigated. Proliferating cell nuclear antigen and desmin expression of podocytes, renal mRNA expression of endothelin (ET-1) and transforming growth factor-beta, blood pressure, and urine albumin excretion were measured. The glomerulosclerosis index was significantly higher in untreated diabetic animals than in the groups that were treated with ACE-i and ET(A)-RB. There were analogous changes in tubulointerstitial damage index. Treatment with either substance comparably lowered urinary albumin excretion in diabetic SHR/N-cp. Podocyte and endothelial cell numbers per glomerulus decreased in untreated diabetic animals; this was prevented by the ACE-i but not by the ET(A)-RB. Glomerular capillary length density was lower in SHR/N-cp, and this was normalized by ACE-i only. Increased expression of desmin and proliferating cell nuclear antigen expression of podocytes in the SHR/N-cp was abrogated by ACE-i but not by ET(A)-RB. Treatment with ACE-i or ET(A)-receptor antagonist resulted in less structural and functional alterations, but the ET(A)-RB was inferior to the ACE-i. This is particularly the case for podocyte changes pointing to angiotensin II-dependent pathomechanisms.

Trandolapril and endothelin antagonist LU-135252 in the treatment of the fructose-induced hypertensive, hyperinsulinemic, hypertriglyceridemic rat.

BACKGROUND: In view of the demonstrated interaction between endothelin and the renin-angiotensin system, the antihypertensive effect of combined therapy with an endothelin antagonist LU-135252 and the angiotensin converting enzyme inhibitor trandolapril, was studied in fructose-induced hypertensive, hyperinsulinemic, hypertriglyceridemic male Sprague-Dawley rats. METHODS: Forty animals were fed a fructose-enriched diet (Tekled, Harlan) for 5 weeks, as follows: group A, fructose only; group B, trandolapril 0.1 mg/kg/day added during the last 2 weeks; group C, LU-135252 100 mg/kg/day added during the last 2 weeks; group D, both trandolapril and LU-135252 added the last 2 weeks. Systolic blood pressure (BP) was measured weekly in conscious rats by the indirect tail-cuff method. Blood samples from a retro-orbital sinus puncture were taken at the beginning of the experiment and after 3 and 5 weeks and examined for insulin and triglyceride concentrations. RESULTS: Systolic BP decreased in group B (trandolapril) from 148.8 +/- 9.8 at 3 weeks to 138.3 +/- 8.7 mm Hg after 5 weeks; in group C (endothelin antagonist) from 155.1 +/- 5.5 to 142.5 +/- 10.6 mm Hg; and in group D (combination) from 154.6 +/- 10.9 to 121.2 +/- 8.9 mm Hg. Triglyceride levels decreased only in the combined trandolapril/endothelin antagonist group from 167.6 +/- 55.3 in the third week to 134.9 +/- 53.7 mg/dL after 5 weeks. Insulin levels decreased only on combination therapy from 7.4 +/- 3.6 to 5.3 +/- 3.8 ng/mL during the same period. The BP decrease was additive compared with the respective individual substances. CONCLUSIONS: The trandolapril/endothelin antagonist combination appears to offer a rational antihypertensive combination that is superior to that of either drug alone. This finding applies to the specific rat model studied in which BP, insulin, and triglycerides were increased by fructose diet.

Endothelial therapy of atherosclerosis and its risk factors.

Atherosclerosis is a chronic systemic disease of the vasculature with an inflammatory component. It accounts for the majority of cardiovascular morbidity and mortality in industrialized countries and its incidence is increasing in developing countries. The impairment of vascular endothelial cell function in atherosclerosis and in conditions associated with increased cardiovascular risk is an important determinant of disease progression. The reduction of endothelium-dependent relaxation in the coronary and systemic circulation in atherosclerosis is in part due to decreased bioavailability of nitric oxide and increased release of oxygen-derived free radicals. Atherosclerosis also increases the formation of vasoconstrictors and growth factors, adhesion of leukocytes, thrombosis, inflammation, cell proliferation, as well as increases in vascular tone. Here we review mechanisms and therapeutic approaches to improve endothelial pathways in atherosclerosis. Restoration of NO bioactivity through pharmacological inhibition of the renin-angiotensin system, statin therapy, or endothelin receptor blockade, ameliorates vascular function in experimental hypercholesterolemia, hypertension and heart failure. These treatments also have therapeutic benefit for patients at risk or with overt atherosclerosis, to reduce vascular and myocardial complications of this disease.

Obesity increases prostanoid-mediated vasoconstriction and vascular thromboxane receptor gene expression.

OBJECTIVES: Vasoconstrictor prostanoids have been implicated in abnormal vasomotion in atherosclerosis and hypertension. METHOD: Using lean and diet-induced obese mice, we investigated whether obesity affects vascular function or expression of genes involved in prostanoid action. RESULTS: In lean C57BL/6J mice, at high concentrations acetylcholine caused endothelium-dependent contractions in the carotid artery but not in the aorta. Endothelium-dependent contractions to acetylcholine were blocked by the non-selective cyclooxygenase (COX) inhibitors indomethacin and meclofenamate, or a prostaglandin H2/thromboxane A2 receptor antagonist, but not by inhibitors of COX-2, thromboxane synthase or cytochrome P450 monooxygenase. Obesity increased endothelium-dependent contractions to acetylcholine in the carotid artery, and prostanoid-mediated vasoconstriction was now present in the aorta. Similarly, contractions to endothelin-1 were largely blocked by meclofenamate and were increased in the aorta of obese mice. Real-time quantitative polymerase chain reaction analysis of the thromboxane receptor gene in the carotid artery revealed a robust upregulation in obese animals (18-fold, 0.05); in comparison, obesity had a less pronounced effect on thromboxane synthase (2.1-fold increase, 0.05), or preproendothelin-1 gene expression (4.2-fold increase, 0.05). CONCLUSIONS: These data demonstrate that obesity augments prostanoid-dependent vasoconstriction and markedly increases vascular thromboxane receptor gene expression. These changes are likely to promote the development of vascular disease, hypertension and thrombosis associated with obesity.

Long-term survival and hemodynamics after endothelin-a receptor antagonism and angiotensin-converting enzyme inhibition in rats with chronic heart failure: monotherapy versus combination therapy.

BACKGROUND: In patients with congestive heart failure (CHF) receiving ACE inhibitors, acute administration of selective endothelin (ET) antagonists additionally improves systemic and cardiac hemodynamics. We investigated, in a rat model of CHF, whether such acute synergistic effects are sustained and accompanied, in the long term, by an additional limitation of left ventricular remodeling or an increase in survival. METHODS AND RESULTS: Rats were subjected to coronary artery ligation and treated for 3 or 9 months with vehicle or with the ACE inhibitor trandolapril (Tr) (0.3 mg/kg(-1) per day(-1)), the ET(A) antagonist LU 135252 (LU, 30 mg/kg(-1) per day(-1)), or their combination starting 7 days after ligation. After 3 months, the combination decreased LV systolic- and end-diastolic pressures (-32% and -80%, respectively) more markedly than Tr (-21% and -61%, respectively) or LU alone (-14% and -48%, respectively). Echocardiographic studies revealed that all treatments limited LV dilatation and increased LV fractional shortening and cardiac index. All treatments equally reduced left ventricular collagen density, whereas only Tr or the combination reduced LV weight. Finally, although LU did not modify long-term survival, Tr and the combination of Tr with LU induced a similar improvement of survival. CONCLUSIONS: In this rat model, long-term combined administration of an ET(A) antagonist and an ACE inhibitor induces additional effects in terms of systemic and cardiac hemodynamics; however, this is not associated with an additional increase in long-term survival.

Endothelin A receptor blockade prevents capillary/myocyte mismatch in the heart of uremic animals.

In the heart of uremic animals and patients, the number of capillaries per volume of myocardium is reduced. Immunohistochemical studies demonstrated increased cardiac endothelin-1 (ET-1) expression in the left ventricle of uremic animals. Therefore, whether treatment with a selective ET(A)-receptor antagonist prevented such capillary-myocyte mismatch was investigated. Twenty-four h after subtotal nephrectomy, rats were left untreated or started on treatment with the ET(A)-receptor antagonist LU 135252 (20 mg/kg per d) and with the angiotensin-converting enzyme (ACE) inhibitor trandolapril (0.3 mg/kg per d), respectively. BP was monitored by telemetry. Myocardial capillary length density was analyzed by stereologic techniques that avoid anisotropy artifacts. In addition, cardiac ET-1 protein and mRNA were measured using immunohistochemistry, in situ hybridization, and quantitative reverse transcription-PCR. Changes in cardiac ET(A)-and ET(B)-PCR. receptor mRNA were measured using reverse transcription-PCR. Fifteen wk after subtotal nephrectomy, significantly reduced left ventricular capillary length density (3307 +/- 535 mm/mm(3)) was found compared with sham-operated controls (3995 +/- 471 mm/mm(3)); this was also seen in animals that were treated with trandolapril (3503 +/- 533 mm/mm(3)) but not in animals that were treated with LU 135252 (3800 +/- 303 mm/mm(3)). The results support a role of ET-1 in the genesis of left ventricular capillary/myocyte mismatch in uremia.