Treatment with aliskiren/amlodipine combination in patients with moderate-to-severe hypertension: a randomised, double-blind, active comparator trial.

AIMS: To assess the extent of reduction in blood pressure (BP) of aliskiren/amlodipine combination therapy compared with amlodipine monotherapy in moderate-to-severe hypertensive patients. METHODS: This was an 8-week multicentre, randomised, double-blind study. After a 1-to 4-week washout period, eligible patients [mean sitting systolic blood pressure (msSBP) ≥ 160 to < 200 mmHg] were randomised to receive a once-daily dose of aliskiren/amlodipine 150/5mg (n = 244) or amlodipine 5 mg (n = 241) for 1 week, followed by up-titration to aliskiren/amlodipine 300/10 mg or amlodipine 10 mg for 7 weeks. Efficacy outcome measures included change from baseline to week 8 endpoint in msSBP (primary endpoint), mean sitting diastolic blood pressure (msDBP), and BP control rate (< 140/90 mmHg). Safety was assessed by monitoring and recording all adverse events (AEs) and laboratory abnormalities. RESULTS: Patients' demographic characteristics were balanced between the two groups, mean baseline BP being 171.0/94.3 mmHg for aliskiren/amlodipine and 171.8/95.6 mmHg for amlodipine. Of 485 randomised patients, 433 (89.3%) completed the study. At week 8 endpoint, combination therapy resulted in significantly greater msSBP/msDBP reductions and BP control rate, compared with monotherapy (all: p ≤ 0.0001). The overall incidence of AEs was similar between the two groups. The most commonly reported AE was peripheral oedema with the incidence lower for combination therapy (14.4%) than for monotherapy (18.3%). CONCLUSION: In this population with considerably elevated BP, use of aliskiren/amlodipine combination showed significantly greater BP reductions and allowed more patients to achieve BP control compared with amlodipine monotherapy, with no additional safety concerns.

Ultrasound guided thrombin injection of pseudoaneurysm of the radial artery after percutaneous coronary intervention.

Thrombin injection is frequently used to occlude iatrogenic pseudoaneurysms in larger vessels, but has never successfully been used in the radial artery location. Here we report the use of this treatment in a patient with radial artery pseudoaneurysm following coronary intervention. After Doppler sonographic visualization of the pseudoaneurysm cavity and its neck, an ultrasound-guided transcutaneous injection of thrombin was carried out. Immediately after the injection, the pseudoaneurysm was completely clotted and Doppler measurement confirmed the stop of blood flow. The result suggests that ultrasound-guided injection of thrombin into a radial artery pseudoaneurysm following coronary intervention is a feasible alternative to surgical intervention.

A novel role for the cyclin-dependent kinase inhibitor p27(Kip1) in angiotensin II-stimulated vascular smooth muscle cell hypertrophy.

Angiotensin II (Ang II) has been shown to stimulate either hypertrophy or hyperplasia. We postulated that the differential response of vascular smooth muscle cells (VSMCs) to Ang II is mediated by the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1), which is abundant in quiescent cells and drops after serum stimulation. Ang II treatment (100 nM) of quiescent VSMCs led to upregulation of the cell-cycle regulatory proteins cyclin D1, Cdk2, proliferating cell nuclear antigen, and Cdk1. p27(Kip1) levels, however, remained high, and the activation of the G1-phase Cdk2 was inhibited as the cells underwent hypertrophy. Overexpression of p27(Kip1) cDNA inhibited serum-stimulated [(3)H]thymidine incorporation compared with control-transfected cells. This cell-cycle inhibition was associated with cellular hypertrophy, as reflected by an increase in the [(3)H]leucine/[(3)H]thymidine incorporation ratio and by an increase in forward-angle light scatter during flow cytometry at 48 hours after transfection. The role of p27(Kip1) in modulating the hypertrophic response of VSMCs to Ang II was further tested by antisense oligodeoxynucleotide (ODN) inhibition of p27(Kip1) expression. Ang II stimulated an increase in [(3)H]thymidine incorporation and the percentage of S-phase cells in antisense ODN-transfected cells but not in control ODN-transfected cells. We conclude that p27(Kip1) plays a role in mediating VSMC hypertrophy. Ang II stimulation of quiescent cells in which p27(Kip1) levels are high results in hypertrophy but promotes hyperplasia when levels of p27(Kip1) are low, as in the presence of other growth factors.

Neurokinin-1 receptor activation induces reactive oxygen species and epithelial damage in allergic airway inflammation.

BACKGROUND: An induction of reactive oxygen species (ROS) is characteristic for inflammation but the exact pathways have not been identified for allergic airway diseases so far. OBJECTIVE: The aim of this study was to characterize the role of the tachykinin NK-1 receptor on ROS production during allergen challenge and subsequent inflammation and remodelling. METHODS: Precision-cut lung slices of ovalbumin (OVA)-sensitized mice were cultivated and ROS-generation in response to OVA challenge (10 microg/mL) was examined by the 2',7'-dichloroflourescein-diacetate method. Long-term ROS effects on epithelial proliferation were investigated by 5-bromo-2'-deoxyuridine incorporation (72 h). In vivo, the results were validated in OVA-sensitized animals which were treated intra-nasally with either placebo, the tachykinin neurokinin 1 (NK-1) receptor antagonist SR 140333 or the anti-oxidant N-acetylcystein (NAC) before allergen challenge. Inflammatory infiltration and remodelling were assessed 48 h after allergen challenge. RESULTS: ROS generation was increased by 3.7-fold, which was inhibited by SR 140333. [Sar(9),Met(11)(O(2))]-Substance P (5 nM) caused a tachykinin NK-1 receptor-dependent fourfold increase in ROS generation. Epithelial proliferation was decreased by 68% by incubation with [Sar(9),Met(11)(O(2))]-SP over 72 h. In-vivo, treatment with SR 140333 and NAC reduced epithelial damage (91.4% and 76.8% vs. placebo, respectively, P<0.01) and goblet cell hyperplasia (67.4% and 50.1% vs. placebo, respectively, P<0.05), and decreased inflammatory cell influx (65.3% and 45.3% vs. placebo, respectively, P<0.01). CONCLUSION: Allergen challenge induces ROS in a tachykinin NK-1 receptor-dependent manner. Inhibition of the tachykinin NK-1 receptor reduces epithelial damage and subsequent remodelling in vivo. Therefore, patients may possibly benefit from treatment regime that includes radical scavengers or tachykinin NK-1 receptor antagonists.

Apoptosis of human macrophages by Flt-4 signaling: implications for atherosclerotic plaque pathology.

BACKGROUND: Neointimal inflammation and angiogenesis are important contributors of progression and destabilization of the atherosclerotic plaque. While the role of vascular endothelial growth factor (VEGF) and its receptors VEGF-R1 (Flt-1) and VEGF-R2 (Flk-1) in this process has clearly been defined, expression of the VEGF-R3 (Flt-4) has only been documented on lymphatic and tumor endothelium. This study examined Flt-4 expression in human atherosclerotic plaque and explored its implications for atherosclerotic disease. METHODS AND RESULTS: Carotid artery thrombendartherectomy specimens from 10 patients with unstable plaque were stained for Flt-4 and its specific growth factors VEGF-C and VEGF-D. Microvascular endothelial cells (MVEC) stained positive for VEGF-C and -D, but not for Flt-4. Interestingly, macrophages within inflammatory perivascular regions coexpressed Flt-4, VEGF-C and VEGF-D. In vitro studies confirmed the expression of Flt-4, VEGF-C and VEGF-D in human monocytes and cultured macrophages. Treatment of macrophages with VEGF-D induced apoptosis as determined by annexin V staining, by immunoblotting of activated caspase 3, and by the ratio of Bcl-2/Bax as well as by DNA fragmentation. Immunohistochemical studies of advanced human carotid atherosclerotic plaque confirmed the coexpression of Flt-4 with activated caspase 3 and TUNEL staining in macrophages, indicating an ongoing apoptotic process. CONCLUSION: Human monocytes/macrophages express VEGF-C and -D and their receptor Flt-4 in vitro and in vivo within advanced atherosclerotic lesions. Flt-4, in turn, mediates monocyte/macrophage apoptosis and may this way alter plaque stability.

Role of exercise cardiogoniometry in coronary artery disease diagnostics.

BACKGROUND: The use of noninvasive diagnostics in coronary artery disease remains underdeveloped. To date, there is no simple and inexpensive method that can lead to a reliable diagnosis. Aside from costly and elaborate imaging techniques, exercise ECG, with its rather moderate sensitivity and specificity, is the main diagnostic method available. METHODS: In this prospective study of 109 patients, the diagnostic value of cardiogoniometry (CGM), a three-dimensional, computer-analyzed vector cardiogram, was determined before and after physical stress, and the results were compared with those obtained from a stress test. We also investigated whether the sensitivity and specificity of the classical bicycle stress test could be increased with the addition of measurements obtained by CGM. Coronary angiography was used as a reference method. RESULTS: CGM had a sensitivity of 39% at rest and 42% after physical stress and a specificity of 63% at rest and 57% after stress. This method was found to be markedly inferior to pre-test probability (sensitivity 53%, specificity 81%), stress ECG (sensitivity 52%, specificity 81%), and resting ECG (sensitivity 50%, specificity 64%). The efficiency of exercise ECG testing was not improved by use of CGM results. CONCLUSION: If CGM is to be established as a viable diagnostic method in daily clinical practice, it must undergo further development.

Cell cycle protein expression in vascular smooth muscle cells in vitro and in vivo is regulated through phosphatidylinositol 3-kinase and mammalian target of rapamycin.

Cell cycle progression represents a key event in vascular proliferative diseases, one that depends on an increased rate of protein synthesis. An increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity is associated with vascular smooth muscle cell proliferation, and rapamycin, which blocks the activity of the mammalian target of rapamycin, inhibits this proliferation in vitro and in vivo. We hypothesized that these 2 molecules converge on a critical pathway of translational regulation that is essential for successful upregulation of cell cycle-regulatory proteins in activated smooth muscle cells. p70(S6) kinase, a target of PI 3-kinase and the mammalian target of rapamycin, was rapidly activated on growth factor stimulation of quiescent coronary artery smooth muscle cells and after balloon injury of rat carotid arteries. The translational repressor protein 4E-binding protein 1 was similarly hyperphosphorylated under these conditions. These events were associated with increases in the protein levels of cyclin B1, cyclin D1, cyclin E, cyclin-dependent kinase 1, cyclin-dependent kinase 2, proliferating cell nuclear antigen, and p21(Cip1) in vivo and in vitro, whereas inhibition of the PI 3-kinase signaling pathway with either rapamycin or wortmannin blocked the upregulation of these cell cycle proteins, but not mRNA, and arrested the cells in vitro before S phase. In contrast to findings in other cell types, growth factor- or balloon injury-induced downregulation of the cell cycle inhibitor p27(Kip1) was not affected by rapamycin treatment. These data suggest that cell cycle progression in vascular cells in vitro and in vivo depends on the integrity of the PI 3-kinase signaling pathway in allowing posttranscriptional accumulation of cell cycle proteins.

Cyclosporine-induced coronary artery constriction--dissociation between thromboxane release and coronary vasospasm.

Cyclosporine influences vascular tone, including that of coronary arteries. But its effect on myocardial prostanoid release, which may contribute to a drug-induced coronary and/or myocardial dysfunction, remains unknown. We used the isolated perfused rat heart to study the effect of cyclosporine on both the mechanical function parameters and myocardial prostanoid release into the effluent by ELISA. Cyclosporine (5 microM) induced an increase of perfusion pressure from 40 +/- 3 to 73 +/- 4 mm Hg within 60 minutes (p < 0.001), reflecting an increase of coronary tone. Cyclosporine did not affect heart rate but contractility (+dp/dtmax) tended to decrease, although not significantly. The drug's effect on coronary tone was rapidly reversible upon withdrawal. Cyclosporine perfusion resulted in an increase of thromboxane B2 liberation from 236 +/- 150 to 1321 +/- 354 pg/ml effluent (p < 0.001), whereas the 6-keto-prostaglandin F1 alpha release was unaffected. The vehicle cremophor did not change any of these parameters. Neither inhibition of myocardial prostanoid formation with acetylsalicylic acid nor thromboxane receptor blockade prevented the cyclosporine-induced increase of perfusion pressure. However, perfusion with nitroglycerin or the voltage-sensitive calcium channel antagonist nifedipine in addition to cyclosporine were able to prevent the increase of perfusion pressure. This is the first time it has been demonstrated that cyclosporine induces an acute release of the prostanoid thromboxane within the myocardium. Despite the resulting imbalance in favor of the vasoconstrictive prostanoid, a dependency of the cyclosporine-induced increase of coronary tone on this imbalance was excluded. Conversely, nitric oxide donation or calcium channel blockade were able to prevent the negative effect of the drug on coronary tone, supporting the concept of endothelium-dependent and/or myogenic mechanism of cyclosporine toxicity on the coronary vascular bed.

Isolation and intravenous injection of murine bone marrow derived monocytes.

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow. Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%± 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%± 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Deazaadenosine prevents leukozyte evasion during acute cardiac allograft rejection by suppression of adhesion molecule expression.

BACKGROUND: In the initial phase after cardiac transplantation, mononuclear cells infiltrate the graft initiating a relevant impulse for rejection. 3-Deazaadenosin (c3Ado), an analog of adenosine, has demonstrated in vitro anti-inflammatory properties. Furthermore, in vivo studies on arteriosclerosis development and septic myocardial dysfunction c3Ado revealed reduced cellular infiltration. In addition ischemia and reperfusion injury could be diminished in a pulmonary animal model. The aim of our study was to investigate the properties of c3Ado to reduce adhesion molecule expression and cellular infiltration in a fully allogeneic cardiac transplant model. METHODS AND RESULTS: Lewis rats were challenged with Wistar-Furth cardiac allografts. Untreated grafts were rejected within 7 days (group 1). In group 2, animals received 2 x 5 mg c3Ado SC per day. Grafts were harvested on days 1, 3, and 6 after transplantation for further examination (n = 4 per group and time point). Immunohistochemical examination revealed significant reduction of graft-infiltrating MHC II positive cells, T-cell receptor positive cells (R73), as well as ED1-positive monocytes and macrophages (P < .01) at days 3 and 6 after transplantation. Adhesion molecule (ICAM-1, VCAM-1) expression on days 1 and 3 after transplantation was almost completely diminished in c3Ado-treated grafts. CONCLUSION: Thus, c3Ado is able to reduce graft infiltration by preventing leukocyte evasion through the suppression of adhesion molecule expression. This may be a novel strategy to protect transplanted organs from early damage after transplantation and extend organ survival after transplantation.

Reversible clopidogrel resistance due to right ventricular myocardial infarction: risk factor of recurrent stent thrombosis?

A 63-year-old male Patient was admitted to the intensive care unit due to acute inferior myocardial infarction with right ventricular dysfunction. He received a loading dose of clopidogrel (600 mg) and aspirin (500 mg) and was immediately revascularized by reopening of the proximal right coronary artery (RCA) and implantation of a bare metal stent. After primary successful intervention the patient suffered from thoracic pain on day 5 of admission. The ECG indicated reinfarction. The proximal RCA was again re-opened by PTCA alone. The following day the patient suffered again from thoracic pain with ST-elevation in the inferior leads, this time complicated by additional total AV-blockade. The angiography showed another time a thrombotic occlusion of the initially implanted stent. He received another intervention with implantation of additional two bare-metal stents, an aortic counter-pulsation and a temporary two-chamber pace maker. Tirofiban was administered for 24 h and the IABP was withdrawn after 60 h. The patient was discharged on Aspirin 300 mg/d, Clopidogrel 150 mg/d and Enoxaparin 40 mg/d. Six weeks later the patient demonstrated an improved right ventricular function (TAPSE 18 mm), liver enzymes were normal, and inhibition of platelet aggregation by clopidogrel (150 mg/d) was sufficient. In conclusion this implies that the reversible "clopidogrel-resistance" might have been due to congestion and reduced metabolism due to right ventricular infarction.

Comparison of in vitro cardiovascular function with in vivo echocardiographic assessment after long-term administration of cyclosporine to rats.

Clinical reports indicate that cyclosporine is able to induce heart failure without rejection after heart transplantation. This supposition is supported by ex vivo animal studies, yet ex vivo studies do not account for the potential of counter-regulatory mechanisms, and the clinical observations seem rare in comparison with the number of patients treated with cyclosporine. We hypothesized that cyclosporine administration to rats would fail to exhibit any effect on myocardial contractility in vivo notwithstanding a negative influence ex vivo. Transthoracic echocardiographic examinations (two-dimensional targeted M-mode tracings) were done in a blinded fashion before and after 1-week treatment of rats (10 or 20 mg/kg/day cyclosporine i.p. vs. vehicle). After excision of the hearts, contractility and changes in coronary tone were determined ex vivo during flow-constant perfusion. Neither cyclosporine nor vehicle treatment resulted in changes of echocardiographic parameters (left ventricular diameter, fractional shortening). The heart rate was significantly increased in the high-dose cyclosporine group. This group showed a significant 38% reduction of contractility during the subsequent perfusion ex vivo, whereas low-dose cyclosporine or vehicle had no effect on myocardial performance. Vasoconstriction did not account for this impairment, because coronary tone was unaltered. Cyclosporine, given in doses used in animal studies, impairs myocardial contractility ex vivo but fails to exhibit any effect on myocardial performance in vivo, possibly because of an increase in sympathetic tone. Considering that the denervated transplanted heart in humans is even sensitized to adrenergic stimuli, our finding makes unlikely a clinical contribution of cyclosporine to failure after orthotopic heart transplantation.

Cell cycle progression: new therapeutic target for vascular proliferative disease.

Entry into and progression of vascular cells through the cell cycle is considered a key event in vascular proliferative diseases. Multiple growth factors and cytokines have been found to regulate vascular cell proliferation. However, the machinery regulating cell cycle represents the "final common pathway" of these signaling cascades and thus provides an attractive therapeutic target for the prevention of vascular proliferative diseases. This review focuses on the current understanding of the regulation of the cell cycle machinery especially as it relates to vascular cell biology and the feasibility of targeting cell cycle for the prevention of restenosis after balloon angioplasty and bypass vein graft disease.

3-deazaadenosine prevents adhesion molecule expression and atherosclerotic lesion formation in the aortas of C57BL/6J mice.

Adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) play an important role during the development of atherosclerosis. 3-Deazaadenosine (c(3)Ado), an adenosine analogue, inhibits endothelial-leukocyte adhesion and ICAM-1-expression in vitro. We hypothesized that c(3)Ado is able to prevent the expression of adhesion molecules and atherosclerotic lesion formation in female C57BL/6J mice. The animals were placed on an atherogenic diet with or without c(3)Ado for 9 weeks. Frozen cross sections of the proximal ascending aorta just beyond the aortic sinus were stained with oil red O, hematoxylin, and elastic van Gieson's stains and were analyzed by computer-aided planimetry for fatty plaque formation and neointimal proliferation. Monoclonal antibodies against CD11b (macrophages), VCAM-1, and ICAM-1 were used for immunohistochemistry. Mice on the atherogenic diet demonstrated multiple (5.4+/-1.6 per animal) lesions covering 3.4+/-2.8% of the endothelium and a marked neointima when compared with control mice (4501+/-775 versus 160+/-38 microm(2), P<0.001). Mice on the cholesterol-rich diet without c(3)Ado showed strong endothelial coexpression of ICAM-1 and VCAM-1. Moreover, there was a 10-fold increase in monocyte accumulation on the endothelial surface (33. 3+/-4.9 versus 3.8+/-1.2, P<0.004). In contrast, in mice treated with c(3)Ado, expression of ICAM-1 and VCAM-1 as well as monocyte adhesion and infiltration were almost completely inhibited. Furthermore, these mice did not show any fatty streak formation or neointima formation (125+/-32 microm(2)). Our results demonstrate that c(3)Ado can inhibit diet-induced fatty streak formation and the expression of endothelial ICAM-1 and VCAM-1 in C57BL/6J mice. This may provide a novel pharmacological approach in the prevention and treatment of atherosclerosis.