Leszek Kalinowski

Nebivolol Reduces Nitroxidative Stress and Restores Nitric Oxide Bioavailability in Endothelium of Black Americans

Background— Alterations in endothelial function may contribute to increased susceptibility of black Americans to cardiovascular disease. The ability to pharmacologically reverse endothelial dysfunction in blacks was tested with nebivolol, a &bgr;1-selective agent with vasodilating and antioxidant properties. Methods and Results— The effects of nebivolol on endothelial nitric oxide (NO), superoxide (O2−), and peroxynitrite concentration (ONOO−) release were studied in human umbilical vein endothelial cells and iliac artery endothelial cells isolated from age-matched black and white donors. Kinetics and concentrations of NO/O2−/ONOO− were measured simultaneously with nanosensors from single cells and shown to have significant interracial differences. The rate of NO release was ≈5 times slower in blacks than in whites (94 versus 505 nmol · L−1 · s−1), whereas the rates of release were faster by ≈2 times for O2− and ≈4 times for ONOO− (22.1 versus 9.4 nmol · L−1 · s−1 for O2− and 810 versus 209 nmol · L−1 · s−1 for ONOO−). Pretreatment with 1.0 to 5.0 &mgr;mol/L nebivolol restored NO bioavailability in endothelial cells from black donors with concurrent reductions in O2− and ONOO− release, similar to levels in the endothelium of whites. The effects of nebivolol were dose-dependent and not observed with atenolol; similar effects were observed with apocynin, an NAD(P)H oxidase inhibitor. Conclusions— Reduced endothelial NO bioavailability in American blacks is mainly due to excessive O2− and ONOO− generation by NAD(P)H and uncoupled endothelial NO synthase. Nebivolol decreased O2− and ONOO− concentrations and restored NO bioavailability in blacks to the level recorded in cells from whites, independently of &bgr;1-selective blockade.

Angiotensin II AT1 Receptor Antagonists Inhibit Platelet Adhesion and Aggregation by Nitric Oxide Release

Abstract—This study investigated the process of nitric oxide (NO) release from platelets after stimulation with different angiotensin II type 1 (AT1)-receptor antagonists and its effect on platelet adhesion and aggregation. Angiotensin II AT1-receptor antagonist–stimulated NO release in platelets was compared with that in human umbilical vein endothelial cells by using a highly sensitive porphyrinic microsensor. In vitro and ex vivo effects of angiotensin II AT1-receptor antagonists on platelet adhesion to collagen and thromboxane A2 analog U46619-induced aggregation were evaluated. Losartan, EXP3174, and valsartan alone caused NO release from platelets and endothelial cells in a dose-dependent manner in the range of 0.01 to 100 &mgr;mol/L, which was attenuated by NO synthase inhibitor NG-nitro-l-arginine methyl ester. The angiotensin II AT1-receptor antagonists had more than 70% greater potency in NO release in platelets than in endothelial cells. The degree of inhibition of platelet adhesion (collagen-stimulated) and aggregation (U46619-stimulated) elicited by losartan, EXP3174, and valsartan, either in vitro or ex vivo, closely correlated with the NO levels produced by each of these drugs alone. The inhibiting effects of angiotensin II AT1-receptor antagonists on collagen-stimulated adhesion and U46619-stimulated aggregation of platelets were significantly reduced by pretreatment with NG-nitro-l-arginine methyl ester. Neither the AT2 receptor antagonist PD123319, the cyclooxygenase synthase inhibitor indomethacin, nor the selective thromboxane A2/prostaglandin H2 receptor antagonist SQ29,548 had any effect on angiotensin II AT1-receptor antagonist–stimulated NO release in platelets and endothelial cells. The presented studies clearly indicate a crucial role of NO in the arterial antithrombotic effects of angiotensin II AT1-receptor antagonists.

Cicletanine stimulates nitric oxide release and scavenges superoxide in endothelial cells.

&NA; Cicletanine ((±)3‐(4‐chlorophenyl)‐1,3‐dihydro‐7‐hydroxy‐6‐methylfuro‐[3,4‐c] pyridine) 3‐(4‐chlorophenyl)‐1,3‐dihydro‐7‐hydroxy‐6‐methylfuro‐[3,4‐c] pyridine) is a novel antihypertensive vasodilator with an incompletely understood mechanism of action. In the studies described here, the release of nitric oxide and superoxide (O2−) stimulated by cicletanine was measured simultaneously in the endothelium of isolated rat aortic rings. Highly sensitive electrochemical nitric oxide and O2− microsensors were placed near the surface of endothelial cells and the kinetics of nitric oxide and O2− release were monitored in situ. The response times for nitric oxide and O2− microsensors were 100 &mgr;s and 50 &mgr;s, respectively, and detection limit was 10‐9 M. Cicletanine stimulated nitric oxide release in aorta endothelium at (micromolar) therapeutic concentrations that were consistent with the concentrations of the compound to induce endothelium‐dependent vasorelaxation in isolated rat aorta. The peak concentration of nitric oxide was 160 ± 8 nM. This concentration was about 70% and was 60% lower as compared with the nitric oxide peak concentration observed after stimulation with receptorindependent agonist (calcium ionophore A23187) and receptor‐dependent agonist (acetylcholine), respectively. However, after administration of cicletanine, only a small concentration of O2− was recorded (peak 3.1 ± 0.2 nM) contrary to a large concentration (27 ± 1.35 nM) observed after stimulation with A23187). Cicletanine not only stimulated nitric oxide release but also was a potent scavenger of O2− at nanomolar level. Both of these effects may contribute to potent vasorelaxation properties of cicletanine and its long‐term therapeutic actions, resulting in cardiovascular tissue protection.

Effect of chronic treatment with the vasopeptidase inhibitor AVE 7688 and ramipril on endothelial function in atherogenic diet rabbits

Cardiovascular disease is the major cause of death in Western nations, although improved possibilities regarding diagnosis and therapy now exist. Endothelial dysfunction is triggered by cardiovascular risk factors such as hypercholesterolaemia, hypertension, adiposity and smoking, contributing to the common endpoint of atherosclerosis. This study examined the pharmacological effects of angiotensin-converting enzyme (ACE) and combined ACE-neutral endopeptidase (NEP) (vasopeptidase) inhibitors on endothelial dysfunction in the model of hyperlipidaemic rabbits. The focus of the study was to assess endothelial function after treatment with the ACE-NEP inhibitor AVE 7688 (30 mg/kg/day) in comparison to the ACE inhibitor (ACE-I) ramipril (1 mg/kg/day). Different parameters, such as endothelial function, blood pressure (BP), expansion of plaques, endothelial nitric oxide (NO) and superoxide (O2 ) release and plasma levels of various lipidaemic parameters were analysed. Control groups consisted of one group fed only with normal diet, one group fed only with atherogenic diet and the direct control group fed with varied diets (six weeks atherogenic diet followed by 12 weeks normal diet). Since for the treatment of atherosclerosis, a change in feeding is absolutely necessary, in the present study, at the start of the treatments with AVE 7688 and ramipril, the rabbits food was changed to a normal diet. At the end of the study, mean arterial blood pressure (MAP) was measured in the anaesthetised animals. The values in standard, atherogenic and varied diet-fed rabbits were around 73±2 mmHg. Angiotensin I (Ang I) given intravenous (i.v.) induced a strong increase in MAP of about 20%. In both the treated groups Ang I-induced BP increase was inhibited. In contrast, i.v. bradykinin led to a strong reduction in MAP in both the treated groups of around 50%. Six weeks feeding with an atherogenic diet in the rabbits induced an enduring endothelial dysfunction despite the food subsequently being changed to a normal chow. All measured parameters indicated a significant favourable effect on endothelial dysfunction as a result of the two treatment regimens. Endothelial function measured in the organ chamber showed somewhat greater improvement in the ACE-NEP treated group than in the ACE-I treated group. The treatment with ramipril, as well as with AVE 7688, restored endothelial function by increasing the ratio of NO to O2- concentration and bioavailability of NO. In this study, a similar protective effect on endothelial function was shown by ACE-NEP inhibition as already seen with ACE inhibitors in an animal model of atherosclerosis.

Altered circadian genes expression in breast cancer tissue according to the clinical characteristics

Breast cancer has a multifactorial etiology. One of the supposed and novel mechanisms is an alteration of circadian gene expression. Circadian genes play a crucial role in many physiological processes. These processes, such as genomic stability, DNA repair mechanism and apoptosis, are frequently disrupted in breast tumors. To assess the significance of circadian gene expression in breast cancer, we carried out an analysis of CLOCK, BMAL1, NPAS2, PER1, PER2, PER3 and CRY1, CRY2, TIMELESS, CSNK1E expression by the use of the quantitative Real-Time PCR technique in tumor tissue and non-tumor adjacent normal tissue sampled from 107 women with a newly diagnosed disease. The obtained data were compared to the clinical and histopathological features. PER1, PER2, PER3, CRY2 were found to be significantly down-expressed, while CLOCK, TIMELESS were over-expressed in the studied tumor samples compared to the non-tumor samples. Only gene expression of CRY1 was significantly down-regulated with progression according to the TNM classification. We found significantly decreased expression of CRY2, PER1, PER2 genes in the ER/PR negative breast tumors compared to the ER/PR positive tumors. Additionally, expression of CRY2, NPAS2 genes had a decreased level in the poorly differentiated tumors in comparison with the well and moderately differentiated ones. Our results indicate that circadian gene expression is altered in breast cancer tissue, which confirms previous observations from various animal and in vitro studies.

Multimodal imaging of the receptor for advanced glycation end-products with molecularly targeted nanoparticles

The receptor for advanced glycation end-products (RAGE) is central to multiple disease states, including diabetes-related conditions such as peripheral arterial disease (PAD). Despite RAGEs importance in these pathologies, there remains a need for a molecular imaging agent that can accurately assess RAGE levels in vivo. Therefore, we have developed a multimodal nanoparticle-based imaging agent targeted at RAGE with the well-characterized RAGE ligand, carboxymethyllysine (CML)-modified human serum albumin (HSA). Methods: A multimodal tracer (64Cu-Rho-G4-CML) was developed using a generation-4 (G4) polyamidoamine (PAMAM) dendrimer, conjugated with both rhodamine and copper-64 (64Cu) chelator (NOTA) for optical and PET imaging, respectively. First, 64Cu-Rho-G4-CML and its non-targeted analogue (64Cu-Rho-G4-HSA) were evaluated chemically using techniques such as dynamic light scattering (DLS), electron microscopy and nuclear magnetic resonance (NMR). The tracers binding capabilities were examined at the cellular level and optimized using live and fixed HUVEC cells grown in 5.5-30 mM glucose, followed by in vivo PET-CT imaging, where the probes kinetics, biodistribution, and RAGE targeting properties were examined in a murine model of hindlimb ischemia. Finally, histological assessment of RAGE levels in both ischemic and non-ischemic tissues was performed. Conclusions: Our RAGE-targeted probe demonstrated an average size of 450 nm, a Kd of 340-390 nM, rapid blood clearance, and a 3.4 times greater PET uptake in ischemic RAGE-expressing hindlimbs than their non-ischemic counterpart. We successfully demonstrated increased RAGE expression in a murine model of hindlimb ischemia and the feasibility for non-invasive examination of cellular, tissue, and whole-body RAGE levels with a molecularly targeted tracer.