Andreia Zago Chignalia

Vascular Smooth Muscle Cell Differentiation to an Osteogenic Phenotype Involves TRPM7 Modulation by Magnesium

Arterial calcification, common in vascular diseases, involves vascular smooth muscle cell (VSMC) transformation to an osteoblast phenotype. Clinical studies suggest that magnesium may prevent this, but mechanisms are unclear. We assessed whether increasing magnesium levels reduce VSMC calcification and differentiation and questioned the role of the Mg2+ transporter, transient receptor potential melastatin (TRPM)7 cation channels in this process. Rat VSMCs were exposed to calcification medium in the absence and presence of magnesium (2.0 to 3.0 mmol/L) or 2-aminoethoxy-diphenylborate (2-APB) (TRPM7 inhibitor). VSMCs from mice with genetically low (MgL) or high-normal (MgH) [Mg2+]i were also studied. Calcification was assessed by von Kossa staining. Expression of osteocalcin, osteopontin, bone morphogenetic protein (BMP)-2, BMP-4, BMP-7, and matrix Gla protein and activity of TRPM7 (cytosol:membrane translocation) were determined by immunoblotting. Calcification medium induced osteogenic differentiation, reduced matrix Gla protein content, and increased expression of the sodium-dependent cotransporter Pit-1. Magnesium prevented calcification and decreased osteocalcin expression and BMP-2 activity and increased expression of calcification inhibitors, osteopontin and matrix Gla protein. TRPM 7 activation was decreased by calcification medium, an effect reversed by magnesium. 2-APB recapitulated the VSMC osteoblastic phenotype in VSMCs. Osteocalcin was increased by calcification medium in VSMCs and intact vessels from MgL but not MgH, whereas osteopontin was increased in MgH, but not in MgL mice. Magnesium negatively regulates vascular calcification and osteogenic differentiation through increased/restored TRPM7 activity and increased expression of anticalcification proteins, including osteopontin, BMP-7, and matrix Gla protein. New molecular insights are provided whereby magnesium could protect against VSMC calcification.

Nicotinamide Adenine Dinucleotide Phosphate Reduced Oxidase 5 (Nox5) Regulation by Angiotensin II and Endothelin-1 Is Mediated via Calcium/Calmodulin-Dependent, Rac-1-Independent Pathways in Human Endothelial Cells

Rationale: Although Nox5 (Nox2 homolog) has been identified in the vasculature, its regulation and functional significance remain unclear. Objectives: We sought to test whether vasoactive agents regulate Nox5 through Ca2+/calmodulin-dependent processes and whether Ca2+-sensitive Nox5, associated with Rac-1, generates superoxide (O2·−) and activates growth and inflammatory responses via mitogen-activated protein kinases in human endothelial cells (ECs). Methods and Results: Cultured ECs, exposed to angiotensin II (Ang II) and endothelin (ET)-1 in the absence and presence of diltiazem (Ca2+ channel blocker), calmidazolium (calmodulin inhibitor), and EHT1864 (Rac-1 inhibitor), were studied. Nox5 was downregulated with small interfering RNA. Ang II and ET-1 increased Nox5 expression (mRNA and protein). Effects were inhibited by actinomycin D and cycloheximide and blunted by diltiazem, calmidazolium and low extracellular Ca2+ ([Ca2+]e). Ang II and ET-1 activated NADPH oxidase, an effect blocked by low [Ca2+]e, but not by EHT1864. Nox5 knockdown abrogated agonist-stimulated O2·− production and inhibited phosphorylation of extracellular signal-regulated kinase (ERK)1/2, but not p38 MAPK (mitogen-activated protein kinase) or SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase). Nox5 small interfering RNA blunted Ang II–induced, but not ET-1–induced, upregulation of proliferating-cell nuclear antigen and vascular cell adhesion molecule-1, important in growth and inflammation. Conclusions: Human ECs possess functionally active Nox5, regulated by Ang II and ET-1 through Ca2+/calmodulin-dependent, Rac-1–independent mechanisms. Nox5 activation by Ang II and ET-1 induces ROS generation and ERK1/2 phosphorylation. Nox5 is involved in ERK1/2-regulated growth and inflammatory signaling by Ang II but not by ET-1. We elucidate novel mechanisms whereby vasoactive peptides regulate Nox5 in human ECs and demonstrate differential Nox5-mediated functional responses by Ang II and ET-1. Such phenomena link Ca2+/calmodulin to Nox5 signaling, potentially important in the regulation of endothelial function by Ang II and ET-1.

Testosterone Induces Vascular Smooth Muscle Cell Migration by NADPH Oxidase and c-Src–Dependent Pathways

Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). The mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10−7 mol/L, 0–120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase–derived ROS and c-Src–dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs.

Conjugated equine estrogen treatment corrected the exacerbated aorta oxidative stress in ovariectomized spontaneously hypertensive rats

OBJECTIVE The increased risk of cardiovascular diseases in postmenopausal women has been linked to the decrease in plasma estrogen levels. Preparation of conjugate equine estrogens (CEE) is one of the most routinely used hormone therapy in postmenopausal women. However, studies on the vascular effects of CEE are still sparse and the mechanism of action is not completely elucidated. In this context, we have determined the effects of CEE in the vascular oxidative stress observed in ovariectomyzed (OVX) spontaneously hypertensive rats (SHR). Mechanisms by which CEE interferes with redox-sensitive pathways and endothelial function were also determined. RESULTS Aortas from OVX rats exhibited increased generation of reactive oxygen species (ROS), NADPH oxidase activity and reduced catalase protein expression, compared to aortas from sham SHR. Endothelium-intact aortic rings from OVX were hyperreactive to NE when compared to Sham aortas. This hyperreactivity was corrected by superoxide dismutase (SOD), catalase, and endothelium removal. Treatment of OVX-SHR with CEE reduced vascular ROS generation, NADPH oxidase activity, enhanced SOD and catalase expression and also corrected the NE-hyperreactivity in aortic rings from OVX-SHR. CONCLUSION Our study indicates a potential benefit of CEE therapy through a mechanism that involves reduction in oxidative stress, improving endothelial function in OVX hypertensive rats.

Testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway with mitochondria-generated reactive oxygen species involvement

Testosterone exerts both beneficial and harmful effects on the cardiovascular system. Considering that testosterone induces reactive oxygen species (ROS) generation and ROS activate cell death signaling pathways, we tested the hypothesis that testosterone induces apoptosis in vascular smooth muscle cells (VSMCs) via mitochondria-dependent ROS generation. Potential mechanisms were addressed. Cultured VSMCs were stimulated with testosterone (10(-7) mol/l) or vehicle (2-12 h) in the presence of flutamide (10(-5) mol/l), CCCP (10(-6) mol/l), mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 3 × 10(-5) mol/l), Z-Ile-Glu(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (Z-IETD-FMK; 10(-5) mol/l), or vehicle. ROS were determined with lucigenin and dichlorodihydrofluorescein; apoptosis, with annexin V and calcein; O2 consumption, with a Clark-type electrode, and procaspases, caspases, cytochrome c, Bax, and Bcl-2 levels by immunoblotting. Testosterone induced ROS generation (relative light units/mg protein, 2 h; 162.6 ± 16 vs. 100) and procaspase-3 activation [arbitrary units, (AU), 6 h; 166.2 ± 19 vs. 100]. CCCP, MnTMPyP, and flutamide abolished these effects. Testosterone increased annexin-V fluorescence (AU, 197.6 ± 21.5 vs. 100) and decreased calcein fluorescence (AU, 34.4 ± 6.4 vs. 100), and O2 consumption (nmol O2/min, 18.6 ± 2.0 vs. 34.4 ± 3.9). Testosterone also reduced Bax-to-Bcl-2 ratio but not cytochrome-c release from mitochondria. Moreover, testosterone (6 h) induced cleavage of procaspase 8 (AU, 161.1 ± 13.5 vs. 100) and increased gene expression of Fas ligand (2(ΔΔCt), 3.6 ± 1.2 vs. 0.7 ± 0.5), and TNF-α (1.7 ± 0.4 vs. 0.3 ± 0.1). CCCP, MnTMPyP, and flutamide abolished these effects. These data indicate that testosterone induces apoptosis in VSMCs via the extrinsic apoptotic pathway with the involvement of androgen receptor activation and mitochondria-generated ROS.

Testosterone induces leucocyte migration by NADPH oxidase-driven ROS- and COX2-dependent mechanisms.

The mechanisms whereby testosterone increases cardiovascular risk are not clarified. However, oxidative stress and inflammation seem to be determinants. Herein, we sought to determine whether exogenous testosterone, at physiological levels, induces leucocyte migration, a central feature in immune and inflammatory responses and the mediating mechanisms. We hypothesized that testosterone induces leucocyte migration via NADPH oxidase (NADPHox)-driven reactive oxygen species (ROS) and cyclooxygenase (COX)-dependent mechanisms. Sixteen-week-old Wistar rats received an intraperitoneal injection (5 ml) of either testosterone (10(-7) mol/l) or saline. Rats were pre-treated with 5 ml of sodium salicylate (SS, non-selective COX inhibitor, 1.25 × 10(-3) mol/l, 1 h prior to testosterone or saline), flutamide (androgen receptor antagonist, 10(-5) mol/l), apocynin (NADPHox inhibitor, 3 × 10(-4) mol/l), N-[2-Cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS398, COX2 inhibitor, 10(-4) mol/l) or saline, 4 h before testosterone or saline administration. Leucocyte migration was assessed 24 h after testosterone administration by intravital microscopy of the mesenteric bed. Serum levels of testosterone were measured by radioimmunoassay. NADPHox activity was assessed in membrane fractions of the mesenteric bed by dihydroethidium (DHE) fluorescence and in isolated vascular smooth muscle cells (VSMC) by HPLC. NADPHox subunits and VCAM (vascular cell adhesion molecule) expression were determined by immunoblotting. Testosterone administration did not change serum levels of endogenous testosterone, but increased venular leucocyte migration to the adventia, NADPHox activity and expression (P < 0.05). These effects were blocked by flutamide. SS inhibited testosterone-induced leucocyte migration (P<0.05). Apocynin and NS398 abolished testosterone-induced leucocyte migration and NADPHox activity (P<0.05). Testosterone induces leucocyte migration via NADPHox- and COX2-dependent mechanisms and may contribute to inflammatory processes and oxidative stress in the vasculature potentially increasing cardiovascular risk.

Influência da circulação extracorpórea sobre as concentrações plasmáticas de atenolol

BACKGROUND Betablockers are used in the treatment of angina pectoris and others ischemic coronary diseases, reducing mortality and cardiovascular events. Atenolol is a hydrophilic betablocker which is characterized by gastrointestinal absorption, small extent of distribution and renal function-dependent elimination. OBJECTIVE The study objective was to determine the inter-individual variability of atenolol in coronary patients. METHODS Plasma atenolol was quantified in six blood samples collected during the preoperative period from seven patients with coronary insufficiency and surgical indication, chronically treated with atenolol PO 25 to 100 mg/day. All patients presented a normal or slightly reduced renal function. RESULTS All enrolled patients presented normal or slightly reduced renal function as a result of age and underlying disease. Atenolol plasma concentrations showed a monoexponential decline, confirming the first-order pharmacokinetics at the doses employed for the control of coronary insufficiency (mean +/- SD): 123 +/- 56, 329 +/- 96, 288 +/- 898, 258 +/- 85, 228 +/- 79 and 182 +/- 73 ng/ml at times zero, 2, 4, 6, 8 and 12h after dose administration. The investigated group showed a small inter-patient variability of atenolol administrated at multiple regimens due to the hydrophilic characteristic of the drug. Furthermore, accumulation of atenolol administered chronically was greater in coronary patients, compared to healthy subjects. CONCLUSION In view of its cardio-selectivity and low-variability, atenolol should be used as the first-choice drug for the treatment of acute coronary syndrome and other cardiovascular diseases.

Disposição cinética do atenolol em pacientes coronarianos submetidos a revascularização do miocárdio

Myocardium ischemia is an important factor of risk for mortality and cardiovascular events in the perioperative period of cardiac and non cardiac surgeries. However, the prophylactic administration of b-blocker agents could reduce these risks. Physiologic changes, occurred during the coronary artery bypass graft (CABG) surgery with cardiopulmonary bypass (CPB), could alter plasma concentration and pharmacokinetics of many drugs. This study investigated the pharmacokinetics of atenolol in patients with unstable angina and without renal dysfunction, submitted to CABG surgery and treated chronically with atenolol PO. For pharmacokinetic analysis, 13 blood samples were collected after doses administrated pre- and post-operatively. Compared to the pre-operative period, it was verified a non-significant reduction in the apparent volume of distribution and plasma clearance after the surgery, remaining unchanged the biological half-life, p>0.05 (NS). A negative linear correlation between plasma clearance and elimination half-life was demonstrated in both periods of the study (r: -0.77 p=0.06, pre-surgery and r: -0.89, p=0.06, post-surgery), while a correlation between volume of distribution and biological half-life was established only before revascularization (r: 0,54 p= 0,03 , pre-surgery and r: 0,09, p=0,03, post-surgery). We suggest that the CABG surgery leads to the normalization of the extension of distribution of atenolol.

Testosterone effects in the mesenteric vascular bed: ROS production, COX activation and leukocyte migration

Coronary arteritis is the fourth most common cause of fatal cardiac disease, after coronary atheroscLerosis, congenital anomalies and coronary dissection. Eosinophilic inflammation of the coronary arteries is extremely rare, involves the major coronaries and occurs as an isolated disease or as part of Churg-Strauss syndrome or Wegeners granulomatosis with involvement of other internal organs. A case of sudden and unexpected death of a healthy young woman is presented. Autopsy revealed eosinophilic inflammation of Left coronary artery with thrombosis of the lumen, causing a fatal cardiac failure. No other pathology was detected. We discuss the importance of performing a full autopsy, including microscopic inspection of the tissues, in order to glean the cause of death and learn about new and rare pathologies.Background. Currently, there are no available methods that can reliably predict when or if an atheroma will rupture. Recent reports suggest cholesterol crystals (CCs), present within the necrotic core, are sharp and can penetrate and disrupt the fibrous cap, contributing to plaque rupture. Our aim is to show CC, normally distributed at random within the necrotic core, often develop a parallel configuration at the site of plaque disruption (PD) which may be a sign of impending plaque rupture. Methods and Results. The coronary arteries of 83 patients who died of acute coronary disease (ACD) were injected with a colored barium gelatin mass. The arteries were dissected, decalcified, cut at 2―3 mm intervals, and all segments mounted for microscopic study. All segments were reviewed to identify PDs and to determine the frequency of parallel cholesterol crystals (P-CCs) at the site of these PDs. There were 215 separate PDs in 83 patients, with 64 (77%) patients showing more than one PD. P-CCs were present in 126 (59%) of all PDs regardless of plaque size or the severity of luminal stenosis and were present with or without luminal thrombosis. Luminal thrombosis was present in 105 PDs (49%), primarily in those segments showing significant luminal stenosis. There was no significant difference in the frequency of luminal thrombosis in those PDs with or without P-CCs. Conclusions. P-CCs are commonly present at the site of PD and may contribute to PD by penetrating and disrupting the fibrous cap. The ability to recognize this parallel configuration may help to identify plaques with impending plaque rupture.Introduction: Electrocardiographic (ECG) changes accompanying lung resection have not been well ☆ Declaration of competing interests. ☆☆ Other Disclos ⁎ Corresponding a 01604. E-mail address: lo 0022-0736/

Reactive Oxygen Species, Oxidative Stress, and Hypertension

– see fro http://dx.doi.org/10.10 investigated previously in a large controlled series of human adults. Thus, our current investigation was undertaken for a better understanding of the ECG changes associated with lung resection. Materials and Methods: Medical records of 117 patients who underwent lung resection (segmentectomy, lobectomy, or pneumonectomy) were reviewed. Their clinical course and ECGs were compared during early, intermediate and late postoperative course (b1 month, 1 month to 1 year and N1 year post-op respectively). Results: Patients in the acute postoperative phase had higher heart rate, increased maximum P-duration and P-dispersion, increased incidence of atrial arrhythmias and frequent ST-T changes. P-vector and QRS-vector were significantly affected after the lung resections; the correlation being most consistent between the anatomical displacements and the QRS-vector in the majority of patients. The axial shifts also demonstrated a characteristic temporal relationship after left pneumonectomy (a leftward deviation in the acute, normal or slight rightward deviation in the intermediate and a rightward deviation in the late postoperative course). The precordial R/S transition is often affected due to the mediastinal shifts and the ECGs in patients after left lung resection may simulate acute anteroseptal myocardial infarction due to a delayed R/S transition. Conclusion: The understanding and recognition of the expected ECG findings after lung resection are imperative to avoid confusing these changes with other acute cardiopulmonary events which would prevent unnecessary further investigational work-up. These ECG changes are often dynamic and may bear a temporal relationship to the dynamic post-surgical changes in the thoracic anatomy.