Aldosterone and angiotensin II synergistically stimulate migration in vascular smooth muscle cells through c-Src-regulated redox-sensitive RhoA pathways

Objective - Synergistic interactions between aldosterone (Aldo) and angiotensin II (Ang II) have been implicated in vascular inflammation, fibrosis, and remodeling. Molecular mechanisms underlying this are unclear. We tested the hypothesis that c-Src activation, through receptor tyrosine kinase transactivation, is critically involved in synergistic interactions between Aldo and Ang II and that it is upstream of promigratory signaling pathways in vascular smooth muscle cells (VSMCs). Methods and Results - VSMCs from WKY rats were studied. At low concentrations (10(-10) mol/L) Aldo and Ang II alone did not influence c-Src activation, whereas in combination they rapidly increased phosphorylation (P<0.01), an effect blocked by eplerenone ( Aldo receptor antagonist) and irbesartan (AT1R blocker). This synergism was attenuated by AG1478 and AG1296 ( inhibitors of EGFR and PDGFR, respectively), but not by AG1024 (IGFR inhibitor). Aldo and Ang II costimulation induced c-Src-dependent activation of NAD(P)H oxidase and c-Src-independent activation of ERK1/2 (P<0.05), without effect on ERK5, p38MAPK, or JNK. Aldo/Ang II synergistically activated RhoA/Rho kinase and VSMC migration, effects blocked by PP2, apocynin, and fasudil, inhibitors of c-Src, NADPH oxidase, and Rho kinase, respectively. Conclusions - Aldo/Ang II synergistically activate c-Src, an immediate signaling response, through EGFR and PDGFR, but not IGFR transactivation. This is associated with activation of redox-regulated RhoA/Rho kinase, which controls VSMC migration. Although Aldo and Ang II interact to stimulate ERK1/2, such effects are c-Src-independent. These findings indicate differential signaling in Aldo-Ang II crosstalk and highlight the importance of c-Src in redox-sensitive RhoA, but not ERK1/2 signaling. Blockade of Aldo/Ang II may be therapeutically useful in vascular remodeling associated with abnormal VSMC migration.

Internal Pudendal Artery from Type 2 Diabetic Female Rats Demonstrate Elevated Endothelin-1-Mediated Constriction

Introduction. Diabetes is a risk factor for female sexual dysfunction (FSD). FSD has several etiologies, including a vasculogenic component that could be exacerbated in diabetes. The internal pudendal artery supplies blood to the vagina and clitoris and diabetes-associated functional abnormalities in this vascular bed may contribute to FSD. Aim. The Goto-Kakizaki (GK) rat is a non-obese model of type 2 diabetes with elevated endothelin-1 (ET-1) activity. We hypothesize that female GK rats have diminished sexual responses and that the internal pudendal arteries demonstrate increased ET-1 constrictor sensitivity. Methods. Female Wistar and GK rats were used. Apomorphine (APO)-mediated genital vasocongestive arousal (GVA) was measured. Functional contraction (ET-1 and phenylephrine) and relaxation (acetylcholine, ACh) in the presence or absence of the ETA receptor antagonist (ET(A)R; atrasentan) or Rho-kinase inhibitor (Y-27632) were assessed in the internal pudendal and mesenteric arteries. Protein expression of ET-1 and RhoA/Rho-kinase signaling pathway was determined in the internal pudendal and mesenteric arteries. Main Outcome Measure. APO-mediated GVAs; contraction and relaxation of internal pudendal and mesenteric arteries; ET-1/RhoA/Rho-kinase protein expression. Results. GK rats demonstrated no APO-induced GVAs. Internal pudendal arteries, but not mesenteric arteries, from GK rats exhibited greater contractile sensitivity to ET-1 compared with Wistar arteries. ETAR blockade reduced ET-1-mediated constriction in GK internal pudendal and mesenteric arteries. Rho-kinase inhibition reduced ET-1-mediated constriction of GK internal pudendal but not mesenteric arteries; however, it had no effect on arteries from Wistar rats. RhoA protein expression was elevated in GK internal pudendal arteries. At the highest concentrations, ACh-mediated relaxation was greater in the GK internal pudendal artery; however, no difference was observed in the mesenteric artery. Conclusions. Female GK rats demonstrate decreased sexual responses that may be because of increased constrictor sensitivity to the ET-1/RhoA/Rho-kinase signaling in the internal pudendal artery. Allahdadi KJ, Hannan JL, Ergul A, Tostes RC, and Webb RC. Internal pudendal artery from type 2 diabetic female rats demonstrate elevated endothelin-1-mediated constriction. J Sex Med 2011;8:2472-2483.

Upregulation of intermediate calcium-activated potassium channels counterbalance the impaired endothelium-dependent vasodilation in stroke-prone spontaneously hypertensive rats

Endothelial dysfunction has been linked to a decrease in nitric oxide (NO) bioavailability and attenuated endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation. The small (SK(Ca)) and intermediate (IK(Ca)) calcium-activated potassium channels play a key role in endothelium-dependent relaxation. Because the repressor element 1-silencing transcription factor (REST) negatively regulates IK(Ca) expression, we hypothesized that augmented REST and decreased IK(Ca) expression contributes to impaired endothelium-dependent vasodilation associated with hypertension. Acetylcholine (ACh) responses were slightly decreased in small mesenteric arteries from male stroke-prone spontaneously hypertensive rats (SHRSPs) versus arteries from Wistar Kyoto (WKY) rats. Incubation with N-nitro-L-arginine methyl ester (L-NAME; 100 mu mol/L) and indomethacin (100 mu mol/L) greatly impaired ACh responses in vessels from SHRSP. lberiotoxin (0.1 mu mol/L), which is a selective inhibitor of large-conductance K(Ca) (BK(Ca)) channels, did not modify EDHF-mediated vasodilation in SHRSP or WKY. UCL-1684 (0.1 mu mol/L.), which is a selective inhibitor of SKCa channels, almost abolished EDHF-mediated vasodilation in WKY and decreased relaxation in SHRSP. 1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole (TRAM-34; 10 mu mol/L) and charybdotoxin (0.1 mu mol/L), which are both IKCa inhibitors, produced a small decrease of EDHF relaxation in WKY but completely abrogated EDHF vasodilation in SHRSP. EDHF-mediated relaxant responses were completely abolished in both groups by simultaneous treatment with UCL-1684 and TRAM-34 or charybdotoxin. Relaxation to SK(Ca)/IK(Ca) channels agonist NS-309 was decreased in SHRSP arteries. The expression of SK(Ca) was decreased, whereas IK(Ca) was increased in SHRSP mesenteric arteries. REST expression was reduced in arteries from SHRSP. Vessels incubated with TRAM-34 (10 mu mol/L) for 24h displayed reduced REST expression and demonstrated no differences in IK(Ca). In conclusion, IK(Ca) channel upregulation, via decreased REST, seems to compensate deficient activity of SK(Ca) channels in the vasculature of spontaneously hypertensive rats. (Translational Research 2009; 154:183-193)

LONGITUDINAL REFERENCE VALUES FOR DUCTUS VENOSUS DOPPLER IN LOW-RISK PREGNANCIES

The aim of this study was to establish normal ranges of blood flow velocities and indices in the fetal ductus venosus (DV) during the second half of normal pregnancy. A Doppler study of 60 healthy pregnant women without fetal pathologies was performed during the second half of pregnancy. The peak systolic velocity (PSV), peak diastolic velocity (PDV), maximum velocity during atrial contraction (VAC), peak systolic velocity/maximum velocity during atrial contraction (S/A ratio), pulsatility index for the vein (PIV), preload index (PLI) and velocity index for the vein (VIV) were calculated from the DV at 4-week intervals. A significant increase in PSV, PDV and VAC was observed from the 20-23(6/7) to the 28-31(6/7) weeks, with stabilization of values until the end of the pregnancy. On the other hand, the study showed a significant decrease for the S/A ratio, PIV, PLI and VIV from the 20-23(6/7) to the 28-31(6/7) weeks and remaining stable from then until term. (E-mail:dralemar@uol.com.br) (C) 2010 World Federation for Ultrasound in Medicine & Biology.

Distribution of fentanyl in the placental intervillous space and in the different maternal and fetal compartments in term pregnant women

Fentanyl is used in obstetrical practice to promote analgesia and anesthesia during labor and in cesarean delivery, with rapid and short-term effects. To determine fentanyl concentrations in maternal plasma, in the placental intervillous space, and in the umbilical artery and vein in term pregnant women. Ten healthy pregnant women underwent epidural anesthesia with fentanyl plus bupivacaine and lidocaine, and fentanyl concentrations were determined in the various maternal and fetal compartments, including the placental intervillous space, which has not been previously studied in the literature. The ratios of fentanyl concentrations in the various maternal and fetal compartments revealed an 86% rate of placental fentanyl transfer. The highest fentanyl concentrations were detected in the placental intervillous space, being 2.19 times higher than in maternal plasma, 2.8 times higher than in the umbilical vein and 3.6 times higher than in the umbilical artery, with no significant differences between the umbilical vein and artery, demonstrating that there was no drug uptake by fetal tissues nor metabolism of the drug by the fetus despite the high rates of placental transfer. The present study demonstrated that the placental intervillous space acted as a site of fentanyl deposit, a fact that may be explained by two hypotheses: (1) the blood collected from the placental intervillous space is arterial and, according to some investigators, the arterial plasma concentrations of the drugs administered to patients undergoing epidural anesthesia are higher than the venous concentrations, and (2) a possible role of P-glycoprotein (P-gp).

UTERINE ULTRASONOGRAPHIC CHANGES DURING ENDOMETRIOSIS TREATMENT: A COMPARISON BETWEEN LEVONORGESTREL-RELEASING INTRAUTERINE DEVICES AND A GONADOTROPIN-RELEASING HORMONE AGONIST

We compared the effects of levonorgestrel-releasing intrauterine devices (LNG-IUD) and a gonadotropin-releasing hormone agonist (GnRHa) on uterine volume, uterine arteries pulsatility index (PI) and endometrial thickness before and after six months of endometriosis treatment. Sixty women aged 18-40 y were allocated randomly to one of two groups: LNG-IUDs were inserted in 30 women, and GnRHa monthly injections were performed on the other 30. All 60 women were submitted to transvaginal 2-D ultrasound scans on the day that the treatment started and then six months later. Measurements of uterine arteries PI, uterine volume and endometrial thickness were performed at both evaluations. The use of LNG-IUDs significantly decreased endometrial thickness (pre = 6.08 +/- 3.00 mm, post = 2.7 +/- 0.98 mm; mean +/- SD), as did the use of GnRHa (pre = 6.96 +/- 3.82 mm, post = 3.23 +/- 2.32 mm). The uterine volume decreased in the GnRHa group (pre = 86.67 +/- 28.38 cm(3), Post = 55.27 +/- 25.52 cm(3)), but not in the LNG-IUD group (pre = 75.77 +/- 20.88 cm(3), post = 75.97 +/- 26.62 cm(3)). Uterine arteries PI increased for both groups; however, the increase was higher in the GnRHa group (0.99 +/- 0.84 vs. 0.38 +/- 0.84, p = 0.007; PI increase in GnRHa and in LNG-IUD groups, respectively). In conclusion, levonorgestrel released directly onto the endometrium by the LNG-IUD induced smaller uterine changes than did the hypoestrogenism induced by GnRHa. Nevertheless, both promoted similar effects on endometrial thickness. (E-mail: wpmartins@gmail.com) (C) 2008 World Federation for Ultrasound in Medicine & Biology.

Distribution of Bupivacaine Enantiomers and Lidocaine and Its Metabolite in the Placental Intervillous Space and in the Different Maternal and Fetal Compartments in Term Pregnant Women

The aim of this study is to determine the concentrations of lidocaine and its metabolite, monoethylglycine xylidide (MEGX), and of the enantiomers of bupivacaine in maternal and fetal compartments. Ten healthy pregnant women were submitted to epidural anesthesia. Drug concentrations were determined in the maternal vein, fetal umbilical artery and vein, and the placental intervillous space. The highest concentrations of the bupivacaine enantiomers lidocaine and of lidocaine and of its MEGX metabolite were detected in maternal plasma and in the placental intervillous space. The placental transfer was 33% for the (+)-(R)-bupivacaine enantiomer and 31% for the (-)-(S)-bupivacaine enantiomer. For lidocaine and its MEGX metabolite, respective placental transfers were 60% and 43%. Lidocaine concentration in the fetal umbilical vein was 1.46 times higher than in the fetal umbilical artery. The highest concentrations of lidocaine and its metabolite and of the enantiomers of bupivacaine were detected in the placental intervillous space. The higher lidocaine concentrations in the fetal umbilical vein than in the fetal umbilical artery suggest that there was tissue uptake of the drug or drug metabolization by the fetus.

Proposal for a Trigonometric Method to Evaluate the Abduction Angle of the Lower Limbs in Neonates

It is difficult to precisely measure articular arc movement in newborns using a goniometer. This article proposes an objective method based on trigonometry for the evaluation of lower limb abduction. With the newborn aligned in the dorsal decubitus position, 2 points are marked at the level of the medial malleolus, one on the sagittal line and the other at the end of the abduction. Using the right-sided line between these 2 points and a line from the medial malleolus to the reference point at the anterior superior iliac spine or umbilical scar, an isosceles triangle is drawn, and half of the inferential abduction angle is obtained by calculating the sine. Twenty healthy full-term newborns comprise the study cohort. Intersubject and intrasubject variability among the abduction angle values (mean [SD], 37 degrees [4]degrees) is low. This method is advantageous because the measurement is precise and because the sine can be used without approximation.

Dorsal root ganglionectomy for the diagnosis of sensory neuropathies. Surgical technique and results

Background: Inflammatory diseases stand out among sensory neuronopathies because, in their active phase, they can be treated with immunosuppressive agents. Immunosuppressive therapy may present severe adverse effects and requires previous inflammatory activity confirmation. Sensory neuronopathies are diagnosed based on clinical and EMG findings. Diagnostic confirmation and identification of inflammatory activity are based on sensory ganglion histopathological examination. We describe the surgical technique used for dorsal root ganglionectomy in patients with clinical/EMG diagnosis of sensory neuronopathies. Methods: The sensory ganglion was obtained from 15 patients through a small T7-T8 hemilaminectomy and foraminotomy to expose the C7 root from its origin to the spinal nerve bifurcation. In 6 patients, the dural cuff supposed to contain the ganglion was resected en bloc; and in 9 patients, the ganglion was obtained through a longitudinal incision of the dural cuff and microsurgical dissection from the ventral and dorsal roots and radicular arteries. All ganglia were histopathologically examined. Results: No ganglion was found in the dural cuff in 2 patients submitted to en bloc removal, and the ganglion was removed in all patients who underwent microsurgical dissection. All but 2 patients that had ganglion examination presented a neuronopathy of nerve cell loss, 3 with mononuclear inflammatory infiltrate. These patients underwent immunosuppressive therapy, and 2 of them presented clinical improvement. No surgical complications were observed. Conclusions: Microsurgical dorsal root ganglionectomy for diagnosing inflammatory sensory ganglionopathies was effective and safe. Although safe, en bloc resection of the proximal dural cuff was not effective for this purpose. (c) 2008 Published by Elsevier Inc.

Comparisons of the release of vasodilator substances from left and right cardiac chambers of the isolated perfused rabbit heart: Implications for intraventricular thrombus formation

Prostacyclin (PgI(2)) and endothelium-derived nitric oxide (EDNO) are produced by the arterial and venous endothelium. In addition to their vasodilator action on vascular smooth muscle, both act together to inhibit platelet aggregation and promote platelet disaggregation. EDNO also inhibits platelet adhesion to the endothelium. EDNO and PgI(2) have been shown to be released from the cultured endocardial cells. In this study, we examined the release of vasoactive substances from the intact endocardium by using isolated rabbit hearts perfused with physiological salt solution (95% O(2)/5% CO(2), T = 37 degrees C). The right and left cardiac chambers were perfused through separate constant-flow perfusion loops (physiological salt solution, 8 ml min(-1)). Effluent from left and right cardiac, separately, was bioassayed on canine coronary artery smooth muscle, which had been contracted with prostaglandin F(2 alpha_)(2 x 10(-6) M) and no change in tension was exhibit. However, addition of calcium ionophore A23187 (10(-6) M) to the cardiac chambers` perfusion line induced vasodilation of the bioassay coronary ring, 61.4 +/- 7.4% versus 70.49 +/- 6.1% of initial prostaglandin F(2 alpha) contraction for the left and right cardiac chambers perfusate, respectively (mean +/- SEM, n = 10, p > 0.05). Production of vasodilator was blocked totally in the left heart but, only partially blocked in the right heart by adding indomethacin (10(-5) M) to the perfusate, respectively, 95.2 +/- 2.2% versus 41.5 +/- 4.8% (mean +/- SEM, n = 10, p < 0.05). 6-Keto prostaglandin F(1 alpha), measured in the endocardial superfusion effluent was also higher for the left cardiac chambers than for the right at the time of stimulation with the A23187, respectively, 25385.88 +/- 5495 pg/ml (n = 8) versus 13,132.45 +/- 1839.82 pg/ml (n = 8), (p < 0.05). These results showed that cyclooxygenase pathway plays major role in generating vasoactive substances for the left cardiac chamber endocardium; while it is not the main pathway for the right ventricular endocardium at which EDNO and PgI(2) Could act together and potentiate their antithrombogenic activities in isolated perfused rabbit heart. This may be an explanation for the intraventricular thrombus mostly seen in left ventricle rather than in right ventricle as a complication of myocardial infarction. (C) 2009 Elsevier Inc. All rights reserved.

Doxycycline Dose-dependently Inhibits MMP-2-Mediated Vascular Changes in 2K1C Hypertension

Hypertension induces vascular alterations that are associated with up-regulation of matrix metalloproteinases (MMPs). While these alterations may be blunted by doxycycline, a non-selective MMPs inhibitor, no previous study has examined the effects of different doses of doxycycline on these alterations. This is important because doxycycline has been used at sub-antimicrobial doses, and the use of lower doses may prevent the emergence of antibiotic-resistant microorganisms. We studied the effects of doxycycline at 3, 10 and 30 mg/kg per day on the vascular alterations found in the rat two kidneyone clip (2K1C) hypertension (n = 20 rats/group). Systolic blood pressure (SBP) was monitored during 4 weeks of treatment. We assessed endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes in the aortic wall was studied, and aortic MMP-2 levels/proteolytic activity were determined by gelatin and in situ zymography, respectively. All treatments attenuated the increases in SBP in hypertensive rats (195.4 +/- 3.9 versus 177.2 +/- 6.2, 176.3 +/- 4.5, and 173 +/- 5.1 mmHg in 2K1C hypertensive rats treated with vehicle, or doxycycline at 3, 10, 30 mg/kg per day, respectively (all p < 0.01). However, only the highest dose prevented 2K1C-induced reduction in endothelium-dependent vasorelaxation (p < 0.05), vascular hypertrophy and increases in MMP-2 levels (all p < 0.05). In conclusion, our results suggest that relatively lower doses of doxycycline do not attenuate the vascular alterations found in the 2K1C hypertension model, and only the highest dose of doxycycline affects MMPs and vascular structure. Our results support the idea that the effects of doxycycline on MMP-2 and vascular structure are pressure independent.

Magnetic Resonance Angiography in Extracranial Giant Cell Arteritis

Background: Noninvasive diagnosis of giant cell arteritis (GCA) remains challenging, particularly with regard to evaluation of extracranial arterial disease. Objectives: The objective of the study was to retrospectively review extracranial involvement in patients with GCA and/or polymyalgia rheumatica (PMR), evaluated with magnetic resonance imaging (MRI), especially 3-dimensional contrast-enhanced magnetic resonance angiography images of the aortic arch and its branches. Methods: Clinical information, biopsy status, and MRI examinations of 28 patients with GCA/PMR were reviewed. Patient images were mixed randomly with 20 normal control images and were independently reviewed by 2 radiologists. Interobserver agreement for detection of arterial stenosis was determined by the k coefficient. Results: Both readers described vascular alterations in keeping with extracranial GCA in 19 of 28 patients (67%) with good interobserver agreement (k = 0.73) and with even higher agreement on diagnosing nonocclusive versus occlusive disease (k = 1.00). The most common lesions were bilateral axillary stenosis or obstructions, observed by both readers in 8 patients (28%). Among the 19 patients with magnetic resonance angiography lesions in the subclavian/axillary arteries, 12 (75%) had biopsy-proven GCA, but only 5 (41%) of these patients had clinical features of large artery disease. Conclusions: In our series review, MRI could provide accurate information on involvement of the aortic arch and its branches in extracranial GCA, depicting different degrees of stenosis. Our analysis also illustrates that occult large artery vasculitis should be considered in patients without biopsy-proven GCA, patients with classic GCA but without clinical signs of large artery disease, and in patients initially diagnosed as having PMR.

Effect of isoflavone extracts from Glycine max on human endothelial cell damage and on nitric oxide production

Objective: In this study, we determined the protective effect of isoflavones from Glycine max on human umbilical vein endothelial cell (ECV304) damage induced by hydrogen peroxide (H(2)O(2)) and on nitric oxide (NO) production. Methods: We studied the regulation of NO synthesis in cultured human endothelial cells by phytoestrogens contained in soy extracts in the presence or absence of ICI 182,780 or N(omega)-nitro-L-arginine methyl esther and determined the protective effect of these isoflavones on ECV304 damage induced by H(2)O(2). Results: We show that soy extracts activate NO synthesis in endothelial cells and protect against cell damage. Conclusions: In conclusion, soy isoflavones markedly protect ECV304 cells against H(2)O(2) damage and promote NO synthesizing. Therefore, these isoflavones call potentially act as an NO promoter and as an antioxidant.

Focal and linear endocardial and epicardial catheter-based cryoablation of normal and infarcted ventricular tissue

Background: This study of a chronic porcine postinfarction model examined whether linear epicardial cryoablation was capable of creating large, homogenous lesions in regions of the myocardium including scarred ventricle. Endocardial and epicardial focal cryolesions were also compared to determine if there were significant differences in lesion characteristics. Methods: Eighty focal endocardial and 28 focal epicardial cryoapplications were delivered to eight normal caprine and four normal porcine ventricular myocardium, and 21 linear cryolesions were applied along the border of infarcted epicardial tissue in a chronic porcine infarct model in six swines. Results: Focal endocardial cryolesions in normal animals measured 9.7 +/- 0.4 mm (length) by 7.3 +/- 1.4 mm (width) by 4.8 +/- 0.2 mm (depth), while epicardial lesions measured 10.2 +/- 1.4 mm (length) by 7.7 +/- 2 mm (width) by 4.6 +/- 0.9 mm (depth); P > 0.05. Linear epicardial cryolesions in the chronic porcine infarct model measured 36.5 +/- 7.8 mm (length) by 8.2 +/- 1.3 mm (width) by 6.0 +/- 1.2 mm (depth). The mean depth of linear cryolesions applied to the border of the infarct scar was 7 +/- 0.7 mm, as measured by magnetic resonance imaging. Conclusions:Cryoablation can create deep lesions when delivered to the ventricular epicardium. Endocardial and epicardial cryolesions created by a focal cryoablation catheter are similar in size and depth. The ability to rapidly create deep linear cryolesions may prove to be beneficial in substrate-based catheter ablation of ventricular arrhythmias.

Holt-Oram syndrome: novel TBX5 mutation and associated anomalous right coronary artery

The Holt-Oram syndrome was confirmed in an asymptomatic 36-year-old man by a novel TBX5-gene mutation (exon 8 acceptor splicing site, c.663-1G greater than A). Computed tomography showed an atrial septal defect and an anomalous right coronary artery crossing between the aorta and pulmonary arteries. Surgery corrected the septal defect and the initial segment of the anomalous vessel was unroofed and enlarged. Anomalous coronary arteries were not previously described in the Holt-Oram syndrome patients and should be added to the list of possible associated cardiac defects.