Anti-endothelial Nitric Oxide Synthase Research Articles

Hypertonic Saline Solution Reduces Microcirculatory Dysfunction and Inflammation in a Rat Model of Brain Death.

Brain death (BD) induces hemodynamic instability with microcirculatory hypoperfusion, leading to increased organ inflammation and dysfunction. This study investigated the effects of 7.5% hypertonic saline solution (HSS) on mesenteric microcirculatory dysfunction and inflammation in a rat model of BD. Male Wistar rats were anesthetized and mechanically ventilated. BD was induced by rapidly inflating an intracranial balloon catheter. The rats were randomly divided into: SH, sham-operated rats subjected to trepanation; NS, rats treated with NaCl 0.9%, 4 mL/kg immediately after BD; T1, rats treated with HSS (NaCl 7.5%, 4 mL/kg) immediately or 60 min after BD, T60. All groups were analyzed 180 min after the start of the experiment. Rats in BD groups presented with a similar hypertensive peak, followed by hypotension. Proportion of perfused small vessels was decreased in the NS group (46%) compared with the SH group (74%, P = 0.0039). HSS restored the proportion of perfused vessels (T1 = 71%, P = 0.0018). The anti-endothelial nitric oxide synthase (eNOS) protein expression significantly increased in rats given HSS (T1, and T60, P = 0.0002). Similar results were observed regarding endothelin-1 (P < 0.0001). Increased numbers of rolling (P = 0.0015) and migrated (P = 0.0063) leukocytes were observed in the NS group compared with the SH group. Rats given HSS demonstrated an overall reduction in leukocyte-endothelial interactions. The ICAM-1 levels increased in the NS group compared with the SH group, and decreased in the HSS-treated groups (P = 0.0002). HSS may improve the density of mesenteric perfused small vessels due to its effects on eNOS and endothelin-1 protein expression, and reduces inflammation by decreasing leukocyte adhesion and migration in a rat model of BD.

Effects of stem cells applications on oxidative stress and apoptosis during implantation

Objective: To investigate the effects of bone marrow derived mesenchymal stem cell (BMSC) application into the rat endometrium on oxidative stress, cell proliferation and apoptosis. Methods: The female rats selected in estrous cycle were divided into three groups (saline, media and BMSC group). The intrauterine and intraperitoneal injections were performed using the saline (200 μL), culture media (200 μL) and 1×106 BMSCs/200 μL culture media, and then they were mated with male rats. On the 7th day of the pregnancy, uterine samples were harvested and dyed with heamatoxylin-eosin histochemically, anti-endothelial nitric oxide synthase and anti-inducible nitric oxide synthase, and anti-proliferating cell nuclear antigen immunohistochemically, with terminal deoxynucleotidyl transferas dUTP nick end labeling for apoptosis. The stainings were evaluated by H-score and the results were analyzed using one-way ANOVA test statistically. Results: It was found that BMSCs increased the endometrial thickness, endometrial epithelium thickness and number of endometrial glands compared to control and sham groups. The intrauterine BMSC application decreased both anti-endothelial nitric oxide synthase and anti-inducible nitric oxide synthase immunoreactivities and the number of apoptotic cells compared to the intraperitoneal applications whereas the immunoreactivity of proliferating cell nuclear antigen was increased. Conclusions: In current study, we define that stem cells do not cause any structural damages. Also they change the distribution of oxidative stress and cell proliferation marker. These findings support the reliability of stem cells in clinical use in the case of infertility.

Optimization of Extraction and Purification of Arctiin from Fructus arctii and Its Protection Against Glucose-Induced Rat Aortic Endothelial Cell Injury

To develop an efficient method for extracting and purifying the active ingredient, arctiin, from Fructus arctii and to investigate the protective effect of arctiin against glucose-induced rat aortic endothelial cell (RAEC) injury was investigated. Using a L9 (34) orthogonal array and two-step column chromatography (with AB-8 macroporous resin) arctiin extraction was optimized using a reflux method with 70% ethanol. The RAECs were then treated with different concentrations of arctiin (1, 10, or 100 μg/ml). The effects of arctiin on cell viability in a high glucose medium, malondialdehyde (MDA) levels, and lactate dehydrogenase were measured using commercially available assays. After extraction, the purity of arctiin reached 95.7%. In rats, arctiin was shown to stimulate the proliferation of RAECs in a high glucose medium in a dose-dependent manner. Exposure of RAECs to high glucose resulted in a significant increase in MDA and release of lactate dehydrogenase. This was accompanied by significant increase in nitric oxide release and expression of antiendothelial nitric oxide synthase. This technique resulted in relatively pure arctiin extraction. Furthermore, the results from this study suggest that arctiin could potentially function as a protector against vascular endothelial cell injury and further investigation is warranted.

A novel approach to the Langendorff technique: preparation of isolated cardiomyocytes and myocardial samples from the same rat heart

Researchers in biomedical sciences must continually re-evaluate their investment in experiments using laboratory animals. Our group is interested in various signalling pathways that underlie physiological and pathophysiological functioning of the mammalian heart. Two important resources for this type of work are isolated cardiomyocytes and homogenized or preserved sections of whole myocardium. In order to improve our experimental approach ethically, we devised an adaptation of the Langendorff whole-heart retrograde perfusion technique that allows the isolation of adult rat ventricular cardiomyocytes and processing/storage of myocardium from the same heart. This could result in a 50% reduction in the number of animals required for certain experiments. We believe that a generalized adoption of this method would represent a meaningful reduction of animal use in our field of research and, furthermore, strengthen data sets by permitting correlation between myocyte function and various parameters of myocardial biochemistry/structure in individual hearts. This approach is of particular relevance for studies of cardiac pathology, given the cost and time involved in generating experimental disease models.

ORIGINAL RESEARCH—BASIC SCIENCE: Effects of ACE Inhibition and Beta-Blockade on Female Genital Structures in Spontaneously Hypertensive Rats

ABSTRACT Introduction and Aim This study evaluated the possible differences between an angiotensin converting enzyme (ACE) inhibitor and a beta-blocker concerning their potential protective role on female external genitalia in spontaneously hypertensive rats (SHR). Main Outcome Measures Morphological changes in the clitoris after antihypertensive treatments. Methods For 6 months, SHR received no treatment; SHR+ramipril (RAM), SHR+atenolol (AT), and control Wistar Kyoto (WKY) rats received no treatment. Clitorises were processed for immunohistochemistry using anti-α-smooth muscle actin (α-SMA), anti-collagen I and III, anti-transforming growth factor β1 (TGFβ1), and anti-endothelial nitric oxide synthase (eNOS) antibodies. Results SHR+RAM and SHR+AT presented significantly lower blood pressure in both groups vs. untreated SHR. Compared with WKY, α-SMA was increased in the arteries and in the cavernous spaces of the clitoris together with a marked increase in wall/lumen ratio in clitoral vessels in untreated SHR. All these alterations were diminished in SHR+AT (P &amp;lt;0.01). SHR+RAM presented differences with respect to SHR+AT in the reduction of these variables. TGFβ1 expression in the vessel wall from the clitoris and collagen I and III deposition in the interstitium from the clitoris in untreated SHR were significantly more (P &amp;lt;0.01) than in WKY. While SHR+AT showed a mild decrease in these variables, SHR+RAM presented a significant reduction (P &amp;lt;0.01) in TGFβ1 expression interstitial fibrosis and in both types of collagens. Positive immunostaining of eNOS in the sinusoidal endothelium from the clitoris was less (P &amp;lt;0.01) in untreated SHR (3.4 ± 1.3%) and SHR+AT (5.1 ± 1.2%) than in SHR+RAM (17.2 ± 1.6%) and WKY (15.9 ± 1.7%). Untreated SHR and SHR+AT presented more surrounding connective tissue at the perineurium in the clitoris (P &amp;lt;0.01) than SHR+RAM. Conclusion ACE inhibition provided a considerable protective role on the female external genitalia structures in SHR by a mechanism that may be, at least in part, independent of the degree of blood pressure lowering.

Role of Scavenger Receptor Class B Type I and Sphingosine 1-Phosphate Receptors in High Density Lipoprotein-induced Inhibition of Adhesion Molecule Expression in Endothelial Cells

We characterized the molecular mechanisms by which high density lipoprotein (HDL) inhibits the expression of adhesion molecules, including vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, induced by sphingosine 1-phosphate (S1P) and tumor necrosis factor (TNF) alpha in endothelial cells. HDL inhibited S1P-induced nuclear factor kappaB activation and adhesion molecule expression in human umbilical vein endothelial cells. The inhibitory HDL actions were associated with nitric-oxide synthase (NOS) activation and were reversed by inhibitors for phosphatidylinositol 3-kinase and NOS. The HDL-induced inhibitory actions were also attenuated by the down-regulation of scavenger receptor class B type I (SR-BI) and its associated protein PDZK1. When TNFalpha was used as a stimulant, the HDL-induced NOS activation and the inhibitory action on adhesion molecule expression were, in part, attenuated by the down-regulation of the expression of S1P receptors, especially S1P(1), in addition to SR-BI. Reconstituted HDL composed mainly of apolipoprotein A-I and phosphatidylcholine mimicked the SR-BI-sensitive part of HDL-induced actions. Down-regulation of S1P(3) receptors severely suppressed the stimulatory actions of S1P. Although G(i/o) proteins may play roles in either stimulatory or inhibitory S1P actions, as judged from pertussis toxin sensitivity, the coupling of S1P(3) receptors to G(12/13) proteins may be critical to distinguish the stimulatory pathways from the inhibitory ones. In conclusion, even though S1P alone stimulates adhesion molecule expression, HDL overcomes S1P(3) receptor-mediated stimulatory actions through SR-BI/PDZK1-mediated signaling pathways involving phosphatidylinositol 3-kinase and NOS. In addition, the S1P component of HDL plays a role in the inhibition of TNFalpha-induced actions through S1P receptors, especially S1P(1).

Localization of nitric oxide synthase type III in the internal thoracic and radial arteries and the great saphenous vein: a comparative immunohistochemical study

Background Endothelial nitric oxide synthase type III is the key enzyme of the nitric oxide production in the vessel wall. In this study the localization of endothelial nitric oxide synthase type III within the wall of the human internal thoracic and radial arteries and the great saphenous vein was investigated. Methods Specimens were harvested from 23 patients undergoing surgical myocardial revascularization and submitted to light and electron microscope analysis using histochemical stainings and immunohistochemistry with specific antibodies anti-endothelial nitric oxide synthase type III, Factor VIII, and α-smooth muscle actin. Results Endothelial nitric oxide synthase type III was evident in the intima of all conduits and, unexpectedly, in the muscle cells of the media of muscular internal thoracic arteries and radial arteries. No endothelial nitric oxide synthase type III expression was found in the media of great saphenous veins. Semiquantitative analysis revealed a higher endothelial nitric oxide synthase type III expression in the wall of internal thoracic artery, particularly at the level of the media. Conclusion Endothelial nitric oxide synthase type III is expressed in the intima of the internal thoracic and radial artery and the great saphenous vein and in the muscle cells of the media of the internal thoracic and radial arteries. However, the internal thoracic artery shows a higher intensity of endothelial nitric oxide synthase type III expression, particularly within the media. The present study provides the first demonstration of the endothelial nitric oxide synthase type III expression at the level of the smooth muscle cells of the tunica media of systemic human arteries and can provide an histologic explanation for the better results of the internal thoracic artery when used for coronary artery bypass grafting.

Enhanced nitric oxide concentrations and expression of nitric oxide synthase in acupuncture points/meridians.

The objectives of this study were to examine the distributions of nitric oxide (NO) in the skin points (acupoints)/meridian regions and determine whether neuronal nitric oxide synthase (nNOS) protein levels were associated with NO concentrations in the areas. Low skin resistance points (LSRP) on the skin surface in response to electrical stimuli were performed in anesthetized adult rats. The skin together with subcutaneous tissue was isolated in meridian regions from PC 2 to 6, BL 36 to 57, CV 3 to 22, and GV 2 to 14. Control skin tissues were obtained in the areas close to related meridians without containing LSRP. Concentrations of nitrite (NO(2)(-)), nitrate (NO(3)(-)), and total NO(2)(-) plus NO(3)(-) (NO(x)(-)) were quantified in the skin tissues, micropunches of brain nuclei, and blood vessels in a blinded fashion. Western blots were also conducted using polyclonal anti-nNOS and anti-endothelial nitric oxide synthase (eNOS) antibody in the skin tissues. NO(x)(-) and NO(3)(-) concentrations were higher (45 +/- 8% and 43 +/- 7% in the CV, 47 +/- 7% and 51 +/- 9% in the BL, and 47 +/- 8% and 45 +/- 6% in the PC) than those in control regions (p < 0.05, n = 6). NO(x)(-) concentrations are 2- to 3-fold greater in skin tissues than those in brain regions and blood vessels (p < 0.05, n = 6-8). nNOS protein levels were consistently increased in the skin regions of BL, PC, and GV meridians compared with their controls (p < 0.05, n = 5-7) but endothelial NO synthase expression was not changed. This is the first evidence showing that NO contents and nNOS expression are consistently higher in the skin acupoints/meridians associated with low electric resistance. The results suggest that enhanced NO in the acupoints/meridians is generated from multiple resources including neuronal NOergic system, and NO might be associated with acupoint/meridian functions including low electric resistance.

Distribution of nitric oxide synthase immunoreactivity in the nervous system and peripheral tissues of Schistosoma mansoni.

The distribution of nitric oxide synthase (NOS) immunoreactivity and putative NOS activity in adult Schistosoma mansoni was analysed using 3 different types of NOS antibodies and NADPH-diaphorase histochemistry. Although potential involvement of the gaseous radical nitric oxide (NO) in host response to infection by schistosomes has been suggested, there is little or no information available regarding the role, or even the presence, of the NO pathway in schistosomes themselves. Here, we demonstrate that antibodies against neuronal NOS (nNOS) and inducible NOS (iNOS) isoforms stain adult worms with distinctive patterns; anti-endothelial NOS (eNOS) shows no selective labelling. nNOS-like immunoreactivity is found in the main nerve cords and the peripheral nervous system. Putative sensory neurons with apical neuronal processes leading to the tegument of male worms are also immunoreactive for nNOS. Anti-iNOS labels a variety of predominantly non-neuronal tissues, showing intense labelling at or near the surface of the worm and in components of the gastrointestinal tract. The distribution of NADPH-diaphorase reactivity (a histochemical marker of NOS), is generally similar to the pattern of NOS immunoreactivity, including labelling of neuronal-like cells as well as developing eggs. These results suggest that an NOS-like enzyme is present in S. mansoni, and indicate potential roles for the different NOS isoforms in neuronal signalling, reproduction and development.

The Role Of Endothelial Nitric Oxide Synthase Expression In The Development Of Pulmonary Hypertension In Chronically Hypoxic Infant Swine

Objective: Our goal was to determine the role of pulmonary endothelial nitric oxide synthase expression in the development of pulmonary hypertension in infants with congenital cyanotic heart disease. Methods: Two groups of 4-week-old piglets were studied. In one group, the piglets were raised in an environment of 10% oxygen from 2 days of age (cyanotic, n = 6), and in the other group the piglets were raised at room air (control, n = 5). Pulmonary hemodynamics were measured in vivo for each animal, and peripheral lung biopsy specimens were obtained for Western blot analysis with the use of antiendothelial nitric oxide synthase antibody and for activity analysis with the use of the tritiated l-arginine assay. Results: The piglets in the chronically hypoxic group had significant increases in mean pulmonary arterial pressure (44.0 ± 3.8 mm Hg vs 14.8 ± 1.2 mm Hg in controls, p = 0.0007) and pulmonary vascular resistance (7272.0 ± 871.1 dyne · cm · sec -5 vs 1844.5 ± 271.2 dyne · cm · sec -5in controls, p = 0.002). These changes in the pulmonary hemodynamics of the hypoxic piglets were accompanied by a twofold increase in the expression of pulmonary endothelial nitric oxide synthase ( p = 0.0043) but no corresponding increase in nitric oxide synthase activity. Conclusions: Raising infant piglets in an environment of 10% oxygen for 4 weeks results in significant pulmonary arterial hypertension accompanied by increased expression of nitric oxide synthase within the lung endothelium. Furthermore, the increased levels of nitric oxide synthase within the lungs of the hypoxic swine were not accompanied by a proportional increase in enzyme activity. These findings suggest that the development of pulmonary hypertension in infants with congenital cyanotic disease is not due to decreased expression of endothelial nitric oxide synthase, but instead may be related to a decreased ability of the enzyme to produce sufficient nitric oxide. (J Thorac Cardiovasc Surg 1998;115:343-50)