trans-Arachidonic acids induce a heme oxygenase-dependent vasorelaxation of cerebral microvasculature

Nitrative stress is an important regulator of vascular tone. We have recently described that trans-arachidonic acids (TAA) are major products of NO(2)(.)-mediated isomerization of arachidonic acid in cell membranes and that nitrative stress increases TAA levels leading to neural microvascular degeneration. In the present study, we explored whether TAA exert acute effects on neuromicrovascular tone and investigated potential mechanisms thereof. TAA induced an endothelium-dependent vasorelaxation of rat brain pial microvasculature. This vasorelaxation was independent of nitric oxide, prostanoids, lipoxygenase products, and CYP(450) metabolite trans-hydroxyeicosatetraenoic acids. However, inhibition of heme oxygenase (using zinc protoporphyrin IX) and of dependent soluble guanylate cyclase (sGC; using ODQ) significantly diminished (by approximately 70%) the TAA-induced vasorelaxation. Consistent with these findings, TAA stimulated heme oxygenase (HO)-2-dependent bilirubin (using siRNA HO-2) and cGMP formation, and the HO product carbon monoxide (using CO-releasing CORM-2) reproduced the sGC-dependent cGMP formation and vasorelaxation. Further exploration revealed that TAA-induced vasorelaxation and bilirubin formation (HO activation) were nearly abrogated by large-conductance calcium-dependent potassium channels (BK(Ca)) (using TEA and iberiotoxin). Opening of BK(Ca) with the selective activator NS1619 induced a concentration-dependent vasorelaxation, which was inhibited by HO and sGC inhibitors. Coimmunoprecipitation suggested a molecular complex interaction between BK(Ca) and HO-2 (but not HO-1). Collectively, these findings identify new properties of TAA, specifically cerebral vasorelaxation through interactive activation of BK(Ca) with HO-2 and, in turn, sGC. Our findings provide new insights into the characterization of nitrative stress-derived TAA products, by showing they can act as acute mediators of nitrative stress on neurovascular tone.     

Isomer-specific effects of conjugated linoleic acid on gene expression in RAW 264.7

Conjugated linoleic acid (CLA) is a mixture of dietary fatty acids that has various beneficial effects including decreasing cancer, atherosclerosis, diabetes and inflammation in animal models. Some controversy exists on the specific isomers of CLA that are responsible for the benefits observed. This study was conducted to examine how different CLA isomers regulate gene expression in RAW 264.7. A mouse macrophage cell line, RAW 264.7, was treated with five different CLA isomers (9E,11E-, 9Z,11E-, 9Z,11Z-, 10E,12Z- and 11Z,13E-CLA). Gene expression microarrays were performed, and several significantly regulated genes of interest were verified by a real-time polymerase chain reaction (PCR). Examination of the biological functions of various significantly regulated genes by the five CLA isomers showed distinct properties. Isomers 9E,11E-, 9Z,11Z-, 10E,12Z- and 11Z,13E-CLA decreased production of proinflammatory cytokines such as interleukin (IL)-1α, IL-1β and IL-6. Many of CLA's effects are believed to be mediated by the fatty acid receptors such as the peroxisome proliferator-activated receptors (PPAR) and retinoid-X-receptors (RXR). Using PPAR and RXR specific antagonists and coactivator recruitment assays, it was evident that multiple mechanisms were responsible for gene regulation by CLA isomers. Coactivator recruitment by CLA isomers showed their distinct properties as selective receptor modulators for PPARγ and RXRα. These studies demonstrate distinct isomer differences in gene expression by CLA and will have important ramifications for determining the potential therapeutic benefit of these dietary fatty acids in prevention of inflammation-related diseases.     

Oxidative stress and increased formation of vasoconstricting F2-isoprostanes in patients with reversible cerebral vasoconstriction syndrome

The pathophysiology of reversible cerebral vasoconstriction syndrome (RCVS) is unknown. Oxidative stress is detrimental to endothelial function and vascular reactivity. We hypothesized that the oxidative stress marker 8-iso-prostaglandin F_2α, which is also a potent vasoconstrictor, might contribute to the pathogenesis of RCVS. Recruited participants included 103 RCVS patients, 53 patients with primary headache with acute severe attacks, and 54 healthy controls. Subjects recruited prior to 2009 were discovery cohort, whereas those after 2009, replication cohort. Urine samples were obtained from all patients at registration and from 79 patients with RCVS again at remission stage. Urine 8-iso-prostaglandin F_2α was analyzed by liquid chromatography-tandem mass spectrometry. Patients with RCVS received magnetic resonance angiography and transcranial color-coded sonography. In RCVS patients, the urine 8-iso-prostaglandin F_2α level was higher than that in the other groups in discovery, replication, and combined cohorts (RCVS, 0.29±0.18; primary headache with acute severe attacks, 0.21±0.19; control, 0.18±0.09 ng/mg creatinine; P<0.001), and it was positively correlated with the flow velocities of major intracranial arteries, especially within the first week of disease onset (middle cerebral artery, Spearman's correlation coefficient [r_s]=0.580, P=0.002; anterior cerebral artery, r_s=0.472, P=0.042; posterior cerebral artery, r_s=0.457, P=0.022; basilar artery, r_s= 0.530, P=0.002). The 8-iso-prostaglandin F_2α level decreased from the ictalto remission stage in RCVS patients (0.31±0.21 vs 0.16±0.10 ng/mg creatinine, P<0.001). 8-Iso-prostaglandin F_2α was higher in patients with RCVS and correlated with the severity of vasoconstrictions. Further studies are required to explore its potential pathogenic role.     

Effects of estrogen on cerebral blood flow and pial microvasculature in rabbits

We tested the hypothesis that intracarotid estrogen infusion increases cerebral blood flow (CBF) in a concentration-dependent manner and direct application of estrogen on pial arterioles yields estrogen receptor-mediated vasodilation. Rabbits of both genders were infused with estrogen via a branch of the carotid artery. Estrogen doses of 20 or 0.05 microg. ml(-1). min(-1) were used to achieve supraphysiological or physiological plasma estrogen levels, respectively. CBF and cerebral vascular resistance were determined at baseline, during the infusion, and 60-min postinfusion, and effects on pial diameter were assessed via a cranial window. Pial arteriolar response to estrogen alone and to estrogen after administration of tamoxifen (10(-7)), an antiestrogen drug that binds to both known estrogen receptor subtypes, was tested. No gender differences were observed; therefore, data were combined for both males and females. Systemic estrogen infusion did not increase regional CBF. Estradiol dilated pial arteries only at concentrations ranging from 10(-4)-10(-7) M (P < or = 0.05). Pretreatment with tamoxifen alone had no effect on arteriolar diameter but inhibited estrogen-induced vasodilation (P < 0.001). Our data suggest that estrogen does not increase CBF under steady-state conditions in rabbits. In the pial circulation, topically applied estradiol at micromolar concentrations dilates vessels. The onset is rapid and dependent on estrogen receptor activation.     

Isomer-specific effects of conjugated linoleic acid (CLA) on adiposity and lipid metabolism

Isomers of conjugated linoleic acid (CLA), unsaturated fatty acids found in ruminant meats and dairy products, have been shown to reduce adiposity and alter lipid metabolism in animal, human, and cell culture studies. In particular, dietary CLA decreases body fat and increases lean body mass in certain rodents, chickens, and pigs, depending on the isomer, dose, and duration of treatment. However, the effects of CLA on human adiposity are conflicting because these studies have used different mixtures and levels of CLA isomers and diverse subject populations. Potential antiobesity mechanisms of CLA include decreased preadipocyte proliferation and differentiation into mature adipocytes, decreased fatty acid and triglyceride synthesis, and increased energy expenditure, lipolysis, and fatty acid oxidation. This review will address the current research on CLA's effects on human and animal adiposity and lipid metabolism as well as potential mechanism(s) responsible for CLA's antiobesity properties.     

Isomer-specific effects of conjugated linoleic acid on proliferative activity of cultured neural progenitor cells

We used forebrain-specific conditional presenilin 1 (PS1) and presenilin 2 (PS2) double knockout mice (dKO mice), which exhibit neurodegenerative disease-like symptoms, including inflammation of the brain and periphery, to investigate whether periodontal and salivary tissues display alterations. Mandibles were dissected for alveolar bone height analysis. Maxillae were fixed and decalcified for histological observation and osteoclast detection. Submandibular glands were fixed for histological observation. The submandibular gland and the gingiva of the mandibular incisor teeth were used to assay inflammatory mediators. At 9 months, the number of osteoclasts had significantly increased in the periodontal ligament and the periodontal tissues exhibited obvious histomorphological abnormalities in the dKO mice compared to the control mice at the same age. Alveolar bone loss in dKO mice increased with age. The salivary tissues in dKO mice exhibited obvious age-dependent histomorphological abnormalities. The levels of the inflammatory mediators IL-1β, TNF-α, and GM-CSF in the submandibular gland and gingiva also increased in an age-dependent manner. These findings suggest that inflammation in the dKO brain could expand to the periphery, including the oral tissue, which could ultimately induce abnormalities in the periodontal and salivary tissues.     

Quantification of F2-isoprostanes as a biomarker of oxidative stress

Oxidant stress has been implicated in a wide variety of disease processes. One method to quantify oxidative injury is to measure lipid peroxidation. Quantification of a group of prostaglandin F(2alpha)-like compounds derived from the nonezymatic oxidation of arachidonic acid, termed the F2-isoprostanes (F2-IsoPs), provides an accurate assessment of oxidative stress both in vitro and in vivo. In fact, in a recent independent study sponsored by the National Institutes of Health (NIH), F2-IsoPs were shown to be the most reliable index of in vivo oxidant stress when compared against other well known biomarkers. This protocol details our laboratory's method to quantify F2-IsoPs in biological fluids and tissues using gas chromatography-mass spectrometry (GC-MS). This procedure can be completed for 12-15 samples in 6-8 h.     

Isomer-specific effects of conjugated linoleic acid on mineralized bone nodule formation from human osteoblast-like cells

Mixed isomers of conjugated linoleic acid (CLA) have been shown to have variable effects on bone formation and resorption in animals. The variable effects of CLA on bone physiology may be due to the different isomers present in common commercial preparations of CLA, and the effects of the predominant individual isomers (9cis,11trans and 10trans,12cis CLA) are not clear. The objective of this study was to determine the effects of individual and mixed isomers of CLA on mineralized bone nodule formation and alkaline phosphatase (ALP) activity in vitro using long-term cultures of SaOS-2 cells. Mineralized bone nodules were stained using the von Kossa method, and ALP activity in cell lysates was measured as a marker of early osteoblast differentiation. The 9cis,11trans isomer increased the number (~4- to 11-fold) and size (~2- to 5-fold) of mineralized bone nodules from 25 to 100 microM, but the 10trans,12cis isomer did not. The increase in mineralized bone nodule formation by 9cis,11trans CLA was accompanied by a variable increase in ALP activity. These results show that the 9cis,11trans isomer of CLA increases the formation of mineralized bone nodules using bone cells of human origin, and provide evidence for isomer-specific effects of CLA on bone health.     

Studies on the stereoselective effects of a novel 5-HT2 receptor antagonist on contractile responses of rat aorta

The effect of the enantiomers of a novel 5-HT2 receptor antagonist, (+/-)-(1R,3S)-1-[2-[4-[3-(p-fluorophenyl)-1-indanyl]-piperazinyl] ethyl]-2-imidazolidinone, was studied on serotonin (5-HT), noradrenaline (NA), potassium (K+), and calcium (Ca2+)-induced contractions in isolated rat thoracic aorta. The enantiomers shifted the 5-HT, NA, K+, and Ca2+ concentration-response curves to the right in a concentration-dependent manner and depressed the maximal contractile responses. The (+)-enantiomer was a far more potent inhibitor of 5-HT-induced contractions than the (-)-enantiomer. The (+)-enantiomer and phentolamine, both at 10(-6) M, had equal inhibitory effects on NA-evoked contractions. The (+)-enantiomer was again more potent inhibiting NA-induced contractions than the (-)-enantiomer. Both enantiomers had an equieffective inhibitory effect on K+ and Ca2(+)-induced contractions. The results show that the 5-HT and alpha-adrenoceptor antagonism of the two enantiomers is stereoselective, the (+)-enantiomer being more potent than the (-)-enantiomer. In contrast the enantiomers had equal, nonstereoselective inhibitory effects on K+ and Ca2(+)-evoked contractions.     

Characterization of the contractile P2Y14 receptor in mouse coronary and cerebral arteries

Extracellular UDP-glucose can activate the purinergic P2Y14 receptor. The aim of the present study was to examine the physiological importance of P2Y14 receptors in the vasculature. The data presented herein show that UDP-glucose causes contraction in mouse coronary and basilar arteries. The EC₅₀ values and immunohistochemistry illustrated the strongest P2Y14 receptor expression in the basilar artery. In the presence of pertussis toxin, UDP-glucose inhibited contraction in coronary arteries and in the basilar artery it surprisingly caused relaxation. After organ culture of the coronary artery, the EC₅₀ value decreased and an increased staining for the P2Y14 receptor was observed, showing receptor plasticity.     

Inosine diphosphatase as a histochemical marker of retinal microvasculature,with special reference to transformation of microglia

Summary Nucleoside diphosphatase (IDPase), localized using inosine diphosphate as substrate, allows the selective staining of blood vessels and cells of vascular origin, such as macrophages and microglia, whereas the neuroglial, the neuronal and the pigment epithelial cells remain unstained. The staining pattern observed in the retina of mouse, rat, cat and monkey are similar; some apparent quantitative differences reflect species differences in the distribution of retinal microvasculature. At the electron-microscopic level, most of the enzyme activity in the blood vessels appears to be located along the outer wall. The cell membrane, parts of the smooth endoplasmic reticulum and the nuclear membrane in the microglial perikarya appear positive; profiles of microglial processes are intensely stained.In the developing eyes of rats and mice, the blood vessels are stainable from the earliest stage of their appearance. An array of amoeboid cells precede the growing blood vessels and spread out over the future vascularized part of the retina. These cells eventually develop characteristic microglial features, and extend many elongated and branched processes between the neuroepithelial cells while remaining in contact with, or in close proximity to, the blood vessels. Intense IDPase activity in the microglial cells, in contrast to the absence of the enzyme in the neuroglial Müller cells, suggests that microglia are involved in phosphate metabolism and indicates functional compartmentalization within the glial tissue lying between the blood retinal barrier and the retinal neurons.     

Measurement of F2-isoprostanes, hydroxyeicosatetraenoic products, and oxysterols from a single plasma sample

Oxidized lipids such as F2-isoprostanes (F2-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), and cholesterol oxidation products (COPs) are widely believed to be involved in multiple diseases. Usually, each product is measured individually in separate blood samples. In this study we describe a method allowing us to measure F2-IsoPs, HETEs, COPs, and arachidonate using a single sample. Plasma (1 ml) samples from healthy volunteers were diluted with heavy isotopic standards, hydrolyzed in alkali with organic solvent, and then subjected to anionic-exchange solid-phase extraction (SPE). After the SPE column was washed, hexane and hexane/ethyl acetate portions were collected and combined for COPs measurement. Thereafter the column was loaded with hexane/ethanol/acetic acid and fractions were collected for total F2-IsoPs, total HETEs, and arachidonate measurement. All compounds in the eluates were measured by gas chromatography-mass spectrometry. The efficiency of SPE and reproducibility for all compounds measured were high. Levels of total F2-IsoPs (0.45+/-0.26 ng/ml (n=157)), total HETEs (34.06+/-16.35 ng/ml (n=21)), total arachidonate (68.36+/-24.45 microg/ml (n=33)), and COPs (7-ketocholesterol, 12.25+/-6.56 ng/ml; 7beta-hydroxycholesterol, 6.32+/-3.46 ng/ml; 7alpha-hydroxycholesterol, 15.06+/-7.06 ng/ml; 24-hydroxycholesterol, 41.39+/-18.22 ng/ml; and 27-hydroxycholesterol, 29.08+/-16.79 ng/ml (n=26)) were recorded in healthy subjects (age range 20 to 66 years; average male to female ratio 1:1).     

Acute and chronic effects of oral enprostil, a synthetic dehydroprostaglandin E2, on uterine contractility

In acute experiments eleven nonpregnant women received a single oral dose of enprostil (prostaglandin E2 analogue) 35-140 mcg. Uterine activity was measured by a microtransducer-tipped Millar catheter. A single, oral dose of enprostil caused a long-lasting contracture response of the uterus. 3 h after enprostil, uterine resting pressure (RP) was still high. In chronic experiments, eleven women with regular menstrual cycles received 35 mcg or 70 mcg BID enprostil and placebo in a crossover, double-blind fashion from cycle day 10 +/- 3 for four weeks, then had a washout period of four weeks followed by another four-week treatment period. An increase of uterine RP after enprostil was dose-dependent. After two weeks of twice-daily administration of enprostil, the baseline RP was lower than after placebo (p less than 0.01) and the increase in RP after the morning enprostil less than that seen on the first day. In eight patients studied, the mean values of plasma progesterone were normal, both during placebo--and enprostil--treated cycles.     

Cellular glycoconjugates and their potential endogenous receptors in the cerebral microvasculature of man: a glycohistochemical study

Protein-carbohydrate interactions are supposed to play a pivotal role in mediation of recognitive interactions, relevant to cellular interactions and transport. The brain microvasculature is the site of numerous cell-cell and cell-matrix recognitive interactions. Assuming carbohydrate-protein interactions play important physiological roles here, then specific carbohydrate-binding proteins should be prominent components of this microvasculature, in addition to the wealth of endogenous glycoconjugates reported by other authors. Human brain and brain tumor microvessels were analyzed histochemically for expression of endogenous sugar-binding proteins using a panel of biotin-conjugated, chemically glycosylated probes with specificities for alpha-/beta-D-galactosides and -D-glucosides, alpha-L-fucosides, alpha-D-mannosides, and beta-D-xylosides, and for expression of endogenous glycoconjugates using a panel of biotin-conjugated plant lectins with specificities for fucoside, galactoside, mannoside and glucoside moieties. Wax-embedded aldehyde- or Bouin-fixed tissues or acetone-fixed frozen sections were examined. Blood-brain barrier function was checked by ascertaining immunohistochemically the extravasation of serum albumin in these tissues. Microvessels in normal human brain tissues (with intact blood-brain barrier) contained abundant endogenous sugar-binding proteins with specificities for beta-galactosides, alpha-mannosides and beta-xylosides, and lesser amounts of proteins binding alpha-galactosides, alpha-fucosides and glucosides. Endogenous glycoconjugates bearing beta-galactoside, alpha-D-mannoside/alpha-D-glucoside or alpha-L-fucoside moieties were also abundant. In brain tumors (with defective blood brain barrier) the microvessels contained altered patterns of endogenous sugar-binding proteins, particularly noticeable in glioblastomas, in which there were notable alterations in galactoside-binding proteins in the microvessels.