Endothelial Nitric Oxide Synthase mRNAs Research Articles

Expression of genes for transport of water and angiogenesis, as well as biochemical biomarkers in Holstein dairy cows during the ovsynch program

Changes in expression of genes associated with angiogenesis and transport of water by cells, as well as biomarkers of oxidative stress were determined at specific times during the ovsynch protocol to synchronize estrus and breed Holstein dairy cows. Blood samples were taken from 82 lactating Holstein cows at the time of the 1st GnRH injection (G1), 7 days later at the time of the PGF2a (PG) injection, and 48 h after the PGF2a treatment when the second injection of GnRH was administered (G2). The serum was analyzed for malondialdehyde (MDA), reduced glutathione (GSH), glutathione peroxidase (GPX), nitric oxide (NO), catalase (CAT), and total antioxidant capacity (TAC). The expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), endothelial nitric oxide synthase (eNOS3), aquaporin 3 (AQP3), and AQP4 mRNAs in peripheral blood mononuclear cells (PBMCs) was analyzed. The number of copies of each of the mRNAs was quantified using qPCR. Pregnancy status was determining at 32 ± 3 days after insemination using an ultrasound “Sonoscape-5V″ model. Receiver operating curves (ROC) were used to assess the sensitivity and specificity of the biochemical parameters in serum to predict establishment of p The expression of MDA, GPX, and Catalase changed (P < 0·05) between G1, PG and G2 phases of the ovsynch protocol with higher levels at PG than at G1 and G2. The highest levels of NO were detected at G2. The ROC analyses identified NO, TAC and CAT as the most sensitive and specific biomarker for pregnancy with areas under the curve being 0.875 (P < 0.0001), 0.843 (P < 0.03), 0.833 (P < 0.017), sensitivity being 75.3, 42.86, and 26.27%, and specificity being 90, 90 and 85% respectively. The expression for VEGF, VEGFR2, eNOS3, AQP3, and AQP4 mRNAs was upregulated at PG compared to G1 and G2 phases of the ovsynch protocol. The results suggest that following the first injection of GnRH, there is an increase in expression of VEGF, VEGFR2, eNOS3, AQP3, and AQP4 mRNAs by the time of the PGF2a injection and then expression decreased. Further, ROC analyses identified increases in NO, TAC and CAT as the most sensitive and specific biomarkers with the greatest potential to predict establishment of pregnancy in Holstein cows.

Perfusion culture of endothelial cells under shear stress on microporous membrane in a pressure-driven microphysiological system

This paper reports perfusion culture of human umbilical vein endothelial cells (HUVECs) on a microporous membrane in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. We designed fluidic culture unit with microporous membrane to culture HUVECs under fluidic shear stress and constructed a perfusion culture model in the PD-MPS platform. Four fluidic culture units were arranged in the microplate-sized device, which enables four-throughput assay for characterization of HUVECs under flow. Medium flow was generated above and below the membrane by sequential pneumatic pressure to apply physiological shear stress to HUVECs. HUVECs exhibited aligned morphology to the direction of the flow with shear stress of 11.5-17.7dyn/cm2 under the flow condition, while they randomly aligned under static culture condition in a 6 well plate. We also observed 3.3- and 5.0-fold increase in the expression levels of the thrombomodulin and endothelial nitric oxide synthase mRNAs, respectively, under the flow condition in the PD-MPS compared to the static culture in 6 well plate. We also observed actin filament aligned to the direction of flow in HUVECs cultured under the flow condition.

Increased dipeptidyl peptidase-4 accelerates chronic stress-related thrombosis in a mouse carotid artery model.

Exposure to chronic psychosocial stress is a risk factor for metabolic cardiovascular disorders. Given that dipeptidyl peptidase-4 (DPP-4) has an important role in human pathobiology, we investigated the role of DPP-4 in stress-related thrombosis in mice, focusing on oxidative stress and the von Willebrand factor (vWF)-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13). Male mice randomly assigned to nonstress and 2-week immobilized-stress groups underwent iron chloride3 (FeCl3)-induced carotid artery thrombosis surgery for morphological and biochemical studies at specific times. On day 14 post-stress/surgery, stress had enhanced the lengths and weights of arterial thrombi, with alterations of plasma DPP-4, plasminogen activation inhibitor-1 and ADAMTS13. The stressed mice had increased levels of vascular cell adhesion molecule-1, intracellular adhesion molecule-1, monocyte chemoattractant protein-1, gp91phox, p22phox, matrix metalloproteinase-2 (MMP-2), MMP-9, cathepsins S and K mRNAs and/or proteins, and reduced levels of endothelial nitric oxide synthase, catalase and superoxide dismutase-1 mRNAs and/or proteins. Stress also accelerated arterial endothelial cell damage. The DPP-4 inhibitor anagliptin ameliorated the stress-induced targeted molecular and morphological changes and thrombosis. In vitro, DPP-4 inhibition also mitigated the alterations in the targeted ADAMTS13 and other oxidative and inflammatory molecules in human umbilical vein endothelial cells in response to H2O2. DPP-4 inhibition appeared to improve the FeCl3-induced thrombosis in mice that received stress, possibly via the improvement of ADAMTS13 and oxidative stress, suggesting that DPP-4 could become a novel therapeutic target for chronic psychological stress-related thrombotic events in metabolic cardiovascular disorders.

Clopidogrel or prasugrel reduces mortality and lessens cardiovascular damage from acute myocardial infarction in hypercholesterolemic male rats

AimsHypercholesterolemia is a hazard for increasing susceptibility of the heart to myocardial infarction (MI) by inducing platelet hyperaggregability. Clopidogrel and prasugrel have documented cardioprotective effects in clinical studies. Herein, we investigated whether clopidogrel and prasugrel could protect against isoproterenol-induced acute MI (A-MI) under hypercholesterolemic conditions in rats. Main methodsDietary hypercholesterolemic rats were subjected to acute doses of isoproterenol. Serum lipids, inflammatory markers, aortic endothelin1 and endothelial nitric oxide synthase (eNOS) mRNAs expression and immunexpression of BCL2 were determined. Key findingsHypercholesterolemic rats showed infiltration of inflammatory cells and reduction in aortic wall thickness, deposition of fibrous tissue between cardiac muscle fibers. Protective doses of prasugrel or clopidogrel for 28 days before A-MI increased survival, amended the ECG parameters -including ST segment elevation- and improved the histopathological picture in hypercholesterolemic rats. This was coupled with reductions in platelet aggregation, creatine kinase-MB activity, endothelin 1, systemic inflammation and cardiac lipid peroxidation and increment in aortic eNOS expression. Clopidogrel and prasugrel groups showed enhanced BCL2 expression in cardiac fibers and aortic wall. SignificancePrasugrel and clopidogrel protected against A-MI via anti-aggregatory and anti-inflammatory effects. These results add to the value of these drugs in correcting cardiovascular dysfunction in patients vulnerable to A-MI after confirmation by appropriate human studies.

Combination treatment with asiaticoside and rapamycin: A new hope for in-stent restenosis

The aim of this study was to investigate and characterize the efficacy and mechanism of action of asiaticoside in combination with rapamycin in the inhibition of in-stent restenosis (ISR). The effects of asiaticoside combined with rapamycin on cell proliferation in vitro were evaluated by MTT assay. The mRNA expression was analyzed by quantitative polymerase chain reaction (qPCR). Enzyme-linked immunosorbent assay (ELISA) was used to confirm protein synthesis. The cell growth inhibition rate in the combination group was significantly higher compared with those in the asiaticoside and rapamycin groups for human aortic fibroblasts (HAFs; 63.50±3.83, 53.06±8.10 and 60.34±4.9%, respectively) and human aortic smooth muscle cells (HASMCs; 33.12±1.35, 26.21±7.59 and 28.27±4.92, respectively; P<0.05). However, for human coronary artery endothelial cells (HCAECs), the cell growth inhibition rates in the combination, asiaticoside and rapamycin groups were 11.09±1.17, 26.22±4.24 and 34.80±2.80%, respectively (P<0.05), as detected by MTT assay. The qPCR assay showed that in the combination group the level of von Willebrand factor (vWF) mRNA was downregulated, while platelet endothelial cell adhesion molecule (PECAM-1) and endothelial nitric oxide synthase (eNOS) mRNAs were upregulated in HCAECs compared with the rapamycin group (P<0.05). Transforming growth factor (TGF)-β1 and TIMP1 mRNAs were downregulated while Smad7 and matrix metalloproteinase 1 (MMP1) mRNAs were upregulated in HAFs compared with the rapamycin and AT groups (P<0.05). The ELISA showed that the type I collagen level was significantly reduced in HASMCs and HAFs (P<0.05). The data suggest that asiaticoside combined with rapamycin may be effective in the reduction of ISR.

Endothelial dysfunction in the early- and late-stage type-2 diabetic Goto-Kakizaki rat aorta

As there are increasing evidences that human diabetes induces cardiovascular dysfunction, we investigated the type-2 diabetes-induced endothelial dysfunction in the early and late-stage Goto-Kakizaki (GK) rat aorta. We performed organ bath studies, and examined the changes in expression levels of muscarinic M(3) receptor, endothelial, inducible, and neuronal nitric oxide synthase (eNOS, iNOS, and nNOS, respectively) mRNAs in the rat aorta utilizing real-time polymerase chain reaction in 12-week-old and 70-week-old GK rats as well as in age-matched Wistar rats. In the 12-week-old GK rat aorta, a significant increase in norepinephrine-induced contraction and a significant decrease in acetylcholine-induced relaxation as well as significant increases in expression levels of muscarinic M(3) receptor and eNOS and a significant decrease in nNOS mRNAs were observed compared to age-matched controls. In the older GK rat aorta, significant decreases in acetylcholine- and nitroglycerine-induced relaxations as well as significant decreases in the expression levels of muscarinic M(3) receptor, eNOS, iNOS, and nNOS mRNAs were observed compared to those in the younger GK rats. In contrast, although significant decreases in acetylcholine and nitroglycerine-induced relaxations were observed, the expression levels of muscarinic M(3) receptor, eNOS, iNOS, and nNOS mRNAs in the older Wistar rats aorta were unchanged, increased, increased and decreased, respectively, compared to the younger Wistar rat aorta. These results indicate that endothelial dysfunction in the rat aorta progresses with age and development of diabetes condition, and that decreased relaxations in the late-stage rat aorta may be due to these alterations.

Cyclohexenonic long-chain fatty alcohol has therapeutic effects on diabetes-induced angiopathy in the rat aorta

We studied the effects of cyclohexenonic long-chain fatty alcohol ( N-hexacosanol) on diabetes-induced angiopathy in the rat aorta. Male Sprague–Dawley rats were divided into 4 groups, a control group and 3 other groups in which diabetes was induced by streptozotocin (50 mg/kg i.p.). Four weeks after the induction of diabetes, the 3 groups received treatment with either vehicle or N-hexacosanol (2 or 8 mg/kg, i.p. every day) for another 4 weeks. To determine the mechanisms of diabetic vascular dysfunction and the effects of N-hexacosanol, we conducted organ bath studies and real-time polymerase chain reaction on muscarinic M 3 receptor, and endothelial and inducible nitric oxide synthase (eNOS and iNOS) mRNAs in the rat aorta. Treatment with N-hexacosanol did not alter the diabetic status, but improved the diabetes-induced hypercontraction produced by norepinephrine and the damaged endothelium-dependent relaxation of the rat aorta induced by acetylcholine. Furthermore, in the diabetic rats, both muscarinic M 3 receptor and iNOS mRNAs were significantly increased, and N-hexacosanol reversed these upregulations. However, the expression of eNOS mRNA showed no change in all groups. These results indicate that N-hexacosanol has beneficial effects on functional dysfunction and reverses the upregulation of muscarinic M 3 receptor and iNOS mRNAs in the diabetic rat aorta.

Differences in nitric oxide production: a comparison of retinal ganglion cells and retinal glial cells cultured under hypoxic conditions

The aim of this study was to compare the effects of hypoxia on nitric oxide synthase (NOS) expression and the production of NO between isolated retinal ganglion cells (RGCs) and retinal glial cells. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the presence of neuronal NOS mRNA, inducible NOS mRNA, and endothelial NOS mRNAs in the two cell types. RGCs and retinal glial cells were separately cultured under hypoxic (10% O 2) or control (20% O 2) conditions. Changes in NOS-mRNA expression were quantified by real-time PCR, and nitrite in the medium was measured up to 96 h of culture. The effects of non-NOS- and iNOS-selective inhibitors on hypoxia-induced release of nitrite in the culture medium were evaluated. RT-PCR revealed the presence of three types of NOSs in the two types of cultured cells. Hypoxic culture conditions significantly changed the expression of all NOS mRNAs in retinal glial cells but not in RGCs. NO production showed significant changes corresponding to those of NOS mRNAs in retinal glial cells but not in RGCs, and both NOS inhibitors significantly reduced hypoxia-induced nitrite release in retinal glial cells. Retinal glial cells but not RGCs may be the major source of NO under hypoxic conditions.

Perturbations in paracrine control of the circulation: role of the endothelial-derived vasomediators, endothelin-1 and nitric oxide.

Central to the control of vascular resistance in the systemic and pulmonary circulation and at the solid organ level is the function of the endothelial-derived vasomediators, endothelin-1 (ET-1) and nitric oxide. Regulation of steady-state levels of ET-1 and endothelial nitric oxide synthase (eNOS) mRNAs represents an early and influential step in their biosynthesis and is highly responsive to exogenous stimuli. ET-1 is expressed from a rapidly inducible promoter to generate a highly labile mRNA. Conversely, the eNOS promoter generates a constitutive level of a very stable mRNA and utilizes posttranscriptional mechanisms to modulate mRNA expression. The response of these genes in models of cellular activation commonly reflects a reciprocal pattern of regulation, namely, transcriptional induction of ET-1 and destabilization of the eNOS mRNA. Elucidating the mechanisms influencing ET-1 and eNOS mRNA is providing novel insight into endothelial gene regulation and providing opportunities for future therapeutic strategies.

Expression of nitric oxide synthases in rat adrenal zona fasciculata cells.

Nitric oxide (NO) synthase (NOS) expression was analyzed in rat adrenal zona fasciculata. Both neuronal NOS and endothelial NOS mRNAs were detected by RT-PCR, immunohistochemistry, and immunoblot analysis. The biochemical characterization of adrenal zona fasciculata NOS enzymatic activity confirmed the presence of a constitutive isoform. In a cell line derived from mouse adrenal cortex, only endothelial NOS expression was detected by both RT-PCR and immunoblot analysis. Nitrate plus nitrite levels in Y1 cell incubation medium were increased in the presence of L-arginine and the calcium ionophore A23187, but not D-arginine, indicating enzymatic activity. Moreover, a low, but significant, conversion of Larginine to L-citrulline, abolished by the NOS inhibitor, N(G)-nitro-L-arginine, was detected in Y1 cells. The effect of L-arginine on pregnenolone production was examined. L-Arginine decreased both basal and ACTH-stimulated pregnenolone production in Y1 cells. The inhibitory effect of L-arginine could be attributed to endogenously generated NO, because it was blocked by N(G)-nitro-L-arginine, and it was mimicked by the addition of a NO donor, diethylenetriamine-NO. An inhibitory effect of NO on pregnenolone production from 22Rhydroxycholesterol and on steroidogenic acute regulatory protein expression was also determined. Taken together, these results suggest that at least part of the adrenal NO could derive from steroidogenic cells and modulate their function.

Expression of Nitric Oxide Synthases in Rat Adrenal Zona Fasciculata Cells

Nitric oxide (NO) synthase (NOS) expression was analyzed in rat adrenal zona fasciculata. Both neuronal NOS and endothelial NOS mRNAs were detected by RT-PCR, immunohistochemistry, and immunoblot analysis. The biochemical characterization of adrenal zona fasciculata NOS enzymatic activity confirmed the presence of a constitutive isoform. In a cell line derived from mouse adrenal cortex, only endothelial NOS expression was detected by both RT-PCR and immunoblot analysis. Nitrate plus nitrite levels in Y1 cell incubation medium were increased in the presence of l-arginine and the calcium ionophore A23187, but not d-arginine, indicating enzymatic activity. Moreover, a low, but significant, conversion of larginine to l-citrulline, abolished by the NOS inhibitor, NG-nitro-l-arginine, was detected in Y1 cells. The effect of l-arginine on pregnenolone production was examined. l-Arginine decreased both basal and ACTH-stimulated pregnenolone production in Y1 cells. The inhibitory effect of l-arginine could be attributed to endogenously generated NO, because it was blocked by NG-nitro-l-arginine, and it was mimicked by the addition of a NO donor, diethylenetriamine-NO. An inhibitory effect of NO on pregnenolone production from 22Rhydroxycholesterol and on steroidogenic acute regulatory protein expression was also determined. Taken together, these results suggest that at least part of the adrenal NO could derive from steroidogenic cells and modulate their function.

Differential and Constitutive Expression of Neuronal, Inducible, and Endothelial Nitric Oxide Synthase mRNAs and Proteins in Pathologically Normal Human Tissues

Nitric oxide (NO) is produced by NO synthases (nNOS, iNOS, and eNOS) expressed in various human tissues and depending on the amount of NO produced in each tissue, the physiological function of NO is determined. However, due to the difficulty in obtaining normal human tissues, little is known about the basal levels of each of the three NOS mRNAsand proteins expressed constitutively in various human tissues. Results of the present study indicate that the basal levels of each of the three NOS mRNAs and proteins expressed in various regions of brain and peripheral tissues are different both in their sizes and in their contents. In Northern blot analysis, two different-sized mRNAs were found for each NOS isozymes: for the nNOS, ∼12 and <12 kb mRNAs; for the iNOS, 4.2 and 4.5 kb mRNAs; for the eNOS, 4.2 and 4.4 kb mRNAs. In the Western blot, several different-sized NOS proteins were detected (∼160, ∼140, and ∼130 kDa for nNOS; ∼130 kDa for iNOS and eNOS) with tissue-specific expression patterns. These differential expression patterns of NOS mRNAs and proteins were caused by alternative splicing in the open-reading frame, and 5′- and/or 3′-untranslated regions of NOS mRNAs. These results suggest that regulation for differential expression of the three NOS genes in various human tissues may occur by alternative splicing of the NOS mRNAs in tissue-specific patterns.

Prenatal hypoxia-ischemia alters expression and activity of nitric oxide synthase in the young rat brain and causes learning deficits

Inhibition of nitric oxide synthase (NOS) is known to possibly impair learning and memory. Our previous studies have demonstrated that prenatal hypoxia-ischemia (HI) decreases NOS expression and NOS activity in the neonatal rat brain. To investigate whether effects of prenatal HI on NOS expression continue and whether prenatal HI affects learning and memory in young rats, NOS expression and NOS activity were determined in the hippocampus of rat brains at 28 days of age following a prenatal HI insult on G17. Performances in the passive avoidance test and the Morris water maze test were also studied in these young rats prior to sampling. Rat fetuses were subjected to either a 30-min prenatal HI insult or a sham operation (SH) on gestation day 17 and rat pups were delivered naturally. Increased locomotor activity was observed in the prenatal HI rats as compared to the SH rats on postnatal days 13 and 15, but not on postnatal days 20 and 30. Prenatal HI affected learning ability in these young rats at 28 days of age, as indicated by a delayed acquisition of passive avoidance and by longer escape latency in the Morris water maze test as compared to the SH group. Prenatal HI did not affect retention of passive avoidance and spatial memory. Concomitant with these learning deficits, expression of neuronal NOS and endothelial NOS mRNAs as well as Ca 2+-depedent NOS activity in the hippocampus of the prenatal HI rat brain were significantly decreased as compared to the SH group. These results suggest that a 30-min prenatal HI insult on gestation day 17 in rats has long-lasting effects on NOS expression and NOS activity in the offspring brain and on learning ability of these young rats. The learning deficit in offspring is possibly associated with the reduction in expression of NOS mRNA and NOS activity in the hippocampus of these animals.

Expression of endothelial factors after arterial injury in the rat.

Endothelin-1 (ET-1) and nitric oxide (NO) are potent vasoactive factors known to play a role in vascular remodeling. This study assessed the temporal expression of endothelial NO synthase (eNOS), preproET-1, and ETA and ETB receptor mRNAs in the rat carotid artery after balloon injury using quantitative competitive reverse transcription-polymerase chain reaction (qcRT-PCR) and the ribonuclease protection assay (RPA). Levels of ET-1 increased sharply after arterial injury, peaking (5.1-fold) at 2 days. This was associated with a dramatic increase in the expression of ETB (63-fold) and ETA (158-fold) receptor mRNA, peaking at days 1 and 2, respectively. Expression of eNOS was not detectable immediately after balloon injury, consistent with complete denudation, but reappeared after day 2 and increased to preinjury levels by day 14. The recovery of eNOS expression mirrored the return of ET-1 and ET receptor expression to baseline levels. The results confirm profound upregulation of the ET system in this model of arterial injury and suggest a critical role for eNOS expression and re-endothelialization in the normalization of ET-1 and ET receptor expression during the recovery phase, events that may be important in long-term arterial patency.

Differential regulation of the constitutive and inducible nitric oxide synthase mRNA by lipopolysaccharide treatment in vivo in the rat.

Endotoxin and cytokines have been reported to have both stimulatory and inhibitory effects on endothelial cell-derived nitric oxide release. The discrepancy may be explained by differential regulation of the endothelial and inducible type of nitric oxide synthase gene expression. This study aimed to investigate the differential effect of lipopolysaccharide treatment in vivo on the three isoforms (endothelial, brain-type, and inducible) of nitric oxide synthase gene expression in the rat. Prospective, controlled, animal trial. Experimental laboratory of a postgraduate medical research institution. Normal, anesthetized rats. Animals were treated with lipopolysaccharide (15 mg/kg iP), saline (1 mL/kg ip), or lipopolysaccharide plus dexamethasone (3 mg/kg ip, 50 mins before lipopolysaccharide administration) in vivo 4 hrs before experimentation. The expression of endothelial, brain-type, and inducible nitric oxide synthase mRNAs was quantified by Northern blot analysis using bovine, rat, and mouse cDNA probes, respectively. An endothelial nitric oxide synthase mRNA was detected at 4.3 kilobase in the heart, lung, and aorta, and a 10-kilobase brain-type nitric oxide synthase mRNA was detected in the brain. The endothelial and brain-type signals were strong in tissues from animals treated with saline, but were reduced by three- to four-fold in tissues from lipopolysaccharide-treated rats as estimated by optical density ratio. The 4.4-kilobase inducible nitric oxide synthase mRNA detected using the murine cDNA probe was absent or negligible in the heart, lung, and brain from saline-treated rats, but was markedly increased in the same tissues from lipopolysaccharide-treated animals. Dexamethasone significantly inhibited lipopolysaccharide-induced inducible nitric oxide synthase mRNA expression, but had no effect on the down-regulation of endothelial and brain nitric oxide synthase mRNAs. Rats treated with lipopolysaccharide in vivo display down-regulation of endothelial nitric oxide mRNA in the heart, lung, and aorta, and brain-type nitric oxide synthase mRNA in the brain There was a parallel up-regulation of inducible nitric oxide synthase mRNA in all tissues except in the aorta. Dexamethasone prevents the induction of inducible nitric oxide synthase mRNA. but has no effect on the down-regulation of endothelial and brain-type nitric oxide synthase mRNAs induced by lipopolysaccharide. Thus, endotoxin regulates constitutive and inducible nitric oxide synthase mRNA differentially.