Production of endothelial nitric oxide synthase (eNOS) over-expressing piglets

Vascular function, vascular structure, and homeostasis are thought to be regulated in part by nitric oxide (NO) released by endothelial cell nitric oxide synthase (eNOS), and NO released by eNOS plays an important role in modulating metabolism of skeletal and cardiac muscle in health and disease. The pig is an optimal model for human diseases because of the large number of important similarities between the genomic, metabolic and cardiovascular systems of pigs and humans. To gain a better understanding of cardiovascular regulation by eNOS we produced pigs carrying an endogenous eNOS gene driven by a Tie-2 promoter and tagged with a V5 His tag. Nuclear transfer was conducted to create these animals and the effects of two different oocyte activation treatments and two different culture systems were examined. Donor cells were electrically fused to the recipient oocytes. Electrical fusion/activation (1 mM calcium in mannitol: Treatment 1) and electrical fusion (0.1 mM calcium in mannitol)/chemical activation (200 μM Thimerosal for 10 min followed by 8 mM DTT for 30 min: Treatment 2) were used. Embryos were surgically transferred to the oviducts of gilts that exhibited estrus on the day of fusion or the day of transfer. Two cloned transgenic piglets were born from Treatment 1 and low oxygen, and another two from Treatment 2 and normal oxygen. PCR, RT-PCR, Western blotting and immunohistochemistry confirmed that the pigs were transgenic, made message, made the fusion protein and that the fusion protein localized to the endothelial cells of placental vasculature from the conceptuses as did the endogenous eNOS. Thus both activation conditions and culture systems are compatible with development to term. These pigs will serve as the founders for a colony of miniature pigs that will help to elucidate the function of eNOS in regulating muscle metabolism and the cardiorespiratory system.     

Sphingosine-1-phosphate promotes the differentiation of human umbilical cord mesenchymal stem cells into cardiomyocytes under the designated culturing conditions

Background  

It is of growing interest to develop novel approaches to initiate differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes. The purpose of this investigation was to determine if Sphingosine-1-phosphate (S1P), a native circulating bioactive lipid metabolite, plays a role in differentiation of human umbilical cord mesenchymal stem cells (HUMSCs) into cardiomyocytes. We also developed an engineered cell sheet from these HUMSCs derived cardiomyocytes by using a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) cell sheet technology.     

Autoinhibition of endothelial nitric oxide synthase (eNOS) in gut smooth muscle by nitric oxide

Nitric oxide in the gut is produced by nNOS in enteric neurons and by eNOS in smooth muscle cells. The eNOS in smooth muscle is activated by vasoactive intestinal peptide (VIP) released from enteric neurons. In the present study, we examined the effect of nitric oxide on VIP-induced eNOS activation in smooth muscle cells isolated from human intestine and rabbit stomach. NOS activity was measured as formation of the 1:1 co-product, l-citrulline from l-arginine. VIP caused an increase in l-citrulline production that was inhibited by NO in a concentration dependent manner (IC(50)~25 microM; maximal inhibition 72% at 100 microM NO). Basal l-citrulline production, however, was unaffected by NO. The effect was not mediated by cGMP/PKG since the PKG inhibitor KT5823 had no effect on eNOS autoinhibition. The autoinhibition was selective for NO since the co-product l-citrulline had no effect on VIP-induced NOS activation. Similar effects were obtained in rabbit gastric and human intestinal smooth muscle cells. The results suggest that NO produced in smooth muscle cells as a result of the activation of eNOS by VIP exerts an autoinhibitory restraint on eNOS thereby regulating the balance of the VIP/cAMP/PKA and NO/cGMP/PKG pathways that regulate the relaxation of gut smooth muscle.     

Endothelial nitric oxide synthase is dynamically expressed during bone marrow stem cell differentiation into endothelial cells

This study was designed to investigate the developmental expression of endothelial nitric oxide synthase (eNOS) during stem cell differentiation into endothelial cells and to examine the functional status of the newly differentiated endothelial cells. Mouse adult multipotent progenitor cells (MAPCs) were used as the source of stem cells and were induced to differentiate into endothelial cells with vascular endothelial growth factor (VEGF) in serum-free medium. Expression of eNOS in the cells during differentiation was evaluated with real-time PCR, nitric oxide synthase (NOS) activity, and Western blot analysis. It was found that eNOS, but no other NOS, was present in undifferentiated MAPCs. eNOS expression disappeared in the cells immediately after induction of differentiation. However, eNOS expression reoccurred at day 7 during differentiation. Increasing eNOS mRNA, protein content, and activity were observed in the cells at days 14 and 21 during differentiation. The differentiated endothelial cells formed dense capillary networks on growth factor-reduced Matrigel. VEGF-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 occurred in these cells, which was inhibited by NOS inhibitor N(G)-nitro-L-arginine methyl ester. In conclusion, these data demonstrate that eNOS is present in MAPCs and is dynamically expressed during the differentiation of MAPCs into endothelial cells in vitro.     

l-Theanine promotes nitric oxide production in endothelial cells through eNOS phosphorylation

Consumption of tea (Camellia sinensis) improves vascular function and is linked to lowering the risk of cardiovascular disease. Endothelial nitric oxide is the key regulator of vascular functions in endothelium. In this study, we establish that l-theanine, a non-protein amino-acid found in tea, promotes nitric oxide (NO) production in endothelial cells. l-theanine potentiated NO production in endothelial cells was evaluated using Griess reaction, NO sensitive electrode and a NO specific fluorescent probe (4-amino-5-methylamino-2',7'-difluororescein diacetate). l-Theanine induced NO production was partially attenuated in presence of l-NAME or l-NIO and completely abolished using eNOS siRNA. eNOS activation was Ca^2 + and Akt independent, as assessed by fluo-4AM and immunoblotting experiments, respectively and was associated with phosphorylation of eNOS Ser 1177. eNOS phosphorylation was inhibited in the presence of ERK1/2 inhibitor, PD-98059 and partially inhibited by PI3K inhibitor, LY-294002 and Wortmanin suggesting PI3K-ERK1/2 dependent pathway. Increased NO production was associated with vasodilation in ex ovo (chorioallantoic membrane) model. These results demonstrated that l-theanine administration in vitro activated ERK/eNOS resulting in enhanced NO production and thereby vasodilation in the artery. The results of our experiments are suggestive of l-theanine mediated vascular health benefits of tea.     

Genotype-dependent expression of endothelial nitric oxide synthase (eNOS) and its regulatory proteins in cultured endothelial cells

DNA polymorphisms in endothelial nitric oxide synthase (eNOS) gene have been shown to be associated with constitutive eNOS expression and coronary artery disease (CAD). In the present study we explored the hypothesis whether genotype-dependent effects can be maintained in vitro during replication, or the effect is conditional on in vivo biological environments. Human umbilical vein endothelial cells (HUVEC) were collected and cultured from 89 normal deliveries of Mexican Americans. The cells were treated with or without cigarette smoking extracts (CSE) and genotypes of eNOS polymorphisms were determined by PCR. We measured the levels of eNOS by ELISA and its binding proteins including heat-shock protein 90 (Hsp-90) and caveolin-1 by Western blotting. The rare C allele for the promoter T786C polymorphism (0.2), and the rare 4 x 27-bp repeat allele in the intron 4 (0.30) were different from those reported in other populations. Yet, the rare T allele in the exon 7 (G894T polymorphism) was similar as others. After four passages in vitro, both the intron 4 and promoter polymorphisms maintained significant effects on eNOS mRNA levels in HUVECs (P < 0.05). However, the effects on eNOS protein and enzyme activity were less consistent. Although primary smokers had significantly lower eNOS protein levels (P < 0.05), the in vitro CSE treatment on cultured HUVECs only resulted in a significant reduction in NO levels as measured by the stable metabolites of nitrite/nitrate (P < 0.001). Neither Hsp-90 nor caveolin-1--important eNOS regulators--appears to mediate the genotypesmoking effects on eNOS expression although HUVECs did produce more Hsp-90 when exposed to CSE. Our study demonstrates that endothelial cells maintain genotype-dependent expression even after the deprivation of in vivo environment. However, the cigarette smoking-genotype interaction may require such in vivo conditions to be manifested.     

Role of endothelial nitric oxide synthase (eNOS) in chronic stress-promoted tumour growth

Accumulating evidence suggests that chronic stress can be a cofactor for the initiation and progression of cancer. Here we evaluated the role of endothelial nitric oxide synthase (eNOS) in stress-promoted tumour growth of murine B16F10 melanoma cell line in C57BL/6 mice. Animals subjected to restraint stress showed increased levels adrenocorticotropic hormone, enlarged adrenal glands, reduced thymus weight and a 3.61-fold increase in tumour growth in respect to no-stressed animals. Tumour growth was significantly reduced in mice treated with the β-antagonist propranolol. Tumour samples obtained from stressed mice displayed high levels of vascular endothelial growth factor (VEGF) protein in immunohistochemistry. Because VEGF can induce eNOS increase, and nitric oxide is a relevant factor in angiogenesis, we assessed the levels of eNOS protein by Western blot analysis. We found a significant increase in eNOS levels in tumour samples from stressed mice, indicating an involvement of this enzyme in stress-induced tumour growth. Accordingly, chronic stress did not promote tumour growth in eNOS(-/-) mice. These results disclose for the first time a pivotal role for eNOS in chronic stress-induced initiation and promotion of tumour growth.     

Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N-monomethyl-L-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation. vascular biology; atherosclerosis; mouse models     

Molecular variation in endothelial nitric oxide synthase gene (eNOS) in western Mediterranean populations

Endothelial nitric oxide synthase (eNOS or NOS3) is the main responsible for nitric oxide (NO) production in vascular system and different polymorphisms have been identified in epidemiological studies. Trying to test the eNOS genetic variation in general populations we studied the 27-bp VNTR in intron 4 and G894T substitution in exon 7 markers in 6 Western Mediterranean populations (3 from Iberian Peninsula, 1 from North Africa, and 2 from Sardinia) and a sample from Ivory Coast. The VNTR frequencies in Western Mediterranean and Ivory Coast fit well into the ranges previously described for Europeans and Sub-Saharans respectively, and a typical African allele has been detected in polymorphic frequencies in the Berber sample. The G894T substitution presents the highest frequencies described for the T allele in the North Mediterranean populations. Linkage disequilibrium is present between both markers in all populations except in the Ivory Coast sample. The variation found for these polymorphisms indicates that they may be a useful tool for population studies even at microgeographical level.     

Sphingosine-1-phosphate promotes barrier-stabilizing effects in human microvascular endothelial cells via AMPK-dependent mechanisms

Breakdown of the endothelial barrier is a critical step in the development of organ failure in severe inflammatory conditions such as sepsis. Endothelial cells from different tissues show phenotypic variations which are often neglected in endothelial research. Sphingosine-1-phosphate (S1P) and AMP-dependent kinase (AMPK) have been shown to protect the endothelium and phosphorylation of AMPK by S1P was shown in several cell types. However, the role of the S1P-AMPK interrelationship for endothelial barrier stabilization has not been investigated. To assess the role of the S1P-AMPK signalling axis in this context, we established an in vitro model allowing real-time monitoring of endothelial barrier function in human microvascular endothelial cells (HMEC-1) and murine glomerular endothelial cells (GENCs) with the electric cell-substrate impedance sensing (ECIS™) system. Following the disruption of the cell barrier by co-administration of LPS, TNF-α, IL-1ß, IFN-γ, and IL–6, we demonstrated self-recovery of the disrupted barrier in HMEC-1, while the barrier remained compromised in GENCs. Under physiological conditions we observed a rapid phosphorylation of AMPK in HMEC-1 stimulated with S1P, but not in GENCs. Consistently, S1P enhanced the basal endothelial barrier in HMEC-1 exclusively. siRNA-mediated knockdown of AMPK in HMEC-1 led to a less pronounced barrier enhancement. Thus we present evidence for a functional role of AMPK in S1P-mediated barrier stabilization in HMEC-1 and we provide insight into cell-type specific differences of the S1P-AMPK-interrelationship, which might influence the development of interventional strategies targeting endothelial barrier dysfunction.     

Functional role of HSP90 complexes with endothelial nitric-oxide synthase (eNOS) and calpain on nitric oxide generation in endothelial cells

Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca(2+)-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca(2+)-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca(2+)-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca(2+)-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.     

Exercise training enhanced myocardial endothelial nitric oxide synthase (eNOS) function in diabetic Goto-Kakizaki (GK) rats

Objective

Exenatide belongs to a new therapeutic class in the treatment of diabetes (incretin mimetics), allowing glucose-dependent glycaemic control in Type 2 diabetes. Randomised controlled trial data suggest that exenatide is as effective as insulin glargine at reducing HbA_1c in combination therapy with metformin and sulphonylureas; with reduced weight but higher incidence of adverse gastrointestinal events. The objective of this study is to evaluate the cost effectiveness of exenatide versus insulin glargine using RCT data and a previously published model of Type 2 diabetes disease progression that is based on the United Kingdom Prospective Diabetes Study; the perspective of the health-payer of the United Kingdom National Health Service.

Methods

The study used a discrete event simulation model designed to forecast the costs and health outcome of a cohort of 1,000 subjects aged over 40 years with sub-optimally-controlled Type 2 diabetes, following initiation of either exenatide, or insulin glargine, in addition to oral hypoglycaemic agents. Sensitivity analysis for a higher treatment discontinuation rate in exenatide patients was applied to the cohort in three different scenarios; (1) either ignored or (2) exenatide-failures excluded or (3) exenatide-failures switched to insulin glargine. Analyses were undertaken to evaluate the price sensitivity of exenatide in terms of relative cost effectiveness. Baseline cohort profiles and effectiveness data were taken from a published randomised controlled trial.

Results

The relative cost-effectiveness of exenatide and insulin glargine was tested under a variety of conditions, in which insulin glargine was dominant in all cases. Using the most conservative of assumptions, the cost-effectiveness ratio of exenatide vs. insulin glargine at the current UK NHS price was -£29,149/QALY (insulin glargine dominant) and thus exenatide is not cost-effective when compared with insulin glargine, at the current UK NHS price.

Conclusion

This study evaluated the relative cost effectiveness of insulin glargine versus exenatide in the management of Type 2 diabetes using a published model. Given no significant difference in glycaemic control and applying the additional effectiveness of exenatide over insulin glargine, with respect to weight loss, and using the current UK NHS prices, insulin glargine was found to be dominant over exenatide in all modelled scenarios. With current clinical evidence, exenatide does not appear to represent a cost-effective treatment option for patients with Type 2 diabetes when compared to insulin glargine.     

Three-dimensional differentiation of adipose-derived mesenchymal stem cells into insulin-producing cells

The aim of this study is to evaluate the collagen/hyaluronic acid (Col/HA) scaffold effect on the differentiation of insulin-producing cells (IPCs) from adipose-derived mesenchymal stem cells (ASCs). In this experimental study, ASCs were cultured and seeded in a Col/HA scaffold (3D culture) and then treated with induction media. After induction, the presence of IPCs was evaluated using gene expression (PDX-1, GLUT-2 and insulin) analysis and immunocytochemistry, while functional maturity was determined by measuring insulin release in response to low- and high-glucose media. The induced IPCs were morphologically similar to pancreatic islet-like cells. Expression of the islet-associated genes PDX-1, GLUT-2 and insulin genes in 3D-cultured cells was markedly higher than the 2D-cultured cells exposure differentiation media. Compared to the 2D culture of ASCs-derived IPCs, the insulin release from 3D ASCs-derived IPCs showed a nearly 4-fold (p?<?0.05) increase when exposed to a high glucose (25 mmol) medium. The percentage of insulin-positive cells in the 3D experimental group showed an approximately 4-fold increase compared to the 2D experimental culture cells. The results of this study demonstrated that the COL/HA scaffold can enhance the differentiation of IPCs from rat ASCs.     

Differential antibacterial activity of nitric oxide from the immunological isozyme of nitric oxide synthase transduced into endothelial cells.

Primary cultures of endothelial cells, grown on the three-dimensional matrix Gelfoam where they take on the morphology of these cells in vivo, were found to phagocytose Staphylococcus aureus and two strains of Escherichia coli. The phagocytosis was independent of opsonization, although once opsonized, these bacteria were phagocytosed by endothelial cells. As cytochalsin D inhibited the internationalization of S. aureus and E. coli, the phagocytosis by endothelial cells appears to be actin-dependent. Transducing the gene for nitric oxide synthase (NOS) II into endothelial cells allowed us to determine the importance of NO(*) in host immunity against these bacteria. While the growth of S. aureus was impeded by NOS II endothelial cells, two strains of E. coli were killed by an NO(*)-dependent pathway. We conclude that endothelial cells have microbicidal mechanisms that are selective for the type of pathogen encountered.     

Vascular and perivascular nitric oxide release and transport: biochemical pathways of neuronal nitric oxide synthase (NOS1) and endothelial nitric oxide synthase (NOS3)

Nitric oxide (NO) derived from nitric oxide synthase (NOS) is an important paracrine effector that maintains vascular tone. The release of NO mediated by NOS isozymes under various O(2) conditions critically determines the NO bioavailability in tissues. Because of experimental difficulties, there has been no direct information on how enzymatic NO production and distribution change around arterioles under various oxygen conditions. In this study, we used computational models based on the analysis of biochemical pathways of enzymatic NO synthesis and the availability of NOS isozymes to quantify the NO production by neuronal NOS (NOS1) and endothelial NOS (NOS3). We compared the catalytic activities of NOS1 and NOS3 and their sensitivities to the concentration of substrate O(2). Based on the NO release rates predicted from kinetic models, the geometric distribution of NO sources, and mass balance analysis, we predicted the NO concentration profiles around an arteriole under various O(2) conditions. The results indicated that NOS1-catalyzed NO production was significantly more sensitive to ambient O(2) concentration than that catalyzed by NOS3. Also, the high sensitivity of NOS1 catalytic activity to O(2) was associated with significantly reduced NO production and therefore NO concentrations, upon hypoxia. Moreover, the major source determining the distribution of NO was NOS1, which was abundantly expressed in the nerve fibers and mast cells close to arterioles, rather than NOS3, which was expressed in the endothelium. Finally, the perivascular NO concentration predicted by the models under conditions of normoxia was paradoxically at least an order of magnitude lower than a number of experimental measurements, suggesting a higher abundance of NOS1 or NOS3 and/or the existence of other enzymatic or nonenzymatic sources of NO in the microvasculature.     

Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells

Endothelial nitric oxide synthase (eNOS) is essential for neovascularization. Here we show that the impaired neovascularization in mice lacking eNOS is related to a defect in progenitor cell mobilization. Mice deficient in eNOS (Nos3(-/-)) show reduced vascular endothelial growth factor (VEGF)-induced mobilization of endothelial progenitor cells (EPCs) and increased mortality after myelosuppression. Intravenous infusion of wild-type progenitor cells, but not bone marrow transplantation, rescued the defective neovascularization of Nos3(-/-) mice in a model of hind-limb ischemia, suggesting that progenitor mobilization from the bone marrow is impaired in Nos3(-/-) mice. Mechanistically, matrix metalloproteinase-9 (MMP-9), which is required for stem cell mobilization, was reduced in the bone marrow of Nos3(-/-) mice. These findings indicate that eNOS expressed by bone marrow stromal cells influences recruitment of stem and progenitor cells. This may contribute to impaired regeneration processes in ischemic heart disease patients, who are characterized by a reduced systemic NO bioactivity.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) regulates endothelial nitric oxide synthase (eNOS) activity and its localization within the human vein endothelial cells (HUVEC) in culture

We have recently demonstrated that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases endothelial nitric oxide synthase (eNOS) phosphorylation, NOS activity, and nitric oxide (NO) synthesis in cultured human umbilical vein endothelial cells (HUVEC), without inducing apoptotic cell death. Although an important factor that regulates eNOS activity is its localization within the cells, little is known about the role of TRAIL in the regulation of eNOS trafficking among cellular compartments and the cytoskeleton involvement in this machinery. Then, we did both quantitative and semi-quantitative evaluations with biochemical assays and immune fluorescence microscopy in the presence of specific inhibitors of NOS activity as well as of cytoskeletal microtubule structures. In our cellular model, TRAIL treatment not only increased NO levels but also caused a time-dependent NO migration of fluorescent spots from the plasma membrane to the inner part of the cells. In unstimulated cells, most of the eNOS was localized at the cell membranes. However, within 10 min following addition of TRAIL, nearly all the cells showed an increased cytoplasm localization of eNOS which appeared co-localized with the Golgi apparatus at a higher extent than in unstimulated cells. These effects were associated to an increased formation of trans-cytoplasm stress fibers with no significant changes of the microtubule network. Conversely, microtubule disruption and Golgi scattering induced with Nocodazole treatment inhibited TRAIL-increased NOS activity, indicating that, on cultured HUVEC, TRAIL ability to affect NO production by regulating eNOS sub-cellular distribution is mediated by cytoskeleton and Golgi complex modifications.