Dual disruption of eNOS and ApoE gene accelerates kidney fibrosis and senescence after injury.

The relationship between cellular senescence and fibrosis in the kidney is being elucidated and we have identified it as therapeutic target in recent studies. Chronic kidney disease has also become a lifestyle disease, often developing on the background of hypertension and dyslipidemia. In this study, we clarify the effect of interaction between these two conditions on kidney fibrosis and senescence. Wild type mice (WT), apolipoprotein E-/- mice (ApoEKO), and endothelial nitric oxide synthase (eNOS)-/- ApoE-/- mice (DKO) were obtained by breeding. Unilateral ureteral obstruction (UUO) was performed on 8-10 week old male mice and the degree of renal tubular injury, fibrosis and kidney senescence were evaluated. DKO manifested elevated blood pressure, higher total cholesterol and lower HDL than WT. DKO showed sustained kidney injury molecule-1 protein expression. Kidney fibrosis was significantly higher in ApoEKO and DKO. mRNA expression of genes related to kidney fibrosis was the highest in DKO. The mRNA expression of Zinc-α2-Glycoprotein and heme oxygenase-1 were significantly decreased in DKO. Furthermore, mRNA expression of p53, p21 and p16 were increased both in ApoEKO and DKO, with DKO being the highest. Senescence associated ß-gal positive tubule area was significantly increased in DKO. Increased DNA damage and target of rapamycin-autophagy spatial coupling compartments (TASCCs) formation was found in DKO. Mice with endothelial dysfunction and dyslipidemia developed kidney fibrosis and accelerated senescence even in young mice after injury. These data highlight the fact managing lifestyle-related diseases from a young age is important for CKD prevention.

Low-intensity pulsed ultrasound therapy promotes recovery from stroke by enhancing angio-neurogenesis in mice in vivo.

Since the treatment window of thrombolytic therapy for stroke is limited, new therapy remains to be developed. We have recently developed low-intensity pulsed ultrasound (LIPUS) therapy to improve cognitive dysfunction in mouse models of vascular dementia and Alzheimer's disease. Here, we further aimed to examine whether our LIPUS therapy improves neurological recovery from ischemic stroke, and if so, to elucidate the mechanisms involved. In a mouse model of middle cerebral artery occlusion (MCAO), we applied LIPUS (32 cycles, 193 mW/cm2) to the whole brain 3 times in the first week (days 1, 3, and 5) after MCAO. We evaluated neurological functions using behavioral tests and performed histological analyses. Furthermore, to elucidate how LIPUS works within the injured brain, we also tested the effects of LIPUS in endothelial nitric oxide synthase (eNOS)-deficient (eNOS-/-) mice. In wild-type mice, the LIPUS therapy markedly improved neurological functions in the tightrope and rotarod tests at 28 days after MCAO. Histological analyses showed that the LIPUS therapy significantly increased the numbers of CD31-positive blood vessels in the perifocal lesion and doublecortin (DCX)-positive neurons in the ischemic striatum, indicating the angio-neurogenesis effects of the therapy. Importantly, these beneficial effects of the LIPUS therapy were totally absent in eNOS-/- mice. No adverse effects of the LIPUS therapy were noted. These results indicate that the LIPUS therapy improves neurological functions after stroke through enhanced neuro-angiogenesis in mice in vivo in an eNOS-dependent manner, suggesting that it could a novel and non-invasive therapeutic option for stroke.

High-salt intake reduces renal tissue levels of inflammatory cytokines in mice.

High salt (HS) intake is usually considered as an aggravating factor to induce inflammatory renal injury. However, the changes in the renal levels of inflammatory cytokines during HS intake is not yet clearly defined. We hypothesize that HS increases renal levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) but decreases interleukin-10 (IL-10; anti-inflammatory cytokine) and these responses exacerbate in NO deficient conditions. Both wild-type (WT) and endothelial NO synthase knockout (eNOSKO) mice (~8 weeks old, n = 6 in each group) were given normal-salt (NS; 0.3% NaCl) and HS (4% NaCl) containing diets for 2 weeks. Systolic blood pressure (SBP) was determined by tail-cuff plethysmography and urine collections were made using metabolic cages. Basal SBP was higher in eNOSKO than WT mice (131 ± 7 vs 117 ± 3 mmHg; p < .05). HS intake for 2 weeks increased SBP in eNOSKO (161 ± 5 mmHg) but not in WT mice. In NS groups, the cytokine levels in renal tissues (measured using ELISA kits and expressed in pg/mg protein) were significantly higher in eNOSKO than WT mice (TNF-α, 624 ± 67 vs. 325 ± 73; IL-6, 619 ± 106 vs. 166 ± 61; IL-10, 6,087 ± 567 vs. 3,929 ± 378). Interestingly, these cytokine levels in HS groups were significantly less both in WT (TNF-α, 114 ± 17; IL-6, 81 ± 14; IL-10, 865 ± 130) and eNOSKO (TNF-α, 115 ± 18; IL-6, 56 ± 7; IL-10, 882 ± 141) mice. These findings indicate that HS induces downregulation of cytokines in the kidney. Such HS-induced reduction in cytokines, particularly TNF-α (a natriuretic agent), would facilitate more salt-retention, and thus, leading to salt-sensitive hypertension in NO deficient conditions.

Hematopoietic Cell-Expressed Endothelial Nitric Oxide Protects the Liver From Insulin Resistance.

OBJECTIVE: Mice genetically deficient in endothelial nitric oxide synthase (Nos3-/-) have fasting hyperinsulinemia and hepatic insulin resistance, indicating the importance of Nos3 (nitric oxide synthase) in maintaining metabolic homeostasis. Although the current paradigm holds that these metabolic effects are derived specifically from the expression of Nos3 in the endothelium, it has been established that bone marrow-derived cells also express Nos3. The aim of this study was to investigate whether bone marrow-derived cell Nos3 is important in maintaining metabolic homeostasis. Approach and Results: To test the hypothesis that bone marrow-derived cell Nos3 contributes to metabolic homeostasis, we generated chimeric male mice deficient or competent for Nos3 expression in circulating blood cells. These mice were placed on a low-fat diet for 5 weeks, a time period which is known to induce hepatic insulin resistance in global Nos3-deficient mice but not in wild-type C57Bl/6 mice. Surprisingly, we found that the absence of Nos3 in the bone marrow-derived component is associated with hepatic insulin resistance and that restoration of Nos3 in the bone marrow-derived component in global Nos3-deficient mice is sufficient to restore hepatic insulin sensitivity. Furthermore, we found that overexpression of Nos3 in bone marrow-derived component in wild-type mice attenuates the development of hepatic insulin resistance during high-fat feeding. Finally, compared with wild-type macrophages, the loss of macrophage Nos3 is associated with increased inflammatory responses to lipopolysaccharides and reduced anti-inflammatory responses to IL-4, a macrophage phenotype associated with the development of hepatic and systemic insulin resistance. CONCLUSIONS: These results would suggest that the metabolic and hepatic consequences of high-fat feeding are mediated by loss of Nos3/nitric oxide actions in bone marrow-derived cells, not in endothelial cells.

Establishing equivalent diabetes in male and female Nos3-deficient mice results in a comparable onset of diabetic kidney injury.

Clinical studies indicate that sex differences exist in susceptibility for developing diabetic kidney disease (DKD), supporting the need to examine both sexes in animal studies of DKD. Streptozotocin (STZ) is commonly used in male mice to induce diabetes and DKD. However, females are not normally included because their sex hormones partially protect them from STZ-induced islet injury and consequent diabetes. To address this issue, we identified a strategy to induce comparable diabetes in male and female mice using STZ and determined whether both sexes develop equivalent renal injury. Male and female mice lacking the gene for endothelial nitric oxide synthase (Nos3-/-) were made diabetic with five or six low-dose STZ injections, respectively. Groups of male and female mice with equivalent hyperglycemia at week 3 after STZ were assessed for DKD at week 8. STZ-treated male and female Nos3-/- mice maintained comparable hyperglycemia between weeks 3 and 8 had an equivalent increase in HbA1c levels and comparable hypertension. Urine albumin/creatinine levels were elevated eightfold in mice of both sexes at week 8, accompanied by an equivalent loss of podocytes. In diabetic males and females, plasma cystatin C levels and glomerular collagen deposition were similarly increased. Kidney mRNA levels of proinflammatory and profibrotic markers and kidney injury molecule-1 (KIM-1) were equally elevated in males and females, indicating comparable kidney injury. This study shows that equivalent diabetes induces a comparable onset of DKD in male and female Nos3-/- mice, demonstrating that it is possible to include males and females together in studies of DKD.

Elevated Neuropeptide Y in Endothelial Dysfunction Promotes Macrophage Infiltration and Smooth Muscle Foam Cell Formation.

Endothelial dysfunction has been linked to vascular inflammation and foam cell formation but the underlying mechanisms still remain unclear. We sought to define the factors inducing inflammation and smooth muscle foam cell formation under endothelial dysfunction using endothelial nitric oxide synthase (eNOS)-deficient mice. Vascular smooth muscle cells (VSMCs) from eNOS-deficient mice displayed increased expression of macrophage-related genes and elevated lipid uptake. Neuropeptide Y (NPY) was upregulated in the aorta from the eNOS-deficient mice and promoted macrophage chemotaxis toward VSMCs while enhancing the activity of matrix metalloproteinase-3. Notably, NPY induced lipid uptake in VSMCs, facilitating smooth muscle foam cell formation, in association with enhanced expression of genes related to modified low-density lipoprotein uptake and macrophages. NPY was augmented by inflammatory pentraxin 3 (PTX3) in VSMCs. PTX3 enhanced macrophage migratory capacity through the NPY/neuropeptide Y receptor axis and this effect was attenuated by pharmacological inhibition with a receptor-specific antagonist. These observations suggest that endothelial dysfunction leads to the elevation of NPY that amplifies vascular inflammation by increasing inflammatory cell chemotaxis and triggers smooth muscle foam cell formation.

Urinary dysfunction in transgenic sickle cell mice: model of idiopathic overactive bladder syndrome.

Voiding abnormalities are common among the sickle cell disease (SCD) population, among which overactive bladder (OAB) syndrome is observed at rates as high as 39%. Although detrusor overactivity is the most common cause of OAB, its molecular pathophysiology is not well elucidated. The nitric oxide (NO) signaling pathway has been implicated in the regulation of lower genitourinary tract function. In the present study, we evaluated the role of the NO signaling pathway in voiding function of transgenic SCD mice compared with combined endothelial and neuronal NO synthase gene-deficient mice, both serving as models of NO deficiency. Mice underwent void spot assay and cystometry, and bladder and urethral specimens were studied using in vitro tissue myography. Both mouse models exhibited increased void volumes; increased nonvoiding and voiding contraction frequencies; decreased bladder compliance; increased detrusor smooth muscle contraction responses to electrical field stimulation, KCl, and carbachol; and increased urethral smooth muscle relaxation responses to sodium nitroprusside compared with WT mice. In conclusion, our comprehensive behavioral and functional study of the SCD mouse lower genitourinary tract, in correlation with that of the NO-deficient mouse, reveals NO effector actions in voiding function and suggests that NO signaling derangements are associated with an OAB phenotype. These findings may allow further study of molecular targets for the characterization and evaluation of OAB.

Lysed Erythrocyte Membranes Promote Vascular Calcification.

BACKGROUND: Intraplaque hemorrhage promotes atherosclerosis progression, and erythrocytes may contribute to this process. In this study we examined the effects of red blood cells on smooth muscle cell mineralization and vascular calcification and the possible mechanisms involved. METHODS: Erythrocytes were isolated from human and murine whole blood. Intact and lysed erythrocytes and their membrane fraction or specific erythrocyte components were examined in vitro using diverse calcification assays, ex vivo by using the murine aortic ring calcification model, and in vivo after murine erythrocyte membrane injection into neointimal lesions of hypercholesterolemic apolipoprotein E-deficient mice. Vascular tissues (aortic valves, atherosclerotic carotid artery specimens, abdominal aortic aneurysms) were obtained from patients undergoing surgery. RESULTS: The membrane fraction of lysed, but not intact human erythrocytes promoted mineralization of human arterial smooth muscle cells in culture, as shown by Alizarin red and van Kossa stain and increased alkaline phosphatase activity, and by increased expression of osteoblast-specific transcription factors (eg, runt-related transcription factor 2, osterix) and differentiation markers (eg, osteopontin, osteocalcin, and osterix). Erythrocyte membranes dose-dependently enhanced calcification in murine aortic rings, and extravasated CD235a-positive erythrocytes or Perl iron-positive signals colocalized with calcified areas or osteoblast-like cells in human vascular lesions. Mechanistically, the osteoinductive activity of lysed erythrocytes was localized to their membrane fraction, did not involve membrane lipids, heme, or iron, and was enhanced after removal of the nitric oxide (NO) scavenger hemoglobin. Lysed erythrocyte membranes enhanced calcification to a similar extent as the NO donor diethylenetriamine-NO, and their osteoinductive effects could be further augmented by arginase-1 inhibition (indirectly increasing NO bioavailability). However, the osteoinductive effects of erythrocyte membranes were reduced in human arterial smooth muscle cells treated with the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide or following inhibition of NO synthase or the NO receptor soluble guanylate cyclase. Erythrocytes isolated from endothelial NO synthase-deficient mice exhibited a reduced potency to promote calcification in the aortic ring assay and after injection into murine vascular lesions. CONCLUSIONS: Our findings in cells, genetically modified mice, and human vascular specimens suggest that intraplaque hemorrhage with erythrocyte extravasation and lysis promotes osteoblastic differentiation of smooth muscle cells and vascular lesion calcification, and also support a role for erythrocyte-derived NO.

The polyphenol resveratrol promotes skeletal growth in mice through a sirtuin 1-bone morphogenic protein 2 longevity axis.

BACKGROUND AND PURPOSE: The polyphenol resveratrol (RSV) exists in high quantities in certain foods (e.g. grapes and nuts). However, the capacity of RSV to confer physiological health benefits and a biological mechanism through which this might occur remains unclear. EXPERIMENTAL APPROACH: Aged, RSV-treated (300 mg·kg-1 ·day-1 ) and genetically modified [endothelial NOS (eNOS-/- )] female mice were assessed using histomorphometric and µCT analysis. Alongside in vivo analysis, molecular siRNA knockdown and pharmacological manipulation of eNOS, BMP2 and sirtuin 1 (SIRT1) and functional cellular assays in an osteoblast cell line panel, explored the mechanism through which RSV might impact overall bone volume. KEY RESULTS: RSV promoted osteoblast activity and bone growth in vivo. RSV dose-dependently and simultaneously increased alkaline phosphatase (ALP) and eNOS levels. Similarly, NO-donor treatment increased ALP, runt homology transcription factor 2, BMP2 and stimulated bone formation, whilst eNOS-deficient mice displayed a bone loss phenotype. Moreover, RSV-induced increase in ALP and BMP2 expression was blocked in eNOS-/- osteoblasts and by BMP-inhibitor noggin. The longevity-linked SIRT1 enzyme was positively regulated by RSV and SIRT1 deletion reduced eNOS, BMP2 and ALP. Like eNOS deletion, loss of SIRT1 blocked RSV-induced osteoblast activity; however, SIRT1 levels remained unchanged in eNOS-/- mice, indicating RSV activation of SIRT1 stimulates BMP2 release via eNOS. This signalling axis is supported by decreased SIRT1, eNOS and BMP2 confirmed in old versus young bone. CONCLUSIONS AND IMPLICATIONS: These findings suggest a new mechanism of action in bone remodelling and the ageing skeleton, where RSV positively impacts bone homeostasis via SIRT1 activation of BMP2.

MicroRNA-214-3p in the Kidney Contributes to the Development of Hypertension.

BACKGROUND: In spite of extensive study, the mechanisms for salt sensitivity of BP in humans and rodent models remain poorly understood. Several microRNAs (miRNAs) have been associated with hypertension, but few have been shown to contribute to its development. METHODS: We examined miRNA expression profiles in human kidney biopsy samples and rat models using small RNA deep sequencing. To inhibit an miRNA specifically in the kidney in conscious, freely moving rats, we placed indwelling catheters to allow both renal interstitial administration of a specific anti-miR and measurement of BP. A rat with heterozygous disruption of the gene encoding endothelial nitric oxide synthase (eNOS) was developed. We used bioinformatic analysis to evaluate the relationship between 283 BP-associated human single-nucleotide polymorphisms (SNPs) and 1870 human miRNA precursors, as well as other molecular and cellular methods. RESULTS: Compared with salt-insensitive SS.13BN26 rats, Dahl salt-sensitive (SS) rats showed an upregulation of miR-214-3p, encoded by a gene in the SS.13BN26 congenic region. Kidney-specific inhibition of miR-214-3p significantly attenuated salt-induced hypertension and albuminuria in SS rats. miR-214-3p directly targeted eNOS. The effect of miR-214-3p inhibition on hypertension and albuminuria was abrogated in SS rats with heterozygous loss of eNOS. Human kidney biopsy specimens from patients with hypertension or hypertensive nephrosclerosis showed upregulation of miR-214-3p; the gene encoding miR-214-3p was one of several differentially expressed miRNA genes located in proximity to human BP-associated SNPs. CONCLUSIONS: Renal miR-214-3p plays a functional and potentially genetic role in the development of hypertension, which might be mediated in part by targeting eNOS.

Direct and specific inhibition of constitutive nitric oxide synthase uniquely regulates brush border membrane Na-absorptive pathways in intestinal epithelial cells.

Pharmacological manipulations of constitutive nitric oxide (cNO) levels have been shown to have variable effects on Na absorption in vivo and in vitro in different tissues. Species differences, untoward in vivo effects (e.g. ENS, blood flow) and pharmacological non-specificity may account for these confounding observations. Thus, to directly and specifically determine the effect of cNO on brush border membrane Na/H exchange (NHE3) and Na-dependent glucose co-transport (SGLT-1), we inhibited cNO synthase (NOS3) with its siRNA in rat small intestinal epithelial cells (IEC-18) in vitro. As expected, intracellular cNO levels were reduced in siRNA NOS3 transfected cells. In these cells, SGLT-1 was significantly reduced compared to control. In contrast, NHE3 was significantly increased in siRNA NOS3 transfected cells. To determine if SGLT-1 changes were secondary to altered Na/K-ATPase, its activity was measured and found to be increased in NOS3 silenced cells. The mechanism of inhibition of SGLT-1 was secondary to diminished affinity of the co-transporter for glucose in NOS3 silenced cells. In contrast, the mechanism of stimulation of NHE3 is by increasing BBM exchanger numbers in siRNA NOS3 cells while the affinity was unaffected. Western blot studies of immunoreactive BBM proteins also confirmed the kinetic studies. All these data indicates that direct and specific inhibition of NOS3 with its siRNA inhibits SGLT-1 while stimulating NHE3 in the BBM. Thus, cNO uniquely and compensatorily regulates BBM NHE3 and SGLT-1 to maintain cellular Na homeostasis and these unique alterations by cNO are mediated by its intracellular 2nd messenger cGMP.

Molecular Profile of Priapism Associated with Low Nitric Oxide Bioavailability.

Priapism is a disorder in which prolonged penile erection persists uncontrollably, potentially leading to tissue damage. Priapism commonly afflicts patient populations with severely low nitric oxide (NO) bioavailability. Because NO is a primary mediator of erection, the molecular mechanisms involved in priapism pathophysiology associated with low NO bioavailability are not well-understood. The objective of this study was to identify dysregulated molecular targets and signaling pathways in penile tissue of a mouse model of low NO bioavailability that have potential relevance to priapism. Neuronal plus endothelial NO synthase double knockout mice (NOS1/3-/-) were used as a model of low NO bioavailability. Priapic-like activity was demonstrated in the NOS1/3-/- mice relative to wild-type (WT) mice by the measurement of prolonged erections following cessation of electrical stimulation of the cavernous nerve. Penile tissue was processed and analyzed by reverse-phase liquid chromatography tandem mass spectrometry. As a result, 1279 total proteins were identified and quantified by spectral counting, 46 of which were down-regulated and 110 of which were up-regulated in NOS1/3-/- versus WT (P < 0.05). Ingenuity Pathway Analysis of differentially expressed proteins revealed increased protein kinase A and G-protein coupled receptor signaling in NOS1/3-/- penises, which represent potential mechanisms contributing to priapism for secondary to low NO bioavailability.

Hepatic dysfunction and thrombocytopenia induced by excess sFlt1 in mice lacking endothelial nitric oxide synthase.

Liver dysfunction is a major problem in patients with severe preeclampsia (PE), hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome, or in patients receiving anti-vascular endothelial growth factor (VEGF) therapy. Excessive soluble fms-like tyrosine kinase 1 (sFlt1) that antagonizes VEGF has been implicated in the pathogenesis of PE. VEGF increases the expression of endothelial nitric oxide synthase (eNOS) and activates it. eNOS polymorphisms that cause reduced NO production are associated with PE. The aim of this study was to clarify the role on hepatic function by excess sFlt1 in the absence of eNOS gene product. We first overexpressed sFlt1 using adenovirus in eNOS -/- and eNOS +/+ mice. Excessive sFlt1 and lack of eNOS synergistically increased plasma levels of liver transaminases, exacerbated infiltration of inflammatory cells, elevated expression levels of cytokines in the liver, and aggravated oxidative stress and coagulation abnormalities. Lack of eNOS in the presence of excess sFlt1 also induced thrombocytopenia, whereas eNOS +/+ mice with excess sFlt1 alone showed no or modest liver phenotype. Taken together, excessive sFlt1 and lack of eNOS synergistically induce hepatic dysfunction and thrombocytopenia, suggesting a novel role for VEGF and nitric oxide signaling in hepatocyte-endothelial cross-talk in health and in liver injury states.

Influence of inflammation and nitric oxide upon platelet aggregation following deposition of diesel exhaust particles in the airways.

BACKGROUND AND PURPOSE: Exposure to nanoparticulate pollution has been implicated in platelet-driven thrombotic events such as myocardial infarction. Inflammation and impairment of NO bioavailability have been proposed as potential causative mechanisms. It is unclear, however, whether airways exposure to combustion-derived nanoparticles such as diesel exhaust particles (DEP) or carbon black (CB) can augment platelet aggregation in vivo and the underlying mechanisms remain undefined. We aimed to investigate the effects of acute lung exposure to DEP and CB on platelet activation and the associated role of inflammation and endothelial-derived NO. EXPERIMENTAL APPROACH: DEP and CB were intratracheally instilled into wild-type (WT) and eNOS-/- mice and platelet aggregation was assessed in vivo using an established model of radio-labelled platelet thromboembolism. The underlying mechanisms were investigated by measuring inflammatory markers, NO metabolites and light transmission aggregometry. KEY RESULTS: Platelet aggregation in vivo was significantly enhanced in WT and eNOS-/- mice following acute airways exposure to DEP but not CB. CB exposure, but not DEP, was associated with significant increases in pulmonary neutrophils and IL-6 levels in the bronchoalveolar lavage fluid and plasma of WT mice. Neither DEP nor CB affected plasma nitrate/nitrite concentration and DEP-induced human platelet aggregation was inhibited by an NO donor. CONCLUSIONS AND IMPLICATIONS: Pulmonary exposure to DEP and subsequent platelet activation may contribute to the reports of increased cardiovascular risk, associated with exposure to airborne pollution, independent of its effects on inflammation or NO bioavailability.

Eph-B4 mediates vein graft adaptation by regulation of endothelial nitric oxide synthase.

OBJECTIVE: Vein graft adaptation is characterized by loss of expression of the tyrosine kinase receptor Eph-B4, the embryonic determinant of venous identity, without increased expression of its ligand ephrin-B2, the embryonic determinant of arterial identity. Endothelial nitric oxide synthase (eNOS) is an important mediator of vessel remodeling. We hypothesized that the mechanism of action of Eph-B4 during vein graft adaptation might be through regulation of downstream eNOS activity. METHODS: Mouse lung endothelial cells were stimulated with ephrin-B2/Fc, without and with preclustering, without and with the eNOS inhibitor Nω-nitro-l-arginine methyl ester hydrochloride or the Eph-B4 inhibitor NVP-BHG712, and assessed by Western blot and immunofluorescence for eNOS and Eph-B4 phosphorylation. Nitric oxide (NO) production was assessed using an NO-specific chemiluminescence analyzer. Cell migration was assessed using a Transwell assay. Human and mouse vein graft specimens were examined for eNOS activity by Western blot, and vessel remodeling was assessed in vein grafts in wild-type or eNOS knockout mice. RESULTS: Ephrin-B2/Fc stimulated both Eph-B4 and eNOS phosphorylation in a bimodal temporal distribution (n = 4; P < .05), with preclustered ephrin-B2/Fc causing prolonged peak Eph-B4 and eNOS phosphorylation as well as altered subcellular localization (n = 4; P < .05). Ephrin-B2/Fc increased NO release (n = 3; P < .01) as well as increased endothelial cell migration (n = 6; P < .05) in an eNOS-dependent fashion. Both human and mouse vein grafts showed increased eNOS phosphorylation compared with normal veins (n = 3; P < .05). Vein grafts from eNOS knockout mice showed less dilation and less wall thickening compared with wild-type vein grafts (n = 7; P < .05). CONCLUSIONS: eNOS is a mediator of vein graft adaptation to the arterial environment. Eph-B4 stimulates eNOS phosphorylation in vitro and may mediate vein graft adaptation by regulation of eNOS activity in vivo.

S-nitrosylation of endogenous protein tyrosine phosphatases in endothelial insulin signaling.

Nitric oxide (NO) exerts its biological function through S-nitrosylation of cellular proteins. Due to the labile nature of this modification under physiological condition, identification of S-nitrosylated residue in enzymes involved in signaling regulation remains technically challenging. The present study investigated whether intrinsic NO produced in endothelium-derived MS-1 cells response to insulin stimulation might target endogenous protein tyrosine phosphatases (PTPs). For this, we have developed an approach using a synthetic reagent that introduces a phenylacetamidyl moiety on S-nitrosylated Cys, followed by detection with anti-phenylacetamidyl Cys (PAC) antibody. Coupling with sequential blocking of free thiols with multiple iodoacetyl-based Cys-reactive chemicals, we employed this PAC-switch method to show that endogenous SHP-2 and PTP1B were S-nitrosylated in MS-1 cells exposed to insulin. The mass spectrometry detected a phenylacetamidyl moiety specifically present on the active-site Cys463 of SHP-2. Focusing on the regulatory role of PTP1B, we showed S-nitrosylation to be the principal Cys reversible redox modification in endothelial insulin signaling. The PAC-switch method in an imaging format illustrated that a pool of S-nitrosylated PTP1B was colocalized with activated insulin receptor to the cell periphery, and that such event was endothelial NO synthase (eNOS)-dependent. Moreover, ectopic expression of the C215S mutant of PTP1B that mimics the active-site Cys215 S-nitrosylated form restored insulin responsiveness in eNOS-ablated cells, which was otherwise insensitive to insulin stimulation. This work not only introduces a new method that explores the role of physiological NO in regulating signal transduction, but also highlights a positive NO effect on promoting insulin responsiveness through S-nitrosylation of PTP1B's active-site Cys215.

The effect of combined inositol supplementation on maternal metabolic profile in pregnancies complicated by metabolic syndrome and obesity.

BACKGROUND: Myoinositol and D-chiroinositol improve insulin resistance in women with obesity and gestational diabetes and in postmenopausal women with metabolic syndrome. We previously reported that offspring born to hypertensive dams lacking endothelial nitric oxide synthase and fed a high-fat diet develop metabolic-like syndrome phenotype. OBJECTIVE: The objective of the study was to investigate the effect of a mixture of myoinositol/D-chiroinositol supplementation during pregnancy on the maternal metabolic profile in pregnancies complicated by the metabolic-like syndrome and obesity using a pregnant mouse model. STUDY DESIGN: Female heterozygous endothelial nitric oxide synthase(-/+) mice with moderate hypertension were placed on a high-fat diet for 4 weeks to induce a metabolic-like syndrome phenotype. Similarly, wild-type C57BL/6 mice were placed on a high-fat diet for 4 weeks to induce a murine obesity model. Mice were then bred with wild-type males. On gestational day 1, dams were randomly allocated to receive either a mixture of myoinositol/D-chiroinositol in water (7.2/0.18 mg/mL, respectively) or water as control (placebo). At term (gestational day 18), maternal weights, systolic blood pressure, and a glucose tolerance test were obtained. Dams were then killed; pups and placentas were weighed and maternal blood collected. Serum levels of metabolic biomarkers relevant to diabetes and obesity (ghrelin, gastric inhibitory peptide, glucagon-like peptide 1, glucagon, insulin, leptin, resistin) were measured by a multiplex enzyme-linked immunosorbent assay. Analysis was done comparing metabolic-like syndrome-myoinositol/D-chiroinositol-treated vs metabolic-like syndrome-nontreated mice and obese-myoinositol/D-chiroinositol-treated vs obese nontreated mice. RESULTS: Mean systolic blood pressure was lower in metabolic-like syndrome pregnant mice treated with myoinositol/D-chiroinositol compared with placebo (P = .04), whereas there was no difference in systolic blood pressure between treated and placebo-treated obese pregnant mice. Pregnant metabolic-like syndrome mice treated with myoinositol/D-chiroinositol showed lower glucose values during the glucose tolerance test and in the area under the curve (myoinositol/D-chiroinositol: 17512.5 ± 3984.4 vs placebo: 29687.14 ± 8258.7; P = .003), but no differences were seen in the obese pregnant mice. Leptin serum levels were lower in the metabolic-like syndrome-myoinositol/D-chiroinositol-treated mice compared with the placebo group (myoinositol/D-chiroinositol: 16985 ± 976.4 pg/dL vs placebo: 24181.9 ± 3128.2 pg/dL, P = .045). No other differences were seen in any of the remaining serum metabolic biomarkers studied in metabolic-like syndrome and in obese pregnant mice. Maternal weight gain was not different in the pregnant metabolic-like syndrome dams, whereas it was lower in the obese myoinositol/D-chiroinositol-treated dams compared with the placebo group (myoinositol/D-chiroinositol: 10.9 ± 0.5 g vs 12.6 ± 0.6 g, P = .04). Fetal and placental weights did not differ between myoinositol/D-chiroinositol-treated and nontreated pregnant dams with metabolic-like syndrome and obesity. CONCLUSION: Combined inositol treatment during pregnancy improves blood pressure, glucose levels at the glucose tolerance test, and leptin levels in pregnant dams with metabolic-like syndrome phenotype but not in obese pregnant dams. In addition, inositol treatment was associated with lower gestational weight gain in the obese but not in the metabolic-like syndrome pregnant dams.

Coagulation Factor Xa and Protease-Activated Receptor 2 as Novel Therapeutic Targets for Diabetic Nephropathy.

OBJECTIVE: The role of hypercoagulability in the pathogenesis of diabetic nephropathy (DN) remains elusive. We recently reported the increased infiltration of macrophages expressing tissue factor in diabetic kidney glomeruli; tissue factor activates coagulation factor X (FX) to FXa, which in turn stimulates protease-activated receptor 2 (PAR2) and causes inflammation. APPROACH AND RESULTS: Here, we demonstrated that diabetes mellitus increased renal FX mRNA, urinary FXa activity, and FX expression in glomerular macrophages. Administration of an oral FXa inhibitor, edoxaban, ameliorated DN with concomitant reductions in the expression of PARs (Par1 and Par2) and of proinflammatory and profibrotic genes. Diabetes mellitus induced PAR2, and lack of Par2 ameliorated DN. FXa or PAR2 agonist increased inflammatory cytokines in endothelial cells and podocytes in vitro. CONCLUSIONS: We conclude that enhanced FXa and PAR2 exacerbate DN and that both are promising targets for preventing DN. Alleviating inflammation is probably more important than inhibiting coagulation per se when treating kidney diseases using anticoagulants.

Glucagon-Like Peptide 1 Receptor Activation Attenuates Platelet Aggregation and Thrombosis.

Short-term studies in subjects with diabetes receiving glucagon-like peptide 1 (GLP-1)-targeted therapies have suggested a reduced number of cardiovascular events. The mechanisms underlying this unexpectedly rapid effect are not known. We cloned full-length GLP-1 receptor (GLP-1R) mRNA from a human megakaryocyte cell line (MEG-01), and found expression levels of GLP-1Rs in MEG-01 cells to be higher than those in the human lung but lower than in the human pancreas. Incubation with GLP-1 and the GLP-1R agonist exenatide elicited a cAMP response in MEG-01 cells, and exenatide significantly inhibited thrombin-, ADP-, and collagen-induced platelet aggregation. Incubation with exenatide also inhibited thrombus formation under flow conditions in ex vivo perfusion chambers using human and mouse whole blood. In a mouse cremaster artery laser injury model, a single intravenous injection of exenatide inhibited thrombus formation in normoglycemic and hyperglycemic mice in vivo. Thrombus formation was greater in mice transplanted with bone marrow lacking a functional GLP-1R (Glp1r(-/-)), compared with those receiving wild-type bone marrow. Although antithrombotic effects of exenatide were partly lost in mice transplanted with bone marrow from Glp1r(-/-) mice, they were undetectable in mice with a genetic deficiency of endothelial nitric oxide synthase. The inhibition of platelet function and the prevention of thrombus formation by GLP-1R agonists represent potential mechanisms for reduced atherothrombotic events.

Ablation of eNOS does not promote adipose tissue inflammation.

Adipose tissue (AT) inflammation is a hallmark characteristic of obesity and an important determinant of insulin resistance and cardiovascular disease; therefore, a better understanding of factors regulating AT inflammation is critical. It is well established that reduced vascular endothelial nitric oxide (NO) bioavailability promotes arterial inflammation; however, the role of NO in modulating inflammation in AT remains disputed. In the present study, 10-wk-old C57BL6 wild-type and endothelial nitric oxide synthase (eNOS) knockout male mice were randomized to either a control diet (10% kcal from fat) or a Western diet (44.9% kcal from fat, 17% sucrose, and 1% cholesterol) for 18 wk (n= 7 or 8/group). In wild-type mice, Western diet-induced obesity led to increased visceral white AT expression of inflammatory genes (e.g., MCP1, TNF-α, and CCL5 mRNAs) and markers of macrophage infiltration (e.g., CD68, ITGAM, EMR1, CD11C mRNAs, and Mac-2 protein), as well as reduced markers of mitochondrial content (e.g., OXPHOS complex I and IV protein). Unexpectedly, these effects of Western diet on visceral white AT were not accompanied by decreases in eNOS phosphorylation at Ser-1177 or increases in eNOS phosphorylation at Thr-495. Also counter to expectations, eNOS knockout mice, independent of the diet, were leaner and did not exhibit greater white or brown AT inflammation compared with wild-type mice. Collectively, these findings do not support the hypothesis that reduced NO production from eNOS contributes to obesity-related AT inflammation.