Impaired eNOS Activity Research Articles

Targeting Neurofibromin 2 (NF2) prevents endothelial dysfunction in obesity: a study in mice and humans

Abstract Introduction The mechanisms underlying endothelial dysfunction (ED) in obesity are poorly understood. Neurofibromin 2 (NF2) is a scaffold-like protein involved in cell growth and survival. However, its role in the vascular endothelium is unknown. Purpose To investigate NF2 function in obesity-related ED. Methods Human aortic endothelial cells (HAECs) were exposed to palmitic acid (PA, 200 uM) or vehicle for 48 hours. Gene silencing of NF2 was performed by small interfering RNA (siRNA). Gene and protein expression were assessed by real time PCR and Western blot, respectively. The interaction of NF2 with endothelial proteins was investigated by co-immunoprecipitation. A constitutive active mutant form of NF2 (Ala518) was employed to study the effects of NF2 gain-of-function. To specifically investigate NF2 role in the vascular endothelium, we generated mice with endothelium-specific deletion of NF2 (NF2 ECKO) by crossing NF2flox/flox mice with tamoxifen-inducible endothelial-specific Cre mice [Cdh5(PAC)-CreERT2]. Endothelium-dependent relaxations to acetylcholine (Ach) were assessed in aortas from NF2 ECKO and wild type (WT) littermates, fed a control and a high fat diet (60 kcal% fat) for 20 weeks. NF2 signalling and endothelial function were also assessed in small visceral fat arteries (VFA) isolated from 18 obese and 18 age-matched healthy subjects undergoing bariatric surgery and cholecystectomy, respectively. Gene in silencing of NF2 by siRNA was performed in VFA from obese patients. Results In HAECs, PA promoted NF2 activation by decreasing its phosphorylation at Ser518. Akt and MYPT-1 were responsible for NF2 dephosphorylation. In PA-treated HAECs, NF2 was mainly found in the plasma membrane as compared to other cell fractions. Among different membrane proteins implicated in endothelial homeostasis, NF2 binds and activates Caveolin 1 (Cav-1), a pivotal repressor of endothelial NO synthase (eNOS). NF2 knockdown in PA-treated HAECs prevented eNOS–Cav-1 interaction, thus preserving eNOS activity and NO levels. By contrast, HAECs expressing the constitutive active mutant form of NF2 displayed reduced eNOS activity. In aortas from obese mice, we found that NF2-Cav-1 interaction was responsible for impaired eNOS activity and ED. Cav-1 gene silencing in NF2-overexpressing aortas prevented ED, thus confirming the direct involvement of Cav-1 in NF2-induced ED. Interestingly, Ach-dependent vasorelaxation was preserved in obese NF2 ECKO mice as compared to WT littermates. Moreover, NO bioavailability was preserved in aortas from NF2 ECKO mice. In VFA from obese patients, NF2 was upregulated, and its activity negatively correlated with Ach-dependent vasorelaxation. Of note, NF2 gene silencing in VFA from obese patients rescued ED. Conclusions In human endothelial cells, mice with endothelium-specific deletion of NF2 and VFA from obese patients, we show that NF2 drives ED by repressing Cav-1. Targeting NF2 may prevent ED in obese patients. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Holcim Stiftung

Activation of APJ Receptors by CMF‐019, but not Apelin, Causes Endothelium‐Dependent Relaxation of Spontaneously Hypertensive Rat Coronary Arteries

Hypertension is a major risk factor for coronary artery disease, but the pathophysiological mechanisms linking these diseases are not completely understood. Apelin is a novel vasoactive adipokine. Receptors for apelin (APJ receptors) are G‐protein‐coupled receptors and are widely expressed throughout the cardiovascular system. APJ receptors can also signal via G‐protein‐independent pathways, including G‐protein‐coupled‐receptor kinase 2 (GRK2), which inhibits nitric oxide synthase (eNOS) activity and nitric oxide (NO) production in endothelial cells. Apelin causes endothelium‐dependent, NO‐mediated relaxation of coronary arteries from normotensive animals, but the effects of activating APJ receptor signaling pathways in hypertensive coronary arteries are unknown. Since endothelium‐dependent relaxations are often blunted in arteries from hypertensive animals, we tested the hypothesis that apelin‐induced relaxation is impaired in coronary arteries from spontaneously hypertensive rats (SHR). Moreover, we hypothesized that endothelium‐dependent relaxations in response to CMF‐019, an APJ receptor G‐protein biased agonist with little effect on GRK2, would be unaffected in SHR coronary arteries. Western blot and RT‐qPCR analysis demonstrated that APJ receptor protein and mRNA expression did not differ between normotensive WKY (Wistar‐Kyoto) and SHR coronary arteries. eNOS protein expression was also similar between SHR and WKY coronary arteries, but GRK2 expression was significantly increased in SHR coronary endothelial cells, as compared to WKY. In myograph studies, apelin (10‐9‐10‐6 M) caused endothelium‐dependent relaxation of isolated WKY coronary arterial rings (pD2= 7.00 ± 0.11; Emax= 54 ± 4% relaxation; n=6), but had no effect in arteries from SHR. By contrast, CMF‐019 (10‐9‐10‐6 M)‐induced relaxation did not differ between normotensive and hypertensive coronary arteries. The relaxation response to CMF‐019 was abolished by the APJ receptor antagonist, F13A (10‐7 M), by removal of the endothelium, and by inhibition of eNOS with nitro‐ L‐arginine (3 x 10‐5 M). Moreover, the GRK2 inhibitor, CMPD 101 (3 x 10‐5 M), partially restored the relaxation response to apelin in SHR coronary arteries (pD2= 6.56 ± 0.22; Emax= 46 ± 6% relaxation; n=6). Immunoblot analysis of SHR coronary endothelial cells demonstrated that CMF‐019 (10‐7 M) significantly increased eNOS phosphorylation whereas apelin (10‐7 M) had no effect. We conclude that apelin failed to cause relaxation in hypertensive coronary arteries, likely due to impaired eNOS activity resulting from APJ receptor signaling via the GRK2 pathway and decreased eNOS phosphorylation. APJ receptor activation by the biased agonist, CMF‐019, caused similar endothelium‐dependent relaxations in WKY and SHR coronary arteries. Thus, APJ receptor‐biased agonists, such as CMF‐019, may be more effective than apelin in causing vasodilation of hypertensive coronary arteries.

Endothelial GTPCH (GTP Cyclohydrolase 1) and Tetrahydrobiopterin Regulate Gestational Blood Pressure, Uteroplacental Remodeling, and Fetal Growth.

[Figure: see text].

Abstract MP14: Endothelial Cullin3 Mutation Causes Decreased Nitric Oxide (NO) Bioavailability And Vascular Dysfunction Through Protein Phosphatase 2A

Mutations in CULLIN3 gene (in-frame deletion of exon 9, termed Cul3Δ9) cause human hypertension (HT) driven by a combination of renal tubular and vascular mechanisms. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE ERT2 mice. The resultant mice (E-Cul3Δ9) developed arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, n=5-7, p<0.05) and a trend towards elevated nighttime blood pressure (peak systolic BP, E-Cul3Δ9 136±3 vs control 128±3 mmHg, n=9-11) that were not associated with any alterations in locomotion, food/water intake or sleep/wake behaviors. No difference was seen in daytime BP. To determine whether vascular remodeling impairs baroreflex function, we performed power spectral analysis. Heart rate (HR), low frequency/high frequency ratio of HR variability, and baroreflex gain were comparable between control and E-Cul3Δ9 mice, suggesting no change in cardiac sympathetic nerve activity. However, low frequency amplitude of arterial pressure variability (16±4 vs 7±2 mmHg 2 , n=5-9, p<0.05) at night was markedly augmented in E-Cul3Δ9 mice, suggesting increased sympathetic activity in vascular tone regulation. Consistently, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (max ACh relaxation: 69% vs 84%, n=5-7, p<0.05) and cerebral resistance basilar artery (41% vs 77%, n=4-6, p<0.05). However, no dilatory impairment in mesenteric resistance artery and no difference in smooth muscle function were observed, suggesting that the effects of Cul3Δ9 are arterial bed specific. Expression of Cul3Δ9 in primary mouse aortic endothelial cells markedly decreased wild type Cul3 protein, phosphorylated eNOS and NO production. Protein phosphatase (PP) 2A, a known Cul3 substrate, dephosphorylates eNOS. Therefore, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor okadaic acid (4nM), but not by a PP1 inhibitor tautomycetin (4nM). Thus, CUL3 mutations in the endothelium may contribute to human HT in part through decreased endothelial NO bioavailability, arterial stiffening and secondary sympathoexcitation.

Abstract P086: Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, And Hypertension

Mutations in CULLIN3 gene (causing in-frame deletion of exon 9, termed Cul3Δ9) cause human hypertension (HT), which is driven by a combination of renal tubular and vascular mechanisms. We have previously shown that disruption of Cullin3 (CUL3) in vascular smooth muscle impairs nitric oxide (NO) signaling and vasodilation through decreased cGMP bioavailability, strongly supporting a role of vascular CUL3 in blood pressure (BP) regulation. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE ERT2 mice. Four weeks after tamoxifen, the resultant mice (E-Cul3Δ9) developed nocturnal HT (Night time peak systolic BP, E-Cul3Δ9: 135±3 vs Control: 124±3 mmHg) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s). No difference was seen in daytime BP. To determine whether vascular remodeling impairs baroreflex function, we performed power spectral analysis. Heart rate (HR), low frequency/high frequency ratio of HR variability, and baroreflex gain were comparable between control and E-Cul3Δ9 mice, suggesting there was no change in cardiac sympathetic nerve activity. However, low frequency amplitude of arterial pressure variability (16±4 vs 7±2 mmHg 2 ) at night was markedly augmented in E-Cul3Δ9 mice, suggesting increased sympathetic activity in vascular tone regulation. Consistently, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%) and cerebral resistance basilar artery (41% vs 77%). No difference in smooth muscle function was observed. Expression of Cul3Δ9 in primary mouse aortic endothelial cells markedly decreased wild type Cul3 protein, phosphorylated eNOS, and NO production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). Thus, CUL3 mutations in the endothelium contribute to human HT in part through decreased NO bioavailability, endothelial dysfunction and secondary sympathoexcitation.

Abstract P069: Microvesicles From Enos-suppressed Endothelial Cells Induce Endothelial Cell Dysfunction

Introduction: Endothelial nitric oxide synthase (eNOS) activity is critical to vascular health. Impaired eNOS activity and diminished NO production are common characteristics of a proatherogenic, dysfunctional endothelial phenotype that is associated with cardiovascular risk factors and disease. Extracellular microvesicles, particularly endothelial cell derived microvesicles (EMVs) represent novel mechanistic mediators of endothelial dysfunction and vascular disease. It is unknown whether eNOS suppression affects EMV number and function. We tested the following hypotheses: 1) eNOS blockade increases EMV release; and 2) EMVs derived from eNOS-suppressed cells adversely affect endothelial cell inflammation, apoptosis and NO production. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with the eNOS inhibitor, L-N G -Nitroarginine methyl ester (L-NAME; 300mM) for 24 h. EMVs (CD144 + ) released into the supernatant from cells treated with L-NAME or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were then treated with either L-NAME-derived or control EMVs for 24 h. To evaluate the role of endocytosis on the endothelial effects of EMVs, HUVECs were pre-incubated (12 h) with EIPA, filipin and chlorpromazine for 2 h, and all experiments repeated. Results: EMV release was markedly higher (~100%; P<0.05) in cells treated with L-NAME compared with control (81±6 vs. 40±7 EMV/μL). L-NAME-generated EMVs induced significantly higher release of IL-6 (38.4±5.1 vs. 21.0±1.9 pg/mL) and IL-8 (38.9±3.5 vs. 27.2±3.1 pg/mL) as well as greater active NF-κB p65 (Ser-536) (9.7±0.7 vs. 6.1±0.6 AU) expression than control EMVs. The expression of activated-caspase-3 was significantly higher in the cells treated with L-NAME (9.5±1.1 vs. 6.4±0.4 AU). Total eNOS (97.1±8.2 vs. 157.5±15.6 AU), activated eNOS (4.9±1.2 vs. 9.1±1.3 AU) and NO production (5.0±0.8 vs. 7.0±0.6 μmol/L) were significantly lower in endothelial cells treated with EMVs from eNOS suppressed cells. Endocytosis blockers mitigated the deleterious endothelial effects of EMVs. Conclusion: eNOS-suppression increases EMV release. Moreover, EMVs from eNOS-suppressed cells increase endothelial cell inflammation and apoptosis and decrease NO production.

Role of thioredoxin-interacting protein in mediating endothelial dysfunction in hypertension

Excessive oxidative stress is a major causative factor of endothelial dysfunction in hypertension. As an endogenous pro-oxidant, thioredoxin-interacting protein (TXNIP) contributes to oxidative damage in various tissues. The present study aimed to investigate the role of TXNIP in mediating endothelial dysfunction in hypertension. In vivo, an experimental model of acquired hypertension was established with two-kidney, one-clip (2K1C) surgery. The expression of TXNIP in the vascular endothelial cells of multiple vessels was significantly increased in hypertensive rats compared with sham-operated rats. Resveratrol, a TXNIP inhibitor, suppressed vascular oxidative damage and increased the expression and activity of eNOS in the aorta of hypertensive rats. Notably, impaired endothelium-dependent vasodilation was effectively improved by TXNIP inhibition in hypertensive rats. In vitro, we observed that Ang II increased the expression of TXNIP in primary human aortic endothelial cells (HAECs) and that TXNIP knockdown by RNA interference alleviated cellular oxidative stress damage and mitigated the impaired eNOS activation and intracellular nitric oxide (NO) production observed in Ang II-treated HAECs. However, inhibiting thioredoxin (TRX) with PX-12 completely blunted the protective effect of silencing TXNIP. In addition, TXNIP knockdown facilitated TRX expression and promoted TRX nuclear translocation to further activate AP1 and REF1. TRX overexpression exhibited favorable effects on eNOS/NO homeostasis in Ang II-treated HAECs. Thus, TXNIP contributes to oxidative stress and endothelial dysfunction in hypertension, and these effects are dependent on the antioxidant capacity of TRX, suggesting that targeting TXNIP may be a novel strategy for antihypertensive therapy.

P0978THE EFFECTS OF CHRONIC DAPAGLIFLOZIN TREATMENT ON THE RENAL MICROVASCULATURE IN A RAT MODEL OF TYPE 2 DIABETES

Abstract Background and Aims Chronic kidney disease (CKD) is an ever-growing concern and CKD associated with diabetes accounted for ∼ 35% of new cases of end-stage renal failure. Clinical trials with insulin-independent sodium-glucose co-transporter 2 (SGLT2) inhibitors not only showed significant improvements in hyperglycemia and thus renal damage, but also reduced the risk of adverse cardiovascular events. Microvessels supply local tissue oxygen demands and are important controllers of regional perfusion. Endothelial dysfunction has been posited to be an important factor driving the pathogenesis of diabetic nephropathy, largely through impaired eNOS activity and thus reduced nitric oxide bioavailability. Thus, we aim to determine the effects of SGLT2 inhibition on vasodilator function in the renal vasculature. We hypothesize that rats chronically treated with dapagliflozin, a SGLT2 inhibitor, improves renal endothelial function and the microvessels are better able to maintain perfusion in response to the inhibition of nitric oxide synthase and prostaglandin blockade. Method Male Zucker fatty diabetic rats (8 wk old), a model of type 2 diabetes, were given daily oral gavage of 1 mg/kg dapagliflozin or its vehicle for up to 22 wks of age (n=6/group). Renal excretory function, glomerular filtration rate (GFR) and indices of renal injury was assessed before treatment and after every 4 wks up until 22 wks of age. GFR was assessed via transcutaneous clearance of FITC-sinistrin. Vasodilator function of the renal microvasculature was assessed via X-ray microangiography. We conducted successive imaging of the microvessels before and during infusion of acetylcholine (ACh) and sodium nitroprusside (SNP) which facilitated the assessment of endothelium dependent and independent dilation respectively. After which, rats were given successive boluses of L-NAME and indomethacin to assess the vasodilatory function of the microvasculature independent of nitric oxide and prostaglandins respectively. These inhibitors were given under the conditions of SNP clamp which allowed for us to titrate the blood pressure to baseline levels. Lastly, we assessed the ability of endothelium hyperpolarizing factors (EDHFs) to maintain microvascular perfusion when SNP infusion ceased and an infusion of ACh commenced while still under the effects of L-NAME and indomethacin. Results As expected, dapagliflozin alleviated hyperglycemia across the treatment period. There was a tendency for dapagliflozin to ameliorate the decline of GFR, although this apparent effect was not statistically significant. Dapagliflozin did not appear to improve indices of renal injury. Treatment with dapagliflozin alleviated polyuria but did not appear to have an impact on urine osmolarity or sodium excretion. The responses (vessel diameter) of renal microvessels to ACh and SNP was greater in dapagliflozin than in vehicle fed rats. The microvessels of vehicle fed rats appeared to undergo relative constriction in response to L-NAME and indomethacin even under the effects of SNP clamp. In contrast, microvessels of dapagliflozin fed rats appeared to be relatively well-perfused after NOS and COX blockade. This suggests that dapagliflozin may improve endothelial dysfunction commonly associated with diabetic nephropathy. Following NOS and COX blockade, ACh was infused in rats to determine the status of vasodilatory function mediated by EDHFs. The microvessels in diabetic rats did not appear to be dilated after infusion of ACh, suggesting that vasodilatory effects of EDHFs on the vasculature is diminished in diabetic rats. Dapagliflozin appeared to improve this effect in that the renal microvessels were dilated even when NOS and COX production was blocked/inhibited. Conclusion Chronic treatment of dapagliflozin may improve endothelial dysfunction and thus retard the progression of diabetic nephropathy in a rat model of type 2 diabetes.

Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening and Hypertension

Mutations in CULLIN3 gene (causing in‐frame deletion of exon 9) cause human hypertension (HT), which is likely to be driven by a combination of renal tubular and vascular mechanisms. We have recently shown that smooth muscle expression of Cul3Δ9 mutant causes vascular dysfunction and HT via augmented RhoA/Rho‐kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo, we bred the conditionally activatable Cul3Δ9 mice with tamoxifen‐inducible Tie2‐CREERT2 mice. The resultant mice (E‐Cul3Δ9) developed nocturnal hypertension (Night time peak systolic BP, E‐Cul3Δ9: 138±3 vs Control: 121±4 mmHg, p<0.01) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, p<0.01). No difference was seen in daytime BP. Nitric oxide synthase (NOS) inhibitor L‐NAME induced smaller increases in nocturnal peak SBP and DBP in E‐Cul3Δ9 mice (15±1 vs 27±3 mmHg, p<0.01), suggesting their NOS activity is low. Of note, E‐Cul3Δ9 mice exhibited impaired endothelial‐dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%, p<0.05) and cerebral resistance basilar artery (41% vs 77%, p<0.01). No difference in smooth muscle function was observed. To determine the molecular mechanisms, we isolated primary aortic endothelial cells from mice carrying the inducible Cul3Δ9 construct and induced Cul3Δ9 expression in vitro using adenovirus carrying Cre recombinase gene. Expression of Cul3Δ9 resulted in marked decreases in wild type Cul3 protein, phosphorylated eNOS, and nitric oxide production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9‐induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). These data define a novel regulatory pathway involving Cullin3/PP2A/phospho‐eNOS in the endothelium. Selective endothelial expression of Cul3Δ9 partially phenocopies the hypertension observed in Cul3Δ9 patients, suggesting that mutations in Cullin‐3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.Support or Funding InformationNIH and AHA

Dynamic changes of angiopoietins and endothelial nitric oxide supply during fluid resuscitation for major gyn-oncological surgery: a prospective observation

BackgroundDespite goal-directed hemodynamic therapy, vascular function may deteriorate during surgery for advanced abdominal tumor masses. Fluid administration has been shown to be associated with distinct changes in serum levels of functional proteins. We sought to determine how serum total protein and angiopoietin (ANG) levels change during major abdominal tumor surgery. In addition, ex vivo endothelial nitric oxide synthase (eNOS) activation as well as NO bioavailability in vivo were assessed.Methods30 patients scheduled for laparotomy for late-stage ovarian or uterine cancer were prospectively included. Advanced hemodynamic monitoring as well as protocol-driven goal-directed fluid optimization were performed. Total serum protein, ANG-1, -2, and soluble TIE2 were determined pre-, intra-, and postoperatively. Phosphorylation of eNOS was assessed in microvascular endothelial cells after incubation with patient serum, and microvascular reactivity was determined in vivo by near-infrared spectroscopy and arterial vascular occlusion.ResultsCardiac output as well as preload gradually decreased during surgery and were associated with a median total fluid intake of 12.8 (9.7–15.4) mL/kg*h and a postoperative fluid balance of 6710 (4113–9271) mL. Total serum protein decreased significantly from baseline (66.5 (56.4–73.3) mg/mL) by almost half intraoperatively (42.7 (36.8–51.5) mg/mL, p < 0.0001) and remained at low level. While ANG-1 showed no significant dilutional change (baseline: 12.7 (11.9–13.9) ng/mL, postop.: 11.6 (10.8 –13.5) ng/mL, p = 0.06), serum levels of ANG-2 were even increased postoperatively (baseline: 2.2 (1.6–2.6) ng/mL vs. postop.: 3.4 (2.3–3.8) ng/mL, p < 0.0001), resulting in a significant shift in ANG-2 to ANG-1 ratio. Ex vivo phosphorylation of eNOS was decreased depending on increased ANG-2 levels and ANG-2/1 ratio (Spearman r = − 0.37, p = 0.007). In vivo, increased ANG-2 levels were associated with impaired capillary recruitment and NO bioavailability (Spearman r = − 0.83, p = 0.01).ConclusionsFluid resuscitation-associated changes in serum vascular mediator profile during abdominal tumor surgery were accompanied by impaired eNOS activity ex vivo as well as reduced NO bioavailability in vivo. Our results may explain disturbed microvascular function in major surgery despite goal-directed hemodynamic optimization.

2162Neurofibromin 2 (NF2) drives obesity-related endothelial dysfunction by targeting Caveolin-1: a study in mice and humans

Abstract Introduction Endothelial dysfunction (ED) is a key underpinning of cardiovascular disease in obesity, but the underlying molecular mechanisms remain elusive. Neurofibromin 2 (NF2) is a scaffold-like protein implicated in various cellular processes, namely growth, differentiation and survival. NF2 is inactivated by Akt-dependent phosphorylation at Ser518, whereas its dephosphorylation by the myosin phosphatase target subunit 1 (MYPT-1) leads to an active conformation. The role of NF2 in obesity-related alterations of endothelial phenotype remains elusive. Purpose To investigate whether NF2 participates to ED in obesity. Methods Human aortic endothelial cells (HAECs) were exposed to palmitic acid (PA, 200 uM) or vehicle for 48 hours. Gene silencing of NF2 was performed by small interfering RNA (siRNA). Protein expression was assessed by Western blot. Nitric oxide (NO) levels were measured by using a colorimetric assay. The interaction of NF2 with endothelial proteins was investigated by co-immunoprecipitation. To specifically determine NF2 role in the endothelium, we generated mice with endothelium-specific deletion of NF2 (NF2 ECKO) by crossing NF2flox/flox mice with tamoxifen-inducible endothelial-specific cre mice [Cdh5(PAC)-CreERT2]. Endothelium-dependent relaxations to acetylcholine (Ach, 10–9 to 10–5 mol/L) were assessed in aortas isolated from male NF2 ECKO and wild type littermates, fed a control (10 kcal% fat) and a high fat diet (60 kcal% fat) for 20 weeks. NF2 signalling and endothelial function were also assessed in small visceral fat arteries (VFA) isolated from 18 obese and 18 age-matched healthy subjects undergoing bariatric surgery and cholecystectomy, respectively. Results Exposure of HAECs to PA decreased NF2 phosphorylation at Ser518, thus leading to an active protein conformation. Blunted NF2 phosphorylation was explained by a reduction of Akt phosphorylation at Ser473 and a concomitant increase of MYPT-1 phosphorylation at Thr696. Pull-down experiments revealed that NF2 binds and activates Caveolin 1 (Cav-1), a pivotal repressor of endothelial NO synthase (eNOS). NF2 knockdown in PA-treated HAECs prevented eNOS–Cav-1 interaction, thus preserving eNOS activity and NO levels. In aortas from obese mice, we found that NF2-Cav-1 interaction was responsible for impaired eNOS activity, reduced NO levels and endothelial dysfunction. By contrast, Ach-dependent vasorelaxation were preserved in obese mice with endothelium-specific deletion of NF2. Moreover, we found that NF2 is activated in VFA from obese patients as compared to healthy controls, and its activity negatively correlated with Ach-dependent vasorelaxation of isolated VFA, as assessed by organ chamber experiments. Conclusions The present findings – obtained in human endothelial cells, conditional mouse models and visceral fat arteries from obese patients – suggest that targeting NF2 may represent a potential therapeutic strategy to prevent ED in patients with obesity.

Abstract 065: Endothelial CULLIN3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and Hypertension

Mutations in CULLIN3 gene (causing in-frame deletion of exon 9) cause human hypertension (HT), which is likely to be driven by a combination of renal tubular and vascular mechanisms. We have recently shown that smooth muscle expression of Cul3Δ9 mutant causes vascular dysfunction and HT via augmented RhoA/Rho-kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE ERT2 mice. The resultant mice (E-Cul3Δ9) developed nocturnal hypertension (Night time peak systolic BP, E-Cul3Δ9: 138±3 vs Control: 121±4 mmHg, p&lt;0.01) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, p&lt;0.01). No difference was seen in daytime BP. Nitric oxide synthase (NOS) inhibitor L-NAME induced smaller increases in nocturnal peak SBP and DBP in E-Cul3Δ9 mice (15±1 vs 27±3 mmHg, p&lt;0.01), suggesting their NOS activity is low. Of note, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%, p&lt;0.05) and cerebral resistance basilar artery (41% vs 77%, p&lt;0.01). No difference in smooth muscle function was observed. To determine the molecular mechanisms, we isolated primary aortic endothelial cells from mice carrying the inducible Cul3Δ9 construct and induced Cul3Δ9 expression in vitro using adenovirus carrying Cre recombinase gene. Expression of Cul3Δ9 resulted in marked decreases in wild type Cul3 protein, phosphorylated eNOS, and nitric oxide production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). These data define a novel regulatory pathway involving Cullin3/PP2A/phospho-eNOS in the endothelium. Selective endothelial expression of Cul3Δ9 partially phenocopies the hypertension observed in Cul3Δ9 patients, suggesting that mutations in Cullin-3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.

Epithelial sodium channels in endothelial cells mediate diet-induced endothelium stiffness and impaired vascular relaxation in obese female mice

ObjectiveMineralocorticoid receptor activation of the epithelial sodium channel in endothelial cells (ECs) (EnNaC) is accompanied by aldosterone induced endothelial stiffening and impaired nitric oxide (NO)-mediated arterial relaxation. Recent data support enhanced activity of the alpha subunit of EnNaC (αEnNaC) mediates this aldosterone induced endothelial stiffening and associated endothelial NO synthase (eNOS) activation. There is mounting evidence that diet induced obesity diminishes expression and activation of AMP-activated protein kinase α (AMPKα), sirtuin 1 (Sirt1), which would be expected to lead to impaired downstream eNOS activation. Thereby, we posited that enhanced EnNaC activation contributes to diet induced obesity related increases in stiffness of the endothelium and diminished NO mediated vascular relaxation by increasing oxidative stress and related inhibition of AMPKα, Sirt1, and associated eNOS inactivation. Materials/MethodsSixteen to twenty week-old αEnNaC knockout (αEnNaC−/−) and wild type littermate (EnNaC+/+) female mice were fed a mouse chow or an obesogenic western diet (WD) containing excess fat (46%) and fructose (17.5%) for 16 weeks. Sodium currents of ECs, endothelial stiffness and NO mediated aortic relaxation were examined along with indices of aortic oxidative stress, vascular remodeling and fibrosis. ResultsEnhanced EnNaC activation-mediated WD-induced increases in sodium currents in isolated lung ECs, increased endothelial stiffness and impaired aortic endothelium-dependent relaxation to acetylcholine (10−9–10−4 mol/L). These abnormalities occurred in conjunction with WD-mediated aortic tissue oxidative stress, inflammation, and decreased activation of AMPKα, Sirt1, and downstream eNOS were substantially mitigated in αEnNaC−/− mice. Importantly, αEnNaC−/− prevented WD induced increases in endothelial stiffness and related impairment of endothelium-dependent relaxation as well as aortic fibrosis and remodeling. However, EnNaC signaling was not involved in diet-induced abnormal expression of adipokines and CYP11b2 in abdominal aortic perivascular adipose tissue. ConclusionThese data suggest that endothelial specific EnNaC activation mediates WD-induced endothelial stiffness, impaired eNOS activation, aortic fibrosis and remodeling through increased aortic oxidative stress and increased inflammation related to a reduction of AMPKα and Sirt 1 mediated eNOS phosphorylation/activation and NO production.

Abstract 121: Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and Hypertension

Mutations in CULLIN3 gene (causing in-frame deletion of exon 9) cause human hypertension (HT), which is likely to be driven by a combination of renal tubular and vascular mechanisms. We have recently shown that smooth muscle expression of Cul3Δ9 mutant causes vascular dysfunction and HT via augmented RhoA/Rho-kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE ERT2 mice. The resultant mice (E-Cul3Δ9) developed nocturnal hypertension (Night time peak systolic BP, E-Cul3Δ9: 138±3 vs Control: 121±4 mmHg, p&lt;0.01) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, p&lt;0.01). No difference was seen in daytime BP. Nitric oxide synthase (NOS) inhibitor L-NAME induced smaller increases in nocturnal peak SBP and DBP in E-Cul3Δ9 mice (15±1 vs 27±3 mmHg, p&lt;0.01), suggesting their NOS activity is low. Of note, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%, p&lt;0.05) and cerebral resistance basilar artery (41% vs 77%, p&lt;0.01). No difference in smooth muscle function was observed. To determine the molecular mechanisms, we isolated primary aortic endothelial cells from mice carrying the inducible Cul3Δ9 construct and induced Cul3Δ9 expression in vitro using adenovirus carrying Cre recombinase gene. Expression of Cul3Δ9 resulted in marked decreases in wild type Cul3 protein, phosphorylated eNOS, and nitric oxide production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). These data define a novel regulatory pathway involving Cullin3/PP2A/phospho-eNOS in the endothelium. Selective endothelial expression of Cul3Δ9 partially phenocopies the hypertension observed in Cul3Δ9 patients, suggesting that mutations in Cullin-3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.

Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and Hypertension

Cullin3 (Cul3) is a critical subunit of the Cul3‐Ring‐Ligase (CRL3) ubiquitin ligase complex that targets proteins for proteasomal degradation. Mutations in CULLIN3 gene (causing in‐frame deletion of exon 9, termed Cul3Δ9) cause hypertension in humans. Evidence suggests that the hypertension in these subjects is likely to be caused by a combination of renal tubular and vascular mechanisms. We have recently shown that smooth muscle expression of Cul3Δ9 causes vascular dysfunction and elevation of blood pressure (BP) via augmented RhoA/Rho‐kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo, we bred the conditionally activatable Cul3Δ9 mice with Tek‐CREERT2 mice specifically expressing tamoxifen‐inducible Cre‐recombinase in the endothelium. The resultant mice (E‐Cul3Δ9) exhibited impaired endothelial‐dependent relaxation in carotid artery (maximal relaxation in response to 30 μM acetylcholine, 69% vs 84% in littermate control mice, n=5, p&lt;0.05, two‐way ANOVA repeated measurements) and in the cerebral basilar artery (maximal relaxation in response to 100 μM acetylcholine, 41% vs 77% in littermate control mice, n=5, p&lt;0.01). No difference in smooth muscle function was observed. Moreover, E‐Cul3Δ9 mice exhibited nocturnal hypertension as determined by radiotelemetry (night time peak systolic BP, 138±3 mmHg n=7 vs 122±4 mmHg n=5, p&lt;0.01). However no difference was seen in daytime pressure. Preliminary data indicated that treatment with nitric oxide synthase (NOS) inhibitor Nω‐Nitro‐L‐arginine methyl ester markedly elevated nocturnal peak SBP in control mice (133±5 mmHg, n=3) but not in E‐Cul3Δ9 mice (143±3 mmHg, n=3), suggesting that NOS activity is low in E‐Cul3Δ9 mice. E‐Cul3Δ9 mice also exhibited arterial stiffening as indicated by elevated pulse wave velocity (3.7±0.3 m/s n=5 vs control mice 2.7±0.1 m/s n=7, p&lt;0.01). To determine the molecular mechanism of endothelial dysfunction, primary aortic endothelial cells were isolated from mice carrying the inducible Cul3Δ9 construct and Cul3Δ9 expression was robustly induced by adenovirus carrying Cre recombinase gene in vitro. Expression of Cul3Δ9 resulted in marked decreases in wild type Cul3 protein, phosphorylated eNOS, and nitric oxide production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which negatively regulates eNOS phosphorylation, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9‐induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). These data define a novel regulatory pathway involving Cullin‐3/PP2A/phospho‐eNOS in the endothelium. Selective endothelial expression of Cul3Δ9 partially phenocopies the hypertension observed in Cul3Δ9 patients, suggesting that mutations in Cullin‐3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.Support or Funding InformationDr. Jing Wu is supported by an American Heart Association Postdoctoral Fellowship (17POST33660685). This project is funded by R01 HL125603 and R01 HL131689 to Dr. Curt Sigmund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ATP2B1 Gene Silencing Increases NO Production Under Basal Conditions Through the Ca2+/calmodulin/eNOS Signaling Pathway in Endothelial Cells.

Emerging epidemiological and experimental evidence has shown that the ATP2B1 gene is associated with blood pressure control. Impaired eNOS activity and NO production may be among the mechanisms involved. However, little is known about how PMCA1, which is encoded by the ATP2B1 gene, regulates the activity of eNOS and NO production. In the present study, we investigated the role of the ATP2B1 gene in regulating eNOS activity and NO production under basal conditions in HUVECs and explored the mechanisms involved. Silencing ATP2B1 gene expression resulted in higher NO production and eNOS activity under basal conditions in HUVECs. Additionally, ATP2B1 gene silencing resulted in enhanced intracellular calcium concentrations compared to that in the negative siRNA-transfected HUVECs. The enhanced eNOS activity mediated by ATP2B1 gene silencing was Ca2+/calmodulin dependent, as verified by the administration of the calcium chelator BAPTA-AM or the calmodulin-specific antagonist W7. Taken together, silencing ATP2B1 gene expression results in higher NO production and eNOS activity under basal conditions in HUVECs. Furthermore, the enhanced eNOS activity induced by ATP2B1 gene silencing may be mediated via higher levels of intracellular Ca2+, and the effect was confirmed to be dependent on the eNOS-calmodulin interaction.

Intravitreal enzyme replacement therapy attenuates retinal disease progression in a canine model of neuronal ceroid lipofuscinosis type 2 (CLN2)

Mineralocorticoid receptor activation of the epithelial sodium channel in endothelial cells (ECs) (EnNaC) is accompanied by aldosterone induced endothelial stiffening and impaired nitric oxide (NO)-mediated arterial relaxation. Recent data support enhanced activity of the alpha subunit of EnNaC (αEnNaC) mediates this aldosterone induced endothelial stiffening and associated endothelial NO synthase (eNOS) activation. There is mounting evidence that diet induced obesity diminishes expression and activation of AMP-activated protein kinase α (AMPKα), sirtuin 1 (Sirt1), which would be expected to lead to impaired downstream eNOS activation. Thereby, we posited that enhanced EnNaC activation contributes to diet induced obesity related increases in stiffness of the endothelium and diminished NO mediated vascular relaxation by increasing oxidative stress and related inhibition of AMPKα, Sirt1, and associated eNOS inactivation.Sixteen to twenty week-old αEnNaC knockout (αEnNaC−/−) and wild type littermate (EnNaC+/+) female mice were fed a mouse chow or an obesogenic western diet (WD) containing excess fat (46%) and fructose (17.5%) for 16 weeks. Sodium currents of ECs, endothelial stiffness and NO mediated aortic relaxation were examined along with indices of aortic oxidative stress, vascular remodeling and fibrosis.Enhanced EnNaC activation-mediated WD-induced increases in sodium currents in isolated lung ECs, increased endothelial stiffness and impaired aortic endothelium-dependent relaxation to acetylcholine (10−9–10−4 mol/L). These abnormalities occurred in conjunction with WD-mediated aortic tissue oxidative stress, inflammation, and decreased activation of AMPKα, Sirt1, and downstream eNOS were substantially mitigated in αEnNaC−/− mice. Importantly, αEnNaC−/− prevented WD induced increases in endothelial stiffness and related impairment of endothelium-dependent relaxation as well as aortic fibrosis and remodeling. However, EnNaC signaling was not involved in diet-induced abnormal expression of adipokines and CYP11b2 in abdominal aortic perivascular adipose tissue.These data suggest that endothelial specific EnNaC activation mediates WD-induced endothelial stiffness, impaired eNOS activation, aortic fibrosis and remodeling through increased aortic oxidative stress and increased inflammation related to a reduction of AMPKα and Sirt 1 mediated eNOS phosphorylation/activation and NO production.

Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and HypertensionΔ

Frame‐shift mutations in Cullin‐3 (causing skipping of exon 9, CUL3Δ9) were previously identified in pseudohypoaldosteronism type II (PHAII) patients that exhibit increased renal NaCl reabsorption, hyperkalaemia, metabolic acidosis and hypertension. The hypertension phenotypes were initially found to be driven by renal tubular mechanisms, but we have recently shown that smooth muscle expression of Cul3Δ9 causes vascular dysfunction and elevation of arterial pressure via augmented RhoA/Rho‐kinase signaling. Whether other extra‐tubular mechanisms contribute remains unknown. We generated a model of selective and inducible expression of CUL3Δ9 in the endothelium using a construct (CAG‐Cul3Δ9), which induces the expression of Cul3Δ9 (and TdTomato reporter) in response to Cre‐recombinase. In primary aortic endothelial cells isolated from CAG‐CUL3Δ9 mice, CUL3Δ9 expression was robustly induced by adenovirus carrying cre recombinase gene. Cul3Δ9 acted in a dominant negative manner by interfering with expression and function of endogenous Cullin‐3, leading to impaired turnover of Cullin‐3 substrate protein phosphatase 2A (PP2A), a marked decrease in phosphorylated eNOS, and reduced nitric oxide (NO) bioavailability. Treatment with PP2A inhibitor Calyculin A (5 nM) rescued CUL3Δ9‐induced impairment of eNOS activity in these cells. Likewise, inhibition of Cullin activity using MLN4924 (1 μM) caused a marked reduction in phosphorylated eNOS and NO production in mouse lung endothelial cells (MLECs), while these changes were prevented by Calyculin A. To test the importance of endothelial Cul3 in vivo, we bred CAG‐Cul3Δ9 mice with Tek‐CREERT2 mice specifically expressing a tamoxifen inducible Cre‐recombinase in the endothelium. The specificity of transgene expression was confirmed by western blot analysis of sorted endothelial and non‐endothelial fractions of the aortas from the resultant mice (termed E‐Cul3Δ9) 4 weeks following tamoxifen injection. E‐Cul3Δ9 mice exhibited impaired endothelial‐dependent relaxation in the carotid artery (relaxation to acetylcholine at 30 μM: 69.9 ± 5.7 % in E‐Cul3Δ9 vs. 85.6 ± 4.3 % in control littermates, p&lt;0.05, mean ± SEM), while no differences were seen in non‐endothelial dependent relaxation. Moreover, E‐Cul3Δ9 mice exhibited nocturnal hypertension as determined by radio telemetry (systolic pressure at 11pm, 143.7 ± 2.8 mmHg E‐Cul3Δ9 vs. 118.5 ± 3.4 control littermates, p&lt;0.01) and arterial stiffening as indicated by elevated pulse wave velocity (4.0 ± 0.5 E‐Cul3Δ9 vs. 2.9 ± 0.2 control littermates, p&lt;0.05). These data define a novel pathway involving Cullin‐3/PP2A/phopho‐eNOS in the regulation of endothelial function. Selective expression of the CUL3Δ9 mutation in the endothelium partially phenocopies the hypertension observed in CUL3Δ9 patients, suggesting that mutations in Cullin‐3 cause human hypertension in part through a vascular mechanism featured by impaired eNOS activity in endothelium.Support or Funding InformationGrants from NIH and AHA

Abstract P158: Cullin3 Regulated Endothelial Function by Modulating eNOS Activity

Pseudohypoaldosteronism type II (PHAII) patients expressing dominant negative cullin3 mutations exhibit increased renal NaCl reabsorption and develop hyperkalaemia, metabolic acidosis and hypertension. It is unclear whether loss of cullin3 function in extra-renal tissues contributes to the hypertensive phenotype. In the vasculature, endothelial Nrf2 stability is tightly regulated by cullin3-based E3 ubiquitin ligase via the redox-sensitive adaptor kelch-like ECH-associated protein 1. In the present study, we found that 24-hour treatment with a pan cullin inhibitor MLN4924 (1 μM) caused a 3-fold increase in Nrf2 protein in mouse lung endothelial cells (MLECs), while tert-butyl hydroperoxide (tBHP, 240 μM) had no effect on Nrf2 level. However, both MLN4924 and tBHP triggered time-dependent accumulation of Nrf2 in the nuclei, which peaked at 40 minutes following treatment. As a result, both treatments induced marked upregulation of antioxidant genes including NAD(P)H quinone oxidoreductase 1, heme oxygenase 1, glutamate cysteine ligase (rate-limiting enzyme in glutathione synthesis), and catalase both in MLECs and primary mouse aortic endothelial cells (MAECs). Of note, MLN4924 upregulated Nox4 expression (1.0 ± 0.15 vs 1.7 ± 0.2) and tBHP upregulated Nox1 (1.0 ± 0.2 vs 4.8 ± 1.1), while MLN4924 and tBHP both markedly increased intracellular superoxide as determined by dihydroethidium staining. In addition, intracellular nitric oxide was decreased by half in MLN4924-treated MLECs. This redox imbalance was likely due to impaired eNOS expression and activation as MLN4924 caused a 25% reduction in total eNOS and a 75% reduction in phosphorylated eNOS, while tBHP lead to a 50% reduction in phosphorylated eNOS with no effect on total eNOS. This suggests that decreased eNOS activation contributed to the oxidative stress induced by these agents. These data imply that suppression of cullin3 in arterial endothelial cells may dampen endothelium-dependent vascular relaxation and contribute to the blood pressure elevation observed in PHAII patients with global loss of cullin3 function. Although cullin3 also negatively regulates Nrf2-mediated antioxidant responses in vascular endothelial cells, this likely occurs as a compensatory mechanism.

Anagliptin increases insulin-induced skeletal muscle glucose uptake via an NO-dependent mechanism in mice.

Recently, incretin-related agents have been reported to attenuate insulin resistance in animal models, although the underlying mechanisms remain unclear. In this study, we investigated whether anagliptin, the dipeptidyl peptidase 4 (DPP-4) inhibitor, attenuates skeletal muscle insulin resistance through endothelial nitric oxide synthase (eNOS) activation in the endothelial cells. We used endothelium-specific Irs2-knockout (ETIrs2KO) mice, which show skeletal muscle insulin resistance resulting from a reduction of insulin-induced skeletal muscle capillary recruitment as a consequence of impaired eNOS activation. In vivo, 8-week-old male ETIrs2KO mice were fed regular chow with or without 0.3% (wt/wt) DPP-4 inhibitor for 8weeks to assess capillary recruitment and glucose uptake by the skeletal muscle. In vitro, human coronary arterial endothelial cells (HCAECs) were used to explore the effect of glucagon-like peptide 1 (GLP-1) on eNOS activity. Treatment with anagliptin ameliorated the impaired insulin-induced increase in capillary blood volume, interstitial insulin concentration and skeletal muscle glucose uptake in ETIrs2KO mice. This improvement in insulin-induced glucose uptake was almost completely abrogated by the GLP-1 receptor (GLP-1R) antagonist exendin-(9-39). Moreover, the increase in capillary blood volume with anagliptin treatment was also completely inhibited by the NOS inhibitor. GLP-1 augmented eNOS phosphorylation in HCAECs, with the effect completely disappearing after exposure to the protein kinase A (PKA) inhibitor H89. These data suggest that anagliptin treatment enhances insulin-induced capillary recruitment and interstitial insulin concentrations, resulting in improved skeletal muscle glucose uptake by directly acting on the endothelial cells via NO- and GLP-1-dependent mechanisms in vivo. Anagliptin may be a promising agent to ameliorate skeletal muscle insulin resistance in obese patients with type 2 diabetes.