Endothelial Function In Diabetic Mice Research Articles

Neuraminidase inhibition improves endothelial function in diabetic mice.

Neuraminidases cleave sialic acids from glycocalyx structures and plasma neuraminidase activity is elevated in type 2 diabetes (T2D). Therefore, we hypothesize circulating neuraminidase degrades the endothelial glycocalyx and diminishes flow-mediated dilation (FMD), whereas its inhibition restores shear mechanosensation and endothelial function in T2D settings. We found that compared with controls, subjects with T2D have higher plasma neuraminidase activity, reduced plasma nitrite concentrations, and diminished FMD. Ex vivo and in vivo neuraminidase exposure diminished FMD and reduced endothelial glycocalyx presence in mouse arteries. In cultured endothelial cells, neuraminidase reduced glycocalyx coverage. Inhalation of the neuraminidase inhibitor, zanamivir, reduced plasma neuraminidase activity, enhanced endothelial glycocalyx length, and improved FMD in diabetic mice. In humans, a single-arm trial (NCT04867707) of zanamivir inhalation did not reduce plasma neuraminidase activity, improved glycocalyx length, or enhanced FMD. Although zanamivir plasma concentrations in mice reached 225.8 ± 22.0 ng/mL, in humans were only 40.0 ± 7.2 ng/mL. These results highlight the potential of neuraminidase inhibition for ameliorating endothelial dysfunction in T2D and suggest the current Food and Drug Administration-approved inhaled dosage of zanamivir is insufficient to achieve desired outcomes in humans.NEW & NOTEWORTHY This work identifies neuraminidase as a key mediator of endothelial dysfunction in type 2 diabetes that may serve as a biomarker for impaired endothelial function and predictive of development and progression of cardiovascular pathologies associated with type 2 diabetes (T2D). Data show that intervention with the neuraminidase inhibitor zanamivir at effective plasma concentrations may represent a novel pharmacological strategy for restoring the glycocalyx and ameliorating endothelial dysfunction.

Neuraminidase-1 inhibition reduces arterial stiffness and improves endothelial function in diabetic mice

Plasma neuraminidase (Neu) activity has been shown to be increased in type 2 diabetes (T2D). The net result of neuraminidase activity is cleavage of sialic acid moieties from structures such as the glycoproteins and proteoglycans found in the glycocalyx, a mesh protruding out from the plasmalemma of all cells. In the vasculature, the glycocalyx participates in multiple functions including the mechanosensation of blood flow by the endothelium and consequently the production of the potent vasodilator and vascular homeostasis regulator, nitric oxide. Notably, endothelial dysfunction in the form of reduced flow-mediated dilation and arterial stiffening are two major vascular abnormalities associated with T2D. Therefore, we hypothesized that inhibition of Neu-1, which is a Neu isoform found in plasma, would improve flow-mediated dilation and reduce vascular stiffness in the db/db mouse model of T2D. To test this hypothesis, db/db mice were implanted subcutaneously with minipumps randomly assigned to deliver a Neu-1 inhibitor (n=10 mice) or vehicle control (n=10 mice) for 4 weeks. All differences reported here are significant at a P<0.05. Results show that compared with db/db controls, Neu-1 inhibition improved flow-mediated dilation in isolated mesenteric arteries as assessed using pressure myography. Neu-1 inhibition also reduced arterial stiffness as measured in vivo via aortic pulse wave velocity and ex vivo via passive pressure diameter curves in isolated femoral and mesenteric arteries. These findings indicate that Neu-1 inhibition can improve endothelial function and decrease arterial stiffness in the setting of T2D and thus support Neu-1 as potential target for amelioration of vascular disease in T2D. The particular mechanisms by which Neu-1 inhibition improves flow-mediated dilation and reduces arterial stiffness remain to be fully elucidated. Supported by: National Institutes of Health grant: R01HL153264 to LM-L and JP. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dietary chlorogenic acid ameliorates oxidative stress and improves endothelial function in diabetic mice via Nrf2 activation.

ObjectivesChlorogenic acid (CGA) is an antioxidant dietary factor. We investigated the effects of CGA on endothelial cell dysfunction in diabetic mice and the mechanistic role of nuclear factor erythroid-related factor 2 (Nrf2) in the antioxidant effect of CGA.MethodsDiabetic (db/db) mice were fed normal chow or chow containing 0.02% CGA for 12 weeks. Human umbilical vein endothelial cells (HUVECs) and mouse aortas were treated with normal or high glucose.ResultsCGA treatment induced upregulation of Nrf2 in HUVECs in a dose-dependent manner. CGA pretreatment prevented reactive oxygen species generation and preserved nitric oxide bioavailability in HUVECs and aortas from wild-type but not Nrf2−/− mice. CGA improved endothelium-dependent relaxation in high glucose-treated aortas from wild-type and db/db mice, but not Nrf2−/− mice. Dietary CGA improved endothelium-dependent relaxation in db/db mice.ConclusionsCGA ameliorates endothelial dysfunction in diabetic mice through activation of the Nrf2 anti-oxidative pathway.

Alk1 haploinsufficiency causes glomerular dysfunction and microalbuminuria in diabetic mice

Endothelial dysfunction has been shown to play an important role in the pathogenesis of glomerular damage during diabetic kidney disease (DKD). As such, a better understanding of the molecular mechanisms involved in glomerular endothelial dysfunctions could provide novel therapeutic strategies for the prevention of DKD. We have previously shown that Alk1/BMP9 signaling plays an important function to maintain vascular integrity in diabetic animals. As such, we evaluated the effects of Alk1 suppression on glomerular endothelial function in diabetic mice. In the present study, we used mice with conditional heterozygote deletion of Alk1 in the endothelium (Alk1ΔEC) to evaluate the role of Alk1 on kidney function during STZ-induced diabetes. DKD was investigated in diabetic control and Alk1ΔEC mice euthanized eight weeks after the onset of diabetes. We showed that Alk1 expression is reduced in the glomeruli of human DKD patients. While renal function was not altered in Alk1ΔEC non-diabetic mice, we showed that Alk1 haploinsufficiency in the glomerular endothelium leads to microalbuminuria, thickening of the glomerular basement membrane, glomerular apoptosis and podocyte loss in diabetic mice. These data suggest that Alk1 is important for the proper function of glomerular endothelial cells and that decreased Alk1 combined with chronic hyperglycemia can impair renal function.

452-P: Exercise-Induced Elevation of MicroRNA-181b Ameliorates Endothelial Dysfunction in Diabetic Mice

Endothelial dysfunction plays an essential role in the pathogenesis of diabetic vascular diseases. Our previous studies showed exercise improves endothelial function in diabetic mice partially through AMPK pathway. MicroRNA 181b (miR-181b) was reported to inhibit NF-κB activity in endothelial cells. However, it is still unknown whether miR-181b is regulated by AMPK signaling and involved in the vasoprotective effect of exercise. The present study investigated the expression of miR-181b in aortas from diabetic mice with/without exercise and reveals a role of the AMPK in exercise-induced miR-181b expression. We first determined the miR-181b level in the aortas of diabetic db/db mice subjected to treadmill exercise (45 min/day for eight weeks). The results showed that exercise significantly increased miR-181b level in the endothelium of mouse aorta compared to the sedentary mice. To test whether the increased blood flow during exercise affects miR-181b expression, we tested the effect of laminar flow on miR-181b expression in Human Umbilical Vein Endothelial Cells (HUVECs). Laminar flow greatly increases miR-181b expression. To understand the mechanism for flow-induced miR-181b expression, we treated HUVECs with AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and found that AICAR-induced miR-181b expression is reversed by AMPK inhibitors. Furthermore, we infected db/db mice with the adenovirus over-expressing miR-181b. MiR-181b overexpression improved endothelial function in db/db mice accompanied by decreased vascular inflammation and increased eNOS expression in mouse arteries. In summary, exercise increases miR-181b level in mouse endothelium probably via shear stress-induced AMPK activation. MiR-181b improves endothelial function in diabetic mice by inhibiting vascular inflammation and increasing eNOS expression. The present results suggest the therapeutic potential of targeting miR-181b against diabetic vasculopathy. Disclosure W. Shang: None. Y. Wang: None. J. Luo: None. X. Tian: None. L. Wang: None. Y. Huang: None. Funding Hong Kong Research Grants Council/Collaborative Research Fund (C4024-16W); Hong Kong Health and Medical Research Fund (05161746)

CTRP13 Preserves Endothelial Function by Targeting GTP Cyclohydrolase 1 in Diabetes.

Endothelial dysfunction plays a crucial role in the progress of diabetic vasculopathy. C1q/tumor necrosis factor-related protein 13 (CTRP13) is a secreted adipokine that can ameliorate atherosclerosis and vascular calcification. However, the role of CTRP13 in regulating endothelial function in diabetes has yet to be explored. In this study, CTRP13 treatment improved endothelium-dependent relaxation in the aortae and mesenteric arteries of both db/db mice and streptozotocin-injected mice. CTRP13 supplement also rescued the impaired endothelium-dependent relaxation ex vivo in the db/db mouse aortae and in high glucose (HG)-treated mouse aortae. Additionally, CTRP13 treatment reduced reactive oxygen species overproduction and improved nitric oxide (NO) production and endothelial NO synthase (eNOS) coupling in the aortae of diabetic mice and in HG-treated human umbilical vein endothelial cells. Mechanistically, CTRP13 could increase GTP cyclohydrolase 1 (GCH1) expression and tetrahydrobiopterin (BH4) levels to ameliorate eNOS coupling. More importantly, CTRP13 rescued HG-induced inhibition of protein kinase A (PKA) activity. Increased PKA activity enhanced phosphorylation of the peroxisome proliferator-activated receptor α and its recruitment to the GCH1 promoter, thus activating GCH1 transcription and, ultimately, endothelial relaxation. Together, these results suggest that CTRP13 preserves endothelial function in diabetic mice by regulating GCH1/BH4 axis-dependent eNOS coupling, suggesting the therapeutic potential of CTRP13 against diabetic vasculopathy.

The targeted inhibition of neuraminidase reverses endothelial glycocalyx degradation and improves endothelial function in type 2 diabetes

Endothelial dysfunction is a hallmark vascular abnormality of type 2 diabetes (T2D) that is characterized by blunted vasodilatory responses to physiological stimuli such as blood flow‐induced shear stress. Previously, we have reported that plasma neuraminidase levels are increased in a mouse model of diet‐induced obesity that is insulin resistant and considered pre‐diabetic. Neuraminidase is an enzyme that degrades the glycocalyx, which is a structure essential for the mechanotransduction of shear stress in the process of flow‐mediated dilation (FMD). Therefore, we hypothesized that circulating neuraminidase is implicated in the degradation of the endothelial glycocalyx and the diminished FMD that are associated with T2D, and that inhibition of circulating neuraminidase activity is an effective means to restore glycocalyx integrity and endothelial function in diabetic mice. In support of this hypothesis, we provide evidence that endothelial function, as assessed by FMD and insulin‐stimulated vasodilation, is blunted in T2D vs. non‐T2D human subjects and that this impairment concurs with elevated plasma neuraminidase activity and increased concentrations of its enzymatic byproduct, sialic acid. We further demonstrate that ex vivo exposure of mouse arteries to neuraminidase significantly reduces FMD. In cultured endothelial cells, we found that neuraminidase sheds sialic acid and Syndecan‐1, which are components of the glycocalyx and that the neuraminidase inhibitor, Zanamivir, attenuates this shedding. Moreover, in an animal model of diabetes (db/db mice), we demonstrate that Zanamivir reduces plasma neuraminidase activity and concomitantly improves femoral artery FMD and vasodilatory responses to acetylcholine and insulin. We also show that endothelial stiffness, assessed with atomic force microscopy using en face aortic samples, is significantly reduced in Zanamivir‐treated compared to placebo‐treated db/db mice. We conclude that inhibition of plasma neuraminidase activity represents a novel therapeutic approach to ameliorate endothelial dysfunction and cardiovascular risk in T2D patients by reversing glycocalyx degradation.Support or Funding InformationNational Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Purple sweet potato color attenuated NLRP3 inflammasome by inducing autophagy to delay endothelial senescence.

Autophagy is a vital negative factor regulating cellular senescence. Purple sweet potato color (PSPC), one type of flavonoid, has been demonstrated to suppress endothelial senescence and restore endothelial function in diabetic mice by inhibiting the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome. However, the roles of autophagy in the inflammatory response during endothelial senescence are unknown. Here, we found that PSPC augmented autophagy to restrict high-glucose-induced premature endothelial senescence. In addition, PSPC administration impaired endothelium aging in diabetic mice by increasing autophagy. Inhibition of autophagy accelerated endothelial senescence, while enhancement of autophagy delayed senescence. Moreover, deactivation of the NLRP3 inflammasome triggered by PSPC was autophagy-dependent. Autophagy receptor microtubule-associated protein 1 light chain 3 and p62 interacted with the inflammasome component NLRP3, suggesting that autophagosomes target the NLRP3 inflammasome and deliver it to the lysosome for degradation. Altogether, PSPC amplified cellular autophagy, subsequently attenuated NLRP3 inflammasome activity and finally delayed endothelial senescence to ameliorate cardiovascular complication. These results suggest a potential therapeutic target in senescence-related cardiovascular diseases.

Abstract P196: Fenofibrate Improves Vascular Contractility and Endothelial Function in Diabetic Mice

Aim: To exam the hypothesis that fenofibrate improves vascular endothelial dysfunction via balancing endothelium-dependent relaxation and constriction of aorta in diabetic mice. Methods: The streptozotocin-induced diabetic mice were treated with fenofibrate (100 mg/Kg/d, 8 weeks). The responses were assessed from serum and aortic parameters, vascular reactivity by wire myograph and aortic protein expression of endothelial nitric oxide synthase (eNOS), peroxisome proliferator-activated receptor α (PPARα), AMP-activated protein kinase-α (AMPKα) and liver kinase B1 (LKB1) and cyclooxygenase-2 (COX-2) in control and diabetic mice given placebo or fenofibrate. Results: Comparing with the control mice (12.2±2.0 μmol/L), serum creatinine increased by 114.8% in diabetic mice (26.2±2.0 μmol/L) and by 66.2% in fenofibrate-treated diabetic mice (21.6±2.8 μmol/L, p<0.01). NO level in aorta was 49.5±8.2 (μmol/g) in diabetic mice and 71.7±5.2 (μmol/g) in fenofibrate-treated diabetic mice (p<0.01). Compared with placebo-treated diabetic group, phosphorylated-eNOS protein in aorta was 3.6±1.0 fold higher (p<0.01) and endothelium-dependent relaxation was increased in fenofibrate-treated diabetic mice (p<0.01). The aorta vasodilation by fenofibrate treatment was reversed with PPARα inhibitor or AMPKα inhibitor. Fenofibrate treatment elevated PPARα expression, subsequently induced LKB1 translocation from nucleus to cytoplasm to activate AMPKα, thus activated eNOS in diabetic aorta (p<0.01). Fenofibrate treatment reduced vascular contractility and expression of COX-2 in aorta from diabetic mice(p<0.01). Prostaglandin E2 and thromboxane A2 were 174.9±16.4 and 128.9±4.4 (pg/ml) in diabetic mice, and reduced to 142.7±14.1 and 105.9±5.7 (pg/ml) in fenofibrate-treated mice (p<0.01). Conclusion: Fenofibrate treatment in diabetic mice normalizes endothelial function by balancing vascular reactivity via increasing NO production and suppressing vasoconstrictor prostaglandin. Our results suggest that implication of fenofibrate as a putative mediator of diabetic vascular complications.

Chronic NaHS treatment decreases oxidative stress and improves endothelial function in diabetic mice.

Hydrogen sulphide (H2S) is endogenously produced in vascular tissue and has anti-oxidant and vasoprotective properties. This study investigates whether chronic treatment using the fast H2S donor NaHS could elicit a vasoprotective effect in diabetes. Diabetes was induced in male C57BL6/J mice with streptozotocin (60 mg/kg daily, ip for 2 weeks) and confirmed by elevated blood glucose and glycated haemoglobin levels. Diabetic mice were then treated with NaHS (100 µmol/kg/day) for 4 weeks, and aortae collected for functional and biochemical analyses. In the diabetic group, both endothelium-dependent vasorelaxation and basal nitric oxide (NO•) bioactivity were significantly reduced ( p < 0.05), and maximal vasorelaxation to the NO• donor sodium nitroprusside was impaired ( p < 0.05) in aorta compared to control mice. Vascular superoxide generation via nicotine adenine dinucleotide phosphate (NADPH) oxidase ( p < 0.05) was elevated in aorta from diabetic mice which was associated with increased expression of NOX2 ( p < 0.05). NaHS treatment of diabetic mice restored endothelial function and exogenous NO• efficacy back to control levels. NaHS treatment also reduced the diabetes-induced increase in NADPH oxidase activity, but did not affect NOX2 protein expression. These data show that chronic NaHS treatment reverses diabetes-induced vascular dysfunction by restoring NO• efficacy and reducing superoxide production in the mouse aorta.

Glycemic Control with Ipragliflozin, a Novel Selective SGLT2 Inhibitor, Ameliorated Endothelial Dysfunction in Streptozotocin-Induced Diabetic Mouse.

Endothelial dysfunction caused by increased oxidative stress is a critical initiator of macro- and micro-vascular disease development in diabetic patients. Ipragliflozin, a selective sodium-glucose cotransporter 2 (SGLT2) inhibitor, offers a novel approach for the treatment of diabetes by enhancing urinary glucose excretion. The aim of this study was to examine whether ipragliflozin attenuates endothelial dysfunction in diabetic mice. Eight-week-old male C57BL/6 mice were treated with streptozotocin (150 mg/kg) by a single intraperitoneal injection to induce diabetes mellitus. At 3 days of injection, ipragliflozin (3 mg/kg/day) was administered via gavage for 3 weeks. Vascular function was assessed by isometric tension recording. Human umbilical vein endothelial cells (HUVEC) were used for in vitro experiments. RNA and protein expression were examined by quantitative RT-PCR (qPCR) and western blot, respectively. Oxidative stress was determined by measuring urine 8-hydroxy-2'-deoxyguanosine (8-OHdG) level. Ipragliflozin administration significantly reduced blood glucose level (P < 0.001) and attenuated the impairment of endothelial function in diabetic mice, as determined by acetylcholine-dependent vasodilation (P < 0.001). Ipragliflozin did not alter metabolic parameters, such as body weight and food intake. Ipragliflozin administration ameliorated impaired phosphorylation of Akt and eNOSSer1177 in the abdominal aorta and reduced reactive oxygen species generation as determined by urinary excretion of 8-OHdG in diabetic mice. Furthermore, qPCR analyses demonstrated that ipragliflozin decreased the expression of inflammatory molecules [e.g., monocyte chemoattractant protein-1 (MCP-1) vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule (ICAM)-1] in the abdominal aorta (P < 0.05). In in vitro studies, incubation with methylglyoxal, one of the advanced glycation end products, significantly impaired phosphorylation of Akt and eNOSSer1177 (P < 0.01) and increased the expression of MCP-1, VCAM-1, and ICAM-1 in HUVEC. Ipragliflozin improved hyperglycemia and prevented the development of endothelial dysfunction under a hyperglycemic state, at least partially by attenuation of oxidative stress.

In vivo inhibition of nuclear factor of activated t cells (NFAT) restores microvascular endothelial function in diabetic mice

In vivo inhibition of nuclear factor of activated t cells (NFAT) restores microvascular endothelial function in diabetic mice

Resveratrol via activation of LKB1-AMPK signaling suppresses oxidative stress to prevent endothelial dysfunction in diabetic mice

ABSTRACTBackground: Resveratrol, a phytoalexin, is reported to activate AMP-activated protein kinase (AMPK) in vascular cells. Whether resveratrol via activation of AMPK improves endothelial dysfunction in diabetes remains unknown. Here, we reported that activation of AMPK is required for resveratrol-induced improvement of endothelial function in diabetic mice. Methods: AMPK phosphorylation and activity, productions of reactive oxygen species, endothelium-dependent/independent relaxation were determined in vitro or in vivo. Results: Exposure of cultured human umbilical vein endothelial cells (HUVECs) to resveratrol activates AMPK by increasing the thr172 phosphorylation and its activities in time/dose-dependent manner. Loss function of liver kinase B1 by siRNA or mutant abolished resveratrol-induced AMPK activation. Incubation of endothelial cells with high glucose (HG) markedly induced oxidative stress of HUVECs, which was abolished by resveratrol in AMPK-dependent manner. In isolated mice aortas, resveratrol, AICAR or apocynin improved endothelium-dependent relaxation impaired by HG. In animal studies, streptozotocin-induced hyperglycemia dramatically increased oxidative stress and caused endothelial dysfunction, which were reversed by administration of resveratrol. Conclusions: We conclude that AMPK activation is required for resveratrol to improve endothelial function in diabetic mice.

Inhibition of miR-200c Restores Endothelial Function in Diabetic Mice Through Suppression of COX-2.

Endothelial dysfunction plays a crucial role in the development of diabetic vasculopathy. Our initial quantitative PCR results showed an increased miR-200c expression in arteries from diabetic mice and patients with diabetes. However, whether miR-200c is involved in diabetic endothelial dysfunction is unknown. Overexpression of miR-200c impaired endothelium-dependent relaxations (EDRs) in nondiabetic mouse aortas, whereas suppression of miR-200c by anti-miR-200c enhanced EDRs in diabetic db/db mice. miR-200c suppressed ZEB1 expression, and ZEB1 overexpression ameliorated endothelial dysfunction induced by miR-200c or associated with diabetes. More importantly, overexpression of anti-miR-200c or ZEB1 in vivo attenuated miR-200c expression and improved EDRs in db/db mice. Mechanistic study with the use of COX-2(-/-) mice revealed that COX-2 mediated miR-200c-induced endothelial dysfunction and that miR-200c upregulated COX-2 expression in endothelial cells through suppression of ZEB1 and increased production of prostaglandin E2, which also reduced EDR. This study demonstrates for the first time to our knowledge that miR-200c is a new mediator of diabetic endothelial dysfunction and inhibition of miR-200c rescues EDRs in diabetic mice. These new findings suggest the potential usefulness of miR-200c as the target for drug intervention against diabetic vascular complications.

O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.

Cardiovascular disease is the primary cause of morbidity and mortality in diabetes, and endothelial dysfunction is commonly seen in these patients. Increased O-linked N-acetylglucosamine (O-GlcNAc) protein modification is one of the central pathogenic features of diabetes. Modification of proteins by O-GlcNAc (O-GlcNAcylation) is regulated by two key enzymes: β-N-acetylglucosaminidase [O-GlcNAcase (OGA)], which catalyzes the reduction of protein O-GlcNAcylation, and O-GlcNAc transferase (OGT), which induces O-GlcNAcylation. However, it is not known whether reducing O-GlcNAcylation can improve endothelial dysfunction in diabetes. To examine the effect of endothelium-specific OGA overexpression on protein O-GlcNAcylation and coronary endothelial function in diabetic mice, we generated tetracycline-inducible, endothelium-specific OGA transgenic mice, and induced OGA by doxycycline administration in streptozotocin-induced type 1 diabetic mice. OGA protein expression was significantly decreased in mouse coronary endothelial cells (MCECs) isolated from diabetic mice compared with control MCECs, whereas OGT protein level was markedly increased. The level of protein O-GlcNAcylation was increased in diabetic compared with control mice, and OGA overexpression significantly decreased the level of protein O-GlcNAcylation in MCECs from diabetic mice. Capillary density in the left ventricle and endothelium-dependent relaxation in coronary arteries were significantly decreased in diabetes, while OGA overexpression increased capillary density to the control level and restored endothelium-dependent relaxation without changing endothelium-independent relaxation. We found that connexin 40 could be the potential target of O-GlcNAcylation that regulates the endothelial functions in diabetes. These data suggest that OGA overexpression in endothelial cells improves endothelial function and may have a beneficial effect on coronary vascular complications in diabetes.

Upregulation of Angiotensin (1-7)-Mediated Signaling Preserves Endothelial Function Through Reducing Oxidative Stress in Diabetes.

Angiotensin-converting enzyme 2 (ACE2)-angiotensin (1-7) [Ang (1-7)]-Mas constitutes the vasoprotective axis and is demonstrated to antagonize the vascular pathophysiological effects of the classical renin-angiotensin system. We sought to study the hypothesis that upregulation of ACE2-Ang (1-7) signaling protects endothelial function through reducing oxidative stress that would result in beneficial outcome in diabetes. Ex vivo treatment with Ang (1-7) enhanced endothelium-dependent relaxation (EDR) in renal arteries from diabetic patients. Both Ang (1-7) infusion via osmotic pump (500 ng/kg/min) for 2 weeks and exogenous ACE2 overexpression mediated by adenoviral ACE2 via tail vein injection (10(9) pfu/mouse) rescued the impaired EDR and flow-mediated dilatation (FMD) in db/db mice. Diminazene aceturate treatment (15 mg/kg/day) activated ACE2, increased the circulating Ang (1-7) level, and augmented EDR and FMD in db/db mouse arteries. In addition, activation of the ACE2-Ang (1-7) axis reduced reactive oxygen species (ROS) overproduction determined by dihydroethidium staining, CM-H2DCFDA fluorescence imaging, and chemiluminescence assay in db/db mouse aortas and also in high-glucose-treated endothelial cells. Pharmacological benefits of ACE2-Ang (1-7) upregulation on endothelial function were confirmed in ACE2 knockout (ACE2 KO) mice both ex vivo and in vitro. We elucidate that the ACE2-Ang (1-7)-Mas axis serves as an important signal pathway in endothelial cell protection in diabetic mice, especially in diabetic human arteries. Endogenous ACE2-Ang (1-7) activation or ACE2 overexpression preserves endothelial function in diabetic mice through increasing nitric oxide bioavailability and inhibiting oxidative stress, suggesting the therapeutic potential of ACE2-Ang(1-7) axis activation against diabetic vasculopathy. Antioxid.

Unconjugated bilirubin mediates heme oxygenase-1-induced vascular benefits in diabetic mice.

Heme oxygenase-1 (HO-1) exerts vasoprotective effects. Such benefit in diabetic vasculopathy, however, remains unclear. We hypothesize that bilirubin mediates HO-1-induced vascular benefits in diabetes. Diabetic db/db mice were treated with hemin (HO-1 inducer) for 2 weeks, and aortas were isolated for functional and molecular assays. Nitric oxide (NO) production was measured in cultured endothelial cells. Hemin treatment augmented endothelium-dependent relaxations (EDRs) and elevated Akt and endothelial NO synthase (eNOS) phosphorylation in db/db mouse aortas, which were reversed by the HO-1 inhibitor SnMP or HO-1 silencing virus. Hemin treatment increased serum bilirubin, and ex vivo bilirubin treatment improved relaxations in diabetic mouse aortas, which was reversed by the Akt inhibitor. Biliverdin reductase silencing virus attenuated the effect of hemin. Chronic bilirubin treatment improved EDRs in db/db mouse aortas. Hemin and bilirubin reversed high glucose-induced reductions in Akt and eNOS phosphorylation and NO production. The effect of hemin but not bilirubin was inhibited by biliverdin reductase silencing virus. Furthermore, bilirubin augmented EDRs in renal arteries from diabetic patients. In summary, HO-1-induced restoration of endothelial function in diabetic mice is most likely mediated by bilirubin, which preserves NO bioavailability through the Akt/eNOS/NO cascade, suggesting bilirubin as a potential therapeutic target for clinical intervention of diabetic vasculopathy.

Effect of short-term polyphenol treatment on endothelial dysfunction and thromboxane A2 levels in streptozotocin-induced diabetic mice.

Diabetes is characterized by the development of endothelial dysfunction, which affects both nitric oxide (NO)-mediated relaxation and endothelium-derived contracting factors, associated with vascular oxidative stress. There is a growing body of evidence suggesting that polyphenols have several beneficial effects, such as antioxidant and anti-inflammatory activities. This study investigated whether short-term treatment with polyphenols (chlorogenic acid (CA), morin (MO), resveratrol (RV)) can improve endothelial dysfunction related to diabetes. Aorta reactivity was determined in organ chambers, and we measured NO production and thromboxane B2 (TXB2; a metabolite of TXA2) from aortas in response to acetylcholine (ACh). Streptozotocin (STZ)-induced diabetic mice (16 weeks) were injected with solvent (ethanol, 10% v/v; intraperitoneally (i.p.)), CA (0.03 mmol/kg/d), MO (0.03 mmol/kg/d), and RV (0.03 mmol/kg/d) for 5 d. The ACh-induced endothelium-dependent relaxation was markedly reduced in rings of STZ-induced diabetic mice compared to controls. The treatment with polyphenols (significantly: MO, tendency: CA and RV) for only 5 d improved the NO components of relaxation, but did not normalize ACh-stimulated NO production. However, polyphenol treatment suppressed the ACh-stimulated level of TXB2 in aortas from STZ-induced diabetic mice. Thus, treatment with polyphenols caused basal NO production and a prompt improvement of the endothelial function in diabetic mice, and this may involve the normalization of TXA2 levels, not NO production, under ACh stimulation.

Inhibition of Bone Morphogenic Protein 4 Restores Endothelial Function in db/db Diabetic Mice

Bone morphogenic protein 4 (BMP4) is involved in the development of endothelial dysfunction in hypertension. This study investigated whether the inhibition of BMP4 signaling improves endothelial function in db/db diabetic mice. Male db/db mice were treated with noggin via osmotic pump infusion (1 µg/[h·kg(-1)]) for 2 weeks. Adenovirus BMP4-short hairpin RNA was introduced via tail vein injection at a dosage of 10(9) pfu/mouse and its effects were examined 7 days after. Vasoreactivity was studied on wire and pressure myograph. Both noggin treatment and adenovirus BMP4-short hairpin RNA transduction improved endothelium-dependent relaxations in aortae and flow-mediated dilatation in mesenteric arteries of db/db mice. Ex vivo treatment with BMP4 inhibitors and adenovirus BMP4-short hairpin RNA rescued the impaired endothelium-dependent relaxations in db/db mouse aortae and reduced reactive oxygen species overproduction determined by dihydroethidium staining, CM-H2DCFDA fluorescence imaging, and chemiluminescence assay in db/db mouse aortae, and also in ex vivo cultured C57BL/6 mouse aortae or primary mouse aortic endothelial cells treated with high glucose. Likewise, activin receptor-like kinase 3 silencing by short hairpin RNA lentivirus improved endothelium-dependent relaxations in db/db mouse aortae accompanied by reactive oxygen species inhibition in endothelial cells. In addition, noggin reduced BMP4 upregulation in high-glucose-treated endothelial cells and in C57BL/6 mouse aortae and in aortae from db/db mice. Inhibition of BMP4/activin receptor-like kinase 3/reactive oxygen species signaling improved endothelial function in diabetic mice through limiting oxidative stress in endothelium. Inhibiting BMP4 cascade can become another potential therapeutic strategy against diabetic vascular dysfunction.

PPARδ Activation Protects Endothelial Function in Diabetic Mice

Recent evidence highlights the therapeutic potential of peroxisome proliferator–activated receptor-δ (PPARδ) agonists to increase insulin sensitivity in diabetes. However, the role of PPARδ in regulating vascular function is incompletely characterized. We investigate whether PPARδ activation improves endothelial function in diabetic and obese mice. PPARδ knockout (KO) and wild-type (WT) mice fed with high-fat diet and db/db mice were used as diabetic mouse models, compared with PPARδ KO and WT mice on normal diet and db/m+ mice. Endothelium-dependent relaxation (EDR) was measured by wire myograph. Flow-mediated vasodilatation (FMD) was measured by pressure myograph. Nitric oxide (NO) production was examined in primary endothelial cells from mouse aortae. PPARδ agonist GW1516 restored EDRs in mouse aortae under high-glucose conditions or in db/db mouse aortae ex vivo. After oral treatment with GW1516, EDRs in aortae and FMDs in mesenteric resistance arteries were improved in obese mice in a PPARδ-specific manner. The effects of GW1516 on endothelial function were mediated through phosphatidylinositol 3-kinase (PI3K) and Akt with a subsequent increase of endothelial nitric oxide synthase (eNOS) activity and NO production. The current study demonstrates an endothelial-protective effect of PPARδ agonists in diabetic mice through PI3K/Akt/eNOS signaling, suggesting the therapeutic potential of PPARδ agonists for diabetic vasculopathy.