Endothelium-dependent Relaxation Of Aorta Research Articles

Pyrrolidine dithiocarbamate ameliorates endothelial dysfunction in thoracic aorta of diabetic rats by preserving vascular DDAH activity.

ObjectiveEndothelial dysfunction plays a pivotal role in the development of diabetic cardiovascular complications. Accumulation of endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) and inhibition of dimethylarginine dimethylaminohydrolase (DDAH) activity have been involved in diabetic endothelial dysfunction. This study was to investigate the effect of pyrrolidine dithiocarbamate (PDTC) on impairment of endothelium-dependent vasodilatation in diabetic rats and its potential mechanism.MethodsDiabetic rats were induced by a single intraperitoneal injection of streptozotocin (60mg/kg), and PDTC (10mg/kg) was given in drinking water for 8 weeks. Blood glucose and serum ADMA concentrations were measured in experimental rats. Recombinant adenovirus encoding human DDAH2 gene were constructed and ex vivo transferred to isolated rat aortas. The maximal relaxation (Emax) and half maximal effective concentration (EC50) of aortic rings response to accumulative concentrations of acetylcholine and vascular DDAH activity were examined before and after gene transfection.ResultsDiabetic rats displayed significant elevations of blood glucose and serum ADMA levels compared to control group (P<0.01). Vascular DDAH activity and endothelium-dependent relaxation of aortas were inhibited, as expressed by the decreased Emax and increased EC50 in diabetic rats compared to control rats (P<0.01). Treatment with PDTC not only decreased blood glucose and serum ADMA concentration (P<0.01) but also restored vascular DDAH activity and endothelium-dependent relaxation, evidenced by the higher Emax and lower EC50 in PDTC-treated diabetic rats compared to untreated diabetic rats (P<0.01). Similar restoration of Emax, EC50 and DDAH activity were observed in diabetic aortas after DDAH2-gene transfection.ConclusionsThese results indicate that PDTC could ameliorate impairment of endothelium-dependent relaxation in diabetic rats. The underlying mechanisms might be related to preservation of vascular DDAH activity and consequent reduction of endogenous ADMA in endothelium via its antioxidant action. This study highlights the therapeutic potential of PDTC in impaired vasodilation and provides a new strategy for treatment of diabetic cardiovascular complications.

Elevated Circulating Trimethylamine N-Oxide Levels Contribute to Endothelial Dysfunction in Aged Rats through Vascular Inflammation and Oxidative Stress.

Vascular endothelial dysfunction, a characteristic of the aging process, is an important risk factor for cardiovascular disease in aging. Although, vascular inflammation and oxidative stress are major contributors to endothelial dysfunction in aging, the underlying mechanisms during the aging process are not fully understood. Accumulating evidence reveals that gut microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) is implicated in the pathogenesis of many cardiovascular diseases. We tested the hypothesis that aging increases circulating TMAO levels, which induce vascular inflammation and oxidative stress, resulting in age-associated endothelial dysfunction. Old (22-mo-old) and young (4-mo-old) Fischer-344 rats were treated without (control) or with 1.0% 3,3-Dimethyl-1-butanol (DMB, an inhibitor of trimethylamine formation) in drinking water for 8 weeks. Compared with young control group, old control group had markedly higher plasma TMAO levels, which were reduced by DMB treatment. Endothelium-dependent relaxation of aorta in response to acetylcholine was impaired in old control group compared with young control group as indicated by decreased maximal relaxation (Emax) and reduced area under the curve (AUC). Emax and AUC were both normalized in old rats treated with DMB. No difference in endothelial-independent relaxation in response to sodium nitroprusside was observed among groups. Molecular studies revealed that old control group exhibits increased expression of proinflammatory cytokines and superoxide production, and decreased expression of endothelial nitric-oxide synthase (eNOS) in the aorta, all of which were restored by DMB treatment. These results suggest that aging increases circulating TMAO levels, which may impair eNOS-derived NO bioavailability by increasing vascular inflammation and oxidative stress, contributing to aging-associated endothelial dysfunction.

Abstract 9: Smooth Muscle Cell Tgfbr2 Deletion in Mice Causes Aortic Hypercontractility and Impaired Endothelium-Dependent Relaxation

Background: Abnormal smooth muscle cell (SMC) TGF-β signaling is proposed as a critical driver in the development of thoracic aortic aneurysms and dissections (TAAD) associated with Marfan and Loeys-Dietz Syndromes as well as nonsyndromic TAAD. However, the mechanisms by which altered SMC TGF-β signaling causes TAAD are poorly understood. Others have proposed that loss of SMC TGF-β signaling causes TAAD by impairing SMC contractility, leading to aortic medial degeneration and dilation. However, mice generated in our lab with deficient SMC TGF-β signaling (due to SMC-specific deletion of the type II TGF-β receptor) have thicker aortic medias and increased mRNA encoding SMC contractile proteins. These observations predict increased contractility. Methods &amp; Results: We addressed this apparent contradiction experimentally by measuring vasomotor function (by tension myography) and contractile protein expression (by immunoblotting) in aortas of mice with normal or deficient SMC TGF-β signaling. Isolated aortic rings from mice with deficient SMC TGF-β signaling showed increased contraction to phenylephrine (Emax: 13.5 mN vs 7.8 mN in controls; p&lt;0.0001; n=19-20) and potassium chloride (Emax: 7.5 mN vs 5.7 mN in controls; p&lt;0.0001; n=19-20). Moreover, levels of smooth muscle myosin heavy chain protein were at least as high in aortas with deficient SMC TGF-β signaling as in control aortas, consistent with their capacity to generate increased contractile force. Surprisingly, aortic segments from mice with deficient SMC TGF-β signaling also had impaired endothelium-dependent relaxation to acetylcholine (Emax: 37% vs 97% in controls; p&lt;0.0001; n=19-20). Endothelium-independent relaxation to sodium nitroprusside was similar between the two groups. CD31 immunostaining of vessel segments revealed equivalent endothelial integrity in both groups. Conclusion: Physiologic SMC TGF-β signaling is an important determinant of both SMC contractility and endothelial function. Disruption of physiologic SMC TGF-β signaling may lead to TAAD through direct effects on SMC as well as through indirect effects on endothelial function. Our results also suggest an unanticipated role for SMC TGF-β signaling in regulating endothelial-mediated vasomotor function.

Selective impairment of blood pressure reduction by endothelial nitric oxide synthase dimer destabilization in mice.

Endothelial dysfunction and oxidative stress are associated with hypertension but whether endothelial superoxide may play a role in the early development of essential hypertension remains uncertain. We investigated whether endothelial nitric oxide synthase (eNOS)-derived endothelial oxidative stress is involved in the regulation of SBP. Wild-type eNOS [mice with endothelium-specific overexpression of bovine endothelial NO-synthase (eNOS-Tg)] or a novel dimer-destabilized eNOS-mutant harboring a partially disrupted zinc-finger [mice with endothelium-specific overexpression of destabilized bovine eNOS destabilized by replacement of Cys 101 to Ala (C101A-eNOS-Tg)] was introduced in C57BL/6 in an endothelial-specific manner. Mice were monitored for aortic endothelium-dependent relaxation, SBP, levels of superoxide and several posttranslational modifications indicating activity and/or increased vascular oxidative stress. Some groups of mice underwent voluntary exercise training for 4 weeks or treatment with the superoxide dismutase mimetic Tempol. C101A-eNOS-Tg showed significantly increased superoxide generation, protein-tyrosine-nitration and eNOS-tyrosine-nitration, eNOS-S-glutathionylation, eNOS phosphorylation and AMP kinase-α phosphorylation at Thr172 in aorta, skeletal muscle, left ventricular myocardium and lung as compared with eNOS-Tg and wild-type controls. Exercise training increased phosphorylation of eNOS at Ser and AMP kinase-α in wild-type. These physiologic adaptations were absent in C101A-eNOS-Tg. Maximal aortic endothelium-dependent relaxation was similar in all strains. C101A-eNOS-Tg displayed normal SBP despite higher levels of eNOS, whereas eNOS-Tg showed significant hypotension. Tempol completely reversed the occurring protein modifications and significantly reduced SBP in C101A-eNOS-Tg but not in wild-type. Oxidative stress generated by endothelial-specific expression of genetically destabilized C101A-eNOS selectively prevents SBP-reducing activity of vascular eNOS, while having no effect on aortic endothelium-dependent relaxation. These data suggest that oxidative stress in microvascular endothelium may play a role for the development of essential hypertension.

The "Rise-Peak-Fall" Pattern of Time Dependency of the Cardiovascular Pleiotropic Effects of Treatment With Low-dose Atorvastatin, Losartan, and a Combination Thereof in Rats.

Treatment with low, subtherapeutic doses of statins and sartans expresses beneficial pleiotropic effects on the arterial wall. The present study explored whether these effects depend on treatment duration. Wistar rats were randomly divided into 4 groups and received low-dose atorvastatin, low-dose losartan, their combination, or saline (control) daily. After 4, 6, 8, and 10 weeks of treatment, the animals were anesthetized, blood samples taken, and hearts and thoracic aortas isolated. Thoracic aorta endothelium-dependent relaxation and parameters of the isolated heart exposed to ischemic-reperfusion injury were assessed along with blood serum parameters and vasoactive genes expression. Low-dose atorvastatin, losartan, and especially their combination showed the characteristic time dependency of all studied parameters (thoracic aorta relaxation, isolated heart parameters, C-reactive protein values, genes encoding endothelial nitric oxide synthase, and CD40). The peak in efficacy was observed after 6 weeks of treatment and subsequently steadily declined. The peak versus control values were significant for all measured parameters. Only a combination of atorvastatin and losartan increased nitric oxide and decreased asymmetric dimethylarginine. A characteristic time-dependent "rise-peak-fall" pattern of the cardiovascular pleiotropic effects of statins and sartans in subtherapeutic low doses was revealed. Evidently, resistance to the explored treatment occurs after a certain period.

Febuxostat, a novel xanthine oxidoreductase inhibitor, improves hypertension and endothelial dysfunction in spontaneously hypertensive rats.

Xanthine oxidase (XO) is an enzyme responsible for the production of uric acid. XO produces considerable amount of oxidative stress throughout the body. To date, however, its pathophysiologic role in hypertension and endothelial dysfunction still remains controversial. To explore the possible involvement of XO-derived oxidative stress in the pathophysiology of vascular dysfunction, by use of a selective XO inhibitor, febuxostat, we investigated the impact of pharmacological inhibition of XO on hypertension and vascular endothelial dysfunction in spontaneously hypertensive rats (SHRs). Sixteen-week-old SHR and normotensive Wistar-Kyoto (WKY) rats were treated with tap water (control) or water containing febuxostat (3 mg/kg/day) for 6 weeks. Systolic blood pressure (SBP) in febuxostat-treated SHR (220 ± 3 mmHg) was significantly (P < 0.05) decreased compared with the control SHR (236 ± 4 mmHg) while SBP in febuxostat-treated WKY was constant. Acetylcholine-induced endothelium-dependent relaxation in aortas from febuxostat-treated SHR was significantly (P < 0.05) improved compared with the control SHR, whereas relaxation in response to sodium nitroprusside was not changed. Vascular XO activity and tissue nitrotyrosine level, a representative indicator of local oxidative stress, were considerably elevated in the control SHR compared with the control WKY, and this increment was abolished by febuxostat. Our results suggest that exaggerated XO activity and resultant increase in oxidative stress in this experimental model contribute to the hypertension and endothelial dysfunction, thereby supporting a notion that pharmacological inhibition of XO is valuable not only for hyperuricemia but also for treating hypertension and related endothelial dysfunction in human clinics.

Human placenta-derived stromal cells decrease inflammation, placental injury and blood pressure in hypertensive pregnant mice.

Pre-eclampsia, the development of hypertension and proteinuria or end-organ damage during pregnancy, is a leading cause of both maternal and fetal morbidity and mortality, and there are no effective clinical treatments for pre-eclampsia aside from delivery. The development of pre-eclampsia is characterized by maladaptation of the maternal immune system, excessive inflammation and endothelial dysfunction. We have reported that detection of extracellular RNA by the Toll-like receptors (TLRs) 3 and 7 is a key initiating signal that contributes to the development of pre-eclampsia. PLacental eXpanded (PLX-PAD) cells are human placenta-derived, mesenchymal-like, adherent stromal cells that have anti-inflammatory, proangiogenic, cytoprotective and regenerative properties, secondary to paracrine secretion of various molecules in response to environmental stimulation. We hypothesized that PLX-PAD cells would reduce the associated inflammation and tissue damage and lower blood pressure in mice with pre-eclampsia induced by TLR3 or TLR7 activation. Injection of PLX-PAD cells on gestational day 14 significantly decreased systolic blood pressure by day 17 in TLR3-induced and TLR7-induced hypertensive mice (TLR3 144-111 mmHg; TLR7 145-106 mmHg; both P<0.05), and also normalized their elevated urinary protein:creatinine ratios (TLR3 5.68-3.72; TLR7 5.57-3.84; both P<0.05). On gestational day 17, aortic endothelium-dependent relaxation responses improved significantly in TLR3-induced and TLR7-induced hypertensive mice that received PLX-PAD cells on gestational day 14 (TLR3 35-65%; TLR7 37-63%; both P<0.05). In addition, markers of systemic inflammation and placental injury, increased markedly in both groups of TLR-induced hypertensive mice, were reduced by PLX-PAD cells. Importantly, PLX-PAD cell therapy had no effects on these measures in pregnant control mice or on the fetuses. These data demonstrate that PLX-PAD cell therapy can safely reverse pre-eclampsia-like features during pregnancy and have a potential therapeutic role in pre-eclampsia treatment.

Antihypertensive effects of oleuropein-enriched olive leaf extract in spontaneously hypertensive rats.

The effects of chronic consumption of oleuropein-enriched (15% w/w) olive leaf extract (OLE) on blood pressure, endothelial function, and vascular oxidative and inflammatory status in spontaneously hypertensive rats (SHR) were evaluated. Ten Wistar Kyoto rats (WKY) and twenty SHR were randomly assigned to three groups: a control WKY group, a control SHR group and a SHR group treated with OLE (30 mg kg(-1)) for 5 weeks. Long-term administration of OLE reduced systolic blood pressure, heart rate, and cardiac and renal hypertrophy. OLE treatment reversed the impaired aortic endothelium-dependent relaxation to acetylcholine observed in SHR. OLE restored aortic eNOS phosphorylation at Ser-1177 and Thr-495 and increased eNOS activity. OLE eliminated the increased aortic superoxide levels, and reduced the elevated NADPH oxidase activity, as a result of reduced NOX-1 and NOX-2 mRNA levels in SHR. OLE reduced the enhanced vascular TLR4 expression by inhibition of mitogen-activated protein kinase (MAPK) signaling with the subsequent reduction of proinflammatory cytokines. In conclusion, OLE exerts antihypertensive effects on genetic hypertension related to the improvement of vascular function as a result of reduced pro-oxidative and pro-inflammatory status.

The Restorative Effects of Eucommia ulmoides Oliver Leaf Extract on Vascular Function in Spontaneously Hypertensive Rats

Eucommia ulmoides Oliv. leaf is a traditional Chinese antihypertensive and antidiabetic medicine. We examined the effects of chronic Eucommia leaf extract (ELE) administration on artery function and morphology in spontaneously hypertensive rats (SHRs). ELE was orally administered via normal diet ad libitum to six-week-old male SHRs at a concentration of 5% for seven weeks. Acetylcholine (ACh)-induced endothelium-dependent relaxation, sodium nitroprusside (SNP)-induced endothelium-independent relaxation, plasma nitric oxide (NO) levels, and media thickness were assessed. ELE significantly improved ACh-induced aortic endothelium-dependent relaxation but did not affect SNP-induced endothelium-independent relaxation in the SHRs, as compared to the animals receiving normal diet. Plasma NO levels and media thickness were significantly increased and decreased, respectively, in the ELE-treated SHRs. Therefore, long-term ELE administration may effectively improve vascular function by increasing plasma NO levels and bioavailability, and by preventing vascular hypertrophy in the SHR aorta.

Abstract 15259: Genetically Engineered eNOS Dimer Destabilization Impairs Blood Pressure Reducing Activity of eNOS in Mice

Endothelial dysfunction and oxidative stress are associated with hypertension but whether endothelial superoxide plays a role in the early development of essential hypertension remains uncertain. In this study we investigated whether eNOS-derived endothelial oxidative stress is involved in the regulation of blood pressure (BP). Normal bovine eNOS (eNOS-Tg) or a novel dimer-destabilized eNOS-mutant harboring a partially disrupted zinc-finger (C101A-eNOS-Tg) was introduced in C57BL/6 mice using the endothelium-specific tie-2 promoter. Mice were monitored for aortic endothelium-dependent relaxation, systolic BP, levels of superoxide and several post-translational protein modifications indicating activity and/or increased vascular oxidative stress. Some groups of mice underwent voluntary exercise training or treatment with SOD mimetic Tempol (1mM). C101A-eNOS-Tg (aorta, skeletal muscle, left ventricular myocardium and lung) showed significantly increased superoxide generation, protein- and eNOS-tyrosine-nitration, eNOS-S-glutathionylation, eNOS1176/79 phosphorylation and Thr172-AMP kinase (AMPKα) phosphorylation as compared to eNOS-Tg and wildtype (WT)-controls. The localization of eNOS-Tg was restricted to endothelium as evidenced by immunohistochemical staining for eNOS and an endothelial-specific marker CD31. Exercise training increased phosphorylation of eNOS and AMPKα in WT while these physiologic adaptations were absent in C101A-eNOS-Tg. Aortic endothelium-dependent relaxation was similar in all strains. In striking contrast, C101A-eNOS-Tg displayed normal BP despite higher levels of eNOS, while eNOS-Tg showed significant hypotension. Tempol completely reversed the occurring protein modifications and significantly reduced BP in C101A-eNOS-Tg but not in WT. We observed that oxidative stress generated by endothelial-specific expression of genetically destabilized C101A- eNOS selectively prevents BP reducing activity of vascular eNOS, while having no effect on aortic endothelial dependent relaxation. Our data suggest that oxidative stress generated by eNOS dimer destabilization contributes to the regulation of blood pressure.

Sulforaphane attenuates the development of atherosclerosis and improves endothelial dysfunction in hypercholesterolemic rabbits.

The aim of the present work was to explore possible protective effects of sulforaphane (SFN) against atherosclerosis development and endothelial dysfunction in hypercholesterolemic rabbits. Rabbits were assigned to three groups of five: group I fed normal chow diet for four weeks, group II fed 1% high cholesterol diet (HCD) and group III fed HCD + SFN (0.25 mg/kg/day). Blood samples were collected for measurement of serum triglycerides (TGs), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), lactate dehydrogenase (LDH) and C-reactive protein (CRP). Aortic malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and total nitrite/nitrate (NOx) were measured. Vascular reactivity and intima/media (I/M) ratio were analyzed. Nuclear factor-kappa B (NF-κB) activation in aortic endothelial cells was identified immunohistochemically. HCD induced significant increases in serum TGs, TC, LDL-C, LDH, and CRP, and aortic MDA and SOD. Moreover, HCD caused significant reductions in serum HDL-C, aortic GSH and NOx. SFN administration significantly decreased HCD-induced elevations in serum TC, LDL-C, CRP, and LDH. while significantly increased HDL-C and GSH levels and normalized aortic SOD and NOx. Additionally, SFN significantly improved rabbit aortic endothelium-dependent relaxation to acetylcholine. Moreover, SFN significantly reduced the elevation in I/M ratio. This effect was confirmed by aortic histopathologic examination. The expression of NF-κB in aortic tissue showed a marked reduction upon treatment with SFN. In conclusion, this study reveals that SFN has the ability to ameliorate HCD-induced atherosclerotic lesions progression and vascular dysfunction, possibly via its lipid-lowering and antioxidant effects and suppression of NF-κB-mediated inflammation.

Antioxidant and anti-inflammatory effects of flavocoxid in high-cholesterol-fed rabbits.

Flavocoxid is a mixed extract containing baicalin and catechin, and it acts as a dual balanced inhibitor of cyclooxygenase-1 (COX-1) and COX-2 peroxidase enzyme activities with a significant inhibition of 5-lipoxygenase (5-LOX) enzyme activity in vitro. Flavocoxid downregulates gene or protein expression of several inflammatory markers and exerts also strong antioxidant activity in several experimental models. Inflammation and oxidative stress contribute in the pathogenesis of atherosclerosis. In the present study, an experimental rabbit model of hypercholesterolemia was developed and the effects of flavocoxid were evaluated. Rabbits were divided into four groups-normal control, high-cholesterol-diet (HCD)-fed group, HCD plus flavocoxid (20mg/kg/day), or HCD plus atorvastatin (10mg/kg/day). Blood samples were collected at the end of the experiment for measuring serum total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), C-reactive protein (CRP), malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD). In addition, the aorta was removed for measurement of antioxidant status, vascular reactivity, and intima/media (I/M) ratio. Elevated levels of serum TC, TGs, LDL-C, and CRP were measured in HCD group. Moreover, HCD caused a significant increase in serum and aortic MDA concomitantly with a reduction in serum and aortic GSH and SOD. Immunohistochemical staining of aortic specimens from HCD-fed rabbits revealed high expression levels of both tumor necrosis factor-alpha (TNF-α) and nuclear factor (NF)-κB. Rabbits in flavocoxid group showed significantly lower levels of serum CRP, serum, and aortic MDA and higher levels of serum HDL-C, serum, and aortic GSH and SOD compared to HCD group. HCD-induced elevations in serum TC and LDL-C did not significantly affected by flavocoxid treatment. Additionally, flavocoxid significantly enhanced rabbit aortic endothelium-dependent relaxation to acetylcholine and decreased the elevated I/M ratio. This effect was confirmed by histopathological examination of the aorta. Moreover, flavocoxid effectively suppresses the release of inflammatory markers. In conclusion, these findings demonstrated that flavocoxid would be useful in preventing oxidative stress, inflammation, and vascular dysfunction induced by HCD.

Antihypertensive effects of probiotics Lactobacillus strains in spontaneously hypertensive rats.

The cardiovascular effects of probiotics Lactobacillus fermentum CECT5716 (LC40), or L. coryniformis CECT5711 (K8) plus L. gasseri CECT5714 (LC9) (1:1) in spontaneously hypertensive rats (SHR) were evaluated. Ten Wistar Kyoto rats (WKY) and 30 SHR were randomly assigned to four groups (n = 10): a control WKY group, a control SHR groups, an SHR group treated with LC40, and an SHR treated with K8/LC9 group for 5 weeks (at a dose of 3.3 × 10(10) colony-forming units/day in drinking water). Long-term administration of probiotics reduced systolic blood pressure. The consumption of K8/LC9 mixture significantly reduced the cardiac and renal hypertrophy. Both groups of probiotics reversed the impaired aortic endothelium-dependent relaxation to acetylcholine observed in SHR. They also abolished the increased aortic superoxide levels by reducing the increased toll-like receptor-4 mRNA levels and NADPH oxidase activity found in SHR. K8/LC9 consumption also increased endothelial nitric oxide synthase phosphorylation. Probiotic treatments induced a change in the cecum microbiota of SHR, with higher counts of the Lactobacillus spp. cluster, and lower counts of Bacteriodes spp. and Clostridium spp. Probiotics exert cardiovascular protective effects in genetic hypertension related to the improvement of vascular pro-oxidative and pro-inflammatory status.

Abstract P183: Myeloid-Derived Suppressor Cells Prevent Cyclosporine A-Induced Hypertension, Endothelial Dysfunction, and Renal Injury in Mice

Cyclosporine A (CsA) is an immunosuppressive drug used to treat focal segmental glomerulosclerosis, reduce autoimmune diseases, and prevent allograft rejection; however a limitation of CsA is that it can induce vascular and renal injury as well as hypertension. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature granulocytes, macrophages, and dendritic cells that suppress pro-inflammatory immune responses by secreting anti-inflammatory cytokines, inhibiting innate and adaptive immune cells, and inducing regulatory T cells (Tregs). We hypothesized that adoptive transfer of MDSCs would prevent CsA-induced vascular and renal injury and hypertension. Daily treatment of male C57BL6/J mice for 1 week with CsA (50 mg/kg/day, i.p. injection) significantly increased systolic blood pressure (Day 7 SBP in mmHg: Con=97±2 vs. CsA=145±3, p&lt;0.05 vs. Con), decreased aortic endothelium-dependent relaxation responses, increased aortic fibronectin levels, increased renal glomerular mesangial expansion, increased renal fibronectin levels, and decreased splenic Treg levels. Adoptive transfer of 1 million MDSCs by i.p. injection on days 1, 4, and 7 partially prevented the CsA-induced rise in systolic blood pressure (CsA+1M MDSCs=120±3 mmHg) and the detrimental vascular and renal effects. However, adoptive transfer of 2 million MDSCs fully prevented the CsA-induced hypertension (CsA+2M MDSCs=105±1 mmHg) and vascular and renal effects. The anti-hypertensive and vascular and renal protective effects of 2 million MDSCs were independent of Treg induction as splenic Treg levels remained significantly decreased. These data suggest that MDSCs can prevent the hypertension and toxicity caused by CsA independent of Tregs and may be a therapeutic target for CsA-treated patients.

Enhanced vasorelaxation effect of endogenous anandamide on thoracic aorta in renal vascular hypertension rats.

Emerging evidence has indicated that anandamide (AEA) is able to stimulate vasorelaxation in both spontaneously hypertensive rats (SHRs) and L-NAME-induced hypertensive rats. Yet it remains unknown whether AEA modulates vasomotion of the aorta in renovascular hypertensive (RVH) rats. The aim of present study is to explore the effect of AEA on the relaxation of thoracic aortas in two-kidney one-clip (2K1C)-induced RVH rats. It is demonstrated that AEA stimulates a pronounced relaxation in the aortas of 2K1C rats compared with sham rats. The enhanced relaxation caused by AEA in aortas from 2K1C rats was diminished in the presence of the cannabinoid receptor-1 (CB1 ) antagonist AM251 and the CB2 receptor antagonist AM630. Likewise, the vasodilation action of AEA was blocked in L-NAME-treated or endothelium-denuded aortas. The Western blot results revealed that the expression of CB1 and CB2 receptors was increased in the 2K1C rat aortas compared with sham rats. The phosphorylation of endothelial nitric oxide synthase (p-eNOS) at the activation site Ser1177 was enhanced in AEA-treated rings from 2K1C rats in both time-dependent anddose-dependentmanners. The augmented p-eNOS expression was inhibited by the co-treatment with AM251 or AM630. Taken together, the present study demonstrated that AEA enhanced endothelium-dependent aortic relaxation through activation of both CB1 and CB2 receptors and P-eNOS/NO pathway in 2K1C rats.

Effects of chronic dietary exposure to monosodium glutamate on feeding behavior, adiposity, gastrointestinal motility, and cardiovascular function in healthy adult rats.

Monosodium glutamate (MSG) is a flavor-enhancer widely used as a food additive. However, its safe dietary concentration and its toxicity, including its possible implication in the recent metabolic syndrome pandemia, is still a controversial issue. Therefore, a deep knowledge of its effects upon regular dietary use is needed. Our aim was to evaluate the effects of chronic exposure to MSG on feeding behavior, abdominal fat, gastrointestinal motility, and cardiovascular function in rats. Two groups of adult male Wistar rats were used: control and treated with MSG (4g/L in drinking water) for 6weeks. Different functional parameters were determined and the histological structure was analyzed in tissues of interest. Compared to control animals, chronic MSG increased water intake but did not modify food ingestion or body weight gain. Neither the abdominal fat volume nor the fat fraction, measured by magnetic resonance imaging, was modified by MSG. Monosodium glutamate did not alter general gastrointestinal motility, but significantly increased the colonic response to mechanical stimulation. It slightly reduced endothelium-dependent relaxation in aorta, without significantly modifying any other cardiovascular parameters. No significant histological alterations were detected in salivary glands, intestinal wall, aorta, heart, and kidney. Chronic treatment with MSG in the adult rat increased water intake. This supports its potential to improve acceptance of low-fat regimens and to increase hydration in the elderly and sportspeople, often at risk of dehydration. Changes in colonic contractility and cardiovascular function could have some long-term repercussions warranting further research.

Attenuation of oxidative stress, inflammation, and endothelial dysfunction in hypercholesterolemic rabbits by allicin.

Allicin, the active substance of garlic, exerts a broad spectrum of pharmacological activities and is considered to have potential therapeutic applications. The present study was designed to investigate the possible beneficial effects of allicin against oxidative stress, inflammation, and endothelial dysfunction in hypercholesterolemic rabbits. Male New Zealand white rabbits were used in this study. Rabbits randomly received 1 of the following treatments: normal chow diet for 4 weeks, 1% high cholesterol diet (HCD), HCD plus allicin (10 mg/kg/day), or HCD plus atorvastatin (10 mg/kg/day). Blood samples were collected at the end of experimental diets for measurement of serum total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), C-reactive protein (CRP), malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD). In addition, the aorta was removed for measurement of vascular reactivity, histopathological changes, intima/media (I/M) ratio, and immunohistochemical staining of both tumor necrosis-alpha (TNF-α) and nuclear factor (NF)-κB. HCD induced significant increases in serum TC, TGs, low-density lipoprotein cholesterol (LDL-C), CRP, and MDA. Moreover, HCD caused significant decrease in serum GSH and SOD. In addition, aortic relaxation response to acetylcholine (ACh) was impaired. Immunohistochemical staining of aortic specimens from HCD-fed rabbits revealed high expression levels of both TNF-α and the oxidant-induced transcription factor, NF-κB. Allicin supplementation significantly decreased serum MDA and CRP, increased serum HDL-C, GSH, and SOD levels while nonsignificantly affecting HCD-induced elevations in serum TC and LDL-C. Additionally, allicin significantly protected against HCD-induced attenuation of rabbit aortic endothelium-dependent relaxation to ACh and elevation in I/M ratio. This effect was confirmed by histopathological examination of the aorta. Moreover, allicin has substantially beneficial effects on aortic expression of TNF-α and NF-κB compared with HCD-fed rabbits. In conclusion, these findings demonstrate that allicin may be useful in reducing oxidative stress, inflammation, vascular dysfunction, and the aortic pathology in hypercholesterolemic rabbits.

Overexpression of MnSOD Does Not Improve Endothelial Function in Hypercholesterolemic Mice

We previously reported that MnSOD‐deficiency does not cause overt vasomotor dysfunction in aged or hypercholesterolemic mice. To determine whether increasing MnSOD protects against vasomotor dysfunction and deleterious molecular changes in atherosclerosis, we crossed hypercholesterolemic mice with mice overexpressing MnSOD to test the hypothesis that lifelong increases in MnSOD protect against vasomotor dysfunction. We used Ldlr‐/‐/ApoB100/100 mice that were wild‐type (MnSOD0/0) or transgenic (MnSODTg/0) and fed a western diet for 6 months. We used organ bath chambers to measure aortic endothelium‐dependent relaxation (relaxation to acetylcholine, Ach), and mRNA from aortic arch was used for qRT‐PCR. Surprisingly, relaxation to Ach was unchanged between MnSOD0/0 and MnSODTg/0 mice, as was relaxation to sodium nitroprusside. As expected, gene expression of MnSOD was significantly increased in the MnSODTg/0 mice compared to MnSOD0/0 mice. Unlike our findings in MnSOD+/‐ mice, however (where Nox2 was increased), Nox2 expression was unchanged between MnSOD0/0 and MnSODTg/0 mice. Importantly, markers of osteogenesis such as Runx2 and BMP2 were unchanged between genotypes. Furthermore, MnSOD overexpression did not alter cellular senescence markers. Collectively, our data support the surprising conclusion that physiologically relevant increases in MnSOD do not significantly affect endothelial function or molecular regulators of histopathological changes in advanced atherosclerosis in hypercholesterolemic mice.

Beneficial effects of calcitriol on hypertension, glucose intolerance, impairment of endothelium-dependent vascular relaxation, and visceral adiposity in fructose-fed hypertensive rats.

Besides regulating calcium homeostasis, the effects of vitamin D on vascular tone and metabolic disturbances remain scarce in the literature despite an increase intake with high-fructose corn syrup worldwide. We investigated the effects of calcitriol, an active form of vitamin D, on vascular relaxation, glucose tolerance, and visceral fat pads in fructose-fed rats. Male Wistar-Kyoto rats were divided into 4 groups (n = 6 per group). Group Con: standard chow diet for 8 weeks; Group Fru: high-fructose diet (60% fructose) for 8 weeks; Group Fru-HVD: high-fructose diet as Group Fru, high-dose calcitriol treatment (20 ng / 100 g body weight per day) 4 weeks after the beginning of fructose feeding; and Group Fru-LVD: high-fructose diet as Group Fru, low-dose calcitriol treatment (10 ng / 100 g body weight per day) 4 weeks after the beginning of fructose feeding. Systolic blood pressure was measured twice a week by the tail-cuff method. Blood was examined for serum ionized calcium, phosphate, creatinine, glucose, triglycerides, and total cholesterol. Intra-peritoneal glucose intolerance test, aortic vascular reactivity, the weight of visceral fat pads, adipose size, and adipose angiotensin II levels were analyzed at the end of the study. The results showed that the fructose-fed rats significantly developed hypertension, impaired glucose tolerance, heavier weight and larger adipose size of visceral fat pads, and raised adipose angiotensin II expressions compared with the control rats. High- and low-dose calcitriol reduced modestly systolic blood pressure, increased endothelium-dependent aortic relaxation, ameliorated glucose intolerance, reduced the weight and adipose size of visceral fat pads, and lowered adipose angiotensin II expressions in the fructose-fed rats. However, high-dose calcitriol treatment mildly increased serum ionized calcium levels (1.44 ± 0.05 mmol/L). These results suggest a protective role of calcitriol treatment on endothelial function, glucose tolerance, and visceral adiposity in fructose-fed rats.

Impact of eNOS-Dependent Oxidative Stress on Endothelial Function and Neointima Formation.

Vascular oxidative stress generated by endothelial NO synthase (eNOS) was observed in experimental and clinical cardiovascular disease, but its relative importance for vascular pathologies is unclear. We investigated the impact of eNOS-dependent vascular oxidative stress on endothelial function and on neointimal hyperplasia. A dimer-destabilized mutant of bovine eNOS where cysteine 101 was replaced by alanine was cloned and introduced into an eNOS-deficient mouse strain (eNOS-KO) in an endothelial-specific manner. Destabilization of mutant eNOS in cells and eNOS-KO was confirmed by the reduced dimer/monomer ratio. Purified mutant eNOS and transfected cells generated less citrulline and NO, respectively, while superoxide generation was enhanced. In eNOS-KO, introduction of mutant eNOS caused a 2.3-3.7-fold increase in superoxide and peroxynitrite formation in the aorta and myocardium. This was completely blunted by an NOS inhibitor. Nevertheless, expression of mutant eNOS in eNOS-KO completely restored maximal aortic endothelium-dependent relaxation to acetylcholine. Neointimal hyperplasia induced by carotid binding was much larger in eNOS-KO than in mutant eNOS-KO and C57BL/6, while the latter strains showed comparable hyperplasia. Likewise, vascular remodeling was blunted in eNOS-KO only. Our results provide the first in vivo evidence that eNOS-dependent oxidative stress is unlikely to be an initial cause of impaired endothelium-dependent vasodilation and/or a pathologic factor promoting intimal hyperplasia. These findings highlight the importance of other sources of vascular oxidative stress in cardiovascular disease. eNOS-dependent oxidative stress is unlikely to induce functional vascular damage as long as concomitant generation of NO is preserved. This underlines the importance of current and new therapeutic strategies in improving endothelial NO generation.