Independent Relaxation Research Articles

Model for type 2 diabetes exhibits changes in vascular function and structure due to vascular oxidative stress and inflammation

ABSTRACT We used a type 2 diabetes rat model produced by a high fat diet (HFD) followed by low dose streptozotocin (STZ) to study diabetic vasculopathy. Animals were evaluated for early vascular structural changes, endothelial function, inflammation, lipid profile and oxidative stress. We used 20 male Sprague-Dawley rats divided equally into control and diabetic groups. Diabetic rats were fed an HFD for 4 weeks, injected intraperitoneally with STZ, then sacrificed at week 15. Aortic endothelial nitric oxide synthase (eNOS), aortic superoxide dismutase (SOD), endothelial-dependent and independent relaxation and contraction, intima-media thickness (IMT), malondialdehyde (MDA) and tumor necrosis factor-alpha (TNF-α) were measured. Histopathological characteristics also were assessed. Diabetic rats exhibited higher fasting blood glucose (FBG), low density lipoprotein, total cholesterol and triglycerides compared to the control group. Aortic endothelium-dependent relaxation due to acetylcholine (ACh) was lower, while aortic endothelium-dependent contraction due to calcium ionophore and endothelium-independent contraction due to phenylephrine (PE) were higher for the diabetic group. eNOS expression was lower in the diabetic group compared to controls. IMT and MDA levels were increased, while SOD activity was decreased in the diabetic group compared to controls. TNF-α was higher in the diabetic group than for controls. Our type 2 diabetes model exhibited endothelial dysfunction associated with early vascular structural changes, dyslipidemia, increased vascular oxidative stress, and inflammation. Therefore, the model is suitable for studying diabetic atherosclerosis.

In-situ dielectric analysis on the responses of vegetable cells to freezing treatment

Abstract An in-situ dielectric measurement was implemented on garlic clove, onion, taro and Chinese yam cells undertaken slow cooling from 2.0 to -11.0 °C. The double-membrane structure of vegetable cell corresponding to cell membrane and tonoplast was identified by splitting up one overlapping β-relaxation into two independent relaxations, and a double–shell model was applied to analyze the two dielectric relaxations based on interface polarization mechanism. The dielectric relaxation time (τ) of vegetable cells were found to respond sensitively to the freezing treatment. An abrupt shift of τ demonstrated the formation of extracellular ice, and the disappearance of the lower-frequency relaxation at the ice crystallization point was attributed to the frozen damage of cell integrity.

Machine perfusion of donor heart with normothermic blood versus hypothermic HTK in preserving coronary endothelium in a porcine model of DCD.

Both machine perfusion (MP) of donor hearts with autologous blood and crystalloid perfusates have advantages and disadvantages. Currently, which of the aforementioned preservation strategies can better preserve the coronary endothelium has not yet been determined. We aim to compare the impact of hypothermic continuous MP with histidine-tryptophan-ketoglutarate (HTK) solution versus normothermic continuous MP with autologous blood on coronary endothelium in a porcine ex vivo model of donation following circulatory death (DCD). DCD pigs underwent circulatory arrest via asphyxiation followed by 30-minute warm ischemia time. Donor hearts were preserved with either hypothermic MP with HTK solution (MP + HTK group; 4 ℃; n=6), or normothermic MP with blood (MP + blood group; 37 ℃; n=6) for 4 hours. After 2-hour ex vivo reperfusion, the assessment of endothelial-dependent (Edep) and -independent (Eind) relaxation of coronary artery, histopathological analysis, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay were performed. Preservation of DCD hearts with MP + Blood strategy significantly improved both Edep and Eind vasorelaxation of coronary artery compared with MP + HTK strategy (maximum relaxation to bradykinin: MP + HTK 80.9%±2.6% vs. MP + Blood 91.9%±1.9%, P<0.001; maximum relaxation to sodium nitroprusside: MP + HTK 97.1%±1.0% vs. MP + Blood 99.8%±0.2%, P<0.05). MP + Blood strategy significantly decreased nitrotyrosine but increased intercellular adhesion molecule-1 immunoreactivity in the coronary artery. The number of TUNEL-positive cells in MP + Blood group were significantly fewer compared with MP + HTK group. Compared with MP + HTK strategy, MP + Blood strategy significantly alleviates coronary endothelial dysfunction during donor heart preservation. This protective effect is associated with the inhibition of apoptosis and nitro-oxidative stress in coronary artery.

Biochanin A, the Most Potent of 16 Isoflavones, Induces Relaxation of the Coronary Artery Through the Calcium Channel and cGMP-dependent Pathway.

The dietary intake of flavonoids seems to be inversely related to cardiovascular mortality. The consumption of isoflavonoids is increasing in the general population, especially due to the use of food supplements and a variety of isoflavonoid-rich foods. However, detailed studies on the vascular influence of individual pure isoflavonoids are mostly missing. For this study, 16 isoflavonoids were initially screened for their vasorelaxant properties on rat aortas. The 2 most potent of them, biochanin A and glycitein, were further tested for the mechanism of action on porcine coronary arteries. They both induced an endothelium independent vascular relaxation, with EC50 below 6 and 17 µM, respectively. Biochanin A, but not glycitein, was able to block the vasoconstriction caused by KCl, CaCl2, serotonin, and U46619 in a dose-dependent manner. Another series of experiments suggested that the major mechanism of action of biochanin A was the inhibition of L-type calcium channels. Moreover, biochanin A in relatively small concentrations (2 - 4 µM) interfered with the cGMP, but not cAMP, pathway in isolated coronary arteries. These results indicate that some isoflavonoids, in particular biochanin A, are able to have vasodilatory effects in micromolar concentrations, which is of potential clinical interest for the management of cardiovascular pathologies.

Progesterone Reduces Endothelial Function Without Increasing Endothelial Mineralocorticoid Receptor Expression in Male C57Bl/6 Mice

Recent work from our lab demonstrated that endothelial cells from women and female mice exhibit higher expression of the mineralocorticoid receptor (ECMR) and identified progesterone as the origin of this sex‐difference in ECMR expression. We also showed that progesterone‐induced increases in ECMR predispose females to obesity‐associated, aldosterone‐mediated endothelial dysfunction and demonstrated that deletion of either progesterone receptor (PR) or endothelial MR protects female mice from aldosterone‐induced endothelial dysfunction. Our preliminary data indicates that endothelial cell PR expression is higher in female mice than in males. However, whether the effects of exogenous progesterone to increase endothelial MR expression is sex‐specific is unknown, a notion that is of clinical importance to the growing population of transgender individuals receiving hormone replacement therapy. We hypothesized that progesterone supplementation increases endothelial MR expression in male mice. We infused male C57bl6 mice with progesterone (0.25mg/day, s.c. osmotic minipump) for 4 weeks. Endothelial and non‐endothelial cells were isolated from aortas to assess MR and PR expression via quantitative real time PCR. Progesterone treatment did not increase ECMR expression (6.37±5.1‐fold from control, Student’s T test, n=9 control, n=7 progesterone) nor in non‐endothelial vascular cells (0.5±0.6‐fold from control). However, a significant increase in PR expression was observed in endothelial cells (6.5±2.6‐fold from control, *p&lt;0.05), that was absent in non‐endothelial vascular cells (0.3±0.4‐fold from control) in response to progesterone. Progesterone supplementation to male mice eliminated the sex discrepancy in endothelial PR expression between male and female mice (5.9±2.4‐fold from male, *p&lt;0.05, n=6) without a correlating change in ECMR expression. Supplementation of human aortic endothelial cells in culture with dihydrotestosterone (10ng/ml) had no effect on ECMR promoter activity (1.2±0.1fold) indicating that testosterone is not the sex specific factor restricting increases in ECMR expression by progesterone in males. We additionally excised thoracic aortas for wire myography. Our results showed that progesterone treatment modestly decreased endothelial mediated relaxation to acetylcholine (2‐way ANOVA with repeated measures, P=0.08) with no significant effect on endothelial independent relaxation to sodium nitroprusside or constriction to phenylephrine compared to control. These data indicate that progesterone‐mediated increases in ECMR expression in females is sex‐specific and does not persist in endothelial cells of male mice, an effect unlikely mediated by testosterone. In addition, endothelial PR expression and endothelial function is sensitive to progesterone treatment in male mice. Therefore, endothelial‐specific PR signaling mechanisms exhibit sex‐specific phenotypes on cardiovascular function, indicating that further study is needed to determine potential risks facing transgender populations on hormone replacement therapies.Support or Funding Information1R01HL130301‐01, 19EIA34760167, Adrenal Center pilot grant, MCG Medical Scholars Program, 1 K99 HL146948‐01A1

Lack of LRP8 triggers the formation of aortic dissection in angiotensin II infused mice

Factor XI-thrombin amplification loop depends on platelets and monocytes and augments angiotensin II (AngII) driven vascular injury in experimental hypertension in mice. Low-density lipoprotein receptor-related protein 8 (LRP8) plays an important role in apolipoprotein E homeostasis and was also found to be important in endothelial barrier stabilization. Moreover, LRP8 is able to form a complex with the platelet glycoprotein Ib alpha (GPIbalpha) which is the main receptor for von Willebrand factor. To explore the role of LRP8 and GPIbalpha in the development of hypertension related to platelet-dependent thrombin generation. We infused LRP8 KO mice with AngII for either 1 or 4 weeks using osmotic minipumps. Blood pressure was recorded using tail cuff measurement. Vascular reactivity was assessed in isolated aortic segments. Leukocyte activation and infiltration were assessed by flow cytometry of aortic tissue and intravital videomicroscopy imaging (IVM). Histology of aortas was performed with sirius red staining. Infusion of AngII for 1 week in LRP8 KO mice altered endothelial dependent and independent vascular relaxation to the same extend as in control mice. Vascular rolling and adherence to the carotid endothelium, visualized by IVM was similar between LRP8 KO and control mice infused with AngII. There were no significant differences in AngII-induced blood pressure increase between the LRP8 KO and control mice. Unexpectedly, LRP8 KO mice demonstrated a drastically increased mortality rate in response to AngII infusion. In the 4 weeks AngII infusion experiments, 80% of the LRP8 KO mice died within the first 10 days of AngII infusion due to aortic dissection complicated by hemorrhage. LRP8 receptor dysfunction lead to the formation of aortic dissection in AngII infused mice. Further studies are needed to better characterize the role of immune cells and platelets in the development of these dissections.

Electronic relaxation dynamics in [Au25(SR)18]-1 (R = CH3, C2H5, C3H7, MPA, PET) thiolate-protected nanoclusters.

We investigate the excited electron dynamics in [Au25(SR)18]-1 (R = CH3, C2H5, C3H7, MPA, PET) [MPA = mercaptopropanoic acid, PET = phenylethylthiol] nanoparticles to understand how different ligands affect the excited state dynamics in this system. The population dynamics of the core and higher excited states lying in the energy range 0.00-2.20 eV are studied using a surface hopping method with decoherence correction in a real-time DFT approach. All of the ligated clusters follow a similar trend in decay for the core states (S1-S6). The observed time constants are on the picosecond time scale (2-19 ps), which agrees with the experimental time scale, and this study confirms that the time constants observed experimentally could originate from core-to-core transitions and not from core-to-semiring transitions. In the presence of higher excited states, R = H, CH3, C2H5, C3H7, and PET demonstrate similar relaxations trends whereas R = MPA shows slightly different relaxation of the core states due to a smaller gap between the LUMO+1 and LUMO+2 gap in its electronic structure. The S1 (HOMO → LUMO) state gives the slowest decay in all ligated clusters, while S7 has a relatively long decay. Furthermore, separate electron and hole relaxations were performed on the [Au25(SCH3)18]-1 nanocluster to understand how independent electron and hole relaxations contribute to the overall relaxation dynamics.

Coligand Effects on the Field-Induced Double Slow Magnetic Relaxation in Six-Coordinate Cobalt(II) Single-Ion Magnets (SIMs) with Positive Magnetic Anisotropy.

Two mononuclear cobalt(II) compounds of formula [Co(dmphen)2(OOCPh)]ClO4·1/2H2O·1/2CH3OH (1) and [Co(dmbipy)2(OOCPh)]ClO4 (2) (dmphen = 2,9-dimethyl-1,10-phenanthroline, dmbipy = 6,6'-dimethyl-2,2'-bipyridine and HOOCPh = benzoic acid) are prepared and magnetostructurally investigated. Each cobalt(II) ion is six-coordinate with a distorted octahedral CoN4O2 environment. The complex cations are interlinked leading to supramolecular chains (1) and pairs (2) that grow along the crystallographic c-axis with racemic mixtures of (Δ,Λ)-Co units. FIRMS allowed us to directly measure the zero-field splitting between the two lowest Kramers doublets, which led to axial anisotropy values of 58.3 cm-1 ≤ D < 60.7 cm-1 (1) and 63.8 cm-1 ≤ D < 64.1 cm-1 (2). HFEPR spectra of polycrystalline samples of 1 and 2 at low temperatures confirm the positive sign of D and provide an estimate of the E/D quotient [0.147/0.187 (1) and 0.052 (2)]. Detailed ac and dc magnetic studies reveal that 1 and 2 are new examples of field-induced single-ion magnets (SIMs) with small transversal anisotropy. CASSCF/NEVPT2 calculations support these results. Two Orbach processes or one Orbach plus a direct relaxation mechanism provide similar agreements with the nonlinear experimental Arrhenius plots at Hdc = 500 and 2500 G for 1. Two independent relaxation processes occur in 2, but in contrast to 1, an observed linear dependence of ln(τ) vs 1/T substantiates Orbach processes against the most widely proposed Raman and direct mechanisms. The analysis of each relaxation process in 2 provided values for Ea and τ0 that are very close to those found for 1, validating the predominant role of the Orbach relaxations in both compounds and, probably, also in other cobalt(II) SIMs. A mechanism based on a spin-phonon coupling is proposed to account for the SIM behavior in 1 and 2 with any Raman or direct processes being discarded.

Differential phosphorylation of the N-terminal extension regulates phytochrome B signaling.

Phytochrome B (phyB) is an excellent light quality and quantity sensor that can detect subtle changes in the light environment. The relative amounts of the biologically active photoreceptor (phyB Pfr) are determined by the light conditions and light independent thermal relaxation of Pfr into the inactive phyB Pr, termed thermal reversion. Little is known about the regulation of thermal reversion and how it affects plants' light sensitivity. In this study we identified several serine/threonine residues on the N-terminal extension (NTE) of Arabidopsis thaliana phyB that are differentially phosphorylated in response to light and temperature, and examined transgenic plants expressing nonphosphorylatable and phosphomimic phyB mutants. The NTE of phyB is essential for thermal stability of the Pfr form, and phosphorylation of S86 particularly enhances the thermal reversion rate of the phyB Pfr-Pr heterodimer in vivo. We demonstrate that S86 phosphorylation is especially critical for phyB signaling compared with phosphorylation of the more N-terminal residues. Interestingly, S86 phosphorylation is reduced in light, paralleled by a progressive Pfr stabilization under prolonged irradiation. By investigating other phytochromes (phyD and phyE) we provide evidence that acceleration of thermal reversion by phosphorylation represents a general mechanism for attenuating phytochrome signaling.

Increased inflammation, oxidative stress and a reduction in antioxidant defense enzymes in perivascular adipose tissue contribute to vascular dysfunction in type 2 diabetes

BackgroundPerivascular adipose tissue (PVAT) surrounds most large blood vessels and plays an important role in vascular homeostasis. The present study was conducted to investigate the contribution of PVAT to vascular dysfunction in a rat model of type 2 diabetes. Material and methodsSeveral in vivo parameters such as lipid profile (total cholesterol and triglyceride systemic levels), fasting glucose levels, glucose tolerance and insulin sensitivity (through glucose and insulin tolerance tests, respectively) were determined in Goto-Kakizaki (GK) diabetic rats and compared with control Wistar rats. At the vascular level, endothelial dependent and independent relaxation and contraction studies were performed in aortic rings in the absence (PVAT−) or in the presence (PVAT+) of thoracic PVAT. We also evaluated vascular oxidative stress and performed western blots, PCR and immunohistochemistry analysis of cytokines and various enzymes in PVAT. ResultsEndothelium-dependent relaxation to acetylcholine, assessed by wire myography, was impaired in GK rats and improved by the antioxidant TEMPOL and by the TLR4 inhibitor, CLI-095 suggesting an increase in oxidative stress and inflammation. In addition, vascular superoxide and peroxynitrite production was increased in the vascular wall of diabetic rats, accompanied by reduced nitric oxide bioavailability. The presence of PVAT had an anticontractile effect in response to phenylephrine in Wistar rats that was lost in GK rats. Western blot and immunohistochemistry analysis revealed that PVAT phenotype shifts, under diabetic conditions, towards a proinflammatory (with increment in CRP, CCL2, CD36), pro-oxidant (increased levels of aldose reductase, and reduced levels of antioxidant deference enzymes) and vasoconstriction state. ConclusionOur data suggest that this rat model of type 2 diabetes is associated with perivascular adipose dysfunction that contributes to oxidative stress, inflammation and endothelial dysfunction.

Glucagon-like peptide-1 increased the vascular relaxation response via AMPK/Akt signaling in diabetic mice aortas

The incretin glucagon-like peptide-1 (GLP-1) elicits direct favorable effects on the cardiovascular system. This study aimed to evaluate the acute effects of GLP-1 on improving aortic endothelial dysfunction in diabetic mice. Additionally, we examined whether GLP-1 elucidated the underlying mechanisms. Using the diabetic mouse models induced by nicotinamide and streptozotocin, we investigated the functional changes in the aorta caused by GLP-1. Organ baths were performed for vascular reactivity in isolated aortic rings, and western blotting was used for protein analysis. The diabetic aortas showed enhanced GLP-1-induced relaxation response and nitric oxide (NO) production. However, the pretreatment of GLP-1 did not significantly change the endothelial-dependent relaxation response to acetylcholine and -independent relaxation response to sodium nitroprusside. On the other hand, the GLP-1-induced relaxation response and NO production were abolished by the endothelial NO synthase inhibitor, GLP-1 receptor antagonist, Akt inhibitor, and AMP-activated protein kinase (AMPK) inhibitor. Finally, in diabetic mice, considerable increases in phosphorylation of Akt and AMPK were found in aortas stimulated with GLP-1, both of which were decreased by pretreatment with the AMPK inhibitor. GLP-1 significantly enhanced endothelial-dependent relaxation in diabetic aortas. The effect may be mediated through activation of the AMPK/Akt pathway via a GLP-1 receptor-dependent mechanism.

EX SITU HEART PERFUSION IMPROVES PRESERVATION OF ENDOTHELIAL FUNCTION IN BOTH DBD AND DCD HEARTS

Purpose Ex situ heart perfusion (ESHP) decreases ischemic time and improves myocardial preservation for transplantation. However, the effects of machine perfusion on endothelial function has not yet been determined. We aimed to compare coronary vasomotor function and endothelial activation following transplantation between hearts donated after circulatory death (DCD) and controls (beating-heart donations) preserved in cold storage or ESHP Methods DCD pigs underwent hypoxic circulatory arrest via asphyxiation followed by 15min of warm ischemia . Both DCD and control hearts were preserved with either 3h of cold storage or 4h of ESHP. Following transplantation, both groups were reperfused for 3h. We assessed coronary artery segments for endothelial-dependent (Edep) and -independent (Eind) relaxation, and sensitivity to vasospasm using a small vessel myograph. Expression of VCAM-1 , and endothelin receptors A (ETa) and B (ETb) were measured with immunoblotting Results Preservation with ESHP partially improved Edep (p=0.098) and significantly enhanced Eind (p=0.004) in controls. DCD hearts showed no differences in Edep (p=0.266), Eind (p=0.182) compared to controls when preserved in cold storage. Preservation of DCD hearts using ESHP significantly improved Edep (p Conclusion ESHP strategies have enabled DCD heart transplantation and expansion of the donor heart pool. Limiting cold ischemia by machine perfusion with a normothermic, oxygenated, and blood-based perfusate seems to limit endothelial activation and dysfunction by decreasing ischemia-reperfusion injury . This may lead to a reduced rate of early graft dysfunction and late development of allograft vasculopathy

Lattice Boltzmann models for micro-tomographic pore-spaces

The lattice Boltzmann method (LBM) is a popular numerical framework to investigate single and multiphase flow though porous media. For estimation of absolute permeability based on micro-tomographic images of a porous medium, the single-relaxation time (SRT) collision model is the most widely-used, although the multiple-relaxation-time (MRT) collision model also has recently acquired wider usage, especially for industrial applications. However, the SRT collision model and a sub-optimal choice of the MRT collision parameters can both lead to permeability predictions that depend on the relaxation parameter, τ. This parametric dependence is nonphysical for Stokes flow in porous media and also leads to much larger number of iterations required for convergence. In this paper, we performed a systematic numerical evaluation of the different sets of relaxation parameters in the D3Q19-MRT model for modeling Stokes flow in 3D microtomographic pore-spaces using the bounceback scheme. These sets of parameters are evaluated from the point of view of accuracy, convergence rate, and an ability to generate parameter-independent permeability solutions. Instead of tuning all six independent relaxation rates that are available in the MRT model, the sets that were analyzed have relaxation rates that depend on one or two independent parameters, namely τ and Λ. We tested elementary porous media at different image resolutions and a random packing of spheres at relatively high resolution. We observe that sets of certain specific relaxation parameters (Sets B, D, or E as listed in Table 2), and τ in the range τ ∈ [1.0, 1.3] can result in best overall accuracy, convergence rate, and parameter-independent permeability predictions.

Abstract P145: Notch3 Mutation is Involved in Pulmonary Vascular Dysfunction and Development of Pulmonary Arterial Hypertension

Notch3 is implicated in CADASIL and pulmonary arterial hypertension (PAH), two diseases characterized by vascular dysfunction and remodelling. Mice harbouring the CADASIL-causing Notch3 mutation (TgNotch3 R169C ) exhibit gain-of-function Notch3 signalling. As increased Notch3 signalling has been demonstrated in several PAH models, we investigated the contribution of R169C mutation to pulmonary vascular dysfunction and PAH development. Briefly, vascular reactivity was studied by wire myography using pulmonary intralobar arteries from 24-week male mice overexpressing either a WT or R169C mutant Notch3 gene. Further mice were exposed to 2 weeks of normoxia or chronic hypobaric hypoxia (10% O 2 ) before assessing PAH development by in vivo haemodynamic measurements. Notch3 effector gene Hes5, implicated in PAH development, was significantly increased in R169C but not WT lung (8.7 fold change vs WT) suggesting increased pulmonary Notch3 activation. Hypercontractility of R169C pulmonary arteries to both 5HT (Emax R169C 146 ± 10% vs WT 92 ± 6%) and ET-1 (Emax R169C 211 ± 15% vs WT 154 ± 32%) was observed; an effect attenuated by ROCK inhibitor Fasudil (5HT Emax R169C + Fas. 117% ± 20%, ET-1 R169C + Fas. 154 ± 11%) (p&lt;0.05). In addition, gene expression for Rho-guanine nucleotide exchange factors LARG and PDZ, which can activate RhoA/ROCK signalling, was upregulated in R169C lung (R169C 33.5 fold vs WT, R169C 18.7 fold vs WT respectively), with no changes to ROCK gene or protein expression. Reduced endothelial-dependent (ACh Emax WT 43 ± 3% vs R169C 36 ± 3%) and -independent relaxation (SNP Emax WT 62 ± 4% vs R169C 57 ± 4%) were also observed in R169C vessels (p&lt;0.05). Chronic hypoxia did not affect systemic haemodynamic parameters. RV systolic pressure increased in hypoxic animals vs normoxic; this was exacerbated in R169C hypoxic animals (WT hypoxic 27.46 ± 0.97 vs WT normoxic 22.76 ± 2.17mmHg, R169C hypoxic 32.18 ± 1.13 mmHg vs R169C normoxic 23.60 ± 1.44) (p&lt;0.05). R169C hypoxic animals also exhibited RV hypertrophy (RV/LV+S) (hypoxic 0.26 ± 0.033 vs normoxic 0.16 ± 0.012). These data demonstrate Notch3 R169C mutation may contribute to pulmonary vascular hypercontractility and PAH susceptibility, and highlight ROCK as a potential mechanism.

Abstract P3005: P66shc Plays a Role in Aldosterone Induced Vascular Remodeling and Dysfunction

Objective: This study sought to investigate whether aldosterone may induce vascular functional and structural alterations in part through p66shc-dependent processes. Design and method: P66shc -/- mice (14 weeks, 6 per group) and age-matched wild type (WT) control mice were treated or not with aldosterone (300 μg /kg/ day) for 4 weeks. Systolic blood pressure (SBP) was measured by tail-cuff method. Endothelium-dependent and independent relaxation was assessed by concentration-response curves to acetylcholine ± L-NAME (100 μM) and sodium nitroprusside (SNP) respectively, in mesenteric arteries pre-contracted with norepinephrine (10 μM). Media-to-lumen ratio (M/L) and cross sectional area (CSA) were evaluated in vessels as pressurized preparations. Reactive oxigen species (ROS) production was evaluated by DHE staining in aorta. Nox 1 expression was evaluated by immunoblotting. Results: SBP was similar in WT and p66shc -/- mice before treatment and was significantly increased after aldosterone treatment in both WT (192.3±4.522 vs 103.3±2.996; p&lt;0.05) and p66shc -/- mice (176.8±5.313 vs 105.3±1.542; p&lt;0.05). However SBP increase was significantly blunted in p66shc -/- mice treated with aldosterone as compared to WT treated mice (176.8±5.313 vs 192.3±4.522, p&lt;0.05). Acetylcholine-induced vasodilation was similar in WT and p66shc -/- mice and was significantly reduced only in WT after treatment. Acetylcholine induced relaxation was blunted by L-NAME in all the groups although at less extent in WT. Endothelium-independent vasodilation was similarly preserved in all the groups. M/L ratio was similar in both WT and p66shc -/- before treatment and was significantly increased by aldosterone only in WT group (p&lt;0.001). CSA was similar in all the groups. Both ROS production and NOX-1 expression were similar in WT and p66shc -/- mice and were significantly increased after treatment only in WT (+20% and +2,5 fold respectively, P&lt;0.05). Conclusions: Despite BP increase, aldosterone induced endothelial dysfunction and vascular remodeling with associated increased oxidative stress in WT but not in p66shc -/- mice. Thus, p66shc may play a role in molecular mechanisms of endothelial dysfunction and vascular remodeling in aldosterone-induced hypertension.

Abstract 052: Rho Kinase and Endoplasmic Reticulum Stress Mediate Peripheral Vascular Dysfunction in a Model of Small Vessel Disease of the Brain

CADASIL, a monogenic condition due to Notch3 mutations is a small vessel disease of the brain resulting in premature vascular dementia and stroke. CADASIL patients have cerebral vascular narrowing, progressive SMCs loss and granular osmiophilic material (GOM) deposition, however it is unclear whether peripheral arteries also exhibit dysfunction. We studied mice harbouring the CADASIL-causing Notch3 mutation (TgNotch3 R169C ), which recapitulates human CADASIL, to evaluate whether peripheral small arteries exhibit vascular dysfunction. Mesenteric arteries (MA) from male TgNotch3 R169C and wildtype (TgNotch3 WT ) mice (6 months old) were investigated. Vascular structure and function were assessed by myography. No changes were observed in blood pressure and cardiac function in CADASIL mice, measured by tail cuff and echocardiography respectively. Expression of Notch3 and target genes was augmented in MA from CADASIL mice ( Notch3 : 2.0±0.5 vs. 6.0±1.3; HeyL: 1.1±0.4 vs. 2.9±0.6, p&lt;0.01). CADASIL mice exhibited impaired endothelium-dependent (Emax 109.9±7.4 vs. 81.3±5.4) and -independent (pD 2 7.8±0.1 vs 6.8±0.3) relaxation and increased reactivity in response to AngII (Emax 33.7±6.8 vs. 72.8±4.4), phenylephrine (Emax: 70.6±7.2 vs. 92.1±4.2), and U46619 (Emax: 123.4±4.4 vs. 215.1±24.4) (p&lt;0.05 vs TgNotch3 WT ); effects attenuated by fasudil (Rho-kinase inhibitor) and 4-PBA (ER stress inhibitor) (p&lt;0.05 vs. TgNotch3 R169C ). MA from CADASIL mice also had hypotrophic remodelling. U46619-induced calcium influx was increased in VSMCs (AUC: 1.3 fold increase) from TgNotch3 R169C and gene expression of Rho GEFs (LARG: 1.1±0.1 vs. 2.1±0.3; PDZ: 0.9±0.1 vs. 2.9±0.4) was increased in MA from TgNotch3 R169C vs. TgNotch3 WT mice (p&lt;0.05). eNOS phosphorylation (Thr 495 ) and BiP expression (ER stress marker) were increased in vessels from CADASIL mice (eNOS: 1.8 fold increase; Bip: 1.7 fold increase; p&lt;0.05 vs. TgNotch3 WT ). In conclusion, our data demonstrated that the vasculopathy associated with Notch3 mutation is also present in peripheral small vessels where the interplay between Notch3, Rho-kinase and ER stress may be important. We identify potential new therapeutic targets in CADASIL, for which there are no disease-specific treatments.

Relaxation phenomena and magnetocaloric effect in the dynamic spin ice Pr2Sn2O7

Spin relaxation and a magnetocaloric effect in a dipolar spin ice Pr2Sn2O7 were investigated by magnetization and alternating susceptibility measurements in Kelvin temperature range and magnetic fields exceeding the estimated value of the saturation field Bsat. An analysis of AC susceptibility revealed existence of two relaxation channels. The calculation of energy spectrum of a single tetrahedron suggests that a fast temperature independent relaxation may be associated with a cross-tunneling process, in which four spins in one tetrahedron are involved. In contrast, a slow relaxation is attributed to a phonon-bottleneck effect. A particle-size analysis identifies a phonon scattering in grain boundaries as a governing mechanism of the phonon bottleneck. Systematic measurements of the magnetization were performed to study magnetocaloric effect in Pr2Sn2O7. A complex analysis of the isothermal change of magnetic entropy ΔSm revealed a dominating effect of magnetocrystalline anisotropy on a magnetocaloric response in an Ising-spin system with a high level of geometrical frustration. The origin of the observed behavior is discussed.

P66SHC KNOCK-OUT PREVENTS ALDOSTERONE INDUCED VASCULAR REMODELING AND DYSFUNCTION

Objective: Recent evidences suggested that aldosterone may induce the activation of p66shc in vascular smooth muscle cells. This in vivo study sought to investigate whether aldosterone may induce functional and structural alterations in part through p66shc-dependent manner. Design and method: P66shc−/− mice (14 weeks, 6 per group) and age-matched wild type (WT) control mice were treated or not with aldosterone (300 μg /kg/ day) for 4 weeks. Systolic blood pressure (SBP) was measured by tail-cuff method. Endothelium-dependent and independent relaxations were assessed by concentration-response curves to acetylcholine (1 nM to 100 μM) ± L-NAME (100 μM) and sodium nitroprusside (SNP) (10 nM to 1 mM) respectively, in mesenteric arteries pre-contracted with norepinephrine (10 μM). Media-to-lumen ratio (M/L) and cross sectional area (CSA) were evaluated on pressurized preparations. Results: SBP was similar in WT and p66shc−/− mice before treatment and was significantly increased after 4 weeks of aldosterone treatment in both WT (192.3 ± 4.522 vs 103.3 ± 2.996; p < 0.05) and p66shc−/− mice (176.8 ± 5.313 vs 105.3 ± 1.542; p < 0.05). However the SBP increase was significantly blunted in p66shc−/− mice treated with aldosterone as compared to WT treated mice (176.8 ± 5.313 vs 192.3 ± 4.522, p < 0.05). Acetylcholine-induced vasodilation was similar in WT and p66shc−/− mice and was significantly reduced only in WT after aldosterone treatment. Acetylcholine induced relaxation was blunted by L-NAME in all the groups although at less extent in WT. This suggests that aldosterone may induce endothelial dysfunction only in this group. Endothelium-independent vasodilation was similarly preserved in all the groups. M/L ratio was similar in both WT and p66shc−/− before treatment and was significantly increased by aldosterone only in WT group (p < 0.001). CSA was similar in all the groups. Conclusions: Despite BP increase, aldosterone induced endothelial dysfunction and vascular remodelling only in WT but not in p66shc−/− mice. Thus, p66shc may be involved in the mechanisms of endothelial dysfunction and vascular remodelling induced by aldosterone in the cardiovascular system.

Cryptostrobin and catechin isolated from Eugenia mattosii D. Legrand leaves induce endothelium-dependent and independent relaxation in spontaneously hypertensive rat aorta.

Considering the therapeutic potential of phenolic compounds, the purpose of the present study was to investigate the mechanisms involved in the relaxation induced by cryptostrobin and catechin, isolated from Eugenia mattosii D. Legrand leaves, in the aorta of spontaneously hypertensive rats (SHR). The thoracic aorta was isolated from SHR and kept in the organ bath system by recording contractile or relaxant responses. The addition of cumulative concentrations of cryptostrobin and catechin induced endothelium-dependent and-independent relaxation in aorta rings from SHR, as well as both compounds were effective in reducing phenylephrine-induced contraction. Pretreatment of aortic rings with Nω-nitro-l-arginine methylester (L-NAME, an inhibitor of nitric oxide synthase) or 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of soluble guanylate cyclase), resulted in a significant change of relaxant effect induced by catechin, and a slight influence on cryptostrobin-induced relaxation. Muscarinic receptor and potassium channels are involved in catechin-induced relaxation as assessed using atropine (a muscarinic receptor antagonist), tetraethylammonium (a non-selective K+ channel blocker) and glibenclamide (an ATP-sensitive K+ channel blocker). Conversely, cryptostrobin, but not catechin, blunted the contraction induced by the addition of phenylephrine in a calcium-free solution. Besides that, cryptostrobin attenuated the contraction of rat aorta rings induced by internal Ca2+ release and external Ca2+ influx. These findings indicated that cryptostrobin and catechin alter vascular smooth muscle reactivity, and this effect may be involved, at least in part, by enhancing the endothelium NO/cGMP pathway and potassium channels activation. In addition, cryptostrobin reduced the phenylephrine, KCl and CaCl2-induced contractions in a calcium-free solution.

A physically based visco-hyperelastic constitutive model for soft materials

Viscoelasticity is an essential mechanical property of soft materials. Many constitutive models have been proposed to capture this mechanical behavior, but few physically based models were developed due to the challenge to incorporate the micro-mechanisms of viscoelasticity into the formulation. In a previous paper, we proposed a constitutive model to characterize the hyperelastic response under different deformation states with only three parameters (Xiang et al., 2018). Based on the above work and the tube theory of polymer dynamics, we develop herein a physically based viscoelastic constitutive model in this paper. We start from a new microscopic picture at the molecular chain scale, and decompose the stress into a hyperelastic part which comes from the elastic ground network (crosslinked network and entanglement network), and a viscous part which is originated from free chains. Utilizing the same scheme from the previous work (Xiang et al., 2018), we are able to decompose the free chains into two parts, the untangled crosslinked network and the entanglement network. The contour length relaxation and disentanglement from the free chain's networks give the viscous behavior. We test VHB 4910 to validate our model. The model is applied to the mechanical behavior of the soft digital materials (DMs). In addition, the model can explain the Shore A index independent relaxation time of DMs and the asymmetry of viscoelasticity under loading and unloading. This viscoelastic model can be used to predict the mechanical response of soft materials.