Endothelial Vasorelaxation Research Articles

Time-restricted feeding improves aortic endothelial relaxation by enhancing mitochondrial function and attenuating oxidative stress in aged mice

Age-related endothelial dysfunction is a pivotal factor in the development of cardiovascular diseases, stemming, at least in part, from mitochondrial dysfunction and a consequential increase in oxidative stress. These alterations are central to the decline in vascular health seen with aging, underscoring the urgent need for interventions capable of restoring endothelial function for preventing cardiovascular diseases. Dietary interventions, notably time-restricted feeding (TRF), have been identified for their anti-aging effects on mitochondria, offering protection against age-associated declines in skeletal muscle and other organs. Motivated by these findings, our study aimed to investigate whether TRF could similarly exert protective effects on endothelial health in the vasculature, enhancing mitochondrial function and reducing oxidative stress. To explore this, 12-month-old C57BL/6 mice were placed on a TRF diet, with food access limited to a 6-h window daily for 12 months. For comparison, we included groups of young mice and age-matched controls with unrestricted feeding. We evaluated the impact of TRF on endothelial function by measuring acetylcholine-induced vasorelaxation of the aorta. Mitochondrial health was assessed using fluororespirometry, and vascular reactive oxygen species (ROS) production was quantified with the redox-sensitive dye dihydroethidium. We also quantified 4-hydroxynonenal (4-HNE) levels, a stable marker of lipid peroxidation, in the aorta using ELISA. Our findings demonstrated that aged mice on a standard diet exhibited significant impairments in aortic endothelial relaxation and mitochondrial function, associated with elevated vascular oxidative stress. Remarkably, the TRF regimen led to substantial improvements in these parameters, indicating enhanced endothelial vasorelaxation, better mitochondrial function, and reduced oxidative stress in the aortas of aged mice. This investigation establishes a vital foundation, paving the way for subsequent clinical research aimed at exploring the cardiovascular protective benefits of intermittent fasting.

The Inhibitory Effect of Trimethylamine (TMA), an Intestinal Bacterial Metabolite, on Endothelial Vasorelaxation in Rat Mesenteric Artery

The effect of the gut microbiota metabolite trimethylamine-(TMA) in isolated vessels is unknown yet. Previously TMAO, the hepatic oxidation product of TMA, at 3 mM has been shown to inhibit endothelium-dependent vasorelaxations of isolated arteries only after 24-hour-interactions. In this study, the effects of TMA (at 1 mM) on endothelium-dependent relaxations with acute (1 or 4 hours) and longer (24 hours) incubation periods were evaluated in superior mesenteric arteries of rat. Acute exposure to TMA of 1 hour significantly inhibited acetylcholine-stimulated endothelium-derived hyperpolarizing (EDH) type relaxations, and this inhibition gradually intensified as the incubation period was prolonged to 4, and 24 hours. The area under the curves (AUCs) of the relaxation-response curves after 1 and 24 hours of TMA incubation were found significantly different compared to each other, whereas similar AUC values were obtained after 4, and 24 hours of incubations. Contractile responses to phenylephrine, and nitric oxide (NO)-mediated relaxations of acetylcholine were similar in arteries before and after pretreatment with TMA for 24 hours. These data indicate that TMA selectively inhibits EDH-type relaxations in rat isolated mesenteric arteries. Although the inhibitory effect of TMA intensifies over time, it appears to be more pronounced during acute incubation periods. The findings strengthen the evidence that TMA is a more toxic metabolite on vascular tone than TMAO.

Chloroquine and cinchonine affect rat vascular smooth muscle tonus through calcium channels - in silico and in vitro approaches.

In the present study, two structurally similar alkaloids from trees of Cinchona genus, chloroquine and cinchonine, were examined for their vasorelaxant effects in amodel of phenylephrine-induced smooth muscle contractions. Potential mechanisms of action associated with endothelial vasorelaxant compounds, voltage-gated Ca2+ channels (LTCCs), and inositol triphosphate receptors were examined in isolated rat aortic rings. Also, an in silico approach was used to predict the activity of the two test compounds. Experimental results revealed that both chloroquine and cinchonine significantly decrease phenylephrine-induced smooth muscle contractions, although to adifferent extent. Evaluated mechanisms of action indicate that endothelium is not involved in the vasorelaxant action of the two tested alkaloids. On the other hand, voltage-gated Ca2+ channels were found to be the dominant way of action associated with the vasorelaxant action of chloroquine and cinchonine. Finally, IP3R is found to have only asmall impact on the observed activity of the tested compounds. Molecular docking studies predicted that chloroquine possesses asignificant activity toward asuitable model of LTCCs, while cinchonine does not. The results of the present study point to the fact that great caution should be paid while administering chloroquine to vulnerable patients, especially those with cardiovascular disorders (Tab. 3, Fig. 3, Ref. 28).

Therapeutic modulation of epigenetic readers rescues obesity-induced vascular dysfunction: role of endothelium and perivascular adipose tissue

Abstract Background/Introduction Bromodomain and extraterminal proteins (BET), particularly BRD4, are epigenetic readers modulating transcriptional programs implicated in inflammation, cancer and renal disease. Therapeutic potential of BRD4 modulation in cardiometabolic disorders remains elusive. Purpose We investigate BRD4-related transcriptional programs and therapeutic modulation in mouse and human models of cardiometabolic disease. Methods Small arteries (100-300 μM) from healthy subjects (n=13) and patients with obesity (n=13) were dissected from visceral fat biopsies collected during elective laparoscopy and mounted on a pressurised myograph to assess the ex-vivo effects of BRD4 inhibition on vascular function. Vasorelaxation to acetylcholine and acetylcholine+L-NAME were evaluated at baseline and after incubation with the BRD4 inhibitor RVX-208. Anti-IL-1β, anti-IL-6 receptor and anti-TNF-α were also employed to assess the relative contribution of distinct BRD4-related pathways. Vasorelaxation was performed in the presence or in the absence of perivascular adipose tissue (PVAT). An experimental mouse model of cardiometabolic disease (high-fat diet+L-NAME supplementation) was orally administered RVX-208 (150 mg/kg) or vehicle for 10 days (n=6 each group) to test in vivo effect of chronic BRD4 inhibition on endothelial and PVAT functions. Vascular and PVAT levels of cytosolic and mitochondrial ROS and nitric oxide were assessed by confocal microscopy; protein and gene expression was measured by Western blot and qPCR. Transcriptional changes upon BRD4 inhibition were investigated by a custom PCR array. Results In patients with obesity, endothelial-dependent vasorelaxation and vascular nitric oxide availability were impaired. ROS levels, as well as TNF-α, IL-1β and IL-6, were increased in the vessel wall and PVAT. In these subjects, RVX-208 substantially attenuated ex-vivo vascular dysfunction. The effect was more pronounced in vessels with intact PVAT, suggesting a restoration of the PVAT anti-contractile phenotype (Figure 1). Distinct ex-vivo modulation of well-established inflammatory pathways showed that the effect observed with BRD4 inhibition was stronger than with anti-IL-1β, anti-IL-6 receptor and anti-TNF-α. In vivo, chronic treatment with RVX-208 restored endothelial vasorelaxation by rescuing PVAT dysfunction. Gene expression profiling in PVAT from cardiometabolic mice unveiled hexokinase-2 (HK2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - as the top downregulated gene by RVX-208 treatment (Figure 2). Conclusions Therapeutic modulation of BRD4 restores cardiometabolic-related vascular dysfunction and PVAT anti-contractile properties. These effects are explained by a suppression of PVAT inflammation (IL-1β, IL-6 and TNF-α), likely driven by HK2 activation. BET inhibitors might represent promising epi-drugs for the prevention and treatment of vascular dysfunction, a hallmark of cardiometabolic disease.

Tcell derived HIV proteins promote oxidative stress, endothelial dysfunction and hypertension via Interleukin 1alpha-mediated mechanisms in mice

While antiretroviral therapy (ART) has extended the life of people living with HIV (PLWH), they experience accelerated development of hypertension and cardiovascular disease (CVD) compared to seronegative individuals. However, the etiology and individual contributions of ART and repressed viral infection remain ill-defined. While ART prevents viral replication, viral reservoirs persist, and viral proteins remain in the circulation leading us to hypothesize that Tcell-derived HIV proteins contribute to endothelial function and hypertension. We utilized a Tg26 transgenic mouse model ubiquitously expressing 7/9 viral proteins. These mice display increased blood pressure (BP) and enhanced neurogenic control of BP (BP response to ganglionic blockade) as well as impaired endothelial vasorelaxation in aorta and mesenteric arteries in both sexes. Bone marrow transplant (BMT) from Tg26 mice into WT (Tg26->WT) increased systolic BP and impaired endothelium dependent relaxation in conduit and resistance vessels (P<0.05), a precursor to hypertension. To identify the immune cell subtype responsible for the CVD, thoracic aortas from WT male and female mice were co-cultured with either WT or Tg26 primary CD3+ Tcells. Vessels exposed to Tg26 Tcells displayed impaired endothelial relaxation versus WT (P<0.05). Vessels incubated in Tg26 CD4+ Tcells, the main target of HIV, also yielded endothelial dysfunction and inhibition of Tcell activation via Abatacept treatment in intact Tg26 mice lowered BP, restored vasorelaxation and reduced sympatho-activation to WT levels in Tg26 mice (P<0.05). A cytokine panel revealed increased production of interleukin 1a (IL-1a) in Tg26 CD3+ T cells (P<0.05). Similarly, Tg26->WT BMT increased plasma IL-1a levels (P<0.1), while Abatacept decreased plasma IL-1a levels in intact Tg26 mice (P<0.05). Treatment with anti-IL-1a antibody abolished Tcell-mediated endothelial dysfunction and primary human microvascular endothelial cells (hMVEC) treated with IL-1a for 24 hours increased ROS mediated DNA damage (8-OHdG), leading us to investigate the source of vascular ROS. Quantitative RT-PCR of the IL-1a exposed hMVEC displayed increases in NADPH oxidase NOX1, NOXO1, and NOX4 while BMT aortas had increased in NOX1, NOXO1, and NOXA1 and inhibition of NOX1 via GKT771 ex vivo restored vasorelaxation. Additionally, anti-IL-1a restored vasorelaxion in intact Tg26 mice. Ultimately, we found that Tcells expressing viral proteins promote the production of IL-1a, increasing endothelial NOX1 and ROS production, leading to endothelial dysfunction, which in addition to sympatho-activation promote hypertension. 1R01HL147639, 21PRE830396, 19EIA34760167 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.

763 ACTIVATION OF SIRT1 ATTENUATES VASCULAR DYSFUNCTION AND THROMBOSIS IN MTHFR DEFICIENCY

Abstract Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/-) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/- mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers.

Protection against angiotensin II-induced hypertension in mice lacking sphingosine kinase 1 in vascular smooth muscle cells

Abstract Background Sphingosine-1-phosphate (S1P), a lipid mediator produced by sphingosine kinases (Sphks), significantly contributes to the maintenance of cardiovascular homeostasis. Mice lacking Sphk1 globally are protected against angiotensin II-induced hypertension, yet, due to pleiotropic action of S1P, it has been challenging to identify a specific cell type responsible for this protection. Purpose The aim of this study was to investigate the role of Sphk1 expressed in vascular smooth muscle cells (VSMCs) in the development of angiotensin II (AngII)-induced hypertension in mice. Methods Male, 12-week-old C57BL/6J wild type (WT) or VSMC-specific, Cre-mediated, Sphk1 knockout (Sphk1VSMC−/−) mice underwent either Sham or AngII (490 ng/min/kg s.c.) treatment for 2 weeks, using a surgically implanted osmotic minipump. Blood pressure was measured by the tail-cuff method. Vascular structure and function were examined ex vivo using wire (vasoconstrictor/vasorelaxant responses) and pressure myography (myogenic tone, structural and elasticity parameters). Gene set enrichment analysis (GSEA) was performed using transcriptomic profiles of the isolated mesenteric arteries (MA, RNASeq). Expression of proteins was analyzed by Western blot. Results Deletion of Sphk1 in VSMCs resulted in 5x and 40x lower expression of Sphk1 mRNA in MA and aorta compared to WT mice respectively. A 2-week infusion of AngII resulted in a development of higher systolic blood pressure (SBP) in WT mice compared to Sphk1VSMC−/− mice (mean SBP ± SEM: 168±4.3 mmHg vs. 132±4.4 mmHg respectively, p<0.05). Studies of ex vivo mesenteric artery (MA) function revealed diminished both, depolarization-induced (in response to KCl), as well as G protein-coupled receptor-elicited (in response to phenylephrine and thromboxane A2 analogue) contraction of MA derived from hypertensive Sphk1VSMC−/− mice compared to WT mice. In the same time, significantly impaired endothelial vasorelaxation was observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice. Moreover, MA from Sphk1VSMC−/− mice were characterized by the significantly reduced myogenic response evoked by intravascular pressure elevation (maximal myogenic response ± SEM: 19.7±1.8% for WT normotensive, 26.2±1.8% for WT AngII vs. 19.0±2.5% for Sphk1VSMC−/− AngII mice), and an increased stiffness compared to WT mice. GSEA found that the lack of Sphk1 gene in VSMCs significantly downregulated molecular pathways related to smooth muscle contraction. In line with transcriptomic studies, significantly reduced level of the Rho-associated protein kinase (Rock) has been observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice using Western blot. Conclusions This study revealed that mice lacking Sphk1 gene in VSMCs are protected against the development of AngII-induced hypertension likely due to the lowered myogenic tone in resistance arteries, which may be mediated by autocrine effects of Sphk1 on Rock proteins. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The National Science Centre, Poland

SIRT1 pharmacological activation rescues vascular dysfunction and prevents thrombosis in MTHFR deficiency

Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here, we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/–) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/– mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers.

Puerarin Alleviates Vascular Cognitive Impairment in Vascular Dementia Rats.

Cerebral ischemia triggers vascular dementia (VD), which is characterized by memory loss, cognitive deficits, and vascular injury in the brain. Puerarin (Pur) represents the major isoflavone glycoside of Radix Puerariae, with verified neuroprotective activity and cardiovascular protective effects. However, whether Pur ameliorates cognitive impairment and vascular injury in rats with permanent occlusion of bilateral common carotid arteries (BCCAO) remains unknown. This work aimed to assess Pur’s effects on BCCAO-induced VD and to dissect the underlying mechanisms, especially examining the function of transient receptor potential melastatin-related 2 (TRPM2) in alleviating cognitive deficits and vascular injuries. Rats with BCCAO developed VD. Pur (50, 100, and 150 mg/kg) dose-dependently attenuated the pathological changes, increased synaptic structural plasticity in the dorsal CA1 hippocampal region and decreased oxidative stress, which eventually reduced cognitive impairment and vascular injury in BCCAO rats. Notably, Pur-improved neuronal cell loss, synaptic structural plasticity, and endothelial vasorelaxation function might be mediated by the reactive oxygen species (ROS)-dependent TRPM2/NMDAR pathway, evidenced by decreased levels of ROS, malondialdehyde (MDA), Bax, Bax/Bcl2, and TRPM2, and increased levels of superoxide dismutase (SOD), Bcl2, and NR2A. In conclusion, Pur has therapeutic potential for VD, alleviating neuronal cell apoptosis and vascular injury, which may be related to the ROS-dependent TRPM2/NMDAR pathway.

Tetrahydroxystilbene glycoside improves endothelial dysfunction and hypertension in obese rats: The role of omentin-1

RationaleHypertension in obesity has become a major threat for public health. Omentin-1, a novel adipokine, is down-regulated in obesity. Tetrahydroxystilbene glycoside (TSG) is the main ingredient extracted from Polygonum multiflorum Thunb (PMT), a traditional Chinese medicinal herb safely used for protecting cardiovascular systems over bimillennium. This study aims to examine (i) the impact of omentin-1 downregulation on obesity-related hypertension in murine models and the underlying mechanisms; (ii) whether tetrahydroxystilbene glycoside (TSG) improved endothelial dysfunction and obesity-associated hypertension via the increase of omentin-1. Methods(TSG-treated) male Zucker diabetic fatty (ZDF) rats and omentin-1 knockout (OMT-/-) mice were used. In vitro, human umbilical vein endothelial cells (HUVECs) and mature adipocytes differentiated from human visceral preadipocyte (HPA-v) were maintained in a co-culture system. ResultsTSG was the main active component of PMT reducing systolic blood pressure and improving endothelial vasodilation. Fortnight-TSG treatment (100 mg/kg/day) increased serum omentin-1 level, also activated Akt/eNOS signaling and enhanced NO bioactivity; decreased expression of NOX2 and p22phox, suppressed production of superoxide and peroxynitrite anion. OMT-/- mice showed elevated blood pressure and impaired endothelial vasorelaxation, whereas hypotensive effect of TSG was blunted. In co-culture system, TSG incubation promoted binding of peroxisome proliferator-activated receptor-γ (PPAR-γ) and Itln-1 promoter in adipocytes, activated Akt/eNOS/NO signaling and attenuated oxidative/nitrative stress in HUVECs. Suppression of Itln-1 with siRNA significantly blocked the protective effect of TSG in vitro. ConclusionsDown-regulation of omentin-1 induces endothelial dysfunction and hypertension in obesity. TSG treatment (at least partially) increases omentin-1 via promoting binding of PPAR-γ and Itln-1 promoter in adipose tissues, subsequently exerts protective effects on endothelial function via activating Akt/eNOS/NO signaling and attenuating oxidative/nitrative stress. These results suggest that TSG could be developed as a promising anti-hypertension agent that protects against endothelial dysfunction and obesity-associated cardiovascular diseases.

Effects of combined exercise training and Chlorella intake on vasorelaxation mediated by nitric oxide in aged mice.

Chronic Chlorella intake and aerobic exercise training reduce arterial stiffness and increase circulating nitric oxide (NO) levels, which has beneficial effects. This study aimed to clarify the combined aortic NO-mediated effects of chronic Chlorella intake and aerobic exercise training on endothelial vasorelaxation in aged mice. In this study, 38-week-old male senescence-accelerated mouse prone 1 (SAMP1) mice were divided into aged sedentary control (Con), aerobic exercise training (AT; voluntary wheel running for 12 weeks), Chlorella intake (CH; 0.5% Chlorella powder in normal diet), and AT and CH combined (AT+CH) groups. Endothelium-dependent vasorelaxation by addition of acetylcholine to the isolated mouse aortic rings was significantly higher in the AT, CH, and AT+CH groups than in the Con group; a significantly greater effect was seen in the AT+CH group than in the AT and CH groups. Similarly, plasma and arterial nitrite/nitrate levels and arterial endothelial NO synthase phosphorylation were significantly higher in the AT, CH, and AT+CH groups than in the Con group; the AT+CH group had higher values than the AT and CH groups. Thus, chronic Chlorella intake combined with aerobic exercise training had pronounced effects on endothelial vasorelaxation in aged mice via an additive increase in arterial NO production. Novelty: Endothelium-dependent vasorelaxation was improved by Chlorella intake and exercise. Chlorella intake and exercise increased arterial Akt/eNOS/NO signaling. This combination approach further improved vasorelaxation via arterial NO production.

Empagliflozin improves endothelial and cardiomyocyte functionin human heart failure with preserved ejection fraction via reduced pro-inflammatory-oxidative pathways and protein kinase Gα oxidation.

Sodium-glucose-cotransporter-2 inhibitors showed favourable cardiovascular outcomes, but the underlying mechanisms are still elusive. This study investigated the mechanisms of empagliflozin in human and murine heart failure with preserved ejection fraction (HFpEF). The acute mechanisms of empagliflozin were investigated in human myocardium from patients with HFpEF and murine ZDF obese rats, which were treated in vivo. As shown with immunoblots and ELISA, empagliflozin significantly suppressed increased levels of ICAM-1, VCAM-1, TNF-α, and IL-6 in human and murine HFpEF myocardium and attenuated pathological oxidative parameters (H2O2, 3-nitrotyrosine, GSH, lipid peroxide) in both cardiomyocyte cytosol and mitochondria in addition to improved endothelial vasorelaxation. In HFpEF, we found higher oxidative stress-dependent activation of eNOS leading to PKGIα oxidation. Interestingly, immunofluorescence imaging and electron microscopy revealed that oxidized PKG1α in HFpEF appeared as dimers/polymers localized to the outer-membrane of the cardiomyocyte. Empagliflozin reduced oxidative stress/eNOS-dependent PKGIα oxidation and polymerization resulting in a higher fraction of PKGIα monomers, which translocated back to the cytosol. Consequently, diminished NO levels, sGC activity, cGMP concentration, and PKGIα activity in HFpEF increased upon empagliflozin leading to improved phosphorylation of myofilament proteins. In skinned HFpEF cardiomyocytes, empagliflozin improved cardiomyocyte stiffness in an anti-oxidative/PKGIα-dependent manner. Monovariate linear regression analysis confirmed the correlation of oxidative stress and PKGIα polymerization with increased cardiomyocyte stiffness and diastolic dysfunction of the HFpEF patients. Empagliflozin reduces inflammatory and oxidative stress in HFpEF and thereby improves the NO-sGC-cGMP-cascade and PKGIα activity via reduced PKGIα oxidation and polymerization leading to less pathological cardiomyocyte stiffness.

Development of Chitosan/Mannitol Microparticles as Delivery System for the Oral Administration of a Spirulina Bioactive Peptide Extract.

Spirulina platensis contains several compounds showing nutritional and therapeutic benefits. Recently, a series of peptides able to reduce the blood pressure level and to enhance the endothelial vasorelaxation was isolated from the hydrolyzed highly water-soluble Spirulina extract (HSE). However, HSE shows critical organoleptic characteristics also having poor intestinal permeability, limiting absorption when orally delivered. This research aims to overcome the critical issues through the encapsulation of HSE in Chitosan/Mannitol—(CM)-based microparticles by spray drying. The produced powders (CM-HSE) showed good process yield (≈70%) and encapsulation efficiency (≈100%) also having good derived flow properties as well as stability up to six months storage. The microparticles constituting the spray-dried powder resulted in an amorphous micrometric state (d50 ≈ 14 µm) able to retain dark colour and unpleasant smell of raw HSE. Moreover, the in vitro permeation study by Franz cell indicated that the engineered microparticles are able to enhance the permeation of HSE through an intestinal biomimetic barrier (551.13 μg/cm2 CM-HSE vs. 315.46 μg/cm2 HSE at 270 min).

Morinda citrifolia (Noni) fruit juice promotes vascular endothelium function in hypertension via glucagon-like peptide-1 receptor-CaMKKβ-AMPK-eNOS pathway.

Morinda citrifolia (Noni) is extensively used in herbal remedies to prevent and treat various diseases, including hypertension. The purpose of this study was to investigate the vascular effects of noni fruit juice and characterize the upstream signaling pathways. We measured the systolic blood pressure, diastolic blood pressure, 24-hr urinary nitric oxide (NO) metabolite excretion, bodyweight (BW), and urine examination in SHR.Cg-Leprcp/NDmcr (SHR/cp) rats after 6 weeks noni juice (15 ml/kg) treatment. Noni juice significantly decreased blood pressure and 24-hr urinary NO metabolite without change of BW or urine volume. Furthermore, the noni juice extract (NJE) promoted endothelial vasorelaxation in rat aorta rings and NO product through an increase in phosphorylation of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVECs). NJE might act on a glucagon like peptide-1 receptor (GLP-1R) via Ca2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ)-AMPK signaling with pretreatment of their inhibitors or antagonist in HUVECs. Deacetylasperulosidic acid (DAA) was an active compound in noni juice to improve NO release through same pathway in HUVECs. These results suggested that noni is a novel dietary plant that probably regulates GLP-1R-induced CaMKKβ-AMPK-eNOS pathway to improve endothelium-dependent relaxation, thus reduce the blood pressure probably via one of its responsible ingredient DAA.

Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

The use of cardiopulmonary bypass (CPB) results in the activation of leukocytes, release of neutrophil extracellular traps (NETs) and severe inflammation. We hypothesize that targeting of circulating cell-free DNA (cfDNA) by DNases might represent a feasible therapeutic strategy to limit CPB-associated side effects. Male Wistar rats (n = 24) underwent CPB with deep hypothermic circulatory arrest (DHCA) and were divided into 3 groups: control (group 1), one i.v. bolus DNase I before CPB start (group 2) and a second DNase I dose before reperfusion (group 3). We found a positive correlation between plasma cfDNA/NETs levels and compromised endothelial vasorelaxation after CPB. DNase I administration significantly diminished plasma cfDNA/NETs levels. Further, a dose-dependent improvement in endothelial function accompanied by significant reduction of circulating intercellular adhesion molecule (ICAM)-1 was observed. Rats of group 3 had significantly reduced plasma IL-6 levels and downregulated expression of adhesion molecules resulting in impaired leukocyte extravasation and reduced MPO activity in lungs. Mechanistically, digestion of NETs by DNase I significantly diminished NETs-dependent upregulation of adhesion molecules in human endothelial cells. Altogether, systemic DNase I administration during CPB efficiently reduced cfDNA/NETs-mediated endothelial dysfunction and inflammation and might represents a promising therapeutic strategy for clinical practice.

Endothelial mTORC1 signaling is required for vascular endothelial function

The mechanistic target of rapamycin complex 1 (mTORC1) that regulates cellular gene and protein programming through its downstream signaling components the S6‐kinase and the ribosomal S6 protein serves as an intracellular energy sensor. Emerging evidence points to mTORC1 signaling as an important regulator of cardiovascular function with implications in hypertension. In this study, we tested the hypothesis that endothelial mTORC1 signaling is critically involved in the regulation of vascular endothelial function and that dysregulation of this pathway contributes to endothelial dysfunction. To test this, we generated mice bearing endothelial‐specific mTORC1 disruption by deleting the gene that encodes Raptor, an essential subunit of the complex. For this, we crossed tamoxifen‐inducible endothelial‐specific Cre mice (TekCreERT2) with mice harboring floxed alleles of the Raptor gene. Upon tamoxifen treatment, TekCreERT2/RaptorF/F mice exhibited ~60% reduction in total Raptor expression in endothelial cells (1.0±0.1 vs 0.4±0.1 AU; p<0.05) with no change in Raptor expression in smooth muscle cells (0.8±0.1 vs 1.0±0.1 AU). Tamoxifen treated TekCreERT2/RaptorF/F mice displayed reduced acetylcholine‐mediated endothelial (Max relax: 61±7 vs 34±11%; p<0.05) but not SNP‐induced smooth muscle vasorelaxation responses (Max relax: 84±2 vs 82±3%) in the aorta compared to tamoxifen‐treated wildtype littermates. To demonstrate the importance of mTORC1 signaling in this reduced acetylcholine‐mediated endothelial response in the aorta, we infected TekCreERT2/RaptorF/F aortic rings with an adenoviral S6‐kinase constitutively active construct (Ad‐S6KCA; 24hrs) to restore mTORC1 signaling. Infection with Ad‐S6KCA improved acetylcholine‐mediated endothelial vasorelaxation responses approaching wildtype control levels (Max relax: 54±6 vs 31±10%; p<0.05). We next assessed the effect of heterozygous deletion of endothelial Raptor in mice (TekCreERT2/RaptorF/+). Aortic rings from TekCreERT2/RaptorF/+ displayed intact acetylcholine‐induced vasorelaxation responses compared to tamoxifen‐treated wildtype controls (Max relax: 50±3 vs 49±8%). We next challenged aortic rings from tamoxifen treated TekCreERT2/RaptorF/+ mice with angiotensin II (100nM; 24hrs) to induce endothelial dysfunction. Angiotensin II incubation reduced acetylcholine‐mediated vasorelaxation responses of aortic rings from wildtype mice compared to vehicle (PBS) (Max relax: 57±4 vs 3±8%; p<0.05). Interestingly, aortic rings of TekCreERT2/RaptorF/+ mice treated with tamoxifen were partially protected from the endothelial dysfunction evoked by angiotensin II (Max relax: 3±8 vs 36±9%; p<0.05) indicating that mTORC1 signaling is critical for the vascular effects of angiotensin II. Our data indicate that endothelial mTORC1 signaling is required for the regulation of endothelial function and highlight the need for coordinated balance of mTORC1 signaling for endothelial homeostasis. We speculate that the endothelial mTORC1 signaling pathway may represent a novel target for cardiovascular risks associated with endothelial dysfunction such as hypertension.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel Potent Decameric Peptide of Spirulina platensis Reduces Blood Pressure Levels Through a PI3K/AKT/eNOS-Dependent Mechanism.

Considered as a superfood of the future, Spirulina platensis matrix has been extensively used because of its beneficial effect on the management of cardiovascular diseases. However, its nutraceutical properties, bioactive compounds, and molecular mechanisms are unknown. Here, we demonstrate that S platensis matrix processed in vitro by simulated gastrointestinal digestion induces direct endothelial nitric oxide (NO)-mediated vasorelaxation of resistance vessels in mice. To gain insight into the bioactive compounds responsible for this effect, we used a complex multistep peptidomic approach to fractionate the crude digest: of the 5 peptide fractions identified (A-E), only fraction E evoked vasorelaxation. High-resolution mass spectrometry-based screening revealed in E the presence of 4 main peptides (SP3-SP6 [spirulina peptides]), of which only SP6 (GIVAGDVTPI) exerted direct endothelium-dependent vasodilation of ex vivo vessels, an effect occurring via a PI3K (phosphoinositide-3-kinase)/AKT (serine/threonine kinase Akt) pathway converging on NO release. In vivo, administration of SP6 evoked a significant hemodynamic effect, reducing blood pressure, an action absent in eNOS (endothelial NO synthase)-deficient mice. Of note, although lower doses of SP6 had no hemodynamic effects, it still enhanced endothelial NO vasorelaxation. Finally, in an experimental model of arterial hypertension, SP6 exerted an antihypertensive effect, improving endothelial vasorelaxation associated with enhanced serum nitrite levels. Based on our results, this novel decameric peptide may extend the possible fields of application for spirulina-derived peptides and could be developed into a promising nonpharmacological approach for the containment of pathologies associated with vascular NO misregulation.

Curcumin vasorelaxation in uterine artery of goat (Capra hircus) is mediated by differential activation of nitric oxide, prostaglandin I2, soluble guanylyl cyclase, and gap junction communication

Background: Curcumin is a principal active constituent of Curcuma longa and has potential therapeutic application in various disease states. In this study, we investigated the mechanism of vasorelaxatory effects of curcumin in middle uterine artery (MUA) of both pregnant (P) and nonpregnant (NP) Capra hircus (Ch). Materials and Methods: The middle uterine arterial rings (MUA) were mounted in an automatic organ bath attached to a PowerLab data acquisition system. We analyzed the effect of curcumin on signaling mediators of endothelium-derived hyperpolarizing factor: nitric oxide (NO), soluble guanylyl cyclase (sGC), prostaglandin I2(PGI2), and myoendothelial gap junctions (MEGJs). Results:The maximal vasorelaxation response to curcumin (1 ρM–100 μM) induced in phenylephrine- precontracted endothelium intact and denuded MUA rings were 42.58%, 25.12% in NP and 55.49%, 12.66%, in P Ch. In the presence of 1H-(1,2,4) oxadiazolo (4,3,-a) quinoxalin-1-one, the maximal curcumin-induced vasorelaxation (CVR) was inhibited to 20.65%, and 15.81% in MUA of NP and P Ch. In the presence of N.-nitro-L-arginine methyl, indomethacin and combination of both CVR were decreased to 20.49%, 12.47%, 12.82% in MUA ring of NP and to 40.60%, 52.55%, 46.53% in MUA ring of P Ch, respectively. The sensitivity of curcumin to MEGJ blockers carbenoxolone, 18β-glycyrrhetinic acid in causing vasorelaxation was 33.18%, 22.61% in MUA rings of NP, and to 15.76%, 18.54% in P Ch. Conclusion: In P Ch, endothelium-dependent vasorelaxation to curcumin is augmented by two-fold as that of MUA of NP. Endothelial vasorelaxation in MUA of NP is mediated through the activation of cyclooxygenase-PGI2-cyclic adenosine monophosphate and endothelial NO synthase (eNOS)-sGC-cyclic guanosine monophosphate (cGMP) signaling with low activation of sGC and MEGJ. In contrast, vasorelaxation to curcumin is mediated through increased activity of sGC and MEGJ with major involvement of eNOS-NO-sGC-cGMP in MUA of P Ch. These studies have strong implications in the therapeutic targeting for hypertensive disorders.

GW29-e0001 Cinnameldehyde Attenuated Endothelial Dysfunction through Nrf2 Activation as a TRPA1 Agonist

Advancing age is characterized with the development of vascular endothelial dysfunction which is the major risk factor for the development of cardiovascular diseases. TRPA1 is involved in lifespan and its agonist cinnameldehyde (CA) mediates endothelial vasorelaxation. Therefore, we hypothesized

A0625 Cinnameldehyde Attenuated Endothelial Dysfunction through Nrf2 Activation as a TRPA1 Agonist

Objectives: Advancing age is characterized with the development of vascular endothelial dysfunction which is the major risk factor for the development of cardiovascular diseases. TRPA1 is involved in lifespan and its agonist cinnameldehyde(CA)mediates endothelial vasorelaxation. Therefore, we hypothesized that TRPA1 is involved in aging-related endothelial dysfunction and CA administration would improve endothelial function. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured until 14th passage and the 6th passage were used as control. Twenty-four months-old male Sprague Dawley (SD) rats were given dietary CA (0.2%) and six months-old rodents were used as young control. Rodent carotid arteries vasorelaxation was detected by wire myograph. TRPA1, Nrf2, UCP2 and its target genes as well as eNOS, p-eNOS were analysed by immunoblotting. Results: Immunoblotting and immunofluorescence confirmed the TRPA1 expression in HUVECs and vascular endothelium. CA(10 μM) promoted Nrf2 nuclear translocation and eNOS phosphorylation, led to the up-regulation of HO-1, GPx-1, NQO-1 and a reduction in ROS production in HUVECs. However, these effects of CA could be reversed by TRPA1 antagonist, HC030031 (10 μM), Nrf2 inhibitor brusatol (40 nM) or UCP2 inhibitor. NO level was found decreased, and enthdothelium dependent relaxation was impaired in carotid arteries of aged rats, but both improved after 12 weeks of CA administration. Immunoblotting showed the expression of TRAP1, Nrf2, UCP2 and p-eNOS significantly decreased in vascular tissue of aged rats but partly restored after CA treatment. Conclusion: TRPA1 may be involved in aging related endothelial dysfunction and CA improved endothelial dependent vasorelaxation through Nrf2 activation as a TRPA1 agonist.