COVID-19 promotes endothelial dysfunction and thrombogenicity: role of proinflammatory cytokines/SGLT2 prooxidant pathway.

BACKGROUND: COVID-19 is associated with an increased risk of cardiovascular complications. Although cytokines have a predominant role in endothelium damage, the precise molecular mechanisms are far from being elucidated. OBJECTIVES: The present study hypothesized that inflammation in patients with COVID-19 contributes to endothelial dysfunction through redox-sensitive SGLT2 overexpression and investigated the protective effect of SGLT2 inhibition by empagliflozin. METHODS: Human plasma samples were collected from patients with acute, subacute, and long COVID-19 (n = 100), patients with non-COVID-19 and cardiovascular risk factors (n = 50), and healthy volunteers (n = 25). Porcine coronary artery endothelial cells (ECs) were incubated with plasma (10%). Protein expression levels were determined using Western blot analyses and immunofluorescence staining, mRNA expression by quantitative reverse transcription-polymerase chain reaction, and the level of oxidative stress by dihydroethidium staining. Platelet adhesion, aggregation, and thrombin generation were determined. RESULTS: Increased plasma levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, monocyte chemoattractant protein-1, and soluble intercellular adhesion molecule-1 were observed in patients with COVID-19. Exposure of ECs to COVID-19 plasma with high cytokines levels induced redox-sensitive upregulation of SGLT2 expression via proinflammatory cytokines IL-1ß, IL-6, and tumor necrosis factor-α which, in turn, fueled endothelial dysfunction, senescence, NF-κB activation, inflammation, platelet adhesion and aggregation, von Willebrand factor secretion, and thrombin generation. The stimulatory effect of COVID-19 plasma was blunted by neutralizing antibodies against proinflammatory cytokines and empagliflozin. CONCLUSION: In patients with COVID-19, proinflammatory cytokines induced a redox-sensitive upregulation of SGLT2 expression in ECs, which in turn promoted endothelial injury, senescence, platelet adhesion, aggregation, and thrombin generation. SGLT2 inhibition with empagliflozin appeared as an attractive strategy to restore vascular homeostasis in COVID-19.

Empagliflozin prevents angiotensin II-induced hypertension related micro and macrovascular endothelial cell activation and diastolic dysfunction in rats despite persistent hypertension: Role of endothelial SGLT1 and 2.

SGLT2 inhibitors (SGLT2i) showed pronounced beneficial effects in patients with heart failure but the underlying mechanisms remain unclear. We evaluated the effect of empagliflozin, selective SGLT2i, on hypertension-induced cardiac and vascular dysfunction. Male Wistar rats received diet with or without empagliflozin (30 mg/kg/day). After 1 week, a hypertensive dose of Ang II (0.4 mg/kg/day) was administered using osmotic mini-pumps for 4 weeks. Systolic blood pressure was determined by sphygmomanometry, the cardiac function by echocardiography and ex vivo (coronary microvascular endothelial cell activation, LV remodeling and fibrosis responses), and the systemic micro and macrovascular endothelial cell activation ex vivo. Empagliflozin treatment did not affect the Ang II-induced hypertensive response. Ang II treatment increased LV mass and induced LV diastolic dysfunction, fibrosis, collagen I and ANP expression, and infiltration of macrophages. In the vasculature, it caused eNOS upregulation in the aorta and down-regulation in mesenteric microvessels associated with increased oxidative stress, ACE, AT1R, VCAM-1, MCP-1, MMP-2, and MMP-9 and collagen I expression, increased endothelial SGLT1 staining in the aorta, mesenteric and coronary microvessels, increased SGLT1 and 2 protein levels in the aorta. All Ang II-induced cardiac and vascular responses were reduced by the empagliflozin treatment. Thus, the SGLT2i effectively attenuated the deleterious impact of Ang II-induced hypertension on target organs including cardiac diastolic dysfunction and remodeling, and endothelial cell activation and pro-atherosclerotic, pro-fibrotic and pro-remodeling responses in macro and microvessels despite persistent hypertension.

Carissa edulis Vahl (Apocynaceae) extract, a medicinal plant of Benin pharmacopoeia, induces potent endothelium-dependent relaxation of coronary artery rings involving nitric oxide.

BACKGROUND: Hypertension is a major cardiovascular risk factor that affects most countries including those of Africa. Although Carissa edulis Vahl, Diodia scandens Sw. and Cleome gynandra L. are traditionally used in Benin as antihypertensive treatments with some efficacy mentioned by the local population, their biological activity on the cardiovascular system remains poorly studied. AIM: The study investigated the vasoreactivity of the plants and assessed the underlying mechanisms using isolated arteries. STUDY DESIGN: Aqueous-ethanolic extracts of aerial parts of C. edulis, D. scandens and C. gynandra were prepared by maceration before being subjected to multi-step liquid-liquid fractionation with solvents of increasing polarity. The vasoreactivity of the extracts and fractions were assessed on isolated porcine coronary artery and rat aorta using organ chambers, the role of nitric oxide (NO) using NG-nitro-L-arginine (NO synthase inhibitor), prostanoids using indomethacin (cyclooxygenases inhibitor) and endothelium-dependent hyperpolarization using TRAM-34 plus UCL 1684 (inhibitors of calcium-dependent K+ channels), and the vascular uptake of polyphenols using Neu reagent. RESULTS: The aqueous-ethanolic crude extract of C. edulis (CECE) induced potent relaxations that were exclusively endothelium-dependent and more pronounced than those to D. scandens and C. gynandra. The n-butanolic fraction of C. edulis (CEBF) was more active than the cyclohexane, dichloromethane, and ethyl acetate fractions. The relaxation induced by CECE and CEBF were inhibited by NG-nitro-L-arginine and affected neither by TRAM-34 plus UCL 1684 nor by indomethacin. CEBF induced sustained endothelium-dependent relaxations for at least 60 min, and inhibited, in a concentration-dependent manner, contractions to KCl, CaCl2, U46619 and serotonin in rings with endothelium. Analysis of CEBF by LCHRMS indicated the presence of polyphenols, terpenes, and alkaloids. Exposure of coronary artery and aorta rings to CEBF caused the accumulation of polyphenols predominantly in the endothelium. CONCLUSION: C. edulis leaf extract induced pronounced endothelium-dependent relaxations and inhibited contractile responses by stimulating the endothelial formation of NO. LCHRMS analysis of the most active fraction, the butanolic fraction, revealed the presence of numerous compounds including polyphenols, terpenes, and alkaloids. The polyphenols of CEBF accumulated preferentially in the endothelium of the arterial wall. Thus, these observations support the folkloric use of C. edulis in hypertension.

Septic shock as a trigger of arterial stress-induced premature senescence: A new pathway involved in the post sepsis long-term cardiovascular complications.

BACKGROUND: Major adverse cardiovascular events among sepsis survivors is an emerging health issue. Because endothelial senescence leads to vascular dysfunction and atherothrombosis, sepsis could be associated to vascular stress-induced premature senescence and thus with long-term cardiovascular events. MATERIALS & METHODS: Adult Wistar male rats were submitted to cecal ligation and puncture, or a SHAM operation. Markers of inflammation, oxidative stress and endothelial senescence were assessed at 3, 7 and 90 days (D), and vascular reactivity was assessed in conductance and resistance vessels at D90. Expression of proteins involved in senescence and inflammation was assessed by Western blot analysis and confocal microscopy, oxidative stress by dihydroethidium probing. RESULTS: Pro-inflammatory endothelial ICAM-1 and VCAM-1 were up-regulated by three-fold in CLP vs. SHAM at D7 and remained elevated at D90. Oxidative stress followed a similar pattern but was detected in the whole vascular wall. Sepsis accelerated premature senescence in aorta vascular tissue as shown by the significant up-regulation of p53 and down-stream p21 and p16 senescent markers at D7, values peaking at D90 whereas the absence of significant variation in activated caspase-3 confirmed p53 as a prime inducer of senescence. In addition, p53 was mainly expressed in the endothelium. Sepsis-induced long-term vascular dysfunction was confirmed in aorta and main mesenteric artery, with a major alteration of the endothelial-dependent nitric oxide pathway. CONCLUSIONS: Septic shock-induced long-term vascular dysfunction is associated with endothelial and vascular senescence. Our model could prove useful for investigating senotherapies aiming at reducing long-term cardiovascular consequences of septic shock.

Angiotensin II-induced upregulation of SGLT1 and 2 contributes to human microparticle-stimulated endothelial senescence and dysfunction: protective effect of gliflozins.

BACKGROUND: Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduced cardiovascular risk in type 2 diabetes patients independently of glycemic control. Although angiotensin II (Ang II) and blood-derived microparticles are major mediators of cardiovascular disease, their impact on SGLT1 and 2 expression and function in endothelial cells (ECs) and isolated arteries remains unclear. METHODS: ECs were isolated from porcine coronary arteries, and arterial segments from rats. The protein expression level was assessed by Western blot analysis and immunofluorescence staining, mRNA levels by RT-PCR, oxidative stress using dihydroethidium, nitric oxide using DAF-FM diacetate, senescence by senescence-associated beta-galactosidase activity, and platelet aggregation by aggregometer. Microparticles were collected from blood of patients with coronary artery disease (CAD-MPs). RESULTS: Ang II up-regulated SGLT1 and 2 protein levels in ECs, and caused a sustained extracellular glucose- and Na+-dependent pro-oxidant response that was inhibited by the NADPH oxidase inhibitor VAS-2780, the AT1R antagonist losartan, sotagliflozin (Sota, SGLT1 and SGLT2 inhibitor), and empagliflozin (Empa, SGLT2 inhibitor). Ang II increased senescence-associated beta-galactosidase activity and markers, VCAM-1, MCP-1, tissue factor, ACE, and AT1R, and down-regulated eNOS and NO formation, which were inhibited by Sota and Empa. Increased SGLT1 and SGLT2 protein levels were observed in the rat aortic arch, and Ang II- and eNOS inhibitor-treated thoracic aorta segments, and were associated with enhanced levels of oxidative stress and prevented by VAS-2780, losartan, Sota and Empa. CAD-MPs promoted increased levels of SGLT1, SGLT2 and VCAM-1, and decreased eNOS and NO formation in ECs, which were inhibited by VAS-2780, losartan, Sota and Empa. CONCLUSIONS: Ang II up-regulates SGLT1 and 2 protein expression in ECs and arterial segments to promote sustained oxidative stress, senescence and dysfunction. Such a sequence contributes to CAD-MPs-induced endothelial dysfunction. Since AT1R/NADPH oxidase/SGLT1 and 2 pathways promote endothelial dysfunction, inhibition of SGLT1 and/or 2 appears as an attractive strategy to enhance the protective endothelial function.

Fluorescent nanocarriers targeting VCAM-1 for early detection of senescent endothelial cells.

Endothelial senescence has been identified as an early event in the development of endothelial dysfunction, a hallmark of cardiovascular disease. This study developed theranostic nanocarriers (NC) decorated with VCAM-1 antibodies (NC-VCAM-1) in order to target cell surface VCAM-1, which is overexpressed in senescent endothelial cells (ECs) for diagnostic and therapeutic purposes. Incubation of Ang II-induced premature senescent ECs or replicative senescent ECs with NC-VCAM-1 loaded with lipophilic fluorescent dyes showed higher fluorescence signals than healthy EC, which was dependent on the NC size and VCAM-1 antibodies concentration, and not observed following masking of VCAM-1. NC loaded with omega 3 polyunsaturated fatty acid (NC-EPA:DHA6:1) were more effective than native EPA:DHA 6:1 to prevent Ang II-induced VCAM-1 and p53 upregulation, and SA-ß-galactosidase activity in coronary artery segments. These theranostic NC might be of interest to evaluate the extent and localization of endothelial senescence and to prevent pro-senescent endothelial responses.

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.

Red Wine Extract Disrupts Th17 Lymphocyte Differentiation in a Colorectal Cancer Context.

SCOPE: Scope: It is well established that immune response and inflammation promote tumoral progression. Immune cells communicate through direct contact or through cytokine secretion, and it is the pro-inflammatory status that will tip the balance toward tumor progression or anti-tumor immunity. It is demonstrated here that a red wine extract (RWE) can decrease inflammation through its action on the inflammasome complex. This study determines whether an RWE could impact other key actors of inflammation, including T helper 17 (Th17) immune cells in particular. METHODS AND RESULTS: Methods and results: Using an RWE containing 4.16 g of polyphenols/liter of wine, it is shown that RWE decreases colorectal cancer cells in vitro and induces a reduction in colorectal tumor growth associated with a decrease in tumor-infiltrating lymphocytes in vivo. The process of T-lymphocyte differentiation in Th17 cells is altered by RWE, as revealed by the decrease in the expression of key actors controlling this process, such as signal transducer and activator of transcription 3 and retinoid acid-related orphan receptor γt. This disruption is associated with an inhibition of inflammatory interleukin 17 secretion. CONCLUSION: The data highlights the major involvement of Th17 immune cells in the biological effects of an RWE.

Preventive Beneficial Effect of an Aqueous Extract of Phyllanthus amarus Schum. and Thonn. (Euphorbiaceae) on DOCA-Salt-Induced Hypertension, Cardiac Hypertrophy and Dysfunction, and Endothelial Dysfunction in Rats.

This study investigated the preventive effect of an aqueous extract of the whole plant of Phyllanthus amarus (AEPA) on blood pressure, cardiac, and endothelial function in the deoxycorticosterone acetate (DOCA) salt-induced hypertensive rat model. Male Wistar rats were assigned into 5 groups receiving either vehicle (control and DOCA salt), DOCA salt combined with AEPA at 100 or 300 mg/kg, or AEPA (100 mg/kg) alone for 5 weeks. In addition, DOCA salt-treated rats were allowed free access to water containing 1% NaCl. Systolic blood pressure, left ventricle parameters, vascular reactivity of primary mesenteric artery rings, the vascular level of oxidative stress, and the level of target proteins were determined, using respectively tail-cuff sphygmomanometry, echocardiography, organ chambers, dihydroethidium staining, and immunofluorescence methods. After 5 weeks, AEPA treatments (100 or 300 mg/kg per day) significantly prevented the increase in systolic blood pressure in DOCA salt-treated rats, respectively, by about 24 and 21 mm Hg, improved cardiac diastolic function, and reduced significantly the increased posterior and septum diastolic wall thickness and the left ventricle mass in hypertensive rats. Moreover, the DOCA salt-induced endothelial dysfunction and the blunted nitric oxide- and endothelium-dependent hyperpolarization-mediated relaxations in primary mesenteric artery were improved after the AEPA treatments. AEPA also reduced the level of vascular oxidative stress and the expression level of target proteins (eNOS, COX-2, NADPH oxidase subunit p22) in DOCA salt rats. Altogether, AEPA prevented hypertension, improved cardiac structure and function, and improved endothelial function in DOCA salt rats. Such beneficial effects seem to be related, at least in part, to normalization of the vascular level of oxidative stress.

Empagliflozin improved systolic blood pressure, endothelial dysfunction and heart remodeling in the metabolic syndrome ZSF1 rat.

BACKGROUND: Empagliflozin (empa), a selective sodium-glucose cotransporter (SGLT)2 inhibitor, reduced cardiovascular mortality and hospitalization for heart failure in patients with type 2 diabetes at high cardiovascular risk independent of glycemic control. The cardiovascular protective effect of empa was evaluated in an experimental model of metabolic syndrome, the obese ZSF1 rat, and its' lean control. METHODS: Lean and obese ZSF1 rats were either non-treated or treated with empa (30 mg/kg/day) for 6 weeks. Vascular reactivity was assessed using mesenteric artery rings, systolic blood pressure by tail-cuff sphygmomanometry, heart function and structural changes by echocardiography, and protein expression levels by Western blot analysis. RESULTS: Empa treatment reduced blood glucose levels from 275 to 196 mg/dl in obese ZSF1 rats whereas normoglycemia (134 mg/dl) was present in control lean ZSF1 rats and was unaffected by empa. Obese ZSF1 rats showed increased systolic blood pressure, and blunted endothelium-dependent relaxations associated with the appearance of endothelium-dependent contractile responses (EDCFs) compared to control lean rats. These effects were prevented by the empa treatment. Obese ZSF1 rats showed increased weight of the heart and of the left ventricle volume without the presence of diastolic or systolic dysfunction, which were improved by the empa treatment. An increased expression level of senescence markers (p53, p21, p16), tissue factor, VCAM-1, SGLT1 and SGLT2 and a down-regulation of eNOS were observed in the aortic inner curvature compared to the outer one in the control lean rats, which were prevented by the empa treatment. In the obese ZSF1 rats, no such effects were observed. The empa treatment reduced the increased body weight and weight of lungs, spleen, liver and perirenal fat, hyperglycemia and the increased levels of total cholesterol and triglycerides in obese ZSF1 rats, and increased blood ketone levels and urinary glucose excretion in control lean and obese ZSF1 rats. CONCLUSION: Empa reduced glucose levels by 28% and improved both endothelial function and cardiac remodeling in the obese ZSF1 rat. Empa also reduced the increased expression level of senescence, and atherothrombotic markers at arterial sites at risk in the control lean, but not obese, ZSF1 rat.

Oral Intake of EPA:DHA 6:1 by Middle-Aged Rats for One Week Improves Age-Related Endothelial Dysfunction in Both the Femoral Artery and Vein: Role of Cyclooxygenases.

In humans, aging is associated with endothelial dysfunction and an increased risk of venous thromboembolism. Although intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at a ratio of 6:1 by old rats improved the endothelial dysfunction in arteries, the impact on veins remains unclear. Eight-month-old male Wistar rats were either untreated or orally administered corn oil, EPA:DHA 1:1, or EPA:DHA 6:1 (500 mg/kg/d) for seven days. Vascular reactivity was studied by myography. In middle-aged femoral artery rings, acetylcholine caused a partial relaxation at low concentrations and a contractile response at high concentrations, whereas in the old femoral vein only a partial relaxation was observed. The EPA:DHA 6:1 treatment blunted the contractile response to acetylcholine in the middle-aged femoral artery and both EPA:DHA 6:1 and 1:1 increased the relaxation to acetylcholine in the old femoral vein. No such effects were observed with corn oil. Both the non-selective cyclooxygenase inhibitor indomethacin and the COX-1 inhibitor SC-560 increased the relaxation to acetylcholine in the middle-aged femoral artery whereas the COX-2 inhibitor NS-398 increased that in the middle-aged femoral vein. In conclusion, our results indicate that aging is associated with an endothelial dysfunction in the femoral artery and vein, which can be improved by EPA:DHA 6:1 treatment-most likely via a cyclooxygenase-dependent mechanism.

Compared Phenolic Compound Contents of 22 Commercial Fruit and Vegetable Juices: Relationship to ex-vivo Vascular Reactivity and Potential in vivo Projection.

The real impact of polyphenol-rich vegetable and fruit juice intake on cardiovascular health remains a matter of controversy. In the present study, rat aorta segments immersed in an organ bath (OB) were used to explore whether the total polyphenol content and/or individual phenolic compound contents of 22 commercial vegetable (n = 3) and fruit juices [(citrus (n = 5), berries (n = 10), apple (n = 2), pineapple (n = 2)] might be associated with vascular tone. Red juices (particularly blackcurrant) and lemon juice caused the most marked vasorelaxation, its amplitude being endothelium dependent or not according to the volume ratio of juice to initial OB solution Vjuice/VOBS). At volume ratios 5% and 10%, both the juice and OB total polyphenol for all juices and total anthocyanin contents for berry juices significantly correlated with aorta vasorelaxation intensity. This was not the case for total or individual flavonols (except kaempferol) or for total or individual flavanols (except epigallocatechin gallate). If one relates our measured concentrations of individual phenolic compounds in OB to what is known about their physiological concentrations, and given our evidenced correlations between compound concentrations and vasorelaxation intensity, kaempferol, epigallocatechin gallate and peonidin-3-O-glucoside seem to emerge as the interesting phenolic compounds likely to be responsible for the potent vasorelaxation observed with fruit juices, and more particularly blackcurrant ones. Clinical investigation is required, however, to confirm our observations.

Angiotensin II-induced redox-sensitive SGLT1 and 2 expression promotes high glucose-induced endothelial cell senescence.

High glucose (HG)-induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co-transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG-induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence-associated beta-galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT-PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2-NBD-glucose. HG increased ECs senescence markers and oxidative stress, down-regulated eNOS expression and NO formation, and induced the expression of VCAM-1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX-4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H2 O2 in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG-induced ECs senescence and SGLT1 and 2 expression. Thus, HG-induced ECs ageing is driven by the local angiotensin system via the redox-sensitive up-regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.

Intake of omega-3 formulation EPA:DHA 6:1 by old rats for 2 weeks improved endothelium-dependent relaxations and normalized the expression level of ACE/AT1R/NADPH oxidase and the formation of ROS in the mesenteric artery.

Omega-3 polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to protect the cardiovascular system, in part, by stimulating the endothelial formation of nitric oxide (NO). EPA:DHA 6:1 has been identified as a potent omega 3 PUFA formulation to induce endothelium-dependent vasorelaxation and activation of endothelial NO synthase (eNOS). This study examined whether intake of EPA:DHA 6:1 (500 mg/kg/day) for 2 weeks improves an established endothelial dysfunction in old rats (20 months old), and, if so, the underlying mechanism was subsequently determined. In the main mesenteric artery rings, an endothelial dysfunction characterized by a blunted NO component, an abolished endothelium-dependent hyperpolarization component, and increased endothelium-dependent contractile responses (EDCFs) are observed in old rats compared to young rats. Age-related endothelial dysfunction was associated with increased vascular formation of reactive oxygen species (ROS) and expression of eNOS, components of the local angiotensin system, senescence markers, and cyclooxygenase-2 (COX-2), and the downregulation of COX-1. The EPA:DHA 6:1 treatment improved the NO-mediated relaxation, reduced the EDCF-dependent contractile response and the vascular formation of ROS, and normalized the expression level of all target proteins in the old arterial wall. Thus, the present findings indicate that a 2-week intake of EPA:DHA 6:1 by old rats restored endothelium-dependent NO-mediated relaxations, most likely, by preventing the upregulation of the local angiotensin system and the subsequent formation of ROS.

Atrial Fibrillation Progression Is Associated with Cell Senescence Burden as Determined by p53 and p16 Expression.

BACKGROUND: Whilst the link between aging and thrombogenicity in atrial fibrillation (AF) is well established, the cellular underlying mechanisms are unknown. In AF, the role of senescence in tissue remodeling and prothrombotic state remains unclear. AIMS: We investigated the link between AF and senescence by comparing the expression of senescence markers (p53 and p16), with prothrombotic and inflammatory proteins in right atrial appendages from patients in AF and sinus rhythm (SR). METHODS: The right atrial appendages of 147 patients undergoing open-heart surgery were harvested. Twenty-one non-valvular AF patients, including paroxysmal (PAF) or permanent AF (PmAF), were matched with 21 SR patients according to CHA2DS2-VASc score and treatment. Protein expression was assessed by tissue lysates Western blot analysis. RESULTS: The expression of p53, p16, and tissue factor (TF) was significantly increased in AF compared to SR (0.91 ± 0.31 vs. 0.58 ± 0.31, p = 0.001; 0.76 ± 0.32 vs. 0.35 ± 0.18, p = 0.0001; 0.88 ± 0.32 vs. 0.68 ± 0.29, p = 0.045, respectively). Expression of endothelial NO synthase (eNOS) was lower in AF (0.25 ± 0.15 vs. 0.35 ± 0.12, p = 0.023). There was a stepwise increase of p53, p16, TF, matrix metalloproteinase-9, and an eNOS progressive decrease between SR, PAF, and PmAF. AF was the only predictive factor of p53 and p16 elevation in multivariate analysis. Conclusions: The study brought new evidence indicating that AF progression is strongly related to human atrial senescence burden and points at a link between senescence, thrombogenicity, endothelial dysfunction and atrial remodeling.

Thrombin Induces Angiotensin II-Mediated Senescence in Atrial Endothelial Cells: Impact on Pro-Remodeling Patterns.

BACKGROUND: Besides its well-known functions in hemostasis, thrombin plays a role in various non-hemostatic biological and pathophysiologic processes. We examined the potential of thrombin to promote premature atrial endothelial cells (ECs) senescence. METHODS AND RESULTS: Primary ECs were isolated from porcine atrial tissue. Endothelial senescence was assessed by measuring beta-galactosidase (SA-ß-gal) activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, protein level by Western blot, and matrix metalloproteinases (MMPs) activity using zymography. Atrial endothelial senescence was induced by thrombin at clinically relevant concentrations. Thrombin induced the up-regulation of p53, a key regulator in cellular senescence and of p21 and p16, two cyclin-dependent kinase inhibitors. Nicotinamide adenine dinucleotide phosphate NADPH oxidase, cyclooxygenases and the mitochondrial respiration complex contributed to oxidative stress and senescence. Enhanced expression levels of vascular cell adhesion molecule (VCAM)-1, tissue factor, transforming growth factor (TGF)-ß and MMP-2 and 9 characterized the senescence-associated secretory phenotype of atrial ECs. In addition, the pro-senescence endothelial response to thrombin was associated with an overexpression of both angiotensin converting enzyme and AT1 receptors and was inhibited by perindoprilat and losartan. CONCLUSIONS: Thrombin promotes premature ageing and senescence of atrial ECs and may pave the way to deleterious remodeling of atrial tissue by a local up-regulation of the angiotensin system and by promoting pro-inflammatory, pro-thrombotic, pro-fibrotic and pro-remodeling responses. Hence, targeting thrombin and/or angiotensin systems may efficiently prevent atrial endothelial senescence.

Effects of a High Fat Meal Associated with Water, Juice, or Champagne Consumption on Endothelial Function and Markers of Oxidative Stress and Inflammation in Young, Healthy Subjects.

Endothelial dysfunction (ED), often linked to hypertriglyceridemia, is an early step of atherosclerosis. We investigated, in a randomized cross-over study, whether high-fat meal (HFM)-induced ED might be reduced by fruit juice or champagne containing polyphenols. Flow-mediated dilatation (FMD) and biological parameters (lipid profile, glycemia, inflammation, and oxidative stress markers) were determined before and two and three hours after the HFM in 17 healthy young subjects (24.6 ± 0.9 years) drinking water, juice, or champagne. Considering the entire group, despite significant hypertriglyceridemia (from 0.77 ± 0.07 to 1.41 ± 0.18 mmol/L, p < 0.001) and a decrease in Low Density Lipoprotein (LDL), the FMD was not impaired. However, the FMD decreased in 10 subjects (from 10.73 ± 0.95 to 8.1 3± 0.86 and 8.07 ± 1.16%; p < 0.05 and p < 0.01; 2 and 3 hours, respectively, after the HFM), without concomitant change in concentration reactive protein or reactive oxygen species, but with an increase in glycemia. In the same subjects, the FMD did not decrease when drinking juice or champagne. In conclusion, HFM can impair the endothelial function in healthy young subjects. Fruit juice, rich in anthocyanins and procyanidins, or champagne, rich in simple phenolic acids, might reduce such alterations, but further studies are needed to determine the underlying mechanisms, likely involving polyphenols.

EPA:DHA 6:1 is a superior omega-3 PUFAs formulation attenuating platelets-induced contractile responses in porcine coronary and human internal mammary artery by targeting the serotonin pathway via an increased endothelial formation of nitric oxide.

At arterial sites of endothelial denudation and dysfunction, activated platelets contribute to vascular injury through the release of potent contracting factors such as serotonin (5-HT). This study evaluated whether omega-3 polyunsaturated fatty acids (PUFAs), known to protect the vascular system, are able to prevent platelets-induced contractile responses in isolated arteries and, if so, to investigate the underlying mechanism and the importance of the omega-3 PUFAs formulation. Porcine coronary arteries (PCA), human internal mammary arteries (IMA) and washed human platelets were prepared and vascular reactivity was studied in organ chambers. In PCA rings, aggregating platelets caused concentration-dependent contractions that were significantly inhibited by the 5-HT2A receptor antagonist ketanserin, and by EPA:DHA 6:1 but not EPA:DHA 1:1 at 0.4% v/v. EPA:DHA 6:1 also prevented the 5-HT-induced contractions but affected only slightly those to the thromboxane A2 analogue U46619. The inhibitory effect of EPA:DHA 6:1 on platelets-induced contractions was not observed in rings without endothelium, and prevented by an eNOS inhibitor but not by inhibitors of endothelium-dependent hyperpolarization. In IMA rings, EPA:DHA 6:1 but not EPA:DHA 1:1 at 0.4% v/v significantly prevented the 5-HT-induced contraction, and induced greater endothelium-dependent relaxations than bradykinin and acetylcholine sensitive to an eNOS inhibitor. EPA:DHA 6:1 strongly inhibits platelets- and 5-HT-induced contractions in PCA rings and those to 5-HT in IMA rings most likely through an increased endothelial formation of NO. These findings suggest that the omega-3 PUFAs EPA:DHA 6:1 formulation may be of interest to prevent platelets-induced vascular injury at arterial sites of endothelial dysfunction.

Urapidil, but not dihydropyridine calcium channel inhibitors, preserves the hypoxic pulmonary vasoconstriction: an experimental study in pig arteries.

Hypoxic pulmonary vasoconstriction (HPV) is a protective mechanism maintaining blood oxygenation by redirecting blood flow from poorly ventilated to well-ventilated areas in the lung. Such a beneficial effect is blunted by antihypertensive treatment with dihydropyridine calcium channel inhibitors. The aim of the present study was to evaluate the effect of urapidil, an antihypertensive agent acting as an α1 adrenergic antagonist and a partial 5-HT1A agonist, on HPV in porcine proximal and distal pulmonary artery rings, and to characterize underlying mechanisms. Rings from proximal and distal porcine pulmonary artery were suspended in organ chambers and aerated with a 95% O2 + 5% CO2 gas mixture. HPV was induced by changing the gas to a 95% N2 + 5% CO2 mixture following a low level of pre-contraction with U46619. Hypoxia induced a contractile response in both proximal and distal pulmonary artery rings. This effect is observed in the presence of a functional endothelium and is inhibited by a soluble guanylyl cyclase inhibitor (ODQ), a NO scavenger (carboxy-PTIO), and by catalase in proximal pulmonary artery rings. The endothelium-dependent HPV is prevented by nicardipine and clevidipine but remained unaffected by urapidil in both proximal and distal pulmonary artery rings. These findings indicate that urapidil, in contrast to nicardipine and clevidipine, preserves the hypoxia-triggered vasoconstriction in isolated pulmonary arteries. They further indicate the involvement of the NO-guanylyl cyclase pathway and H2 O2 in HPV. Further research is warranted to determine the potential clinical relevance of the preserved hypoxia-induced pulmonary vasoconstriction by urapidil.

Tambulin is a major active compound of a methanolic extract of fruits of Zanthoxylum armatum DC causing endothelium-independent relaxations in porcine coronary artery rings via the cyclic AMP and cyclic GMP relaxing pathways.

BACKGROUND: Zanthoxylum armatum DC (Z. armatum), belonging to Rutaceae family, has been traditionally used for the treatment of various diseases such as hypertension, abdominal pain, headache, fever, high altitude sickness, diarrhea, dysentery, and as a tonic, condiment, and an anthelmintic treatment. HYPOTHESIS: The present study aims to evaluate the vasorelaxant effect of a methanolic extract of the fruits of Z. armatum, isolate the active components and characterize the underlying mechanism. STUDY DESIGN: A methanolic extract of fruits of Z. armatum was prepared and its vasorelaxant effect was studied using porcine coronary artery rings. Thereafter, the methanolic extract was analyzed, and a major compound was isolated and its structure elucidated (tambulin). Different pharmacological tools were used to characterize the vasorelaxant effect of tambulin. RESULTS: The methanolic extract and the isolated tambulin caused similar endothelium-independent relaxations of porcine coronary artery rings with and without endothelium indicating a direct relaxing effect at the vascular smooth muscle. Tambulin did not affect the relaxation curves to the endothelium-dependent vasodilators, bradykinin and the calcium ionophore A23187 in rings with endothelium. Tambulin (1 µM) slightly but significantly shifted leftwards the concentration-relaxation curve to the endothelium-independent vasodilators, sodium nitroprusside (SNP), forskolin (FC) and isoproterenol but not those to soluble guanylyl cyclase activators (YC-1 and BAY 41-2272) and K+ channel openers (levcromakalim and 1-EBIO). Pretreatment with tambulin inhibited, in a concentration-dependent manner, contractions to KCl, serotonin (5-HT), CaCl2 and U46619 in coronary artery rings without endothelium. Both the protein kinase A (H-89, 10 µM) and the protein kinase G (Rp-8-br-cyclic GMPS, 30 µM) inhibitors significantly reduced relaxations to tambulin in coronary artery rings without endothelium. CONCLUSION: The present findings indicate that tambulin isolated from Z. armatum (fruits) is a major active principle inducing vasorelaxation through a direct effect at the vascular smooth muscle and involving both the cyclic AMP and/or cyclic GMP relaxing pathways.