In Vitro Impact of Pro-Senescent Endothelial Microvesicles on Isolated Pancreatic Rat Islets Function

BackgroundIschemia-driven islet isolation procedure is one of the limiting causes of pancreatic islet transplantation. Ischemia-reperfusion process is associated with endothelium dysfunction and the release of pro-senescent microvesicles. We investigated whether pro-senescent endothelial microvesicles prompt islet senescence and dysfunction in vitro.Material and methodsPancreatic islets were isolated from male young rats. Replicative endothelial senescence was induced by serial passaging of primary porcine coronary artery endothelial cells, and microvesicles were isolated either from young passage 1 (P1) or senescent passage 3 (P3) endothelial cells. Islet viability was assessed by fluorescence microscopy, apoptosis by flow cytometry, and Western blot. Function was assessed by insulin secretion and islet senescence markers p53, p21, and p16 by Western blot. Microvesicles were stained by the PKH26 lipid fluorescent probe and their islet integration assessed by microscopy and flow cytometry.ResultsRegardless of the passage, half microvesicles were integrated in target islets after 24 hours incubation. Insulin secretion significantly decreased after treatment by senescent microvesicles (P3: 1.7 ± 0.2 vs untreated islet: 2.7 ± 0.2, P < .05) without altering the islet viability (89.47% ± 1.69 vs 93.15% ± 0.97) and with no significant apoptosis. Senescent microvesicles significantly doubled the expression of p53, p21, and p16 (P < .05), whereas young microvesicles had no significant effect.ConclusionPro-senescent endothelial microvesicles specifically accelerate the senescence of islets and alter their function. These data suggest that islet isolation contributes to endothelial driven islet senescence.     

Reversal of granulocyte adherence to nylon fibers using local anesthetic agents: possible application to filtration leukapheresis

The effects of the cationic anesthetic agents tetracaine and lidocaine on granulocyte function, morphology, and adherence to nylon fibers were studied in an attempt to improve current methods of granulocyte collection by filtration leukapheresis (FL). When dissolved in acid- citrate-dextrose (ACD) plasma, these drugs significantly increased granulocyte elution from the fibers in a dose-related fashion. Granulocytes exposed to tetracaine and lidocaine remained more than 95% viable, retained normal bactericidal capacity after the drugs were washed from the cells, and had preserved membrane integrity, as evidenced by the normal ultrastructural appearance of tetracaine- exposed cells and an absence of leakage of lysozyme or lactic dehydrogenase. Granulocytes eluted with the anesthetic agents were rounded in shape with a reduction in the number of filopodial cytoplasmic projections and a relative absence of cytoplasmic vacuolization when compared to granulocytes eluted with ACD plasma alone. Dose-related inhibition of phagocytosis and adherence, which was largely reversible after washing the granulocytes, was noted. Greater than 95% of the lidocaine could be removed from the eluate with a single centrifugation and resuspension, indicating that granulocytes prepared by FL with anesthetic-enhanced elution could be potentially transfusable.     

Platelets inhibit the induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells

We have investigated the role of platelets in regulating the hemostatic and vasomotor properties of vascular smooth muscle. Experiments were performed to examine the effect of the releasate from activated platelets on the production of nitric oxide from interleukin-1 beta (IL- 1 beta)-treated cultured rat aortic smooth muscle cells. Treatment of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite in the culture media and in marked elevation of intracellular cyclic guanosine monophosphate (GMP) levels. The releasate from collagen-aggregated platelets blocked the IL-1 beta- mediated production of nitrite and the accumulation of cyclic GMP in smooth muscle cells in a platelet number-dependent manner. In functional assays, the perfusates from columns containing IL-1 beta- treated smooth muscle cells relaxed detector blood vessels without endothelium and the addition of IL-1 beta-treated smooth muscle cells to suspensions of platelets inhibited their thrombin-induced aggregation. The simultaneous treatment of smooth muscle cells with IL- 1 beta and the platelet releasate abolished both the vasorelaxing activities of the perfusates and the inhibition of platelet aggregation. Platelet releasates treated with a neutralizing antibody to platelet-derived growth factor (PDGF) failed to block IL-1 beta- induced nitric oxide production by the smooth muscle cells, as measured by both biochemical and functional assays. The platelet releasate from a patient with gray platelet syndrome likewise failed to block IL-1 beta-induced nitrite release by smooth muscle cells. These results demonstrate that platelets downregulate the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells through the release of PDGF. This effect may represent a novel mechanism by which platelets regulate vasomotor tone and thrombus formation at sites of vascular injury.     

Selective anticancer effects of a synthetic flavagline on human Oct4-expressing cancer stem-like cells via a p38 MAPK-dependent caspase-3-dependent pathway

Cancer stem cells (CSCs) are considered as the initiators of the carcinogenic process and are therefore emerging targets for innovative anticancer therapies. In order to evaluate the anticancer chemopreventive activity of flavagline derivatives, we used the pluripotent teratocarcinomal cell as a model of Oct4-expressing cancer stem-like cell and determined the underlying cellular and molecular mechanisms induced by a synthetic flavagline. We precisely investigated the effects of the flavagline derivative FL3 on the human embryonal carcinoma (EC) cell line NT2/D1 and compared the responses to those of a normal more restrictive pluripotent stem cell line (i.e. BJ fibroblast cell line). FL3 selectively inhibited the proliferation of NT2/D1 cells by inducing G₁ phase cell cycle arrest in a dose-dependent manner. Moreover, FL3 treatment specifically triggered apoptosis in association with an induction of the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and caspase-3 activation followed by a drastic downregulation of the master regulator of stemness Oct4. Forced inhibition of p38 MAPK activity by the specific pharmacological inhibitor SB203580 or by p38 MAPK gene knockdown using small-interfering RNA (siRNA) counteracted the effects of FL3, demonstrating that its chemopreventive action is related to growth inhibition and a p38-dependent caspase-3-dependent induction of apoptosis in Oct4-expressing CSCs. This study also shows that FL3 selectively kills poorly differentiated and highly aggressive carcinomal cells, but has little effect on normal stem-like cells. Thus FL3 offers great promise for cancer treatment since it is able to target the carcinogenic process without affecting normal cells.     

Chronic treatment with progesterone but not medroxyprogesterone acetate restores the endothelial control of vascular tone in the mesenteric artery of ovariectomized rats

OBJECTIVE: To examine whether chronic administration of the natural hormone progesterone or a synthetic progestogen, medroxyprogesterone acetate, to ovariectomized rats affects the endothelial control of arterial tone in the isolated mesenteric artery. DESIGN: Sham-operated rats received a daily subcutaneous injection of solvent (sesame oil), whereas ovariectomized rats received either sesame oil, progesterone (22 mg kg/day), or medroxyprogesterone acetate (22 mg kg/day) for 4 weeks, according to their respective group. RESULTS: Phenylephrine-induced contractions were significantly increased (about 200% at 10 microM) by N-nitro-L-arginine, a nitric oxide synthase inhibitor, in intact mesenteric arterial rings from the sham-operated but not from the ovariectomized group. The progesterone but not the medroxyprogesterone treatment restored the potentiating effect of N-nitro-L-arginine on phenylephrine-induced contraction (about 180% at 10 microM). Contractions to phenylephrine were not affected by the combination of charybdotoxin plus apamin, two inhibitors of endothelium-derived hyperpolarizing factor-mediated responses, in all groups. Acetylcholine induced endothelium-dependent relaxations, which were partially inhibited by N-nitro-L-arginine and abolished by the combination of N-nitro-L-arginine plus charybdotoxin and apamin, in all groups. Acetylcholine induced similar charybdotoxin and apamin-sensitive hyperpolarizations in intact mesenteric artery segments from all groups. CONCLUSIONS: Chronic administration of progesterone, but not medroxyprogesterone, to ovarictomized rats restores the endothelium-dependent attenuation of contractile responses to phenylephrine in mesenteric arterial rings through the endothelial formation of nitric oxide. Thus, an enhancement of the protective effect of endothelial cells on the arterial wall might contribute to the beneficial effect of certain progestogen-containing preparations during hormonal treatment.     

Red wine polyphenols induce EDHF-mediated relaxations in porcine coronary arteries through the redox-sensitive activation of the PI3-kinase/Akt pathway

Red wine polyphenolic compounds (RWPCs) are potent inducers of endothelium-dependent relaxations of coronary arteries, which involve both nitric oxide and endothelium-derived hyperpolarizing factor (EDHF). The EDHF-mediated relaxation to RWPCs is critically dependent on the formation of reactive oxygen species by a flavin-dependent enzyme. The aim of the present study was to determine the role of redox-sensitive protein kinases including p38 MAPK, ERK1/2 and PI3-kinase/Akt in RWPCs-induced EDHF-mediated relaxation. Porcine coronary artery rings were suspended in organ chambers for measurement of changes in isometric tension. Confluent cultures of porcine coronary artery endothelial cells were used to determine the phosphorylation level of p38 MAPK, ERK1/2 and Akt by Western blot analysis. All experiments were performed in the presence of indomethacin and Nomega-nitro-L-arginine. RWPCs caused pronounced endothelium-dependent relaxations, which were significantly reduced by wortmannin and LY294002, two inhibitors of PI3-kinase, and not affected by PD98059 (an inhibitor of ERK1/2 kinase kinase) and SB203580 (an inhibitor of p38 MAPK). In contrast, wortmannin did not affect relaxations to bradykinin or levcromakalim. RWPCs elicited within minutes a sustained and concentration-dependent phosphorylation of p38 MAPK, ERK1/2 and Akt in endothelial cells. The phosphorylation of Akt in response to RWPCs was abolished by wortmannin and LY294002, and by the membrane-permeant analogue of superoxide dismutase Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin. The present findings demonstrate that RWPCs cause EDHF-mediated relaxations of coronary arteries; these responses are critically dependent on the redox-sensitive activation of the PI3-kinase/Akt pathway in endothelial cells.     

The progestin levonorgestrel induces endothelium-independent relaxation of rabbit jugular vein via inhibition of calcium entry and protein kinase C: role of cyclic AMP

The progestin and oestrogen component of oral contraceptives have been involved in the development of venous thromboembolic events in women. In the present study we determined the vasoactive effects of sex steroids used in oral contraceptives in isolated preconstricted rabbit jugular veins in the presence of diclofenac and examined the underlying mechanisms. The natural hormone progesterone, the synthetic progestins levonorgestrel, 3-keto-desogestrel, gestodene and chlormadinone acetate, and the synthetic estrogen 17 alpha-ethinyloestradiol induced concentration-dependent relaxations of endothelium-intact veins constricted with U46619. Levonorgestrel also inhibited constrictions evoked by either a high potassium (K(+)) solution or phorbol myristate acetate (PMA) in the absence and presence of extracellular calcium (Ca(2+)). In addition, levonorgestrel depressed contractions evoked by Ca(2+) and reduced (45)Ca(2+) influx in depolarized veins. Relaxations to levonorgestrel in U46619-constricted veins were neither affected by the presence of the endothelium nor by the inhibitor of soluble guanylyl cyclase, NS2028, but were significantly improved either by the selective cyclic AMP phosphodiesterase inhibitor rolipram or in the absence of diclofenac, and decreased by the protein kinase A inhibitor, Rp-8-CPT-cAMPS. Rolipram also potentiated relaxations to levonorgestrel in PMA-constricted veins in the presence, but not in the absence of extracellular Ca(2+). Levonorgestrel increased levels of cyclic AMP and inhibited PMA-induced activation of protein kinase C in veins. These findings indicate that levonorgestrel caused endothelium-independent relaxations of jugular veins via inhibition of Ca(2+) entry and of protein kinase C activation. In addition, the cyclic AMP effector pathway contributes to the levonorgestrel-induced relaxation possibly by depressing Ca(2+) entry.     

Angiotensin‐converting enzyme inhibition increases glucose‐induced insulin secretion in response to acute restraint

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Role of gender and estrogen receptors in the rat aorta endothelium-dependent relaxation to red wine polyphenols

Regular intake of moderate amounts of beverages rich in polyphenols such as red wine is associated with a protective effect on the vascular system, in part, by increasing the endothelial formation of nitric oxide (NO), a major vasoprotective factor. Since estrogens are potent inducers of NO formation and polyphenols have been shown to have phytoestrogen properties, we determined whether estrogen receptors mediate the stimulatory effect of red wine polyphenols (RWPs) on the endothelial formation of NO using isolated rat aortic rings and cultured endothelial cells.RWPs caused endothelium-dependent relaxations, which were more pronounced in the aorta of female than male rats. Increased relaxations were also observed to acetylcholine but not to sodium nitroprusside. Relaxations to RWPs were abolished by nitro l-arginine and MnTMPyP, markedly reduced by polyethyleneglycol-catalase and wortmannin, and not affected by the estrogen antagonist ICI 182,780 in aortic rings from males and females. eNOS expression was higher in aortic sections of female than male rats. RWPs caused the phosphorylation of Akt and eNOS in endothelial cells, which was unaffected by ICI 182,780.Thus, RWPs cause redox-sensitive PI3-kinase/Akt-dependent NO-mediated relaxations, which are more pronounced in the aorta of female than male rats; an effect most likely due to the increased expression level of eNOS rather than activation of estrogen receptors.     

GROWTH HORMONE ATTENUATES MYOCARDIAL FIBROSIS IN RATS WITH CHRONIC PRESSURE OVERLOAD-INDUCED LEFT VENTRICULAR HYPERTROPHY

• 1 The role of growth hormone (GH) in cardiac remodelling and function in chronic and persistent pressure overload‐induced left ventricular hypertrophy has not been defined. The aim of the present study was to assess short‐term GH treatment on left ventricular function and remodelling in rats with chronic pressure overload‐induced hypertrophy.
• 2 Twenty‐six weeks after induction of ascending aortic stenosis (AAS), rats were treated with daily subcutaneous injections of recombinant human GH (1 mg/kg per day; AAS‐GH group) or saline (AAS‐P group) for 14 days. Sham‐operated animals served as controls. Left ventricular function was assessed by echocardiography before and after GH treatment. Myocardial fibrosis was evaluated by histological analysis.
• 3 Before GH treatment, AAS rats presented similar left ventricular function and structure. Treatment of rats with GH after the AAS procedure did not change bodyweight or heart weight, both of which were higher in the AAS groups than in the controls. After GH treatment, posterior wall shortening velocity (PWSV) was lower in the AAS‐P group than in the control group. However, in the AAS‐GH group, PWSV was between that in the control and AAS‐P groups and did not differ significantly from either group. Fractional collagen (% of total area) was significantly higher in the AAS‐P and AAS‐GH groups compared with control (10.34 ± 1.29, 4.44 ± 1.37 and 1.88 ± 0.88%, respectively; P < 0.05) and was higher still in the AAS‐P group compared with the AAS‐GH group.
• 4 The present study has shown that short‐term administration of GH to rats with chronic pressure overload‐induced left ventricular hypertrophy induces cardioprotection by attenuating myocardial fibrosis.