Extracellular vesicles as biomarkers and biovectors in primary aldosteronism

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Glycosylation with O-linked β-N-acetylglucosamine (O-GlcNAc) induces vascular dysfunction via production of superoxide anion/reactive oxygen species

Overproduction of superoxide anion (•O2-) and O-linked β-N-acetylglucosamine (O-GlcNAc)-modification in the vascular system are contributors to endothelial dysfunction. This study tested the hypothesis that increased levels of O-GlcNAc-modified proteins contribute to •O2- production via activation of NADPH oxidase, resulting in impaired vasodilation. Rat aortic segments and vascular smooth muscle cell (VSMCs) were incubated with vehicle (methanol) or PUGNAc (100 µM). PUGNAc produced a time-dependent increase in O-GlcNAc levels in VSMC and decreased endothelium-dependent relaxation, which was prevented by apocynin and Tiron, suggesting that •O2- contributes to endothelial dysfunction under augmented O-GlcNAc levels. Aortic segments incubated with PUGNAc also exhibited increased levels of ROS, assessed by dihydroethidium fluorescence, and augmented •O2- production, determined by lucigenin-enhanced chemiluminescence. Additionally, PUGNAc treatment increased Nox1 and Nox4 protein expression in aorta and VSMCs. Translocation of p47phox subunit from the cytosol to the membrane was greater in aortas incubated with PUGNAc. VSMCs displayed increased p22phox protein expression after PUGNAc incubation, suggesting that NADPH oxidase is activated in conditions where O-GlcNAc protein levels are increased. In conclusion, O-GlcNAc levels reduce endothelium-dependent relaxation by overproduction of •O2- via activation of NADPH oxidase. This may represent an additional mechanism by which augmented O-GlcNAc levels impair vascular function

Chemerin receptor blockade improves vascular function in diabetic obese mice via redox-sensitive- and Akt-dependent pathways

Chemerin and its G protein-coupled receptor [chemerin receptor 23 (ChemR23)] have been associated with endothelial dysfunction, inflammation, and insulin resistance. However, the role of chemerin on insulin signaling in the vasculature is still unknown. We aimed to determine whether chemerin reduces vascular insulin signaling and whether there is interplay between chemerin/ChemR23, insulin resistance, and vascular complications associated with type 2 diabetes (T2D). Molecular and vascular mechanisms were probed in mesenteric arteries and cultured vascular smooth muscle cells (VSMCs) from C57BL/6J, nondiabetic lean db/m, and diabetic obese db/db mice as well as in human microvascular endothelial cells (HMECs). Chemerin decreased insulin-induced vasodilatation in C57BL/6J mice, an effect prevented by CCX832 (ChemR23 antagonist) treatment. In VSMCs, chemerin, via oxidative stress- and ChemR23-dependent mechanisms, decreased insulin-induced Akt phosphorylation, glucose transporter 4 translocation to the membrane, and glucose uptake. In HMECs, chemerin decreased insulin-activated nitric oxide signaling. AMP-activated protein kinase phosphorylation was reduced by chemerin in both HMECs and VSMCs. CCX832 treatment of db/db mice decreased body weight, insulin, and glucose levels as well as vascular oxidative stress. CCX832 also partially restored vascular insulin responses in db/db and high-fat diet-fed mice. Our novel in vivo findings highlight chemerin/ChemR23 as a promising therapeutic target to limit insulin resistance and vascular complications associated with obesity-related diabetes

Upregulation of Nrf2 and decreased redox signaling contribute to renoprotective effects of chemerin receptor blockade in diabetic mice

Chemerin, acting through its receptor ChemR23, is an adipokine associated with inflammatory response, glucose and lipid metabolism and vascular function. Although this adipokine has been associated with the development and progression of kidney disease, it is not clear whether the chemerin/ChemR23 system plays a role in renal function in the context of diabetes. Therefore, we sought to determine whether ChemR23 receptor blockade prevents the development and/or progression of diabetic nephropathy and questioned the role of oxidative stress and Nrf2 in this process. Renal redox state and function were assessed in non-diabetic lean db/m and diabetic obese db/db mice treated with vehicle or CCX832 (ChemR23 antagonist). Renal reactive oxygen species (ROS) production, which was increased in diabetic mice, was attenuated by CCX832. This was associated with an increase in Nox 4 expression. Augmented protein oxidation in db/db mice was not observed when mice were treated with CCX832. CCX832 also abrogated impaired Nrf2 nuclear activity and associated downregulation in antioxidants expression in kidneys from db/db mice. Our in vivo findings highlight the role of the redox signaling and Nrf2 system as renoprotective players during chemerin receptor blockade in diabetic mice. The chemerin/ChemR23 system may be an important target to limit renal dysfunction associated with obesity-related diabetes

Reduced lymphatic reserve in heart failure with preserved ejection fraction

Background: Microvascular dysfunction plays an important role in the pathogenesis of heart failure with preserved ejection fraction (HFpEF). However, no mechanistic link between systemic microvasculature and congestion, a central feature of the syndrome, has yet been investigated. Objectives: This study aimed to investigate capillary–interstitium fluid exchange in HFpEF, including lymphatic drainage and the potential osmotic forces exerted by any hypertonic tissue Na+ excess. Methods: Patients with HFpEF and healthy control subjects of similar age and sex distributions (n = 16 per group) underwent: 1) a skin biopsy for vascular immunohistochemistry, gene expression, and chemical (water, Na+, and K+) analyses; and 2) venous occlusion plethysmography to assess peripheral microvascular filtration coefficient (measuring capillary fluid extravasation) and isovolumetric pressure (above which lymphatic drainage cannot compensate for fluid extravasation). Results: Skin biopsies in patients with HFpEF showed rarefaction of small blood and lymphatic vessels (p = 0.003 and p = 0.012, respectively); residual skin lymphatics showed a larger diameter (p = 0.007) and lower expression of lymphatic differentiation and function markers (LYVE-1 [lymphatic vessel endothelial hyaluronan receptor 1]: p < 0.05; PROX-1 [prospero homeobox protein 1]: p < 0.001) compared with control subjects. In patients with HFpEF, microvascular filtration coefficient was lower (calf: 3.30 [interquartile range (IQR): 2.33 to 3.88] l × 100 ml of tissue–1 × min–1 × mm Hg–1 vs. 4.66 [IQR: 3.70 to 6.15] μl × 100 ml of tissue–1 × min–1 × mm Hg–1; p < 0.01; forearm: 5.16 [IQR: 3.86 to 5.43] l × 100 ml of tissue–1 × min–1 × mm Hg–1 vs. 5.66 [IQR: 4.69 to 8.38] μl × 100 ml of tissue–1 × min–1 × mm Hg–1; p > 0.05), in keeping with blood vascular rarefaction and the lack of any observed hypertonic skin Na+ excess, but the lymphatic drainage was impaired (isovolumetric pressure in patients with HFpEF vs. control subjects: calf 16 ± 4 mm Hg vs. 22 ± 4 mm Hg; p < 0.005; forearm 17 ± 4 mm Hg vs. 25 ± 5 mm Hg; p < 0.001). Conclusions: Peripheral lymphatic vessels in patients with HFpEF exhibit structural and molecular alterations and cannot effectively compensate for fluid extravasation and interstitial accumulation by commensurate drainage. Reduced lymphatic reserve may represent a novel therapeutic target

TNF-α induces vascular insulin resistance via positive modulation of PTEN and decreased Akt/eNOS/NO signaling in high fat diet-fed mice

Abstract\ud \ud Background\ud High fat diet (HFD) induces insulin resistance in various tissues, including the vasculature. HFD also increases plasma levels of TNF-α, a cytokine that contributes to insulin resistance and vascular dysfunction. Considering that the enzyme phosphatase and tension homologue (PTEN), whose expression is increased by TNF-α, reduces Akt signaling and, consequently, nitric oxide (NO) production, we hypothesized that PTEN contributes to TNF-α-mediated vascular resistance to insulin induced by HFD. Mechanisms underlying PTEN effects were determined.\ud \ud \ud Methods\ud Mesenteric vascular beds were isolated from C57Bl/6J and TNF-α KO mice submitted to control or HFD diet for 18 weeks to assess molecular mechanisms by which TNF-α and PTEN contribute to vascular dysfunction.\ud \ud \ud Results\ud Vasodilation in response to insulin was decreased in HFD-fed mice and in ex vivo control arteries incubated with TNF-α. TNF-α receptors deficiency and TNF-α blockade with infliximab abolished the effects of HFD and TNF-α on insulin-induced vasodilation. PTEN vascular expression (total and phosphorylated isoforms) was increased in HFD-fed mice. Treatment with a PTEN inhibitor improved insulin-induced vasodilation in HFD-fed mice. TNF-α receptor deletion restored PTEN expression/activity and Akt/eNOS/NO signaling in HFD-fed mice.\ud \ud \ud Conclusion\ud TNF-α induces vascular insulin resistance by mechanisms that involve positive modulation of PTEN and inhibition of Akt/eNOS/NO signaling. Our findings highlight TNF-α and PTEN as potential targets to limit insulin resistance and vascular complications associated with obesity-related conditions.This work was supported by grants from Fundação de Amparo à Pesquisa\ud do Estado de São Paulo (FAPESP 2013/08216-2-CRID), Coordenação de Aper‑\ud feiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de\ud Desenvolvimento Científico e Tecnológico (CNPq), Brazil

Role of chemerin/ChemR23 system on vascular insulin signaling in C57BL/6J and db/db mice

Chemerin e seu receptor (ChemR23) têm sido amplamente associados à disfunção endotelial, inflamação e resistência à insulina. No entanto, é ainda desconhecido se chemerin influencia diretamente a sinalização da insulina na vasculatura. A hipótese deste estudo é de que chemerin diminui a sinalização vascular da insulina, e que o uso de antagonista de ChemR23 (CCX 832) em um modelo de diabetes do tipo 2 relacionado à obesidade melhora as respostas vasculares a insulina. Mecanismos moleculares e vasculares foram investigados em artérias mesentéricas e células de músculo liso vascular em cultura (CMLV) de camundongos C57BL/6J, db/m (controles, não obesos, não diabéticos) e db/db (diabéticos, obesos), assim como em células endoteliais (CE) de humanos em cultura. Nossos resultados mostraram que chemerin diminui a vasodilatação induzida por insulina em camundongos C57BL/6J, efeito mediado por ChemR23, PI3K/Akt e estresse oxidativo. Em CMLV, chemerin, através de mecanismos dependentes de estresse oxidativo e ChemR23, diminui a fosforilação de IRS-1, PI3K e Akt e a translocação de GLUT4 para a membrana, induzidas por insulina. Chemerin também diminui a captação de glicose induzida por insulina via estresse oxidativo e ativação de AMPK e PI3K/Akt. Em CE, chemerin diminui a sinalização de óxido nítrico (NO) ativada pela insulina, novamente via ChemR23, estresse oxidativo e PI3K/Akt. CCX 832 diminui a massa corporal (sem alterar a ingestão de ração), os níveis de insulina e glicose (sem alterar a tolerância à glicose) e estresse oxidativo em aorta e rim de camundongos db/db. CCX 832 restaura parcialmente a disfunção vascular observada em camundongos db/db, sem modificar parâmetros estruturais destas artérias. Adicionalmente, CCX 832 diminui marcadores pró-inflamatórios em tecido adiposo perivascular (PVAT) e melhora a sinalização da insulina em aorta de camundongos db/db. Nossos achados destacam o sistema chemerin/ChemR23 como um novo e promissor alvo terapêutico para limitar a resistência à insulina e as complicações vasculares associadas ao diabetes relacionado à obesidade.Chemerin and its G protein-coupled receptor (ChemR23) have been associated with endothelial dysfunction, inflammation and insulin resistance. Whether chemerin directly influences insulin signaling in the vasculature is unknown. We hypothesized that chemerin impairs vascular insulin signaling in obesity-related type 2 diabetes, effect that would be improved by the novel ChemR23 antagonist (CCX 832). Molecular and vascular mechanisms were probed in mesenteric arteries and cultured vascular smooth muscle cells (VSMC) from C57BL/6J, non-diabetic lean db/m and diabetic obese db/db mice as well as in human microvascular endothelial cells (EC). Chemerin decreased insulin-induced vasodilatation in C57BL/6J mice, effect mediated by ChemR23, PI3K/Akt and oxidative stress. In VSMC, chemerin, via oxidative stress- and ChemR23-dependent mechanisms, decreased insulin-induced IRS-1, PI3K and Akt phosphorylation, GLUT4 translocation to the membrane. In addition, chemerin decreases insulin-induced glucose uptake via oxidative stress and AMPK and PI3K/Akt activation. In EC, chemerin decreased insulin-activated nitric oxide (NO) signaling via ChemR23, oxidative stress and PI3K/Akt signaling pathway. CCX 832 decreases body weight (without altering food intake), insulin and glucose levels (without altering glucose tolerance) and oxidative stress in aorta and kidney from db/db mice. CCX 832 partially restored vascular dysfunction in db/db mice without modifying structural parameters. Additionally, CCX 832 decreases proinflammatory markers in perivascular adipose tissue (PVAT) and improves insulin signaling in aorta from db/db mice. Our findings highlight chemerin/ChemR23 system as a promising new therapeutic target to limit insulin resistance and vascular complications associated with obesity-related diabetes

Effects of the adipokine chemerin on the vascular reactivity: analysis in the rat aorta

Embora seja na obesidade onde se observa hipertrofia e hiperplasia dos adipócitos e aumento da síntese e liberação de adipocinas, condição associada com resistência à insulina e disfunção endotelial, é de suma importância entender os efeitos biológicos de adipocinas, mais especificamente da adipocina chemerin, em condições não patológicas. Os mecanismos pelos quais as citocinas liberadas pelo tecido adiposo podem interferir na função vascular ainda não estão totalmente esclarecidos. Além disso, praticamente não se conhecem os efeitos da citocina/adipocina chemerin sobre a função vascular. Levando-se em consideração que o receptor para chemerin está presente no músculo liso vascular e no endotélio, este trabalho avaliou a atividade biológica e celular desta adipocina sobre a vasculatura de animais não obesos. Investigou-se os efeitos produzidos por esta citocina na reatividade vascular, bem como os mecanismos pelos quais ela modifica a função vascular em animais não obesos. A hipótese deste trabalho é que chemerin aumenta a reatividade vascular a estímulos constritores de endotelina-1 (ET-1) e fenilefrina (PhE) e diminui a vasodilatação induzida pela acetilcolina (ACh) e nitroprussitao de sódio (NPS). Nossos objetivos específicos incluíram determinar: 1) se chemerin promove alterações na reatividade vascular; 2) se as alterações de reatividade vascular promovidas por chemerin são mediadas por modificações da função das células endoteliais ou células de músculo liso vascular; 3) quais vias de sinalização (foco na via das MAPKs) estão sendo modificadas por chemerin e como elas contribuem para as alterações de reatividade vascular produzidas por esta citocina. Nosso estudo demonstrou que a adipocina chemerin possui atividade biológica e celular em aortas de ratos não obesos. Chemerin aumentou respostas vasculares a estímulos contráteis (ET-1 e PhE), atuando tanto no endotélio quanto diretamente em células do músculo liso vascular. O aumento da resposta a estímulos contráteis à ET-1 e PhE foi mediado pela via MEK-ERK1/2, COX-1 e COX-2 e aumento da expressão dos receptores para ET-1, ETA e ETB. Além disso, esta adipocina diminuiu a vasodilatação induzida pela ACh, por meio do desacoplamento da eNOS e aparente envolvimento de estresse oxidativo, e pelo NPS, através de ação sobre a guanilato ciclase. Nossos estudos poderão contribuir para um melhor entendimento sobre o papel dos fatores liberados pelo tecido adiposo visceral sobre a função vascular e, consequentemente, sobre as alterações vasculares presentes na obesidade e patologias associadas.Although hypertrophy and hyperplasia of adipocytes as well as increased synthesis and release of adipokines are commonly observed in obesity, a condition associated with insulin resistance and endothelial dysfunction, it is extremely important to understand the biological effects of adipokines, or more specifically of the adipokine chemerin, in non-pathological conditions,. The mechanisms by which cytokines released by the adipose tissue may interfere with vascular function are not yet fully understood. Furthermore, the effects of the cytokine/adipokine chemerin on vascular function are not known. Considering that the chemerin receptor is expressed by vascular smooth muscle and endothelial cells, this study investigated the effects produced by this cytokine in vascular reactivity, as well as the mechanisms by which it modifies vascular function in non-obese animals. Our working hypothesis is that chemerin enhances vascular reactivity to constrictor stimuli, such as endothelin-1(ET-1) and phenylephrine (Phe), and decreases the vasodilation induced by acetylcholine (ACh) and sodium nitroprussiate (SNP). Our specific aims were to determine: 1) whether chemerin induces changes in vascular reactivity, 2) if the alterations of vascular reactivity induced by chemerin are mediated by changes in the function of endothelial cells or vascular smooth muscle cells, 3) which signaling pathways (focus on the MAPKs pathway) are being modified by chemerin and how they contribute to changes in vascular reactivity produced by this cytokine. Our study showed that the adipokine chemerin has biological and cellular activity in aortas from non-obese rats. Chemerin increased vascular responses to contractile stimuli (ET-1 and PhE), producing effects both in the endothelial and vascular smooth muscle cells. The increased contractile responses to ET-1 and PhE were mediated via activation of MEK-ERK1/2, COX-1 and COX-2 and increased expression of the ETA and ETB receptors. Furthermore, this adipokine reduced the vasodilation induced by ACh via eNOS uncoupling and oxidative stress, and by SNP, via effects in the enzyme guanylate cyclase. Our studies may contribute to a better understanding of the role of factors released by the visceral adipose tissue on vascular function and, consequently, on the vascular lesions in obesity and obesity-associated diseases

TNF-α induces vascular insulin resistance via positive modulation of PTEN and decreased Akt/eNOS/NO signaling in high fat diet-fed mice

Abstract Background High fat diet (HFD) induces insulin resistance in various tissues, including the vasculature. HFD also increases plasma levels of TNF-α, a cytokine that contributes to insulin resistance and vascular dysfunction. Considering that the enzyme phosphatase and tension homologue (PTEN), whose expression is increased by TNF-α, reduces Akt signaling and, consequently, nitric oxide (NO) production, we hypothesized that PTEN contributes to TNF-α-mediated vascular resistance to insulin induced by HFD. Mechanisms underlying PTEN effects were determined. Methods Mesenteric vascular beds were isolated from C57Bl/6J and TNF-α KO mice submitted to control or HFD diet for 18 weeks to assess molecular mechanisms by which TNF-α and PTEN contribute to vascular dysfunction. Results Vasodilation in response to insulin was decreased in HFD-fed mice and in ex vivo control arteries incubated with TNF-α. TNF-α receptors deficiency and TNF-α blockade with infliximab abolished the effects of HFD and TNF-α on insulin-induced vasodilation. PTEN vascular expression (total and phosphorylated isoforms) was increased in HFD-fed mice. Treatment with a PTEN inhibitor improved insulin-induced vasodilation in HFD-fed mice. TNF-α receptor deletion restored PTEN expression/activity and Akt/eNOS/NO signaling in HFD-fed mice. Conclusion TNF-α induces vascular insulin resistance by mechanisms that involve positive modulation of PTEN and inhibition of Akt/eNOS/NO signaling. Our findings highlight TNF-α and PTEN as potential targets to limit insulin resistance and vascular complications associated with obesity-related conditions

Chemerin Regulates Crosstalk Between Adipocytes and Vascular Cells Through Nox

Adipocytes produce adipokines, including chemerin, a chemoattractant that mediates effects through its ChemR23 receptor. Chemerin has been linked to endothelial dysfunction and vascular injury in pathological conditions, such as obesity, diabetes mellitus, and hypertension. Molecular mechanisms underlying this are elusive. Here we assessed whether chemerin through redox-sensitive signaling influences molecular processes associated with vascular growth, apoptosis, and inflammation. Human microvascular endothelial cells and vascular smooth muscle cells were stimulated with chemerin (50 ng/mL). Chemerin increased generation of reactive oxygen species and phosphorylation of mitogen-activated protein kinases, effects that were inhibited by ML171, GKT137831 (Nox inhibitors), and N-acetylcysteine (reactive oxygen species scavenger). Chemerin increased mRNA expression of proinflammatory mediators in vascular cells and increased monocyte-to-endothelial cell attachment. In human vascular smooth muscle cells, chemerin induced phosphorylation of mitogen-activated protein kinases and stimulated proliferation (increased proliferating cell nuclear antigen expression [proliferation marker] and BrdU incorporation [proliferation assay]). Chemerin decreased phosphatidylinositol 3-kinase/protein kinase B activation and increased TUNEL-positive human vascular smooth muscle cells. In human microvascular endothelial cells, chemerin reduced endothelial nitric oxide synthase activity and nitric oxide production. Adipocyte-conditioned medium from obese/diabetic mice (db/db), which have elevated chemerin levels, increased reactive oxygen species generation in vascular smooth muscle cells, whereas adipocyte-conditioned medium from control mice had no effect. Chemerin actions were blocked by CCX 832, a ChemR23 inhibitor. Our data demonstrate that chemerin, through Nox activation and redox-sensitive mitogen-activated protein kinases signaling, exerts proapoptotic, proinflammatory, and proliferative effects in human vascular cells. These findings elucidate some molecular mechanisms through chemerin, which is increased in obesity, whereby adipocytes may influence vascular function. We identify chemerin as a novel vasoactive adipokine, which may be important in obesity-related vascular injury