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To evaluate whether the chronic effect of photobiomodulation therapy (PBM) on systolic arterial pressure (SAP) from two kidneys one clip (2 K-1C) hypertension animal models can cause a hypotensive effect. Serum levels of nitric oxide were also analyzed and the assessment of lipid peroxidation of the thoracic aorta artery. Male Wistar rats were used. Hypertensive animals (2 K-1C) with Systolic arterial pressure (SAP) greater than or equal to 160 mmHg were used. Systolic arterial pressure (SAP) was determined by the tail plethysmography technique. Normotensive (2 K) and hypertensive (2 K-1C) rats were treated to PBM for 4 weeks using a laser whose irradiation parameters were: red wavelength (λ) = 660 nm: operating continuously; 56 s per point (3 points) spot size = 0.0295 cm2; average optical power of 100 mW; energy of 5.6 J per point; irradiance of 3.40 W/cm2; fluency of 190 J/cm2 per point. The application was on the animals tails, at 3 different points simultaneously, in contact with the skin. To assess serum nitrite and nitrate (NOx) levels, blood collection was performed after chronic PBM treatment, 24 h after the last laser application. The evaluation of the lipid peroxidation of the thoracic aorta artery was performed by measuring the concentration of hydroperoxide by the FOX method. Chronic photobiomodulation therapy (PBM) by red laser (660 nm) can induce a hypotensive effect in 64% of 2 K-1C hypertensive animals, which we say responsive animals. There was no difference in serum NO levels 24 h after the last red laser application, between treated and non-treated groups. Aortic rings from 2 K-1C hypertensive animals present a higher lipid peroxidation. The chronic PBM treatment by red laser decreased aortic rings lipid peroxidation in hypertensive responsive groups, compared to control. our results indicate that chronic PBM made by red laser has an important hypotensive effect in renovascular hypertensive models, by a mechanism that involves decrease in oxidative stress from vascular beds.

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Resumo Fundamento A formação de células espumosas ocorre devido ao aumento em lipoproteína plasmática de baixa densidade (LDL) e desregulação da inflamação, sendo importante para o desenvolvimento da aterosclerose. Objetivo Avaliar o perfil do fator de necrose tumoral alfa (TNF-α) e da interleucina-6 (IL-6) no método de formação da célula espumosa existente, otimizando esse protocolo. Métodos A LDL foi isolada, oxidada e marcada com sonda de isotiocianato de fluoresceína (FITC). As células espumosas foram geradas de célula derivada de monócitos humanos THP-1 e incubadas na ausência (controle) ou presença de FITC-ox-LDL (10, 50, 100, 150 ou 200 μg/mL), por 12, 24, 48 ou 72 horas. A FITC-ox-LDL na célula foi quantificada por microscopia. O ensaio de imunoabsorção enzimática foi avaliado para quantificar a IL-6 e o TNF-α, com um p <0,05 considerado significativo. Resultados Todas as concentrações de FITC-ox-LDL testadas apresentaram fluorescência mais alta em comparação com o controle, demonstrando maior acúmulo de lipoproteínas nas células. Quanto mais alta a concentração de FITC-ox-LDL, maior a produção de TNF-α e IL-6. A produção de IL-6 pelas células espumosas foi detectada até o valor de 150 µg/mL da LDL máxima de estímulo. Concentrações acima de 50 μg/mL de LDL estimularam maior liberação de TNF-α comparado ao controle. Conclusões Nosso modelo contribui para o entendimento da liberação de IL-6 e TNF-α em resposta a várias concentrações de ox-LDL usando o método otimizado para a formação de células espumosas.

Abstract Background The formation of foam cells occurs due to the increase in low-density plasma lipoprotein (LDL) and dysregulation of inflammation, which is important for the development of atherosclerosis. Objective To evaluate the profile of tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6) in the existing foam cell formation method, optimizing this protocol. Methods The LDL was isolated, oxidized, and labeled with a Fluorescein isothiocyanate (FITC) probe. Foam cells were generated from THP-1 human monocyte-derived cells and incubated in the absence (control) or presence of FITC-ox-LDL (10, 50, 100, 150, or 200 μg/mL), for 12, 24, 48, or 72 hours. The accumulated FITC-ox-LDL in the cell was quantified by microscopy. The enzyme-linked immunosorbent assay was evaluated to quantify the IL-6 and TNF-α, with p < 0.05 considered significant. Results All the FITC-ox-LDL concentrations tested showed a higher fluorescence when compared to the control, showing a greater accumulation of lipoprotein in cells. The higher the concentration of FITC-ox-LDL, the greater the production of TNF-α and IL-6. The production of IL-6 by foam cells was detected up to the value of 150 µg/mL of the maximum stimulus for LDL. Concentrations above 50 μg/mL LDL stimulated greater release of TNF-α compared to control. Conclusions Our model contributes to the understanding of the release of IL-6 and TNF-α in response to different concentrations of ox-LDL, using an optimized method for the formation of foam cells.

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BACKGROUND: The formation of foam cells occurs due to the increase in low-density plasma lipoprotein (LDL) and dysregulation of inflammation, which is important for the development of atherosclerosis. OBJECTIVE: To evaluate the profile of tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6) in the existing foam cell formation method, optimizing this protocol. METHODS: The LDL was isolated, oxidized, and labeled with a Fluorescein isothiocyanate (FITC) probe. Foam cells were generated from THP-1 human monocyte-derived cells and incubated in the absence (control) or presence of FITC-ox-LDL (10, 50, 100, 150, or 200 µg/mL), for 12, 24, 48, or 72 hours. The accumulated FITC-ox-LDL in the cell was quantified by microscopy. The enzyme-linked immunosorbent assay was evaluated to quantify the IL-6 and TNF-α, with p < 0.05 considered significant. RESULTS: All the FITC-ox-LDL concentrations tested showed a higher fluorescence when compared to the control, showing a greater accumulation of lipoprotein in cells. The higher the concentration of FITC-ox-LDL, the greater the production of TNF-α and IL-6. The production of IL-6 by foam cells was detected up to the value of 150 µg/mL of the maximum stimulus for LDL. Concentrations above 50 µg/mL LDL stimulated greater release of TNF-α compared to control. CONCLUSIONS: Our model contributes to the understanding of the release of IL-6 and TNF-α in response to different concentrations of ox-LDL, using an optimized method for the formation of foam cells.

FUNDAMENTO: A formação de células espumosas ocorre devido ao aumento em lipoproteína plasmática de baixa densidade (LDL) e desregulação da inflamação, sendo importante para o desenvolvimento da aterosclerose. OBJETIVO: Avaliar o perfil do fator de necrose tumoral alfa (TNF-α) e da interleucina-6 (IL-6) no método de formação da célula espumosa existente, otimizando esse protocolo. MÉTODOS: A LDL foi isolada, oxidada e marcada com sonda de isotiocianato de fluoresceína (FITC). As células espumosas foram geradas de célula derivada de monócitos humanos THP-1 e incubadas na ausência (controle) ou presença de FITC-ox-LDL (10, 50, 100, 150 ou 200 µg/mL), por 12, 24, 48 ou 72 horas. A FITC-ox-LDL na célula foi quantificada por microscopia. O ensaio de imunoabsorção enzimática foi avaliado para quantificar a IL-6 e o TNF-α, com um p <0,05 considerado significativo. RESULTADOS: Todas as concentrações de FITC-ox-LDL testadas apresentaram fluorescência mais alta em comparação com o controle, demonstrando maior acúmulo de lipoproteínas nas células. Quanto mais alta a concentração de FITC-ox-LDL, maior a produção de TNF-α e IL-6. A produção de IL-6 pelas células espumosas foi detectada até o valor de 150 µg/mL da LDL máxima de estímulo. Concentrações acima de 50 µg/mL de LDL estimularam maior liberação de TNF-α comparado ao controle. CONCLUSÕES: Nosso modelo contribui para o entendimento da liberação de IL-6 e TNF-α em resposta a várias concentrações de ox-LDL usando o método otimizado para a formação de células espumosas.

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The aim of this study was to evaluate the participation of nitric oxide (NO) in the hypotensive and vasorelaxation effect induced by PBM using an aluminum gallium arsenide (AlGaAs) diode laser (660 nm). Male Wistar rats were treated with the inhibitor of nitric oxide synthase (L-NAME). A red laser (660 nm; 63 J/cm2; 56 s/point) was applied to the abdominal region at six different points. Thoracic aorta was dissected for vascular reactivity study, and a laser (660 nm; 96 J/cm2; 56 s) was applied after incubation with the NO donor DETA-NO, PBS, or hydroxicobalamin. Endothelial cells (HUVEC) were treated with DETA-NO or CuSO4, and then, PBM (63 J/cm2) was applied, and the nitric oxide was detected. Hypertensive L-NAME rats did not exhibit a decrease in blood pressure after PBM. PBM promoted vasodilation in the aorta isolated from normotensive rats, and less effect in the aorta of L-NAME rats and the addition of the NO donor, DETA-NO, promoted greater vasodilation by PBM in the aorta of L-NAME rats. In endothelial cells, an increase in NO, after PBM, was detected; however, with the addition of CuSO4, which catalyzes the decomposition of NO storage, there was no detection of NO after PBM. The results of this study demonstrate that the hypotensive and vasodilatory effect of PBM with a red laser at 660 nm is modulated by the release of nitric oxide from the storage.

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Abstract Background: The endothelium is a monolayer of cells that extends on the vascular inner surface, responsible for the modulation of vascular tone. By means of the release of nitric oxide (NO), the endothelium has an important protective function against cardiovascular diseases. Objective: Verify if cis- [Ru(bpy)2(NO2)(NO)](PF6)2 (BPY) improves endothelial function and the sensibility of conductance (aorta) and resistance (coronary) to vascular relaxation induced by BPY. Methods: Normotensive (2K) and hypertensive (2K-1C) Wistar rats were used. For vascular reactivity study, thoracic aortas were isolated, rings with intact endothelium were incubated with: BPY(0.01 to10 µM) and concentration effect curves to acetylcholine were performed. In addition, cumulative concentration curves were performed to BPY (1.0 nM to 0.1 µM) in aortic and coronary rings, with intact and denuded endothelium. Results: In aorta from 2K-1C animals, the treatment with BPY 0.1µM increased the potency of acetylcholine-induced relaxation and it was able to revert the endothelial dysfunction. The presence of the endothelium did not modify the effect of BPY in inducing the relaxation in aortas from 2K and 2K-1C rats. In coronary, the endothelium potentiated the vasodilator effect of BPY in vessels from 2K and 2K-1C rats. Conclusion: Our results suggest that 0.1 µM of BPY is able to normalize the relaxation endothelium dependent in hypertensive rats, and the compound BPY induces relaxation in aortic from normotensive and hypertensive rats with the same potency. The endothelium potentiate the relaxation effect induced by BPY in coronary from normotensive and hypertensive rats, with lower effect on coronary from hypertensive rats.

Resumo Fundamento: O endotélio é uma monocamada de células que se estende sobre a superfície interna vascular, responsável pela modulação do tônus vascular. Por meio da liberação de óxido nítrico (NO), o endotélio tem uma função protetora importante contra doenças cardiovasculares. Objetivo: Verificar se o cis- [Ru (BPY)2 (NO2) (NO)] (PF6) 2 (BPY) melhora a função endotelial e a sensibilidade da condutância (aorta) e da resistência (coronária) ao relaxamento vascular induzido por BPY. Métodos: Foram utilizados ratos Wistar normotensos (2K) e hipertensos (2K-1C). Para o estudo de reatividade vascular, as aortas torácicas foram isoladas, os anéis com endotélio intacto foram incubados com: BPY (0,01 a 10 µM) e se realizaram curvas de efeito de concentração para acetilcolina. Adicionalmente, foram feitas curvas de concentração cumulativas para BPY (1,0 nM a 0,1 µM) nos anéis aórticos e coronários, com endotélio intacto e nu. Resultados: Na aorta de animais 2K-1C, o tratamento com BPY 0,1 µM aumentou a potência do relaxamento induzido pela acetilcolina e foi capaz de reverter a disfunção endotelial. A presença do endotélio não modificou o efeito da BPY na indução do relaxamento em aortas de ratos 2K e 2K-1C. Na coronária, o endotélio potencializou o efeito vasodilatador do BPY em vasos de ratos 2K e 2K-1C. Conclusão: Nossos resultados sugerem que 0,1 µM de BPY é capaz de normalizar o relaxamento dependente do endotélio em ratos hipertensos, e o composto BPY induz relaxamento na aorta de ratos normotensos e hipertensos com a mesma potência. O endotélio potencializa o efeito de relaxamento induzido pela BPY em coronárias de ratos normotensos e hipertensos, com menor efeito em coronárias de ratos hipertensos.

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BACKGROUND:: The endothelium is a monolayer of cells that extends on the vascular inner surface, responsible for the modulation of vascular tone. By means of the release of nitric oxide (NO), the endothelium has an important protective function against cardiovascular diseases. OBJECTIVE:: Verify if cis- [Ru(bpy)2(NO2)(NO)](PF6)2 (BPY) improves endothelial function and the sensibility of conductance (aorta) and resistance (coronary) to vascular relaxation induced by BPY. METHODS:: Normotensive (2K) and hypertensive (2K-1C) Wistar rats were used. For vascular reactivity study, thoracic aortas were isolated, rings with intact endothelium were incubated with: BPY(0.01 to10 µM) and concentration effect curves to acetylcholine were performed. In addition, cumulative concentration curves were performed to BPY (1.0 nM to 0.1 µM) in aortic and coronary rings, with intact and denuded endothelium. RESULTS:: In aorta from 2K-1C animals, the treatment with BPY 0.1µM increased the potency of acetylcholine-induced relaxation and it was able to revert the endothelial dysfunction. The presence of the endothelium did not modify the effect of BPY in inducing the relaxation in aortas from 2K and 2K-1C rats. In coronary, the endothelium potentiated the vasodilator effect of BPY in vessels from 2K and 2K-1C rats. CONCLUSION:: Our results suggest that 0.1 µM of BPY is able to normalize the relaxation endothelium dependent in hypertensive rats, and the compound BPY induces relaxation in aortic from normotensive and hypertensive rats with the same potency. The endothelium potentiate the relaxation effect induced by BPY in coronary from normotensive and hypertensive rats, with lower effect on coronary from hypertensive rats. FUNDAMENTO:: O endotélio é uma monocamada de células que se estende sobre a superfície interna vascular, responsável pela modulação do tônus vascular. Por meio da liberação de óxido nítrico (NO), o endotélio tem uma função protetora importante contra doenças cardiovasculares. OBJETIVO:: Verificar se o cis- [Ru (BPY)2 (NO2) (NO)] (PF6) 2 (BPY) melhora a função endotelial e a sensibilidade da condutância (aorta) e da resistência (coronária) ao relaxamento vascular induzido por BPY. MÉTODOS:: Foram utilizados ratos Wistar normotensos (2K) e hipertensos (2K-1C). Para o estudo de reatividade vascular, as aortas torácicas foram isoladas, os anéis com endotélio intacto foram incubados com: BPY (0,01 a 10 µM) e se realizaram curvas de efeito de concentração para acetilcolina. Adicionalmente, foram feitas curvas de concentração cumulativas para BPY (1,0 nM a 0,1 µM) nos anéis aórticos e coronários, com endotélio intacto e nu. RESULTADOS:: Na aorta de animais 2K-1C, o tratamento com BPY 0,1 µM aumentou a potência do relaxamento induzido pela acetilcolina e foi capaz de reverter a disfunção endotelial. A presença do endotélio não modificou o efeito da BPY na indução do relaxamento em aortas de ratos 2K e 2K-1C. Na coronária, o endotélio potencializou o efeito vasodilatador do BPY em vasos de ratos 2K e 2K-1C. CONCLUSÃO:: Nossos resultados sugerem que 0,1 µM de BPY é capaz de normalizar o relaxamento dependente do endotélio em ratos hipertensos, e o composto BPY induz relaxamento na aorta de ratos normotensos e hipertensos com a mesma potência. O endotélio potencializa o efeito de relaxamento induzido pela BPY em coronárias de ratos normotensos e hipertensos, com menor efeito em coronárias de ratos hipertensos.

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PURPOSE: Verify if sodium nitroprusside (SNP) is able to improve endothelial function and if this effect is independent of nitric oxide (NO) release of the compound. METHODS: Normotensive (2K) and hypertensive (2K-1C) wistar rats were used. Intact endothelium aortas were placed in a myograph and incubated with SNP: 0.1nM; 1nM or 10nM during 30min. Cumulative concentration-effect curves for acetylcholine (Ach) were realized to measure the relaxing capacity. Intracellular NO were measured (by DAF-2DA probe) in HUVEC treated with SNP 0.1nM or DETA/NO 0.1µM. The detection of intracellular superoxide radical (O2•-) was obtained by using DHE probe. RESULTS: Treatment of 2K-1C aortic rings with SNP (0.1; 1.0 and 10nM) improved endothelium dependent relaxation induced by acetylcholine. This improvement induced by SNP was verified at the concentration of 0.1nM, which does not release NO, suggesting that this effect was not induced due to NO release by SNP compound. Besides, we show that the cell treatment with 0.1nM of SNP decreased the fluorescence intensity to DHE in cells stimulated with angiotensin II. These results indicate that SNP decreases the concentration of O2•- in HUVEC cells. CONCLUSIONS: The SNP at a concentration that does not release NO inside the cells is able to attenuate endothelial dysfunction. DRUGS AND CHEMICALS: Acetylcholine (Ach) (PubChem CID:6060); angiotensin II human (Ang II) (PubChem CID: 16211177); diethylenetriamine/nitric oxide (DETA-NO) (PubChem CID 4518); dihydroethidium (DHE) (PubChem CID: 128682); phenylephrine (Phe) (PubChem CID: 5284443); sodium nitroprusside (SNP) (PubChem CID: 11963579); Thiazolyl Blue Tetrazolium Bromide (MTT) (PubChem CID: 64965); 4,5-diaminofluorescein diacetate (DAF-2DA); 4-hidroxy-Tempo (Tempol) (PubChem CID: 137994), were purchased from Sigma-Aldrich (St. Louis, MO, USA).

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