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1.
J Cosmet Dermatol ; 22(3): 958-968, 2023 Mar.
Article in English | MEDLINE | ID: mdl-36250215

ABSTRACT

Alternative in vitro methods are important, as there is a call to ban the use of animals in cosmetics research. AIM: To suggest the expansion of the use of in vitro safety techniques recommended by the OECD guidelines and to propose the use of the automation of the in vitro mammalian micronucleus test method by flow cytometry to assess the genotoxic potential of Centella asiatica, Horse Chestnut, Witch Hazel, Blend, Ecoblend, and Caffeine extracts due to their widespread use in commercial products. METHODS: Flow cytometer analysis was performed using the Accuri™ C6 equipment and analyzed using the FlowJo software. Cytotoxicity tests followed OECD 129 guidelines and Phototoxicity followed OECD/GD 432 guidelines. RESULTS: The results showed that the cytotoxicity assay presented a decrease in cell viability when cells were exposed to Centella asiatica from a concentration of 5.0%, horse chestnut 2.5%, Witch hazel 2.5%, Blend 3.13%, and Caffeine 3%, while Ecoblend at the tested concentrations did not show cytotoxicity. In the phototoxicity test, the samples at the tested concentrations showed a PIF <2 being considered potentially non-phototoxic. Finally, in the genotoxicity automated assay, samples were considered potentially non-genotoxic. CONCLUSION: In vitro methods are of paramount importance for the development of pre-clinical tests and the use of test automation helps to reduce the time for analysis and dissemination of results, being a determining factor for the prospect of new compounds.


Subject(s)
Caffeine , Cosmetics , Animals , Flow Cytometry/methods , Micronucleus Tests/methods , DNA Damage , Mammals
2.
Neurotox Res ; 40(6): 1653-1663, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36342586

ABSTRACT

Excessive levels of dopamine in the synaptic cleft, induced by cocaine for example, activates dopaminergic receptors, mainly D1R, D2R, and D3R subtypes, contributing to neurotoxic effects. New synthetic 1-[(2,3-dihydro-1-benzofuran-2-yl)methyl]piperazine derivatives (the LINS01 compounds), designed as histaminergic receptor (H3R) ligands, are also dopaminergic receptor ligands, mainly D2R and D3R. This study aims to evaluate the neurotoxicity of these new synthetic LINS01 compounds (LINS01003, LINS01004, LINS01011, and LINS01018), as well as to investigate their protective potential on a cocaine model of dopamine-induced neurotoxicity using SH-SY5Y cell line culture. Neurotoxicity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and automated cell counting with fluorescent dyes (acridyl orange and propidium iodide) assays. Concentration-response curves (CRCs) were performed for all LINS compounds and cocaine using MTT assay. The results show that LINS series did not decrease cell viability after 48h of exposure-except for 100 µM LINS01018, which was discontinued from the study. Likewise, MTT, LDH, and fluorescent dyes staining showed no difference is cell viability for LINS compounds at 10 µM. When incubated with 2.5 mM cocaine (lethal concentration 50) for 48h, 10 µM of each LINS compound, metoclopramide (D2R antagonist) and haloperidol (D2R/D3R antagonist), ameliorated cocaine-induced neurotoxicity. However, only metoclopramide, haloperidol, and LINS01011 compound significantly decreased LDH released in the culture medium, suggesting that this new synthetic compound presents a more robust effect. This preliminary in vitro neurotoxicity study suggests that LINS01 compounds are not neurotoxic, and that they play a promising role in preventing cocaine-induced neurotoxicity.


Subject(s)
Cocaine , Neuroblastoma , Humans , Cocaine/toxicity , Dopamine , Haloperidol/pharmacology , Metoclopramide , Piperazine , Fluorescent Dyes , Cell Culture Techniques
3.
Mol Cell Biochem ; 476(4): 1849-1859, 2021 Apr.
Article in English | MEDLINE | ID: mdl-33469821

ABSTRACT

Luteolin is a flavonoid with antioxidant properties already demonstrated in studies related to inflammation, tumor, and cardiovascular processes; however, there are no available information regarding its antioxidant effects at the venous endothelial site. We investigated the effects of luteolin (10, 20, and 50 µmol/L) in cultures of rat venous endothelial cells. Nitric oxide (NO) and reactive oxygen species (ROS) were analyzed by fluorimetry; 3-nitrotyrosine (3-NT) residues were evaluated by immunofluorescence, and prostacyclin (PGI2) release was investigated by colorimetry. Intracellular NO levels were significantly enhanced after 10 min of luteolin incubation, with a parallel decrease in ROS generation. These results were accompanied by a significant reduction in the expression of 3-NT residues and enhanced PGI2 rates. Therefore, luteolin is effective in reducing ROS thereby improving NO availability in venous endothelial cells. Besides, luteolin-induced decrease in 3-NT residues may correlate with the enhancement in endothelial PGI2 bioavailability. These findings suggest the future application of this flavonoid as a protective agent by improving endothelial function in several circulatory disorders related to venous insufficiency.


Subject(s)
Antioxidants/pharmacology , Endothelium, Vascular/metabolism , Luteolin/pharmacology , Venae Cavae/metabolism , Animals , Nitric Oxide/metabolism , Rats , Reactive Oxygen Species/metabolism
4.
Inflamm Res ; 68(10): 845-855, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31218444

ABSTRACT

INTRODUCTION: Carboxypeptidase M (CPM) is a glycosylphosphatidylinositol anchored enzyme that plays an important role in the kallikrein-kinin system (KKS). CPM catalytic domain hydrolyzes Arg from C-terminal peptides (i.e., bradykinin and kallidin), generating des-Arg-kinins, the agonists of B1 receptor (B1R). It is known that CPM and kinin B1R are co-localized in the plasma membrane microdomains, where they interact with each other, facilitating receptor signaling. AIMS: We hypothesized here that this CPM-B1R interaction could also affect the activity of the enzyme. METHODS: Thus, in this work, we evaluated the impact of B1R presence or absence on CPM activity and expression, using primary culture of microvascular endothelial cells from wild-type, kinin B1R knockout mice (B 1 -/- ), and transgenic rats overexpressing B1 receptor exclusively in the endothelium. In addition, HEK293T cells, as wells as B 1 -/- primary culture of endothelial cells, both transfected with B1R, were also used. RESULTS: CPM expression and activity were downregulated in cells of knockout mice compared to control and this reduction was rescued after B1R transfection. Cells overexpressing B1R presented higher levels of CPM mRNA, protein, and activity. This profile was reverted by pre-incubation with the B1R antagonist, R715, in highly expressing receptor cells. CONCLUSIONS: Our data show that kinin B1R positively modulates both CPM expression and activity, suggesting that CPM-B1R interaction in membrane microdomains might affect enzyme activity, beyond interfering in receptors signaling. This work highlights the interactions among different components of KKS and contributes to a better understanding of its patho-physiological role.


Subject(s)
Endothelial Cells/metabolism , Metalloendopeptidases/metabolism , Receptor, Bradykinin B1/metabolism , Animals , Cells, Cultured , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Humans , Lung/cytology , Metalloendopeptidases/genetics , Mice, Inbred C57BL , Mice, Knockout , Rats, Sprague-Dawley , Rats, Transgenic , Receptor, Bradykinin B1/genetics
5.
Mediators Inflamm ; 2018: 8597361, 2018.
Article in English | MEDLINE | ID: mdl-30116155

ABSTRACT

BACKGROUND/AIMS: We have previously shown that low birth weight (LBW) rats exposed to intrauterine malnutrition have an impaired lung inflammatory response and reduced levels of inflammatory mediators; however, circulating leptin levels were not increased. We evaluated long leptin receptor isoform (ObRb) expression in lung endothelial cells from low birth weight rats and examined its role in the production of lipid mediators and cytokines. METHODS: Lung endothelial cells were obtained from normal birth weight (NBW) rats or LBW rats subjected to intrauterine malnutrition. These cells were stimulated with leptin (10 ng/mL), LPS (lipopolysaccharide, 1 µg/mL), or leptin plus LPS. Six hours after stimulation, the production of inflammatory mediators (PGE2, LTB4, IL-1ß, and IL-6) was evaluated using commercial ELISA kits, and Western blotting was performed to investigate p38MAPK, NF-κB, and ObRb expression. RESULTS: Leptin increased IL-1ß levels in only cells from the NBW group, whereas LPS increased PGE2 and LTB4 levels in cells from both groups; leptin addition potentiated lipid mediator production induced by LPS in the NBW group. LPS enhanced the production of IL-1ß and IL-6 in only endothelial cells from NBW rats. Leptin receptor expression was decreased (63%) in endothelial cells from LBW rats. None of the stimuli increased NF-κB or p38 signaling pathway expression in cells from LBW rats. CONCLUSION: These results suggest that intrauterine malnutrition compromises leptin receptor expression and cytokine production in pulmonary endothelial cells stimulated by LPS; these effects seem to involve the NF-κB and p38MAPK signaling pathways.


Subject(s)
Endothelial Cells/metabolism , Lung/cytology , Malnutrition , Maternal Nutritional Physiological Phenomena , Receptors, Leptin/metabolism , Animals , Birth Weight , Cytokines/metabolism , Female , Inflammation , Leptin/metabolism , Lipids/chemistry , Lipopolysaccharides , Macrophages/metabolism , Male , NF-kappa B/metabolism , Pregnancy , Pregnancy, Animal , Rats , Rats, Wistar , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism
6.
PeerJ ; 6: e4688, 2018.
Article in English | MEDLINE | ID: mdl-29713565

ABSTRACT

BACKGROUND: Acetylation alters several protein properties including molecular weight, stability, enzymatic activity, protein-protein interactions, and other biological functions. Our previous findings demonstrating that diacetyl/peroxynitrite can acetylate L-lysine, L-histidine, and albumin in vitro led us to investigate whether diacetyl-treated rats suffer protein acetylation as well. METHODS: Wistar rats were administered diacetyl daily for four weeks, after which they were sacrificed, and their lung proteins were extracted to be analysed by Nano-LC-MS/MS (Q-TOF). A C18 reversed-phase column and gradient elution with formic acid/acetonitrile solutions from 2 to 50% over 150 min were used to separate the proteins. Protein detection was performed using a microTOF-Q II (QTOF) equipped with captive source and an electrospray-ionization source. The data from mass spectrometry were processed using a Compass 1.7 and analyzed using Protein Scape, software that uses Mascot algorithms to perform protein searches. RESULTS: A set of 3,162 acetylated peptides derived from 351 acetylated proteins in the diacetyl-treated group was identified. Among them, 23 targeted proteins were significantly more acetylated in the diacetyl-treated group than in the PBS control. Protein acetylation of the group treated with 540 mg/kg/day of diacetyl was corroborated by Western blotting analysis. CONCLUSIONS: These data support our hypothesis that diacetyl exposure in animals may lead to the generation of acetyl radicals, compounds that attach to proteins, affecting their functions and triggering adverse health problems.

7.
Exp Cell Res ; 362(1): 188-194, 2018 01 01.
Article in English | MEDLINE | ID: mdl-29162537

ABSTRACT

The role of the vascular endothelium in modulating the arterial system has been widely investigated, but poorly explored at the venous site. In the present work, primary cultures of venous endothelium from rat Vena Cava (VC) and Portal Vein (PV) were established, characterized and analyzed according to their growth pattern and ability to produce nitric oxide (NO) and prostanoids (PGF2 α and PGI2), at basal state and after stimulation with Angiotensin II (Ang II, 1µmol/L). Basal NO was detected in all examined cells in culture. Pre-incubation with Ang II increased NO production in cells from VC (but not in PV cultures), through activation of both AT1 and AT2 receptors. Both cultures exhibited detectable levels of PGF2 α at resting conditions, which were similarly enhanced by Ang II. Basal PGI2 levels were higher in PV, but increased after Ang II treatment in VC, with no further effect on PV cells. We conclude that endothelial cells from VC and PV exhibit important properties and react to Ang II, probably influencing the whole circulatory system. This experimental cell model gives support to further studies concerning intracellular pathways of the venous endothelium, analyzed in separate from the vascular smooth muscle wall.


Subject(s)
Angiotensin II/pharmacology , Endothelium, Vascular/drug effects , Portal Vein/drug effects , Animals , Cells, Cultured , Endothelial Cells/drug effects , Endothelium, Vascular/metabolism , Male , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Nitric Oxide/metabolism , Portal Vein/metabolism , Rats , Rats, Wistar
8.
Protein Pept Lett ; 22(12): 1111-6, 2015.
Article in English | MEDLINE | ID: mdl-26446563

ABSTRACT

Kinins are important vasoactive peptides, but the role of the B1 receptor subtype in the vascular control is poorly understood. This study analyzed the nitric oxide (NO) release, L-arginine (L-Arg) uptake and the expression of the cationic amino acid transporter (CAT) -1 in endothelial cells obtained from B1 receptor knockout (B1-/-) and wild type (WT) mice. NO production was assessed through a fluorescent dye in living cells stimulated with acetylcholine. L-Arg uptake was determined indirectly in the culture medium by HPLC, in the presence or absence of the CAT-1 blocker N-ethylmaleimide (NEM). CAT-1 mRNA levels and protein expression were determined by qPCR and western blot, respectively. NO release was significantly reduced in B1-/- when compared to WT cells. This result was accompanied by a decreased rate in the L-Arg uptake by B1-/- cells. Incubation with NEM impaired the L-Arg uptake in WT, but had no effect in B1-/- cells. Protein expression and mRNA levels for CAT-1 were reduced in B1-/- in comparison to WT cells. These findings suggest an important role of the endothelial B1 receptor in the vascular control by interfering with CAT-1 expression, L-Arg uptake and NO release.


Subject(s)
Arginine/metabolism , Endothelial Cells/metabolism , Nitric Oxide/metabolism , Receptor, Bradykinin B1/genetics , Receptor, Bradykinin B1/metabolism , Animals , Cationic Amino Acid Transporter 1/genetics , Cationic Amino Acid Transporter 1/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout
9.
J Strength Cond Res ; 29(5): 1349-58, 2015 May.
Article in English | MEDLINE | ID: mdl-25546444

ABSTRACT

The study's purpose was to compare the response of performing 1, 3, and 5 sets on measures of performance and muscle hypertrophy. Forty-eight men, with no weight training experience, were randomly assigned to one of the 3 training groups, 1 SET, 3 SETS, 5 SETS, or control group. All training groups performed 3 resistance training sessions per week for 6 months. The 5 repetition maximum (RM) for all training groups increased in the bench press (BP), front lat pull down (LPD), shoulder press (SP), and leg press (LP) (p ≤ 0.05), with the 5 RM increases in the BP and LPD being significantly greater for 5 SETS compared with the other training groups (p ≤ 0.05). Bench press 20 RM in the 3-SET and 5-SET groups significantly increased with the increase being significantly greater than the 1-SET group and the 5-SET group increase being significantly greater than the 3-SET group (p ≤ 0.05). LP 20 RM increased in all training groups (p ≤ 0.05), with the 5-SETS group showing a significantly greater increase than the 1-SET group (p ≤ 0.05). The 3-SET and 5-SET groups significantly increased elbow flexor muscle thickness (MT) with the 5-SET increase being significantly greater than the other 2 training groups (p ≤ 0.05). The 5-SET group significantly increased elbow extensor MT with the increase being significantly greater than the other training groups (p ≤ 0.05). All training groups decreased percent body fat, increased fat-free mass, and vertical jump ability (p ≤ 0.05), with no differences between groups. The results demonstrate a dose-response for the number of sets per exercise and a superiority of multiple sets compared with a single set per exercise for strength gains, muscle endurance, and upper arm muscle hypertrophy.


Subject(s)
Muscle Strength/physiology , Muscle, Skeletal/anatomy & histology , Muscle, Skeletal/physiology , Physical Endurance/physiology , Resistance Training/methods , Weight Lifting/physiology , Adaptation, Physiological , Adult , Arm , Humans , Hypertrophy , Male , Resistance Training/statistics & numerical data , Time Factors , Young Adult
10.
Rev Bras Ginecol Obstet ; 36(8): 353-8, 2014 Aug.
Article in Portuguese | MEDLINE | ID: mdl-25184348

ABSTRACT

PURPOSE: To evaluate the prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae in women undergoing assisted reproduction in a public reference service in the midwestern region of Brazil. METHODS: A cross-sectional study was conducted on 340 women aged from 20 to 47 years with a history of infertility, undergoing assisted reproduction techniques. Infections with Chlamydia trachomatis and Neisseria gonorrhoeae identified in urine specimens by PCR, and the profile of infertility were analyzed. We used the χ(2) test or Fisher's exact test to evaluate the association between infection and variables. RESULTS: The prevalence of Chlamydia trachomatis infection was 10.9%, and Neisseria gonorrhoeae co-infection was observed in 2 cases. Women infected with Chlamydia trachomatis had more than 10 years of infertility (54.1%; p<0.0001). The tubal factor was the main cause in infected cases (56.8%; p=0.047). Tubal occlusion was found in 67.6% of cases with positive infection (p=0.004). CONCLUSION: There was an association of tubal obstruction with infection by Chlamydia trachomatis and Neisseria gonorrhoeae, reinforcing the need for effective strategies for an early detection of sexually transmitted diseases, especially in asymptomatic women of childbearing age.


Subject(s)
Chlamydia Infections/complications , Chlamydia Infections/epidemiology , Chlamydia trachomatis , Gonorrhea/complications , Gonorrhea/epidemiology , Infertility, Female/microbiology , Adult , Cross-Sectional Studies , Female , Hospitals, Public , Humans , Prevalence , Reproductive Techniques, Assisted , Retrospective Studies
11.
Rev. bras. ginecol. obstet ; 36(8): 353-358, 08/2014. tab
Article in Portuguese | LILACS | ID: lil-720501

ABSTRACT

OBJETIVO: Avaliar a prevalência de infecção por Chlamydia trachomatis e Neisseria gonorrhoeae em mulheres submetidas à reprodução assistida em um serviço público de referência da região Centro-Oeste do Brasil. MÉTODOS: Estudo transversal com 340 mulheres com idade entre 20 e 47 anos, histórico de infertilidade, submetidas às técnicas de reprodução assistida. Foram analisadas as infecções por Chlamydia trachomatis e Neisseria gonorrhoeae detectadas em amostras de urina pela técnica de PCR e o perfil da infertilidade. Utilizou-se o teste do χ2 ou o teste exato de Fisher para avaliar a associação entre a infecção e as variáveis. RESULTADOS: Observou-se prevalência de 10,9% das mulheres com infecção por Chlamydia trachomatis, sendo que houve coinfecção por Neisseria gonorrhoeae em 2 casos. Mulheres infectadas por Chlamydia trachomatis apresentaram mais de 10 anos de infertilidade (54,1%; p<0,0001). O fator tubário foi a principal causa nos casos com infecção (56,8%; p=0,047). A obstrução tubária foi encontrada em 67,6% dos casos com infecção positiva (p=0,004). CONCLUSÃO: Houve associação da obstrução tubária com a infecção por Chlamydia trachomatis e Neisseria gonorrhoeae, reforçando a necessidade de estratégias efetivas para detecção precoce das doenças sexualmente transmissíveis, principalmente em mulheres assintomáticas em idade fértil. .


PURPOSE: To evaluate the prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae in women undergoing assisted reproduction in a public reference service in the midwestern region of Brazil. METHODS: A cross-sectional study was conducted on 340 women aged from 20 to 47 years with a history of infertility, undergoing assisted reproduction techniques. Infections with Chlamydia trachomatis and Neisseria gonorrhoeae identified in urine specimens by PCR, and the profile of infertility were analyzed. We used the χ2 test or Fisher's exact test to evaluate the association between infection and variables. RESULTS: The prevalence of Chlamydia trachomatis infection was 10.9%, and Neisseria gonorrhoeae co-infection was observed in 2 cases. Women infected with Chlamydia trachomatis had more than 10 years of infertility (54.1%; p<0.0001). The tubal factor was the main cause in infected cases (56.8%; p=0.047). Tubal occlusion was found in 67.6% of cases with positive infection (p=0.004). CONCLUSION: There was an association of tubal obstruction with infection by Chlamydia trachomatis and Neisseria gonorrhoeae, reinforcing the need for effective strategies for an early detection of sexually transmitted diseases, especially in asymptomatic women of childbearing age. .


Subject(s)
Adult , Female , Humans , Chlamydia trachomatis , Chlamydia Infections/complications , Chlamydia Infections/epidemiology , Gonorrhea/complications , Gonorrhea/epidemiology , Infertility, Female/microbiology , Cross-Sectional Studies , Hospitals, Public , Prevalence , Reproductive Techniques, Assisted , Retrospective Studies
12.
Rev. bras. med. esporte ; 19(4): 280-286, jul.-ago. 2013. graf, tab
Article in Portuguese | LILACS | ID: lil-686660

ABSTRACT

INTRODUÇÃO: Estudos comparando modelos de periodização em sequências de treinamento resistido (TR) realizadas do menor para o maior grupo muscular sobre as alterações musculares em indivíduos destreinados em TR são escassos. OBJETIVO: Comparar o efeito da periodização ondulatória (PO) e da periodização linear (PL) sobre a força máxima e hipertrofia muscular em uma sequência de execução dos exercícios dos menores para os maiores grupamentos musculares. MÉTODOS: Vinte e nove homens não experientes em (TR) foram distribuídos aleatoriamente em três grupos: PO (n =10), PL (n = 13) e grupo controle (GC, n = 9). Os indivíduos realizaram o teste de uma repetição máxima (1RM) nos exercícios rosca bíceps (RB), rosca tríceps (RT), puxada aberta (PA) e supino reto (SR), contração isométrica voluntária máxima (CIVM) e espessura muscular (EM) para flexores de cotovelo (FC) e extensores de cotovelo (EC) antes e após o período de 12 semanas de treinamento. O grupo PO variou o volume e a intensidade do treinamento diariamente, e o grupo PL a cada quatro semanas. O GC não realizou TR. Foi realizada uma ANOVA de dois caminhos com medidas repetidas, e cálculo do tamanho do efeito (TE), nas cargas obtidas para analisar o efeito do tratamento sobre as variações pré- e pós-período de TR. RESULTADOS: Os principais achados do estudo foram: 1) o grupo PO apresentou maior TE para 1RM dos exercícios RT e RB e para EM dos FC e EC quando comparado ao grupo PL; 2) não houve diferença no TE para os exercícios SR e PA que finalizavam a sessão. CONCLUSÃO: Ambos os modelos de periodização adotados foram eficientes para promover aumentos de força e hipertrofia muscular. Contudo, segundo o cálculo do TE, a PO promoveu maior incremento da força máxima nos exercícios que iniciaram a sessão e hipertrofia muscular.


INTRODUCTION: Studies comparing periodization models in sequences that begin with small muscle group and progressed toward large muscle group in untrained subjects in resistance training are scarce. OBJECTIVE: The purpose of this study was to compare the effects of ondulatory periodization and linear periodization models on maximum strength and muscular hypertrophy in a muscle group increasing exercise sequence. METHODS: Twenty-nine men with no experience in RT were randomly assigned into three groups: ondulatory periodization (OP, n = 10), linear periodization (LP, n = 13), and control group (CG, n = 9). The individuals performed 1RM tests in four exercises: biceps curl (BC), triceps extension (TE), lat pull down (LPD) and bench press (BP) and evaluations of maximum voluntary isometric contraction (MVIC), muscle thickness of elbow flexors (EF) and elbow extensors (EE) before and after the 12 weeks of training were carried out. The OP group varied in volume and intensity on a daily basis, while LP group varied every four weeks. The CG did not perform ST. A two-way ANOVA with repeated measures and the effect size (ES) were used to analyze muscle thickness, 1RM load improvement in each of the four exercises and the MVIC between groups. RESULTS: The major findings of this study were: 1) OP showed major ES for 1RM of BC and TE and for muscle thickness of EF and EE when compared with LP. 3) The ES data did not show significant differences for BP and LPD which finished the training session. CONCLUSIONS: We conclude that both periodization models were efficient at improving strength gains and muscular growth. However, ES data show that OP promotes major gains in strength for exercises that are positioned at the beginning of the session and hypertrophy.

13.
Exp Cell Res ; 319(8): 1102-10, 2013 May 01.
Article in English | MEDLINE | ID: mdl-23416243

ABSTRACT

Endothelial cells from microvasculature are directly involved in a large number of vascular diseases; however, culture of these cells is problematic, since most methodologies employ proteolytic enzymes or mechanical techniques, leading to cell damage and contamination of endothelial cultures with other cellular types. Besides, primary cultured cells have a short life span in vitro and undergo replicative senescence after 3-4 passages, limiting long-term studies. In the present work we report the generation of a spontaneously immortalized endothelial culture obtained from mice pulmonary capillaries. Firstly, primary (third passage) and immortalized (100th) cultures were established. Further, monoclonal populations were obtained by serial dilutions from immortalized cultures. Cells were analyzed according to: (1) morphological appearance, (2) expression of specific endothelial markers by fluorescent staining [von Willebrand Factor (vWF), endothelial nitric oxide synthase (eNOS), angiotensin converting enzyme (ACE) and Ulex europaeus (UEA-1)] and by flow cytometry (endoglin, VE-cadherin and VCAM-1), and (3) release of nitric oxide (NO), assessed by the specific fluorescent dye DAF-2 DA, and prostacyclin (PGI2), quantified by enzyme immune assay. In both cultures cells grew in monolayers and presented cobblestone appearance at confluence. Positive staining for vWF, eNOS, ACE and UEA-1 was detected in cloned as well as in early-passage cultured cells. Similarly, cultures presented equal expressions of endoglin, VE-cadherin and VCAM-1. Values of NO and PGI2 levels did not differ between cultures. From these results we confirm that the described spontaneously immortalized endothelial cell line is capable of unlimited growth and retains typical morphological and functional properties exhibited by primary cultured cells. Therefore, the endothelial cell line described in the present study can become a suitable tool in the field of endothelium research and can be useful for the investigation of production of endothelial mediators, angiogenesis and inflammation.


Subject(s)
Endothelial Cells/cytology , Microcirculation , Primary Cell Culture/methods , Animals , Cell Line, Transformed , Cell Proliferation , Cell Separation/methods , Cell Shape , Cell Transformation, Neoplastic/pathology , Endothelial Cells/pathology , Endothelial Cells/physiology , Flow Cytometry , Lung/blood supply , Male , Mice , Mice, Inbred C57BL , Microcirculation/physiology
14.
J Strength Cond Res ; 26(5): 1389-95, 2012 May.
Article in English | MEDLINE | ID: mdl-22516910

ABSTRACT

The aim of this study was to investigate the effects of nonlinear periodized (NLP) and linear periodized (LP) resistance training (RT) on muscle thickness (MT) and strength, measured by an ultrasound technique and 1 repetition maximum (1RM), respectively. Thirty untrained men were randomly assigned to 3 groups: NLP (n = 11, age: 30.2 ± 1.1 years, height: 173.6 ± 7.2 cm, weight: 79.5 ± 13.1 kg), LP (n = 10, age: 29.8 ± 1.9 years, height: 172.0 ± 6.8 cm, weight: 79.9 ± 10.6 kg), and control group (CG; n = 9, age: 25.9 ± 3.6 years, height: 171.2 ± 6.3 cm, weight: 73.9 ± 9.9 kg). The right biceps and triceps MT and 1RM strength for the exercises bench press (BP), lat-pull down, triceps extension, and biceps curl (BC) were assessed before and after 12 weeks of training. The NLP program varied training biweekly during weeks 1-6 and on a daily basis during weeks 7-12. The LP program followed a pattern of intensity and volume changes every 4 weeks. The CG did not engage in any RT. Posttraining, both trained groups presented significant 1RM strength gains in all exercises (with the exception of the BP in LP). The 1RM of the NLP group was significantly higher than LP for BP and BC posttraining. There were no significant differences in biceps and triceps MT between baseline and posttraining for any group; however, posttraining, there were significant differences in biceps and triceps MT between NLP and the CG. The effect sizes were higher in NLP for the majority of observed variables. In conclusion, both LP and NLP are effective, but NLP may lead to greater gains in 1RM and MT over a 12-week training period.


Subject(s)
Muscle Strength , Muscle, Skeletal/anatomy & histology , Muscle, Skeletal/physiology , Resistance Training/methods , Adult , Arm/physiology , Humans , Male , Muscle, Skeletal/diagnostic imaging , Ultrasonography , Young Adult
15.
Peptides ; 32(10): 2116-21, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21945423

ABSTRACT

To investigate the venoconstrictor effect of angiotensin II (Ang II) in spontaneously hypertensive rats (SHR), we used preparations of mesenteric venular beds and the circular muscle of the portal veins. Vessels were tested with Ang II in the presence or absence of losartan, PD 123319, HOE 140, L-NAME, indomethacin, or celecoxib. In the mesenteric venular bed of SHR, the effect of Ang II (0.1 nmol) was nearly abolished by losartan and enhanced by HOE 140, indomethacin, and celecoxib, while PD123319 and L-NAME had no effect. In portal vein preparations, cumulative-concentration response curves (CCRC) to Ang II (0.1-100 nmol/L) exhibited a lower maximal response (E(max)) in SHR compared to Wistar rats. AT(1) receptor expression was similar in the two strains, while AT(2) receptor levels were lower in SHR portal veins when compared to Wistar. In SHR portal veins, losartan shifted the CCRC to Ang II to the right, while indomethacin and HOE 140 increased the E(max) to Ang II. PD 123319, celecoxib, and L-NAME had no effect. Taken together, our results suggest that Ang II-induced venoconstriction in SHR is mediated by activation of AT(1) receptors and this effect may be counterbalanced by kinin B(2) receptor and COX metabolites. Furthermore, our data indicate that there are different cellular and molecular mechanisms involved in the regulation of venous tonus of normotensive and hypertensive rats. These differences probably reflect distinct factors that influence arterial and venous bed in hypertension.


Subject(s)
Angiotensin II/pharmacology , Hypertension/physiopathology , Mesenteric Veins/drug effects , Mesenteric Veins/physiology , Portal Vein/drug effects , Portal Vein/physiology , Vasoconstriction/drug effects , Animals , Male , Mesenteric Veins/anatomy & histology , Portal Vein/anatomy & histology , Rats , Rats, Inbred SHR , Rats, Wistar
16.
Peptides ; 32(8): 1700-5, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21704095

ABSTRACT

Kinin B(1) and B(2) receptors play an essential role in inflammatory process and cardiovascular homeostasis. The present study investigated the vascular reactivity and nitric oxide (NO) generation in the isolated mesenteric arteriolar bed from B(1) (B(1)(-/-)) and B(2) receptor (B(2)(-/-)) knockout mice. Endothelial-dependent relaxation was significantly decreased in arterioles from both B(1)(-/-) and B(2)(-/-) in comparison to wild type (WT) mice, with no differences for endothelial-independent relaxating or vasoconstrictor agents. Plasmatic and vascular NO production were markedly reduced in both B(1)(-/-) and B(2)(-/-). In contrast, in the presence of l-arginine, Ca(2+) and co-factors for the enzyme, NO synthase activity was higher in homogenates of mesenteric vessels of B(1)(-/-) and B(2)(-/-). The present study demonstrated that targeted deletion of B(1) or B(2) receptor gene in mice induces important alterations in the vascular reactivity of resistance vessels and NO metabolism. The severe impairment in the endothelial-mediated vasodilation accompanied by decreased NO bioavailability, despite the augmented NOS activity, strongly indicates an exacerbation of NO inactivation in B(1)(-/-) and B(2)(-/-) vessels. The present data provide valuable information in order to clarify the relevance of kinin receptors in regulating vascular physiology and may point to new approaches regarding its correlation with endothelial dysfunction, oxidative stress and NO availability.


Subject(s)
Endothelium, Vascular/metabolism , Nitric Oxide/metabolism , Receptor, Bradykinin B1/metabolism , Receptor, Bradykinin B2/metabolism , Animals , Arginine/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Nitric Oxide Synthase/metabolism , Receptor, Bradykinin B1/genetics , Receptor, Bradykinin B2/genetics
17.
J Strength Cond Res ; 24(11): 2962-9, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20938358

ABSTRACT

The purpose of this study was to examine the influence of exercise order on strength and muscle volume (MV) after 12 weeks of nonlinear periodized resistance training. The participants were randomly assigned into 3 groups. One group began performing large muscle group exercises and progressed to small muscle group exercises (LG-SM), whereas another group started with small muscle group exercises and advanced to large muscle group exercises (SM-LG). The exercise order for LG-SM was bench press (BP), machine lat pull-down (LPD), triceps extension (TE), and biceps curl (BC). The order for the SM-LG was BC, TE, LPD, and BP. The third group did not exercise and served as a control group (CG). Training frequency was 2 sessions per week with at least 72 hours of rest between sessions. Muscle volume was assessed at baseline and after 6 weeks and 12 weeks of training by ultrasound techniques. One repetition maximum strength for all exercises was assessed at baseline and after 12 weeks of training. Effect size data demonstrated that differences in strength and MV were exhibited based on exercise order. Both training groups demonstrated greater strength improvements than the CG, but only BP strength increased to a greater magnitude in the LG-SM group as compared with the SM-LG. In all other strength measures (LPD, TE, and BC), the SM-LG group showed significantly greater strength increases. Triceps MV increased in the SM-LG group; however, biceps MV did not differ significantly between the training groups. In conclusion, if an exercise is important for the training goals of a program, then it should be placed at the beginning of the training session, regardless of whether or not it is a large muscle group exercise or a small muscle group exercise.


Subject(s)
Muscle Strength/physiology , Muscle, Skeletal/physiology , Resistance Training/methods , Adipose Tissue/physiology , Adult , Body Height/physiology , Body Weight/physiology , Humans , Male , Muscle, Skeletal/anatomy & histology
18.
Hypertension ; 51(3): 689-95, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18212275

ABSTRACT

Angiotensin-converting enzyme (ACE) is an ectoprotein able to modulate the activity of a plethora of compounds, among them angiotensin I and bradykinin. Despite several decades of research, new aspects of the mechanism of action of ACE have been elucidated, expanding our understanding of its role not only in cardiovascular regulation but also in different areas. Recent findings have ascribed an important role for ACE/kinin B(2) receptor heterodimerization in the pharmacological properties of the receptor. In this work, we tested the hypothesis that this interaction also affects ACE enzymatic activity. ACE catalytic activity was analyzed in Chinese hamster ovary cell monolayers coexpressing the somatic form of the enzyme and the receptor coding region using as substrate the fluorescence resonance energy transfer peptide Abz-FRK(Dnp)P-OH. Results show that the coexpression of the kinin B(2) receptor leads to an augmentation in ACE activity. In addition, this effect could be blocked by the B(2) receptor antagonist icatibant. The hypothesis was also tested in endothelial cells, a more physiological system, where both proteins are naturally expressed. Endothelial cells from genetically ablated kinin B(2) receptor mice showed a decreased ACE activity when compared with wild-type mice cells. In summary, this is the first report showing that the ACE/kinin B(2) receptor interaction modulates ACE activity. Taking into account the interplay among ACE, ACE inhibitors, and kinin receptors, we believe that these results will shed new light into the arena of the controversial search for the mechanism controlling these interactions.


Subject(s)
Peptidyl-Dipeptidase A/metabolism , Receptor, Bradykinin B2/metabolism , Animals , Bradykinin/analogs & derivatives , Bradykinin/pharmacology , Bradykinin B2 Receptor Antagonists , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Female , Mice , Mice, Inbred C57BL , Mice, Knockout , Ovary/drug effects , Ovary/metabolism , Ovary/pathology , Receptor, Bradykinin B2/genetics , Transfection
19.
Int Immunopharmacol ; 8(2): 293-9, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18182243

ABSTRACT

This study characterized pharmacologically the functional responses to agonists angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label at the N-terminal (TOAC1-AngII and TOAC0-BK) and internal (TOAC3-AngII and TOAC3-BK) positions of these vasoactive peptides. Affinity constants of the ligands for AT1 and B2 receptors were evaluated in vitro by binding assays and biological effects by extracellular acidification rates and in vivo by blood pressure responses. In contrast to internally labeled analogues (TOAC3-AngII or TOAC3-BK), the TOAC1-AngII and TOAC0-BK derivatives dose-dependently increased the extracellular acidification rate in adherent cultured Chinese hamster ovary (CHO) cells expressing AT1 or B2 receptors, respectively. In addition, TOAC(1)-AngII induced an increase in blood pressure when injected intravenously in awaken rats although with a potency four times smaller when compared to native AngII. Similarly to BK, TOAC0-BK dose-dependently decreased blood pressure when injected intra-arterially in rats with a lower potency when compared to the native peptide. On the contrary, TOAC3-AngII or TOAC3-BK did not provoke any alteration in blood pressure levels. In summary, our results confirmed that the insertion of TOAC-probe in the N-terminal region of peptides does not significantly modify the affinity or biological activity in vitro and in vivo conditions and could be an important tool to evaluate peptide-receptor interaction mechanism. Conversely, possibly due to the unique bend-inducing property of the cyclic TOAC probe, its insertion at position 3 in both AngII and BK structures seems to restrict the interaction and the activation of the AT1 and B2 receptors.


Subject(s)
Angiotensin II/analogs & derivatives , Bradykinin/analogs & derivatives , Cyclic N-Oxides/pharmacology , Angiotensin II/pharmacology , Animals , Blood Pressure/drug effects , Bradykinin/pharmacology , CHO Cells , Cricetinae , Cricetulus , Male , Rats , Rats, Wistar
20.
Hypertension ; 50(4): 756-61, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17664391

ABSTRACT

We investigated whether angiotensin II infusion modulates in vivo the kinin B1 receptor expression and the mechanisms involved in this process. We also evaluated the role of the B1 receptor activation in aorta. Wistar rats received 400 ng/kg per minute of angiotensin II or saline (control rats) infusion during 14 days through an osmotic minipump. To investigate the role of superoxide anion in B1 receptor expression, rats received a reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor in the drinking water during 14 days (60 mg/L of apocynin) simultaneously with angiotensin II infusion. Angiotensin II induced B1 receptor expression in the aorta and increased significantly systolic blood pressure, superoxide anion, and the nuclear factor kappaB activity. Apocynin treatment did not alter the blood pressure levels of angiotensin II rats and reduced the B1 receptor expression, superoxide anion generation, and nuclear factor kappaB activity to similar levels of the control rats. Vascular reactivity studies in isolated aorta reveal that B1 receptor agonist promoted endothelium-dependent dilation and increased the NO generation in aorta of angiotensin II rats. NO synthase inhibitor and B1 receptor antagonist inhibited the vasodilation and NO generation, which were not affected by B2 receptor antagonist or indomethacin. These results provide evidence that angiotensin II induces B1 receptor expression in aorta by superoxide anion generation, via reduced nicotinamide-adenine dinucleotide phosphate oxidase, concomitant to nuclear factor kappaB activation. We have also shown that B1 receptor agonist causes endothelium-dependent vasodilation through NO production in aortic rings, suggesting that the B1 receptor expression could be related with the vascular tonus control of angiotensin II rats.


Subject(s)
Angiotensin II/physiology , Aorta, Thoracic/metabolism , Receptor, Bradykinin B1/metabolism , Acetophenones/pharmacology , Angiotensin II/administration & dosage , Animals , Aorta, Thoracic/physiology , Blood Pressure/physiology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Hypertension/metabolism , Hypertension/physiopathology , Infusion Pumps, Implantable , Male , NADPH Oxidases/metabolism , NF-kappa B/metabolism , Nitric Oxide/metabolism , Rats , Rats, Wistar , Superoxides/metabolism
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