Three-dimensional imaging of mitochondrial cristae complexity using cryo-soft X-ray tomography.

Mitochondria are dynamic organelles that change morphology to adapt to cellular energetic demands under both physiological and stress conditions. Cardiomyopathies and neuronal disorders are associated with structure-related dysfunction in mitochondria, but three-dimensional characterizations of the organelles are still lacking. In this study, we combined high-resolution imaging and 3D electron density information provided by cryo-soft X-ray tomography to characterize mitochondria cristae morphology isolated from murine. Using the linear attenuation coefficient, the mitochondria were identified (0.247 ± 0.04 µm-1) presenting average dimensions of 0.90 ± 0.20 µm in length and 0.63 ± 0.12 µm in width. The internal mitochondria structure was successfully identified by reaching up the limit of spatial resolution of 35 nm. The internal mitochondrial membranes invagination (cristae) complexity was calculated by the mitochondrial complexity index (MCI) providing quantitative and morphological information of mitochondria larger than 0.90 mm in length. The segmentation to visualize the cristae invaginations into the mitochondrial matrix was possible in mitochondria with MCI ≥ 7. Altogether, we demonstrated that the MCI is a valuable quantitative morphological parameter to evaluate cristae modelling and can be applied to compare healthy and disease state associated to mitochondria morphology.

Rapamycin activates TGF receptor independently of its ligand: implications for endothelial dysfunction.

Rapamycin, the macrolide immunosuppressant and active pharmaceutic in drug-eluting stents (DES), has a well-recognized antiproliferative action that involves inhibition of the mTOR pathway after binding to the cytosolic protein FKBP12. TGF receptor-type I (TGFRI) spontaneous activation is inhibited by the association with FKBP12. We hypothesized that rapamycin, in addition to inhibition of mTOR signaling, activates TGFRI independent of TGFß. Human umbilical vein endothelial cells (HUVECs) were treated with rapamycin (10 nmol/l) and/or TGFß RI kinase inhibitor (TGFRIi, 100 nmol/l) for 24 h. Rapamycin induced SMAD phosphorylation (SMAD1, SMAD2, and SMAD5) and PAI-1 up-regulation, which was specifically abrogated by SMAD2 knockdown. TGFRIi efficiently blocked phosphorylation of SMAD2, but not SMAD1/5. Interestingly, the inhibitor did not alter cell proliferation arrest induced by rapamycin. Active TGFß secretion was not affected by the treatment. Neutralizing TGFß experiments did not influence SMAD2 phosphorylation or PAI-1 expression indicating that activation of this pathway is independent of the ligand. In addition, rapamycin induction of endothelial-to-mesenchymal transition (EndMT) was potentiated by IL-1ß and efficiently blocked by TGFRIi. In vivo, the prothrombogenic effects of rapamycin and up-regulation of PAI-1 in murine carotid arteries were reduced by TGFRIi treatment. In conclusion, we provide evidence that rapamycin activates TGF receptor independent of its ligand TGFß, in concert with promotion of PAI-1 expression and changes in endothelial phenotype. These undesirable effects, the prothrombogenic state, and activation of EndMT are SMAD2-dependent and independent of the therapeutic rapamycin-induced cell proliferation arrest.

Shear stress-induced Ang II AT1 receptor activation: G-protein dependent and independent mechanisms.

Mechanotransduction enables cells to sense and respond to stimuli, such as strain, pressure and shear stress (SS), critical for maintenance of cardiovascular homeostasis or pathological states. The angiotensin II type 1 receptor (AT1R) was the first G protein-coupled receptor described to display stretch-induced activation in cardiomyocytes independent of its ligand Ang II. Here, we assessed whether SS (15 dynes/cm(2), 10 min), an important mechanical force present in the cardiovascular system, activates AT1R independent of its ligand. SS induced extracellular signal-regulated kinase (ERK) activation, used as a surrogate of AT1R activation, in Chinese hamster ovary cells expressing the AT1R (CHO+AT1) but not in wild type cells (CHO). AT1R dependent SS-induced ERK activation involves Ca(2+) inflow and activation of Gαq since Ca(2+) chelator EGTA or Gαq-specific inhibitor YM-254890 decreased SS-induced ERK activation. On the other hand, the activation of JAK-2 and Src, two intracellular signaling molecules independent of G protein activation, were not differently modulated in the presence of AT1R. Also, ERK activation by SS was observed in CHO cells expressing the mutated AT1R DRY/AAY, which has impaired ability to activate Gαq dependent intracellular signaling. Altogether we provided evidence that SS activates AT1R in the absence of its ligand by both a G protein-dependent and -independent pathways. The biological relevance of these observations deserves to be further investigated since the novel mechanisms described extend the knowledge of the activation of GPCRs independent of its traditional ligand.

Thioredoxin interacting protein genetic variation is associated with diabetes and hypertension in the Brazilian general population.

OBJECTIVE: To investigate the relationship between TXNIP polymorphisms, diabetes and hypertension phenotypes in the Brazilian general population. METHODS: Five hundred seventy-six individuals randomly selected from the general urban population according to the MONICA-WHO project guidelines were phenotyped for cardiovascular risk factors. A second, independent, sample composed of 487 family-trios from a different site was also selected. Nine TXNIP polymorphisms were studied. The potential association between TXNIP variability and glucose-phenotypes in children was also explored. TXNIP expression was quantified by real-time PCR in 53 samples from human smooth muscle cells primary culture. RESULTS: TXNIP rs7211 and rs7212 polymorphisms were significantly associated with glucose and blood pressure related phenotypes. In multivariate logistic regression models the studied markers remained associated with diabetes even after adjustment for covariates. TXNIP rs7211 T/rs7212 G haplotype (present in approximately 17% of individuals) was significantly associated to diabetes in both samples. In children, the TXNIP rs7211 T/rs7212 G haplotype was associated with fasting insulin concentrations. Finally, cells harboring TXNIP rs7212 G allele presented higher TXNIP expression levels compared with carriers of TXNIP rs7212 CC genotype (p=0.02). CONCLUSION: Carriers of TXNIP genetic variants presented higher TXNIP expression, early signs of glucose homeostasis derangement and increased susceptibility to chronic metabolic conditions such as diabetes and hypertension. Our data suggest that genetic variation in the TXNIP gene may act as a "common ground" modulator of both traits: diabetes and hypertension.

A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures.

beta-Galactosidase (beta-Gal) activity is a widely accepted biomarker to detect senescence both in situ and in vitro. A cytochemical assay based on production of a blue-dyed precipitate that results from the cleavage of the chromogenic substrate X-Gal is commonly used. Blue and nonblue cells are counted under the microscope and a semiquantitative percentage of senescent cells can be obtained. Here, we present a quantitative, fast, and easy to use chemiluminescent assay to detect senescence. The Galacton chemiluminescent method used to detect the prokaryotic beta-Gal reporter enzyme in transfection studies was adapted to assay mammalian beta-Gal. The assay showed linear production of luminescence in a time- and cell-number-dependent manner. The chemiluminescent assay showed significant correlation with the cytochemical assay in detecting replicative senescence (Pearson r=0.8486, p<0.005). Moreover, the chemiluminescent method (Galacton) also detected stress-induced senescence in cells treated with H2O2 similar to the cytochemical assay (X-Gal) (Galacton: control 25,207.3+/-6548.6, H2O2 52,487.4+/-16,284.9, p<0.05; X-Gal: control 41.31+/-7.0%, H2O2 92.97+/-2.8%, p<0.01). Thus, our method is well suited to the detection of replicative and stress-induced senescence in cell culture.

Local TAT-p27Kip1 fusion protein inhibits cell proliferation in rat carotid arteries.

INTRODUCTION: p27(Kip1) is a cyclin kinase inhibitor that induces cell cycle arrest. In this study, the efficacy of fusion protein TAT- p27(Kip1) to inhibit cell proliferation in rat perivascular injured carotid arteries was tested. METHODS: The cDNA of p27(Kip1) and GFP (green fluorescein protein) fused to the TAT epitope, which allows cell penetration, yielded TAT-p27 (Kip1) and TAT-GFP fusion proteins. In vitro biological activity on cell proliferation was evaluated by [(3)H] thymidine DNA incorporation in rabbit aortic endothelial cells (REC). An in vivo model used a silicone collar filled with saline positioned around the carotid vessel for 14 days to produce an increased adventitia cross-sectional area. RESULTS: TAT-p27(Kip1) inhibited REC proliferation in vitro using either 100, 200, and 500 nM compared to control (88.2 +/- 4.4, 81.3 +/- 7, 71.9 +/- 4.2 vs. 100 +/- 6.7%, N = 3, respectively, p < 0.05). This response was stable for purified proteins stored at -20*C for at least 23 days. In vivo , TAT-p27(Kip1) solution reduced adventitia cross-sectional area in a dose-dependent manner compared to TAT-GFP (area in mm(2) - TAT-p27(Kip1): 200 nM, 0.160 +/- 0.018; 500 nM, 0.050 +/- 0.005 vs. TAT-GFP: 500 nM, 0.595 +/- 0.066 vs. the contralateral: 0.047 +/- 0.005, N = 7, p < 0.01). CONCLUSION: Taken together, these results provide evidence that TAT-p27(Kip1) can inhibit vascular cells proliferation. It is the first successful demonstration that the cell permeable TAT-p27(Kip1) has potential as a vascular anti-proliferative agent.

Dynamic regulation of MTHFR mRNA expression and C677T genotype modulate mortality in coronary artery disease patients after revascularization.

INTRODUCTION: A large body of evidence links plasma homocysteine (Hcy) concentrations and cardiovascular disease. A common MTHFR polymorphism (C677T) leads to a variant with reduced activity and associated with increased Hcy levels. Coronary surgery precipitates a significant and sustained increase in the blood concentrations of Hcy and elevated levels of plasma Hcy have been associated to saphenous vein (SV) graft disease after CABG. However, the effects of MTHFR genotypes in the incidence of cardiovascular events after CABG have not been investigated prospectively. Here, we investigate whether MTHFR gene variants are associated with an increased cardiovascular risk in individuals submitted to CABG. We also propose a molecular mechanism to explain our findings. METHODS: We performed MTHFR C677T genotypes in 558 patients with two or three vessel-disease and normal left ventricular function prospectively followed in the MASS II Trial, a randomized study to compare treatments for multivessel CAD and preserved left ventricle function. Follow-up time was 5 years. Survival curves were calculated with the Kaplan-Meier method, and evaluated with the log-rank statistic. We assessed the relationship between baseline variables and the composite end-point of death, myocardial infarction and refractory angina using a Cox proportional hazards survival model. Finally, using an ex-vivo organ culture we have reproduced the arterialization of SV implants by culturing human SV either under venous hemodynamic condition (flow: 5 mL/min; no pressure) or arterial hemodynamic condition (flow: 50 mL/min; pressure: 80 mm Hg) for 1 day. MTHFR gene expression was quantified by real time RT-PCR in 15 SV from different individuals in both experimental conditions. RESULTS: There were no significant differences among individuals within each genotype group for baseline clinical characteristics. A statistically significant association between the TT genotype, associated with increased serum levels of Hcy, and cardiovascular mortality after 5 years was verified (p=0.007) in individuals submitted to CABG surgery. In addition, MTHFR TT genotype was still significantly associated with a 4.4 fold increased risk in cardiovascular outcomes (p=0.01) even after adjustment of a Cox multivariate model for age, sex, hypertension, diabetes, LDL, HDL, triglycerides, and number of diseased vessels in this population. Finally, a significant reduction in MTHFR gene expression was demonstrated in human SV when submitted to an arterial hemodynamic condition (p=0.02). CONCLUSIONS: There is a dynamic regulation of MTHFR gene expression during the arterialization process of human saphenous vein grafts resulting in lower levels of gene expression when in an arterial hemodynamic condition. In addition, the C677T MTHFR functional variant is associated with a worse outcome in individuals submitted to CABG. Taken together, these data suggest an important role of Hcy metabolism in individuals after CABG.

Ação inibitória da Interleucina - 1ß sobre a proliferação de células musculares lisas cultivadas a partir de veias safenas humanas / Inhibitory action of the Interleukin 1ß over the cellular proliferation of smooth muscle cells cultivated from human saphenous veins

INTRODUÇAO: A veia safena é um enxerto coronário eficiente. Porém, sua patência pode ser limitada por desenvolvimento de aterosclerose. Estudos experimentais "ex vivo", por nós realizados anteriormente, demonstraram apoptose (ensaio de TUNEL) em veias safenas humanas cultivadas sob pressão arterial por 24 horas. Nessas veias safenas, a expressão gênica da Interleucina-1ß avaliada por RT-PCR em tempo real, também mostrou-se elevada. Não há ainda consenso sobre a ação moduladora das citocinas sobre proliferação/apoptose das células musculares lisas das veias safenas. OBJETIVO: Avaliar a influência da Interleucina -1ß na proliferação inicial de cultura de células primárias de músculo liso de veia safena humana. MÉTODO: Foram cultivadas células primárias de músculo liso de seis diferentes veias safenas humanas (em triplicata). O meio de cultura foi o DMEM, suplementado com 10 por cento de soro fetal bovino. O grupo controle não recebeu Interleucina - 1ß. Nos demais grupos, as células cultivadas receberam, respectivamente, 0,1; 1; 10 e 100 ng/mL de Interleucina - 1ß. A proliferação celular foi avaliada através da quantificação de timidina triciada [ H], incorporada às células recém-proliferadas. RESULTADOS: O tratamento com Interleucina - 1ß diminuiu a proliferação celular, a saber: Grupo controle (sem Interleucina - 1ß): definiu-se esse grupo como apresentando 100±4,5 por cento de proliferação celular. Nos demais grupos, a quantidade de Interleucina - 1ß administrada e a proliferação celular aferida foram, respectivamente, 0,1 ng/mL:112±0,7 por cento; 1 ng/mL:83,8±4,7 por cento; 10 ng/mL:69,1±3,8 por cento; 100 ng/mL:67,3±10,9 por cento; (p<0,01). CONCLUSÕES: Estes resultados indicam que a administração de quantidade crescente de Interleucina - 1ß inibe a proliferação de células primárias de músculo liso, cultivadas a partir de veias safenas humanas. Isso sugere que o processo de apoptose, observado já em fase precoce (um dia) de exposição do enxerto venoso a regime pressórico arterial, pode estar relacionado à ação dessa citocina.

Alterações estruturais e moleculares (cDNA) precoces em veias safenas humanas cultivadas sob regime pressórico arterial / Precocious structural and molecular (cDNA) changes in the human saphenous veins cultivated under arterial hemodynamic conditions

OBJETIVO: A veia safena (VS) empregada na revascularização do miocárdio (RM) fica submetida a estresse tênsil elevado e contínuo. Sua resposta adaptativa à nova condição hemodinâmica pode predispor à oclusão do enxerto. Este trabalho visou verificar as alterações estruturais precoces e moleculares (cDNA) entre VS humanas submetidas a baixo regime pressórico versus condições hemodinâmicas sistêmicas. MÉTODO: Quarenta segmentos de VS foram cultivados "ex-vivo" sob condição hemodinâmica venosa (CHV) (sem pressão, fluxo:5 ml/min) e sob condição hemodinâmica arterial (CHA) (pressão: 80mmHg, fluxo:50mL/min). Foram analisadas: viabilidade celular (coloração MTT), densidade celular (coloração hoechst 33258) e apoptose (ensaio TUNEL), antes e um, dois e quatro dias após o procedimento. Determinamos alvos moleculares alterados precocemente nas veias cultivadas sob condição arterial, através da análise "cDNA microarray" de segmentos das VS. A busca desses alvos foi realizada através de pool homogeneizado do RNA desses segmentos venosos, interagindo por homologia em lâmina contendo 16000 genes humanos pré-determinados (Agilent Technologies slide). Os genes com expressão alterada foram certificados por PCR em tempo real, em veias de 16 diferentes indivíduos. RESULTADOS: Houve diminuição gradual da densidade celular e da viabilidade tecidual nas VS cultivadas mediante CHA, enquanto nenhuma alteração ocorreu quando a veia foi cultivada até quatro dias na CHV. No grupo sob CHA houve sinais de processo apoptótico celular (TUNEL-positivo) já a partir do 1° dia de cultivo, o que não ocorreu no outro grupo. A densidade celular das veias sob regime arterial, decorridas 24h de cultivo, era similar à das amostras frescas das mesmas, mas inúmeras células já apresentavam indícios de processo apoptótico. Os alvos moleculares mais alterados(de acordo com o PCR em tempo real) e selecionados para pesquisa foram o Oncogene 3 e a Interleucina 1ß. A expressão do Oncogene 3 estava elevada em 11 (68,7 por cento) das veias cultivadas sob regime arterial, enquanto observou-se aumento da expressão da Interleucina 1ß em nove (56,2 por cento) desses segmentos venosos (p<0,05). CONCLUSÃO: O modelo de estudo "ex vivo" permitiu mimetizar os eventos iniciais sofridos "in vivo" pela VS utilizada na RM. No grupo CHA houve perda de viabilidade precoce das células (apoptose) e elevação significativa nas expressões gênicas do Oncogene 3 e da Interleucina 1ß. O seguimento em longo prazo desses...

Interaction of a non-peptide agonist with angiotensin II AT1 receptor mutants.

To identify residues of the rat AT1A angiotensin II receptor involved with signal transduction and binding of the non-peptide agonist L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-isobutyl-3-thienyl]phenyl]methyl]imidazol[4,5,6]-pyridine) we have performed ligand binding and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G1961, G196W, and D278E. Compound L-162,313 displaced [125I]-Sar1,Leu8-AngII from the mutants G196I and G196W with IC50 values similar to that of the wild-type. The affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. In inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G1961 mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain of the AT1 receptor are involved in the binding of the non-peptide ligand, or in a general receptor activation phenomenon that involves conformational modifications for a preferential binding of agonists or antagonists.