Ester Tobías

Contraction of human hepatic stellate cells activated in culture: A role for voltage-operated calcium channels

BACKGROUND/AIMS Voltage-operated calcium channels are essential for the regulation of vascular tone and are potential targets for vasodilating agents. They regulate calcium entry and thereby cell contraction in vascular cell types. Hepatic stellate cells in the activated phenotype have contractile properties and could participate in the regulation of sinusoidal blood flow. Thus, this study was aimed at investigating the presence of voltage-operated calcium channels in human hepatic stellate cells activated in culture and the effects of their stimulation on intracellular calcium concentration ([Ca2+]i) and cell contractility. METHODS Binding studies using [3H]-nitrendipine were performed to demonstrate the presence of voltage-operated calcium channels. Voltage-operated calcium channels were stimulated by causing cell membrane depolarization either by electrical field stimulation or extracellular high potassium. [Ca2+]i and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method with an epifluorescence microscope coupled to a charge-coupled device-imaging system. RESULTS Binding studies demonstrated the existence of voltage-operated calcium channels in human activated hepatic stellate cells (7.1+/-1.4x10(4) sites/cell with a Kd of 2.1+/-0.1 nM). Both electrical field stimulation and potassium chloride-induced cell depolarization resulted in a marked and prolonged increase in [Ca2+]i followed by intense cell contraction. The degree of cell contraction correlated with the intensity of calcium peaks. Removal of extracellular calcium or preincubation of cells with nitrendipine, a specific antagonist of voltage-operated calcium channels, completely blocked the effects on [Ca2+]i and cell contraction, whereas preincubation of cells with BayK-8644, a specific agonist of voltage-operated calcium channels, increased calcium peaks and contraction. CONCLUSION Activated human hepatic stellate cells have a large number of voltage-operated calcium channels, the activation of which is associated with an increase in [Ca2+]i followed by marked cell contraction. Voltage-operated calcium channels probably play an important role in the regulation of activated hepatic stellate cells contractility.

Molecular basis of reduced birth weight in smoking pregnant women: mitochondrial dysfunction and apoptosis

In utero exposure of fetuses to tobacco is associated with reduced birth weight. We hypothesized that this may be due to the toxic effect of carbon monoxide (CO) from tobacco, which has previously been described to damage mitochondria in non‐pregnant adult smokers. Maternal peripheral blood mononuclear cells (PBMCs), newborn cord blood mononuclear cells (CBMCs) and placenta were collected from 30 smoking pregnant women and their newborns and classified as moderate and severe smoking groups, and compared to a cohort of 21 non‐smoking controls. A biomarker for tobacco consumption (cotinine) was assessed by ELISA (enzyme‐linked immunosorbent assay). The following parameters were measured in all tissues: mitochondrial chain complex IV [cytochrome c oxidase (COX)] activity by spectrophotometry, mitochondrial DNA levels by reverse transcription polymerase chain reaction, oxidative stress by spectrophotometric lipid peroxide quantification, mitochondrial mass through citrate synthase spectrophotometric activity and apoptosis by Western blot parallelly confirmed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) assay in placenta. Newborns from smoking pregnant women presented reduced birth weight by 10.75 percent. Materno‐fetal mitochondrial and apoptotic PBMC and CBMC parameters showed altered and correlated values regarding COX activity, mitochondrial DNA, oxidative stress and apoptosis. Placenta partially compensated this dysfunction by increasing mitochondrial number; even so ratios of oxidative stress and apoptosis were increased. A CO‐induced mitotoxic and apoptotic fingerprint is present in smoking pregnant women and their newborn, with a lack of filtering effect from the placenta. Tobacco consumption correlated with a reduction in birth weight and mitochondrial and apoptotic impairment, suggesting that both could be the cause of the reduced birth weight in smoking pregnant women.

Mitochondrial disturbances in HIV pregnancies.

Background:Mitochondrial consequences from foetal exposure to HIV infection and antiretrovirals could be further investigated. Objective:The main objective of this study was to evaluate maternofoetal mitochondrial disturbances in HIV infection and antiretroviral administration in human pregnancies as the aetiopathogenic basis of suboptimal perinatal-clinical features. Design:Cross-sectional, prospective, observational, exploratory and controlled study. Methods:Clinical/epidemiological data of 35 HIV-infected pregnant women and 17 controls were collected. Mitochondrial DNA (mtDNA) and RNA (mtRNA) content (real time-PCR), enzymatic activities and content (spectrophotometry) were measured in leucocytes. Genetic-functional, maternofoetal and molecular-clinical correlations were assessed. Results:Birth weight was lower in infants from HIV-infected mothers compared with controls. MtDNA values were slightly decreased in HIV cases, although not reaching statistical significance. MtRNA values were lower in HIV-infected mothers. Similarly, binary complex II+III enzymatic activity decreased to 50% in both HIV-infected mothers (44.45 ± 3.77%) and their infants (48.79 ± 3.41%) (P = 0.001 and P < 0.001). Global CI+III+IV enzymatic activity was lower in HIV-infected mothers and infants (90.43 ± 2.39% and 51.16 ± 9.30%) (P < 0.005 and P < 0.05). MtDNA content correlated with function in mothers and infants. Maternofoetal parameters correlated at genetic and functional levels. Conclusion:HAART toxicity caused mitochondrial damage in HIV-infected pregnant women and their newborns, being present at a genetic and functional level with a maternofoetal correlation.

Mitochondrial impact of human immunodeficiency virus and antiretrovirals on infected pediatric patients with or without lipodystrophy.

We determined the mitochondrial status of a group of HIV-infected children, some with body fat abnormalities (BFA). We included 24 controls, 16 HIV-infected untreated, 26 HIV-infected treated, 6 BFA-untreated, and 21 BFA-treated patients. Genetic, translational, and functional mitochondrial values were measured. As compared with controls, mitochondrial DNA depletion and a reduction in functionality were found in BFA groups.

Mitochondrial evolution in HIV-infected children receiving first- or second-generation nucleoside analogues.

Background:Highly active antiretroviral therapy (HAART) and HIV-related mitochondrial toxicity lead to several adverse effects and have become a major issue, especially in children. The main goal in the treatment of HIV-infected children is to maximize cost-effectiveness while minimizing toxicity. We aimed to study the evolution of mitochondrial parameters over time in children receiving different types antiretroviral regimens. Methods:We followed-up 28 HIV-infected children receiving HAART including either first-generation nucleoside reverse transcriptase inhibitors (1gNRTIs; didanosine, zidovudine, or stavudine; n = 15) or second-generation NRTIs (2gNRTIs; the remaining drugs; n = 13) for a period of 2 years for their immunovirological and mitochondrial status, and compared these subjects with a group of untreated HIV-infected patients (n = 10) and uninfected controls (n = 27). We measured T-lymphocyte CD4+ content (flow cytometry), viral load (real-time polymerase chain reaction), and lactate levels (spectrophotometry); we assessed mtDNA content (real-time polymerase chain reaction), mitochondrial protein levels (Western blot), oxidative stress, mitochondrial mass, and electron transport chain function (spectrophotometry) in peripheral blood mononuclear cells. Results:At the second time point, lactate levels were significantly higher in children on 1gNRTIs compared with those receiving 2gNRTIs (1.28 ± 0.08 vs. 1.00 ± 0.07 mmol/L, respectively; P = 0.022). MtDNA content was similar among all HIV-infected groups and significantly lower than in healthy controls at baseline. Oxidative stress tended to increase over time in all the groups, with no differences among them. However, a significant decrease in cytochrome c oxidase activity was found over time in HIV-infected patients; this decline was greater in the 1gNRTIs group. Conclusions:HIV infection and the use of 1gNRTIs caused greater mitochondrial damage than 2gNRTIs over time. The higher lactate levels and the significant decrease observed in cytochrome c oxidase activity argue against the use of 1gNRTIs in HIV-infected children when an alternative is available, in accordance with international recommendations.

Study of oxidative, enzymatic mitochondrial respiratory chain function and apoptosis in perinatally HIV-infected pediatric patients.

Abstract Mitochondrial toxicity in perinatally human immunodeficiency virus (HIV)-infected pediatric patients has been scarcely investigated. Limited data are available about HIV or antiretroviral (ARV)-mediated mitochondrial damage in this population group, specifically, regarding oxygen consumption and apoptosis approach. We aimed to elucidate whether a given mitochondrial DNA depletion is reflected at downstream levels, to gain insight on the pathology of HIV and highly active antiretroviral therapy (HAART) in perinatally HIV-infected pediatric patients. We studied 10 healthy control participants and 20 perinatally HIV-infected pediatric patients (10 under ARV treatment and 10 off treatment). We determined mitochondrial mass, subunits II and IV of complex IV, global and specific mitochondrial enzymatic and oxidative activities, and apoptosis from peripheral blood mononuclear cells. Global oxygen consumption was significantly compromised in HIV-infected untreated patients, compared to the control group (0.76 ± 0.01 versus 1.59 ± 0.15; P = 0.014). Apoptosis showed a trend to increase in untreated patients as well. The overall complex (C) CI-III-IV activity of the mitochondrial respiratory chain (MRC) was significantly decreased in HIV-infected treated patients with respect to the control group (1.52 ± 0.38 versus 6.38 ± 1.53; P = 0.02). No statistically significant differences were found between untreated and HAART-treated patients. These findings suggest the pathogenic role of both HIV and HAART in mitochondrial dysfunction in vertical infection. The abnormalities in mitochondrial genome may be downstream reflected through a global alteration of the MRC. Mitochondrial impairment associated with HIV and HAART was generalized, rather than localized, in this series of perinatally HIV-infected patients.

Mitochondrial DNA disturbances and deregulated expression of oxidative phosphorylation and mitochondrial fusion proteins in sporadic inclusion body myositis.

Sporadic inclusion body myositis (sIBM) is one of the most common myopathies in elderly people. Mitochondrial abnormalities at the histological level are present in these patients. We hypothesize that mitochondrial dysfunction may play a role in disease aetiology. We took the following measurements of muscle and peripheral blood mononuclear cells (PBMCs) from 30 sIBM patients and 38 age- and gender-paired controls: mitochondrial DNA (mtDNA) deletions, amount of mtDNA and mtRNA, mitochondrial protein synthesis, mitochondrial respiratory chain (MRC) complex I and IV enzymatic activity, mitochondrial mass, oxidative stress and mitochondrial dynamics (mitofusin 2 and optic atrophy 1 levels). Depletion of mtDNA was present in muscle from sIBM patients and PBMCs showed deregulated expression of mitochondrial proteins in oxidative phosphorylation. MRC complex IV/citrate synthase activity was significantly decreased in both tissues and mitochondrial dynamics were affected in muscle. Depletion of mtDNA was significantly more severe in patients with mtDNA deletions, which also presented deregulation of mitochondrial fusion proteins. Imbalance in mitochondrial dynamics in muscle was associated with increased mitochondrial genetic disturbances (both depletion and deletions), demonstrating that proper mitochondrial turnover is essential for mitochondrial homoeostasis and muscle function in these patients.

Myostatin and insulin-like growth factor-1 in hypertensive heart disease: a prospective study in human heart donors.

Objective: The physiopathological mechanisms implicated in hypertensive heart disease are multi-factorial, including myocyte hypertrophy, apoptosis and myocardial remodelling. In this process, some hormonal and local growth factors have a regulatory influence. The aim of this study was to evaluate the potential role of myostatin and insulin-like growth factor-1 (IGF-1) myocardial expression in the development of hypertensive-induced cardiac damage. Methods: Samples of human myocardium tissue from organ donors were prospectively collected and classified according to the presence of hypertension, alcohol consumption, other causes of myocardial damage and the presence of structural cardiomyopathy (CMP). Myocardial samples were studied by immunohistochemistry and myostatin, and IGF-1 myocardial expression was evaluated in all the different groups of donors. Hypertensive donors were compared to other groups. Results: A total of 66 heart samples from human donors were collected: 33 donors had no previous or present history of hypertension and 33 donors presented defined hypertension. Donors with hypertension presented higher myocyte cell and nuclear hypertrophy and showed similar myostatin myocardial expression as controls, but lower IGF-1 myocardial expression. Myostatin expression was significantly higher in hypertensive donors with CMP compared to non-hypertensive healthy donors. The presence of CMP of diverse origin (alcoholic, valve and coronary) also significantly increased myostatin myocardial expression. Conclusion: The presence of hypertension significantly decreases IGF-1 myocardial expression. Myostatin myocardial expression increases in the presence of structural CMP either of hypertensive or other origin. These effects open the possibility of modulating hypertensive-induced cardiac damage.

Insulin-like growth factor 1 myocardial expression decreases in chronic alcohol consumption

BackgroundAlcoholic cardiomyopathy (CMP) is one of the major complications of chronic excessive alcohol consumption. The pathogenic mechanisms implicated are diverse, inducing functional and structural changes in the myocardium. Insulin-like Growth Factor 1 (IGF-1) plays an important role in modulating the cell cycle, and helps the differentiation and proliferation of cardiac tissue inhibiting apoptosis. Experimental studies have suggested the role of IGF-1 in alcohol-induced cardiac damage. The aim of the present study was to determine the effect of chronic alcohol consumption on IGF-1 myocardial expression and to compare this expression in cases of hypertension and other cardiac diseases.MethodsWe studied heart samples from human organ donors: 10 healthy donors, 16 with hypertension, 23 with chronic alcohol consumption and 7 with other causes of cardiac disease. IGF-1 myocardial expression was evaluated with a specific immunohistochemistry assay using a semi-quantitative method.ResultsA significant decrease in IGF-1 myocardial expression was observed comparing all the cases included with control donors. This decrease in IGF-1 myocardial expression was significantly lower in the group of donors with chronic alcohol consumption compared to controls. On group evaluation according to the presence of CMP, donors with chronic alcohol consumption without CMP presented significantly lower IGF-1 expression than controls, whereas donors with chronic alcohol consumption with CMP showed a downward trend without achieving significance.ConclusionsChronic alcohol consumption significantly reduces IGF-1 myocardial expression. This decrease induced by alcohol is partially compensated in the presence of structural myocardial damage.

Mitochondrial and apoptotic in vitro modelling of differential HIV-1 progression and antiretroviral toxicity

OBJECTIVES Ex vivo analysis of mitochondrial function may reveal HIV progression and the impact of ART. We propose a mitochondrial and apoptotic in vitro model using Jurkat T cells incubated with plasma. The objectives of this study were to evaluate mitochondrial and apoptotic lesions in this model in relation to HIV progression, and to assess the effect of >1 year of standard non-thymidine-containing therapy. METHODS This was a cross-sectional comparison among three age- and gender-matched groups (n = 19 × 3): healthy non-HIV-infected participants, HIV-infected long-term non-progressors (LTNPs) and standard antiretroviral-naive chronically infected patients [standard progressors (Sps)], longitudinally evaluated before (Sp1) and after (Sp2) >1 year of efavirenz + tenofovir + emtricitabine therapy. We analysed mitochondrial DNA content by RT-PCR, mitochondrial function by spectrophotometry, mitochondrial protein synthesis by western blot analysis, mitochondrial dynamics by western blot analysis (MFN2), apoptotic transition pore formation by western blot analysis (VDAC-1) and mitochondrial membrane potential and annexin V/propidium iodide fluorescence by flow cytometry. RESULTS There was a decreasing non-significant trend towards lower mitochondrial parameters for HIV-infected values with respect to uninfected control reference values. HIV progression (LTNP versus Sp1) was associated with decreased mitochondrial genetic, functional and translational parameters, which partially recovered after treatment intervention (Sp2). Mitochondrial fusion showed a trend to decrease non-significantly in Sp patients compared with LTNP patients, especially after therapy. All apoptotic parameters showed a trend to increase in Sp1 with respect to LTNP, followed by recovery in Sp2. CONCLUSIONS We proposed an in vitro model for mitochondrial and apoptotic assessment to test the effects of HIV infection and its therapy, resembling in vivo conditions. This model could be useful for clinical research purposes.