Josep M. Nicolás

Angiotensin II induces contraction and proliferation of human hepatic stellate cells

BACKGROUND & AIMS Circulating levels of angiotensin II (ANGII), a powerful vasoconstrictor factor, are frequently increased in chronic liver diseases. In these conditions, hepatic stellate cells (HSCs) proliferate and acquire contractile properties. This study investigated the presence of receptors for ANGII and the effects of ANGII in human HSCs activated in culture. METHODS The presence of ANGII receptors was assessed by binding studies. The effects of ANGII on intracellular calcium concentration ([Ca(2+)](i)), cell contraction, and cell proliferation were also assessed. RESULTS Binding studies showed the presence of ANGII receptors of the AT1 subtype. ANGII elicited a marked dose-dependent increase in [Ca(2+)](i) and cell contraction. Moreover, ANGII stimulated DNA synthesis and increased cell number. All these effects were totally blocked by losartan and reduced by nitric oxide donors or prostaglandin E(2). The effects of ANGII were barely detectable in quiescent cells (2 days in culture), suggesting that phenotypic transformation of HSCs is associated with a marked increase in the effects of ANGII. CONCLUSIONS ANGII induces contraction and is mitogenic for human-activated HSCs by acting through AT1 receptors. These results suggest that activated HSCs are targets of the vasoconstrictor action of ANGII in the intrahepatic circulation.

Activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin II.

BACKGROUND & AIMS The renin-angiotensin system plays an important role in hepatic fibrogenesis. In other organs, myofibroblasts accumulated in damaged tissues generate angiotensin II, which promotes inflammation and extracellular matrix synthesis. It is unknown whether myofibroblastic hepatic stellate cells, the main hepatic fibrogenic cell type, express the renin-angiotensin system and synthesize angiotensin II. The aim of this study was to investigate whether quiescent and activated human hepatic stellate cells contain the components of the renin-angiotensin system and synthesize angiotensin II. METHODS Hepatic stellate cells were freshly isolated from normal human livers (quiescent hepatic stellate cells) and from human cirrhotic livers (in vivo activated hepatic stellate cells). Culture-activated hepatic stellate cells were used after a second passage of quiescent hepatic stellate cells. Angiotensinogen, renin, and angiotensin-converting enzyme were assessed by quantitative polymerase chain reaction. Angiotensin II production was assessed by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS Quiescent hepatic stellate cells barely express the renin-angiotensin system components--angiotensinogen, renin, and angiotensin-converting enzyme--and do not secrete angiotensin II. In contrast, both in vivo activated hepatic stellate cells and culture-activated hepatic stellate cells highly express active renin and angiotensin-converting enzyme and secrete angiotensin II to the culture media. Mature angiotensin II protein is also detected in the cytoplasm of in vivo activated and culture-activated hepatic stellate cells. Growth factors (platelet-derived growth factor and epidermal growth factor) and vasoconstrictor substances (endothelin-1 and thrombin) stimulate angiotensin II synthesis, whereas transforming growth factor-beta and proinflammatory cytokines have no effect. Vasodilator substances markedly attenuate the effect of endothelin-1. CONCLUSIONS After activation, human hepatic stellate cells express the components of the renin-angiotensin system and synthesize angiotensin II. These results suggest that locally generated angiotensin II could participate in tissue remodeling in the human liver.

Non-invasive ventilation after extubation in hypercapnic patients with chronic respiratory disorders: randomised controlled trial

BACKGROUND Non-invasive ventilation can prevent respiratory failure after extubation in individuals at increased risk of this complication, and enhanced survival in patients with hypercapnia has been recorded. We aimed to assess prospectively the effectiveness of non-invasive ventilation after extubation in patients with hypercapnia and as rescue therapy when respiratory failure develops. METHODS We undertook a randomised controlled trial in three intensive-care units in Spain. We enrolled 106 mechanically ventilated patients with chronic respiratory disorders and hypercapnia after a successful spontaneous breathing trial. We randomly allocated participants by computer to receive after extubation either non-invasive ventilation for 24 h (n=54) or conventional oxygen treatment (n=52). The primary endpoint was avoidance of respiratory failure within 72 h after extubation. Analysis was by intention to treat. This trial is registered with clinicaltrials.gov, identifier NCT00539708. FINDINGS Respiratory failure after extubation was less frequent in patients assigned non-invasive ventilation than in those allocated conventional oxygen therapy (8 [15%] vs 25 [48%]; odds ratio 5.32 [95% CI 2.11-13.46]; p<0.0001). In patients with respiratory failure, non-invasive ventilation as rescue therapy avoided reintubation in 17 of 27 patients. Non-invasive ventilation was independently associated with a lower risk of respiratory failure after extubation (adjusted odds ratio 0.17 [95% CI 0.06-0.44]; p<0.0001). 90-day mortality was lower in patients assigned non-invasive ventilation than in those allocated conventional oxygen (p=0.0146). INTERPRETATION Early non-invasive ventilation after extubation diminished risk of respiratory failure and lowered 90-day mortality in patients with hypercapnia during a spontaneous breathing trial. Routine implementation of this strategy for management of mechanically ventilated patients with chronic respiratory disorders is advisable. FUNDING IDIBAPS, CibeRes, Fondo de Investigaciones Sanitarias, European Respiratory Society.

Automatic control of tracheal tube cuff pressure in ventilated patients in semirecumbent position: A randomized trial*

Objective:The aspiration of subglottic secretions colonized by bacteria pooled around the tracheal tube cuff due to inadvertent deflation (<20 cm H2O) of the cuff plays a relevant role in the pathogenesis of ventilator-associated pneumonia. We assessed the efficacy of an automatic, validated device for the continuous regulation of tracheal tube cuff pressure in preventing ventilator-associated pneumonia. Design:Prospective randomized controlled trial. Setting:Respiratory intensive care unit and general medical intensive care unit. Patients:One hundred and forty-two mechanically ventilated patients (age, 64 ± 17 yrs; Acute Physiology and Chronic Health Evaluation II score, 18 ± 6) without pneumonia or aspiration at admission. Interventions:Within 24 hrs of intubation, patients were randomly allocated to undergo continuous regulation of the cuff pressure with the automatic device (n = 73) or routine care of the cuff pressure (control group, n = 69). Patients remained in a semirecumbent position in bed. Measurements and Main Results:The primary end point variable was the incidence of ventilator-associated pneumonia. Main causes for intubation were decreased consciousness (43, 30%) and exacerbation of chronic respiratory diseases (38, 27%). Cuff pressure <20 cm H2O was more frequently observed in the control than the automatic group (45.3 vs. 0.7% determinations, p < .001). However, the rate of ventilator-associated pneumonia with clinical criteria (16, 22% vs. 20, 29%) and microbiological confirmation (11, 15% vs. 10, 15%), the distribution of early and late onset, the causative microorganisms, and intensive care unit (20, 27% vs. 16, 23%) and hospital mortality (30, 41% vs. 23, 33%) were similar for the automatic and control groups, respectively. Conclusions:Cuff pressure is better controlled with the automatic device. However, it did not result in additional benefits to the semirecumbent position in preventing ventilator-associated pneumonia.

Human hepatic stellate cells show features of antigen‐presenting cells and stimulate lymphocyte proliferation

Following cell activation, hepatic stellate cells (HSCs) acquire proinflammatory and profibrogenic properties. We investigated whether activated HSCs also display immune properties. Here we show that cultured human HSCs express membrane proteins involved in antigen presentation, including members of the HLA family (HLA-I and HLA-II), lipid-presenting molecules (CD1b and CD1c), and factors involved in T-cell activation (CD40 and CD80). Exposure of HSCs to proinflammatory cytokines markedly up-regulates these molecules. Importantly, cells freshly isolated from human cirrhotic livers (in vivo activated HSCs) highly express HLA-II and CD40, suggesting that HSCs can act as antigen-presenting cells (APCs) in human fibrogenesis. We also explored whether human HSCs can efficiently process exogenous antigens. Activated HSCs internalize low- and high-molecular-weight dextran and transferrin, indicating that they can perform fluid-phase and receptor-mediated endocytosis. Moreover, HSCs can perform phagocytosis of macromolecules because they internalize latex particles as well as bacteria. Interestingly, both culture-activated and in vivo activated HSCs express high levels of CD68, a protein involved in antigen trafficking. Finally, we studied whether HSCs modulate T-lymphocyte proliferation. In basal conditions, coculture of irradiated HSCs barely induces allogeneic T-lymphocyte proliferation. However, cytokine-stimulated HSCs stimulate the allogeneic T-lymphocyte response in an HLA-II-dependent manner. In conclusion, human activated HSCs express molecules for antigen presentation, internalize macromolecules, and modulate T-lymphocyte proliferation. These results suggest that HSCs may play a role in the immune function of the liver.

The Effect of Controlled Drinking in Alcoholic Cardiomyopathy

Alcoholic cardiomyopathy is a complication of long-standing alcohol abuse and is related to a persons total lifetime dose of ethanol (1, 2). The malady is not uncommon; in developed countries, it accounts for 3% to 40% of all cases of dilated cardiomyopathy (3-6). In our previous studies, one third of asymptomatic alcoholic persons showed some evidence of cardiac dysfunction (1, 2, 7). Although the natural course of chronic alcoholic cardiomyopathy is not precisely defined, abstinence from alcohol has been reported to improve cardiac function, or at least halt its deterioration (8-10). The treatment of alcoholic cardiomyopathy is generally assumed to require abstinence (11), and any further alcohol intake is thought to be deleterious. Yet abstinence is not always possible, and a sizable proportion of persons treated for alcoholism report that they are able to drink moderately (12). We therefore designed a 4-year prospective cohort study of patients with alcoholic cardiomyopathy. Although total abstinence was urged for all patients at the initial interview and on subsequent occasions, fewer than one third stopped drinking completely. A similar number maintained a controlled drinking pattern, and the remainder continued to abuse alcohol. We evaluated the effect of controlled drinking, defined as a daily intake of 20 to 60 g of alcohol per day (one to four standard drinks), in patients with alcoholic cardiomyopathy. Methods Patient Selection All patients were younger than 60 years of age and reported consuming more than 100 g of ethanol per day for at least 10 years. Over a 6-year period, 51 alcoholic patients were admitted to the cardiology unit or the emergency department of Hospital Clnic, Barcelona, Spain, with signs or symptoms of heart failure (New York Heart Association [NYHA] functional class II to IV) (13), left ventricular ejection fraction of 0.5 or less, and no evidence of any heart disease other than dilated cardiomyopathy. Alcoholic cardiomyopathy was diagnosed in all of these patients, and they were consecutively recruited for the study. Fifteen patients who presented to the outpatient alcoholism unit during the same period for assistance in terminating their dependence on alcohol, displayed cardiac enlargement on radiography, and were found to have alcoholic cardiomyopathy (left ventricular ejection fraction < 0.5) were also included in the study. The cardiothoracic index was abnormally large in all patients ( 0.5). In addition, all but 4 of the patients had an end-diastolic diameter greater than 2 standard deviations above that of a group of healthy volunteers. Of the 66 alcoholic persons initially evaluated, 11 were excluded. Five had severe withdrawal symptoms, 1 reported cocaine use, and 1 was infected with HIV. Two patients with evidence of coronary artery disease were not considered good candidates for the study. Two patients had such severe alcoholic cardiomyopathy (ejection fraction < 0.1) that they died shortly after admission. Thus, a total of 55 patients with alcoholic cardiomyopathy remained in the study. No patients objected to inclusion, and all gave informed consent for the various procedures. The Institutional Review Board of Hospital Clnic approved the study protocol. All patients were white men of Spanish descent who lived with their families in or around Barcelona and had histories of stable employment. Most were skilled laborers or office workers, and none were indigent. As part of our standardized program designed to terminate dependence on alcohol, all patients were repeatedly urged to become abstinent. The proportion of patients who stopped drinking was similar to that reported by others (12, 14). To assess the normal variability of cardiac function over time, we also evaluated 10 healthy men (median age, 45 years [range, 35 to 45 years]) who reported drinking less than 20 g of ethanol per day. It is important to note that alcoholic beverages are part of Spanish culture and that total abstention from alcohol is uncommon. The controls were studied at the beginning and end of the study in the same manner as the men with alcoholism. Clinical Studies One of two physicians obtained a detailed history of ethanol intake for each patient using repeated interviews and a validated structured questionnaire (15, 16). In our experience, intra- and interobserver reliability of this type of assessment are greater than 90%. Data were confirmed by consultation with family members. Minor discrepancies in alcohol intake were found in five cases (9%), which were resolved by further consultation with the patients and their families. Frequency and amount of daily ethanol intake since patients began drinking habitually were recorded. Life events, such as marriage, military service, and work posts, were used as anchor points to help recollection (time-line follow-back method [15]). The total lifetime dose of ethanol was estimated by multiplying the amount of ethanol consumed per day by the number of years of each alcohol intake period multiplied by 365 and adding the total amounts ingested during these different periods (1, 7). Withdrawal symptoms were evaluated according to the Clinical Institute for Withdrawal Assessment Scale (17). Laboratory and Nutritional Studies Blood samples for laboratory tests were obtained the day after admission. Overall nutrition was assessed in terms of the proportion of actual weight to ideal weight. The lean body mass and muscular area of the arm were calculated from the circumference of the upper nondominant arm and the thickness of the tricipital skin fold, respectively. The fatty area of the arm, calculated from the thickness of the tricipital fold, was considered indicative of total body fat (1, 18). Body mass index was determined as the body weight relative to the square of the body height (kg/m2). Patients were considered to have caloric malnutrition if their body weight was less than 80% of their ideal weight or if the calculated lean body mass was more than 10% below the control value. Protein malnutrition was diagnosed when patients had abnormally low values for three of the following: hemoglobin, lymphocyte count, total protein level, albumin level, prealbumin level, retinol-binding protein level, or transferrin level (18, 19). Hepatic ultrasonography was performed in all patients. Percutaneous needle biopsies of the liver were performed when serum aminotransferase levels remained elevated to more than twice the normal values for more than 2 months or when ultrasonography showed liver abnormalities. Cardiac Studies Past and present signs and symptoms of heart failure were evaluated, and NYHA functional class was determined according to the Goldman activity scale (13). Three days after admission, when results of tests for blood alcohol were uniformly negative, chest radiography, conventional electrocardiography, bidimensional echocardiography (Toshiba SS-10 instrument, Otawara, Japan), and technetium-99 radionuclide angiocardiography (Picker-Dyna 4-15 -camera, Haifer, Israel) were performed. End-diastolic and end-systolic diameters, the shortening fraction, and the mass of the left ventricle were measured according to the standards of the American Society of Echocardiography (20). Values for left ventricular ejection fraction were obtained by using isotopic angiography, which is reported to be a reliable method (1, 2, 21). The personnel who performed and evaluated these tests had no knowledge of the alcoholic history of the patients. To rule out ischemic heart disease, patients underwent a treadmill electrocardiographic test. At the end of the first year and annually thereafter for up to 4 years, interviews, laboratory tests, and clinical examinations were repeated for all patients. Ethanol consumption, pharmacologic treatment, and incidental disease were recorded at each interview. At the end of the first year of follow-up, patients were classified into one of four groups according to level of alcohol consumption: 1) complete abstinence; 2) controlled drinking of 20 to 60 g of ethanol per day, a range commonly accepted as moderate [22]; 3) an indeterminate range of 60 to 80 g of ethanol per day; and 4) alcohol abuse, defined as more than 80 g of ethanol per day. For patients who died during follow-up, death was classified as cardiologic or noncardiologic. Statistical Analysis We used standard statistical methods from the SPSS Statistical Analysis System, version 9.0 (SPSS, Inc., Chicago, Illinois) (23). In each group of patients, we used the paired t-test when comparing variables from different time periods. Role of the Funding Sources The funding sources had no role in the design, analysis, or interpretation of the study or in the decision to submit the results for publication. Results Clinical Characteristics Patients with alcoholic cardiomyopathy ranged in age from 31 to 59 years. Alcoholic beverages were generally consumed continuously as part of everyday life, and no binge drinking was reported. Patients most often drank wine, beer, or brandy and less frequently drank anisette, whiskey, or gin. The mean daily ethanol intake was 208 g (range, 100 to 310 g) over 26.6 years (Table 1). The mean total lifetime dose of ethanol (26.1 kg/kg of body weight [range, 12 to 42 kg/kg]) was similar to that observed in previous studies (1, 2). Most of the patients (75%) had smoked one to two packs of cigarettes per day since the second decade of their lives. None of the patients used illicit drugs. Table 1. Baseline Epidemiologic, Nutritional, and Clinical Characteristics of 55 Alcoholic Men with Cardiomyopathy Nutritional Status and Laboratory Data Nutritional and laboratory variables are shown in Table 1. Six patients exhibited mild caloric malnutrition, and 4 had slight protein malnutrition. Of the 28 patients who had liver biopsy, 3 had normal livers, 17 had cirrhosis, 6 had fatty liver, and 2 had mild alcoholic hepatitis. Among the 17 patients with cirrhosis,

Atrophy of the corpus callosum in chronic alcoholism

To determine the prevalence of corpus callosum atrophy in chronic alcoholics and its relationship to cognitive function and brain atrophy, a prospective clinicoradiologic study was carried out in 28 right-handed male patients with chronic alcoholism and 14 age- and sex-matched right-handed control subjects. Clinical evaluation, neuropsychological testing and measurement of the midsagittal corpus callosum area and thickness (genu, truncus and splenium), as well as the frontal lobe index and the width of the cortical sulci on T1- and T2-weighted magnetic resonance images were performed. Compared to controls, alcoholics had significantly decreased corpus callosum area and thickness, mainly in the genu. Two-thirds had a corpus callosum area 2 SD below the mean of the control group. The sagittal area of the corpus callosum body correlated negatively with the degree of frontal and cortical atrophies (r = -0.5579 and -0.6853, respectively p < 0.01, both). Alcoholics with corpus callosum atrophy exhibited impairment of visual and logical memories (p < 0.05 both) and those with reduced thickness of the genu showed impairment of frontal lobe tasks (p < 0.05). The reduction of corpus callosum indices (age-corrected) also correlated with the total lifetime dose of ethanol consumed (r = 0.6107, p < 0.001), but was not related to nutritional status or electrolyte imbalance. Atrophy of the corpus callosum is common among alcoholic patients and may reflect the severity and pattern of cortical damage. The degree of this atrophy also correlated with the severity of ethanol intake.

Functional status and quality of life 12 months after discharge from a medical ICU in healthy elderly patients: a prospective observational study

IntroductionLong-term outcomes of elderly patients after medical ICU care are little known. The aim of the study was to evaluate functional status and quality of life of elderly patients 12 months after discharge from a medical ICU.MethodsWe prospectively studied 112/230 healthy elderly patients (≥65 years surviving at least 12 months after ICU discharge) with full functional autonomy without cognitive impairment prior to ICU entry. The main diagnoses at admission using the Acute Physiology and Chronic Health Evaluation III (APACHE III) classification diagnosis and length of ICU stay and ICU scores (APACHE II, Sepsis-related Organ Failure Assessment (SOFA) and OMEGA) at admission and discharge were collected. Comprehensive geriatric assessment included the presence of the main geriatric syndromes and the application of Lawton, Barthel, and Charlson Indexes and Informant Questionnaire on Cognitive Decline to evaluate functionality, comorbidity and cognitive status, respectively. The EuroQol-5D assessed quality of life. Data were collected at baseline, during ICU and ward stay and 3, 6 and 12 months after hospital discharge. Paired or unpaired T-tests compared differences between groups (continuous variables), whereas the chi-square and Fisher exact tests were used for comparing dichotomous variables. For variables significant (P ≤ 0.1) on univariate analysis, a forward multiple regression analysis was performed.ResultsOnly 48.9% of patients (mean age: 73.4 ± 5.5 years) were alive 12 months after discharge showing a significant decrease in functional autonomy (Lawton and Barthel Indexes) and quality of life (EuroQol-5D) compared to baseline status (P < 0.001, all). Multivariate analysis showed a higher Barthel Index and EQ-5D vas at hospital discharge to be associated factors of full functional recovery (P < 0.01, both). Thus, in patients with a Barthel Index ≥ 60 or EQ-5D vas ≥40 at discharge the hazard ratio for full functional recovery was 4.04 (95% CI: 1.58 to 10.33; P = 0.005) and 6.1 (95% CI: 1.9 to 19.9; P < 0.01), respectively. Geriatric syndromes increased after ICU stay and remained significantly increased during follow-up (P < 0.001).ConclusionsThe survival rate of elderly medical patients 12 months after discharge from the ICU is low (49%), although functional status and quality of life remained similar to baseline in most of the survivors. However, there was a two-fold increase in the prevalence of geriatric syndromes.

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.

Human myofibroblastic hepatic stellate cells express Ca2+-activated K+ channels that modulate the effects of endothelin-1 and nitric oxide

BACKGROUND/AIMS High-conductance Ca(2+)-activated K(+) (BK(Ca)) channels modulate the effects of vasoactive factors in contractile cells. It is unknown whether hepatic stellate cells (HSCs) contain BK(Ca) channels and what their role in the regulation of HSCs contractility is. METHODS The presence of BK(Ca) channels in HSCs was assessed by the patch-clamp technique. The functional role of BK(Ca) channels was investigated by measuring intracellular calcium concentration ([Ca(2+)](i)) and cell contraction in individual cells after stimulation with endothelin-1 in the presence or absence of specific modulators of BK(Ca) channels. RESULTS BK(Ca) channels were detected by patch-clamp in most of the activated HSCs studied. Incubation of cells with iberiotoxin, a BK(Ca) channel blocker, increased both the sustained phase of [Ca(2+)](i) elicited by endothelin-1 and the number of cells undergoing contraction, while the use of NS1619, a BK(Ca) channel opener, induced opposite effects. Stimulation of HSCs with S-nitroso-N-acetyl-penicillamine (SNAP), a nitric oxide (NO)-donor, increased the opening of BK(Ca) channels and reduced the effects of endothelin-1. Conversely, iberiotoxin abolished the inhibitory effect of SNAP on endothelin-induced [Ca(2+)](i) increase and cell contraction. CONCLUSIONS Activated human HSCs contain BK(Ca) channels that modulate the contractile effect of endothelin-1 and mediate the inhibitory action of NO.