Cobalt chloride postconditioning as myoprotective therapy in cardiac ischemia-reperfusion.

Since damage induced by ischemia-reperfusion (I/R) involves alterations in Ca2+ homeostasis and is reduced by ischemic postconditioning (IP) and that CoCl2 can trigger changes resembling the response to a hypoxic event in normoxia and its blockade on Ca2+ current in heart muscle, our aim was to evaluate CoCl2 as an IP therapeutic tool. Mechanic and energetic parameters of isolated and arterially perfused male Wistar rat heart ventricles were simultaneously analyzed in a model of I/R in which 0.23 mmol/L CoCl2 was introduced upon reperfusion and kept or withdrawn after 20 min or introduced after 20 min of reperfusion. The presence of CoCl2 did not affect diastolic pressure but increased post-ischemic contractile recovery, which peaked at 20 min and decreased at the end of reperfusion. This decrease was prevented when CoCl2 was removed at 20 min of reperfusion. Total heat release increased throughout reperfusion, while economy increased between 15 and 25 min. No effect was observed when CoCl2 was introduced at 20 min of reperfusion. In addition, both the area under the contracture curve evoked by 10 mmol/L caffeine-36 mmol/L Na+ and the contracture tension relaxation rate were higher with CoCl2.Furthermore, CoCl2 decreased the number of arrhythmias during reperfusion and the ventricular damaged area. The presence of CoCl2 in reperfusion induces cardioprotection consistent with the improvement in cellular calcium handling. The use of CoCl2 constitutes a potential cardioprotective tool of clinical relevance.

Angiotensin II type 1 receptor is involved in hypertension and vascular alterations caused by environmental toxicant hexachlorobenzene.

Environmental hexachlorobenzene (HCB) increases blood pressure (BP) in female rats, causing alterations in arterial structure and function. Here we study the role of Angiotensin II receptor type 1 (AT1) in HCB-induced hypertension through the use of AT1 antagonist losartan. HCB-treated male rats showed a 22.7% increase in BP which was prevented by losartan. Losartan blocked HCB-induced changes in arterial morphology (decreased aorta cell number and increased wall thickness). Losartan also prevented HCB-induced alterations in artery relaxation by acetylcholine and nitroprusside but not the reduction in the maximum contraction by phenylephrine. Losartan rescued arterial molecular alterations caused by HCB (i.e. an increase in TGF-ß1 and AT1 expression and a decrease in eNOS expression and nitrite levels) and reduced hydrogen sulfide plasma concentration. In conclusion: in this work we demonstrate that AT1 activity is involved in HCB effects on the vascular system leading to hypertension.

Coronary Intimal Thickening Begins in Fetuses and Progresses in Pediatric Population and Adolescents to Atherosclerosis.

The prevalence of coronary intimal thickening (IT) was assessed in fetuses and pediatric population. We studied the coronary arteries of 63 hearts obtained from fetuses, infants, children, and adolescents, deceased from noncardiac disease or trauma. Histomorphometric analysis, planimetry, and immunohistochemical studies were conducted. Intimal thickening consisted of proliferation of smooth muscle cells and scarce monocytes embedded in amorphous deposits within the internal elastic membrane (IEM). Intermingled lesions of intimal hyperplasia and parietal nonstenotic plaques were also observed. Intimal thickening was found in 10% of 20 fetuses, in 33.3% of 18 infants, 73.3% of 15 children, and 100% of 10 adolescents. A significant correlation (r = 0.671, P < 0.001) was found between the extent of IT and age. The IEM was duplicated or interrupted in 43% of patients, showing a positive correlation with the degree of IT (P = 0.01). Intimal thickening was predominantly found near bifurcation sites in the left anterior descending coronary artery (55.6%) and in zones free of bifurcation in the right coronary artery (75%). In conclusion, the prevalence and extension of IT lesions are higher at older ages within a young population. Intimal thickening may be regarded as the first event occurring in coronary preatherosclerosis, preceding lipid deposition.

Productos intermedios de la cocaína: la pasta base o "paco" / Intermediate products of cocaine: the base paste or "paco"

El uso indebido de drogas se ha convertido en un grave problema a nivel mundial. En los últimos años, en nuestro país se ha incrementado en más del 200% el consumo de pasta base de cocaína (paco). A pesar de que el paco es un producto intermedio en la obtención de cocaína, y que muchos de sus efectos son atribuibles al contenido de esa droga, su consumo produce un cuadro clínico claramente distinto al observado en los consumidores de clorhidrato de cocaína, lo cual puede estar relacionado con su impureza. Sin perjuicio del gran impacto social producido por el consumo de paco, poco se sabe sobre su composición química y menos aún sobre sus efectos crónicos en los distintos órganos ni sobre su fisiopatología. Si bien existe material de autopsia de adictos al paco, los hallazgos están contaminados por la coexistencia en un mismo paciente de múltiples tóxicos. Urge la formación de grupos multidisciplinarios, con moderna tecnología para enfrentar este gravísimo flagelo. (AU)

Drug abuse has become a serious problem worldwide. In recent years, in our country the consumption of cocaine base paste (Paco) has increased by more than 200%. Despite of the fact that Paco is an intermediate product in the manufacture of cocaine, and that many of its effects are attributable to its content, its consumption produces a clearly different clinical picture than that observed in cocaine hydrochloride users, which It may be related to the impurity of this drug. Without prejudice to the great social impact produced by the consumption of this drug, little is known about its chemical composition and even less about its chronic effects on the different organs or its pathophysiology. Although there is an autopsy material for drug addicts, the findings are contaminated by the coexistence of multiple toxins in the same patient. The formation of multidisciplinary groups is urgent, with modern technology to face this very serious scourge. (AU)

Prevalencia del polimorfismo del nucleótido simple rs4704963 (T > C) del gen Early B-Cell Factor 1 en una población de pacientes diabéticos tipo 2 con obesidad / Prevalence of Early B-Cell Factor 1 Gene rs4704963 Single Nudeotide Polymorphism (T>C) in a Population of Type 2 Diabetic Patients with Obesity

RESUMEN Introducción: En estudios previos, se determinó para una población con agravamiento de la diabetes tipo 2 con obesidad (DBT+Ob) que sufría estrés una prevalencia del polimorfismo de nucleótido único (SNP) rs4704963 (T > C) del gen Early B-Cell Factor 1 (EBF1) del 16,5%. Objetivos: Determinar la prevalencia de este SNP en pacientes con DBT+ Ob que acuden al Hospital Ramos Mejía de la Ciudad Autónoma de Buenos .Aires y establecer si dicho polimorfismo se asocia con el estrés o la ocurrencia de eventos coronarios agudos. Material y métodos: Se llevó a cabo un estudio observacional, prospectivo, sobre prevalencia del polimorfismo en 53 pacientes con DBT+Ob e índice de masa corporal (IMC) entre 28 y 41, atendidos en el citado hospital en un período de 15 meses. A cada paciente se le computó una escala de estrés percibido, además de evaluarlo mediante la escala de acontecimientos vitales estresantes. Para el análisis estadístico, se realizaron las pruebas de Chi cuadrado y se calcularon los odds ratio (OR). Resultados: La población evaluada (53 pacientes) tuvo una media de edad de 60,2 ±9,77 años; 47,2% fueron hombres. De ellos, 8 individuos (15,1%) presentaron el SNP y todos fueron heterocigotas. Quince sujetos (28,3%) tuvieron síndrome isquémico agudo (SIA) y de estos solo uno (6,6%) tenía el SNP No se halló relación estadísticamente significativa entre la presencia del SNP y la aparición de SIA (p = 0,282). Catorce pacientes (26,4%) presentaron estrés crónico moderado o grave, y no hubo relación entre este hallazgo y la presencia del SNP (p = 0,979). Conclusiones: La prevalencia del SNP rs4704963 (T > C) del gen EBF1 en la población de DBT+Ob estudiada fue del 15,1% y no se halló relación estadísticamente significativa del SNP con el estrés ni con el SIA.

ABSTRACT Background: Previous studies established that in a population with exacerbation of type 2 diabetes with obesity (DBT+Ob) suffering from stress, the prevalence of early B-Cell Factor 1 (EBF1) gene rs4704963 single nucleotide polymorphism (SNP) (T>C) is 16.5%. Objectives: The aim of this study was to determine the prevalence of this SNP in patients with DBT+Ob attending Hospital Ramos Mejía of the Autonomous City of Buenos Aires and to ascertain whether this polymorphism is associated with stress or acute coronary events. Methods: An observational, prospective study on the prevalence of rs4704963 SNP was performed in 53 patients with DBT+Ob and body mass index between 28 and 41, seen in Hospital Ramos Mejía for a period of 15 months. Each patient was evaluated with a stressful life events scale and a perceived stress scale. The chisquare test and odds ratio (OR) were used for statistical analysis. Results: A total of 53 patients were included in the study. Mean population age was 60.2±9.77 years and 47.2% were men. Among these patients, 8 (15.1%) presented SNP and all were heterozygous. Fifteen patients (28.3%) had acute ischemic syndrome (AIS), and among these, only one (6.6%) had SNP No statistically significant relationship was found between the presence of SNP and AIS (p=0.282). Fourteen patients (26.4%) presented moderate or severe chronic stress, and there was no relationship between this finding and the presence of SNP (p=0.979). Conclusions: The prevalence of EBF1 gene rs4704963 SNP (T>C) in the DBT+Ob population was 15.1%. No statistically significant association was found between SNP and stress or AIS.

Environmental pollutant hexachlorobenzene induces hypertension in a rat model.

Hexachlorobenzene (HCB) is a dioxin-like environmental pollutant, widely distributed in the environment. New research links exposure to high levels of persistent organic environmental toxicants to cardiovascular disease, however little is known about the effect of HCB on vascular function and on blood pressure. The purpose of the present study was to evaluate biochemical and cardiovascular changes resulting from subchronic HCB exposure. Adult female Sprague-Dawley rats were treated with vehicle or HCB (5 or 500 mg/kg b.w) for 45 days. Systolic blood pressure (BP), recorded by tail cuff plethysmography, was significantly increased at 35, 40 and 45 days of 500 mg/kg HCB-treatment. HCB (500 mg/kg) increased arterial thickness, while both 5 and 500 mg/kg HCB decreased proliferating cell nuclear antigen (PCNA) protein levels and cellular nuclei in abdominal aortas indicating a hypertrophic process. Also, aortas from both groups of HCB-treated rats presented higher sensitivity to noradrenalin (NA) and a significant decrease in maximum contractile response. Arteries from 500 mg/kg HCB-treated rats showed a significant increase in the levels of transforming growth factor-ß1 (TGF-ß1) mRNA and angiotensin II type1 receptor (AT1), and a significant decrease in estrogen receptor alpha (ERα), endothelial nitric oxidide synthase (eNOS) protein expression and deiodinase II (DII) mRNA levels. In conclusion, we have demonstrated for the first time that subchronic HCB administration significantly increases BP and alters associated cardiovascular parameters in rats. In addition, HCB alters the expression of key vascular tissue molecules involved in BP regulation, such as TGF-ß1, AT1, ERα, eNOS and DII.

Metabolic Changes Following Perinatal Asphyxia: Role of Astrocytes and Their Interaction with Neurons.

Perinatal Asphyxia (PA) represents an important cause of severe neurological deficits including delayed mental and motor development, epilepsy, major cognitive deficits and blindness. The interaction between neurons, astrocytes and endothelial cells plays a central role coupling energy supply with changes in neuronal activity. Traditionally, experimental research focused on neurons, whereas astrocytes have been more related to the damage mechanisms of PA. Astrocytes carry out a number of functions that are critical to normal nervous system function, including uptake of neurotransmitters, regulation of pH and ion concentrations, and metabolic support for neurons. In this work, we aim to review metabolic neuron-astrocyte interactions with the purpose of encourage further research in this area in the context of PA, which is highly complex and its mechanisms and pathways have not been fully elucidated to this day.

Long lasting cerebellar alterations after perinatal asphyxia in rats.

The developing brain may be particularly vulnerable to injury before, at and after birth. Among possible insults, hypoxia suffered as a consequence of perinatal asphyxia (PA) exhibits the highest incidence levels and the cerebellar circuitry appears to be particularly susceptible, as the cellular makeup and the quantity of inputs change quickly during days and weeks following birth. In this work, we have used a murine model to induce severe global PA in rats at the time of birth. Short-term cerebellar alterations within this PA model have been previously reported but whether such alterations remain in adulthood has not been conclusively determined yet. For this reason, and given the crucial cerebellar role in determining connectivity patterns in the brain, the aim of our work is to unveil long-term cerebellum histomorphology following a PA insult. Morphological and cytological neuronal changes and glial reaction in the cerebellar cortex were analyzed at postnatal 120 (P120) following injury performed at birth. As compared to control, PA animals exhibited: (1) an increase in molecular and granular thickness, both presenting lower cellular density; (2) a disarrayed Purkinje cell layer presenting a higher number of anomalous calbindin-stained cells. (3) focal swelling and marked fragmentation of microtubule-associated protein 2 (MAP-2) in Purkinje cell dendrites and, (4) an increase in glial fibrillary acidic protein (GFAP) expression in Bergmann cells and the granular layer. In conclusion, we demonstrate that PA produces long-term damage in cellular histomorphology in rat cerebellar cortex which could be involved in the pathogenesis of cognitive deficits observed in both animals and humans.

Neuroprotective effects of hypothermia on synaptic actin cytoskeletal changes induced by perinatal asphyxia.

Cerebral hypoxia-ischemia damages synaptic proteins, resulting in cytoskeletal alterations, protein aggregation and neuronal death. In the previous works, we have shown neuronal and synaptic changes in rat neostriatum subjected to hypoxia that leads to ubi-protein accumulation. Recently, we also showed that, changes in F-actin organization could be related to early alterations induced by hypoxia in the Central Nervous System. However, little is known about effective treatment to diminish the damage. The main aim of this work is to study the effects of birth hypothermia on the actin cytoskeleton of neostriatal post-synaptic densities (PSD) in 60 days olds rats by immunohistochemistry, photooxidation and western blot. We used 2 different protocols of hypothermia: (a) intrahypoxic hypothermia at 15°C and (b) post-hypoxia hypothermia at 32°C. Consistent with previous data at 30 days, staining with phalloidin-Alexa(488) followed by confocal microscopy analysis showed an increase of F-actin fluorescent staining in the neostriatum of hypoxic animals. Correlative photooxidation electron microscopy confirmed these observations showing an increment in the number of mushroom-shaped F-actin staining spines in neostriatal excitatory synapses in rats subjected to hypoxia. In addition, western blot revealed ß-actin increase in PSDs in hypoxic animals. The optic relative density measurement showed a significant difference between controls and hypoxic animals. When hypoxia was induced under hypothermic conditions, the changes observed in actin cytoskeleton were blocked. Post-hypoxic hypothermia showed similar answer but actin cytoskeleton modifications were not totally reverted as we observed at 15°C. These data suggest that the decrease of the body temperature decreases the actin modifications in dendritic spines preventing the neuronal death.

Transforming growth factor ß1 and coronary intimal hyperplasia in pediatric patients with congenital heart disease.

BACKGROUND: Congenital heart defects or the process of their repair leads to an increased risk for adult cardiovascular disease compared with the general population. Intimal hyperplasia is a preatherosclerotic lesion that may be produced as a consequence of transforming growth factor ß1 (TGF-ß1) pathway activation. We studied the presence of intimal hyperplasia in arteries from a pediatric population with congenital heart disease (CHD) and TGF-ß1 expression to enlighten its possible role in the genesis of these lesions. METHODS: Coronary arteries from 10 controls and 98 CHD patients (54% cyanotic type, 32% surgically repaired) were stained, and the presence and degree of intimal thickening were analyzed. The expression of TGF-ß1 was studied by immunohistochemistry. RESULTS: The difference between the presence of coronary intimal hyperplasia in patients with cyanotic (35; 66.1%) and noncyanotic CHD (29; 64.3%) was not significant. However, surgically repaired CHD presented a higher rate of coronary intimal hyperplasia (80%) than did the group without surgical intervention (47.3%), P = 0.0002. The immunostaining for TGF-ß1 analyzed in samples of patients with cyanotic and noncyanotic CHD showed no significant differences. However, TGF-ß1 expression was more intense on the intimal layer of patients with surgically repaired CHD than on that of those without surgery (intimal area positive for TGF-ß1, 50.43% vs 15.91%, respectively; Mann-Whitney U test P = 0.0005). CONCLUSION: The high incidence of intimal hyperplasia in patients with surgically repaired CHD is correlated with TGF-ß1 expression and may contribute to the development of atherosclerotic coronary artery disease in CHD patients.

Long-chain acyl-CoA synthetase 4 is regulated by phosphorylation.

Long chain acyl CoA synthetase 4 (Acsl4) is a key enzyme in steroidogenesis. It participates in steroid synthesis through of arachidonic acid release and Steroidogenic Acute Regulatory protein (StAR) induction. Acsl4 prefers arachidonic acid as substrate and acts probably as a homodimer. In steroidogenic cells, it has been demonstrated that Acsl4 is a high turnover protein located mainly in mitochondrial-associated membrane fraction (MAM) bound to other proteins and that it is newly synthesized by hormone stimulation. The synthesis of Acsl4 constitutes an early step in steroidogenesis. In the steroid synthesis process, activation of kinases plays a very important role. For this reason, the aim of this work was to study Acsl4 as a possible phosphoprotein and try to elucidate the role of its phosphorylation. We have determined for the first time that Acsl4 is a phosphoprotein whose phosphorylation is hormone-dependent. We also demonstrated that Acsl4 acts effectively as a dimer and that phosphorylation occurs after dimer formation. Studies in vitro demonstrated that Acsl4 is a substrate of both PKA and PKC and its phosphorylation by these kinases regulates its activity.

New enzymes involved in the mechanism of action of epidermal growth factor in a clonal strain of Leydig tumor cells.

The studies presented herein were designed to investigate the effect of mouse epidermal growth factor (mEGF) on arachidonic acid (AA) release in a clonal strain of cultured murine Leydig cells (designed MA-10). In MA-10 cells, mEGF promotes AA release and metabolism to lipoxygenated products to induce the steroidogenic acute regulatory (StAR) protein. However, the mechanism by which mEGF releases AA in these cells is not totally elucidated. We show that mEGF produces an increment in the mitochondrial AA content in a short-term incubation (30 min). This AA is released by the action of a mitochondrial acyl-CoA thioesterase (Acot2), as demonstrated in experiments in which Acot2 was down or overexpressed. This AA in turn regulates the StAR protein expression, indirect evidence of its metabolism to lipoxygenated products. We also show that mEGF induces the expression (mRNA and protein) of Acot2 and an acyl-CoA synthetase that provides the substrate, arachidonyl-CoA, to Acot2. This effect is also observed in another steroidogenic cell line, the adrenocortical Y1 cells. Taken together, our results show that: 1) mEGF can induce the generation of AA in a specific compartment of the cells, i.e. the mitochondria; 2) mEGF can up-regulate acyl-CoA synthetase and Acot2 mRNA and protein levels; and 3) mEGF-stimulated intramitochondrial AA release leads to StAR protein induction.

An arachidonic acid generation/export system involved in the regulation of cholesterol transport in mitochondria of steroidogenic cells.

Recent studies demonstrated the importance of the mitochondrial ATP in the regulation of a novel long-chain fatty acid generation/export system in mitochondria of diabetic rat heart. In steroidogenic systems, mitochondrial ATP and intramitochondrial arachidonic acid (AA) generation are important for steroidogenesis. Here, we report that mitochondrial ATP is necessary for the generation and export of AA, steroid production and steroidogenic acute regulatory protein induction supported by cyclic 3'-5'-adenosine monophosphate in steroidogenic cells. These results demonstrate that ATP depletion affects AA export and provide new evidence of the existence of the fatty acid generation and export system involved in mitochondrial cholesterol transport.

cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells.

We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces cholesterol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol transport in isolated mitochondria from nonstimulated cells. The effect of arachidonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accumulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regulation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role.