Elena Constantinescu

Changes in oxidative balance in rat pericytes exposed to diabetic conditions

Recent data indicate that the oxidative stress plays an important role in the pathogenesis of diabetes and its complications such as retinopathy, nephropathy and accelerated atherosclerosis. In diabetic retinopathy, it was demonstrated a selective loss of pericytes accompanied by capillary basement membrane thickening, increased permeability and neovascularization. This study was designed to investigate the role of diabetic conditions such as high glucose, AGE‐Lysine, and angiotensin II in the modulation of antioxidant enzymes activities, glutathione level and reactive oxygen species (ROS) production in pericytes. The activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total glutathione (GSH) was measured spectrophotometrically. The production of ROS was detected by spectrofluorimetry and fluorescence microscopy after loading the cells with 2′‐7′ dichlorofluoresceine diacetate; as positive control H2O2 was used. Intracellular calcium was determined using Fura 2 AM assay. The results showed that the cells cultured in high glucose alone, do not exhibit major changes in the antioxidant enzyme activities. The presence of AGE‐Lys or Ang II induced the increase of SOD activity. Their combination decreased significantly GPx activity and GSH level. Athree times increase in ROS production and a significant impairment of intracellular calcium homeostasis was detected in cells cultured in the presence of the three pro‐diabetic agents used. In conclusion, our data indicate that diabetic conditions induce in pericytes: (i) an increase of ROS and SOD activity, (ii) a decrease in GPx activity and GSH level, (iii) a major perturbation of the intracellular calcium homeostasis. The data may explain the structural and functional abnormalities of pericytes characteristic for diabetic retinopathy.

Effects of ageing on carbonyl stress and antioxidant defense in RBCs of obese Type 2 diabetic patients.

In this study we investigated the effects of ageing on the carbonyl stress (protein carbonyls and 4‐hydroxy‐2‐nonenal groups) and glutathione antioxidant defense in red blood cells (RBCs) of obese Type 2 diabetic patients with/without hypertensive complications. To this purpose the following methods were used: spectrophotometry (protein carbonyls, glutathione and glutathione peroxidase assays), immunofluorescence (4‐hydroxy‐2‐nonenal localization), western blotting (immunodetection of carbonylated proteins). The results showed that compared to RBCs of healthy subjects, in obese Type 2 diabetics, ageing is associated with: (i) an increase in the concentration and expression of carbonylated proteins, a marker of oxidative stress; (ii) a decrease of both non‐enzymatic and enzymatic endogenous glutathione defenses; (iii) a severely disturbed oxidant/antioxidant balance when obesity was associated with hypertension. The simultaneous insults of high blood pressure, obesity, and diabetes conducted to the highest carbonyl strss, exposure of 4‐hydroxy‐2‐nonenal Michel adducts at the outer leaflet of RBCs plasmalemma, and the lowest glutathione antioxidant potential, particularly in elderly patients. These results can explain the gradual age‐dependent diminishment of the detoxification potenital of RBCs that at the old age can not overcome the deleterious effects of the high systemic oxidative stress.

Advanced Glycation End Products, Oxidative Stress and Metalloproteinases are altered in the Cerebral Microvasculature during Aging

Biological aging is associated with an increased incidence of cerebrovascular disease. Recent findings indicate that oxidative stress promoting age-related changes of cerebral circulation are involved in neurodegenerative disorders such as Alzheimers disease (AD) and Parkinsons disease. The aim of this study was to evaluate the contribution of cerebral microvessels to the oxidative stress during brain aging, by: (i) assessment of precursors for advanced glycation end products (AGE) formation, (ii) activities of antioxidant enzymes, namely superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione disulfide reductase (GR), and (iii) the activities of metalloproteinases (MMPs), MMP-2 and MMP-9, involved in synaptogenesis and memory consolidation. The experiments were performed on two groups of male Wistar rats: 15 young (3-6 months old) and 15 aged (18-24 months old) animals. The cerebral microvessels were isolated by mechanical homogenization, the concentration of protein carbonyls and the activity of antioxidant enzymes were evaluated by spectrophotometry, and gelatin SDS-PAGE zymography was employed to evaluate MMP-2 and MMP-9 activities. The results showed that, by comparison with young rats, aged brain microvessels contain: (i) approximately 106 % increase of protein carbonyls production; (ii) approximately 68% higher GPx activity, unmodified activities of SOD and GR; (iii) approximately 30% diminishment in MMP-2 activity, and the specific occurrence of MMP-9 enzyme. The data suggest that the age-related changes of microvessels could increase the propensity for cerebral diseases and might represent, at least in part, a prerequisite for the deterioration of mental and physical status in the elderly.

The hyperlipemic hamster ‐ a model for testing the anti‐atherogenic effect of amlodipine

Male Golden Syrian hamsters were subjected to a hyperlipemic diet. At intervals ranging from 2 to 14 weeks, the animals were examined for changes in serum constituents and structural modifications of lesion‐prone areas: the cardiac valves, coronary arteries and aortic arch. Serum was characterized by a gradual increase in cholesterol, triglycerides and a decrease in total peroxyl‐radical trapping potential. The sequence of modifications of the endothelial cells, smooth muscle cells, and migrating plasma monocytes as well as of the extracellular matrix were established. Amlodipine treatment of hyperlipemic hamster was assessed. Amlodipine exhibited an athero‐protective effect, acting as antioxidant, reducing the LDL uptake by the vessel wall and consequently, limiting the size and extent of lesioned areas. The hyperlipemic hamster is a reliable model to unravel the cellular alterations leading to atheroma formation, and for testing the effect of drugs in this process.

A Hyperlipidemic Diet Induces Structural Changes in Cerebral Blood Vessels

The cerebrovascular pathology is an important contributor to the death rate presently. Hyperlipidemia , an established risk factor for cardiovascular diseases, is also incriminated in the neurodegenerative disorders. The aim of this study was to evaluate the effect of a hyperlipidemic (HL) diet on the morphology of the cerebral vessels and on the amyloid deposition in the HL hamster, an accepted model of atherosclerosis. Hamsters fed a HL diet were tested periodically for serum parameters and sacrificed after 3 and 6 months. The methods used were: paraffin embedding, thioflavin S amyloid staining, fluorescence and electron microscopy (EM). Increased serum cholesterol and triglycerides characterized the HL hamsters. The carotid arteries developed fatty streaks after 3 months and atherosclerotic plaques after 6 months HL diet. The brain cortex comprised irregularly shaped microvessels with large perivascular spaces, enlarged endothelial cells (EC) and occasionally a lumen full of lipoprotein particles. The thioflavin S reaction revealed a discreet staining of the capillaries walls; the EC cytoplasm and basal lamina contained a fibrillar material, with a pattern similar to an incipient amyloid deposit. Some large meningeal vessels from animals with serum cholesterol over 1000 mg/dl presented an intense autofluorescence in the adventitia; EM examination identified lipid-loaded perivascular cells in these areas. In conclusion, the detected morphological changes induced by the HL diet could represent a serious impairment for the normal brain function. These data may contribute to the better understanding of the risks of hyperlipidemia for the mental health, and its reversal could become a new therapeutic approach for neurodegenerative disorders.

Immunological detection of an analogue of the erythroid protein 4.1 in endothelial cells.

Endothelial cells (EC) of arterial and venous origin were investigated by indirect immunofluorescence and immunoautoradiography for the presence of red cell membrane 4.1-like protein. By immunofluorescence, EC exhibited a relatively uniform fluorescent staining sometimes of a reticular pattern, distributed over the entire cell. All controls were negative. Immunoblot analysis of EC revealed a cross reactive band of a molecular weight comparable to that of the erythrocyte band 4.1. These findings indicate that endothelial cells of arterial and venous origin express a polypeptide immunologically related to the erythrocyte protein 4.1, which may play an important role in membrane-cytoskeleton interactions.

Ceruloplasmin and oxidized LDL colocalize in atherosclerotic lesions of hamster

Epidemiological studies show that the risk for cardiovascular diseases increases with increasing levels of free-copper in plasma. It is known that intact ceruloplasmin (CP), the major protein transporter of copper in human plasma, oxidizes low density lipoproteins (LDL) in vitro. Our aim was to study the interaction between LDL and CP in vitro and in vivo, in an animal model of diet-induced atherosclerosis. In order to visualize the pathway of LDL into the arterial wall, human native LDL was labeled with fluorescent DiI and injected into male, Golden Syrian hyperlipemic hamsters. In vitro results demonstrated that slightly degraded CP has a significant oxidation potential against LDL at neutral pH. In vivo, after 24 hours circulation, LDL-DiI was taken up by the enlarged intima and fatty streaks of the arterial wall. Immunohistochemical localization of oxidized LDL and CP revealed their presence in the same areas of the arteries that take up LDL-DiI. Co-localization of LDL and CP in the enlarged intima of pro-atherosclerotic areas might explain the possible copper-induced oxidation process that might occur after native LDL is taken-up from the blood, transcytosed through the endothelium and accumulated in focalized deposits.

Arterial smooth muscle cells dysfunction in hyperglycaemia and hyperglycaemia associated with hyperlipidaemia : from causes to effects

Abstract Given the important role of smooth muscle cells in arterial wall dysfunction in diabetes, as well as in diabetes associated with accelerated atherosclerosis, we provide a brief review of the recent achievements in identification of signalling molecules underlying their altered cellular responses, and examine the consequences of these pathological insults on smooth muscle cells properties. The original results emerging from the Golden Syrian hamster model (rendered diabetic or simultaneously hyperlipidaemic-diabetic) and from human aortic smooth muscle cells cultured in 25 mM glucose (to mimic diabetic condition) or sera of obese type 2 diabetic patients (to mimic the metabolic syndrome condition) are presented in this context. We conclude this review with several open issues disclosed by the most recent literature that deserve essential attention for targeting the translational medicine.