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Biomedical subjects

N Wiernsperger

Publications and source records attributed to N Wiernsperger.

At least 37 records · Page 2Linked to original sources
PubMed1998

In vivo effect of 8-epi-PGF2alpha on retinal circulation in diabetic and non-diabetic rats.

Retinal hemodynamic responses to a F2-isoprostane, 8-epi-PGF2alpha, were quantitated in vivo in non-diabetic and diabetic rats using a video fluorescein angiography system. Vascular diameters and retinal mean circulation time were determined before and after 5 microl intra-vitreous injection of 8-epi-PGF2alpha (10(-5) to 10(-3) M), 10(-4) M 8-epi-PGF2alpha, + 10(-3) M SQ29,548 or 10(-3) M LCB2853 (two inhibitors of TXA2 receptor), 10(4) M 9beta-PGF2alpha, or the carrier in non-diabetic animals. Diabetic rats received either 8-epi-PGF2alpha 10(-4) M, or the carrier. Compared to control animals, diabetic rats presented in the basal state a venous vasodilation (P<0.01), without modification of retinal mean circulation time or blood flow. After intravitreous injection of 8-epi-PGF2alpha, a significant arterial vasoconstriction was observed in control but not in diabetic animals. This vasoconstriction was concomitant with increased retinal mean circulation time in control but not in diabetic rats, inducing an impaired reduction of blood flow. No vasoconstriction was observed after injection of either the carrier, 9beta-PGF2alpha or the isoprostane associated to the inhibitors of TXA2 receptors. This is the first direct observation that the isoprostane 8-iso-PGF2alpha is a potent vasoconstricting agent in the retina. It occurs at the arterial but not venous level, and is likely mediated through a TXA2-like receptor. Differences observed between control and diabetic animals suggest altered adaptative mechanisms toward vasoconstrictor substances (such as isoprostanes) in diabetic rats.

Angiography↗
PubMed1998

Fatty acid composition of phospholipids and neutral lipids from human diabetic small arteries and veins by a new TLC method.

It has been suggested that lipid peroxidation of polyunsaturated fatty acids (PUFA) may play a role in the pathogenesis of diabetic complications. To test this hypothesis, we aimed to compare PUFA composition of small arteries and veins (< 500 microm diameter) obtained from diabetic or non-diabetic Guadeloupean patients undergoing arterio-venous shunt surgery before renal dialysis. Small forearm subcutaneous vessels were analysed by a new TLC method which involved inclusion of vascular biopies directly in alveoles made in the TLC gel and lyophilization onto the plate. The TLC plate was then chromatographed and lipids were both extracted and eluted during this step. Fatty acid composition of phospholipid and neutral lipid fractions were determined. Similar fatty acid composition was obtained for arteries and veins from diabetic or non-diabetic subjects. In phospholipids from diabetic vessels, major changes consisted of a 20% decrease of arachidonic acid (20:4 n-6), a 40% decrease of its elongation product 22:4 n-6 and 30% increase of 18:2 n-6. In neutral lipids, 20:4 n-6 was also diminished by 60% whereas oleic acid increased by 15%. This loss of arachidonic acid in small diabetic vessels suggests impaired delta6-desaturase forming 20:4 n-6 or alternatively increased peroxide formation, in the vascular wall of small vessels in diabetic patients.

Adipose Tissue↗
PubMed1998

The formation of tubular structures by endothelial cells is under the control of fibrinolysis and mechanical factors.

This study highlights the importance of several factors involved in the formation of capillary-like structure formation (CLS) using Human Umbilical Vein Endothelial Cells (HUVEC) and Bovine Retinal Endothelial Cells (BREC) cultured on fibrin gels. The fibrin concentration inducing (CLS) was 0.5 mg/ml for HUVEC and 8 mg/ml for BREC. The high fibrin concentration required for the latter cells appeared necessary to counterbalance the extensive fibrinolysis of the gel by the BREC. Fibrin degradation products measured in the culture media showed that fibrin degradation was mandatory but not sufficient for CLS formation. Fibrin degradation acted in concert with the mechanical, concentration dependent properties of the gels to induce CLS. For example, HUVEC did not form CLS on a rigid fibrin of 8 mg/ml in spite of fibrinolysis. As cell reorganisation occurred, the fibrin was disrupted (HUVEC) or pleated (BREC) giving indirect proof of the development of mechanical forces. During CLS formation, an increasing amount of latent TGFbeta1 was measured in the medium (1000-1700 pg/ml). The active form of TGFbeta1 was not, however, detected and the addition of anti-TGF-beta1 antibody to the medium did not influence the formation of the CLS network. Yet, added activated TGF-beta1 led to the formation of less organised structures, that were completely abolished by the concomitant addition of the same anti-TGF-beta1 antibody. Thus, it is likely that TGF-beta1 secreted by the endothelial cells remained in its latent form. In conclusion, a balance between the mechanical properties of fibrin and the fibrinolytic activity of each cell type may regulate CLS formation in our models. We think that the high fibrinolitic activity of the BREC may represent a defense mechanism to protect the retina against thrombosis-induced damage in vivo.

Journal Article↗
PubMed1997

Action of metformin on erythrocyte membrane fluidity in vitro and in vivo.

The lipid domains of the cell membrane are believed to be one of the sites where biguanides exert their antihyperglycemic effect. We have examined the effects of metformin on the membrane fluidity of intact erythrocytes in vivo and in vitro. Membrane fluidity was measured by monitoring changes in the anisotropy of the fluorescent probe 6-antroyloxystearic acid (6-AS). The erythrocyte membranes from patients with non-insulin dependent diabetes mellitus treated with metformin were more fluid than those from non-insulin dependent diabetes mellitus patients treated by diet or healthy controls. There was no correlation between membrane fluidity and the plasma lipids or the parameters of metabolic control, suggesting that the high fluidity is an effect of metformin itself. Incubation of erythrocytes from healthy controls and diabetic patients treated by diet or glibenclamide with metformin in vitro confirmed that metformin increases the fluidity of erythrocyte membranes. In vitro metformin did not alter the fluidity of membranes from diabetic patients treated with metformin, perhaps because the basal high fluidity due to their in vivo interaction with plasma metformin could be increased no further. Since insulin appears to be required for the antihyperglycemic effect of metformin, the effect of insulin on membrane fluidity was also evaluated. Insulin generally had a small fluidizing effect on erythrocytes in vitro. The fluidizing action of both insulin and metformin could represent a membrane event common to the hormone and drug leading to additive or synergistic effects in vivo.

Adult↗
PubMed1997

Advanced glycation end products induce specific glycoprotein alterations in retinal microvascular cells.

In order to investigate the mechanisms involved in diabetic retinopathy, we studied the effects of advanced glycosylation end products (AGE) on retinal microvascular cell glycoproteins. Bovine retinal pericytes (BRP) and endothelial cells (BREC) were incubated in the presence of AGE-modified albumin and cell glycoproteins analyzed by lectin affinoblotting and metabolic radiolabeling with sugar precursors. Selective modifications in the glycoprotein sugar chains were observed mainly in BREC and for a 210 kDa membrane glycoprotein. Indeed, a 40% decrease of alpha(2,3) sialic acid, beta(1,3) galactose or alpha(1,6) fucose content was observed without significant protein amount changes. These glycoprotein alterations were related to the concentration of AGE. Neither BRP nor BREC glycoproteins were modified when cells were incubated with high glucose or fructose concentrations. These results suggest a new diabetic pathogenic mechanism in which a protein post-translational modification, in this case glycation, could modify another post-translational process such as the enzymatic glycosylation.

Animals↗
PubMed1997

Growth modulation of retinal microvascular cells by early and advanced glycation products.

To investigate the possible implication of non-enzymatic glycosylation in the etiopathogenesis of the diabetic retinopathy, we studied the effect of early and advanced glycation products on the growth of retinal microvascular cells. Glucose modified products were obtained by incubating bovine serum albumin or fetal bovine serum with 0.5 M glucose for 10 (early glycation products: EG-BSA and EG-FBS, respectively) or 60 days (advanced glycation end products: AGE-BSA and AGE-FBS, respectively). Cell growth was assessed by cell counting and DNA content determination. EG-BSA or AGE-BSA significantly decreased pericyte proliferation after 8 days of culture (33 and 13% inhibition, respectively). Concerning endothelial cells, EG-BSA reduced proliferation to 40% whereas AGE-BSA increased it to 156% after 4 days of culture. The glucose-treated sera didn't exhibit the same growth effects, neither the EG-FBS nor the AGE-FBS significantly affected endothelial cell proliferation. Only the AGE-FBS showed a significant inhibitory effect on pericyte proliferation (40% inhibition). We conclude that retinal microvascular cell growth in vitro could be differently modulated by early and advanced glycation products. The inhibitory effect of AGEs observed on pericyte growth, suggests that glycoxidation could be implicated in the pericyte loss observed in diabetic retinopathy.

Animals↗
PubMed1997

Effects of insulin and the combination of insulin plus metformin (glucophage) on microvascular reactivity in control and diabetic hamsters.

The purpose of this study was to determine the in vivo microvascular reactivity of arterioles (mean internal diameter range: 16.0 to 106.4 microm) and venules (mean internal diameter range: 24.0 to 117.3 microm) in the hamster cheek pouch to insulin and to the mixture insulin + metformin. Experiments were performed using an intravital microscope coupled to a closed-circuit TV system and a videotape. The TV monitor display was used to obtain arteriolar and venular internal diameter measurements by an image-shearing device. The studied drugs were applied topically, added to the superfusion solution, to avoid systemic effects that would complicate the analysis of the results. In control animals (glycemia 7.7 +/- 0.4 mmol/L), application of insulin (10 to 500 microU/mL/min) evoked vasodilatation in a dose-dependent fashion in arterioles (4.9 +/- 3.2% to 50.9 +/- 6.5%, smallest and largest concentration, respectively, values expressed in percent of the initial diameter as mean +/- SE) and venules (-2.1 +/- 3.1% to 14.3 +/- 5.1%), decreased and finally abolished the spontaneous vasomotion frequency (from 9.5 +/- 0.3 cycles per minute [cpm] to 0.0 +/- 0.0 cpm) and amplitude (from 8.6 +/- 0.3 to 0.0 +/- 0.0 microm). Addition of metformin, 0.2 mg/mL/min, did not significantly change either the observed vasodilatation in arterioles and venules or the vasomotion frequency and amplitude curves. Two types of diabetic hamsters were studied: severely diabetic, induced with three intraperitoneal injections of streptozotocin, diluted in physiological saline, 50 mg/kg/dose, given in three consecutive days, and mildly diabetic, induced by a single dose of streptozotocin. All diabetic animals were studied four weeks after the onset of diabetes and no specific treatment for diabetes was given. In severely diabetic hamsters (glycemia 18.0 +/- 2.2 mmol/L), application of insulin, in the same concentration range, evoked a significantly reduced vasodilatation in arterioles as compared with control animals (5.9 +/- 1.3% to 18.9 +/- 3.5%) and did not change the vasodilatation observed in the venules (5.9 +/- 1.4% to 21.3 +/- 2.5%). In these preparations no spontaneous arteriolar vasomotion could be detected. Addition of metformin did not significantly improve the impaired vasodilatation. In mildly diabetic hamsters (glycemia 12.1 +/- 0.8 mmol/L), application of insulin, in the same concentration range, evoked vasodilatation, in a dose-dependent fashion, equivalent to the one observed in control animals, in arterioles (3.1 +/- 2.5% to 53.4 +/- 10.0%) and venules (7.1 +/- 3.0% to 29.9 +/- 4.8%) and also reduced the vasomotion frequency (from 10.1 +/- 0.3 to 0.1 +/- 0.1 cpm) and amplitude (from 9.2 +/- 0.6 to 0.2 +/- 0.2 microm). Addition of metformin tended to increase the observed arteriolar dilatation (6.6 +/- 3.0% to 67.8 +/- 5.5%), did not change the venular dilatation (6.7 +/- 4.8% to 28.0 +/- 3.3%), and tended to preserve vasomotion frequency and amplitude. These experiments show that (1) insulin has a direct dilatatory effect on arterioles and venules; (2) the vasodilatation evoked by insulin is impaired in severe diabetes, and (3) no significant abnormality could be detected on microvascular reactivity in mild diabetes. Further addition of metformin helped to maintain the spontaneous arteriolar vasomotion even during moderate vasodilatation and tended to augment the arteriolar dilatation evoked by insulin in mildly diabetic animals.

Administration, Topical↗
PubMed1997

Effects of metformin on lactate uptake and gluconeogenesis in the perfused rat liver.

To directly assess the effects of the biguanide, metformin, on hepatic gluconeogenesis, it was added at high therapeutic levels (90 microg/ml) to the medium perfusing an isolated rat liver. Lactate (1 mg/min) was infused simultaneously along with [14C]lactate with or without [3H]lactate. [6-(3)H]glucose was added at the beginning of the perfusion in studies where [3H]lactate was not infused. Glucose levels decreased relative to control studies (metformin dose = 0) and lactate concentrations increased in this closed system. Quantitative analysis of the relationship between labeled glucose and lactate indicated that the flux of carbon from lactate to glucose and CO2 was halved, whereas reflux from glucose to lactate increased by approximately 80%. This was corroborated by measurement of labeled lactate extraction as well as glucose, CO2, and lactate production across the liver. Glycogen content of the liver fell by 60% relative to control and was greater for the gluconeogenic pathway. These data are consistent with an inhibitory action of metformin on gluconeogenesis, which is due to a primary inhibition of hepatic lactate uptake.

Animals↗
PubMed1997

Involvement of cell-cell interactions in the pathogenesis of diabetic retinopathy.

Retinopathy is a severely disabling complication of diabetes mellitus whose underlying mechanisms are still obscure. The key question is why retinal microvessels are so reactive to the diabetic environment, whereas other microvessels show no evidence of alteration. The answer could lie in the particular structure and location of retinal microvessels since they are composed of, and surrounded by, various types of cells, thereby favouring cell-cell interactions which occur between cells of the capillary wall itself but also with circulating blood cells and retinal neural cells. In the retinal capillary wall, pericytes are in close relation with underlying endothelial cells, and both cell types have close contacts with the capillary basement membrane. Adhesion molecules and cell surface glycoconjugates appear to be the main mediators of interactions between circulating blood cells and capillary endothelial cells, whereas growth factors seem to play a major role in interactions between glial and capillary wall cells in the retina. Biochemical dysfunctions observed in diabetes, such as glycation of proteins and enhanced oxidative stress, could modify these cell-cell and cell-matrix interactions, thereby disturbing the complex cellular organization in which retinal microvessels are embedded. The aim of this review was to provide an overall, nonexhaustive description of some types of cellular interactions that may underlie the pathogenic mechanisms involved in flow and growth changes leading to diabetic retinopathy.

Animals↗
PubMed1996

Description of an in vitro angiogenesis model designed to test antiangiogenic molecules.

Angiogenesis is involved in numerous pathologies. Studies with in vitro models allow the description and analysis of the different steps involved in this process under defined culture conditions. We describe a controllable and reproducible in vitro model. We assessed the usefulness of this model with two different cell lines: human umbilical vein endothelial cells and bovine retinal endothelial cells. These cells reorganize themselves and change their phenotypes within 24 h after seeding under our culture conditions (low human serum percentage, defined cell density, fibrin matrix) to form 'capillary-like structures' (CLS) in vitro. We showed that, depending on the cell line used, the fibrinolytic activity of the cells was a determining factor which could induce or prevent the formation of the CLS. Inhibitors of angiogenesis can be tested using such a model.

Angiogenesis Inducing Agents↗
PubMed1996

Docosahexaenoic acid is a major n-3 polyunsaturated fatty acid in bovine retinal microvessels.

The aim of this study was to purify microvessels from bovine retina and also to cultivate bovine retinal endothelial cells (BRECs) or intramural pericytes, to determine their fatty acid composition. Microvessels were obtained after Dounce homogenization of the retina followed by centrifugation on albumin cushion and finally microvessels in the pellet were trapped on a 100-microns nylon filter. Contamination of microvessel preparations by neuronal tissue, assessed after both microscopic examination and western blotting with a monoclonal antibody raised against rhodopsin, was minor. In the entire bovine retina, docosahexaenoic acid (DHA) represented 23.3% of the total fatty acids and there was about three times less arachidonic acid (AA) (8.2%) than DHA. In contrast, DHA and AA levels were almost equivalent in the retinal microvessels with approximately 10% of total fatty acids. When compared with intact microvessels, the DHA proportion of confluent monolayers of both BRECs or pericytes in primary cultures dropped to approximately 2% of the total fatty acids, whereas AA was unchanged. Culture medium supplementation with unesterified DHA (10 microM) restored the DHA proportion of BRECs close to the microvascular value at the expense of linoleic acid without affecting AA very much. In contrast, DHA supplementation in pericytes increased the DHA proportion of these cells at the expense of AA. In conclusion, DHA of intact microvessels represented 10% of the total fatty acids, which was close to the AA proportion. Mild DHA supplementation of BRECs or pericytes in primary cultures restored their DHA proportion to the original microvessel value. This high percentage of polyunsaturated fatty acids in retinal microvessels should allow us to test the hypothesis that oxidation products derived from these fatty acids may be involved in the pathogenic process leading to diabetic retinopathy.

Animals↗
PubMed1996

The antidiabetic drug metformin elevates receptor tyrosine kinase activity and inositol 1,4,5-trisphosphate mass in Xenopus oocytes.

Although metformin is an important antidiabetic, its mechanism of action is still unknown. To study its mechanism, we examined metformin stimulation of insulin action on the Xenopus oocyte. Similar to therapeutic concentrations, maximal stimulation of insulin-induced meiotic cell division was achieved at about 1-10 microg/ml (or 7.7-77 /microM) metformin. An equivalent concentration of metformin was required to elevate receptor tyrosine kinase activity (in whole cells or a membrane-cortex preparation) and, through this tyrosine kinase activation, inositol 1,4,5-trisphosphate (IP3) production. With whole cells, the preincubation time for metformin stimulation of insulin action (approximately 1 h) was equivalent to the time required for metformin to maximize tyrosine phosphorylation and raise IP3, levels. With the membrane-cortex preparation, metformin was active within minutes; thus, metformin may act at an intracellular site. Since metformin can increase IP3, mass, we prevented elevation of calcium by prior microinjection of a calcium chelator or heparin (a drug that inhibits IP3 binding to the IP3 receptor). Both the chelator and heparin blocked metformin stimulation of insulin action on whole cells. Since microinjection of IP3, also stimulates insulin action, metformin may stimulate insulin action by elevation of intracellular calcium in addition to activation of the receptor tyrosine kinase.

Animals↗
PubMed1996

Interaction of glucose and metformin with isolated red cell membrane.

Isolated human erythrocyte membranes (red blood cell (RBC) ghosts) were incubated with glucose at 5, 10, 20 and 100 mmol/l concentrations, with insulin (0.01 to 200 mU/l) and metformin (CAS 657-24-9) 0.5 up to 50.0 mumol/l). Binding studies with 14C-glucose and subsequent gel electrophoresis revealed 60% of the radioactivity around ban 4.2-4.5 at 5 mmol/l, whereas a random distribution of radioactivity over all protein bands of the RBC membrane was found at 20 mmol/l concentration after incubation for 30 min or 48 h. Metformin does not bind covalently to RBC membranes, however, after photochemical linkage of 14C-metformin via the aminoreactive linker azidophenylglyoxal the highest radioactivity (21%) was counted in the range of band 4.2-4.5. In parallel with an increase of order parameters of 5-doxyl-stearic acid the thiol status of the membranes decreases as determined by monobromobimane fluorescence. 20 and 100 mmol/l concentrations of glucose decrease the reactivity of membrane thiols towards bromobimane significantly to 73 and 62% of the controls. Concomitantly, membrane fluidity at polar sites is diminished as measured by order parameters of spin label 5-doxyl stearic acid. In RBC membranes pretreated with 20 mmol/l glucose the decreased fluorescence is significantly raised again by insulin and metformin. This effect is even more pronounced, if insulin and metformin are incubated together. Reaction of membrane thiols with a maleimido spin label detects modification in the ratio of mobile and immobilized spin label populations in the electron paramagnetic resonance signal under the above conditions, indicative of conformational changes of membrane proteins.

Bridged Bicyclo Compounds↗
PubMed1995

Metformin induces an agonist-specific increase in albumin production by primary cultured rat hepatocytes.

Metformin (MET) is known to increase several biological effects of insulin (INS), but there is no information concerning its direct effects on protein synthesis. We studied the action of MET on albumin production by primary cultures of freshly isolated rat hepatocytes, alone or in combination with various agonists: INS, IGF-1, EGF, thyroxin, and dexamethasone. While having no effect alone, MET in vitro potentiates the effects of INS, IGF-1, and EGF. When this increasing effect toward INS was studied over a broad concentration range, MET appeared to improve low-acting INS levels and to intensify the maximal INS effects. In contrast, MET did not change the production of albumin stimulated by thyroxin or dexamethasone. Animals chronically pretreated with MET in vivo showed a higher yield of isolated hepatocytes, better attachment, and especially higher viability after liver perfusion and during cell culture. This may largely explain why basal albumin rates were higher than in in vitro-treated cells. The effect of MET in the presence of the agonists exhibited the same agonist-specificity as in vitro. Our data provide new insights into the pharmacology of MET by showing that hepatic protein synthesis is increased by MET and INS. From the specificity of action of MET towards INS, IGF-1, and EGF (but not thyroxin or dexamethasone), we hypothesize that this biguanide may act on intracellular pathways located between membrane receptors and sites of branching in the signaling cascades shared by these agonists.

Albumins↗
PubMed1995

The effects of metformin on the capillary permeability to albumin in women patients with cyclic edema.

An experimental work has suggested the efficacy of metformin, an oral antidiabetic agent, on capillary permeability. This agent has been tested in 10 women patients with cyclic edema, 7 of them being obese. The capillary permeability to albumin studied by an isotope test derived from Landis's method was initially increased. After a mean of six-weeks of treatment the albumin retention and an index demonstrating the interstitial protein elimination through the lymph route were significantly improved. Concomitantly, the swelling feelings were reduced in 8 cases and the lower limb edema had decreased or disappeared in 8 of 9 patients who initially presented these symptoms. The effects of metformin are beneficial and have been observed to be independent of glycemic change. The results suggest a special effect of this agent on the microcirculation. Nevertheless, a controlled study is required.

Adult↗