Paul Urios

Effect of an aldose reductase inhibitor on type IV collagen production by human endothelial cells cultured in high glucose

SummaryDiabetic microangiopathy is characterized by a thickening of capillary basement membranes associated with type IV collagen accumulation. An increase in type IV collagen content of the aortic wall is also observed in macroangiopathy. In order to analyse the importance of the polyol pathway in the development of the collagen metabolism alterations seen in diabetic angiopathy and their prevention by aldose reductase inhibitors, we have studied the effects of sorbinil on the high glucose-induced stimulation of type IV collagen biosynthesis in human umbilical vein endothelial cells. Primary cultures were exposed to high glucose (16.7 mmol/l), with and without 0.11 mmol/l sorbinil, for 3 or 6 days after beginning of confluence. We measured the soluble type IV collagen secreted into the culture medium and the insoluble type IV collagen accumulated in the extracellular matrix and cells, by ELISA. We also studied [14C]proline incorporation into the newly synthesized collagenous and total proteins in the culture supernatant and in the extracellular matrix and cell fraction. High glucose decreased the number of cells and increased the amount of type IV collagen in the culture supernatant and in the extracellular matrix and cell fraction. It also increased proline incorporation into the newly synthesized collagenous and total proteins in the culture supernatant and in the extracellular matrix and cell fraction. Sorbinil corrected all these high glucose-induced alterations. The corrective effects of sorbinil on the proliferation and on type IV collagen metabolism of endothelial cells cultured in high glucose may be attributed to prevention of polyol pathway dysregulation.

Cyclic guanosylmonophosphate urinary excretion in parasympathicomimetic or parasympatholytic syndromes induced by reserpine or diphemanil-methylsulfate

Parasympathetic hyperactivity is found in some infants presenting faint episodes and could be responsible of certain Sudden Infant Death Syndrome cases. Therefore it was interesting to look for a noninvasive biochemical indicator of parasympathetic activity. A parasympaticomimetic syndrome associated with muscarinic receptor stimulation, which has been followed during 48 h, was obtained in the awake rat by reserpine injection (6.25 mg/kg at T0 and T24h), and a model of prolonged parasympatholytic syndrome, by administration of diphemanil-methylsulfate (DPMS), a muscarinic receptor inhibitor, in drinking water (mean daily dosis: 150 mg/kg). Significant bradycardia and tachycardia were respectively observed. In the reserpine-treated rats we found significantly increased cyclic guanosylmonophosphate (cGMP) urinary excretion between T24h and T48h, when compared with vehicle-treated controls (+87% in one experiment, +135% in the other, when expressed in pmol/microg creatinine); norepinephrine urinary excretion between T24h and T48h was decreased (-44%); the increase in cGMP urinary excretion was not significantly modified by the NO-synthase inhibitor, L-nitroarginine-methyl-ester. In the DPMS-treated rats, we observed a significantly decreased cGMP (-20%) and increased norepinephrine urinary excretion (+61%). Thus cGMP excretion varied in opposite directions in the reserpine- and DPMS-treated rats. The link between these modifications in cGMP excretion and muscarinic receptor stimulation or blockade has still to be fully demonstrated. Urinary cGMP excretion could be tested as screening parameter in infants at risk of faint episodes associated with bradycardia.

A flavonoid fraction purified from Rutaceae aurantiae (DaflonR) inhibiting AGE formation, reduces urinary albumin clearance and corrects hypoalbuminemia in normotensive and hypertensive diabetic rats

AIM Advanced glycation endproducts (AGEs) have been shown to contribute to alteration of glomerular permselectivity to proteins in diabetes. Oxidative stress is required for AGE formation. Therefore we studied the effect of an antioxidant micronized purified flavonoid fraction (MPFF, Daflon(R) 500 mg), on urinary albumin clearance in diabetic rats. METHODS Hyperglycaemia was induced by streptozotocin 55 mg/kg IM at days 0 and 7 in normotensive Wistar rats (NWR, diabetes duration 5 months) or hypertensive Wistar Kyoto rats (SHR, diabetes duration 2 months). MPFF was administered at 300 mg/kg/day, from day -2 until sacrifice. RESULTS After 5 months of diabetes in NWR, MPFF reduced albumin clearance from 729±92 to 392±60 nl/min/kg, p<0.01, and restored albuminemia from 20.4±0.9 to 24.0±1 g/l, p<0.05; albumin fractional clearance was significantly diminished in the flavonoid-treated diabetic rats (0.360±0.037‰ versus 1.335±0.430‰ in the diabetic controls, p<0.001); MPFF did not significantly modify blood glucose and plasma fructosamine levels. After 2 months of diabetes in SHR, MPFF reduced albumin clearance from 243±121 to 101±47 nl/min/kg, p<0.05, and restored albuminemia from 21.1±1.6 to 26.7±2.2 g/l (p<0.05); MPFF also decreased plasma fluorescence characteristic of AGEs (p<0.02). Besides hesperetin, a main metabolite of MPFF recovered in plasma, inhibited in vitro the formation of the crosslinking AGE pentosidine in collagen incubated with high glucose (p<0.001). CONCLUSION Our results confirm the role of glycoxidative stress in diabetic nephropathy. MPFF might be useful as complementary treatment for preventing diabetic microangiopathy.

Skin collagen pentosidine and fluorescence in diabetes were predictors of retinopathy progression and creatininemia increase already 6years after punch-biopsy.

OBJECTIVE Advanced glycation end products (AGEs) of collagens appear to contribute to microvascular complications in diabetes. Do high concentrations of AGEs in skin collagen predict accelerated progression of these complications after 6 years and indicate the need for tighter anti-diabetic treatment? DESIGN AND METHODS We measured two AGE parameters in collagen extracted from skin punch-biopsies: pentosidine and fluorescence at 370/440nm, as markers and predictors of microvascular complications, in 30 patients with diabetes (14 type-1, 16 type-2) without renal insufficiency, and in age- and gender-matched normoglycemic controls, followed at Hôtel-Dieu in Paris. RESULTS At the time of biopsy, marked increases in pentosidine (p=0.0014) and fluorescence (p=0.0001) expressed per collagen hydroxyproline, were found in the patients with diabetes versus the controls. A significant effect of age was found for pentosidine, but not fluorescence, measurements in the normoglycemic controls. Therefore pentosidine but not fluorescence results were corrected for age in the patients. Pentosidine and fluorescence were correlated with diabetes duration. Fluorescence was significantly dependent on retinopathy presence and score in type-1 and type-2 diabetes, whereas pentosidine was not. Fluorescence was correlated with microalbuminuria only in type-1 diabetes. Neither fluorescence nor pentosidine were correlated with creatininemia. Already six years after biopsy, retinopathy score progression and creatininemia increase were significantly correlated with initial pentosidine and fluorescence measurements. CONCLUSIONS These AGEs are good predictors of progression of microvascular complications and appear to be pathogenic. High skin concentrations of AGEs should induce tighter anti-diabetic treatment.

Unexpected elevation of pentosidine formation in collagen incubated with glucose by low concentrations of the AGE-inhibitor aminoguanidine.

To the Editor: In diabetes, protein modification by hyperglycaemia leads to Amadori products like HbA1c, which in turn can be oxidised to form advanced glycation end products (AGE), and particularly pentosidine [1]. Pentosidine crosslinks peptidic chains, alters physicochemical properties of proteins and contributes to the development of diabetic complications. Skin collagen pentosidine levels, adjusted for age, have been shown to correlate with the severity of complications in Type 1 diabetic patients [2]. More recently, when adjusted for age and diabetes duration, they were significantly associated with nephropathy and neuropathy [3]. Aminoguanidine (pimagedine) inhibits advanced glycation by preventing conversion of Amadori products into AGE [4]. It was tested successfully in experimental models of diabetes, reducing proteinuria and retinopathy, and improving nerve conduction velocity [5]. While screening various inhibitors of AGE formation in collagen incubated with glucose, we used aminoguanidine as reference. We observed that, at aminoguanidine concentrations higher than 18 mmol/l, aminoguanidine reduced pentosidine formation as expected, but increased it significantly at lower concentrations. This was confirmed in different experiments. Insoluble collagen from bovine Achilles tendon (9 mg/ml, Sigma, St Louis, Mo., USA) was incubated in 200 mmol/l sodium phosphate buffer, pH=7.4, with or without 250 mmol/l glucose, in the absence or presence of aminoguanidine hydrochloride (obtained from equimolar amounts of aminoguanidine bicarbonate [Sigma] and HCl in 400 mmol/l phosphate buffer) at concentrations ranging from 1.6 mmol/l to 89 mmol/l. This was done for 28 days at 37 °C with toluene (10 μl/1 ml). Each condition was repeated in quadruplicates. After incubation, collagen was washed, lyophilised and submitted to acid hydrolysis under vacuum. Pentosidine was measured by HPLC [1], hydroxyproline was determined colorimetrically [6] and the pentosidine : hydroxyproline molar ratio was calculated. Specific advanced glycation by pentosidine (SAGP) was evaluated as follows: (sample pentosidine:hydroxyproline – pentosidine:hydroxyproline of control without glucose) × 100/ (pentosidine:hydroxyproline of collagen + glucose – pentosidine:hydroxyproline of collagen without glucose). Results were presented as means ± SEM. Statistical comparisons were performed by analysis of variance. Four experiments were conducted: a preliminary experiment (y) and three further experiments a, b, c. In experiment c, EDTA (Sigma) and diethylene triamine pentaacetic acid (DTPA) (Acros Organics, Geel, Belgium) were tested at 10 and 36.8 mmol/l, to compare the activity of chelators with that of aminoguanidine, which is reported to possess chelating activity [7]. In experiment y, in the presence of glucose alone, the pentosidine hydroxyproline ratio was 31.6±1.7 pmol/μmol; 40 mmol/l aminoguanidine reduced SAGP to 24±4% of this collagen + glucose control (p<0.001), but 8 mmol/l aminoguanidine unexpectedly increased SAGP to 172±5% (p<0.001). This paradoxical observation was confirmed in experiments a and b, using extended aminoguanidine concentration scales. Results are shown in Figure 1. The maximum paradoxical increase in SAGP was observed with 8 mmol/l aminoguanidine. Increases were found at concentrations from 1.6 mmol/l to 18 mmol/l. However, at 40 mmol/l and 89 mmol/l aminoguanidine, the inhibitory effect of aminoguanidine was confirmed (33±3%, p<0.001, and 31±5%, p<0.001, at 40 mmol/l; 3±1%, p<0.001, and 2±1%, p<0.001 at 89 mmol/l; in experiments a and b respectively). In experiment c, we found a significant reduction of SAGP in the presence of EDTA (34±12%, p<0.001, and 25±3%, p<0.001, at 10 mmol/l and 36.8 mmol/l respectively) and a marked fall in the presence of DTPA (5±1%, p<0.001, and 6±3%, p<0.001). We presented a preliminary abstract of our observations at the International Symposium on Maillard Reaction in Kumamoto [8]. Since then, paradoxical increases in pentosidine formation after ribose glycation of bovine serum albumin have been observed using a novel pyridoxamine derivative and various AGE inhibitors [9]. Formation of AGE from glucose, but not from pentose, needs oxygen and trace metal ions [7]. Under our conditions of glycation by glucose, pentosidine is formed in the presence of air and trace amounts of metal ions. As DTPA traps these, pentosidine formation is almost completely inhibited. Aminoguanidine prevents pentosidine formation at high concentrations above 18 mmol/l through two possible mechanisms: dicarbonyl trapping and chelating activity [4, 7]. At concentrations lower than 18 mmol/l, the unexpected increase in pentosidine formation may result from a reaction of aminoguanidine with (i) auto-oxidation products of glucose (arabinose or glyoxal [10]), and (ii) with two accessible lysines and/or hydroxylysines at a suitable distance to form pentosidine. In the presence of oxygen and a few free metal ions, this could take place as long as aminoguanidine chelating activity is not complete. Indeed its apparent binding constant for copper is approximately 2 mmol/l [7]. Increased pentosidine formation from glucose in albumin, in the presence of metal ions and 5 mmol/l aminoguanidine (+21%), was recently reported [9], with pentosidine formation inhibited by 25 mmol/l aminoguanidine. Our model of AGE formation in high glucose appears to mimic in vivo conditions in the extracellular matrix of diabetic patients and in some cells with high glucose, such as endothelial cells, exaggerating however the glucose level. Interestingly, aminoguanidine was tested in clinical trials, significantly decreasing circulating LDL levels by 28% after 1 Diabetologia (2004) 47:959–961

Correlations between urinary excretion of catecholamines and electrocardiographic parameters of vagal hyperreactivity in infants with fainting spells. Implication of sympathetic hypotonia

BACKGROUND Vagal hyperreactivity (VHR) is a frequent etiology of infant fainting spells; but it is sometimes difficult to diagnose. A biochemical test would therefore be useful, especially as the oculocardiac reflex (OCR) test innocuity is not absolute. AIMS To evaluate urinary excretions of norepinephrine, epinephrine and dopamine as markers for vagal hyperreactivity. METHODS During check-up of 55 infants from 0.5 to 11 months of age, for discomfort episodes, including OCR and Holter recording, 24h urinary assays of total norepinephrine, epinephrine and dopamine were carried out to evaluate sympathetic activity. RESULTS Epinephrine and norepinephrine urinary excretions were negatively correlated with VHR intensity, as measured by the OCR ECG parameters: RRmax, % cardiac deceleration and minimal frequency; dopamine excretion was not. When RRmax(OCR) was greater or equal to 800 ms, epinephrine urinary excretion tended to be less or equal to 9 nmol/mmol creatinine and norepinephrine excretion less or equal to 190 nmol/mmol creatinine. CONCLUSION A delay in maturation of the sympathetic system and/or adrenomedullary glands with low secretion of norepinephrine and epinephrine inducing a desequilibrium of the sympathetic/parasympathetic balance may contribute to the fainting spells observed with VHR. Epinephrine and norepinephrine urinary excretions may provide informative complementary noninvasive markers for VHR.