Miklós Káplár

Cerebrovascular Reactivity and Reserve Capacity in Type II Diabetes Mellitus

The aim of the study was to test the hypothesis that cerebrovascular reserve capacity and cerebrovascular reactivity are impaired in patients suffering from non insulin-dependent diabetes mellitus. We also intended to investigate factors which may influence resting cerebral blood flow velocity and cerebrovascular reserve capacity. A total of 28 patients suffering from type II diabetes mellitus and 20 healthy control subjects were studied. Based on diabetes duration patients were divided into two groups: subjects with > 10 years and those with < or = 10 years disease duration. Middle cerebral artery mean blood flow velocities were measured at rest and after intravenous administration of 1g acetazolamide. Cerebrovascular reactivity and reserve capacity were calculated. Blood glucose, insulin, glycosylated hemoglobin, hemostatic factors (fibrinogen, alpha-2 macroglobulin and von Willebrand factor antigen) were determined. Cerebrovascular reactivity and reserve capacity values were compared between the two diabetic subgroups and controls. Correlations between laboratory parameters and cerebrovascular reserve were investigated by linear regression analysis. Resting cerebral blood flow velocity was similar in controls and in the two diabetic subgroups. Cerebrovascular reactivity was elevated for a shorter time in patients with > 10 years disease duration than in controls and short-term diabetic patients. Cerebrovascular reserve capacity was lower in the long-term diabetes group (means +/- SD: 39.6 +/- 20.7%) than in patients with < or = 10 years disease duration (63.3 +/- 17.4%, p < 0.02 after Bonferroni correction). Cerebrovascular reserve capacity was inversely related to the duration of the disease (R = 0.53, p < 0.003). None of the determined laboratory factors had any relation with resting cerebral blood flow and cerebrovascular reserve capacity. The vasodilatory ability of cerebral arterioles is diminished in long-standing type II diabetes mellitus.

The possible association of in vivo leukocyte-platelet heterophilic aggregate formation and the development of diabetic angiopathy

Circulating leukocyte-platelet heterophilic aggregates produce procoagulant, oxidative and mitogenic substances, and can cause microembolism in capillaries as well as acute arterial thrombosis. Our aim was to determine if there was any difference in the number of circulating heterophilic aggregates between diabetic patients and controls, if the formation of aggregates correlated with the actual HgbA1c level, duration of diabetes and postprandial rise in serum glucose level, with different vascular complications and whether decreasing postprandial serum glucose had any effect on heterophilic aggregate formation. The number of circulating heterophilic aggregates was measured in 90 diabetic patients (Type 1, 29; Type 2, 61) and in 23 control subjects by a flow-cytometric assay, and the result was given as percentage of the respective leukocyte subsets. There was no significant difference in lymphocyte-platelet and neutrophil-platelet aggregate number in patients and controls; however, there was a significant difference in the percentage of monocyte-platelet aggregates between the diabetic and control group (Type 1, 43.0 - 17.8; Type 2, 34.9 - 12.5; control, 24.6 - 8.2; P < 0.01 and P < 0.5, respectively). Patients with proliferative retinopathy and nephropathy showed the highest number of monocyte-platelet aggregates. No significant correlation was, however, found with HgbA1c. In Type 2 diabetes a non-significant, but remarkable, tendency between elevation of postprandial serum glucose levels and platelet-monocyte aggregate formation was observed and acarbose seemed to be effective in decreasing both. This study provides further support that heterophilic aggregates might have role in the pathogenesis of diabetic vascular complications.

THE ASSOCIATION OF REDUCED ENDOTHELIUM DERIVED RELAXING FACTOR-NO PRODUCTION WITH ENDOTHELIAL DAMAGE AND INCREASED IN VIVO PLATELET ACTIVATION IN PATIENTS WITH DIABETES MELLITUS

The role of reduced endothelial production of EDRF-NO in the pathogenesis of diabetic angiopathy has received much attention, however, most of the rather conflicting data were gained from animal experiments. Limited human experience seems to be available in insulin dependent diabetes, calling attention to decreased EDRF-NO production. Hereby the clinical, as well as laboratory investigation (urinary and serum nitrate/nitrite, lipid peroxidation, glucometabolic parameters, endothelial and in vivo platelet activation markers, etc.) of 35 non-insulin dependent (NIDDM) and 15 insulin dependent diabetics (IDDM) patients are given. Urinary and serum nitrate/nitrite concentrations were proven to be reduced in both patients groups. This change was independent of diabetes duration, presence of macroangiopathy, coronary heart disease and the glucometabolic parameters, however, correlation was registered with lipid peroxidation (total antioxidant status). An inverse correlation of nitrate/nitrite excretion with endothelial markers (von Willebrand factor, soluble thrombomodulin) was documented in NIDDM, this correlation was much stronger in IDDM. Moreover, in IDDM patients reduced nitrate/nitrite excretion was strongly associated with elevated plasmatic beta-thromboglobulin levels. The data presented here support to the hypothesis, that EDRF-NO production is reduced in diabetes and this reduction seems to correlate with endothelial damage. In IDDM the decreased nitrate/nitrite excretion may also lead to increased in vivo platelet activation, which suggests that the reduced amount of EDRF-NO might play a role in the pathogenesis of angiopathy in IDDM.

Increased levels of platelet activation markers are positively associated with carotid wall thickness and other atherosclerotic risk factors in obese patients

The role of platelets in the development of atherosclerosis and obesity-related prothrombotic state is still under investigation. In this cross-sectional cohort study, we measured the levels of different platelet activation markers and evaluated their relationship with carotid intima-media thickness (IMT) along with other atherosclerotic risk factors in obese patients with or without atherosclerotic co-morbidities. We enrolled 154 obese patients, including 98 with either hypertension, type 2 diabetes mellitus or dyslipidaemia, 56 without these co-morbidities and 62 age- and sex-matched healthy controls. Platelet P-selectin expression and the number of platelet-derived microparticles (PMPs) were measured by flow cytometry; soluble P-selectin levels were analysed by ELISA and Thr715Pro P-selectin polymorphism was determined by PCR-RFLP. Carotid IMT was examined by ultrasonography. The levels of platelet activation parameters were significantly elevated in all obese subjects with increased carotid IMT compared to healthy controls. There was no effect of Thr715Pro genotype on soluble P-selectin levels in obese individuals contrary to normal subjects. Significant and positive association was revealed between carotid IMT and platelet P-selectin (p<0.0001), soluble P-selectin (p=0.039) and PMP (p=0.0001) levels. After adjusting for multiple variables, independent association was found between soluble P-selectin and fibrinogen (p=0.007), PMP levels and body mass index (p<0.0001) as well as platelet P-selectin and carotid IMT (p=0.012) plus plasminogen activator inhibitor-1 (p=0.009). In conclusion, P-selectin and PMP levels showed positive associations with abnormal carotid IMT and other risk factors in obesity suggesting a critical role of enhanced platelet reactivity in atherosclerotic wall alteration.

Effects of rs769217 and rs1001179 polymorphisms of catalase gene on blood catalase, carbohydrate and lipid biomarkers in diabetes mellitus

Abstract Oxidative stress and deficiency of the enzyme catalase, which is the primary scavenger of the oxidant H2O2, may contribute to diabetes. The current study examined two polymorphisms in the catalase gene, −262C>nT in the promoter and 111C>T in exon 9, and their effects on blood catalase activity as well as on concentrations of blood glucose, haemoglobin A1c, triglyceride, cholesterol, HDL, LDL, ApoA-I and ApoB. Subjects were type-1 and type-2 diabetics. We evaluated PCR-single strand conformational polymorphism for 111C>T and PCR-restriction fragment length polymorphism for − 262C>T. TT genotype frequency of 111C>T polymorphism was increased in type-1 diabetes. Type-2 diabetics with the CC or CT genotypes had decreased catalase and increased glucose, hemoglobinA1c and ApoB. Type-2 diabetics who have TT genotype in −262C>T may have elevated risk for diabetes complications; these patients had the lowest mean catalase and HDL, as well as the highest glucose, haemoglobin A1c, cholesterol and ApoB.

Changes in Cerebral Blood Flow Detected by SPECT in Type 1 and Type 2 Diabetic Patients

Although macrovascular complications are typical for type 2 diabetes mellitus (T2DM), cerebral microvascular damage develops both in type 1 diabetes mellitus (T1DM) and T2DM. Color Doppler ultrasound is widely used for the examination of large- and medium-sized arteries, whereas SPECT and MRI are capable of identifying disturbances in the circulation of microvessels. Former studies using semiquantitative methods showed reduced reactivity and reserve capacity of cerebral vessels in both T1DM and T2DM patients. Our aim was to investigate whether there was any difference in the effects of the 2 types of diabetes mellitus on the global or regional cerebral blood flow, influenced by microvascular damage. Methods: In our study, the circulation and reserve capacity of cerebral arteries was examined using 99mTc-hexamethylpropylene amine oxime SPECT. A total of 17 individuals with T1DM and 43 individuals with T2DM were involved in the study. Results: Both basal and acetazolamide-challenged brain circulation were significantly lower in T2DM patients than in T1DM patients. We did not find a significant difference in the reserve capacity. However, the circulation of the frontal and occipital lobes changed differently in the 2 groups. The ratio of the circulation of the frontal and occipital lobes was significantly reduced both in basal and in acetazolamide-stimulated states in T2DM patients, independently of age (P < 0.0005 and P < 0.017), showing a greater relative decrease in the circulation of the frontal lobe in T2DM patients. Conclusion: There was a significant association between basal brain circulation and age, body mass index, and high-density lipoprotein (HDL), whereas acetazolamide-stimulated circulation showed a significant association with serum triglyceride and HDL.

Increased leukocyte-platelet adhesion in chronic myeloproliferative disorders with high platelet counts.

The heterophilic adhesions between monocytes and platelets may result in the modification of both platelet and monocyte function. This mutual modification includes a greater activation of platelets with increased production of PDGF and other metabolites as well as an enhanced tissue factor expression of monocytes with greater activity in the circulation. The heterophilic aggregation has been well documented during extracorporal circulation, haemodialysis and in diabetic retinopathy. Here we provide evidence that there is significant increase of monocyte–platelet aggregates in disorders associated with high platelet counts, such as chronic myeloproliferative disorders. The presence of these heterophilic aggregates may contribute to the vascular complications observed frequently in polycythaemia vera and essential thrombocythaemia.The heterophilic adhesions between monocytes and platelets may result in the modification of both platelet and monocyte function. This mutual modification includes a greater activation of platelets with increased production of PDGF and other metabolites as well as an enhanced tissue factor expression of monocytes with greater activity in the circulation. The heterophilic aggregation has been well documented during extracorporal circulation, haemodialysis and in diabetic retinopathy. Here we provide evidence that there is significant increase of monocyte-platelet aggregates in disorders associated with high platelet counts, such as chronic myeloproliferative disorders. The presence of these heterophilic aggregates may contribute to the vascular complications observed frequently in polycythaemia vera and essential thrombocythaemia.

Effect of C111T polymorphism in exon 9 of the catalase gene on blood catalase activity in different types of diabetes mellitus

Hydrogen peroxide plays a major role in the pathomechanism of diabetes mellitus and its main regulator is enzyme catalase. The blood catalase and the C111T polymorphism in exon 9 was examined in type 1, type 2 and gestational diabetes mellitus. Compared to the control group (104.7 ± 18.5 MU/l) significantly decreased (p < 0.001) blood catalase activities were detected in type 2 (71.2 ± 14.6 MU/l), gestational (68.5 ± 12.2 MU/l) diabetes mellitus and without change in type 1 (102.5 ± 26.9 MU/l). The blood catalase decreased (p = 0.043) with age for type 2 diabetics and did not change (p>0.063) for type 1, gestational diabetic patients and controls. Blood catalase showed a weak association with hemoglobin A1c for type 1 diabetic patients (r = 0.181, increasing). The mutant T allele was increased in type 1 and gestational diabetes mellitus, and CT+TT genotypes showed decreased blood catalase activity for type 1 and increased activities for type 2 diabetic patients. The C111T polymorphism may implicate a very weak effect on blood catalase activity in different types of diabetes mellitus.

Investigation of Thr715Pro P-selectin gene polymorphism and soluble P-selectin levels in type 2 diabetes mellitus

Increased levels of soluble P-selectin (sP-selectin) have been shown in a number of different disorders, e.g. diabetes mellitus (DM) and cardiovascular disease (CVD). Several studies have attempted to demonstrate the association of the most intensively examined variant of P-selectin gene polymorphism (Thr715Pro) with sP-selectin levels in healthy subjects and in CVD, but contradictory data have been reported. To clarify the effect of Pro715 allele on the sP-selectin levels in type 2 DM, we analysed this polymorphism in diabetic patients and compared these data with sP-selectin levels. Type 2 DM patients (n = 119), 48 BMImatched non diabetic individuals - consisting mostly of overweight subjects - and 57 healthy volunteers were included in the study. TheThr715Pro polymorphism was analysed by PCR-RFLP, while sP-selectin levels were measured by ELISA. Significantly elevated sP-selectin levels were found in both DM and in overweight subjects compared to healthy controls. We confirmed previous reports that in healthy Pro715 allele carriers lower sPselectin levels could be measured; however, this difference was only significant in case of lean subjects. No significant difference was detected in sP-selectin level among DM and overweight individuals according to this genotype. However, significant difference was observed in sP-selectin levels in older DM patients compared to younger ones, but these levels were not accounted for by the Thr715Pro polymorphism. We suggest that in type 2 DM individuals, the significantly elevated sP-selectin levels are not due to the Thr715Pro P-selectin gene polymorphism.

No correlation between impairment of cerebrovascular reserve capacity and electrophysiologically assessed severity of neuropathy in noninsulin-dependent diabetes mellitus.

INTRODUCTION Microvascular abnormalities have an important role in the most frequent neurological complications of diabetes mellitus: neuropathy and cerebrovascular disorders. Severity of neuropathy as well as of cerebral microvascular damage can be quantitatively evaluated by instrumental methods like nerve conduction studies and transcranial Doppler. In the present study, we investigated whether a correlation exists between the severity of peripheral neuropathy and the impairment of cerebrovascular reserve capacity (CRC) in 20 patients with Type 2 diabetes mellitus. METHODS CRC was measured by transcranial Doppler and defined as the maximal percentage increase in blood flow velocity in the middle cerebral artery within 20 min after an intravenous dose of 1000 mg of acetazolamide. Nerve conduction studies of the median, ulnar, peroneal, and sural nerves were performed. Severity of neuropathy was scored based on conduction velocities, amplitudes, and distal latencies. RESULTS There was no correlation between the neuropathic score and CRC (R= .003, P= .99). Neither CRC nor the neuropathic score correlated significantly with age, duration of diabetes, and serum values of HbA(1c), glucose, insulin, von Willebrand factor, and alpha(2) - macroglobulin. Severity of neuropathy but not CRC correlated with microalbuminuria (R= .47, P= .038 and R= .14, P= .54). Improper treatment reflected by HbA(1c) >10% was associated with significantly more severe albuminuria, higher actual blood glucose level, higher von Willebrand factor activity, and marginally higher neuropathic score (21 vs. 13, P=.096), but was not associated with CRC (44% vs. 42%, P= .81). When duration of diabetes was dichotomized to 15 years and less or over 15 years, CRC was significantly smaller (35% vs. 50%, P= .036) and neuropathy was more severe in the subgroup with longer diabetes duration (19 vs. 11.5 points, P= .07). CONCLUSIONS Although both CRC and peripheral nerve function are affected more severely in patients with long-lasting Type 2 diabetes mellitus, damage in the cerebrovascular system and in the long peripheral nerves occur independently. As in diabetes mellitus pathological changes in autonomic and large peripheral nerves develop simultaneously, decreased CRC in diabetic patients might be predominantly due to structural changes of resistance arteries or to metabolic than to neurogenic factors.