Andres Arend

Vitamin D reduces deposition of advanced glycation end-products in the aortic wall and systemic oxidative stress in diabetic rats

AIMS Vitamin D may have an important role in reducing the risk of cardiovascular disease. Advanced glycation end-products (AGEs) such as Nε-(carboxymethyl)lysine (CML), have been implicated in diabetic vascular complications via oxidative stress-mediated pathways. We investigated the potential protective effect of vitamin D on CML accumulation in the diabetic aortic wall. To test the effects of vitamin D on systemic oxidative stress we also assessed liver oxidative stress index (OSI) and serum total antioxidant capacity (TAC). METHODS Male Wistar rats were assigned to three groups: control, untreated diabetes, and diabetes+cholecalciferol. Diabetes was induced by streptozotocin, followed by oral administration of cholecalciferol (500 IU/kg) for 10 weeks in the treatment group. Aortic CML accumulation was determined by ELISA and immunohistochemical assays. OSI was assessed by measuring TAC and the level of total peroxides in the liver and serum using colorimetric assays. RESULTS Untreated diabetes was associated with significantly elevated CML levels in the aortic wall (19.5 ± 3.3 vs 10.2 ± 4.7 ng/mL), increased liver OSI (6.8 ± 1.9 vs 3.1 ± 0.7), and reduced serum TAC (0.4 ± 0.1 vs 0.8 ± 0.3 mmol Trolox/L), in comparison with the control group. Cholecalciferol significantly blocked the accumulation of CML in the aortic wall (10.4 ± 8.4 vs 19.5 ± 3.3 ng/mL), decreased liver OSI (4.2 ± 1.4 vs 6.8 ± 1.9), and improved serum TAC (1.0 ± 0.2 vs 0.4 ± 0.1 mmol Trolox/L), compared with the untreated diabetic group. CONCLUSIONS Streptozotocin-diabetes resulted in increased deposition of AGEs and increased oxidative stress in the serum and liver. Vitamin D supplementation may provide significant protection against oxidative stress-mediated vascular complications in diabetes.

Effect of vitamin D on aortic remodeling in streptozotocin-induced diabetes

BackgroundDiabetes mellitus is associated with micro- and macrovascular complications and increased cardiovascular risk. Elevated levels of serum asymmetric dimethylarginine (ADMA) may be responsible for endothelial dysfunction associated with diabetes-induced vascular impairment. Vitamin D may have potential protective effects against arterial stiffening. This study aimed to examine both the effects of diabetes on the functional/structural properties of the aorta and the endothelial function and the effects of vitamin D supplementation.MethodsMale Wistar rats (n = 30) were randomly assigned to control untreated, diabetic untreated, and diabetic + cholecalciferol groups. Diabetes was induced by intraperitoneal injection of streptozotocin, followed by oral administration of cholecalciferol (500 IU/kg) for 10 weeks in the treatment group. Aortic pulse wave velocity (PWV) was recorded over a mean arterial pressure (MAP) range of 50 to 200 mmHg using a dual pressure sensor catheter. Intravenous infusion of phenylephrine and nitroglycerine was used to increase and decrease MAP, respectively. Serum 25-hydroxyvitamin D [25(OH)D] levels were measured using a radioimmune assay. ADMA levels in serum were measured by enzyme-linked immunoassay. Aortic samples were collected for histomorphometrical analysis.ResultsPWV up to MAP 170 mmHg did not reveal any significant differences between all groups, but in diabetic rats, PWV was significantly elevated across MAP range between 170 and 200 mmHg. Isobaric PWV was similar between the treated and untreated diabetic groups, despite significant differences in the levels of serum 25(OH)D (493 ± 125 nmol/L vs 108 ± 38 nmol/L, respectively). Serum levels of ADMA were similarly increased in the treated and untreated diabetic groups, compared to the control group. The concentration and integrity of the elastic lamellae in the medial layer of the aorta was impaired in untreated diabetic rats and improved by vitamin D supplementation.ConclusionPWV profile determined under isobaric conditions demonstrated differential effects of uncontrolled diabetes on aortic stiffness. Diabetes was also associated with elevated serum levels of ADMA. Vitamin D supplementation did not improve the functional indices of aortic stiffness or endothelial function, but prevented the fragmentation of elastic fibers in the aortic media.

Deletion of the CCK2 receptor gene reduces mechanical sensitivity and abolishes the development of hyperalgesia in mononeuropathic mice

Previous studies suggest that cholecystokinin (CCK) is implicated in the modulation of pain sensitivity and the development of neuropathic pain. We used CCK2 receptor deficient (CCK2 −/−) mice and assessed their mechanical sensitivity using Von Frey filaments, as well as the development and time course of mechanical hyperalgesia in a model of neuropathic pain. We found that CCK2 −/− mice displayed mechanical hyposensitivity, which was reversed to the level of wild‐type animals after administration of naloxone (0.1–10 mg/kg). On the other hand, injection of L‐365260 (0.01–1 mg/kg), an antagonist of CCK2 receptors, decreased dose‐dependently, mechanical sensitivity in wild‐type mice. The mechanism of reduced mechanical sensitivity in CCK2 −/− mice may be explained by changes in interactions between CCK and opioid systems. Indeed, CCK2 −/− mice natively expressed higher levels of lumbar CCK1, opioid δ and κ receptors. Next, we found that CCK2 −/− mice did not develop mechanical hyperalgesia in the Bennetts neuropathic pain model. Induction of neuropathy resulted in decrease of lumbar pro‐opiomelanocortin (POMC) gene expression in wild‐type mice, but increase of POMC expression in CCK2 −/− mice. In addition, induction of neuropathy resulted in further increase of opioid δ receptor in CCK2 −/− mice. Gene expression results indicate up‐regulation of opioid system in CCK2 −/− mice, which apparently result in decreased neuropathy score. Our study suggests that not only pain sensitivity, but also mechanical sensitivity and the development of neuropathic pain are regulated by antagonistic interactions between CCK and opioid systems.

Bone formation zones in heterotopic ossifications: histologic findings and increased expression of bone morphogenetic protein 2 and transforming growth factors beta2 and beta3.

Heterotopic ossifications (HOs) formed after total endoprosthetic replacement of the hip joint were collected during revision surgery (n = 7). Tissues collected during regular hip arthroplasty (n = 12) were used as reference. Histomorphometric analysis was performed for assessment of bone formation activity in HOs and reference bone. HOs were dissected with histological guidance into three zones: formed bone, zone of active bone formation, and zone with fibrous connective and fibrocartilagineous tissue. Relative expression of the mRNA for bone morphogenetic protein 2 (BMP-2), transforming growth factor ß2 (TGF-ß2), and TGF-ß3 was determined by reverse-transcription polymerase chain reaction relative to ß-actin. Expression of all three growth factors was higher than in orthotopic bone. Similarly, the osteoid surface density was increased in HOs. The levels of all growth factors were higher in the zone of active bone formation or remodeling than in the zone of formed bone. In matured HOs, the osteoid surface density as well as mRNA levels were lower, although still significantly raised, indicating that bone formation slows down after 2 years. Immunohistochemical analysis demonstrated the presence of TGF-ß1, TGF-ß2, TGF-ß3, and BMP-2 proteins in the zone of bone formation. We conclude that bone formation after heterotopic bone induction is initially intense, slows down within 2 years, and thereupon continues as active remodeling mainly on the border of HO. Our data indicate that BMP-2, TGF-ß2, and TGF-ß3 are involved in bone formation in HO.

Complete glutathione system in probiotic Lactobacillus fermentum ME-3

There is much information about glutathione (GSH) in eukaryotic cells, but relatively little is known about GSH in prokaryotes. Without GSH and glutathione redox cycle lactic acid bacteria (LAB) cannot protect themselves against reactive oxygen species. Previously we have shown the presence of GSH in Lactobacillus fermentum ME-3 (DSM14241). Results of this study show that probiotic L. fermentum ME-3 contains both glutathione peroxidase and glutathione reductase. We also present that L. fermentum ME-3 can transport GSH from environment and synthesize GSH. This means that it is characterized by a complete glutathione system: synthesis, uptake and redox turnover ability that makes L. fermentum ME-3 a perfect protector against oxidative stress. To our best knowledge studies on existence of the complete glutathione system in probiotic LAB strains are still absent and glutathione synthesis in them has not been demonstrated.

Trans-Resveratrol Alone and Hydroxystilbenes of Rhubarb (Rheum rhaponticum L.) Root Reduce Liver Damage Induced by Chronic Ethanol Administration : a Comparative Study in Mice

The hepatoprotective effects and pharmacokinetics of trans‐resveratrol and hydroxystilbenes of the garden rhubarb (Rheum rhaponticum L., R. rhaponticum) root ethanol extract were studied.

Male mice with deleted Wolframin (Wfs1) gene have reduced fertility

BackgroundWolfram Syndrome (WS) is an autosomal recessive disorder characterised by non-autoimmune diabetes mellitus, optic atrophy, cranial diabetes insipidus and sensorineural deafness. Some reports have described hypogonadism in male WS patients. The aim of our study was to find out whether Wfs1 deficient (Wfs1KO) male mice have reduced fertility and, if so, to examine possible causes.MethodsWfs1KO mice were generated by homologous recombination. Both Wfs1KO and wild type (wt) male mice were mated with wt female mice. The number of litters and the number of pups were counted and pregnancy rates calculated. The motility and morphology of the sperm and the histology of testes were analysed. Serum testosterone and FSH concentrations were also measured.ResultsThe pregnancy rate in wt females mated with Wfs1KO males was significantly lower than in the control group (15% vs. 32%; p < 0.05), but there was no significant difference in litter size. Analysis of male fertility showed that, in the Wfs1KO group, eight males out of 13 had pups whereas in the control group all 13 males had at least one litter. Sperm motility was not affected in Wfs1KO mice, but Wfs1KO males had less proximal bent tails (14.4 +/- 1.2% vs. 21.5 +/- 1.3 p < 0.05) and less abnormal sperm heads (22.8 +/- 1.8 vs. 31.5 +/- 3.5, p < 0.05) than wt males. Testes histology revealed significantly reduced number of spermatogonia (23.9 +/- 4.9 vs. 38.1 +/- 2.8; p < 0.05) and Sertoli cells (6.4 +/- 0.5 vs. 9.2 +/- 1.0; p < 0.05) in Wfs1KO mice. Serum testosterone and FSH concentrations did not differ between the two groups.ConclusionThe impaired fertility of Wfs1KO male mice is most likely due to changes in sperm morphology and reduced number of spermatogenic cells. The exact mechanism through which the Wfs1 gene influences sperm morphology needs to be clarified in further studies.

Bone tissue content of TGF-2 changes with time in human heterotopic ossification after total hip arthroplasty

Transforming growth factor beta isoforms (TGF-β1, TGF-β2, and TGF-β3) most likely play a role in bone physiology, but little is known about their relative importance in normal as well as in heterotopic bone. This study focused on possible differences in the localization and relative content of different TGF beta isoforms in heterotopic ossifications (HO) by comparing HOs, which have developed less than 17 months (immature HOs) with those developed 3–9 years (mature HOs). The HOs were harvested after total hip arthroplasty (THA) during revision surgery. The HO samples were decalcified, embedded in paraffin and sectioned. Azan staining was used to evaluate histological structure of the ossifications and immunohistochemical analysis was performed to estimate the localization of three TGF beta isoforms in the HOs. Comparison of different TGF beta isoforms in the immature and the mature ossifications showed that the content of TGF-β2 was decreased by almost three times in the mature HO as compared to the immature HO (p = 0.0064). The proportions of other isoforms in HOs did not differ significantly. This study shows that the relative importance of TGF betas change with HO development.

Low-dose radiation modifies the progression of chronic renal failure.

Previous studies have indicated that the application of low dose radiation to an arterial ligation has the potential to subsequently reduce or eliminate restenosis caused by smooth muscle cell proliferation. Sufficient kidney irradiation causes a radiation nephropathy and often leads to renal failure. In order to evaluate the effect of low-dose irradiation on the kidney we hypothesized that this particular therapy modifies renal injury in rats with renal ablation and subsequently slows the rate of the progression. For further clarification of the effect of irradiation at low doses, we determined proliferating cell nuclear antigen (PCNA) and monocyte chemoattractant protein-1 (MCP-1) expression in remnant kidneys after low-dose radiation. Adult Wistar rats (n = 10) were studied during the two weeks after renal ablation. The left kidney was irradiated 24 hours after an operation in anaesthetised animals with 3 Grey in a single dose. Ablated rats without irradiation (n = 9) served as nephrectomized animals group. Rats without surgery and without radiation (n = 10) served as healthy controls. Renal damage was assessed using the following parameters: urine protein excretion rate (UprotV, mg/day), awake systolic blood pressure (SBP, mm Hg), serum creatinine (SCr, micromol/l). The indirect immunofluorescence method was used for the detection of PCNA and MCP-1 expression. Glomerular and tubular immunostaining was scored semiquantitatively. Numerous PCNA positive cells and MCP-1 expression were present in the glomerulus and tubulointerstitium in nephrectomized rat kidneys. Low-dose radiation application was associated with a significant reduction in PCNA and low MCP-1 expression. This study shows that the application of low-dose irradiation has the potential to modify the progression of chronic renal failure in rats.

Lipoic Acid Prevents Suppression of Connective Tissue Proliferation in the Rat Liver Induced by n–3 PUFAs

As previously shown, dietary n–3 polyunsaturated fatty acids (n–3 PUFAs) suppress connective tissue proliferation in the rat liver wound concurrent with an elevated level of lipid peroxidation. The present study was undertaken to investigate the influence of α-lipoic acid (LA), a natural anti-oxidant, on these effects of n–3 PUFAs. Rats were fed with a commercial pellet diet (control group) or with diets enriched with 10% of sunflower oil (n–6 group) or 10% of fish oil (n–3 group) for 8 weeks followed by addition of LA to the same diets for 10 days. Then a liver thermic wound was induced and the administration of LA was continued for 6 days. The proliferation of the connective tissue, the level of lipid peroxidation and their peroxidizability and the content of prostaglandins E2 and F2α were measured in the liver wounds. LA prevented the suppression of connective tissue proliferation in the healing wound induced by n–3 PUFAs, avoided the increase in peroxidation of lipids, reduced peroxidizability of lipids and modulated the decrease in PGE2 and PGF2α. The results indicate that dietary LA may prevent the suppression of liver wound healing induced by n–3 PUFAs.