Features of angioarchitectonics and structure of the aortic wall of white rats in the early stages of experimental diabetes mellitus

Background. Atherosclerotic cardiovascular diseases, as well as coronary heart disease, cerebrovascular disease, and peripheral artery disease (probably caused by atherosclerosis), are the leading cause of disability and mortality in people with diabetes. Objective. The purpose of our study was to determine the histostructural features and morphometric analysis of the components of the aortic wall and its hemomicrocirculatory bed after 2 and 4 weeks of streptozotocin-induced diabetes mellitus. Methods. The samples for the histology were the sections of the wall of the ascending part, the aortic arch, and the descending part of the aorta of 26 mature white male rats weighing 100 - 160 g. For morphometric examination, a series of images of the aortic wall was taken using a Meiji MT4300 LED microscope with an x40 objective, x10 eyepiece. Results and conclusion. The measurements were carried out using the ImageJ software. The development of micro- and macroangiopathies in experimental animals with 8-week streptozotocin-induced diabetes mellitus was histologically proved. Statistical analysis revealed a significant difference of all morphometric parameters of both - the components of the aortic wall and the vessels of its hemomicrocirculatory bed after 4 weeks of experimental diabetes in comparison with the norm, control, and the 2-nd week of the experiment. An explicit dependency of the severity of destructive changes in the wall of the aorta and links of its hemomicrocirculatory bed of vessels on the duration of the experiment was determined.

Hypoglycemic effect of alloxan and thymulin both diluted in wistars rats with degeneration of beta cells islets of Langerhans

Diabetic animals induced by alloxan show severe hyperglycemia and intense catabolism characterized by the absence of insulin. Therefore, the objective of this study is to assess whether the alloxan 6CH, is able to reverse or mitigate the changes promoted by diabetes mellitus, as well as assess the effects of thymulin. In biological tests male Wistar rats were used induced to experimental diabetes by the administration of alloxan (iv 42 mg / kg). The sample comprised four groups (n = 4): G1 – control without the induction of diabetes, G2 - diabetic without treatment, G3 - diabetic treated with thymulin 12CH and G4 - treated with alloxan 6CH. The data were statistically analyzed by ANOVA followed by Tukey-Kramer test (p < 0.05). After treatment for 40 days slight decrease of glucose in animals treated with alloxan (502 ± 28) mg/dl and thymulin (500 ± 10) mg/dl was observed compared with untreated animals (563 ± 23)mg/dl. Remained unchanged feed intake and water, however, significant decrease of body weight in diabetic group (96 ± 21)g was observed compared to animals treated with alloxan (27 ± 23)g and thymulin (20 ± 16)g, fact not observed when the last two groups are compared with the control (5.1 ± 3.9)g. Significant reduction in the percentage of lymphocytes in diabetic animals (44.8 ± 2.4)% and increase in the group treated with thymulin (12CH) (83.3 ± 4.5)% was checked, when compared to the others. Animals treated with alloxan and thymulin showed clinical improvement. Based on these findings it is concluded that alloxan and thymulin improve the general state of the animal, and suggest inhibition of strong catabolism observed in diabetic animals without treatment.

The effect of Galega officinalis L. extract on the content of the advanced glycation end products and their receptors in rat leukocytes under experimental diabetes mellitus

Background. Diabetes mellitus intensifies non-enzymatic glycosylation (glycation) of biomolecules under conditions of chronic hyperglycemia and facilitates accumulation of advanced glycation end products. Disorders of the cells of various tissues are caused by binding of advanced glycation end products to the corresponding receptors, the level of receptors for advanced glycation end products increases under conditions of hyperglycemia. The interaction between receptors for advanced glycation end products and advanced glycation end products leads to the formation of excessive reactive oxygen species, changes in intracellular signaling, gene expression, increased secretion of pro-inflammatory cytokines and contributes to the development of diabetic complications. The search for factors of natural origin that will slow down the development of specific complications of diabetes, determines the feasibility of studies of the corrective ability of biologically active substances isolated from medicinal plants for the process of glycation of proteins in diabetes. Materials and methods. Experimental diabetes mellitus was induced by intraperitoneal administration of streptozotocin. Separation of blood leukocytes was performed in Ficoll density gradient. To determine the extent of advanced glycation end products and receptor for advanced glycation end products in leukocyte immunoperoxidase labeling was performed. Results. A decrease in the content of advanced glycation end products in leukocy­tes under conditions of experimental diabetes mellitus was found. The obtained data indicate a possible contravention of glucose uptake by leukocytes in the studied pathology. At the same time, an increase in exposure to the receptor for advanced glycation end products leukocyte membranes in response to chronic hyperglycemia has been demonstrated. The ability of alkaloid free fraction of Galega officinalis extract to reduce the content of receptors for end products of glycation on the membranes of immunocompetent cells in diabetic animals has been confirmed, which may be due to the presence of biologically active substances with hypoglycemic action in its composition. Conclusion. Corrective effect of alkaloid free fraction of Galega officinalis L. extract on the content of receptor for advanced glycation end products in diabetes mellitus is mediated by its normalizing effect on carbohydrate metabolism.

HISTOLOGICAL AND MORPHOMETRIC ASPECTS OF STRUCTURE OF AORTIC WALL AND ITS HEMOMICROCIRCULATORY BED IN LATE STAGES OF EXPERIMENTAL DIABETES MELLITUS

According to the World Health Organization, cardiovascular diseases and diabetes mellitus occupy a significant niche in the structure of diseases with high disability and mortality impact and pose major healthcare and social issues. It should be stressed that 50-80% of fatal cases in patients with diabetes are associated with cardiovascular diseases. The purpose of this study is to determine the histostructural characteristics and to perform morphometric analysis of the components of the aortic wall and its hemomicrocirculatory bed of the aorta in 6 and 8 weeks of streptozotocin-induced diabetes mellitus. The material for the histological study included the sections of the wall of the ascending aorta, the aortic arch, and the descending aorta taken from 26 sexually mature white male rats weighing 100 - 160 g. For morphometric examination, a series of photos of the aortic wall was taken using a Meiji MT4300 LED microscope with an x40 objective, x10 ocular. The measurements were carried out using the Image J software. The development of micro - and macroangiopathies in experimental animals with 8-week streptozotocin-induced diabetes mellitus was histologically confirmed. Statistical analysis revealed a significant difference in all morphometric parameters of the components of the aortic wall and the vessels of its hemomicrocirculatory bed after 8 weeks of experimental diabetes as compared with the normal values, control values, and in values obtained at the 6-week period of the experiment. The study has demonstrated clear dependence between the severity of destructive changes in the aortic wall and sections of its hemomicrocirculatory bed and the duration of the experiment.

Mitochondrial Antioxidant SkQ1 Has a Beneficial Effect in Experimental Diabetes as Based on the Analysis of Expression of microRNAs and mRNAs for the Oxidative Metabolism Regulators

Diabetes mellitus and related complications are among the most important problems of the world-leading healthcare systems. Despite their priority, molecular and genetic aspects of diabetes pathogenesis are poorly understood; however, the involvement of oxidative stress in this process is undoubted. Rats with experimental diabetes induced by the intraperitoneal injection of alloxan were subjected to the antioxidant pre-therapy with a series of mitochondria-targeted 10-(6’-plastoquinonyl)decyltriphenylphosphonium (SkQ1) injections and analyzed for the expression of mRNAs and microRNAs by real-time quantitative polymerase chain reaction to identify potential predictors of diabetes. Animals that received SkQ1 before diabetes induction demonstrated lower blood glucose levels compared to the diabetic animals not subjected to the therapy. SkQ1 caused changes in the mRNA levels of genes involved in the cellular defense against free radicals, which indicates a beneficial effect of the pre-therapy. Moreover, similar changes were observed on the epigenetic level, as the microRNA expression patterns not only proved the SkQ1 efficacy but also correlated with the expression levels of their mRNA targets. Oxidative stress and macromolecule damage by free radicals are determining factors in diabetes, which suggests that strategies aimed at restoring the antioxidant status of the cell can be beneficial. Mitochondria-targeted antioxidant SkQ1 demonstrates positive effects on several levels, from the normalization of the blood glucose content to genetic and epigenetic changes. Our results can serve as a basis for the development of novel therapeutic and diagnostic strategies.

Pharmacological blockade of histone methyltransferase SETD7 restores angiogenic response in experimental diabetes

Background Peripheral artery disease (PAD) is highly prevalent in patients with diabetes and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in diabetic patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in the ischemic limb of diabetic patients. Mono-methylation of histone 3 at lysine 4 (H3K4me1) - a specific epigenetic signature induced by the methyltransferase SETD7 - favours a chromatin active and open state thus enabling the gene transcription. Purpose To investigate whether SETD7-dependent epigenetic changes modulate post-ischemic vascularization in experimental diabetes. Methodology Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 20 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro angiogenic assays like migration assay & tube formation assay were performed. Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor called (R)-PFI-2. T1D mice (streptozotocin-induced diabetes) was orally treated with (R)-PFI-2 and with vehicle for 21 days and followed by induction of hindlimb ischemia. Blood flow recovery was analyzed at 30 minutes, 7 and 14 days by laser doppler imaging. Gastrocnemius muscle samples from patients with and without T2D were employed to translate our experimental findings. Results RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with an increase of H3K4-mono-methylation levels as well as with impaired endothelial cell migration and tube formation. Of interest, both gene silencing (SETD7-siRNA) and pharmacological blockade of SETD7 by (R)-PFI-2 rescued hyperglycemia-induced impairment of angiogenic properties in HAECs. RNA-seq in HG-treated HAECs with and without SETD7 depletion unveiled an array of differentially expressed genes, which were mainly involved in blood vessel growth and angiogenic response. Among dysregulated genes, Chromatin immunoprecipitation (ChIP) assays showed that SETD7 specifically mono-methylates H3K4m1 in proximity of Semaphorin-3G (SEMA3G) promoter, thus regulating its expression. Treatment of T1D mice with (R)-PFI-2 improved blood flow reperfusion at 14 days as compared to vehicle-treated animals. Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients. Conclusion Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Zurich