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ObjectiveIn diabetes, vascular dysfunction is characterized by impaired endothelial function due to increased oxidative stress. Empagliflozin, as a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), offers a novel approach for the treatment of type 2 diabetes by enhancing urinary glucose excretion. The aim of the present study was to test whether treatment with empagliflozin improves endothelial dysfunction in type I diabetic rats via reduction of glucotoxicity and associated vascular oxidative stress.MethodsType I diabetes in Wistar rats was induced by an intravenous injection of streptozotocin (60 mg/kg). One week after injection empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 7 weeks. Vascular function was assessed by isometric tension recording, oxidative stress parameters by chemiluminescence and fluorescence techniques, protein expression by Western blot, mRNA expression by RT-PCR, and islet function by insulin ELISA in serum and immunohistochemical staining of pancreatic tissue. Advanced glycation end products (AGE) signaling was assessed by dot blot analysis and mRNA expression of the AGE-receptor (RAGE).ResultsTreatment with empagliflozin reduced blood glucose levels, normalized endothelial function (aortic rings) and reduced oxidative stress in aortic vessels (dihydroethidium staining) and in blood (phorbol ester/zymosan A-stimulated chemiluminescence) of diabetic rats. Additionally, the pro-inflammatory phenotype and glucotoxicity (AGE/RAGE signaling) in diabetic animals was reversed by SGLT2i therapy.ConclusionsEmpagliflozin improves hyperglycemia and prevents the development of endothelial dysfunction, reduces oxidative stress and improves the metabolic situation in type 1 diabetic rats. These preclinical observations illustrate the therapeutic potential of this new class of antidiabetic drugs.

Aims: Oxidative stress is involved in the development of cardiovascular disease. There is a growing body of evidence for a crosstalk between different enzymatic sources of oxidative stress. With the present study, we sought to determine the underlying crosstalk mechanisms, the role of the mitochondrial permeability transition pore (mPTP), and its link to endothelial dysfunction. Results: NADPH oxidase (Nox) activation (oxidative burst and translocation of cytosolic Nox subunits) was observed in response to mitochondrial reactive oxygen species (mtROS) formation in human leukocytes. In vitro, mtROS-induced Nox activation was prevented by inhibitors of the mPTP, protein kinase C, tyrosine kinase cSrc, Nox itself, or an intracellular calcium chelator and was absent in leukocytes with p47phox deficiency (regulates Nox2) or with cyclophilin D deficiency (regulates mPTP). In contrast, the crosstalk in leukocytes was amplified by mitochondrial superoxide dismutase (type 2) (MnSOD +/ -) deficiency. In vivo, increases in blood pressure, degree of endothelial dysfunction, endothelial nitric oxide synthase (eNOS) dysregulation/uncoupling (e.g., eNOS S-glutathionylation) or Nox activity, p47phox phosphorylation in response to angiotensin-II (AT-II) in vivo treatment, or the aging process were more pronounced in MnSOD +/ -mice as compared with untreated controls and improved by mPTP inhibition by cyclophilin D deficiency or sanglifehrin A therapy. Innovation: These results provide new mechanistic insights into what extent mtROS trigger Nox activation in phagocytes and cardiovascular tissue, leading to endothelial dysfunction. Conclusions: Our data show that mtROS trigger the activation of phagocytic and cardiovascular NADPH oxidases, which may have fundamental implications for immune cell activation and development of AT-II-induced hypertension. Antioxid. Redox Signal. 20, 247-266.

The aim of this study was to investigate the neurochemical coding of myenteric neurons in the guinea pig gastric corpus by using immunohistochemical methods. Antibodies and antisera against calbindin (CALB), calretinin (CALRET), choline acetyltransferase (ChAT), calcitonin gene-related peptide (CGRP), dopamine beta-hydroxylase (DBH), beta-endorphin (ENK), neuropeptide Y (NPY), neuron-specific enolase (NSE), nitric oxide synthase (NOS), protein gene product 9.5 (PGP), parvalbumin (PARV), serotonin (5-HT), somatostatin (SOM), substance P (SP), tyrosine hydroxylase (TH), and vasoactive intestinal peptide (VIP) were used. Double- and triple-labeling studies revealed colocalization of certain transmitters and enabled the identification of distinct subpopulations of gastric enteric neurons. NPY/VIP/NOS/ENK were present in 28% of all neurons, whereas 11% had NPY/VIP/DBH/ChAT; NOS-only neurons made up 2% of the population. The combination SP/ChAT/ENK occurred in 21% of the population, whereas SP/ChAT/ENK/CALRET and SP/CHAT/SOM/ +/- CALRET was identified in 5% and 6% of all cells, respectively. 5-HT-containing neurons comprised 2% of all cells and could be further classified by the presence of additional antigens as 5-HT/SP/(ChAT) or 5-HT/VIP/(ChAT). Approximately 21% of all neurons contained only ChAT with no additional antigen present and are referred to as ChAT/-. Gastric myenteric ganglion cells were not immunoreactive for CALB, PARV, CGRP, or TH. The results of this study indicate that gastric myenteric neurons can be characterized on the basis of different chemical coding. Neurochemical coding of corpus myenteric neurons revealed some similarities and significant differences in comparison with other regions of the gut. These differences might reflect adaptation of enteric nerves according to regional specialization and the distinct functions of the proximal stomach as a gastric reservoir.

Several different monoclonal and polyclonal antibodies to choline acetyltransferase (ChAT) were screened to identify effective antibodies for immunocytochemical marking of cholinergic neurons in the enteric nervous system. Excellent immunohistochemical results were obtained with two of the antibodies in the myenteric plexus of the guinea pig stomach and small intestine. One was a mouse monoclonal antibody designated B3.9B3, and the second was a rabbit polyclonal antibody referred to as Peptide 3. Both antibodies clearly stained neuronal cell bodies as well as nerve fibers to the muscle layers and fibers encircling stained and unstained cell bodies. Cell counts indicated that approximately 64% (21.0 +/- 8.6 cells/ganglion) of gastric myenteric neurons are ChAT positive. Pelvic ganglia and the inferior mesenteric ganglia were examined as controls. Strong labeling of the majority of neurons was found in the pelvic ganglia, whereas few immunoreactive cells were apparent in the predominantly noradrenergic inferior mesenteric ganglion. Lack of effective antibodies to enteric neuronal ChAT has hampered progress in the study of the neurophysiology of cholinergic neurons in the digestive tract. Application of the B3.9B3 and Peptide 3 antibodies now promises to facilitate investigation of this important subset of enteric neurons.