Peter Rösen

Endothelial relaxation is disturbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant

SummaryIncreased oxidative stress has been suggested to contribute to disturbances in the regulation of coronary flow and the increased cardiac risk in diabetes mellitus. Using the isolated perfused heart of streptozotocin-diabetic rats our study shows that basal and maximal coronary flow (tested by infusion of sodium nitroprusside) are not altered in diabetes, but that 5-hydroxytryptamine (5-HT) stimulated endothelium-dependent increase in coronary flow becomes progressively impaired. This defect of the endothelium-dependent vasodilatation was prevented by perfusion of the hearts with Superoxide dismutase and pretreatment of the diabetic rats with tocopherol-acetate. Morphological studies also revealed that pretreatment with tocopherol-acetate was cardioprotective, and largely prevented severe alterations of myocardial structure typically observed after a diabetes duration of 3 months; deterioration and fragmentation of myofilament bundles were seen less, and the numbers of areas of focal necrosis and of contraction bands were clearly reduced. In contrast to untreated diabetic hearts the autonomic nerve fibers detected by catecholamine fluorescence were running in parallel in hearts of tocopherol-treated diabetic rats, and the amount of catecholamines was not different from that of healthy control rats. Trichrome staining and immunohistochemical staining of collagen I and III showed a dramatic increase in the number and the size of deposits of collagen fibers at precapillary locations in the diabetic hearts which were significantly reduced by anti-oxidative treatment. These findings demonstrate that oxidative stress may not only play a major role in the impairment of endothelium-dependent regulation of coronary flow, but also in the development of perivascular fibrosis and severe changes of the autonomic nerves and contractile system in myocardium.

Use of electrochemical impedance measurements to monitor beta-adrenergic stimulation of bovine aortic endothelial cells.

Abstract Due to the high permeability of endothelial cell layers derived from macrovascular vessels, precise determination of their barrier function towards ion movement requires refined experimental techniques. We thus cultured bovine aortic endothelial cells (BAEC) directly on thin gold-film electrodes and measured the electrochemical impedance to study their passive electrical properties in general and during β-adrenergic stimulation. Impedance spectra (10–2·106 Hz) of confluent cell monolayers revealed that the electrical characteristics of the cells can be modelled by a simple resistor-capacitor parallel network. Under control conditions the overall resistance of confluent BAEC monolayers was 3.6±0.6 Ω·cm2 (n=30) and the capacitance was 0.6±0.1 µF/cm2. Both quantities are discussed with respect to morphological characteristics of these cells. Stimulation of BAECs with the synthetic β-adrenoceptor agonist isoproterenol leads to a concentration-dependent, highly specific increase of the cell layer resistance characterized by a concentration for half-maximal response (EC50) of 0.3±0.1 µM. The cell layer capacitance, however, remained unaffected. Using impedance measurements at a single frequency, we analysed the response of BAECs to treatment with isoproterenol in comparison with several chemically unrelated compounds known to stimulate the adenosine 3’,5’-cyclic monophosphate (cAMP)-dependent signal transduction cascade. These studies confirmed that the enhancement of the cell layer resistance after β-adrenergic stimulation is mediated by an increase in intracellular cAMP.

Induction of apoptosis by high proinsulin and glucose in cultured human umbilical vein endothelial cells is mediated by reactive oxygen species

Summary There is much evidence that diabetes and hyperglycaemia contribute to the impairment of endothelial function and induce severe changes in the proliferation, the adhesive and synthetic properties of endothelial cells. Induction of apoptosis could represent one mechanism to prevent the new accumulation of those vascular defects and to allow generation of vascular endothelium. In this study, we demonstrate that high concentrations of glucose or proinsulin induce apoptosis in human umbilical endothelial cells by three independent methods (DNA fragmentation, fluorescence activated cell sorting analysis, and morphology). The number of apoptotic cells was increased by glucose (30 mmol/l or proinsulin (100 nmol/l) from less than 10 % to about 30 %. Activation of protein kinase C (PKC) largely prevented the induction of apoptosis, whereas inhibition of PKC further increased the number of apoptotic cells. Similar changes as induced by glucose were also observed after incubation of the cells with the non-metabolisable 3-0-methylglucose. These findings indicate that hyperglycaemic conditions stimulate the induction of apoptosis in endothelial cells by a mechanism which is independent from the formation of diacylglycerol and the activation of PKC. The induction of apoptosis by the non-metabolisable glucose suggests that formation of oxygen derived radicals by autoxidative processes is involved and may lead to an activation of transcription factors such as nuclear transcription factor-ϰB (NF-ϰB) transferring the activation signal into the nucleus and leading to changes in gene expression necessary for induction of apoptosis. [Diabetologia (1998) 41: 249–256]

Role of oxygen derived radicals for vascular dysfunction in the diabetic heart: Prevention by α-tocopherol?

The evidence that the generation of reactive oxygen intermediates (ROI) plays an important role for the increased cardiovascular risk in diabetes is summarised. In addition to the well known parameters of oxidative stress as lipid hydroperoxides and thiobarbituric acid substances (TBARS), recent observations indicate that isoprostanes which can be taken as a more specific parameter of oxidative, are generated in higher amounts by diabetic patients. This increased formation of isoprostanes can be inhibited by an instalment of a close metabolic control or the supplementation with tocopherol. The cause for the elevated oxidative stress is not yet fully understood, however the autoxidation of glucose, the formation of advanced glycation endproducts and the activation of NADPH-oxidase seem to be relevant processes. Since ROI are able to quench nitric oxide and to inhibit the synthesis of prostacyclin, the antithrombotic, vasodilating and antiatherosclerotic properties of endothelium are impaired in diabetes. Additionally, the balance of endothelial mediators released by endothelium is shifted to angiotensin II and endothelin, compounds which enhance the proliferation of smooth muscle cells and may limit the coronary reserve of myocardium. The activation of the transcription factor NF-κB by glucose and its autoxidative products is regarded as a key event in the transformation of the vasculature in diabetes. Epidemiological observations and very recent clinical studies underlie the impact of ROI for the development of cardiovascular complications in diabetes and suggest that an antioxidative treatment might be helpful to reduce the cardiac risk in diabetes.

Influence of diabetes on cardiac nitric oxide synthase expression and activity.

UNLABELLED There is some evidence that the endothelium dependent vasodilatation of coronary arteries is impaired in both types of diabetes. The underlying mechanisms are not yet clear, in particular whether this defect is caused by a direct effect of diabetes on the activity and the expression of nitric oxide synthases (NOS) or indirectly by an enhanced inactivation of nitric oxide. METHODS To study this question we determined the activity (conversion of L-arginine to citrulline) and the mRNAs encoding the isoforms of NOS (using polymerase chain reaction after reverse transcription of the mRNAs into cDNAs by reverse transcriptase) in hearts of streptozotocin diabetic rats and in rat heart endothelial cells (RHEC). The formation of reactive oxygen intermediates (ROI) was measured by the dichloro-dihydro-fluorescein method. RESULTS The activity of total NOS and the amounts of mRNAs encoding ecNOS and iNOS were dependent on the duration of diabetes. After a diabetes duration of 4 to 6 weeks both the total activity as well as the mRNAs encoding ecNOS and iNOS were elevated. A reduction of NOS activity and the amounts of mRNAs of ecNOS and iNOS was only seen after a diabetes duration longer than 20 weeks, a time at which a loss of endothelium has been described. In RHEC, high glucose (22 mM) and H(2)O(2) (100 microM) were able to increase the mRNA encoding ecNOS, but not iNOS. This increase in ecNOS mRNA was inhibited by lipoic acid (1 microM). In addition, high glucose (22 and 30 mM) led to an enhanced formation of ROI and to activation of the transcription NF kappa B. CONCLUSION These observations suggest that diabetes causes a temporary increase in NOS activity and ecNOS mRNA in the rat heart which is presumably the consequence of an enhanced oxidative stress exerted by hyperglycaemia. Together with previously published observations, our data suggest that the impairment of endothelium dependent vasodilatation in rat heart is not the consequence of a reduced activity and expression of NOS, but is caused by an enhanced inactivation of nitric oxide by ROI.

Metformin delays the manifestation of diabetes and vascular dysfunction in Goto-Kakizaki rats by reduction of mitochondrial oxidative stress.

This study was undertaken to test the hypothesis that hyperglycaemia induces the generation of reactive oxygen species (ROS) by mitochondria and that the oxidative stress thereby exerted is diminished by treatment with metformin. As a parameter of mitochondrial ROS formation, the activity of mitochondrial aconitase activity was determined using Goto–Kakizaki (GK) rats as model of type 2 diabetes.

Evidence for succinate production by reduction of fumarate during hypoxia in isolated adult rat heart cells

It has been demonstrated that perfusion of myocardium with glutamic acid or tricarboxylic acid cycle intermediates during hypoxia or ischemia, improves cardiac function, increases ATP levels, and stimulates succinate production. In this study isolated adult rat heart cells were used to investigate the mechanism of anaerobic succinate formation and examine beneficial effects attributed to ATP generated by this pathway. Myocytes incubated for 60 min under hypoxic conditions showed a slight loss of ATP from an initial value of 21 +/- 1 nmol/mg protein, a decline of CP from 42 to 17 nmol/mg protein and a fourfold increase in lactic acid production to 1.8 +/- 0.2 mumol/mg protein/h. These metabolite contents were not altered by the addition of malate and 2-oxoglutarate to the incubation medium nor were differences in cell viability observed; however, succinate release was substantially accelerated to 241 +/- 53 nmol/mg protein. Incubation of cells with [U-14C]malate or [2-U-14C]oxoglutarate indicates that succinate is formed directly from malate but not from 2-oxoglutarate. Moreover, anaerobic succinate formation was rotenone sensitive. We conclude that malate reduction to succinate occurs via the reverse action of succinate dehydrogenase in a coupled reaction where NADH is oxidized (and FAD reduced) and ADP is phosphorylated. Furthermore, by transaminating with aspartate to produce oxaloacetate, 2-oxoglutarate stimulates cytosolic malic dehydrogenase activity, whereby malate is formed and NADH is oxidized. In the form of malate, reducing equivalents and substrate are transported into the mitochondria where they are utilized for succinate synthesis.

Regulation of endothelial permeability by β-adrenoceptor agonists : contribution of β1- and β2-adrenoceptors

The barrier function of cultured, macrovascular endothelial cells derived from bovine aorta was analyzed using confluent monolayers of cells and measuring the exchange of fluorescein dextrans of different molecular masses. The effects of beta-adrenoceptor agonists with different selectivity for beta 1- and beta 2-adrenoceptors (AR) were investigated. Formoterol, a novel high-affinity agonist for beta 2-AR recently introduced in the treatment of bronchial asthma, showed a significant reduction of cell permeability with subnanomolar concentrations, whereas the catecholamines (-)-isoproterenol and (-)-norepinephrine only showed significant effects with micromolar concentrations. In order to elucidate if this difference in potential to regulate cell permeability is related to appropriate changes in the selectivity and affinity of the agonists for beta 2 AR, we investigated the beta AR-coupled adenylate cyclase (AC) in membranes from endothelial cells and compared AC stimulation with the binding of agonists to the receptors using [125I](-)-iodopindolol as radioligand. beta-Adrenoceptors revealed to be closely coupled to AC as assessed by a similar magnitude of effects by receptor agonists in comparison to GTP analogues and direct stimulants of AC activity. AC activity was increased by formoterol in parallel to its receptor occupancy of beta 2AR with nanomolar concentrations which were 50-fold higher than those used for the regulation of cell permeability indicating the existence of spare receptors. In contrast to formoterol, the catecholamines (-)-isoproterenol and (-)-norepinephrine stimulated AC activity through both beta 1AR and beta 2AR. From the overproportional high contribution of beta 1AR to AC stimulation (42%) in comparison to its low fraction (13%) in receptor binding we calculated that beta 1AR is 3-4-fold more effectively coupled to AC than beta 2 AR.

Subcellular distribution of malate-aspartate cycle intermediates during normoxia and anoxia in the heart

The subcellular distribution of adenine nucleotides, phosphocreatine and intermediates of the malate-aspartate cycle was investigated in adult rat heart myocytes under normoxia and anoxia. Cytosolic and mitochondrial concentrations of metabolites were determined by a fractionation method using digitonin. Under normoxia, cytosolic/mitochondrial gradients were found for ATP (c/m = 4), AMP (c/m less than 0.01), citrate (c/m = 0.5), aspartate (c/m = 3), glutamate (c/m = 2), while phosphocreatine and glutamine were confined to the cytosolic space. No gradients were found for malate and 2-oxoglutarate. The results show that the transport of electrons from the cytosol into the mitochondria is supported by the glutamate gradient and by a high glutamate/aspartate ratio inside the mitochondria (Glu/Asp = 15) which is maintained by the energy-dependent Glu-Asp exchange across the mitochondrial membrane. Under anoxia, cytosolic glutamate is transaminated with pyruvate, yielding alanine and 2-oxoglutarate, which is oxidized to succinate inside the mitochondria and leaves the cell. The data indicate that stimulation of transamination is caused by a mass action effect following a decrease in cytosolic 2-oxoglutarate which may be due to succinate-2-oxoglutarate exchange across the mitochondrial membrane. Inhibition of the energy-dependent inward transport of glutamate may support this process.

Sympathetic autonomic neuropathy in the heart of the spontaneous diabetic BB rat

There is evidence that autonomic nerves are disturbed in spontaneous diabetic BB rats indicating a peripheral diabetic autonomic neuropathy. For histofluorescent visualization of catecholamine containing nerve fibres in myocardium the method of De La Torre was applied. For electron microscopy, tissue specimens of ventricular and atrial myocardium were prepared. In myocardium of BB rats diabetic for 28 weeks morphometric quantification of catecholamine histofluorescence revealed a loss of fluorescent nerve fibres and varicosities of 57% from 1.18 +/- 0.17 to 0.51 +/- 0.18 per cent of the total field area (p < 0.009). Ultrastructurally, morphometric quantification of the density of axon profiles containing synaptic vesicles in transverse sectioned atrial myocardium showed a decrease of 42% (p < 0.021) in the diabetic group. In the diabetic nerves, axoplasmic lysis and a reduction in the Schwann cell envelope were prominent. Morphologic changes of nerves in ventricles were comparable to the findings in atrial tissue. It is concluded that a diabetic autonomic neuropathy of intramural sympathetic nervous tissue occurs in myocardium of spontaneous diabetic BB rats despite insulin substitution.