Gene transfer of superoxide dismutase isoforms reverses endothelial dysfunction in diabetic rabbit aorta

Increased production of oxygen free radicals is an important mechanism of endothelial dysfunction in diabetes mellitus. Our goal was to test whether adenovirus (Ad)-mediated gene transfer of copper/zinc (CuZn) or manganese superoxide dismutase (Mn SOD) improves relaxation of diabetic vessels. The aortas from 9 alloxan-induced diabetic mellitus (DM) and 16 control rabbits were used. Control and DM rings were transduced ex vivo with Ad vectors encoding Mn SOD (AdMn SOD), CuZn SOD (AdCuZn SOD), beta-galactosidase (Ad(beta)gal), or diluents. In the absence of gene transfer, SOD activity was significantly increased in DM aortas. Transgene expression in DM AdCuZn SOD and DM AdMn SOD-transduced vessels was confirmed by Western blot analysis and by increased SOD activity (DM AdCuZn SOD, 76.2 +/- 9.3; DM AdMn SOD, 65.2 +/- 4.8; P < 0.05 vs. DM Ad(beta)gal; 50.9 +/- 4.4 U/mg protein). Superoxide production was increased in DM Ad(beta)gal-transduced aorta and relaxations to acetylcholine were impaired in these vessels. Gene transfer of CuZn SOD and Mn SOD corrected both of these defects. Thus Ad-mediated gene transfer CuZn and Mn SOD to the diabetic aorta improves endothelium-dependent relaxation.     

L-Arginine does not improve endothelium-dependent relaxation inin vitro Watanabe rabbit thoracic aorta

Summary The findings that even a singlein vitro addition of L-arginine is able to normalize endothelium function in cerebral vessel from diet-induced hypercholesterolemic rabbits prompted us to investigate if similar results could be obtained on Watanabe rabbits thoracic aorta, in which we previously demonstrated low content of the amino acid.L-Arginine (1 mM) preincubated for 45 minutes before the addition of drugs for studing endothelium-dependent vascular relaxation, did not modify the effect of acetylcholine on aortic isolated preparations. The lack of any effect by L-arginine indicates that the amino acid deficiency is not main cause of the impairment of endothelium function. The muscarinic receptor functionality affected by atherosclerotic process and/or the increased synthesis of EDCFs could account for the reduced endothelium-dependent relaxation.     

Endothelial dysfunction does not require loss of endothelial nitric oxide synthase

Whereas altered nitric oxide (NO.) formation from endothelial nitric oxide synthase (NOS) causes impaired vascular reactivity in a number of cardiovascular diseases, questions remain regarding how endothelial injury results in impaired NO. formation. It is unknown if loss of NOS expression or activity is required or if other factors are involved. Detergent treatment has been used to induce endothelial dysfunction. Therefore, NOS and NO. synthesis were characterized in a rat heart model of endothelial injury and dysfunction induced by the detergent Triton X-100. Cardiac NO. formation was directly measured by electron paramagnetic resonance spectroscopy. NOS activity was determined by the L-[(14)C]arginine conversion assay. Western blots and immunohistology were applied to define the amounts of NOS present in heart tissue before and after Triton treatment. Immunoelectron microscopy was performed to assess intracellular NOS distribution. A short bolus of Triton X-100, 0.25%, abolished responses to histamine and calcium ionophore while preserving response to nitroprusside. Complete blockade of NO. generation occurred after Triton treatment, but NOS activity assayed with addition of exogenous substrate and cofactors was unchanged, and identical 135-kDa NOS bands were seen on Western blots, indicating that NOS was not removed from the heart or structurally damaged by Triton. Immunohistochemistry showed no change in NOS localization after Triton treatment, and immunoelectron microscopy revealed similar NOS distribution in the plasma membrane and intracellular membranes. These results demonstrate that the endothelial dysfunction was due to decreased NO. synthesis but was not caused by loss or denaturation of NOS. Thus endothelial dysfunction due to mild endothelial membrane injury may occur in the presence of active NOS and is triggered by loss of NOS substrates or cofactors.     

Characterization of endothelial thromboxane receptors in rabbit aorta

An increased synthesis of thromboxane (TX) A(2) is associated with a number of cardiovascular diseases including atherosclerosis, unstable angina and hypertension. We previously identified a subgroup of NZW rabbits in which isolated arteries failed to contract to the TX agonists, U46619 or I-BOP. In vascular smooth muscle membranes, there was a significant decrease in TX receptors, termed TP. These rabbits are referred to as vTP- and those with the TP receptor are called vTP+. Because TP receptors are expressed in some types of endothelial cells, the present study was designed to determine whether functional TP receptors are present in endothelial cells cultured from aortas of vTP+ and vTP- rabbits. Radioligand binding studies were performed with (125)I-BOP. Aortic endothelial cells from vTP+ rabbits exhibited specific and saturable binding. In contrast, in endothelial preparations from vTP- rabbit aortas, no measurable binding to (125)I-BOP was detected. Using an anti-TP receptor antibody, we compared the amount of receptor expressed in endothelial cell lysates obtained from vTP+ and vTP- rabbits. Consistent with the results observed radioligand binding assays, the expression of TP receptor protein was decreased in vTP- compared to vTP+ endothelial cells. An in vitro wound healing assay was used on confluent monolayers of endothelial cells. In the untreated vTP+ cells, the area of the scratch was completely closed by 30 h. In the vTP+ cells treated with U46619 (3 microM), the rate of closure of the scratch area was reduced with approximately 12% of the scratch area remaining at 30 h. Pretreatment with the TP receptor antagonist, SQ 29548 (10 microM) prevented the inhibitory effect of U46619. The rate of closure of the scratch in the vTP- was not altered by U46619. In a separate study, U46619 (3 microM) increased the release of 6-keto PGF(1alpha), the stable metabolite of prostacyclin, in vTP+ but not vTP- endothelial cells. Pretreatment with SQ29548 (10 microM) or the cyclooxygenase inhibitor, indomethacin (10 microM) blocked the increase in vTP+ endothelial cells. In vascular reactivity studies in aortas from vTP+ rabbits, removal of the endothelium enhanced the vasoconstrictor response to U46619 indicating that activation of endothelial TP receptors may modulate vascular tone via the release of the vasodilator, prostacyclin. The results of this study suggest an important role for endothelial TP receptors in modulating vascular function.     

Characterization of endothelial thromboxane receptors in rabbit aorta

An increased synthesis of thromboxane (TX) A₂ is associated with a number of cardiovascular diseases including atherosclerosis, unstable angina and hypertension. We previously identified a subgroup of NZW rabbits in which isolated arteries failed to contract to the TX agonists, U46619 or I-BOP. In vascular smooth muscle membranes, there was a significant decrease in TX receptors, termed TP. These rabbits are referred to as vTP− and those with the TP receptor are called vTP+. Because TP receptors are expressed in some types of endothelial cells, the present study was designed to determine whether functional TP receptors are present in endothelial cells cultured from aortas of vTP+ and vTP− rabbits. Radioligand binding studies were performed with ¹²⁵I-BOP. Aortic endothelial cells from vTP+ rabbits exhibited specific and saturable binding. In contrast, in endothelial preparations from vTP− rabbit aortas, no measurable binding to ¹²⁵I-BOP was detected. Using an anti-TP receptor antibody, we compared the amount of receptor expressed in endothelial cell lysates obtained from vTP+ and vTP− rabbits. Consistent with the results observed radioligand binding assays, the expression of TP receptor protein was decreased in vTP− compared to vTP+ endothelial cells. An in vitro wound healing assay was used on confluent monolayers of endothelial cells. In the untreated vTP+ cells, the area of the scratch was completely closed by 30 h. In the vTP+ cells treated with U46619 (3 μM), the rate of closure of the scratch area was reduced with approximately 12% of the scratch area remaining at 30 h. Pretreatment with the TP receptor antagonist, SQ 29548 (10 μM) prevented the inhibitory effect of U46619. The rate of closure of the scratch in the vTP− was not altered by U46619. In a separate study, U46619 (3 μM) increased the release of 6-keto PGF_1α, the stable metabolite of prostacyclin, in vTP+ but not vTP− endothelial cells. Pretreatment with SQ29548 (10 μM) or the cyclooxygenase inhibitor, indomethacin (10 μM) blocked the increase in vTP+ endothelial cells. In vascular reactivity studies in aortas from vTP+ rabbits, removal of the endothelium enhanced the vasoconstrictor response to U46619 indicating that activation of endothelial TP receptors may modulate vascular tone via the release of the vasodilator, prostacyclin. The results of this study suggest an important role for endothelial TP receptors in modulating vascular function.     

Sewage sludge does not induce genotoxicity and carcinogenesis

Through a series of experiments, the genotoxic/mutagenic and carcinogenic potential of sewage sludge was assessed. Male Wistar rats were randomly assigned to four groups: Group 1 - negative control; Group 2 - liver carcinogenesis initiated by diethylnitrosamine (DEN; 200 mg/kg i.p.); Group 3 and G4-liver carcinogenesis initiated by DEN and fed 10,000 ppm or 50,000 ppm of sewage sludge. The animals were submitted to a 70% partial hepatectomy at the 3^rd week. Livers were processed for routine histological analysis and immunohistochemistry, in order to detect glutathione S-transferase positive altered hepatocyte foci (GST-P⁺ AHF). Peripheral blood samples for the comet assay were obtained from the periorbital plexus immediately prior to sacrificing. Polychromatic erythrocytes (PCEs) were analyzed in femoral bone-marrow smears, and the frequencies of those micronucleated (MNPCEs) registered. There was no sewage-sludge-induced increase in frequency of either DNA damage in peripheral blood leucocytes, or MNPCEs in the femoral bone marrow. Also, there was no increase in the levels of DNA damage, in the frequency of MNPCEs, and in the development of GST-P AHF when compared with the respective control group.     

Dietary L-arginine restores aspirin-induced endothelial dysfunction in rat aorta

L-Arginine induced elevation of the vascular prostanoid led us to think that the risk of coronary spasm may increase in L-arginine consumers when they are subjected to cyclooxygenase inhibitors and this limits the therapeutic value of aspirin. So the aim was to investigate the interaction of aspirin and dietary L-arginine in male rats. Animals were divided into four groups and fed with normal food. The first group received tap water while the second, third and fourth groups were subjected daily to aspirin (8.6 mg/kg), L-arginine (143 mg/kg) and aspirin + L-arginine combination in their drinking water respectively for 7 days. Vasomotor responses were recorded in the aortic rings suspended for isometric-force recordings. Aspirin treatment significantly reduced the dilation to acetylcholine and sodium nitroprusside. Attenuated phenylephrine contractility was associated with normal acetylcholine response in L-arginine group. Addition of L-arginine to aspirin treatment completely prevented aspirin-induced endothelial dysfunction but defective response to sodium nitroprusside persisted. Dietary L-arginine without affecting maximal dilation to acetylcholine significantly increased the share of dilator prostanoid which appears to resist aspirin. These results demonstrated that dietary L-arginine increases dilator prostaoid in rat aortic rings. Contrary to our expectation, co-administered L-arginine protected aspirin induced endothelial dysfunction and ruled out the limitation of aspirin use in L-arginine consumers.     

In vivo exposure of human lymphocytes to technetium-99m in nuclear medicine patients does not induce detectable genetic effects.

Recent reports have demonstrated that exposure of nuclear medicine patients to thallium-201 does not result in a detectable increase in mutation at the hprt locus in human lymphocytes. In an effort to study further the potential genetic effects of medical exposures to low dose radiation, we have examined chromosome aberrations and mutations in peripheral blood lymphocytes from nuclear medicine patients exposed to clinical doses of technetium-99m. Our results show that there is no exposure-related increase in chromosomal damage; furthermore, the data do not confirm earlier reports of exposure-related increases in mutations induced by technetium-99m.     

SAA does not induce cytokine production in physiological conditions

SAA has been shown to have potential proinflammatory properties in inflammatory diseases such as atherosclerosis. These include induction of tumor necrosis factor α, interleukin-6, and monocyte chemoattractant protein 1 in vitro. However, concern has been raised that these effects might be due to use of recombinant SAA with low level of endotoxin contaminants or its non-native forms. Therefore, physiological relevance has not been fully elucidated. In this study, we investigated the role of SAA in the production of inflammatory cytokines. Stimulation of mouse monocyte J774 cells with lipid-poor recombinant human SAA and purified SAA derived from cardiac surgery patients, but not ApoA-I and ApoA-II, elicited pro-inflammatory cytokines like granulocyte colony stimulating factor (G-CSF). However, HDL-associated SAA failed to stimulate production of these cytokines. Using neutralizing antibodies against toll like receptor (TLR) 2 and 4, we could evaluate that TLR 2 is responsible for G-CSF production by lipid-poor SAA. To confirm these data in vivo, we expressed mouse SAA in SAA deficient C57BL/6 mice using an adenoviral vector. G-CSF was identically expressed in SAA-Adenoviral infected mice as well as in control null-Adenoviral mice at the early time points (4–8 h) and could not be detected in plasma 24 h after infection when plasma SAA levels were maximally elevated, indicating that adenoviral vector rather than SAA affected G-CSF levels. Taken together, our findings suggest that lipid-poor SAA, but not HDL-associated SAA, stimulates G-CSF production and this stimulation is mediated through TLR 2 in J774 cells. However, its physiological role in vivo remains ambiguous.     

Coronary-active bovine heart proteins induce relaxation in the rabbit aorta]

Seven cardioactive polypeptides have been identified in the precardiac and auricular regions of the bovine heart. Those polypeptides were studied for their action on the function of isolated preparations of the vessel. It has been found that compounds isolated from the precardiac region induce different (20-55%) relaxation of smooth muscles in a strip of the aorta of rabbit. The possibility of formation of endogenic cardioactive compounds in model experiments imitating processing is studied. The data obtained permit assuming and alternatively explaining existence of multiple forms of the mentioned compounds as a result of partial proteolytic splitting of larger molecules.     

Interferon gamma does not induce antiviral resistance in T lymphocytes

We compared the activity of human recombinant alpha and gamma interferons (IFNs) on normal T lymphocytes and various T cell lines. IFN gamma, unlike IFN alpha, did not promote the antiviral state in these cells, or induce the activity of 2'-5' oligoadenylate synthetase. The lack of antiviral effect was observed using an RNA virus (VSV) and a DNA virus (HSV, type 1) as challenger viruses.     

Barium responsiveness of the rat aorta and femoral artery during pregnancy

The barium responses of isolated aortic strips and femoral arteries from non-pregnant and pregnant rats were investigated. Barium caused concentration-related increases in tension of vessels from both pregnant and non-pregnant rats. The concentration-response curves of femoral arteries from non-pregnant and 3 week pregnant rats were not different; however contractility and slopes of concentration-response lines for thracic aortas from 1, 2 and 3 week pregnant rats were significantly less than those of aortas from non-pregnant rats. In addition, barium caused rhythmic contractions to develop in both femoral arteries and aortas of 3 week pregnant rats more frequently than vessels from non-pregnant rats. Rhythmic contractions did not develop in aortas from 3 week pregnant rats rats in calcium-free Krebs. Since the effects of barium on the electrical and mechanical activity of various muscles have been postulated to be similar to and/or dependent on calcium, these results may indicate that changes in calcium sensitivity of vascular smooth muscle occur during pregnancy. Such changes may contribute to the blood flow redistribution and other cardiovascular adaptations of pregnancy.     

Interferon activity is not detected in blastocyst secretions and does not induce decidualization in mice.

Mouse trophoblast cells synthesized and secreted proteins during the peri-implantation period, some in the molecular size range of alpha interferons (IFN-alpha), known mediators of the maternal recognition of pregnancy in sheep and cows. However, conditioned media samples containing secreted proteins from Day-5 mouse blastocysts or from trophoblast outgrowths did not contain detectable levels of antiviral activity indicative of IFN. In addition, it was not possible to induce a decidual reaction in suitably sensitized uteri with intraluminal instillation of IFN-alpha. The results indicate that IFNs are probably not involved in the maternal recognition of pregnancy at the time of implantation in the mouse.     

alpha-IFN treatment does not induce Ki-ras expression in hairy-cell leukemia patients

alpha-Interferon (IFN) is effective in the treatment of hairy-cell leukemia (HCL), but the treatment is sometimes over a long period. Biological changes such as the increase of tumorigenicity can occur rapidly in vivo as a result of beginning this treatment; an increase in c-Ki-ras oncogene expression has also been observed. In order to determine whether the findings observed in vitro would be duplicated in an in vivo system, we decided to analyze the Ki-ras RNA and protein levels in the lymphocytes of three HCL patients, compared with these levels in seven normal donors and one non-treated HCL patients. Ki-ras was not activated by IFN, at least not in lymphocytes. Therefore, the data suggest that the drug could be used for long-term therapy with relatively low risk to the patients.     

Interleukin 2 does not induce phosphatidylinositol hydrolysis in activated T cells

Hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerol and myoinositol-1,4,5-trisphosphate is thought to be a primary event in the activation of cells by some growth factors, mitogenic lectins, and oncogenes. The mechanism whereby interleukin 2 (IL 2) binding to its receptor on activated T lymphocytes leads to cell proliferation has not been determined. Because the mitogenic has not been determined. Because the mitogenic action of IL 2 resembles that of some growth factors, the possible role of phosphatidylinositol breakdown in the activation of T cells by IL 2 was examined. In human or murine IL 2-sensitive cells, incubation with IL 2 did not alter the rate of turnover of phosphatidylinositol, phosphatidylinositol-5-phosphate, phosphatidylinositol-4,5-bisphosphate, or phosphatidylcholine in 32PO4-loaded cells. IL 2 also did not alter either the isotopic labeling of diacylglycerol or [3H]arachidonic acid release from cells. In addition, IL 2 did not alter the rate of formation of the phosphatidylinositol breakdown products myoinositol-1,4,5-trisphosphate, myoinositol-1,4-bisphosphate, or myoinositol-1-phosphate. In contrast, under similar conditions, IL 2 induced significant increases in [3H]thymidine incorporation and cell proliferation. Mitogenic lectins such as concanavalin A and phytohemagglutinin gave significant changes in isotopic labeling of phosphoinositols, diacylglycerols, and phosphatidylinositols, indicating that phosphatidylinositol hydrolysis induced by mitogenic lectins was detectable in the assay systems. IL 2, in contrast to other growth factors, does not appear to signal cells by increasing phosphatidylinositol breakdown.     

Cyclic AMP does not inhibit A23187-induced prostaglandin biosynthesis in cultured rabbit renal microvascular endothelial cells.

We have previously shown that cultured rabbit renal preglomerular microvascular endothelial cells have the ability to synthesize a number of common prostaglandins. In the present study we have examined whether endogenous cyclic AMP is involved in the regulation of PGI2 and PGE2 biosynthesis in these cultured cells. Isoproterenol and forskolin produced an increase in cyclic AMP accumulation in these cells but had no effect on PGI2 or PGE2 biosynthesis either in the presence or absence of A23187. Similar results were noted in the presence of 3-isobutyl-1-methylxanthine, a cyclic AMP-phosphodiesterase inhibitor. These studies suggested that endogenous cyclic AMP does not regulate the biosynthesis of PGI2 or PGE2 in cultured renal preglomerular microvascular endothelial cells either under basal or A23187-stimulated condition. They further suggested that the effect of 3-isobutyl-1-methylxanthine on prostaglandin biosynthesis in these cultured cells was not secondary to its effects on phosphodiesterase.     

Muramyl dipeptide does not induce slow-wave sleep or fever in rats

The synthetic muramyl dipeptide, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), is reported to increase slow-wave sleep and body temperature in cats, rabbits, and squirrel monkeys. The present study examined the ability of MDP to induce sleep and fever in rats. MDP was administered IP at 50, 250 and 500 micrograms/kg. Sleep and body temperature were monitored for 12 hr. MDP failed to affect the duration of wakefulness, S1, S2, or total (S1 + S2) slow-wave sleep. There was also no change in the latency to the first episode of S2 sleep. In contrast, rapid-eye-movement (REM) sleep was significantly suppressed for the first 6 hr after 250 and 500 microgram/kg doses of MDP. There was, however, a rebound increase in REM sleep after the initial period of suppression which resulted in no overall change in the amount of REM sleep. Body temperature was unaffected by MDP. Thus, we conclude that MDP has neither sleep-promoting nor pyrogenic actions in the rat when administered systemically at doses reported to be effective in several other species.