Biomedical subjects

F Squadrito

Publications and source records attributed to F Squadrito.

At least 19 recordsLinked to original sources

PubMed2003

Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse.

AIMS/HYPOTHESIS: We studied the gene therapy efficacy of diabetes-associated wound healing disorder with an adeno-associated virus (AAV) vector expressing the 165-amino acid isoform of human vascular endothelial growth factor-A (VEGF-A) by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ db+/db+ mice and their normal littermates ( db+/+m). METHODS: Animals were randomized to receive intradermally into the wound edges either rAAV-LacZ (a control gene), or rAAV-VEGF165. Animals were killed on different days (7 and 14 days after skin injury) and wounded skin tissues were used for gene marker studies, histological evaluation and immunohistochemistry, and wound breaking strength analysis. Furthermore we studied the VEGF mature protein in the wounds. RESULTS: We found that AAV vectors are highly efficient for gene transfer to the mouse skin, displaying an exquisite tropism for the panniculus carnosus by using the beta-galactosidase activity assay. We confirmed the increased expression of the angiogenic factor at day 7 by measuring the wound content of the mature protein. Delivery of VEGF165 to incisional skin wounds of diabetic mice resulted in a remarkable induction of new vessel formation with consequent improvement in the wound healing process. The rAAV-VEGF165 gene improved wound healing in diabetic mice through the stimulation of angiogenesis, reepithelization, synthesis and maturation of extracellular matrix. Moreover the recombinant AAV encoding the human VEGF165 increased the breaking strength of the wound and enhanced the wound content of VEGF. CONCLUSION/INTERPRETATION: Our study suggests that VEGF gene transfer might represent a new approach to treat wound healing disorders associated with diabetes.

Angiogenesis Inducing Agents↗

PubMed2002

Recombinant AAV vector encoding human VEGF165 enhances wound healing.

Delivery of therapeutic genes represents an appealing possibility to accelerate healing of wounds that are otherwise difficult to treat, such as those in patients with metabolic disorders or infections. Experimental evidence indicates that in such conditions potentiation of neo-angiogenesis at the wound site might represent an important therapeutic target. Here we explore the efficacy of gene therapy of wound healing with an adeno-associated virus (AAV) vector expressing the 165 amino acid isoform of vascular endothelial growth factor-A (VEGF-A). By gene marker studies, we found that AAV vectors are highly efficient for gene transfer to the rat skin, displaying an exquisite tropism for the panniculus carnosus. Gene expression from these vectors is sustained and persistent over time. Delivery of VEGF165 to full thickness excisional wounds in rats resulted in remarkable induction of new vessel formation, with consequent reduction of the healing time. Histological examination of treated wounds revealed accelerated remodeling of epidermis and dermis, with formation of a thick granular layer, containing numerous newly formed capillaries, as well as vessels of larger size. These data underline the importance of neo-angiogenesis in the healing process and indicate that VEGF gene transfer might represent a novel approach to treat wound healing disorders.

Animals↗

PubMed2001

Oxidative stress causes nuclear factor-kappaB activation in acute hypovolemic hemorrhagic shock.

Nuclear Factor kappaB (NFkappaB) is an ubiquitous rapid response transcription factor involved in inflammatory reactions and exerts its action by expressing cytokines, chemokines, and cell adhesion molecules. We investigated the role of NF-kappaB in acute hypovolemic hemorrhagic (Hem) shock. Hem shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min (bleeding period) until mean arterial blood pressure (MAP) fell and stabilized within the range of 20-30 mmHg. Hemorrhagic shocked rats died in 26.3 +/- 2.1 min following the discontinuance of bleeding, experienced a marked hypotension (mean arterial blood pressure = 20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (200 +/- 15 pg/ml, 20 min after the end of bleeding). Furthermore, aortas taken 20 min after bleeding from hemorrhagic shocked rats showed a marked hypo-reactivity to phenylephrine (PE; 1nM to 10 microM) compared with aortas harvested from sham shocked rats. Hem shocked rats also had increased levels of TNF-alpha mRNA in the liver (15-20 min after the end of bleeding) and enhanced plasma levels of 2,5-dihydroxybenzoic acid (2,5-DHBA; 6 +/- 2.2 microm), 2,3-dihydroxybenzoic acid (2,3-DHBA; 13 +/- 2.1 microm), both studied to evaluate OH(*) production. Electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus 10 min after the end of hemorrhage and remained elevated until the death of animals. Western blot analysis suggested that the levels of inhibitory IkappaBalpha protein in the cytoplasm became decreased at 5 min after the end of bleeding. IRFI-042, a vitamin E analogue (20 mg/kg intraperitoneally 2 min after the end of bleeding), inhibited the loss of IkappaBalpha protein from the cytoplasm and blunted the increase in NF-kappaB binding activity. Furthermore IRFI-042 increased survival time (117.8 +/- 6.51 min; p <.01) and survival rate (vehicle = 0% and IRFI-042 = 80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-alpha, reduced plasma TNF-alpha (21 +/- 4.3 pg/ml), and restored to control values the hypo-reactivity to PE. Our results suggest that acute blood loss (50% of the estimated total blood volume over a period of 20 min) causes early activation of NF-kappaB, likely through an increased production of reactive oxygen species. This experiment indicates that NF-kappaB-triggered inflammatory cascade becomes early activated during acute hemorrhage even in the absence of resuscitation procedures.

Animals↗

PubMed2001

Nuclear factor-kappaB as a target of cyclosporin in acute hypovolemic hemorrhagic shock.

BACKGROUND: Cyclosporin is an immunosuppressive drug that blocks Nuclear Factor kappaB (NF-kappaB) activation. We investigated the role of NF-kappaB in acute hypovolemic hemorrhagic (Hem) shock and the effects of cyclosporin in this model of experimental shock. METHODS: Hem shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min (bleeding period) until mean arterial blood pressure (MAP) fell and stabilized within the range of 20-30 mmHg. Two minutes after bleeding cessation, animals received intravenously cyclosporin (1 mg kg(-1)) or its vehicle. Survival rate and survival time were evaluated for 120 min after bleeding was discontinued. Plasma TNF-alpha levels were investigated at different time points after bleeding cessation. Moreover we investigated levels of TNF-alpha mRNA in the liver, vascular reactivity, liver NF-kappaB binding activity and levels of the inhibitory protein IkappaBalpha in the cytoplasm. RESULTS: Hemorrhagic shocked rats died in 27+/-6 min following the cessation of bleeding, experienced a marked hypotension (mean arterial blood pressure=20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (208+/-22 pg ml(-1), 20 min after the end of bleeding). Furthermore, aortas taken 20 min after bleeding from hemorrhagic shocked rats showed a marked hypo-reactivity to phenylephrine (PE: 1 nM-10 microM) compared with aortas harvested from sham shocked rats. Hem shocked rats also had increased levels of TNF-alpha mRNA in the liver (15-20 min after the end of bleeding). Electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus 10 min after the end of hemorrhage and remained elevated until the death of animals. Western blot analysis suggested that the levels of inhibitory protein IkappaBalpha in the cytoplasm decreased at 5 min after the end of bleeding. Cyclosporin inhibited the loss of IkappaBalpha protein from the cytoplasm and prevented NF-kappaB binding activity in the nucleus. Furthermore, cyclosporin increased survival time (118+/-7 min; P<0.01) and survival rate (vehicle=0% and cyclosporin=80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-alpha, reduced plasma TNF-alpha (28+/-7 pg ml(-1)), and restored to control values the hypo-reactivity to PE. CONCLUSIONS: Our results suggest that acute blood loss (50% of the estimated total blood volume over a period of 20 min) causes early activation of NF-kappaB which triggers an inflammatory cascade leading to a fatal outcome. Cyclosporin blocks NF-kappaB activation and protects against hypovolemic hemorrhagic shock.

Animals↗

PubMed2001

Effects of simvastatin treatment on sICAM-1 and sE-selectin levels in hypercholesterolemic subjects.

This study was performed to determine whether the levels of soluble intercellular adhesion molecule-1 (sICAM-l) and soluble endothelial molecule-1 (sE-selectin) were elevated in subjects with hypercholesterolemia who presented with no other risk factors or evidence of atherosclerosis. The effects of administration of an HMG-CoA reductase inhibitor on the serum levels of these molecules were also examined. Forty hypercholesterolemic subjects (HCh) (19 males and 21 females), without hypertension or cardiovascular disease, received placebo for 4 weeks. The patients were then randomized in two groups; 20 of them (simvastatin group) were treated with simvastatin (20 mg/day) and the other 20 (placebo group) continued placebo administration. After 12 and 24 weeks of either simvastatin or placebo treatment, sICAM-1 and sE-selectin levels were measured. The same parameters were measured in 20 control subjects (C) with normal cholesterol levels, matched for sex and age. HCh had sICAM-1 basal values higher than C (352.4+/-57.9 ng/ml versus 114.9+/-89.6 ng/ml; P<0.001); however, sE-selectin basal values were not different in the two groups. No correlation was observed between HCh sICAM-1 levels and cholesterol levels (total and low-density lipoprotein). Furthermore, cholesterol-lowering treatment with simvastatin did not significantly diminish sICAM-1 levels. Our findings would support the hypothesis that patients with isolated hypercholesterolemia and without clinical atherosclerosis may be silent carriers of arterial subendothelial inflammation, expressed as an increase of sICAM-1.

Adult↗

PubMed2001

The phytoestrogen alpha-zearalenol reverses endothelial dysfunction induced by oophorectomy in rats.

It has been shown recently that alpha-zearalenol, a resorcyclic acid lactone, prevents bone loss in a rat model of postmenopausal bone loss. We have therefore investigated the effects of this phytoestrogen on endothelial dysfunction induced by estrogen deficiency in rats. Female mature Sprague-Dawley rats underwent a bilateral oophorectomy (OVX rats). Sham-operated animals (sham OVX rats) were used as controls. Three weeks after surgery, animals were randomized to the following treatments: alpha-zearalenol (1 mg/kg/day, i.m., for 4 weeks), 17beta-estradiol (20 microg/kg/day, i.m., for 4 weeks), or their vehicle (100 microl, i.m., of cottonseed oil). Two other groups of rats were treated with alpha-zearalenol or 17beta-estradiol plus the pure estrogen receptor antagonist ICI 182780 (2.5 mg/kg/day, i.m., for 4 weeks). Mean arterial blood pressure (MAP), heart rate (HR), total plasma cholesterol, plasma estradiol, and plasma alpha-zearalenol were studied. We also investigated endothelial-dependent (acetylcholine, 10 nM to 10 microM) and endothelial-independent (sodium nitroprusside, 15 nM to 30 nM) relaxation of aortic rings, as well as N(G)-methyl-L-arginine (L-NMA: 10 to 100 microM)-induced vasoconstriction and calcium-dependent nitric oxide synthase (cNOS) activity in homogenates of lungs taken from both sham OVX rats and OVX rats. Untreated OVX rats had, compared with sham OVX animals, unchanged body weight, MAP, HR, and plasma cholesterol. In contrast oophorectomy reduced plasma estradiol levels (OVX, 2 +/- 0.5 pg/ml; sham OVX, 35 +/- 6 pg/ml), impaired endothelial-dependent relaxation and blunted L-NMA-induced contraction (L-NMA 100 microM: sham OVX, 2.7 +/- 0.3 g/mg tissue; OVX, 1.3 +/- 0.1 g/mg tissue). Moreover OVX rats showed a reduced calcium-dependent NO synthase (cNOS) activity. Treatment with alpha-zearalenol or with 17beta-estradiol reverted the endothelial dysfunction and increased cNOS activity in lung homogenates. These effects were abolished by the pure estrogen receptor antagonist ICI 182780. Our data suggest that alpha-zearalenol improves endothelial-dependent relaxation in OVX rats through an estrogen receptor-mediated effect.

Acetylcholine↗

PubMed2001

Serotonin, norepinephrine and dopamine involvement in the antidepressant action of hypericum perforatum.

Hypericum perforatum is considered an effective alternative to the synthetic antidepressants in the treatment of mild-to-moderate depression. Recently, we showed that the effects on neurotransmitter contents in different brain regions of laboratory animals are more evident after administration of hypericum extracts containing a higher concentration of flavonoids, thus suggesting that these compounds are important in the antidepressant action of hypericum perforatum. We studied the effects of Ph-50, a hypericum extract standardized to flavonoids (50%) and containing 0.3% hypericin and 4.5% hyperforin on brain serotonin content, norepinephrine and dopamine by a high-performance liquid chromatography method in discrete brain areas (cortex, diencephalon and brainstem) in male Sprague-Dawley rats. Moreover, we evaluated the effects of Ph-50 alone or in association with sulpiride (a dopamine receptor antagonist), metergoline (a serotonin receptor antagonist) and 6-hydroxydopamine (6-OH-DA, destroying norepinephrine-containing neurons) using a forced-swimming test in the rat. Hypericum extract (Ph-50; 250-500 mg/kg) with acute oral administration enhanced serotonin, norepinephrine and dopamine content in the brain and reduced the immobility time of rats in the forced-swimming test. Sulpiride, metergoline and 6-OH-DA significantly increased the period of immobility in the forced-swimming test for the rats receiving hypericum extract (Ph-50). The results indicate that the neurotransmitters studied could be involved in the anti-immobility effects of hypericum, and suggest that its antidepressant action is probably mediated by serotonergic, noradrenergic and dopaminergic system activation.

Animals↗

PubMed2001

Raxofelast, a hydrophilic vitamin E-like antioxidant, stimulates wound healing in genetically diabetic mice.

BACKGROUND: Impaired wound healing is a well-documented phenomenon in experimental and clinical diabetes. Emerging evidence favors the involvement of free radicals in the pathogenesis of diabetes-related healing deficit. This study assessed the effect of systemic administration of raxofelast, a protective membrane antioxidant agent, on wound healing by using healing-impaired (db/db) mice. METHODS: The wound healing effect of raxofelast was investigated by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ db+/db+ mice and their healthy littermates (db+/+m). Animals were then randomized to the following treatment: raxofelast (15 mg/kg/d intraperitoneally) or its vehicle (dimethyl sulfoxide/sodium chloride 0.9%, 1:1, vol/vol). The animals were killed on different days, and the wounded skin tissues were used for histologic evaluation and for analysis of malondialdehyde (MDA) level and myeloperoxidase (MPO) activity, wound breaking strength, and collagen content. RESULTS: Diabetic mice showed delayed wound healing together with low collagen content, breaking strength, and increased MDA levels and MPO activity when compared with their healthy littermates. The administration of raxofelast did not modify the process of wound repair in healthy (db/+) mice, but significantly improved impaired wound healing in diabetic mice through the stimulation of angiogenesis, reepithelialization, synthesis, and maturation of extracellular matrix. Furthermore, raxofelast treatment significantly reduced MDA levels, MPO activity, and increased the breaking strength and collagen content of the wound. CONCLUSIONS: The current study provides evidence that raxofelast restores wound healing to nearly normal levels in experimental diabetes-impaired wounds and suggests that an increased lipid peroxidation in diabetic mice may have a role in determining a defect of wound repair.

Animals↗

PubMed2001

Reduction of carbon tetrachloride-induced rat liver injury by IRFI 042, a novel dual vitamin E-like antioxidant.

Carbon tetrachloride (CCl4 )-induced hepatotoxicity is likely the result of a CCl4 -induced free radical production which causes membrane lipid peroxidation and activation of transcription factors regulating both the TNF-alpha gene and the early-immediate genes involved in tissue regeneration. IRFI 042 is a novel vitamin E-like compound having a masked sulphydryl group in the aliphatic side chain. We studied the effect of IRFI 042 on CCl4 -induced liver injury. Liver damage was induced in male rats by an intraperitoneal injection of CCl4 (1 ml/kg in vegetal oil). Serum alanine aminotransferase (ALT) activity, liver malondialdehyde (MAL), hydroxyl radical formation (OH*), calculated indirectly by a trapping agent, hepatic reduced glutathione (GSH) concentration, plasma TNF-alpha, liver histology and hepatic mRNA levels for TNF-alpha were evaluated 48 h after CCl4 administration. Hepatic vitamin E (VE) levels were evaluated, in a separate group of animals, 2 h after CCl4 injection. A control group with vitamin E (100 mg/kg) was also treated in order to evaluate the differences versus the analogue treated groups. Intraperitoneal injection of carbon tetrachloride produced a marked increase in serum ALT activity (CCl4 = 404.61 +/- 10.33 U/L; Controls= 28.54 +/- 4.25 U/L), liver MAL (CCl4 = 0.67 +/- 0.16 nmol/mg protein; Controls= 0.13 +/- 0.06 nmol/mg protein), OH(7) levels assayed as 2,3-DHBA (CCl4 = 8.73 +/- 1.46 microM; Controls= 0.45 +/- 0.15 microM) and 2,5-DHBA (CCl4 = 24.61 +/- 3.32 microM; Controls= 2.75 +/- 0.93 microM), induced a severe depletion of GSH (CCl4 = 3.26 +/- 1.85 micromol/g protein; Controls= 17.82 +/- 3.13 micromol/g protein) and a marked decrease in VE levels (CCl4 = 5.67 +/- 1.22 nmol/g tissue; Controls= 13.47 +/- 3.21 nmol/g tissue), caused liver necrosis, increased plasma TNF-alpha levels (CCl4 = 57.36 +/- 13.24 IU/ml; Controls= 7.26 +/- 2.31 IU/ml) and enhanced hepatic mRNA for TNF-alpha (CCl4 = 19.22 +/- 4.38 a.u.; Controls= 0.76 +/- 0.36 a.u.). IRFI 042 (100 mg/kg, 30 min after CCl4 injection) blunted liver MAL (0.32 +/- 0.17 nmol/mg protein), decreased the serum levels of ALT (128.71 +/- 13.23 U/L), and restored the hepatic concentrations of VE (9.52 +/- 3.21 nmol/g tissue), inhibited OH* production (2,3-DHBA= 3.54 +/- 1.31 microM; 2,5-DHBA= 7.37 +/- 2.46 microM), restored the endogenous antioxidant GSH (12.77 +/- 3.73 mmol/g protein) and improved histology. Furthermore IRFI 042 treatment suppressed plasma TNF-alpha concentrations (31.47 +/- 18.25 IU/ml) and hepatic TNF-alpha mRNA levels (11.65 +/- 3.21 a.u.). The acute treatment with vitamin E failed to exert any protective effect against CCl4 -induced hepatotoxicity. These investigations suggest that IRFI 042 treatment may be of benefit during free radical-mediated liver injury.

Alanine Transaminase↗

PubMed2001

Signal transduction events in Chinese hamster ovary cells expressing human CD14; effect of endotoxin desensitization.

Previous studies suggest that endotoxin (LPS) stimulation of CD14 receptors may be coupled to heterotrimeric G proteins. However, characterization of the G protein-coupled signaling pathways is incomplete. Also, specific changes in the transduction pathways occur in a phenomenon known as LPS tolerance or desensitization induced by prior exposure to LPS. In the present study, we examined potential CD14-dependent G protein-coupled signaling events in response to LPS, and changes in signaling in these pathways during LPS desensitization in Chinese Hamster Ovary (CHO) cells. LPS stimulated inhibitory kappa B alpha (IkappaB alpha) degradation and p38 phosphorylation in CHO cells transfected with human CD14 receptor (CHO-CD14), but not in CHO cells transfected with vector only. However, activation of these signaling events diverged early in the signal transduction pathways. Pretreatment with pertussis toxin, which inactivates inhibitor G protein (G alpha i) function, significantly inhibited LPS-induced p38 phosphorylation, but not LPS-induced IkappaB alpha degradation. Mastoparan, a putative G alpha i agonist, synergized with LPS to induce p38 phosphorylation. Thus, LPS stimulation of p38 phosphorylation is, in part, G alpha i coupled, whereas IkappaB alpha degradation is not. In subsequent studies, CHO-CD14 cells were desensitized by prior LPS exposure. LPS-desensitized cells exhibited augmented IkappaB alpha content and were refractory to LPS-induced IkappaB alpha degradation and p38 phosphorylation. Pretreatment with cycloheximide, a protein synthesis inhibitor, prevented the effect of LPS desensitization on augmenting cellular IkappaB alpha content and its refractoriness to LPS-induced degradation. However, cycloheximide pretreatment did not prevent impaired p38 phosphorylation in desensitized cells. IkappaB alpha upregulation in LPS tolerance may occur through increased synthesis and/or induction of protein that suppress IkappaB alpha degradation. The latter protein synthesis-dependent mechanisms may be distinct from mechanismis inhibiting p38 phosphorylation in tolerance. These findings suggest that LPS tolerance induces CD14-dependent signaling alterations in G alpha i-coupled pathways leading to mitogen-activated (MAP) kinase activation as well as G alpha i-independent pathways inducing IkappaB alpha degradation.

Animals↗

PubMed2001

Cardiovascular effects of raloxifene hydrochloride.

Raloxifene hydrochloride binds to the estrogen receptor and shows tissue-selective effects; thus, it belongs to a class of drugs recently described as selective estrogen receptor modulators (SERMs). Tissue selectivity of raloxifene may be achieved through several mechanisms: the ligand structure, interaction of the ligand with different receptor subtypes in various tissues, and intracellular events after ligand binding. Raloxifene has estrogen-agonist effects on bone and lipids and estrogen antagonist effects on the breast and uterus. In addition to its well established effects on osteoporosis, recent preclinical and clinical findings suggest that raloxifene also possesses beneficial effects on the cardiovascular system. These findings indicated that raloxifene may have cardioprotective properties without an increased risk of cancer or other side effects. Raloxifene has been shown to reduce total and low-density lipoprotein cholesterol concentrations in plasma, an effect similar to that produced by estrogens. Unlike estrogens, however, raloxifene does not increase high-density lipoprotein cholesterol and triglyceride levels in plasma. Endothelium is thought to play an important role in the genesis of atherosclerosis. Several lines of evidence suggest that an intervention with endothelial function might influence the progression of coronary disease and the incidence of cardiovascular events. Raloxifene increases the nitric oxide/endothelin-1 ratio, and improves endothelium-dependent vasomotion in post-menopausal women to the same extent as estrogens. Furthermore, in two randomized trials on post-menopausal women raloxifene reduced homocysteine levels, another independent risk factor for the development of cardiovascular disease. Although estrogens remain the drugs of choice in the hormonal therapy of most postmenopausal women, raloxifene may represent and alternative in women who are at risk of coronary artery disease.

Animals↗

PubMed2001

Randomized, double-blind, placebo-controlled study on effects of raloxifene and hormone replacement therapy on plasma no concentrations, endothelin-1 levels, and endothelium-dependent vasodilation in postmenopausal women.

The endothelium is thought to play an important role in the genesis of atherosclerosis, and several lines of evidence suggest that the effect of an intervention on endothelial function might predict its involvement in coronary disease progression and in the rate of cardiovascular events. Estrogen has direct effects on the blood vessel wall, indicating that vascular endothelium may play a key role in the cardiovascular protective effects of hormone replacement therapy (HRT). Raloxifene relaxes coronary arteries in vitro by an estrogen receptor-dependent and NO-dependent mechanism, thus suggesting that this selective estrogen receptor modulator could also have beneficial effects on endothelial function. This study compared the effects of HRT and raloxifene on NO products, endothelin-1 plasma levels, and endothelium-dependent vasodilatation in postmenopausal women. Healthy postmenopausal women (n=90) were enrolled in a double-blind, randomized, placebo-controlled, 6-month trial. Women were randomly assigned to receive continuous HRT (1 mg 17beta-estradiol combined with 0.5 mg norethisterone acetate), raloxifene (60 mg/d), or placebo for 6 months. Flow-mediated endothelium-dependent vasodilation of the brachial artery, plasma NO concentrations, and endothelin levels were measured at baseline and after 6 months of therapy. The mean baseline level of NO breakdown products was 26.5+/-10.7 micromol/L and increased to 36.3+/-11.4 micromol/L after 6 months of treatment with raloxifene. The mean baseline plasma endothelin level was 17.3+/-8.9 pg/mL and decreased to 11.5+/-2.1 pg/mL after 6 months of treatment with the selective estrogen receptor modulator. The mean baseline ratio of NO (breakdown products) to endothelin was also significantly increased at the end of treatment with raloxifene. Postmenopausal women treated with HRT had similar changes in plasma nitrites/nitrates and endothelin levels as well as in the ratio of NO to endothelin. In contrast, these markers of endothelial function did not change in the placebo-treated women. Flow-mediated endothelium-dependent vasodilation of the brachial artery was 8.3+/-2.1% at baseline and increased to 12.3+/-2.1% after 6 months of treatment with raloxifene. HRT also caused a significant and similar increase in flow-mediated endothelium-dependent vasodilation. No change in flow-mediated vasodilation was observed in the participants treated with placebo. We conclude that raloxifene therapy and HRT influence endothelial function and improve flow-mediated endothelium-dependent vasodilation to a comparable extent in healthy postmenopausal women at least after a 6-month treatment period. However, further investigation is warranted to enhance our understanding of the mechanisms of the effect of raloxifene on vascular function and to determine whether its effect on endothelial function may contribute to the reduction in cardiovascular-related morbidity and mortality.

Aged↗

PubMed2001

Inhibition of lipid peroxidation restores impaired vascular endothelial growth factor expression and stimulates wound healing and angiogenesis in the genetically diabetic mouse.

Impaired wound healing is a well-documented phenomenon in experimental and clinical diabetes. Experimental evidence suggests that a defect in vascular endothelial growth factor (VEGF) regulation might be associated with wound-healing disorders. We studied the involvement of lipid peroxidation in the pathogenesis of altered VEGF expression in diabetes-related healing deficit by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ db+/ db+ mice and their normal (db+/+m) littermates. Animals were then randomized to the following treatment: raxofelast (15 mg.kg(-1).day(-1) i.p.), an inhibitor of lipid peroxidation, or its vehicle (DMSO/NaCl 0.9%, 1:1 vol: vol). The animals were killed on different days (3, 6, and 12 days after skin injury), and the wounded skin tissues were used for histological evaluation, for analysis of conjugated dienes (CDs), as an index of lipid peroxidation and wound breaking strength. Furthermore, we studied the time course of VEGF mRNA expression throughout the skin-repair process (3, 6, and 12 days after skin injury), by means of reverse transcriptase-polymerase chain reaction, as well as the mature protein in the wounds. Diabetic mice showed impaired wound healing with delayed angiogenesis, low breaking strength, and increased wound CD content when compared with their normal littermates. In healthy control mice, a strong induction of VEGF mRNA was found between day 3 and day 6 after injury, while no significant VEGF mRNA expression was observed at day 12 after injury. In contrast, VEGF mRNA levels, after an initial increase (day 3), were significantly lower in diabetic mice than in normal littermates, and light induction of VEGF mRNA expression was also present at day 12 after injury. Similarly, the wound content of the angiogenic factor was markedly changed in diabetic mice. Administration of raxofelast did not modify the process of wound repair in normal mice, but significantly improved the impaired wound healing in diabetic mice through the stimulation of angiogenesis, re-epithelization, and synthesis and maturation of extracellular matrix. Moreover, raxofelast treatment significantly reduced wound CD levels and increased the breaking strength of the wound. Lastly, the inhibition of lipid peroxidation restored the defect in VEGF expression during the process of skin repair in diabetic mice and normalized the VEGF wound content. The current study provides evidence that lipid peroxidation inhibition restores wound healing to nearly normal levels in experimental diabetes-impaired wounds and normalizes the defect in VEGF regulation associated with diabetes-induced skin-repair disorders.

Animals↗

PubMed2001

Effect of cross-tolerance between endotoxin and TNF-alpha or IL-1beta on cellular signaling and mediator production.

Endotoxin [lipopolysaccharide (LPS)] tolerance suppresses macrophage/monocyte proinflammatory-mediator production. This phenomenon also confers cross-tolerance to other stimuli including tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta. Post-receptor convergence of signal transduction pathways might occur after LPS, IL-1beta, and TNF-alpha stimulation. Therefore, it was hypothesized that down-regulation of common signaling molecules induces cross-tolerance among these stimuli. LPS tolerance and cross-tolerance were examined in THP-1 cells. Phosphorylation of MAP kinases and degradation of inhibitor kappaBalpha (IkappaBalpha) DNA binding of nuclear factor-kappaB (NF-kappaB), and mediator production were examined. In naive cells, LPS, TNF-alpha, and IL-1beta induced IkappaBalpha degradation, kinase phosphorylation, and NF-kappaB DNA binding. LPS stimulation induced production of TNF-alpha or TxB2 and degradation of IRAK. However, neither TNF-alpha nor IL-1beta induced IRAK degradation or stimulated TNF-alpha or TxB2 production in naive cells. Pretreatment with each stimulus induced homologous tolerance to restimulation with the same agonist. LPS tolerance also suppressed LPS-induced TxB2 and TNF-alpha production. LPS pretreatment induced cross-tolerance to TNF-alpha or IL-1beta stimulation. Pretreatment with TNF-alpha induced cross-tolerance to LPS-induced signaling events and TxB2 production. Although pretreatment with IL-1beta did not induce cross-tolerance to LPS-induced signaling events, it strongly inhibited LPS TNF-alpha and TxB2 production. These data demonstrate that IL-1beta induces cross-tolerance to LPS-induced mediator production without suppressing LPS-induced signaling to MAP kinases or NF-kappaB activation.

Culture Media, Conditioned↗

PubMed2000

Neuroprotective effects of Ginkgo biloba extract in brain ischemia are mediated by inhibition of nitric oxide synthesis.

We studied the effects of pre-treatment (15 days) with oral administration of Ginkgo biloba extract (Ph-Gb 37.5-150 mg/kg) on brain malonildialdehyde (MDA), brain edema, brain nitrite and nitrate and delayed neuronal death following transient cerebral ischemia in the Mongolian gerbil. Survival was not modified, however, pre-treatment with Ginkgo biloba significantly and in a dose-dependent way reduced post-ischemic brain MDA levels and post-ischemic brain edema. Delayed neuronal death in the CA1 of the hippocampus was attenuated by the highest dose of the extract. Increase of nitrite and nitrate was observed after cerebral ischemia in the hippocampus and it was dose-dependently reduced in animals pretreated with Ph-Gb, thus suggesting that neuroprotective effects of Ginkgo biloba may be due to an inhibitory action on nitric oxide formation.

Administration, Oral↗

PubMed2000

The reduction of myocardial damage and leukocyte polymorphonuclear accumulation following coronary artery occlusion by the tyrosine kinase inhibitor tyrphostin AG 556.

We investigated the effects of tyrophostin AG 556, a tyrosine kinase inhibitor, on the phenomenon of leukocyte accumulation during ischaemia and reperfusion of the myocardium. Male anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5 h reperfusion (MI/R). Sham myocardial ischaemia-reperfusion rats (Sham MI/R) were used as controls. Myocardial necrosis, myocardial myeloperoxidase activity (MPO), serum creatinine phosphokinase activity (CPK) serum Tumor Necrosis Factor (TNF-alpha) and Interleukin 6 (IL-6), cardiac intercellular adhesion molecule-1 (ICAM-1) and TNF-alpha expression and myocardial contractility (left ventricle dP/dt(max)) were evaluated. Myocardial ischaemia plus reperfusion in untreated rats produced marked myocardial necrosis, increased serum CPK activity (196.5 +/- 19 U/100 ml, at the end of reperfusion) and myeloperoxidase activity (MPO, a marker of leukocyte accumulation) both in the area-at-risk (4.5 +/- 0.5 U/g/tissue) and in necrotic area (8.2 +/- 1.2 U/g/tissue), reduced myocardial contractility (1,706 +/- 52 mmHg/s, at the end of reperfusion) and induced a marked increase in the serum levels of TNF-alpha (1,950 +/- 97 pg/ml, at 1 h of reperfusion) and IL-6 (998 +/- 16 U/ml, at the end of reperfusion). Finally, myocardial ischaemia-reperfusion injury also increased cardiac mRNA for TNF-alpha and ICAM-1 in the myocardium-at risk. Tyrphostin AG 556 (0.5, 1 and 2 mg/kg subcutaneously 5 min after the onset of reperfusion) lowered myocardial necrosis and myeloperoxidase activity in the area-at-risk (1.5 +/- 0.2 U/g/tissue, following the highest dose) and in necrotic area (2.9 +/- 0.3 U/g/tissue following the highest dose), decreased serum CPK activity (96 +/- 9 U/100 ml, at the end of reperfusion), lowered serum TNF-alpha and IL-6, increased myocardial contractility (2,096 +/- 88 mmHg s, at the end of reperfusion) and reduced cardiac mRNA levels for TNF-alpha and ICAM-1. The present data suggest that tyrosine kinase inhibitors protect against myocardial ischaemia-reperfusion injury by reducing leukocyte accumulation to the ischaemic myocardium.

Animals↗

PubMed2000

IRFI 042, a novel dual vitamin E-like antioxidant, inhibits activation of nuclear factor-kappaB and reduces the inflammatory response in myocardial ischemia-reperfusion injury.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a ubiquitous rapid response transcription factor involved in inflammatory reactions which exerts its effect by expressing cytokines, chemokines, and cell adhesion molecules. Oxidative stress causes NF-kappaB activation. IRFI 042 is a novel dual vitamin E-like antioxidant and we, therefore, investigated its ability to protect the heart from oxidative stress and to halt the inflammatory response in a model of myocardial ischaemia-reperfusion injury. METHODS: Anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5-h reperfusion (MI/R). Sham myocardial ischaemia rats (sham-operated rats) were used as controls. Myocardial necrosis, cardiac output, cardiac and plasma vitamin E levels, myocardial malondialdehyde (MAL), cardiac SOD activity and elastase content (an index of leukocyte infiltration), hydroxyl radical (OH&z.ccirf;) formation, cardiac amount of mRNA codifying for ICAM-1 (evaluated by the means of reverse transcriptase polymerase chain reaction) and ICAM-1 immunostaining in the at-risk myocardium were investigated. NF-kappaB activation and the inhibitory protein of NF-kappaB, I-kappaBalpha, were also studied in at-risk myocardium by using electrophoretic mobility shift assay (EMSA) and Western blot analysis, respectively. RESULTS: The ischaemia-reperfusion model produced wide heart necrosis (area at risk-necrotic area=52+/-5%; necrotic area-left ventricle=28+/-3%), increased cardiac MAL, an index of lipid peroxidation (area at risk=62.5+/-3.9 nmol/g tissue; necrotic area=80.3+/-5.1 nmol/g tissue), induced tissue neutrophil infiltration, and caused a marked decrease in endogenous antioxidants. Furthermore, myocardial ischaemia plus reperfusion caused in the area at risk peak message for ICAM-1 at 3 h of reperfusion and increased cardiac ICAM-1 immunostaining at 5 h of reperfusion. NF-kappaB activation was also evident at 0.5 h of reperfusion and reached its maximum at 2 h of reperfusion. I-kappaBalpha was markedly decreased at 45 min of occlusion; peak reduction was observed at 1 h of reperfusion and thereafter it was gradually restored. Intraperitoneal administration of IRFI 042 (5, 10, 20 mg/kg, 5 min after reperfusion) reduced myocardial necrosis expressed as a percentage either of the area at risk (18+/-4%) or the total left ventricle (11+/-2%), and improved cardiac output. This treatment also limited membrane lipid peroxidation in the area at risk (MAL=14.3+/-2.5 nmol/g tissue) and in the necrotic area (MAL=26.5+/-3.7 nmol/g tissue), restored the endogenous antioxidants vitamin E and superoxide dismutase, and inhibited detrimental hydroxyl radical formation. Finally, IRFI 042 blocked the activation of NF-kappaB, reduced cardiac ICAM-1 expression, and blunted tissue elastase content, an index of leukocytes accumulation at the site of injury. CONCLUSIONS: Our data suggest that IRFI 042 is cardioprotective during myocardial infarction by limiting reperfusion-induced oxidative stress and by halting the inflammatory response.

Analysis of Variance↗

PubMed2000

Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats.

BACKGROUND: We investigated the effect of genistein, a phytoestrogen derived from a soy diet with a flavonoid chemical structure, on endothelial dysfunction induced by estrogen deficiency in rats. METHODS: Female mature Sprague-Dawley rats were subjected to a bilateral ovariectomy (OVX rats). Sham-operated animals (Sham OVX rats) were used as controls. Three weeks after surgery animals were randomized to the following treatments: genistein (0.2 mg/kg/day, s.c. for 4 weeks), 17 beta-estradiol (20 micrograms/kg/day, s.c. for 4 weeks) or their respective vehicles. Mean arterial blood pressure (MAP), heart rate (HR), total plasma cholesterol, plasma estradiol, plasma genistein levels and uterine weights were studied. Furthermore, we investigated acetylcholine (ACh 10 nM-10 microM) and sodium nitroprusside: (SN 15-30 nM) induced relaxation of aortic rings as well as NG-L-arginine (L-NMA: 10-100 microM) induced vasoconstriction in phenylephrine precontracted aortic segments and calcium-dependent nitric oxide synthase (cNOS) activity in homogenates of lungs taken from both sham OVX and OVX rats. RESULTS: Untreated OVX rats had, compared with sham OVX animals, unchanged body weight, MAP, HR and plasma cholesterol. In contrast ovariectomy impaired endothelial responses, blunted L-NMA induced contraction (L-NMA 100 microM: Sham OVX = 2.1 +/- 0.2 g/mg tissue; OVX = 1.7 +/- 0.4 g/mg tissue) and reduced cNOS activity. Treatment with 17 beta-estradiol increased the hormone plasma levels, reverted the endothelial dysfunction and increased cNOS activity in lung homogenates. Genistein supplementation enhanced the circulating levels of the phytoestrogen and affected NOS activity and endothelial dysfunction to the same extent. CONCLUSIONS: Our data suggest that genistein and 17 beta-estradiol show overlapping effects on experimental endothelial dysfunction.

Acetylcholine↗