The influence of the polar head and the hydrophobic chain on the skin penetration enhancement effect of poly(ethylene glycol) derivatives.

The effect of a homologue series of nonionic surfactants, namely poly(ethylene glycol) (PEG) fatty acid esters, differing in oxyethylene (PEG 8, PEG 12, and PEG 40) and fatty acid (stearate, mono and di-laurate, and mono and di-oleate) chain lengths, on in vitro skin permeability of ketoprofen (KTP) vehicled in plasters was investigated. The drug diffusion through hairless mouse skin as well as the effect of the surfactant type and strength was studied by Franz diffusion cells and ATR-FTIR spectroscopy. The use of PEG stearate series revealed that the surfactant with the largest polar head, namely PEG 40, was ineffective in enhancing the skin permeation of KTP, independently of the plaster concentrations. The effect of the hydrophobic chain was investigated only by using the shortest oxyethylene chains. The experimental results revealed that the oxyethylene chain length of surfactants appeared to be more influent than the alkyl chain. The prediction of the absorption enhancing capability of these PEG derivatives appeared related to the vehicle other than the proper combination of the number of ethylene oxide groups and alkyl groups.

The carbonyl scavenger carnosine ameliorates dyslipidaemia and renal function in Zucker obese rats.

The metabolic syndrome is a risk factor that increases the risk for development of renal and vascular complications. This study addresses the effects of chronic administration of the endogenous dipeptide carnosine (ß-alanyl-L-histidine, L-CAR) and of its enantiomer (ß-alanyl-D-histidine, D-CAR) on hyperlipidaemia, hypertension, advanced glycation end products, advanced lipoxidation end products formation and development of nephropathy in the non-diabetic, Zucker obese rat. The Zucker rats received a daily dose of L-CAR or D-CAR (30 mg/kg in drinking water) for 24 weeks. Systolic blood pressure was recorded monthly. At the end of the treatment, plasma levels of triglycerides, total cholesterol, glucose, insulin, creatinine and urinary levels of total protein, albumin and creatinine were measured. Several indices of oxidative/carbonyl stress were also measured in plasma, urine and renal tissue. We found that both L- and D-CAR greatly reduced obese-related diseases in obese Zucker rat, by significantly restraining the development of dyslipidaemia, hypertension and renal injury, as demonstrated by both urinary parameters and electron microscopy examinations of renal tissue. Because the protective effect elicited by L- and D-CAR was almost superimposable, we conclude that the pharmacological action of L-CAR is not due to a pro-histaminic effect (D-CAR is not a precursor of histidine, since it is stable to peptidic hydrolysis), and prompted us to propose that some of the biological effects can be mediated by a direct carbonyl quenching mechanism.

Selective estrogen receptor-alpha agonist provides widespread heart and vascular protection with enhanced endothelial progenitor cell mobilization in the absence of uterotrophic action.

The beneficial effects of estrogens on the cardiovascular system are associated with adverse effects on reproductive tissues. On the basis of previous work indicating a major role for estrogen receptor (ER)-alpha in maintaining cardiovascular health, we evaluated the tissue selectivity of the ER alpha-selective agonist propyl pyrazole triol (PPT) compared with 17beta-estradiol (E2) in vivo. Four weeks postovariectomy, equimolar doses of PPT and E2 were administered to rats in subcutaneous implants for 5 d. Both treatments restored rapid vasorelaxation of aortic tissue to estrogenic agents and prevented coronary hyperresponsiveness to angiotensin II in isolated heart preparations. Accordingly, multiple endpoints of myocardial ischemia-reperfusion injury exacerbated by ovariectomy returned to baseline following treatment. These protective effects were linked to increased in vivo levels of endothelial progenitor cells (EPCs). Human EPC function was enhanced in vitro after PPT treatment. In sharp contrast to E2, PPT treatment had no effect on uterine weight and histomorphology except for vessel density, and failed to up-regulate classic estrogen target genes. Dissection of the effects on vascular reactivity and uterine morphology was also observed following increased exposure to PPT at a higher dose for longer time. These data provide the first in vivo evidence for tissue-specific ER alpha activation. By conferring cardiovascular protection dissected from unwanted uterotrophic effects, ER alpha-selective agonists may represent a potential safer alternative to natural hormones.

Changes in sympathetic activity in prion neuroinvasion.

Prion diseases are neurodegenerative diseases affecting humans and animals in which the infectious agent or prion is PrP(res), a protease-resistant conformer of the cell protein PrP. The natural transmission route of prion diseases is peripheral infection, with the lymphoreticular system (LRS) and peripheral nerves being involved in animal models of scrapie neuroinvasion and human prion diseases. To study the effects of PrP neuroinvasion on sympathetic nerve function, we measured plasma catecholamine levels, blood pressure, heart rate, and PrP tissue levels in intraperitoneally or intracerebrally infected mice. The results indicate a specific alteration in sympathetic nerve function because the levels of noradrenaline (but not adrenaline) were increased in the animals infected peripherally (but not in those infected intracerebrally) and correlated with increased blood pressure. These findings confirm that prion neuroinvasion uses the sympathetic nervous system to spread from the periphery to the central nervous system after invading the LRS.

Synthesis of human renalase1 in Escherichia coli and its purification as a FAD-containing holoprotein.

Renalase is a protein ubiquitous in vertebrates, which has been proposed to modulate blood pressure and heart rate, and whose downregulation might result in hypertension. Despite its potential relevance for human health, the biochemical characterization of renalase is still lacking, possibly due to difficulties in obtaining it in recombinant form. By expressing two different gene constructs, we found that the major isoform of human renalase, renalase1, is mainly produced in Escherichia coli in inclusion bodies. However, by optimizing the expression conditions, significant amounts of soluble products were obtained. Both soluble renalase forms have been purified to homogeneity exploiting their N-terminal His-tag. Linking of the protein of interest to the SUMO protein did not improve solubility, but yielded untagged renalase1 after proteolytic processing of the fusion product. The two recombinant renalase forms displayed the same molecular properties. They bind equimolar amounts of FAD and appear to be correctly folded by various criteria. The procedures for the production and isolation of recombinant renalase1 here reported are expected to boost the much awaited biochemical studies on this remarkable protein.

A sensitive and specific precursor ion scanning approach in liquid chromatography/electrospray ionization tandem mass spectrometry to detect methylprednisolone acetate and its metabolites in rat urine.

A new, simple, sensitive and specific liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method in precursor ion scanning (PIS) mode has been developed for the rapid detection of methylprednisolone acetate (MPA) and its metabolites in rat urine. A suitable product ion specific for methylprednisolone (MP) and MPA was selected after a fragmentation study on 20 (cortico)steroids at different collision energies (5-40 eV). Urine samples were simply treated with acetonitrile then dried in a SpeedVac system. The method was validated and compared with other PIS methods for detecting corticosteroids in human urine. It was more sensitive, with limit of detection (LOD) and lower limit of quantitation (LLOQ), respectively, of 5 and 10 ng mL(-1). The method was applied for the analysis of rat urine collected before and after (24, 48, 72 h) intra-articular (IA) injection of a marketed formulation of MPA (Depo-Medrol(R)). MS/MS acquisitions were taken at different collision energies for the precursor ions of interest, detected in PIS mode, to verify the MP-related structure. Six different metabolites were detected in rat urine, and their chemical structures were assigned with a computational study.

Pretreatment with tetrandrine has protective effects against isoproterenol-induced myocardial infarction in rabbits.

Tetrandrine, the active principle of Stephania tetrandra radix extracts, has broad pharmacological activity, including effects on the cardiovascular system: it has been shown to reduce the size of acute myocardial infarction in rats undergoing coronary vessel ligation and to improve heart lesions in the constriction/reperfusion model by means of mechanisms involving peroxidation, calcium antagonism and coagulation. The aim of this study was to investigate whether tetrandrine has anti-infarction, antioxidant and anticoagulant effects in rabbits treated with isoproterenol, a drug capable of causing peroxide generation, calcium overload and coagulation alterations, and inducing myocardial infarction. The results showed that pretreatment with tetrandrine protects against the myocardial injuries caused by isoproterenol. It counteracted the appearance of myocardial necrotic lesions and ischemic electrocardiographic modifications, such as ST segment alterations, prevented the appearance of the plasma cardiac necrosis markers c-troponin I and myoglobin, lowered malondialdehyde levels, and prolonged partial thromboplastin time. The protective effects of tetrandrine can be attributed to its antioxidant action in lowering peroxide levels and its ability to counteract coagulating activity. Tetrandrine seems to offer full protection against myocardial infarction experimentally induced by the non-invasive treatment of rabbits with isoprotenerol.

Interaction between ticlopidine or warfarin or cardioaspirin with a highly standardized deterpened Ginkgo biloba extract (VR456) in rat and human.

Ginkgo biloba is available in Europe as an over-the-counter drug and it is reported to cause hemorrhage when co-administered with other anti-platelet agents. We set out to study the interactions of ticlopidine with Ginkgo biloba extract or VR456, a new highly standardized deterpened extract from Ginkgo biloba leaves. Male Wistar rats were used to study the effects of ticlopidine (50-100 mg/kg/day), given alone and in combination for 5 days with Ginkgo biloba extract (50 mg/kg/day) or VR456 (50 mg/kg/day), on bleeding time and ex vivo ADP-induced platelet aggregation measurements. In addition, human studies were performed with the compounds under investigation. Combined treatment of ticlopidine and undeterpened Ginkgo biloba extract increased anti-platelet effect and prolonged the bleeding time in the rat. On the contrary, the combination treatment of ticlopidine and VR456 increased anti-platelet effect but did not prolong bleeding time. Moreover, daily administration of 360 mg of VR456 for 14 days to ticlopidine-treated humans did not highlight any unwanted effect and did not alter PT/INR and PTT parameters. Same results have been also obtained in warfarin or in cardioaspirin-treated patients. These data point out the clear role played by the terpenoid, PAF-antagonist fraction of Ginkgo biloba extract in affecting bleeding risk in anticoagulant-treated subjects and suggest VR456 as a possible option treatment in geriatric people subjected to anticoagulant treatment where the use of standard Ginkgo biloba extracts are discouraged.

Effect of hydrogen sulphide-donating sildenafil (ACS6) on erectile function and oxidative stress in rabbit isolated corpus cavernosum and in hypertensive rats.

OBJECTIVE To study the effect of the H(2)S-donating derivative of sildenafil (ACS6) compared to sildenafil citrate and sodium hydrosulphide (NaHS) on relaxation, superoxide formation and NADPH oxidase and type 5 phosphodiesterase (PDE5) expression in isolated rabbit cavernosal tissue and smooth muscle cells (CSMCs), and in vivo on indices of oxidative stress induced with buthionine sulphoximine (BSO). MATERIALS AND METHODS Relaxation was studied in an organ bath in response to carbachol and after incubation with interleukin-1beta for 12 h. CSMCs were incubated with tumour-necrosis factor-alpha or the thromboxane A(2) (TXA(2)) analogue, U46619, with or with no sildenafil citrate, ACS6 or NaHS for 16 h. Superoxide formation and the expression of p47(phox) (an active subunit of the NADPH oxidase complex) and PDE5 protein was then assessed using Western blotting. Rats were also treated with BSO (with or with no sildenafil citrate or ACS6) for 7 days; cavernosal cGMP, cAMP, glutathionine and plasma TXA(2) and 8-isoprostane F(2alpha) was measured by enzyme-linked immunosorbent assay. RESULTS ACS6 and sildenafil citrate relaxed cavernosal smooth muscle equipotently; NaHS alone had little effect at up to 100 microm. The formation of superoxide and expression of p47(phox) and PDE5 was reduced by ACS6, sildenafil citrate and NaHS (order of potency: ACS6 > sildenafil citrate > NaHS). The effects of ACS6 were blocked by inhibitors of protein kinase A (PKA) and PKG. In rats treated with BSO, both ASC6 and sildenafil citrate reduced the increased plasma levels of TXA(2) and 8-isoprostane F(2alpha) but increased cGMP, cAMP and glutathionine levels in corpus cavernosum. CONCLUSIONS By virtue of a dual action on PKA and PKG activation, ACS6 not only promotes erection, acutely, but might also have a long-term beneficial effect through inhibition of oxidative stress and downregulation of PDE5.

Anti-ischemic activity and endothelium-dependent vasorelaxant effect of hydrolysable tannins from the leaves of Rhus coriaria (Sumac) in isolated rabbit heart and thoracic aorta.

The aim of this work was to investigate the cardioprotective activity of hydrolysable gallotannins from Rhus coriaria L. leaves extract (RCLE) in isolated rabbit heart preparations, submitted to low-flow ischemia/reperfusion damage. RCLE induces a dose-dependent normalization of coronary perfusion pressure (CPP), reducing left ventricular contracture during ischemia, and improving left ventricular developed pressure and the maximum rate of rise and fall of left ventricular pressure at reperfusion. Creatinine kinase (CK) and lactate dehydrogenase (LDH) outflow were significantly reduced during reperfusion. In parallel there was a rise in the release of the cytoprotective 6-ketoprostaglandin F (1alpha) (6-keto-PGF (1alpha)) and a decrease of tumor necrosis factor-alpha (TNF-alpha), both significant only at the highest RCLE concentrations (150-500 microg/mL). The vasorelaxant activity of RCLE was studied in isolated rabbit aorta rings precontracted with norepinephrine (NE) with and without endothelium. The vasorelaxation induced by RCLE was predominantly endothelium-dependent as demonstrated by the loss of RCLE vasorelaxant ability in i) de-endothelized rings and ii) in intact aortic rings after pretreatment with NG-monomethyl- L-arginine (L-NMMA) and 1 H-[1.2.4]oxadiazolo[4.3- A]quinoxalin-1-one (ODQ). The inhibition of vasorelaxation in intact rings by indomethacin (INDO) demonstrates the ability of RCLE to modulate the coronary endothelium cyclooxygenase (COX) pathway. The K-ATP channel antagonist glibenclamide (GLIB) was ineffective. The antioxidant activity of RCLE, investigated in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) model and in living cell systems (rat erythrocytes), was stronger than that of gallic acid, ascorbic acid and trolox. The structure of its main bioactive constituents, profiled by HPLC-ESI-HR-S, comprised a mixture of polygalloylated D-glucopyranose with different degrees of galloylation and 3- O-methylgallic acid. The cardiovascular protective effect of RCLE seems to be due to an interplay of different factors: COX pathway activation, TNF-alpha inhibition, endothelial nitric oxide synthase (eNOS) activation, and free radical and ROS scavenging.

Plasma cardiac necrosis markers C-troponin I and creatine kinase, associated with increased malondialdehyde levels, induced in rabbits by means of 5-aminolevulinic acid injection.

A number of papers have described high levels of 5-aminolevulinic acid in cases of heart damage due to acute myocardial infarction, acute intermittent porphyria or chronic kidney failure, but it is not known whether the heart damage is directly associated with 5-aminolevulinic acid. The aim of this study was to verify whether such an association exists by injecting rabbits with 5-aminolevulinic acid and searching for the appearance of cardiac necrosis markers and histological heart alterations, and investigate whether the cardiotoxic activity of 5-aminolevulinic acid may involve peroxidation by seeking the presence of the peroxide marker malondialdehyde. The administration of 5-aminolevulinic acid led to the appearance of c-troponin I and creatine kinase, induced histological heart alterations and increased the malondialdehyde levels. The plasma levels of malondialdehyde and cardiac necrosis markers were also measured after the injection of 5-aminolevulinic acid in combination with the daunorubicin agent inducing peroxidation. The combined administration very significantly increased the plasma levels of 5-aminolevulinic acid, malondialdehyde, and the cardiac necrosis markers c-troponin I and creatine kinase. It therefore seems that there is a close relationship between altered 5-aminolevulinic acid levels, malondialdehyde and cardiac necrosis markers, which is attributable to the capacity of 5-aminolevulinic acid to generate toxic oxygen species that damage the heart. High plasma 5-aminolevulinic acid levels should be considered a factor contributing to cardiotoxicity and to the appearance of cardiac necrosis markers.

Anti-inflammatory and cardioprotective activities of synthetic high-density lipoprotein containing apolipoprotein A-I mimetic peptides.

Apolipoprotein A-I (apoA-I) mimetic peptides may represent an alternative to apoA-I for large-scale production of synthetic high-density lipoproteins (sHDL) as a therapeutic agent. In this study, the cardioprotective activity of sHDL made with either L37pA peptide or its d-stereoisomer, D37pA, was compared to sHDL made with apoA-I. The peptides were reconstituted with palmitoyl-oleoyl-phosphatidylcholine, which yielded sHDL particles comparable to apoA-I sHDL in diameter, molecular weight, and alpha-helical content. Pretreatment of endothelial cells with either peptide sHDL reduced tumor necrosis factor alpha-stimulated vascular cell adhesion molecule-1 expression to the same extent as apoA-I sHDL. In an isolated rat heart model of ischemia/reperfusion (I/R) injury, L37pA and D37pA sHDL significantly reduced postischemic cardiac contractile dysfunction compared to the saline control, as indicated by a 49.7 +/- 6.4% (L37pA; P < 0.001) and 53.0 +/- 9.1% (D37pA; P < 0.001) increase of left ventricular-developed pressure (LVDP) after reperfusion and by a 45.4 +/- 3.4% (L37pA; P < 0.001) and 49.6 +/- 2.6% (D37pA; P < 0.001) decrease of creatine kinase (CK) release. These effects were similar to the 51.3 +/- 3.0% (P < 0.001) increase of LVDP and 51.3 +/- 3.0 (P < 0.001) reduction of CK release induced by apoA-I sHDL. Consistent with their cardioprotective effects, all three types of sHDL particles mediated an approximate 20% (P < 0.001) reduction of cardiac tumor necrosis factor alpha (TNFalpha) content and stimulated an approximate 35% (P < 0.05) increase in postischemic release of prostacyclin. In summary, L37pA and D37pA peptides can form sHDL particles that retain a similar level of protective activity as apoA-I sHDL on the endothelium and the heart; thus, apoA-I mimetic peptides may be useful therapeutic agents for the prevention of cardiac I/R injury.

Combined simvastatin-manidipine protect against ischemia-reperfusion injury in isolated hearts from normocholesterolemic rats.

This study investigated whether oral simvastatin and manidipine interact in protecting the perfused rat heart from ischemia-reperfusion damage. Simvastatin (0.3 to 3 mg/kg) and manidipine (1 to 10 mg/kg) were given orally singly or together to normocholesterolemic rats once a day for seven consecutive days. At the end of treatment, systolic blood pressure and heart rate were measured in conscious rats, and the lipid profile and other biochemical markers, such as thromboxane B(2), nitrite/nitrates and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)) were determined in the plasma. Hearts were then isolated, perfused with Krebs-Henseleit, and subjected to low flow ischemia-reperfusion injury. Post-ischemic recovery of left ventricular function was measured as left ventricular developed pressure and left ventricular end-diastolic pressure. Creatine kinase, lactate dehydrogenase, tumor necrosis factor-alpha and 6-keto-PGF(1alpha) were measured in the heart effluents. In conscious animals, simvastatin alone increased plasma 6-keto-PGF(1alpha) release while manidipine alone reduced systolic blood pressure with a slight sympathetic reflex increase in heart rate, and increased plasma nitrite/nitrates. The combined treatment produced the same effects, but significantly more marked, and accompanied by a significant reduction of thromboxane B(2). Combined treatment was also significantly more effective than the single drugs in protecting the hearts from ischemia-reperfusion injury, with inhibition of creatine kinase, lactate dehydrogenase and tumor necrosis factor-alpha, and enhancement of 6-keto-PGF(1alpha) during reperfusion. These data show that simvastatin and manidipine interact positively in protecting the rat heart from ischemia-reperfusion injury, possibly through increased prostaglandin and nitric oxide formation by the vascular endothelial cells.

A carnosine analog mitigates metabolic disorders of obesity by reducing carbonyl stress.

Sugar- and lipid-derived aldehydes are reactive carbonyl species (RCS) frequently used as surrogate markers of oxidative stress in obesity. A pathogenic role for RCS in metabolic diseases of obesity remains controversial, however, partly because of their highly diffuse and broad reactivity and the lack of specific RCS-scavenging therapies. Naturally occurring histidine dipeptides (e.g., anserine and carnosine) show RCS reactivity, but their therapeutic potential in humans is limited by serum carnosinases. Here, we present the rational design, characterization, and pharmacological evaluation of carnosinol, i.e., (2S)-2-(3-amino propanoylamino)-3-(1H-imidazol-5-yl)propanol, a derivative of carnosine with high oral bioavailability that is resistant to carnosinases. Carnosinol displayed a suitable ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile and was determined to have the greatest potency and selectivity toward α,ß-unsaturated aldehydes (e.g., 4-hydroxynonenal, HNE, ACR) among all others reported thus far. In rodent models of diet-induced obesity and metabolic syndrome, carnosinol dose-dependently attenuated HNE adduct formation in liver and skeletal muscle, while simultaneously mitigating inflammation, dyslipidemia, insulin resistance, and steatohepatitis. These improvements in metabolic parameters with carnosinol were not due to changes in energy expenditure, physical activity, adiposity, or body weight. Collectively, our findings illustrate a pathogenic role for RCS in obesity-related metabolic disorders and provide validation for a promising new class of carbonyl-scavenging therapeutic compounds rationally derived from carnosine.

Anti-inflammatory and gastrointestinal effects of a novel diclofenac derivative.

S-diclofenac (2-[(2,6-dichlorophenyl)amino]benzeneacetic acid 4-(3H-1,2,dithiol-3-thione-5-yl)phenyl ester; ACS 15) is a novel molecule comprising a hydrogen sulfide (H2S)-releasing dithiol-thione moiety attached by an ester linkage to diclofenac. S-diclofenac administration inhibited lipopolysaccharide-induced inflammation (as evidenced by reduced lung and liver myeloperoxidase activity) and caused significantly less gastric toxicity than diclofenac. S-diclofenac did not affect blood pressure or heart rate of the anesthetized rat. S-diclofenac administration downregulated expression of genes encoding enzymes which synthesize nitric oxide, prostanoids, and H2S; reduced plasma IL-1beta/TNF-alpha; and elevated plasma IL-10. Reduced liver NF-kappaB p65 and AP-1/c-fos DNA-binding activity was also observed. These effects were mimicked in large part by a combination of diclofenac plus an H2S-releasing moiety (ADT-OH). Incubation of S-diclofenac (100 microM) with rat plasma or liver homogenate caused a time-dependent release of H2S, which was inhibited by sodium fluoride (10 mM). Administration of S-diclofenac (47.2 micromol/kg, i.p.) to conscious rats significantly increased plasma H2S concentration (at 45 min and 6 h). We propose that H2S release from S-diclofenac in vivo contributes to the observed effects.

Synthetic high density lipoproteins for the treatment of myocardial ischemia/reperfusion injury.

Synthetic high density lipoproteins (sHDL) are discoidal lipoprotein particles made of an apolipoprotein and a phospholipid, which mimic most, if not all, of the atheroprotective properties of plasma HDL, including stimulation of reverse cholesterol transport (RCT), prevention of endothelial dysfunction, and inhibition of lipid oxidation. sHDL are currently under development as a novel treatment for atherosclerotic cardiovascular disease. A number of preclinical studies have demonstrated the ability of single or multiple injections of sHDL to induce the regression of atherosclerotic plaques and prevent arterial restenosis. In the first phase II trial in patients with acute coronary syndromes, a short-term treatment with sHDL containing the disulfide-linked dimer of the apolipoprotein A-IMilano variant (A-IM/A-IM) caused a remarkable reduction of atheroma volume. sHDL also display a direct cardioprotective activity in ex vivo and in vivo models of myocardial ischemia/reperfusion (I/R) injury, and may become a useful adjunctive therapy to improve clinical outcomes in patients with acute coronary syndromes or undergoing coronary procedures.

Asymmetric dimethylarginine (ADMA) induces vascular endothelium impairment and aggravates post-ischemic ventricular dysfunction in rats.

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide (NO) inhibitor recognized as an independent risk factor for endothelial dysfunction and coronary heart diseases. This study investigated whether ADMA (10 mg/kg day for 14 days) affected endothelial function and aggravated post-ischemic ventricular dysfunction in the perfused rat heart. Systolic blood pressure and heart rate, plasma levels of ADMA and nitrite/nitrate were measured in vehicle- and ADMA-treated rats. Perfused hearts were submitted to global ischemia-reperfusion and vascular endothelial dysfunction was examined with angiotensin II in coronary vessels and aortic rings. Endothelial NO synthase (eNOS) and angiotensin-converting enzyme (ACE) mRNA expression in aortic and cardiac tissues were measured. ADMA-treated rats had higher systolic blood pressure (1.3-fold, P<0.01) and slower heart rate (16%, P<0.05) than controls. Plasma ADMA rose (1.9-fold, P<0.01) and nitrite/nitrate concentration decreased 59% (P<0.001). Ventricular contraction (stiffness) increased significantly, with worsening of post-ischemic ventricular dysfunction. In preparations from ADMA-treated rats the coronary vasculature's response to angiotensin II was almost doubled (P<0.01) and the maximal vasorelaxant effect of acetylcholine in aortic rings was significantly lower than in preparations from vehicle-treated rats. In cardiac and aortic tissues eNOS mRNA and ACE mRNA levels were similar in controls and ADMA-treated rats. The increased plasma levels of ADMA presumably cause endothelial dysfunction because of a deficiency in NO production, which also appears involved in the aggravation of myocardial ischemia-reperfusion injury.

Regulatory landscape of AGE-RAGE-oxidative stress axis and its modulation by PPARγ activation in high fructose diet-induced metabolic syndrome.

BACKGROUND: The AGE-RAGE-oxidative stress (AROS) axis is involved in the onset and progression of metabolic syndrome induced by a high-fructose diet (HFD). PPARγ activation is known to modulate metabolic syndrome; however a systems-level investigation looking at the protective effects of PPARγ activation as related to the AROS axis has not been performed. The aim of this work is to simultaneously characterize multiple molecular parameters within the AROS axis, using samples taken from different body fluids and tissues of a rat model of HFD-induced metabolic syndrome, in the presence or absence of a PPARγ agonist, Rosiglitazone (RGZ). METHODS: Rats were fed with 60% HFD for the first half of the treatment duration (21 days) then continued with either HFD alone or HFD plus RGZ for the second half. RESULTS: Rats receiving HFD alone showed metabolic syndrome manifestations including hypertension, dyslipidemia, increased glucose levels and insulin resistance, as well as abnormal kidney and inflammatory parameters. Systolic blood pressure, plasma triglyceride and glucose levels, plasma creatinine, and albuminuria were significantly improved in the presence of RGZ. The following molecular parameters of the AROS axis were significantly upregulated in our rat model: carboxymethyl lysine (CML) in urine and liver; carboxyethyl lysine (CEL) in urine; advanced glycation end products (AGEs) in plasma; receptor for advanced glycation end products (RAGE) in liver and kidney; advanced oxidation protein products (AOPP) in plasma; and 4-hydroxynonenal (HNE) in plasma, liver, and kidney. Conversely, with RGZ administration, the upregulation of AOPP and AGEs in plasma, CML and CEL in urine, RAGE in liver as well as HNE in plasma and liver was significantly counteracted/prevented. CONCLUSIONS: Our data demonstrate (i) the systems-level regulatory landscape of HFD-induced metabolic syndrome involving multiple molecular parameters, including HNE, AGEs and their receptor RAGE, and (ii) attenuation of metabolic syndrome by PPARγ modulation.

Inhibition of MMP-2 activation and release as a novel mechanism for HDL-induced cardioprotection.

High density lipoproteins (HDL) protect the heart against ischemia/reperfusion (I/R) injury, and matrix metalloproteinase-2 (MMP-2) directly contributes to cardiac contractile dysfunction after I/R. To investigate the possible involvement of MMP-2 inhibition in HDL-mediated cardioprotection, isolated rat hearts underwent 20 min of low-flow ischemia and 30 min of reperfusion. Plasma-derived and synthetic HDL attenuated the I/R-induced cardiac MMP-2 activation and release in a dose-dependent way. The attenuation of I/R-induced MMP-2 activation by HDL correlated with the reduction of post-ischemic contractile dysfunction and cardiomyocyte necrosis. These results indicate prevention of MMP-2 activation as a novel mechanism for HDL-mediated cardioprotection.

Angiotensin II type 1 receptor antagonism improves endothelial vasodilator function in L-NAME-induced hypertensive rats by a kinin-dependent mechanism.

OBJECTIVE: This study was designed to investigate the ability of a chronic blockade of angiotensin II type 1 receptors with losartan to reverse the endothelial dysfunction present in N-nitro-L-arginine methyl ester (L-NAME)-treated hypertensive rats and the possible dependence of this effect on bradykinin B2-receptor activation. METHODS: Rats treated with L-NAME alone (60 mg/kg per day for 8 weeks) or with L-NAME + losartan, L-NAME + icatibant (a bradykinin B2-receptor antagonist) and L-NAME + losartan + icatibant were studied. Losartan, icatibant or losartan + icatibant were co-administered with L-NAME during the last 4 weeks of the experiment. Endothelial nitric oxide synthase gene expression in aortic tissues, plasma nitrite/nitrate concentrations, the relaxant effect of acetylcholine on norepinephrine-precontracted aortic rings and 6-keto-PGF1alpha release from aortic rings were used as markers of the endothelial function. RESULTS: Rats treated with L-NAME alone and L-NAME + icatibant showed, as compared with untreated animals, a clear-cut increase in systolic blood pressure and a decrease of all the markers of endothelial function evaluated. In L-NAME-rats, administration of losartan reduced the systolic blood pressure and restored endothelial nitric oxide synthase gene expression, plasma nitrite/nitrate levels, the relaxant activity of acetylcholine on aortic rings and the generation of 6-keto-PGF1alpha from the aortic tissues. Co-administration of icatibant with losartan blunted the stimulatory effect of losartan on the markers of endothelial function evaluated. CONCLUSION: These results demonstrated that losartan is capable of reversing the endothelial vasodilator dysfunction in L-NAME-induced hypertensive rats, and that the beneficial effect of losartan is mediated by bradykinin B2-receptor activation.