Ophthalmic wild olive (ACEBUCHE) oil nanoemulsions exert oculoprotective effects against oxidative stress induced by arterial hypertension.

Oxidative stress plays a key role in several systemic and ocular diseases, including hypertensive eye diseases. In this context, we previously showed that oral administration of wild olive (acebuche, ACE) oil from Olea europaea var. sylvestris can counteract ocular damage secondary to arterial hypertension by modulating excess reactive oxygen species (ROS) produced by the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Therefore, this work describes the development of an ACE oil-based formulation for ocular administration as a local therapy to counteract hypertension-related oxidative damage. Specifically, ACE oil nanoemulsions (NEs) were successfully produced and characterized, exhibiting appropriate features for ophthalmic administration, including a nanometer size (<200 nm), moderate negative ZP, adequate osmolality and pH, and colloidal stability in biorelevant fluids. Likewise, the NEs presented a shear thinning behavior, especially convenient for ocular instillation. In vivo evaluation was performed through either intravitreal injection or topical ophthalmic administration in mice with hypertension induced via administration of Nω-nitro-L-arginine-methyl-ester (L-NAME). Both routes of administration reduced hypertensive morphological alterations and demonstrated a noticeable antioxidant effect thanks to the reduction of the activity/expression of NADPH oxidase in cornea and retina. Thus, an ACE oil ophthalmic formulation represent a promising therapy for ocular pathologies associated with arterial hypertension.

Limonin ameliorates cardiovascular dysfunction and remodeling in hypertensive rats.

AIMS: Limonin is a tetracyclic triterpenoid isolated from citrus fruits. Here, the effects of limonin on cardiovascular abnormalities in nitric oxide-deficient rats induced by Nω-Nitrol-arginine methyl ester (L-NAME) were explored. MAIN METHODS: Male Sprague Dawley rats were given L-NAME (40 mg/kg, drinking water) for 3 weeks and then treated daily with polyethylene glycol (vehicle), limonin (50 or 100 mg/kg) or telmisartan (10 mg/kg) for two weeks. KEY FINDINGS: Limonin (100 mg/kg) markedly reduced L-NAME-induced hypertension, cardiovascular dysfunction and remodeling in rats (P < 0.05). Increases in systemic angiotensin-converting enzyme (ACE) activity and angiotensin II (Ang II) and a reduction in circulating ACE2 were restored in hypertensive rats treated with limonin (P < 0.05). Reductions in antioxidant enzymes and nitric oxide metabolites (NOx) and increases in oxidative stress components induced by L-NAME were relieved by limonin treatment (P < 0.05). Limonin suppressed the increased expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in cardiac tissue and circulating TNF-α in rats that received L-NAME (P < 0.05). Changes in Ang II receptor type I (AT1R), Mas receptor (MasR), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) and NADPH oxidase subunit 2 (gp91phox) protein expression in cardiac and aortic tissue were normalized by limonin (P < 0.05). SIGNIFICANCE: In conclusion, limonin ameliorated L-NAME-induced hypertension, cardiovascular dysfunction and remodeling in rats. These effects were relevant to restorations of the renin-angiotensin system, oxidative stress and inflammation in NO-deficient rats. The molecular mechanisms are associated with the modulation of AT1R, MasR, NF-ĸB and gp91phox protein expression in cardiac and aortic tissue.

Hydrogen suppresses oxidative stress by inhibiting the p38 MAPK signaling pathway in preeclampsia.

BACKGROUND: Hypertensive disorders complicating pregnancy (HDCP) are one of the most serious medical disorders during pregnancy. OBJECTIVES: To investigate the effects of hydrogen on the mitogen-activated protein kinase (MAPK) signaling pathway in preeclampsia (PE). MATERIAL AND METHODS: The N(omega)-nitro-L-arginine methyl ester (L-NAME)-induced PE model with Sprague Dawley (SD) rats was employed. An inhibitor of MAPK signaling pathways (SB203580) was used as a p38 MAPK inhibitor. The SD rats were randomized into 5 groups: non-pregnant (NP); normal pregnancy (P); pregnancy + L-NAME (L); pregnancy + L-NAME + hydrogen-rich saline (LH); and pregnancy + L-NAME + hydrogen-rich saline + SB203580 (LHS). The pregnancies were terminated on day 22 of gestation, and the placentas and kidneys were microscopically inspected. Tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß) and malondialdehyde (MDA) levels were assessed. The mean systolic blood pressure (SBP) and level of proteinuria were recorded. The p38 MAPK mRNA expression and p-p38 MAPK protein levels were measured using real-time polymerase chain reaction (RT-PCR) and western blot, respectively. RESULTS: It was found that hydrogen-rich saline (LH group) decreased placental MDA, proteinuria, TNF-α, and IL-1ß levels in the placental tissues compared with the L group (all p < 0.05). Additionally, hydrogen-rich saline (LH group) treatment significantly decreased the p38 MAPK mRNA expression and p-p38 MAPK protein levels compared with the L group (p < 0.05). The p38 MAPK inhibitor SB203580 (LHS group) further decreased the p38 MAPK mRNA expression and p-p38 MAPK protein levels compared with the LH group (p < 0.05). CONCLUSIONS: Hydrogen can decrease the reactive oxygen species (ROS) content and inhibit the MAPK pathway. The protective effect of hydrogen may be associated with the inhibition of the p38 MAPK signaling pathway.

Evaluation of the Effect of the Fibroblast Growth Factor Type 2 (FGF-2) Administration on Placental Gene Expression in a Murine Model of Preeclampsia Induced by L-NAME.

The abnormal implantation of the trophoblast during the first trimester of pregnancy precedes the appearance of the clinical manifestations of preeclampsia (PE), which is a hypertensive disorder of pregnancy. In a previous study, which was carried out in a murine model of PE that was induced by NG-nitro-L-arginine methyl ester (L-NAME), we observed that the intravenous administration of fibroblast growth factor 2 (FGF2) had a hypotensive effect, improved the placental weight gain and attenuated the fetal growth restriction, and the morphological findings that were induced by L-NAME in the evaluated tissues were less severe. In this study, we aimed to determine the effect of FGF2 administration on the placental gene expression of the vascular endothelial growth factor (VEGFA), VEGF receptor 2 (VEGFR2), placental growth factor, endoglin (ENG), superoxide dismutase 1 (SOD1), catalase (CAT), thioredoxin (TXN), tumor protein P53 (P53), BCL2 apoptosis regulator, Fas cell surface death receptor (FAS), and caspase 3, in a Sprague Dawley rat PE model, which was induced by L-NAME. The gene expression was determined by a real-time polymerase chain reaction using SYBR green. Taking the vehicle or the L-NAME group as a reference, there was an under expression of placental VEGFA, VEGFR2, ENG, P53, FAS, SOD1, CAT, and TXN genes in the group of L-NAME + FGF2 (p < 0.05). The administration of FGF2 in the murine PE-like model that was induced by L-NAME reduced the effects that were generated by proteinuria and the increased BP, as well as the response of the expression of genes that participate in angiogenesis, apoptosis, and OS. These results have generated valuable information regarding the identification of molecular targets for PE and provide new insights for understanding PE pathogenesis.

The possible role of nitric oxide signaling and NMDA receptors in allopurinol effect on maximal electroshock- and pentylenetetrazol-induced seizures in mice.

Allopurinol, a uric-acid-lowering medication, has shown its efficacy in several studies suggesting that allopurinol can be prescribed as adjunctive cure meant for intractable epilepsy. The exact mechanism of allopurinol is still unknown. This study evaluates allopurinol's effect on seizure threshold, seizure incidence, and mortality rate in mice models. Moreover, the possible involvement of nitric oxide (NO) pathway and N-methyl-D-aspartate (NMDA) receptors are investigated. To evaluate the effect of allopurinol on seizure, we used the pentylenetetrazole (PTZ)-induced seizure along with maximal electroshock (MES)-induced seizure. To assess the underlying mechanism behind the allopurinol activity, we used nitric oxide synthase (NOS) substrate (L-arginine), NOS inhibitors (L-NAME, aminoguanidine, 7-nitroindazole), and NMDA receptor antagonist (MK-801). Intraperitoneal allopurinol administration at a dose of 50 mg/kg in mice showed a significant (p < 0.001) anti-convulsant activity in the PTZ-induced seizure. Even though pre-treatment with L-Arginine (60 mg/kg) potentiates allopurinol's anti-convulsant effect in the PTZ-induced seizure, pre-treatment with L-NAME (10 mg/kg), aminoguanidine (100 mg/kg), and 7-nitroindazole (30 mg/kg) reversed the anti-convulsant effect of allopurinol in the PTZ-induced seizure. In addition, pre-treatment with MK-801 also decreased the anti-convulsant effect of allopurinol in the PTZ-induced seizure. While allopurinol at a dose of 50 mg/kg and 100 mg/kg did not induce protection against seizure incidence in the MES-induced seizure, it revealed a remarkable effect in reducing the mortality rate in the MES-induced seizure. Allopurinol increases the seizure threshold in PTZ-induced seizure and enhances the survival rate in MES-induced seizure. Allopurinol exerts its anti-convulsant effect, possibly through targeting NO pathway and NMDA receptors.

Evaluation of Oxidative Stress Biomarkers, Pro-Inflammatory Cytokines, and Histological Changes in Experimental Hypertension, Dyslipidemia, and Type 1 Diabetes Mellitus.

The present study aims to compare the oxidative stress biomarkers, pro-inflammatory cytokines, and histological changes induced by three cardiovascular risk factors, namely, hypertension, dyslipidemia, and type 1 diabetes mellitus. Hypertension was induced with 40 mg/kg body weight (b.w.) of N omega-nitro-L-arginine-methyl (L-NAME) administered orally. Dyslipidemia was induced by the administration of a diet with a high cholesterol (2%) content. Diabetes mellitus was induced by intraperitoneal administration of a single dose of streptozocin (65 mg/kg). Malondialdehyde (MDA) and total oxidative status (TOS) are increased by all three cardiovascular risk factors (up to 207%). The indirect assessment of NO synthesis (NOx) is observed to be reduced after L-NAME administration (43%), and dyslipidemia induction (16%), while type 1 diabetes mellitus is associated with the highest levels of NOx (increased 112%). Hypertension, dyslipidemia, and type 1 diabetes reduced the total antioxidative capacity (TAC) and total thiol (SH) levels (up to 57%). The values of evaluated pro-inflammatory cytokines, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß), assessed from the ascending aorta were elevated by all three cardiovascular risk factors, with the highest levels induced by type 1 diabetes mellitus (up to 259%). The histopathological examination of the ascending and descending aorta revealed reversible pro-atherogenic changes consisting of the accumulation of lipid droplets in the subendothelial connective tissue on rats with hypertension and dyslipidemia. Irreversible pro-atherogenic changes consisting of a reduction of the specific elasticity of the arteries were observed in rats with type 1 diabetes mellitus. Type 1 diabetes mellitus demonstrates an alteration of the oxidative stress parameters, the elevation of tissue levels of the pro-inflammatory cytokines and causing irreversible pro-atherogenic changes on the aortic wall.

Maternal vitamin D deficiency reduces docosahexaenoic acid, placental growth factor and peroxisome proliferator activated receptor gamma levels in the pup brain in a rat model of preeclampsia.

BACKGROUND: Preeclampsia is a pregnancy disorder characterized with abnormal placental angiogenesis. Vitamin D and long chain polyunsaturated fatty acids (LCPUFA) play a crucial role in pregnancy and are required for normal placental and fetal growth and development. This study reports the effect of maternal vitamin D on LCPUFA levels in the mother and offspring brain fatty acid levels and angiogenic markers in a rat model of preeclampsia. METHODS: Female rats were divided into four groups from pre-pregnancy to pregnancy, viz Control; Preeclampsia (PE); Vitamin D deficient with PE (VDD-PE) and Vitamin D supplemented with PE (VDS-PE). Preeclampsia was induced by administering l-nitroarginine methyl ester (L-NAME) at the dose of 50 mg/kg body weight/day from day 14 to day 19 of gestation. Dams were sacrificed at d20 of gestation to collect dam blood, placenta and pup brain. LCPUFA levels from dam plasma, erythrocytes and placenta and its transcription factor peroxisome proliferator activated receptor gamma (PPAR-g) from placenta were estimated. Pup brain LCPUFA levels, angiogenic factors vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) and transcription factor hypoxia inducible factor (Hif-1α) and PPAR-g were also estimated. RESULTS: Maternal vitamin D status influences fatty acid levels. Placental PPAR-g levels were lower in the VDD-PE group as compared to the VDS-PE groups (p < 0.01). In the offspring brain, both PE and VDD-PE group showed lower levels of DHA (p < 0.05 for both) while saturated fatty acids (SFA) levels in the VDD-PE group were higher as compared to the control group (p < 0.05). VDD-PE group also showed lower levels of PlGF and PPAR-g (p < 0.01 and p < 0.05, respectively) in the pup brain while vitamin D supplementation demonstrated levels similar to control. CONCLUSION: This study for the first time demonstrates that maternal vitamin D status influences LCPUFA metabolism and angiogenesis in the offspring brain.

Electrocardiographic Abnormalities in Hypertension Models

Abstract Background: Hypertensive condition can lead to abnormalities in heart structure and electrical activity. The electrocardiogram (ECG) is a recording of the electrical activity of the heart and widely used to diagnose and detect heart problem. Objective: We conducted a comparative ECG analysis between two hypertension models (L-NAME and SHR) and their controls (Wistar and Wistar-Kyoto) at six and 15 th week of age. Methods: Blood pressure was measured at the end of the 15 th week, and electrocardiography was performed at six and 15 weeks of age in anaesthetized rats. Data normality was confirmed by Kolmogorov-Smirnov test followed by unpaired Student's t-test and the Mann-Whitney for parametric and non-parametric data, respectively. Results are expressed as mean ± SD. The accepted level of significance was set at p < 0.05. Results: L-NAME exhibited prolongation of JT and QT intervals and SHR showed a decrease in heart rate when compared to Wistar-Kyoto and L-NAME. Wistar-Kyoto exhibited short PR interval with increased QRS complex, and only QT prolongation at 15 weeks compared to Wistar. Conclusions: All the hypertension models used in this study featured an increase in blood pressure. However, while SHR showed cardiac dysfunction, L-NAME exhibited changes in ventricular performance. These results may guide future studies on different types and models of hypertension.

Vitexin ameliorates preeclampsia phenotypes by inhibiting TFPI-2 and HIF-1α/VEGF in a l-NAME induced rat model.

Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality with few safe, effective, and minimally invasive therapeutics. Inflammation, oxidative stress, and angiogenic imbalance have been reported to contribute to PE pathogenesis. Vitexin (VI) possesses various pharmacological activities including the potent regulation of the above biological processes in different conditions. This study aims to investigate whether VI has therapeutic potential to PE and the underlying mechanisms. Sprague-Dawley pregnant rats pretreated with or without VI were fed with l-NAME-containing water to induce experimental PE. Results showed that VI decreased high systolic blood pressure and urinary protein in PE rats time- and dose-dependently. Meanwhile, VI of higher dosage (45, 60 mg/kg) corrected abnormal pregnancy outcomes, including low pup weight and low pups/placenta ratio. In addition, VI of high dosage (60 mg/kg) decreased sFlt-1, increased PlGF and alleviated oxidative stress both in blood and placental samples compared with nontreated PE group. Furthermore, VI alleviated placental TFPI-2, HIF 1α, and VEGF in PE rats. In short, the present study suggests that the inhibition of placental TFPI-2 and HIF-1α/VEGF might be one of the potential mechanisms underlying the protective effects of VI to experimental PE induced by l-NAME.

The Probiotic Lactobacillus fermentum Prevents Dysbiosis and Vascular Oxidative Stress in Rats with Hypertension Induced by Chronic Nitric Oxide Blockade.

SCOPE: Whether the probiotic Lactobacillus fermentum CECT5716 (LC40) ameliorates hypertension in rats with chronic nitric oxide (NO) synthase inhibition is tested. METHODS AND RESULTS: Rats are randomly divided into four different groups and treated for 4 weeks: a) vehicle (control), b) vehicle plus NG -nitro-l-arginine methyl ester (l-NAME; 50 mg 100 mL-1 in drinking water), c) LC40 (109 colony-forming units d-1 by gavage), and d) LC40 plus l-NAME. l-NAME induces gut dysbiosis, characterized mainly by an increased Fimicutes/Bacteroidetes (F/B) ratio and reduced Bifidobacterium content, increased Th17 cells and reduced Treg in mesenteric lymph nodes (MLN), increased aortic Th17 infiltration and reactive oxygen species, reduced aortic endothelium-dependent relaxant response to acetylcholine, and hypertension. LC40 prevents gut dysbiosis, alters the Th17/Treg balance in MLN, vascular oxidative stress, and inflammation, slightly improves endothelial dysfuncion but do not inhibit the development of l-NAME-induced hypertension. CONCLUSION: Chronic LC40 treatment, in this model of chronic inhibition of NO synthesis, reduces early events involved in atherosclerosis development, such as vascular oxidative stress and pro-inflammatory status, as a result of prevention of gut dysbiosis and immune changes in MLN, but not hypertension, confirming the critical role of NO in the antihypertensive effects of LC40 in genetic hypertension.

Erythropoietin Protects the Kidney by Regulating the Effect of TNF-α in L-NAME-Induced Hypertensive Rats.

BACKGROUND/AIMS: Hypertension is the leading cause of death worldwide. Chronic high blood pressure induces inflammation. Tumor necrosis factor (TNF)-α plays a major role in inflammation and also depresses the synthesis of erythropoietin, which exerts protective effects on tissue; however, the mechanism is still unclear. We investigated the protective effect of erythropoietin against tissue damage caused by hypertension in the kidney and whether this effect was suppressed by TNF-α. METHODS: First, we detected the optimum chronic dose for darbepoetin-α (Depo), which is a long-acting erythropoietin analog for rats. We separated 60 female adult rats into 6 groups: control, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), L-NAME+Depo, L-NAME+Remicade (an anti-TNF-α antibody), L-NAME+Depo+Remicade, Depo, and control. After 1 month of treatment, we measured cardiovascular parameters, took blood samples, sacrificed the rats, and removed kidneys for analyses. RESULTS: The apoptotic index and the plasma and kidney mRNA levels of TNF-α increased in the L-NAME group and decreased in all other treatment groups. Macrophage accumulation increased in the L-NAME and L-NAME+Remicade groups, while it decreased in the Depo group. The mRNA abundance of TNF receptor 1 (TNFR1) decreased slightly in the Depo group and TNFR2 increased significantly in the same group. CONCLUSION: Erythropoietin protects kidney tissue against hypertension by preventing the apoptotic effects of TNF-α by blocking macrophage accumulation, decreasing TNF-α levels, and switching the TNF-α receptors from the apoptotic receptor TNFR1 to the proliferative receptor TNFR2.

Lactacystin-Induced Model of Hypertension in Rats: Effects of Melatonin and Captopril.

Lactacystin is a proteasome inhibitor that interferes with several factors involved in heart remodelling. The aim of this study was to investigate whether the chronic administration of lactacystin induces hypertension and heart remodelling and whether these changes can be modified by captopril or melatonin. In addition, the lactacystin-model was compared with NG-nitro-l-arginine-methyl ester (L-NAME)- and continuous light-induced hypertension. Six groups of three-month-old male Wistar rats (11 per group) were treated for six weeks as follows: control (vehicle), L-NAME (40 mg/kg/day), continuous light (24 h/day), lactacystin (5 mg/kg/day) alone, and lactacystin with captopril (100 mg/kg/day), or melatonin (10 mg/kg/day). Lactacystin treatment increased systolic blood pressure (SBP) and induced fibrosis of the left ventricle (LV), as observed in L-NAME-hypertension and continuous light-hypertension. LV weight and the cross-sectional area of the aorta were increased only in L-NAME-induced hypertension. The level of oxidative load was preserved or reduced in all three models of hypertension. Nitric oxide synthase (NOS) activity in the LV and kidney was unchanged in the lactacystin group. Nuclear factor-kappa B (NF-κB) protein expression in the LV was increased in all treated groups in the cytoplasm, however, in neither group in the nucleus. Although melatonin had no effect on SBP, only this indolamine (but not captopril) reduced the concentration of insoluble and total collagen in the LV and stimulated the NO-pathway in the lactacystin group. We conclude that chronic administration of lactacystin represents a novel model of hypertension with collagenous rebuilding of the LV, convenient for testing antihypertensive drugs or agents exerting a cardiovascular benefit beyond blood pressure reduction.

Garcinia mangostana pericarp extract protects against oxidative stress and cardiovascular remodeling via suppression of p47phox and iNOS in nitric oxide deficient rats.

Nω-Nitro-l-arginine methyl ester (l-NAME)-induced hypertension and cardiovascular remodeling are associated with oxidative stress and inflammation. Garcinia mangostana Linn., has been reported to have antioxidant and anti-inflammatory properties. This study investigated whether G. mangostana pericarp extract (GME) could prevent l-NAME-induced hemodynamic alterations, cardiovascular remodeling, oxidative stress and inflammation in rats. Male Sprague-Dawley rats were given 40mg/kg/day of l-NAME in drinking water to induce hypertension, and were simultaneously treated with GME at a dose of 200mg/kg/day. Rats that received l-NAME for five weeks had high blood pressure, left ventricular hypertrophy and thickening of aortic wall. Vascular superoxide production, plasma malondialdehyde (MDA), and plasma tumor necrosis factor alpha (TNF-α) were significantly increased in l-NAME-hypertensive rats (p<0.05). This was consistent with up-regulation of the p47phox NADPH oxidase subunit and iNOS protein expression in aortic tissues (p<0.05). Low levels of plasma nitric oxide metabolites were observed in l-NAME hypertension. GME prevented the development of hypertension and cardiovascular remodeling induced by l-NAME with reduced oxidative stress and inflammation. These data suggest that GME had a protective effect against l-NAME-induced hypertension and cardiovascular remodeling via suppressing p47phox NADPH oxidase subunit and iNOS protein expression resulting in enhancing NO bioavailability.

Oral administration of veratric acid, a constituent of vegetables and fruits, prevents cardiovascular remodelling in hypertensive rats: a functional evaluation.

In our previous studies, veratric acid (VA) shows beneficial effect on hypertension and its associated dyslipidaemia. In continuation, this study was designed to investigate the effect of VA, one of the major benzoic acid derivatives from vegetables and fruits, on cardiovascular remodelling in hypertensive rats, primarily assessed by functional studies using Langendorff isolated heart system and organ bath system. Hypertension was induced in male albino Wistar rats by oral administration of N ω -nitro-l-arginine methyl ester hydrochloride (l-NAME) (40 mg/kg body weight (b.w.)) in drinking water for 4 weeks. VA was orally administered at a dose of 40 mg/kg b.w. l-NAME-treated rats showed impaired cardiac ventricular and vascular function, evaluated by Langendorff isolated heart system and organ bath studies, respectively; a significant increase in the lipid peroxidation products such as thiobarbituric acid-reactive substances and lipid hydroperoxides in aorta; and a significant decrease in the activities of superoxide dismutase, catalase, glutathione peroxidase and levels of GSH, vitamin C and vitamin E in aorta. Fibrotic remodelling of the aorta and heart were assessed by Masson's Trichrome staining and Van Gieson's staining, respectively. In addition, l-NAME rats showed increased heart fibronectin expression assessed by immunohistochemical analysis. VA supplementation throughout the experimental period significantly normalised cardiovascular function, oxidative stress, antioxidant status and fibrotic remodelling of tissues. These results of the present study conclude that VA acts as a protective agent against hypertension-associated cardiovascular remodelling.

Heme oxygenase suppresses markers of heart failure and ameliorates cardiomyopathy in L-NAME-induced hypertension.

Heart failure and related cardiac complications remains a great health challenge. We investigated the effects of upregulating heme-oxygenase (HO) on myocardial histo-pathological lesions, proinflammatory cytokines/chemokines, oxidative mediators and important markers of heart failure such as osteopontin and osteoprotergerin in N(ω)-nitro-l-arginine methyl ester (L-NAME)-induced hypertension. Treatment with the HO-inducer, heme-arginate improved myocardial morphology in L-NAME hypertensive rats by attenuating subendocardial injury, interstitial fibrosis, mononuclear-cell infiltration and cardiomyocyte hypertrophy. These were associated with the reduction of several inflammatory/oxidative mediators including chemokines/cytokines such as macrophage inflammatory protein-1 alpha (MIP-1α), macrophage chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1ß, endothelin-1, 8-isoprostane, nitrotyrosine, and aldosterone. Similarly, heme-arginate abated the elevated levels of extracellular matrix/remodeling proteins including transforming-growth factor beta (TGF-ß1) and collagen-IV in the myocardium. These were accompanied by significant reduction of proteins of heart failure such as osteopontin and osteoprotegerin. Interestingly, the cardio-protective effects of heme-arginate were associated with the potentiation of adiponectin, atrial-natriuretic peptide (ANP), HO-1, HO-activity, cyclic gnanosine monophosphate (cGMP) and the total-anti-oxidant capacity, whereas the HO-inhibitor, chromium-mesoporphyrin nullified the effects of heme-arginate, exacerbating inflammatory injury and oxidative insults. We conclude that heme-arginate therapy protects myocardial damage by potentiating the HO-adiponectin-ANP axis, which in turn suppressed the elevated levels of aldosterone, pro-inflammatory chemokines/cytokines, mononuclear-cell infiltration and oxidative stress, with concomitant reduction of extracellular matrix/remodeling proteins and heart failure proteins. These data suggest a cardio-protective role of the HO system against L-NAME-induced hypertension that could be explored in the design of novel strategies against cardiomyopathy.

Chlorella pyrenoidosa ameliorated L-NAME-induced hypertension and cardiorenal remodeling in rats.

PURPOSE: Hypertension is one of the main factors causing cardiovascular diseases. The aim of the study is to investigate the effects of Chlorella pyrenoidosa on blood pressure and cardiorenal remodeling in rats with N (ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced endothelial dysfunction. METHODS: Rats were fed a diet containing L-NAME (40 mg/kg) with or without chlorella (4 or 8 %) for 5 weeks. We found that chlorella retarded the development of hypertension and cardiorenal remodeling during the 5-week experimental period. RESULTS: Although there was no difference in NO( x ) levels or plasma arginine concentrations, plasma and tissues ACE activities were significantly lower in the chlorella groups than in the L-NAME group. Moreover, tissue tumor necrosis factor-α concentrations and renal CYP4A expression were also lower in the chlorella group. CONCLUSION: These results suggest that chlorella might ameliorate the elevation of blood pressure and show cardiorenal-protective effects in nitric oxide-deficient rats, and one possible mechanism might be mediated by its ACE inhibitory activity.

El sistema renina angiotensina y el estrés oxidativo en un modelo experimental de preeclampsia en ratas / The renin angiotensin system and the oxidative stress in a animal model of preeclampsia in rats

La preeclampsia es un síndrome exclusivo de la gestación humana y responsable de una alta morbimortalidad perinatal, cuyas manifestaciones incluyen: hipertensión arterial, proteinuria y edema. Un mecanismo postulado en la fisiopatología de la preeclampsia, es la reducción de la perfusión placentaria y el desarrollo del síndrome clínico materno ocasionado por la liberación de factores placentarios que afectan la regulación de la presión arterial y la función renal. Uno de los factores que ocasiona el trastorno endotelial son las especies reactivas de oxígeno, el incremento de elementos vasoactivos, así como la disminución de agentes vasorelajantes como el óxido nítrico. Todas estas alteraciones vasculares conducen no sólo a la hipertensión sino también a la disfunción renal. Debido a la importancia del papel del óxido nítrico y su desregulación en la preeclampsia, en el presente trabajo se caracterizó un modelo experimental de preeclampsia que resulta de la inhibición de la síntesis de óxido nítrico mediante la administración de L-NAME a ratas preñadas y no preñadas. En el mismo se evaluó el estatus oxidativo y, el papel del sistema renina angiotensina en la contribución de la disfunción renal. Los resultados demuestran el papel primordial del óxido nítrico y su desregulación en este modelo de preeclampsia experimental. En efecto, se demostró que el tratamiento durante siete días con L-NAME incrementó la presión arterial media, aumentó la sensibilidad vascular, inhibió la actividad de la sintasa del óxido nítrico renal y redujo el guanilil monofosfato cíclico urinario. La disfunción endotelial renal en este modelo experimental se manifiesto por proteinuria, incremento de la creatinina plasmática, disminución de la excreción urinaria de sodio, potasio y creatinina, así como, evidencia morfológica de endoteliosis glomerular. Al caracterizar el papel de las enzimas antioxidantes renales se encontró una reducción significativa de la actividad de las mismas, y un incremento de la peroxidación lipídica asociada a una elevada concentración de agentes pro-oxidantes. Nuestro modelo experimental constituye una buena aproximación a la preeclampsia humana y no un efecto inespecífico del L-NAME, ya que aún cuando la inhibición crónica de la síntesis de óxido nítrico en ratas no preñadas induce un incremento de la presión arterial media, proteinuria, reducción de la actividad de la sintasa del óxido nítrico renal y de la excreción urinaria de guanilil monofosfato cíclico similar a las ratas preñadas, los efectos sobre la proteinuria, las acciones morfológicas renales, la excreción urinaria de sodio, potasio y creatinina, y sobre el sistema renina angiotensina son específicos de la preeclampsia experimental en ratas. Así, se demostró que en las ratas preñadas tratadas con L-NAME la actividad de la enzima convertidora de angiotensina plasmática, los niveles de renina plasmática, y la aldosterona amniótica se encuentran marcadamente disminuidos, cuando se comparan con las ratas preñadas normotensas. Estos hallazgos sugieren que la preeclampsia experimental se caracteriza por la supresión de los componentes circulantes del sistema renina angiotensina, que podrían ser responsables del desbalance entre los sistemas vasoconstrictores y vasodilatadores observados en la preeclampsia, así como de algunos de los signos de la preeclampsia, similar a lo que ocurre en la mujer embarazada hipertensa. Por otra parte, al evaluar la contribución del estrés oxidativo en el daño renal en la preeclampsia experimental, se demostró una disminución de la actividad de las enzimas antioxidantes renales. Asimismo, la disminución de la actividad de la glutatión peroxidasa plasmática y la tendencia a la reducción en la glutatión peroxidasa en el líquido amniótico, con el simultáneo incremento de los valores de las sustancias que reaccionan con el ácido tiobarbitúrico (TBARS) plasmático, sugiriéndose que la desregulación generalizada y renal está asociada a una baja protección oxidativa durante la preeclampsia, que favorece a la insuficiencia renal. El incremento temprano del estrés oxidativo placentario juega un papel fundamental en la disfunción endotelial generalizada y el daño renal en la preeclampsia. Debido a ello, nos planteamos que el tratamiento temprano con antioxidantes o con desacoplantes de la NAD (P) H oxidasa podría interrumpir el proceso de este síndrome. Efectivamente, el tratamiento crónico con un compuesto que mimetiza a la superóxido dismutasa, el tempol, o el desacoplante del ensamblaje de la NAD(P)H oxidasa (apocinina), fueron capaces de reducir significativamente la hipertensión inducida por el L-NAME. Igualmente, ambos compuestos fueron capaces de prevenir la proteinuria y la reducción de la actividad de las enzimas antioxidantes renales estudiadas. En conclusión, la preeclampsia experimental inducida por la inhibición crónica de la síntesis de óxido nítrico en ratas preñadas, reproduce los signos clásicos de la preeclampsia humana, y se acompaña de la desregulación del sistema renina angiotensina y de disfunción renal. Esto nos permite aseverar que este modelo experimental de preeclampsia constituye una buena aproximación a la preeclampsia humana. Se demuestra que el daño renal encontrado en este modelo experimental se asocia a una disminución de los mecanismos antioxidantes renales, que lleva a un incremento del estrés oxidativo, y a una reducción de la protección de la función renal. Estos resultados indican que la sobreproducción de especies reactivas de oxígeno tanto placentaria como renal, son causa fundamental de la disfunción endotelial generalizada y del daño renal. Finalmente, la inhibición del estrés oxidativo mediante el uso de agentes antioxidantes como el tempol o la apocinina, pudiese ser una de las posibles estrategias terapéuticas en el tratamiento de la hipertensión inducida por el embarazo humano y abre nuevos horizontes en el tratamiento de este síndrome.

Mice lacking macrophage 12/15-lipoxygenase are resistant to experimental hypertension.

In mouse arteries, Alox15 [leukocyte-type 12/15-lipoxygenase (LO)] is assumed to regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids that mediate the endothelium-dependent relaxations to AA and acetylcholine (ACh). We used Alox15(-/-) mice, made by targeted disruption of the Alox15 gene, to characterize its role in the regulation of blood pressure and vascular tone. Systolic blood pressures did not differ between wild-type (WT) and Alox15(-/-) mice between 8-12 wk of age, but Alox15(-/-) mice exhibited resistance toward both N(G)-nitro-L-arginine-methyl ester (L-NAME)- and deoxycorticosterone acetate (DOCA)/high-salt-induced hypertension. ACh relaxed mesenteric arteries and abdominal aortas of WT and Alox15(-/-) mice to an identical extent. The LO inhibitor nordihydroguaiaretic acid attenuated the ACh relaxations by 35% in arteries from both WT and Alox15(-/-) mice. Reverse-phase HPLC analysis of [(14)C]AA metabolites in aorta and peritoneal macrophages (PM) revealed differences. Unlike PM, aorta tissue did not produce detectable amounts of 15-hydroxyeicosatetraenoic acid. Although Alox15 mRNA was detected in aorta, high-resolution gel electrophoresis with immunodetection revealed no Alox15 protein expression. Unlike aorta, Alox15 protein was detected in PM, intestine, fat, lung, spleen, and skin from WT, but not Alox15(-/-), mice. Injection of WT PM, a primary source of Alox15 protein, into Alox15(-/-) mice abolished their resistance toward L-NAME-induced hypertension. On the other hand, WT mice acquired resistance to L-NAME-induced hypertension after depletion of macrophages by clodronate injection. These studies indicate that Alox15 is involved in development of experimental hypertension by altering macrophage functions but not via synthesis of the vasoactive LO metabolites in mouse arteries.

Sildenafil citrate decreases sFlt-1 and sEng in pregnant l-NAME treated Sprague-Dawley rats.

OBJECTIVES: We have previously shown that sildenafil citrate improves various fetal outcomes in pregnant, L-NAME treated, Sprague-Dawley rats. We therefore aimed to identify which component/s of this diverse pathophysiologic cascade is/are improved by this drug. STUDY DESIGN: This study is a sub-analysis of plasma samples obtained in a previous study in which 24 pregnant Sprague-Dawley dams were divided into three groups (n=8) i.e. the control group (CON), the experimental control group (PRE) where the pre-eclampsia-like symptoms were induced using l-NAME, and the experimental group (SCT) where the pre-eclampsia-like symptoms were once again induced using L-NAME but these animals were treated with sildenafil citrate. On gestation day 20 blood samples were collected in heparin-coated tubes and plasma samples were then analysed for specific variables using commercially available kits for rats. RESULTS: There was a significant increase in the plasma levels of soluble fms-like tyrosine kinase1 (sFlt-1) in the PRE group (1228.80±116.29 pg/ml) when compared to the CON (774.91±26.81 pg/ml) and SCT (698.98±20.78 pg/ml) groups, respectively (p<0.001). The plasma levels of soluble endoglin (sEng) were significantly decreased in the SCT group (149.47±3.72 ng/ml) when compared to the CON (178.52±5.33 ng/ml) and PRE (183.44±8.294 ng/ml) groups, respectively (p<0.01). Plasma nitric oxide and l-arginine levels showed a decreasing trend in the PRE groups when compared to the control (CON) and treated (SCT) groups, respectively. CONCLUSION: Sildenafil citrate reduces the plasma levels of anti-angiogenic factors, sFlt-1 and sEng, in pre-eclamptic (L-NAME induced) Sprague-Dawley rats and may therefore be responsible for the reduction in blood pressure and proteinuria as well as the improved fetal outcomes noted in an earlier study.

Pharmacologically induced ischemia-reperfusion syndrome in the rat small intestine.

BACKGROUND: Reports of ischemia-reperfusion (I/R) injury in vivo describe experiments in which lesions are induced by the physical procedure of clamping, (I/RIC). We compare this procedure with a pharmacologic technique in which I/R injury is induced by drug superfusion (I/RID). MATERIALS AND METHODS: We used rat intestine to determine whether the responses provoked by I/RIC, such as changes in reactive oxygen species (ROS) and nitric oxide levels, are also provoked by I/RID. To this end, rats were treated with allopurinol, SOD, catalase, L-NAME, and L-arginine. In both I/R models ischemia was maintained for 60 min, followed by 30 min of reperfusion. RESULTS: In both ischemia models, we observed significant differences in Evans blue (vascular permeability) and LDH (tissue injury) concentrations during the reperfusion period compared with the control group. I/RIC always induced greater injury. However, proportionally, the degree of protection was similar in the two models for the different treatments assayed. This indicates that the pathophysiologic mechanisms are the same. CONCLUSIONS: Our I/RID model induces a significant intestinal alteration during the reperfusion period and, also in general terms, this alteration is prevented or worsened in a similar and proportional way to that observed when using the classic I/RIC model. The I/RID model helps to explain the development and evolution of pathologies characterized by the induction of intermittent vasospasms that produce transitory reductions in vascular perfusion, which in turn can generate ROS though an I/R mechanism.