Interaction between mild hypercholesterolemia, HDL-cholesterol levels, and angiotensin II in intimal hyperplasia in mice

Two month old C57BL/6 mice were placed on three different diets: 1) normal diet (NC; 0.025% cholesterol), 2) hypercholesterolemic Western-type diet (HC-W; 0.2% cholesterol), and 3) hypercholesterolemic Paigen-type diet (HC-P; 1.25% cholesterol plus 0.5% cholic acid). At 6 months of age, the animals underwent ligation of the left carotid artery and were randomly assigned to vehicle (PBS, subcutaneous) or angiotensin II (Ang II; 1.4 mg/kg/day, subcutaneous) treatment for 4 weeks. Low density lipoprotein-cholesterol levels were similarly increased in both HC diets (NC, 4 +/- 3 mg/dl; HC-W, 123 +/- 17 mg/dl; HC-P, 160 +/- 14 mg/dl). However, the levels of high density lipoprotein-cholesterol (HDL-C) were reduced only in animals fed the HC-P diet (NC, 82 +/- 6 mg/dl; HC-W, 79 +/- 7 mg/dl; HC-P, 58 +/- 7 mg/dl). In Ang II-treated mice, carotid artery ligation induced intimal smooth muscle cell proliferation to a similar extent in NC- and HP-W-fed animals. However, a significantly larger intimal area developed in ligated vessels from Ang II-treated mice fed the HC-P diet (3.6-fold higher than in Ang II-treated NC mice). Together, these results show the accelerating effect of mild hypercholesterolemia, reduced HDL-C levels, and Ang II on intimal hyperplasia after carotid artery ligation in mice.     

Altered cardiovascular responses to chronic angiotensin II infusion in aged rats

In this work we determined by telemetry the cardiovascular effects produced by Ang II infusion on blood pressure (BP) and heart rate (HR) in aged rats. Male Wistar aged (48-52 weeks) and young (12 weeks) rats were used. Ang II (6 microg/h, young, n=6; aged, n=6) or vehicle (0.9% NaCl 1 microl/h, young, n=4; aged, n=5) were infused subcutaneously for 7 days, using osmotic mini-pump. The basal diurnal and nocturnal BP values were higher in aged rats (day: 98+/-0.3 mm Hg, night: 104+/-0.4 mm Hg) than in the young rats (day: 92+/-0.2 mm Hg, night: 99+/-0.2 mm Hg). In contrast, the basal diurnal and nocturnal HR values were significantly smaller in the aged rats. Ang II infusion produced a greater increase in the diurnal BP in the aged rats (Delta MAP=37+/-1.8 mm Hg) compared to the young ones (Delta MAP=30+/-3.5 mm Hg). In contrast, the nocturnal MAP increase was similar in both groups (young rats; Delta MAP=22+/-3.0 mm Hg, aged rats; Delta MAP=24+/-2.6 mm Hg). During Ang II infusion HR decreased transiently in the young rats. An opposite trend was observed in the aged rats. Ang II infusion also inverted the BP circadian rhythm, in both groups. No changes in HR circadian rhythm were observed. These differences suggest that the aging process alters in a different way Ang II-sensitive neural pathways involved in the control of autonomic activity.     

Renal nuclear angiotensin II receptors in normal and hypertensive rats

Accumulation of Angiotensin II (Ang II) in the kidneys of hypertensive rats infused chronically with Ang II occurs by AT1 receptor mediated internalization of Ang II, which may interact with intracellular targets, including nuclear binding sites. The aims of this study were to determine if kidney cell nuclei have specific Ang II binding sites and if chronic infusion of Ang II (70 ng/min; n=9) influences the nuclear Ang II binding capacity. Kidneys were harvested from control and Ang II infused rats and the renal cortexes were homogenized to obtain crude membrane preparations and nuclear fractions. Ang II binding sites were measured with a single point assay by incubating each fraction with 10 nM 125I-Sar-Ile-Ang II in the absence (total binding sites) or presence of either 2.5 M Sar-Leu-Ang II or 25 microM losartan to detect specific AT or AT1 binding sites. Both fractions exhibited specific Ang II binding sites that were displaced by both saralasin and losartan. In control rats, crude membrane preparations had 792 +/- 218 and the nuclear fraction had 543 +/- 222 fmol/mg protein AT1 receptors. AT1 receptor levels in membrane (885 +/- 170 fmol/mg protein) and nuclear fractions (610 +/- 198 fmol/mg protein) were not significantly different in Ang II infused rats. These data support the presence of nuclear Ang II receptors predominantly of the AT1 subtype in renal cells. Chronic Ang II infusion did not alter overall Ang II receptor densities.     

beta1 antagonist and beta2 agonist, celiprolol, restores the impaired endothelial dependent and independent responses and decreased TNFalpha in rat with type II diabetes

The effect of beta antagonists in the diabetic vascular lesion is controversial. We investigated the effect of celiprolol hydrochloride, a beta1 antagonist and mild beta2 agonist, on the lesions and function in type II male Otsuka Long-Evans Tokushima Fatty (OLETF) diabetic rats. OLETF rats were fed regular chow with or without atenolol (25 mg/kg/day) or celiprolol (100 mg/kg/day) treatment (group DM, no treatment; group DM-a, atenolol treatment; group DM-c, celiprolol treatment), and treatment was continued for 31 days. Separately, normoglycemic control rats, LETO, were prepared as group C. On day 3, endothelial cells of the right internal carotid artery were removed by balloon injury, and the rats were evaluated 4 weeks after balloon injury. The plasma glucose and lipid levels were unchanged throughout the treatment period. Intimal thickening was observed in the right carotid artery in the DM and DM-a groups; however, little thickening was observed in those of DM-c rats. Acetylcholine-induced NO-dependent relaxation in arteries was improved in DM-c rats compared with DM and DM-a rats (maximum relaxation DM 30.8+/-4.5, DM-a 37.4+/-3.9, DM-c 48.8+/-4.6%, *P<0.05 vs. DM for DM-c rats). Tone-related basal NO release and acetylcholine-induced NO-dependent relaxation in the arteries and plasma NO(x) (sum of NO(2)(-) and NO(3)(-)) were greater in DM-c and C groups than in DM and DM-a groups. The serum TNFalpha levels did not increase in DM-c rats compared with those of the DM or DM-a groups, and were comparable with those of group C. CONCLUSION: In conclusion, Celiprolol improves endothelial function in the arteries of OLETF rats, and further restore it 4 weeks after endothelial denudation in the arteries of OLETF rats. NO and O(2)(-) may have a role in the important underlying mechanisms by reducing the TNFalpha levels.     

Effects of TH-142177 on angiotensin II-induced proliferation, migration and intracellular signaling in vascular smooth muscle cells and on neointimal thickening after balloon injury.

We investigated the effects of TH-142177 (N-n-butyl-N-[2'-(1-H-tetrazole-5-yl) biphenyl-4-yl]-methyl-(N-carboxy methyl-benzylamino)-acetamide), a novel selective antagonist of angiotensin II type 1-receptor (AT1-R) on angiotensin II (AII)-induced proliferation and migration of vascular smooth muscle cells (VSMC), and on neointimal formation in the rat carotid artery after balloon injury, and on the intracellular signaling by the stimulation of AT1-R. High affinity AII receptor sites were detected in rat VSMC by the use of [125I]Sar1,Ile8-AII. TH-142177 and losartan competed with [125I]Sar1,Ile8-AII for the binding sites in VSMC in a monophasic manner, although PD123177, a selective antagonist of angiotensin II type 2-receptor (AT2-R), had little inhibitory effect, demonstrating the predominant existence of AT1-R in rat VSMC. TH-142177 prevented AII-induced DNA synthesis and migration, with a significant inhibition of 74 and 55%, respectively, at the concentration of 100 nM. AII-induced activation of p21ras, mitogen-activated protein kinase (p42MAPK and p44MAPK), and protein kinase C was significantly (50-78%) inhibited by TH-142177 (100 nM), suggesting that the activation of these enzymes is mediated through the stimulation of AT1-R. Balloon-injured left carotid arteries in rats showed extensive neointimal thickening, and TH-142177 (3 mg/kg) brought out a marked decrease in the neointimal thickening after balloon injury. In conclusion, TH-142177 inhibited AII-induced proliferation and migration of rat VSMC and neointimal formation in the carotid artery after balloon injury, and these effects may be related, in part, to the suppression of ras, p42MAPK and p44MAPK, and protein kinase C activities through the blockade of AT1-R. Thus, TH-142177 may have therapeutic potential for the treatment of vascular diseases such as atherosclerosis and restenosis.     

Peripheral administration of AT1 receptor blockers and pressor responses to central angiotensin II and sodium.

Central administration of AT1 receptor blockers prevents salt-sensitive hypertension and inhibits progression of CHF. We investigated in Wistar rats the effectiveness of peripheral administration of two AT1 receptor blockers, losartan and embusartan, in exerting central AT1 receptor blockade. Losartan or embusartan at doses of 30 and 100 mg/kg were administered subcutaneously (s.c.) as a single dose, or one dose daily for 6 days. The BP responses to intracerebroventricular (i.c.v.) injection of Ang II, i.c.v. infusion of Na+-rich aCSF (0.3 M NaCl), and intravenous (i.v.) injection of Ang II were then measured. Losartan or embusartan at 30 and 100 mg/kg both inhibited the BP increases induced by i.c.v. Ang II and, to a lesser extent, by Na+-rich aCSF. After a single dose, this inhibition was more pronounced for losartan. However, after 6 days of treatment, there were no significant differences between the effects of losartan and embusartan. Losartan and embusartan blocked responses to Ang II i.v. to a similar extent. These results indicate that results from single-dose studies may not reflect the chronic steady-state, and that during chronic treatment both AT1 receptor blockers are similarly effective in inhibiting AT1 receptors in the central nervous system, when assessed by pressor responses to acute increases in CSF Na+ or CSF Ang II.     

Estrogen prevents intestinal inflammation after trauma-hemorrhage via downregulation of angiotensin II and angiotensin II subtype I receptor

Although angiotensin II (Ang II) plays a key role in development of organ ischemia-reperfusion injury, it remains unclear whether it is involved in development of intestinal injury following trauma-hemorrhage (T-H). Studies have shown that 17beta-estradiol (E2) administration following T-H improves small intestinal blood flow; however, it is unclear whether Ang II plays a role in this E2-mediated salutary effect. Male Sprague-Dawley rats underwent laparotomy and hemorrhagic shock (removal of 60% total blood volume, fluid resuscitation after 90 min). At onset of resuscitation, rats were treated with vehicle, E2, or E2 and estrogen receptor antagonist ICI 182,780 (ICI). A separate group of rats was treated with Ang II subtype I receptor (AT1R) antagonist losartan. At 24 h after T-H, plasma Ang II, IL-6, TNF-alpha, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-3 levels, myeloperoxidase (MPO) activity, and AT1R expression were determined. T-H significantly increased plasma and intestinal Ang II, IL-6, TNF-alpha levels, intestinal ICAM-1, CINC-1, CINC-3 levels, MPO activity, and AT1R protein compared with shams. E2 treatment following T-H attenuated increased intestinal MPO activity, Ang II level, and AT1R protein expression. ICI administration abolished the salutary effects of E2. In contrast, losartan administration attenuated increased MPO activity without affecting Ang II and AT1R levels. Thus Ang II plays a role in producing small intestine inflammation following T-H, and the salutary effects of E2 on intestinal inflammation are mediated in part by Ang II and AT1R downregulation.     

Endothelin-1 induces contraction of human and Australian possum gallbladder in vitro

This study examined the role of the renin-angiotensin and vasopressin systems on systolic blood pressure (SBP) variability following subarachnoid haemorrhage (SAH) in conscious rats. Animals received no treatment, the angiotensin II AT1 receptor antagonist, losartan, or the vascular vasopressin receptor antagonist, AVPX. SAH resulted in a transient sympathetic activation as estimated from the increase in the mid-frequency oscillations of SBP (3.2 +/- 0.8 mm Hg2, 3 hours after the injury vs. 1.3 +/- 0.3 mm Hg2 in control conditions, p < 0.01). On the second and fourth day following SAH, a marked elevation in the low-frequency component of SBP was observed (7.1 +/- 1.0 mm Hg2 on day 2 vs. 2.6 +/- 0.3 mm Hg2 in control conditions, p < 0.001 and 6.3 +/- 1.1 mm Hg2 on day 4 vs. 2.6 +/- 0.3 mm Hg2 in control conditions, p < 0.01). Pre-treatment with losartan prevented the acute rise in the mid-frequency oscillations in SBP and partially reduced the low-frequency component observed at 2 and 4 days. Administration of AVPX on the second and fourth day following SAH normalised the elevated low-frequency oscillations in SBP. This study indicates that the modifications in SBP variability observed in the early and delayed stage after subarachnoid haemorrhage involve angiotensin II. Vasopressin seems to be implicated in the delayed development of low-frequency fluctuations of SBP.     

Angiotensin-(3-7) pressor effect at the rostral ventrolateral medulla

Ang-(3-7) is a fragment of the renin-angiotensin system that can be derived both from Ang II or Ang-(1-7). In the present study we determined the cardiovascular effects produced by angiotensin-(3-7) [Ang-(3-7)] microinjection into the rostral ventrolateral medulla (RVLM), a key region for the control of sympathetic drive to the periphery. RVLM microinjection of Ang-(3-7) (20, 40 or 80 ng) in male Wistar rats anesthetized with urethane produced significant increases in MAP (19+/-3.8 mm Hg, n=5; 16+/-1.6 mm Hg, n=15 and 11+/-1.2 mm Hg, n=4, respectively) as compared to saline (4+/-0.7 mm Hg, n=6). These alterations were similar to that induced by Ang-(1-7) (14+/-1.3 mm Hg, 40 ng; n=12) and Ang II (17+/-2.3 mm Hg, 40 ng; n=7). Microinjection of losartan (AT(1) receptor antagonist, 100 pmol) or A779 (selective Mas receptor antagonist, 100 pmol) did not alter the pressor effect caused by Ang-(3-7). Microinjection of an Ang-(3-7) analogue, d-Ala(7)-Ang-(3-7) (100 pmol), completely abolished the pressor effect caused by Ang-(3-7). These results suggest that Ang-(3-7) may be an additional peptide of the RAS to act as neuromodulator, at least at the RVLM. Further, the Ang-(3-7) pressor effect is not mediated by the interaction with AT(1) or the Ang-(1-7), Mas, receptors.     

Effects of captopril and angiotensin II receptor blockers (AT1, AT2) on myocardial ischemia-reperfusion induced infarct size

The renin-angiotensin system (RAS) plays a major role in regulating the cardiovascular system, and disorders of the RAS contribute largely to the cardiac pathophysiology, including myocardial ischemia-reperfusion (MI/R) injury. Two subtypes of angiotensin II (Ang II) receptors have been defined on the basis of their differential pharmacological properties. The current study was undertaken to address the question as to whether the inhibition of the angiotensin converting enzyme (ACE) by captopril and the AT1 and AT2 receptor blockers losartan and PD123319 modulate MI/R-induced infarct size in an in vivo rat model. To produce necrosis, a branch of the descending left coronary artery was occluded for 30 min followed by two hours of reperfusion. ECG changes, blood pressure, and heart rate were measured during the experiment. Captopril (3 mg/kg), losartan (2 mg/kg), and PD123319 (20 μg/kg/min) were given in an IV 10 min before ischemia and were continued during the ischemic period. The infarcted area was measured by TTC staining. The volume of infarct and the risk zone was determined by planimetry. Compared to the control group (55.62±4.00%) both captopril and losartan significantly reduced the myocardial infarct size (30.50±3.26% and 37.75±4.44%), whereas neither PD123319 nor PD123319+losartan affected the infarct size volume (46.50±3.72% and 54.62±2.43%). Our data indicates that captopril and losartan exert cardioprotective activity after an MI/R injury. Also, infarct size reduction by losartan was halted by a blockade of the AT2 receptor. Therefore, the activation of AT2 receptors may be potentially protective and appear to oppose the effects mediated by the AT1 receptors.     

Evaluation of a prostacyclin analog, iloprost, and a thromboxane A2 receptor antagonist, daltroban, in experimental intimal hyperplasia.

This study evaluated the efficacy of a prostacyclin analog, iloprost, and a thromboxane A2 receptor antagonist, daltroban, as inhibitors of experimental intimal hyperplasia. The vascular injury model used is based on an endothelial injury induced by a brief infusion of air into an isolated segment of the common carotid artery in the rat. Iloprost and daltroban were administered by continuous IV infusion for two weeks. The infusion rates were 0.1 micrograms/kg/min for iloprost and 0.1 mg/kg/hr for daltroban; these dosing rates are associated with significant alterations in eicosanoid-related pharmacologic effects. The animals were sacrificed at two weeks and the carotid arteries fixed in situ for light microscopy. The myointimal thickening was measured as the intima to media area (I/M) ratio. The control animals developed marked intimal thickening, with an I/M ratio of 0.76 +/- 0.12 (mean +/- SEM; N = 7). There was no inhibition of intimal hyperplasia (P greater than 0.05) after either iloprost (I/M ratio: 1.04 +/- 0.13; N = 8) or daltroban (I/M ratio: 0.70 +/- 0.04; N = 6). It is concluded that neither of these two modulators of eicosanoid activity, iloprost and daltroban, inhibit intimal hyperplasia following experimental endothelial injury.     

Effects of angiotensin II type 1 receptor blockade on the oxidative stress in spontaneously hypertensive rat tissues

The aim of this work was to investigate the production of oxidative damage in homogenized kidney, liver and brain of spontaneously hypertensive rats (SHR), as well as the involvement of angiotensin (Ang) II in this process. Groups of 12-week-old SHR and Wistar Kyoto rats (WKY) were given 10 mg/kg/day losartan in the drinking water during 14 days. Other groups of WKY and SHR without treatment were used as controls. The production of thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) and the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (Gpx) were determined. No significant difference in TBARS was observed between untreated SHR or WKY rats; GSH content was lower in the liver but higher in the brain of SHR compared to WKY rats. In tissues from the SHR group, SOD and Gpx activities were reduced, whereas CAT activity was slightly increased in kidney. TBARS levels did not change in WKY rats after losartan administration, but were reduced in SHR liver and brain. Losartan treatment decreased GSH content in WKY kidney, but increased GSH in SHR liver. The activity of the antioxidant enzymes was not modified by losartan in WKY rats; however, their activities increased in tissues from treated SHR. The lower activity of antioxidant enzymes in tissues from hypertensive rats compared to those detected in normotensive controls, indicates oxidative stress production. Ang II seems to play no role in this process in normotensive animals, although AT1 receptor blockade in SHR enhances the enzymatic activity indicating that Ang II is implicated in oxidative stress generation in the hypertensive animals.     

Vascular response to angiotensin II is exaggerated through an upregulation of AT1 receptor in AT2 knockout mice

Blood pressure is elevated and pressor response to angiotensin II (Ang II) is exaggerated in AT2 null mice. The purpose of the present study was to elucidate the mechanism for the increased responsiveness to Ang II in the mice. The contraction of aortic strips generated by Ang II was significantly greater in the AT2 gene-deleted mice than the control, which was completely abolished by AT1 antagonist losartan. The aortic content of AT1 receptor was significantly increased (P < 0.05, n = 5) in the AT2 null mice (212 +/- 58.2 fmol/mg protein) compared with the control (98.2 +/- 55.9 fmol/mg protein). While both AT1 and AT2 mRNAs were expressed in the aorta of the control mice, only AT1 mRNA was expressed in the AT2 knockout mice. The expression of AT1 mRNA in the AT2 knockout mice was significantly higher (1.5-fold, P < 0.05, n = 5) than that in the control. The present study clearly demonstrated that the increased vascular reactivity to Ang II in AT2 knockout mice is at least partly due to an increased vascular AT1 receptor expression and suggested that AT2 counteracts AT1-mediated vascular action of Ang II through downregulation of AT1 receptor by a crosstalk between these receptors by some as yet unknown mechanisms.     

Effects of losartan, a nonpeptide angiotensin II receptor antagonist, on cardiac hypertrophy and the tissue angiotensin II content in spontaneously hypertensive rats.

Losartan, a recently developed nonpeptide angiotensin II (Ang II) receptor antagonist, was administered orally to 10-week-old spontaneously hypertensive rats (SHR) for 2 weeks. Cardiac weight and tissue Ang II, as well as plasma renin activity (PRA) and Ang II, were determined. Treatment with Losartan (10 mg/kg per day) lowered blood pressure markedly. Losartan reduced significantly the left ventricular weight by 11% compared with control rats. The left ventricular Ang II content was lowered by Losartan (18.6 +/- 0.9 pg/tissue; 21.9 +/- 0.9 pg/tissue, control, p less than 0.05), whereas PRA and plasma Ang II concentration were increased by the treatment. With the control and Losartan-treated animals, there was a significant positive correlation between the left ventricular weight and the tissue Ang II content (r = 0.563, p less than 0.05). These results provide evidence that cardiac tissue Ang II, rather than circulating Ang II, plays an important role in the pathophysiology of left ventricular hypertrophy of this animal model of human hypertension.     

Angiotensin II induces nephrin dephosphorylation and podocyte injury: role of caveolin-1

Nephrin, an important structural and signal molecule of podocyte slit-diaphragm (SD), has been suggested to contribute to the angiotensin II (Ang II)-induced podocyte injury. Caveolin-1 has been demonstrated to play a crucial role in signaling transduction. In the present study, we evaluated the role of caveolin-1 in Ang II-induced nephrin phosphorylation in podocytes. Wistar rats-receiving either Ang II (400 ng/kg/min) or normal saline (via subcutaneous osmotic mini-pumps, control) were administered either vehicle or telmisartan (3 mg/kg/min) for 14 or 28 days. Blood pressure, 24-hour urinary albumin and serum biochemical profile were measured at the end of the experimental period. Renal histomorphology was evaluated through light and electron microscopy. In vitro, cultured murine podocytes were exposed to Ang II (10⁻⁶ M) pretreated with or without losartan (10⁻⁵ M) for variable time periods. Nephrin and caveolin-1 expression and their phosphorylation were analyzed by Western-blotting and immunofluorescence. Caveolar membrane fractions were isolated by sucrose density gradient centrifugation, and then the distribution and interactions between Ang II type 1 receptor (AT1), nephrin, C-terminal Src kinase (Csk) and caveolin-1 were evaluated using Western-blotting and co-immunoprecipitation. Podocyte apoptosis was evaluated by cell nucleus staining with Hoechst-33342.Ang II-receiving rats displayed diminished phosphorylation of nephrin but enhanced glomerular/podocyte injury and proteinuria when compared to control rats. Under control conditions, podocyte displayed expression of caveolin-1 in abundance but only a low level of phospho moiety. Nonetheless, Ang II stimulated caveolin-1 phosphorylation without any change in total protein expression. Nephrin and caveolin-1 were co-localized in caveolae fractions. AT1 receptors and Csk were moved to caveolae fractions and had an interaction with caveolin-1 after the stimulation with Ang II. Transfection of caveolin-1 plasmid (pEGFPC3-cav-1) significantly increased Ang II-induced nephrin dephosphorylation and podocyte apoptosis. Furthermore, knockdown of caveolin-1 expression (using siRNA) inhibited nephrin dephosphorylation and prevented Ang II-induced podocyte apoptosis. These findings indicate that Ang II induces nephrin dephosphorylation and podocyte injury through a caveolin-1-dependent mechanism.     

Angiotensin II via AT1 receptor accelerates arterial thrombosis in renovascular hypertensive rats.

Although there are some in vitro evidence that angiotensin II (Ang II) may promote thrombosis, there is still no data concerning effect of Ang II on arterial thrombus formation. In the present study we have investigated the influence of Ang II on electrically induced arterial thrombosis in a common carotid artery of renovascular hypertensive rats. Furthermore, we examined if Ang II effect is mediated via AT1 receptor. We measured some coagulation and fibrinolytic parameters at the same time. Since platelets play crucial role in the initiation of arterial thrombosis their contribution in the mode of Ang II action was also determined. Intravenous infusion of Ang II caused significant increase in arterial thrombus weight, which was reversed by losartan, selective AT1 receptor antagonist. The prothrombotic effect of Ang II was accompanied by increase in haemostatic and decrease in fibrinolytic potential of rat plasma. While number of data has clearly demonstrated that Ang II can augment human platelets aggregation, at least in rats, platelets were not involved in the mechanism of Ang II action. Our study shows that Ang II via AT1 receptor accelerates arterial thrombosis in renovascular hypertensive rat, therefore may be considered as a risk factor of myocardial infarction or stroke.     

A new inorganic vasodilator, trans-[Ru(NO)(NH(3))(4)(POEt)(3)](PF(6))(3): hypotensive effect of endothelium-dependent and -independent vasodilators in different hypertensive animals models.

The hypotensive effect of RuNO was investigated in acute and chronic hypertensive rats, as well as in normotensive rats. Acute hypertension rats were used with 30% increase on basal BP (phenylephrine, angiotensin II (Ang II), N(G)-nitro-L-arginine methyl ester (L-NAME), and adult spontaneously hypertensive rats (SHR) (basal BP 168 +/- 3 mm Hg) were used as models for chronic hypertension. Rats were implanted with catheters (iv/ia) for BP measurements and for in bolus administration of RuNO, sodium nitroprusside (SNP), and acetylcholine (Ach) (10, 20, 40 nmol/kg, iv). The principal findings of this study were: (i) The hypotensive response to RuNO was 150% higher in acutely (phenylephrine and Ang II) and chronically (SHR) hypertensive rats than in normotensive rats, except in the case of L-NAME-induced hypertension (deltaMAP = 10 +/- 1.4 mm Hg). Chronic SHR showed 60% increase (deltaMAP = 19 +/- 0.8 mm Hg) in the effect compared to normotensive rats. (ii) The hypotensive response to SNP was lower (60%) in hypertensive rats than in normotensive rats, when compared to RuNO. However, the responses were similar in L-NAME-induced hypertension (deltaMAP = 30 +/- 2 mm Hg). (iii) The vasodilator response to Ach was increased in rats with Ang II-induced hypertension (deltaMAP = 53 +/- 1 mm Hg) and in SHR (deltaMAP = 67 +/- 3 mm Hg). RuNO response was more potent than SNP in hypertensive models and the increment in relation to normotensive was observed in the phenylephrine- and L-NAME-treated rats. This response could be correlated to the different endothelial dysfunction present in each model.     

Effect of angiotensin AT2 receptor stimulation on vascular cyclic GMP production in normotensive Wistar Kyoto rats

In the present study in normotensive Wistar Kyoto rats (WKY), we investigated whether any angiotensin II (ANG II) increases in vascular cyclic GMP production were via stimulation of AT(2) receptors. Adult WKY were infused for 4h with ANG II (30 ng/kg per min, i.v.) or vehicle (0.9% NaCl, i.v.) after pretreatment with (1) vehicle, (2) losartan (100 mg/kg p.o.), (3) PD 123319 (30 mg/kg i.v.), (4) losartan+PD 123319, (5) icatibant (500 microg/kg i.v.), (6) L-NAME (1 mg/kg i.v.), (7) minoxidil (3 mg/kg i.v.). Mean arterial blood pressure (MAP) was continuously monitored, and plasma ANG II and aortic cyclic GMP were measured at the end of the study. ANG II infusion over 4h raised MAP by a mean of 13 mmHg. This effect was completely prevented by AT(1) receptor blockade. PD 123319 slightly attenuated the pressor effect induced by ANG II alone (123.4+/-0.8 versus 130.6+/-0.6) but did not alter MAP in rats treated simultaneously with ANG II + losartan (113+/-0.6 versus 114.3+/-0.8). Plasma levels of ANG II were increased 2.2-3.7-fold by ANG II infusion alone or ANG II in combination with the various drugs. The increase in plasma ANG II levels was most pronounced after ANG II+losartan treatment but absent in rats treated with losartan alone. Aortic cyclic GMP levels were not significantly changed by either treatment. Our results demonstrate that the AT(2) receptor did not contribute to the cyclic GMP production in the vascular wall of normotensive WKY.     

Angiotensin II stimulates intercellular adhesion molecule-1 via an AT1 receptor/nuclear factor-kappaB pathway in brain microvascular endothelial cells

Microvascular changes in the brain are significant causes of cerebral edema and ischemia injury. A number of studies suggest that angiotensin (Ang) II may be involved in the initiation and regulation of processes occurring in brain ischemia. We recently reported that Ang II injures brain microvascular endothelial cells (BMEC) partially via stimulating intercellular adhesion molecule-1 (ICAM-1) expression. However, the signaling cascade leading to Ang II-induced ICAM-1 expression in BMEC was unclear. The present study tested the hypothesis that Ang II induces ICAM-1 expression via an AT1 receptor/nuclear factor-kappaB (NF-kappaB) pathway in BMEC. Ang II directly stimulated the expression of ICAM-1 mRNA and protein in primary cultured BMEC. Ang II treatment also resulted in the degradation of IkappaBalpha and increase of NF-kappaB p65 subunit in the nucleus as well as the DNA binding activity of nuclear NF-kappaB. These effects were abolished by pretreatment with the selective AT1 receptor antagonists, losartan and compound EXP-2528, or losartan plus the AT2 receptor antagonist PD123319, but not by PD123319 alone. Moreover, there were no significant differences between the losartan and losartan plus PD123319 groups. These findings indicate that Ang II-induced ICAM-1 upregulation in brain microvascular endothelial cells may be mediated via an AT1 receptor/NF-kappaB pathway.     

Upregulation of LOX-1 expression in aorta of hypercholesterolemic rabbits: modulation by losartan

Angiotensin-II (Ang-II) enhances the modification of LDL and the expression of its lectin-like receptor (LOX-1) by activating type 1 (AT(1)) receptors. This study was designed to determine the effect of hypercholesterolemia on LOX-1 expression in aorta and its modulation by the AT(1) receptor blocker losartan. Male New Zealand White rabbits were fed regular chow (Control group), chow with 1% cholesterol and 4% peanut oil (HC-diet group), or 1% cholesterol and 4% peanut oil diet plus losartan (25 mg/kg/day) (Losartan + HC-diet group) for 10 weeks. Animal body weight, serum cholesterol levels, and arterial blood pressure were measured. Aortic intimal thickening was quantitated in H&E-stained segments. LOX-1 expression in aortas was examined by immunohistochemistry and semi-quantitative RT-PCR. High-cholesterol diet did not affect body weight, but induced hypercholesterolemia and extensive intimal thickening. Aortas of rabbits in the control group showed a modest LOX-1 expression in the endothelium. Aortic intimal proliferation in HC-diet group was associated with a marked increase in LOX-1 expression (protein and mRNA) in the endothelium and neointima. Treatment with losartan attenuated aortic intimal proliferation and markedly decreased the enhanced LOX-1 expression. Thus high-cholesterol diet induces the upregulation of LOX-1 expression in neointima of aortas of rabbits. Treatment with losartan, an AT(1) blocker, markedly decreases this enhanced LOX-1 expression.