V. Anania

Cardiovascular Phenotype of a Mouse Strain With Disruption of Bradykinin B2-Receptor Gene

BACKGROUND To evaluate the role of kinins in the regulation of cardiovascular function, we studied the phenotype of a mouse strain with disruption of the bradykinin B2-receptor gene (Bk 2r-/-). METHODS AND RESULTS Under basal conditions, tail-cuff blood pressure was higher in Bk2r-/- than in wild-type Bk2r+/+ and heterozygous Bk2r+/- mice (124+/-1 versus 109+/-1 and 111+/-2 mm Hg, respectively; P<.01 for both comparisons), a difference that was confirmed by measurements of intra-arterial blood pressure in unanesthetized mice. Heart weight was greater in Bk2r-/- than in Bk2r+/+ and Bk2r+/- mice (505+/-10 versus 449+/-12 and 477+/-10 mg/100 g body wt, P<.05). Chronic blockade of B2-receptors by Icatibant (50 nmol/100 g body wt twice a day S.C.) or inhibition of nitric oxide synthase by nitro-L-arginine-methyl ester (0.14 mmol/100 g body wt orally) increased the blood pressure of Bk2r+/+ to the levels of Bk2r-/- mice. Compared with the wild-type strain, both Bk2r-/- and Bk2r+/- mice showed exaggerated vasopressor responses to angiotensin II. In addition, chronic administration of an angiotensin AT1-receptor antagonist reduced the basal blood pressure of Bk2r-/- by 21+/-3 mmHg (P<.05) to the levels of Bk2r+/+. No difference was detected between strains as far as plasma renin activity and the expression of renin and AT1-receptor genes are concerned. Chronic salt loading (0.84 mmol/g chow for 15 days) increased the blood pressure of Bk2r-/- and Bk2r+/- by 34+/-3 and 14+/-6 mm Hg, respectively, whereas it was ineffective in Bk2r+/+. CONCLUSIONS Our results suggest that a normally functioning B2-receptor is essential for the maintenance of cardiovascular homeostasis in mice. Dysfunction of the kallikrein-kinin system could contribute to increase blood pressure levels by leaving the activity of vasoconstrictor agents unbalanced.

Modulation by α- and γ-tocopherol and oxidized low-density lipoprotein of apoptotic signaling in human coronary smooth muscle cells∗

Apoptosis may play an important role in atherogenesis. Oxidized low-density lipoprotein (oxLDL) promotes apoptosis in the arterial wall in addition to several other proatherogenic effects. Tocopherol supplements have been suggested to protect against coronary heart disease (CHD) in epidemiological studies. The effects of oxLDL and alpha- and gamma-tocopherol on apoptotic signaling pathways are poorly understood. Thus, the goal of the study was to investigate these pathways in the presence of copper-oxidized LDL and tocopherols in human coronary smooth muscle cells (SMC). We showed that oxLDL-mediated apoptosis, assessed by DNA fragmentation, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, and caspase activation stimulated several transcription factors and proapoptotic dynamic movements of the Bcl-2 family proteins through the mitogen-activated protein kinase (MAPK) and Jun kinase pathways. alpha-Tocopherol and gamma-tocopherol significantly reduced these molecular events and cell death effectors caspase-3 and -8. Under our experimental conditions, alpha-tocopherol was significantly more effective than gamma-tocopherol, and oxLDL-mediated apoptosis increased c-Jun, cyclic AMP-responsive element-binding, Ets-like element kinase-dependent 7, and activating transcription factor-2 proteins as well as nuclear activity of the activated protein-1 complex in human coronary SMC. Moreover, our results demonstrate that tocopherols may exert their antiatherogenic effects at least in part via reduction of the MAPK and JunK cascade together with a protective profile of apoptotic genes of the Bcl-2 family. These data are consistent with the beneficial effects of tocopherols on atherogenesis seen in experimental studies and on CHD in epidemiological surveys.

Regulation of bradykinin B2‐receptor expression by oestrogen

1 Tissue kallikrein is overexpressed in the kidney of female rats, this sexual dimorphism being associated with a greater effect of early blockade of bradykinin B2‐receptors on female blood pressure phenotype. We evaluated the effect of ovariectomy and oestradiol benzoate (50 μg kg−1 every two days for two weeks) on the vasodepressor response to intra‐arterial injection of bradykinin (150–900 ng kg−1) and on the expression of bradykinin B2‐receptors. 2 Ovariectomy reduced the magnitude of the vasodepressor response to bradykinin and unmasked a secondary vasopressor effect. Oestrogen replacement restored the vasodepressor response to bradykinin in ovariectomized rats. 3 The vasodepressor responses to sodium nitroprusside (3–18 μg kg−1), acetylcholine (30–600 ng kg−1), desArg9‐bradykinin (150–900 ng kg−1) or prostaglandin E2 (30–600 ng kg−1) were significantly reduced by ovariectomy. Oestrogen restored to normal the responses to desArg9‐bradykinin, acetylcholine and prostaglandin E2, but not that to sodium nitroprusside. 4 B2‐receptor mRNA levels were decreased by ovariectomy in the aorta and kidney and they were restored to normal levels by oestrogen. Neither ovariectomy nor oestradiol affected receptor expression in the heart and uterus. 5 These results indicate that oestrogen regulates B2‐receptor gene expression and function. Since kinins exert a cardiovascular protective action, reduction in their vasodilator activity after menopause might contribute to the increased risk of pathological cardiovascular events. Conversely, the cardioprotective effects of oestrogen replacement might be, at least in part, mediated by activation of the kallikrein‐kinin system.

Evidence for oxidative activation of c-Myc-dependent nuclear signaling in human coronary smooth muscle cells and in early lesions of Watanabe heritable hyperlipidemic rabbits: protective effects of vitamin E.

BackgroundOxidized LDL (oxLDL) promotes atherogenesis, and antioxidants reduce lesions in experimental models. OxLDL-mediated effects on c-Myc are poorly characterized, and those on c-Myc nuclear pathways are completely unknown. c-Myc stimulates smooth muscle cell (SMC) proliferation and could be involved in atherosclerosis. We investigated the early effects of oxLDL and &agr;-tocopherol on c-Myc, its binding partner Max, and the carboxy-terminal domain–binding factors activator protein-2 and elongation 2 factor in human coronary SMCs. We also investigated whether 9-week treatment of Watanabe heritable hyperlipidemic (WHHL) rabbits with diet-enriched &agr;-tocopherol reduces c-Myc expression and oxLDL in the left coronary artery. Methods and ResultsOxLDL enhanced c-Myc/Max expression and transcription by cotransfection assay and the nuclear activities of E2F and activator protein-2 by binding shift and supershift in coronary SMCs. &agr;-Tocopherol significantly reduced these molecular events. Furthermore, &agr;-tocopherol reduced early lesions, SMC density, and the immunohistochemical presence of c-Myc, which colocalized with oxLDL/foam cells in the coronaries of WHHL rabbits. ConclusionsWe provide the first evidence that oxLDL and &agr;-tocopherol may influence c-Myc activation and several c-Myc–dependent signaling pathways in human coronary SMCs. The observation that in vivo, an antioxidant reduces both c-Myc and oxLDL in early coronary lesions of rabbits is consistent with, but does not prove, the hypothesis that c-Myc–dependent factors activated by oxidative processes contribute to atherogenesis and coronary heart disease.

Early Blockade of Bradykinin B2-Receptors Alters the Adult Cardiovascular Phenotype in Rats

We evaluated whether long-term inhibition of bradykinin B2-receptors by the long-acting antagonist Hoe 140 (D-Arg,[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin) affects the blood pressure of normotensive rats. Neither Hoe 140 (at 75 nmol/d for 8 weeks) nor its vehicle altered systolic pressure of adult rats on a normal or high sodium intake. In further experiments, pairs of Hoe 140-treated rats were mated and their offspring maintained on Hoe 140 and a normal sodium diet. Controls were given vehicle instead of Hoe 140. At 9 weeks of age, rats given Hoe 140 during prenatal and postnatal phases of life showed greater systolic pressures, heart rates, and body weights than controls (122 +/- 1 versus 113 +/- 1 mm Hg, 444 +/- 6 versus 395 +/- 8 beats per minute, 258 +/- 7 versus 213 +/- 3 g, respectively, P < .01), whereas urinary creatinine excretion was reduced (1.13 +/- 0.05 versus 1.36 +/- 0.04 mumol/100 g body wt in controls, P < .05). The difference in blood pressure (confirmed by direct intra-arterial measurement) persisted after 20 days of dietary sodium loading, whereas it was nullified by sodium restriction. In additional experiments, the offspring of untreated rats received Hoe 140 or vehicle from 2 days to 11 weeks of age. At this stage, systolic pressure and body weight were significantly greater in Hoe 140-treated rats compared with controls, and heart rate was similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Hoe 140, a bradykinin B2‐receptor antagonist, on renal function in conscious normotensive rats

1 . The present study was designed to determine if endogenous kinins are involved in the regulation of arterial blood pressure and renal function in conscious rats given deoxycorticosterone enantate (DOC, 25 mg kg−1, s.c., weekly) or vehicle for two weeks. 2 . The bradykinin B2‐receptor antagonist, d‐Arg[Hyp3,Thi5,d‐Tic7,Oic8]‐bradykinin (Hoe 140), at a dose of 300 μg kg−1, s.c., blocked the hypotensive effect of 300 ng kg−1 bradykinin i.a., but it did not alter the blood pressure lowering action of 300 ng kg−1 acetylcholine or prostaglandin E2. Inhibition of the response to bradykinin persisted up to 6 h after the administration of Hoe 140. 3 . Administration of 300 μg kg−1 Hoe 140 s.c. four times a day did not alter mean blood pressure, renal blood flow, or renal function in rats given DOC‐vehicle. However, it decreased urinary volume by 70% (from 48.2 ± 3.8 to 14.3 ± 3.7 ml 24 h−1, P < 0.01) and urinary secretion of sodium by 54% (from 1.02 ± 0.05 to 0.47 ± 0.16 mmol 24 h−1, P < 0.01) and potassium by 30% (from 2.93 ± 0.15 to 2.04 ± 0.15 mmol 24 h−1, P < 0.05) in DOC‐treated rats. Mean blood pressure, glomerular filtration rate and total renal blood flow remained unchanged. 4 . Our results suggest that endogenous kinins play a role in the regulation of renal excretion of water and sodium in the presence of elevated levels of DOC.

Effect of endothelin on regional hemodynamics and renal function in awake normotensive rats

Summary: The effects of endothelin on regional hemodynamics and renal function were studied in awake normotensive rats. Intravenous injection of endothelin (700 pmol/kg) transiently lowered mean blood pressure (from 108 ± 2 to 84 ± 2 mm Hg, p < 0.01), due to a reduction in total vascular resistance (38 ± 1%, p < 0.01), and increased stroke volume (29 ± 5%, p < 0.01) and heart rate (from 399 ± 18 to 447 ± 18 bpm, p < 0.05); mesenteric and renal blood flow was reduced (37 ± 13, p < 0.05 and 63 ± 5%, p < 0.01), whereas carotid blood flow was increased (78 ± 5%, p < 0.01). This effect was followed by long-lasting hypertension due to increased total vascular resistance (112 ± 19%, p < 0.01); stroke volume, mesenteric, and renal blood flow were reduced (34 ± 5, 41 ± 4, and 58 ± 4%, respectively, p < 0.05) and carotid blood flow returned to basal levels. Bilateral nephrectomy enhanced the initial hypotensive effect. Pretreatment with nifedipine blocked the hypertensive effect, whereas bilateral nephrectomy did not. A subpressor dose of endothelin (70 pmol/kg) had no effect on stroke volume, mesenteric blood flow, glomerular filtration rate, and plasma renin activity; carotid blood flow was transiently increased (48 ± 16%, p < 0.05), then returned to basal levels; renal blood flow decreased (22 ± 6 and 15 ± 4% at 30 s and 10 min, respectively p < 0.05). Our data show a heterogeneity in the response of regional vascular beds to endothelin, the renal and mesenteric compartments being very sensitive to its vasoconstricting action. Endothelin, acting as paracrine or endocrine hormone, might have an important role in the pathogenesis of high blood pressure.

Kallikrein-Kinin System and Blood Pressure Sensitivity to Salt

We evaluated the blood pressure response to chronic salt loading in a rat strain inbred for low urinary kallikrein excretion. Low-kallikrein rats showed greater systolic blood pressure values (130 +/- 1 versus 114 +/- 2 mm Hg in controls; P < .05) at 9 weeks of age. Systolic blood pressure was increased after 10 days of dietary sodium loading in the low-kallikrein group and remained unchanged in controls (153 +/- 1 versus 112 +/- 2 mm Hg, P < .01). In additional experiments, blood pressure sensitivity to salt was tested in low-kallikrein rats receiving a chronic infusion of rat glandular kallikrein (1.7 micrograms/day per 100 g body weight, IV) or vehicle. Systolic blood pressure of vehicle-treated rats was increased by salt loading (from 138 +/- 1 to 158 +/- 2, 153 +/- 1, and 145 +/- 2 mm Hg at 5, 10, and 15 days, respectively; P < .01), while it remained unchanged in the kallikrein-treated group (from 136 +/- 2 to 146 +/- 5, 140 +/- 2, and 134 +/- 4 mm Hg at 5, 10, and 15 days, respectively; P = NS). Urinary kallikrein excretion was increased by kallikrein infusion (from 13.6 +/- 1.4 to 17.8 +/- 2.1 nanokatals per 24 hours; P < .01). Plasma immunoreactive kallikrein levels were higher in the kallikrein-treated group (66.4 +/- 4.4 versus 57.7 +/- 1.4 ng/mL in vehicle-treated rats; P < .05). On normal sodium diet, the ratio of kidney weight to body weight was lower in low-kallikrein rats (329 +/- 5 versus 370 +/- 8 mg/100 g body weight in controls; P < .01). This difference was associated with a decreased number of glomeruli per unit square area and increased width of Bowmans space. These results indicate that kallikrein replacement prevents the exaggerated blood pressure increase observed in rats with a genetically determined defect in urinary kallikrein excretion. Histological abnormalities are present at different levels in the nephron, and they may be functionally related to the altered cardiovascular and renal phenotype of this strain.

Chronic kinin receptor blockade induces hypertension in deoxycorticosterone-treated rats.

1 The contribution of endogenous kinins to the regulation of blood pressure and renal function of Wistar rats was evaluated by use of the new B2‐receptor antagonist, Hoe 140, (d‐Arg[Hyp3,Thi5,d‐Tic7,Oic8]‐bradykinin). 2 Neither Hoe 140 (4 μg h−1 s.c, for 6 weeks), nor vehicle altered systolic blood pressure (SBP, tail‐cuff plethysmography) or renal function in rats, under normal conditions. 3 Chronic administration of deoxycorticosterone (DOC, 25 mg kg−1 s.c., weekly) increased SBP slightly only after 6 weeks (from 124 ± 2 to 133 ± 3 mmHg, P < 0.05). An earlier and greater rise in SBP (P < 0.01) occurred when DOC was combined with chronic infusion of Hoe 140 (from 125 ± 1 to 154 ± 3, P < 0.01). The hypertensive effect of Hoe 140 was confirmed by direct measurement of mean blood pressure (143 ± 2 vs 122 ± 2 mmHg in controls, P < 0.01). 4 DOC caused an initial fall, followed by a transitory increase in urinary volume and sodium excretion; thereafter, both parameters returned to baseline. The initial antidiuretic and antinatriuretic effects were enhanced by Hoe 140 (P < 0.05). 5 Urinary prostaglandin E2 excretion was increased by DOC (from 106 ±3 to 153 ±4 ng 24 h−1, P < 0.01) and this effect was prevented by Hoe 140 (from 95 ± 3 to 104 ± 3 ng 24 h−1, NS). By contrast, the high urinary vasopressin excretion and suppressed plasma renin activity found in DOC‐treated rats were not altered by Hoe 140. 6 These data suggest that endogenous kinins play an important role in the regulation of arterial blood pressure under conditions of mineralocorticoid excess.

Brain kinins are responsible for the pressor effect of intracerebroventricular captopril in spontaneously hypertensive rats.

The role of the brain kallikrein-kinin system in the regulation of arterial blood pressure of normotensive and spontaneously hypertensive rats was evaluated. Intracerebroventricular administration of the kinin antagonist [DArg0]Hyp3-Thi5,8[DPhe7]bradykinin caused no change in mean blood pressure in Wistar-Kyoto, Sprague-Dawley, or spontaneously hypertensive rats. The antagonist proved to be very potent in blocking the pressor effect of intracerebroventricular bradykinin (32 +/- 3 vs. 3 +/- 1 mm Hg, p less than 0.01). It was specific, as the pressor effect induced by other unrelated peptides was similar during the infusion of either vehicle or kinin antagonist (angiotensin II, 25 +/- 4 vs. 26 +/- 2 mm Hg; prostaglandin E2, 48 +/- 3 vs. 47 +/- 8 mm Hg; norepinephrine, 17 +/- 2 vs. 18 +/- 2 mm Hg; leucine-enkephaline, 15 +/- 2 vs. 16 +/- 1 mm Hg; neurotensin, 18 +/- 2 vs. 19 +/- 1 mm Hg; substance P, 19 +/- 2 vs. 19 +/- 2 mm Hg). Intracerebroventricular administration of 1 mg captopril, an inhibitor of kininase II (one of the enzymes responsible for kinin degradation), caused no change in mean blood pressure in normotensive rats, whereas it increased mean blood pressure by 44 +/- 9 mm Hg (p less than 0.01) in spontaneously hypertensive rats. This increase in mean blood pressure was blocked and then reversed into a hypotensive effect (22 +/- 6 mm Hg, p less than 0.05) during the infusion of kinin antagonist. Our data suggest that the pressor effect induced by intracerebroventricular captopril is due to a transient elevation in endogenous brain kinin levels, supporting the hypothesis that the brain kallikrein-kinin system plays a role in the central regulation of blood pressure in spontaneously hypertensive rats.