Increased aortic stiffness assessed by pulse wave velocity in apolipoprotein E-deficient mice

Atherosclerosis develops and progresses spontaneously in apolipoprotein E-knockout (apoE-KO) mice. A direct consequence of atherosclerosis is an increase in vascular stiffness. Pulse wave velocity (PWV) has been used to assess the stiffness of large vessels and was found to be increased in patients with atherosclerosis. In the present study, aortic stiffness was assessed by PWV in 4- and 13-mo-old apoE-KO mice and age-matched controls (C57BL/6J). In 13-mo-old apoE-KO mice with extensive atherosclerotic lesions in the aorta (61 +/- 4%), PWV increased significantly (3.8 +/- 0.2 m/s) compared with controls (2.9 +/- 0.2 m/s). Endothelial nitric oxide (EDNO)-mediated vasorelaxation in response to ACh was markedly diminished in the aortic rings isolated from 13-mo-old apoE-KO mice compared with age-matched controls. In contrast, in 4-mo-old apoE-KO mice with only moderate atherosclerotic lesions in the aorta (23 +/- 5%), there were no significant changes in PWV and EDNO-mediated relaxation compared with controls. Blood pressure was not different among the four groups of mice. There were no significant differences in endothelium-independent vascular responses to sodium nitroprusside among different groups investigated. Histological evaluation revealed focal fragmentation of the elastic laminae in the aortic walls of 13-mo-old apoE-KO mice. These results demonstrate for the first time that aortic stiffness determined by PWV increases in 13-mo-old apoE-KO mice. Endothelial dysfunction and elastic destruction in vascular wall caused by atherosclerosis may have contributed.     

Synergistic effect of angiotensin II and nitric oxide synthase inhibitor in increasing aortic stiffness in mice

Although they are implicated on their own as risk factors for cardiovascular disease, the potential link between nitric oxide (NO) deficiency, ANG II, and vascular stiffening has not been tested before. We evaluated the role of chronic ANG II treatment and NO deficiency, alone and in combination, on aortic stiffness in mice and tested parameters contributing to increases in active or passive components of vascular stiffness, including blood pressure, vascular smooth muscle contractility, and extracellular matrix components. Untreated (control) mice and mice treated with a NO synthase (NOS) inhibitor [N(omega)-nitro-L-arginine methyl ester (L-NAME), 0.5 g/l] were implanted with osmotic minipumps delivering ANG II (500 ng.kg(-1).min(-1)) for 28 days. Aortic stiffness was then measured in vivo by pulse wave velocity (PWV) and ex vivo by load-strain analysis to obtain values of maximal passive stiffness (MPS). Blood pressure and aortic contractility ex vivo were measured. ANG II treatment or NOS inhibition with L-NAME did not independently increase vascular stiffness; however, the combined treatments worked synergistically to increase PWV and MPS. The combined treatments of ANG II + L-NAME also significantly increased aortic wall collagen content while decreasing elastin. These novel results suggest that NO deficiency and ANG II act synergistically to increase aortic stiffness in mice predominantly via changes in aortic wall collagen/elastin ratio.     

Angiotensin II attenuates functional hyperemia in the mouse somatosensory cortex

We investigated whether angiotensin II (ANG II), a peptide that plays a central role in the genesis of hypertension, alters the coupling between synaptic activity and cerebral blood flow (CBF), a critical homeostatic mechanism that assures adequate cerebral perfusion to active brain regions. The somatosensory cortex was activated by stroking the facial whiskers in anesthetized C57BL/6J mice while local CBF was recorded by laser-Doppler flowmetry. Intravenous ANG II infusion (0.25 mug.kg-1.min-1) increased mean arterial pressure (MAP) from 82 +/- 2 to 102 +/- 3 mmHg (P < 0.05) without affecting resting CBF (P > 0.05). ANG II attenuated the CBF increase produced by whisker stimulation by 65% (P < 0.05) but did not affect the response to hypercapnia or to neocortical application of the nitric oxide donor S-nitroso-N-acetyl penicillamine (P > 0.05). The effect of ANG II on functional hyperemia persisted if the elevation in MAP was offset by controlled hemorrhage or prevented by topical application of the peptide to the activated cortex. ANG II did not reduce the amplitude of the P1 wave of the field potentials evoked by whisker stimulation (P > 0.05). Infusion of phenylephrine increased MAP (P > 0.05 from ANG II) but did not alter the functional hyperemic response (P > 0.05). The data suggest that ANG II alters the coupling between CBF and neural activity. The mechanisms of the effect are not related to the elevation in MAP and/or to inhibition of the synaptic activity evoked by whisker stimulation. The imbalance between CBF and neural activity induced by ANG II may alter the homeostasis of the neuronal microenvironment and contribute to brain dysfunction during ANG II-induced hypertension.     

An Interaction of Renin-Angiotensin and Kallikrein-Kinin Systems Contributes to Vascular Hypertrophy in Angiotensin II-Induced Hypertension: In Vivo and In Vitro Studies

The kallikrein-kinin and renin-angiotensin systems interact at multiple levels. In the present study, we tested the hypothesis that the B1 kinin receptor (B1R) contributes to vascular hypertrophy in angiotensin II (ANG II)–induced hypertension, through a mechanism involving reactive oxygen species (ROS) generation and extracellular signal-regulated kinase (ERK1/2) activation. Male Wistar rats were infused with vehicle (control rats), 400 ng/Kg/min ANG II (ANG II rats) or 400 ng/Kg/min ANG II plus B1 receptor antagonist, 350 ng/Kg/min des-Arg⁹-Leu⁸-bradykinin (ANGII+DAL rats), via osmotic mini-pumps (14 days) or received ANG II plus losartan (10 mg/Kg, 14 days, gavage - ANG II+LOS rats). After 14 days, ANG II rats exhibited increased systolic arterial pressure [(mmHg) 184±5.9 vs 115±2.3], aortic hypertrophy; increased ROS generation [2-hydroxyethidium/dihydroethidium (EOH/DHE): 21.8±2.7 vs 6.0±1.8] and ERK1/2 phosphorylation (% of control: 218.3±29.4 vs 100±0.25]. B1R expression was increased in aortas from ANG II and ANG II+DAL rats than in aortas from the ANG II+LOS and control groups. B1R antagonism reduced aorta hypertrophy, prevented ROS generation (EOH/DHE: 9.17±3.1) and ERK1/2 phosphorylation (137±20.7%) in ANG II rats. Cultured aortic vascular smooth muscle cells (VSMC) stimulated with low concentrations (0.1 nM) of ANG II plus B1R agonist exhibited increased ROS generation, ERK1/2 phosphorylation, proliferating-cell nuclear antigen expression and [H3]leucine incorporation. At this concentration, neither ANG II nor the B1R agonist produced any effects when tested individually. The ANG II/B1R agonist synergism was inhibited by losartan (AT1 blocker, 10 µM), B1R antagonist (10 µM) and Tiron (superoxide anion scavenger, 10 mM). These data suggest that B1R activation contributes to ANG II-induced aortic hypertrophy. This is associated with activation of redox-regulated ERK1/2 pathway that controls aortic smooth muscle cells growth. Our findings highlight an important cross-talk between the DABK and ANG II in the vascular system and contribute to a better understanding of the mechanisms involved in vascular remodeling in hypertension.     

Angiotensin II-induced hypertrophy is potentiated in mice overexpressing p22phox in vascular smooth muscle

Increased reactive oxygen species (ROS) are implicated in several vascular pathologies associated with vascular smooth muscle hypertrophy. In the current studies, we utilized transgenic (Tg) mice (Tg(p22smc)) that overexpress the p22(phox) subunit of NAD(P)H oxidase selectively in smooth muscle. These mice have a twofold increase in aortic p22(phox) expression and H(2)O(2) production and thus provide an excellent in vivo model in which to assess the effects of increased ROS generation on vascular smooth muscle cell (VSMC) function. We tested the hypothesis that overexpression of VSMC p22(phox) potentiates angiotensin II (ANG II)-induced vascular hypertrophy. Male Tg(p22smc) mice and negative littermate controls were infused with either ANG II or saline for 13 days. Baseline blood pressure was not different between control and Tg(p22smc) mice. ANG II significantly increased blood pressure in both groups, with this increase being slightly exacerbated in the Tg(p22smc) mice. Baseline aortic wall thickness and cross-sectional wall area were not different between control and Tg(p22smc) mice. Importantly, the ANG II-induced increase in both parameters was significantly greater in the Tg(p22smc) mice compared with control mice. To confirm that this potentiation of vascular hypertrophy was due to increased ROS levels, additional groups of mice were coinfused with ebselen. This treatment prevented the exacerbation of hypertrophy in Tg(p22smc) mice receiving ANG II. These data suggest that although increased availability of NAD(P)H oxidase-derived ROS is not a sufficient stimulus for hypertrophy, it does potentiate ANG II-induced vascular hypertrophy, making ROS an excellent target for intervention aimed at reducing medial thickening in vivo.     

Heart rate-associated mechanical stress impairs carotid but not cerebral artery compliance in dyslipidemic atherosclerotic mice

The cardiac cycle imposes a mechanical stress that dilates elastic carotid arteries, while shear stress largely contributes to the endothelium-dependent dilation of downstream cerebral arteries. In the presence of dyslipidemia, carotid arteries stiffen while the endothelial function declines. We reasoned that stiffening of carotid arteries would be prevented by reducing resting heart rate (HR), while improving the endothelial function would regulate cerebral artery compliance and function. Thus we treated or not 3-mo-old male atherosclerotic mice (ATX; LDLr(-/-):hApoB(+/+)) for 3 mo with the sinoatrial pacemaker current inhibitor ivabradine (IVA), the β-blocker metoprolol (METO), or subjected mice to voluntary physical training (PT). Arterial (carotid and cerebral artery) compliance and endothelium-dependent flow-mediated cerebral dilation were measured in isolated pressurized arteries. IVA and METO similarly reduced (P < 0.05) 24-h HR by ≈15%, while PT had no impact. As expected, carotid artery stiffness increased (P < 0.05) in ATX mice compared with wild-type mice, while cerebral artery stiffness decreased (P < 0.05); this paradoxical increase in cerebrovascular compliance was associated with endothelial dysfunction and an augmented metalloproteinase-9 (MMP-9) activity (P < 0.05), without changing the lipid composition of the wall. Reducing HR (IVA and METO) limited carotid artery stiffening, but plaque progression was prevented by IVA only. In contrast, IVA maintained and PT improved cerebral endothelial nitric oxide synthase-dependent flow-mediated dilation and wall compliance, and both interventions reduced MMP-9 activity (P < 0.05); METO worsened endothelial dysfunction and compliance and did not reduce MMP-9 activity. In conclusion, HR-dependent mechanical stress contributes to carotid artery wall stiffening in severely dyslipidemic mice while cerebrovascular compliance is mostly regulated by the endothelium.     

Adenosine A1-receptor knockout mice have a decreased blood pressure response to low-dose ANG II infusion

Adenosine, acting on A(1)-receptors (A(1)-AR) in the nephron, increases sodium reabsorption, and also increases renal vascular resistance (RVR), via A(1)-ARs in the afferent arteriole. ANG II increases blood pressure and RVR, and it stimulates adenosine release in the kidney. We tested the hypothesis that ANG II-infused hypertension is potentiated by A(1)-ARs' influence on Na(+) reabsorption. Mean arterial pressure (MAP) was measured by radiotelemetry in A(1)-AR knockout mice (KO) and their wild-type (WT) controls, before and during ANG II (400 ng·kg(-1)·min(-1)) infusion. Baseline MAP was not different between groups. ANG II increased MAP in both groups, but on day 12, MAP was lower in A(1)-AR KO mice (KO: 128 ± 3 vs. 139 ± 3 mmHg, P < 0.01). Heart rates were significantly different during days 11-14 of ANG II. Basal sodium excretion was not different (KO: 0.15 ± 0.03 vs. WT: 0.13 ± 0.04 mmol/day, not significant) but was higher in KO mice 12 days after ANG II despite a lower MAP (KO: 0.22 ± 0.03 vs. WT: 0.11 ± 0.02 mmol/day, P < 0.05). Phosphate excretion was also higher in A(1)-AR KO mice on day 12. Renal expression of the sodium-dependent phosphate transporter and the Na(+)/glucose cotransporter were lower in the KO mice during ANG II treatment, but the expression of the sodium hydrogen exchanger isoform 3 was not different. These results indicate that the increase in blood pressure seen in A(1)-AR KO mice is lower than that seen in WT mice but was increased by ANG II nonetheless. The presence of A(1)-ARs during a low dose of ANG II-infusion limits Na(+) and phosphate excretion. This study suggests that A(1)-AR antagonists might be an effective antihypertensive agent during ANG II and volume-dependent hypertension.     

Nitrite supplementation reverses vascular endothelial dysfunction and large elastic artery stiffness with aging

We tested the hypothesis that short-term nitrite therapy reverses vascular endothelial dysfunction and large elastic artery stiffening with aging, and reduces arterial oxidative stress and inflammation. Nitrite concentrations were lower (P < 0.05) in arteries, heart, and plasma of old (26-28 month) male C57BL6 control mice, and 3 weeks of sodium nitrite (50 mg L(-1) in drinking water) restored nitrite levels to or above young (4-6 month) controls. Isolated carotid arteries of old control mice had lower acetylcholine (ACh)-induced endothelium-dependent dilation (EDD) (71.7 ± 6.1% vs. 93.0 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (P < 0.05 vs. young), and sodium nitrite restored EDD (95.5 ± 1.6%) by increasing NO bioavailability. 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), a superoxide dismutase (SOD) mimetic, apocynin, a nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, and sepiapterin (exogenous tetrahydrobiopterin) each restored EDD to ACh in old control, but had no effect in old nitrite-supplemented mice. Old control mice had increased aortic pulse wave velocity (478 ± 16 vs. 332 ± 12 AU, P < 0.05 vs. young), which nitrite supplementation lowered (384 ± 27 AU). Nitrotyrosine, superoxide production, and expression of NADPH oxidase were ～100-300% greater and SOD activity was ～50% lower in old control mice (all P < 0.05 vs. young), but were ameliorated by sodium nitrite treatment. Inflammatory cytokines were markedly increased in old control mice (P < 0.05), but reduced to levels of young controls with nitrite supplementation. Short-term nitrite therapy reverses age-associated vascular endothelial dysfunction, large elastic artery stiffness, oxidative stress, and inflammation. Sodium nitrite may be a novel therapy for treating arterial aging in humans.     

ANG II increases TIMP-1 expression in rat aortic smooth muscle cells in vivo

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tissue remodeling processes. TIMP-1 is the main native inhibitor of MMPs and it contributes to the development of tissue fibrosis. It is known that ANG II plays a fundamental role in vascular remodeling. In this study, we investigated whether ANG II modulates TIMP-1 expression in rat aortic smooth muscle cells. In vitro, ANG II induces TIMP-1 mRNA expression in a dose-dependent manner. The maximal increase in TIMP-1 expression was present after 3 h of ANG II stimulation. The ANG II increase in TIMP-1 expression was mediated by the ANG type 1 receptors because it was blocked by losartan. The increase in TIMP-1 expression was present after the first ANG II treatment, whereas repeated treatments (3 and 5 times) did not modify TIMP-1 expression. In vivo, exogenous ANG II was administered to Sprague-Dawley rats (200 ng. kg(-1). min(-1) sc) for 6 and 25 days. Control rats received physiological saline. After treatment, systolic blood pressure was significantly higher (P < 0.01), whereas plasma renin activity was suppressed (P < 0.01), in ANG II-treated rats. ANG II increased TIMP-1 expression in the aorta of ANG II-treated rats both at the mRNA (P < 0.05) and protein levels as evaluated by Western blotting (P < 0.05) and/or immunohistochemistry. Neither histological modifications at the vascular wall nor differences in collagen content in the tunica media were present in both the ANG II- and saline-treated groups. Our data demonstrate that ANG II increases TIMP-1 expression in rat aortic smooth muscle cells. In vivo, both short- and long-term chronic ANG II treatments increase TIMP-1 expression in the rat aorta. TIMP-1 induction by ANG II in aortic smooth muscle cells occurs in the absence of histological changes at the vascular wall.     

Sex differences in the development of angiotensin II-induced hypertension in conscious mice

Sex has an important influence on blood pressure (BP) regulation. There is increasing evidence that sex hormones interfere with the renin-angiotensin system. Thus the purpose of this study was to determine whether there are sex differences in the development of ANG II-induced hypertension in conscious male and female mice. We used telemetry implants to measure aortic BP and heart rate (HR) in conscious, freely moving animals. ANG II (800 ng.kg(-1).min(-1)) was delivered via an osmotic pump implanted subcutaneously. Our results showed baseline BP in male and female mice to be similar. Chronic systemic infusion of ANG II induced a greater increase in BP in male (35.1 +/- 5.7 mmHg) than in female mice (7.2 +/- 2.0 mmHg). Gonadectomy attenuated ANG II-induced hypertension in male mice (15.2 +/- 2.4 mmHg) and augmented it in female mice (23.1 +/- 1.0 mmHg). Baseline HR was significantly higher in females relative to males (630.1 +/- 7.9 vs. 544.8 +/- 16.2 beats/min). In females, ANG II infusion significantly decreased HR. However, the increase in BP with ANG II did not result in the expected decrease in HR in either intact male or gonadectomized mice. Moreover, the slope of the baroreflex bradycardia to phenylephrine was blunted in males (-5.6 +/- 0.3 to -2.9 +/- 0.5) but not in females (-6.5 +/- 0.5 to -5.6 +/- 0.3) during infusion of ANG II, suggesting that, in male mice, infusion of ANG II results in a resetting of the baroreflex control of HR. Ganglionic blockade resulted in greater reduction in BP on day 7 after ANG II infusion in males compared with females (-61.0 +/- 8.9 vs. -36.6 +/- 6.6 mmHg), suggesting an increased contribution of sympathetic nerve activity in arterial BP maintenance in male mice. Together, these data indicate that there are sex differences in the development of chronic ANG II-induced hypertension in conscious mice and that females may be protected from the increases in BP induced by ANG II.     

Chronic angiotensin II infusion attenuates the renal sympathoinhibitory response to acute volume expansion

In this study the hypothesis was tested that chronic infusion of ANG II attenuates acute volume expansion (VE)-induced inhibition of renal sympathetic nerve activity (SNA). Rats received intravenous infusion of either vehicle or ANG II (12 ng. kg(-1). min(-1)) for 7 days. ANG II-infused animals displayed an increased contribution of SNA to the maintenance of mean arterial pressure (MAP) as indicated by ganglionic blockade, which produced a significantly (P < 0.01) greater decrease in MAP (75 +/- 3 mmHg) than was observed in vehicle-infused (47 +/- 8 mmHg) controls. Rats were then anesthetized, and changes in MAP, mean right atrial pressure (MRAP), heart rate (HR), and renal SNA were recorded in response to right atrial infusion of isotonic saline (20% estimated blood volume in 5 min). Baseline MAP, HR, and hematocrit were not different between groups. Likewise, MAP was unchanged by acute VE in vehicle-infused animals, whereas VE induced a significant bradycardia (P < 0.05) and increase in MRAP (P < 0.05). MAP, MRAP, and HR responses to VE were not statistically different between animals infused with vehicle vs. ANG II. In contrast, VE significantly (P < 0.001) reduced renal SNA by 33.5 +/- 8% in vehicle-infused animals but was without effect on renal SNA in those infused chronically with ANG II. Acutely administered losartan (3 mg/kg iv) restored VE-induced inhibition of renal SNA (P < 0.001) in rats chronically infused with ANG II. In contrast, this treatment had no effect in the vehicle-infused group. Therefore, it appears that chronic infusion of ANG II can attenuate VE-induced renal sympathoinhibition through a mechanism requiring AT(1) receptor activation. The attenuated sympathoinhibitory response to VE in ANG II-infused animals remained after arterial barodenervation and systemic vasopressin V(1) receptor antagonism and appeared to depend on ANG II being chronically increased because ANG II given acutely had no effect on VE-induced renal sympathoinhibition.     

Xenogenic macrophage immunization reduces atherosclerosis in apolipoprotein E knockout mice

Atherosclerosis is a complex chronic inflammatory disease in which macrophages play a critical role, and the intervention of the inflammatory process in atherogenesis could be a therapeutic strategy. In this study, we investigated the efficacy of xenogenic macrophage immunization on the atherosclerotic lesion formation in a model of murine atherosclerosis. Apolipoprotein E knockout (apoE-KO) mice were repeatedly immunized with formaldehyde-fixed cultured human macrophages (phorbol ester-stimulated THP-1 cells), using human serum albumin as a control protein or HepG2 cells as human control cells, once a week for four consecutive weeks. The vehicle phosphate-buffered saline was injected in the nonimmunized controls. THP-1 immunization induced antibodies that are immunoreactive with mouse macrophages. Although the plasma lipid levels were unchanged by the immunization, the atherosclerotic lesion area in the aortic root was significantly reduced by >50% in 16-wk-old THP-1-immunized apoE-KO mice compared with that in control mice. THP-1 immunization reduced in vivo macrophage infiltration, reduced in vitro macrophage adhesion, and changed cytokine production by macrophages to the antiatherogenic phenotype. Xenogenic macrophage immunization protects against the development of atherosclerosis in apoE-KO mice by modulating macrophage function in which antibodies induced by the immunization are likely to be involved. This method is a novel and potentially useful cell-mediated immune therapeutic technique against atherosclerosis. antibody; THP-1 cells     

Central stiffening in adulthood linked to aberrant aortic remodeling under suboptimal intrauterine conditions

This study examined perturbed aortic development and subsequent wall stiffening as a link to later cardiovascular disease. Placental insufficiency was induced in pregnant guinea pigs at midgestation by uterine artery ligation. Near term, fetuses were killed and defined as normal birth weight (NBW), low birth weight (LBW), and intrauterine growth restricted (IUGR). Offspring were classified according to birth weight and killed in adulthood. Collagen and elastin content of aortas were analyzed using Sirius red and orcein staining, respectively. Immunofluorescence was used for detection of α-actin and nonmuscle myosin heavy chain (MHC-B), a marker of synthetic-type vascular smooth muscle cells (VSMCs). Ex vivo generation of length-tension curves was performed with aortic rings from adult offspring. Relative elastic fiber content was decreased by 10% in LBW and 14% in IUGR compared with NBW fetuses. In adulthood, relative elastic fiber content was 51% lower in LBW vs. NBW, and the number of elastic laminae adjusted for wall thickness was 25% lower in LBW (P < 0.01). The percent area stained for MHC-B was sixfold higher in LBW vs. NBW fetuses (P < 0.0001) and threefold higher in LBW vs. NBW adult offspring (P < 0.05). The increase in MHC-B in LBW offspring concurred with a 41% increase in total collagen content and a 33 and 56% increase in relative and total α-actin content, respectively (P < 0.05). Thus aortic wall stiffening in adulthood can be traced to altered matrix composition established under suboptimal intrauterine conditions that is amplified postnatally by the activity of synthetic VSMCs.     

Microvascular rarefaction and decreased angiogenesis in rats with fetal programming of hypertension associated with exposure to a low-protein diet in utero

In hypertension, increased peripheral vascular resistance results from vascular dysfunction with or without structural changes (vessel wall remodeling and/or microvascular rarefaction). Humans with lower birth weight exhibit evidence of vascular dysfunction. The current studies were undertaken to investigate whether in utero programming of hypertension is associated with in vivo altered response and/or abnormal vascular structure. Offspring of Wistar dams fed a normal (CTRL) or low (LP)-protein diet during gestation were studied. Mean arterial blood pressure response to ANG II was significantly increased, and depressor response to sodium nitroprusside (SNP) infusions significantly decreased in male LP adult offspring relative to CTRL. No arterial remodeling was observed in male LP compared with CTRL offspring. Capillary and arteriolar density was significantly decreased in striated muscles from LP offspring at 7 and 28 days of life but was not different in late fetal life [day 21 of gestation (E21)]. Angiogenic potential of aortic rings from LP newborn (day of birth, P0) was significantly decreased. Striated muscle expressions (Western blots) of ANG II AT(1) receptor subtype, endothelial nitric oxide synthase, angiopoietin 1 and 2, Tie 2 receptor, vascular endothelial growth factor and receptor, and platelet-derived growth factor C at E21 and P7 were unaltered by antenatal diet exposure. In conclusion, blood pressure responses to ANG II and SNP are altered, and microvascular structural changes prevail in this model of fetal programming of hypertension. The capillary rarefaction is absent in the fetus and appears in the neonatal period, in association with decreased angiogenic potential. The study suggests that intrauterine protein restriction increases susceptibility to postnatal factors resulting in microvascular rarefaction, which could represent a primary event in the genesis of hypertension.     

Superoxide-lowering therapy with TEMPOL reverses arterial dysfunction with aging in mice

To test the hypothesis that the antioxidant enzyme superoxide dismutase (SOD) mimetic TEMPOL improves arterial aging, young (Y, 4-6 months) and old (O, 26-28 months) male C57BL6 mice received regular or TEMPOL-supplemented (1mM) drinking water for 3 weeks (n = 8 per group). Aortic superoxide was 65% greater in O (P < 0.05 vs. Y), which was normalized by TEMPOL. O had large elastic artery stiffening, as indicated by greater aortic pulse wave velocity (aPWV, 508 ± 22 vs. 418 ± 22 AU), which was associated with increased adventitial collagen I expression (P < 0.05 vs. Y). TEMPOL reversed the age-associated increases in aPWV (434 ± 21 AU) and collagen in vivo, and SOD reversed the increases in collagen I in adventitial fibroblasts from older rats in vitro. Isolated carotid arteries of O had impaired endothelial function as indicated by reduced acetylcholine-stimulated endothelium-dependent dilation (EDD) (75.6 ± 3.2 vs. 94.5 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (L-NAME) associated with decreased endothelial NO synthase (eNOS) expression (P < 0.05 vs. Y). TEMPOL restored EDD (94.5 ± 1.4%), NO bioavailability and eNOS in O. Nitrotyrosine and expression of NADPH oxidase were ~100-200% greater, and MnSOD was ~75% lower in O (P < 0.05 vs. Y). TEMPOL normalized nitrotyrosine and NADPH oxidase in O, without affecting MnSOD. Aortic pro-inflammatory cytokines were greater in O (P < 0.05 vs. Y) and normalized by TEMPOL. Short-term treatment of excessive superoxide with TEMPOL ameliorates large elastic artery stiffening and endothelial dysfunction with aging, and this is associated with normalization of arterial collagen I, eNOS, oxidative stress, and inflammation.     

Role of superoxide anion in regulating pressor and vascular hypertrophic response to angiotensin II

Our purpose was to address the role of NAPDH oxidase-derived superoxide anion in the vascular response to ANG II. Blood pressure, aortic superoxide anion, 3-nitrotyrosine, and medial cross-sectional area were compared in wild-type mice and in mice that overexpress human superoxide dismutase (hSOD). The pressor response to ANG II was significantly less in hSOD mice. Superoxide anion levels were increased twofold in ANG II-treated wild-type mice but not in hSOD mice. 3-Nitrotyrosine increased in aortic endothelium and adventitia in wild-type but not hSOD mice. In contrast, aortic medial cross-sectional area increased 50% with ANG II in hSOD mice, comparable to wild-type mice. The lower pressor response to ANG II in the mice expressing hSOD is consistent with a pressor role of superoxide anion in wild-type mice, most likely because it reacts with nitric oxide. Despite preventing the increase in superoxide anion and 3-nitrotyrosine, the aortic hypertrophic response to ANG II in vivo was unaffected by hSOD.     

Angiotensin II-induced reduction in exercise capacity is associated with increased oxidative stress in skeletal muscle

Angiotensin II (ANG II)-induced oxidative stress has been known to be involved in the pathogenesis of cardiovascular diseases. We have reported that the oxidative stress in skeletal muscle can limit exercise capacity in mice (16). We thus hypothesized that ANG II could impair the skeletal muscle energy metabolism and limit exercise capacity via enhancing oxidative stress. ANG II (50 ng·kg(-1)·min(-1)) or vehicle was infused into male C57BL/6J mice for 7 days via subcutaneously implanted osmotic minipumps. ANG II did not alter body weight, skeletal muscle weight, blood pressure, cardiac structure, or function. Mice were treadmill tested, and expired gases were analyzed. The work to exhaustion (vertical distance × body weight) and peak oxygen uptake were significantly decreased in ANG II compared with vehicle. In mitochondria isolated from skeletal muscle, ADP-dependent respiration was comparable between ANG II and vehicle, but ADP-independent respiration was significantly increased in ANG II. Furthermore, complex I and III activities were decreased in ANG II. NAD(P)H oxidase activity and superoxide production by lucigenin chemiluminescence were significantly increased in skeletal muscle from ANG II mice. Treatment of ANG II mice with apocynin (10 mmol/l in drinking water), an inhibitor of NAD(P)H oxidase activation, completely inhibited NAD(P)H oxidase activity and improved exercise capacity, mitochondrial respiration, and complex activities in skeletal muscle. ANG II-induced oxidative stress can impair mitochondrial respiration in skeletal muscle and limit exercise capacity.     

Lack of microsomal prostaglandin E synthase-1 reduces cardiac function following angiotensin II infusion

Our laboratory previously reported that inducible PGE(2) synthase, mPGES-1, contributes to micromolar production of PGE(2) in neonatal ventricular myocytes in vitro, which stimulates their growth. We therefore hypothesized that mPGES-1 contributes to cardiac hypertrophy following angiotensin II (ANG II) infusion. To test this hypothesis, we used 10- to 12-wk-old mPGES-1 knockout mice (mPGES-1 KO) and C57Bl/6 control mice infused for 8 wk with either 1.4 mg · kg(-1) · day(-1) ANG II or vehicle subcutaneously. Blood pressure [systolic blood pressure (SBP)] was measured throughout the study, and cardiac function was assessed by M-mode echocardiography at baseline and at 8 wk of infusion. At the conclusion of the study, immunohistochemistry was used to evaluate collagen fraction, myocyte cross-sectional area (MCSA), and apoptosis. At baseline, there was no difference in SBP between mPGES-1 KO mice and C57BL/6 controls. ANG II infusion increased SBP to similar levels in both strains. In control mice, infusion of ANG II increased MCSA and posterior wall thickness at diastole (PWTd) but had little effect on cardiac function, consistent with compensatory hypertrophy. In contrast, cardiac function was worse in mPGES-1 KO mice after ANG II treatment. Ejection fraction declined from 76.2 ± 2.7 to 63.3 ± 3.4% after ANG II, and left ventricular dimension at systole and diastole increased from 1.29 ± 0.02 to 1.78 ± 0.15 mm and from 2.57 ± 0.03 to 2.90 ± 0.13 mm, respectively. Infusion of ANG II increased both the LV-to-body weight and the mass-to-body weight ratios to a similar extent in both strains. However, PWTd increased by a lesser extent in KO mice, suggesting an impaired hypertrophic response. ANG II infusion increased collagen staining similarly in both strains, but TdT-dUTP nick end labeling staining was greater in mPGES-1 KO mice. Overall, these results are consistent with a beneficial effect for mPGES-1 in the maintenance of cardiac function in ANG II-dependent hypertension.     

Enhanced renal expression of preproendothelin mRNA during chronic angiotensin II hypertension

The purpose of this study was to determine the role of endothelin in mediating the renal hemodynamic and arterial pressure changes observed during chronic ANG II-induced hypertension. ANG II (50 ng x kg(-1) x min(-1)) was chronically infused into the jugular vein by miniosmotic pump for 2 wk in male Sprague-Dawley rats with and without endothelin type A (ET(A))-receptor antagonist ABT-627 (5 mg x kg(-1) x day(-1)) pretreatment. Arterial pressure increased in ANG II rats compared with control rats (149 +/- 5 vs. 121 +/- 6 mmHg, P < 0.05, respectively). Renal expression of preproendothelin mRNA was increased by approximately 50% in both the medulla and cortex of ANG II rats. The hypertensive effect of ANG II was completely abolished in rats pretreated with the ET(A)-receptor antagonist (114 +/- 5 mmHg, P < 0.05). Glomerular filtration rate was decreased by 33% in ANG II rats, and this response was attenuated in rats pretreated with ET(A)-receptor antagonist. These data indicate that activation of the renal endothelin system by ANG II may play an important role in mediating chronic renal and hypertensive actions of ANG II.     

Role of alpha1beta1-integrin in arterial stiffness and angiotensin-induced arterial wall hypertrophy in mice

We examined the arterial phenotype of mice lacking alpha(1)-integrin (alpha(1)(-/-)) at baseline and after 4 wk of ANG II or norepinephrine (NE) administration. Arterial mechanical properties were determined in the carotid artery (CA). Integrin expression, MAPK kinases, and focal adhesion kinase (FAK) were assessed in the aorta. No change in arterial pressure was observed in alpha(1)(-/-) mice. Elastic modulus-wall stress curves were similar in alpha(1)(-/-) and alpha(1)(+/+) animals, indicating no change in arterial stiffness. The rupture pressure was lower in alpha(1)(-/-) mice, demonstrating decreased mechanical strength. Lack of alpha(1)-integrin was accompanied by an increase in beta(1)-, alpha(v)-, and alpha(5)-integrins but no change in alpha(2)-integrin. ANG II increased medial cross-sectional area of the CA in alpha(1)(+/+), but not alpha(1)(-/-), mice, whereas equivalent pressor doses of NE did not produce a significant increase in either group. In alpha(1)(+/+) mice, ANG II induced alpha(1)-integrin expression and smooth muscle cell (SMC) hypertrophy in the CA in association with increased aortic expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain and phosphorylation of ERK1/2, p38 MAPK, and FAK. ANG II did not induce SMC hypertrophy or phosphorylation of p38 MAPK and FAK in alpha(1)(-/-) mice. A functional anti-alpha(1)-integrin antibody inhibited in vitro the ANG II-induced phosphorylation of FAK and p38 MAPK. In conclusion, alpha(1)(-/-) mice exhibit a reduced mechanical strength at baseline and a lack of ANG II-induced SMC hypertrophy. These results emphasize the importance of alpha(1)beta(1)-integrin in p38 MAPK and FAK phosphorylation during vascular hypertrophy in response to ANG II.