Thu-Phuc Nguyen-Huu

Genetic Ace2 deficiency accentuates vascular inflammation and atherosclerosis in the ApoE knockout mouse.

Rationale: Angiotensin-converting enzyme (ACE)2 opposes the actions of angiotensin (Ang) II by degrading it to Ang 1-7. Objective: Given the important role of Ang II/Ang 1-7 in atherogenesis, we investigated the impact of ACE2 deficiency on the development of atherosclerosis. Methods and Results: C57Bl6, Ace2 knockout (KO), apolipoprotein E (ApoE) KO and ApoE/Ace2 double KO mice were followed until 30 weeks of age. Plaque accumulation was increased in ApoE/Ace2 double KO mice when compared to ApoE KO mice. This was associated with increased expression of adhesion molecules and inflammatory cytokines, including interleukin-6, monocyte chemoattractant protein-1, and vascular cell adhesion molecule-1, and an early increase in white cell adhesion across the whole aortae on dynamic flow assay. In the absence of a proatherosclerotic (ApoE KO) genotype, ACE2 deficiency was also associated with increased expression of these markers, suggesting that these differences were not an epiphenomenon. ACE inhibition prevented increases of these markers and atherogenesis in ApoE/ACE2 double KO mice. Bone marrow macrophages isolated from Ace2 KO mice showed increased proinflammatory responsiveness to lipopolysaccharide and Ang II when compared to macrophages isolated from C57Bl6 mice. Endothelial cells isolated from Ace2 KO mice also showed increased basal activation and elevated inflammatory responsiveness to TNF-&agr;. Similarly, selective inhibition of ACE2 with MLN-4760 also resulted in a proinflammatory phenotype with a physiological response similar to that observed with exogenous Ang II (10−7 mol/L). Conclusions: Genetic Ace2 deficiency is associated with upregulation of putative mediators of atherogenesis and enhances responsiveness to proinflammatory stimuli. In atherosclerosis-prone ApoE KO mice, these changes potentially contribute to increased plaque accumulation. These findings emphasize the potential utility of ACE2 repletion as a strategy to reduce atherosclerosis.

Role of the Sympathetic Nervous System in Schlager Genetically Hypertensive Mice

Early studies indicate that the hypertension observed in the Schlager inbred mouse strain may be attributed to a neurogenic mechanism. In this study, we examined the contribution of the sympathetic nervous system in maintaining hypertension in the BPH/2J mouse and used c-Fos immunohistochemistry to elucidate whether neuronal activation in specific brain regions was associated with waking blood pressure. Male hypertensive (BPH/2J; n=14), normotensive (BPN/3J; n=18), and C57/Bl6 (n=5) mice were implanted with telemetry devices, and after 10 days of recovery, recordings of blood pressure, heart rate, and locomotor activity were measured to determine circadian variation. Mean arterial pressure was higher in BPH/2J than in BPN/3J or C57/Bl6 mice (P<0.001), and BPH/2J animals showed exaggerated day-night differences (17±2 versus 6±1 mm Hg in BPN/3J or +8±2 mm Hg in C57/Bl6 mice; P<0.001). Acute sympathetic blockade with pentolinium (7.5 mg/kg IP) during the active and inactive phases reduced blood pressure to comparable levels in BPH/2J and BPN/3J mice. The number of c-Fos–labeled cells was greater in the amygdala (+180%; P<0.01), paraventricular nucleus (+110%; P<0.001), and dorsomedial hypothalamus (+48%; P<0.001) in the active (hypertensive) phase in BPH/2J compared with BPN/3J mice. The level of neuronal activation was mostly similar in these regions in the inactive phase. Of all of the regions studied, neuronal activation in the medial amygdala, as detected by c-Fos, was highly correlated to mean arterial pressure (r=0.98). These findings indicate that the hypertension is largely attributable to sympathetic nervous system activity, possibly generated through greater levels of arousal regulated by neurons located in the medial amygdala.

A Novel Interaction Between Sympathetic Overactivity and Aberrant Regulation of Renin by miR-181a in BPH/2J Genetically Hypertensive Mice

Genetically hypertensive mice (BPH/2J) are hypertensive because of an exaggerated contribution of the sympathetic nervous system to blood pressure. We hypothesize that an additional contribution to elevated blood pressure is via sympathetically mediated activation of the intrarenal renin–angiotensin system. Our aim was to determine the contribution of the renin–angiotensin system and sympathetic nervous system to hypertension in BPH/2J mice. BPH/2J and normotensive BPN/3J mice were preimplanted with radiotelemetry devices to measure blood pressure. Depressor responses to ganglion blocker pentolinium (5 mg/kg IP) in mice pretreated with the angiotensin-converting enzyme inhibitor enalaprilat (1.5 mg/kg IP) revealed a 2-fold greater sympathetic contribution to blood pressure in BPH/2J mice during the active and inactive period. However, the depressor response to enalaprilat was 4-fold greater in BPH/2J compared with BPN/3J mice, but only during the active period (P=0.01). This was associated with 1.6-fold higher renal renin messenger RNA (mRNA; P=0.02) and 0.8-fold lower abundance of micro-RNA-181a (P=0.03), identified previously as regulating human renin mRNA. Renin mRNA levels correlated positively with depressor responses to pentolinium (r=0.99; P=0.001), and BPH/2J mice had greater renal sympathetic innervation density as identified by tyrosine hydroxylase staining of cortical tubules. Although there is a major sympathetic contribution to hypertension in BPH/2J mice, the renin–angiotensin system also contributes, doing so to a greater extent during the active period and less during the inactive period. This is the opposite of the normal renin–angiotensin system circadian pattern. We suggest that renal hyperinnervation and enhanced sympathetically induced renin synthesis mediated by lower micro-RNA-181a contributes to hypertension in BPH/2J mice.

Angiotensin Type 1A Receptors in C1 Neurons of the Rostral Ventrolateral Medulla Modulate the Pressor Response to Aversive Stress

The rise in blood pressure during an acute aversive stress has been suggested to involve activation of angiotensin type 1A receptors (AT1ARs) at various sites within the brain, including the rostral ventrolateral medulla. In this study we examine the involvement of AT1ARs associated with a subclass of sympathetic premotor neurons of the rostral ventrolateral medulla, the C1 neurons. The distribution of putative AT1AR-expressing cells was mapped throughout the brains of three transgenic mice with a bacterial artificial chromosome-expressing green fluorescent protein under the control of the AT1AR promoter. The overall distribution correlated with that of the AT1ARs mapped by other methods and demonstrated that the majority of C1 neurons express the AT1AR. Cre-recombinase expression in C1 neurons of AT1AR-floxed mice enabled demonstration that the pressor response to microinjection of angiotensin II into the rostral ventrolateral medulla is dependent upon expression of the AT1AR in these neurons. Lentiviral-induced expression of wild-type AT1ARs in C1 neurons of global AT1AR knock-out mice, implanted with radiotelemeter devices for recording blood pressure, modulated the pressor response to aversive stress. During prolonged cage-switch stress, expression of AT1ARs in C1 neurons induced a greater sustained pressor response when compared to the control viral-injected group (22 ± 4 mmHg for AT1AR vs 10 ± 1 mmHg for GFP; p < 0.001), which was restored toward that of the wild-type group (28 ± 2 mmHg). This study demonstrates that AT1AR expression by C1 neurons is essential for the pressor response to angiotensin II and that this pathway plays an important role in the pressor response to aversive stress.

Renin-angiotensin and sympathetic nervous system contribution to high blood pressure in Schlager mice.

Objective Schlager hypertensive (BPH/2J) mice have been suggested to have high blood pressure (BP) due to an overactive sympathetic nervous system (SNS), but the contribution of the renin–angiotensin system (RAS) is unclear. In the present study, we examined the cardiovascular effects of chronically blocking the RAS in BPH/2J mice. Methods Schlager normotensive (BPN/3J, n = 6) and BPH/2J mice (n = 8) received the angiotensin AT1A-receptor antagonist losartan (150 mg/kg per day) in drinking water for 2 weeks. Pre-implanted telemetry devices were used to record mean arterial pressure (MAP), heart rate (HR) and locomotor activity. Results MAP was reduced by losartan treatment in BPN/3J (−23 mmHg, P < 0.01) and in BPH/2J mice (−25 mmHg, P < 0.001), whereas HR was increased. Losartan had little effect on initial pressor responses to feeding or to stress, but did attenuate the sustained pressor response to cage-switch stress. During the active period, the hypotension to sympathetic blockade with pentolinium was greater in BPH/2J than BPN/3J (suggesting neurogenic hypertension), but was not affected by losartan. During the inactive period, a greater depressor response to pentolinium was observed in losartan-treated animals. Conclusion The hypotensive actions of losartan suggest that although the RAS provides an important contribution to BP, it contributes little, if at all, to the hypertension-induced or the greater stress-induced pressor responses in Schlager mice. The effects of pentolinium suggest that the SNS is mainly responsible for hypertension in BPH/2J mice. However, the RAS inhibits sympathetic vasomotor tone during inactivity and prolongs sympathetic activation during periods of adverse stress, indicating an important sympatho-modulatory role.

Cardiovascular reactivity and neuronal activation to stress in Schlager genetically hypertensive mice.

Schlager inbred hypertensive mice (BPH/2J) have been suggested to have high blood pressure (BP) due to an overactive sympathetic nervous system (SNS). The brain nuclei associated with the hypertension are also those involved in the integration of the cardiovascular responses to stress. Therefore, in the present study, we hypothesize that BPH/2J mice likely have a greater response to stress that is associated with greater neuronal activation in the limbic system, hypothalamus and medulla in regions known to regulate sympathetic activity. Male hypertensive BPH/2J and normotensive BPN/3J mice were implanted with telemetry devices and exposed to dirty cage-switch, an acute model of aversive stress. Stress exposure caused a 60% greater pressor response in BPH/2J compared with BPN/3J mice and an increase in activity, by contrast the level of tachycardia was less in BPH/2J mice. Stress-induced cardiovascular responses were also associated with greater neuronal activation, as detected by c-Fos expression, in BPH/2J compared with BPN/3J mice in the medial nucleus of the amygdala (MeAm), dorsomedial hypothalamus (DMH) (P<0.001) and marginally in the rostral ventrolateral medulla (RVLM; P=0.7). These findings suggest that hypertension in the BPH/2J mice is associated with greater sympathetic vasomotor responses to central pathways mediating the arousal responses to acute aversive stress in particular the amygdala, hypothalamus and rostral ventrolateral medulla.

Endothelial cationic amino acid transporter-1 overexpression can prevent oxidative stress and increases in arterial pressure in response to superoxide dismutase inhibition in mice.

Oxidative stress may play an important role in the pathogenesis of hypertension. The aim of our study is to examine whether increased expression of the predominant endothelial l‐arginine transporter, cationic amino acid transporter‐1 (CAT1), can prevent oxidative stress‐induced hypertension.

GABAA receptor dysfunction contributes to high blood pressure and exaggerated response to stress in Schlager genetically hypertensive mice.

Objective: Schlager BPH/2J hypertensive mice have high blood pressure (BP) likely due to overactivity of the sympathetic nervous system regulated by neurons in amygdala-hypothalamic pathways. These areas are normally under tonic inhibition by GABA containing neurons that may be deficient in Schlager hypertensive mice as suggested by microarray analysis. In the present study, cardiovascular effects of chronic activation of GABAA receptors were examined in BPH/2J mice. Methods: Male normotensive BPN/3J and hypertensive BPH/2J mice were administered diazepam in drinking water for 7 days. BP, heart rate and locomotor activity were recorded by telemetry. Results: Diazepam (2.5 mg/kg) reduced BP of BPN/3J mice during the night-time by −7.1 ± 2.0 mmHg (P = 0.001) but had no effect in BPH/2J mice (+2 ± 2 mmHg) and no effect on heart rate or locomotor activity in either strain. Diazepam reduced the responses to restraint stress in BPN/3J mice by 20% (P = 0.01) and there was no association between Fos-immunoreactive neurons and neurons expressing GABAA receptors or neuropeptide Y in the medial amygdala and paraventricular nucleus of the hypothalamus. By contrast diazepam had no effect on the pressor response to stress in BPH/2J mice and ∼50% of stress-activated neurons in these regions also expressed GABAA receptors and ∼45% were neuropeptide Y-containing. Conclusion: These findings show that BPH/2J mice are resistant to the effects of diazepam and suggest that GABAA receptor dysfunction in BPH/2J mice may be contributing to the neurogenic hypertension by not suppressing arousal-induced sympathetic activation within amygdala and hypothalamic nuclei.

Major Contribution of the Medial Amygdala to Hypertension in BPH/2J Genetically Hypertensive Mice

BPH/2J mice are recognized as a neurogenic model of hypertension primarily based on overactivity of the sympathetic nervous system and greater neuronal activity in key autonomic cardiovascular regulatory brain regions. The medial amygdala (MeAm) is a forebrain region that integrates the autonomic response to stress and is the only region found to have greater Fos during the night and daytime in BPH/2J compared with BPN/3J mice. To determine the contribution of the MeAm to hypertension, the effect of neuronal ablation on blood pressure (BP) was assessed in BPH/2J (n=7) and normotensive BPN/3J mice (n=7). Mice were preimplanted with radiotelemetry devices to measure 24-hour BP and cardiovascular responses to stress, before and 1 to 3 weeks after bilateral lesions of the MeAm. Baseline BP was 121±4 mm Hg in BPH/2J and 101±2 mm Hg in BPN/3J mice (Pstrain<0.001). MeAm lesions reduced BP by 11±2 mm Hg in BPH/2J mice (Plesion<0.001) but had no effect in BPN/3J mice. The hypotensive effect of lesions in BPH/2J mice was similar during both day and night, suggesting that the MeAm has tonic effects on BP, but the pressor response to stress was maintained in both strains. Midfrequency BP power was attenuated in both strains (Plesion<0.05) and the depressor responses to pentolinium after enalaprilat pretreatment was attenuated after lesions in BPH/2J mice (Plesion<0.001; n=3). These findings indicate that the MeAm provides a tonic contribution to hypertension in BPH/2J mice, which is independent of its role in stress reactivity or circadian BP influences.

Cardiovascular role of angiotensin type1A receptors in the nucleus of the solitary tract of mice

AIMS The nucleus of the solitary tract (NTS) is important for cardiovascular regulation and contains angiotensin type 1A (AT1A) receptors. To assess its function, we examined the effect of expressing in AT1A receptors in the NTS of mice lacking these receptors. METHODS AND RESULTS Bilateral microinjections of lentivirus expressing AT1A receptors (AT1Av mice, n = 6) or green fluorescent protein (GFPv, n = 8, control) under the control of the PRSx8 promotor were made into the NTS of AT1A receptors null mice (AT1A(-/-)). Telemetry devices recorded blood pressure (BP), heart rate (HR), and locomotor activity. Expression of AT1A receptors in the NTS increased BP by 11.2 ± 4 mmHg (P < 0.05) at 2 and 3 weeks, whereas GFPv mice remained at pre-injection BP. Ganglion blockade reduced BP to similar levels pre- and post-transfection in GFPv and AT1Av mice. Greater pressor responses to cage-switch stress were observed following AT1A receptors expression (+18 ± 2 mmHg pre- to +24 ± 2 mmHg post-virus, P < 0.05) with similar stress-induced pressor responses pre- and post-virus in GFPv mice. Pressor responses to restraint stress pre- and post-virus were similar in AT1Av but were 20% less post-GFPv (P < 0.001). The lack of attenuation in BP to restraint was associated with four-fold greater Fos-expression in AT1A receptors mice. AT1A receptors expression in the NTS did not alter baroreflex gain differently between groups. CONCLUSION The results suggest that transfection of AT1A receptors on neurons in the NTS elevates BP independent of the SNS and pressor responses to aversive stimuli are associated with greater Fos-expression in forebrain regions. This study suggests a novel mechanism by which the NTS may modulate MAP in the long-term via AT1A receptors.