Daian Chen

Role of Angiotensin II Type 1A Receptors in Cardiovascular Reactivity and Neuronal Activation After Aversive Stress in Mice

We determined whether genetic deficiency of angiotensin II Type 1A (AT1A) receptors in mice results in altered neuronal responsiveness and reduced cardiovascular reactivity to stress. Telemetry devices were used to measure mean arterial pressure, heart rate, and activity. Before stress, lower resting mean arterial pressure was recorded in AT1A−/− (85±2 mm Hg) than in AT1A+/+ (112±2 mm Hg) mice; heart rate was not different between groups. Cage-switch stress for 90 minutes elevated blood pressure by +24±2 mm Hg in AT1A+/+ and +17±2 mm Hg in AT1A−/− mice (P<0.01), and heart rate increased by +203±9 bpm in AT1A+/+ and +121±9 bpm in AT1A−/− mice (P<0.001). Locomotor activation was less in AT1A−/− (3.0±0.4 U) than in AT1A+/+ animals (6.0±0.4 U), but differences in blood pressure and heart rate persisted during nonactive periods. In contrast to wild-type mice, spontaneous baroreflex sensitivity was not inhibited by stress in AT1A−/− mice. After cage-switch stress, c-Fos immunoreactivity was less in the paraventricular (P<0.001) and dorsomedial (P=0.001) nuclei of the hypothalamus and rostral ventrolateral medulla (P<0.001) in AT1A−/− compared with AT1A+/+ mice. Conversely, greater c-Fos immunoreactivity was observed in the medial nucleus of the amygdala, caudal ventrolateral medulla, and nucleus of the solitary tract (P<0.001) of AT1A−/− compared with AT1A+/+ mice. Greater activation of the amygdala suggests that AT1A receptors normally inhibit the degree of stress-induced anxiety, whereas the lesser activation of the hypothalamus and rostral ventrolateral medulla suggests that AT1A receptors play a key role in autonomic cardiovascular reactions to acute aversive stress, as well as for stress-induced inhibition of the baroreflex.

Type 1 angiotensin receptors on macrophages ameliorate IL-1 receptor–mediated kidney fibrosis

In a wide array of kidney diseases, type 1 angiotensin (AT1) receptors are present on the immune cells that infiltrate the renal interstitium. Here, we examined the actions of AT1 receptors on macrophages in progressive renal fibrosis and found that macrophage-specific AT1 receptor deficiency exacerbates kidney fibrosis induced by unilateral ureteral obstruction (UUO). Macrophages isolated from obstructed kidneys of mice lacking AT1 receptors solely on macrophages had heightened expression of proinflammatory M1 cytokines, including IL-1. Evaluation of isolated AT1 receptor-deficient macrophages confirmed the propensity of these cells to produce exaggerated levels of M1 cytokines, which led to more severe renal epithelial cell damage via IL-1 receptor activation in coculture compared with WT macrophages. A murine kidney crosstransplantation concomitant with UUO model revealed that augmentation of renal fibrosis instigated by AT1 receptor-deficient macrophages is mediated by IL-1 receptor stimulation in the kidney. This study indicates that a key role of AT1 receptors on macrophages is to protect the kidney from fibrosis by limiting activation of IL-1 receptors in the kidney.

Expression of angiotensin type 1A receptors in C1 neurons restores the sympathoexcitation to angiotensin in the rostral ventrolateral medulla of angiotensin type 1A knockout mice.

In adult mice we determined whether expression of angiotensin II (Ang II) type 1A receptors (AT1ARs) in C1 neurons mediates the excitation of the rostral ventrolateral medulla (RVLM) by Ang II. Blood pressure, heart rate, and sympathetic nerve activity were measured in anesthetized, artificially ventilated wild-type (n=15) and AT1AR knockout (AT1A−/−; n=9) mice. Microinjection of Ang II (50 nL of 0.1 to 1.0 mmol/L) into the RVLM induced a dose-related, sympathetically mediated pressor response (maximum of 17±2 mm Hg) in wild-type mice. These microinjections had no effect in AT1A−/− mice. Endogenous AT1Rs occur on catecholaminergic C1 neurons in the RVLM. We induced AT1AR or green fluorescent protein expression in C1 neurons of AT1A−/− mice through bilateral microinjection of replication-deficient lentiviruses, with transgene expression under the control of a phox2 transcription factor binding promoter (PRSx8) (Lv-PRSx8-AT1A, n=10, and Lv-PRSx8-GFP, n=5). Transgene expression was observed in a significant proportion of RVLM C1 neurons. In anesthetized Lv-PRSx8-AT1A injected mice, unilateral RVLM microinjection of Ang II (50 nL of 1 mmol/L) increased blood pressure (17±4 mm Hg) and sympathetic nerve activity (155±32%). No response to Ang II occurred in Lv-PRSx8-GFP microinjected mice. These results show that Ang II–mediated excitation of RVLM neurons in adult mice depends on the AT1AR with little or no effect of type 1B or 2 receptors. Expression of the AT1AR predominantly in C1 catecholamine neurons restores the response to Ang II in the AT1A−/− mouse and demonstrates that these neurons are sympathoexcitatory in the mouse.

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.

Central Neural Regulation of Cardiovascular Function by Angiotensin: A Focus on the Rostral Ventrolateral Medulla

Angiotensin II acts through specific receptors to alter several brain functions including fluid and electrolyte control, neuroendocrine function and autonomic efferent activity. This review discusses one brain site, the rostral ventrolateral medulla, where the actions of angiotensin II have been intensively studied. The rostral ventrolateral medulla plays a critical role in the generation and regulation of sympathetic activity to the cardiovascular system and hence is important for blood pressure control. Current evidence indicates that angiotensin II activates neurons in the rostral ventrolateral medulla via the AT1A receptor. In some models of cardiovascular disease, blockade of AT1 receptors in the rostral ventrolateral medulla reduces sympathetic nerve activity and blood pressure suggesting that overactivity of the angiotensin system in this nucleus may play a role in the maintenance of high blood pressure.

Blood pressure reactivity to emotional stress is reduced in AT1A-receptor knockout mice on normal, but not high salt intake

Pharmacological evidence suggests that angiotensin II type 1 (AT1) receptors are involved in the regulation of cardiovascular response to emotional stress and reinforcing effect of dietary salt on this response. In this study, we examined the effect of genetic deletion of AT1A receptors on the cardiovascular effects of stress and salt in mice. AT1A receptor knockout (AT1A−/−) and wild-type (AT1A+/+) mice were implanted with telemetry devices and placed on a normal (0.4%) or high (3.1%) salt diet (HSD). Resting blood pressure (BP) in AT1A−/− mice (84±3 mm Hg) was lower than in AT1A+/+ mice (107±2 mm Hg). Negative emotional (restraint) stress increased BP by 33±3 mm Hg in AT1A+/+ mice. This response was attenuated by 40% in AT1A−/− mice (18±3 mm Hg). Conversely, the BP increase caused by food presentation and feeding was similar in AT1A−/− (25±3 mm Hg) and AT1A+/+ mice (26±3 mm Hg). HSD increased resting BP by 14±4 mm Hg in AT1A−/− mice without affecting it significantly in AT1A+/+ mice. Under these conditions, the pressor response to restraint stress in AT1A−/− mice (30±3 mm Hg) was no longer different from that in wild-type animals (28±3 mm Hg). The BP response to feeding was not altered by HSD in either AT1A−/− or AT1A+/+ mice (25±2 and 27±3 mm Hg, respectively). These results indicate that AT1A receptor deficiency leads to a reduction in BP reactivity to negative emotional stress, but not feeding. HSD can selectively reinforce the cardiovascular response to negative stress in AT1A−/− mice. However, there is little interaction between AT1A receptors, excess dietary sodium and feeding-induced cardiovascular arousal.

Vascular Type 1A Angiotensin II Receptors Control BP by Regulating Renal Blood Flow and Urinary Sodium Excretion

Inappropriate activation of the type 1A angiotensin (AT1A) receptor contributes to the pathogenesis of hypertension and its associated complications. To define the role for actions of vascular AT1A receptors in BP regulation and hypertension pathogenesis, we generated mice with cell-specific deletion of AT1A receptors in smooth muscle cells (SMKO mice) using Loxp technology and Cre transgenes with robust expression in both conductance and resistance arteries. We found that elimination of AT1A receptors from vascular smooth muscle cells (VSMCs) caused a modest (approximately 7 mmHg) yet significant reduction in baseline BP and exaggerated sodium sensitivity in mice. Additionally, the severity of angiotensin II (Ang II)-dependent hypertension was dramatically attenuated in SMKO mice, and this protection against hypertension was associated with enhanced urinary excretion of sodium. Despite the lower BP, acute vasoconstrictor responses to Ang II in the systemic vasculature were largely preserved (approximately 80% of control levels) in SMKO mice because of exaggerated activity of the sympathetic nervous system rather than residual actions of AT1B receptors. In contrast, Ang II-dependent responses in the renal circulation were almost completely eliminated in SMKO mice (approximately 5%-10% of control levels). These findings suggest that direct actions of AT1A receptors in VSMCs are essential for regulation of renal blood flow by Ang II and highlight the capacity of Ang II-dependent vascular responses in the kidney to effect natriuresis and BP control.

Stimulation of Angiotensin Type 1A Receptors on Catecholaminergic Cells Contributes to Angiotensin-Dependent Hypertension

Hypertension contributes to multiple forms of cardiovascular disease and thus morbidity and mortality. The mechanisms inducing hypertension remain unclear although the involvement of homeostatic systems, such as the renin–angiotensin and sympathetic nervous systems, is established. A pivotal role of the angiotensin type 1 receptor in the proximal tubule of the kidney for the development of experimental hypertension is established. Yet, other systems are involved. This study tests whether the expression of angiotensin type 1A receptors in catecholaminergic cells contributes to hypertension development. Using a Cre-lox approach, we deleted the angiotensin type 1A receptor from all catecholaminergic cells. This deletion did not alter basal metabolism or blood pressure but delayed the onset of angiotensin-dependent hypertension and reduced the maximal response. Cardiac hypertrophy was also reduced. The knockout mice showed attenuated activation of the sympathetic nervous system during angiotensin II infusion as measured by spectral analysis of the blood pressure. Increased reactive oxygen species production was observed in forebrain regions, including the subfornical organ, of the knockout mouse but was markedly reduced in the rostral ventrolateral medulla. These studies demonstrate that stimulation of the angiotensin type 1A receptor on catecholaminergic cells is required for the full development of angiotensin-dependent hypertension and support an important role for the sympathetic nervous system in this model.

The Kidney and Hypertension: Lessons From Mouse Models

The pathogenesis of hypertension is multi-factorial, involving many of the systems contributing to blood pressure homeostasis including the vasculature, kidneys, central, and sympathetic nervous systems, along with various hormonal regulators. However, over the years, many studies have indicated a predominant importance of the kidney in blood pressure homeostasis and hypertension. This work has established the notion that the kidney is a key determinant of the chronic level of intra-arterial pressure playing a major role in the pathogenesis of hypertension. Therefore, this review will focus on recent work using genetically modified mouse models addressing the role of the kidney in hypertension. In particular, human genetic studies of Mendelian disorders with major impact on blood pressure homeostasis have provided powerful evidence for a role of the kidney in hypertension. Of the approximately 20 genes identified as causal in these disorders, virtually all have an effect on the control of solute transport in the kidney. As such, we have especially focused on generation of mouse models addressing the nature of these specific molecular defects in nephron function that produce an alteration in blood pressure.

Impact of Angiotensin Type 1A Receptors in Principal Cells of the Collecting Duct on Blood Pressure and Hypertension

The main actions of the renin–angiotensin system to control blood pressure (BP) are mediated by the angiotensin type 1 receptors (AT1Rs). The major murine AT1R isoform, AT1AR, is expressed throughout the nephron, including the collecting duct in both principal and intercalated cells. Principal cells play the major role in sodium and water reabsorption. Although aldosterone is considered to be the dominant regulator of sodium reabsorption by principal cells, recent studies suggest a role for direct actions of AT1R. To specifically examine the contributions of AT1AR in principal cells to BP regulation and the development of hypertension in vivo, we generated inbred 129/SvEv mice with deletion of AT1AR from principal cells (PCKO). At baseline, we found that BPs measured by radiotelemetry were similar between PCKOs and controls. During 1-week of low-salt diet (<0.02% NaCl), BPs fell significantly (P<0.05) and to a similar extent in both groups. On a high-salt (6% NaCl) diet, BP increased but was not different between groups. During the initial phase of angiotensin II–dependent hypertension, there was a modest but significant attenuation of hypertension in PCKOs (163±6 mm Hg) compared with controls (178±2 mm Hg; P<0.05) that was associated with enhanced natriuresis and decreased alpha epithelial sodium channel activation in the medulla of PCKOs. However, from day 9 onward, BPs were indistinguishable between groups. Although effects of AT1AR on baseline BP and adaptation to changes in dietary salt are negligible, our studies suggest that direct actions of AT1AR contribute to the initiation of hypertension and epithelial sodium channel activation.