Chronic Knockdown of the NTS AT1R Increases Blood Inflammatory-Endothelial Progenitor Cells Ratio and Exacerbates Hypertension in the SHR

Abstract

AT1 receptor subtype a (AT1R1a) expression is increased in the nucleus of the solitary tract (NTS) in Spontaneously Hypertensive Rat (SHR) compared to Wistar Kyoto (WKY) controls. However, the chronic role of AT1Ra in the NTS for cardiovascular control is not well understood. In this study, we investigated the hypothesis that the NTS AT1Ra is involved in neural regulation of the peripheral inflammatory status, and linked with hypertension. Transduction of brain neuronal cultures with AAV2-AT1R-shRNA resulted in a 72% decrease in AT1Ra mRNA, and attenuated AngII-induced increase in ERK1/2 phosphorylation and neuronal firing. Specific NTS microinjection of AAV2-AT1R-shRNA vector in the SHR resulted in a ~30 mmHg increase in the mean arterial pressure (MAP) compared to control vector injected animals (Sc-shRNA: 154±4; AT1R-shRNA: 183±10 mmHg), and induced a resetting of the baroreflex control of heart rate to higher MAP. In addition, AAV2-AT1R-shRNA-treated SHRs exhibited a 74% decrease in circulating endothelial progenitor cells (EPC, CD90⁺, CD4⁻/5⁻/8⁻), and a 300% increase in the circulating inflammatory cells (IC) including CD4⁺/CD8⁺, CD45⁺/3⁺ T lymphocytes, and macrophages (CD68⁺). As a result, the EPC/IC ratio was decreased by 8~15 fold in the AT1R-shRNA-treated SHR. However, identical injection of AAV2-AT1R-shRNA into the NTS of WKY had no effect on MAP and ICs. These observations suggest that increased expression of the AT1Ra in SHR NTS may present a counter-hypertensive mechanism involving inflammatory/angiogenic cells.

Introduction

The renin-angiotensin system (RAS) is intrinsic to the brain and plays a critical role in the control of blood pressure (BP) and body fluid homeostasis. Angiotensin II (AngII) is a potent vasoconstrictor, a major culprit in genesis of hypertension, and coordinates a range of interrelated functions such as blood volume and arterial pressure¹. This multi-functional peptide produces its effect predominantly through the AT1 receptors (AT1R). Circulating AngII also exerts its actions on BP control and body fluid homeostasis, through stimulation of AT1R in the circumventricular organs lacking a blood-brain barrier, thereby activating hypothalamic and brain stem sites, and contributing to sympathoexcitation and the hypertensive response².

The nucleus of solitary tract (NTS) is a primary sensory nucleus involved in processing of sensory information from visceral afferent nerves including the cardiovascular receptors. The medial NTS is particularly important in receiving and processing baroreceptor inputs from the aortic arch and carotid sinus regions. Studies have revealed an unequivocal effect of AngII in this brain area in regulation of synaptic transmission and baroreceptor sensitivity³. For example, AngII can trigger nitric oxide release from the endothelium which can depress the baroreflex function³. However, the effects of AngII on BP regulation in the NTS remain a subject of debate, as microinjection of AngII into the medial NTS can result in either a depressor or a pressor effect, dependent on the experimental design^{4, 5}. In addition, microinjection of AngII into the NTS induces an inconsistent effect on heart rate ^{3, 6}. None the less, all these effects are mediated by AT1R since they can be attenuated by blockade of AT1R^{3, 7}.

Recent evidence suggested an association between the neurogenic hypertension and an inflammatory condition, wherein the proinflammatory cytokines (PIC), such as the tumor necrosis factor-alpha (TNF-α), contribute to the hypertensive effect⁸. In support of this, AngII infusion increases PIC, and RAS blockade attenuates circulating and tissue levels of cytokines in cardiovascular diseases². Consistent with this view is the evidence of the involvement of T cell activation in AngII-induced hypertension⁹. In addition, AngII and AT1R expression is increased in the brain cardioregulatory areas, including the NTS, of the SHR compared to its normotensive Wistar Kyoto (WKY) control^{10, 11}. Taken together, these observations suggest that the brain AngII and AT1R may be involved in the development of peripheral inflammation and vasculature damage, compounding the condition of hypertension. Thus, we hypothesize that increased AT1R expression in the NTS affects BP and influences inflammatory status in the circulation. We tested this hypothesis by chronic knockdown NTS AT1R by AAV2-AT1R-shRNA mediated gene transfer.

Materials and Methods

An expanded Methods section is available in the Online Data Supplement at http://hyper.ahajournals.org.

Results

Characterization of AAV2-AT1R-shRNA

There are two subtypes of AT1R expressed in the NTS: AT1Ra and AT1Rb. We decided to target AT1Ra gene since our RT- quantitative PCR result showed that more than 98% AT1R in the NTS is AT1Ra, and AT1Ra mRNA levels were significantly increased in adult SHR compared age-matched normotenisve WKY controls (Figure S1). A small hairpin RNA targeted AT1Ra was cloned into an AAV vector under the control of human U6 promoter (Figure 1A). Infection of SHR brain neuronal cultures with the AAV2-AT1R-shRNA showed a robust transduction as evidenced by GFP expression (Figure 1B). This transduction resulted in a 72% decrease in the AT1Ra mRNA levels compared with AT1Ra mRNA levels in control vector, AAV2-Sc-shRNA, transduced neurons, while there was no significant change in AT1Ra mRNA levels between AAV2-Sc-shRNA treated neurons and PBS treated control (Figure 1C). In addition, AAV2-AT1R-shRNA transduction abolished ERK1/2 phosphorylation induced by AngII (Figure 1D). Furthermore, AT1R-shRNA infection diminished AngII induced increase in action potential frequency (APF, Figure 1E). These observations confirmed the efficacy of the AAV2- AT1R-shRNA on AT1Ra knockdown.

Figure 1. Characterization of AAV2-AT1R-shRNA vector in SHR brain neurons.

A. Diagram of AAV2-AT1R-shRNA (top) and control vector, AAV2-Sc-shRNA (bottom). A GFP reporter gene was used to visualize vector delivery. B. GFP gene expression in the brain neuronal cultures 5 days following AAV2-AT1R-shRNA infection. C. AAV2-AT1R-shRNA decreases AT1Ra mRNA level in neuronal cultures. D. AT1Ra knockdown abolishes activation of ERK1/2 phosphorylation by AngII (100 nmol/L). Left panel is a representative Western blot showing phosphorylated (P-) and total (T-) ERK1/2, and right panel shows quantification of P-ERK bands that have been normalized with T- ERK1/2. Data are mean ± SEM, *P < 0.05 (n=4) compared with control. E. Effect of AT1R-shRNA pretreatment on AngII mediated increase of chronotropic activity in SHR brain neurons. Top panels are representative tracings of recordings. Middle panels are distribution plots of APF in basal and by AngII treatment (100 nmol/L) in neurons pretreated with indicated viral vector. Lines match corresponding APF point in basal with associated APF response to AngII. Bottom bar graph represents the responses to AngII, as normalized to basal level expressed as mean response ± SEM. (* p<0.05, n=4).

AT1Ra knockdown increases MAP and alters baroreflex in the SHR

Transduction of SHR NTS with AAV2-AT1R-shRNA resulted in an increase in mean arterial pressure (MAP) compared to AAV2-Sc-shRNA treated rats. This increase was first apparent 28 days, reached peak values 49 days following microinjection and was maintained until the termination of the protocol (Sc-shRNA: 154±4; AT1R-shRNA: 183±10 mmHg). In contrast to SHR, an identical microinjection of AAV2-AT1R-shRNA in WKY rats did not affect MAP (Figure 2A). No significant changes were observed in heart rate (HR) between AT1Ra knockdown rats and control animals in both SHR and WKY rats (Figure 2A). RT-quantitative PCR showed that a modest but insignificant decrease in the AT1Ra mRNA levels was observed 7 days following AT1R-shRNA transduction. This decrease became significant by 21 days (42%), reached almost maximal levels at 35 days (~45%) and was maintained until the end of the study (50%) (Figure 2B). However, AT1Rb mRNA level did not change by chronic AT1Ra knockdown (data not shown). Baroreflex function assay in the SHR showed that NTS-AT1R knockdown triggered a shift in the MAP-HR curve upward and to the right associated with significant increases in the operating point, maximal gain, lower plateau, upper plateau, saturation and threshold levels. However, the averaged gain from the linear part of the slope was unchanged by AT1R-shRNA (Figure 2C, S1). Spectral analysis indicated that the sympathetic vasomotor drive (VLF) was significantly increased in the AT1Ra knockdown SHR (Figure S2).

Figure 2. Effect of NTS AT1Ra knockdown on MAP and baroreflex.

A. The MAP is increased in the NTS knockdown SHR (*, P<0.05), but not WKY rats; there is no significantly changes in HR between AT1Ra knockdown rats and control animals in both SHR and WKY rats (n=6/group); B: AT1Ra mRNA levels in the SHR NTS in differing time course following AAV2-AT1R-shRNNA injection; C. averaged sigmoidal function curves derived from baroreflex tests of AAV2-AT1R-shRNA injection SHR and control animals (n=6/group). For each curve, operating point (diamond), threshold (square), and saturation (circle) are represented as mean ± SEM.

AT1Ra knockdown heightened the cardiac hypertrophy in the SHR

Animals were sacrifice at the end of the experiments and body weight to heart weight (HW/BW) ratio was measured. Significant increases in HW/BW were observed in the AT1R knockdown SHR (AT1R-shRNA: 0.44±0.01%; Sc-shRNA: 0.38±0.01%; P<0.05), but not in WKY rats (AT1R-shRNA: 0.33±0.01%; Sc-shRNA: 0.32±0.01%, P>0.05) comparing to their controls. Hematoxylin and eosin staining showed an increase in cardiomyocyte diameter in the AT1Ra knockdown SHR compared to control rats (AT1R-shRNA: 2.55±0.17; Sc-shRNA: 1.73±0.09 µm; P<0.05) (Figure S3).

AAV2-AT1R-shRNA vector primarily targets the NTS neurons

Animals were euthanized at the end of the experiment. Coronal sections of brainstem containing the NTS were used for immunostaining using antibodies specific for neuronal nucleus (NeuN), astrocyte (GFAP) and microglia (Iba1) to identify AT1R-shRNA transduced cell type (s). We observed that GFP, a reporter gene in AAV2-AT1R-shRNA viral vector, was primarily co-localized with NeuN, but not with GFAP and Iba1 positive cells (Figure 3).

Figure 3. Transduction of AAV2-AT1R-shRNA in the NTS.

A. a representative micrograph showed GFP reporter gene expression in the NTS, indicating successful transduction of AAV2-AT1R-shRNA vector in the NTS. CC-central canal. AP-area postrema. B-D: immunosating using antibody specific for neurons (NeuN, red), astrocyte (GFAP, red) and microglia (Iba1, red), and merged with GFP. White arrowheads indicated the co-localization of GFP with NeuN.

RT-quantitative PCR showed that AT1Ra mRNA levels were ~50% less in the NTS of AAV2-AT1R-shRNA treated SHR and WKY rats compared to their control vector administration animals. However, no changes in the AT1Ra mRNA were observed in other brain areas such as the PVN (Figure S4).

AT1Ra knockdown increases ICs and decreases endothelial progenitor cells (EPCs) in the blood of SHR

Circulating EPC and IC levels in mononuclear cells fractions were analyzed using Fluorescence Activated Cell Sorting (FACS). We selected CD90⁺ (CD4/5/8⁻) as a marker for rat EPCs based on previously published reports¹². We observed a 74% decrease in EPCs; a 300% increase in CD4⁺+CD8⁺, a 325% increase in CD45/3⁺, and a 235% increase in CD68⁺ in the ATR knockdown SHR (all P<0.05). This resulted in 8–15 fold decrease in circulating EPC/ICs ratio (Figure 4). There were no significant changes were observed between AT1Ra knockdown WKY rats and control animals (Figure S5).

Figure 4. AT1Ra knockdown decreases circulating EPCs/ICs ratio in SHR.

The values in Sc-shRNA injected group were assigned to unit 1, and the data in AT1Ra knockdown group were presented as a ratio over their respective control. AT1Ra knockdown resulted in a significant decrease in EPCs (CD90⁺, CD4/CD5/CD8⁻), and increases in ICs including CD4⁺/CD8⁺ lymphocyte, T lymphocyte (CD45/3⁺) and macrophages (CD68⁺). The ratio of EPC /ICs is decreased by 8~15 fold compared to control group (Mean ±SEM, n=6/group).

Discussion

The present study examines the physiological outcome of chronic knockdown of the AT1Ra in the NTS and reveals several novel findings: (i) decreased NTS AT1Ra mRNA in the SHR results in an elevation in MAP and the sympathetic vasomotor drive, and the baroreflex resetting to higher levels of arterial pressure, altering the operating range of MAP without changing the buffering capacity of the baroreflex. These data indicate that AT1Ra plays an important role in the maintenance of the set point of arterial pressure, and that its upregulation in the NTS has a restraining influence on chronic MAP in the SHR. (ii) The NTS AT1Ra knockdown-dependent elevation in BP is associated with changes in the circulating EPCs and ICs. This suggests a communication between the NTS and cell types involved with vascular repair and inflammation in hypertension.

Although the role of the AT1R in cardiovascular function has been extensively investigated, most studies were carried out in an acute experimental situation and with use of anesthetized animals^{13, 14}. Here, we demonstrate a profound effect of a specific and chronic AT1Ra downregulation in the NTS of conscious SHR, which resulted in an elevation in MAP, and caused baroreflex resetting in the SHR exclusively. Considering that the AT1Ra upregulation in the NTS occurred only in the adult SHR, and that knockdown of the AT1Ra did not affect the MAP in normotensive WKY rats, we conclude that the elevated AT1Ra expression in the NTS of SHR may be a compensatory mechanism of hypertension, and has a beneficial inhibiting effect to further BP elevation, as we had recently proposed¹⁵. The ‘restraining’ influence of certain types of NTS neurons on chronic arterial pressure has previously been reported¹⁵. For example, the expression of the phosphatidylinositide 3-kinases (PI3K), a key enzyme involved in AngII-mediated neuronal signaling, is significantly increased in both the NTS and PVN of SHR compared to WKY rats^{15, 16}. However, chronic neuronal blockade of PI3K in the NTS is pro-hypertensive¹⁵, while it is anti-hypertensive in the PVN of SHR¹⁶. Similarly, our recent studies demonstrate that prorenin receptor (PRR), a newest member of brain RAS, is elevated in the NTS and supraoptic nucleus (SON) of SHR compared to normotensive WKY rats. Chronic knockdown of the PRR in the SHR NTS induces pressor effect¹⁷, while causes depresser effect in the SON¹⁸. The opposing effects of activation of NTS vs. PVN/SON are generally attributed to the notion that the NTS is considered an ‘inhibitory’ cardioregulatory nucleus, as its activation (by baro-input for example) reduces the arterial pressure, while the activation of the ‘excitatory’ cardioregulatory nuclei such as PVN/SON increases the arterial pressure¹⁹. Thus we believe that similar to the PI3K and PRR, upregulation of AT1Ra in certain neuronal types within the NTS, may promote compensatory activation of the NTS, as the system attempts to restore the cardiovascular homeostasis and prevent even higher BP development.

How is the NTS AT1Ra involved in BP regulation? One of the mechanisms may involve neural regulation of the activity of certain immune organs such as the bone marrow and spleen. Recent evidence has demonstrated a direct neuronal connection between the brain cardioregulatory areas and the bone marrow^20–22. As we have previously shown, the AngII-mediated effects in the brain cardioregulatory areas results in the dysfunctional brain-bone marrow communication, which contributes to the development and establishment of hypertension by elevating the circulating ICs and downregulating the EPC levels^{21, 22}. Similarly, direct sympathetic innervations of the spleen and the central AngII-dependent increase in the splenic sympathetic nerve activity are directly related to the enhanced splenic immune response, characterized by elevated IC levels^{23, 24}. On the other hand, stimulation of the vagus nerve exerts anti-inflammatory effects by decreasing the levels of the inflammatory cytokines and suppressing the activation of ICs²⁵. This is relevant for the present study, considering that AngII is a well known contributor to the dysfunctional vagal reflex in hypertension²⁵. Therefore, dysfunctional AT1Ra signaling in the NTS may be directly involved in disruption of autonomic nervous system-immune system communication and induces hypertension.

The brain also exhibits heightened inflammatory status in the hypertensive animal models, and the inflammatory processes in the brain cardioregulatory regions are linked with modulation of the autonomic nervous system and increased BP^{20, 22, 26}. The NTS itself is characterized by accumulation of different ICs, as well as by elevated cytokine levels in the SHR, which may influence the NTS neuronal activity and induce sympathoexcitation²². Increased expression of the NTS AT1Ra in the SHR may serve as a counter-hypertensive mechanism and attempts to decrease the sympathetic drive. Chronic knockdown the NTS AT1Ra increases the sympathetic vasomotor drive. In turn, this increased sympathetic outflow may directly influence the activity of the bone marrow activity²², as well as the spleen^{9, 23, 24}, contributing to the decrease in the EPCs and increase in the ICs, as previously described²¹, which may subsequently contribute to BP elevation. Although we do not present direct evidence in present study to demonstrate that increase in ICs precedes BP changes and induces BP elevation, all existing evidences suggest that inflammation may occur earlier and is critical in the initiation and establishment of hypertension: (i) studies led by Drs. Harrison’s and Abboud’s groups have indicated that increased inflammatory precedes hypertensive state^{9, 27}; (ii) Our recent studies have shown a time-dependent increase in bone marrow derived ICs and decrease in EPCs in AngII infusion rat model; Inhibition of brain ROS which attenuates high BP also corrects this EPCs/ICs imbalance. These changes in ICs and EPCs in AngII model of hypertension seems to be centrally-mediated since increase in BP by chronic phenylephrine infusion fails to influence EPCs/ICs balance²¹ and (iii) our unpublished data shows significant increase in ICs in prehypertensive SHR. Furthermore, increasing evidence demonstrates the role of macrophages and T cells in the development and establishment of hypertension. Mice lacking the T lymphocytes do not develop hypertension and the associated vascular dysfunction, and genetic deletion of macrophages markedly reduces experimental hypertension⁹. However, our present study does not establish whether the correction of the EPC/IC imbalance would attenuate the increase in BP, and presently renders the suggested connection between the NTS AT1Ra knockdown, the increase in BP, and the EPC/IC imbalance purely correlative. Further investigation is warranted in support of this hypothesis.

Taken together, knockdown of the NTS AT1Ra increases sympathetic outflow to the periphery, which results in further elevation of BP in the SHR. This is associated with increases the circulating macrophages and T cells and decrease of the EPC levels. This may further increase the inflammatory status and compromise the reparative capacity of the vasculature damaged by hypertension. Ultimately, these synergistic effects of the dysfunctional autonomic nervous system and overactive immune system may lead to deterioration in hypertension, as previously suggested^{9, 27}.

Perspectives

The present study reveals a novel role for AT1Ra in the NTS of SHR in modifying peripheral inflammation and restraining the BP. Future studies should seek to determine the modulatory factors that enhance the AT1Ra effect. Such information might allow the development of new tools for specific activation of NTS AT1Ra, which may provide a powerful intervention to lower arterial pressure chronically and perhaps decrease hypertension-dependent peripheral inflammation.

Novelty and Significance.

1) What Is New?

Chronic knockdown of the NTS AT1Ra in the conscious SHR elevates BP and shifts the set point of arterial pressure to a higher level. This increase in BP is accompanied by decrease in EPCs and an increase in ICs, including the macrophages and T lymphocytes in the blood.

2) What Is Relevant?

The NTS AT1Ra participates in the regulation of sympathetic nervous system and it is involved in the control of set point of arterial pressure.

3) Summary

Chronic knockdown of the NTS AT1Ra in the SHR increases neuronal activity and sympathetic outflow, leading to increased BP. This increase in BP is accompanied by decreased EPCs and increased ICs, which may further contribute to the hypertensive phenotype. These data suggest that increased AT1Ra in the NTS of SHR is a compensatory mechanism of hypertension; as it acts to inhibit further BP elevation.

Abbreviation

AngII

angiotensin II

AT1R

AT1 receptors

BP

blood pressure

BW

body weight

EPCs

endothelial progenitor cells

FACS

fluorescence activated cell sorting

HW

heart weight

ICs

inflammatory cells

MAP

mean arterial pressure

NTS

nucleus of the solitary tract

NE

norepinephrine

PVN

paraventricular nucleus

PI3K

phosphatidylinositide 3-kinases

PIC

proinflammatory cytokines

SHR

Spontaneously Hypertensive Rat

SON

supraoptic nucleus

RAS

renin-angiotensin system

TNF-α

tumour necrosis factor-alpha

AVP

vasopressin

WKY

Wistar Kyoto