Interleukin 17A Contributes to Blood-Brain Barrier Disruption of Hypothalamic Paraventricular Nucleus in Rats With Myocardial Infarction.

BACKGROUND: Elevated inflammatory cytokines in the periphery have been identified as active contributors to neuroinflammation and sympathetic overactivity in heart failure (HF). Yet, the exact mechanisms by which these cytokines breach the blood-brain barrier (BBB) to exert their effects on the brain remain elusive. Interleukin 17A has been linked to BBB disruption in various neurologic disorders, and its levels were significantly augmented in circulation and the brain in HF. The present study aimed to determine whether the BBB integrity was compromised within the hypothalamic paraventricular nucleus (PVN), and if so, whether interleukin 17A contributes to BBB disruption in myocardial infarction-induced HF. METHODS AND RESULTS: Male Sprague-Dawley rats underwent coronary artery ligation to induce HF or sham surgery. Some HF rats received bilateral PVN microinjections of an interleukin 17 receptor A small interfering RNA or a scrambled small interfering RNA adeno-associated virus. Four weeks after coronary artery ligation, the permeability of the BBB was evaluated by intracarotid injection of fluorescent dyes (fluorescein isothiocyanate-dextran 10 kDa+rhodamine-dextran 70 kDa). Compared with sham-operated rats, HF rats exhibited an elevated extravasation of fluorescein isothiocyanate-dextran 10 kDa within the PVN but not in the brain cortex. The plasma interleukin 17A levels were positively correlated with fluorescein isothiocyanate 10 kDa extravasation in the PVN. The expression of caveolin-1, a transcytosis marker, was augmented, whereas the expression of tight junction proteins was diminished in HF rats. Interleukin 17 receptor A was identified within the endothelium of PVN microvessels. Treatment with interleukin 17 receptor A small interfering RNA led to a significant attenuation of fluorescein isothiocyanate 10 kDa extravasation in the PVN and reversed expression of caveolin-1 and tight junction-associated proteins in the PVN. CONCLUSIONS: Collectively, these data indicate that BBB permeability within the PVN is enhanced in HF and is likely attributable to increased interleukin 17A/interleukin 17 receptor A signaling in the BBB endothelium, by promoting caveolar transcytosis and degradation of tight junction complexes.

Reducing brain TACE activity improves neuroinflammation and cardiac function in heart failure rats.

Tumor necrosis factor (TNF)-α converting enzyme (TACE) is a key metalloprotease mediating ectodomain shedding of a variety of inflammatory mediators, substrates, and growth factors. We previously reported that TACE-mediated production of TNF-α in the hypothalamic paraventricular nucleus (PVN) contributes to sympathetic excitation in heart failure (HF). Here, we sought to determine whether central interventions in TACE activity attenuate neuroinflammation and improve cardiac function in heart failure. Myocardial infarction-induced HF or sham-operated (SHAM) rats were treated with bilateral paraventricular nucleus microinjection of a TACE siRNA or a 4-week intracerebroventricular (ICV) infusion of the TACE inhibitor TAPI-0. Compared with SHAM rats, scrambled siRNA-treated HF rats had higher TACE levels in the PVN along with increased mRNA levels of TNF-α, TNF-α receptor 1 and cyclooxygenase-2. The protein levels of TNF-α in cerebrospinal fluid and phosphorylated (p-) NF-κB p65 and extracellular signal-regulated protein kinase (ERK)1/2 in the PVN were also elevated in HF rats treated with scrambled siRNA. The expression of these inflammatory mediators and signaling molecules in the PVN of HF rats were significantly attenuated by TACE siRNA. Interestingly, the mRNA level of TNF-α receptor 2 in the PVN was increased in HF treated with TACE siRNA. Moreover, sympathetic excitation, left ventricular end-diastolic pressure, pulmonary congestion, and cardiac hypertrophy and fibrosis were reduced by PVN microinjection of TACE siRNA. A 4-week treatment with intracerebroventricular TAPI-0 had similar effects to ameliorate these variables in HF rats. These data indicate that interventions suppressing TACE activity in the brain mitigate neuroinflammation, sympathetic activation and cardiac dysfunction in HF rats.

Brain Interleukin-17A contributes to neuroinflammation and cardiac dysfunction in rats with myocardial infarction.

Proinflammatory cytokines produced outside the central nervous system can act in the brain to promote sympathetic activation that contributes to the progression of heart failure (HF). Interleukin (IL)-17A, a key inflammatory regulator which orchestrates immune responses to promote chronic inflammation, has been implicated in the pathophysiology of HF. We previously reported that IL-17A acts within the brain, particularly in the hypothalamic paraventricular nucleus (PVN), to increase expression of inflammatory mediators and, consequently, sympathetic outflow. The present study sought to determine whether IL-17A levels are elevated in a rat model of HF induced by myocardial infarction and, if so, whether increased expression of IL-17A in the brain itself contributes to neuroinflammation and cardiac dysfunction in this disease setting. Male SD rats underwent coronary artery ligation (CL) to induce HF or sham operation (SHAM). Compared with SHAM rats, HF rats exhibited significantly increased IL-17A levels in plasma, beginning within 1 week with a peak increase at 4 weeks after CL. IL-17A levels in cerebrospinal fluid (CSF) were also increased in HF rats and correlated with IL-17A levels in the plasma. The mRNA expression of IL-17A and its receptor IL-17RA, but not IL-17RC, was markedly upregulated in the PVN of HF when compared with SHAM rats. Genetic knockdown of IL-17RA by bilateral PVN microinjections of an IL-17RA siRNA AAV virus attenuated mRNA expression of proinflammatory cytokines and chemokines, and ameliorated sympathetic activation and cardiac function in HF rats. These data indicate that elevated expression of IL-17A in the brain in HF contributes to the excessive central inflammatory state and cardiac dysfunction in HF. Interventions to suppress IL-17A/IL-17RA axis in the brain have the potential for treating HF.

Voluntary Exercise Eliminates Maternal Gestational Hypertension-Induced Hypertensive Response Sensitization to Postweaning High-Fat Diet in Male Adult Offspring.

BACKGROUND: Exercise has profound effects on cardiovascular function and metabolism in both physiological and pathophysiological states. The present study tested whether voluntary exercise would protect male offspring against maternal gestational hypertension-induced hypertensive response sensitization elicited by post-weaning high-fat diet (HFD). METHODS AND RESULTS: On low-lard-fat diet, offspring of both normotensive and hypertensive dams had comparable resting blood pressure, but HFD feeding elicited an enhanced increase in blood pressure (ie, hypertensive response sensitization) in sedentary offspring of hypertensive dams when compared with sedentary offspring of normotensive dams. The HFD fed sedentary offspring of hypertensive dams displayed greater sympathetic activity, enhanced pressor responses to centrally administered ANG II (angiotensin II) or leptin, and greater mRNA expression of proinflammatory cytokines, leptin, and a marker of blood-brain barrier leakage in the hypothalamic paraventricular nucleus. The enhanced blood pressure and central sympathetic activity in HFD-fed sedentary offspring of hypertensive dams were significantly reduced by exercise but fell only to levels comparable to HFD-fed exercising offspring of normotensive dams. HFD-induced increases in plasma IL-6 (interleukin-6) and sympathetic activity and greater pressor responses to central TNF (tumor necrosis factor)-α in offspring from both normotensive and hypertensive dams were also maintained after exercise. Nevertheless, exercise had remarkably beneficial effects on metabolic and autonomic function, brain reactivity to ANG II and leptin and gene expression of brain prohypertensive factors in all offspring. CONCLUSIONS: Voluntary exercise plays a beneficial role in preventing maternal hypertension-induced hypertensive response sensitization, and that this is associated with attenuation of enhanced brain reactivity and centrally driven sympathetic activity.

Loss of the Protective Effect of Estrogen Contributes to Maternal Gestational Hypertension-Induced Hypertensive Response Sensitization Elicited by Postweaning High-Fat Diet in Female Offspring.

Background A recent study conducted in male offspring demonstrated that maternal gestational hypertension (MHT) induces hypertensive response sensitization (HTRS) elicited by postweaning high-fat diet (HFD). In this study, we investigated the sensitizing effect of MHT on postweaning HFD-induced hypertensive response in female rat offspring and assessed the protective role of estrogen in HTRS. Methods and Results The results showed that MHT also induced a sensitized HFD-elicited hypertensive response in intact female offspring. However, compared with male offspring, this MHT-induced HTRS was sex specific in that intact female offspring exhibited an attenuated increase in blood pressure. Ovariectomy significantly enhanced the HFD-induced increase in blood pressure and the pressor response to centrally administered angiotensin II or tumor necrosis factor-α in offspring of normotensive dams, which was accompanied by elevated centrally driven sympathetic activity, upregulated mRNA expression of prohypertensive components, and downregulated expression of antihypertensive components in the hypothalamic paraventricular nucleus. However, when compared with HFD-fed ovariectomized offspring of normotensive dams, the MHT-induced HTRS and pressor responses to centrally administered angiotensin II or tumor necrosis factor-α in HFD-fed intact offspring of MHT dams were not potentiated by ovariectomy, but the blood pressure and elicited pressor responses as well as central sympathetic tone remained higher. Conclusions The results indicate that in adult female offspring MHT induced HTRS elicited by HFD. Estrogen normally plays a protective role in antagonizing HFD prohypertensive effects, and MHT compromises this normal protective action of estrogen by augmenting brain reactivity and centrally driven sympathetic activity.

Predator Scent-Induced Sensitization of Hypertension and Anxiety-like Behaviors.

Post-traumatic stress disorder (PTSD), an anxiety-related syndrome, is associated with increased risk for cardiovascular diseases. The present study investigated whether predator scent (PS) stress, a model of PTSD, induces sensitization of hypertension and anxiety-like behaviors and underlying mechanisms related to renin-angiotensin systems (RAS) and inflammation. Coyote urine, as a PS stressor, was used to model PTSD. After PS exposures, separate cohorts of rats were studied for hypertensive response sensitization (HTRS), anxiety-like behaviors, and changes in plasma levels and mRNA expression of several components of the RAS and proinflammatory cytokines (PICs) in the lamina terminalis (LT), paraventricular nucleus (PVN), and amygdala (AMY). Rats exposed to PS as compared to control animals exhibited (1) a significantly greater hypertensive response (i.e., HTRS) when challenged with a slow-pressor dose of angiotensin (ANG) II, (2) significant decrease in locomotor activity and increase in time spent in the closed arms of a plus maze as well as general immobility (i.e., behavioral signs of increased anxiety), (3) upregulated plasma levels of ANG II and interleukin-6, and (4) increased expression of message for components of the RAS and PICs in key brain nuclei. All the PS-induced adverse effects were blocked by pretreatment with either an angiotensin-converting enzyme antagonist or a tumor necrosis factor-α inhibitor. The results suggest that PS, used as an experimental model of PTSD, sensitizes ANG II-induced hypertension and produces behavioral signs of anxiety, probably through upregulation of RAS components and inflammatory markers in plasma and brain areas associated with anxiety and blood pressure control.

Transforming Growth Factor-α Acts in Hypothalamic Paraventricular Nucleus to Upregulate ERK1/2 Signaling and Expression of Sympathoexcitatory Mediators in Heart Failure Rats.

Activation of epidermal growth factor receptor (EGFR) tyrosine kinase is associated with increased extracellular signal-regulated kinase (ERK) 1/2 signaling in the hypothalamic paraventricular nucleus (PVN), which contributes to the sympathetic excitation in heart failure (HF). Transforming growth factor (TGF)-α is a major endogenous ligand for EGFR. The present study sought to determine whether TGF-α increases in the PVN in HF and promotes the activation of EGFR to increase ERK1/2 activity. Male rats received bilateral PVN microinjections of an EGFR siRNA or a scrambled siRNA followed by an intracerebroventricular (ICV) injection of TGF-α or vehicle one week later. In rats pretreated with the scrambled siRNA, ICV TGF-α increased phosphorylated (p-) EGFR and upregulated the expression of p-ERK1/2 and mRNA levels of proinflammatory cytokines (PICs) and renin-angiotensin system (RAS) components in the PVN, when compared with the untreated age-matched control rats. These responses to ICV TGF-α were significantly attenuated in rats pretreated with the EGFR siRNA. Furthermore, bilateral PVN microinjection of a TGF-α siRNA in HF rats significantly decreased the elevated levels of TGF-α, p-EGFR, p-ERK1/2 and the mRNA expression of PICs and RAS components in the PVN, compared with the HF rats treated with a scrambled siRNA. The TGF-α siRNA-treated HF rats also exhibited lower plasma norepinephrine levels and improved peripheral manifestations of HF. These data suggest that TGF-α expression is upregulated in the PVN in HF and induces the activation of EGFR-mediated ERK1/2 signaling to augment the inflammation and RAS activity that drives sympathetic excitation in HF.

IL (Interleukin)-17A Acts in the Brain to Drive Neuroinflammation, Sympathetic Activation, and Hypertension.

[Figure: see text].

Maternal Angiotensin II-Induced Hypertension Sensitizes Postweaning High-Fat Diet-Elicited Hypertensive Response Through Increased Brain Reactivity in Rat Offspring.

Background Prenatal and postnatal insults can induce a physiological state that leaves offspring later in life vulnerable to subsequent challenges (stressors) eliciting cardiometabolic diseases including hypertension. In this study, we investigated whether maternal angiotensin II-induced hypertension in rats sensitizes postweaning high-fat diet (HFD)-elicited hypertensive response and whether this is associated with autonomic dysfunction and altered central mechanisms controlling sympathetic tone in offspring. Methods and Results When eating a low-lard-fat diet, basal mean arterial pressure of male offspring of normotensive or hypertensive dams were comparable. However, HFD feeding significantly increased mean arterial pressure in offspring of normotensive and hypertensive dams, but the elevated mean arterial pressure induced by HFD was greater in offspring of hypertensive dams, which was accompanied by greater sympathetic tone and enhanced pressor responses to centrally administrated angiotensin II or leptin. HFD feeding also produced comparable elevations in cardiac sympathetic activity and plasma levels of angiotensin II, interleukin-6, and leptin in offspring of normotensive and hypertensive dams. Reverse transcriptase polymerase chain reaction analyses in key forebrain regions implicated in the control of sympathetic tone and blood pressure indicated that HFD feeding led to greater increases in mRNA expression of leptin, several components of the renin-angiotensin system and proinflammatory cytokines in offspring of hypertensive dams when compared with offspring of normotensive dams. Conclusions The results indicate that maternal hypertension sensitized male adult offspring to HFD-induced hypertension. Increased expression of renin-angiotensin system components and proinflammatory cytokines, elevated brain reactivity to pressor stimuli, and augmented sympathetic drive to the cardiovascular system likely contributed.

Silencing Epidermal Growth Factor Receptor in Hypothalamic Paraventricular Nucleus Reduces Extracellular Signal-regulated Kinase 1 and 2 Signaling and Sympathetic Excitation in Heart Failure Rats.

Activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling in cardiovascular regulatory regions of the brain contributes to sympathetic excitation in myocardial infarction (MI)-induced heart failure (HF) by increasing brain renin-angiotensin system (RAS) activity, neuroinflammation, and endoplasmic reticulum (ER) stress. The mechanisms eliciting brain ERK1/2 signaling in HF are still poorly understood. We tested the involvement of the epidermal growth factor receptor (EGFR) which, upon activation, stimulates ERK1/2 activity. Adult male Sprague-Dawley rats received bilateral microinjections of a lentiviral vector encoding a small interfering RNA (siRNA) for EGFR, or a scrambled siRNA, into the hypothalamic paraventricular nucleus (PVN), a recognized source of sympathetic overactivity in HF. One week later, coronary artery ligation was performed to induce HF. Four weeks later, the EGFR siRNA-treated HF rats, compared with the scrambled siRNA-treated HF rats, had lower mRNA and protein levels of EGFR, lower levels of phosphorylated (p-) EGFR and p-ERK1/2 and lower mRNA levels of the inflammatory mediators TNF-α, IL-1ß and cyclooxygenase-2, the RAS components angiotensin-converting enzyme and angiotensin II type 1a receptor and the ER stress markers BIP and ATF4 in the PVN. They also had lower plasma and urinary norepinephrine levels and improved peripheral manifestations of HF. Additional studies revealed that p-EGFR was increased in the PVN of HF rats, compared with sham-operated control rats. These results suggest that activation of EGFR in the PVN triggers ERK1/2 signaling, along with ER stress, neuroinflammation and RAS activity, in MI-induced HF. Brain EGFR may be a novel target for therapeutic intervention in MI-induced HF.

An injectable microparticle formulation for the sustained release of the specific MEK inhibitor PD98059: in vitro evaluation and pharmacokinetics.

PD98059 is a reversible MEK inhibitor that we are investigating as a potential treatment for neurochemical changes in the brain that drive neurohumoral excitation in heart failure. In a rat model that closely resembles human heart failure, we found that central administration of PD98059 inhibits phosphorylation of ERK1/2 in the paraventricular nucleus of the hypothalamus, ultimately reducing sympathetic excitation which is a major contributor to clinical deterioration. Studies revealed that the pharmacokinetics and biodistribution of PD98059 match a two-compartment model, with drug found in brain as well as other body tissues, but with a short elimination half-life in plasma (approximately 73 min) that would severely limit its potential clinical usefulness in heart failure. To increase its availability to tissues, we prepared a sustained release PD98059-loaded PLGA microparticle formulation, using an emulsion solvent evaporation technique. The average particle size, yield percent, and encapsulation percent were found to be 16.73 µm, 76.6%, and 43%, respectively. In vitro drug release occurred over 4 weeks, with no noticeable burst release. Following subcutaneous injection of the microparticles in rats, steady plasma levels of PD98059 were detected by HPLC for up to 2 weeks. Furthermore, plasma and brain levels of PD98059 in rats with heart failure were detectable by LC/MS, despite expected erratic absorption. These findings suggest that PD98059-loaded microparticles hold promise as a novel therapeutic intervention countering sympathetic excitation in heart failure, and perhaps in other disease processes, including cancers, in which activated MAPK signaling is a significant contributing factor. Graphical abstract.

TNF-α-induced sympathetic excitation requires EGFR and ERK1/2 signaling in cardiovascular regulatory regions of the forebrain.

Peripherally or centrally administered TNF-α elicits a prolonged sympathetically mediated pressor response, but the underlying molecular mechanisms are unknown. Activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in cardiovascular regions of the brain has recently been recognized as a key mediator of sympathetic excitation, and ERK1/2 signaling is induced by activation of epidermal growth factor receptor (EGFR) tyrosine kinase activity. The present study examined the role of EGFR and ERK1/2 signaling in the sympathetic response to TNF-α. In urethane-anesthetized rats, intracarotid artery injection of TNF-α increased phosphorylation of EGFR and ERK1/2 in the subfornical organ (SFO) and the hypothalamic paraventricular nucleus (PVN); upregulated the gene expression of excitatory mediators in SFO and PVN; and increased blood pressure (BP), heart rate (HR), and renal sympathetic nerve activity (RSNA). A continuous intracerebroventricular infusion of the selective EGFR tyrosine kinase inhibitor AG1478 or the ERK1/2 inhibitor PD98059 significantly attenuated these responses. Bilateral PVN microinjections of TNF-α also increased phosphorylated ERK1/2 and the gene expression of excitatory mediators in PVN, along with increases in BP, HR, and RSNA, and these responses were substantially reduced by prior bilateral PVN microinjections of AG1478. These results identify activation of EGFR in cardiovascular regulatory regions of the forebrain as an important molecular mediator of TNF-α-driven sympatho-excitatory responses and suggest that EGFR activation of the ERK1/2 signaling pathway plays an essential role. These mechanisms likely contribute to sympathetic excitation in pathophysiological states like heart failure and hypertension, in which circulating and brain TNF-α levels are increased.NEW & NOTEWORTHY Proinflammatory cytokines contribute to the augmented sympathetic nerve activity in hypertension and heart failure, but the central mechanisms involved are largely unknown. The present study reveals that TNF-α transactivates EGFR in the subfornical organ and the hypothalamic paraventricular nucleus to initiate ERK1/2 signaling, upregulate the gene expression of excitatory mediators, and increase sympathetic nerve activity. These findings identify EGFR as a gateway to sympathetic excitation and a potential target for intervention in cardiovascular disease states.

An Injectable Microparticle Formulation Provides Long-Term Inhibition of Hypothalamic ERK1/2 Activity and Sympathetic Excitation in Rats with Heart Failure.

Sympathetic excitation contributes to clinical deterioration in systolic heart failure (HF). Significant inhibition of hypothalamic paraventricular nucleus (PVN) ERK1/2 signaling and a subsequent reduction of plasma norepinephrine (NE) levels in HF rats were achieved 2 weeks after a single subcutaneous injection of PD98059-loaded polymeric microparticles, without apparent adverse events, while blank microparticles had no effect. Similar reductions in plasma NE, a general indicator of sympathetic excitation, were previously achieved in HF rats by intracerebroventricular infusion of PD98059 or genetic knockdown of PVN ERK1/2 expression. This study presents a clinically feasible therapeutic approach to the central abnormalities contributing to HF progression.

Brain TACE (Tumor Necrosis Factor-α-Converting Enzyme) Contributes to Sympathetic Excitation in Heart Failure Rats.

TNF-α (tumor necrosis factor-α) is initially synthesized as a transmembrane protein that is cleaved by TACE (TNF-α-converting enzyme) to release soluble TNF-α. The elevated level of TNF-α in the brain and circulation in heart failure (HF) suggests an increase in the TACE-mediated ectodomain shedding process. The present study sought to determine whether TACE is upregulated in cardiovascular/autonomic brain regions like subfornical organ and hypothalamic paraventricular nucleus in rats with ischemia-induced HF and whether TACE plays a role in TNF-α-driven sympathetic excitation. We found that TACE was expressed throughout the subfornical organ and paraventricular nucleus, with significantly higher levels in HF than in sham-operated (Sham) rats. Intracerebroventricular injection of recombinant TACE induced a mild increase in blood pressure, heart rate, and renal sympathetic nerve activity that peaked at 15 to 20 minutes in both Sham and HF rats. HF rats had a secondary prolonged increase in these variables that was prevented by the TNF-α inhibitor SPD304. Intracerebroventricular administration of the TACE inhibitor TNF-alpha protease inhibitor 1 decreased blood pressure, heart rate, and renal sympathetic nerve activity in Sham and HF rats, with an exaggerated reduction in heart rate and renal sympathetic nerve activity in the HF rats. Direct microinjection of TACE or TNF-alpha protease inhibitor 1 into paraventricular nucleus or subfornical organ of Sham and HF rats elicited blood pressure, heart rate, and renal sympathetic nerve activity responses similar to intracerebroventricular TACE or TNF-alpha protease inhibitor 1. Intracerebroventricular infusion of Ang II (angiotensin II) and IL (interleukin)-1ß increased TACE expression in subfornical organ and paraventricular nucleus of normal rats. These data suggest that a TACE-mediated increase in soluble TNF-α in the brain contributes to sympathetic excitation in HF.

Stress-Induced Sensitization of Angiotensin II Hypertension Is Reversed by Blockade of Angiotensin-Converting Enzyme or Tumor Necrosis Factor-α.

BACKGROUND: Post-traumatic stress disorder (PTSD) is characterized by a disordered stress response and associated with increased cardiovascular disease risk. The present study investigated whether angiotensin (Ang) II-elicited hypertensive response is sensitized in a model of PTSD and whether inhibition of angiotensin-converting enzyme (ACE) or tumor necrosis factor (TNF)-α prior to PTSD blocks this sensitization of Ang II hypertension. METHODS: The resident-intruder paradigm was used to model PTSD. Each intruder rat (male Sprague-Dawley) was given normal drinking water or was pretreated with either an ACE inhibitor (captopril) or a TNF-α inhibitor (pentoxifylline) in the drinking water for 2 weeks. Subsequently, they were exposed to a different resident (male Long-Evans) for 2 hours on 3 days with each session separated by 1 day and then received a subcutaneous infusion of Ang II for 2 weeks. RESULTS: The stressed rats had a significantly enhanced hypertensive response to the Ang II infusion (stressed Δ40.2 ± 3.9 mm Hg vs. unstressed Δ20.5 ± 4.5 mm Hg) and an upregulation of mRNA or protein expression of renin-angiotensin system (RAS) and proinflammatory cytokine (PIC) components and of a microglial marker in the lamina terminalis and hypothalamic paraventricular nucleus when compared with unstressed control rats. Both the sensitized hypertensive response and enhanced gene and protein expression were blocked by pretreatment with either ACE (Δ21.3 ± 3.9 mm Hg) or TNF-α inhibitor (Δ21.4 ± 2.6 mm Hg). CONCLUSIONS: The results indicate that upregulation of the brain RAS and PICs produced by severe stress contributes to traumatic-induced sensitization of hypertensive response to Ang II, and disorders such as PTSD may predispose individuals to development of hypertension.

Sex differences in the central and peripheral manifestations of ischemia-induced heart failure in rats.

Sex differences in the presentation, outcome, and responses to treatment of systolic heart failure (HF) have been reported. In the present study, we examined the effect of sex on central neural mechanisms contributing to neurohumoral excitation and its peripheral manifestations in rats with HF. Male and female Sprague-Dawley rats underwent coronary artery ligation (CL) to induce HF. Age-matched rats served as controls. Ischemic zone and left ventricular function were similar 24 h and 4 wk after CL. Female rats with HF had a lower mortality rate and less hemodynamic compromise, pulmonary congestion, and right ventricular remodeling 4 wk after CL. Plasma angiotensin II (ANG II), arginine vasopressin (AVP), and norepinephrine levels were increased in HF rats in both sexes, but AVP and norepinephrine levels increased less in female rats. In the hypothalamic paraventricular nucleus, a key cardiovascular-related nucleus contributing to neurohumoral excitation in HF, mRNA levels for the proinflammatory cytokines tumor necrosis factor-α and interleukin-1ß as well as cyclooxygenase-2 and the ANG II type 1a receptor were increased in HF rats of both sexes, but less so in female rats. Angiotensin-converting enzyme 2 protein levels increased in female HF rats but decreased in male HF rats. mRNA levels of AVP were lower in female rats in both control and HF groups compared with the respective male groups. Activation of extracellular signal-regulated protein kinases 1 and 2 increased similarly in both sexes in HF. The results suggest that female HF rats have less central neural excitation and less associated hemodynamic compromise than male HF rats with the same degree of initial ischemic cardiac injury. NEW & NOTEWORTHY Sex differences in the presentation and responses to treatment of heart failure (HF) are widely recognized, but the underlying mechanisms are poorly understood. The present study describes sex differences in the central nervous system mechanisms that drive neurohumoral excitation in ischemia-induced HF. Female rats had a less intense central neurochemical response to HF and experienced less hemodynamic compromise. Sex hormones may contribute to these differences in the central and peripheral adaptations to HF.

Angiotensin II Type 1a Receptors in the Subfornical Organ Modulate Neuroinflammation in the Hypothalamic Paraventricular Nucleus in Heart Failure Rats.

Inflammation in the hypothalamic paraventricular nucleus (PVN) contributes to neurohumoral excitation and its adverse consequences in systolic heart failure (HF). The stimuli that trigger inflammation in the PVN in HF are not well understood. Angiotensin II (AngII) has pro-inflammatory effects, and circulating levels of AngII increase in HF. The subfornical organ (SFO), a circumventricular structure that lacks an effective blood-brain barrier and senses circulating AngII, contains PVN-projecting neurons. We hypothesized that activation of AngII type 1a receptors (AT1aR) in the SFO induces neuroinflammation downstream in the PVN. Male rats received SFO microinjections of an adeno-associated virus carrying shRNA for AT1aR, a scrambled shRNA, or vehicle. One week later, some rats were euthanized to confirm the transfection potential and knockdown efficiency of the shRNA. Others underwent coronary artery ligation to induce HF or a sham coronary artery ligation (Sham). Four weeks later, HF rats that received the scrambled shRNA had increased mRNA in SFO and PVN for AT1aR, inflammatory mediators and indicators of neuronal and glial activation, increased plasma levels of AngII, tumor necrosis factor-α, norepinephrine and arginine vasopressin, and impaired cardiac function, compared with Sham rats that received scrambled shRNA. The central abnormalities were ameliorated in HF rats that received AT1aR shRNA, as were plasma norepinephrine and vasopressin. Sham rats that received AT1aR shRNA had reduced SFO AT1aR mRNA but no other changes compared with Sham rats that received scrambled shRNA. The results suggest that activation of AT1aR in the SFO upregulates the neuroinflammation in the PVN that contributes to neurohumoral excitation in HF.

Blood-borne interleukin-1ß acts on the subfornical organ to upregulate the sympathoexcitatory milieu of the hypothalamic paraventricular nucleus.

We previously reported that microinjection of the proinflammatory cytokine interleukin-1ß (IL-1ß) into the subfornical organ (SFO) elicits a pressor response accompanied by increases in inflammation and renin-angiotensin system (RAS) activity in the SFO and hypothalamic paraventricular nucleus (PVN). The present study sought to determine whether blood-borne IL-1ß induces similar neurochemical changes in the SFO and PVN and, if so, whether increased inflammation and RAS activity at the SFO level orchestrate the sympathoexcitatory response to circulating IL-1ß. In urethane-anesthetized male Sprague-Dawley rats, intravenous injection of IL-1ß (500 ng) increased blood pressure, heart rate, renal sympathetic nerve activity, and mRNA for angiotensin-converting enzyme, angiotensin II type 1a receptor, cyclooxygenase-2, tumor necrosis factor-α, and IL-1ß, as well as the tumor necrosis factor-α p55 receptor and the IL-1 receptor, in the SFO and PVN. Pretreatment with SFO microinjections of the angiotensin II type 1a receptor blocker losartan (1 µg), the angiotensin-converting enzyme inhibitor captopril (1 µg), or the cyclooxygenase-2 inhibitor NS-398 (2 µg) attenuated expression of these excitatory mediators in the SFO and downstream in the PVN and the IL-1ß-induced pressor responses. An SFO lesion minimized the IL-1ß-induced expression of inflammatory and RAS components as well as c-Fos, an indicator of neuronal excitation, in the PVN. These studies demonstrate that circulating IL-1ß, which increases in cardiovascular disorders such as hypertension and heart failure, acts on the SFO to increase inflammation and RAS activity in the SFO and PVN and that intervening in these neurochemical processes in the SFO can significantly reduce the sympathetic response.

TNF-α receptor 1 knockdown in the subfornical organ ameliorates sympathetic excitation and cardiac hemodynamics in heart failure rats.

In systolic heart failure (HF), circulating proinflammatory cytokines upregulate inflammation and renin-angiotensin system (RAS) activity in cardiovascular regions of the brain, contributing to sympathetic excitation and cardiac dysfunction. Important among these is the subfornical organ (SFO), a forebrain circumventricular organ that lacks an effective blood-brain barrier and senses circulating humors. We hypothesized that the tumor necrosis factor-α (TNF-α) receptor 1 (TNFR1) in the SFO contributes to sympathetic excitation and cardiac dysfunction in HF rats. Rats received SFO microinjections of a TNFR1 shRNA or a scrambled shRNA lentiviral vector carrying green fluorescent protein, or vehicle. One week later, some rats were euthanized to confirm the accuracy of the SFO microinjections and the transfection potential of the lentiviral vector. Other rats underwent coronary artery ligation (CL) to induce HF or a sham operation. Four weeks after CL, vehicle- and scrambled shRNA-treated HF rats had significant increases in TNFR1 mRNA and protein, NF-κB activity, and mRNA for inflammatory mediators, RAS components and c-Fos protein in the SFO and downstream in the hypothalamic paraventricular nucleus, along with increased plasma norepinephrine levels and impaired cardiac function, compared with vehicle-treated sham-operated rats. In HF rats treated with TNFR1 shRNA, TNFR1 was reduced in the SFO but not paraventricular nucleus, and the central and peripheral manifestations of HF were ameliorated. In sham-operated rats treated with TNFR1 shRNA, TNFR1 expression was also reduced in the SFO but there were no other effects. These results suggest a key role for TNFR1 in the SFO in the pathophysiology of systolic HF.NEW & NOTEWORTHY Activation of TNF-α receptor 1 in the subfornical organ (SFO) contributes to sympathetic excitation in heart failure rats by increasing inflammation and renin-angiotensin system activity in the SFO and downstream in the hypothalamic paraventricular nucleus. Cytokine receptors in the SFO may be a target for central intervention in cardiovascular conditions characterized by peripheral inflammation.

Endoplasmic reticulum stress increases brain MAPK signaling, inflammation and renin-angiotensin system activity and sympathetic nerve activity in heart failure.

We previously reported that endoplasmic reticulum (ER) stress is induced in the subfornical organ (SFO) and the hypothalamic paraventricular nucleus (PVN) of heart failure (HF) rats and is reduced by inhibition of mitogen-activated protein kinase (MAPK) signaling. The present study further examined the relationship between brain MAPK signaling, ER stress, and sympathetic excitation in HF. Sham-operated (Sham) and HF rats received a 4-wk intracerebroventricular (ICV) infusion of vehicle (Veh) or the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 10 µg/day). Lower mRNA levels of the ER stress biomarkers GRP78, ATF6, ATF4, and XBP-1s in the SFO and PVN of TUDCA-treated HF rats validated the efficacy of the TUDCA dose. The elevated levels of phosphorylated p44/42 and p38 MAPK in SFO and PVN of Veh-treated HF rats, compared with Sham rats, were significantly reduced in TUDCA-treated HF rats as shown by Western blot and immunofluorescent staining. Plasma norepinephrine levels were higher in Veh-treated HF rats, compared with Veh-treated Sham rats, and were significantly lower in the TUDCA-treated HF rats. TUDCA-treated HF rats also had lower mRNA levels for angiotensin converting enzyme, angiotensin II type 1 receptor, tumor necrosis factor-α, interleukin-1ß, cyclooxygenase-2, and NF-κB p65, and a higher mRNA level of IκB-α, in the SFO and PVN than Veh-treated HF rats. These data suggest that ER stress contributes to the augmented sympathetic activity in HF by inducing MAPK signaling, thereby promoting inflammation and renin-angiotensin system activity in key cardiovascular regulatory regions of the brain.