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.

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.

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.

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.

Leptin Mediates High-Fat Diet Sensitization of Angiotensin II-Elicited Hypertension by Upregulating the Brain Renin-Angiotensin System and Inflammation.

Obesity is characterized by increased circulating levels of the adipocyte-derived hormone leptin, which can increase sympathetic nerve activity and raise blood pressure. A previous study revealed that rats fed a high-fat diet (HFD) have an enhanced hypertensive response to subsequent angiotensin II administration that is mediated at least, in part, by increased activity of brain renin-angiotensin system and proinflammatory cytokines. This study tested whether leptin mediates this HFD-induced sensitization of angiotensin II-elicited hypertension by interacting with brain renin-angiotensin system and proinflammatory cytokine mechanisms. Rats fed an HFD for 3 weeks had significant increases in white adipose tissue mass, plasma leptin levels, and mRNA expression of leptin and its receptors in the lamina terminalis and hypothalamic paraventricular nucleus. Central infusion of a leptin receptor antagonist during HFD feeding abolished HFD sensitization of angiotensin II-elicited hypertension. Furthermore, central infusion of leptin mimicked the sensitizing action of HFD. Concomitant central infusions of the angiotensin II type 1 receptor antagonist irbesartan, the tumor necrosis factor-α synthesis inhibitor pentoxifylline, or the inhibitor of microglial activation minocycline prevented the sensitization produced by central infusion of leptin. RT-PCR analysis indicated that either HFD or leptin administration upregulated mRNA expression of several components of the renin-angiotensin system and proinflammatory cytokines in the lamina terminalis and paraventricular nucleus. The leptin antagonist and the inhibitors of angiotensin II type 1 receptor, tumor necrosis factor-α synthesis, and microglial activation all reversed the expression of these genes. The results suggest that HFD-induced sensitization of angiotensin II-elicited hypertension is mediated by leptin through upregulation of central renin-angiotensin system and proinflammatory cytokines.

Inhibition of Brain Mitogen-Activated Protein Kinase Signaling Reduces Central Endoplasmic Reticulum Stress and Inflammation and Sympathetic Nerve Activity in Heart Failure Rats.

Mitogen-activated protein kinase (MAPK) signaling and endoplasmic reticulum (ER) stress in the brain have been implicated in the pathophysiology of hypertension. This study determined whether ER stress occurs in subfornical organ and hypothalamic paraventricular nucleus in heart failure (HF) and how MAPK signaling interacts with ER stress and other inflammatory mediators. HF rats had significantly higher levels of the ER stress biomarkers (glucose-regulated protein 78, activating transcription factor 6, activating transcription factor 4, X-box binding protein 1, P58(IPK), and C/EBP homologous protein) in subfornical organ and paraventricular nucleus, which were attenuated by a 4-week intracerebroventricular infusion of inhibitors selective for p44/42 MAPK (PD98059), p38 MAPK (SB203580), or c-Jun N-terminal kinase (SP600125). HF rats also had higher mRNA levels of tumor necrosis factor-α, interleukin-1ß, cyclooxygenase-2, and nuclear factor-κB p65, and a lower mRNA level of IκB-α, in subfornical organ and paraventricular nucleus, compared with SHAM rats, and these indicators of increased inflammation were attenuated in the HF rats treated with the MAPK inhibitors. Plasma norepinephrine level was higher in HF rats than in SHAM rats but was reduced in the HF rats treated with PD98059 and SB203580. A 4-week intracerebroventricular infusion of PD98059 also improved some hemodynamic and anatomic indicators of left ventricular function in HF rats. These data demonstrate that ER stress increases in the subfornical organ and paraventricular nucleus of rats with ischemia-induced HF and that inhibition of brain MAPK signaling reduces brain ER stress and inflammation and decreases sympathetic excitation in HF. An interaction between MAPK signaling and ER stress in cardiovascular regions of the brain may contribute to the development of HF.

Central Renin-Angiotensin System Activation and Inflammation Induced by High-Fat Diet Sensitize Angiotensin II-Elicited Hypertension.

Obesity has been shown to promote renin-angiotensin system activity and inflammation in the brain and to be accompanied by increased sympathetic activity and blood pressure. Our previous studies demonstrated that administration of a subpressor dose of angiotensin (Ang) II sensitizes subsequent Ang II-elicited hypertension. The present study tested whether high-fat diet (HFD) feeding also sensitizes the Ang II-elicited hypertensive response and whether HFD-induced sensitization is mediated by an increase in renin-angiotensin system activity and inflammatory mechanisms in the brain. HFD did not increase baseline blood pressure, but enhanced the hypertensive response to Ang II compared with a normal-fat diet. The sensitization produced by the HFD was abolished by concomitant central infusions of either a tumor necrosis factor-α synthesis inhibitor, pentoxifylline, an Ang II type 1 receptor blocker, irbesartan, or an inhibitor of microglial activation, minocycline. Furthermore, central pretreatment with tumor necrosis factor-α mimicked the sensitizing action of a central subpressor dose of Ang II, whereas central pentoxifylline or minocycline abolished this Ang II-induced sensitization. Real-time quantitative reverse transcription-polymerase chain reaction analysis of lamina terminalis tissue indicated that HFD feeding, central tumor necrosis factor-α, or a central subpressor dose of Ang II upregulated mRNA expression of several components of the renin-angiotensin system and proinflammatory cytokines, whereas inhibition of Ang II type 1 receptor and of inflammation reversed these changes. The results suggest that HFD-induced sensitization of Ang II-elicited hypertension is mediated by upregulation of the brain renin-angiotensin system and of central proinflammatory cytokines.

Activation of central PPAR-γ attenuates angiotensin II-induced hypertension.

Inflammation and renin-angiotensin system activity in the brain contribute to hypertension through effects on fluid intake, vasopressin release, and sympathetic nerve activity. We recently reported that activation of brain peroxisome proliferator-activated receptor (PPAR)-γ in heart failure rats reduced inflammation and renin-angiotensin system activity in the hypothalamic paraventricular nucleus and ameliorated the peripheral manifestations of heart failure. We hypothesized that the activation of brain PPAR-γ might have beneficial effects in angiotensin II-induced hypertension. Sprague-Dawley rats received a 2-week subcutaneous infusion of angiotensin II (120 ng/kg per minute) combined with a continuous intracerebroventricular infusion of vehicle, the PPAR-γ agonist pioglitazone (3 nmol/h) or the PPAR-γ antagonist GW9662 (7 nmol/h). Angiotensin II+vehicle rats had increased mean blood pressure, increased sympathetic drive as indicated by the mean blood pressure response to ganglionic blockade, and increased water consumption. PPAR-γ mRNA in subfornical organ and hypothalamic paraventricular nucleus was unchanged, but PPAR-γ DNA-binding activity was reduced. mRNA for interleukin-1ß, tumor necrosis factor-α, cyclooxygenase-2, and angiotensin II type 1 receptor was augmented in both nuclei, and hypothalamic paraventricular nucleus neuronal activity was increased. The plasma vasopressin response to a 6-hour water restriction also increased. These responses to angiotensin II were exacerbated by GW9662 and ameliorated by pioglitazone, which increased PPAR-γ mRNA and PPAR-γ DNA-binding activity in subfornical organ and hypothalamic paraventricular nucleus. Pioglitazone and GW9662 had no effects on control rats. The results suggest that activating brain PPAR-γ to reduce central inflammation and brain renin-angiotensin system activity may be a useful adjunct in the treatment of angiotensin II-dependent hypertension.

Proinflammatory cytokines upregulate sympathoexcitatory mechanisms in the subfornical organ of the rat.

Our previous work indicated that the subfornical organ (SFO) is an important brain sensor of blood-borne proinflammatory cytokines, mediating their central effects on autonomic and cardiovascular function. However, the mechanisms by which SFO mediates the central effects of circulating proinflammatory cytokines remain unclear. We hypothesized that proinflammatory cytokines act within the SFO to upregulate the expression of excitatory and inflammatory mediators that drive sympathetic nerve activity. In urethane-anesthetized Sprague-Dawley rats, direct microinjection of tumor necrosis factor (TNF)-α (25 ng) or interleukin (IL)-1ß (25 ng) into SFO increased mean blood pressure, heart rate, and renal sympathetic nerve activity within 15 to 20 minutes, mimicking the response to systemically administered proinflammatory cytokines. Pretreatment of SFO with microinjections of the angiotensin II type-1 receptor blocker losartan (1 µg), angiotensin-converting enzyme inhibitor captopril (1 µg) or cyclooxygenase-2 inhibitor NS-398 (2 µg) attenuated those responses. Four hours after the SFO microinjection of TNF-α (25 ng) or IL-1ß (25 ng), mRNA for angiotensin-converting enzyme, angiotensin II type-1 receptor, TNF-α and the p55 TNF-α receptor, IL-1ß and the IL-1R receptor, and cyclooxygenase-2 had increased in SFO, and mRNA for angiotensin-converting enzyme, angiotensin II type-1 receptor, and cyclooxygenase-2 had increased downstream in the hypothalamic paraventricular nucleus. Confocal immunofluorescent images revealed that immunoreactivity for the p55 TNF-α receptor and the IL-1 receptor accessory protein, a subunit of the IL-1 receptor, colocalized with angiotensin-converting enzyme, angiotensin II type-1 receptor-like, cyclooxygenase-2, and prostaglandin E2 EP3 receptor immunoreactivity in SFO neurons. These data suggest that proinflammatory cytokines act within the SFO to upregulate the expression of inflammatory and excitatory mediators that drive sympathetic excitation.

Central SDF-1/CXCL12 expression and its cardiovascular and sympathetic effects: the role of angiotensin II, TNF-α, and MAP kinase signaling.

The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptors are expressed by neurons and glial cells in cardiovascular autonomic regions of the brain, including the hypothalamic paraventricular nucleus (PVN), and contribute to neurohumoral excitation in rats with ischemia-induced heart failure. The present study examined factors regulating the expression of SDF-1 in the PVN and mechanisms mediating its sympatho-excitatory effects. In urethane anesthetized rats, a 4-h intracerebroventricular (ICV) infusion of angiotensin II (ANG II) or tumor necrosis factor-α (TNF-α) in doses that increase mean blood pressure (MBP) and sympathetic drive increased the expression of SDF-1 in PVN. ICV administration of SDF-1 increased the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK), JNK, and p38 MAPK in PVN, along with MBP, heart rate (HR), and renal sympathetic nerve activity (RSNA), but did not affect total p44/42 MAPK, JNK, and p38 MAPK levels. ICV pretreatment with the selective p44/42 MAPK inhibitor PD98059 prevented the SDF-1-induced increases in MBP, HR, and RSNA; ICV pretreatment with the selective JNK and p38 MAPK inhibitors attenuated but did not block these SDF-1-induced excitatory responses. ICV PD98059 also prevented the sympatho-excitatory response to bilateral PVN microinjections of SDF-1. ICV pretreatment with SDF-1 short-hairpin RNA significantly reduced ANG II- and TNF-α-induced phosphorylation of p44/42 MAPK in PVN. These findings identify TNF-α and ANG II as drivers of SDF-1 expression in PVN and suggest that the full expression of their cardiovascular and sympathetic effects depends upon SDF-1-mediated activation of p44/42 MAPK signaling.

Subfornical organ mediates sympathetic and hemodynamic responses to blood-borne proinflammatory cytokines.

Proinflammatory cytokines play an important role in regulating autonomic and cardiovascular function in hypertension and heart failure. Peripherally administered proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), act on the brain to increase blood pressure, heart rate, and sympathetic nerve activity. These molecules are too large to penetrate the blood-brain barrier, and so the mechanisms by which they elicit these responses remain unknown. We tested the hypothesis that the subfornical organ (SFO), a forebrain circumventricular organ that lacks a blood-brain barrier, plays a major role in mediating the sympathetic and hemodynamic responses to circulating proinflammatory cytokines. Intracarotid artery injection of TNF-α (200 ng) or IL-1ß (200 ng) dramatically increased mean blood pressure, heart rate, and renal sympathetic nerve activity in rats with sham lesions of the SFO (SFO-s). These excitatory responses to intracarotid artery TNF-α and IL-1ß were significantly attenuated in SFO-lesioned (SFO-x) rats. Similarly, the increases in mean blood pressure, heart rate, and renal sympathetic nerve activity in response to intravenous injections of TNF-α (500 ng) or IL-1ß (500 ng) in SFO-s rats were significantly reduced in the SFO-x rats. Immunofluorescent staining revealed a dense distribution of the p55 TNF-α receptor and the IL-1 receptor accessory protein, a subunit of the IL-1 receptor, in the SFO. These data suggest that SFO is a predominant site in the brain at which circulating proinflammatory cytokines act to elicit cardiovascular and sympathetic responses.

Differential regulation of EHD3 in human and mammalian heart failure.

Electrical and structural remodeling during the progression of cardiovascular disease is associated with adverse outcomes subjecting affected patients to overt heart failure (HF) and/or sudden death. Dysfunction in integral membrane protein trafficking has long been linked with maladaptive electrical remodeling. However, little is known regarding the molecular identity or function of these intracellular targeting pathways in the heart. Eps15 homology domain-containing (EHD) gene products (EHD1-4) are polypeptides linked with endosomal trafficking, membrane protein recycling, and lipid homeostasis in a wide variety of cell types. EHD3 was recently established as a critical mediator of membrane protein trafficking in the heart. Here, we investigate the potential link between EHD3 function and heart disease. Using four different HF models including ischemic rat heart, pressure overloaded mouse heart, chronic pacing-induced canine heart, and non-ischemic failing human myocardium we provide the first evidence that EHD3 levels are consistently increased in HF. Notably, the expression of the Na/Ca exchanger (NCX1), targeted by EHD3 in heart is similarly elevated in HF. Finally, we identify a molecular pathway for EHD3 regulation in heart failure downstream of reactive oxygen species and angiotensin II signaling. Together, our new data identify EHD3 as a previously unrecognized component of the cardiac remodeling pathway.