UBR1 Promotes Sex-Dependent ACE2 Ubiquitination in Hypertension.

Background: Angiotensin (Ang)-II impairs the function of the antihypertensive enzyme ACE2 by promoting its internalization, ubiquitination and degradation thus contributing to hypertension. However, few ACE2 ubiquitination partners have been identified and their role in hypertension remains unknown. Methods: Proteomics and bioinformatic analysis were used to identify ACE2 ubiquitination partners in the brain, heart, and kidney from Ang-II-infused C57BL6/J mice from both sexes and validated the interaction between UBR1 and ACE2 in cells. Central and peripheral UBR1 knockdown was then performed in male mice to investigate its role in the maintenance of hypertension. Results: Proteomics analysis from hypothalamus identified UBR1 as a potential E3 ligase promoting ACE2 ubiquitination. Enhanced UBR1 expression, associated with ACE2 reduction, was confirmed in various tissues from hypertensive male mice and human samples. Treatment of endothelial and smooth muscle cells with testosterone, but not 17ß-estradiol, confirmed a sex-specific regulation of UBR1. In vivo silencing of UBR1 using chronic administration of small interference RNA resulted in the restoration of ACE2 levels in hypertensive males. A transient decrease in blood pressure following intracerebroventricular, but not systemic, infusion was also observed. Interestingly, UBR1 knockdown increased the brain activation of Nedd4-2, an E3 ligase promoting ACE2 ubiquitination and reduced expression of SGK1, the kinase inactivating Nedd4-2. Conclusions: These data demonstrate that UBR1 is a novel ubiquitin ligase targeting ACE2 in hypertension. UBR1 and Nedd4-2 E3 ligases appear to work synergistically to ubiquitinate ACE2. Targeting of these ubiquitin ligases may represent a novel strategy to restore ACE2 compensatory activity in hypertension.

Emerging Role of Kinin B1 Receptor in Persistent Neuroinflammation and Neuropsychiatric Symptoms in Mice Following Recovery from SARS-CoV-2 Infection.

Evidence suggests that patients with long COVID can experience neuropsychiatric, neurologic, and cognitive symptoms. However, these clinical data are mostly associational studies complicated by confounding variables, thus the mechanisms responsible for persistent symptoms are unknown. Here we establish an animal model of long-lasting effects on the brain by eliciting mild disease in K18-hACE2 mice. Male and female K18-hACE2 mice were infected with 4 × 103 TCID50 of SARS-CoV-2 and, following recovery from acute infection, were tested in the open field, zero maze, and Y maze, starting 30 days post infection. Following recovery from SARS-CoV-2 infection, K18-hACE2 mice showed the characteristic lung fibrosis associated with SARS-CoV-2 infection, which correlates with increased expression of the pro-inflammatory kinin B1 receptor (B1R). These mice also had elevated expression of B1R and inflammatory markers in the brain and exhibited behavioral alterations such as elevated anxiety and attenuated exploratory behavior. Our data demonstrate that K18-hACE2 mice exhibit persistent effects of SARS-CoV-2 infection on brain tissue, revealing the potential for using this model of high sensitivity to SARS-CoV-2 to investigate mechanisms contributing to long COVID symptoms in at-risk populations. These results further suggest that elevated B1R expression may drive the long-lasting inflammatory response associated with SARS-CoV-2 infection.

Nedd4-2 up-regulation is associated with ACE2 ubiquitination in hypertension.

AIMS: Angiotensin-converting enzyme 2 (ACE2) is a critical component of the compensatory renin-angiotensin system that is down-regulated during the development of hypertension, possibly via ubiquitination. However, little is known about the mechanisms involved in ACE2 ubiquitination in neurogenic hypertension. This study aimed at identifying ACE2 ubiquitination partners, establishing causal relationships and clinical relevance, and testing a gene therapy strategy to mitigate ACE2 ubiquitination in neurogenic hypertension. METHODS AND RESULTS: Bioinformatics and proteomics were combined to identify E3 ubiquitin ligases associated with ACE2 ubiquitination in chronically hypertensive mice. In vitro gain/loss of function experiments assessed ACE2 expression and activity to validate the interaction between ACE2 and the identified E3 ligase. Mutation experiments were further used to generate a ubiquitination-resistant ACE2 mutant (ACE2-5R). Optogenetics, blood pressure telemetry, pharmacological blockade of GABAA receptors in mice expressing ACE2-5R in the bed nucleus of the stria terminalis (BNST), and capillary western analysis were used to assess the role of ACE2 ubiquitination in neurogenic hypertension. Ubiquitination was first validated as leading to ACE2 down-regulation, and Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) was identified as a E3 ligase up-regulated in hypertension and promoting ACE2 ubiquitination. Mutation of lysine residues in the C-terminal of ACE2 was associated with increased activity and resistance to angiotensin (Ang)-II-mediated degradation. Mice transfected with ACE2-5R in the BNST exhibited enhanced GABAergic input to the paraventricular nucleus (PVN) and a reduction in hypertension. ACE2-5R expression was associated with reduced Nedd4-2 levels in the BNST. CONCLUSION: Our data identify Nedd4-2 as the first E3 ubiquitin ligase involved in ACE2 ubiquitination in Ang-II-mediated hypertension. We demonstrate the pivotal role of ACE2 on GABAergic neurons in the maintenance of an inhibitory tone to the PVN and the regulation of pre-sympathetic activity. These findings provide a new working model where Nedd4-2 could contribute to ACE2 ubiquitination, leading to the development of neurogenic hypertension and highlighting potential novel therapeutic strategies.

Cardiomyocyte-specific deletion of TLR4 attenuates angiotensin II-induced hypertension and cardiac remodeling.

Toll-like receptor 4 (TLR4) is an integral factor in the initiation of the innate immune response and plays an important role in cardiovascular diseases such as hypertension and myocardial infarction. Previous studies from our lab demonstrated that central TLR4 blockade reduced cardiac TLR4 expression, attenuated hypertension, and improved cardiac function. However, the contribution of cardiac specific TLR4 to the development of hypertension and cardiac remodeling is unknown. Therefore, we hypothesized that cardiomyocyte specific knockdown of TLR4 would have beneficial effects on hypertension, cardiac hypertrophy, and remodeling. To test this hypothesis, cardiomyocyte-specific TLR4 knockdown (cTLR4KO) mice were generated by crossing floxed TLR4 mice with Myh6-Cre mice, and subjected to angiotensin II (Ang II, 1 µg/kg/min or vehicle for 14 days) hypertension model. Blood pressure measurements using radio telemetry revealed no differences in baseline mean arterial pressure between control littermates and cTLR4KO mice (103 ± 2 vs. 105 ± 3 mmHg, p > 0.05). Ang II-induced hypertension (132 ± 2 vs. 151 ± 3 mmHg, p < 0.01) was attenuated and cardiac hypertrophy (heart/body weight; 4.7 vs. 5.8 mg/g, p < 0.01) was prevented in cTLR4KO mice when compared with control mice. In addition, the level of myocardial fibrosis was significantly reduced, and the cardiac function was improved in cTLR4KO mice infused with Ang II. Furthermore, cardiac inflammation, as evidenced by elevated gene expression of TNF, IL-6, and MCP-1 in the left ventricle, was attenuated in cTLR4KO mice infused with Ang II. Together, this data revealed a protective role for cardiomyocyte-specific deletion of TLR4 against Ang II-induced hypertension and cardiac dysfunction through inhibition of proinflammatory cytokines.

Kinin B1 Receptor Mediates Bidirectional Interaction between Neuroinflammation and Oxidative Stress.

Hypertension is associated with increased expression of kinin B1 receptors (B1R) and increased levels of pro-inflammatory cytokines within the neurons. We previously reported that angiotensin II (Ang II) upregulates B1R expression and can induce neuroinflammation and oxidative stress in primary hypothalamic neurons. However, the order in which B1R activation, neuroinflammation, and oxidative stress occur has not yet been studied. Using primary hypothalamic neurons from neonatal mice, we show that tumor necrosis factor (TNF), lipopolysaccharides (LPS), and hydrogen peroxide (H2O2) can upregulate B1R expression and increase oxidative stress. Furthermore, our study shows that B1R blockade with R715, a specific B1R antagonist, can attenuate these effects. To further confirm our findings, we used a deoxycorticosterone acetate (DOCA)-salt model of hypertension to show that oxidative stress is upregulated in the hypothalamic paraventricular nucleus (PVN) of the brain. Together, these data provide novel evidence that relationship between oxidative stress, neuroinflammation, and B1R upregulation in the brain is bidirectional, and that B1R antagonism may have beneficial effects on neuroinflammation and oxidative stress in various disease pathologies.

Dopamine receptor 3: A mystery at the heart of cardiac fibrosis.

Dopamine receptors have been extensively studied in the mammalian brain and spinal cord, as dopamine is a vital determinant of bodily movement, cognition, and overall behavior. Thus, dopamine receptor antagonist antipsychotic drugs are commonly used to treat multiple psychiatric disorders. Although less discussed, these receptors are also expressed in other peripheral organ systems, such as the kidneys, eyes, gastrointestinal tract, and cardiac tissue. Consequently, therapies for certain psychiatric disorders which target dopamine receptors could have unidentified consequences on certain functions of these peripheral tissues. The existence of an intrinsic dopaminergic system in the human heart remains controversial and debated within the literature. Therefore, this review focuses on literature related to dopamine receptors within cardiac tissue, specifically dopamine receptor 3 (D3R), and summarizes the current state of knowledge while highlighting areas of research which may be lacking. Additionally, recent findings regarding crosstalk between D3R and dopamine receptor 1 (D1R) are examined. This review discusses the novel concept of understanding the role of the loss of function of D3R may play in collagen accumulation and cardiac fibrosis, eventually leading to heart failure.

COVID-19 Infection Enhances Susceptibility to Oxidative Stress-Induced Parkinsonism.

BACKGROUND: Viral induction of neurological syndromes has been a concern since parkinsonian-like features were observed in patients diagnosed with encephalitis lethargica subsequent to the 1918 influenza pandemic. Given the similarities in the systemic responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with those observed after pandemic influenza, there is a question whether a similar syndrome of postencephalic parkinsonism could follow coronavirus disease 2019 infection. OBJECTIVE: The goal of this study was to determine whether prior infection with SARS-CoV-2 increased sensitivity to a mitochondrial toxin known to induce parkinsonism. METHODS: K18-hACE2 mice were infected with SARS-CoV-2 to induce mild-to-moderate disease. After 38 days of recovery, mice were administered a non-lesion-inducing dose of the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and euthanized 7 days later. Subsequent neuroinflammation and substantia nigra pars compacta (SNpc) dopaminergic (DA) neuron loss were determined and compared with SARS-CoV-2 or MPTP alone. RESULTS: K18-hACE2 mice infected with SARS-CoV-2 or MPTP showed no SNpc DA neuron loss after MPTP. In mice infected and recovered from SARS-CoV-2 infection, MPTP induced a 23% or 19% greater loss of SNpc DA neurons than SARS-CoV-2 or MPTP, respectively (P < 0.05). Examination of microglial activation showed a significant increase in the number of activated microglia in both the SNpc and striatum of the SARS-CoV-2 + MPTP group compared with SARS-CoV-2 or MPTP alone. CONCLUSIONS: Our observations have important implications for long-term public health, given the number of people who have survived SARS-CoV-2 infection, as well as for future public policy regarding infection mitigation. However, it will be critical to determine whether other agents known to increase risk for PD also have synergistic effects with SARS-CoV-2 and are abrogated by vaccination. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Kinin B1R Activation Induces Endoplasmic Reticulum Stress in Primary Hypothalamic Neurons.

The endoplasmic reticulum (ER) is a key organelle involved in homeostatic functions including protein synthesis and transport, and the storage of free calcium. ER stress potentiates neuroinflammation and neurodegeneration and is a key contributor to the pathogenesis of neurogenic hypertension. Recently, we showed that kinin B1 receptor (B1R) activation plays a vital role in modulating neuroinflammation and hypertension. However, whether B1R activation results in the progression and enhancement of ER stress has not yet been studied. In this brief research report, we tested the hypothesis that B1R activation in neurons contributes to unfolded protein response (UPR) and the development of ER stress. To test this hypothesis, we treated primary hypothalamic neuronal cultures with B1R specific agonist Lys-Des-Arg9-Bradykinin (LDABK) and measured the components of UPR and ER stress. Our data show that B1R stimulation via LDABK, induced the upregulation of GRP78, a molecular chaperone of ER stress. B1R stimulation was associated with an increased expression and activation of transmembrane ER stress sensors, ATF6, IRE1α, and PERK, the critical components of UPR. In the presence of overwhelming ER stress, activated ER stress sensors can lead to oxidative stress, autophagy, or apoptosis. To determine whether B1R activation induces apoptosis we measured intracellular Ca2+ and extracellular ATP levels, caspases 3/7 activity, and cell viability. Our data show that LDABK treatment does increase Ca2+ and ATP levels but does not alter caspase activity or cell viability. These findings suggest that B1R activation initiates the UPR and is a key factor in the ER stress pathway.

The Actin Bundling Protein Fascin-1 as an ACE2-Accessory Protein.

We have previously shown that angiotensin-converting enzyme 2 (ACE2), an enzyme counterbalancing the deleterious effects of angiotensin type 1 receptor activation by production of vasodilatory peptides Angiotensin (Ang)-(1-9) and Ang-(1-7), is internalized and degraded in lysosomes following chronic Ang-II treatment. However, the molecular mechanisms involved in this effect remain unknown. In an attempt to identify the accessory proteins involved in this effect, we conducted a proteomic analysis in ACE2-transfected HEK293T cells. A single protein, fascin-1, was found to differentially interact with ACE2 after Ang-II treatment for 4 h. The interactions between fascin-1 and ACE2 were confirmed by confocal microscopy and co-immunoprecipitation. Overexpression of fascin-1 attenuates the effects of Ang-II on ACE2 activity. In contrast, downregulation of fascin-1 severely decreased ACE2 enzymatic activity. Interestingly, in brain homogenates from hypertensive mice, we observed a significant reduction of fascin-1, suggesting that the levels of this protein may change in cardiovascular diseases. In conclusion, we identified fascin-1 as an ACE2-accessory protein, interacting with the enzyme in an Ang-II dependent manner and contributing to the regulation of enzyme activity.

Hypothalamic kinin B1 receptor mediates orexin system hyperactivity in neurogenic hypertension.

Brain orexin system hyperactivity contributes to neurogenic hypertension. We previously reported upregulated neuronal kinin B1 receptor (B1R) expression in hypertension. However, the role of central B1R activation on the orexin system in neurogenic hypertension has not been examined. We hypothesized that kinin B1R contributes to hypertension via upregulation of brain orexin-arginine vasopressin signaling. We utilized deoxycorticosterone acetate (DOCA)-salt hypertension model in wild-type (WT) and B1R knockout (B1RKO) mice. In WT mice, DOCA-salt-treatment increased gene and protein expression of orexin A, orexin receptor 1, and orexin receptor 2 in the hypothalamic paraventricular nucleus and these effects were attenuated in B1RKO mice. Furthermore, DOCA-salt- treatment increased plasma arginine vasopressin levels in WT mice, but not in B1RKO mice. Cultured primary hypothalamic neurons expressed orexin A and orexin receptor 1. B1R specific agonist (LDABK) stimulation of primary neurons increased B1R protein expression, which was abrogated by B1R selective antagonist R715 but not by the dual orexin receptor antagonist, ACT 462206, suggesting that B1R is upstream of the orexin system. These data provide novel evidence that B1R blockade blunts orexin hyperactivity and constitutes a potential therapeutic target for the treatment of salt-sensitive hypertension.

Loss of Function in Dopamine D3 Receptor Attenuates Left Ventricular Cardiac Fibroblast Migration and Proliferation in vitro.

Evidence suggests the existence of an intracardiac dopaminergic system that plays a pivotal role in regulating cardiac function and fibrosis through G-protein coupled receptors, particularly mediated by dopamine receptor 3 (D3R). However, the expression of dopamine receptors in cardiac tissue and their role in cardiac fibroblast function is unclear. In this brief report, first we determined expression of D1R and D3R both in left ventricle (LV) tissue and fibroblasts. Then, we explored the role of D3R in the proliferation and migration of fibroblast cell cultures using both genetic and pharmaceutical approaches; specifically, we compared cardiac fibroblasts isolated from LV of wild type (WT) and D3R knockout (D3KO) mice in response to D3R-specific pharmacological agents. Finally, we determined if loss of D3R function could significantly alter LV fibroblast expression of collagen types I (Col1a1) and III (Col3a1). Cardiac fibroblast proliferation was attenuated in D3KO cells, mimicking the behavior of WT cardiac fibroblasts treated with D3R antagonist. In response to scratch injury, WT cardiac fibroblasts treated with the D3R agonist, pramipexole, displayed enhanced migration compared to control WT and D3KO cells. Loss of function in D3R resulted in attenuation of both proliferation and migration in response to scratch injury, and significantly increased the expression of Col3a1 in LV fibroblasts. These findings suggest that D3R may mediate cardiac fibroblast function during the wound healing response. To our knowledge this is the first report of D3R's expression and functional significance directly in mouse cardiac fibroblasts.

Kinin B1 Receptor Mediates Renal Injury and Remodeling in Hypertension.

Despite many readily available therapies, hypertensive kidney disease remains the second most prevalent cause of end-stage renal disease after diabetes, and continues to burden patient populations and escalate morbidity and mortality rates. Kinin B1 receptor (B1R) activation has been shown to have a role in the development of hypertension, one of the major etiologies for chronic kidney disease. However, the role of B1R in hypertension induced renal injury and remodeling remains unexplored. Using a DOCA-salt-induced hypertensive mouse model, we investigated whether B1R deficiency reduces hypertensive renal injury and fibrosis. To further recognize the translational role of B1R, we examined the expression of B1R and its correlation with collagen deposition in renal biopsies from control and hypertensive kidney disease patients. Our data indicates that renal B1R expression was upregulated in the kidneys of DOCA-salt hypertensive mice. Genetic ablation of B1R protected the mice from DOCA-salt-induced renal injury and fibrosis by preventing inflammation and oxidative stress in the kidney. Cultured human proximal tubular epithelial cells expressed B1R and stimulation of B1R with an agonist resulted in increased oxidative stress. In human kidney biopsy samples, we found that the B1R immunoreactivity was not only significantly increased in hypertensive patients compared to normotensive patients, but also there is a positive correlation between B1R expression and renal fibrosis levels. Taken together, our results identify a critical role of B1R in the development of inflammation and fibrosis of the kidney in hypertension.

Activation of Kinin B1R Upregulates ADAM17 and Results in ACE2 Shedding in Neurons.

Angiotensin converting enzyme 2 (ACE2) is a critical component of the compensatory axis of the renin angiotensin system. Alterations in ACE2 gene and protein expression, and activity mediated by A Disintegrin And Metalloprotease 17 (ADAM17), a member of the "A Disintegrin And Metalloprotease" (ADAM) family are implicated in several cardiovascular and neurodegenerative diseases. We previously reported that activation of kinin B1 receptor (B1R) in the brain increases neuroinflammation, oxidative stress and sympathoexcitation, leading to the development of neurogenic hypertension. We also showed evidence for ADAM17-mediated ACE2 shedding in neurons. However, whether kinin B1 receptor (B1R) activation has any role in altering ADAM17 activity and its effect on ACE2 shedding in neurons is not known. In this study, we tested the hypothesis that activation of B1R upregulates ADAM17 and results in ACE2 shedding in neurons. To test this hypothesis, we stimulated wild-type and B1R gene-deleted mouse neonatal primary hypothalamic neuronal cultures with a B1R-specific agonist and measured the activities of ADAM17 and ACE2 in neurons. B1R stimulation significantly increased ADAM17 activity and decreased ACE2 activity in wild-type neurons, while pretreatment with a B1R-specific antagonist, R715, reversed these changes. Stimulation with specific B1R agonist Lys-Des-Arg9-Bradykinin (LDABK) did not show any effect on ADAM17 or ACE2 activities in neurons with B1R gene deletion. These data suggest that B1R activation results in ADAM17-mediated ACE2 shedding in primary hypothalamic neurons. In addition, stimulation with high concentration of glutamate significantly increased B1R gene and protein expression, along with increased ADAM17 and decreased ACE2 activities in wild-type neurons. Pretreatment with B1R-specific antagonist R715 reversed these glutamate-induced effects suggesting that indeed B1R is involved in glutamate-mediated upregulation of ADAM17 activity and ACE2 shedding.

Kinin B1 receptor: A target for neuroinflammation in hypertension.

Kinins are a family of oligopeptides of the kallikrein-kinin system that act as potent vasoactive hormones and inflammatory mediators. The bioactive kinins mainly consist of bradykinin and kallidin, and their metabolites des-Arg9-bradykinin and des-Arg10-kallidin. Physiological effects of kinins are mediated by activation of highly selective G-protein coupled kinin B1 and B2 receptors. Growing evidence suggests that B1 receptor activation mediates diverse physiological and pathological features of cardiovascular diseases. However, studies are limited regarding the impact of B1 receptor mediated neuroinflammation on the development of hypertension and other cardiovascular diseases. Given the potential role for B1 receptor activation in immune cell infiltration, microglia activation, and cytokine production within the central nervous system, B1 receptor mediated signaling cascades might result in elevated neuroinflammation. In this review, we will discuss the potential pro-inflammatory role of B1 receptor activation in hypertension. A better understanding of B1 receptor inflammatory signaling may lead to the development of therapeutics that target B1 receptors to treat neurogenic hypertension.

Kinin B1 Receptor Blockade Prevents Angiotensin II-induced Neuroinflammation and Oxidative Stress in Primary Hypothalamic Neurons.

Neuroinflammation has become an important underlying factor in many cardiovascular disorders, including hypertension. Previously we showed that elevated angiotensin II (Ang II) and angiotensin II type I receptor (AT1R) expression levels can increase neuroinflammation leading to hypertension. We also found that kinin B1 receptor (B1R) expression increased in the hypothalamic paraventricular neurons resulting in neuroinflammation and oxidative stress in neurogenic hypertension. However, whether there are any potential interactions between AT1R and B1R in neuroinflammation is not clear. In the present study, we aimed to determine whether Ang II-mediated effects on inflammation and oxidative stress are mediated by the activation of B1R in mouse neonatal primary hypothalamic neuronal cultures. Gene expression and immunostaining revealed that both B1R and AT1R are expressed on primary hypothalamic neurons. Ang II stimulation significantly increased the expression of B1R, decreased mitochondrial respiration, increased the expression of two NADPH oxidase subunits (Nox2 and Nox4), increased the oxidative potential, upregulated several proinflammatory genes (IL-1ß, IL-6, and TNFα), and increased NF-kB p65 DNA binding activity. These changes were prevented by pretreatment with the B1R-specific peptide antagonist, R715. In summary, our study demonstrates a causal relationship between B1R expression after Ang II stimulation, suggesting a possible cross talk between AT1R and B1R in neuroinflammation and oxidative stress.

Aging influences cerebrovascular myogenic reactivity and BK channel function in a sex-specific manner.

AIMS: The myogenic reactivity of the middle cerebral arteries (MCA) protects the brain by altering the diameter in response to changes in lumen pressure. Large conductance potassium (BK) channels are known to regulate the myogenic reactivity, yet, it is not clear how aging alters the myogenic reactivity via the BK channel in males and females. Thus, we hypothesize that age-associated changes in BK channel subunits modulate the myogenic reactivity in a sex-specific manner. METHODS AND RESULTS: We used vascular reactivity, patch-clamp, and biochemical methods to measure myogenic reactivity, BK channel function, and expression, respectively in cerebral vessels of adult and aged male and female Sprague Dawley rats. Our results suggest that aging and ovariectomy (OVX) exaggerated the myogenic reactivity of MCA in females but attenuated it in males. Aging induced outward eutrophic remodelling in females but inward hypertrophic remodelling in males. Aging decreased total, Kv, BK channel currents, and spontaneous transient outward currents (STOC) in vascular smooth muscle cells isolated from females, but not in males. Aging increased BKα subunit mRNA and protein both in males and females. However, aging decreased BKß1 subunit protein and mRNA in females only. In males, BKß1 mRNA is increased, but protein is decreased. Iberiotoxin-induced MCA constriction is lower in aged females but higher in aged males. Activation of BKα (10 µM NS1619) and BKß1 (10 µM S-Equol) subunits failed to increase STOCs and were unable to decrease the myogenic reactivity of MCA in aged female but not in aged male rats. OVX decreased, but chronic supplementation of oestradiol restored BK channel expression and function. CONCLUSION: Overall our results suggest that aging or OVX-associated downregulation of the BKß1 expression and function in females results in exaggerated myogenic reactivity of MCA. However, age-associated increase in BK channel function in males attenuated myogenic reactivity of MCA.

Glutamatergic neurons of the paraventricular nucleus are critical contributors to the development of neurogenic hypertension.

KEY POINTS: Recurrent periods of over-excitation in the paraventricular nucleus (PVN) of the hypothalamus could contribute to chronic over-activation of this nucleus and thus enhanced sympathetic drive. Stimulation of the PVN glutamatergic population utilizing channelrhodopsin-2 leads to an immediate frequency-dependent increase in baseline blood pressure. Partial lesions of glutamatergic neurons of the PVN (39.3%) result in an attenuated rise in blood pressure following Deoxycorticosterone acetate (DOCA)-salt treatment and reduced index of sympathetic activity. These data suggest that stimulation of PVN glutamatergic neurons is sufficient to cause autonomic dysfunction and drive the increase in blood pressure during hypertension. ABSTRACT: Neuro-cardiovascular dysregulation leads to increased sympathetic activity and neurogenic hypertension. The paraventricular nucleus (PVN) of the hypothalamus is a key hub for blood pressure (BP) control, producing or relaying the increased sympathetic tone in hypertension. We hypothesize that increased central sympathetic drive is caused by chronic over-excitation of glutamatergic PVN neurons. We tested how stimulation or lesioning of excitatory PVN neurons in conscious mice affects BP, baroreflex and sympathetic activity. Glutamatergic PVN neurons were unilaterally transduced with channelrhodopsin-2 using an adeno-associated virus (CamKII-ChR2-eYFP-AAV2) in wildtype mice (n = 7) to assess the impact of acute stimulation of excitatory PVN neurons selectively on resting BP in conscious mice. Stimulation of the PVN glutamatergic population resulted in an immediate frequency-dependent (2, 10 and 20 Hz) increase in BP from baseline by ∼9 mmHg at 20 Hz stimulation (P < 0.001). Additionally, in vGlut2-cre mice glutamatergic neurons of the PVN were bilaterally lesioned utilizing a cre-dependent caspase (AAV2-flex-taCASP3-TEVp). Resting BP and urinary noradrenaline (norepinephrine) levels were then recorded in conscious mice before and after DOCA-salt hypertension. Partial lesions of glutamatergic neurons of the PVN (39.3%, P < 0.05) resulted in an attenuated rise in BP following DOCA-salt treatment (P < 0.05 at 7 day time point, n = 8). Noradrenaline levels as an index of sympathetic activity between the lesion and wildtype groups showed a significant reduction after DOCA-salt treatment in the lesioned animals (P < 0.05). These experiments suggest that stimulation of PVN glutamatergic neurons is sufficient to cause autonomic dysfunction and drive the increase in BP.

Excessive Glutamate Stimulation Impairs ACE2 Activity Through ADAM17-Mediated Shedding in Cultured Cortical Neurons.

The excitotoxicity of glutamate plays an important role in the progression of various neurological disorders via participating in inflammation and neuronal damage. In this study, we identified the role of excessive glutamate stimulation in the modulation of angiotensin-converting enzyme type 2 (ACE2), a critical component in the compensatory axis of the renin-angiotensin system (RAS). In primary cultured cortical neurons, high concentration of glutamate (100 µM) significantly reduced the enzymatic activity of ACE2. The elevated activity of ADAM17, a member of the 'A Disintegrin And Metalloprotease' (ADAM) family, was found to contribute to this glutamate-induced ACE2 down-regulation. The decrease of ACE2 activity could be prevented by pre-treatment with antagonists targeting ionotropic glutamate receptors. In addition, the glutamate-induced decrease in ACE2 activity was significantly attenuated when the neurons were co-treated with MitoTEMPOL or blockers that target oxidative stress-mediated signaling pathway. In summary, our study reveals a strong relationship between excessive glutamate stimulation and ADAM17-mediated impairment in ACE2 activity, suggesting a possible cross-talk between glutamate-induced excitotoxicity and dysregulated RAS.

Kinin B1 Receptor Promotes Neurogenic Hypertension Through Activation of Centrally Mediated Mechanisms.

Hypertension is associated with increased activity of the kallikrein-kinin system. Kinin B1 receptor (B1R) activation leads to vasoconstriction and inflammation. Despite evidence supporting a role for the B1R in blood pressure regulation, the mechanisms by which B1R could alter autonomic function and participate in the pathogenesis of hypertension remain unidentified. We sought to explore whether B1R-mediated inflammation contributes to hypertension and investigate the molecular mechanisms involved. In this study, we tested the hypothesis that activation of B1R in the brain is involved in the pathogenesis of hypertension, using the deoxycorticosterone acetate-salt model of neurogenic hypertension in wild-type and B1R knockout mice. Deoxycorticosterone acetate-salt treatment in wild-type mice led to significant increases in B1R mRNA and protein levels and bradykinin levels, enhanced gene expression of carboxypeptidase N supporting an increase in the B1R ligand, associated with enhanced blood pressure, inflammation, sympathoexcitation, autonomic dysfunction, and impaired baroreflex sensitivity, whereas these changes were blunted or prevented in B1R knockout mice. B1R stimulation was further shown to involve activation of the ASK1-JNK-ERK1/2 and NF-κB pathways in the brain. To dismiss potential developmental alterations in knockout mice, we further used B1R blockade selectively in the brain of wild-type mice. Supporting the central origin of this mechanism, intracerebroventricular infusion of a specific B1R antagonist, attenuated the deoxycorticosterone acetate-salt-induced increase in blood pressure in wild-type mice. Our data provide the first evidence of a central role for B1R-mediated inflammatory pathways in the pathogenesis of deoxycorticosterone acetate-salt hypertension and offer novel insights into possible B1R-targeted therapies for the treatment of neurogenic hypertension.

Reliability modelling of redundant safety systems without automatic diagnostics incorporating common cause failures and process demand.

Redundant safety systems are commonly used in the process industry to respond to hazardous events. In redundant systems composed of identical units, Common Cause Failures (CCFs) can significantly influence system performance with regards to reliability and safety. However, their impact has been overlooked due to the inherent complexity of modelling common cause induced failures. This article develops a reliability model for a redundant safety system using Markov analysis approach. The proposed model incorporates process demands in conjunction with CCF for the first time and evaluates their impacts on the reliability quantification of safety systems without automatic diagnostics. The reliability of the Markov model is quantified by considering the Probability of Failure on Demand (PFD) as a measure for low demand systems. The safety performance of the model is analysed using Hazardous Event Frequency (HEF) to evaluate the frequency of entering a hazardous state that will lead to an accident if the situation is not controlled. The utilisation of Markov model for a simple case study of a pressure protection system is demonstrated and it is shown that the proposed approach gives a sufficiently accurate result for all demand rates, durations, component failure rates and corresponding repair rates for low demand mode of operation. The Markov model proposed in this paper assumes the absence of automatic diagnostics, along with multiple stage repair strategy for CCFs and restoration of the system from hazardous state to the "as good as new" state.