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.

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.

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.

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.

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.

Clinical Relevance and Role of Neuronal AT1 Receptors in ADAM17-Mediated ACE2 Shedding in Neurogenic Hypertension.

RATIONALE: Neurogenic hypertension is characterized by an increase in sympathetic activity and often resistance to drug treatments. We previously reported that it is also associated with a reduction of angiotensin-converting enzyme type 2 (ACE2) and an increase in a disintegrin and metalloprotease 17 (ADAM17) activity in experimental hypertension. In addition, while multiple cells within the central nervous system have been involved in the development of neurogenic hypertension, the contribution of ADAM17 has not been investigated. OBJECTIVE: To assess the clinical relevance of this ADAM17-mediated ACE2 shedding in hypertensive patients and further identify the cell types and signaling pathways involved in this process. METHODS AND RESULTS: Using a mass spectrometry-based assay, we identified ACE2 as the main enzyme converting angiotensin II into angiotensin-(1-7) in human cerebrospinal fluid. We also observed an increase in ACE2 activity in the cerebrospinal fluid of hypertensive patients, which was correlated with systolic blood pressure. Moreover, the increased level of tumor necrosis factor-α in those cerebrospinal fluid samples confirmed that ADAM17 was upregulated in the brain of hypertensive patients. To further assess the interaction between brain renin-angiotensin system and ADAM17, we generated mice lacking angiotensin II type 1 receptors specifically on neurons. Our data reveal that despite expression on astrocytes and other cells types in the brain, ADAM17 upregulation during deoxycorticosterone acetate-salt hypertension occurs selectively on neurons, and neuronal angiotensin II type 1 receptors are indispensable to this process. Mechanistically, reactive oxygen species and extracellular signal-regulated kinase were found to mediate ADAM17 activation. CONCLUSIONS: Our data demonstrate that angiotensin II type 1 receptors promote ADAM17-mediated ACE2 shedding in the brain of hypertensive patients, leading to a loss in compensatory activity during neurogenic hypertension.

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.

Brain ACE2 overexpression reduces DOCA-salt hypertension independently of endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress was previously reported to contribute to neurogenic hypertension while neuronal angiotensin-converting enzyme type 2 (ACE2) overexpression blunts the disease. To assess which brain regions are important for ACE2 beneficial effects and the contribution of ER stress to neurogenic hypertension, we first used transgenic mice harboring a floxed neuronal hACE2 transgene (SL) and tested the impact of hACE2 knockdown in the subfornical organ (SFO) and paraventricular nucleus (PVN) on deoxycorticosterone acetate (DOCA)-salt hypertension. SL and nontransgenic (NT) mice underwent DOCA-salt or sham treatment while infected with an adenoassociated virus (AAV) encoding Cre recombinase (AAV-Cre) or a control virus (AAV-green fluorescent protein) to the SFO or PVN. DOCA-salt-induced hypertension was reduced in SL mice, with hACE2 overexpression in the brain. This reduction was only partially blunted by knockdown of hACE2 in the SFO or PVN, suggesting that both regions are involved but not essential for ACE2 regulation of blood pressure (BP). DOCA-salt treatment did not increase the protein levels of ER stress and autophagy markers in NT mice, despite a significant increase in BP. In addition, these markers were not affected by hACE2 overexpression in the brain, despite a significant reduction of hypertension in SL mice. To further assess the role of ER stress in neurogenic hypertension, NT mice were infused intracerebroventricularlly with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, during DOCA-salt treatment. However, TUDCA infusion failed to blunt the development of hypertension in NT mice. Our data suggest that brain ER stress does not contribute to DOCA-salt hypertension and that ACE2 blunts neurogenic hypertension independently of ER stress.

Brain angiotensin-converting enzyme type 2 shedding contributes to the development of neurogenic hypertension.

RATIONALE: Overactivity of the brain renin-angiotensin system is a major contributor to neurogenic hypertension. Although overexpression of angiotensin-converting enzyme type 2 (ACE2) has been shown to be beneficial in reducing hypertension by transforming angiotensin II into angiotensin-(1-7), several groups have reported decreased brain ACE2 expression and activity during the development of hypertension. OBJECTIVE: We hypothesized that ADAM17-mediated ACE2 shedding results in decreased membrane-bound ACE2 in the brain, thus promoting the development of neurogenic hypertension. METHODS AND RESULTS: To test this hypothesis, we used the deoxycorticosterone acetate-salt model of neurogenic hypertension in nontransgenic and syn-hACE2 mice overexpressing ACE2 in neurons. Deoxycorticosterone acetate-salt treatment in nontransgenic mice led to significant increases in blood pressure, hypothalamic angiotensin II levels, inflammation, impaired baroreflex sensitivity, and autonomic dysfunction, as well as decreased hypothalamic ACE2 activity and expression, although these changes were blunted or prevented in syn-hACE2 mice. In addition, reduction of ACE2 expression and activity in the brain paralleled an increase in ACE2 activity in the cerebrospinal fluid of nontransgenic mice after deoxycorticosterone acetate-salt treatment and were accompanied by enhanced ADAM17 expression and activity in the hypothalamus. Chronic knockdown of ADAM17 in the brain blunted the development of hypertension and restored ACE2 activity and baroreflex function. CONCLUSIONS: Our data provide the first evidence that ADAM17-mediated shedding impairs brain ACE2 compensatory activity, thus contributing to the development of neurogenic hypertension.

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.

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.

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.

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.

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.

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.

Central TNF inhibition results in attenuated neurohumoral excitation in heart failure: a role for superoxide and nitric oxide.

This study examined the effect of central tumor necrosis factor-alpha (TNF) blockade on the imbalance between nitric oxide and superoxide production in the paraventricular nucleus (PVN) and ventrolateral medulla (VLM), key autonomic regulators, and their contribution to enhanced sympathetic drive in mice with congestive heart failure (CHF). We also used a TNF gene knockout (KO) mouse model to study the involvement of TNF in body fluid homeostasis and sympathoexcitation in CHF. After implantation of intracerebroventricular (ICV) cannulae, myocardial infarction (MI) was induced in wild-type (WT) and KO mice by coronary artery ligation. Osmotic mini-pumps were implanted into one set of WT + MI/Sham mice for continuous ICV infusion of Etanercept (ETN), a TNF receptor fusion protein, or vehicle (VEH). Gene expressions of neuronal nitric oxide synthase (NOS) and angiotensin receptor-type 2 were reduced, while those of inducible NOS, Nox2 homologs, superoxide, peroxynitrite and angiotensin receptor-type 1 were elevated in the brainstem and hypothalamus of MI + VEH. Plasma norepinephrine levels and the number of Fos-positive neurons were also increased in the PVN and VLM in MI + VEH. MI + ETN and KO + MI mice exhibited reduced oxidative stress, reduced sympathoexcitation and an improved cardiac function. These changes in WT + MI were associated with increased sodium and fluid retention. These results indicate that elevated TNF in these autonomic regulatory regions of the brain alter the production of superoxide and nitric oxide, contributing to fluid imbalance and sympathoexcitation in CHF.

ACE2/ANG-(1-7)/Mas pathway in the brain: the axis of good.

The last decade has seen the discovery of several new components of the renin-angiotensin system (RAS). Among them, angiotensin converting enzyme-2 (ACE2) and the Mas receptor have forced a reevaluation of the original cascade and led to the emergence of a new arm of the RAS: the ACE2/ANG-(1-7)/Mas axis. Accordingly, the new system is now seen as a balance between a provasoconstrictor, profibrotic, progrowth axis (ACE/ANG-II/AT(1) receptor) and a provasodilatory, antifibrotic, antigrowth arm (ACE2/ANG-(1-7)/Mas receptor). Already, this simplistic vision is evolving and new components are branching out upstream [ANG-(1-12) and (pro)renin receptor] and downstream (angiotensin-IV and other angiotensin peptides) of the classical cascade. In this review, we will summarize the role of the ACE2/ANG-(1-7)/Mas receptor, focusing on the central nervous system with respect to cardiovascular diseases such as hypertension, chronic heart failure, and stroke, as well as neurological diseases. In addition, we will discuss the new pharmacological (antagonists, agonists, activators) and genomic (knockout and transgenic animals) tools that are currently available. Finally, we will review the latest data regarding the various signaling pathways downstream of the Mas receptor.

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays.

Angiotensin-converting enzyme 2 (ACE2) is a component of the renin-angiotensin system, and its expression and activity have been shown to be reduced in cardiovascular diseases. Enzymatic activity of ACE2 is commonly measured by hydrolysis of quenched fluorescent substrates in the absence or presence of an ACE2-specific inhibitor, such as the commercially available inhibitor DX600. Whereas recombinant human ACE2 is readily detected in mouse tissues using 1 µM DX600 at pH 7.5, the endogenous ACE2 activity in mouse tissues is barely detectable. We compared human, mouse, and rat ACE2 overexpressed in cell lines for their sensitivity to inhibition by DX600. ACE2 from all three species could be inhibited by DX600, but the half maximal inhibitory concentration (IC(50)) for human ACE2 was much lower (78-fold) than for rodent ACE2. Following optimization of pH, substrate concentration, and antagonist concentration, rat and mouse ACE2 expressed in a cell line could be accurately quantified with 10 µM DX600 (>95% inhibition) but not with 1 µM DX600 (<75% inhibition). Validation that the optimized method robustly quantifies ACE2 in mouse tissues (kidney, brain, heart, and plasma) was performed using wild-type and ACE2 knockout mice. This study provides a reliable method for measuring human, as well as endogenous ACE2 activity in rodents. Our data underscore the importance of validating the effect of DX600 on ACE2 from each particular species at the experimental conditions employed.