SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein.

Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson's disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson's disease in COVID-19 infected individuals, and a potential therapeutic avenue for intervention.

Chronic Renin-Angiotensin System Activation Induced Neuroinflammation: Common Mechanisms Underlying Hypertension and Dementia?

Hypertension is a major risk factor for the pathogenesis of vascular dementia and Alzheimer's disease. Chronic activation of the renin-angiotensin system (RAS) contributes substantially to neuroinflammation. We propose that neuroinflammation arising from chronic RAS activation can initiate and potentiate the onset of hypertension and related dementia. Neuroinflammation induced by chronic activation of the RAS plays a key role in the pathogenesis of dementia. Increased levels of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and transforming growth factor (TGF)-ß have been reported in brain tissue of vascular dementia patients and animal models of vascular dementia induced by either angiotensin II infusion or transverse aortic coarctation. It is proposed that neuronal cell death and synaptic dysfunction induced by neuroinflammation lead to cognitive impairment in dementia. The neuroprotective RAS pathway, regulated by angiotensin-converting enzyme 2 (ACE2) which converts angiotensin II into angiotensin-(1-7), can attenuate hypertension and dementia. Furthermore, the use of anti-hypertensive medications in preventing dementia or cognitive decline in hypertensive patients and animal models of dementia have mostly been beneficial. Current evidence suggests a strong link between RAS induced neuroinflammation and the onset of hypertension and dementia, which warrants further investigation. Strategies to counteract an overactive RAS and enhance the neuroprotective arm of the RAS may help prevent or improve cognitive impairment associated with hypertension.

Stimulation of Angiotensin Converting Enzyme 2: A Novel Treatment Strategy for Diabetic Nephropathy.

Despite current therapies for diabetic nephropathy, many patients continue to progress to end-stage renal disease requiring renal replacement therapy. While the precise mechanisms underlying diabetic nephropathy remain to be determined, it is well established that chronic activation of the renin angiotensin aldosterone system (RAAS) plays a substantial role in the pathogenesis of diabetic nephropathy. Angiotensin converting enzyme 2 (ACE2), the enzyme responsible for activating the reno-protective arm of the RAAS converts angiotensin (Ang) II into Ang 1-7 which exerts reno-protective effects. Chronic RAAS activation leads to kidney inflammation and fibrosis, and ultimately lead to end-stage kidney disease. Currently, angiotensin converting enzyme inhibitors and Ang II receptor blockers are approved for renal fibrosis and inflammation. Targeting the reno-protective arm of the RAAS should therefore, provide further treatment options for kidney fibrosis and inflammation. In this review, we examine how targeting the reno-protective arm of the RAAS can ameliorate kidney inflammation and fibrosis and rescue kidney function in diabetic nephropathy. We argue tissue ACE2 stimulation provides a unique and promising therapeutic approach for diabetic nephropathy.

Endothelial-specific overexpression of cationic amino acid transporter-1 prevents loss of kidney function in heart failure.

Heart failure (HF) is associated with impaired L-arginine transport. In the present study, we tested the hypothesis that augmented L-arginine transport prevents the loss of kidney function in HF. Renal function was assessed in wildtype mice (WT), transgenic mice with HF (dilated cardiomyopathy, DCM) and double transgenic mice (double transgenic mice with DCM and CAT-1 overexpression, HFCAT-1) with HF and endothelial-specific overexpression of the predominant L-arginine transporter, cationic amino acid transporter-1 (CAT-1) (n=4-8/group). Cardiac function was assessed via echocardiography and left ventricular catheterisation. Renal function was assessed via quantification of albuminuria and creatinine clearance. Plasma nitrate and nitrite levels together with renal fibrosis and inflammatory markers were also quantified at study end. Albumin/creatinine ratio was two-fold greater in DCM mice than in WT mice (P=0.002), and tubulointerstitial and glomerular fibrosis were approximately eight- and three-fold greater, respectively, in DCM mice than in WT mice (P≤0.02). Critically, urinary albumin/creatinine ratio and tubulointerstitial and glomerular fibrosis were less in HFCAT-1 mice than in DCM mice (P<0.05). Renal CAT-1 expression and plasma nitrate and nitrite levels were less in DCM mice compared with WT (P≤0.03) but was greater in HFCAT-1 mice than in DCM mice (P≤0.009). Renal expression of IL-10 was less in DCM mice compared with WT (P<0.001) but was greater in HFCAT-1 mice compared with DCM mice (P=0.02). Our data provide direct evidence that augmented L-arginine transport prevents renal fibrosis, inflammation and loss of kidney function in HF.

Impaired l-arginine-nitric oxide pathway contributes to the pathogenesis of resistant hypertension.

The precise mechanisms underlying resistant hypertension remain elusive. Reduced nitric oxide (NO) bioavailability is frequently documented in chronic kidney disease, obesity, diabetes and advanced age, all of which are risk factors for resistant hypertension. Sympathetic overactivity and chronic activation of the renin-angiotensin system are salient features of resistant hypertension. Interestingly, recent data indicate that renal sympathetic overactivity can reduce the expression of neuronal nitric oxide synthase in the paraventricular nucleus. Reduced NO levels in the paraventricular nucleus can increase sympathetic outflow and this can create a vicious cycle contributing to resistant hypertension. Angiotensin II can reduce l-arginine transport and hence NO production. Reduced NO levels may reduce the formation of angiotensin 1-7 dampening the cardio-protective effects of the renin-angiotensin system contributing to resistant hypertension. In addition, interleukin-6 (IL-6) is demonstrated to be independently associated with resistant hypertension, and IL-6 can reduce NO synthesis. Despite this, NO levels have not been quantified in resistant hypertension. Findings from a small proof of concept study indicate that NO donors can reduce blood pressure in patients with resistant hypertension but more studies are required to validate these preliminary findings. In the present paper, we put forward the hypothesis that reduced NO bioavailability contributes substantially to the development of resistant hypertension.

Serelaxin attenuates renal inflammation and fibrosis in a mouse model of dilated cardiomyopathy.

NEW FINDINGS: What is the central question of this study? The aim was to determine the renoprotective effects of serelaxin in the setting of chronic heart failure. What are the main findings and its importance? Our data indicate that serelaxin can reduce renal fibrosis and inflammation in experimental heart failure. Currently, there are no effective treatments to rescue renal function in heart failure patients, and our data suggest that serelaxin might have the potential to reduce renal fibrosis and inflammation in heart failure. ABSTRACT: Serelaxin has been demonstrated to attenuate renal fibrosis and inflammation in cardiorenal disease. In the present study, we tested the hypothesis that serelaxin can prevent the decline in renal function in dilated cardiomyopathy (DCM) by targeting renal fibrosis and inflammation. Male transgenic mice with DCM (n = 16) and their wild-type littermates (WT; n = 20) were administered either vehicle or serelaxin (500 µg kg-1  day-1 ; subcutaneous minipumps; 8 weeks). Cardiac function was assessed via echocardiography before and during the eighth week of serelaxin treatment. Renal function and inflammation as well as cardiac and renal fibrosis were assessed at the end of the study. Serelaxin had minimal effect on cardiac function (P ≥ 0.99). Tubulointerstitial and glomerular fibrosis were ∼3-fold greater in vehicle-treated DCM mice compared with vehicle-treated WT mice (P ≤ 0.001). Renal mRNA expression of Tnfα and Il1α were ∼4- and ∼3-fold greater, respectively, in vehicle-treated DCM mice compared with vehicle-treated WT mice (P ≤ 0.05). Tubulointerstitial and glomerular fibrosis were 46 and 45% less, respectively, in serelaxin-treated DCM mice than in vehicle-treated DCM mice (P ≤ 0.01). Renal cortical mRNA expression of Tnfα and Il1α were 56 and 58% less, respectively, in the former group compared with the latter (P ≤ 0.05). The urinary albumin:creatinine ratio was ∼3-fold greater in vehicle-treated DCM mice compared with vehicle-treated WT mice (P = 0.02). The urinary albumin:creatinine ratio was not significantly different between vehicle-treated DCM mice and serelaxin-treated DCM mice (P = 0.38). These data suggest that serelaxin can attenuate renal fibrosis and inflammation and has the potential to exert renoprotective effects in DCM.

N-Acetylcysteine Attenuates the Development of Renal Fibrosis in Transgenic Mice with Dilated Cardiomyopathy.

Mechanisms underlying the renal pathology in cardiorenal syndrome (CRS) type 2 remain elusive. We hypothesised that renal glutathione deficiency is central to the development of CRS type 2. Glutathione precursor, N-acetylcysteine (NAC;40 mg/kg/day; 8 weeks) or saline were administered to transgenic mice with dilated cardiomyopathy (DCM) and wild-type (WT) controls. Cardiac structure, function and glutathione levels were assessed at the end of this protocol. Renal fibrosis, glutathione content, expression of inflammatory and fibrotic markers, and function were also evaluated. In both genotypes, NAC had minimal effect on cardiac glutathione, structure and function (P ≥ 0.20). In NAC treated DCM mice, loss of glomerular filtration rate (GFR), tubulointerstitial and glomerular fibrosis and renal oxidised glutathione levels were attenuated by 38%, 99%, 70% and 52% respectively, compared to saline treated DCM mice (P ≤ 0.01). Renal expression of PAI-1 was greater in saline treated DCM mice than in WT mice (P < 0.05). Renal PAI-1 expression was less in NAC treated DCM mice than in vehicle treated DCM mice (P = 0.03). Renal IL-10 expression was greater in the former cohort compared to the latter (P < 0.01). These data indicate that normalisation of renal oxidized glutathione levels attenuates PAI-1 expression and renal inflammation preventing loss of GFR in experimental DCM.

Pharmacological hypothesis: Nitric oxide-induced inhibition of ADAM-17 activity as well as vesicle release can in turn prevent the production of soluble endothelin-converting enzyme.

Endothelin-1 (ET-1) and nitric oxide (NO) are two highly potent vasoactive molecules with opposing effects on the vasculature. Endothelin-converting enzyme (ECE) and nitric oxide synthase (NOS) catalyse the production of ET-1 and NO, respectively. It is well established that these molecules play a crucial role in the initiation and progression of cardiovascular diseases and have therefore become targets of therapy. Many studies have examined the mechanism(s) by which NO regulates ET-1 production. Expression and localization of ECE-1 is a key factor that determines the rate of ET-1 production. ECE-1 can either be membrane bound or be released from the cell surface to produce a soluble form. NO has been shown to reduce the expression of both membrane-bound and soluble ECE-1. Several studies have examined the mechanism(s) behind NO-mediated inhibition of ECE expression on the cell membrane. However, the precise mechanism(s) behind NO-mediated inhibition of soluble ECE production are unknown. We hypothesize that both exogenous and endogenous NO, inhibits the production of soluble ECE-1 by preventing its release via extracellular vesicles (e.g., exosomes), and/or by inhibiting the activity of A Disintegrin and Metalloprotease-17 (ADAM17). If this hypothesis is proven correct in future studies, these pathways represent targets for the therapeutic manipulation of soluble ECE-1 production.

The Cardiovascular and Neurotoxic Effects of the Venoms of Six Bony and Cartilaginous Fish Species.

Fish venoms are often poorly studied, in part due to the difficulty in obtaining, extracting, and storing them. In this study, we characterize the cardiovascular and neurotoxic effects of the venoms from the following six species of fish: the cartilaginous stingrays Neotrygon kuhlii and Himantura toshi, and the bony fish Platycephalus fucus, Girella tricuspidata, Mugil cephalus, and Dentex tumifrons. All venoms (10-100 µg/kg, i.v.), except G. tricuspidata and P. fuscus, induced a biphasic response on mean arterial pressure (MAP) in the anesthetised rat. P. fucus venom exhibited a hypotensive response, while venom from G. tricuspidata displayed a single depressor response. All venoms induced cardiovascular collapse at 200 µg/kg, i.v. The in vitro neurotoxic effects of venom were examined using the chick biventer cervicis nerve-muscle (CBCNM) preparation. N. kuhlii, H. toshi, and P. fucus venoms caused concentration-dependent inhibition of indirect twitches in the CBCNM preparation. These three venoms also inhibited responses to exogenous acetylcholine (ACh) and carbachol (CCh), but not potassium chloride (KCl), indicating a post-synaptic mode of action. Venom from G. tricuspidata, M. cephalus, and D. tumifrons had no significant effect on indirect twitches or agonist responses in the CBCNM. Our results demonstrate that envenoming by these species of fish may result in moderate cardiovascular and/or neurotoxic effects. Future studies aimed at identifying the molecules responsible for these effects could uncover potentially novel lead compounds for future pharmaceuticals, in addition to generating new knowledge about the evolutionary relationships between venomous animals.

High-Fiber Diet and Acetate Supplementation Change the Gut Microbiota and Prevent the Development of Hypertension and Heart Failure in Hypertensive Mice.

BACKGROUND: Dietary intake of fruit and vegetables is associated with lower incidence of hypertension, but the mechanisms involved have not been elucidated. Here, we evaluated the effect of a high-fiber diet and supplementation with the short-chain fatty acid acetate on the gut microbiota and the prevention of cardiovascular disease. METHODS: Gut microbiome, cardiorenal structure/function, and blood pressure were examined in sham and mineralocorticoid excess-treated mice with a control diet, high-fiber diet, or acetate supplementation. We also determined the renal and cardiac transcriptome of mice treated with the different diets. RESULTS: We found that high consumption of fiber modified the gut microbiota populations and increased the abundance of acetate-producing bacteria independently of mineralocorticoid excess. Both fiber and acetate decreased gut dysbiosis, measured by the ratio of Firmicutes to Bacteroidetes, and increased the prevalence of Bacteroides acidifaciens. Compared with mineralocorticoid-excess mice fed a control diet, both high-fiber diet and acetate supplementation significantly reduced systolic and diastolic blood pressures, cardiac fibrosis, and left ventricular hypertrophy. Acetate had similar effects and markedly reduced renal fibrosis. Transcriptome analyses showed that the protective effects of high fiber and acetate were accompanied by the downregulation of cardiac and renal Egr1, a master cardiovascular regulator involved in cardiac hypertrophy, cardiorenal fibrosis, and inflammation. We also observed the upregulation of a network of genes involved in circadian rhythm in both tissues and downregulation of the renin-angiotensin system in the kidney and mitogen-activated protein kinase signaling in the heart. CONCLUSIONS: A diet high in fiber led to changes in the gut microbiota that played a protective role in the development of cardiovascular disease. The favorable effects of fiber may be explained by the generation and distribution of one of the main metabolites of the gut microbiota, the short-chain fatty acid acetate. Acetate effected several molecular changes associated with improved cardiovascular health and function.

Stimulating the Activity of Amyloid-Beta Degrading Enzymes: A Novel Approach for the Therapeutic Manipulation of Amyloid-Beta Levels.

Alzheimer's disease is a debilitating neurological disease placing significant burden on health care budgets around the world. It is widely believed that accumulation of amyloid-beta (Aß) in the brain is a key event that initiates neurodegeneration, thus the clearance of Aß from brain could be a key therapeutic strategy. Aß exists in an equilibrium in healthy individuals, and recent research would suggest that dysfunction in the clearance pathways is the driving force behind its accumulation. One mechanism of clearance is proteolytic degradation by enzymes, and increasing the expression of these enzymes in animal models of Alzheimer's disease has indeed shown promising results. This approach could be challenging to translate into the clinic given the likely need for genetic manipulation. We hypothesize that stimulating the activity of these enzymes (as opposed to increasing expression) through pharmacological agents will enhance degradation or at least prevent amyloid deposition, and is therefore another potentially novel avenue to manipulate Aß levels for therapeutic purposes. We discuss the recent research supporting this hypothesis as well as possible drawbacks to this approach.

Canopy Venom: Proteomic Comparison among New World Arboreal Pit-Viper Venoms.

Central and South American pitvipers, belonging to the genera Bothrops and Bothriechis, have independently evolved arboreal tendencies. Little is known regarding the composition and activity of their venoms. In order to close this knowledge gap, venom proteomics and toxin activity of species of Bothriechis, and Bothrops (including Bothriopsis) were investigated through established analytical methods. A combination of proteomics and bioactivity techniques was used to demonstrate a similar diversification of venom composition between large and small species within Bothriechis and Bothriopsis. Increasing our understanding of the evolution of complex venom cocktails may facilitate future biodiscoveries.

Effects of Dietary l-Arginine on Nitric Oxide Bioavailability in Obese Normotensive and Obese Hypertensive Subjects.

Obesity related hypertension is a major risk factor for resistant hypertension. We do not completely understand the mechanism(s) underlying the development of obesity related hypertension which hinders the development of novel treatment strategies for this condition. Data from experimental studies and small clinical trials indicate that transport of l-arginine, the substrate for nitric oxide (NO), and subsequent NO production are reduced in obesity induced hypertension. Reduced NO bioavailability can induce hypertension via multiple mechanisms. Mirmiran et al. recently analyzed data from a large population study and found that the association between dietary l-arginine and serum nitrate and nitrite was weakened in obese hypertensive subjects compared to obese normotensives. These data suggest that l-arginine dependent NO production is impaired in the former group compared to the latter which may represent a novel mechanism contributing to hypertension in the setting of obesity.

Role of Renal Oxidative Stress in the Pathogenesis of the Cardiorenal Syndrome.

Renal dysfunction and heart failure commonly co-exist; it is termed the cardiorenal syndrome (CRS). This combination of renal and cardiac impairment presents a substantial clinical challenge and is associated with adverse prognosis. The pathogenesis of the CRS is complex, including chronic activation of the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system, together with reduced renal perfusion. Chronic activation of the RAAS can impair mitochondrial function, and increase mitochondrial derived oxidative stress which in turn can lead to renal injury and sodium and water retention. For example, it has been shown that exogenous Ang II augments renal mitochondrial oxidative stress, reduces GFR and induces albuminuria in rats with heart failure. Administration of Ang II also augmented renal mitochondrial dysfunction in aged mice. Current treatments for CRS, including angiotensin-converting enzyme inhibitors, exert limited renal protection if any at all. Therefore, novel treatments particularly those that can target renal mechanisms downstream to chronic activation of the renal renin-angiotensin system are likely to exert renoprotection in the setting of CRS.

N-acetylcysteine attenuates the development of cardiac fibrosis and remodeling in a mouse model of heart failure.

Oxidative stress plays a central role in the pathogenesis of heart failure. We aimed to determine whether the antioxidantN-acetylcysteine can attenuate cardiac fibrosis and remodeling in a mouse model of heart failure. Minipumps were implanted subcutaneously in wild-type mice (n = 20) and mice with cardiomyopathy secondary to cardiac specific overexpression of mammalian sterile 20-like kinase 1 (MST-1;n = 18) to administerN-acetylcysteine (40 mg/kg per day) or saline for a period of 8 weeks. At the end of this period, cardiac remodeling and function was assessed via echocardiography. Fibrosis, oxidative stress, and expression of collagen types I andIIIwere quantified in heart tissues. Cardiac perivascular and interstitial fibrosis were greater by 114% and 209%, respectively, inMST-1 compared to wild type (P ≤ 0.001). InMST-1 mice administeredN-acetylcysteine, perivascular and interstitial fibrosis were 40% and 57% less, respectively, compared to those treated with saline (P ≤ 0. 03). Cardiac oxidative stress was 119% greater inMST-1 than in wild type (P < 0.001) andN-acetylcysteine attenuated oxidative stress inMST-1 by 42% (P = 0.005). These data indicate thatN-acetylcysteine can blunt cardiac fibrosis and related remodeling in the setting of heart failure potentially by reducing oxidative stress. This study provides the basis to investigate the role ofN-acetylcysteine in chronic heart failure.

N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin.

Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 µM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer's disease.