Glycyrrhizin through liquorice intake modulates ACE2 and HMGB1 levels-A pilot study in healthy individuals with implications for COVID-19 and ARDS.

BACKGROUND: Glycyrrhizin, an active component of liquorice root extract, exhibits antiviral and immunomodulatory properties by direct inhibition of the pro-inflammatory alarmin HMGB1 (High-mobility group box 1). OBJECTIVE: The aim of this study was to explore the role of liquorice intake on the viral entry receptor ACE2 (angiotensin-converting enzyme 2) and the immunoregulatory HMGB1 in healthy individuals and to explore HMGB1 expression in coronavirus disease 2019 (COVID-19) or non-COVID-19 in ARDS (acute respiratory distress syndrome patients). MATERIAL AND METHODS: This study enrolled 43 individuals, including hospitalised patients with i) acute respiratory distress syndrome (ARDS) due to COVID-19 (n = 7) or other underlying causes (n = 12), ii) mild COVID-19 (n = 4) and iii) healthy volunteers (n = 20). Healthy individuals took 50 g of liquorice (containing 3% liquorice root extract) daily for 7 days, while blood samples were collected at baseline and on day 3 and 7. Changes in ACE2 and HMGB1 levels were determined by Western blot analysis and enzyme-linked immunosorbent assay, respectively. Additionally, HMGB1 levels were measured in hospitalised COVID-19 patients with mild disease or COVID-19 associated acute respiratory distress syndrome (ARDS) and compared with a non-COVID-19-ARDS group. RESULTS: Liquorice intake significantly reduced after 7 days both cellular membranous ACE2 expression (-51% compared to baseline levels, p = 0.008) and plasma HMGB1 levels (-17% compared to baseline levels, p<0.001) in healthy individuals. Half of the individuals had a reduction in ACE2 levels of at least 30%. HMGB1 levels in patients with mild COVID-19 and ARDS patients with and without COVID-19 were significantly higher compared with those of healthy individuals (+317%, p = 0.002), but they were not different between COVID-19 and non-COVID-19 ARDS. CONCLUSIONS: Liquorice intake modulates ACE2 and HMGB1 levels in healthy individuals. HMGB1 is enhanced in mild COVID-19 and in ARDS with and without COVID-19, warranting evaluation of HMGB1 as a potential treatment target and glycyrrhizin, which is an active component of liquorice root extract, as a potential treatment in COVID-19 and non-COVID-19 respiratory disease.

Pro-oxidative priming but maintained cardiac function in a broad spectrum of murine models of chronic kidney disease.

AIMS: Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular events and exhibit myocardial changes including left ventricular (LV) hypertrophy and fibrosis, overall referred to as 'uremic cardiomyopathy'. Although different CKD animal models have been studied for cardiac effects, lack of consistent reporting on cardiac function and pathology complicates clear comparison of these models. Therefore, this study aimed at a systematic and comprehensive comparison of cardiac function and cardiac pathophysiological characteristics in eight different CKD models and mouse strains, with a main focus on adenine-induced CKD. METHODS AND RESULTS: CKD of different severity and duration was induced by subtotal nephrectomy or adenine-rich diet in various strains (C57BL/6J, C57BL/6 N, hyperlipidemic C57BL/6J ApoE-/-, 129/Sv), followed by the analysis of kidney function and morphology, blood pressure, cardiac function, cardiac hypertrophy, fibrosis, myocardial calcification and inflammation using functional, histological and molecular techniques, including cardiac gene expression profiling supplemented by oxidative stress analysis. Intriguingly, despite uremia of variable degree, neither cardiac dysfunction, hypertrophy nor interstitial fibrosis were observed. However, already moderate CKD altered cardiac oxidative stress responses and enhanced oxidative stress markers in each mouse strain, with cardiac RNA sequencing revealing activation of oxidative stress signaling as well as anti-inflammatory feedback responses. CONCLUSION: This study considerably expands the knowledge on strain- and protocol-specific differences in the field of cardiorenal research and reveals that several weeks of at least moderate experimental CKD increase oxidative stress responses in the heart in a broad spectrum of mouse models. However, this was insufficient to induce relevant systolic or diastolic dysfunction, suggesting that additional "hits" are required to induce uremic cardiomyopathy. TRANSLATIONAL PERSPECTIVE: Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular adverse events and exhibit myocardial changes, overall referred to as 'uremic cardiomyopathy'. We revealed that CKD increases cardiac oxidative stress responses in the heart. Nonetheless, several weeks of at least moderate experimental CKD do not necessarily trigger cardiac dysfunction and remodeling, suggesting that additional "hits" are required to induce uremic cardiomyopathy in the clinical setting. Whether the altered cardiac oxidative stress balance in CKD may increase the risk and extent of cardiovascular damage upon additional cardiovascular risk factors and/or events will be addressed in future studies.

Renal denervation reduces atrial remodeling in hypertensive rats with metabolic syndrome.

Atrial fibrillation (AF) is highly prevalent in hypertensive patients with metabolic syndrome and is related to inflammation and activation of the sympathoadrenergic system. The multi-ligand Receptor-for-Advanced-Glycation-End-products (RAGE) activates inflammation-associated tissue remodeling and is regulated by the sympathetic nervous system. Its counterpart, soluble RAGE (sRAGE), serves as anti-inflammatory decoy receptor with protective properties. We investigated the effect of sympathetic modulation by renal denervation (RDN) on atrial remodeling, RAGE/sRAGE and RAGE ligands in metabolic syndrome. RDN was performed in spontaneously hypertensive obese rats (SHRob) with metabolic syndrome compared with lean spontaneously hypertensive rats (SHR) and with normotensive non-obese control rats. Blood pressure and heart rate were measured by telemetry. The animals were killed 12 weeks after RDN. Left atrial (LA) and right atrial (RA) remodeling was assessed by histological analysis and collagen types. Sympathetic innervation was measured by tyrosine hydroxylase staining of atrial nerve fibers, RAGE/sRAGE, RAGE ligands, cytokine expressions and inflammatory infiltrates were analyzed by Western blot and immunofluorescence staining. LA sympathetic nerve fiber density was higher in SHRob (+44%) versus controls and reduced after RDN (-64% versus SHRob). RAGE was increased (+718%) and sRAGE decreased (- 62%) in SHRob as compared with controls. RDN reduced RAGE expression (- 61% versus SHRob), significantly increased sRAGE levels (+162%) and induced a significant decrease in RAGE ligand levels in SHRob (- 57% CML and - 51% HMGB1) with reduced pro-inflammatory NFkB activation (- 96%), IL-6 production (- 55%) and reduced inflammatory infiltrates. This led to a reduction in atrial fibrosis (- 33%), collagen type I content (- 72%), accompanied by reduced LA myocyte hypertrophy (- 21%). Transfection experiments on H9C2 cardiomyoblasts demonstrated that RAGE is directly involved in fibrosis formation by influencing cellular production of collagen type I. In conclusion, suppression of renal sympathetic nerve activity by RDN prevents atrial remodeling in metabolic syndrome by reducing atrial sympathetic innervation and by modulating RAGE/sRAGE balance and reducing pro-inflammatory and pro-fibrotic RAGE ligands, which provides a potential therapeutic mechanism to reduce the development of AF.

Renal Denervation Prevents Atrial Arrhythmogenic Substrate Development in CKD.

BACKGROUND: In patients with chronic kidney disease (CKD), atrial fibrillation (AF) is highly prevalent and represents a major risk factor for stroke and death. CKD is associated with atrial proarrhythmic remodeling and activation of the sympathetic nervous system. Whether reduction of the sympathetic nerve activity by renal denervation (RDN) inhibits AF vulnerability in CKD is unknown. METHODS: Left atrial (LA) fibrosis was analyzed in samples from patients with AF and concomitant CKD (estimated glomerular filtration rate [eGFR], <60 mL/min per 1.73 m2) using picrosirius red and compared with AF patients without CKD and patients with sinus rhythm with and without CKD. In a translational approach, male Sprague Dawley rats were fed with 0.25% adenine (AD)-containing chow for 16 weeks to induce CKD. At week 5, AD-fed rats underwent RDN or sham operation (AD). Rats on normal chow served as control. After 16 weeks, cardiac function and AF susceptibility were assessed by echocardiography, radiotelemetry, electrophysiological mapping, and burst stimulation, respectively. LA tissue was histologically analyzed for sympathetic innervation using tyrosine hydroxylase staining, and LA fibrosis was determined using picrosirius red. RESULTS: Sirius red staining demonstrated significantly increased LA fibrosis in patients with AF+CKD compared with AF without CKD or sinus rhythm. In rats, AD demonstrated LA structural changes with enhanced sympathetic innervation compared with control. In AD, LA enlargement was associated with prolonged duration of induced AF episodes, impaired LA conduction latency, and increased absolute conduction inhomogeneity. RDN treatment improved LA remodeling and reduced LA diameter compared with sham-operated AD. Furthermore, RDN decreased AF susceptibility and ameliorated LA conduction latency and absolute conduction inhomogeneity, independent of blood pressure reduction and renal function. CONCLUSIONS: In an experimental rat model of CKD, RDN inhibited progression of atrial structural and electrophysiological remodeling. Therefore, RDN represents a potential therapeutic tool to reduce the risk of AF in CKD, independent of changes in renal function and blood pressure.

Myopathic Cardiac Genotypes Increase Risk for Myocarditis.

Impairments in certain cardiac genes confer risk for myocarditis in children. To determine the extent of this association, we performed genomic sequencing in predominantly adult patients with acute myocarditis and matched control subjects. Putatively deleterious variants in a broad set of cardiac genes were found in 19 of 117 acute myocarditis cases vs 34 of 468 control subjects (P = 0.003). Thirteen genes classically associated with cardiomyopathy or neuromuscular disorders with cardiac involvement were implicated, including >1 associated damaging variant in DYSF, DSP, and TTN. Phenotypes of subjects who have acute myocarditis with or without deleterious variants were similar, indicating that genetic testing is necessary to differentiate them.

Cathepsin A contributes to left ventricular remodeling by degrading extracellular superoxide dismutase in mice.

In the heart, the serine carboxypeptidase cathepsin A (CatA) is distributed between lysosomes and the extracellular matrix (ECM). CatA-mediated degradation of extracellular peptides may contribute to ECM remodeling and left ventricular (LV) dysfunction. Here, we aimed to evaluate the effects of CatA overexpression on LV remodeling. A proteomic analysis of the secretome of adult mouse cardiac fibroblasts upon digestion by CatA identified the extracellular antioxidant enzyme superoxide dismutase (EC-SOD) as a novel substrate of CatA, which decreased EC-SOD abundance 5-fold. In vitro, both cardiomyocytes and cardiac fibroblasts expressed and secreted CatA protein, and only cardiac fibroblasts expressed and secreted EC-SOD protein. Cardiomyocyte-specific CatA overexpression and increased CatA activity in the LV of transgenic mice (CatA-TG) reduced EC-SOD protein levels by 43%. Loss of EC-SOD-mediated antioxidative activity resulted in significant accumulation of superoxide radicals (WT, 4.54 µmol/mg tissue/min; CatA-TG, 8.62 µmol/mg tissue/min), increased inflammation, myocyte hypertrophy (WT, 19.8 µm; CatA-TG, 21.9 µm), cellular apoptosis, and elevated mRNA expression of hypertrophy-related and profibrotic marker genes, without affecting intracellular detoxifying proteins. In CatA-TG mice, LV interstitial fibrosis formation was enhanced by 19%, and the type I/type III collagen ratio was shifted toward higher abundance of collagen I fibers. Cardiac remodeling in CatA-TG was accompanied by an increased LV weight/body weight ratio and LV end diastolic volume (WT, 50.8 µl; CatA-TG, 61.9 µl). In conclusion, CatA-mediated EC-SOD reduction in the heart contributes to increased oxidative stress, myocyte hypertrophy, ECM remodeling, and inflammation, implicating CatA as a potential therapeutic target to prevent ventricular remodeling.

Apolipoprotein C3 induces inflammation and organ damage by alternative inflammasome activation.

NLRP3-inflammasome-driven inflammation is involved in the pathogenesis of a variety of diseases. Identification of endogenous inflammasome activators is essential for the development of new anti-inflammatory treatment strategies. Here, we identified that apolipoprotein C3 (ApoC3) activates the NLRP3 inflammasome in human monocytes by inducing an alternative NLRP3 inflammasome via caspase-8 and dimerization of Toll-like receptors 2 and 4. Alternative inflammasome activation in human monocytes is mediated by the Toll-like receptor adapter protein SCIMP. This triggers Lyn/Syk-dependent calcium entry and the production of reactive oxygen species, leading to activation of caspase-8. In humanized mouse models, ApoC3 activated human monocytes in vivo to impede endothelial regeneration and promote kidney injury in an NLRP3- and caspase-8-dependent manner. These data provide new insights into the regulation of the NLRP3 inflammasome and the pathophysiological role of triglyceride-rich lipoproteins containing ApoC3. Targeting ApoC3 might prevent organ damage and provide an anti-inflammatory treatment for vascular and kidney diseases.

Sympathoadrenergic suppression improves heart function by upregulating the ratio of sRAGE/RAGE in hypertension with metabolic syndrome.

Receptors-for-Advanced-Glycation-End-products (RAGE) activate pro-inflammatory programs mediated by carboxymethyllysine (CML) and high-mobility-group-box1 protein (HMGB1). The soluble isoform sRAGE neutralizes RAGE-ligands preventing cardiovascular complications in conditions associated with increased sympathetic activation like hypertension and diabetes. The effects of sympathetic modulation on RAGE/sRAGE-balance and end-organ damage in metabolic syndrome on top of hypertension remains unknown. We hypothesized that increased sympathoadrenergic activity might lead to an unfavourable RAGE/sRAGE regulation. Renal denervation (RDN) was used to modulate sympathetic activation in obese spontaneously hypertensive rats (SHRobRDN) versus sham-operated obese spontaneously hypertensive rats (SHRob), their hypertensive lean controls (SHR) and non-hypertensive controls. Cardiac fibrosis was assessed by histological analysis and sRAGE/RAGE and ligand levels by Western blotting. Levels of CML and HMGB1 were highest in SHRob and were significantly lowered by RDN in serum (-44% and -45%) and myocardium (-25% and -52%). Myocardial RAGE was increased in SHR (+72% versus controls) and in SHRob (+68% versus SHR) while sRAGE decreased (-50% in SHR versus controls and -51% in SHRob versus SHR). RDN reduced myocardial RAGE expression. (-20%) and increased sRAGE levels in heart (+80%) and serum (+180%) versus sham-operated SHRob. Myocardial fibrosis correlated inversely with myocardial sRAGE content (r = -0.79; p = .004; n = 10). Myocardial sRAGE shedding active A-Disintegrin-And-Metalloprotease-10 (ADAM-10) was decreased in SHR (-33% versus controls) and in SHRob (-54% versus SHR), and was restored after RDN (+129% versus SHRob). Serum ADAM-10 activity was also decreased in SHRob (-66% versus SHR) and restored after RDN (+150% versus SHRob). In vitro, isoproterenol induced a ß1-adrenergic receptor mediated increase of RAGE expression in splenocytes (+200%) and decreased sRAGE secretion of splenocytes and cardiac fibroblasts (-50% and -49%) by ß2-adrenergic receptor stimulation mediated suppression of ADAM-10 activity. In conclusion, sympathetic activity affects sRAGE/RAGE-balance, which can be suppressed through sympathetic modulation by RDN, preventing RAGE-induced cardiac damage in hypertension with metabolic syndrome.

Suppressed MMP-9 Activity in Myocardial Infarction-Related Cardiogenic Shock Implies Diminished Rage Degradation.

BACKGROUND: Receptor for advanced glycation end products (RAGE) and its cleavage fragment soluble RAGE (sRAGE) are opposite players in inflammation. Enhanced monocytic RAGE expression and decreased plasma sRAGE levels are associated with higher mortality in infarction-related cardiogenic shock. Active matrix metalloproteinase-9 (MMP-9) has been implied in RAGE ectodomain cleavage and subsequently sRAGE shedding in vitro. We investigated MMP-9 activity in myocardial infarction-induced cardiogenic shock with regard to RAGE/sRAGE regulation. METHODS AND RESULTS: We determined MMP-9 serum activity by zymography and tissue inhibitor of matrix metalloproteinases (TIMP-1) expression by Western blot and correlated it to RAGE/sRAGE data in patients with cardiogenic shock after acute myocardial infarction (CS, n = 30), in patients with acute myocardial infarction without shock (AMI, n = 20) and in healthy volunteers (n = 20).MMP-9 activity is increased in AMI (P = 0.02 versus controls), but significantly decreased in CS with lowest levels in non-survivors (n = 13, P = 0.02 versus AMI). In all patients, MMP-9 activity correlated inversely with RAGE expression on circulating monocytes (r = -0.57; P = 0.0001; n = 50).TIMP-1 levels showed an inverse regulation in comparison to active MMP-9 with significantly decreased levels in AMI as compared with controls (P = 0.02 versus controls) and highest levels in non-survivors of CS (P <0.001 versus AMI). CONCLUSIONS: Serum MMP-9 activity is increased in acute myocardial infarction, but markedly suppressed in cardiogenic shock. Maintaining MMP-9 activity could be a therapeutic target to limit RAGE-induced deleterious inflammation in cardiogenic shock.

Role of receptor for advanced glycation end products in cardiogenic shock.

OBJECTIVES: Activation of the receptor for advanced glycation end products by its ligands promotes inflammatory processes and tissue injury. The available evidence suggests that soluble forms of receptor for advanced glycation end products circulating in the plasma may neutralize the ligand-mediated damage by acting as a decoy. Thus, it is hypothesized that receptor for advanced glycation end products expression might be deleterious, whereas soluble receptor for advanced glycation end products might be beneficial in cardiogenic shock. However, until now, no data exist regarding the role of soluble receptor for advanced glycation end products and receptor for advanced glycation end products in humans with cardiogenic shock complicating myocardial infarction. DESIGN: Prospective observational cohort study. SETTING: Intensive critical care unit of a university hospital. PATIENTS: Forty patients with cardiogenic shock complicating acute myocardial infarction, 20 age-matched patients with acute uncomplicated myocardial infarction and, 20 age-matched healthy volunteers. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Monocytic receptor for advanced glycation end products expression assessed by flow cytometry was significantly increased in cardiogenic shock nonsurvivors (137.02±7.48 mean fluorescence intensity; n=13) compared to survivors (67.80±8.33 mean fluorescence intensity; n=17; p<.001). Conversely, nonsurvivors had significantly decreased plasma soluble receptor for advanced glycation end products levels (79.87±10.62 arbitrary units; n=13; p=.004) compared to survivors (127.65±10.52 arbitrary units; n=17) as assessed by Western blotting. Receptor for advanced glycation end products expression and soluble receptor for advanced glycation end products levels were determined as independent predictors for 28-day mortality in cardiogenic shock confirmed by receiver-operator characteristics and multivariate analysis (receptor for advanced glycation end products: area under the curve, 0.943±0.05; p<.001; soluble receptor for advanced glycation end products: area under the curve, 0.815±0.08; p<.01). Both receptor for advanced glycation end products>103.6 mean fluorescence intensity or soluble receptor for advanced glycation end products<76.88 arbitrary units independently predicted a 27.87-fold (p<.001) and a 3.97-fold (p=.019) increase in 28-day mortality in cardiogenic shock. CONCLUSIONS: Enhanced monocytic receptor for advanced glycation end products expression and decreased plasma soluble receptor for advanced glycation end products levels play a central role in patients with cardiogenic shock associated with proinflammatory and destroying pathways, resulting in an enhanced 28-day mortality-rate. Receptor for advanced glycation end products and soluble receptor for advanced glycation end products may be prognostic biomarkers for survival in cardiogenic shock and might represent a novel therapeutic target in cardiogenic shock.