Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection.

BACKGROUND: SARS-CoV-2 is associated with an increased risk of venous and arterial thrombosis, but the underlying mechanism is still unclear. METHODS: We performed a cross-sectional analysis of platelet function in 25 SARS-CoV-2 and 10 healthy subjects by measuring Nox2 (NADPH oxidase 2)-derived oxidative stress and thromboxane B2, and investigated if administration of monoclonal antibodies against the S protein (Spike protein) of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the S protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation. RESULTS: Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and thromboxane B2 biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared with controls; both effects were lowered by Nox2 and TLR4 (Toll-like receptor 4) inhibitors. Two hours after administration of monoclonal antibodies, a significant inhibition of platelet activation was observed in patients with SARS-CoV-2 compared with untreated ones. In vitro study showed that S protein per se did not elicit platelet activation but amplified the platelet response to subthreshold concentrations of agonists and functionally interacted with platelet TLR4. A docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains; furthermore, immunoprecipitation and immunofluorescence showed S protein-TLR4 colocalization in platelets from SARS-CoV-2. Plasma from patients with SARS-CoV-2 enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF (tumor necrosis factor) α inhibitor; this effect was recapitulated by an in vitro study documenting that TNFα alone promoted platelet activation and amplified the platelet response to S protein via p47phox (phagocyte oxidase) upregulation. CONCLUSIONS: The study identifies 2 TLR4-dependent and independent pathways promoting platelet-dependent thrombus growth and suggests inhibition of TLR4. or p47phox as a tool to counteract thrombosis in SARS-CoV-2.

Role of Dickkopf-3 in Blood Pressure Regulation in Mice and Hypertensive Rats.

BACKGROUND: Dkk3 (Dickkopf-3) is a secreted glycoprotein known for its proapoptotic and angiogenic activity. The role of Dkk3 in cardiovascular homeostasis is largely unknown. Remarkably, the Dkk3 gene maps within a chromosome segment linked to the hypertensive phenotype in spontaneously hypertensive rats (SHR). METHODS: We used Dkk3-/- mice or stroke-resistant (sr) and stroke-prone (sp) SHR to examine the role of Dkk3 in the central and peripheral regulation of blood pressure (BP). We used lentiviral expression vector to rescue Dkk3 in knockout mice or to induce Dkk3 overexpression or silencing in SHR. RESULTS: Genetic deletion of Dkk3 in mice enhanced BP and impaired endothelium-dependent acetylcholine-induced relaxation of resistance arteries. These alterations were rescued by restoring Dkk3 expression either in the periphery or in the central nervous system (CNS). Dkk3 was required for the constitutive expression of VEGF (vascular endothelium growth factor), and the action of Dkk3 on BP and endothelium-dependent vasorelaxation was mediated by VEGF-stimulated phosphatidylinositol-3-kinase pathway, leading to eNOS (endothelial NO synthase) activation both in resistance arteries and the CNS. The regulatory function of Dkk3 on BP was confirmed in SHR stroke-resistant and SHR stroke-prone in which was blunted in both resistance arteries and brainstem. In SHR stroke-resistant, lentiviral expression vector-induced Dkk3 expression in the CNS largely reduced BP, whereas Dkk3 knock-down further enhanced BP. In SHR stroke-prone challenged with a hypersodic diet, lentiviral expression vector-induced Dkk3 expression in the CNS displayed a substantial antihypertensive effect and delayed the occurrence of stroke. CONCLUSIONS: These findings demonstrate that Dkk3 acts as peripheral and central regulator of BP by promoting VEGF expression and activating a VEGF/Akt (protein kinase B)/eNOS hypotensive axis.

Mitophagy modulation for the treatment of cardiovascular diseases.

BACKGROUND: Defects of mitophagy, the selective form of autophagy for mitochondria, are commonly observed in several cardiovascular diseases and represent the main cause of mitochondrial dysfunction. For this reason, mitophagy has emerged as a novel and potential therapeutic target. METHODS: In this review, we discuss current evidence about the biological significance of mitophagy in relevant preclinical models of cardiac and vascular diseases, such as heart failure, ischemia/reperfusion injury, metabolic cardiomyopathy and atherosclerosis. RESULTS: Multiple studies have shown that cardiac and vascular mitophagy is an adaptive mechanism in response to stress, contributing to cardiovascular homeostasis. Mitophagy defects lead to cell death, ultimately impairing cardiac and vascular function, whereas restoration of mitophagy by specific compounds delays disease progression. CONCLUSIONS: Despite previous efforts, the molecular mechanisms underlying mitophagy activation in response to stress are not fully characterized. A comprehensive understanding of different forms of mitophagy active in the cardiovascular system is extremely important for the development of new drugs targeting this process. Human studies evaluating mitophagy abnormalities in patients at high cardiovascular risk also represent a future challenge.

Atrial natriuretic peptide stimulates autophagy/mitophagy and improves mitochondrial function in chronic heart failure.

Mitochondrial dysfunction, causing increased reactive oxygen species (ROS) production, is a molecular feature of heart failure (HF). A defective antioxidant response and mitophagic flux were reported in circulating leucocytes of patients with chronic HF and reduced ejection fraction (HFrEF). Atrial natriuretic peptide (ANP) exerts many cardiac beneficial effects, including the ability to protect cardiomyocytes by promoting autophagy. We tested the impact of ANP on autophagy/mitophagy, altered mitochondrial structure and function and increased oxidative stress in HFrEF patients by both ex vivo and in vivo approaches. The ex vivo study included thirteen HFrEF patients whose peripheral blood mononuclear cells (PBMCs) were isolated and treated with αANP (10-11 M) for 4 h. The in vivo study included six HFrEF patients who received sacubitril/valsartan for two months. PBMCs were characterized before and after treatment. Both approaches analyzed mitochondrial structure and functionality. We found that levels of αANP increased upon sacubitril/valsartan, whereas levels of NT-proBNP decreased. Both the ex vivo direct exposure to αANP and the higher αANP level upon in vivo treatment with sacubitril/valsartan caused: (i) improvement of mitochondrial membrane potential; (ii) stimulation of the autophagic process; (iii) significant reduction of mitochondrial mass-index of mitophagy stimulation-and upregulation of mitophagy-related genes; (iv) reduction of mitochondrial damage with increased inner mitochondrial membrane (IMM)/outer mitochondrial membrane (OMM) index and reduced ROS generation. Herein we demonstrate that αANP stimulates both autophagy and mitophagy responses, counteracts mitochondrial dysfunction, and damages ultimately reducing mitochondrial oxidative stress generation in PBMCs from chronic HF patients. These properties were confirmed upon sacubitril/valsartan administration, a pivotal drug in HFrEF treatment.

MST1 mediates doxorubicin-induced cardiomyopathy by SIRT3 downregulation.

Heart failure is a major side effect of doxorubicin (DOX) treatment in patients with cancer. However, the mechanisms underlying the development of DOX-induced heart failure need to be addressed. This study aims to test whether the serine/threonine kinase MST1, a major Hippo pathway component, contributes to the development of DOX-induced myocardial injury. C57BL/6J WT mice and mice with cardiomyocyte-specific dominant-negative MST1 (kinase-dead) overexpression received three weekly injections of DOX, reaching a final cumulative dose of 18 mg/kg. Echocardiographic, histological and biochemical analyses were performed six weeks after the first DOX administration. The effects of MST1 inhibition on DOX-induced cardiomyocyte injury were also tested in vitro. MST1 signaling was significantly activated in cardiomyocytes in response to DOX treatment in vitro and in vivo. Wild-type (WT) mice treated with DOX developed cardiac dysfunction and mitochondrial abnormalities. However, these detrimental effects were abolished in mice with cardiomyocyte-specific overexpression of dominant-negative MST1 (DN-MST1) or treated with XMU-MP-1, a specific MST1 inhibitor, indicating that MST1 inhibition attenuates DOX-induced cardiac dysfunction. DOX treatment led to a significant downregulation of cardiac levels of SIRT3, a deacetylase involved in mitochondrial protection, in WT mice, which was rescued by MST1 inhibition. Pharmacological inhibition of SIRT3 blunted the protective effects of MST1 inhibition, indicating that SIRT3 downregulation mediates the cytotoxic effects of MST1 activation in response to DOX treatment. Finally, we found a significant upregulation of MST1 and downregulation of SIRT3 levels in human myocardial tissue of cancer patients treated with DOX. In summary, MST1 contributes to DOX-induced cardiomyopathy through SIRT3 downregulation.

Oleuropein, a Component of Extra Virgin Olive Oil, Improves Liver Steatosis and Lobular Inflammation by Lipopolysaccharides-TLR4 Axis Downregulation.

Gut-dysbiosis-induced lipopolysaccharides (LPS) translocation into systemic circulation has been suggested to be implicated in nonalcoholic fatty liver disease (NAFLD) pathogenesis. This study aimed to assess if oleuropein (OLE), a component of extra virgin olive oil, lowers high-fat-diet (HFD)-induced endotoxemia and, eventually, liver steatosis. An immunohistochemistry analysis of the intestine and liver was performed in (i) control mice (CTR; n = 15), (ii) high-fat-diet fed (HFD) mice (HFD; n = 16), and (iii) HFD mice treated with 6 µg/day of OLE for 30 days (HFD + OLE, n = 13). The HFD mice developed significant liver steatosis compared to the controls, an effect that was significantly reduced in the HFD + OLE-treated mice. The amount of hepatocyte LPS localization and the number of TLR4+ macrophages were higher in the HFD mice in the than controls and were lowered in the HFD + OLE-treated mice. The number of CD42b+ platelets was increased in the liver sinusoids of the HFD mice compared to the controls and decreased in the HFD + OLE-treated mice. Compared to the controls, the HFD-treated mice showed a high percentage of intestine PAS+ goblet cells, an increased length of intestinal crypts, LPS localization and TLR4+ expression, and occludin downregulation, an effect counteracted in the HFD + OLE-treated mice. The HFD-fed animals displayed increased systemic levels of LPS and zonulin, but they were reduced in the HFD + OLE-treated animals. It can be seen that OLE administration improves liver steatosis and inflammation in association with decreased LPS translocation into the systemic circulation, hepatocyte localization of LPS and TLR4 downregulation in HFD-induced mouse model of NAFLD.

Polymorphic variants at NDUFC2, encoding a mitochondrial complex I subunit, associate with cardiac hypertrophy in human hypertension.

BACKGROUND: A dysfunction of NADH dehydrogenase, the mitochondrial Complex I (CI), associated with the development of left ventricular hypertrophy (LVH) in previous experimental studies. A deficiency of Ndufc2 (subunit of CI) impairs CI activity causing severe mitochondrial dysfunction. The T allele at NDUFC2/rs11237379 variant associates with reduced gene expression and impaired mitochondrial function. The present study tested the association of both NDUFC2/rs11237379 and NDUFC2/rs641836 variants with LVH in hypertensive patients. In vitro studies explored the impact of reduced Ndufc2 expression in isolated cardiomyocytes. METHODS: Two-hundred-forty-six subjects (147 male, 59.7%), with a mean age of 59 ± 15 years, were included for the genetic association analysis. Ndufc2 silencing was performed in both H9c2 and rat primary cardiomyocytes to explore the hypertrophy development and the underlying signaling pathway. RESULTS: The TT genotype at NDUFC2/rs11237379 associated with significantly reduced gene expression. Multivariate analysis revealed that patients carrying this genotype showed significant differences for septal thickness (p = 0.07), posterior wall thickness (p = 0.008), RWT (p = 0.021), LV mass/BSA (p = 0.03), compared to subjects carrying either CC or CT genotypes. Patients carrying the A allele at NDUFC2/rs641836 showed significant differences for septal thickness (p = 0.017), posterior wall thickness (p = 0.011), LV mass (p = 0.003), LV mass/BSA (p = 0.002) and LV mass/height2.7(p = 0.010) after adjustment for covariates. In-vitro, the Ndufc2 deficiency-dependent mitochondrial dysfunction caused cardiomyocyte hypertrophy, pointing to SIRT3-AMPK-AKT-MnSOD as a major underlying signaling pathway. CONCLUSIONS: We demonstrated for the first time a significant association of NDUFC2 variants with LVH in human hypertension and highlight a key role of Ndufc2 deficiency-dependent CI mitochondrial dysfunction on increased susceptibility to cardiac hypertrophy development.

1,5-disubstituted-1,2,3-triazoles counteract mitochondrial dysfunction acting on F1FO-ATPase in models of cardiovascular diseases.

The compromised viability and function of cardiovascular cells are rescued by small molecules of triazole derivatives (Tzs), identified as 3a and 3b, by preventing mitochondrial dysfunction. The oxidative phosphorylation improves the respiratory control rate in the presence of Tzs independently of the substrates that energize the mitochondria. The F1FO-ATPase, the main candidate in mitochondrial permeability transition pore (mPTP) formation, is the biological target of Tzs and hydrophilic F1 domain of the enzyme is depicted as the binding region of Tzs. The protective effect of Tz molecules on isolated mitochondria was corroborated by immortalized cardiomyocytes results. Indeed, mPTP opening was attenuated in response to ionomycin. Consequently, increased mitochondrial roundness and reduction of both length and interconnections between mitochondria. In in-vitro and ex-vivo models of cardiovascular pathologies (i.e., hypoxia-reoxygenation and hypertension) were used to evaluate the Tzs cardioprotective action. Key parameters of porcine aortic endothelial cells (pAECs) oxidative metabolism and cell viability were not affected by Tzs. However, in the presence of either 1 µM 3a or 0.5 µM 3b the impaired cell metabolism of pAECs injured by hypoxia-reoxygenation was restored to control respiratory profile. Moreover, endothelial cells isolated from SHRSP exposed to high-salt treatment rescued the Complex I activity and the endothelial capability to form vessel-like tubes and vascular function in presence of Tzs. As a result, the specific biochemical mechanism of Tzs to block Ca2+-activated F1FO-ATPase protected cell viability and preserved the pAECs bioenergetic metabolism upon hypoxia-reoxygenation injury. Moreover, SHRSP improved vascular dysfunction in response to a high-salt treatment.

Progressive stages of dysmetabolism are associated with impaired biological features of human cardiac stromal cells mediated by the oxidative state and autophagy.

Cardiac stromal cells (CSCs) are the main players in fibrosis. Dysmetabolic conditions (metabolic syndrome-MetS, and type 2 diabetes mellitus-DM2) are strong pathogenetic contributors to cardiac fibrosis. Moreover, modulation of the oxidative state (OxSt) and autophagy is a fundamental function affecting the fibrotic commitment of CSCs, that are adversely modulated in MetS/DM2. We aimed to characterize CSCs from dysmetabolic patients, and to obtain a beneficial phenotypic setback from such fibrotic commitment by modulation of OxSt and autophagy. CSCs were isolated from 38 patients, stratified as MetS, DM2, or controls. Pharmacological modulation of OxSt and autophagy was obtained by treatment with trehalose and NOX4/NOX5 inhibitors (TREiNOX). Flow-cytometry and real-time quantitative polymerase chain reaction (RT-qPCR) analyses showed significantly increased expression of myofibroblasts markers in MetS-CSCs at baseline (GATA4, ACTA2, THY1/CD90) and after starvation (COL1A1, COL3A1). MetS- and DM2-CSCs displayed a paracrine profile distinct from control cells, as evidenced by screening of 30 secreted cytokines, with a significant reduction in vascular endothelial growth factor (VEGF) and endoglin confirmed by enzyme-linked immunoassay (ELISA). DM2-CSCs showed significantly reduced support for endothelial cells in angiogenic assays, and significantly increased H2 O2 release and NOX4/5 expression levels. Autophagy impairment after starvation (reduced ATG7 and LC3-II proteins) was also detectable in DM2-CSCs. TREiNOX treatment significantly reduced ACTA2, COL1A1, COL3A1, and NOX4 expression in both DM2- and MetS-CSCs, as well as GATA4 and THY1/CD90 in DM2, all versus control cells. Moreover, TREiNOX significantly increased VEGF release by DM2-CSCs, and VEGF and endoglin release by both MetS- and DM2-CSCs, also recovering the angiogenic support to endothelial cells by DM2-CSCs. In conclusion, DM2 and MetS worsen microenvironmental conditioning by CSCs. Appropriate modulation of autophagy and OxSt in human CSCs appears to restore these features, mostly in DM2-CSCs, suggesting a novel strategy against cardiac fibrosis in dysmetabolic patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

SIRT1 pharmacological activation rescues vascular dysfunction and prevents thrombosis in MTHFR deficiency.

Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here, we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/-) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/- mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers.

The Role of Autophagy in the Heart.

Autophagy is an evolutionarily conserved mechanism by which cytoplasmic elements are degraded intracellularly. Autophagy has also emerged as a major regulator of cardiac homeostasis and function. Autophagy preserves cardiac structure and function under baseline conditions and is activated during stress, limiting damage under most conditions. It reduces injury and preserves cardiac function during ischemia. It also reduces chronic ischemic remodeling and mediates the cardiac adaptation to pressure overload by restricting misfolded protein accumulation, mitochondrial dysfunction, and oxidative stress. Impairment of autophagy is involved in the development of diabetes and aging-induced cardiac abnormalities. Autophagy defects contribute to the development of cardiac proteinopathy and doxorubicin-induced cardiomyopathy. However, massive activation of autophagy may be detrimental for the heart in certain stress conditions, such as reperfusion injury. In this review, we discuss recent evidence supporting the important role of autophagy and mitophagy in the regulation of cardiac homeostasis and adaptation to stress.

Inhibition of PKCθ Improves Dystrophic Heart Phenotype and Function in a Novel Model of DMD Cardiomyopathy.

Chronic cardiac muscle inflammation and subsequent fibrotic tissue deposition are key features in Duchenne Muscular Dystrophy (DMD). The treatment of choice for delaying DMD progression both in skeletal and cardiac muscle are corticosteroids, supporting the notion that chronic inflammation in the heart plays a pivotal role in fibrosis deposition and subsequent cardiac dysfunction. Nevertheless, considering the adverse effects associated with long-term corticosteroid treatments, there is a need for novel anti-inflammatory therapies. In this study, we used our recently described exercised mdx (ex mdx) mouse model characterised by accelerated heart pathology, and the specific PKCθ inhibitor Compound 20 (C20), to show that inhibition of this kinase leads to a significant reduction in the number of immune cells infiltrating the heart, as well as necrosis and fibrosis. Functionally, C20 treatment also prevented the reduction in left ventricle fractional shortening, which was typically observed in the vehicle-treated ex mdx mice. Based on these findings, we propose that PKCθ pharmacological inhibition could be an attractive therapeutic approach to treating dystrophic cardiomyopathy.

Neurotoxic Effect of Doxorubicin Treatment on Cardiac Sympathetic Neurons.

Doxorubicin (DOXO) remains amongst the most commonly used anti-cancer agents for the treatment of solid tumors, lymphomas, and leukemias. However, its clinical use is hampered by cardiotoxicity, characterized by heart failure and arrhythmias, which may require chemotherapy interruption, with devastating consequences on patient survival and quality of life. Although the adverse cardiac effects of DOXO are consolidated, the underlying mechanisms are still incompletely understood. It was previously shown that DOXO leads to proteotoxic cardiomyocyte (CM) death and myocardial fibrosis, both mechanisms leading to mechanical and electrical dysfunction. While several works focused on CMs as the culprits of DOXO-induced arrhythmias and heart failure, recent studies suggest that DOXO may also affect cardiac sympathetic neurons (cSNs), which would thus represent additional cells targeted in DOXO-cardiotoxicity. Confocal immunofluorescence and morphometric analyses revealed alterations in SN innervation density and topology in hearts from DOXO-treated mice, which was consistent with the reduced cardiotropic effect of adrenergic neurons in vivo. Ex vivo analyses suggested that DOXO-induced denervation may be linked to reduced neurotrophic input, which we have shown to rely on nerve growth factor, released from innervated CMs. Notably, similar alterations were observed in explanted hearts from DOXO-treated patients. Our data demonstrate that chemotherapy cardiotoxicity includes alterations in cardiac innervation, unveiling a previously unrecognized effect of DOXO on cardiac autonomic regulation, which is involved in both cardiac physiology and pathology, including heart failure and arrhythmias.

Mitochondrial a Kinase Anchor Proteins in Cardiovascular Health and Disease: A Review Article on Behalf of the Working Group on Cellular and Molecular Biology of the Heart of the Italian Society of Cardiology.

Second messenger cyclic adenosine monophosphate (cAMP) has been found to regulate multiple mitochondrial functions, including respiration, dynamics, reactive oxygen species production, cell survival and death through the activation of cAMP-dependent protein kinase A (PKA) and other effectors. Several members of the large family of A kinase anchor proteins (AKAPs) have been previously shown to locally amplify cAMP/PKA signaling to mitochondria, promoting the assembly of signalosomes, regulating multiple cardiac functions under both physiological and pathological conditions. In this review, we will discuss roles and regulation of major mitochondria-targeted AKAPs, along with opportunities and challenges to modulate their functions for translational purposes in the cardiovascular system.

Impact of chronic use of heat-not-burn cigarettes on oxidative stress, endothelial dysfunction and platelet activation: the SUR-VAPES Chronic Study.

Tobacco habit still represents the leading preventable cause of morbidity and mortality worldwide. Heat-not-burn cigarettes (HNBCs) are considered as an alternative to traditional combustion cigarettes (TCCs) due to the lack of combustion and the absence of combustion-related specific toxicants. The aim of this observational study was to assess the effect of HNBC on endothelial function, oxidative stress and platelet activation in chronic adult TCC smokers and HNBC users. The results showed that both HNBC and TCC display an adverse phenotype in terms of endothelial function, oxidative stress and platelet activation. Future randomised studies are strongly warranted to confirm these data.

Hippo Deficiency Leads to Cardiac Dysfunction Accompanied by Cardiomyocyte Dedifferentiation During Pressure Overload.

RATIONALE: The Hippo pathway plays an important role in determining organ size through regulation of cell proliferation and apoptosis. Hippo inactivation and consequent activation of YAP (Yes-associated protein), a transcription cofactor, have been proposed as a strategy to promote myocardial regeneration after myocardial infarction. However, the long-term effects of Hippo deficiency on cardiac function under stress remain unknown. OBJECTIVE: We investigated the long-term effect of Hippo deficiency on cardiac function in the presence of pressure overload (PO). METHODS AND RESULTS: We used mice with cardiac-specific homozygous knockout of WW45 (WW45cKO), in which activation of Mst1 (Mammalian sterile 20-like 1) and Lats2 (large tumor suppressor kinase 2), the upstream kinases of the Hippo pathway, is effectively suppressed because of the absence of the scaffolding protein. We used male mice at 3 to 4 month of age in all animal experiments. We subjected WW45cKO mice to transverse aortic constriction for up to 12 weeks. WW45cKO mice exhibited higher levels of nuclear YAP in cardiomyocytes during PO. Unexpectedly, the progression of cardiac dysfunction induced by PO was exacerbated in WW45cKO mice, despite decreased apoptosis and activated cardiomyocyte cell cycle reentry. WW45cKO mice exhibited cardiomyocyte sarcomere disarray and upregulation of TEAD1 (transcriptional enhancer factor) target genes involved in cardiomyocyte dedifferentiation during PO. Genetic and pharmacological inactivation of the YAP-TEAD1 pathway reduced the PO-induced cardiac dysfunction in WW45cKO mice and attenuated cardiomyocyte dedifferentiation. Furthermore, the YAP-TEAD1 pathway upregulated OSM (oncostatin M) and OSM receptors, which played an essential role in mediating cardiomyocyte dedifferentiation. OSM also upregulated YAP and TEAD1 and promoted cardiomyocyte dedifferentiation, indicating the existence of a positive feedback mechanism consisting of YAP, TEAD1, and OSM. CONCLUSIONS: Although activation of YAP promotes cardiomyocyte regeneration after cardiac injury, it induces cardiomyocyte dedifferentiation and heart failure in the long-term in the presence of PO through activation of the YAP-TEAD1-OSM positive feedback mechanism.

Trehalose, a natural disaccharide, reduces stroke occurrence in the stroke-prone spontaneously hypertensive rat.

Cerebrovascular disease, a frequent complication of hypertension, is a major public health issue for which novel therapeutic and preventive approaches are needed. Autophagy activation is emerging as a potential therapeutic and preventive strategy toward stroke. Among usual activators of autophagy, the natural disaccharide trehalose (TRE) has been reported to be beneficial in preclinical models of neurodegenerative diseases, atherosclerosis and myocardial infarction. In this study, we tested for the first time the effects of TRE in the stroke-prone spontaneously hypertensive rat (SHRSP) fed with a high-salt stroke permissive diet (JD). We found that TRE reduced stroke occurrence and renal damage in high salt-fed SHRSP. TRE was also able to decrease systolic blood pressure. Through ex-vivo studies, we assessed the beneficial effect of TRE on the vascular function of high salt-fed SHRSP. At the molecular level, TRE restored brain autophagy and reduced mitochondrial mass, along with the improvement of mitochondrial function. The beneficial effects of TRE were associated with increased nuclear translocation of TFEB, a transcriptional activator of autophagy. Our results suggest that TRE may be considered as a natural compound efficacious for the prevention of hypertension-related target organ damage, with particular regard to stroke and renal damage.

Von Willebrand factor with increased binding capacity is associated with reduced platelet aggregation but enhanced agglutination in COVID-19 patients: another COVID-19 paradox?

Patients with Coronavirus-associated disease-2019 (COVID-19) display alterations of the hemostatic system and the presence of a prothrombotic status frequently leading to vascular complications. However, the impact of COVID-19 on platelet activity, aggregation and agglutination still needs to be clarified. We measured total levels of von Willebrand factor (vWF) and vWF binding to the platelet glycoprotein (Gp) complex (GPIb-IX-V), in a cohort of COVID-19 patients admitted to the intensive care unit of our Institution. Moreover, we evaluated platelet aggregation in response to agonists (ADP, collagen, arachidonic acid) and platelet agglutination in response to ristocetin. We found that levels of vWF antigen and the active form of vWF binding to platelets (vWF:RCo), were markedly increased in these patients. These results were associated with higher agglutination rates induced by ristocetin, thereby indirectly indicating an increased capability of vWF to bind to platelets. Conversely, we found that platelet aggregation in response to both ADP and collagen was lower in COVID-19 patients compared to healthy volunteers. This study shows that COVID-19 is associated with increased vWF-induced platelet agglutination but reduced platelet responsivity to aggregation stimuli. Our findings have translational relevance since platelet adhesion to vWF may represent a marker to predict possible complications and better delineate therapeutic strategies in COVID-19 patients.

Renal arteries denervation: from the treatment of resistant hypertension to the treatment of atrial fibrillation.

Renal denervation (RDN) is a therapeutic strategy for patients with uncontrolled arterial hypertension characterized by considerable fluctuations during its progression. After initial strong enthusiasm, the procedure came to an abrupt halt following the publication of the Symplicity HTN-3 study results. The results of recently published studies highlight the reduction in blood pressure values after RDN and justify the inclusion in the Guidelines of new recommendations for the use of RDN in clinical practice, in selected patients. Additionally, RDN findings are summarized in view of other potential indications such as atrial fibrillation. Six prospective, randomized studies are presented that evaluated RDN as an adjunct therapy to pulmonary vein isolation for the treatment of atrial fibrillation. In five studies, patients had uncontrolled hypertension despite therapy with three antihypertensive drugs. The analysis of these studies showed that RDN reduced the recurrence of atrial fibrillation (AF) by 57% compared to patients with pulmonary vein isolation (PVI) only. Modulation of the autonomic nervous system by RDN has been shown not only to reduce blood pressure but also to have an antiarrhythmic effect in symptomatic AF patients when the strategy is combined with PVI, thus opening up new therapeutic scenarios.

Low-grade endotoxaemia enhances artery thrombus growth via Toll-like receptor 4: implication for myocardial infarction.

AIMS: Low-grade endotoxaemia is detectable in human circulation but its role in thrombosis is still unclear. METHODS AND RESULTS: We measured serum lipopolysaccharide (LPS) concentration, soluble P-selectin (sP-selectin), a marker of platelet activation, and zonulin, a marker of gut permeability, in peripheral circulation, coronary thrombi, and intracoronary blood of patients with ST-elevation myocardial infarction (STEMI, n = 50) and stable angina (SA) (n = 50), respectively, and in controls (n = 50). Experimental study was carried out in mice to assess if Escherichia coli-LPS (E. coli-LPS) possess thrombotic property. Coronary thrombi from STEMI showed higher concentrations of LPS, sP-selectin vs. intracoronary blood of SA and peripheral blood of controls (P < 0.001). Zonulin was higher in STEMI compared to the other two groups [4.57 (3.34-5.22); 2.56 (0.41-4.36); 1.95 (1.22-2.65) ng/mL; P < 0.001] and correlated with LPS (Rs = 0.585; P < 0.001). Escherichia coli DNA was positive in 34% of STEMI vs. 12% of SA and 4% of controls (P < 0.001). In a subgroup of 12 STEMI, immunohistochemical analysis of coronary thrombi showed positivity for leucocyte Toll-like receptor 4 (TLR4), cathepsin G, and LPS from E. coli in 100%, 80%, and 25% of samples, respectively. E. coli-LPS injected in mice to reach LPS concentrations like those detected in coronary thrombi was associated with enhanced artery thrombosis and platelet activation, an effect blunted by TLR4 inhibitor co-administration. In vitro study demonstrated that LPS from E. coli enhanced platelet aggregation via TLR4-mediated leucocyte cathepsin G activation. CONCLUSION: ST-elevation myocardial infarction patients disclose an enhanced gut permeability that results in LPS translocation in human circulation and eventually thrombus growth at site of artery lesion via leucocyte-platelet interaction.