CD73 on T Cells Orchestrates Cardiac Wound Healing After Myocardial Infarction by Purinergic Metabolic Reprogramming.

BACKGROUND: T cells are required for proper healing after myocardial infarction. The mechanism of their beneficial action, however, is unknown. The proinflammatory danger signal ATP, released from damaged cells, is degraded by the ectonucleotidases CD39 and CD73 to the anti-inflammatory mediator adenosine. Here, we investigate the contribution of CD73-derived adenosine produced by T cells to cardiac remodeling after ischemia/reperfusion and define its mechanism of action. METHODS: Myocardial ischemia (50 minutes followed by reperfusion) was induced in global CD73-/- and CD4-CD73-/- mice. Tissue injury, T-cell purinergic signaling, cytokines, and cardiac function (magnetic resonance tomography at 9.4 T over 4 weeks) were analyzed. RESULTS: Changes in functional parameters of CD4-CD73-/- mice were identical to those in global CD73 knockouts (KOs). T cells infiltrating the injured heart significantly upregulated at the gene (quantitative polymerase chain reaction) and protein (enzymatic activity) levels critical transporters and enzymes (connexin43, connexin37, pannexin-1, equilibrative nucleoside transporter 1, CD39, CD73, ecto-nucleotide pyrophosphatase/phosphodiesterases 1 and 3, CD157, CD38) for the accelerated release and hydrolysis of ATP, cAMP, AMP, and NAD to adenosine. It is surprising that a lack of CD39 on T cells (from CD39-/- mice) did not alter ATP hydrolysis and very likely involves pyrophosphatases (ecto-nucleotide pyrophosphatase/phosphodiesterases 1 and 3). Circulating T cells predominantly expressed A2a receptor (A2aR) transcripts. After myocardial infarction, A2b receptor (A2bR) transcription was induced in both T cells and myeloid cells in the heart. Thus, A2aR and A2bR signaling may contribute to myocardial responses after myocardial infarction. In the case of T cells, this was associated with an accelerated secretion of proinflammatory and profibrotic cytokines (interleukin-2, interferon-γ, and interleukin-17) when CD73 was lacking. Cytokine production by T cells from peripheral lymph nodes was inhibited by A2aR activation (CGS-21680). The A2bR agonist BAY 60-6583 showed off-target effects. The adenosine receptor agonist NECA inhibited interferon-γ and stimulated interleukin-6 production, each of which was antagonized by a specific A2bR antagonist (PSB-603). CONCLUSIONS: This work demonstrates that CD73 on T cells plays a crucial role in the cardiac wound healing process after myocardial infarction. The underlying mechanism involves a profound increase in the hydrolysis of ATP/NAD and AMP, resulting primarily from the upregulation of pyrophosphatases and CD73. We also define A2bR/A2aR-mediated autacoid feedback inhibition of proinflammatory/profibrotic cytokines by T cell-derived CD73.

A survey of membrane receptor regulation in valvular interstitial cells cultured under mechanical stresses.

Degenerative valvular diseases have been linked to the action of abnormal forces on valve tissues during each cardiac cycle. It is now accepted that the degenerative behavior of valvular cells can be induced mechanically in vitro. This approach of in vitro modeling of valvular cells in culture constitutes a powerful tool to study, characterize, and develop predictors of heart valve degeneration in vivo. Using such in vitro systems, we expect to determine the exact signaling mechanisms that trigger and mediate propagation of degenerative signals. In this study, we aim to uncover the role of mechanosensing proteins on valvular cell membranes. These can be cell receptors and triggers of downstream pathways that are activated upon the action of cyclical tensile strains in pathophysiological conditions. In order to identify mechanosensors of tensile stresses on valvular interstitial cells, we employed biaxial cyclic strain of valvular cells in culture and quantitatively evaluated the expression of cell membrane proteins using a targeted protein array and interactome analyses. This approach yielded a high-throughput screening of all cell surface proteins involved in sensing mechanical stimuli. In this study, we were able to identify the cell membrane proteins which are activated during physiological cyclic tensile stresses of valvular cells. The proteins identified in this study were clustered into four interactomes, which included CC chemokine ligands, thrombospondin (adhesive glycoproteins), growth factors, and interleukins. The expression levels of these proteins generally indicated that cells tend to increase adhesive efforts to counteract the action of mechanical forces. This is the first study of this kind used to comprehensively identify the mechanosensitive proteins in valvular cells.

N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro.

Myocardial infarction (MI) leads to loss and degradation of contractile cardiac tissue followed by sterile inflammation of the myocardium through activation and recruitment of innate and adaptive cells of the immune system. Recently, it was shown that cardiac myosin binding protein-C (cMyBP-C), a protein of the cardiac sarcomere, is degraded following MI, releasing a predominant N-terminal 40-kDa fragment (C0C1f) into myocardial tissue and the systemic circulation. We hypothesized that early release of C0C1f contributes to the initiation of inflammation and plays a key role in recruitment and activation of immune cells. Therefore, we investigated the role of C0C1f on macrophage/monocyte activation using both mouse bone marrow-derived macrophages and human monocytes. Here we demonstrate that C0C1f leads to macrophage/monocyte activation in vitro. Furthermore, C0C1f induces strong upregulation of pro-inflammatory cytokines (interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and interleukin-1ß (IL-1ß)) in cultured murine macrophages and human monocytes, resulting in a pro-inflammatory phenotype. We identified the toll-like receptor 4 (TLR4), toll-like receptor 2 (TLR2), and Advanced Glycosylation End Product-Specific Receptor (RAGE) as potential receptors for C0C1f whose activation leads to mobilization of the NFκB signaling pathway, a central mediator of the pro-inflammatory signaling cascade. Thus, C0C1f appears to be a key player in the initiation of inflammatory processes and might also play an important role upon MI.

The intracellular renin-angiotensin system: Friend or foe. Some light from the dopaminergic neurons.

The renin-angiotensin system (RAS) is one of the oldest hormone systems in vertebrate phylogeny. RAS was initially related to regulation of blood pressure and sodium and water homeostasis. However, local or paracrine RAS were later identified in many tissues, including brain, and play a major role in their physiology and pathophysiology. In addition, a major component, ACE2, is the entry receptor for SARS-CoV-2. Overactivation of tissue RAS leads several oxidative stress and inflammatory processes involved in aging-related degenerative changes. In addition, a third level of RAS, the intracellular or intracrine RAS (iRAS), with still unclear functions, has been observed. The possible interaction between the intracellular and extracellular RAS, and particularly the possible deleterious or beneficial effects of the iRAS activation are controversial. The dopaminergic system is particularly interesting to investigate the RAS as important functional interactions between dopamine and RAS have been observed in the brain and several peripheral tissues. Our recent observations in mitochondria and nucleus of dopaminergic neurons may clarify the role of the iRAS. This may be important for the developing of new therapeutic strategies, since the effects on both extracellular and intracellular RAS must be taken into account, and perhaps better understanding of COVID-19 cell mechanisms.

G Protein-Coupled Estrogen Receptor Protects From Angiotensin II-Induced Increases in Pulse Pressure and Oxidative Stress.

Our previous work showed that the G protein-coupled estrogen receptor (GPER) is protective in the vasculature and kidneys during angiotensin (Ang) II-dependent hypertension by inhibiting oxidative stress. The goal of the current study was to assess the impact of GPER deletion on sex differences in Ang II-induced hypertension and oxidative stress. Male and female wildtype and GPER knockout mice were implanted with radiotelemetry probes for measurement of baseline blood pressure before infusion of Ang II (700 ng/kg/min) for 2 weeks. Mean arterial pressure was increased to the same extent in all groups, but female wildtype mice were protected from Ang II-induced increases in pulse pressure, aortic wall thickness, and Nox4 mRNA. In vitro studies using vascular smooth muscle cells found that pre-treatment with the GPER agonist G-1 inhibited Ang II-induced ROS and NADP/NADPH. Ang II increased while G-1 decreased Nox4 mRNA and protein. The effects of Ang II were blocked by losartan and Nox4 siRNA, while the effects of G-1 were inhibited by adenylyl cyclase inhibition and mimicked by phosphodiesterase inhibition. We conclude that during conditions of elevated Ang II, GPER via the cAMP pathway suppresses Nox4 transcription to limit ROS production and prevent arterial stiffening. Taken together with our previous work, this study provides insight into how acute estrogen signaling via GPER provides cardiovascular protection during Ang II hypertension and potentially other diseases characterized by increased oxidative stress.

Transcription Factor 7-like 2 Mediates Canonical Wnt/ß-Catenin Signaling and c-Myc Upregulation in Heart Failure.

BACKGROUND: How canonical Wnt/ß-catenin signals in adult hearts, especially in different diseased states, remains unclear. The proto-oncogene, c-Myc, is a Wnt target and an early response gene during cardiac stress. It is not clear whether c-Myc is activated or how it is regulated during heart failure. METHODS AND RESULTS: We investigated canonical Wnt/ß-catenin signaling and how it regulated c-Myc expression in failing hearts of human ischemic heart disease, idiopathic dilated cardiomyopathy, and murine desmin-related cardiomyopathy. Our data demonstrated that canonical Wnt/ß-catenin signaling was activated through nuclear accumulation of ß-catenin in idiopathic dilated cardiomyopathy, ischemic heart disease, and murine desmin-related cardiomyopathy when compared with nonfailing controls and transcription factor 7-like 2 (TCF7L2) was the main ß-catenin partner of the T-cell factor (TCF) family in adult hearts. We further revealed that c-Myc mRNA and protein levels were significantly elevated in failing hearts by real-time reverse transcription polymerase chain reaction, Western blotting, and immunohistochemical staining. Immunoprecipitation and confocal microscopy further showed that ß-catenin interacted and colocalized with TCF7L2. More importantly, chromatin immunoprecipitation confirmed that ß-catenin and TCF7L2 were recruited to the regulatory elements of c-Myc. This recruitment was associated with increased histone H3 acetylation and transcriptional upregulation of c-Myc. With lentiviral infection, TCF7L2 overexpression increased c-Myc expression and cardiomyocyte size, whereas shRNA-mediated knockdown of TCF7L2 suppressed c-Myc expression and cardiomyocyte growth in cultured neonatal rat cardiomyocytes. CONCLUSIONS: This study indicates that TCF7L2 mediates canonic Wnt/ß-catenin signaling and c-Myc upregulation during abnormal cardiac remodeling in heart failure and suppression of Wnt/ß-catenin to c-Myc axis can be explored for preventing and treating heart failure.

The neutral sphingomyelinase-2 is involved in angiogenic signaling triggered by oxidized LDL.

Capillaries of the external part of the normal arterial wall constitute the vasa vasorum network. In atherosclerotic lesions, neovascularization occurs in areas of intimal hyperplasia where it may promote plaque expansion, and intraplaque hemorrhage. Oxidized LDL that are present in atherosclerotic areas activate various angiogenic signaling pathways, including reactive oxygen species and the sphingosine kinase/sphingosine-1-phosphate pathway. We aimed to investigate whether oxidized LDL-induced angiogenesis requires neutral sphingomyelinase-2 activation and the neutral sphingomyelinase-2/sphingosine kinase-1 pathway. The role of neutral sphingomyelinase-2 in angiogenic signaling was investigated in Human Microvascular Endothelial Cells (HMEC-1) forming capillary tube on Matrigel and in vivo in the Matrigel plug assay in C57BL/6 mice and in the chicken chorioallantoic membrane model. Low concentration of human oxidized LDL elicits HMEC-1 capillary tube formation and neutral sphingomyelinase-2 activation, which were blocked by neutral sphingomyelinase-2 inhibitors, GW4869 and specific siRNA. This angiogenic effect was mimicked by low concentration of C6-Ceramide and was inhibited by sphingosine kinase-1 inhibitors. Upstream of neutral sphingomyelinase-2, oxidized LDL-induced activation required LOX-1, reactive oxygen species generation by NADPH oxidase and p38-MAPK activation. Inhibition of sphingosine kinase-1 blocked the angiogenic response and triggered HMEC-1 apoptosis. Low concentration of oxidized LDL was angiogenic in vivo, both in the Matrigel plug assay in mice and in the chorioallantoic membrane model, and was blocked by GW4869. In conclusion, low oxLDL concentration triggers sprouting angiogenesis that involves ROS-induced activation of the neutral sphingomyelinase-2/sphingosine kinase-1 pathway, and is effectively inhibited by GW4869.