A targetable pathway in neutrophils mitigates both arterial and venous thrombosis.

Arterial and venous thrombosis constitutes a major source of morbidity and mortality worldwide. Long considered as distinct entities, accumulating evidence indicates that arterial and venous thrombosis can occur in the same populations, suggesting that common mechanisms are likely operative. Although hyperactivation of the immune system is a common forerunner to the genesis of thrombotic events in both vascular systems, the key molecular control points remain poorly understood. Consequently, antithrombotic therapies targeting the immune system for therapeutics gain are lacking. Here, we show that neutrophils are key effectors of both arterial and venous thrombosis and can be targeted through immunoregulatory nanoparticles. Using antiphospholipid antibody syndrome (APS) as a model for arterial and venous thrombosis, we identified the transcription factor Krüppel-like factor 2 (KLF2) as a key regulator of neutrophil activation. Upon activation through genetic loss of KLF2 or administration of antiphospholipid antibodies, neutrophils clustered P-selectin glycoprotein ligand 1 (PSGL-1) by cortical actin remodeling, thereby increasing adhesion potential at sites of thrombosis. Targeting clustered PSGL-1 using nanoparticles attenuated neutrophil-mediated thrombosis in APS and KLF2 knockout models, illustrating the importance and feasibility of targeting activated neutrophils to prevent pathological thrombosis. Together, our results demonstrate a role for activated neutrophils in both arterial and venous thrombosis and identify key molecular events that serve as potential targets for therapeutics against diverse causes of immunothrombosis.

Correction: The thromboprotective effect of traditional Chinese medicine Tongji 2 granules is dependent on anti-inflammatory activity by suppression of NF-κB pathways.

[This corrects the article DOI: 10.1371/journal.pone.0241607.].

Myeloid Krüppel-like factor 2 is a critical regulator of metabolic inflammation.

Substantial evidence implicates crosstalk between metabolic tissues and the immune system in the inception and progression of obesity. However, molecular regulators that orchestrate metaflammation both centrally and peripherally remains incompletely understood. Here, we identify myeloid Krüppel-like factor 2 (KLF2) as an essential regulator of obesity and its sequelae. In mice and humans, consumption of a fatty diet downregulates myeloid KLF2 levels. Under basal conditions, myeloid-specific KLF2 knockout mice (K2KO) exhibit increased feeding and weight gain. High-fat diet (HFD) feeding further exacerbates the K2KO metabolic disease phenotype. Mechanistically, loss of myeloid KLF2 increases metaflammation in peripheral and central tissues. A combination of pair-feeding, bone marrow-transplant, and microglial ablation implicate central and peripheral contributions to K2KO-induced metabolic dysfunction observed. Finally, overexpression of myeloid KLF2 protects mice from HFD-induced obesity and insulin resistance. Together, these data establish myeloid KLF2 as a nodal regulator of central and peripheral metabolic inflammation in homeostasis and disease.

GPCRs and Insulin Receptor Signaling in Conversation: Novel Avenues for Drug Discovery.

Type 2 diabetes is a major health issue worldwide with complex metabolic and endocrine abnormalities. Hyperglycemia, defects in insulin secretion and insulin resistance are classic features of type 2 diabetes. Insulin signaling regulates metabolic homeostasis by regulating glucose and lipid turnover in the liver, skeletal muscle and adipose tissue. Major treatment modalities for diabetes include the drugs from the class of sulfonyl urea, Insulin, GLP-1 agonists, SGLT2 inhibitors, DPP-IV inhibitors and Thiazolidinediones. Emerging antidiabetic therapeutics also include classes of drugs targeting GPCRs in the liver, adipose tissue and skeletal muscle. Interestingly, recent research highlights several shared intermediates between insulin and GPCR signaling cascades opening potential novel avenues for diabetic drug discovery.

Distinct roles of resident and nonresident macrophages in nonischemic cardiomyopathy.

Nonischemic cardiomyopathy (NICM) resulting from long-standing hypertension, valvular disease, and genetic mutations is a major cause of heart failure worldwide. Recent observations suggest that myeloid cells can impact cardiac function, but the role of tissue-intrinsic vs. tissue-extrinsic myeloid cells in NICM remains poorly understood. Here, we show that cardiac resident macrophage proliferation occurs within the first week following pressure overload hypertrophy (POH; a model of heart failure) and is requisite for the heart's adaptive response. Mechanistically, we identify Kruppel-like factor 4 (KLF4) as a key transcription factor that regulates cardiac resident macrophage proliferation and angiogenic activities. Finally, we show that blood-borne macrophages recruited in late-phase POH are detrimental, and that blockade of their infiltration improves myocardial angiogenesis and preserves cardiac function. These observations demonstrate previously unappreciated temporal and spatial roles for resident and nonresident macrophages in the development of heart failure.

cAMP assays in GPCR drug discovery.

GPCRs are a major family of cell surface receptors and the most druggable protein targets in modern medicine. Recent elucidation of crystal structures of a vast number of GPCRs eludes to the finer details of biased agonism or functional selectivity of many of these receptors and warrants a better understanding of their biological effects as well as therapeutic potential. Receptor function is measured in terms desensitization/resensitization, which provides insights on receptor activation and differential coupling to various G proteins. This review article presents thoughts on the potential of GPCR desensitization assay/cAMP assay, a highly sensitive and versatile platform for high-throughput assays for elucidating novel functions of many orphan GPCRs. Besides, these assays also are very sensitive to screen for agonists, partial agonists, or antagonists and provide a simplified system for novel drug discovery for Gs-coupled GPCRs and understanding their physiological implications.

Proinflammatory Cytokines Mediate GPCR Dysfunction.

Proinflammatory reaction by the body occurs acutely in response to injury that is considered primarily beneficial. However, sustained proinflammatory cytokines observed with chronic pathologies such as metabolic syndrome, cancer, and arthritis are detrimental and in many cases is a major cardiovascular risk factor. Proinflammatory cytokines such as interleukin-1, interleukin-6, and tumor necrosis factor α (TNFα) have long been implicated in cardiovascular risk and considered to be a major underlying cause for heart failure (HF). The failure of the anti-TNFα therapy for HF indicates our elusive understanding on the dichotomous role of proinflammatory cytokines on acutely beneficial effects versus long-term deleterious effects. Despite these well-described observations, less is known about the mechanistic underpinnings of proinflammatory cytokines especially TNFα in pathogenesis of HF. Increasing evidence suggests the existence of an active cross-talk between the TNFα receptor signaling and G-protein-coupled receptors such as ß-adrenergic receptor (ßAR). Given that ßARs are the key regulators of cardiac function, the review will discuss the current state of understanding on the role of proinflammatory cytokine TNFα in regulating ßAR function.

Transcription Factor KLF2 in Dendritic Cells Downregulates Th2 Programming via the HIF-1α/Jagged2/Notch Axis.

UNLABELLED: The adaptive immune response is tightly regulated by complex signals in dendritic cells (DCs). Although Th2 polarization is dictated by defined functional DC subsets, the molecular factors that govern the amplitude of these responses are not well understood. Krüppel-like factor 2 (KLF2) is a transcription factor that negatively regulates the activation of numerous immune cells in response to stimuli. Here, we demonstrate that suppression of KLF2 in conditioned DCs preferentially amplifies Th2 responses in two model systems, one of which is a prototypical intracellular pathogen and the other an allergen. This elevation in Th2 responses was dependent on contact-mediated Notch signaling in vitro and in vivo A deficiency of KLF2 increased the expression of Notch ligand Jagged2 via hypoxia-inducible factor 1α (HIF-1α), which led to Th2 amplification. Our results revealed a novel circuit in DCs for Th2 polarization that is governed by KLF2. IMPORTANCE: Dendritic cells are the key element that bridges innate and adaptive immunity. A complex and not-well-understood area in dendritic cell biology is the regulatory network that predetermines or moderates their function to shape the adaptive immune response. Our study for the first time demonstrates that KLF2, a transcription factor, conditions dendritic cells to regulate Th2 responses via a Jagged2/Notch axis. Downregulation of KLF2 expression in dendritic cells may provide a beneficial effect for treatment of diseases such as obesity or parasitic infections but may be deleterious in the case of invasion by intracellular pathogens. Strategies to tune KLF2 may be useful for future therapeutic approaches to particular diseases of mankind.

Gßγ-independent recruitment of G-protein coupled receptor kinase 2 drives tumor necrosis factor α-induced cardiac ß-adrenergic receptor dysfunction.

BACKGROUND: Proinflammatory cytokine tumor necrosis factor-α (TNFα) induces ß-adrenergic receptor (ßAR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. METHODS AND RESULTS: Two different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNFα showed that TNFα alone is sufficient to mediate ßAR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling ßAR desensitization independent of sympathetic overdrive. TNFα-mediated ßAR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in ß2AR-overexpressing human embryonic kidney 293 cells showed significant ßAR desensitization, GRK2 upregulation, and recruitment to the ßAR complex following TNFα. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated ßAR phosphorylation and GRK2 recruitment on TNFα. Furthermore, TNFα-mediated ßAR phosphorylation was not blocked with ßAR antagonist propranolol. Additionally, TNFα administration in transgenic mice with cardiac overexpression of Gßγ-sequestering peptide ßARK-ct could not prevent ßAR desensitization or cardiac dysfunction showing that GRK2 recruitment to the ßAR is Gßγ independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNFα-mediated ßAR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNFα-mediated loss in contractility, showing that TNFα-induced ßAR desensitization is GRK2 dependent. CONCLUSIONS: TNFα-induced ßAR desensitization is mediated by GRK2 and is independent of Gßγ, uncovering a hitherto unknown cross-talk between TNFα and ßAR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.

miRNA-548c: a specific signature in circulating PBMCs from dilated cardiomyopathy patients.

High fidelity genome-wide expression analysis has strengthened the idea that microRNA (miRNA) signatures in peripheral blood mononuclear cells (PBMCs) can be potentially used to predict the pathology when anatomical samples are inaccessible like the heart. PBMCs from 48 non-failing controls and 44 patients with relatively stable chronic heart failure (ejection fraction of ≤ 40%) associated with dilated cardiomyopathy (DCM) were used for miRNA analysis. Genome-wide miRNA-microarray on PBMCs from chronic heart failure patients identified miRNA signature uniquely characterized by the downregulation of miRNA-548 family members. We have also independently validated downregulation of miRNA-548 family members (miRNA-548c & 548i) using real time-PCR in a large cohort of independent patient samples. Independent in silico Ingenuity Pathway Analysis (IPA) of miRNA-548 targets shows unique enrichment of signaling molecules and pathways associated with cardiovascular disease and hypertrophy. Consistent with specificity of miRNA changes with pathology, PBMCs from breast cancer patients showed no alterations in miRNA-548c expression compared to healthy controls. These studies suggest that miRNA-548 family signature in PBMCs can therefore be used to detect early heart failure. Our studies show that cognate networking of predicted miRNA-548 targets in heart failure can be used as a powerful ancillary tool to predict the ongoing pathology.

Phosphoinositide 3-kinase γ inhibits cardiac GSK-3 independently of Akt.

Activation of cardiac phosphoinositide 3-kinase α (PI3Kα) by growth factors, such as insulin, or activation of PI3Kγ downstream of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors stimulates the activity of the kinase Akt, which phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). We found that PI3Kγ inhibited GSK-3 independently of the insulin-PI3Kα-Akt axis. Although insulin treatment activated Akt in PI3Kγ knockout mice, phosphorylation of GSK-3 was decreased compared to control mice. GSK-3 is activated when dephosphorylated by the protein phosphatase 2A (PP2A), which is activated when methylated by the PP2A methyltransferase PPMT-1. PI3Kγ knockout mice showed increased activity of PPMT-1 and PP2A and enhanced nuclear export of the GSK-3 substrate NFATc3. GSK-3 inhibits cardiac hypertrophy, and the hearts of PI3Kγ knockout mice were smaller compared to those of wild-type mice. Cardiac overexpression of a catalytically inactive PI3Kγ (PI3Kγ(inact)) transgene in PI3Kγ knockout mice reduced the activities of PPMT-1 and PP2A and increased phosphorylation of GSK-3. Furthermore, PI3Kγ knockout mice expressing the PI3Kγ(inact) transgene had larger hearts than wild-type or PI3Kγ knockout mice. Our studies show that a kinase-independent function of PI3Kγ could directly inhibit GSK-3 function by preventing the PP2A-PPMT-1 interaction and that this inhibition of GSK-3 was independent of Akt.

G-protein coupled receptor resensitization-appreciating the balancing act of receptor function.

G-protein coupled receptors (GPCRs) are seven transmembrane receptors that are pivotal regulators of cellular responses including vision, cardiac contractility, olfaction, and platelet activation. GPCRs have been a major target for drug discovery due to their role in regulating a broad range of physiological and pathological responses. GPCRs mediate these responses through a cyclical process of receptor activation (initiation of downstream signals), desensitization (inactivation that results in diminution of downstream signals), and resensitization (receptor reactivation for next wave of activation). Although these steps may be of equal importance in regulating receptor function, significant advances have been made in understanding activation and desensitization with limited effort towards resensitization. Inadequate importance has been given to resensitization due to the understanding that resensitization is a homeostasis maintaining process and is not acutely regulated. Evidence indicates that resensitization is a critical step in regulating GPCR function and may contribute towards receptor signaling and cellular responses. In light of these observations, it is imperative to discuss resensitization as a dynamic and mechanistic regulator of GPCR function. In this review we discuss components regulating GPCR function like activation, desensitization, and internalization with special emphasis on resensitization. Although we have used ß-adrenergic receptor as a proto-type GPCR to discuss mechanisms regulating receptor function, other GPCRs are also described to put forth a view point on the universality of such mechanisms.

Regulation of ß-adrenergic receptor function: an emphasis on receptor resensitization.

G protein-coupled receptors are the largest family of cell surface receptors regulating multiple cellular processes. ß-adrenergic receptor (ßAR) is a prototypical member of GPCR family and has been one of the most well studied receptors in determining regulation of receptor function. Agonist activation of ßAR leads to conformational change resulting in coupling to G protein generating cAMP as secondary messenger. The activated ßAR is phosphorylated resulting in binding of ß-arrestin that physically interdicts further G protein coupling leading to receptor desensitization. The phosphorylated ßAR is internalized and undergoes resensitization by dephosphorylation mediated by protein phosphatase 2A in the early endosomes. Although desensitization and resensitization are two sides of the same coin maintaining the homeostatic functioning of the receptor, significant interest has revolved around understanding mechanisms of receptor desensitization while little is known about resensitization. In our current review we provide an overview on regulation of ßAR function with a special emphasis on receptor resensitization and its functional relevance in the context of fine tuning receptor signaling.

Inhibition of protein phosphatase 2A activity by PI3Kγ regulates ß-adrenergic receptor function.

Phosphoinositide 3-kinase γ (PI3Kγ) is activated by G protein-coupled receptors (GPCRs). We show here that PI3Kγ inhibits protein phosphatase 2A (PP2A) at the ß-adrenergic receptor (ßAR, a GPCR) complex altering G protein coupling. PI3Kγ inhibition results in significant increase of ßAR-associated phosphatase activity leading to receptor dephosphorylation and resensitization preserving cardiac function. Mechanistically, PI3Kγ inhibits PP2A activity at the ßAR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A) on serine residues 9 and 93, resulting in enhanced binding to PP2A. Indeed, enhanced phosphorylation of ß2ARs is observed with a phosphomimetic I2PP2A mutant that was completely reversed with a mutant mimicking dephosphorylated state. siRNA depletion of endogenous I2PP2A augments PP2A activity despite active PI3K resulting in ß2AR dephosphorylation and sustained signaling. Our study provides the underpinnings of a PI3Kγ-mediated regulation of PP2A activity that has significant consequences on receptor function with broad implications in cellular signaling.