Peroxisome proliferator-activated receptor δ (PPARδ) is a critical regulator of energy metabolism in the heart. Here, we propose a mechanism that integrates two deleterious characteristics of heart failure, hypoxia and a metabolic shift toward glycolysis, involving the microRNA cluster miR-199a∼214 and PPARδ. We demonstrate that under hemodynamic stress, cardiac hypoxia activates DNM3os, a noncoding transcript that harbors the microRNA cluster miR-199a∼214, which shares PPARδ as common target. To address the significance of miR-199a∼214 induction and concomitant PPARδ repression, we performed antagomir-based silencing of both microRNAs and subjected mice to biomechanical stress to induce heart failure. Remarkably, antagomir-treated animals displayed improved cardiac function and restored mitochondrial fatty acid oxidation. Taken together, our data suggest a mechanism whereby miR-199a∼214 actively represses cardiac PPARδ expression, facilitating a metabolic shift from predominant reliance on fatty acid utilization in the healthy myocardium toward increased reliance on glucose metabolism at the onset of heart failure.

Although aberrant reactivation of embryonic gene programs is intricately linked to pathological heart disease, the transcription factors driving these gene programs remain ill-defined. Here we report that increased calcineurin/Nfat signalling and decreased miR-25 expression integrate to re-express the basic helix-loop-helix (bHLH) transcription factor dHAND (also known as Hand2) in the diseased human and mouse myocardium. In line, mutant mice overexpressing Hand2 in otherwise healthy heart muscle cells developed a phenotype of pathological hypertrophy. Conversely, conditional gene-targeted Hand2 mice demonstrated a marked resistance to pressure-overload-induced hypertrophy, fibrosis, ventricular dysfunction and induction of a fetal gene program. Furthermore, in vivo inhibition of miR-25 by a specific antagomir evoked spontaneous cardiac dysfunction and sensitized the murine myocardium to heart failure in a Hand2-dependent manner. Our results reveal that signalling cascades integrate with microRNAs to induce the expression of the bHLH transcription factor Hand2 in the postnatal mammalian myocardium with impact on embryonic gene programs in heart failure.

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase C␥2, which bears similarity to the signaling pathway evoked by T-and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3K␣ or -␤ strongly and selectively suppressed GPVI-induced Ca 2؉ mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase C␥2 by PI3K␣/␤. That PI3K␣ and -␤ have a non-redundant function in GPVIinduced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca 2؉ increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3K␣/␤ was blocked or p85␣ was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3K␦ or -␥. Furthermore, PI3K␣/␤, but not PI3K␥, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3K␣ and -␤ isoforms are required for full GPVI-dependent platelet Ca 2؉ signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3K␣ an interesting new target for anti-platelet therapy.Exposed collagen in a damaged vessel wall activates platelets via their immunoglobulin family receptor, glycoprotein VI (GPVI), 3 by using a complex signal transduction pathway, which is reminiscent to the pathway employed by immune receptors in T and B cells (1, 2). In platelets, tyrosine phosphorylation of the Fc receptor ␥-chain, linked to GPVI via Src family kinases, leads to a cascade of protein phosphorylation events, cumulating in the activation of phospholipase C␥2 (PLC␥2). This key effector enzyme triggers many downstream events, including production of inositol 1,4,5-trisphosphate (InsP 3 ), mobilization of cytosolic Ca 2ϩ , activation of integrin ␣ IIb ␤ 3 , secretion of platelet granules loaded with autocrine-stimulating agents (ADP and ATP), and exposure of negatively charged phosphatidylserine (PS) at the platelet surface to ensure coagulation (1, 3, 4). All these responses are potently triggered by GPVI ligands, which, besides collagen, include collagen-related peptides and the snake venom convulxin (5-7).One of the GPVI-induced signaling events contributing to PLC␥2 activation is activation of the protein/lipid kinase, phosphoinositide 3-kinase (PI3K) in both human and mouse platelets (8 -11). Evidence for this role ca...

Background-Monocytes are cellular components of wound repair, arteriogenesis, and atherogenesis. Vascular endothelial growth factor (VEGF)-A and placental growth factor recruit monocytes to sites of arteriogenesis via stimulation of VEGF receptor-1 (VEGFR-1). The chemotactic response of monocytes to VEGF-A is attenuated in individuals with diabetes mellitus (DM). This VEGF resistance correlates with impaired collateral growth. The aim of this study is to elucidate the molecular basis of VEGF resistance and impaired monocyte response in DM. Methods and Results-Phosphorylation of Akt, p38, and extracellular signal-regulated kinase 1/2 (ERK1/2) could be stimulated with either placental growth factor-1 or VEGF-A in monocytes from non-DM but not DM individuals. In contrast, formyl-methionyl-leucyl-phenylalanine caused a comparable activation of these molecules in both DM and non-DM monocytes. Baseline phosphorylation of Akt, p38, and ERK1/2 was significantly elevated in monocytes from DM compared with non-DM subjects. Of note, H 2 O 2 activated Akt, p38, and ERK1/2 in non-DM monocytes ex vivo. Protein tyrosine phosphatases had stronger oxidative modifications in monocytes from DM than from non-DM individuals, which reflects functional protein tyrosine phosphatase inhibition, similar to that seen after H 2 O 2 challenge. Overall, protein tyrosine phosphatase and protein tyrosine phosphatase-1B activity were reduced in DM monocytes. DM monocytes revealed higher expression of the receptor for advanced glycation end products. Stimulation with advanced glycation end products ligands resulted in activation of non-DM monocytes and inhibition of VEGFR-1-mediated chemotaxis. The elevated baseline phosphorylation/activation of Akt, p38, and ERK1/2 in DM monocytes likely causes the resistance to further stimulation with specific stimuli such as VEGF-A, revealing a molecular explanation of the DM-related signal transduction defect. Conclusions-We propose that elevated advanced glycation end products expression and increased oxidative stress in diabetic monocytes lead to activation of VEGFR-1-related signaling pathways and to desensitization of VEGFR-1 responses. These data establish VEGF resistance as a novel molecular concept for DM-related cellular dysfunction. Key Words: diabetes mellitus Ⅲ monocytes Ⅲ signal transduction Ⅲ vascular endothelial growth factor receptor-1 G rowth factors are potent and crucial mediators of vascular growth processes, including angiogenesis and arteriogenesis. 1,2 Both processes can enhance regional blood flow and restore impaired tissue function. Monocytes contribute to arteriogenesis by recruitment to the growing vessel wall. [3][4][5] Moreover, angiogenesis is monocyte dependent in the context of wound healing. 6 Editorial see p 104 Clinical Perspective on p 159The family of vascular endothelial growth factor (VEGF) and its receptors (VEGFR) are mediators of angiogenesis and arteriogenesis in both embryonic development and adult life. 1,4,7 VEGFR-2 mediates crucial functions of endothelial cel...

Summary. Background: Interaction of murine Gas6 with the platelet Gas6 receptors Tyro3, Axl and Mer (TAM) plays an important role in arterial thrombus formation. However, a role for Gas6 in human platelet activation has been questioned. Objective: To determine the role of Gas6 in human and murine platelet activation and thrombus formation. Methods and Results: Gas6 levels appeared to be 20-fold higher in human plasma than in platelets, suggesting a predominant role of plasma-derived Gas6. Human Gas6 synergizes with ADP-P2Y 12 by enhancing and prolonging the phosphorylation of Akt. Removal of Gas6 from plasma impaired ADP-induced platelet aggregation. Under flow conditions, absence of human Gas6 provoked gradual platelet disaggregation and integrin a IIb b 3 inactivation. Recombinant human Gas6 reversed the effects of Gas6 removal. In mouse blood, deficiency in Gas6 or in one of the TAM receptors led to reduced thrombus formation and increased disaggregation, which was completely antagonized by external ADP. In contrast, collagen-induced platelet responses were unchanged by the absence of Gas6 in both human and mouse systems. Conclusions: The ADP-P2Y 12 and Gas6-TAM activation pathways synergize to achieve persistent a IIb b 3 activation and platelet aggregation. We postulate a model of thrombus stabilization in which plasma Gas6, by signaling via the TAM receptors, extends and enhances the platelet-stabilizing effect of autocrine ADP, particularly when secretion becomes limited.

Objective-To elucidate the downstream mechanisms of vascular endothelial growth factor receptor 2 (VEGFR2), a key receptor in angiogenesis, which has been associated with atherosclerotic plaque growth and instability. Methods and Results-By using a yeast-2-hybrid assay, we identified A Disintegrin And Metalloprotease 10 (ADAM10) as a novel binding partner of VEGFR2. ADAM10 is a metalloprotease with sheddase activity involved in cell migration; however, its exact function in endothelial cells (ECs), angiogenesis, and atherosclerosis is largely unknown. For the first time to our knowledge, we show ADAM10 expression in human atherosclerotic lesions, associated with plaque progression and neovascularization. We demonstrate ADAM10 expression and activity in ECs to be induced by VEGF; also, ADAM10 mediates the ectodomain shedding of VEGFR2. Furthermore, VEGF induces ADAM10-mediated cleavage of vascular endothelium (VE)-cadherin, which could increase vascular permeability and facilitate EC migration. Indeed, VEGF increases vascular permeability in an ADAM10-and ADAM17-dependent way; inhibition of ADAM10 reduces EC migration and chemotaxis. Conclusion-These data provide the first evidence of ADAM10 expression in atherosclerosis and neovascularization. Key Words: angiogenesis Ⅲ atherosclerosis Ⅲ endothelial function Ⅲ growth factors Ⅲ metalloproteinases A ngiogenesis is associated with tumor growth, metastasis, and growth and instability of atherosclerotic plaques. The latter contribute to plaque rupture and its clinical complications. 1 Expression of the major angiogenic factor, vascular endothelial growth factor (VEGF) A, increases during atherogenesis. In endothelial cells (ECs), VEGF exerts its stimulatory effect mostly via VEGF receptor (VEGFR) 2 (kinase insert domain receptor [KDR]/fetal liver kinase-1 ) and is a potent angiogenic mediator of EC migration, proliferation, and survival. 2 VEGF binding induces VEGFR2 dimerization, followed by (auto)phosphorylation on tyrosine residues. Activation of VEGFR2 induces signaling via phospholipase C (PLC)␥, activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 pathway, and EC proliferation. Furthermore, activation of p38 -mitogen-activated protein kinase or phosphatidylinositol 3-kinase (PI3K) leads to EC migration, survival, and increased permeability. 2,3 See accompanying article on page 2087In addition to angiogenesis, VEGF and its receptors are associated with development of atherosclerosis, plaque angiogenesis, and plaque instability. Recently, it was suggested that VEGF may induce a more vulnerable plaque phenotype by promoting leukocyte recruitment. 4 6 The neutralizing VEGF antibody bevacizumab (Avastin) is an effective anticancer therapy that inhibits tumor angiogenesis; however, it is associated with an increase in cardiovascular adverse effects based on thrombosis and plaque instability. 7 Unraveling downstream VEGFR signaling mechanisms involved in angiogenesis and atherosclerosis will identify novel therapeutic...

BACKGROUND Heart rate follows a diurnal variation, and slow heart rhythms occur primarily at night.

Heart failure is preceded by ventricular remodeling, changes in left ventricular mass, and myocardial volume after alterations in loading conditions. Concentric hypertrophy arises after pressure overload, involves wall thickening, and forms a substrate for diastolic dysfunction. Eccentric hypertrophy develops in volume overload conditions and leads wall thinning, chamber dilation, and reduced ejection fraction. The molecular events underlying these distinct forms of cardiac remodeling are poorly understood. Here, we demonstrate that miR-148a expression changes dynamically in distinct subtypes of heart failure: while it is elevated in concentric hypertrophy, it decreased in dilated cardiomyopathy. In line, antagomir-mediated silencing of miR-148a caused wall thinning, chamber dilation, increased left ventricle volume, and reduced ejection fraction. Additionally, adeno-associated viral delivery of miR-148a protected the mouse heart from pressure-overload-induced systolic dysfunction by preventing the transition of concentric hypertrophic remodeling toward dilation. Mechanistically, miR-148a targets the cytokine co-receptor glycoprotein 130 (gp130) and connects cardiomyocyte responsiveness to extracellular cytokines by modulating the Stat3 signaling. These findings show the ability of miR-148a to prevent the transition of pressure-overload induced concentric hypertrophic remodeling toward eccentric hypertrophy and dilated cardiomyopathy and provide evidence for the existence of separate molecular programs inducing distinct forms of myocardial remodeling.