Search

There is significant variability in the efficacy and safety of oral P2Y12 inhibitors, which are used to prevent ischemic outcomes in common diseases such as coronary and peripheral arterial disease and stroke. Clopidogrel, a prodrug, is the most used oral P2Y12 inhibitor and is activated primarily after being metabolized by a highly polymorphic hepatic cytochrome CYP2C219 enzyme. Loss-of-function genetic variants in CYP2C219 are common, can result in decreased active metabolite levels and increased on-treatment platelet aggregation, and are associated with increased ischemic events on clopidogrel therapy. Such patients can be identified by CYP2C19 genetic testing and can be treated with alternative therapy. Conversely, universal use of potent oral P2Y12 inhibitors such as ticagrelor or prasugrel, which are not dependent on CYP2C19 for activation, has been recommended but can result in increased bleeding. Recent clinical trials and meta-analyses have demonstrated that a precision medicine approach in which loss-of-function carriers are prescribed ticagrelor or prasugrel and noncarriers are prescribed clopidogrel results in reducing ischemic events without increasing bleeding risk. The evidence to date supports CYP2C19 genetic testing before oral P2Y12 inhibitors are prescribed in patients with acute coronary syndromes or percutaneous coronary intervention. Clinical implementation of such genetic testing will depend on among multiple factors: rapid availability of results or adoption of the concept of performing preemptive genetic testing, provision of easy-to-understand results with therapeutic recommendations, and seamless integration in the electronic health record.

Background: ADAR1 (adenosine deaminase acting on RNA-1)-mediated adenosine to inosine (A-to-I) RNA editing plays an essential role for distinguishing endogenous from exogenous RNAs, preventing autoinflammatory ADAR1 also regulates cellular processes by recoding specific mRNAs, thereby altering protein functions, but may also act in an editing-independent manner. The specific role of ADAR1 in cardiomyocytes and its mode of action in the heart is not fully understood. To determine the role of ADAR1 in the heart, we used different mutant mouse strains, which allows to distinguish immunogenic, editing-dependent, and editing-independent functions of ADAR1., Methods: Different Adar1 -mutant mouse strains were employed for gene deletion or specific inactivation of ADAR1 enzymatic activity in cardiomyocytes, either alone or in combination with Ifih1 (interferon induced with helicase C domain 1) or Irf7 (interferon regulatory factor 7) gene inactivation. Mutant mice were investigated by immunofluorescence, Western blot, RNAseq, proteomics, and functional MRI analysis., Results: Inactivation of Adar1 in cardiomyocytes resulted in late-onset autoinflammatory myocarditis progressing into dilated cardiomyopathy and heart failure at 6 months of age. Adar1 depletion activated interferon signaling genes but not NFκB (nuclear factor kappa B) signaling or apoptosis and reduced cardiac hypertrophy during pressure overload via induction of Irf7 . Additional inactivation of the cytosolic RNA sensor MDA5 (melanoma differentiation-associated gene 5; encoded by the Ifih1 gene) in Adar1 mutant mice prevented activation of interferon signaling gene and delayed heart failure but did not prevent lethality after 8.5 months. In contrast, compound mutants only expressing catalytically inactive ADAR1 in an Ifih1 -mutant background were completely normal. Inactivation of Irf7 attenuated the phenotype of Adar1 -deficient cardiomyocytes to a similar extent as Ifih1 depletion, identifying IRF7 as the main mediator of autoinflammatory responses caused by the absence of ADAR1 in cardiomyocytes., Conclusions: Enzymatically active ADAR1 prevents IRF7-mediated autoinflammatory reactions in the heart triggered by endogenous nonedited RNAs. In addition to RNA editing, ADAR1 also serves editing-independent roles in the heart required for long-term cardiac function and survival.

Rationale: Cardiac involvement and hypotension dominate the prognosis of light-chain amyloidosis (AL). Evidence suggests that there is also peripheral vascular involvement in AL but its prognostic significance is unknown. Objective: To evaluate vascular dysfunction in patients with AL as a potential future area of intervention, we assessed the prognostic utility of flow-mediated dilatation (FMD), a marker of vascular reactivity, which is augmented under conditions of hypotension and autonomic dysfunction. Methods and Results: We prospectively evaluated 115 newly diagnosed untreated AL patients in whom FMD was measured. FMD in AL patients was significantly higher than age-, sex- and risk factors-matched controls (4.0% versus 2.32%; P =0.006) and comparable with control groups at lower cardiovascular risk ( P >0.1). Amyloidosis patients presented increased plasma and exhaled markers of the NO pathway while their FMD significantly correlated with augmented sustained vasodilatation after sympathetic stimulation. Increased FMD (≥4.5%) was associated with early mortality (hazard ratio, 4.36; 95% CI, 1.41-13.5; P =0.010) and worse survival (hazard ratio, 2.11; 95% CI, 1.17-3.82; P =0.013), even after adjustment for Mayo stage, nerve involvement and low systolic blood pressure. This finding was confirmed in a temporal validation AL cohort (n=55; hazard ratio, 4.2; 95% CI, 1.45-12.3; P =0.008). FMD provided significant reclassification value over the best prognostic model (continuous Net Reclassification Index, 0.61; P =0.001). Finally, better hematologic response was associated with lower posttreatment FMD. Conclusions: FMD is relatively increased in AL and independently associated with inferior survival with substantial reclassification value. Reactive vasodilation merits further investigation as a novel risk biomarker in AL.Visual Overview: An online visual overview is available for this article.

Medicine is at the crossroads of expanding disciplines. Prompt adaptation of medicine to each rapidly advancing research field, bridging bench to bedside, is a key step toward health improvement. Cardiovascular disease still ranks first among the mortality causes in the Western world, indicating a poor adaptation rate of cardiovascular medicine, albeit the gigantic scientific breakthroughs of this century. This urges the cardiovascular research field to explore novel concepts with promising prognostic and therapeutic potential. This review attempts to introduce the newly emerging field of epitranscriptome (or else known as RNA epigenetics) to cardiovascular researchers and clinicians summarizing its applications on health and disease. The traditionally perceived, intermediate carrier of genetic information or as contemporary revised as, occasionally, even the final product of gene expression, RNA, is dynamically subjected to >140 different kinds of chemical modifications determining its fate, which may profoundly impact the cellular responses and thus both health and disease course. Which are the most prevalent types of these RNA modifications, how are they catalyzed, how are they regulated, which role may they play in health and disease, and which are the implications for the cardiovascular medicine are few important questions that are discussed in the present review.

Background: The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and functionally important endothelial lncRNAs., Methods: Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression., Results: A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18 , SMAD6 , and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding., Conclusion: MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function., (© 2017 The Authors.)

Rationale: Circular RNAs (circRNAs) are noncoding RNAs generated by back splicing. Back splicing has been considered a rare event, but recent studies suggest that circRNAs are widely expressed. However, the expression, regulation, and function of circRNAs in vascular cells is still unknown., Objective: Here, we characterize the expression, regulation, and function of circRNAs in endothelial cells., Methods and Results: Endothelial circRNAs were identified by computational analysis of ribo-minus RNA generated from human umbilical venous endothelial cells cultured under normoxic or hypoxic conditions. Selected circRNAs were biochemically characterized, and we found that the majority of them lacks polyadenylation, is resistant to RNase R digestion and localized to the cytoplasm. We further validated the hypoxia-induced circRNAs cZNF292, cAFF1, and cDENND4C, as well as the downregulated cTHSD1 by reverse transcription polymerase chain reaction in cultured endothelial cells. Cloning of cZNF292 validated the predicted back splicing of exon 4 to a new alternative exon 1A. Silencing of cZNF292 inhibited cZNF292 expression and reduced tube formation and spheroid sprouting of endothelial cells in vitro. The expression of pre-mRNA or mRNA of the host gene was not affected by silencing of cZNF292. No validated microRNA-binding sites for cZNF292 were detected in Argonaute high-throughput sequencing of RNA isolated by cross-linking and immunoprecipitation data sets, suggesting that cZNF292 does not act as a microRNA sponge., Conclusions: We show that the majority of the selected endothelial circRNAs fulfill all criteria of bona fide circRNAs. The circRNA cZNF292 exhibits proangiogenic activities in vitro. These data suggest that endothelial circRNAs are regulated by hypoxia and have biological functions., (© 2015 American Heart Association, Inc.)

Objectives: The masking effect (ME) is present in masked hypertensive patients; however, both normotensive and hypertensive individuals may show a similar phenomenon. Previous studies have shown that ME has been associated with left ventricular hypertrophy and microalbuminuria in treated hypertensive patients. The aim of our study was to evaluate the association between the magnitude of systolic ME and the extent of common carotid artery intima-media thickness (CCA-IMT) development in normotensive individuals and untreated masked hypertensive and hypertensive patients., Participants and Methods: A total of 1154 individuals underwent 24 h ambulatory blood pressure (BP) monitoring and carotid artery ultrasonographic measurements. The final study population included 360 patients with systolic ME (daytime systolic BP higher than office systolic BP). The participants were divided into three groups according to office and daytime BP values: normotensives, masked hypertensives, and hypertensives., Results: Masked hypertensives presented significantly higher systolic ME (-14.6 mmHg) than their normotensive (-8.2 mmHg) and hypertensive (-9.5 mmHg) counterparts. However, systolic ME was associated significantly with CCA-IMT only in the group of masked hypertensives (r=-0.399, P<0.001). The multivariate linear regression analyses showed significant and independent associations of CCA-IMT with the following factors: age (B=0.028, 95% confidence interval: 0.001-0.055; P=0.044) and systolic ME (B=-0.034, 95% confidence interval: -0.066 to -0.003; P=0.034). A 10 mmHg decrease in systolic ME correlated to an increase of 0.034 mm in the CCA-IMT., Conclusion: Systolic ME was associated significantly with CCA-IMT values in masked hypertensives. Both normotensive and hypertensive participants have failed to show similar associations.

Rationale: The recently discovered chemokine CXC motif ligand 16 (CXCL16) is highly expressed in atherosclerotic lesions and is a potential pathogenic mediator in coronary artery disease., Objective: The aim of this study was to test the role of CXCL16 on platelet activation and vascular adhesion, as well as the underlying mechanism and signaling pathway., Methods and Results: Reverse-transcriptase polymerase chain reaction, Western blotting, confocal microscopy, and flow cytometry revealed that CXCL16-specific receptor, CXC motif receptor 6, is highly expressed in platelets. According to flow cytometry and confocal microscopy, stimulation of platelets with CXCL16 induced platelet degranulation, integrin α(IIb)β(3) activation, and shape change. CXCL16 increased Akt phosphorylation (Thr(308)/Ser(473)), an effect abrogated by phosphatidylinositide 3-kinase inhibitors wortmannin (100 nmol/L) and LY294002 (25 µmol/L). The phosphatidylinositide 3-kinase inhibitors and Akt inhibitor SH-6 (20 µmol/L) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin α(IIb)β(3) activation, and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXC motif receptor 6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro after high arterial shear stress (2000(-s)) and to injured vascular wall in vivo after carotid ligation. CXCL16-induced stimulation of platelet adhesion again was prevented by phosphatidylinositide 3-kinase and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P(2)Y(1) (MRS2179, 100 µmol/L) and especially P(2)Y(12) (Cangrelor, 10 µmol/L) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotid ligation in vivo., Conclusions: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXC motif receptor 6-dependent phosphatidylinositide 3-kinase/Akt signaling and paracrine activation, suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.

Objective: Hyperlipidemia is associated with platelet hyperactivity. In the present study, we evaluated the binding of oxidized low-density lipoprotein (oxLDL) on the surface of circulating platelets in patients with stable coronary artery disease and acute coronary syndromes and its possible association with platelet activation. Furthermore, the role of oxLDL binding on platelet adhesion to collagen and endothelial cells in vitro as well as after carotid ligation in mice was investigated., Methods and Results: Using flow cytometry, patients with acute coronary syndromes (n=174) showed significantly enhanced oxLDL binding compared with patients with stable coronary artery disease (n=182; P=0.007). Platelet-bound oxLDL positively correlated with the degree of platelet activation (expression of P-selectin and activated fibrinogen receptor; P<0.001 for both). Plasma oxLDL was increased in patients with acute coronary syndromes compared with stable angina pectoris patients. Preincubation of isolated platelets with oxLDL, but not with native LDL, resulted in enhanced platelet adhesion to collagen and activated endothelial cells under high shear stress in vitro, as well as after carotid ligation in C57BL/6J mice and apolipoprotein E(-/-) mice fed a high cholesterol diet., Conclusions: Increased platelet-bound oxLDL in patients with acute coronary syndromes may play an important role in atherothrombosis, thus providing a potential future therapeutic target.

Objective: To investigate the role of junctional adhesion molecule A (JAM-A) on adhesion and differentiation of human CD34(+) cells into endothelial progenitor cells., Methods and Results: Tissue healing and vascular regeneration is a multistep process requiring firm adhesion of circulating progenitor cells to the vascular wall and their further differentiation into endothelial cells. The role of JAM-A in platelet-mediated adhesion of progenitor cells was investigated by adhesion assays in vitro and with the help of intravital fluorescence microscopy in mice. Preincubation of human CD34(+) progenitor cells with soluble JAM-A-Fc (sJAM-A-Fc) resulted in significantly decreased adhesion over immobilized platelets or inflammatory endothelium under high shear stress in vitro and after carotid ligation in vivo or ischemia/reperfusion injury in the microcirculation of mice. Human CD34(+) cells express JAM-A, as defined by flow cytometry and Western blot analysis. JAM-A mediates differentiation of CD34(+) cells to endothelial progenitor cells and facilitates CD34(+) cell-induced reendothelialization in vitro. Pretreatment of human CD34(+) cells with sJAM-A-Fc resulted in increased neointima formation 3 weeks after endothelial denudation in the carotid arteries of nonobese diabetic/severe combined immunodeficient mice., Conclusions: These results indicate that the expression of JAM-A on CD34(+) cells mediates adhesion to the vascular wall after injury and differentiation into endothelial progenitor cells, a mechanism potentially involved in vascular regeneration. Human CD34(+) cells express JAM-A, mediating their interaction with platelets and endothelial cells. Specifically, JAM-A expressed on human CD34(+) progenitor cells regulates their adhesion over immobilized platelets or inflammatory endothelium under high shear stress in vitro and after carotid ligation in vivo or ischemia/reperfusion injury in the microcirculation of mice. Moreover, it mediates differentiation of CD34(+) cells to endothelial progenitor cells and facilitates reendothelialization.

Background: Peripheral homing of progenitor cells in areas of diseased organs is critical for tissue regeneration. The chemokine stromal cell-derived factor-1 (SDF-1) regulates homing of CD34+ stem cells. We evaluated the role of platelet-derived SDF-1 in adhesion and differentiation of human CD34+ cells into endothelial progenitor cells., Methods and Results: Adherent platelets express substantial amounts of SDF-1 and recruit CD34+ cells in vitro and in vivo. A monoclonal antibody to SDF-1 or to its counterreceptor, CXCR4, inhibits stem cell adhesion on adherent platelets under high arterial shear in vitro and after carotid ligation in mice, as determined by intravital fluorescence microscopy. Platelets that adhere to human arterial endothelial cells enhance the adhesion of CD34+ cells on endothelium under flow conditions, a process that is inhibited by anti-SDF-1. During intestinal ischemia/reperfusion in mice, anti-SDF-1 and anti-CXCR4, but not isotype control antibodies, abolish the recruitment of CD34+ cells in microcirculation. Moreover, platelet-derived SDF-1 binding to CXCR4 receptor promotes platelet-induced differentiation of CD34+ cells into endothelial progenitor cells, as verified by colony-forming assays in vitro., Conclusions: These findings imply that platelet-derived SDF-1 regulates adhesion of stem cells in vitro and in vivo and promotes differentiation of CD34+ cells to endothelial progenitor cells. Because tissue regeneration depends on recruitment of progenitor cells to peripheral vasculature and their subsequent differentiation, platelet-derived SDF-1 may contribute to vascular and myocardial regeneration.

Purpose of Review: The response to injury model in the development of atherosclerosis is broadly accepted by the scientific audience. Platelets are generally not believed to be involved in the initiation of atherosclerosis. New data imply, however, that the response to injury model is too simple for a complete understanding of the inflammatory disease atherosclerosis. The involvement of platelets in the initiation of atherosclerotic lesion formation is critical in directing the atherosclerotic process into regeneration or ongoing vascular injury., Recent Findings: Platelets internalize oxidized phospholipids and promote foam cell formation. Platelets also recruit circulating blood cells including progenitor cells to the vessel, that are able to differentiate into foam cells or endothelial cells depending on conditions. Platelets express various scavenger receptors that are able to regulate LDL-uptake. LDL-laden platelets are internalized by adherent progenitor cells that in turn differentiate into macrophages and foam cells., Summary: An expanding body of evidence continues to build on the role of platelets as initial actors in the development of atherosclerotic lesions. Platelets bind to leukocytes, endothelial cells, and circulating progenitor cells and initiate monocyte transformation into macrophages. Therefore platelets regulate the initiation, development and total extent of atherosclerotic lesions.

Objective: Thrombotic events and immunoinflammatory processes take place next to each other during vascular remodeling in atherosclerotic lesions. In this study we investigated the interaction of platelets with dendritic cells (DCs)., Methods and Results: The rolling of DCs on platelets was mediated by PSGL-1. Firm adhesion of DCs was mediated through integrin alphaMbeta2 (Mac-1). In vivo, adhesion of DCs to injured carotid arteries in mice was mediated by platelets. Pretreatment with soluble GPVI, which inhibits platelet adhesion to collagen, substantially reduced recruitment of DCs to the injured vessel wall. In addition, preincubation of DCs with sJAM-C significantly reduced their adhesion to platelets. Coincubation of DCs with platelets induced maturation of DCs, as shown by enhanced expression of CD83. In the presence of platelets, DC-induced lymphocyte proliferation was significantly enhanced. Moreover, coincubation of DCs with platelets resulted in platelet phagocytosis by DCs, as verified by different cell phagocytosis assays. Finally, platelet/DC interaction resulted in apoptosis of DCs mediated by a JAM-C-dependent mechanism., Conclusions: Recruitment of DCs by platelets, which is mediated via CD11b/CD18 (Mac-1) and platelet JAM-C, leads to DC activation and platelet phagocytosis. This process may be of importance for progression of atherosclerotic lesions.

Objective: Thrombus formation after atherosclerotic plaque rupture critically involves the platelet collagen receptor glycoprotein (GP) VI. We investigated the impact of EXP3179, an active metabolite of the angiotensin II type 1 (AT1)-receptor antagonist Losartan (LOS) on GPVI-dependent platelet activation., Methods and Results: EXP3179 and LOS but not EXP3174--the major AT1-receptor blocking metabolite of LOS--dose-dependently inhibited collagen-I (P<0.01) and GPVI-dependent platelet aggregation (P<0.01) analyzed by optical aggregometry. Platelet activation was further determined by flow cytometry measuring the expression of platelet PAC-1, an epitope of the activated fibrinogen-receptor complex. EXP3179 and LOS inhibited collagen-I (P<0.01) and GPVI-dependent PAC-1 expression (P<0.01). EXP3179 and LOS but not EXP3174 decreased the adhesion of GPVI-receptor expressing Chinese hamster ovarian cells on collagen-I under arterial shear conditions determined by flow chamber analysis (P<0.01 and P<0.05). EXP3179 also reduced human atherosclerotic plaque material-induced platelet aggregation (P<0.01) in vitro and murine platelet adhesion after acute vessel injury in vivo as determined by intravital microscopy (P<0.01)., Conclusion: EXP3179 acts as a specific inhibitor of the platelet collagen receptor GPVI independent of AT1-receptor antagonism. Further investigations may clarify its individual potential as a novel pharmacological approach to specifically inhibit atherothrombotic events by GPVI-receptor blockade.