Michael Bode

Regulation of tissue factor gene expression in monocytes and endothelial cells: Thromboxane A2 as a new player

Tissue factor (TF) is the primary activator of the coagulation cascade. Under normal conditions, endothelial cells (ECs) and blood cells, such as monocytes, do not express TF. However, bacterial lipopolysaccharide (LPS) induces TF expression in monocytes and this leads to disseminated intravascular coagulation during endotoxemia and sepsis. A variety of stimuli induce TF expression in ECs in vitro, although it is unclear how much TF is expressed by the endothelium in vivo. LPS induction of TF gene expression in monocytic cells and ECs is mediated by various intracellular signaling pathways and the transcription factors NF-ĸB, AP-1 and Egr-1. In contrast, vascular endothelial cell growth factor (VEGF) induces TF gene expression in ECs via the transcription factors NFAT and Egr-1. Similarly, oxidized phospholipids (such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine) induce TF expression in ECs and possibly monocytes via NFAT and Egr-1. Thromboxane A2 (TXA2) can now be added to the list of stimuli that induce TF gene expression in both monocytes and ECs. Interestingly, inhibition of the TX-prostanoid (TP) receptor also reduces TF expression in with tumor necrosis factor (TNF)-α stimulated ECs and LPS stimulated monocytes, which suggests that TP receptor antagonist may be useful in reducing pathologic TF expression in the vasculature and blood.

Protective and pathological roles of tissue factor in the heart.

UNLABELLED Tissue factor (TF) is expressed in the heart where it is required for haemostasis. High levels of TF are also expressed in atherosclerotic plaques and likely contribute to atherothrombosis after plaque rupture. Indeed, risk factors for atherothrombosis, such as diabetes, hypercholesterolaemia, smoking and hypertension, are associated with increased TF expression in circulating monocytes, microparticles and plasma. Several therapies that reduce atherothrombosis, such as statins, ACE inhibitors, beta-blockers and anti-platelet drugs, are associated with reduced TF expression. In addition to its haemostatic and pro-thrombotic functions, the TF : FVIIa complex and downstream coagulation proteases activate cells by cleavage of protease-activated receptors (PARs). In mice, deficiencies in either PAR-1 or PAR-2 reduce cardiac remodelling and heart failure after ischaemia-reperfusion injury. This suggests that inhibition of coagulation proteases and PARs may be protective in heart attack patients. In contrast, the TF/thrombin/PAR-1 pathway is beneficial in a mouse model of Coxsackievirus B3-induced viral myocarditis. We found that stimulation of PAR-1 increases the innate immune response by enhancing TLR3-dependent IFN-β expression. Therefore, inhibition of the TF/thrombin/PAR-1 pathway in patients with viral myocarditis could have detrimental effects. CONCLUSION The TF : FVIIa complex has both protective and pathological roles in the heart.

Regulation of ankyrin repeat and suppressor of cytokine signalling box protein 4 expression in the immortalized murine endothelial cell lines MS1 and SVR: a role for tumour necrosis factor alpha and oxygen.

During vascular development, endothelial cells are exposed to a variety of rapidly changing factors, including fluctuating oxygen levels. We have previously shown that ankyrin repeat and suppressor of cytokine signalling box protein 4 (ASB4) is the most highly differentially expressed gene in the vascular lineage during early differentiation and is expressed in the embryonic vasculature at a time when oxygen tension is rising because of the onset of placental blood flow. To further our understanding of the regulation of ASB4 expression in endothelial cells, we tested the effect of various stressors for their ability to alter ASB4 expression in the immortalized murine endothelial cell lines MS1 and SVR. ASB4 expression is decreased during hypoxic insult and shear stress, whereas it is increased in response to tumour necrosis factor alpha (TNF‐α). Further investigation indicated that nuclear factor kappa B (NF‐κB) is the responsible transcription factor involved in the TNF‐α‐induced upregulation of ASB4, placing ASB4 downstream of NF‐κB in the TNF‐α signalling cascade and identifying it as a potential regulator for TNF‐αs numerous functions associated with inflammation, angiogenesis and apoptosis. Copyright

Protease-Activated Receptor 1 Enhances Poly I:C Induction of the Antiviral Response in Macrophages and Mice

The coagulation cascade is activated during viral infections as part of the host defense system. Coagulation proteases activate cells by cleavage of protease-activated receptors (PARs). Recently, we reported that the activation of PAR-1 enhanced interferon (IFN)β and CXCL10 expression in cardiac fibroblasts and in the hearts of mice infected with Coxsackievirus B3. In this study, we used the double-stranded RNA mimetic polyinosinic:polycytidylic acid (poly I:C) to induce an antiviral response in macrophages and mice. Activation of PAR-1 enhanced poly I:C induction of IFNβ and CXCL10 expression in the murine macrophage cell line RAW264.7, bone-marrow derived mouse macrophages (BMM) and mouse splenocytes. Next, poly I:C was used to induce a type I IFN innate immune response in the spleen and plasma of wild-type (WT) and PAR-1-/- mice. We found that poly I:C treated PAR-1-/- mice and WT mice given the thrombin inhibitor dabigatran etexilate exhibited significantly less IFNβ and CXCL10 expression in the spleen and plasma than WT mice. These studies suggest that thrombin activation of PAR-1 contributes to the antiviral response in mice.

Prominent R wave in ECG lead V1 predicts improvement of left ventricular ejection fraction after cardiac resynchronization therapy in patients with or without left bundle branch block.

BACKGROUND QRS morphology on postprocedural ECG indicating posterolateral left ventricular pacing may be predictive of response to cardiac resynchronization therapy (CRT). OBJECTIVE The purpose of this study was to assess whether a positive vector in V1 and/or negative vector in lead I on the first postprocedural ECG, suggesting posterolateral capture from CRT, correlates with improvement in left ventricular ejection fraction (LVEF). METHODS A retrospective chart review was conducted on all patients who underwent CRT implantation at our institution between April 2008 and December 2011. Biventricular (BiV) paced QRS morphology was defined as R/S ≥1 in V1 and/or R/S ≤ 1 in lead I. The primary outcome was improvement of LVEF ≥7.5%. The χ(2) and t tests were used for analysis. RESULTS Of 68 patients, 49 (72%) met our BiV paced QRS morphology criteria. Thirty-four of these 49 patients (69%) had improvement in LVEF. Of the 19 patients who did not meet our criteria, 17 (89%) did not have an improvement in LVEF (sensitivity 94%, specificity 53%, χ(2) = 19.04, P < .0001). The average LVEF improvement in patients who met our BiV paced QRS morphology criteria was significantly greater than in those who did not (14.27% vs 2.63%, P = .0001). Preprocedural left bundle branch block was not a predictor of echocardiographic response. CONCLUSION Our results highlight the importance of periprocedural ECG analysis to optimize response to CRT. Moreover, patients without left bundle branch block still benefited from CRT if they met our BiV paced morphology criteria. This suggests that postprocedural left ventricular activation as reflected on the ECG may supersede the baseline conduction delay.

Regulation of ASB4 expression in the immortalized murine endothelial cell lines MS1 and SVR: a role for TNF-α and oxygen

During vascular development, endothelial cells are exposed to a variety of rapidly changing factors, including fluctuating oxygen levels. We have previously shown that ankyrin repeat and suppressor of cytokine signalling box protein 4 (ASB4) is the most highly differentially expressed gene in the vascular lineage during early differentiation and is expressed in the embryonic vasculature at a time when oxygen tension is rising because of the onset of placental blood flow. To further our understanding of the regulation of ASB4 expression in endothelial cells, we tested the effect of various stressors for their ability to alter ASB4 expression in the immortalized murine endothelial cell lines MS1 and SVR. ASB4 expression is decreased during hypoxic insult and shear stress, whereas it is increased in response to tumour necrosis factor alpha (TNF‐α). Further investigation indicated that nuclear factor kappa B (NF‐κB) is the responsible transcription factor involved in the TNF‐α‐induced upregulation of ASB4, placing ASB4 downstream of NF‐κB in the TNF‐α signalling cascade and identifying it as a potential regulator for TNF‐αs numerous functions associated with inflammation, angiogenesis and apoptosis. Copyright

A combined deficiency of tissue factor and PAR-4 is associated with fatal pulmonary hemorrhage in mice

INTRODUCTION Mice with a complete absence of tissue factor (TF) die during embryonic development whereas mice with low levels of TF (Low-TF mice) survive to adulthood. Low-TF mice exhibit spontaneous hemorrhage in various organs, including the lung. In contrast, mice can survive without protease-activated receptor (PAR)-4, which is the major thrombin receptor on mouse platelets. We determined the effect of combining a deficiency PAR-4 (primary hemostasis) with a deficiency in TF (secondary hemostasis) on embryonic development and survival of adult mice. MATERIALS AND METHODS Low-TF mice (mTF-/-, hTF+/+) were crossed with PAR-4-/- mice to generate heterozygous mice (mTF+/-, hTF+/-, PAR-4+/-). These mice were intercrossed to generate Low-TF mice lacking PAR-4. Mice surviving to wean were genotyped and survival was monitored for 6months. RESULTS We observed the expected number of Low-TF,PAR-4-/- mice at wean indicating survival in utero and after birth. However, an absence of PAR-4 was associated with premature death of all Low-TF,PAR-4-/- mice in the 6month observational period. This compares with 40% death of the Low-TF,PAR-4+/+ mice (p=0.003). Low-TF,PAR-4+/- mice had an intermediate phenotype with 55% of the mice dying within 6months. The primary cause of mortality of Low-TF,PAR-4-/- mice was pulmonary hemorrhage. CONCLUSIONS Low-TF,PAR-4-/- mice survive into adulthood, but combining a deficiency of primary hemostasis (PAR-4 deficiency) with secondary hemostasis (low levels of TF) leads to premature death primarily due to pulmonary hemorrhage.

The factor Xa inhibitor rivaroxaban reduces cardiac dysfunction in a mouse model of myocardial infarction

INTRODUCTION Rivaroxaban selectively inhibits factor Xa (FXa), which plays a central role in blood coagulation. In addition, FXa activates protease-activated receptor-2 (PAR-2). We have shown that PAR-2-/- mice exhibit less cardiac dysfunction after cardiac injury. MATERIAL AND METHODS Wild-type (WT) and PAR-2-/- mice were subjected to left anterior descending artery (LAD) ligation to induce cardiac injury and heart failure. Mice received either placebo or rivaroxaban chow either starting at the time of surgery or 3 days after surgery and continued up to 28 days. Cardiac function was measured by echocardiography pre-surgery and 3, 7 and 28 days after LAD ligation. We also measured anticoagulation, intravascular thrombi, infarct size, cardiac hypertrophy and inflammation at various times. RESULTS Rivaroxaban increased the prothrombin time and inhibited the formation of intravascular thrombi in mice subjected to LAD ligation. WT mice receiving rivaroxaban immediately after surgery had similar infarct sizes at day 1 as controls but exhibited significantly less impairment of cardiac function at day 3 and beyond compared to the placebo group. Rivaroxaban also inhibited the expansion of the infarct at day 28. Rivaroxaban did not significantly affect the expression of inflammatory mediators or a neutrophil marker at day 2 after LAD ligation. Delaying the start of rivaroxaban administration until 3 days after surgery failed to preserve cardiac function. In addition, rivaroxaban did not reduce cardiac dysfunction in PAR-2-/- mice. CONCLUSIONS Early administration of rivaroxaban preserves cardiac function in mice after LAD ligation.

Integrating basic science in academic cardiology training: two international perspectives on a common challenge

Political bodies and professional societies acknowledge that translational research benefits from researchers trained in both, clinical medicine and basic science. Yet, few physicians undergoing clinical training in cardiology seek this dual career (Milewicz et al. J Clin Invest 125:3742–3747, 2015). The reasons are likely manifold, but with cardiology having become increasingly interventional and facing economic pressure, how much attention, credit, and encouragement is given to physicians interested in basic cardiovascular science? Having studied and worked in hospitals and laboratories, in both Germany and the USA, we aim to compare in this article how basic science education is currently integrated into cardiology training at German and US university hospitals, from medical school to more advanced career stages. By doing so, we hope to provide some outside perspectives to young physicians and decision makers alike, that may inspire changes to curricula in the respective countries and around the world.

Regulation of ankyrin repeat and suppressor of cytokine signalling box protein 4 expression in the immortalized murine endothelial cell lines MS1 and SVR: a role for tumour necrosis factor alpha and oxygen: REGULATION OF ASB4 EXPRESSION IN ENDOTHELIAL CELL LINES

During vascular development, endothelial cells are exposed to a variety of rapidly changing factors, including fluctuating oxygen levels. We have previously shown that ankyrin repeat and suppressor of cytokine signalling box protein 4 (ASB4) is the most highly differentially expressed gene in the vascular lineage during early differentiation and is expressed in the embryonic vasculature at a time when oxygen tension is rising because of the onset of placental blood flow. To further our understanding of the regulation of ASB4 expression in endothelial cells, we tested the effect of various stressors for their ability to alter ASB4 expression in the immortalized murine endothelial cell lines MS1 and SVR. ASB4 expression is decreased during hypoxic insult and shear stress, whereas it is increased in response to tumour necrosis factor alpha (TNF‐α). Further investigation indicated that nuclear factor kappa B (NF‐κB) is the responsible transcription factor involved in the TNF‐α‐induced upregulation of ASB4, placing ASB4 downstream of NF‐κB in the TNF‐α signalling cascade and identifying it as a potential regulator for TNF‐αs numerous functions associated with inflammation, angiogenesis and apoptosis. Copyright