Peter Leenders

Genetic and pharmacological modifications of thrombin formation in apolipoprotein e-deficient mice determine atherosclerosis severity and atherothrombosis onset in a neutrophil-dependent manner.

Background Variations in the blood coagulation activity, determined genetically or by medication, may alter atherosclerotic plaque progression, by influencing pleiotropic effects of coagulation proteases. Published experimental studies have yielded contradictory findings on the role of hypercoagulability in atherogenesis. We therefore sought to address this matter by extensively investigating the in vivo significance of genetic alterations and pharmacologic inhibition of thrombin formation for the onset and progression of atherosclerosis, and plaque phenotype determination. Methodology/Principal Findings We generated transgenic atherosclerosis-prone mice with diminished coagulant or hypercoagulable phenotype and employed two distinct models of atherosclerosis. Gene-targeted 50% reduction in prothrombin (FII−/WT:ApoE−/−) was remarkably effective in limiting disease compared to control ApoE−/− mice, associated with significant qualitative benefits, including diminished leukocyte infiltration, altered collagen and vascular smooth muscle cell content. Genetically-imposed hypercoagulability in TMPro/Pro:ApoE−/− mice resulted in severe atherosclerosis, plaque vulnerability and spontaneous atherothrombosis. Hypercoagulability was associated with a pronounced neutrophilia, neutrophil hyper-reactivity, markedly increased oxidative stress, neutrophil intraplaque infiltration and apoptosis. Administration of either the synthetic specific thrombin inhibitor Dabigatran etexilate, or recombinant activated protein C (APC), counteracted the pro-inflammatory and pro-atherogenic phenotype of pro-thrombotic TMPro/Pro:ApoE−/− mice. Conclusions/Significance We provide new evidence highlighting the importance of neutrophils in the coagulation-inflammation interplay during atherogenesis. Our findings reveal that thrombin-mediated proteolysis is an unexpectedly powerful determinant of atherosclerosis in multiple distinct settings. These studies suggest that selective anticoagulants employed to prevent thrombotic events may also be remarkably effective in clinically impeding the onset and progression of cardiovascular disease.

Activated Protein C Protects Against Myocardial Ischemia/ Reperfusion Injury via Inhibition of Apoptosis and Inflammation

OBJECTIVE In spite of major advances in reperfusion therapy for patients presenting with acute coronary syndrome, long-term morbidity is still substantial. A limitation of initial treatment of myocardial ischemia is the lack of prevention of ischemia/reperfusion (I/R) injury. Activated protein C (APC), a crucial mediator in the coagulation process, plays a prominent role in the crosstalk between coagulation and inflammation and provides cytoprotective effects via inhibition of apoptosis and inflammation in several human and animal studies. METHODS AND RESULTS APC was administered in an animal model for myocardial I/R. APC largely inhibited early myocardial I/R injury after varying reperfusion times, an effect that was absent on administration of heparin, a nonspecific anticoagulant agent. The protective effects of APC were absent in case of absence or blockade of protease activated receptor-1 (PAR-1), indicating a critical role for PAR-1 in this process. Furthermore, we showed a strong antiapoptotic effect of APC in the early phase of reperfusion combined with an antiinflammatory effect at an early stage (IL-6), as well as at a later stage (leukocyte infiltration). CONCLUSIONS APC exerts strong protective effects on early myocardial I/R injury, primarily via inhibition of apoptosis and inflammation, which are regulated via PAR-1.

In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy

In vivo (molecular) imaging of the vessel wall of large arteries at subcellular resolution is crucial for unraveling vascular pathophysiology. We previously showed the applicability of two-photon laser scanning microscopy (TPLSM) in mounted arteries ex vivo. However, in vivo TPLSM has thus far suffered from in-frame and between-frame motion artifacts due to arterial movement with cardiac and respiratory activity. Now, motion artifacts are suppressed by accelerated image acquisition triggered on cardiac and respiratory activity. In vivo TPLSM is performed on rat renal and mouse carotid arteries, both surgically exposed and labeled fluorescently (cell nuclei, elastin, and collagen). The use of short acquisition times consistently limit in-frame motion artifacts. Additionally, triggered imaging reduces between-frame artifacts. Indeed, structures in the vessel wall (cell nuclei, elastic laminae) can be imaged at subcellular resolution. In mechanically damaged carotid arteries, even the subendothelial collagen sheet (approximately 1 microm) is visualized using collagen-targeted quantum dots. We demonstrate stable in vivo imaging of large arteries at subcellular resolution using TPLSM triggered on cardiac and respiratory cycles. This creates great opportunities for studying (diseased) arteries in vivo or immediate validation of in vivo molecular imaging techniques such as magnetic resonance imaging (MRI), ultrasound, and positron emission tomography (PET).

Active site inhibited factor VIIa attenuates myocardial ischemia/reperfusion injury in mice

Summary.  Background: Inhibition of specific coagulation pathways such as the factor VIIa‐tissue factor complex has been shown to attenuate ischemia/reperfusion (I/R) injury, but the cellular mechanisms have not been explored. Objectives: To determine the cellular mechanisms involved in the working mechanism of active site inhibited factor VIIa (ASIS) in the protection against myocardial I/R injury. Methods: We investigated the effects of a specific mouse recombinant in a mouse model of myocardial I/R injury. One hour of ischemia was followed by 2, 6 or 24 h of reperfusion. Mouse ASIS or placebo was administered before and after induction of reperfusion. Results: ASIS administration reduced myocardial I/R injury by more than 40% at three reperfusion times. Multiplex ligation dependent probe amplification (MLPA) analysis showed reduced mRNA expression in the ischemic myocardium of CD14, TLR‐4, interleukin‐1 (IL‐1) receptor‐associated kinase (IRAK) and IκBα upon ASIS administration, indicative of inhibition of toll‐like receptor‐4 (TLR‐4) and subsequent nuclear factor‐κB (NF‐κB) mediated cell signaling. Levels of nuclear activated NF‐κB and proteins influenced by the NF‐κB pathway including tissue factor (TF) and IL‐6 that were increased after I/R, were attenuated upon ASIS administration. After 6 and 24 h of reperfusion, neutrophil infiltration into the area of infarction was decreased upon ASIS administration. There was, however, no evidence of an effect of ASIS on apoptosis (Tunel staining and MLPA analysis). Conclusions: We conclude that the diminished amount of myocardial I/R injury after ASIS administration is primarily due to attenuated inflammation‐related lethal I/R injury, probably mediated through the NF‐κB mechanism.

Endogenous hedgehog expression contributes to myocardial ischemia-reperfusion-induced injury.

The developmentally important hedgehog (Hh) pathway is activated in ischemic tissue, and exogenously administered Sonic hedgehog (Shh) supports tissue repair after cardiac ischemia. Hence, it is currently assumed that the endogenous increase in Shh during ischemia serves a beneficial role in limiting cardiac tissue damage. To prove or refute this hypothesis, we treated mice with the smoothened (Smo) inhibitor cyclopamine to block the Hh pathway during myocardial ischemia and reperfusion. The experimental induction of myocardial ischemia resulted in activation of the Hh pathway and hallmark features of myocardial damage, such as left ventricular dilatation and reduced cardiac output. Unexpectedly, cyclopamine treatment ameliorated left ventricular dilatation and cardiac output. As the beneficial effect of exogenous Shh was suggested to depend on reduced apoptosis, increased vascularization, and reduced fibrosis, we subsequently assessed the effect of cyclopamine on these processes. Vascularization was similar in cyclopamine-treated and control-treated animals, but increased apoptosis and reduced fibrosis were observed in the cyclopamine-treated animals. Thus, Hh seems to exert a dualistic action in cardiac ischemia in which high exogenous levels are able to foster tissue repair and endogenous Hh seems to be deleterious.

Peripheral Vascular Alterations During Experimental Heart Failure in the Rat Do They Exist

Structural changes of the peripheral vascular component as seen during hypertension and atherosclerosis have been suggested during heart failure but have never been reported. Therefore, we studied possible structural alterations in the peripheral vasculature in an experimental model of heart failure, induced by ligation of the left coronary artery in rats. Large conduit and resistance-type arteries were excised at 1, 3, 5, and 12 weeks after myocardial infarct induction (MI) or sham surgery. Vessel dimensions (medial cross-sectional area [CSA], internal and external diameters, and media-to-lumen ratios) as well as medial collagen and elastin volume fractions were measured by computerized morphometry. The hydroxyproline assay was used to determine collagen and elastin content biochemically. In separate groups of animals, peripheral tissue flows were measured by using radioactive microspheres 5 and 12 weeks after MI. To evaluate the effects of the degree of heart failure, the animals of the 12-week group (n = 10) were subdivided into groups of moderate (< 45% infarct size) and large (> 45% infarct size) infarction. At all time points, body weights of sham-operated and MI rats were comparable. Lung weights of infarcted animals were increased proportionally to infarct size. No major changes in vessel dimensions were seen at the earlier time points. Twelve weeks after coronary artery ligation, significantly smaller CSAs were observed in several large conduit arteries such as the thoracic aorta, carotid artery, and superior mesenteric artery. These changes coincided with reductions in both internal and external diameters. In contrast, internal and external diameters of mesenteric and pulmonary resistance arteries were increased after 12 weeks of coronary artery ligation.(ABSTRACT TRUNCATED AT 250 WORDS)

Monitoring platelet dependent thrombin generation in mice

Calibrated automated thrombin generation assay was adapted to measure thrombin generation in platelet rich plasma from mice. Vena cava phlebotomy appeared the best technique for blood sampling. The concentration-effect curves of tissue factor and platelet count have been determined. Corn trypsin inhibitor 2μM inhibits contact activation effectively. Recombinant human thrombomodulin does not inhibit thrombin generation in mouse plasma but activated protein C (20nM) does. Thrombin generation was dose dependently diminished by low molecular weight heparin and increased by high concentrations of exogenous factor VIII i.e. the assay can detect both hypo- and hypercoagulability.

Percutaneous implantation of a new intracoronary stent in pigs

Sixty-two self-expanding parallel wire stainless steel stents were implanted in normal coronary arteries of 31 young pigs using a newly developed delivery system. In 57 of 62 procedures, the percutaneous coronary implant of the stent was successful; five stents were released in side branches. Implants remained in place for a few hours to 6 months. In spite of correct sizing, two stents migrated out of the coronary arteries. Seven pigs died prematurely; in six of them death might be stent-related. Although no anticoagulant and antiplatelet aggregation drugs were administered during the follow-up period, at autopsy thrombi were observed in only seven arteries (nonobstructive in four of seven arteries). All arteries except for three were patent; these three vessels occluded probably due to oversizing of the stent. Complete neointimal coverage was found within 3 weeks. Important hyperplasia was not seen. It was concluded that coronary implantation of this stent usually was easy. Obstructive thrombus formation was rather uncommon despite the absence of chronic anticoagulant and antiplatelet aggregation therapy. Hyperplasia was rare.

Structural adaptation to ischemia in skeletal muscle : effects of blockers of the renin-angiotensin system

Objective To investigate the effects of long-term treatment with blockers of the renin–angiotensin system on capillarization and growth of fibers in ischemic hind-limb muscles and in muscles under normal growth conditions. Methods Ischemia was induced by partial ligation of the left common iliac artery. Results Ischemia resulted in a significant increase in capillary and fiber density in the soleus muscle, a significant decrease in mean fiber size and a decrease in muscle cross-sectional area after 4 weeks compared with the contralateral nonischemic muscle. Ischemia also significantly decreased the muscle body weight ratio of the left soleus muscle. We observed no significant effect on total number of capillaries and capillary: fiber ratio, suggesting that ischemia did not result in an increase in capillarization in this muscle. Treatments with subhypotensive and with hypotensive doses of the angiotensin converting enzyme (ACE) inhibitor benazeprilat, the angiotensin (Ang) II AT1 antagonist valsartan, or the Ang II AT2 antagonist PD 123 319 for 4 weeks did not influence any of the above-described changes in the normal and ischemic muscles and treatment effects were also independent of the degree of reduction of blood pressure. Conclusion Treatments with an ACE inhibitor and with Ang II receptor antagonists in dose ranges that moderately lower blood pressure do not influence vessel density and any of the other structural adaptations after hind-limb ischemia. Administrations of ACE inhibitors and Ang II AT1 antagonists may therefore be adequate and beneficial therapies under ischemic conditions, such as in the treatment of hypertension complicated by intermittent claudication, for which treatment must not increase ischemia.

Effects of angiotensin II on cardiac function and peripheral vascular structure during compensated heart failure in the rat

UNLABELLED The present experiments were designed to test the hypothesis that the activation of the renin-angiotensin system during compensated heart failure may have adverse effects on cardiac function and change the peripheral vascular structure. ANG II (250 ng/kg/min) or saline (0.9% NaCl) were infused in myocardial-infarcted and sham-operated rats. After 2 weeks, cardiac function and peripheral vascular changes were investigated. RESULTS ANG II infusion reduced baseline cardiac index in sham rats but did not further reduce this index in ANG II-infused MI rats. Total peripheral resistance was similarly increased in ANG II-infused infarcted and sham rats, and also plasma ANG II concentrations were comparable. ANG II elevated systolic blood pressure by approximately 70 mm Hg in sham rats and increased the medial cross-sectional area of the superior mesenteric artery by 33%. However, ANG II infusions in MI rats resulted in only a minor increase in blood pressure, whereas the cross-sectional area of the superior mesenteric artery did not change. ANG II infusion had no effect on vessel dimensions of the resistance arteries of the pulmonary and mesenteric vascular bed of either group. Calculated ED50 and peak pressor response to acute ANG II injections were comparable in all groups, confirming the presence of functionally intact AT1 receptors. The increases in plasma atrial natriuretic peptide (ANP) and nitric oxide (NO) synthase activity (estimated by aortic cyclic GMP concentrations) were higher in ANG II-infused MI rats than in ANG II-infused sham rats. CONCLUSION ANG II infusion in rats with and without MI has comparable negative effects on cardiac function but has different effects on blood pressure and vascular structure. The concomitant increases in plasma ANP and NO synthase activity in ANG II-infused MI rats suggest that the growth stimulatory and hypertensive actions of ANG II in sham rats may be counter-regulated by activation of inhibitory neurohumoral systems such as ANP or NO in MI rats.