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Myocardial infarction ( MI) induces a complex inflammatory immune response, followed by the remodelling of the heart muscle and scar formation. The rapid regeneration of the blood vessel network system by the attraction of hematopoietic stem cells is beneficial for heart function. Despite the important role of chemokines in these processes, their use in clinical practice has so far been limited by their limited availability over a long time-span in vivo. Here, a method is presented to increase physiological availability of chemokines at the site of injury over a defined time-span and simultaneously control their release using biodegradable hydrogels. Two different biodegradable hydrogels were implemented, a fast degradable hydrogel ( FDH) for delivering Met- CCL5 over 24 hrs and a slow degradable hydrogel ( SDH) for a gradual release of protease-resistant CXCL12 (S4V) over 4 weeks. We demonstrate that the time-controlled release using Met- CCL5- FDH and CXCL12 (S4V)- SDH suppressed initial neutrophil infiltration, promoted neovascularization and reduced apoptosis in the infarcted myocardium. Thus, we were able to significantly preserve the cardiac function after MI. This study demonstrates that time-controlled, biopolymer-mediated delivery of chemokines represents a novel and feasible strategy to support the endogenous reparatory mechanisms after MI and may compliment cell-based therapies. [ABSTRACT FROM AUTHOR]

Abstract: Tissue factor pathway inhibitor (TFPI) is the main inhibitor of tissue factor (TF)-mediated coagulation. In atherosclerotic plaques TFPI co-localizes with TF, where it is believed to play an important role in attenuating TF activity. Findings in animal models such as TFPI knockout models and gene transfer models are consistent on the role of TFPI in arterial thrombosis as they reveal an active role for TFPI in attenuating arterial thrombus formation. In addition, ample experimental evidence exists indicating that TFPI has inhibitory effects on both smooth muscle cell migration and proliferation, both which are recognized as important pathological features in atherosclerosis development. Nonetheless, the clinical relevance of these antithrombotic and atheroprotective effects remains unclear. Paradoxically, the majority of clinical studies find increased instead of decreased TFPI antigen and activity levels in atherothrombotic disease, particularly in atherosclerosis and coronary artery disease (CAD). Increased TFPI levels in cardiovascular disease might result from complex interactions with established cardiovascular risk factors, such as hypercholesterolemia, diabetes and smoking. Moreover, it is postulated that increased TFPI levels reflect either the amount of endothelial perturbation and platelet activation, or a compensatory mechanism for the increased procoagulant state observed in cardiovascular disease. In all, the prognostic value of plasma TFPI in cardiovascular disease remains to be established. The current review focuses on TFPI in clinical studies of asymptomatic and symptomatic atherosclerosis, coronary artery disease and ischemic stroke, and discusses potential atheroprotective actions of TFPI. [Copyright &y& Elsevier]

Abstract: Vitamin K-antagonists (VKA) are the most widely used anti-thrombotic drugs with substantial efficacy in reducing risk of arterial and venous thrombosis. Several lines of evidence indicate, however, that VKA inhibit not only post-translational activation of vitamin K-dependent coagulation factors but also synthesis of functional extra-hepatic vitamin K-dependent proteins thereby eliciting undesired side-effects. Vascular calcification is one of the recently revealed side-effects of VKA. Vascular calcification is an actively regulated process involving vascular cells and a number of vitamin K-dependent proteins. Mechanistic understanding of vascular calcification is essential to improve VKA-based treatments of both thrombotic disorders and atherosclerosis. This review addresses vitamin K-cycle and vitamin K-dependent processes of vascular calcification that are affected by VKA. We conclude that there is a growing need for better understanding of the effects of anticoagulants on vascular calcification and atherosclerosis. [Copyright &y& Elsevier]

Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where, together with fibrin and platelets, the bacteria initiate the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin could be used to detect S. aureus in endocarditic vegetations via noninvasive fluorescence or positron emission tomography (PET) imaging. These prothrombin derivatives bound staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor. [ABSTRACT FROM AUTHOR]

The article discusses a study on the use of gadolinium-labeled quantum dots (QDs) as a contrast agent for molecular magnetic resonance imaging (MRI). The study used in vivo longitudinal and transverse rate relaxation rate mapping to compare longitudinal and transverse relaxation rate-based contrast improvement, proton visibility and changes induced by QDs targeted to the angiogenic vasculature of tumors in mice. Under the study, cyclic tripeptide asparagine-glycine-arginine (cNGR) was used to label paramagnetic gadolinium chelates (pQDs).

Background: Endothelin-1 (ET-1), a long-acting paracrine mediator, is implicated in cardiovascular diseases but clinical trials with ET-receptor antagonists were not successful in some areas. We tested whether the quasi-irreversible receptor-binding of ET-1 (i) limits reversing effects of the antagonists and (ii) can be selectively dissociated by an endogenous counterbalancing mechanism. Methodology/Principal findings: In isolated rat mesenteric resistance arteries, ETA-antagonists, endothelium-derived relaxing factors and synthetic vasodilators transiently reduced contractile effects of ET-1 but did not prevent persistent effects of the peptide. Stimuli of peri-vascular vasodilator sensory-motor nerves such as capsaicin not only reduced but also terminated long-lasting effects of ET-1. This was prevented by CGRP-receptor antagonists and was mimicked by exogenous calcitonin gene-related peptide (CGRP). Using 2-photon laser scanning microscopy in vital intact arteries, capsaicin and CGRP, but not ETA-antagonism, were observed to promote dissociation of pre-existing ET-1/ETA-receptor complexes. Conclusions: Irreversible binding and activation of ETA-receptors by ET-1 (i) occur at an antagonist-insensitive site of the receptor and (ii) are selectively terminated by endogenously released CGRP. Hence, natural stimuli of sensory-motor nerves that stimulate release of endogenous CGRP can be considered for therapy of diseases involving ET-1. [ABSTRACT FROM AUTHOR]

Objectives: We aimed to investigate whether early thrombus formation can be visualized with in vivo magnetic resonance imaging (MRI) by the use of a novel bimodal α2-antiplasmin–based contrast agent (CA). Background: Thrombus formation plays a central role in several vascular diseases. During the early phases of thrombus formation, activated factor XIII (FXIIIa) covalently cross-links α2-antiplasmin to fibrin, indicating the potential of α2-antiplasmin–based CAs in the detection of early thrombus formation. Methods: A bimodal CA was synthesized by coupling gadolinium-diethylene triamine pentaacetic acid and rhodamine to an α2-antiplasmin–based peptide. For the control CA, a glutamine residue essential for cross-linking was replaced by alanine. In vitro-generated thrombi were exposed to both CAs and imaged by MRI and 2-photon laser-scanning microscopy. Immunohistochemistry was performed on human pulmonary thromboemboli sections to determine the presence of α2-antiplasmin and FXIII in different thrombus remodeling phases. In vivo feasibility of the CA in detecting early thrombus formation specifically was investigated with MRI. Results: In vitro–generated thrombi exposed to the α2-antiplasmin–based CA showed hyperintense magnetic resonance signal intensities at the thrombus edge. No hyperintense signal was observed when we used the α2-antiplasmin–based CA in the presence of FXIII inhibitor dansylcadaverine nor when we used the control CA. Two-photon laser-scanning microscopy demonstrated that the α2-antiplasmin–based CA bound to fibrin. Immunohistochemistry demonstrated substantial α2-antiplasmin staining in fresh compared with lytic and organized thrombi. The administration of CA in vivo within seconds after inducing thrombus formation increased contrast-to-noise ratios (CNRs 2.28 ± 0.39, n=6) at the site of thrombus formation compared with the control CA (CNRs −0.14 ± 0.55, p = 0.003, n = 6) and α2-antiplasmin–based CA administration 24 to 48 h after thrombus formation (CNRs 0.11 ± 0.23, p = 0.006, n = 6). Conclusions: A bimodal CA was developed, characterized, and validated. Our results showed that this bimodal CA enabled noninvasive in vivo magnetic resonance visualization of early thrombus formation. [Copyright &y& Elsevier]

The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26–34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1. [ABSTRACT FROM AUTHOR]

Platelet factor 4 (CXCL4) is a chemokine abundantly stored in platelets. Upon injury and during atherosclerosis, CXCL4 is transported through the vessel wall where it modulates the function of vascular smooth muscle cells (VSMCs) by affecting proliferation, migration, gene expression and cytokine release. Variant CXCL4L1 is distinct from CXCL4 in function and expression pattern, despite a minor three-amino acid difference. Here, the effects of CXCL4 and CXCL4L1 on the phenotype and function of human VSMCs were compared in vitro. VSMCs were found to constitutively express CXCL4L1 and only exogenously added CXCL4 was internalized by VSMCs. Pre-treatment with heparin completely blocked CXCL4 uptake. A role of the putative CXCL4 receptors CXCR3 and DARC in endocytosis was excluded, but LDL receptor family members appeared to be involved in the uptake of CXCL4. Incubation of VSMCs with both CXCL4 and CXCL4L1 resulted in decreased expression of contractile marker genes and increased mRNA levels of KLF4 and NLRP3 transcription factors, yet only CXCL4 stimulated proliferation and calcification of VSMCs. In conclusion, CXCL4 and CXCL4L1 both modulate gene expression, yet only CXCL4 increases the division rate and formation of calcium-phosphate crystals in VSMCs. CXCL4 and CXCL4L1 may play distinct roles during vascular remodeling in which CXCL4 induces proliferation and calcification while endogenously expressed CXCL4L1 governs cellular homeostasis. The latter notion remains a subject for future investigation. [ABSTRACT FROM AUTHOR]

Atherosclerosis is characterized by chronic inflammation of the arterial wall due to chemokine-driven mononuclear cell recruitment. Activated platelets can synergize with chemokines to exacerbate atherogenesis; for example, by deposition of the chemokines platelet factor-4 (PF4, also known as CXCL4) and RANTES (CCL5), triggering monocyte arrest on inflamed endothelium. Homo-oligomerization is required for the recruitment functions of CCL5, and chemokine heteromerization has more recently emerged as an additional regulatory mechanism, as evidenced by a mutual modulation of CXCL8 and CXCL4 activities and by enhanced monocyte arrest resulting from CCL5-CXCL4 interactions. The CCL5 antagonist Met-RANTES reduces diet-induced atherosclerosis; however, CCL5 antagonism may not be therapeutically feasible, as suggested by studies using Ccl5-deficient mice which imply that direct CCL5 blockade would severely compromise systemic immune responses, delay macrophage-mediated viral clearance and impair normal T cell functions. Here we determined structural features of CCL5-CXCL4 heteromers and designed stable peptide inhibitors that specifically disrupt proinflammatory CCL5-CXCL4 interactions, thereby attenuating monocyte recruitment and reducing atherosclerosis without the aforementioned side effects. These results establish the in vivo relevance of chemokine heteromers and show the potential of targeting heteromer formation to achieve therapeutic effects. [ABSTRACT FROM AUTHOR]

Objective: Matrix γ-carboxyglutamate (Gla) protein (MGP) is a vitamin K-dependent protein and a strong inhibitor of vascular calcification. Vitamin K deficiency leads to inactive uncarboxylated MGP (ucMGP), which accumulates at sites of arterial calcification. We hypothesized that as a result of ucMGP deposition around arterial calcification, the circulating fraction of ucMGP is decreased. Here we report on the development of an ucMGP assay and the potential diagnostic utility of monitoring serum ucMGP levels. Methods and Results: An ELISA-based assay was developed with which circulating ucMGP can be determined. Serum ucMGP levels were measured in healthy subjects (n = 165) and in four patient populations; patients who underwent angioplasty (n = 30), patients with aortic stenosis (n = 25), hemodialysis patients (n = 52), and calciphylaxis patients (n = 10). All four patient populations had significantly lower ucMGP levels. In angioplasty patients and in those with aortic stenosis, some overlap was observed with the control population. However, in the hemodialysis and calciphylaxis populations, virtually all subjects had ucMGP levels below the normal adult range. Conclusion: Serum ucMGP may be used as a biomarker to identify those at risk for developing vascular calcification. This assay may become an important tool in the diagnosis of cardiovascular calcification. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Tissue factor (TF) plays an important role in hemostasis, inflammation, angiogenesis, and the pathophysiology of atherosclerosis and cancer. In this article we uncover a mechanism in which protein S. which is well known as the cofactor of activated protein C, specifically inhibits TF activity by promoting the interaction between full-length TF pathway inhibitor (TFPI) and factor Xa (FXa). The stimulatory effect of protein S on FXa inhibition by TFPI is caused by a 10-fold reduction of the Ki of the FXa/TFPI complex, which decreased from 4.4 nM in the absence of protein S to 0.5 nM in the presence of protein S. This decrease in K, not only results in an acceleration of the feedback inhibition of the TF-mediated coagulation pathway, but it also brings the TFPI concentration necessary for effective FXa inhibition well within range of the concentration of TFPI in plasma. This mechanism changes the concept of regulation of TF-induced thrombin formation in plasma and demonstrates that protein S and TFPI act in concert in the inhibition of TF activity. Our data suggest that protein S deficiency not only increases the risk of thrombosis by impairing the protein C system but also by reducing the ability of TFPI to down-regulate the extrinsic coagulation pathway. [ABSTRACT FROM AUTHOR]

Human matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein that binds Ca2+ ions and that is involved in the prevention of vascular calcification. MGP is a 10.6-kD protein (84 amino acids) containing five γ-carboxyglutamic acid (Gla) residues and one disulfide bond. Studies of the mechanism by which MGP prevents calcification of the arterial media are hampered by the low solubility of the protein (<10 μg/mL). Because of solubility problems, processing of a recombinantly expressed MGP-fusion protein chimera to obtain MGP was unsuccessful. Here we describe the total chemical synthesis of MGP by tBoc solid-phase peptide synthesis (SPPS) and native chemical ligation. Peptide Tyr1-Ala53 was synthesized on a derivatized resin yielding a C-terminal thioester group. Peptide Cys54-Lys84 was synthesized on Lys-PAM resin yielding a C-terminal carboxylic acid. Subsequent native chemical ligation of the two peptides resulted in the formation of a native peptide bond between Ala53 and Cys54. Folding of the 1-84-polypeptide chain in 3 M guanidine (pH 8) resulted in a decrease of molecular mass from 10,605 to 10,603 (ESI-MS), representing the loss of two protons because of the formation of the Cys54-Cys60 internal disulfide bond. Like native MGP, synthetic MGP had the same low solubility when brought into aqueous buffer solutions with physiological salt concentrations, confirming its native like structure. However, the solubility of MGP markedly increased in borate buffer at pH 7.4 in the absence of sodium chloride. Ca2+-binding to MGP was confirmed by analytical HPLC, on which the retention time of MGP was reduced in the presence of CaCl2. Circular dichroism studies revealed a sharp increase in α-helicity at 0.2 mM CaCl2 that may explain the Ca2+-dependent shift in high-pressure liquid chromatography (HPLC)-retention time of MGP. In conclusion, facile and efficient chemical synthesis in combination with native chemical ligation yielded MGP preparations that can aid in unraveling the mechanism by which MGP prevents vascular calcification. [ABSTRACT FROM AUTHOR]

The use of extracorporeal life support (ECLS) devices has significantly increased in the last decades. Despite medical and technological advancements, a main challenge in the ECLS field remains the complex interaction between the human body, blood, and artificial materials. Indeed, blood exposure to artificial surfaces generates an unbalanced activation of the coagulation cascade, leading to hemorrhagic and thrombotic events. Over time, several anticoagulation and coatings methods have been introduced to address this problem. This narrative review summarizes trends, advantages, and disadvantages of anticoagulation and coating methods used in the ECLS field. Evidence was collected through a PubMed search and reference scanning. A group of experts was convened to openly discuss the retrieved references. Clinical practice in ECLS is still based on the large use of unfractionated heparin and, as an alternative in case of contraindications, nafamostat mesilate, bivalirudin, and argatroban. Other anticoagulation methods are under investigation, but none is about to enter the clinical routine. From an engineering point of view, material modifications have focused on commercially available biomimetic and biopassive surfaces and on the development of endothelialized surfaces. Biocompatible and bio-hybrid materials not requiring combined systemic anticoagulation should be the future goal, but intense efforts are still required to fulfill this purpose. [ABSTRACT FROM AUTHOR]

The integrin αIIbβ3 is the most abundant integrin on platelets. Upon platelet activation, the integrin changes its conformation (inside-out signalling) and outside-in signalling takes place leading to platelet spreading, platelet aggregation and thrombus formation. Bloodsucking parasites such as mosquitoes, leeches and ticks express anticoagulant and antiplatelet proteins, which represent major sources of lead compounds for the development of useful therapeutic agents for the treatment of haemostatic disorders or cardiovascular diseases. In addition to hematophagous parasites, snakes also possess anticoagulant and antiplatelet proteins in their salivary glands. Two snake venom proteins have been developed into two antiplatelet drugs that are currently used in the clinic. The group of proteins discussed in this review are disintegrins, low molecular weight integrin-binding cysteine-rich proteins, found in snakes, ticks, leeches, worms and horseflies. Finally, we highlight various oral antagonists, which have been tested in clinical trials but were discontinued due to an increase in mortality. No new αIIbβ3 inhibitors are developed since the approval of current platelet antagonists, and structure-function analysis of exogenous disintegrins could help find platelet antagonists with fewer adverse side effects. [ABSTRACT FROM AUTHOR]