Trapped in the Matrix: Neutrophil Extracellular Traps (NETs) and Fibrin in Wound Healing

The aim of this thesis was to investigate the effect of fibrin, NETs and the induction of NETosis, in wound healing. To achieve this, we created in vitro models to study the formation of NETs by several inducers, such as PMA, LPS, S. aureus, E. coli and N. meningitidis. Furthermore, we studied the role of NETs and extracellular DNA in sepsis and thrombosis. A diabetic rat model was used to study the effect of fibrin on wound healing

In vitro induction of NETosis: Comprehensive live imaging comparison and systematic review

__Background__ Multiple inducers of in vitro Neutrophil Extracellular Trap (NET) formation (NETosis) have been described. Since there is much variation in study design and results, our aim was to create a systematic review of NETosis inducers and perform a standardized in vitro study of NETosis inducers important in (cardiac) wound healing. __Methods__ In vitro NETosis was studied by incubating neutrophils with PMA, living and dead bacteria (S. aureus and E. coli), LPS, (activated) platelets (supernatant), glucose and calcium ionophore Ionomycin using 3-hour periods of time-lapse confocal imaging. __Results__ PMA is a consistent and potent inducer of NETosis. Ionomycin also consistently resulted in extrusion of DNA, albeit with a process that differs from the NETosis process induced by PMA. In our standardized experiments, living bacteria were also potent inducers of NETosis, but dead bacteria, LPS, (activated) platelets (supernatant) and glucose did not induce NETosis. __Conclusion__ Our systematic review confirms that there is much variation in study design and results of NETosis induction. Our experimental results confirm that under standardized conditions, PMA, living bacteria and Ionomycin all strongly induce NETosis, but real-time confocal imaging reveal different courses of events

Effects of diabetes mellitus on fibrin clot structure and mechanics in a model of acute neutrophil extracellular traps (Nets) formation

Subjects with diabetes mellitus (DM) have an increased risk of arterial thrombosis, to which changes in clot structure and mechanics may contribute. Another contributing factor might be an increased formation of neutrophil extracellular traps (NETs) in DM. NETs are mainly formed during the acute phase of disease and form a network within the fibrin matrix, thereby influencing clot properties. Previous research has shown separate effects of NETs and DM on clot properties, therefore our aim was to study how DM affects clot properties in a model resembling an acute phase of disease with NETs formation. Clots were prepared from citrated plasma from subjects with and without DM with the addition of NETs, induced in neutrophils by S. aureus bacteria or phorbol myristate acetate (PMA). Structural parameters were measured using scanning electron microscopy, mechanical properties using rheology, and sensitivity to lysis using a fluorescence-based fibrinolysis assay. Plasma clots from subjects with DM had significantly thicker fibers and fewer pores and branch points than clots from subjects without DM. In addition, fibrinolysis was significantly slower, while mechanical properties were similar between both groups. In conclusion, in a model of acute NETs formation, DM plasma shows prothrombotic effects on fibrin clots.BN/Gijsje Koenderink La

Evolution and Development of Ventricular Septation in the Amniote Heart

<div><p>During cardiogenesis the epicardium, covering the surface of the myocardial tube, has been ascribed several functions essential for normal heart development of vertebrates from lampreys to mammals. We investigated a novel function of the epicardium in ventricular development in species with partial and complete septation. These species include reptiles, birds and mammals. Adult turtles, lizards and snakes have a complex ventricle with three cava, partially separated by the horizontal and vertical septa. The crocodilians, birds and mammals with origins some 100 million years apart, however, have a left and right ventricle that are completely separated, being a clear example of convergent evolution. In specific embryonic stages these species show similarities in development, prompting us to investigate the mechanisms underlying epicardial involvement. The primitive ventricle of early embryos becomes septated by folding and fusion of the anterior ventricular wall, trapping epicardium in its core. This folding septum develops as the horizontal septum in reptiles and the anterior part of the interventricular septum in the other taxa. The mechanism of folding is confirmed using DiI tattoos of the ventricular surface. Trapping of epicardium-derived cells is studied by transplanting embryonic quail pro-epicardial organ into chicken hosts. The effect of decreased epicardium involvement is studied in knock-out mice, and pro-epicardium ablated chicken, resulting in diminished and even absent septum formation. Proper folding followed by diminished ventricular fusion may explain the deep interventricular cleft observed in elephants. The vertical septum, although indistinct in most reptiles except in crocodilians and pythonidsis apparently homologous to the inlet septum. Eventually the various septal components merge to form the completely septated heart. In our attempt to discover homologies between the various septum components we aim to elucidate the evolution and development of this part of the vertebrate heart as well as understand the etiology of septal defects in human congenital heart malformations.</p></div

Septum formation in the mouse.

<p>(<b>A, B</b>) Almost transverse sections of the same embryo showing WT1+ epicardial cells in the folding septum (FS,→) at ED 10.5, Fig. B is more apically located. (<b>C, D</b>) epicardial cells in FS of wildtype mouse at ED 12.5 and (<b>E</b>) present in the inlet septum underneath the posterior AV cushion. Note: WT1 staining of mesenchyme in septal OFT cushion is unrelated to epicardial cells. (<b>F-H</b>) Podoplanin mutant with diminutive PEO, presents with sparse epicardium lining the pericardial cavity (<b>F, G</b>) and with an underdeveloped septum lacking EPDCs in both FS (<b>G</b>) and inlet septum (IS) (<b>H</b>). (<b>I-L</b>) Immunostained for Tbx5 in a wild type mouse ED 14.5, four levels from anterior-posterior. (<b>I</b>) Tbx5 in LV trabeculations but not in the RV close to the outflow tract; core of septum is negative. (<b>J-L</b>) More posteriorly located sections, trabeculations in RV belonging to the inlet part become positive for Tbx5. (<b>M-P</b>) Four positions of a 3D Amira reconstruction of ED 10.5. The epicardial cushion in pink (*), the folding septum in dark blue and the inlet septum in light blue. Endocardial cushions in green and the AVC myocardium in yellow. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106569#pone.0106569.s004" target="_blank">Fig. S3</a> for animated 3D. Abbrev. AVC atrioventricular cushions; FS folding septum; IS inlet septum; LV left ventricle; M mitral orifice; RV right ventricle; OFT outflow tract cushion; T tricuspid orifice, • interventricular communication, + septal band.</p

Development of chicken septum.

<p>(<b>A</b>) epicardium (→) infolding, located between OFT and AVcanal. (<b>B</b>) In situ hybridisation showing weakly positive Tbx5 of the RV and negative OFT with boundary (arrow). The atria are strongly positive. (<b>C</b>) more posterior section of the same embryo through folding septum (FS), the stronger left sided expression is evident, as is the septal band (+); boundary (> <) indicates FS. (<b>D-G</b>) 3D reconstruction with septum components and epicardial cushion. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106569#pone.0106569.s003" target="_blank">figure S2</a> for full animation and Fig. 6 for underlying sections, explaining the various components. (<b>H-L</b>) PEO quail-chicken chimeras. (<b>H, I</b>) anterior quail PEO(+liver) transplant, quail endothelial cells are exclusively present in FS and anterior free wall (<b>J-L</b>) posterior PEO (+liver) transplant with quail vascular profiles in IS (<b>J, K</b>) and right face of tricuspid orifice (<b>L</b>), but not in FS. (<b>K</b>) Several quail cells (arrows) in septal band (+), but FS does not harbor quail cells and remains negative (<b>K, L</b>). (<b>M-P</b>) DiI marking at HH17 of anterior myocardium surviving until HH28 and 31. (<b>M</b>) parts of the DiI patch (arrow) after survival to HH28 on left, (<b>N</b>) DiI on the right face and (<b>O</b>) DiI near the apex. (<b>P</b>) DiI inside the septum at HH31. Abbrev. as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106569#pone-0106569-g002" target="_blank">Fig 2</a>. Others: AVC atrioventricular cushions; LA/LV left atrium and ventricle; RA/RV right atrium and ventricle; + septal band.</p

Chicken embryo HH27.

<p>Provides 6 sections of a serially sectioned chicken embryo (HH27) to demonstrate the merging of the folding and inlet components before septation is finished. From this embryo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106569#pone-0106569-g003" target="_blank">Fig 3D</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106569#pone.0106569.s003" target="_blank">Fig. S2</a> have been reconstructed. Similar series served as basis for the other species depicted in Fig S1, S3 and S4. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106569#pone-0106569-g006" target="_blank"><b>Fig 6</b></a><b>.A</b> is most cranial, showing the epicardial cushion (*) at a level between outflow tract (OFT) and the right (RA) and left atria (LA) with the AV cushions in between. The cranial cap of the left ventricle (LV) is grazed in the section. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106569#pone-0106569-g006" target="_blank"><b>Fig 6</b></a><b>.B</b> and <b>C</b> give the cranial extension of the folding septum (FS) with the epicardial cushion (*) located between the outflow tract and the fused AV cushions (AVC). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106569#pone-0106569-g006" target="_blank"><b>Fig 6</b></a><b>.D</b> shows the FS bordering the interventricular foramen. It is evident that the core of the folding septum is lined on the left and right side by many trabeculations. <b>Fig. 6.E</b> The AV cushions are attached to the flanks of the inlet septum where also the tip of the septal OFT cushion is found (arrow). The inlet (IS) and folding components have fused and constitute the floor of the interventricular foramen. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106569#pone-0106569-g006" target="_blank"><b>Fig 6</b></a><b>.E, F</b> The right AV junction is present in the RV immediately above the arrow and can be traced upstream in Fig F and downstream in Fig. D. Note the close relationship to the IS. The folding septum becomes less compact and the trabeculations become more conspicuous. Abbrev. AVC atrioventricular cushions; FS folding septum; IS inlet septum; LA left atrium; LV left ventricle; RA right atrium; RV right ventricle; OFT outflow tract cushion with its proximal tip indicated by arrow in Fig. F.</p

Reptile cardiac development.

<p>(<b>A, B</b>) The epicardial cushion (*) is located between OFT and AV cushions. (<b>C, D</b>) cardiac troponin I (cardiac muscle) and RALDH2 (epicardial cells) stainings show folding septum (arrow, asterisk). (<b>E</b>) 3D reconstruction in an anterior view, the epicardial patches are depicted in pink. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106569#pone.0106569.s002" target="_blank">Figure S1</a> 1 for full animation. (<b>F</b>) right sided view of the septum, folding (FS) and inlet (IS) septum are depicted in shades of blue. For further colors see legend to Fig. 5E. (<b>G</b>) Scanning electron microscopy of anterior inner face, note communication between the three cava. The folding (syn. horizontal) septum is out of view. (<b>H</b>) A sharp decline of Tbx5 mRNA expression (arrow) between cavum dorsale and OFT. (<b>I</b>) Sharp boundary at muscular OFT (inside of dotted line) and wall of cavum pulmonale (outside dotted line), but the tip of trabeculations in the cavum dorsale stain strongly (arrows). (<b>J</b>) Section downstream of Fig C, showing sharp decline of Tbx5 protein expression at folding septum. (<b>K</b>) Section more to the apex of J, showing uniform immunostaining for Tbx5. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106569#pone-0106569-g001" target="_blank">Fig 1</a>, others: AVC: AV cushions; ca, cp, cv: cavum arteriosum, pulmonale and venosum; L left AV orifice; OFT outflow tract cushions; R right AV orifice; →: infolding; * epicardium and EPDCs; • position of cavum venosum in 3D reconstruction of Fig F.</p

Evolution and septation of the heart.

<p>A. Evolution of hearts in higher vertebrates. Archosaurs (crocodilians, birds) and mammals independently evolved complete ventricular septation. Birds and mammals have lost either a left (lAo) or right (rAo) aorta. The horizontal (hs) and vertical septum (vs) are schematically indicated, together with the pulmonary trunk (Pt). The evolutionary tree is based on ref (2). B. Septum components in the human heart. Right face of the septum in a human heart after opening the right ventricle (RV), with inlet and folding components. Dissection line of the RV free wall in pink. Abbreviations: FS folding septum, IS inlet septum; MB moderator band; Pu pulmonary semilunar valve leaflets; SB septal band; TV anterior tricuspid valve leaflet with chordae tendineae (arrows) connected to SB and IS.; VIF ventriculo-infundibular fold. Fig. courtesy dr. L. Houyel.</p

Index for the terminology used.

<p>The numbers refer to the ^superscripts in the Table.</p>1<p>. Combinations of aortic sac, truncus, conus and bulbus have been used to describe this segment. Conus and bulbus are usually myocardial, whereas aortic sac and truncus refer mostly to the vascular part. Septation from the vascular, semilunar valve and intracardiac levels is interchangeably referred to as either aorto-pulmonary septum or outflow tract (OFT) septum. The endocardial cushions in the proximal intracardiac part myocardialize through induction by neural crest cells forming the aorto-pulmonary or OFT septum. The distal part of the cushions is remodelled into semilunar valves that are separated by fibrous tissue between the orifices of the great arteries. In reptiles the aorto-pulmonary septum is branched and separates the two aortae and the pulmonary trunk. In mammals the distinction between proximal and distal endocardial cushions is inconspicuous.</p>2.<p>Bulboventricular fold, synonymous with the primary fold, between outflow and inlet portion of the primitive ventricle.</p>3.<p>Anterior (positional), primary (time-related) and folding (mechanistic, new in this paper) septum are synonymously used. The bulboventricular fold extends apically over the anterior surface of the heart and deepens to enclose epicardium and subepicardial tissue, thus forming an anteriorly located folding septum. The folding septum is considered to be homologous to the reptilian horizontal septum, which is also called the muscular ridge (see for further synonyms ref 13).</p>4.<p>The apical trabecular septum develops from the coalescence of many trabeculations and does not show a clear demarcation with the folding septum or the inlet septum.</p>5.<p>The inlet septum in early stages of eventually completely septated hearts and in some reptiles (presence is species-dependent) is a dense muscular structure on the posterior wall of the ventricle without an infolding mechanism. In the current study we have clearly shown that the anterior margin of the inlet septum with the folding septum is formed by the septal band or trabecula septomarginalis. In earlier literature the septal band has been described as the posterior margin of the primary (or folding) septum.</p>6.<p>The superior and inferior atrioventricular endocardial cushions fuse in the midline. In the central part the cushions are remodelled into fibrous (membranous) tissue that becomes part of the fibrous heart skeleton. Part of this forms the membranous septum which is located between right atrium and outflow of the left ventricle (atrio-ventricular component) and the remainder between RV and LV (interventricular component).This tissue is obliquely embedded in both the atrial and ventricular septa and as such is sometimes referred to as atrioventricular septum.</p><p>Index for the terminology used.</p