VEGFR2 blockade reduces intraplaque haemorrhage and enhances plaque stability by augmentation of plaque neovessel maturation

<p>Immature plaque neovessels contribute to atherosclerotic plaque instability and intraplaque haemorrhage by leaking erythrocytes and leukocytes in the plaque. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), together with the angiopoietin-Tie2 system, regulates the maturation of  neovessels..</p> <p>We have previously shown that murine vein graft lesions exhibit massive plaque neovascularization and that leaky vessels and intraplaque haemorrhage contribute to lesion growth</p

TGF<i>β</i>1 experiments.

<p>(A) The sprouting percentage (red curve), the angiogenesis level (blue curve), and the fibrinolysis percentage (green curve), are plotted against changes in the initial concentration of fibrin-bound latent-TGF<i>β</i>1 (relative units). The sprouting percentage is the percentage of simulations (out of a 100 simulations) that have an angiogenesis level larger than zero. The angiogenesis level is a measure that simultaneously reflects sprout depth and sprout count, and the mean angiogenesis level is taken over all simulations that actually formed sprouts. The fibrinolysis percentage is the percentage of the initial fibrin lattice sites that are invaded by the endothelial cells at MCS 6000. (B) Addition of active TGF<i>β</i>1 has a biphasic effect on sprout formation in our model. The sprouting frequency increases for the addition of low doses of TGF<i>β</i>1, but global degradation of the complete endothelial cell monolayer prevents sprout formation at high doses of TGF<i>β</i>1. Error bars are the standard deviation of 100 runs.</p

Model validation experiments.

<p>The sprouting percentage (red curve), the angiogenesis level (blue curve), and the fibrinolysis percentage (green curve), are plotted against changes in (A) the initial concentration of fibrin-bound plasminogen (relative units), (B) the decay rate of uPAR (MCS⁻¹), and (C) the decay rate of PAI-1 (MCS⁻¹). The sprouting percentage is the percentage of simulations (out of a 100 simulations) that have an angiogenesis level larger than zero. The angiogenesis level is a measure that simultaneously reflects sprout depth and sprout count, and the mean angiogenesis level is calculated over all simulations that actually formed sprouts. The fibrinolysis percentage is the percentage of the initial fibrin lattice sites that are invaded by the endothelial cells at MCS 6000. (D) Blocking PAI-1 activity increased endothelial sprouting in 3D fibrin matrices in a biphasic manner. hMVECs were seeded confluently on top of 3D fibrin matrices. Subsequently, the hMVECs were stimulated with the combination of FGF-2/TNF<i>α</i> (bT) with or without 100 U/ml trasylol, 25 ug/ml anti-uPAR antibody H2, control mIgG or anti-PAI-1 antibody MAI-2 (n = 4 independent donors, each in triplicate). 7 days after seeding and stimulation with FGF-2/TNF<i>α</i>, tube length was quantified by using Optimas software and expressed as mm/cm² with error bars expressing standard error of the mean. For statistical analysis a one-way ANOVA with Bonferroni post-hoc test was used. * indicates P < 0.05. Error bars of panels A-C are the standard deviation of 100 runs.</p

Additional file 1: Figure S1. of TGFβ1-induced SMAD2/3 and SMAD1/5 phosphorylation are both ALK5-kinase-dependent in primary chondrocytes and mediated by TAK1 kinase activity

Expression of ALK1, ALK2, ALK3 and ALK5 mRNA in primary bovine cartilage and chondrocytes. a With the use of qPCR, expression of ALK1, ALK2, ALK3 and ALK5 was measured in both freshly isolated cartilage explants and in primary chondrocytes after 1 week of cell culture in DMEM/F12 supplemented with 10% non-heat-inactivated FCS without passage. All four ALKs were readily detected in both groups, but expression of all the receptors was higher in freshly isolated tissue. For calculations of the -ΔCt, two reference genes were used: bGapdh and bRps14. (PDF 2065 kb

Schematic overview of plasmin and TGF<i>β</i>1 interactions.

<p>Plasminogen (PLG) reversibly binds fibrin, forming fibrin-bound plasminogen (<i>F</i>_PLG). <i>F</i>_PLG is converted by cell-bound uPAR (arrow 1) to fibrin-bound plasmin (<i>F</i>_PLS). <i>F</i>_PLS degrades fibrin. Latent-TGF<i>β</i>1 (LTGF) binds fibrin reversibly. Fibrin-bound latent-TGF<i>β</i>1 (<i>F</i>_LTGF) is activated and released by <i>F</i>_PLS (arrow 2), resulting in active, diffusive TGF<i>β</i>1 and free fibrin. Active TGF<i>β</i>1 induces production of uPAR (arrow 3). Cells secrete (<i>s</i>) PAI-1 (PAI), which inhibits uPAR activity (arrow 4). The gray, dotted lines indicate diffusion of proteins and curved, gray lines indicate decay.</p

CPM parameters.

<p>CPM parameters.</p

Overview of the binding and conversion reactions of plasminogen and latent-TGF<i>β</i>1 in relation to fibrin.

<p>Plasminogen (PLG) and latent-TGF<i>β</i>1 (LTGF) do not compete for binding with fibrin, thus fibrin can be unbound (F), bound solely by plasminogen (<i>F</i>_PLG), bound by solely latent-TGF<i>β</i>1 (<i>F</i>_LTGF), or by both (<i>F</i>_PLG,LTGF). Plasminogen reversible binds fibrin (reactions 1A and 1B). Latent-TGF<i>β</i>1 also reversible binds fibrin (reactions 2A, 2B, and 2C). Latent-TGF<i>β</i>1 is released from fibrin by plasmin into the active form (TGF, reactions 3A, 3B, and 3C). Fibrin-bound plasminogen can be converted to fibrin-bound plasmin, either without (<i>F</i>_PLS, reaction 4A) or with (<i>F</i>_{PLS, LTGF}, reaction 4B) co-binding of latent-TGF<i>β</i>1. Reactions 5A and 5B represent fibrinolysis, which can result in the release of latent-TGF<i>β</i>1 (reaction 5B).</p

Spontaneous ‘uPAR-rich’ cell selection in the monolayer by a uPAR-plasmin-TGF<i>β</i>1 positive feedback loop.

<p>All cells in the model (A) express the same level of uPAR (the uPAR concentration in the cells is indicated by the red color) at initialization of a simulation. Local changes in fibrin-cell contact can increase local plasmin concentration (B), resulting in degradation of fibrin and release of active TGF<i>β</i>1 (C). TGF<i>β</i>1 can stimulate uPAR expression (D). The positive feedback loop selects ‘uPAR-rich’ cells in the monolayer (E), with a few cells having high level (red color) and most cells having low levels (blue color).</p

<i>In vitro</i> and <i>in silico</i> model setup.

<p>The <i>in vitro</i> model of Koolwijk <i>et al</i>. [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006239#pcbi.1006239.ref001" target="_blank">1</a>] can be studied with phase contrast views of the monolayer as used throughout this paper (A) or with cross-sections of the matrix after fixation and histological staining (B; see, e.g., Ref. [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006239#pcbi.1006239.ref001" target="_blank">1</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006239#pcbi.1006239.ref039" target="_blank">39</a>]; not used in this paper). A schematic illustration of the <i>in vitro</i> model (C) is shown in the middle, with a monolayer of endothelial cells (blue) that form capillary-like tubes in a fibrin matrix (yellow). Images of an <i>in silico</i> simulation that represents a cross-section of the <i>in vitro</i> model are shown on the right. Endothelial cells and fibrin (D) are modeled with the CPM, the uPAR concentration of cells (E) is modeled with an ODE equation, and a PDE system represents the concentrations of all forms of fibrin (F) and TGF<i>β</i>1 (G).</p

In vitro angiogenesis on HMW and LMW fibrin matrices.

<p>(A) Scanning electron microscopic analysis of a HMW fibrin network and (B) of a LMW fibrin network. The high molecular weight (HMW) form of fibrin has a more open network structure than the low molecular weight (LMW) form of fibrin, which has denser fibers [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006239#pcbi.1006239.ref003" target="_blank">3</a>]. Bars represent 1 <i>μ</i>m. (C) Representative top views of vascular ingrowth in unfragmented fibrin; (D) in HMW fibrin; and (E) in LMW fibrin. Experiments were performed as previously described [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006239#pcbi.1006239.ref001" target="_blank">1</a>]; independent replicates given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006239#pcbi.1006239.s003" target="_blank">S2 Fig</a>. Bars represent 500 <i>μ</i>m.</p