Mobilisation und Funktion von Endothelialen Progenitorzellen (EPC) durch Aktivierung des angeborenen Immunsystems

In allen Stadien der Atherosklerose tragen Endotheliale Progenitorzellen (EPC) möglicherweise protektiv zur Endothelrestauration bei. Zugleich ist das angeborene Immunsystem eng in proatherosklerotischen Entzündungsprozessen involviert. Die Bedeutung der Aktivierung des angeborenen Immunsystems speziell über die pattern-recognition-receptors (PRR) retinoic acid inducible gene-I (RIG-I), melanoma differentiation-associated gene-5 (MDA-5) und toll-like receptor-3 (TLR-3) auf die Funktion und Mobilisation von EPC war Gegenstand der hier vorgelegten Dissertationsarbeit. In vitro führt eine spezifische Stimulation der genannten Rezeptoren jeweils zu einer Veränderung des Rezeptorexpressionsmuster. Dies führt zu vermehrter Apoptose (TLR-3, MDA-5), oxidativem Stress (alle Rezeptoren) und im Falle von TLR-3 zu einer verminderten Migration und Proliferation. In vivo kommt es durch spezifische MDA-5 und TLR-3 Stimulation zusätzlich zu einer Erhöhung der EPC in den drei Kompartimenten Knochenmark, peripheres Blut und Milz. In vivo zeigen TLR-3 Knockout Modelle, dass die Erhöhung der EPC im peripheren Blut und der Milz direkt durch eine TLR-3 Aktivierung vermittelt wird. Im atherosklerotischen ApoE Knockout Tiermodell zeigt sich nach TLR-3 Stimulation eine EPC Erhöhung im peripheren Blut, nicht aber in der Milz. Die Diskrepanz zwischen einem erhöhten EPC Pool einerseits bei gleichzeitig beeinträchtigter Endothelfunktion andererseits kann durch ein vermindertes EPC Regenerationspotential erklärt werden, was in den in vivo Transfusionsexperimenten mit TLR-3 stimulierten Wildytp EPC und TLR-3 defizienten EPC gezeigt werden konnte. Zusammenfassend konnte gezeigt werden, dass EPC die PRR TLR-3, MDA-5 und RIG-I exprimieren und somit ebenso wie Endothelzellen als immunkompetent anzusehen sind. Eine spezifische Stimulation von TLR-3 und MDA-5 führt zu einer Erhöhung der EPC Zahl bei gleichzeitig proatherosklerotischen Effekten. Dies führt, wie am Beispiel von TLR-3 gezeigt werden konnte, zu einem in vivo verminderten Regenerationspotential

Proinflammatory Stimulation of Toll-Like Receptor 9 with High Dose CpG ODN 1826 Impairs Endothelial Regeneration and Promotes Atherosclerosis in Mice.

BACKGROUND:Toll-like receptors (TLR) of the innate immune system have been closely linked with the development of atherosclerotic lesions. TLR9 is activated by unmethylated CpG motifs within ssDNA, but also by CpG motifs in nucleic acids released during vascular apoptosis and necrosis. The role of TLR9 in vascular disease remains controversial and we sought to investigate the effects of a proinflammatory TLR9 stimulation in mice. METHODS AND FINDINGS:TLR9-stimulation with high dose CpG ODN at concentrations between 6.25 nM to 30 nM induced a significant proinflammatory cytokine response in mice. This was associated with impaired reendothelialization upon acute denudation of the carotid and increased numbers of circulating endothelial microparticles, as a marker for amplified endothelial damage. Chronic TLR9 agonism in apolipoprotein E-deficient (ApoE-/-) mice fed a cholesterol-rich diet increased aortic production of reactive oxygen species, the number of circulating endothelial microparticles, circulating sca-1/flk-1 positive cells, and most importantly augmented atherosclerotic plaque formation when compared to vehicle treated animals. Importantly, high concentrations of CpG ODN are required for these proatherogenic effects. CONCLUSIONS:Systemic stimulation of TLR9 with high dose CpG ODN impaired reendothelialization upon acute vascular injury and increased atherosclerotic plaque development in ApoE-/- mice. Further studies are necessary to fully decipher the contradictory finding of TLR9 agonism in vascular biology

Proinflammatory response by concentration dependent TLR9 activation in acute vascular injury.

<p>WT mice were subjected to an electric denudation of the left carotid artery as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146326#pone.0146326.g001" target="_blank">Fig 1</a> and received repetitive s.c. injections of 0.625nM (n = 7), 6.25nM (n = 7), 18nM (n = 6), 30nM CpG ODN (n = 7) or vehicle (n = 7) over 7 days. Splenic CD3 positive lymphocytes from 18nM and 30nM CpG ODN treated mice expressed more IFN-γ and RANTES compared to vehicle controls (a). TLR9 activation by 6.25nM, 18nM and 30nM CpG ODN significantly impaired reendothelialization in WT mice compared to vehicle controls (b). Further, stimulation with 18nM and 30nM CpG ODN elevated levels of circulating CD11b positive cells (c). Data are presented as mean±SEM. Statistical analysis was performed using ANOVA (Holm-Sidak's multiple comparisons test). *p<0.05, **p<0.001, p***<0.0001.</p

TLR9 stimulation augments atherosclerosis.

<p>ApoE^-/- mice were fed a cholesterol rich diet for 7 weeks and treated 3 times a week with 18nM CpG ODN s.c. for 6 weeks (a). Chronic stimulation of TLR9 in ApoE^-/- mice led to a significant increase in aortic atherosclerotic plaque size compared to vehicle treated mice (b, quantitative and representative Oil-red O image; n = 9). MOMA-2 stained atherosclerotic plaque were similar compared to vehicle treated mice (c, quantification and representative image). Data are presented as mean±SEM. Statistical analysis was performed using 2-tailed, unpaired students t-test. Scale bar 200μm in b and c. **p<0.001.</p

CpG ODN stimulation in acute vascular injury.

<p>WT mice were injected with PBS (n = 4) or 3-30nM CpG ODN (n = 2) i.v., respectively. Activation of TLR9 with 30nM CpG ODN led to the highest plasma IL-6 concentrations in mice compared to vehicle controls (a). For the acute injury, WT mice (n = 12–13) were subjected to an electric denudation of the left carotid artery and received repetitive injections of 30nM CpG ODN or vehicle over 7 days (b). Reendothelialization was greatly impaired in TLR9-stimulated mice compared to vehicle controls (c, representative Evan’s blue stained photomicrograph; and d, quantitative). The number of circulating EMPs (e, n = 5) and the number of circulating sca-1/flk-1 positive cells (f, n = 5) were significantly increased in ODN treated mice. Gating strategy for FACS analysis is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146326#pone.0146326.s003" target="_blank">S3 Fig</a>. Data are presented as mean±SEM. Statistical analysis was performed using 2-tailed, unpaired students t-test. Scale bar 200μm in c. *p<0.05, **p<0.001, ***p<0.0001.</p

Concentration dependent plaque development by TLR9 stimulation.

<p>ApoE^-/- mice were fed a cholesterol rich diet for 8 weeks and treated 3 times a week s.c. with 0.625nM (n = 7) or 18nM CpG ODN (n = 7), or vehicle (n = 8) in the last 7 weeks. Chronic TLR9 stimulation with high concentrations of CpG ODN in ApoE^-/- mice led to a significant increase in aortic atherosclerotic plaque size compared to low dose CpG ODN and vehicle treated mice (a). Immunohistochemical staining demonstrates increased TLR9 expression in aortic plaques of 18nM CpG ODN treated mice (b, representative image). Differential blood analysis shows increased CD3 and CD3/CD8 positive cells, while CD3/CD4 and neutrophil granulocytes (CD11b⁺/Ly6C⁺/CD115^- cells) are reduced (c). TLR9 expression is induced in B-cells, T-helper cells, and non-classical monocytes (CD11b⁺/Ly6C⁺ low/CD115⁺) are by CpG ODN, while it is decreased in neutrophil granulocytes (d). Data are presented as mean±SEM. Statistical analysis was performed using Kruskal-Wallis test (Dunn’s multiple comparison test). *p<0.05, **p<0.001, ***p<0.0001, ˚p<0.05 18nM to 0.625nM CpG ODN.</p