AIMS: Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS: MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION: Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.

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AIMS: Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS: MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION: Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.

Die Migration und Proliferation vaskulärer Zellen spielt eine entscheidende Rolle in der Pathogenese atherosklerotischer Gefäßwandveränderungen und trägt zudem in hohem Maße zu restenosebedingten Komplikationen interventioneller Therapien der Atherosklerose bei. Dies bedingt ein großes Interesse an pharmakologischen Interventionsmöglichkeiten zur Prävention / Therapie atherosklerotischer und restenotischer vaskulärer Läsionen. Dabei kommen neben lokal applizierbaren Substanzgruppen insbesondere Pharmaka in Betracht, die bereits Anwendung zur Behandlung metabolischer Risikofaktoren kardiovaskulärer Erkrankungen finden. Hierzu gehören insbesondere die oralen Antidiabetika vom Typ der Thiazolidindeone, die als Liganden für den "Peroxisome Proliferator-Activated Receptor gamma" (PPARg) agieren. PPARs sind eine Gruppe neuer Regulatoren der Genexpression, für die in den vergangenen Jahren eine Reihe vaskulärer Wirkungen nachgewiesen wurden. Wir konnten zeigen, daß die Proliferation und Migration von Gefäßmuskelzellen durch PPARg-Liganden gehemmt wird. Untersuchungen zu den beteiligten Signalübertragungsschritten ergaben, daß die pharmakologische Aktivierung von PPARg in Gefäßmuskelzellen insbesondere die durch die Mitogen-aktivierten Protein Kinasen ERK1/2 vermittelte Signaltransduktion beeinflußt. Diesbezüglich haben wir nachgewiesen, daß PPARg in Gefäßmuskelzellen die mitogene Signaltransduktion via ERK1/2 MAPK -> Elk-1 -> c-fos und die chemotaktische Signalübertragung via ERK1/2 MAPK -> Ets-1 -> Matrixmetalloproteinase-9 hemmt. Wir konnten ferner zeigen, daß PPARg-Liganden die Endothelzellmigration hemmen, die durch die Neovaskularisation atherosklerotischer Plaques und der damit verbundenen erhöhten Vulnerabilität einer Plaqueruptur eine Rolle in der Pathobiologie der Atherosklerose spielt. Diese migrationshemmende Wirkung der PPARg-Liganden basiert vermutlich auf einer Inhibition der für die Endothelzellmigration erforderlichen Signaltransduktion über den PI3 Kinase -> Akt -> eNOS Pathway. Die Inhibition dieses Signalwegs könnte die Folge der von uns beobachteten PPARg-Ligand-induzierten Expression der Phosphatase PTEN sein, die den PI3K -> Akt Signalweg negativ reguliert und die Aktivierung und Phosphorylierung von Akt inhibiert. Somit haben PPARg-aktivierende Liganden eine wichtige Funktion in der Behandlung der metabolischen Hauptrisikofaktoren kardiovaskulärer Erkrankungen, spielen aber gleichzeitig eine vermutlich ebenso wichtige Rolle in der Protektion atherosklerotischer und restenosebedingter Gefäßwandveränderungen durch direkte vaskuläre Effekte. / Migration and proliferation of vascular cells not only play an important role in the pathogenesis of atherosclerotic lesion formation, but also contribute to restenosis after therapeutic angioplasty. Therefore, pharmacological strategies for the prevention and/or treatment of atherosclerotic and restenotic vascular lesions are of great clinical interest. This involves substances that can be locally administered via stents, as well as agents that are already in clinical use for the treatment of metabolic risk factors. Among the latter, antidiabetic thiazolidinediones which function as ligands for the "peroxisome proliferator-activated receptor gamma" (PPARg), have been identified as promising drugs to target vascular lesion formation. PPARs constitute a group of novel regulators of gene expression, that exert several vascular effects. We report that vascular smooth muscle cell proliferation and migration is inhibited by PPARg-ligands. Investigating the signalling steps that are involved, we find that pharmacological activation of PPARg interferes with signal transduction through the mitogen-activated protein kinases ERK1/2 in vascular smooth muscle cells. We demonstrate that PPARg inhibits mitogenic signal transduction via ERK1/2 MAPK -> Elk-1 -> c-fos and also blocks chemotactic signalling through the ERK1/2 MAPK -> Ets-1 -> matrix metalloproteinase-9 pathway in vascular smooth muscle cells. We also showed that PPARg-ligands inhibit endothelial cell migration, which participates in the neovascularization of atherosclerotic plaques, thereby contributing to plaque destabilization and increased risk of plaque hemorrhage. This antimigratory action of PPARg-ligands results from an inhibition of signal transduction via PI3 Kinase -> Akt -> eNOS, a pathway that is crucial for endothelial cell migration. Since we observed a PPARg-ligand-induced upregulation of PTEN, a phosphatase that negatively regulates the PI3K -> Akt signalling pathway, this might constitute the mechanism by which PPARg-ligands inhibit endothelial cell migration. In conclusion, PPARg-activating ligands may provide a dual benefit in cardiovascular disease by ameliorating metabolic risk factors, as well as protecting the vasculature from atherosclerotic and restenotic alterations through direct vascular effects.