P602The lysosomal transfer of LDL/cholesterol from macrophages into vascular smooth muscle cells results in their phenotypic alteration

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Abstract

The local interaction of macrophages [MP] with vascular smooth muscle cells [VSMC] is known to play a key role in atherosclerotic plaque development and plaque destabilization. The majority of foam cells within the atherosclerotic lesion are thought to be derived from macrophages. However, it is well known that VSMC give rise to a significant number of lipid-laden cells, too. While the mechanism of conversion of macrophages into foam cells has been investigated extensively, the mechanism of lipid uptake by VSMC is less clear.

To study VSMC-derived foam cell formation an in-vitro co-culture model of lipid-loaded MP and VSMC was established.

MP were exposed to fluorescence-labelled acetylated LDL [FL-acLDL] prior to co-culture with VSMC. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MP had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol [FL-Chol] these complexes were transferred during co-culture, as well, and resulted in cholesterol positive lipid droplet formation in VSMC. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter [FP] to mark early endosomes, no co-localisation between Rab5a-FP and the transported Fl-acLDL within VSMC was detected implying a mechanism independent of phagocytosis. Next, expression of LAMP1-FP, marking all lysosomes in VSMC, revealed that the FL-acLDL was located in non-acidic lysosomes. MP infected with a virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Next, the consequences of the lipid transport were tested. Resuce of the acLDL-loaded VSMC from co-culture and subsequent analysis revealed an increase in phagocytotic activity. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat specific primers rendered induction of genes typical for MP (CD68 and Mac2) and downregulation of the cholesterol sensitive HMG-CoA reductase.

Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MP into VSMC in-vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell like cell. This way VSMC may loose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.

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