Disruption of hedgehog signalling in ApoE − /− mice reduces plasma lipid levels, but increases atherosclerosis due to enhanced lipid uptake by macrophages

Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, Maastricht, The Netherlands.
The Journal of Pathology (Impact Factor: 7.43). 08/2007; 212(4):420-8. DOI: 10.1002/path.2193
Source: PubMed


Embryonic pathways are often re-expressed in adult pathology. Here we investigated the role of the morphogen hedgehog (hh), which we found to be re-expressed in atherosclerotic plaques. Male ApoE - /- mice were treated for 12 weeks with an anti-hh antibody (5E1) or a control IgG (1E6) starting at the age of 6 or 18 weeks. Inhibition of hh signalling induced a significant increase in total plaque area in the aortic arch, a result of an increase (54% and 36%, respectively) in the area of advanced plaques (atheromata). In mice treated with anti-hh, plaques contained large (18-35% > ctrl), lipid-filled, sometimes multinucleated macrophage foam cells. Plasma cholesterol levels decreased after anti-hh treatment. In bone marrow-derived macrophages, foam cell formation was enhanced after inhibition of hh signalling. Anti-hh treatment caused a 54-75% increase in early oxLDL uptake (10-240 min), which was scavenger receptor-mediated. After 3-24 h of oxLDL incubation, intense Oil red O staining as well as increased amounts of cholesterol esters were present in these macrophages after anti-hh treatment. Activation of the HH-signalling cascade by recombinant Shh induced a decrease in oxLDL uptake. Here we show that the hh-signalling pathway is one of the morphogenic pathways that regulate plasma lipid levels and atherosclerosis development and progression.

10 Reads
  • Source
    • "Foam cell induction Cells were plated in 24-well tissue culture plates (Costar) at a density of 1 9 10 5 cells/ml, when reaching sub-confluence, the macrophages were incubated with oxLDL (50 lg/ ml) for 24 h (Beckers et al. 2007), after that, the medium was aspirated and cells were rinsed twice with 0.01 M PBS (Boster, Wuhan, China). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Macrophage-derived foam cell formation elicited by oxidized low-density lipoprotein (oxLDL) is the hallmark of early atherogenesis. Detection of foam cell formation is conventionally practiced by Oil Red O (ORO) staining of lipid-laden macrophages. Other methods include 1,1'-dioctadecyl-3,3,3'3'-tetra-methylindocyanide percholorate (DiI)-labeled oxLDL (DiI-oxLDL) uptake and Nile Red staining. The purpose of the present study is to report an optimized method for assessing foam cell formation in cultured macrophages by ORO staining and DiI-oxLDL uptake. After incubation with oxLDL (50 μg/ml) for 24 h, the macrophages were fixed, stained with ORO for just 1 min, pronounced lipid droplets were clearly observed in more than 90% of the macrophages. To test the in vivo applicability of this method, lesions (or foam cells) of cryosections of aortic sinus or primary mouse peritoneal macrophages from ApoE deficient mice fed a high cholesterol diet were successfully stained. In another set of experiments, treatment of macrophages with DiI-oxLDL (10 μg/ml) for 4 h resulted in significant increase in oxLDL uptake in macrophages as demonstrated by confocol microscopy and flow cytometry. We conclude that the optimized ORO staining and fluorescent labeled oxLDL uptake techniques are very useful for assessing intracellular lipid accumulation in macrophages that are simpler and more rapid than currently used methods.
    Full-text · Article · Nov 2010 · Cytotechnology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The aim of the present study was to evaluate the expression of hedgehog (Hh) signaling molecules and the chemotactic activity of Sonic hedgehog (Shh) in monocytes from control (CTR) and diabetic patients with or without coronary artery disease (CAD). Previously several studies demonstrated that exogenous administration of Shh can induce angiogenesis and accelerate repair of ischemic myocardium and skeletal muscles. Blood samples were collected from (1) CTR (n = 25); (2) patients with stable CAD without diabetes mellitus (CAD-DM, n = 10); and (3) with stable CAD with DM (CAD+DM, n = 15). Monocytes were isolated by Percoll gradient and subjected to PCR and chemotaxis analysis. Hh signaling molecules were expressed in human monocytes, and Shh-induced monocyte chemotaxis. Shh-stimulated migration of monocytes from CTR measured 172.5 +/- 90% and a maximal stimulation was observed at Shh concentration of 1 microg/ml. However, Shh failed to induce migration of monocytes from CAD+DM (94.3 +/- 27%, P < 0.001 vs. CTR). The impaired response to Shh was associated with strong transcriptional upregulation of the receptor Ptc, while expression of downstream molecules was not altered. Moreover, Ptc is strongly expressed in macrophages of human aortic atherosclerotic plaque. Thus, Shh is a potent chemoattractant for monocytes and it activates classical signaling pathways related to migration. The Shh signaling was negatively affected by DM which might be involved in the pathogenesis of DM-related complications.
    Full-text · Article · Aug 2009 · Archiv für Kreislaufforschung
  • [Show abstract] [Hide abstract]
    ABSTRACT: Steroidal alkaloids from Veratrum californicum (Durand) are known to exert teratogenic effects (e.g., cyclopia, holoprosencephaly) by blocking the Hedgehog (Hh) signaling pathway, which plays a considerable role in embryonic development and organogenesis. Most surprisingly, recent studies demonstrate that this complex signaling network is active even in the healthy adult organism, where it seems to control important aspects of basic metabolism and interorgan homeostasis. Abnormal activation of Hh signaling, however, can lead to the development of different tumors, psoriasis, and other diseases. This review provides an overview of how the principle teratogenic and hazardous constituent of Veratrum californicum, cyclopamine, interferes with Hh signaling and can potentially serve as a beneficial therapeutic for different tumors and psoriasis.
    No preview · Article · Aug 2009 · Planta Medica
Show more