Download Alma Zernecke - Rudolf-Virchow

Alma Zernecke
E-mail: [email protected]
Phone: +49(0)931 31 80373
Fax:
+49(0)931 31 83255
http://www.rudolf-virchow-zentrum.de/forschung/zernecke.html
Atherosclerosis, with its clinical manifestations of myocardial infarction, stroke and peripheral artery disease, is
imminently becoming the leading cause of death worldwide. Inflammation has emerged as a crucial force driving
the initiation and progression of atherosclerotic lesion formation. Initiated by the activation and dysfunction of
endothelial cells, leukocyte subsets are recruited and accumulate in atherosclerotic lesions. Details are emerging
regarding the involvement of different leukocyte subpopulations in the pathology of this disease. While mononuclear cells found in the lesions are predominantly comprise monocyte-derived macrophages, which transform
into foam cells characteristic for fatty-streak lesions, T-lymphocytes and dendritic cells have also been revealed
in close proximity. Moreover, immune responses are described to participate in all phases of atherosclerosis,
and pro-atherogenic and atheroprotective cytokines and T cell subpopulations have been defined. The delicately
adjusted two-edged immune balance and the exact function of these cell types remain elusive to date.
The non-random attraction of mononuclear
cells to specific tissue targets is governed
by sequential, and also mutually overlapping steps in the interaction with the vessel wall: namely rolling interactions, followed by integrin-dependent arrest and
chemokine-triggered transendothelial diapedesis. We previously identified several
adhesion molecules and chemokines/receptors as well as their regulation that are important in the accumulation of leukocytes
at sites of inflammation.
Recently, we could show that the transmembrane adhesive glycoprotein junctional
adhesion molecule A (JAM-A) is not only
expressed as a cellular variant, but is also
released after cleavage, predominantly by
the disintegrin and metalloproteinase
(ADAM) 17. Functionally, soluble JAM-A inhibited the transendothelial migration of
isolated neutrophils in vitro, and decreased
the recruitment of neutrophils in a murine
air pouch (Koenen et al.). In addition, we
addressed the function of JAM-C, another
member of the immunoglobulin superfamily, in leukocyte recruitment. Treatment
with a blocking anti-JAM-C antibody inhibited the accumulation of monocytes in
carotid arteries and reduced neointima
formation after arterial injury in Apolipoprotien-E deficient (Apoe-/-) mice (Shagdarsuren et al.). Moreover, in this study we
could unravel that JAM-C, in contrast to
findings in human platelets, was not expressed by murine platelets.
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Fig. 1:
Reduced numbers of Gr1low monocytes in cx3cr1-deficient mice under steady-state conditions. Flow
cytometric analysis of blood monocytes and comparison between monocyte population size of wt,
cx3cr1gfp/+ and cx3cr1gfp/gfp mice (left panel). cx3cr1-deficient atheromas show changes in cellular
composition. Irradiated Apoe-/- recipients of indicated bone marrow cells were subjected to high-fat diet
for 12 weeks, after which their plaque content was analyzed. cx3cr1/GFP expression is shown in green;
DAPI staining in blue (right panels).
Activated platelets contribute to the exacerbation of atherogenesis; for example, by
depositing the chemokines platelet factor4 (PF4, also known as CXCL4) and RANTES
(CCL5), triggering monocyte arrest on inflamed endothelium. Homo-oligomerization
is required for the recruitment functions of
CCL5, and chemokine heteromerization has
more recently emerged as an additional
regulatory mechanism, as evidenced by an
enhanced monocyte arrest resulting from
CCL5-CXCL4 interactions. We have deter-
mined the structural features of CCL5-CXCL4
heteromers and designed stable peptide inhibitors that specifically disrupt pro-inflammatory CCL5-CXCL4 interactions. This
results in attenuating monocyte recruitment and reducing atherosclerosis, establishing the in vivo relevance of chemokine
heteromers in inflammatory leukocyte recruitment (Koenen et al.).
Besides their function in leukocyte recruitment, chemokines are also important
in controlling the survival and cell homeo-
stasis of different cell subpopulations.
For example, we could show that absence
of the receptor cx3cr1 resulted in reduced
monocyte survival. In this study, we used
knock-in mice that carry a targeted replacement of the cx3cr1 gene by a gene
encoding green fluorescent protein in
order to visualize monocytes/macrophages
by their GFP-expression. cx3cr1 is a chemokine receptor with a single ligand,
the membrane-tethered chemokine CX3CL1
(fractalkine). All blood monocytes express
cx3cr1, but its expression levels differ between the two main subsets, with human
CD16+ and murine Gr1low monocytes being
CX3CR1high. In mice, the absence of either
cx3cr1 or cx3cl1 resulted in a significant reduction of Gr-1low blood monocyte levels
under both steady-state (Fig. 1) and inflammatory conditions. Apoe-/- mice transplanted with cx3cr1-deficent bone marrow
displayed a diminished accumulation of
plaque cx3cr1+ cells (Fig. 1) and were protected against development of atherosclerosis. Introduction of a Bcl2 transgene to
enforce the survival of monocytes restored
the wild-type phenotype, suggesting that
the CX3C-axis provides an essential survival
signal (Landsman et al.). These findings
provided a mechanistic explanation for the
role played by the previously recognized
function of the cx3cr1 chemokine family in
atherogenesis.
Chemokines are induced in the context
of inflammation and in the course of apoptosis, as previously observed for the induction of CXCL12. This chemokine and
its receptor CXCR4 have been implicated
in mechanisms counteracting apoptosis.
Apoptosis involves shedding of membranous microvesicles called apoptotic
bodies. We could now show that endothelial cell-derived apoptotic bodies
are generated during the development
of atherosclerosis, and convey paracrine
alarm signals to recipient vascular cells,
inducing CXCL12 expression. This is mediated by microRNA-126, enriched in apoptotic bodies, repressing the negative regulator of G-protein signaling, RGS16, and
unlocking CXCR4 to trigger an auto-regulatory feedback loop that increases production of CXCL12. Transfer of apoptotic bodies, microRNA-126, or local intraluminal
exposure of carotid arteries of Apoe-/- mice
to endothelial AB isolated from miR-126+/+
but not miR-126-/- mice limited atherosclerosis (Fig. 2) and conferred features
of plaque stability (Zernecke et al.),
highlighting the important functions of
microRNAs in atherosclerosis.
Less is known about the recruitment and
function of T cells and dendritic cell subsets in atherosclerosis. By targeting specific chemokines/cytokines and their receptors in Apoe-/- mice, we will address the
functions of different immune cell subpopulations in atherosclerosis. A particular
focus will not only be on their interactions
at sites of inflammation, but also within
lymphatic tissue, and their role in shaping specialized immune responses that
control the development of atherosclerosis. Furthermore, we will investigate the localization of these cells in the
vessel wall and their routes of entry during
lesion formation.
Given the remarkable role of adaptive
and innate immunity in atherosclerosis,
targeting of its cellular constituents and
understanding the complex equilibrium
and interplay between immune cell subpopulations that contribute to the process
of atherosclerosis will be important to
identify new therapeutic approaches for
treating this disease.
Fig. 2:
PBS or apoptotic bodies protect against early atherosclerotic lesion formation. Apoe-/- mice were fed a
high-fat diet for 6 weeks and injected twice weekly with PBS or AB. Representative images of aortic
roots stained by Oil-red-O to detect atherosclerotic lesion areas.
Extramural Funding
DFG (SFB688, TP A12, ZE 827/1-1,
ZE827/4-1)
Selected Publications
Koenen, R.R., Pruessmeyer, J., Soehnlein, O., Fraemohs, L., Zernecke, A.,
Schwarz, N., Reiss, K., Sarabi, A., Lindbom, L., Hackeng, T.M., and Weber, C.
(2009) Regulated release and functional
modulation of junctional adhesion
molecule A by disintegrin metalloproteinases. Blood, 113, 4799-809.
Shagdarsuren, E., Djalali-Talab, Y., Aurrand-Lions, M., Bidzhekov, K., Liehn,
E.A., Imhof, B.A., Weber, C., and Zernecke, A. (2009) Importance of junctional adhesion molecule-C for neointimal
hyperplasia
and
monocyte
recruitment in atherosclerosis-prone
mice. Arterioscler Thromb Vasc Biol,
29, 1161-3.
Koenen, R.R., von Hundelshausen, P.,
Nesmelova, I.V., Zernecke, A., Liehn,
E.A., Sarabi, A., Kramp, B.K., Piccinini,
A.M., Paludan, S.R., Kowalska, M.A.,
Kungl, A.J., Hackeng, T.M., Mayo, K.H.,
and Weber, C. (2009) Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice. Nat Med, 15,
97-103.
Landsman, L.*, Bar-On, L.*, Zernecke,
A.*, Kim, K.W., Krauthgamer, R.,
Shagdarsuren, E., Lira, S.A., Weissmann,
I.L., Weber, C., and Jung, S. (2009)
cx3cr1 is required for monocyte homeostasis and atherogenesis by promoting
cell survival. Blood, 113, 963-72. *equal
contribution.
Zernecke, A., Bidzhekov, K., Noels, H.,
Shagdarsuren, E., Gan, L., Denecke, B.,
Hristov, M., Köppel, T., Nazari Jahantigh,
M., Lutgens, E., Wang, S., Olson, E.N.,
Schober, A., and Weber, C. (2009) Delivery of microRNA-126 apoptotic bodies
induces CXCL12-dependent vascular protection . Science Sign, 2; ra 81.
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