Dendritic cells in neointima formation after rat carotid balloon injury
coordinated expression withanti-apoptotic Bcl-2 and HSP47 in arterial repair
Gerhard Bauriedel, MD, FACC*,*,
Alexander Jabs, BS*,
Dirk Skowasch, MD*,
Randolph Hutter, MD ,
Juan J. Badimon, PhD, FACC,
Valentin Fuster, MD, PhD, FACC,
Ulrich Welsch, MD, PhD and
Berndt Lüderitz, MD, FACC*
* Department of Medicine–Cardiology, University of Bonn, Bonn, Germany
The Zena and Michael A. Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, New York, USA
Institute of Anatomy, University of Munich, MunichGermany
View larger version (33K):
[in a new window]
|
Figure 1 Bar graph demonstrating mean area (A) and cellularity (B) of each vascular compartment during time course. Open bars = neointima; hatched bars = media; solid bars = adventitia. *p < 0.05 vs. baseline, as indicated. Of note, medial cellularity decreased by 29% (p = 0.002) at 48 h compared with baseline and recovered (p = 0.001) at day 4 (not marked). Uninjured control segments (C) did not show neointima formation or altered structure of media and adventitia.
|
|
View larger version (86K):
[in a new window]
|
Figure 2 Photomicrographs of early neointima formation, focused on the luminal surface region. (A) Palisade-like alignment of single cells (arrows) along the internal elastic lamina (IEL) at day 4 (trichrome staining). (B) Strong cell-bound OX-62 signaling restricted to early neointima contrasts to unlabeled media at day 7. (C) Magnified detail of neointima at day 7 demonstrating consistent colocalization of OX-62 (dark blue) and S100 (red) in neointimal dendritic cells (DC) (no nuclear counterstaining). (D) Transmission electron microscopy identification of two DCs extending along the IEL at day 7. Note their long dendritic processes and veils, the lobed nucleus, and prominent tubulovesicular network. (E) Transmission electron microscopy detection of apoptosis located in basal neointima close to IEL. Apoptotic shrinkage and detached anchorage from surrounding extracellular matrix are indicated by condensed cytoplasm and pericellular region markedly less dense than adjacent matrix. (F) Representative hyperplastic neointima at day 28. Note the distinct residual OX-62 labeling along the luminal surface, while the vast majority of cells located in basal regions are negative. (G) Transmission electron microscopy image of representative hyperplastic neointima at day 28 that predominantly comprises cells with SMC appearance and apparently shows no apoptosis. Luminal neointima reveals perpendicular alignment of the cells and loose extracellular matrix compared with basal regions. (H) Intense  -smooth muscle actin immunoreactivity of the neointima and weak staining of medial SMCs at day 28. Bar = 30 µm (A to C, F to H); bar = 5 µm (D, E).
|
|
View larger version (95K):
[in a new window]
|
Figure 3 Dendritic cells in early neointima formation. (A) Bar graph demonstrating percentage of neointimal OX-62+ dendritic cells (DC) after angioplasty. Open bars = neointima. *p < 0.05 versus value at seven days. (B) Staining of DCs in rat spleen as positive control for OX-62. Bar = 30 µm (B).
|
|
View larger version (109K):
[in a new window]
|
Figure 4 Photomicrographs of specific B-cell lymphoma 2 protein (Bcl-2) (A to C) and heat shock protein 47 (HSP47) immuno-staining (D to F) in neointima formation from day 4 to day 28 after injury. (A) Strongly cell-bound signaling of Bcl-2 in incipient neointima formation at day 4 (arrow). (B) Luminal prevalence of Bcl-2-positive cells and declining expression in basal regions at day 14. (C) Double staining for OX-62 (dark blue) and Bcl-2 (red) in neointima at day 28 demonstrating persistent luminal prevalence of Bcl-2+ dendritic cells (no nuclear counterstaining). (D) Luminal cells adhering to the internal elastic lamina exhibit strong HSP47 labeling at day 4. (E) Heat shock protein 47 signals are maximal at the luminal surface and continuously decrease to basal regions at day 28. (F) Double staining for OX-62 (dark blue) and HSP47 (red) in neointima at day 14. Note dendritic cell-bound neointimal HSP47 expression at the luminal border and sparse HSP47 immunoreactivity without presence of dendritic cells in deep neointimal areas (no nuclear counterstaining). Bar = 30 µm (A to F).
|
|
View larger version (22K):
[in a new window]
|
Figure 5 Bar graph demonstrating mean expression of B-cell lymphoma 2 protein (Bcl-2) (A) and of heat shock protein 47 (HSP47) (B) in each vascular compartment during time course. Open bars = neointima; hatched bars = media; solid bars = adventitia. *p < 0.05 versus value at four days, as indicated for neointima; p < 0.05 versus baseline, as indicated for adventitia.
|
|
View larger version (106K):
[in a new window]
|
Figure 6 Photomicrographs of transmural response to injury, focused on adventitial remodeling. (A) Intense -smooth muscle actin immunoreactivity in the media and outer adventitia at day 4; also perivascular microvessels of the vasa vasorum are positive (arrows). (B) Transmission electron microscopy detail of hypocellular inner adventitia at day 7 illustrating two cells that simultaneously display end-stage apoptotic shrinkage (arrows); see large regions of non-structured matrix (*). (C) Transmission electron microscopy detail of hypercellular outer adventitia at day 7 showing several viable cells with fibroblast-like appearance embedded in dense extracellular matrix. Also, see in the endothelial cell lining a perivascular capillary (*). (D) No signals of OX-62+ dendritic cells are found in the media (M) and the broad adventitia at day 4, in contrast to intense OX-62 immuno-staining along the luminal surface. (E) Cell-bound B-cell lymphoma 2 protein signals are exclusively found in the outer adventitia (arrows) at day 4. While demarcation from perivascular connective tissue is not always possible, inner adventitia (*), M, and lumen (L) can be easily distinguished. (F) Transmural heat shock protein 47 expression pattern at day 4. Note the distinct immuno-staining exclusively located in the outer adventitia. Bar = 50 µm (A, D to F); bar = 5 µm (B, C).
|
|
|
