Blockade of Angio-Associated Migratory Cell Protein Inhibits Smooth Muscle Cell Migration and Neointima Formation in Accelerated Atherosclerosis
Felix Vogt, MD*,
Alma Zernecke, MD ,
Marie Beckner, PhD ,
Nicole Krott, MSc*, ,
Anja-Katrin Bosserhoff, PhD||,
Rainer Hoffmann, MD*,
Marc A.M.J. Zandvoort, PhD#,
Thomas Jahnke, MD¶,
Malte Kelm, MD*,
Christian Weber, MD and
Rüdiger Blindt, MD*,*
* Department of Cardiology, RWTH Aachen University, Aachen, Germany
Interdisciplinary Center of Clinical Research (IZKF) "BIOMAT" within the faculty of Medicine, RWTH Aachen University, Aachen, Germany
Institute of Molecular Cardiovascular Research, RWTH Aachen University, Aachen, Germany
Department of Pathology, Pittsburgh University Medical Center, Pittsburgh, Pennsylvania
|| Institute of Pathology, University of Regensburg, Regensburg, Germany
¶ Department of Diagnostic Radiology, University Clinics Schleswig-Holstein, Campus Kiel, Germany
# Department of Biophysics, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, the Netherlands.
View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 1 Expression and Subcellular Localization of AAMP
(A) Two-photon laser-scanning microscopy of sparse smooth muscle cells (left) detecting intensified angio-associated migratory cell protein (AAMP) distribution (green) at the cellular membrane (arrows), whereas dense cells (right) contain less AAMP without distinctive distribution patterns. (B) Subcellular fractioning: the cellular membrane contains >50% of total cellular AAMP; as expected by fluorescence microscopy, the cellular nuclei did not contain AAMP. (C) Flow cytometric analysis of AAMP expression treated or untreated with lysis buffer confirmed the results of the subcellular fractioning experiments (red: nonimmune serum, green: without Tween, blue: with Tween). (D) Computed AAMP protein sequence analysis revealed a potential transmembrane domain between amino acids 322 and 345. FITC = fluorescein isothiocyanate.
|
|
View larger version (39K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 2 Phenotype-Related AAMP Expression in SMCs and the Effect of AAMP on SMC Migration
(A) Western blot analysis of angio-associated migratory cell protein (AAMP) expression in human smooth muscle cells (hSMCs) and rat smooth muscle cells (rSMCs). (B) Blockade of AAMP by an inhibitory recombinant AAMP-antibody (anti-rAAMP-ab) reduced the migratory potential of medial and of neointimal rSMCs in modified transwell chambers. (C) The migratory activity of hSMCs and rSMCs after AAMP-sense vector-transfection was increased compared with controls. (D) The AAMP knockdown by small interfering ribonucleic acid (siRNA) alleviated hSMC migration compared with controls. (E) Five-bromodeoxyuridine (BrdU) assays showed no effect of anti-rAAMP-ab treatment, vector transfection, and siRNA treatment on SMC proliferation. GFP = green fluorescent protein.
|
|
View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 3 RhoA Activity Is Reduced by AAMP Blockade
(A) The siRNA knockdown reduced AAMP content in the membrane fraction but not in the cytosolic fraction. (B) The RhoA activity assay: in the membrane fraction, siRNA and anti-rAAMP-ab treatment greatly reduced RhoA activity, whereas activated RhoA expression was elevated after AAMP overexpression. In the cytosolic fraction, expression of inactive RhoA was not affected by anti-rAAMP-ab treatment, vector transfection, or AAMP siRNA treatment. Abbreviations as in Figure 2.
|
|
View larger version (35K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 4 Increased AAMP Expression in Murine Carotid Arteries After Wire-Induced Injury
(A) Photomicrographs of angio-associated migratory cell protein (AAMP)–stained and nonimmune serum control murine sections. (B) Respective pixel intensity analysis. NI = neointima.
|
|
View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 5 Increased AAMP Expression in Balloon-Injured Porcine Coronary Arteries
(A) Photomicrographs of angio-associated migratory cell protein (AAMP)–stained and nonimmune serum control porcine sections. (B) Respective pixel intensity analysis. (C) Vessel wall protein lysate analysis of AAMP-expression. NI = neointima.
|
|
View larger version (49K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 7 AAMP Blockade Reduces Wire-Induced NI Formation and SMC Density in ApoE–/– Mice
(A) Mice intraperitoneally treated with nonimmune serum developed normal neointimal hyperplasia, whereas in the anti-rAAMP-ab–treated group, neointimal area was significantly reduced. (B) Histomorphometrical assessment revealed a marked reduction of neointimal hyperplasia in the anti-rAAMP-ab–treated group, whereas medial areas did not differ. (C and D) One week after anti-rAAMP-ab treatment, neointimal SMC density represented by  -SMA staining was reduced compared with control. After 4 weeks, anti-rAAMP-ab treatment did not reduce neointimal SMC density compared with nonimmune serum. (E) Vascular cell proliferation analysis revealed similar proliferation indexes (percent Ki67-positive nuclei) in the neointima (NI) at 1 and 4 weeks after injury in both groups. Abbreviations as in Figures 2 and 6.
|
|
|
