Charakterisierung des proinflammatorischen Phänotyps neointimaler glatter Gefäßmuskelzellen nach Gefäßverletzung

Leukocyte recruitment is cruicial for the response to vascular injury in spontaneous and accelerated atherosclerosis. Whereas the mechanisms of leukocytes to endothelium or matrix-bound platelets have been characterized, less is known about the proadhesive role of smooth muscle cells that get exposed to the bloodstream after endothelial denudation. In laminar flow assay neointimal rat SMCs supported a higher arrest of monocytes and "memory" T lymphocytes than medial SMCs. The enhanced arrest was dependent of both, P-Selektin and VLA-4, as demonstrated by pretreatment with blocking antibodies. The increase in monocyte arrest on neointimal SMCs was triggered by the CXC chemokine GRO-alpha and fractalkine, wheras blocking MCP-1 or the SDF-1alpha receptor CXCR4 on monocytes did not significantly affect monocyte arrest. Enhanced VLA-4 dependent memory T cell arrest, however, was reduced by blockade of CXCR4, indicating mediation by SDF-1alpha. To provide a molecular basis for the difference in the adhesive potential of the distinct SMC subtypes, neointimal and medial, we investigated expression of relevant adhesion molecules and chemokines by real-time RT-PCR and flow cytometry. The functional phenotype of neointimal SMCs was paralleled by a constitutively enhanced expression of P-selectin, GRO-alpha, SDF-1alpha;, fractalkine and MCP-1. Unlike fractalkine, GRO-alpha; and SDF-1alpha; MCP-1 – albeit upregulated in neointimal SMCs – was not immobilized on the surface of neointimal SMCs. Increased expression of P-selectin in neointimal SMCs was associated with enhanced NF-kappaB activity, as demonstrated by immunofluorescence staining for nuclear p65 in vitro. Immunoprecipitation revealed enhanced IKK activity in neointimal SMCs, implicating a regulation of the neointimal phenotype via NF-kappaB pathways. To ultimately demonstrate the direct contribution of the enhanced constitutive NF-kappaB activity to the neointimal phenotype, adenovirus-mediated transfer of the NF-kappaB inhibitor IkappaB-alpha; was performed. Inhibition of NF-kappaB activation resulted in marked reduction of increased leukocyte arrest under flow conditions. Furthermore P-Selektin expression in neointimal SMCs was confirmed in vivo in a atherogenic mouse model of vascular injury. alpha-Actin+ VE-cadherin-cells microdissected from the neointima two weeks after injury could be identified as source for P-selectin by RT-PCR. In conclusion, in this study we have identified a NF-kappaB mediated proinflammatory phenotype of neointimal SMCs that is characterized by increased expression of P-selectin and chemokines and thereby effectively supports leukocyte recruitment. We could show that in vitro blockade of NF-kappaB activation can alter the functional phenotype of SMCs and moreover identify critical genes targeted by NF-kappaB, resulting in a better understanding of the cellular response after vascular injury.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2008

Author dc:creator

• Hagemann, Ute

Contributors dc:contributor

• Weber, Christian

• info:eu-repo/classification/ddc/610
• Restenose
• Medizin
• Adhäsionsmoleküle
• glatte Gefäßmuskelzellen
• Chemokine
• restenosis
• inflammation
• smooth muscle cells
• chemokines

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112632%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A50070