Background:
After vascular injury, vascular smooth muscle cells (SMCs) switch from a differentiated contractile state to synthetic de-differentiated phenotype which contributes to the pathophysiology of restenosis. Experimental data generated by our lab indicate that TGF-β downregulates contractile proteins and stimulates migration. To understand how TGF-β promotes SMC phenotypic switch in injured arteries, we performed an Affymetrix Array analysis and identified Lymphocyte Specific Protein-1 (LSP1) among other upregulated genes. LSP1 is known to play a role in neutrophil extravasation, however the role of LSP1 within SMCs is unknown. We hypothesize that LSP1 contributes to SMC pathophysiological behavior through changes in cell architecture and migration
in-vivo
and
in-vitro.
Methods and Results:
After carotid artery angioplasty, male Sprague-Dawley rats were sacrificed at 3, 7, and 14 days after injury for immunohistochemistry. Immunofluorescence staining revealed a unique upregulation of LSP1 within the neointima, media, and adventitia at 7 and 14 days, but not at 3 days after injury. Confocal images revealed that the LSP1 positive cells minimally express α-SMA (Pierson’s Coefficient, r=.017). Additional characterization experiments using immune cell markers CD3 and CD45 show no co-localization with LSP1 positive cells. To mimic the
in-vivo
neointimal cells and vascular injury induced de-differentiation
in-vitro
, rat A10 cells were treated with solvent or PDGF-bb (10 ng/mL). Quantitative RT-PCR demonstrated an upregulation of LSP1 mRNA after 24 hrs of PDGF-BB stimulation. Using Western Blotting, we confirm an upregulation of LSP1 protein after 48 hrs of PDGF-BB stimulation. Lastly, we performed nuclear and cytoplasmic fractionation followed by Western Blotting which demonstrated that LSP1 is remained within cytoplasmic fraction of the A10 cell after treatment with PDGF-BB.
Conclusion:
These results demonstrate that LSP1 is increased
in-vivo
after balloon injury, and
in-vitro
after PDGF-BB stimulation. Experiments to characterize the identity of these LSP1 cells
in-vivo
are in process, with future
in-vitro
experiments to focus on the role of LSP1 phosphorylation as a part of cytoskeletal remodeling and cellular migration.