1. Macrophage depletion reduces monocyte chemotactic protein-1 and transforming growth factor-β1 in healing rat vein grafts 
Randal A Wolff, PhD, Jeffrey J Tomas, BS, Debra A Hullett, PhD, V.Emily Stark, MS, Nico van Rooijen, PhD, John R Hoch, MD 
Journal of Vascular Surgery 
Volume 39, Issue 4, Pages (April 2004)
DOI: /j.jvs

2. Fig 1
Immunohistochemistry of monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-β1 (TGF-β1) from animals treated with Lip-Clod, Vector, or phosphate-buffered saline solution (PBS). Top, Immunohistochemical (3,3′-diaminobenzidine) stain of spleen macrophages shows minimal ED1-positive (macrophages and monocytes) staining of the spleen section from animals treated with Lip-Clod. Middle and bottom, Vein graft cross-sections stained for MCP-1 and TGF-β, respectively. Sections from animals treated with Lip-Clod show minimal MCP-1 and TGF-β staining compared with the Vector and PBS control animals. These photomicrographs also show reduced intimal hyperplasia and reduced hyperproliferation of the adventitia with Lip-Clod treatment. Sections were counterstained with hematoxylin. Parallel staining with a control antibody was used as a negative control (not shown). (Original magnification, ×200.)
Journal of Vascular Surgery  , DOI: ( /j.jvs )

3. Fig 2
Lip-Clod attenuates intimal hyperplasia. Compared with either control group, neointimal area is significantly smaller in the Lip-Clod group at both 1 and 4 weeks. The internal elastic lamina was traced to define the border separating the neointima from the media and adventitia. Area was derived by computerized morphometry. Error bars represent SEM. PBS, Phosphate-buffered saline solution.
Journal of Vascular Surgery  , DOI: ( /j.jvs )

4. Fig 3
Lip-Clod and reduces neointimal and total cellularity. At 1 week, number of neointimal cells per neointimal area (top) and number of total cells per total cross-section (bottom) are significantly reduced or approach significant reduction with Lip-Clod treatment compared with control treatments. At 4 weeks, Lip-Clod treatment significantly reduces neointimal and total cross-section cellularity compared with treatment with phosphate-buffered saline solution (PBS). In addition, Lip-Clod treatment reduces total cross-section cellularity compared with Vector treatment at 4 weeks. Absence of infiltrating macrophages contributes to the reduction of cell numbers in the Lip-Clod group. Hematoxylin-stained nuclei were counted, and values were derived with computerized image analysis.
Journal of Vascular Surgery  , DOI: ( /j.jvs )

5. Fig 4
Total and neointimal monocyte chemotactic protein-1 (MCP-1) expression is significantly decreased in Lip-Clod–treated grafts at 1 week compared with grafts treated with phosphate-buffered saline solution (PBS). Lip-Clod treatment versus PBS at 1 week showed a significant reduction in total and neointimal MCP-1 staining, whereas the reduction in neointimal staining versus Vector approached significance. At 4 weeks, the MCP-1 level in the Lip-Clod group remained near the same low level seen at 1 week. The levels in both the Vector and PBS groups decreased, so that at 4 weeks there was no significant difference among the groups in MCP-1 protein expression across the entire section or in the neointima. A mean value for each graft was derived from three sections taken from the perianastomotic region of the vein graft. Results were analyzed with analysis of variance. Error bars represent SEM; n ≥5 for each treatment or time point.
Journal of Vascular Surgery  , DOI: ( /j.jvs )

6. Fig 5
Total and neointimal transforming growth factor-β (TGF-β) expression is significantly decreased in Lip-Clod–treated grafts at 1 week compared with grafts treated with phosphate-buffered saline solution (PBS). Lip-Clod treatment versus PBS at 1 week showed a significant reduction in total and neointimal TGF-β staining. Lip-Clod treatment versus Vector showed a significant reduction in total area positive for TGF-β, and approached significance for reduction in neointimal area positive for TGF-β. There was no difference in TGF-β total or neointimal protein expression among the three treatments at 4 weeks. A mean value for each graft was derived from three sections taken from the perianastomotic region of the vein graft. Results were analyzed with analysis of variance. Error bars represent SEM; n ≥ 5 for each treatment or time point.
Journal of Vascular Surgery  , DOI: ( /j.jvs )

7. Fig 6
Enzyme-linked immunoassay of whole-graft lysates shows a reduction in transforming growth factor-β (TGF-β) expression with Lip-Clod treatment, but no change in monocyte chemotactic protein-1 (MCP-1) expression. A, No difference in MCP-1 expression is seen among the three treatments at 1 week. At 4 weeks MCP-1 expression is highest in the phosphate-buffered saline solution (PBS) group and lowest in the Vector group, but these differences did not reach statistical significance. B, At 1 week TGF-β expression is significantly reduced with Lip-Clod versus PBS. At 4 weeks the reduction in TGF-β expression is significant versus both control groups.
Journal of Vascular Surgery  , DOI: ( /j.jvs )

8. Fig 7
Grafts from animals treated with liposomally encapsulate Lip-Clod have a higher level of mRNA for monocyte chemotactic protein-1 (MCP-1) than do animals treated with phosphate-buffered saline solution (PBS), but no change in mRNA for TGF-β. Grafts were homogenized in Trizol, and mRNA was extracted and subjected to reverse transcription to cDNA. Copy number of gene of interest and housekeeping gene were determined with quantitative polymerase chain reaction, on the basis of threshold cycle of standards with known copy numbers.
Journal of Vascular Surgery  , DOI: ( /j.jvs )