Macrophage depletion reduces monocyte chemotactic protein-1 and transforming growth factor-beta1 in healing rat vein grafts.
OBJECTIVE
We previously showed that treatment with liposomally encapsulated dichloromethylene bisphosphonate reduces intimal hyperplasia development and macrophage accumulation in a rat epigastric vein to femoral artery model of intimal hyperplasia. Our objective in this study was to determine the effect of liposomally encapsulated dichloromethylene bisphosphonate on the expression of two cytokines essential to neointimal development, monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-beta1 (TGF-beta).


METHODS
We injected rats both 2 days preoperatively and 2 weeks postoperatively with liposomally encapsulated dichloromethylene bisphosphonate (Lip-Clod), liposomally encapsulated phosphate-buffered saline solution (Vector), or phosphate-buffered saline solution (PBS), and harvested the grafts at 1 and 4 weeks. In the perianastomotic region, MCP-1 and TGF-beta protein expression in the total graft cross-section and in the neointima was determined with immunohistochemistry. In whole-graft lysates, MCP-1 and TGF-beta protein were determined with an enzyme-linked immunosorbent assay, and messenger RNA expression was determined with reverse transcription quantitative polymerase chain reaction.


RESULTS
Lip-Clod treatment reduced intimal hyperplasia when compared with Vector or PBS treatment. These reductions were significant (P<.05) at both time points. When compared with the PBS treatment, at 1 week but not at 4 weeks Lip-Clod reduced both MCP-1 and TGF-beta protein (P< or =.01 and P< or =.006) in the perianastomotic region of vein grafts. In whole-graft lysates, no significant difference was seen in MCP-1 protein at either time point; however, TGF-beta protein expression was significantly reduced at both 1 and 4 weeks (P=.02 and P=.004). Message analysis in whole-graft lysates at 1 week showed that MCP-1 message expression increased in the Lip-Clod group compared with the PBS group (P=.02), but no significant differences among groups for TGF-beta message levels. Results with Vector were often intermediate to results with Lip-Clod and PBS.


CONCLUSIONS
The major effect of Lip-Clod treatment on TGF-beta and MCP-1 protein levels in the perianastomotic region is observed at 1 week, and macrophage depletion with Lip-Clod inhibits graft neointimal hyperplasia and TGF-beta protein expression in whole-graft lysates at 1 and 4 weeks. These results support the concept that the infiltrating macrophages contribute a significant portion of the cytokines that facilitate intimal hyperplasia and that reducing these cytokines early after grafting influences the development of intimal hyperplasia at later time points.


CONCLUSIONS
All vascular surgeons have patients who have undergone a technically satisfying vein graft, only to have the bypass fail during the first year due to perianastomotic intimal hyperplasia (IH). We hypothesize that vein graft IH is analogous to aberrant wound healing. Central to wound healing is the recruitment of macrophages with their cytokines. This work raises the question whether clinical strategies designed to either decrease macrophages or the cytokines released by macrophages at the time of vein graft placement will be efficacious for limiting the development of IH.
Wolff
Randal A
RA
Department of Surgery, University of Wisconsin Hospital and Clinics, Veterans Affairs Medical Center, Madison 53792, USA.
Tomas
Jeffrey J
JJ
Hullett
Debra A
DA
Stark
V Emily
VE
van Rooijen
Nico
N
Hoch
John R
JR
D000818:Animals; D000963:Antimetabolites / Q000008:administration & dosage / Q000276:immunology; D001807:Blood Vessel Prosthesis; D018932:Chemokine CCL2 / Q000096:biosynthesis*; D004002:Clodronic Acid / Q000008:administration & dosage / Q000276:immunology; D006965:Hyperplasia; D007165:Immunosuppression Therapy / Q000379:methods*; D008081:Liposomes; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology; D008297:Male; D023421:Models, Animal; D051381:Rats; D011917:Rats, Inbred Lew; D016212:Transforming Growth Factor beta / Q000096:biosynthesis*; D053773:Transforming Growth Factor beta1; D017539:Tunica Intima / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D014945:Wound Healing / Q000187:drug effects / Q000502:physiology
D000963:Antimetabolites; D018932:Chemokine CCL2; D008081:Liposomes; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D004002:Clodronic Acid