Antisense to transforming growth factor-beta1 messenger RNA reduces vein graft intimal hyperplasia and monocyte chemotactic protein 1.

BACKGROUND: Autogenous vein grafts are commonly used for arterial reconstructive procedures. Their success is limited by the development of intimal hyperplasia (IH), a fibroproliferative disease that predisposes the grafts to occlusive stenosis. Mesenchymal cell proliferation and the deposition of an extracellular matrix characterize neointimal development. Increasing evidence suggests that, regardless of blood vessel type, IH results from complex interactions among vessel wall cells, infiltrating leukocytes, and cytokines. Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with powerful effects on inflammatory cell chemotaxis; smooth muscle cell, fibroblast, and endothelial cell proliferation; and extracellular matrix synthesis. METHODS: Epigastric vein to common femoral artery interposition grafts were placed in male Lewis rats and harvested at 1, 2, 4, and 12 weeks after surgery. We used replication-defective adenoviruses to deliver a control reporter gene for the enzyme beta-galactosidase (Ad-GAL), empty virus (Ad-CMVpLpA), or the sequence encoding the antisense strand of TGF-beta1 (Ad-AST). The vein graft was transduced passively in medium containing 10 7 plaque-forming units per milliliter of Ad-GAL, Ad-CMVpLpA, or Ad-AST for 20 minutes at room temperature. The adenovirus-treated grafts were compared with grafts treated with medium without virus (sham). RESULTS: The Ad-GAL control grafts showed beta-galactosidase activity from 3 days to 4 weeks. Twenty percent of cells were positive out to 2 weeks, at which time the number of cells positive for beta-galactosidase activity began to decline. Treatment with Ad-AST resulted in a significant reduction vs sham, Ad-CMVpLpA, and Ad-GAL in TGF-beta1 messenger RNA, total TGF-beta1 protein, and bioactive TGF-beta1 protein. Neointimal area was significantly reduced in the Ad-AST group vs Ad-GAL at 4 weeks, vs Ad-CMVpLpA at 4 and 12 weeks, and vs sham at 2 and 4 weeks. The medial/adventitial layer was significantly thicker in the Ad-AST group than the Ad-GAL group at 12 weeks. In addition, we studied the effect of Ad-AST on monocyte chemotactic protein 1 (MCP-1). Although the reduction in TGF-beta1 resulted in a reduction of MCP-1 messenger RNA in whole-graft homogenates and MCP-1 protein-positive staining in histologic sections from the perianastomotic region, no reduction in the number of ED1-positive cells (monocytes and macrophages) was observed. CONCLUSIONS: Perioperative antisense TGF-beta1 treatment of the vein to be used in arterial reconstructions resulted in a prolonged diminution of IH; this emphasizes the importance of TGF-beta1 in neointimal thickening and indicates that ex vivo gene therapy can reduce the vessel's predisposition to IH. CLINICAL RELEVANCE: The main cause of occlusion and graft failure after peripheral and cardiac arterial reconstruction is IH. The study of the mechanisms and mediators of IH, including TGF-beta1, should lead to future gene therapies to prevent or limit IH. The clinical effect of such treatments would be enormous, because they would increase graft longevity, thereby enhancing quality of life and enabling patients to live without the threat of limb loss or recurrent heart attack.

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. CONCLUSION: 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. CLINICAL RELEVANCE: 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.

Beta-galactosidase-tagged adventitial myofibroblasts tracked to the neointima in healing rat vein grafts.

OBJECTIVE: Myofibroblasts are present transiently in normal healing wounds. However, they have been found to persist in the stroma of neoplasms, fibrotic conditions and other pathological settings. In rat vein grafts, we have observed the prolonged presence of myofibroblasts. Our aim was to determine the origin of myofibroblasts in vein grafts. METHODS: Epigastric vein to femoral artery grafts were microsurgically placed in male Lewis rats and harvested. Neointimal development, cellular death and proliferation, and cell phenotypes were analyzed using immunohistochemistry and light and electron microscopy. To follow cellular movement in the vessel wall, vein grafts were transfected with replication-defective adenovirus containing the gene encoding beta-galactosidase (n = 50), and harvested at 1, 2, 3, 4, 5, 6, 7, 14 and 28 days. Grafts were analyzed after X-gal staining. RESULTS: Myofibroblasts were detected in the outer adventitia at 4 days, in the media at 1 week and in the developing neointima at 2 weeks. Cells tagged using adenoviral beta-galactosidase demonstrated adventitia to neointima cell migration. CONCLUSIONS: Although there may be other sources of myofibroblasts in this model, the adventitia has been shown to be an origin of myofibroblasts which subsequently migrate through the vessel wall to the neointima during graft remodeling and contribute to neointimal formation.

17 beta-estradiol attenuates intimal hyperplasia and macrophage accumulation with a reduction in monocyte chemoattractant protein 1 expression in a vein graft model.

OBJECTIVE: Autogenous vein grafts are commonly used for arterial reconstructive procedures. Their success is limited by the development of intimal hyperplasia, a fibroproliferative disease that predisposes the grafts to occlusive stenosis. Our goal was to assess whether 17 beta-estradiol (E(2)) inhibits vein graft intimal hyperplasia coincident with a reduction in monocyte chemoattractant protein 1 (MCP-1) expression and macrophage accumulation. METHOD: Male Lewis rats were implanted with time-release pellets that contained 0.5 mg E(2) (E5 group) or placebo (PL group). Epigastric vein to common femoral artery interposition grafts were harvested at 2, 4, 8, and 12 weeks after surgery. We assessed macrophage/monocytes numbers, proliferating cell nuclear antigen, MCP-1, and transforming growth factor-beta1 with use of immunohistochemistry. MCP-1 message expression was quantified by real-time polymerase chain reaction. RESULTS: The time-release pellets raised the serum E(2) level to greater than 250 pg/mL on the day of surgery. Serum E(2) level declined to 43 +/- 13 pg/mL by 4 weeks and to baseline by 6 weeks. We found that the neointimal area ratio was reduced significantly in the E5 group at 2 and 4 weeks (45%, P <.05, and 68%, P < 0.05, respectively) relative to that in the PL group. The number of proliferating cells was reduced in the E5 group. There was a significant attenuation of MCP-1 expression and of the number of macrophages accumulating in the graft with E(2) treatment. Furthermore, MCP-1 messenger ribonucleic acid expression was also significantly attenuated in the E5 group at 4 weeks when compared to the PL group. There was no significant difference between the two groups in the expression of transforming growth factor-beta1. CONCLUSIONS: E(2) treatment reduces vein-graft intimal hyperplasia coincident with a reduction in MCP-1 expression, macrophage accumulation, and cell proliferation.