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Ann Thorac Surg 2004;77:1580-1585
© 2004 The Society of Thoracic Surgeons

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Original article: cardiovascular

Local application of rapamycin inhibits neointimal hyperplasia in experimental vein grafts

^a Departments of Cardiac Surgery and Pathology, Innsbruck University, Innsbruck, Austria

Accepted for publication October 2, 2003.

^* Address reprint requests to Dr Schachner, Innsbruck University Hospital, Department of Cardiac Surgery, Anichstrasse 35, A-6020 Innsbruck, Austria
e-mail: thomas.schachner{at}uibk.ac.at

	Abstract

Top Abstract Introduction Material and methods Results Comment Conclusion Acknowledgments References

 
BACKGROUND: Rapamycin is an immunosuppressive agent which also exhibits marked antiproliferative properties. Rapamycin coated stents have been demonstrated to suppress restenosis in experimental and clinical studies of percutaneous coronary catheter intervention. We investigated whether rapamycin can reduce neointima formation in a mouse model of vein graft disease.

METHODS: C57BL6J mice underwent interposition of the inferior vena cava from isogenic donor mice into the common carotid artery using a previously described cuff technique. In the treatment group, 100 µg or 200 µg of rapamycin was applied locally in pluronic gel. The control group did not receive local treatment. Grafts were harvested at 1, 2, 4, and 6 weeks and underwent morphometric analysis as well as immunohistochemical analysis.

RESULTS: In grafted veins without treatment (controls), median intimal thickness was 9.6 (6.4 to 29)µm, 11.9 (7.9 to 39.9)µm, 46.6 (12.4 to 57.7)µm, and 57.5 (32.5 to 71.1)µm after 1, 2, 4, and 6 weeks, respectively. Treatment with 100 µg or 200 µg rapamycin showed a dose dependant reduction of intimal thickness. In the 200 µg rapamycin treatment group the intimal thickness was 4.3 (3.4 to 5.6)µm, 3.8 (3.2 to 6.3)µm, 17.1 (4.8 to 63)µm, and 33.9 (11.3 to 80.3)µm after 1, 2, 4, and 6 weeks, respectively. This difference of intimal thickness of 200 µg treated animals compared with controls was statistically significant at 1 and 2 weeks. Immunohistochemically the reduction of intimal thickness was associated with a decreased amount of infiltration of CD-8 positive cells and a decreased amount of metallothionein positive cells in the rapamycin treated grafts.

CONCLUSIONS: We conclude that perivascular application of rapamycin inhibits neointimal hyperplasia of vein grafts in a mouse model. These results suggest that rapamycin may have a therapeutic potential for the treatment of vein graft disease.

	Introduction

Top Abstract Introduction Material and methods Results Comment Conclusion Acknowledgments References

 
Occlusion of the graft lumen is the main complication in the follow-up of patients after aortocoronary vein bypass grafting. This occurs in up to 15% in the first year, and increases up to 50% occluded veins after 10 years [1]. Of the patent vein grafts, 7% show signs of degeneration after 1 year and 77% after 10 years, respectively [2]. Neointimal hyperplasia, which develops immediately after grafting, is the most important early change of the grafted vein. Although not solely responsible for the graft failure, this neointimal process is to be regarded as the guiding track easing the development of fatal sclerotic changes [3].

Rapamycin is a macrolide antibiotic with marked immunosuppressive and antiproliferative properties [4–8]. The drug inhibits the progression of the cells from the G₁ to the S phase within the mitotic cycle. One key action is that the drug forms a complex with FK506 binding protein (FKBP); the resulting complex inhibits the mammalian target of rapamycin (mTOR). The mTOR is activated, via autophosphorylation, by cytokine or growth factor-induced signals. The activated mTOR activates through phosphorylation other kinases, thus intervening in the complex process of cell cycle regulation [9]. Another well-established effect of rapamycin in this context is the inhibition of vascular smooth muscle cell (VSMC) migration [10–12] and reversion of chronic graft vascular disease in a cardiac allograft model [13]. The drug is clinically used in transplantation medicine [14–16] and as a coating of coronary artery stents [17–19].

To study the effects of rapamycin on neointimal hyperplasia in vein grafts we chose a mouse model that was developed at our institutions [20], and aimed to investigate its effect on neointimal hyperplasia using a surgically feasible application method. Pluronic-127 gel (BASF, Germany) has been demonstrated to be an effective carrier for a drug and does not influence neointimal hyperplasia in this animal model [21].

	Material and methods

Top Abstract Introduction Material and methods Results Comment Conclusion Acknowledgments References

 
Mice and vein grafting
Three month-old male C57BL/6J mice were purchased from Harlan-Winkelmann (Borchen, Germany). They were maintained at 24°C and received food and water ad libitum. All procedures were performed according to protocols approved by the Austrian Ministry of Science according to Section 8 of the law on animal experiments and all animals were treated according to the Guide for the Use and Care of Laboratory Animals, published by the National Institutes of Health (National Institutes of Health publication No. 85 to 23, revised 1985).

Donor mice were anesthetized with pentobarbital sodium (50 mg/kg intramuscularly [IM]). Atropine sulfate (1 mg/kg IM) was administered for cardiorespiratory stabilization. A median abdominal incision was made and 0.5 mL saline solution containing 100 U/mL of heparin was injected into the inferior vena cava. With two lateral thoracotomies the anterior thoracic cage was removed. The inferior vena cava was separated from surrounding fat. After an incision of the right atrium the graft was washed with saline solution containing 100 U/mL heparin. The vena cava was removed and stored in cold Ringer's lactate solution until grafting.

The recipient mice were anesthetized as described above. Through a median cervical incision, after resection of the right sterno-cleido-mastoid muscle, the right common carotid artery was exposed and mobilized from the bifurcation as far as possible towards the proximal and distal ends. The vessel was ligated with two 8/0 silk sutures and divided. The proximal and distal stumps of the carotid artery were passed through polyethylene cuffs with an inside diameter of 0.5 mm (Portex LTD, London, UK). These cuffs were prepared with a length of 1 mm and a handlelike extension of 1 mm. The vessel was occluded and fixed to the extension of the cuff with Yasargil micro clamps. After removing the ligature the artery was everted over the cuff body and fixed to the cuff with an 8/0 silk suture. Both the proximal and distal stumps were prepared the same way. The inferior vena cava from the donor mouse was interposed between the carotid artery cuffs by pulling the end of the vein over the everted and cuffed parts of the artery fixing it with 8/0 silk sutures. After removing the Yasargil clamps good blood flow and pulsations had to be observed for a successful operation.

Rapamycin application and tissue preparation
In the treatment groups 100 µg and 200 µg of rapamycin (Wyeth, Collegeville, PA) were applied in the perivascular spaces of the grafted vein. As a carrier we used 0.1 mL of 20% Pluronic-F 127 gel. The control group did not receive local treatment. The wound was closed with a 5/0 Vicryl (Ethicon, Inc, Somerville, NJ) running suture.

For histologic analysis the animals underwent autopsy 1, 2, 4, and 6 weeks postoperatively. The grafts were perfusion fixed with 4% phosphate-buffered formaldehyde via puncture of the left ventricle, as previously described [20]. The interposed vein segments were cut out at the cuff ends and fixed with 4% phosphate-buffered formaldehyde. Due to the small size of the grafts, they were embedded in a piece of mouse liver tissue. A suitable sized liver fragment was cut off the organ and incised orthogonally to the cut surface. The vein was placed into this incision, strictly taking care of a correct 90° angle to the anticipated cutting plane of the microtome. The incision was closed by a single mattress suture to prevent loss of the graft by shrinking due to formalin fixation. Consecutively the compounds were formalin fixed and paraffin embedded.

Histology and lesion quantification
Sections (4-µm thick) were cut and were Elastica van Gieson stained for measurement of the intimal thickness (IT). Digital photomicrographs of all vessels were taken using a SONY DSC-70 camera (SONY Corp, Japan) with a resolution of 2048 · 1586 pixels with a color depth of 24 bits per pixel in RGB. The microscope used was a Zeiss Axioplan (Zeiss, Germany) with plan flourit optics with a photoadapter. For getting an overview picture, a 10x magnification objective was used. The zoom objective of the camera was adjusted to full zoom, and afterwards reduced by 3 microsteps. For measurements, a 20x magnification objective was used with the same camera adjustment. All photographs were saved in JPEG format. The measurements were done using OPTIMAS 5.0 image analysis software (MediaCybernetics, Silver Spring, MD) on an IBM compatible PC. For reproducibility reasons the overview pictures were used to mark the exact locations where the thickness measurements were taken. The intimal thickness measurements were performed by two experienced observers (T.M., A.O.). For achieving a reproducible result, the cross sections of the veins were divided in four quadrants. In each quadrant, three measurements were made. The median value of all measurements was regarded as representative for the intimal thickness.

Immunohistochemistry
Immunohistochemistry was carried out on paraffin embedded sections of animals 4 weeks postoperatively. The following primary antibodies were used: mouse anti-CD-8 (clone C8/144B IgG1-kappa; Dako Inc, Glostrup, Denmark) for detection of T-lymphocytes; mouse antimetallothionein (clone E9 IgG1-kappa; Dako Inc, Glostrup, Denmark); mouse anti smooth muscle actin (SMA) (IgG1, Biogenex Inc, San Ramon, CA), mouse anti platelet derived growth factor receptor alpha (PDGFR) (Santa Cruz Biotechnology, CA). The sections were visualized using NexES IHC automatic immunohistochemical stainer (Ventana Medical Systems, Tucson, AZ) with diaminobenzidine basic kit (Ventana Medical Systems, Tucson, AZ).

The results were quantified by a pathologist (A.T.) by counting the number of positively staining cells in medium power fields (200x magnification; 0.747 mm²/field). The number of counted cells was extrapolated to 1.0 or 0.2 mm², respectively. Staining intensity was not quantified, but only intensities at least two times stronger than background have been taken in consideration. Since cellular borders of migrated myofibroblasts were hardly ever to be defined, the total area of SMA positive patches were evaluated. In the case of metallothionein expression in the neointima, only a description analysis was performed, since the staining was not confined to cells but to the matrix.

Statistical analysis
The SPSS software (SPSS 10.0; SPSS, Chicago, IL) for Windows was used for statistical analysis. Neointimal thickness is given as median and range. Comparisons of histologic measurements of the intimal thickness and of immunohistochemistry were made by the Mann-Whitney U test. Results were considered statistically significant at p values of less than 0.05.

	Results

Top Abstract Introduction Material and methods Results Comment Conclusion Acknowledgments References

 
The median intimal thickness in native veins was 1.6 (1.3 to 2.4)µm. Neointimal hyperplasia developed in all vein grafts (Fig 1). In grafted veins without treatment (controls, n = 6 or 7 at each point of time) median intimal thickness was 9.6 (6.4 to 29)µm, 11.9 (7.9 to 39.9)µm, 46.6 (12.4 to 57.7)µm, and 57.5 (32.5 to 71.1)µm after 1, 2, 4, and 6 weeks, respectively. Veins treated with 100 µg rapamycin (n = 6 or 7 at each point of time) showed a trend towards reduction of intimal thickness with 7.4 (1.7 to 17.7)µm, 5.8 (2.4 to 18)µm, 42.2 (12.4 to 65)µm, and 48.3 (18.9 to 75.3)µm after 1 week (p = 0.29 vs controls), 2 weeks (p = 0.04 vs controls), 4 weeks (p = 1 vs controls), and 6 weeks (p = 0.48 vs controls). The difference to controls, however, reached statistical significance only at 2 weeks postoperatively. Application of 200 µg rapamycin (n = 6 or 7 at each point of time) led to a reduced intimal thickness of 4.3 (3.4 to 5.6)µm, 3.8 (3.2 to 6.3)µm, 17.1 (4.8 to 63)µm and 33.9 (11.3 to 80.3)µm after 1 week (p = 0.002 vs controls), 2 weeks (p = 0.002 vs controls), 4 weeks (p = 0.26 vs controls), and 6 weeks (p = 0.18 vs controls). This was a reduction of neointimal thickness in rapamycin (200 µg) treated vein grafts of 55%, 68%, 63%, and 41% after 1, 2, 4, and 6 weeks, respectively, compared with controls. The difference to controls was statistically significant at 1 and 2 weeks.

View larger version (30K): [in this window] [in a new window]   Fig 1. Reduction of neointimal hyperplasia in vein grafts by perivascular treatment with rapamycin. = control; = rapamycin 100 µg; = rapamycin 200 µg.

View larger version (117K): [in this window] [in a new window]   Fig 2. (Upper a and b) Quantitative differences in the distribution of CD-8 positive T cells in the adventitia of rapamycin (200 µg)-treated (a) animals and controls (b) at 4 weeks postoperatively (200 fold magnification, immunoperoxidase stain). Note the positively stained cells in the lower part of the left figure (rapamycin-treated animal). (Lower a and b) Immunohistochemical staining for metallothionein in rapamycin (200 µg)-treated (a) animals and controls (b) at 4 weeks postoperatively (40 fold magnification, immunoperoxidase stain). Note the large patch in the neointima of the rapamycin- treated animal.

The metallothionein positivity corresponded to the thickness of the neointima and was patchier in the rapamycin treated cases. The number of metallothionein positive cells (mainly histioctyes) in the adventitia and the media was significantly decreased in rapamycin cases compared with controls (Fig 2 and Table 1).

Immunohistochemical staining with anti SMA antibody showed a clear expression in the neointima and adventitia, whereas rarely positive cells were found in the media. In the rapamycin treated group the total area of SMA positive cells was slightly increased compared with controls; however, this difference was not statistically significant. The number of SMA positive cells in the adventitia of both groups did not differ (Table 1).

	Comment

Top Abstract Introduction Material and methods Results Comment Conclusion Acknowledgments References

 
In our study we investigated the effect of perivascularly applied rapamycin on the development of neointimal hyperplasia in experimental vein grafts. We chose rapamycin to test the effect of its antiproliferative properties and of local immunosuppression on the pathogenesis of vein graft disease. Hirko and colleagues [22] demonstrated a reduced intimal thickness and decreased inflammatory cell infiltration in canine vein grafts after orally applied cyclosporine therapy.

With the use of pluronic gel some prolonging of drug contact to the graft could be reached but one disadvantage of this method, however, is the limited release time of the gel. As an example, Fulton and colleagues [23] found that after 5 days, 80% of antisense oligonucleotides to proliferating cell nuclear antigen (PCNA) were released from pluronic gel.

In our experiment we could demonstrate an inhibitory effect of rapamycin on the development of neointimal hyperplasia. This effect was dose dependent and reached statistical significance at concentrations of 200 µg in 0,1 mL gel, where the intimal thickness was reduced by 50% after 1 week and by almost 70% after 2 weeks compared with controls. Suzuki and colleagues [24] found, after 4 weeks, a 50% reduction of in stent stenosis with rapamycin coated stents compared with bare metal stents in pigs. In human coronary arteries rapamycin coated stents showed significantly less neointimal hyperplasia than bare metal stents [18].

The immunohistochemical staining with anti CD8 antibody revealed the presence of cytotoxic T–lymphocytes in the majority of control cases. On the contrary, they were found in the minority of rapamycin treated veins. In the control group the average amount of CD-8 positive cells was about five times more than in the rapamycin (200 µg) treated group. Inflammation plays an important role in the development of vein graft neointimal hyperplasia. An early infiltration of the vein graft with monocytes and macrophages is usually found [25]. T-lymphocytes, which accumulate in stenotic aortocoronary saphenous vein grafts, are thought to initiate and modulate immune reactions that lead to the progression of graft arteriosclerosis [26]. In accordance with these results we found infiltration of the grafted veins with CD-8 positive lymphocytes, which was reduced in veins treated with rapamycin.

Various growth factors contribute to the development of neointimal hyperplasia, one of which is platelet-derived growth factor (PDGF) [3, 27]. The PDGF is a positive regulator of the cell cycle. It stimulates migration and proliferation and it inhibits apoptosis. There are two different protein subunits that form either homodimers (PDGF-AA, PDGF-BB) or heterodimers (PDGF-AB). There are two corresponding receptors (PDGFR, PDGFRß). All PDGF isoforms can bind to PDGFR whereas PDGF-AA and PDGF-AB cannot bind to PDGFRß [27]. In native human saphenous veins PDGFR expression is less than expression of PDGFRß [28]. In the balloon injury model of the carotid artery in rats, Sirois and colleagues [29] found that both PDGFR and PDGFRß were overexpressed in the media and in the neointima 2 weeks after injury. The PDGFR blockade inhibited intimal hyperplasia in balloon injury models [30, 31] thus suggesting a role in vein graft neointimal hyperplasia too. We found a strong presence of PDGFR positive cells in controls as well as in rapamycin treated veins. The number of PDGFR positive cells in rapamycin treated vein grafts was about half of the number of PDGFR positive cells in controls. However, this difference did not reach statistical significance.

Metallothionein is a highly conserved, ubiquitously expressed low molecular weight protein. The expression of metallothionein is induced by heavy metals, heat, inflammation, and other stress conditions. Kang and colleagues [32] demonstrated that metallothionein inhibits ischemia reperfusion injury in the mouse heart. Metallothioneins can protect against oxidative damage by scavenging harmful oxygen radicals, which can be generated by activated leukocytes [33]. It has been shown that overexpression of metallothionein stimulates cellular multiplication and that downregulation of metallothionein by antisense oligomers has a growth inhibitory effect [34]. In our study we found a four times higher amount of metallothionein positive cells in controls compared with rapamycin treated vein grafts. This could be interpreted as a sign of reduced cellular growth activity and a reduced cellular stress reaction in rapamycin treated vein grafts.

Alpha smooth muscle actin is associated with the contractile apparatus, and it is decreased in neointimal SMCs compared with medial SMCs [35]. Cultured media SMCs from porcine coronary arteries show a strong expression of alpha smooth muscle actin. Cultured adventitial fibroblasts from porcine coronary arteries initially show no expression of alpha smooth muscle actin but they develop its expression within 10 days in culture, which is interpreted as myofibroblastic transformation [36]. Wolf and colleagues [37] have demonstrated that growing coronary artery collaterals show a weak expression of alpha smooth muscle actin in the neointima, whereas after remodeling into mature and thicker vessels the neointima contained more alpha smooth muscle actin. In our experiments rapamycin treated vein grafts showed about 30% higher density of smooth muscle actin positive cells than controls. These findings would be consistent with remodeling of the vein graft towards a more contractile phenotype under the treatment with rapamycin; however, this difference was not statistically significant.

	Conclusion

Top Abstract Introduction Material and methods Results Comment Conclusion Acknowledgments References

 
We conclude that perivascular application of rapamycin substantially inhibits neointimal hyperplasia of vein grafts in a mouse model. This reduction of vein graft disease is associated with a decreased amount of infiltration of CD-8 positive cells and a decreased amount of metallothionein in the rapamycin treated grafts. These results suggest that rapamycin may have a therapeutic potential for the treatment of vein graft disease.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Conclusion Acknowledgments References

 
This work was, in part, supported by a grant from the Fund for Research Development at Innsbruck University Hospital and from SKWB Schoeller Bank.

	References

Top Abstract Introduction Material and methods Results Comment Conclusion Acknowledgments References

 

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