Reduction In Intimal Thickening Research Articles

Prevention of vein graft intimal hyperplasia with photochemical tissue passivation

Saphenous vein is the conduit of choice for bypass grafting. Saphenous vein grafts have poor long-term patency rates because of intimal hyperplasia (IH) and subsequent accelerated atherosclerosis. One of the primary triggers of IH is endothelial injury resulting from excessive dilation of the vein after exposure to arterial pressures. Photochemical tissue passivation (PTP) is a technology that cross-links adventitial collagen by a light-activated process, which limits dilation by improving vessel compliance. The objective of this study was to investigate whether PTP limits the development of IH in a rodent venous interposition graft model. PTP is accomplished by coating venous adventitia with a photosensitizing dye and exposing it to light. To assess the degree of collagen cross-linking after PTP treatment, a biodegradation assay was performed. Venous interposition grafts were placed in the femoral artery of Sprague-Dawley rats. Rats were euthanized after 4weeks, and intimal thickness was measured histologically. Vein dilation at the time of the initial procedure was also measured. Time to digestion was 63± 7minutes for controls, 101± 2.4minutes for rose bengal (RB), and 300± 0minutes for PTP (P< .001 PTP vs control). A total of 37 animals underwent the procedure: 12 PTP, 12 RB only, and 13 untreated controls. Dilation of the graft after clamp release was 99% for control, 65% for RB only, and 19% for PTP-treated (P< .001 PTP vs control). Intimal thickness was 77± 59μm in controls, 60± 27μm in RB only, and 33± 28μm in PTP-treated grafts. There was a statistically significant 57% reduction in intimal thickness after treatment with PTP compared with untreated controls (P= .03). PTP treatment of venous interposition grafts in a rat model resulted in significant collagen cross-linking, decreased vessel compliance, and significant reduction in IH.

Prevention of Neointimal Hyperplasia by Local Application of Lentiviral Vectors Encoding Pin1 shRNA in Pluronic F127.

Inhibition of intimal hyperplasia plays an important role in preventing restenosis. Previously, we reported the provocative role of Pin1 in regulating vascular smooth muscle cell (VSMC) proliferation. Here we intended to identify whether locally delivered lentivirus-mediated siPin1 via pluronic F127 (PF127) could inhibit neointimal formation and further explore the potential mechanisms thereof. In vitro studies revealed that lentivirus-mediated siPin1 dispersed in PF127 suppressed proliferation and induced senescence in VSMCs. Reduction of Pin1 expression resulted in a decrease of phospho-Akt (p-Akt) expression level in VSMCs. Reactivation of Akt phosphorylation overcame the siPin1-mediated senescence. In a rat wire injury model, periadventitial delivery of lentivirus-mediated siPin1 via PF127 produced inhibition of intimal hyperplasia 14 days after injury without evidence for toxicity. Furthermore, the reduction of intimal thickness was associated with a decreased amount of PCNA positive cells, decreased telomerase activity and shortened telomere length. Therefore, these results suggest that PF127 delivery of lentivirus-mediated siPin1 to artery may have a therapeutic potential for the treatment of restenosis.

Role of the VEGF 936 gene polymorphism and VEGF-A levels in the late-term arteriovenous fistula thrombosis in patients undergoing hemodialysis.

Vascular access is vital for hemodialysis patients. A major factor that facilitates arteriovenous (AV) fistula failure is stenosis and thrombosis due to intimal hyperplasia developing in the venous segment of AV fistula. It has been reported that VEGF accelerated re-endothelialization, reduction in intimal thickening, and/or mural thrombus formed in the injured vascular structures. In this study, we aimed to identify the effect of the VEGF 936 gene polymorphism and vascular endothelial growth factor-A (VEGF-A) levels in the late period of AV fistula loss in hemodialysis patients. The study was carried out with a patient group of 42 individuals who experienced two or more fistula thrombosis in the late period after the AV fistula operation and also a control group of 38 patients who have not had any AV fistula thrombosis history for 3 years or more. All participants were assessed for VEGF-936C/T gene polymorphism and VEGF-A levels. VEGF-936C/T genotypes were determined in the large proportion in the control group (31.6 %), while VEGF-936C/C genotypes were determined in a large proportion in the patient group (90.5 %). Individuals carrying the VEGF-936C/C genotype had an increased risk of 5.54 for getting AV fistula thrombosis. The VEGF-A levels of patient group (27.3 ± 43.5 pg/ml) were significantly lower than those of the control group (70.7 ± 53.1 pg/ml). There is an increased risk of AV fistula thrombosis in individuals carrying the VEGF-936C/C genotype. The other renal replacement modalities should be considered in patients with this genotype. As a result, it will be possible to prevent the morbidity and mortality due to fistula failure.

Attenuation of Transplant Arteriosclerosis by Oral Feeding of MHC Encoding Chitosan-DNA Nanoparticles Is Mediated by Humoral Immunity

Purpose One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the allo-immune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation. Methods and Materials We performed fully allogeneic mouse abdominal aortic transplantats using C57BL/6 (H2 b ) mice as donors and CBA.J (H2 k ) mice as recipients Aortic grafts were analyzed by histology and morphometry on day 30 after transplantation, levels of circulating alloantibodies were detected by FACS analysis. Results Pre-treatment of recipient mice with chitosan-DNA nanoparticles encoding for K b , one of the MHC-I molecules of the donor, resulted in a significant reduction of intimal proliferation compared to untreated controls. When Ovalbumin was fed instead of K b encoding nanoparticles (K b -NP) or Balb/c (H2 d ) grafts were used instead of C57BL/6 (H2 b ) grafts as antigen controls, both groups showed no reduction of intimal thickness indicating an antigen-specific mechanism. In addition, analysis of peripheral blood of the transplanted mice showed significant suppression of alloantibody formation in the K b -NP fed group compared to all other allogeneic transplanted groups suggesting modulation of the humoral immune response. Conclusions These results demonstrate the potential of chitosan-DNA nanoparticles to induce K b -specific tolerance and to reduce the development of transplant arteriosclerosis.

Antioxidant Role of Vitamin E in Atherogenesis induced by Hyperfibrinogenemia

We used an experimental model of atherogenesis to evaluate the effect of vitamin E on oxidative stress induced by hyperfibrinogenemia (HF) and the possible normalization of oxidative stress markers. The following variables were studied: nitric oxide (NO), L-citrulline, superoxide dismutase (SOD) activity and regression of histopathological lesions in the thoracic aorta. The study was performed in 36 Wistar rats that were divided into three groups of 12 rats each: A, control group; B, HF for 90 days; C, HF for 90 days + vitamin E. Hyperfibrinogenemia was induced by the injection of epinephrine (0.1 ml/day/rat) during 90 days. The dose of vitamin E was 2 mg/day/rat during 75 days. We measured the plasma levels of fibrinogen (mg/dl), NO (uM) and L-citrulline (mM); SOD activity (U/ml) was assayed in red cell lysates using spectrophotometry. The histopathological sections of the thoracic aorta were examined using light microscopy (LM). Statistical analysis was performed using MANOVA and Fisher’s test; a p value <0.05 was considered statistically significant. Rats in group B had a significant increase in fibrinogen levels B (407±8.9 mg/dl) compared to groups A (203±9 mg/dl) and C (191.58±17.79 mg/dl) (p<0.001). We observed a significant decrease in NO in group B (13.73±1.76 uM) versus groups A (23.58±0.08 uM) and C (26.64±3.65 uM) (p<0.001). L-citrulline increased significantly in groups B (4.99±0.18 mM) and C (6.60±0.16 mM) compared to group A (3.03±0.13 mM) (p<0.001). SOD activity was greater in groups B (251.67±10.34 U/ml) and C (304.75±10.43 U/ml) versus group A (139.44±4.74 U/ml) (p<0.001). Light microscopic examination revealed the presence of endothelial denudation, intimal thickening and vessel wall protrusion in group B (90%), while recovery of endothelial denudation and a 50% reduction in intimal thickening was observed in group C (p<0.001). High SOD activity might be insufficient to prevent abnormalities in the oxidative stress pathway induced by HF. Vitamin E would stop the chain reaction initiated by free radicals and thus decrease the superoxide anion, stimulating the bioavailability of NO with normalization of fibrinogen plasma levels.

Local Application of Rapamycin Inhibits Vein Graft Restenosis in Rabbits

objective The aim of this study was to investigate whether local application of rapamycin reduced neointimal formation in a rabbit model of venous disease. Methods Each rabbit (n = 30; 2.5–3.5 kg) received a treated and a control graft. For the treated graft, 0.3 rapamycin mg was applied locally in Pluronic gel. The control graft received only the Pluronic gel. Grafts were harvested at 28 days for morphometric, immunohistochemical, and flow cytometry analysis. Results In the control group, the intimal thickness was 63.72 ± 14.0 μm; in treated group, it was 77.76 ± 14.9 μm ( P < .05). Immunohistochemically, proliferation cell nuclear antigen–positive cells were present in the control group and in the treatment group but not in normal external jugular veins. The control group showed much stronger expression than the treatment group ( P < .05). Flow cytometry showed, among the control group, decreased G 0G 1-stage cells and increased S/G 2M-stage cells. Among the treatment group, S/G 2M stage cells were decreased compared with the control. The progression indexes of the control and treatment groups were 29.3 ± 7.15 and 20.1 ± 9.48, respectively, a remarkable decrease ( P < .05). Proliferating cells in the control group were apparently inhibited by rapamycin. The treatment group showed positive staining for P27 kip1, but neither the control group nor the normal external jugular veins showed positive results ( P < .05). The degree of reduction in intimal thickness and inhibition of proliferating cells in the treatment group correlated with the expression of P27 kip1. Conclusions We observed that perivascular application of rapamycin inhibited neointimal hyperplasia of vein grafts in a rabbit model, an effect that appeared to result from increased P27 kip1 expression.

Everolimus Inhibits Monocyte/Macrophage Migration in Vitro and Their Accumulation in Carotid Lesions of Cholesterol-Fed Rabbits

Monocytes/macrophages recruited into the arterial wall during atherogenesis are crucial in the initiation and progression of atherosclerosis and play a fundamental role in the destabilization process that is the main causal event of acute coronary syndromes. In the present study, we investigated the effect of the mammalian target of rapamycin inhibitor everolimus on macrophage accumulation within carotid lesions elicited by perivascular collar placement in cholesterol-fed rabbits. Everolimus (1.5 mg/kg given 1 day before collaring followed by 1 mg/kg/day for 14 days, administered by oral gavage) markedly decreased lesion macrophage content as compared with vehicle control (-65%; p < 0.01). This effect was associated with a reduction in intimal thickening and occurred in the absence of changes in plasma cholesterol concentrations. To gain insights on the potential mechanism(s) underlying this effect, we investigated the influence of everolimus on chemoattractant-induced migration of human monocytes in vitro. Pretreatment with therapeutic concentrations of everolimus (10 nM) significantly lowered monocyte chemotaxis in response to various chemotactic factors (i.e., monocyte chemoattractant protein-1/CCL2, fractalkine/CX3CL1, interleukin-8/CXCL8, complement fragment 5a, or N-formyl-Met-Leu-Phe) without inducing monocyte cell death. These results suggest that everolimus may favorably influence the atherosclerotic process by affecting the recruitment of monocytes into early lesions.

Editorial Comment: Heart Transplantation, Cardiac Allograft Vasculopathy and Proliferation Signal Inhibitors

Editorial Comment: Heart Transplantation, Cardiac Allograft Vasculopathy and Proliferation Signal Inhibitors

Gene transfer using the mature form of VEGF-D reduces neointimal thickening through nitric oxide-dependent mechanism

Gene transfer to the vessel wall using vascular endothelial growth factors (VEGFs) has shown therapeutic potential for the treatment of restenosis. In this study, we evaluated the effect of catheter-mediated adenoviral (Ad) gene transfer of the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) in balloon-denuded cholesterol-fed rabbit aorta. AdLacZ was used as a control. Transduced VEGF-D(DeltaNDeltaC) mRNA was detectable in the arterial wall with RT-PCR at 6, 14 and 28 days. Gene transfer efficiency as detected with X-gal staining 6 days after the AdLacZ transduction was 1.91 +/- 1.32% in intima. AdVEGF-D(DeltaNDeltaC) gene transfer led to 52% reduction in intima/media ratio (I/M) as compared to the AdLacZ controls at 14 days time point. At 6 days there were no differences in I/M, but the number of macrophages in the vessel wall was 85% lower in the AdVEGF-D(DeltaNDeltaC) group as compared to the controls. The therapeutic effect was no longer detectable 28 days after the gene transfer. The therapeutic effect of VEGF-D(DeltaNDeltaC) was nitric oxide (NO)-dependent as the feeding of NO synthase inhibitor, L-NAME, blocked the reduction in intimal thickening. It is concluded that AdVEGF-D(DeltaNDeltaC) gene transfer reduces intimal thickening and macrophage influx into the vessel wall in balloon-denuded rabbit aortas.

The effect of tyrphostin AG-556 on intimal thickening in a mouse model of arterial injury

Background. Inflammation has been shown to play an important role in promoting the response to arterial injury and proinflammatory cytokines, such as tumor necrosis factor (TNF) alpha, are candidate mediators. AG-556 is a tyrosine kinase inhibitor proven to be effective in a model of multiple sclerosis-like syndrome in mice due to its immunomodulating effect. In the current study, we investigated the effect of the tyrphostin AG-556 on neointimal thickening and cytokine profile in a model of arterial injury in the mouse. Methods. Injury was induced by external cuff placement on the left femoral artery of wild-type C57BL/6 mice. AG-556 dissolved in DMSO was injected intraperitoneally daily to the injured mice in a dosage of 2 mg/mouse. Control mice received DMSO injections. Histological analysis was carried out to assess neointimal formation. Splenocytes were cultured in the absence and presence of a mitogen for evaluation of thymidine incorporation and cytokine production. Results. AG-556 treatment significantly attenuated intimal thickening (43,000 ± 17,000 μm 2; n = 11) when compared to DMSO administration (286,000 ± 127,000 μm 2; n = 10; P < 0.05). Basal interferon-gamma production by splenocytes from AG-556-treated mice was increased by approximately 20-fold in comparison with levels in DMSO-treated animals, whereas Con-A induced secretion of the cytokine was similar between both groups. Levels of TNF-alpha, IL-4 and IL-10 in the culture supernatant from treated and non-treated animals did not differ significantly. Conclusion. The tyrosine kinase inhibitor AG-556 may have a role in the reduction of intimal thickening. The effect could be mediated via an immune modulating effect involving a significant increase in the smooth muscle cell inhibitory cytokine IFN-gamma.

Mycophenolate mofetil inhibits intimal hyperplasia and attenuates the expression of genes favouring smooth muscle cell proliferation and migration

Aim Intimal hyperplasia remains the leading cause of late graft failure following heart transplantation. The immunosuppressive drug mycophenolate mofetil has been shown to inhibit the development of intimal hyperplasia. This study aimed to assess the efficacy of a combination of mycophenolate mofetil, calcineurin inhibition, and sirolimus on the development of intimal hyperplasia. Methods Male Sprague-Dawley rats received mycophenolate mofetil (30 mg/kg per day) and either tacrolimus (0.1 mg/kg per day), cyclosporine (5 mg/kg per day), or sirolimus (0.05 mg/kg per day) and were compared to an untreated control group. All animals underwent left common carotid artery balloon angioplasty. Morphometric analysis was performed on representative transverse sections, and intima medial ratios calculated at 2 weeks. Profibrotic gene expression was assessed with competitive RT-PCR at 2 weeks for metalloproteinase-2, metalloproteinase-9, TIMP-1, collagen III, and TGF-β. Sections were stained with sirius red, and extracellular matrix deposition was quantified. Results Mycophenolate mofetil in combination with rapamycin was associated with the greatest reduction in intimal thickening (intima medial ratio 0.79; range 0.45–0.86), compared to its combination with either cyclosporine (1.41; range 1.06–1.68, P < .02) or tacrolimus (0.93; range 0.81–1.37, P < .05) and controls (1.47; range 1.02–2.04, P < .005). Mycophenolate mofetil and rapamycin significantly inhibited all profibrotic genes studied compared to controls ( P < .01) but there were no differences between tacrolimus and cyclosporine. Mycophenolate mofetil and sirolimus significantly attenuated extracellular matrix deposition compared to tacrolimus and cyclosporin ( P < .023). Conclusion The benefits of mycophenolate mofetil in combination with sirolimus are preferential over those with cyclosporine or tacrolimus. Randomised trials are warranted to assess if mycophenolate mofetil should be an alternative agent to calcineurin-inhibitors when used in combination with sirolimus.

Perivascular application of C-type natriuretic peptide attenuates neointimal hyperplasia in experimental vein grafts.

C-type natriuretic peptide (CNP), which is produced by vascular endothelial cells, exhibits anti-proliferative and anti-inflammatory effects. Cytotoxic T-lymphocytes may be involved in vein graft disease. Attenuation of vein graft disease necessitates a remodelling of the arterialized vein towards a more contractile phenotype which is characterized, among other factors, by the calponin amount. We investigated the effects of perivascularly applied CNP in a mouse model of vein graft disease. 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, 10(-6)mol/l of CNP were applied locally in pluronic gel. The control group did not receive local treatment. Grafts were harvested at 1, 2, 4, and 8 weeks and underwent morphometric analysis as well as immunohistochemical analysis. In grafted veins without treatment (controls) median intimal thickness was 10 (6-29), 12 (8-40)microm, was 47 (12-58), and 79 (62-146)microm after 1, 2, 4 and 8 weeks, respectively. In the treatment groups, which received 10(-6)mol/l of CNP, the intimal thickness was 5 (3-6), 6 (4-15), 32 (5-54), and 43 (39-70)microm after 1, 2, 4 and 8 weeks, respectively. This reduction of intimal thickness was significant at 1, 2 and 8 weeks. Immunohistochemically, the reduction of intimal thickness was associated with a decreased infiltration of CD-8 positive cells and an increased amount of calponin in the CNP-treated grafts. We conclude that perivascular application of CNP inhibits neointimal hyperplasia of vein grafts in a mouse model. These results suggest that CNP may have a therapeutic potential for the prevention of vein graft disease.

Differential effects of modern immunosuppressive agents on the development of intimal hyperplasia.

Modern immunosuppressive agents such as tacrolimus and rapamycin are claimed to be associated with a reduction in vascular narrowing, a central feature of chronic rejection. This study assesses the effect of cyclosporine, tacrolimus and rapamycin on the development of intimal thickening, fibrosis-associated genes and deposition of extracellular matrix (ECM) proteins in a model of intimal hyperplasia. Male Sprague-Dawley rats received either no treatment or 5 mg/kg cyclosporine, 0.1 mg/kg tacrolimus or 0.05 mg/kg rapamycin. Animals underwent left common carotid balloon angioplasty, and intima medial ratios, pro-fibrotic gene expression and ECM accumulation were calculated at 14 and 28 days. Cyclosporine was associated with increased intimal thickening compared to controls ( P < 0.004). Tacrolimus had no effect on intimal thickening, whilst rapamycin significantly inhibited intimal thickening at both 14 and 28 days ( P < 0.004 and P < 0.026, respectively). All groups significantly inhibited matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, transforming growth factor (TGF)-beta and collagen III expression at 14 days ( P < 0.001), but increased ECM deposition. However, rapamycin marginally reduced ECM deposition compared to cyclosporine ( P < 0.06). Treatment with cyclosporine was associated with worsening of vascular narrowing, whilst rapamycin showed a beneficial reduction in intimal thickening. Treatment with all immunosuppressive agents resulted in increased ECM deposition. Rapamycin may halt the progression of vascular narrowing compared to both cyclosporine and tacrolimus.

Effects of the combination of rapamycin with tacrolimus or cyclosporin on experimental intimal hyperplasia.

Allograft vasculopathy remains the leading cause of late allograft failure following transplantation and can be inhibited by the antiproliferative drug rapamycin. This study assessed the efficacy of combining rapamycin therapy with calcineurin inhibition. Male Sprague-Dawley rats received rapamycin 0.05 mg/kg daily and either tacrolimus 0.1 mg/kg or cyclosporin 5 mg/kg daily, and findings were compared with those in an untreated control group. Animals underwent left common carotid artery balloon angioplasty; the artery was explanted after 2 weeks. Morphometric analysis was performed on transverse sections and the intima : media ratio was calculated. Profibrotic gene expression was measured with competitive reverse transcriptase-polymerase chain reaction at 14 and 28 days. Proliferation was determined with proliferating cell nuclear antigen at 14 and 28 days. Extracellular matrix deposition was quantified with Sirius red. The combination of rapamycin and tacrolimus was associated with the greatest reduction in intimal thickening. Furthermore, treatment with rapamycin and tacrolimus significantly attenuated extracellular matrix deposition compared with rapamycin and cyclosporin (P < 0.02). The effects of rapamycin in combination with tacrolimus were better than those observed with rapamycin and cyclosporin.

Oral everolimus inhibits in-stent neointimal growth.

Rapamycin (sirolimus)-eluting stents are associated with reduced restenosis rates in animal studies and initial human trials. The present study evaluated whether orally administered everolimus (a macrolide of the same family as sirolimus) inhibits in-stent neointimal growth in rabbit iliac arteries. New Zealand white rabbits were randomized to everolimus 1.5 mg/kg per day starting 3 days before stenting and reduced to 1 mg/kg per day from days 14 to 28 (group 1), everolimus 1.5 mg/kg given 1 day before stenting followed by 0.75 mg/kg per day for 28 days (group 2), or matching placebo for each group. Drugs were administered by oral gavage. Stents were deployed in both iliac arteries, and arteries were harvested 28 days after stenting. Group 1 everolimus-treated rabbits experienced weight loss and anorexia, which resolved after the everolimus dose was lowered on day 14. Group 2 animals were healthy for the duration of everolimus dosing. Both everolimus treatment groups significantly reduced in-stent neointimal growth (46% reduction and 42% reduction in intimal thickness in groups 1 and 2, respectively). In group 2 everolimus-treated animals, the neointima was healed or healing, characterized by stent struts covered by a thin neointima, overlying endothelial cells, and only small foci of fibrin. Scanning electron microscopy showed >80% stent surface endothelialization in group 2 everolimus-treated rabbits. Oral everolimus suppresses in-stent neointimal growth in the rabbit iliac artery. At a dose of 1.5 mg/kg given 1 day before stenting followed by 0.75 mg/kg per day for 28 days, everolimus was well tolerated and was associated with significant neointimal healing.

Gene Transfer of Heme Oxygenase-1 and Carbon Monoxide Delivery Inhibit Chronic Rejection

The hallmark of chronic rejection is the occlusion of the artery lumen by intima hyperplasia as a consequence of leukocyte infiltration and vascular smooth muscle cell (VSMC) migration and proliferation. Heme oxygenase-1 (HO-1) is a tissue protective molecule which degrades heme into carbon monoxide (CO), free iron and biliverdin. We analyzed the effects of HO-1 gene transfer into the vessel wall using an adenoviral vector (AdHO-1) and of CO delivery in a model of chronic allogeneic aorta rejection in rats. Carbon monoxide treatment was achieved by a new pharmacological approach in transplantation using methylene chloride (MC), which releases CO after degradation. AdHO-1-mediated gene transfer into aorta endothelial cells (ECs) or CO delivery resulted in a significant reduction in intimal thickness compared to untreated or noncoding adenovirus-treated controls. Aortas transduced with AdHO-1 or treated with CO showed a reduction in the number of leukocytes as well as in the expression of adhesion molecules, costimulatory molecules and cytokines, with the gene transfer treatment displaying a more pronounced effect than the CO treatment. Conversely, CO inhibited VSMC accumulation in the intima more efficiently than AdHO-1 treatment. Gene transfer of HO-1 and pharmacological manipulation of CO are novel approaches to the analysis and treatment of chronic rejection.

Vascular 06

Aims: Allograft vasculopathy remains the leading cause of late graft failure following heart transplantation. The antiproliferative drug sirolimus has been shown to inhibit the development of intimal hyperplasia, a central feature of allograft vasculopathy. This study aims to assess the efficacy of dual combination of sirolimus and calcineurin inhibition on the development of intimal hyperplasia, reflective of modern clinical immunosuppressive practice. Methods: Male Sprague-Dawley rats were assigned to receive sirolimus (0.05 mg kg−1 per day) and either tacrolimus (0.1 mg kg−1 per day) or cyclosporin (5 mg kg−1 per day) and compared to an untreated control group. All animals underwent left common carotid artery balloon angioplasty and were explanted at 2 weeks. Morphometric analysis was performed on representative transverse sections and intima medial ratios calculated using computer-aided planimetry. Pro-fibrotic gene expression was assessed with competitive RT-PCR at 2 weeks for metalloproteinase-2, metalloproteinase-9, TIMP-1, collagen III and TGF-β. Sections were stained with picro-sirius red and extracellular matrix deposition was quantified using an image intensifier. Results: Sirolimus in combination with tacrolimus was associated with the greatest reduction in intimal thickening, median intima medial ratio 0.68 (range 0.36–0.94) compared to treatment with sirolimus and both cyclosporin and tacrolimus, 1.12 (range 1.06–1.68, P < 0.032) and untreated controls, 1.47 (1.02–2.04, P < 0.005). Furthermore, treatment with sirolimus and tacrolimus significantly attenuated extracellular matrix deposition compared to sirolimus and cyclosporin (P < 0.023). Conclusion: The synergistic benefits of sirolimus in combination with tacrolimus are preferential over those observed with cyclosporin. Randomized trials are warranted to assess if tacrolimus should be the primary calcineurin-inhibitor of choice when used in combination with sirolimus.

Endoluminal smooth muscle cell seeding limits intimal hyperplasia

Purpose: Intimal hyperplasia is one of the main responses of the vascular wall to injury. In the current study, we tested the hypothesis that endoluminal seeding of host syngeneic vascular cells could limit intimal hyperplasia induced by either mechanical deendothelialization or chronic allograft rejection in rat aorta. Methods: An experimental model of in situ seeding of syngeneic endothelial cells, smooth muscle cells (SMCs), and fibroblasts (FIBs) was used in mechanically deendothelialized and allografted aortas. In a preliminary study, the ability of the three cell types (n = 5 per group) to seed on the deendothelialized luminal surface of the aortic wall was evaluated after 2 days, with the use of fluorescent PKH as marker. In the first model, the abdominal aorta of Lewis rats was deendothelialized (n = 6) or deendothelialized and seeded with either SMCs (n = 6) or FIBs (n = 6) before flow was restored. In the allograft model, aortas were harvested from dark agouti rats and orthotopically grafted in Lewis receivers, directly (n = 6) or after deendothelialization. Deendothelialization was performed alone (n = 6) or associated with the seeding of similar host (Lewis) syngeneic SMCs (n = 6) or FIBs (n = 6). Results were evaluated at 2 months with histologic and morphometric methods. Results: SMCs and FIBs were able to adhere in situ to the deendothelialized aortic wall, whereas endothelial cells were not. In mechanically deendothelialized aortas, the seeding of syngeneic SMCs led to a significant reduction in intimal thickness compared with deendothelialized aortas or FIB-seeded aortas (26.9 ± 1.7 μm vs 55.5 ± 1.7 and 56.7 ± 1.7 μm, respectively), and a lower nuclear content (382.2 ± 35.7 μm2 vs 779.6 ± 65.9 and 529.6 ± 24.3 μm2, respectively) of neointima. After SMC seeding, intimal hyperplasia was richer in elastin, whereas after FIB seeding it was richer in collagen. In allografts, the seeding of syngeneic SMC led to a significant reduction in intimal thickness compared with control aortas, deendothelialized aortas, or FIB-seeded aortas (31.6 ± 1.1 μm vs 88.55 ± 2.8, 74.6 ± 2.9, and 85.7 ± 2.6 μm, respectively), and a reduced nuclear content of the neointima (444.9 ± 23.4 μm2 vs 1529.1 ± 116, 972.3 ± 50, and 645.2 ± 32.4 μm2, respectively). Differences observed in the extracellular matrix composition were equivalent to those observed in the mechanically deendothelialized model. Conclusions: Our results suggest that endoluminal seeding of syngeneic SMCs can be effective in reducing intimal hyperplasia both in a deendothelialization model and in arterial allografts. SMC and FIB endoluminal seeding led to a significatively different accumulation of extracellular matrix in the intima. (J Vasc Surg 2001;34:707-15.)

BHUx: A Patent Polyherbal Formulation to Prevent Atherosclerosis

Ayurvedic medicine is a time-tested system of medicine which has been in clinical use for centuries in India. Being a time-tested system, it has an edge over other existing systems of health management, especially for dealing with chronic disorders such as coronary artery disease, which is of a complex multi-etiological nature. Recently, we have shown that BHUx, a patented polyherbal formulation consisting of the aqueous fraction of five medicinal plants of the ayurvedic system, has significant anti-inflammatory properties through inhibition of cyclooxygenase-2 and lipoxygenase-15. Here we have investigated its effect on diet-induced atherosclerosis in albino rabbits. BHUx was given orally for 3 months to rabbits pre-treated with an atherogenic diet for 3 months. After 6 months, the dorsal aorta was processed for histological studies for calcium and collagen content. The results demonstrated a remarkable reduction in intimal thickening in the treated animals. In addition, there was less calcification at the intima–medial interface and increased intensity of collagen cap on the surface along with an increase in survival, compared with the sham control. We suggest that BHUx is a potent, multi-factorial formulation against atherosclerosis.

THE USE OF MHC CLASS II-DEFICIENT MICE TO DETERMINE THE ROLE OF DIRECT VS INDIRECT ANTIGEN PRESENTATION IN THE PATHOGENESIS OF CHRONIC REJECTION

14 The relative contributions of direct vs indirect donor alloantigen presentation to recipients' T cells on the development of chronic rejection (CR) is as yet ambiguous. It has been argued that while direct allorecognition may play an important role in initiating acute cellular rejection (ACR); CR on the contrary, is predominantly an outcome of donor allopeptides being presented in the context of recipient MHC. Given that CR is one of the two most common causes for late organ allograft failure, it is somewhat imperative that the prevailing conundrum concerning its pathogenesis be resolved. To address unequivocally this issue, we transplanted aortas (AO) from C57B1/6 Aβb (H2b, MHC class II-1-)→naive C3H (H2k) recipients (Group V; n=6). It was reasoned that due to lack of MHC class II expression in the donor tissue, the antigen (Ag) presentation would be primarily indirect allowing us to study its role in the development of CR. Additionally, the role of direct Ag presentation in the pathogenesis of this lesion was also studied by transplanting AO from naive C3H→C57Bl/6 Aβb recipients (Group VI; n=6). Controls (n=6/group) included Tx of AO from C3H → C3H (Group 1), C57Bl/6 Aβb→C57Bl/6 Aβb (Group II), C3H→C57Bl/6 WT (Group III) and C57Bl/6 WT→C3H (Group IV). As published previously, these aortic allografts undergo self-resolving ACR with spontaneous acceptance, eventually developing changes pathognomonic of CR by d30 post-Tx. At the time of sacrifice (d30-40 post-Tx), the grafts were harvested for immunohistochemical analysis and the sera and splenocytes were also recovered for the determination of α-donor antibody and proliferative responses by complement-dependent microcytotoxicity and MLR assays respectively. Unlike that from Groups I and II, AO harvested from animals in Groups III and IV had marked circumferential intimal thickening due largely to proliferation of α-smooth muscle actin✦ (α-smA✦) cells. Additionally, both the α-donor and α-third party proliferative responses and the titers of cytotoxic antibodies remained comparable in animals in Groups III and IV. On the contrary, AO obtained from the majority of Group V animals exhibited marked reduction in intimal thickening which was largely eccentric and patchy with preservation of elastic membranes; this finding was associated with unmitigated α-donor proliferative and humoral responses. Interestingly, despite the lack of α-donor proliferative and cytotoxic antibody responses, circumferential intimal thickening of moderate severity was witnessed in the majority of aortic allografts harvested from Group VI recipients. Taken together, these data suggest that despite preservation of α-donor immune responses, presentation of Ag by indirect pathway alone does not precipitate the fulminant development of CR indicating that perhaps both pathways of Ag presentation are required for optimal immune activation. More importantly, the presence of moderate CR in recipients which lack α-donor cellular and humoral responses underscore the importance of numerous immune and non-immune pathways which operate in concert towards the development of this lesion.