Smooth muscle cells from the anastomosed artery are the major precursors for neointima formation in both artery and vein grafts.

Accumulation of smooth muscle cells (SMC) results in neointima formation in injured vessels. Two graft models consisting of vein and artery grafts were created by anastomosing common carotid arteries to donor vessels. To identify the origin of the neointima cells from anastomosed arteries, we use Wnt1-Cre/reporter mice to label and track SMCs in the common carotid artery. The contribution of SMCs in the neighboring arteries to neointima formation was studied. On evaluating the artery grafts after 1 month, >90 % of the labeled neointima cells were found to have originated from the anastomosing host arteries. Most of the neointima cells were also smooth muscle α-actin positive (SMA-α(+)) and expressed the smooth muscle myosin heavy chain (SMMHC), the SMC terminal differentiation marker. In vein grafts, about 60 % SMA-α-positive cells were from anastomosing arteries. Bone marrow cells did not contribute to neointima SMCs in vein grafts, but did co-stain with markers of inflammatory cells. Wnt1 expression was not detected in the neointima cells in the vein or artery grafts, or the injured femoral arteries. Neointima SMCs showed the synthetic phenotype and were positively labeled with BrdU in vitro and in vivo. Treatment with the IGF-1 receptor inhibitor suppressed SMC proliferation and neointima formation in vein grafts. Our results indicate that SMCs from the neighboring artery are predominantly present in the neointima formed in both vein and artery grafts and that Wnt1-Cre mice can be used to explore the role of SMCs originating from neighboring vessels in vascular remodeling.

Flow patterns and preferred sites of intimal thickening in bypass-grafted arteries .

AIM: In bypass-grafted arteries, anastomotic intimal hyperplasia develops more markedly at the distal junction than the proximal one. It is likely that it arises from the difference in flow patterns at these two sites. Therefore we have studied the relationship between the flow patterns and precise locations of wall thickening specific to the particular vessel. METHODS: In total 30 bypass-grafting procedures were carried out on the femoral arteries of dogs with 10 autologous common carotid arteries and 20 saphenous veins. The vessels were harvested at 3 months after operation, and precise locations of intimal thickening and characteristics of the flow such as flow patterns and distributions of fluid velocity and wall shear stress were studied in detail. RESULTS: At the proximal anastomosis, a large recirculation zone was formed only at the inlet of the partially or totally occluded host artery, whereas at the distal anastomosis, it was formed at both the floor of the host artery and the toe of the bypass in most vessels, and the former was connected to the latter, extending the region of disturbed flow to lateral walls of the host artery. Wall thickening was found mainly in these regions occupied with slow secondary and recirculation flows where wall shear stress was very low. CONCLUSION: The flow at the distal anastomosis is more disturbed and complex than that at the proximal anastomosis. This difference in flow pattern that determine the region of low wall shear stress might explain why intimal hyperplasia develop more markedly at the distal junction.

Molecular mechanism of remodeling of autologous artery graft interposed to vein in rabbit.

Our previous study found that the artery interposed to vein did not develop atherosclerosis but rather underwent atrophic remodeling in hyperlipidemic rabbits, suggesting that local hemodynamic load was another important determinant for the development of atherosclerosis. This study focused on the cellular and molecular changes in autologous artery grafts derived from rabbits fed with high lipid diet for 1, 2, 4, 8, and 12 weeks. Thickness, area of vessel wall, and lumen area were measured and analyzed on the grafted common carotid artery (GCCA) interposed to vein and on the right common carotid artery. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling. Both elastin and collagen of GCCA were identified by the method of double stains of elastin and collagen. Reverse transcription polymerase chain reaction was used to observe matrix metalloproteinases (MMPs) mRNA expression changes in the examined arteries. The lumen area increased gradually in control common carotid artery and remained unchanged in GCCA 3 months later, since the surgery and the start of high lipid diet, while significantly increased apoptosis was evidenced from inner to outer part of GCCA. Collagen content decreased gradually and elastic fibers remained unchanged in GCCA. At 1 week after operation, the mRNA expression of MMP(2) and MMP(9) increased significantly and returned to baseline thereafter. The artery interposed to a vein underwent atrophy, characterized by increased apoptosis in the vessel wall from intima to adventitia, possibly due to low shear stress circumference and reduced vessel collagen resulting from postsurgical upregulated MMP(2) and MMP(9) expression.

Genetic susceptibility of the arterial wall is an important determinant of atherosclerosis in C57BL/6 and FVB/N mouse strains.

OBJECTIVE: How genetic variations among inbred mouse strains translate into differences in atherosclerosis susceptibility is of significant interest for the development of new therapeutic strategies. The objective of the present study was to examine whether genetically controlled arterial wall properties influence atherosclerosis susceptibility in FVB/N (FVB) and C57BL/6 (B6) apolipoprotein E knockout (apoE(-/-)) mouse strains. METHODS AND RESULTS: Common carotid artery segments from B6 apoE(-/-), F1 apoE(-/-), and FVB apoE(-/-) mice were transplanted to hybrid F1 apoE(-/-) mice, which can accept grafts from both parental strains without adaptive immune responses. The mice were fed a high-fat diet, and atherosclerosis was induced in the transplanted artery segments by placement of a perivascular constrictive collar. Artery segments from B6 apoE(-/-) mice developed much larger atherosclerotic lesions than artery segments from FVB or F1 apoE(-/-) mice. No differences in aortic arch atherosclerosis of the recipient mice were observed between groups. CONCLUSIONS: Genetically controlled factors acting at the level of the arterial wall are important determinants of atherosclerosis susceptibility in FVB apoE(-/-) and B6 apoE(-/-) mice.

Microsurgically induced pure arterial aneurysm model in rats.

INTRODUCTION: The aim of the study was to develop a reliable and reproducible arterial aneurysm model for microsurgical training and further research with dimensions comparable to those encountered in aneurysms in humans. METHODS: The arterial aneurysm models were created microsurgically at the bifurcation of the abdominal aorta using a graft of the carotid artery in 20 Wistar rats. RESULTS: The aneurysms were created successfully and no complications occurred. The average volume of this arterial aneurysm model was 35.19±5.64 mm (3). The time required to create this kind of aneurysm was 192±14.4 min. The central zone of blood inflow into the aneurysm was not affected by any thrombus formation. CONCLUSION: The presented model at the aortic bifurcation in the rat is reliable and immediately available for microsurgical and technical training as well as for scientific studies on aneurysms. Since this kind of model also reproduces arterial aneurysms, basic techniques such as suturing and microtechniques needed for the dissection and repair of vessels can be taught during its creation.

Long-term survival of composite hemiface/mandible/tongue allografts correlates with multilineage chimerism development in the lymphoid and myeloid compartments of recipients.

BACKGROUND: To maximize aesthetic and functional outcomes for complex craniofacial defects, application of composite tissue allografts opens unique one-stage reconstructive option. We have assessed role of different components of the hemiface/mandible/tongue allograft on induction and maintenance of donor-specific chimerism, in correlation with allograft survival. METHODS: Twenty-two composite hemiface/mandible/tongue transplantations were performed in three groups: group 1 (n=8)-isotransplantations between Lewis (LEW) rats (RT1)-served as controls. Group 2 (n=8)-allograft rejection controls-hemiface/mandible/tongue transplants performed across major histocompatibility complex (MHC) barrier between semiallogeneic LEW-Brown-Norway (RT1) donors and LEW (RT1) recipients without immunosuppression. Allografts in group 3 (n=6) received tapered cyclosporine A monotherapy. Assessments included monitoring of rejection, flow cytometry for donor-specific chimerism of major histocompatibility complex class I (RT1) antigen, immunohistochemistry for engraftment of donor-origin cells into lymphoid organs and bone marrow (BM) compartment, and histology for hemiface/mandible/tongue architecture. RESULTS: Isograft controls survived indefinitely; in allografts without treatment, rejection started within 5 to 7 days. Treated hemiface/mandible/tongue allotransplants survived up to 385 days, without signs of rejection or graft loss. Flap angiography confirmed intact vascularity, and computer tomography scan and histology confirmed bone viability. Donor-specific chimerism at day 125 was present for T cells (3.3% CD4/RT1, 1.1% CD8/RT1) and B cells (6.7% CD45RA/RT1). Engraftment of donor-origin cells was confirmed into BM compartment and lymphoid organs of recipients. CONCLUSIONS: We introduced a new multitissue model of composite hemiface/mandible/tongue allograft containing lymphoid and vascularized BM components. Long-term allograft survival correlated with development and maintenance of donor-specific chimerism in lymphoid organs and BM compartment.

A new carotid artery transplantation model of rats.

To establish a murine carotid artery transplantation model for the study of the chronic rejection, 80 rats were divided into two groups, an allotransplant (ACI-Lewis) group and an isotransplant (Lewis-Lewis) group (control group). The donor carotid artery and the recipient carotid artery were anastomosed by using a polyethylene cuff (internal diameter: 0.7 mm, length: 3 mm).The pathological changes of carotid artery transplant were observed 14, 28 and 56 days after the transplantation. The results showed that the model was successfully established in 95% of the animals. The chronic rejection-associated arteriosclerosis was induced 28 days after the transplantation. The new chronic rejection model of carotid artery by using cuff technique caused fewer traumas and was easy to make. The pathological changes of the transplant mimicked the chronic rejection-associated arteriosclerosis found in human transplant.

Wild-type apo A-I and apo A-I(Milano) gene transfer reduce native and transplant arteriosclerosis to a similar extent.

Apolipoprotein (apo) A-I(Milano) is an apo A-I mutant characterized by a cysteine for arginine substitution at position 173. Apo A-I(Milano) carriers have much less atherosclerosis than expected from their low plasma high-density lipoprotein cholesterol levels, suggesting that this mutant may have superior atheroprotective properties. Here, we compare the effect of hepatocyte-directed gene transfer of wild-type human apo A-I and human apo A-I(Milano) on endothelial progenitor cell (EPC) biology and on the progression of native atherosclerosis and allograft vasculopathy in C57BL/6 apo E(-/-) mice. Human apo A-I and apo A-I(Milano) transfer resulted in an equivalent increase of EPC number and function as well as EPC incorporation and endothelial regeneration in allografts and inhibited the progression of native atherosclerosis and allograft vasculopathy to a similar extent. In conclusion, the current head-to-head comparison indicates that human apo A-I(Milano) transfer is not superior compared to wild-type human apo A-I transfer.

A new carotid artery transplantation model of rats / 华中科技大学学报（医学）（英德文版）

To establish a murine carotid artery transplantation model for the study of the chronic rejection, 80 rats were divided into two groups, an allotransplant (ACI-Lewis) group and an isotransplant (Lewis-Lewis) group (control group). The donor carotid artery and the recipient carotid artery were anastomosed by using a polyethylene cuff (internal diameter: 0.7 mm, length: 3 mm).The pathological changes of carotid artery transplant were observed 14, 28 and 56 days after the transplantation. The results showed that the model was successfully established in 95% of the animals. The chronic rejection-associated arteriosclerosis was induced 28 days after the transplantation. The new chronic rejection model of carotid artery by using cuff technique caused fewer traumas and was easy to make. The pathological changes of the transplant mimicked the chronic rejection-associated arteriosclerosis found in human transplant.

Cryografts implantation in human circulation would ensure a physiological transition in the arterial wall energetics, damping and wave reflection.

Each artery conduces blood (conduit function, CF) and smoothes out the pulsatility (buffering function, BF), while keeping its wall protected against the high oscillations of the pulse waves (damping function, xi). These functions depend on each segment viscoelasticity and capability to store and dissipate energy. When a graft/prosthesis is implanted, the physiological gradual transition in the viscoelasticity and functionality of adjacent arterial segments is disrupted. It remains to be elucidated if the cryografts would allow keeping the physiological biomechanical transition. The aim of this study was to evaluate the cryografts capability to reproduce the functional, energetic and reflection properties of patients' arteries and fresh homografts. Common carotid's pressure, diameter and wall-thickness were recorded in vivo (15 patients) and in vitro (15 cryografts and 15 fresh homografts from donors). Calculus: elastic (Epd) and viscous (Vpd) indexes, CF, BF, dissipated (WD) and stored (WPS) energy and xi. The graft-patient's artery matching was evaluated using the reflection coefficient (Gamma) and reflected power (WGamma). Cryografts did not show differences in Epd, Vpd, BF, CF, WD, WPS, and xi, in respect to fresh homografts and patients' arteries, ensuring a reduced Gamma and WGamma. Cryografts could be considered as alternatives in arterial reconstructions since they ensure the gradual transition of patients' arteries biomechanical and functional behavior.

[Biocompatibility of decellularized canine carotid artery allograft cross-linked by carbodiimide].

UNLABELLED: OBJECTIVE Crosslink decellularized canine carotid artery allograft by EDC [1-3-(dimethylamino)propyl-3-ethylcarbodiimide methiodide] and evaluate the biocompatibility of it. METHODS: Use the multi-step detergent-enzyme method to construct decellularized canine carotid artery allograft and cross-link it by EDC with the weight ratio of decellularized artery to EDC 1:1 and 1:2. Evaluate the biocompatibility of it by the cytotoxical MTT test and the rat subdermal bury test. RESULTS: Decellularized canine carotid artery cross-linked by EDC has a lower degradation rate treated by collagenase type II, the result of MTT test show that the EDC cross-linked decellularized artery has no cytotoxity and the rat subdermal bury test show that crosslinking greatly enhance the ability of decellularize artery to resist the enzyme degradation and lower the immune reaction. The more the artery was cross-linked , the more effects it has. CONCLUSIONS: Decellularized canine carotid artery cross-linked by EDC has fairly good biocompatibility and ability to resist the collagenase degradation.

Implantation of decellularized small-caliber vascular xenografts with and without surface heparin treatment.

Heparin treatment of decellularized xenografts has been reported to reduce graft thrombogenicity. However, little is known about the in vivo comparison of heparin-treated with non-heparin-treated xenografts, especially for small-caliber vascular implants. We implanted either a heparin-treated or a non-heparin-treated canine carotid artery as bilateral carotid xenograft in rabbits (n = 24). Small-caliber xenografts (3 approximately 4 mm) were decellularized by enzymatic and detergent extraction and were further covalently linked with heparin. During implantation, thrombosis rate was 4% in the heparin-treated xenografts and 25% in the non-heparin-treated xenografts after 3 weeks (P < 0.05). After 6 months, it was 8 versus 58%, respectively (P < 0.01). Both heparin-treated and non-heparin-treated xenografts harvested at the end of 3 and 6 months showed a satisfactory cellular reconstruction of either smooth muscle cells or endothelial cells. These results indicate that heparin treatment of the small-caliber decellularized xenograft reduces the in vivo thrombogenicity. Both heparin-treated and non-heparin-treated xenografts seem to undergo a similar cellular remodeling process up to 6 months.

Orally administered penicillamine is a potent inhibitor of neointimal and medial thickening in porcine saphenous vein-carotid artery interposition grafts.

OBJECTIVE: In patients who have undergone coronary artery bypass grafting, blood copper levels are elevated for 6 weeks after surgery. Copper is an established risk factor for cardiovascular disease and atherogenesis and promotes oxidative stress, lipid oxidation, cell proliferation, and matrix formation, all components of vein graft disease. This project therefore examined the effect of the copper chelator penicillamine on saphenous vein graft thickening in a pig model. METHODS: Saphenous vein-carotid artery interposition grafts were carried out in Landrace pigs. Penicillamine (10 mg/kg once daily, n = 8) was administered orally incorporated into small amounts of mashed potato for 1 month (n = 8 controls). Vein grafts were then excised and fixed at 100 mm Hg, histologic sections were prepared, and morphometry and measurement of proliferating cell nuclear antigen count were carried out. In vitro studies on the effect of copper or penicillamine on human vascular smooth muscle cell replication was carried out with bromodeoxyuridine incorporation. RESULTS: Administration of penicillamine had a potent inhibitory effect on both neointimal and medial thickness and proliferating cell nuclear antigen count but elicited a marked increase in luminal area and reduced serum copper concentrations. In vitro, copper augmented vascular smooth muscle cell proliferation, an effect blocked by penicillamine. Penicillamine alone also inhibited in vitro vascular smooth muscle cell replication. CONCLUSION: The administration of penicillamine reduces vein graft thickening and promotes positive remodeling through negation of copper-induced cell proliferation. Copper chelators may therefore be therapeutically useful in preventing late vein graft failure in patients undergoing reconstructive arterial surgery.

Evaluation of compliance and stiffness of decellularized tissues as scaffolds for tissue-engineered small caliber vascular grafts using intravascular ultrasound.

This study evaluated the compliance and stiffness of decellularized canine common carotid artery as well as decellularized canine ureter and compared it with that of polytetrafluoroethylene, elastin gel combined with polylactic acid tube, and canine common carotid artery. To calculate the compliance and stiffness, internal diameters and cross-sectional areas were measured according to changes in the intraluminal pressures using intravascular ultrasound in a closed circuit system equipped with a syringe pump. The pressure-area curve, stiffness parameter beta, and diameter compliance were evaluated. Canine common carotid artery and decellularized canine common carotid artery, as well as decellularized canine ureter, showed a compliant response, a J-shaped curve. However, the latter evidenced different characteristics in the low pressure range. Although the cross-sectional area of the elastin gel combined with polylactic acid tube showed some changes, it did not present a J-shape curve. Polytetrafluoroethylene exhibited a noncompliant response.The results in this study have shown that the compliance in the decellularized matrices was maintained after cell extraction, which demonstrated the importance of the remaining matrix structure in the mechanical properties of decellularized tissue. A clear difference between the decellularized matrices and synthetic materials was noted in terms of the compliance, even in materials composed of relatively elastic materials.

Shear stress contributes to t-PA mRNA expression in human endothelial progenitor cells and nonthrombogenic potential of small diameter artificial vessels.

Seeding endothelial progenitor cells (EPCs) onto the surface of vascular grafts has been proved to be a promising strategy to improve nonthrombogenic potentials of small diameter artificial vessels. Here, we investigated whether in vitro shear stress modulates the tissue-type plasminogen activator (t-PA) secretion and mRNA expression in human EPCs and improves patency of the EPC-seeded polyurethane small diameter vascular grafts implanted in the canine carotid artery in vivo. In vitro shear stress, in a dose-dependent manner, increased t-PA secretion and mRNA expression of human EPCs. The in vivo implantation of EPC-seeded vascular grafts remained highly patent in shear stress pretreatment compared with stationary condition. The present findings demonstrate for the first time that in vitro shear stress can enhance t-PA secretion and gene expression in human EPCs, which contributes to improvement in nonthrombogenic potentials of EPC-seeded small diameter artificial vessels with maintenance of in vivo highly patency rate.

Successful type II endoleak closure by subclavian-to-carotid artery transposition after stent-graft placement of a distal aortic arch aneurysm.

Endovascular stent-graft placement has become a safe and effective treatment modality for various diseases of the distal aortic arch as well as of the descending aorta. However, its effectiveness may be limited by various kinds of endoleaks resulting in persistent or recurrent perfusion of the aneurysm sac. Subsequently, systemic pressurization leads to expansion of the aneurysm sac, exposing the patient to a recurrent risk of aneurysm rupture. We report on the case of a 57-year-old male who underwent emergency stent-graft placement in March 2001 due to a contained rupture of a distal aortic arch aneurysm involving the origin of the left subclavian artery. Due to the emergency condition, a subclavian-to-carotid artery transposition had not been performed prior to stent-graft placement. During follow-up the patient developed a type II endoleak originating from the left subclavian artery with consecutive enlargement of the aneurysm sac. The endoleak was successfully treated by subclavian-to-carotid artery transposition.

Enxertos vasculares homólogos e heterólogos conservados em glicerina na fleboplastia da jugular em eqüinos / Arterial homograft and venous heterograft conserved in glycerin in the phleboplasty of the jugular in equines

Doze eqüinos foram divididos aleatoriamente em dois grupos de seis animais (grupos I e II), com a finalidade de estudar a compatibilidade tecidual e a propriedade de indução de trombos de dois tecidos biológicos conservados em glicerina a 98 por cento. Esses tecidos foram usados na restauração da jugular externa e se constituíram de artéria carótida comum homóloga (ACCHo), no grupo I, e veia jugular externa heteróloga (VJEHe), no grupo II. Para a restauração, utilizaram-se duas técnicas de anastomose da jugular, sendo, no grupo I, a técnica de bypass e, no grupo II, a substituição de um segmento da jugular esquerda por meio de anastomose vascular término-terminal. Para avaliar a trombogênese local e a histocompatibilidade, foram realizados exames clínicos, hematológicos, ecoDopplercardiográficos e histológicos dos segmentos vasculares enxertados. Os segmentos foram colhidos após 45 dias da avaliação pós-operatória, tendo a jugular direita como testemunha para os exames histológicos. Ambos os tecidos foram compatíveis quando implantados nos eqüinos, sem processo inflamatório acentuado, indicativo de rejeição. A técnica de bypass não foi eficiente na restauração da jugular, ocorrendo trombose obliterante dos enxertos de ACCHo. A substituição completa do segmento da jugular por VJEHe pode ser viável para o restabelecimento do fluxo sangüíneo da jugular de eqüinos, desde que se mantenha a igualdade dos diâmetros entre o enxerto e o vaso receptor.

Investigation about origin and spreading of neoendothelium in autologous arterial vessel replacement.

INTRODUCTION: In many fields of surgery an autologous arterial vessel replacement is used. Because of trauma and insufficient nutrition, a loss of complete endothelium in the replaced vessel segment and at the anastomoses can be observed within a few hours. The origin and the spread of the neoendothelium are unknown. Our study uses Von-Willebrand-protein, a product of endothelium cells, as a marker to obtain new information about the origin and spread of the new endothelial cells. MATERIALS AND METHODS: Seventy Wistar rats, seven groups of 10 animals each, were operated. A four-millimetre-long segment of the common carotid artery was isolated and reinserted. After a varying length of time (between directly after the operation and six months after), the common carotid artery including bifurcation was isolated after cardioperfusion. Carotid arteries were embedded and cross-sections were stained with hematoxylineosin, Verhoeff's tissue elastin stain and immunohistochemical anti-Von-Willebrand-factor-antibody. The complete vessel was divided into nine points of measurement on each side, three points at each anastomosis and three points in transplanted segment. There the number of positive endothelial cells was rated. RESULTS: Twenty-four hours after the operation no further endothelium was detectable in autologous transplant. Stainings eight days after operation contained Von-Willebrand-factor-positive cells in luminal cell layers. Near to the anastomosis excessive myointimal hyperplasia was detectable. Also, eight days after operation, some positive endothelial cells in adventitia were seen near to the anastomosis in small vessel lumina. Four weeks after operation, the luminal endothelium was completely regenerated and the luminal endothelial layer was confluent. Eight days after the operation regeneration in lateral regions was faster than in the region of the transplant. CONCLUSION: In our experiment regeneration in lateral regions was faster than in the region of the transplant. Early staining in adventitia and proof of newly formed vasa vasora in adventitia may be a sign of a possible migration from adventitia to luminal area.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: The effects of rapamycin in combination with tacrolimus were better than those observed with rapamycin and cyclosporin.