Improved Patency of ePTFE Grafts as a Hemodialysis Access Site by Seeding Autologous Endothelial Cells Expressing Fibulin-5 and VEGF.

Small caliber synthetic vascular grafts used for dialysis access sites have high failure rates due to neointima formation and thrombosis. Seeding synthetic grafts with endothelial cells (ECs) provides a biocompatible surface that may prevent graft failure. We tested the use of ePTFE grafts seeded with autologous ECs expressing fibulin-5 and vascular endothelial growth factor (VEGF), as a dialysis access site in a porcine model. We connected the carotid arteries and jugular veins of 12 miniature pigs using 7-mm ePTFE grafts; five grafts were seeded with autologous venous ECs modified to express fibulin-5 and VEGF, and seven unseeded grafts were implanted at the same location and served as controls. At 6 months, after completion of angiography, the carotid arteries and jugular veins with the connecting grafts were excised and fixed. Autologous EC isolation and transduction and graft seeding were successful in all animals. At 3 months, 4 of 5 seeded grafts and 3 of 7 control grafts were patent. At 6 months, 4 of 5 (80%) seeded grafts and only 2 of 7 (29%) control grafts were patent. Seeding ePTFE vascular grafts with genetically modified ECs improved long term small caliber graft patency. The biosynthetic grafts offer a novel therapeutic modality for vascular access in hemodialysis.

Apoptosis, proliferation, and morphology during vein graft remodeling in rabbits.

Neo-intima development and atherosclerosis limit the long-term use of vein grafts for revascularization of ischemic tissues. Recently, studies have confirmed that proliferating cell nuclear antigen (PCNA) plays an important role in cell proliferation. Our research confirmed that 28 days after vein transplantation, PCNA expression increases significantly. Using rabbits, rather than rodents, for a more representative model of human vein grafts, we aimed to establish a time course of changes in cell proliferation and apoptosis using morphometric and immunohistochemical analyses, western blot, terminal deoxynucleotidyl transferase dUTP nick end labeling, and transmission electron microscopy (TEM). The external jugular veins of 42 healthy purebred male New Zealand white rabbits were grafted onto their common carotid arteries. The rabbits were divided into seven groups, with vein grafts being harvested before surgery, and at 1, 3, 7, 14, 28, and 90 days afterwards. The extent of stenosis and apoptosis, PCNA protein levels, and TEM morphology were subsequently examined. Intimal thickness was slightly decreased 1 day following surgery, but then increased continuously until the 90th day. Western blot and immunohistochemistry both indicated lowered PCNA expression on day 1, although levels subsequently increased, peaking at 7 days post-surgery. After surgery, apoptosis was lowest on day 7, and remained low thereafter. TEM revealed signs of apoptosis as vein graft restenosis progressed. Proliferation and apoptosis co-occurred following grafting, indicating that both processes were involved in vein graft remodeling. Apoptosis levels were highest between days 1 and 3 after surgery, whereas proliferation culminated on the 7th day.

Chronic implantation of intravascular cardioverter defibrillator in a canine model: device stability, vascular patency, and anchor histology.

INTRODUCTION: A percutaneously placed implantable intravascular defibrillator (PICD) has been developed with a right ventricular (RV) single-coil lead and titanium electrodes in the superior vena cava (SVC) and the inferior vena cava (IVC). This study evaluated implant techniques, device stability, and anchor histology of the PICD over 9 months in a canine model. METHODS: Twenty-four hounds (wt = 30-55 kg) were anesthetized and a custom sheath introduced into the right femoral vein. The PICD was advanced over a wire and positioned with the titanium electrodes (cathodes) in the SVC and the IVC. A nitinol anchor secured the device in the jugular. The RV lead was positioned in the RV apex and screwed into place. The catheters, wires, and sheath were removed with an average implant time of 14 minutes. In one group of animals (n = 13), serial venograms were performed at 7 days, 14 days, and 28 days. In a second group (n = 6) and third group (n = 5), venograms were also performed at 90 days and 270 days, respectively. Six canines were sacrificed and anchor histologic examination done at 90 days. RESULTS: All implants were successful with no surgical complications observed. Devices (N = 24) remained appropriately positioned with no anchor migration. Histology at 90 days showed 98% endothelialization of the anchor. Venograms revealed patent IVC and jugular veins in all animals at every time point examined. CONCLUSIONS: The PICD can be rapidly and chronically implanted in animals. Long-term intravascular defibrillator placement is feasible in a canine model.

[Pharmacokinetic evaluation of danshensu with in vivo microdialysis in freely moving rat's jugular vein].

OBJECTIVE: To investigate the pharmacokinetics of Danshensu with in vivo microdialysis in freely moving rat's jugular vein. METHOD: three days after a microdialysis probe introducer was implanted into the jugular vein, a microdialysis probe was introduced to the blood vessel, and began to sample following a single intravenous injection (40 mg x kg(-1)) or a single oral dose (40 mg x kg(-1)) of Danshensu. All the samples were analyzed with HPLC. The concentration of Danshensu in blood were calculated according to the recovery of microdialysis probe and the concentration in dialysates. Pharmacokinetic parameters were than calculated with the concentration-time curve. RESULT: For intravenous administration, t(1/2 zeta) = (69.62 +/- 33.42) min, AUC(0-infinity) = (3416.24 +/- 779.80) min x mg x L(-1), MRT(0-infinity) = (38.15 +/- 8.61) min, and for oral administration, Cmax = (7.42 +/- 3.08) mg x L(-1), tmax = (31.50 +/- 8.57) min, t(1/2 zeta) = (83.25 +/- 37.35) min, AUC(0-infinity) = (793.19 +/- 101.32) min x mg x L(-1), MRT(0-infinity) = (125.89 +/- 58.27) min. The oral bioavailability of Danshensu F = 22.56%. CONCLUSION: In vivo microdialysis in freely moving rat's jugular vein is a useful tool to obtain a complete set of free drug concentrations to determine reliable pharmacokinetic parameters.

Preclinical testing of tissue-engineered heart valves re-endothelialized under simulated physiological conditions.

BACKGROUND: The in vivo regeneration capacity of decellularized heart valve grafts is still controversial. The aim of this study was to evaluate function, morphological changes, and cellular composition of decellularized versus re-endothelialized ovine pulmonary valves (PV) after implantation into lambs for 1 or 3 months. METHODS AND RESULTS: PV (n=21) were decellularized using detergents. Twelve PV were repopulated with autologous jugular veins endothelial cells (ECs) in a dynamic pulsatile bioreactor under simulated physiological conditions. Morphological evaluation before implantation included histological stainings (H&E, Movat-pentachrome, von-Kossa, DAPI), immunostainings (anti-perlecan, anti-eNOS, anti-procollagen-I, anti-SM-alpha-actin), electron microscopy (EM), and DNA extraction. Decellularization led to cell-free scaffolds with preserved extracellular matrix (ECM) including basement membrane. Reseeded PV (n=5) were completely covered with ECs expressing endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). The function of orthotopically implanted decellularized and re-endothelialized PV (n=7, each) was analyzed after 1 and 3 months by echocardiography and revealed no differences in competence between both groups. A confluent EC monolayer expressing eNOS/vWF was only found in re-endothelialized PV but not in decellularized PV, whereas the valve matrices were comparable repopulated with interstitial cells expressing SM-alpha-actin and procollagen-I. More thrombotic and neointima formations were observed in decellularized PV. No signs of calcification were detected in both PV types. CONCLUSIONS: In vitro re-endothelialization of detergent-decellularized valves with autologous ECs under simulated physiological conditions significantly improves total EC valve coverage 3 months after implantation, whereas the valve repopulation with interstitial cells in vivo occurs most likely by cell migration inside the scaffold.

In vitro differences between venous and arterial-derived smooth muscle cells: potential modulatory role of decorin.

OBJECTIVE: We analyzed the phenotypic and functional differences between venous and arterial smooth muscle cells (SMC) and the role of decorin in modulating these differences. METHODS AND RESULTS: SMC were isolated from the jugular veins and carotid arteries of male white New Zealand rabbits. Venous SMC demonstrated increased proliferation (2-fold, p<0.001), migration (1.7-fold, p<0.001), and collagen synthesis (4-fold, p<0.001), with decreased adhesion to collagen and fibronectin (1.2-fold, p<0.01) compared to arterial SMC. Higher levels of gelatinase activity (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase (TIMP) were also observed in venous SMC. Venous SMC demonstrated increased expression of SMemb and decreased expression of SM1--markers of a dedifferentiated and differentiated phenotype, respectively. Arterial SMC produced increased levels of the inhibitory proteoglycan, decorin, compared to venous SMC. Conditioned medium from arterial SMC (ASMC-CM) significantly decreased DNA synthesis, collagen synthesis, and gelatinase activity in venous SMC. Removal of decorin from ASMC-CM by immunoprecipitation significantly reversed the inhibitory effects of ASMC-CM on venous SMC proliferation and collagen synthesis but did not affect gelatinase activities. CONCLUSION: Venous SMC are more dedifferentiated and demonstrate increased proliferative and synthetic capacity than arterial SMC. Differential decorin expression between arterial and venous SMC contributes to these differences in biologic behavior. Venous SMC properties may contribute to accelerated atherosclerosis in venous bypass grafts.

Rapid and efficient vascular transport of arginine polymers inhibits myointimal hyperplasia.

BACKGROUND: We recently discovered that short polymers of arginine efficiently translocate across the cytoplasmic membrane independent of the basic amino acid transporter. We evaluated the kinetics and biological effects of heptamers of L-arginine and D-arginine (L-R7 and D-R7, respectively) in vascular cells. We assessed the effects of these peptides on the NO synthesis pathway and vascular cell proliferation. METHODS AND RESULTS: Human umbilical vein endothelial cell and rabbit vascular segments were incubated in medium containing biotin-labeled L-R7 or D-R7. Both polymers rapidly translocated through the vessel wall and into the vascular cells in a dose- and time-dependent fashion. At a dose of 10 micromol/L for 30 minutes, 100% of the endothelial cells showed evidence of cytoplasmic and nuclear localization of the peptides. To evaluate the biological effects of the polymer translocation on myointimal formation, rabbit jugular vein segments were incubated with polymers (10 micromol/L, 30 minutes) or vehicle before arterial interposition grafting. Planimetric measurement 28 days after surgery revealed that L-R7 and D-R7 substantially reduced myointimal formation compared with the control condition (intima/media ratio: control 1. 50.5, L-R7 0.40.2, and D-R7 0.80.2; P:<0.05). Furthermore, basal nitrate and nitrite production from L-R7-treated grafts was significantly higher than that from both control and D-R7-treated veins. Studies in vitro of cultured vascular smooth muscle cells revealed that both polymers also exhibit an NO-independent inhibition of vascular smooth muscle cell proliferation. CONCLUSIONS: Short polymers of arginine have the unique ability of vascular cell translocation, and they also have direct biological effects. These attributes are potentially useful in treating myointimal hyperplasia.

Comparison of three myofibroblast cell sources for the tissue engineering of cardiac valves.

The objective of this study was to evaluate the capacity of three clinically useful tissue sources: tricuspid valve leaflet (TVL), carotid artery (CA), and jugular vein (JV), to generate myofibroblasts for potential use in a tissue-engineered cardiac valve replacement. Tissue biopsies of clinically appropriate sizes obtained from juvenile sheep were used for this work. Cells obtained from all three tissue sources exhibited a myofibroblast phenotype in vitro, as demonstrated by their immunoreactivity with antibodies directed against vimentin, alpha-smooth muscle actin, fibronectin, and chondroitin sulfate. Protein synthesis characteristics were defined for the key extracellular matrix components: collagen, glycosaminoglycans, and elastin. Among the three sources, JV generated the highest numbers of cells, and JV cells produced the largest amount of collagen per cell. These data suggest that venous tissue, with its relative ease of accessibility, may generate myofibroblasts potentially useful for the interstitial cellular component of a tissue-engineered cardiac valve.

Murine model of neointimal formation and stenosis in vein grafts.

OBJECTIVE: Previous studies have suggested that neointimal formation, a central cause of vein graft stenosis, has several potential cell sources. It was hypothesized that neointimal cells arise primarily from the cells of the vein graft. METHODS AND RESULTS: This study investigated vein graft neointimal cell origins using a model of vein-to-artery cross-transplantation between transgenic Rosa26 mice (constitutive expression of bacterial beta-galactosidase marker gene) and wild-type mice. Vein-originating cells survived and make a major contribution to neointimal formation within the vein graft, mostly adjacent to the lumen/endothelium, suggesting an intimate association with endothelial cells. Cross-transplantation of veins from thrombomodulin promoter-driven beta-galactosidase reporter transgenic mice to wild-type arteries demonstrated survival of vein graft endothelial cells. Neointimal thickening was greater at the proximal and, to a lesser extent, distal ends, in comparison to the middle of the graft. By contrast, arterial grafts had almost no neointimal formation throughout the graft. The relative neointimal wall thickness is much greater in this model compared with other murine and larger-species vein graft models, even showing near-occlusive stenosis of the perianastomotic region. CONCLUSIONS: Vein graft neointimal cells arise predominantly from vein-derived cells, suggesting clinical relevance of stenosis-inhibiting therapies directed at the vein graft.

Pressurization facilitates adenovirus-mediated gene transfer into vein graft.

We investigated whether application of non-distending hydrostatic pressure facilitates gene transfer into vein grafts. An external jugular vein was placed in a chamber with 100 microl adenovirus solution at a titer of 10(10) pfu/ml and was pressurized to up to 8 atm above ambient pressure for 10 min. Histochemical analysis demonstrated a positive transgene expression in all layers of the vessel wall. Gene transfer with 8 atm pressurization resulted in an approximately 50 times higher transgene expression than that without pressurization. Under 8 atm pressurization, the efficiency of gene transfer reached a plateau at 7.5 min. The application of hydrostatic pressure may improve the effectiveness of intraoperative genetic engineering of vein grafts.

Controlled periadventitial administration of verapamil inhibits neointimal smooth muscle cell proliferation and ameliorates vasomotor abnormalities in experimental vein bypass grafts.

OBJECTIVE: Inhibition of early myointimal proliferation may improve longterm patency of vein grafts, but the clinical use of many experimental drugs is limited by systemic toxicity. To determine whether this goal can be achieved by low-dose targeted drug administration, we constructed a polymeric system delivering verapamil and evaluated the effects on local and downstream vein graft morphology, neointimal smooth muscle cell proliferation, and vasomotor function. METHODS: Ethylene-vinyl acetate polymeric delivery systems were constructed, containing 2% verapamil by weight. These are flexible, biocompatible, and nonbiodegradable matrices, delivering the drug at a rate of 10 micrograms/day. The autologous external jugular vein was used to create a carotid artery bypass graft in hypercholesterolemic (n = 22) rabbits. Verapamil-containing matrices (n = 12) or plain polymers (control, n = 10) were wrapped around the proximal third of the veins after reperfusion. Graft vasomotor function was evaluated and was also compared with function of an additional group of normocholesterolemic vein grafts (n = 8). RESULTS: Twenty-eight days after grafting, intimal index (intima/media thickness ratio) was 31% lower, neointima/original lumen surface ratio was 26% lower, and residual luminal area was 71% greater (4.00 +/- 1.2 mm2 versus 2.34 +/- 0.9 mm2, all p < 0.01) under verapamil matrices compared with control grafts. Neointimal smooth muscle cell content was reduced from 45.4% to 28.2%, and net neointimal smooth muscle cell thickness was reduced by 47% (30 microns vs 15.8 microns, both p < 0.01). Verapamil-treated segments distal to the matrices also showed significantly lower neointimal smooth muscle cell density and increased lumen size. Sensitivity to serotoin and vasomotor responses to serotonin, norepinephrine, and sodium nitroprusside in distal segments were significantly lower in verapamil-treated grafts than in controls. CONCLUSIONS: Periadventitial controlled administration of verapamil below 1% of the systemic dose effectively inhibits myointimal hyperplasia in vein grafts. Local polymeric drug delivery may be readily applicable to coronary revascularization operations.

In vitro-lined endothelium: initial integrity and ultrastructural events.

BACKGROUND: The early fate of in vitro-endothelialized prosthetic vascular grafts was assessed in the nonhuman primate. METHODS: Each of 17 male chacma baboons received a control and a confluently endothelialized 4 mm polytetrafluoroethylene graft in femoro-femoral positions (8.2 +/- 0.8 cm). All experimental grafts were precoated with fibrinolytically inhibited fibrin glue and lined with cultured autologous endothelial cells (EC) from the external jugular vein. The average time period needed to obtain first-passage mass-cultures sufficient for preconfluent graft endothelialization was 19.8 +/- 5.2 days. Before implantation in vitro-lined grafts were kept in culture for another 16.1 +/- 4.3 days to achieve complete confluence and maturation of the EC cytoskeleton. RESULTS: After 9 days of implantation, endothelial-lined grafts still showed a confluent endothelium that was free of any fibrin deposits. However, the EC density was significantly lower than at implantation (39.7 +/- 7.6 x 10(3) versus 59.9 +/- 8.5 x 10(3) EC/cm2; p < 0.05), and occasional 10-microns-wide intercellular gaps with adherent platelets and leukocytes were visible. Transmission electron microscopy showed leukocytes and cell debris in the underlying fibrin glue. After 4 weeks of implantation, the endothelium of experimental prostheses had regained a high cell density (72.7 +/- 10.5 x 10(3) EC/cm2) with a mature and well-differentiated morphologic appearance. At both observation periods, the surface of control grafts showed a wide range from fibrin deposits to an amorphous protein coverage containing spread platelets. CONCLUSIONS: The endothelium of in vitro-endothelialized vascular prostheses remains confluent after implantation and is nonthrombogenic in spite of a moderate initial cell loss.

Endothelial seeding of venous prostheses.

To evaluate the potential benefit of endothelial seeding of venous prostheses, 20 dogs were subjected to iliocaval reconstruction with expanded polytetrafluoroethylene grafts protected by an arteriovenous fistula. Grafts seeded with enzymatically derived endothelial cells were compared with control grafts that were sham seeded with culture medium and blood. Five seeded and seven sham-seeded grafts remained patient and were perfusion fixed in situ 4 to 6 weeks after operation. Specimens were examined by light, scanning, and transmission electron microscopy. No statistical difference in early patency was noted. The mean thrombus-free surface area was 80% in the seeded and 71% in the sham-seeded group. Light microscopy of these areas revealed a monocellular layer lining the lumen in all grafts. Scanning electron microscopy demonstrated a thin cellular lining covering 50% to 100% of the specimens' surface area in four of the five seeded and five of seven sham-seeded grafts. Transmission electron microscopy revealed these cells to exhibit characteristics typical of endothelial cells. The subcellular layer was equally thin in both groups. Early patency rates were not benefited by endothelial seeding of grafts placed in the venous system. Seeding of grafts with enzymatically derived endothelial cells provides a good endothelial cover with a thin subendothelial layer but not to a greater extent than does sham seeding of the venous prostheses.

Mast cell infiltration: a possible mechanism for vein graft vasospasm.

Mast cell infiltration of the arterial wall has been demonstrated in atherosclerotic vessels and implicated in coronary artery spasm. Spasm of vein bypass grafts has also been reported. In this study we performed vein bypass grafting of the carotid arteries in rabbits and examined the grafts for the presence of mast cells. We also determined vein graft vasoreactivity to histamine, to assess whether mediators of mast cells may have a functional role in vivo. In the control veins no mast cells were identified in 80 high-power fields (400X). In the vein bypass grafts an average of 2.6 +/- 0.8 (p = 0.01) mast cells were identified in the same number of high-power fields. Isometric tension studies of control vein and vein bypass grafts treated with histamine resulted in sigmoid dose-response curves. The ED50 for control vein was 4.69 +/- 0.63 X 10(-5) mol/L. Compared with control vein, the vein bypass grafts showed a rightward shift in the dose-response curve to histamine (ED50 11.6 +/- 1.7 X 10(-5) mol/L, p = 0.01). The histaminergic response in both vessels was blocked by the H1 receptor antagonist pyrilamine (10(-7) mol/L) and was not altered by the H2 receptor antagonist cimetidine (10(-5) mol/L). The decreased sensitivity of vein bypass grafts to histamine suggests receptor down-regulation and is possibly the result of increased histamine in the vein bypass grafts. The presence of mast cells and histamine receptors, as well as altered histamine sensitivity, in vein bypass grafts suggests that infiltration by these cells may contribute to vein bypass graft vasospasm.

All-trans-retinoic acid decreases cell proliferation and increases apoptosis in an animal model of vein bypass grafting.

BACKGROUND: We have previously demonstrated a decrease in intimal hyperplasia in vein bypass grafts from animals treated with all-trans-retinoic acid (atRA). The purpose of this study was to examine the effect of atRA on proliferation and apoptosis rates in healing vein bypass grafts. METHODS: Interposition jugular vein bypass grafts were placed in the carotid artery of 30 New Zealand white rabbits. Animals received either atRA (10 mg/kg/d) or vehicle (corn oil) for a period of 2 weeks. Animals were killed at 3, 7, or 28 days after graft placement after having received 3 doses of 5-bromo-2'-¿Deoxyuridine (BRDU, 35 Mg/KG). Animals Were Perfusion Fixed, And Vein Grafts Were Prepared For Immunohistochemistry By Using Antibodies To Brdu, Proliferating Cell Nuclear Antigen, And Bcl-XL. Apoptosis Was Measured By Using The Tunel Assay. Histologic Sections Were Analyzed By A Pathologist Blinded To The Study, And An Index Of Positively Stained Cells Was Generated For Each Layer Of The Vein Graft Wall. RESULTS: All-trans-retinoic acid reduced the proliferation index in the neointima of vein grafts during the first week after surgery. Apoptotic rates were higher in the intima of vein grafts from animals treated with atRA, which could not be explained by changes in bcl-xl expression. No differences were noted in the media or adventitia between the groups. CONCLUSIONS: atRA decreased cell proliferation and increased apoptosis in the intima of healing vein bypass grafts. These effects contribute to decreased intimal hyperplasia, which has been previously noted.

Phenotypic reversion of smooth muscle cells in hybrid vascular prostheses.

Our purpose was to evaluate whether or not and when phenotypic modulation of smooth muscle cells (SMCs) in hybrid vascular prostheses preincorporated with SMCs occurs upon implantation. Two types of hybrid vascular grafts incorporated with vascular cells derived from canine jugular veins were prepared: grafts containing a collagen gel layer covered with an endothelial monolayer at the luminal surface (Model I graft) and those containing an endothelial monolayer and SMC multilayer (Model II graft). They were bilaterally implanted into carotid arteries of the same dogs from which the cells had been harvested for 2 wk (n = 3) and 12 wk (n = 3). The time-dependent changes in populations of three SMC phenotypes (synthetic, intermediate, and contractile) in the neoarterial layers were quantified by morphometric evaluation using a transmission electron microscope in hybrid vascular grafts. Before implantation, all the SMCs were of the synthetic phenotype. In Model II grafts at 2 wk, synthetic and intermediate SMCs were dominant especially in the luminal layer. On the other hand, neoarterial layers at 12 wk were dominated by contractile SMCs, which were evenly distributed throughout the entire neoarterial tissues. A markedly delayed phenotypic reversion was noted for the Model I grafts at 12 wk. In the hybrid grafts, during about 3 mo of implantation, neoarterial SMCs transformed from the synthetic to the contractile phenotypes, which was promoted by SMC incorporation.

Endothelial cell seeding of prosthetic vascular grafts: early experimental studies with cultured autologous canine endothelium.

Thirteen adult dogs underwent thoracoabdominal bypass operations with 6-mm, double-velour Dacron grafts 25 to 30 cm long. Experimental grafts were seeded with cultured autologous endothelial cells (n = 7). Unseeded grafts served as controls (n = 6). Endothelial cells were harvested from external jugular vein segments using 0.1% trypsin and 0.5% collagenase solutions. Grafts were studied at weeks 2 and 4. Endothelial cell coverage of experimental graft surfaces after two weeks was 60% to 70%, and approximately 80% after four weeks. Immunofluorescence using factor VIII-related antigen confirmed the graft's inner surface to be endothelium. Endothelial cell coverage in control grafts occurred as pannus ingrowth, and never exceeded more than 10% of the conduit surface. Generation of an early endothelial surface in prosthetic grafts is possible in a canine model using cultured autologous cells.

Effects of two novel non-peptide antagonists at the rabbit bradykinin B2 receptor.

Large species differences have been previously observed in the pharmacology of bradykinin (BK) B2 receptor antagonists. We investigated the effect of two novel non-peptide antagonists, compound 9 (a benzodiazepine peptidomimetic related to icatibant) and the thiosemicarbazide bradyzide on the rabbit B2 receptor (contractility of the jugular vein, competition of [3H]BK binding to a B2 receptor-green fluorescent protein (B2R-GFP) conjugate, subcellular distribution of B2R-GFP). While compound 9 is about 9000-fold less potent than icatibant, it shares with the latter peptide drug a selective, insurmountable and largely irreversible antagonist behavior against BK and the capacity to translocate B2R-GFP from the membrane into the cells. Bradyzide, reportedly very potent at rodent B2 receptors, was a competitive and reversible antagonist of moderate potency at the rabbit B2 receptor (contractility pA2 6.84, binding competition IC50 5 nM). The C-terminal region of icatibant, reproduced by compound 9, is likely to be important in the non-equilibrium behavior of icatibant. Bradyzide, a non-peptide antagonist developed on different structural grounds, is competitive at the rabbit B2 receptor.

Degradation of a supporting prosthesis can optimize arterialization of autologous veins.

In a previous study, we implanted autologous vein grafts in the carotid artery of rabbits supported by a compliant, biodegradable prosthesis to prevent vein wall damage due to the higher arterial pressure. We showed that such a supporting prosthesis indeed reduces damage to these vein grafts and allows for more regular and gradual arterialization than that afforded by unsupported vein grafts. To evaluate the influence of the rate of biodegradation of such a supporting prosthesis on the process of arterialization of autologous vein grafts, we implanted vein grafts supported with prostheses, which degrade within 3 weeks (group I), 6 weeks (group II), or 3 months (group III), into the carotid artery of rabbits, and then evaluated them up to 6 weeks after implantation. At 6 weeks, the group I vein grafts showed a thinner vein wall than did the adjacent artery during dilatation. In group II, the vein wall thickness and luminal diameter had completely adjusted to that of the adjacent carotid artery. The group III vein grafts showed a significantly thinner vein wall in the absence of dilatation. All supported vein grafts showed regular longitudinally oriented and, in some areas, circularly oriented cell layers, together with thin elastic laminae, which were most pronounced in group II. We conclude that a supporting, compliant prosthesis can stimulate, regulate, and optimize the arterialization of autologous vein grafts in rabbits. If the rate of degradation is carefully chosen, the radius and wall thickness of the vein graft can completely adjust to that of the adjacent artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular remodeling of depopulated bovine ureter used as an arteriovenous graft in the canine model.

BACKGROUND: One of the greatest challenges in hemodialysis access surgery is improving the durability of prosthetic grafts caused by structural deterioration. The depopulated bovine ureter SynerGraft (SG) (CryoLife, Inc) is a tissue-engineered vascular graft processed to remove the xenograft cells while maintaining an unfixed connective tissue matrix capable of autologous cell repopulation by the recipient. STUDY DESIGN: Nineteen 6-mm diameter bovine ureter SG conduits were implanted in 12 dogs as arteriovenous grafts between the carotid artery and jugular vein (n = 11) or between the femoral artery and vein (n = 8). Performance of these biologic conduits was compared with that of 15 IMPRA (Bard) ePTFE grafts implanted in 9 dogs, including 9 arteriovenous grafts between the carotid artery and jugular vein and 6 femoral artery to femoral vein grafts. After 14 days, the grafts were accessed once weekly. Histologic and immunohistochemical analyses were performed on grafts explanted between 10 to 60 weeks. RESULTS: The 6- and 12-month primary patency rates of the bovine SG were 72.6% and 58.6%, respectively, compared with 6- and 12-month primary patency for ePTFE conduits of 57.4% and 57.4%, respectively. None of the bovine SG grafts became infected, but synthetic conduits became infected within 54 days of implantation. At 10 weeks, bovine ureter SG conduit showed fibroblast cell migration and proliferation with incorporation into the surrounding subcutaneous tissue, and elongated cells expressing the contractile protein smooth muscle actin were also observed. After 24 weeks, procollagen synthesis was demonstrated in the fully colonized graft matrix. The ePTFE grafts had no evidence of cellular ingrowth and an absence of endothelium. CONCLUSIONS: The bovine SG was appropriately remodeled to its host environment through an organized process of recellularization and neovascularization. The absence of infection, similar patency rates, and cell repopulation of the matrix warrant further investigation.