Development and evaluation of a novel decellularized vascular xenograft.

Although autogenous saphenous vein remains the standard for coronary and infrapopliteal bypass, many patients do not have a suitable vein. Attempts at developing a small-caliber vascular graft have failed largely due to occlusion, neointimal hyperplasia, or aneurismal degradation. We have designed and characterized a novel small-caliber vascular xenograft that may overcome these failure modes. To reduce immune reactions, porcine common carotid arteries were decellularized by enzymatic and detergent treatments. Histology and electron microscopic examination showed complete removal of cellular components while the extracellular matrix structure remained intact. To reduce thrombogeneity, decellularized vascular grafts were covalently linked with heparin. The efficiency of heparin linkage was demonstrated with toluidine blue staining and the antithrombogeneity of the heparin-treated grafts was demonstrated with a clot time test. Mechanical testing of the graft was performed. Decellularized-heparin-treated grafts were similar in compliance to fresh vessels and burst testing showed grafts to withstand pressures exceeding 10 times physiologic blood pressure. There was no difference in suture retention strength between fresh vessels and decellularized-heparin-treated grafts. Decellularized, heparinized grafts were implanted in dogs as carotid artery bypass grafts and showed smooth muscle cells densely populating the wall, and endothelial cells lining the lumen by two months. This study provides a new strategy to develop a small-caliber vascular graft with excellent mechanical properties, antithrombogeneity, and tissue compatibility.

Surgical intervention for complications caused by femoral artery catheterization in pediatric patients.

PURPOSE: This study evaluated the risk factors and surgical management of complications caused by femoral artery catheterization in pediatric patients. METHODS: From January 1986 to March 2001, the hospital records of all children who underwent operative repairs for complications caused by femoral artery catheterization were reviewed. A prospective cardiac data bank containing 1674 catheterization procedures during the study period was used as a means of determining risk factors associated with iatrogenic femoral artery injury. RESULTS: Thirty-six operations were performed in 34 patients (age range, 1 week-17.4 years) in whom iatrogenic complications developed after either diagnostic or therapeutic femoral artery catheterizations during the study period. Non-ischemic complications included femoral artery pseudoaneurysms (n = 4), arteriovenous fistulae (n = 5), uncontrollable bleeding, and expanding hematoma (n = 4). Operative repairs were performed successfully in all patients with non-ischemic iatrogenic femoral artery injuries. In contrast, ischemic complications occurred in 21 patients. Among them, 14 patients had acute femoral ischemia and underwent surgical interventions including femoral artery thrombectomy with primary closure (n = 6), saphenous vein patch angioplasty (n = 6), and resection with primary anastomosis (n = 2). Chronic femoral artery occlusion (> 30 days) occurred in seven patients, with symptoms including either severe claudication (n = 4) or gait disturbance or limb growth impairment (n = 3). Operative treatments in these patients included ileofemoral bypass grafting (n = 5), femorofemoral bypass grafting (n = 1), and femoral artery patch angioplasty (n = 1). During a mean follow-up period of 38 months, no instances of limb loss occurred, and 84% of children with ischemic complications eventually gained normal circulation. Factors that correlated with an increased risk of iatrogenic groin complications that necessitated surgical intervention included age younger than 3 years, therapeutic intervention, number of catheterizations (>or= 3), and use of 6F or larger guiding catheter. CONCLUSION: Although excellent operative results can be achieved in cases of non-ischemic complications, acute femoral occlusion in children younger than 2 years often leads to less satisfactory outcomes. Operative intervention can provide successful outcome in children with claudication caused by chronic limb ischemia. Variables that correlated with significant iatrogenic groin complications included a young age, therapeutic intervention, earlier catheterization, and the use of a large guiding catheter.

Colonic necrosis subsequent to catheter-directed thrombin embolization of the inferior mesenteric artery via the superior mesenteric artery: a complication in the management of a type II endoleak.

The optimal management of endoleaks after endovascular repair of abdominal aortic aneurysms remains to be established. In this report, we describe a persistent side-branch, or type II, endoleak 1 year after endograft implantation treated with catheter-directed embolization of the aneurysm sac and the inferior mesenteric artery via the superior mesenteric artery, with embolization agents including thrombin, lipiodol, and gelfoam powder. Shortly after the embolization procedure, colonic necrosis developed in the patient, manifested by peritonitis, which necessitated a partial colectomy. This case underscores the devastating complication of colonic ischemia as a result of catheter-directed embolization of the inferior mesenteric artery in the management of an endoleak.

Antitumor activity of antisense clusterin oligonucleotides is improved in vitro and in vivo by incorporation of 2'-O-(2-methoxy)ethyl chemistry.

Phosphorothioate (P=S) antisense oligonucleotides (ASO) targeting the cell survival gene clusterin synergistically enhance castration- and chemotherapy-induced apoptosis in prostate cancer xenografts. This study compares efficacy, tissue half-lives, and toxicity of P=S clusterin ASO to third-generation backbone 2'-O-(2-methoxy)ethyl (2'MOE) ribose-modified clusterin ASO. Northern analysis quantified changes in clusterin mRNA levels in human PC-3 cells and tumors. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay measured effects of combined clusterin ASO plus paclitaxel on PC-3 cell growth. Athymic mice bearing PC-3 tumors were treated with paclitaxel plus either P=S clusterin ASO, 2'-MOE clusterin ASO, or mismatch control oligonucleotides for 28 days. Weekly body weights and serum parameters were measured to assess toxicity. Tissue half-life of P=S and 2'-MOE ASO in PC-3 tumors was assessed using capillary gel electrophoresis (CGE). Both 2'-MOE and P=S ASO decreased clusterin mRNA levels in a dose-dependent and sequence-specific manner. 2'-MOE ASO more potently suppressed clusterin mRNA (80 versus 40% at 500 nM) compared with P=S ASO. IC(50) of paclitaxel was equally reduced (50--75%) by both compounds. In vivo tissue half-life was significantly longer for 2'-MOE-modified ASO than for P=S ASO (5 versus 0.5 days). Using CGE, >90% of detected 2'-MOE ASO in tumor tissue was full length. Weekly administration of 2'-MOE clusterin ASO was equivalent to daily P=S clusterin ASO in enhancing paclitaxel efficacy in vivo. 2'-MOE-modified ASO potently suppressed clusterin expression and prolonged tissue half-lives with no additional side effects. These results support the use of 2'-MOE-modified ASO over conventional P=S ASO by potentially increasing potency and allowing longer dosing intervals in clinical trials.

Effects of nicotine and cotinine on porcine arterial endothelial cell function.

UNLABELLED: BACKGROUND. There has been a significant amount of research on the effects of nicotine on vascular biology; however, little is known about the effects of cotinine, the metabolic product of nicotine. This study used a novel vascular perfusion system to study the effects of nicotine and cotinine on the vascular endothelial cell function. METHODS: Porcine common carotid arteries were cultured in a novel vascular perfusion system with nicotine or cotinine or as controls. After 24 h, vessels were precontracted with norepinephrine and subsequently relaxed with acetylcholine. Vessel diameters were recorded and analyzed. After culture, samples were taken for en face, immunohistochemistry, and RT-PCR for eNOS. Porcine coronary arteries were incubated as controls or with nicotine or cotinine and tested on a myograph system to measure contraction and relaxation. RESULTS: Porcine carotid arteries treated with nicotine and cotinine showed a 27.2% and a 41.2% reduction in endothelial-dependent relaxation, respectively, as compared to control vessels (P<0.05). Rings of coronary arteries treated with nicotine relaxed similarly to control rings while cotinine-treated rings failed to relax to endothelial-dependent stimulation. RT-PCR for eNOS mRNA showed a 23. 2 and a 24.1% reduction in eNOS expression for nicotine- and cotinine-treated vessels, respectively (P<0.01). Additionally, immunohistochemical staining for eNOS showed less dense staining on nicotine- and cotinine-treated vessels as compared to controls. En face preparations showed normal endothelial cell morphology in all groups, but cell density decreased slightly in vessels treated with nicotine and cotinine. CONCLUSION: These results indicate that cotinine may have even more effect on the impairment of endothelial-dependent vasorelaxation than nicotine for the regulation of vessel tone in porcine carotid and coronary arteries.

A simple physiologic pulsatile perfusion system for the study of intact vascular tissue.

Perfusion vascular culture models may provide a useful link between cell culture models and animal culture models by allowing a high level of control over important parameters while maintaining physiologic structure. The purpose of this study was to develop and test a new vascular culture system for pulsatile perfusion culture of intact vascular tissue. The system generates a pulsatile component of flow by means of a cam-driven syringe and a peristaltic pump and compliance chamber. Cams were designed, constructed and tested to simulate canine femoral and common carotid artery flows. The mean pressure was adjusted between 60 and 200 mmHg without significantly affecting flow rate, flow waveform, or the pressure waveform. Porcine common carotid artery segments were cultured in this pulsatile perfusion system. The viability of vascular segments was tested after various culture times with a functional assay that demonstrated both smooth muscle cell and endothelial cell response to vasomotor challenge.

Effects of homocysteine on smooth muscle cell proliferation in both cell culture and artery perfusion culture models.

BACKGROUND: Hyperhomocysteinemia is associated with increased risk for vascular disease. However, the pathogenic mechanisms of homocysteine are largely unknown. We evaluated the effects of homocysteine on smooth muscle cell (SMC) and endothelial cell proliferation in cell culture and on SMC proliferation of balloon angioplasty-injured arteries in a perfusion culture model. METHODS: Human and pig SMCs and endothelial cells were cultured with variable amounts of homocysteine for 72 h and the total cells were counted using a hemocytometer. Fresh pig carotid arteries were harvested from a local slaughterhouse and cultured in a newly designed artery perfusion culture system. Five groups of arteries (six per group) were cultured for 48 h under different conditions: normal control, balloon angioplasty injury alone, and injury with three different doses of homocysteine. Vessel viability was evaluated. SMC proliferation was assayed by bromodeoxyuridine (BrdU) DNA labeling. RESULTS: At concentrations equivalent to those in human hyperhomocysteinemia, homocysteine significantly stimulated both cultured human and pig SMC proliferation with a dose-dependent effect, while it inhibited cultured endothelial cell growth. Perfusion-cultured pig carotid arteries remained contractile in response to norepinephrine and relaxant to nitroglycerine, and viable cells were also isolated from the cultured arteries. SMC proliferation (BrdU index) showed significant differences among the groups. SMC proliferation was stimulated by vascular injury and further enhanced by homocysteine in a dose-dependent manner. The proliferative response occurred strongly on the luminal side of the vessel wall, with the effects tapering toward the adventitia. CONCLUSIONS: Homocysteine had a mitogenic effect on vascular SMCs and a cytotoxic effect on endothelial cells. This differential effect of homocysteine on vascular cells may represent a pathogenic mechanism of vascular lesion formation in patients with hyperhomocysteinemia.

A new perfusion culture system used to study human vein.

BACKGROUND: Cell culture studies, ring studies, and indirect physiologic studies are the predominant models used to study human vascular tissue. Such studies are limited in their capacity to permit physiologic single-factor changes or to provide the proper mechanical stress or extracellular matrix present in normal tissues. We present a newly devised organ culture system that addresses these issues and permits survival of intact segments of human vascular tissue in a perfused environment. Our experience culturing human saphenous vein with this system is detailed. METHODS: Perfusion culture chambers were designed and constructed in our laboratory. Excess saphenous vein segments were collected from coronary artery bypass graft cases at our hospital and then mounted into our perfusion culture system for 0, 24, 48, 72, or 96 h. Vasomotor assays, hematoxylin and eosin staining, bromodeoxyuridine staining, and factor VIII staining were performed to assess tissue survival. RESULTS: A total of 24 veins were cultured. Average vessel length was 5 cm. The vessels contracted and relaxed the following amounts: time 0 (6.7% contraction, 5.0% relaxation), 24 h (5.7%, 5.3%), 48 h (5.2%, 2.8%), 72 h (4.8%, 5.3%), 96 h (4.8%, 3.8%). Hematoxylin and eosin staining, bromodeoxyuridine staining, and factor VIII staining support the viability of the tissue segments. CONCLUSION: A new perfusion organ culture system has been devised that permits survival of intact human venous tissue for periods up to 96 h. Studies that permit physiologic single-factor changes along with precise control of the hemodynamic environment are possible with this system.

Pharmacokinetic properties of several novel oligonucleotide analogs in mice.

Biophysical and pharmacokinetic properties of five analogs of ISIS 3082, a 20-mer phosphorothioate oligodeoxynucleotide that inhibits the expression of mouse intercellular adhesion molecule 1, were evaluated. Compared to the parent compound, ISIS 3082, the 2'-propoxy modified phosphodiester, ISIS 9044 and the 2'-propoxy phosphorothioate, ISIS 9045, had greater affinity for complementary RNA and were more lipophilic. A chimeric oligonucleotide comprised of 2'-propoxy diester wings and a phosphorothioate deoxy center (ISIS 9046) had equal affinity. It was also more lipophilic than ISIS 3082, but less so than the other 2'-propoxy modified analogs. The two analogs with 5'-lipophilic conjugates, ISIS 9047 (5'-octadecylamine) and ISIS 8005 (5'-(2'-O-hexylamino-carbonyl-oxycholesterol) were more lipophilic than ISIS 3082 (3- and 7-fold, respectively) but had similar affinity for complementary RNA. Binding of ISIS 3082 to bovine serum albumin was salt-dependent and, at physiological concentration (320 mOsmol), the dissociation constant (Kd) was 140 microM. Similarly, the 2'-propoxy phosphodiester, ISIS 9044, displayed salt-dependent bovine serum albumin binding, but not binding was measurable at physiological salt conditions. In contrast, the more lipophilic phosphorothioate analogs displayed much higher affinity to bovine serum albumin at 320 mOsmol than ISIS 3082. After bolus injection to mice, the initial volumes of distribution of the more lipophilic phosphorothioate analogs, ISIS 9045, ISIS 9047 and ISIS 8005, were less and the initial clearance from plasma was slower than ISIS 3082. The pharmacokinetics of the other analogs was similar to ISIS 3082. Distribution of ISIS 3082 into peripheral tissues was similar to that reported for other phosphorothioates with liver and kidney accumulating the highest fraction of the dose. The only modification to markedly influence distribution was the very lipophilic cholesterol conjugate (ISIS 8005), which increased substantially the fraction of the dose accumulated by the liver. Little intact drug was found in urine or feces for any analog, and the patterns of metabolites suggested that for all analogs the principal metabolic pathway was due to 3'-exonuclease activity. The metabolism of ISIS 3082 was similar to that reported for other phosphorothioates. After 2 hr, most of the radioactivity in plasma represented metabolites but, in tissues, intact ISIS 3082 was present for much longer periods of time and metabolites accumulated more slowly. The 24-hr exposure to ISIS 3082 of liver and kidney was 20.7 and 67.9 microM/hr, respectively. The rates of metabolism in plasma, liver and kidney of the two 5'-conjugates, ISIS 9047 and ISIS 8005, were similar to ISIS 3082, as was the pattern of metabolism. The rate of metabolism of ISIS 9044 (2'-propoxy phosphodiester oligonucleotide) was much more rapid in liver and plasma, but surprisingly much slower in the kidney. ISIS 9045 (full 2-propoxy phosphorothioate) was much more stable than ISIS in all tissues, the enhanced stability of ISIS 9045 resulted in increased exposure of liver and kidney to the drug, whereas the exposure of the liver to the two more lipophilic analogs, ISIS 9047 and ISIS 8005, was greater because a higher fraction of the dose was distributed to the liver. The exposure of the kidney to ISIS 9044 was also greater than that to ISIS 3082 due to the surprising stability of the drug in the kidney.