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J C Stanley

Publications and source records attributed to J C Stanley.

At least 55 records · Page 3Linked to original sources
PubMed1996

Systemic delivery of the interleukin-1 receptor antagonist protein using a new strategy of direct adenoviral-mediated gene transfer to skeletal muscle capillary endothelium in the isolated rat hindlimb.

Current gene therapy strategies using adenoviral vectors to target the lung or liver have been complicated by an acute inflammatory response that can result in loss of transgene expression as well as tissue injury and necrosis. Skeletal muscle comprises 40% of total body weight; it possesses a high density, accessible capillary network that is resistant to injury and thus may be a logical target for adenoviral vectors. We hypothesized that adenoviral transduction of the rat skeletal muscle capillary bed during vascular isolation would achieve efficient gene transfer sufficient to achieve systemic serum levels of a recombinant protein without significant tissue injury. During vascular isolation of the hindleg, a replication-incompetent adenovirus (Ad) encoding for either the marker gene, human placental alkaline phosphatase (hpAP), or interleukin-1 receptor antagonist (IL-1ra) was infused and subsequently flushed from the circulation after a 30-min dwell period. Gene transfer over a 10(9)-10(12) particle/ml range to the gastrocnemius capillary endothelium and muscle fibers was highly efficient and titer-dependent, reaching maximum transduction rates of 71 +/- 7% and 25 +/- 5%, respectively, 5 days after gene transfer (n = 3-8 rats/group, p < 0.05). hpAP transgene expression was barely detectable at 14 days. No significant tissue injury or necrosis of the skeletal muscle was observed at 5 and 14 days, and distant organ gene transfer was minimal or absent. Gastrocnemius muscle from rats (n = 4) given Ad-IL-1ra had 241 +/- 66 pg IL-1ra/mg protein at 5 days, while those given Ad-hpAP, negative control (n = 3) had 35 +/- 14 pg IL-1ra/mg protein (p < 0.05). Ad-IL-1ra rats (n = 4) had serum levels of 185 +/- 20 pg/ml IL-1ra at 5 days whereas Ad-hpAP control rats (n = 5) had no IL-1ra detectable (p < 0.0001). Athymic rats given Ad-IL-1ra (n = 6) had serum levels of 493 +/- 62 pg/ml IL-1ra 14 days after transduction, and IL-1ra was detected for up to 98 days. Sera from Ad-IL-1ra athymic rats significantly inhibited IL-1 beta-induced (1 ng/ml) prostaglandin E2 (PGE2) production from cultured endothelial cells by 82 +/- 2% (p < 0.001). Thus, this gene transfer strategy is the first to result in substantial transduction of both skeletal muscle capillary endothelium and fibers, sufficient to achieve pharmacologic levels of IL-1ra. Although no acute tissue injury or necrosis was observed, persistence of transgene expression in athymic rats suggests that loss of expression in normal rats was by an immune-mediated mechanism.

Adenoviridae↗
PubMed1996

PKH26 and 125I-PKH95: characterization and efficacy as labels for in vitro and in vivo endothelial cell localization and tracking.

PKH26, a fluorescent cell label, and PKH95, a 125 I-radioactive cell label, are both potentially valuable endothelial cell labels because they bind irreversibly within cell membranes. These labels would be particularly well suited to tract transplanted endothelial cells in vivo. However, no previous studies documenting lack of transfer of the label to unlabeled endothelial cells, as well as the effect of the label on endothelial cell function, have been undertaken. The purpose of this study was to determine the optimal method of endothelial cell (EC) labeling with PKH26 and PKH95, whether significant to EC-to-EC transfer of the label occurs, the effects of these labels on EC proliferation and membrane function, and the feasibility of using these labels for long-term quantitative EC tracking in vivo. Canine ECs in confluent monolayers or in cell suspension were labeled by exposure to 1.0 or 5.0 microM PKH26 for 1, 3, or 5 min. Cell viability was determined by trypan blue exclusion. The percentage of cells labeled and their fluorescence intensity were determined in a fluorescent-activated cell sorter (FACS). Effect of the label on cell function was assessed by measuring EC proliferation rates as well as intercellular adhesion molecule (ICAM) expression before and after induction with tumor necrosis factor (TNF). To determine if transfer of the label occurs, both labeled and nonlabeled ECs were grown in coculture and subjected to FACS analysis. For in vivo cell tracking, doubly labeled ECs were injected into the femoral artery of rat hind-limbs, and whole-body tissue analysis was undertaken to determine labeled-EC distribution at 60 days. Endothelial cells were labeled with equal efficacy as monolayers or in suspension. Labeling had no effect on EC proliferation rates nor on TNF-induced upregulation of ICAM expression. Coculture experiments revealed no significant label transfer to nonlabeled ECs. In vivo cell tracking studies documented that 8% of label remained within the skeletal muscle capillaries at 60 days after injection. PKH26 and PKH95 labels incorporate stably into EC membranes, do not alter endothelial cell function, and provide a precise means for quantitative EC tracking and histologic localization both in vitro and in vivo. These labels should prove to be very useful for studies of endothelial cell biology and transplantation.

Animals↗
PubMed1996

A [+18RGD] protamine variant for nontoxic and effective reversal of conventional heparin and low-molecular-weight heparin anticoagulation.

Protamine sulfate reversal of heparin anticoagulation causes adverse side effects. Additionally, protamine sulfate is relatively ineffective at reversing factor Xa inhibition caused by low-molecular weight heparin (LMWH, Enoxaparin). Previously, a +18 compound partially reversed heparin and LMWH with minimal toxicity. In the present study, a new +18 protamine-like variant, [+18RGD], with an added RGD sequence [acetyl-EA(R2A2R2A)4R2GRGDSPA-amide], was compared to a previously developed compound, [+18BE,Acetyl-EAA-(K2A2K2A)4K2-Amide] and standard protamine [Prot +21] regarding the reversal of conventional unfractionated heparin (Hep) and LMWH. These three agents were given at 1 mg per 100 IU activity of Hep or LMWH rapidly over 10 sec. Hemodynamic toxicity was based on maximum declines in blood pressure, heart rate, cardiac output, and oxygen consumption over the first 5 min after reversal (calculated as a total toxicity score, TTS). The more negative the TTS, the more toxic the agent. Degrees of toxicity (TTS) of [+18RGD], [+18BE],and[Prot +21] for reversal of Hep were -1.19, -2.00, and -7.32, respectively; and for reversal of LMWH they were -2.85, -3.98,and -6.17, respectively. These differences were significant for Hep (P < 0.01) and approached significance for LMWH (P = 0.07). Maximum hemodynamic perturbations paralleled the TTS pattern. [+18RGD] provided equal reversal efficacy to [Prot +21] for Hep, with a statistically significant (P < 0.05) lessening of platelet count declines (Plt 27, -46, and -55%, respectively). [+18RGD] improved reversal efficacy for LMWH, at 3, 10, and 30 min following its administration. At 3 min, antifactor Xa reversal was 72, 40, and 30%, respectively, for [+18RGD], [+18BE], and [Prot +21]; [+18RGD] effects were significantly better (P < 0.01). [+18RGD] reversed both Hep and LMWH anticoagulation with minimal toxicity. Such a compound should decrease clinical complications attending the use of standard protamine for reversal of conventional heparin or LMWH anticoagulation.

Amino Acid Sequence↗
PubMed1996

Tissue plasminogen activator increases canine endothelial cell proliferation rate through a plasmin-independent, receptor-mediated mechanism.

BACKGROUND: Tissue plasminogen activator (tPA) is elevated in cancer patients and is thought to promote tumor angiogenesis by facilitating endothelial cell migration through plasmin-mediated degradation of extracellular matrix. Due to the presence of an epidermal growth factor (EGF)-finger domain in the tPA A-chain and the existence of an endothelial cell (EC) receptor that binds this domain, it was hypothesized that tPA has a direct receptor-mediated effect on EC proliferation, independent of plasmin. METHODS AND RESULTS: Using cultured canine ECs, tPA (7.25 microg/ml, approximately 107 nM) increased proliferation as much as 50 and 170% in the absence and presence of growth factors, respectively. tPA-induced increases in EC proliferation occurred independent of plasmin generation, as the plasmin inhibitor, aprotinin (10 microg/ml) did not inhibit tPA-induced proliferation. However, tPA-induced proliferation was inhibited dose-dependently to a maximum of 78% using a monoclonal antibody against the tPA EGF-finger domain. This antibody, known to inhibit tPA binding to its receptor, did not inhibit tPA-induced plasmin generation. To investigate the role of potential signal transduction pathways, ECs were exposed to lavendustin A, a tyrosine kinase inhibitor, at 33.5 microM (IC50 for basic fibroblast growth factor). Lavendustin A did not inhibit tPA-induced EC proliferation. However, Rp-cAMP, an inhibitor of cAMP-dependent kinases, specifically inhibited tPA-induced EC proliferation in a dose-dependent manner (IC50 = 50.5 microM). Pertussis toxin at maximal concentrations for this system (0.5 ng/ml) did not inhibit tPA-induced EC proliferation. CONCLUSION: These results lend support to the hypothesis that tPA may have a direct receptor-mediated effect on EC proliferation and that this effect occurs independent of plasmin and may be dependent upon protein kinase A activity.

Animals↗
PubMed1996

Effects of differing rates of protamine reversal of heparin anticoagulation.

BACKGROUND: Protamine sulfate reversal of heparin anticoagulation may be associated with adverse cardiovascular side effects. The purpose of this study was to determine whether diminished systemic oxygen consumption and hemodynamic changes were more likely to accompany rapid versus slow protamine administration. METHODS: Fifteen patients undergoing abdominal aortic aneurysm resection in a prospective randomized double-blinded study received intravenous protamine (1.5 mg/kg) rapidly during a 3-minute period (group I, n = 7) or slowly during a 15-minute period (group II, n = 8). Systemic oxygen consumption (VO2) and hemodynamic parameters were assessed for up to 20 minutes after protamine administration began. RESULTS: Blood pressure declines (millimeters of mercury) were greatest in group I with rapid protamine administration (-19 systolic and -9 diastolic) compared with group II with slow protamine administration (-12 systolic and -1 diastolic). Heart rate fell markedly in both groups I and II. Cardiac output (CO) declined in group I at virtually all time periods. Similar CO declines in group II occurred 10 minutes after protamine infusion had begun and persisted for 3 minutes after protamine administration was complete. Maximum VO2 decreases were -16% (60 seconds into protamine infusion) and -13% (1.5 minutes after protamine infusion) in groups I and II, respectively, with statistically significant declines (p < 0.05) occurring only in group I compared with baseline values. Statistically significant differences (p < 0.01), however, were found when mean declines during and after protamine infusion were compared with controls for both CO and VO2 in both groups. CONCLUSIONS: Significant declines in systemic VO2 and hemodynamic perturbations accompany protamine reversal of heparin anticoagulation during aortic surgery. Rapid protamine administration increases the magnitude of these adverse responses.

Aged↗
PubMed1996

Vascular surgery in the United States: workforce issues. Report of the Society for Vascular Surgery and the International Society for Cardiovascular Surgery, North American Chapter, Committee on Workforce Issues.

The Committee on Workforce Issues of the Society for Vascular Surgery (SVS) and the North American Chapter, International Society for Cardiovascular Surgery (NA-ISCVS) generated data on the numbers and trends of operations performed and workforce requirements to provide surgical care of patients with vascular disease. Data for analysis were obtained from The National Center for Health Statistics-National Hospital Discharge Survey and questionnaires sent to 2732 vascular surgeons. The data showed that SVS/NA-ISCVS and regional vascular society (RVS) surgeons performed 51% of 583,000 vascular procedures undertaken in the United States in 1992. This represents a 24% increase over the 41% reported in 1985 by similarly defined surgeons. Analysis of 1992 index cases documented that SVS/NA-ISCVS and RVS surgeons accounted for 80% of 31,000 aortoiliofemoral bypasses, 68% of 46,000 aortic aneurysmectomies, 64% of 91,000 carotid endarterectomies, and 72% of 98,000 angioaccess procedures. The mean numbers of vascular operations performed in 1992 by SVS/NA-ISCVS and RVS surgeons were 144 and 100, respectively. These procedures represented 64% and 39% of the total surgical caseload of SVS/NA-ISCVS and RVS surgeons, respectively. American Board of Surgery (ABS)-certified vascular surgeons performed a mean of 171 vascular operations in 1992. Other surgeons, including ABS-certified general surgeons, appear to be performing fewer vascular operations. The latter fact and the increasing incidence of vascular disease in an expanding elderly population supports a continued need for vascular surgery specialists. Evolving technology and new health care delivery systems, however, may lessen the need for surgical care of these patients. Continued assessments of workforce activity will allow better definition of changing vascular surgery needs.

Humans↗
PubMed1996

Retroviral-mediated transduction of endothelial cells with the lac Z gene impairs cellular proliferation in vitro and graft endothelialization in vivo.

PURPOSE: An endothelialized lumen within a synthetic graft that expresses recombinant proteins with anticoagulant or antiproliferative activity has the potential to improve graft function. However, preliminary data suggest that genetic modification of endothelial cells (ECs) impairs their proliferation. The purpose of this investigation was to test the hypothesis that retroviral transduction of cultured ECs with the lac Z gene encoding for beta-galactosidase would decrease EC proliferation in vitro and graft endothelialization in vivo. METHODS: In vitro studies compared canine EC proliferation over a 14-day period among early-passage ECs (two and three) and late-passage ECs (six and nine) transduced with the BAG vector (containing the lac Z gene and the neomycin resistance gene), ECs transduced with the neomycin resistance gene only, the nontransduced ECs. In vivo canine studies assessed endothelialization of expanded polytetrafluoroethylene thoracoabdominal grafts seeded with autologous lac Z-transduced ECs (n = 7) or nontransduced ECs (n = 3) compared with that of nonseeded grafts (n = 3). Histochemical staining and DNA polymerase chain reaction was used 6 weeks after implantation to detect the presence of the lac Z gene in the grafts' cellular linings and perigraft tissues. Endothelialization was assessed by light microscopy and electron microscopy. RESULTS: Proliferation of late-passage lac Z-transduced ECs in vitro was significantly decreased compared with neomycin resistance-transduced ECs or nontransduced ECs. Among early-passage ECs smaller but significant decreases in proliferation were noted among lac Z-transduced cells compared with nontransduced cells. Six of seven expanded polytetrafluoroethylene grafts seeded with transduced ECs showed lac Z gene expression. Lac Z gene expression was not found on grafts seeded with nontransduced ECs or nonseeded grafts. The endothelialized luminal surface area was significantly less in grafts seeded with lac Z-transduced ECs compared with grafts seeded with nontransduced ECs. CONCLUSIONS: Retroviral-mediated transduction of canine ECs with the lac Z gene encoding for beta-galactosidase impairs EC proliferation in vitro and the ability of transduced ECs to form a confluent EC monolayer on the luminal surface of synthetic grafts in vivo.

Animals↗
PubMed1996

Nitric oxide reversibly inhibits the migration of cultured vascular smooth muscle cells.

Augmentation of nitric oxide (NO) production in vivo decreases lesions in a variety of models of arterial injury, and inhibition of NO synthase exacerbates experimental intimal lesions. Both vascular smooth muscle cell (VSMC) proliferation and migration contribute to lesion formation. Although NO inhibits VSMC proliferation, its effects on VSMC migration are unknown. To test the hypothesis that NO inhibits VSMC migration independent of inhibition of proliferation, we examined migration of rat aortic VSMCs after wounding of a confluent culture in the presence of chemical donors of NO. Hydroxyurea was used to eliminate any confounding effect of NO on proliferation. Three NO donors, diethylamine NONOate, spermine NONOate, and S-nitrosoglutathione, exhibited concentration-dependent inhibition of both number of migrating VSMCs and maximal distance migrated. Inhibition of migration was also seen with 8-Br-cGMP, suggesting that activation of guanylate cyclase may play a role in mediating the antimigratory effects of NO. Migration resumed after removal of NO donors, as evidenced by an increase in distance migrated. Measurement of VSMC protein synthesis and mitochondrial respiration indicated that inhibition of migration by NO donors was not due to metabolic cytostasis. These findings indicate that NO reversibly inhibits VSMC migration independent of proliferation or cytotoxicity, a novel mechanism by which both endogenous and pharmacological NO may alter vascular pathology.

Animals↗
PubMed1996

Cerebral oximetry in patients undergoing carotid endarterectomy under regional anesthesia.

BACKGROUND AND PURPOSE: Near-infrared spectroscopy is a technique that can potentially monitor changes in cerebral oxygenation. There are at present limited clinical data regarding the value of this technology in relating neurological outcome to cerebrovascular hemoglobin oxygen saturation (ScO2). This investigation reports changes in ScO2 due to carotid cross-clamping during carotid endarterectomy in awake patients. METHODS: ScO2 was monitored in 38 adult patients undergoing 41 carotid endarterectomies under regional anesthesia. Ipsilateral and contralateral hemispheres were monitored simultaneously during 36 operations, with ipsilateral monitoring alone in the remaining 5 operations. RESULTS: No significant difference was detected between ipsilateral and contralateral ScO2 during preclamp or postclamp periods. Carotid cross-clamping caused a statistically significant (P < .01) decrease in the ipsilateral ScO2, which decreased from 71.8 +/- 6.91% to 65.8 +/- 8.2%, while the contralateral ScO2 remained stable at 70.5 +/- 7.5% and 70.3 +/- 7.9%. The change in ipsilateral ScO2 ranged from +2.6% to -28.6% of the preclamp value. The difference between ipsilateral and contralateral ScO2 during cross-clamping was statistically significant (P < .001). The duration of cross-clamping was 39 +/- 11 minutes (range, 18 to 89 minutes). The decrease in ipsilateral ScO2 was highly variable from patient to patient and did not correlate with the duration of cross-clamping. CONCLUSIONS: These results suggest that carotid artery occlusion causes a statistically significant but variable decrease in ScO2 in the majority of patients. Data in this investigation provide a range of ScO2 values that was not associated with a clinically detectable neurological dysfunction.

Adult↗
PubMed1995

Direct effects of protamine sulfate on myocyte contractile processes. Cellular and molecular mechanisms.

BACKGROUND: Administration of the arginine-rich, highly charged protamine (PROT) molecule has been associated with episodes of acute left ventricular (LV) dysfunction. The objective of the present study was to test the hypothesis that PROT has direct effects on isolated LV myocyte contractile processes and sarcolemmal transduction systems. METHODS AND RESULTS: Exposure of porcine LV myocytes (n = 305) to 40 micrograms/mL PROT (reflecting a dose of 2.5 mg/kg) decreased basal contractile function and beta-adrenergic responsiveness. For example, myocyte percent shortening was 4.3 +/- 0.1% in control myocytes and decreased to 2.8 +/- 0.2% in the presence of 40 micrograms/mL PROT (P < .05). Myocyte percent shortening was 9.3 +/- 0.7% after beta-adrenergic receptor stimulation (isoproterenol; 25 nmol/L) and was significantly reduced in the presence of 40 micrograms/mL PROT (5.7 +/- 0.7%, P < .05). PROT reduced myocyte responsiveness to forskolin (100 mumol/L), which directly activates adenylate cyclase, by > 40% from forskolin. In addition, PROT abolished the inotropic effects of ouabain on myocyte contractile function. To determine contributory mechanisms for the effects of PROT on myocyte sarcolemmal systems, beta-receptor- and cardiac glycoside-binding characteristics were determined in sarcolemmal preparations. beta-receptor binding was 175 +/- 10 fmol/mg and was reduced to 140 +/- 6 fmol/mg in the presence of PROT (P < .05). Ouabain receptor binding was 7.1 pmol/mg and decreased to 2.6 +/- 0.4 pmol/mg in the presence of PROT. In addition, cAMP production after stimulation with isoproterenol and forskolin was significantly blunted in the presence of PROT. Variants of the PROT moelcule were constructed by specific amino acid substitutions and deletions, which provided a means to vary charge as well as structure. Substitution of arginine with lysine in the PROT peptide sequence ameliorated the negative effects on myocyte contractile processes; despite identical overall charge (21+). However, a PROT variant with an 18+ charge but different amino acid sequence induced significant negative effects on myocyte function and inotropic responsiveness. Thus, the effects of PROT on myocyte contractile processes are not due simply to the high positive charge of the molecule. To further establish that PROT can contribute to changes in LV function in the clinical setting, fluorescein-labeled PROT was circulated in antegradely perfused rabbit hearts. Microscopic examination revealed that PROT could traverse the vascular compartment of the myocardium and come in direct contact with the myocyte. CONCLUSIONS: The unique findings from the present study suggest that a fundamental contributory mechanisms for the changes in LV function observed after protamine administration may be the direct effect of unbound protamine on myocyte contractile processes.

Adrenergic beta-Agonists↗
PubMed1995

Nitric oxide inhibition of endothelial cell mitogenesis and proliferation.

BACKGROUND: Endothelial cell (EC) proliferation is essential in vascular repair after injury to the vessel wall. Impaired EC proliferation may be an important factor contributing to vessel wall disease. Nitric oxide (NO) inhibits proliferation of many cells, including smooth muscle cells (SMC). We tested the hypothesis that NO inhibits EC proliferation and DNA synthesis. METHODS: Cultured canine venous ECs were treated with NO donors: S-nitroso-N-acetylpenicillamine (SNAP), S-nitroso-glutathione (GSNO), or spermine NONOate (SP NO). Proliferation was determined by cell counts after 48 hours. Parallel proliferation studies were done with rat aortic SMC. ECs synchronized in S phase were treated with the NO donor diethylamine NONOate (DEA NO), and DNA synthesis was measured as the incorporation of tritiated thymidine. A NO antagonist, cPTIO, was used to reverse the effects of DEA NO: RESULTS: Concentration-dependent (1 to 100 mmol/L) inhibition of EC proliferation (11% to 71% inhibition; p < 0.05) was seen with SNAP. Similar inhibition of proliferation was noted with the NO donors GSNO and SP NO and in SMC treated with SNAP. DEA NO caused concentration-dependent (0.1 to 1 mmol/L) inhibition of EC DNA synthesis (39% to 85% inhibition; p < 0.05), which was reversed by cPTIO. CONCLUSIONS: NO inhibits proliferation and mitogenesis of cultured ECs. This may occur in certain pathologic states, where production of NO in plaques and diseased vessels impedes reendothelialization, thus contributing to adverse thrombotic and vasospastic events.

Animals↗
PubMed1995

Inhibition of aortic aneurysm development in blotchy mice by beta adrenergic blockade independent of altered lysyl oxidase activity.

PURPOSE: This study was designed to define the effects of beta-adrenergic blockade on aortic lysyl oxidase (LO), an enzyme responsible for elastin and collagen cross-linking, and aneurysm formation in the blotchy mouse. It was hypothesized that beta-blockade would inhibit the development of aneurysms because of its hemodynamic effect rather than a direct effect on LO activity. METHODS: Three groups of mice were studied: group I--normal littermates of blotchy mice; group II--untreated blotchy mice; group III--blotchy mice given either propranolol, atenolol, or nadolol. Data from the three different beta blocker-treated animals, group III, were statistically identical and were combined for analysis. The study was concluded when the mice were 4 months of age. At that time systolic blood pressure, heart rate, and aortic diameters were measured, and the entire aorta from each mouse was subjected to a bioassay for LO activity. RESULTS: Group I normal mice had an aortic arch diameter of 0.10 +/- 0.02 cm. Group II blotchy mice developed aortic arch aneurysms with a diameter of 0.21 +/- 0.03 cm. In Group III, beta blockade reduced the aortic arch diameter in blotchy mice to 0.11 +/- 0.03 cm. Mean heart rate in group III beta-blocked mice was reduced 25% compared with group I normal mice, and 18% compared with group II untreated blotchy mice. Blood pressures were similar in all three groups. Group II blotchy mice exhibited approximately half of the aortic LO activity (2.43 +/- 0.57 cpm/micrograms protein) noted in group I normal mice (5.82 +/- 1.06 cpm/micrograms protein). Aortic LO activity in group III blotchy mice remained low (2.09 +/- 0.85 cpm/micrograms protein) despite administration of beta-blockers. CONCLUSIONS: This is the first study to document an actual decrease in the level of aortic LO activity in blotchy mouse. beta-Blockade inhibits development of aortic aneurysms in blotchy mice. This is associated with a reduction in heart rate, but not by alterations in LO activity.

Animals↗
PubMed1995

Effective and less toxic reversal of low-molecular weight heparin anticoagulation by a designer variant of protamine.

PURPOSE: This investigation assessed protamine reversal of heparin anticoagulation by formation of a protamine-heparin alpha-helix by use of a new designer-variant protamine [+18BE] that was made from an existing protamine variant [+18B] whose non-alpha-helix-forming amino acid proline (P) was replaced by an alpha-helix-forming glutamic acid (E). The rate of administration of the new [+18BE] variant protamine on efficacy and toxicity in comparison to that of [+21] standard protamine and [+18B] was also studied. METHODS: Acetyl-EAA(K2A2K2A)4K2-Amide [+18BE] was administered intravenously in a 1:1 dose to low-molecular-weight heparin (LMWH)-anticoagulated (intravenous 150 IU antifactor Xa/kg) dogs over 10 seconds or 3 minutes (n = 7, each group). Reversal efficacy was documented by measuring activated clotting time, thrombin clotting time, antifactor Xa, and antifactor IIa. Toxicity was defined by measuring systemic blood pressure, heart rate, cardiac output, pulmonary artery pressure, and oxygen consumption. Measurements were made at baseline, after administration of LMWH, before its reversal, and for 30 minutes thereafter. Results were compared with those after LMWH reversal with [+21] standard protamine and the [+18B] variant. A total toxicity score (TTS) was calculated for each compound from maximal declines in blood pressure, heart rate, cardiac output, and oxygen consumption. RESULTS: LMWH anticoagulation reversal was significantly (p < 0.01) less toxic over 10 seconds and 3 minutes with the [+18BE] designer variant (TTS -2.3, -2.2) compared with the [+21] standard protamine (TTS -6.4, -7.2). Percent LMWH reversal at 3 minutes revealed [+18BE] to have antifactor Xa activity as high as 91%, compared with 68% for protamine [+21], when given over 3 minutes (p < 0.05). CONCLUSIONS: This investigation documents that a new designer variant of protamine [+18BE] has superior efficacy compared with [+21] standard protamine for reversal of LMWH anticoagulation and that this occurs with a highly favorable toxicity profile.

Amino Acid Sequence↗
PubMed1995

Effects of retroviral-mediated tissue plasminogen activator gene transfer and expression on adherence and proliferation of canine endothelial cells seeded onto expanded polytetrafluoroethylene.

PURPOSE: Seeding prosthetic arterial grafts with genetically modified endothelial cells (ECs) has the potential to substantially improve graft function. However, preliminary applications suggest that grafts seeded with retrovirally transduced ECs yield a significantly lower percent surface coverage than those seeded with nontransduced ECs. The objective of this study was to test the hypothesis that canine ECs transduced with the human tissue plasminogen activator (tPA) gene would have a lower rate of adherence to pretreated expanded polytetrafluoroethylene (ePTFE) both in vitro and in vivo and that they would proliferate at a slower rate on pretreated ePTFE in vitro. METHODS: Early passage ECs derived from canine external jugular vein were transduced with the retroviral MFG vector containing the gene for human tPA. ECs exposed to media alone served as controls. Iodine 125-labeled ECs were seeded in vitro onto ePTFE graft segments pretreated with canine whole blood, fibronectin (50 micrograms/ml), or media alone, and the percent of ECs adherent at 1 hour were determined (n = 3). Additional tPA-transduced and -nontransduced ECs were grown for 10 days on either fibronectin (50 micrograms/ml)-pretreated ePTFE wafers or tissue culture plastic pretreated with gelatin (1%) or fibronectin (50 micrograms/ml), and the EC proliferation rates were determined (n = 3). Furthermore, 125I-labeled ECs were seeded onto fibronectin (50 micrograms/ml)-pretreated ePTFE graft segments implanted as carotid and femoral artery interposition grafts (n = 3). The grafts were harvested after 1 hour, and the percent of ECs adherent was determined. RESULTS: Human tPA was detected by immunohistochemical staining in 61% +/- 5% of the transduced ECs and was expressed at 35.4 +/- 12.9 ng/hr/10(6) cells. Fibronectin and whole blood pretreatment of the ePTFE grafts led to greater EC adherence in vitro than did media alone (90.9% +/- 5.3% vs 77.8% +/- 5.8% vs 4.7% +/- 1.1%, p < or = 0.05). No significant difference in the rates of adherence or proliferation was seen in vitro between the transduced and nontransduced ECs. No significant difference in proliferation was found for the transduced ECs on the three matrices tested in vitro. In contrast, adherence of the transduced ECs in vivo was significantly lower than that of nontransduced ECs (64.7% +/- 2.1% vs 73.7% +/- 4.1%, p < or = 0.05) 1 hour after implantation. CONCLUSIONS: Lower rates of surface endothelialization by genetically modified ECs in vivo do not appear to be due to an impaired capacity to initially adhere or proliferate on the synthetic graft but may result from decreased adherence after exposure to in vivo hemodynamic forces.

Animals↗
PubMed1995

Gadolinium-enhanced magnetic resonance angiography of abdominal aortic aneurysms.

PURPOSE: The objective of this study was to assess the usefulness of gadolinum-enhanced magnetic resonance angiography (MRA) for defining anatomic features relevant to performing aortic surgery for aneurysmal disease. METHODS: Anatomic data defined by MRA, including abdominal aortic aneurysm (AAA) size and character, as well as the status of the celiac, mesenteric, renal, and iliac arteries, were correlated with angiography, ultrasonography, computed tomography, or operative data in 43 patients. Five MRA sequences were obtained in an hour-long examination optimized for aortoiliac, splanchnic, and renal artery imaging at 1.5 T in a body coil. Four of the sequences were performed during or after infusion of gadolinium to improve image quality. RESULTS: MRA correctly defined the maximum aneurysm diameter, as well as its proximal and distal extent in all patients. MRA detected 33 of 35 significant stenoses among 153 splanchnic, renal, or iliac branches examined (sensitivity = 94% and specificity = 98%). MRA did not resolve the degree of aortic branch stenotic disease sufficiently to precisely grade its severity. MRA did not reliably define the status of the inferior mesenteric artery, lumbar arteries or internal iliac arteries. One ruptured AAA and one inflammatory AAA were correctly diagnosed by MRA. No patient had a contrast reaction or contrast-induced renal toxicity related to administration of gadolinium. CONCLUSION: Gadolinium-enhanced MRA of AAA provides appropriate, essential anatomic information for aortic reconstructive surgery in a 1-hour examination devoid of contrast-related renal toxicity or catheterization-related complications attending conventional arteriography.

Adult↗
PubMed1995

Retroviral vector-mediated transfer and expression of human tissue plasminogen activator gene in human endothelial and vascular smooth muscle cells.

PURPOSE: Enhancement of the fibrinolytic activity of vascular cells by tissue plasminogen activator (tPA) gene transfer has considerable clinical potential. However, it is unknown whether greater constitutive expression of the tPA gene might increase plasminogen activator inhibitor-1 (PAI-1) secretion, which could negate expected increases in fibrinolytic activity that accompany greater tPA protein production. The objective of this investigation was to determine whether transduction of human endothelial cells (EC) and vascular smooth muscle cells (SMC) with a retroviral vector containing the human tPA gene would increase tPA production and what effect this would have on endogenous PAI-1 secretion and subsequent fibrinolytic activity. METHODS: Cultivated human EC and SMC either were transduced with a murine leukemia retroviral vector (MFG) containing the human tPA gene and, in the case of controls, the lacZ gene, or they were exposed to media alone. On days 14 and 28 after transduction, supernatent tPA antigen and PAI-1 antigen levels were measured by ELISA, and supernatent tPA activity was quantitated with a spectrolyse tPA/PAI assay. RESULTS: Southern and Northern blot analyses documented integration and transcription of the tPA gene in both EC and SMC. Greater tPA antigen production occurred in MFG-tPA-transduced EC and SMC compared with nontransduced or MFG-lac Z-transduced cells (p < 0.05). The tPA activity increased in transduced human saphenous vein EC (up to 5.1-fold) and human iliac artery EC (up to 4.7-fold), but no increased tPA activity occurred in transduced SMC, compared with nontransduced or MFG-lac Z-transduced cells (p < 0.05). PAI-1 antigen was unchanged in transduced SMC but decreased in MFG-tPA-transduced EC (p < 0.05). PAI-1 mRNA was unchanged in the transduced EC and SMC compared with nontransduced cells, suggesting that posttranslational events may have caused the changes in EC PAI-1. CONCLUSIONS: This investigation demonstrated that MFG-mediated tPA gene transfer into human EC resulted in a significant increase in tPA activity. Enhancement of adult human EC fibrinolytic activity by transfer of the human tPA gene has not been previously reported and represents a necessary finding in the development of this gene therapy technology for the prevention of thrombotic complications of vascular disease.

Blotting, Northern↗
PubMed1995

Pediatric renovascular hypertension: a thirty-year experience of operative treatment.

PURPOSE: This study was undertaken to characterize the changing operative treatment of pediatric renovascular hypertension and subsequent outcomes in a 30-year experience at a single institution. METHODS: Clinical data were analyzed on 57 pediatric patients, 24 girls and 33 boys, ranging in age from 10 months to 17 years, who underwent operations for renovascular hypertension from 1963 to 1993 at the University of Michigan. Renal artery disease included atypical medial-perimedial dysplasia, often with secondary intimal fibroplasia (88%), and inflammatory mural fibrosis (12%). Abdominal aortic narrowings affected 15 patients. Data were categorized into three chronologic eras (I:1963-1972, II:1973-1980, and III:1981-1993) to allow identification of therapeutic trends. RESULTS: Primary surgical procedures were undertaken 74 times. Ex vivo reconstruction was necessary once. Primary operations included aortorenal bypass with autogenous vein grafts (n = 26) or internal iliac artery grafts (n = 7); iliorenal bypass with vein grafts (n = 2); renal artery resection beyond the stenosis and reimplantation into the aorta (n = 10), the main renal artery (n = 2), an adjacent segmental renal artery (n = 3), or the superior mesenteric artery (n = 3); renal artery resection and reanastomosis (n = 3); focal renal arterioplasty (n = 2); operative dilation (n = 7); splenorenal bypass (n = 2); and primary nephrectomy (n = 7). Among 23 primary operations performed in era I, 56.5% were aortorenal bypasses with vein grafts, but in era III this form of revascularization represented only 3% of 33 primary operations. No reimplantations were performed in era I, whereas reimplantations accounted for 51.5% of era III procedures. Thirteen patients underwent staged or concomitant aortic reconstructions with thoracoabdominal aortoaortic bypass grafts (n = 5) or patch aortoplasty (n = 8). Fourteen patients underwent a total of 20 secondary operations, including seven secondary nephrectomies. Operative therapy benefited 98% of these children: hypertension was cured in 45 (79%), improved in 11 (19%), and unchanged in one (2%). There were no operative deaths. CONCLUSIONS: Contemporary surgical management emphasizes direct reimplantation of main renal arteries into the aorta, reimplantation of segmental arteries into adjacent renal arteries, patch aortoplasty for associated abdominal aortic coarctations, and single-stage revascularizations. Pediatric patients with renovascular hypertension clearly benefit from carefully executed operative therapy.

Adolescent↗