Imaging vascular thrombosis with 99mTc-labeled fibrin alpha-chain peptide.

UNLABELLED: An agent that permits scintigraphic detection of chronic deep venous thrombosis (DVT) or pulmonary embolism (PE) would be a welcome addition to the armamentarium of nuclear medicine. Because fibrin is the integral part of each clot, old or fresh, we hypothesized that a 99mTc-labeled fibrin alpha-chain N-terminal peptide, Gly-Pro-Arg-Pro-Pro, that binds to the C-terminal portion of the gamma-chain of fibrin can detect DVT and PE. METHODS: The peptide was modified to Gly-Pro-Arg-Pro-Pro-Aba-Gly-Gly-(D)-Ala-Gly to permit efficient binding of 99mTc (99mTc-TP 850). The stability of the peptide was examined in vitro as well as in vivo. The ability of the agent to bind to rabbit, dog, and human fibrin and to inhibit adenosine diphosphate-induced platelet aggregation was examined. Blood clearance and 3-h tissue distribution were studied. DVT was induced in 8 rabbits using a stimulating electrode and in 2 rabbits by inserting a thrombin-soaked suture. PE was induced in 6 additional rabbits by introducing tantalum-impregnated blood clots into the right atrium, and the rabbits were radiographed to locate the emboli. 99mTc-TP 850 was then injected through a lateral ear vein, and each rabbit was imaged for up to 3 h. The rabbits were then killed, the heart and lungs were dissected and radiographed and the clots were harvested so that clot-to-blood radioactivity ratios could be determined. RESULTS: The peptide analog permitted efficient incorporation of 99mTc, which was stable in vitro and in vivo. The blood clearance was biphasic, with an alpha phase half-life of approximately 4 min (20%) and a beta phase half-life of approximately 13 min (88%). The mean binding of 99mTc-TP 850 to human, dog, and rabbit fibrin was 46% +/- 2%, 60% +/- 3%, and 56% +/- 2.5%, respectively, and the inhibitory concentration of 50% for dog and rabbit platelet aggregation was 236 pm and 167 pm, respectively. All clots, including 24-h-old pulmonary emboli, were delineated. The radioactivity associated with clots varied from 0.01 to 0.09 %ID/g, with clot-to-blood radioactivity ratios ranging from 1.2 to 12.0. However, 48-h-old pulmonary emboli had lysed and were seen neither by radiography nor by scintigraphy. CONCLUSION: A fibrin alpha-chain, N-terminal peptide that binds to the C-terminal portion of the gamma-chain of fibrin has been modified and labeled with 99mTc. The resultant peptide is stable in vitro and in vivo; binds to human, dog, and rabbit fibrin in large quantities; and inhibits platelet aggregation. The peptide clears rapidly from the blood and delineates experimental DVT and PE in rabbits. This agent is worthy of further investigation.

Effects of angioplasty balloon inflation time on arterial contractions and mechanics.

The purpose of this study was to characterize and compare the immediate effects of short and long angioplasty inflation times on arterial contractions and passive mechanics and thereby determine if there is a potential advantage obtained by increasing the duration of balloon inflation. In each of 10 nonatherosclerotic New Zealand rabbits, one external iliac artery was dilated for 20 seconds, and the contralateral artery was dilated for 2 minutes. Although angioplasty stretched the arteries 27% and 30% for the short and long dilations respectively, the pre- and post-angioplasty arteriographic diameters were not different. Both short and long dilations had equal effects on passive biomechanics: circumferential wall stress was increased (P less than .01); wall thickness was decreased (P less than .01); the incremental elastic modulus was increased (P less than .01). In vitro studies of arterial rings demonstrated that maximal active contractile force in response to KCl (70 mM) was significantly (P less than .05) less for dilated arteries than for undilated arteries. More importantly, maximal active force after the 2-minute dilations was significantly (P less than .05) less than after the 20-second dilations. These results suggest that, when dilating normal arteries, increasing angioplasty balloon inflation time from 20 seconds to 2 minutes offers no mechanical advantage but produces more smooth muscle cell dysfunction, which may reduce vasospasm and restenosis after angioplasty.

Magnetic resonance chemical shift imaging and spectroscopy of atherosclerotic plaque.

An in vivo magnetic resonance imaging (MRI) technique for identification and characterization of atherosclerotic plaque was assessed in animal and human models. Atherosclerosis was induced in the abdominal aorta of four rabbits by a combination of balloon denudation and a high cholesterol diet. In vivo conventional spin-echo and fat/water suppressed images of the rabbit aortae were obtained at 1.5 T. Chemical shift imaging (CSI) was achieved using a hybridization of selective excitation and modified Dixon techniques. These techniques were then used to obtain images of atherosclerotic lesions in the carotid arteries of four patients prior to endarterectomy. The MRI results were corroborated by histologic and high-resolution proton MR spectroscopic (8.5 T) analysis of rabbit aorta, human carotid endarterectomy, and six additional human superficial femoral and iliac atherectomy specimens. All animal and human lesions were classified as either fatty streaks or fibrotic plaque. When compared to conventional spin-echo images, fat suppression by CSI substantially improved the measured contrast-to-noise ratio between plaque and vessel lumen, and enhanced its discrimination from periadventitial fat. In contrast, water suppression eliminated visualization of plaque due to the negligible amount of isotropic (liquid-like) signal from the immobilized lesion lipids. Magnetic resonance spectroscopy corroborated the CSI results by demonstrating broad, ill-defined fat resonances characteristic of nonmobile lipids in both human and rabbit atherosclerotic lesions. These findings indicate that in vivo MRI of plaque is technically feasible and can be markedly improved using chemical shift imaging.

Fatty liver. Chemical shift phase-difference and suppression magnetic resonance imaging techniques in animals, phantoms, and humans.

In vitro animal and human models were used to evaluate the potential of chemical shift magnetic resonance imaging (MRI) for assessing fatty liver. Phantoms of varying fat content were created from mayonnaise-agar preparations. Fatty liver was induced in eight rats by feeding them ethanol for three to six weeks (36% of total calories), whereas eight control rats were fed a normal diet. T1-weighted in-phase and opposed-phase MR images were obtained of the phantoms animals, and 28 human subjects. Additional images obtained in animals included long TR images with in-phase and opposed-phase technique, and hybrid chemical shift water and fat suppression. The rats were killed and histologic status was graded blindly by a hepatopathologist as normal, mild, moderate, or severe fatty change, for correlation with MR grading. Quantitative analysis of MR images included fat signal fraction for animals, and relative signal decrease between in-phase and opposed-phase images for phantom and human data. Phantom in-phase signal increased linearly with respect to fat content, whereas opposed-phase signal decreased linearly. MRI and histologic grading of rat livers were highly correlated, especially when based on water suppression images (r = 0.91, P = .0001). Opposed-phase images were also highly correlated, while fat suppression images were less effective. There was no overlap between MR-derived fat fractions for control (2.6%-5.7%) versus ethanol-fed rats (7.7%-17.9%, P = .0002). Human liver considered to be fatty by visual inspection (n = 8) had higher relative signal decrease than nonfatty liver (n = 22) (P less than .001). Phantom, animal, and human data demonstrate that comparison of T1-weighted in-phase and opposed-phase images is both practical and sensitive in the detection and grading of fatty liver.

Endothelin-1 increases arterial sensitivity to 5-hydroxytryptamine.

Rings of rabbit abdominal aorta were pretreated with endothelin-1 (ET-1) or vehicle and then isometric contractions to cumulative additions of 5-hydroxytryptamine (5-HT) were recorded. ET-1 at concentrations of 0.4, 1 and 2 nM produced contractions of 7, 15 and 35% of maximal potassium chloride-induced contractions, respectively. Pretreatment of aortic rings with these concentrations of ET-1 increased (P less than 0.01) arterial sensitivity (EC50) to 5-HT 1.9-, 2.4- and 3.5-fold, respectively, without affecting peak 5-HT-induced force. Acetylcholine-induced, endothelium-dependent aortic relaxations were not affected by pretreatment with ET-1-pretreated but not untreated aortic rings. These results suggest that ET-1 could promote arterial vasospasm by sensitizing the artery to 5-HT released from platelets.

Use of Ferrum in MRI of lung parenchyma and pulmonary embolism.

MRI of lung parenchyma and pulmonary embolism (PE) remains challenging. "Ferrum," a ferric hydroxide sucrose complex used clinically for iron deficiency anemia for more than 40 years, was investigated as a negative MRI contrast agent in five rabbits bearing experimental PE as well as in five normal volunteers. Clots were prepared by spontaneous coagulation of 0.1 ml In-111 labeled autologous red blood cells and introduced through the jugular vein. Scintigraphic imaging permitted anatomical localization of PE in vivo and thereby served as a control for MR imaging. MRI was performed on a 1.5 T GE Signa scanner before and after induction of PE, and before and after the injection of Ferrum. T1-weighted images were obtained continuously for up to 90 min using varying doses of Ferrum. In five normal human volunteers, a single dose of 100 mg each was administered. T1- and T2-weighted spin-echo and gradient-echo images of lung parenchyma were repeatedly obtained before and after agent administration. In rabbit, Ferrum remained in circulation for several hours where it shortened both T1 and T2 of blood, improving the contrast between PE and lung parenchyma (i.e., intravascular compartment). A dose of 3 mg/kg was enough to increase the contrast-to-noise ratio (CNR) between PE and lung parenchyma by almost three fold, substantially improving lesion detectability. CNR increased up to five-fold when the dose was increased up to 20 mg/kg at which point CNR reached a plateau. In humans, T2-weighted spin-echo sequence appeared to be most sensitive to changes in signal-to-noise ratio (SNR) of normal lung parenchyma. Within 60 min after injection of 100 mg of iron, SNR dropped by 34% (p < .025). However, 24 hr later, SNR returned to almost normal. Ferrum increased the contrast between PE and lung parenchyma in the rabbit and decreased the parenchymal SNR in humans in nontoxic doses. These results suggest that Ferrum is worthy of further investigation of PE imaging in humans.

MR imaging of pulmonary parenchyma and emboli by paramagnetic and superparamagnetic contrast agents.

Using experimentally induced pulmonary emboli in an animal model, three intravenously administered contrast agents, Gd-DTPA-albumin microspheres (8-15 microns, 0.2 M particles/mg protein, 39-106 micrograms Gd/mg, 50 mg/ml), Gd-DTPA-liposomes (15-30 microns, 130 micrograms/mg lipid, 6 mg Gd/ml) and superparamagnetic ferrosome, (60 nm, 100 mM iron and 20 mg lipid/ml) were examined for MR imaging. Gd-DTPA entrapped in lung capillaries did not enhance the signal intensity of lung parenchyma, but liposomes (5 ml) served as better Gd-DTPA carriers and increased the parenchymal signal intensity by up to a factor of 2.3. However, neither agent improved delineation of pulmonary emboli. Ferrosome decreased the intensity of lung parenchyma, improving detectability of pulmonary emboli by several factors.

Ultrasonic evaluation of erythrocyte aggregation dynamics.

The dynamics of aggregation and disaggregation of blood of varying hematocrit in oscillatory flow in a distensible horizontal tube was determined by measuring the developing echo intensity of the blood samples with a 10 MHz B-mode ultrasonic scanner. Early aggregation could be detected within 10 sec. of stoppage of flow. The rate of echo intensity buildup and thus, presumably the rate of aggregation when flow was stopped was inversely related to hematocrit, as was the rate of echo intensity reduction when flow was resumed. Polycythemic blood of 60% hematocrit showed no echo intensity increase over 5 min. Increasing the shear stress when flow was resumed resulted in rapid decreases in aggregation. In all cases, disaggregation following flow resumption was faster than aggregation following flow stoppage.

Endothelial cell seeding on prosthetic surfaces.

Once thought to be a monolayer of passive cells lining the vasculature, endothelial cells are now known to be important regulators of normal vascular physiology. Unfortunately, these critically important cells are destroyed or removed by interventional and surgical procedures performed to recanalize or bypass vascular obstructions. The loss of these cells contributes to thrombosis and restenosis, the major complications observed after angioplasty, stent deployment, and prosthetic graft implantation. One approach to preventing these complications is the placement of endothelial cells on stents or prosthetic grafts prior to their placement in vivo in the hope that these cells will, after growth and maturation, release the factors necessary to inhibit thrombosis and intimal thickening. The purpose of this review paper is to provide an overview of the physiologic functions of normal and dysfunctional endothelial cells, and to discuss experiments in which endothelial cells have been placed on metallic stents and prosthetic grafts.

Pathogenesis of atherosclerosis.

Cardiovascular disease is the leading cause of death in the United States. According to numbers compiled by the American Heart Association, nearly one of every two Americans dies of cardiovascular disease. For example, in 1987, 976,706 (46%) of the estimated 2,127,000 deaths recorded in the United States were attributable to diseases of the heart and blood vessels [1]. Most of these deaths can be attributed to atherosclerosis and its ensuing complications. The pathogenesis of atherosclerosis is not completely understood. Nevertheless, the purpose of this review is to provide an overview of how an atherosclerotic lesion might develop on the basis of our current understanding. This overview will focus on one hypothesis of atherosclerosis development, the modified response-to-injury hypothesis. Several additional hypotheses will be described briefly. These descriptions can serve as a framework on which researchers can build a more complete understanding of the processes involved in this complicated, multifactorial disease.

Placement of endothelial cells on the luminal surface of denuded arteries in vitro and in vivo.

PURPOSE: Experiments were performed to determine if the percutaneous placement of endothelial cells on denuded arterial surfaces is feasible. MATERIALS AND METHODS: For in vitro adhesion assays, rabbit microvascular endothelial cells were stained with a fluorescent marker and placed on the luminal surface of disks of denuded rabbit aorta. At varying times thereafter, the nonadherent cells were removed, and the adherent cells were quantitated with use of fluorescence microscopy. For in vivo studies, angioplasty was performed on external iliac arteries in five rabbits, and a double-balloon catheter, positioned at the dilatation site, was used to deliver fluorescent rabbit microvascular endothelial cells. Ten minutes (n = 2), 1 hour (n = 2), 1 day (n = 1), or 3 days (n = 1) after cell placement, the number of fluorescent cells remaining on each artery was determined. RESULTS: In vitro rabbit microvascular endothelial cell attachment was (a) serum-dependent, peaking with media containing 25% autologous serum; (b) time-dependent, peaking at 30 minutes; and (c) cell density-dependent. In vivo rabbit microvascular endothelial cell attachment was (a) noncircumferential, (b) appeared to be gravity-dependent, and (c) appeared unchanged over 3 days with respect to number of cells per cross-section and length of artery having endothelium. CONCLUSIONS: Percutaneous delivery of endothelial cells onto denuded arterial surfaces with use of optimal conditions is feasible and these cells remain adherent for at least 3 days.

Vascular smooth muscle contraction and relaxation: pathways and chemical modulation.

The purpose of this review has been to summarize the information learned in the past few years regarding the mechanisms responsible for smooth muscle contraction and relaxation and to relate this information to the vasoconstrictor and vasodilator agents used by the interventional radiologist. Because this review can only provide an overview of what is known about these processes, the reader is directed to the references cited below. This reference list contains review articles that will provide in-depth information about the topics presented herein.

Phorbol amplification of serotonin-induced arterial contractions is endothelium dependent.

This study determined the effect of phorbol pretreatment on serotonin [5-hydroxytryptamine (5-HT)]-induced contractions of rabbit abdominal aorta. Rings of aorta pretreated with 12-O-tetradecanoylphorbol 13-acetate (TPA, 10(-7) M) contracted more strongly (P less than 0.01) than rings pretreated with the vehicle dimethyl sulfoxide (0.0007%), but only at low 5-HT concentrations (10(-8) to 10(-7) M). A 10-min exposure to TPA (10(-7) M) increased (P less than 0.01) 5-HT contractions (10(-7) M) from 23.7 +/- 2.6 to 42.1 +/- 4.5%. This amplification required an intact endothelium and the use of a biologically active phorbol. Pretreatment with TPA (10(-7) M) reduced (P less than 0.01) acetylcholine-induced relaxations from 56.6 +/- 6.6 to 20.0 +/- 13.8%. Changes in guanosine 3',5'-cyclic monophosphate-mediated relaxation are not involved in this decrease, since relaxations to sodium nitroprusside (10(-9) to 10(-8) M) were not altered by TPA pretreatment. These results suggest that activation of protein kinase C in endothelial cells may produce arterial supersensitivity to 5-HT by decreasing the release of endothelium-derived relaxing factor(s) and/or by increasing the release of endothelium-derived contracting factor(s).

Drug delivery into the arterial wall: a time-course study with use of a lipophilic dye.

PURPOSE: One potential approach to the prevention of restenosis after angioplasty is to deliver antiproliferative agents directly to the angioplasty site. The purpose of this study was to determine the time course of drug penetration into the media of the balloon-dilated artery. MATERIALS AND METHODS: Balloon angioplasty of the left and right iliac arteries was performed once for 1 minute in each of five rabbits. A double-balloon catheter was then positioned at the site of angioplasty, and the fluorescent dye PKH26 (molecular weight, 961) was delivered under pressure to simulate drug delivery. Afterward, the arteries were removed and dye penetration into the media was measured on frozen cross sections by epifluorescence microscopy. RESULTS: Delivery of the dye was performed for periods ranging from 5 to 50 minutes at a mean pressure of 189 mm Hg. The depth of dye penetration (D, micrometers) was directly related to dye perfusion time (T, minutes) (D = 0.348T + 11.958, r = 0.496, P < .01). This equation predicts complete medial dye penetration in 81 minutes assuming an average intima-media thickness (40 microns). CONCLUSION: This study demonstrates that PKH26 can be delivered to the media of the dilated artery. However, the time required to obtain complete penetration may limit the utility of this double-balloon catheter approach to drug delivery.

Resistance of freshly adherent endothelial cells to detachment by shear stress is matrix and time dependent.

PURPOSE: The placement of endothelial cells on the surfaces of arteries immediately after vascular interventions has the potential to limit restenosis by inhibiting intimal thickening and by stimulating arterial enlargement. Because such re-endothelialization is dependent on rapid formation of strong endothelial cell-matrix interactions, experiments were performed to identify the extracellular matrix that provided endothelial cells with the greatest resistance to detachment by a shear stress in the least amount of time. MATERIALS AND METHODS: Rabbit microvascular endothelial cells were plated onto glass slides coated with collagen, laminin, vitronectin, or fibronectin. After allowing 5-45 minutes for cell adhesion, each slide was placed in a parallel plate chamber, and the number of cells present before and after exposure of the cells to shear stresses (1-25 dynes/cm2) were counted. RESULTS: Endothelial cell retention to the matrix-coated slides was time and matrix dependent. The percentages of endothelial cells retained after adhesion times of 5, 15, 30, and 45 minutes followed by exposure to 15 dynes/cm2 were 9%, 20%, 32%*, and 38%* for collagen; 7%, 20%, 36%*, and 49%* for laminin; 35%, 47%, 62%, and 76%* for vitronectin; and 64%, 58%, 71%, and 78% for fibronectin, respectively (*P < .05 versus 5 minutes adhesion). Similar results were obtained for lower and higher shear stresses, indicating that cell retention was independent of shear stress above 1 dyne/cm2. CONCLUSIONS: The resistance of freshly adherent endothelial cells to detachment by shear stress is matrix- (fibronectin approximately equal to vitronectin > laminin approximately equal to COL) and time-dependent. Fibronectin provided the greatest cell retention in the least amount of time.

Atherosclerotic rabbit iliac arteries: comparison of balloon angioplasty and laser-assisted balloon angioplasty.

The effects of balloon angioplasty (BA) and laser-assisted balloon angioplasty (LABA) on arteries were compared. Atherosclerosis was induced in the iliac arteries of New Zealand White rabbits by means of balloon denudation and a diet supplemented with 1% cholesterol and 3% peanut oil. Six weeks later, one iliac artery was dilated with a 2.5- or 3.0-mm-diameter balloon. The contralateral iliac artery was treated with a 1.5-mm-diameter laser probe heated with 6 W of argon laser energy, and then BA was performed. Four weeks later, the mean luminal diameter of the LABA-treated arteries was smaller than that of the BA-treated arteries (BA, 1.57 mm +/- 0.15; LABA, 0.82 mm +/- 0.19; P less than .01). This restenosis was due to greater intimal fibrocellular proliferation (intimal area: BA, 0.83 mm2 +/- 0.16; LABA, 1.41 mm2 +/- 0.26; P less than .05). The LABA-treated arteries produced less potassium chloride-induced maximal force (P less than .01) and had smaller incremental elastic moduli (P less than .05) than did the BA-treated arteries. LABA is not the treatment of choice for small-caliber arteries, in which thermal injury to the arterial wall would be significant.

Immediate and long-term effects of angioplasty-balloon dilation on normal rabbit iliac artery.

The present study was performed to determine the immediate (hours) and long-term (28-day) effects of angioplasty-balloon dilatation on arterial wall diameter, histology, response to vasoconstrictors, and passive mechanics. Dilated left iliac arteries of New Zealand rabbits were compared to control, right iliac arteries. In the immediate studies, dilation increased arteriographic diameter by 32%, denuded the endothelium, stretched and may have lysed smooth muscle cells, decreased arterial wall thickness, and increased passive stress and incremental elastic modulus. The dilated arteries failed to contract to norepinephrine or potassium chloride. In contrast, 28 days after dilation, arteriographic diameter had returned to normal, an intimal thickening had formed, passive stress and the incremental elastic modulus had decreased to below normal, and the arteries contracted, but to only 53% to 67% of control. Angioplasty had no long-term effect on arterial sensitivity (EC50) to potassium chloride or serotonin but did produce a decreased sensitivity (6X) to norepinephrine. These studies demonstrate that the effects of dilatation of the normal artery are partially reversible, suggest that restenosis after angioplasty is more likely to be due to intimal proliferation than increased arterial stiffness, and suggest that vasospasm of arteries after long-term recovery from the dilation is more likely to be mediated by serotonin than by norepinephrine.

Amplification of serotonin-induced arterial contractions with thrombin.

The purpose of this study was to determine if exposure of arteries to thrombin increases arterial sensitivity to serotonin and could thereby promote vasospasm. Rings of rabbit abdominal aorta, with endothelium intact or removed, were pretreated with vehicle or thrombin (2.5 U/mL). Thereafter, arterial contractions in response to cumulative additions of serotonin were recorded. Arterial contractions were expressed as a percentage of maximal potassium chloride-induced contractions. Arterial sensitivity was expressed as the EC50, the concentration of serotonin that produced a half-maximal contraction. Exposure of aortic rings to thrombin alone produced contractions of 11% and 22% for rings with and without endothelium, respectively. Removal of the endothelium had no effect on maximal serotonin-induced contractions (93% with endothelium vs 98% without). In contrast, maximal serotonin-induced contractile force was increased after pretreatment with thrombin (107% vs 121% for rings with and without endothelium, respectively; P less than .01). Arterial sensitivity to serotonin was not affected by the removal of endothelium. However, thrombin pretreatment increased (P less than .01) arterial sensitivity to serotonin 2.6-fold and 4.7-fold for rings with and without endothelium, respectively. The results of this study demonstrate that arterial contractions due to serotonin are amplified by thrombin and suggest that vasospasm may be more likely to occur at sites in arteries where the endothelium is absent and both thrombin and serotonin are present.