Nitric oxide inhibition increases matrix metalloproteinase-9 expression by rat aortic smooth muscle cells in vitro.

OBJECTIVE: The hypothesis to be tested was that diminished bioavailable nitric oxide (NO) affects matrix metalloproteinase (MMP) expression and activation in vascular smooth muscle cells (SMCs). METHODS: Cultivated rat aortic SMCs (RA-SMCs) were exposed to increasing concentrations of L-N-monomethyl arginine (L-NMMA), a nonselective inhibitor of NO synthase, in the presence of proinflammatory cytokines (50 ng/mL interleukin [IL]-1beta, 50 ng/mL interferon-gamma, and 30 microg/mL lipopolysaccharide). Nitrite and nitrate, two of the final end products of NO metabolism, were measured in media collected at 48 hours with the use of the Saville assay (n = 4). MMP activity was measured with 1% gelatin zymography (n = 4). In separate experiments in which 2 ng/mL of IL-1beta and L-NMMA was used, MMP protein and messenger RNA (mRNA) levels were determined with Western blot analysis (n = 3) and semiquantitative reverse transcriptase-polymerase chain reaction (n = 3), respectively. Data were analyzed with nonparametric analysis of variance. RESULTS: Increasing concentrations of the NO synthase inhibitor L-NMMA caused a dose-dependent decrease (P <.05) in nitrite and nitrate production by RA-SMCs after cytokine exposure. Zymography documented an early dosedependent increase (P <.05 compared with cytokines alone) in 92-kd MMP activity, with no significant changes in 72-kd MMP activity after treatment with L-NMMA (P >.05 compared with cytokines alone). Reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that the addition of L-NMMA to IL-1beta-stimulated RA-SMCs led to significant increases in MMP-9 mRNA (n = 3, P <.01 for 1.0 mmol/L L-NMMA) and MMP-9 protein levels (n = 3, P <.05), respectively. No differences in MMP-2 mRNA or protein levels were demonstrated. CONCLUSIONS: Inhibition of cytokine-induced NO expression in RA-SMCs is associated with a selective, dose-dependent increase in MMP-9 expression and synthesis. These findings suggest that alterations in local NO synthesis may influence MMP-9-dependent vessel wall damage.

Regulation of lysyl oxidase by interferon-gamma in rat aortic smooth muscle cells.

Lysyl oxidase is an essential catalyst for the cross-linking of extracellular collagen and elastin. Abnormalities in lysyl oxidase activity may contribute to the pathogenesis of arterial diseases characterized by abnormal matrix remodeling. This study tested the hypothesis that interferon (IFN)-gamma, a proinflammatory cytokine present in aortic aneurysm and arteriosclerotic plaque rupture, downregulates lysyl oxidase gene expression in rat aortic smooth muscle cells. Steady-state lysyl oxidase mRNA levels decreased in a concentration- and time-dependent manner to 30% of control levels after 24 hours of treatment with IFN-gamma. Cell layer lysyl oxidase activity decreased in parallel with the observed changes in steady-state mRNA. Nuclear runoff studies suggested that transcriptional regulation was responsible for at least 40% of the observed downregulation. mRNA decay studies suggested that IFN-gamma also decreased lysyl oxidase mRNA half-life from 9 to 6 hours. Downregulation of lysyl oxidase by IFN-gamma did not appear to require new protein synthesis. This study documents that IFN-gamma downregulates lysyl oxidase gene expression in rat aortic smooth muscle cells by transcriptional and posttranscriptional mechanisms. If similar regulation occurs in vivo, it is possible that IFN-gamma-mediated changes in lysyl oxidase may contribute to arterial diseases characterized by abnormal extracellular matrix.

Transforming growth factor-beta 1 increases lysyl oxidase enzyme activity and mRNA in rat aortic smooth muscle cells.

PURPOSE: This investigation was designed to test the hypothesis that transforming growth factor-beta 1 (TGF-beta 1) regulates lysyl oxidase secretion from vascular smooth muscle cells. Lysyl oxidase is an enzyme that catalyzes an essential step in collagen and elastin cross-linking in the extracellular matrix, and TGF-beta 1 has been implicated in the pathogenesis of restenosis after vascular injury. The effect of TGF-beta 1 on lysyl oxidase in vascular smooth muscle cells has not been previously defined. METHODS: Rat aortic smooth muscle cells were grown in culture to confluence. Cells in passage 2 to 6 were incubated for 24 hours in media containing 0.1, 0.5, 1.0, or 10.0 ng/ml of TGF-beta 1. Lysyl oxidase activity in the media was quantitated with a tritium-release bioassay against an insoluble 3H-labeled aortic clastin substrate. Northern blot analyses were performed to determine steady-state levels of lysyl oxidase mRNA in the smooth muscle cells. RESULTS: Lysyl oxidase activity in the media increased 1.5-fold above control levels after exposure to 10 ng/ml of TGF-beta 1 (p < 0.01). This increase in lysyl oxidase activity was associated with a concentration-dependent increase in steady-state levels of lysyl oxidase mRNA, being 4.3- and 6.2-fold above control levels after exposure to 1 and 10 ng/ml TGF-beta 1, respectively (p < 0.01). The observed increase in steady-state lysyl oxidase mRNA after exposure to TGF-beta 1 was also time-dependent over the 24-hour experimental period. CONCLUSIONS: TGF-beta 1 appears to regulate lysyl oxidase in cultured rat aortic smooth muscle cells. Increases in lysyl oxidase activity may be one of the mechanisms by which TGF-beta 1 contributes to arterial restenosis after vascular injury.

Isolation, cloning, and characterisation of a trp homologue from squid (Loligo forbesi) photoreceptor membranes.

The invertebrate phototransduction system is a valuable model of the ubiquitous inositol lipid signalling system. Taking advantage of the ability to obtain relatively large amounts of retinal material from the cephalopod eye, partial protein sequence data were obtained for a 92-kDa component isolated from a detergent-insensitive cytoskeletal fraction of a squid retinal microvillar membrane preparation. Degenerate oligonucleotides, designed on the basis of these sequence data, were used to isolate a full-length cDNA, encoding the 92-kDa component, using both cDNA library screening and 5'-rapid amplification of cDNA ends (5'-RACE) techniques. Comparison of the amino acid sequence encoded by this cDNA with entries in the OWL composite protein sequence database reveals greatest sequence similarity with the products of the Drosophila trp and trpl genes. Greatest variation from the Drosophila Trp protein is seen in the carboxyl-terminal region, which is considerably truncated in the squid protein and which accounts for most of the substantial difference in molecular weight seen between these proteins. This variation may be significant as the carboxyl-terminal domain has been shown to be in the regulation of several ligand-gated channels. The carboxyl-terminal domain has been expressed and shown to interact with calmodulin in a calcium-dependent fashion, thereby supporting this hypothesis. The likely occurrence of other homologues in a variety of systems suggests that this is a novel and important family of regulated ion channels involved in calcium signalling.

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.

Legal and economic challenges to personal autonomy: a view from the United States of America.

Steadily and surely, commonly accepted notions of individual rights and the place of the individual in society are subject to increasing scrutiny. This article critically analyses legal and economic parameters which directly affect both personal autonomy and the role of government, interpreted through its higher level state and federal judiciary, on the public.

Platelet reactivity in vivo in dogs with arterial prostheses seeded with endothelial cells.

This study was designed to assess platelet activity in vivo with vascular prostheses seeded with endothelial cells to determine the time course for development of thromboresistance and to test the ability of prostheses to produce prostacyclin. Sixteen dogs were randomly allocated to receive seeded (experimental group) or unseeded (control group) velour Dacron aortic prostheses. Serial measurements of platelet survival were performed to assess platelet interaction with prostheses in vivo, and platelet serotonin was monitored as an index of platelet release in vivo. After placement of prostheses, dogs in the experimental group had rapid normalization of platelet survival, with most having normal platelet survival at 4 to 8 weeks after surgery. In contrast, most control animals had reduced platelet survival throughout the 12 week period of study. Significant differences between groups in mean platelet survival were noted at 8 weeks after surgery (p less than .005) and in mean platelet serotonin at 12 weeks after surgery (p less than .05). Luminal surface production of 6-keto-PGF1 alpha from seeded prostheses was similar to aortic production and significantly greater (p less than .05) than that of control prostheses. Gross thrombus was present on 6.0 +/- 3.4% of the prosthetic surface in experimental animals in comparison to 26.6 +/- 19.2% in controls (p less than .005). The results of these studies document accelerated nonreactivity with platelets of seeded prostheses due to rapid coverage with endothelium possessing a normal ability to produce prostacyclin.

Effect of different aspirin doses on arterial thrombosis after canine carotid endarterectomy: a scanning electron microscope and indium-111-labeled platelet study.

Although it is widely accepted that aspirin inhibits platelet aggregation in arterial thrombosis, the appropriate dosage of aspirin remains quite controversial. The purpose of this study was to determine the effect of different doses of aspirin (0.5 mg/kg vs. 10 mg/kg) on mural thrombus formation after carotid endarterectomy. Eighteen hours after oral aspirin administration, 20 endarterectomies were performed on mongrel dogs with the use of the operating microscope. Blood flow was then restored for 3 hours and the vessels were prepared for investigation with the scanning electron microscope. Ten endarterectomies were also performed on unmedicated dogs as controls. Five minutes before vessel unclamping, autologous indium-111-labeled platelets were administered intravenously, and the endarterectomized portions of the vessels were studied with a gamma counter system after harvesting. Group 1, the control group, revealed extensive mural thrombus consisting of platelet aggregates, fibrin, red blood cells, and white blood cells. Six of the 10 vessels in Group 2, premedicated with 0.5 mg of aspirin per kg, demonstrated varying amounts of mural thrombus. Group 3 (10 vessels), premedicated with 10 mg of aspirin per kg, revealed a platelet monolayer completely covering the exposed vessel wall media, with scattered white blood cells and infrequent fine fibrin strands overlying the platelet surface. The mean (+/- SD) radioactivity per group expressed as counts/minute/mm2 was: Group 1--2055.3 +/- 1905.5, log = 7.253 +/- 0.926; Group 2--1235.6 +/- 1234.3, log = 6.785 +/- 0.817; Group 3--526 +/- 433.06, log = 5.989 +/- 0.774.(ABSTRACT TRUNCATED AT 250 WORDS)

Early versus delayed heparin reversal after carotid endarterectomy in the dog. A scanning electron microscopy study.

The present study investigates the hematological reaction to arterial injury during the first 10 minutes after endarterectomy in dogs to determine if heparin reversal during this early period predisposes to thrombus formation. Known platelet physiology would predict that heparinization during this early period would be useful to allow a fibrin-free platelet monolayer to form. After systemic heparinization (145 mu/kg) of the experimental animals, 42 endarterectomies were performed. Blood flow was then resumed for specific periods of time, and the vessels were prepared for scanning electron microscopy. Group 1 vessels (from the unheparinized control group) revealed mural thrombus formation after 10 minutes of blood flow. Group 2 vessels revealed the progressive formation of a fibrin-free platelet monolayer after 2, 5, or 10 minutes of blood flow resumption under systemic heparinization. Group 3 arteries, harvested at 10 minutes, underwent immediate (within 1 to 2 minutes after resumption of flow) heparin reversal with protamine sulfate, and demonstrated numerous patches of fibrin covering the platelet monolayer. Group 4 arteries, studied after 3 hours of blood flow, also underwent immediate heparin reversal. Two of these seven specimens had clumps of fibrin overlying the platelet monolayer. The Group 5 vessels had heparin reversal at 10 minutes, and demonstrated no fibrin overlying the platelet monolayer after 3 hours of blood flow. This study demonstrates the formation of a fibrin-free platelet monolayer over the endarterectomized vessel wall within 10 minutes of resumption of flow under systemic heparinization. These findings suggest that heparin may safely be reversed following a carotid endarterectomy if one awaits the initial critical 10 minutes of blood flow.

Enhanced patency of small-diameter, externally supported Dacron iliofemoral grafts seeded with endothelial cells.

Fourteen adult foxhounds underwent bilateral iliofemoral bypasses with externally supported, knitted Dacron grafts measured 4 mm in internal diameter and 10 cm in length. These conduits were preclotted with 10 ml of blood mixed with 0.5 ml of culture medium. Autologous endothelial cells, enzymatically derived from external jugular veins, were added to blood and medium used to preclot one graft in each dog. The other, unseeded graft served as a control. Grafts were anastomosed, end to end, to the iliac and femoral arteries. All dogs received dipyridamole, 50 mg twice a day for 4 days preoperatively, and aspirin, 5 grains four times a day for 1 day preoperatively. Both drugs were continued 14 days after operation. Grafts were removed from three dogs at 2 and 4 weeks and from four dogs at 8 and 16 weeks. All grafts were patent at 2 weeks during drug administration. Cumulative patency rates beyond 2 weeks were 73% in 11 seeded grafts and 27% in 11 control grafts, a statistically significant difference (P = 0.03). Seeded grafts were completely surfaced with a monolayer of endothelium between 2 and 4 weeks. Small-graft patency appeared related to evolution of endothelial surfaces, the development of which was clearly facilitated by seeding with autologous endothelium.

Expanded polytetrafluoroethylene vascular prostheses seeded with enzymatically derived and cultured canine endothelial cells.

Thirteen adult dogs underwent thoracoabdominal bypass operations with 6 or 10 mm expanded polytetrafluoroethylene (PTFE) grafts 25 to 30 cm long. Eight experimental grafts were seeded with cultured autologous endothelial cells prior to implantation. Five unseeded grafts served as controls. Endothelial cells, derived from external jugular vein segments using 0.1% trypsin and 0.5% collagenase solutions, were cultivated for 14 days prior to seeding. Grafts were studied at 2 and 4 weeks after implantation. Endothelial linings in control grafts were restricted to limited anastomotic pannus ingrowths, never exceeding 10% of the graft surface. Experimental grafts demonstrated an endothelial surface coverage averaging 64% and 91% at 2 and 4 weeks, respectively. Generation of an early lining of endothelium in expanded PTFE grafts is possible in a canine model using cultured autologous cells.

The effect of heparin reversal after carotid endarterectomy in the dog. A scanning electron microscopy study.

The purpose of this study was to determine reversal of heparin, immediately after carotid endarterectomy would have an adverse effect on the thrombogenicity of the endarterectomized vessel wall. After systemic heparinization, unilateral common carotid endarterectomies were performed under the operating microscope on 14 dogs. Half of the animals were given protamine sulfate to reverse the heparin. Three hours after resumption of blood flow, these arteries, as well as contralateral vessels used as controls for fixation technique, were perfused with glutaraldehyde and prepared for scanning electron microscopy (SEM). Thrombin clotting times were measured throughout the experiments. Sections of the endarterectomized portions viewed by SEM showed nearly total coverage of the exposed collagen of the media with flattened platelets. There were scattered leukocytes, but few erythrocytes, little fibrin, and no true thrombus. There were no difference between the animals that received heparin reversal and those that did not. A group of five additional arteries underwent the same procedure except that no heparin was given. As expected, large amount of thrombus had formed within the lumina of these control vessels by 3 hours. Since previous studies suggest that arterial thrombosis usually occurs within 3 hours of endothelial injury, the authors conclude that total reversal of heparin does not increase thrombogenicity of the endarterectomized vessel. This suggests that heparin may be safely reversed in patients to help maintain postoperative hemostasis.

Isolation of adult canine venous endothelium for tissue culture.

In order to provide autologous adult endothelial cells for the production of cell-lined artificial vascular prostheses, we have developed a method for harvesting large numbers of cells with minimal contamination by other cellular types. In this technique, the vein is incubated in trypsin-EDTA solution followed by collagenase and the endothelial cells flushed off with a stream of culture medium. With care and appropriate timing, the endothelium can be selectively removed leaving the underlying basal lamina intact.

Immediate seeding of enzymatically derived endothelium in Dacron vascular grafts. Early experimental studies with autologous canine cells.

Twenty-eight adult dogs underwent thoracoabdominal bypass with 6-mm double-velour Dacron grafts. The experimental grafts were seeded immediately prior to implantation with enzymatically harvested endothelial cells. Endothelial cells were procured from autologous external jugular vein segments using 0.1% trypsin and 0.5% collagenase solutions. Unseeded grafts served as controls. The grafts were studied from one to 28 days after implantation. The seeded grafts exhibited greater than 80% uniform luminal coverage with endothelial cells at day 28. Immunofluorescent microscopy was used to confirm the presence of endothelium. The experimental grafts studied at the two time periods of one to seven days and 14 to 28 days had 94.6% and 88.5% clot-free surfaces, respectively. The control grafts studied during similar periods had 81.0% and 40.1% clot-free surfaces. These differences were significant at the 14- to 28-day period.

The circumoval precipitin reaction on Schistosoma mansoni eggs as seen by scanning electron microscopy.

The circumoval precipitin reaction on Schistosoma mansoni eggs was examined for the first time by scanning electron microscopy. The precipitates appeared as layered segmenters, and apparent fusion of adjacent precipitates was observed. This suggests that the single precipitates seen by light microscopy may on occasion be a collection of several precipitates exuding from nearby pores. The precipitates appeared on all areas of the egg surface except the spine.