Interleukin 1 and lipopolysaccharide induce an inhibitor of tissue-type plasminogen activator in vivo and in cultured endothelial cells.

Human IL-1, recombinant murine IL-1 and E. coli LPS were found to be potent inducers of plasminogen activator (PA)-inhibitor activity, both in vivo, in rats, as well as in cultured human endothelial cells. In vivo, LPS rapidly and dose-dependently (0.01-1,000 micrograms/kg) increased plasma PA-inhibitor activity. Infusion of IL-1 into rats resulted in a small but significant increase in PA-inhibitor activity in rat plasma. Likewise, in cultured human umbilical vein endothelial cells, LPS and IL-1 induced increased synthesis of PA-inhibitor. We suggest that the induced rat plasma inhibitor might be of endothelial origin.

An endothelial storage granule for tissue-type plasminogen activator.

In previous studies we have shown that, after stimulation by a receptor ligand such as thrombin, tissue-type plasminogen activator (tPA) and von Willebrand factor (vWf) will be acutely released from human umbilical vein endothelial cells (HUVEC). However, the mechanisms involved in the secretion of these two proteins differ in some respects, suggesting that the two proteins may be stored in different secretory granules. By density gradient centrifugation of rat lung homogenates, a particle was identified that contained nearly all tPA activity and antigen. This particle had an average density of 1.11-1.12 g/ml, both in Nycodenz density gradients and in sucrose density gradients. A similar density distribution of tPA was found for a rat endothelial cell line and for HUVEC. After thrombin stimulation of HUVEC to induce tPA secretion, the amount of tPA present in high-density fractions decreased, concomitant with the release of tPA into the culture medium and a shift in the density distribution of P-selectin. vWf, known to be stored in Weibel-Palade bodies, showed an identical distribution to tPA in Nycodenz gradients. In contrast, the distribution in sucrose gradients of vWf from both rat and human lung was very different from that of tPA, suggesting that tPA and vWf were not present in the same particle. Using double-immunofluorescence staining of HUVEC, tPA- and vWf-containing particles showed a different distribution by confocal microscopy. The distribution of tPA also differed from the distribution of tissue factor pathway inhibitor, endothelin-1, and caveolin. By immunoelectronmicroscopy, immunoreactive tPA could be demonstrated in small vesicles morphologically different from the larger Weibel-Palade bodies. It is concluded that tPA in endothelial cells is stored in a not-previously-described, small and dense (d = 1.11-1.12 g/ml) vesicle, which is different from a Weibel-Palade body.

A guide to murine coagulation factor structure, function, assays, and genetic alterations.

Murine blood coagulation factors and function are quite similar to those of humans. Because of this similarity and the adaptability of mice to genetic manipulation, murine coagulation factors and inhibitors have been extensively studied. These studies have provided significant insights into human hemostasis. They have also provided useful experimental models for evaluation of the pathophysiology and treatment of thrombosis. This review contains recommendations for obtaining, processing and assaying mouse blood hemostatic components, and it summarizes the extensive literature on murine coagulation factor structure and function, assays and genetic alteration. It is intended to be a convenient reference source for investigators of hemostasis and thrombosis.

No effect of C-reactive protein on early atherosclerosis development in apolipoprotein E*3-leiden/human C-reactive protein transgenic mice.

OBJECTIVE: C-reactive protein (CRP) has been associated with risk of cardiovascular disease. It is not clear whether CRP is causally involved in the development of atherosclerosis. Mouse CRP is not expressed at high levels under normal conditions and increases in concentration only several-fold during an acute phase response. Because the dynamic range of human CRP is much larger, apolipoprotein E*3-Leiden (E3L) transgenic mice carrying the human CRP gene offer a unique model to study the role(s) of CRP in atherosclerosis development. METHODS AND RESULTS: Atherosclerosis development was studied in 15 male and 15 female E3L/CRP mice; E3L transgenic littermates were used as controls. The mice were fed a hypercholesterolemic diet to induce atherosclerosis development. Cholesterol exposure did not differ between E3L/CRP and E3L mice. Plasma CRP levels were on average 10.2+/-6.5 mg/L in male E3L/CRP mice, 0.2+/-0.1 mg/L in female E3L/CRP mice, and undetectable in E3L mice. Quantification of atherosclerosis showed that lesion area in E3L/CRP mice was not different from that in E3L mice. CONCLUSIONS: This study demonstrates that mildly elevated levels of CRP in plasma do not contribute to the development of early atherosclerosis in hypercholesterolemic E3L/CRP mice.

Purification and properties of rat alpha 2 acute-phase macroglobulin.

Alpha 2 acute-phase macroglobulin was isolated from plasma of turpentine-injected rats. In the method conditions known to damage the biological activities of alpha 2 macroglobulin are avoided. The procedure successively involves: rivanol precipitation, concanavalin A-Sepharose chromatography and ion-exchange chromatography on DEAE-cellulose. Proteolytic activities were minimized throughout the purification. Thus alpha 2 macroglobulin was obtained in a 20% yield and was pure by biochemical and immunological criteria. Its molecular weight appeared to be 760 000 and it consisted of four subunits (Mr 190 000). The protein has an A1cm 1% = 8.8 and an isoelectric point = 4.8. The amino acid and carbohydrate compositions were determined. Our preparations bound 1 molecule of trypsin or 1 molecule of plasmin/molecule of alpha 2 macroglobulin. Kinetic parameters for alpha 2 macroglobulin-bound trypsin and plasmin were determined and compared with those of free trypsin and plasmin using butoxycarbonyl-L-valylglycyl-L-arginine-2-naphthylamide and benzoyl-L-arginine ethylester as substrates.

Binding of unmodified low-density lipoproteins to human fibroblasts. An investigation by immunoelectron microscopy.

The bindinf of unmodified low density lipoproteins to the plasma membrane of fibroblasts was studied at the ultrastructural level. The bound low density lipoprotein was visualized by an indirect immunoperoxidase technique, with the use of an antiserum against apoprotein B. Immunoreactive regions representing bound apoprotein B were found on the plasma membrane, in indented regions with a diameter of 0.15--0.30 micrometer and a fuzzy coat on the cytoplasmic side. Fibroblasts from a patient homozygous for hyperlipoproteinaemia type IIa showed no immunoreactive material in the indented regions. The specific 125I-labelled low density lipoprotein binding to these homozygous fibroblasts was 7% compared to control fibroblasts.

Evidence for the presence of two different fibrinolytic inhibitors in human endothelial cell conditioned medium.

In human umbilical artery and vein endothelial cell conditioned medium fibrinolytic inhibitors have been detected by two different techniques. A fast-acting inhibitor of tissue-type plasminogen activator (t-PA) and urokinase has been detected and quantified by its capacity to neutralize the above-mentioned plasminogen activators in a kinetic assay. By reverse fibrin autography after SDS-polyacrylamide gel electrophoresis a fibrinolytic inhibitor can be detected with a molecular mass of 52 kDa. The mutual relationship between these two inhibitors was studied. Neutralization of the fast-acting inhibitor by t-PA results in the formation of a complex with a molecular mass of 100 kDa. The t-PA added to endothelial cell conditioned medium in excess of the fast-acting inhibitor is fully stable. However, the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography is not affected by complete neutralization of the fast-acting inhibitor, and removal of the formed complexes by immune adsorption with immobilized anti-t-PA IgG. This suggests that the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography does not react with t-PA. Moreover, endothelial cell conditioned medium that is depleted of the fast-acting inhibitor does not show lysis resistance when directly applied to the reverse fibrin autography indicator gel (without previous electrophoresis), although the inhibitor is still present in the zymogram after SDS-polyacrylamide gel electrophoresis. This suggests that the inhibitor is induced by the SDS treatment. Heating the endothelial cell conditioned medium for 15 min at 70 degrees C fully destroys the fast-acting inhibitory activity, but leaves the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography unaffected. Moreover, at least one additional fibrinolytic inhibitor is detected in the zymogram after SDS-polyacrylamide gel electrophoresis. We conclude that the fast-acting inhibitor is not the same as the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography; the latter inhibitor is not operational in endothelial cell conditioned medium, but is induced by SDS-polyacrylamide gel electrophoresis.

Effects of LDL, HDL and their combination on the endogenous cholesterol synthesis in monocyte macrophages.

It is commonly assumed that the low density lipoprotein (LDL) degradation in extra-hepatic cells proceeds partly by way of the high-affinity LDL uptake process. In addition, it has been suggested that extra-hepatic LDL catabolism proceeds partly by way of preferential uptake and subsequent degradation of LDL by phagocytic scavenger cells. In order to study the effect of LDL and high density lipoprotein (HDL) on cholesterol metabolism in such scavenger cells, cultured human or pig monocytes were incubated with varying amounts of human or pig LDL, HDL and LDL/HDL mixtures, and the incorporation of [14C]acetate into cholesterol was measured. The addition of LDL suppressed endogenous cholesterol synthesis. Incubation of monocytes with LDL/HDL mixtures reduced the total amount of de novo synthesized cholesterol to the same extent as incubation with LDL alone. Our results suggest that in cultured monocyte macrophages HDL does not influence endogenous cholesterol synthesis, even in the presence of LDL.

Simvastatin improves disturbed endothelial barrier function.

BACKGROUND: Recent clinical trials have established that inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) reduce the risk of acute coronary events. These effects of statins cannot be fully explained by their lipid-lowering potential. Improved endothelial function may contribute to the positive effects of statin treatment. METHODS AND RESULTS: In the present study, we report that simvastatin reduces endothelial barrier dysfunction, which is associated with the development of atherosclerosis. Treatment of human umbilical vein endothelial cells for 24 hours with 5 micromol/L simvastatin reduced the thrombin-induced endothelial barrier dysfunction in vitro by 55+/-3%, as assessed by the passage of peroxidase through human umbilical vein endothelial cell monolayers. Similar effects were found on the thrombin-induced passage of (125)I-LDL through human aortic endothelial cell monolayers. This reduction in barrier dysfunction by simvastatin was both dose and time dependent and was accompanied by a reduction in the thrombin-induced formation of stress fibers and focal adhesions and membrane association of RhoA. Simvastatin treatment had no effect on intracellular cAMP levels. In Watanabe heritable hyperlipidemic rabbits, treatment for 1 month with 15 mg/kg simvastatin reduced vascular leakage in both the thoracic and abdominal part of the aorta, as evidenced by the Evans blue dye exclusion test. The decreased permeability was not accompanied by a reduction of oil red O-stainable atherosclerotic lesions. CONCLUSIONS: These data show that simvastatin, in a relatively high concentration, improves disturbed endothelial barrier function both in vitro and in vivo. The data also support the beneficial effects of simvastatin in acute coronary events by mechanisms other than its lipid-lowering effect.

Increased levels of soluble vascular cell adhesion molecule 1 are associated with risk of cardiovascular mortality in type 2 diabetes: the Hoorn study.

Membrane-bound vascular cell adhesion molecule 1 (VCAM-1) allows the tethering and rolling of monocytes and lymphocytes as well as firm attachment and transendothelial migration of leukocytes. Soluble forms of VCAM (sVCAM-1) may serve as monitors of increased expression of membrane-bound VCAM-1 and thus may reflect progressive formation of atherosclerotic lesions. Levels of sVCAM-1 have been found to be increased among type 2 diabetic as compared with nondiabetic subjects. To study the association of plasma sVCAM-1 concentration and risk of cardiovascular and all-cause mortality among nondiabetic and diabetic subjects, we investigated an age-, sex-, and glucose-tolerance-stratified sample (n = 631) of a population-based cohort aged 50-75 years that was followed prospectively. Plasma levels of sVCAM-1 were determined in frozen -70 degrees C baseline samples. After 7.4 years (mean) of follow-up, 107 (17%) subjects had died (42 of cardiovascular causes). In the entire group, increased sVCAM-1 levels were significantly associated with increased risk of cardiovascular mortality (relative risks [RRs] per 100 ng/ml sVCAM-1 increase, 1.10 [1.05-1.15] after adjustment for age, sex, and glucose tolerance status). This RR was somewhat diminished by further adjustment for the presence of hypertension and cardiovascular disease; levels of total, HDL, and LDL cholesterol and homocysteine; the presence of microalbuminuria (a putative marker of endothelial dysfunction); levels of von Willebrand factor (a marker of endothelial dysfunction) and C-reactive protein (a marker of low-grade inflammation); and estimates of glomerular filtration rate. However, the RR remained statistically significant. The RR among type 2 diabetic subjects was 1.13 (1.07-1.20) per 100 ng/ml sVCAM-1 increase after adjustment for age and sex, which was somewhat higher but not significantly different from the RR in nondiabetic subjects (P value for interaction term, 0.12). Further adjustment for other risk factors gave similar results. In conclusion, levels of sVCAM-1 are independently associated with the risk of cardiovascular mortality in type 2 diabetic subjects and therefore might be useful for identifying subjects at increased cardiovascular risk. Increased plasma sVCAM-1 levels may reflect progressive formation of atherosclerotic lesions, or sVCAM-1 itself may have bioactive properties related to cardiovascular risk. Our data, however, argue against the hypotheses of sVCAM-1 levels simply being a marker of endothelial dysfunction, of low-grade inflammation, or of an impaired renal function.

Hormone replacement therapy.

Hormone replacement therapy is increasingly being used for purposes unrelated to the alleviation of menopausal symptoms, such as the prevention of osteoporosis and cardiovascular disease. Clinical trials, however, suggest that the one drug/many purposes concept may be too optimistic. The availability of new estrogen-like compounds and the discovery of a second estrogen receptor have opened new possibilities for more specific drug development.

Polylysine as a vehicle for extracellular matrix-targeted local drug delivery, providing high accumulation and long-term retention within the vascular wall.

We present the first steps in the elaboration of an approach of extracellular matrix-targeted local drug delivery (ECM-LDD), designed to provide a high concentration, ubiquitous distribution, and long-term retention of a drug within the vessel wall after local intravascular delivery. The approach is based on the concept of a bifunctional drug comprising a "therapeutic effector" and an "affinity vehicle," which should bind to an abundant component of the vessel wall. The aim of the present study was to select molecules suitable for the role of affinity vehicles for ECM-LDD and to study their intravascular delivery and retention ex vivo and in an animal model. By use of fluorescence microscopy, the following molecules were selected on the basis of strong binding to cross sections of human vessels: protamine, polylysine, polyarginine, a glycosaminoglycan-binding peptide from vitronectin, and a synthetic dendrimer. With polylysine as a prototypic affinity vehicle, we showed that after intravascular delivery, polylysine was concentrated throughout a luminal layer of the vascular wall to an extremely high concentration of 20 g/L and was retained therein for at least 72 hours of perfusion without noticeable losses. Low molecular weight (fluorescein) and high molecular weight (hirudin) compounds could be chemically conjugated to polylysine and were retained in the vessel wall after intravascular delivery of the conjugates. In conclusion, by use of the ECM-LDD method, an extremely high concentration and long-term retention of locally delivered drug can be reached. Polycationic molecules can be considered as potential affinity vehicles for ECM-LDD.

The lipid composition and localization of free and esterified cholesterol in different types of xanthomas.

The lipid composition of 17 xanthomatous lesions from 16 patients with different types of hyperlipoproteinaemia was analyzed. The relative amounts of free cholesterol, cholesterolesters, and phospholipids in xanthomas obtained from patients who subsequently showed regression of their remaining type of xanthomas were different in comparison with xanthomas which did not show regression. One tendinous xanthoma contained more than 50% free cholesterol and resembled in lipid composition the gruel plaques found in atherosclerotic lesions. The relative amounts of free cholesterol, cholesterolesters, and phospholipids in regressing xanthomas were similar to those of fatty streaks found in arteries of children between the ages of 5-10 yr. Histochemical studies using an enzymatic method demonstrated that free and esterified cholesterol were located in focal areas of xanthomatous tissue. It is concluded that the physical state of lipids in xanthomatous lesions can vary remarkably and plays an important role with regard to the possibility of regression.

Localization of lipase-like immunoreactivity in porcine adipose, aortic and myocardial tissue.

Lipase has been purified from pig adipose tissue and antibodies have been produced in rabbit. By indirect immunofluorescence and immunoenzyme techniques lipase-like immunoreactivity was demonstrated in the intima of pig aorta, in the endothelial cells of the myocardium and in plasma membranes of adipocytes and skeletal muscle cells. Lipase activity in extracts of some of these tissues was inhibited by the addition of anti-lipase antibodies. At least part of the immunoreactivity in the examined tissues is due to active lipases.

Progression and regression of atherosclerosis in APOE3-Leiden transgenic mice: an immunohistochemical study.

Apolipoprotein E3-Leiden (APOE3-Leiden) transgenic mice develop hyperlipidemia and are highly susceptible to diet-induced atherosclerosis. We have studied the progression and regression of atherosclerosis using immunohistochemistry. Female transgenic mice were fed a moderate fat diet to study atherosclerosis over a longer time period. Fatty streaks arose in the intima and consisted of lipid filled macrophages which differed in origin. All macrophages expressed the macrophage scavenger receptor while two thirds expressed sialoadhesin and were positive for an antibody recognizing marginal zone macrophages (MOMA-1). All macrophages were negative for the scavenger receptor MARCO and 50% were positive for CD4. Small fatty streaks contained CD-3 positive T-lymphocytes which were for more than 70% CD4-positive. ICAM-1 was positive both in atherosclerotic and control mice. In early plaques, fibrosis was observed on the luminal and medial site of the foam cells while smooth muscle cells were only observed in the fibrous cap. To study regression, we used a high fat, high cholesterol diet to rapidly induce atherosclerosis (14 weeks). The animals were then fed normal chow. Subsequently, atherosclerosis was assayed over time (4, 8, 16 weeks). Cholesterol levels dropped in 4 weeks to control levels. The animals did not show a significantly decrease in plaque size over time. but the percentage macrophages was significantly smaller in the animals after 4 weeks. In conclusion, the APOE3-Leiden mouse is a useful model to study the progression and regression of atherosclerosis.

Raman spectroscopy for quantifying cholesterol in intact coronary artery wall.

The chemical composition of vascular lesions, an important determinant of plaque progression and rupture, can not presently be determined in vivo. Prior studies have shown that Raman spectroscopy can accurately quantify the amounts of major lipid classes and calcium salts in homogenized coronary artery tissue. This study determines how the relative cholesterol content, which is calculated from Raman spectra collected at the luminal surface of an artery, is related to its depth in an intact arterial wall. Raman spectra of human atherosclerotic plaques were measured after thin tissue layers were successively placed on them. From these spectra, relative cholesterol contents were calculated and used to determine how cholesterol signal strength is attenuated by overlaying tissue. Then, intact artery samples (n = 13) were examined spectroscopically, sectioned and stained specifically for cholesterol. Images of these sections were digitized, and image intensities were related to cholesterol content. These cholesterol amounts were weighed appropriately for depth into the tissue and area-integrated for comparison with spectroscopy results. A decaying exponential curve was fit to the layer study data (r2 = 0.97) and showed that approximately 300 microm of tissue attenuates cholesterol signals by 50%. In intact plaques, the spectroscopically-determined cholesterol amounts correlated strongly and linearly with those determined by digital microscopy (r2 = 0.94). With Raman spectroscopy techniques, the cholesterol content of a lesion can be determined by properly accounting for its depth into an arterial wall. Our results suggest that chemical concentrations in an artery wall could be mapped throughout its thickness, possibly by combining Raman spectroscopy methods with other techniques.

On the role of calcium in the acute release of tissue-type plasminogen activator and von Willebrand factor from the rat perfused hindleg region.

The involvement of calcium in the release of tissue-type plasminogen activator (t-PA) and von Willebrand Factor (vWF) from vascular endothelial cells was studied ex vivo using a rat hindleg perfusion system. By adding either platelet-activating factor or bradykinin to the perfusing Tyrode solution, a rapid release of t-PA and vWF was induced. Extracellular calcium was required for the acute release of both glycoproteins as this release was totally abolished in the presence of EGTA. The calcium ionophore A-23187 induced (Ca-dependently) the release of both proteins, suggesting that Ca-influx was also sufficient to induce release. The absence of an effect of the calcium L-type channel blockers, verapamil and diltiazem, and of the calcium channel agonist BAY K-8644, suggested that endothelial voltage-operated calcium channels were not involved in release. Trifluoperazine, a calmodulin antagonist, significantly inhibited the induced release of t-PA and vWF, while the "intracellular calcium antagonist" TMB-8 had no effect. Lanthanum chloride (200 microM) inhibited the induced release of t-PA but not that of vWF. Our results suggest that Ca2+ influx is essential for the release of t-PA and vWF from the perfused rat hindleg.

The simultaneous acute release of tissue-type plasminogen activator and von Willebrand factor in the perfused rat hindleg region.

In perfused rat hindlegs, platelet-activating factor and bradykinin induced the acute release of both tissue-type plasminogen activator (t-PA) and von Willebrand Factor (vWF). The time course of release was similar for both proteins, and the amounts of t-PA and vWF released under various conditions were closely correlated. Release of both t-PA and vWF required extracellular calcium, and could be induced by the calcium ionophore A-23187. Protein synthesis was not required for release to occur. Phorbol myristate acetate also induced release of t-PA and vWF, though with a different time course; DDAVP was inactive. The results suggest that the release of t-PA, and that of vWF, are closely linked at the cellular level.

Protein synthesis inhibition by cycloheximide does not affect the acute release of tissue-type plasminogen activator.

The acute release of tissue-type plasminogen activator (t-PA) was studied in perfused rat hindlegs. Pretreatment of rats with the protein syNthesis inhibitor cycloheximide (2 mg/kg) at 1, 3 or 5 h prior to perfusion of rat hindlegs did not influence the amount of t-PA released by platelet-activating factor (20 nM) or bradykinin (1 microM). The amount of t-PA activity that could be extracted from hindleg skeletal muscle was not decreased by cycloheximide pretreatment though it was decreased in lung extracts. The in vivo release of t-PA was not affected by cycloheximide pretreatment. The data suggest that the acute release of t-PA from vascular endothelial cells does not require ongoing protein synthesis, but that acutely released t-PA is derived from a stable endothelial storage pool.

The role of cyclic nucleotides in the release of tissue-type plasminogen activator and von Willebrand factor.

The modulation of the induced acute release of tissue-type plasminogen activator (t-PA) and of von Willebrand factor (vWF) by compounds affecting cyclic nucleotide levels was studied, using an isolated rat hindleg perfusion system. Platelet-activating factor (PAF; 5 nM) or bradykinin (0.8 microM) were used to induce release of t-PA and vWF. The guanylate cyclase activators sodium nitroprusside and atrial natriuretic factor reduced the induced release of t-PA and vWF. Release was not affected by inhibiting nitric oxide production with NG-nitro-L-arginine. The effects of nitroprusside and atrial natriuretic factor could not be reproduced by infusion of 8-bromo-cGMP. The adenylate cyclase activator forskolin had no effect on bradykinin-induced release of t-PA and vWF, reduced PAF-induced t-PA release, but potentiated PAF-induced vWF release. These modulatory effects were only partially mimicked by infusion of 8-bromo-cAMP. None of the compounds tested was able to induce the release of t-PA or of vWF in the absence of stimulation by bradykinin or platelet-activating factor. Cyclic nucleotides can thus modulate, but not induce, the acute release of t-PA and vWF from perfused rat hindlegs.