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Aims: To study whether microalbuminuria, endothelial dysfunction and low-grade inflammation are associated with the presence and progression of diabetic retinopathy. Methods: Patients with Type 2 diabetes (n = 328) attending a diabetes clinic were followed for 10 years and examined annually during the last 7 years. Retinopathy was assessed after pupillary dilatation by direct ophthalmoscopy (baseline) and two-field 60°fundus photography (follow-up). Urinary albumin excretion, and markers of endothelial function (von Willebrand factor, tissue-type plasminogen activator, soluble E-selectin (sE-selectin), and soluble vascular cell adhesion molecule 1) and inflammatory activity (C-reactive protein and fibrinogen) were determined. Results: The prevalence of retinopathy was 33.8%. The median diabetes duration at baseline was 7 years (interquartile range 2-12 years). The highest tertiles of baseline urinary albumin excretion and glycated haemoglobin (HbA1c) were associated with prevalent retinopathy: odds ratio (OR) 95% confidence interval (CI) 2.80 (1.44-5.46) and 2.19 (1.11-4.32), respectively. Progression of retinopathy occurred in 188 patients. The second and third tertiles of baseline sE-selectin were associated with progression of retinopathy [1.44 (1.04-2.01) and 1.61 (1.19-2.18)] but not independently of HbA1c. None of the other markers was significantly associated with the presence or progression of retinopathy. High baseline HbA1c was significantly associated with progression of retinopathy: 1.65 (1.21-2.25). Conclusions: In this population of patients with Type 2 diabetes who attended a diabetes clinic, there was some evidence for a role of endothelial dysfunction in the progression of retinopathy. We could not demonstrate a role for low-grade inflammation. Our study emphasizes the importance of glycaemic control in the development and progression of retinopathy. © 2007 The Authors.

Increased plasma levels of fibrinogen are associated with a higher risk of cardiovascular disease. It has been suggested that lipid levels may influence the fibrinogen levels by a mechanism other than the synthesis rate, for example a decreased clearance rate. We performed a pilot study to explore this possibility. Twelve male Wistar rats were fed for four weeks with a control low fat/low cholesterol diet, a high fat/high cholesterol diet, and a high fat/high cholesterol diet with an additional 0.5% cholic acid. Labeled 125I fibrinogen was injected, and blood was sampled repeatedly. From the plasma radioactivity of the samples, fibrinogen halflife time was calculated for each animal. Our results suggest that plasma lipids lengthen the fibrinogen halflife times, although the differences were not statistically significant in this small study. Our final conclusion from this study is that lipids may have an effect on the turnover rate of fibrinogen and possibly affect fibrinogen levels through this mechanism.

SummaryEstrogen therapy may increase the risk of arterial thromboembolism, at least in the short term. In a randomized, double-blind and placebocontrolled study in 25 healthy postmenopausal women (52.5 ± 2.8 years), we therefore examined the short-term effect of unopposed estrogen on the fasting and fat-load-stimulated plasma levels of total factor VII versus active factor VII. Plasma total factor VII was measured by use of a chromogenic assay; plasma active FVII by a recently developed method using truncated tissue factor. As compared to placebo, 8 weeks of oral 17β-estradiol (2 mg daily) increased the mean fasting and postprandial plasma levels of total factor VII by 17 and 21% points, respectively (both P < 0.01), but did not affect the fasting and/or postprandial plasma levels of active factor VII (mean change both 0.05 ng/mL; P > 0.35). Furthermore, the change in the fasting level of total factor VII after therapy was not associated with the change in the fasting level of active factor VII (r = 0.27; P = 0.21). These findings argue against the idea that elevated levels of total factor VII underlie an increased risk of arterial thromboembolism in postmenopausal women using unopposed estrogen replacement.

Several clinical studies have demonstrated an inverse relationship between circulating levels of estrogen and tissue-type plasminogen activator (t-PA). The present study was designed to test the hypothesis that estrogen lower plasma levels of t-PA by increasing its clearance from the bloodstream. 17α-Ethinyl estradiol (EE) treatment resulted in a significant increase in the clearance rate of recombinant human t-PA in mice (0.46 mL/min in treated mice v 0.32 mL/min in controls; P < .01). The clearance of endogenous, bradykinin-released t-PA in rats was also significantly increased after EE treatment (area under the curve [AUC], 24.9 ng/mL min in treated animals v 31.9 ng/mL · min in controls; P < .05). Two distinct t-PA clearance systems exist in vivo: the low-density lipoprotein receptor-related protein (LRP) on liver parenchymal cells and the mannose receptor on mainly liver endothelial cells. Inhibition of LRP by intravenous injection of receptor-associated protein (RAP) as a recombinant fusion protein with Salmonella japonicum glutathione S-transferase (GST) significantly retarded t-PA clearance in control mice (from 0.41 to 0.25 mL/min; n = 5, P < .001) and EE-treated mice (from 0.66 to 0.35 mL/min; n = 5, P < .005), but did not eliminate the difference in clearance capacity between the 2 experimental groups. Similar results were obtained in mice in which LRP was inhibited via overexpression of the RAP gene in liver by adenoviral gene transduction. In contrast, administration of mannan, a mannose receptor antagonist, resulted in identical clearances (0.22 mL/min in controls and 0.24 mL/min in EE-treated mice). Northern blot analysis showed a 6-fold increase in mannose receptor mRNA expression in the nonparenchymal liver cells of EE-treated mice, whereas the parenchymal LRP mRNA levels remained unchanged. These findings were confirmed at the protein level by ligand blotting and Western blotting analysis. Our results demonstrate that EE treatment results in increased plasma clearance rate of t-PA via induction of the mannose receptor and could explain for the inverse relationship between estrogen status and plasma t-PA concentrations as observed in humans. Chemicals/CAS: Estradiol Congeners; Ethinyl Estradiol, 57-63-6; Lectins, C-Type; mannose receptor; Mannose-Binding Lectins; Receptors, Cell Surface; Recombinant Proteins; Tissue Plasminogen Activator, EC 3.4.21.68

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.

Summary The thrombolytic and antithrombotic effects of tissue-type plasminogen activator (t-PA) depend on its concentration in the blood. The mannose receptor is one of the receptors that mediate the rapid clearance of t-PA from the blood by the liver (Noorman and Rijken, Fibrinolysis & Proteolysis 1997; 11: 173–186). We hypothesized that by blocking the binding of t-PA to this receptor it might be possible to decrease the clearance of t-PA and thereby increase the plasma concentration of endogenous t-PA and exogenous t-PA. Inhibitors of the t-PA-mannose receptor interaction may thus be useful drugs in thrombolytic and antithrombotic therapy. We developed monoclonal antibodies against the human mannose receptor. These antibodies are able to inhibit binding of t-PA to the mannose receptor. By use of the antibodies it was shown that the mannose receptor is expressed by few human cell types. The expression of the mannose receptor on macrophages is highly regulated and depends on the type of macrophage activation. The expression of the mannose receptor in the liver is limited to endothelial cells and Kupffer cells. We developed high affinity ligands of the mannose receptor that are able to inhibit the plasma clearance of therapeutic t-PA doses by maximally 60% in the rat. Therapeutical concentrations of the low affinity mannose receptor ligand and antithrombotic agent dextran partially inhibit t-PA plasma clearance (33%) and thereby increase endogenous t-PA plasma concentrations by 20–60% in the rat. Thus mannose receptor inhibitors can be considered as a new tool to increase the t-PA concentration in blood and thereby increase its thrombolytic and antithrombotic effects.

SummaryWe studied thirteen male-to-female (M→F) and ten female-to-male (F→M) transsexuals who, for four months, received cross-sex treatment with, respectively, ethinylestradiol and cyproterone acetate, and with testosterone esters. We assessed the effects of treatment on plasma levels of tissue-type plasminogen activator (tPA), von Willebrand factor (vWF), vWF-propeptide (vWF:AgII) and bigendothelin-1 (big-ET-1), four proteins that are markers of endothelial cell functioning. We also measured urokinase-type PA (uPA) and plasminogen activator inhibitor-type 1 (PAI-1), which may not be endothelium-derived but share major clearance pathways with tPA.In M→F transsexuals, mean plasma levels of tPA (minus 4.4 ng/ml), big-ET-1 (minus 0.8 pg/ml), uPA (minus 0.5 ng/ml) and PAI-1 (minus 26 ng/ml) decreased (all Ps ≤0.02). The level of vWF increased (plus 24%; P = 0.005), while vWF:AgII did not change (P = 0.49).In F→M transsexuals, levels of big-ET-1 increased (plus 0.4 pg/ml; P = 0.02), while tPA, uPA and PAI-1 did not change (all Ps >0.25). In this group vWF decreased (minus 14%; P = 0.06), but vWF:AgII did not change (P = 0.38).Estrogens and androgens have clear effects on plasma levels of endothelial marker proteins. The mechanisms behind these effects are complex and appear to involve both altered secretion (big-ET-1) and processing and/or clearance (vWF and possibly tPA). Therefore, effects of hormones on the levels of endothelial marker proteins do not necessarily reflect changes in endothelial cell functioning, at least with regard to changes in vWF level associated with the oral administration of high doses of ethinylestradiol and cyproterone acetate to healthy men and the parenteral administration of testosterone to healthy women.

The acute release of t-PA was studied in vitro in human endothelial cells from different origin. It proved possible to enhance the amounts of t-PA which can be released by increasing t-PA synthesis, both in vitro, and in vivo in rats. These data suggest the feasibility to induce and (or) enhance acute release of t-PA in man. Chemicals/CAS: bradykinin, 58-82-2, 5979-11-3; butyric acid, 107-92-6, 156-54-7, 461-55-2; retinoic acid, 302-79-4; thrombin, 9002-04-4; tissue plasminogen activator, 105913-11-9

The aim of the present study was to investigate whether hypothyroidism would affect the components of the rat plasma fibrinolytic system. Hypothyroidism was induced by feeding rats a thiouracil-containing diet for 2 weeks. During this period, half of the animals received subcutaneous injections of L-thyroxine (20 μg/kg daily) to restore thyroid hormone levels. Groups of euthyroid rats were similarly treated with L-thyroxine. Plasma tissue-type PA (t-PA) activity was not affected by thyroid status. Plasma urokinase-type PA (u-PA) activity was significantly decreased by the thiouracil-containing diet; however, this effect was not abolished by L-thyroxine. Plasminogen, α2-antiplasmin and fibrinogen were not affected by hypothyroidism. In the hypothyroid group, plasma PAI activity was significantly increased; no such increase was found in animals simultaneously receiving L-thyroxine. Plasma PAI activity correlated with T3 (r = -0.586; P < 0.005) and cholesterol (r = +0.737; P < 0.001); in multiple regression analysis, however, the correlation between PAI activity and T3 was not significant. The increase in plasma PAI activity during hypothyroidism may thus not be a direct effect of the thyroid hormone, but an indirect one through some other, possibly cholesterol related, component. Chemicals/CAS: cholesterol, 57-88-5; levothyroxine, 51-48-9; plasminogen activator inhibitor, 105844-41-5; thiouracil, 141-90-2; tissue plasminogen activator, 105913-11-9; urokinase, 139639-24-0; Cholesterol, 57-88-5; Plasminogen Inactivators; Thiouracil, 141-90-2; Thyroxine, 7488-70-2; Tissue Plasminogen Activator, EC 3.4.21.68; Triiodothyronine, 6893-02-3; Urinary Plasminogen Activator, EC 3.4.21.73

Hyperlipidaemia was induced in rats by feeding a diet enriched with butter (40% w/w) and cholesterol (5% w/w), and supplemented with thiouracil (0.3% w/w). As has been demonstrated previously, this treatment induces increased plasma levels of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) activity. In the present study plasma levels of von Willebrand factor (vWF) were also found to be increased. The tissue concentration t-PA and its clearance from the blood were compared in the hyperlipidaemic rats and in normolipidaemic control rats. t-PA activity and antigen were measured in lung, heart, kidney and liver after extraction with acid acetate-buffer or with KSCN-buffer, and were found to be similar in the tissue extracts of hyperlipidaemic and control rats. Plasma t-PA clearance was measured in both groups of rats in vivo, while the efficacy of the liver in extracting t-PA was analysed using an ex vivo liver perfusion system. The in vivo clearance study showed that the half-life of t-PA was significantly prolonged in hyperlipidaemic rats (83 ± 9 s) compared with control rats (58 ± 6 s). In the liver perfusion experiments, the extraction of t-PA from the perfusate was smaller in the hyperlipidaemic group (48 ± 5%) than in the control rats (60 ± 8%). Together the data suggest that the increased plasma level of t-PA in hyperlipidaemic rats may be due to decreased liver clearance.

Pharmacological modulation of the in vivo induction of plasminogen activator inhibitor type-1 (PAI-1) synthesis was studied in rats using the induction of PAI-1 by endotoxin as a model system. Both the cyclooxygenase inhibitors acetylsalicylic acid and indomethacin enhanced PAI-1 induction. The combined cyclooxygenase-lipoxygenase inhibitor, BW755C, dose-dependently inhibited induction. Since five other lipoxygenase inhibitors, a phospholipase inhibitor, an inhibitor of leukotriene formation and dexamethasone had no effect on the endotoxin-induced increase in PAI-1 synthesis, the effect of BW755C could not be ascribed to its known pharmacological properties. In addition, induction of PAI was enhanced by isobutyl-methylxanthine, a phosphodiesterase inhibitor, but not, however, by other phosphodiesterase inhibitors, or by forskolin or N(G)-nitro-L-arginine, suggesting an effect of isobutyl-methylxanthine other than through cyclic nucleotides. Heparin and hirudin had no effect either. Overall, the data showed that the induction of PAI-1 synthesis by endotoxin in vivo can be up- and down-regulated pharmacologically, but the mechanisms involved remain elusive. Chemicals/CAS: 2 (3,4 dihydro 3,4 dioxo 1 naphthylamino)benzoic acid methyl ester, 114832-13-2; 3 [3 tert butylthio 1 (4 chlorobenzyl) 5 isopropyl 2 indolyl] 2,2 dimethylpropionic acid, 118414-82-7; 3 amino 1 (3 trifluoromethylphenyl) 2 pyrazoline, 66000-40-6; acetylsalicylic acid, 493-53-8, 50-78-2, 53663-74-4, 53664-49-6, 63781-77-1; cycloheximide, 642-81-9, 66-81-9; dexamethasone, 50-02-2; diltiazem, 33286-22-5, 42399-41-7; forskolin, 66575-29-9; ginkgolide B, 15291-77-7, 99796-69-7; heparin, 37187-54-5, 8057-48-5, 8065-01-8, 9005-48-5; hirudin, 8001-27-2; indometacin, 53-86-1, 74252-25-8, 7681-54-1; isobutylmethylxanthine, 28822-58-4; mepyramine maleate, 59-33-6; n [3 (3 phenoxyphenyl)allyl]acetohydroxamic acid, 106328-57-8; n(g) nitroarginine methyl ester, 50903-99-6; naloxone, 357-08-4, 465-65-6; phentolamine mesylate, 65-28-1; phentolamine, 50-60-2, 73-05-2; plasminogen activator inhibitor, 105844-41-5; propranolol, 13013-17-7, 318-98-9, 3506-09-0, 4199-09-1, 525-66-6; verapamil, 152-11-4, 52-53-9; 1-Methyl-3-isobutylxanthine, 28822-58-4; Aspirin, 50-78-2; BW-755C, 66000-40-6; Indomethacin, 53-86-1; Lipopolysaccharides; Plasminogen Activator Inhibitor 1; Tissue Plasminogen Activator, EC 3.4.21.68

SummaryAged rats are more susceptible to endotoxin-induced effects, including microthrombosis and platelet aggregation, than are young rats. To investigate whether changes in the fibrinolytic system might be involved, we investigated the fibrinolytic activity in plasma euglobulin fractions and tissues (lung and heart) of young (6-months old) and aged (24-months old) rats under baseline conditions and after challenge with endotoxin. Aged rats had lower plasma levels of tissue-type plasminogen activator (t-PA) and of urokinase-type PA (u-PA) activity. PA inhibitor (PAI) activity was higher in the plasma of aged rats, as was t-PA activity in lung and heart.Rats were treated with either a low dose (1 μg/kg) or a high dose (10 mg/kg) of endotoxin. Both treatments induced a transient phase of increased blood fibrinolytic activity, as evidenced by higher levels of tissue-type plasminogen activator (t-PA) activity and decreased levels of PA inhibitor (PAI) activity. Over time, the fibrinolytic activity decreased, probably due to increased levels of PA inhibitor.Both the early increase in t-PA activity, and the subsequent increase in PAI activity, were more pronounced in the aged rats, as compared with the younger rats, after the high dose of endotoxin. The aged rats also responded to an injection of interleukin-1β or tumor necrosis factor-α with a larger increase of PAI activity than did the younger rats.Together the data suggest that, compared to young rats, aged rats have a decreased base-line plasma fibrinolytic activity, while their fibrinolytic system is more responsive to challenge by endotoxin and cytokines.

SummaryThe influence of changes in liver blood flow on the clearance of rt-PA was studied both in healthy subjects and in a perfused rat liver model. Liver blood flow in healthy subjects was documented indirectly by the clearance of indocyanine green (ICG). Exercise reduced liver blood flow on average by 57% with a 95% confidence interval (95% Cl) ranging from 51% to 62% (n = 5) and increased plasma levels of rt-PA activity (after an i. v. infusion of 18 mg of rt-PA over 120 min) by 119% (95% Cl, 58% - 203%) and rt-PA antigen by 91% (95% Cl, 30% - 140%). In the perfused rat liver model it was shown that halving or doubling of the physiological flow rate of a perfusate, containing rt-PA caused a proportional change in the clearance of rt-PA, while the extraction of rt-PA by the liver remained similar. In conclusion, liver blood flow is a major determinant of the clearance of rt-PA. This may have important implications for dosage of rt-PA in patients with myocardial infarction.

The effects on fibrinolytic components of pentoxifylline (Trental), of its first metabolite BL 194 (penthydroxyfylline), and of its analogue HWA 448 (torbafylline) were studied in rats. BL 194, though not pentoxifylline and HWA 448, significantly enhanced the induced release by platelet-activating factor (PAF) of tissue-type plasminogen activator (tPA) from isolated perfused rat hindlegs. In contrast, the simultaneously induced release of von Willebrane factor (vWF) was reduced by BL 194. The effect of BL 194 on PAF-induced release of tPA and vWF could be mimicked by isobutyl-methylxanthine (IBMX), an inhibitor of phosphodiesterases. In vivo, BL 194 and pentoxifylline did not affect baseline levels of plasma tPA and PA inhibitor activity, nor did these compounds affect the in vivo induction of tPA release by PAF. Similarly, the induction by endotoxin of PA inhibitor activity was not influenced by pentoxifylline or BL 194. By its opposite effects on tPA and vWF release. BL 194 might favrorably influence the thrombotic balance.

SummaryThe 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 εM) 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.

Plasmin inhibition by rat tissue extracts were compared with rat plasma plasmin inhibition. Nine different tissues were extracted with an acid acetate-buffer (lung, heart, brain, adrenal, aorta, skeletal muscle, spleen and liver) or with KSCN-buffer (kidney), and antiplasmin activity was evaluated by a spectrophotometric method. All tissue extracts inhibited plasmin dose-dependently; the amount of plasmin inhibited by a fixed amount of extract depended upon the tissue. In rat plasma, antiplasmin activity was acid-labile, but acid treatment did not affect the plasmin inhibitory effect of tissues extracts. Chemicals/CAS: antiplasmin, 9049-68-7; plasmin, 9001-90-5, 9004-09-5; Antiplasmin; Plasmin, EC 3.4.21.7; Tissue Extracts

Stimulation of the isolated perfused rat hindleg vascular bed with various concentrations of endothelin-1 or endothelin-3 resulted in the acute release of tissue-type plasminogen activator (t-PA) and of von Willebrand factor (vWF). The release of both endothelial cell products required the presence of extracellular calcium; the release of vWF by endothelin was instantly inhibited by disodium EDTA. The calcium antagonists nifedipine, diltiazem and verapamil partially inhibited endothelin-induced t-PA release, but had no effect on endothelin-induced vWF release.

Summary Sprague-Dawley rats were treated for 2 weeks with diets containing either 40% butter plus 5% cholesterol (B group), or 0.3% thiouracil (T group), or both butter-cholesterol and thiouracil (BT group). The activity of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (UK) was evaluated in euglobulin fractions, and PA inhibitor (PAI) activity in citrated plasma. The t-PA activity was increased in the BT group, compared with controls, but unchanged in the other experimental groups. PAI activity was increased in all treatment groups, but more pronouncedly in the BT group. Euglobulin UK activity did not differ between groups when assayed spectrophotometrically, but showed a decrease on fibrin autography in the BT group after dextran sulphate precipitation. The correlation between plasma triglycerides (TGs) and fibrinolytic variables (t-PA, UK, PAI) was different in the various treatment groups, suggesting that not only the TG levels, but also the way of producing these levels was of importance.

SummaryIn perfused rat hindlegs, platelet-activating factor and bradyki-nin 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.

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. Conclusion - 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. © 2005 American Heart Association, Inc. Chemicals / CAS: C reactive protein, 9007-41-4; cholesterol, 57-88-5; Apolipoprotein E3; Apolipoproteins E; Biological Markers; C-Reactive Protein, 9007-41-4; Cholesterol, 57-88-5

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.

Objective: To study the effects of combined hormone replacement therapy on markers of endothelial function and inflammatory activity. Design: Prospective, randomized, controlled study. Setting: Academic hospital. Patient(s): Healthy postmenopausal women with an intact uterus. Intervention(s): For the first 12 months, the hormone replacement therapy group (n = 14) received oral E2, 1 mg daily, sequentially combined with 5 or 10 mg of dydrogesterone. Thereafter, they received oral E2, 2 mg daily, sequentially combined with 10 mg of dydrogesterone. The control group (n = 13) received no treatment. Data were collected at baseline and at 3, 12, and 15 months. Main Outcome Measure(s): Parameters of endothelial function and inflammatory activity. Result(s): During 12 months of follow-up, we observed decreases of 15% in plasma levels of endothelin-1, of 21% in soluble thrombomodulin, of 14% in von Willebrand factor, and of 12% in clottable fibrinogen in the hormone replacement therapy group compared with the control group. There was a 5% decrease in soluble E-selectin levels. All significant changes were observed by 3 months and sustained after 15 months. Brachial artery flow-mediated vasodilatation and C-reactive protein levels did not change significantly. Conclusion(s): Long-term combined hormone replacement therapy with E2 and dydrogesterone in healthy women was associated with sustained improvement in some aspects of endothelial function and in clottable fibrinogen levels.

SummaryThe effect of compounds increasing intracellular adenosine 3’:5’-cyclic monophosphate [cAMP]i levels (prostacyclin, isoproterenol, forskolin, cholera toxin), and of the cAMP analogs 8-bromo-cAMP and dibutyryl-cAMP, on the regulated secretion (acute release) of tissue-type plasminogen activator (tPA) and von Willebrand factor (vWF) was studied in cultured human umbilical vein endothelial cells (HUVEC).Prostacyclin, isoproterenol and forskolin, which increased [cAMP]i in HUVEC, and the cell-permeant cAMP analog 8-bromo-cAMP induced dose- and time-dependent secretion of tPA and vWF. The extent of vWF and tPA release correlated with [cAMP]i, and was increased by the phosphodiesterase inhibitor isobutylmethylxanthine.In contrast to thrombin, the cAMP-elevating agents did not increase the intracellular calcium concentration [Ca2+]i in HUVEC. At sub-maximal concentrations, the effects of thrombin and prostacyclin were additive.Our results show that an increase in [cAMP]i resulted in regulated secretion (acute release) of tPA and vWF from HUVEC, without the concomitant increase in [Ca2+]i which is, in HUVEC, essential for thrombin-induced regulated secretion to occur. cAMP-induced secretion represents a novel mechanism for causing regulated secretion of tPA and vWF from endothelial cells.

The synthesis, storage and regulated secretion (acute release) of tissue-type plasminogen activator (tPA) were studied in cultured rat heart endothelial (RHE) cells. In this study, special attention was paid to the correspondence between tPA metabolism in RHE cells in vitro and tPA metabolism in rats in vivo. RHE cells synthetized tPA, as shown by a spectrophotometric activity assay, by fibrin autography and by an ELISA for tPA antigen. The synthesis of tPA was strongly enhanced by phorbol myristate acetate, by all-trans-retinoic acid, by 8-bromoadenosine 3':5' cyclic- monophosphate (8-br-cAMP) and by compounds that enhance cAMP synthesis, such as cholera toxin and forskolin. Urokinase-type plasminogen activator and plasminogen activator inhibitor were not detected. tPA was constitutively secreted by RHE cells, but was also present in an intracellular pool of small dense granules, from which it could be acutely released by stimulating the cells with thrombin or the calcium ionophore A-23187. This release was maximal during the first minutes after stimulation. In all these aspects of tPA metabolism, RHE cells closely resembled rat endothelial cells in vivo. It is concluded that cultured RHE rat heart endothelial cells constitute a relevant in vitro model for studying endothelial tPA metabolism.

Abstract In this study, we investigated the role of Ca 2+ and G proteins in thrombin-induced acute release (regulated secretion) of tissue-type plasminogen activator (TPA) and von Willebrand factor (vWF), using a previously described system of primary human umbilical vein endothelial cells (HUVECs). The acute release of TPA and vWF, as induced by α-thrombin, was almost zero after chelation of Ca 2+ i , showing that an increase in [Ca 2+ ] i was required. It did not matter whether the increase in [Ca 2+ ] i came from an intracellular or extracellular Ca 2+ source. Thrombin-induced release of TPA and vWF already started at low [Ca 2+ ] i , around 100 nmol/L. Half-maximal release was found at a [Ca 2+ ] i of 261 nmol/L for TPA and at 222 nmol/L for vWF. The Ca 2+ signal was transduced to calmodulin, as calmodulin inhibitors inhibited TPA and vWF release. The Ca 2+ ionophore ionomycin dose dependently released vWF; half-maximal vWF release occurred at a [Ca 2+ ] i of 311 nmol/L. In contrast, no TPA release was found at all below a [Ca 2+ ] i of 500 nmol/L. Thus, below 500 nmol/L [Ca 2+ ] i , an increase in [Ca 2+ ] i alone was sufficient to induce vWF release but not sufficient to induce TPA release. Protein kinase C did not appear to be involved in TPA or vWF release, as neither an activator nor an inhibitor of protein kinase C significantly influenced release. Inhibition of phospholipase A 2 also did not reduce thrombin-induced TPA and vWF release. The involvement of G proteins was studied by using both saponin-permeabilized and intact cells. GDP-β-S, which inhibits heterotrimeric and small G proteins, significantly inhibited thrombin-induced vWF and TPA release from permeabilized cells. AlF 4 − , which activates heterotrimeric G proteins, induced TPA and vWF release in both intact and permeabilized HUVECs. Preincubation of HUVECs with pertussis toxin significantly inhibited thrombin-induced vWF release, due to inhibition of thrombin-induced Ca 2+ influx. Pertussis toxin did not affect ionomycin-induced release. The inhibitory effect of pertussis toxin was less obvious in thrombin-induced TPA release, because it was counterbalanced by a positive effect of the toxin on TPA release. Thus, both inhibitory and stimulatory (pertussis toxin–sensitive) G proteins were involved in TPA release. Therefore, thrombin-induced acute release of TPA and vWF differed in two respects. First, below a [Ca 2+ ] i of 500 nmol/L, an increase in Ca 2+ was sufficient for vWF release but not for TPA release. Second, pertussis toxin–sensitive G proteins were differentially involved in acute TPA and vWF release.

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.

The monocrotaline pyrrole (MCTP)-treated rat is a useful model for the study of certain chronic pulmonary vascular diseases. A single, i.v. administration of a low dose of MCTP causes pneumotoxicity, pulmonary vascular remodeling, sustained increases in pulmonary arterial pressure, and right ventricular hypertrophy in rats. The pulmonary vascular lesions are characterized by endothelial cell alterations, platelet and fibrin microvascular thrombosis, pulmonary edema, and thickening of the intimal and medial layers of the vessel wall. These lesions suggest that some dysfunction of the hemostatic system occurs in the lungs of rats treated with MCTP. We evaluated the concentrations of two adhesion proteins, cellular fibronectin (cFn) and von Willebrand factor (vWF), in the plasma of rats treated with MCTP. We hypothesized that changes in these factors occur along with markers of pneumotoxicity and ventricular hypertrophy and that such changes might contribute to the genesis of the vascular lesions. Enzyme-linked immunosorbent assays were used to measure cFn and vWF concentrations in the plasma of rats after MCTP treatment. Rats treated with a single, i.v. injection of 3.5 mg MCTP/kg body weight had delayed and progressive lung injury characterized at 5 days post-treatment by increases in the lung-to-body weight ratio and in lactate dehydrogenase activity and protein concentration in cell-free bronchoalveolar lavage fluid (BALF). Values for these markers were further increased at 8 days and reached a plateau thereafter. The number of nucleated cells within the BALF was increased at 8 and 14 days. Right ventricular hypertrophy, an indirect marker of pulmonary hypertension, was evident at 14 days. The cFn concentration was increased in plasma of rats at 8 and 14 days after treatment with MCTP. There was no difference between the vWF concentration in plasma of rats treated with MCTP and those treated with vehicle at any time. We conclude that an increase in plasma cFn concentration occurs prior to the onset of right ventricular hypertrophy and that this change is consistent with a role for cFn in the genesis of vascular remodeling and pulmonary hypertension in the MCTP-treated rat. The lung vascular injury and pulmonary hypertension in this model were not reflected in altered vWF concentration in the plasma.

The process of acute release of tissue-type plasminogen activator (tPA) is important in locally speeding up fibrinolysis. Using a sensitive enzyme-linked immunosorbent assay for tPA, we investigated the acute release of tPA from cultured human umbilical vein endothelial cells. The addition of thrombin (0.003 to 3 NIH U/mL) caused the dose-dependent release of noncomplexed, enzymatically active tPA into the medium. The amount of tPA released into the medium by thrombin was similar to the difference in the amounts of tPA present in extracts from thrombin-treated cells and control cells. The process of acute release of tPA was complete in 1 minute, whereas the concomitant release of von Willebrand factor into the medium was slightly slower (maximum after 3 minutes). By increasing (c.q. decreasing) tPA synthesis, it was found that the amount of tPA constitutively secreted, the amount acutely released, and the amount in cell extracts were increased (c.q. decreased) to the same extent. The same relation was found in vivo. When rats were pretreated with cholera toxin or retinoic acid to increase tPA synthesis, plasma levels of tPA were increased, whereas acute release of tPA, as induced by bradykinin, was increased to the same extent. Acutely released tPA and constitutively secreted tPA were liberated from different pathways in human umbilical vein endothelial cells; tPA had, relative to the in vivo situation, a short residence time in the acutely releasable pathway.

In 20 tissue samples from human brain tumours the concentrations were measured of (1) total plasminogen activator activity, (2) tissue-type plasminogen activator (t-PA) activity, (3) urokinase-type plasminogen activator (u-PA) activity, and (4) t-PA antigen. Most tumours contained a considerable amount of t-PA, but a high interindividual and in a few cases even an intra-individual variability was observed. A weak but significant negative correlation was found between t-PA concentration and the oedema/tumour ratio, as calculated from the preoperative computerized tomography (CT) brain scanning. No correlation was found with u-PA activity. It is concluded that t-PA and u-PA are probably not important factors in the production of peritumoral cerebral oedema, but a correlation between locally different amounts of t-PA or u-PA and the locally different extent of surrounding oedema has not yet been excluded.

Groups of five rats were fed for sixteen weeks a slightly deficient diet, supplemented with 0–40% of a dried preparation of the blue-green alga Spirulina as a protein source. Control groups were fed a normal rat diet. No significant differences between groups were found in food intake, growth rate or carbon dioxide production. All animals remained apparently healthy, and had similar organ weights. The study suggests taht Spirulina may be used as a protein source in rat diets.

Plasminogen activator (PA) and PA inhibitor (PAI) antigen, activity, and mRNA were analyzed in the three layers of rat aorta, and the effect of endotoxin on PA and PAI was studied. All PA activity in aorta was identified as tissue-type PA (TPA) activity; no urokinase-type PA was detected. In the tunica adventitia TPA activity, TPA antigen, and TPA mRNA were detected, whereas in the tunica media TPA antigen and TPA mRNA, but no TPA activity, were found. PAI activity was detected in the tunica media, explaining the absence of TPA activity in this layer. Removal of the endothelial cells had no effect on TPA antigen and PAI activity in intima-media preparations. Also, similar amounts of PAI-1 mRNA were found in intima-media preparations, irrespective of the presence or absence of the intima. Immunohistochemical staining showed that TPA immunoreactivity was present in all three layers of the aorta, whereas PAI-1 immunoreactivity was found in medial smooth muscle cells but not in endothelial cells. After endotoxin treatment, TPA activity was decreased in extracts of the total aorta and of the adventitia, although TPA antigen and TPA mRNA were unchanged. PAI-1 mRNA was strongly increased in the tunica adventitia and in the tunica media, as was PAI activity in the tunica media. Thus, endotoxin decreased TPA activity by increasing the synthesis of PAI-1; TPA was unaffected. Our observations in rat aorta differ from observations in mouse aorta and in rat carotid artery, and they caution against extrapolation from one tissue (or species) to another. Chemicals/CAS: plasminogen activator inhibitor, 105844-41-5; tissue plasminogen activator, 105913-11-9; urokinase, 139639-24-0; Endotoxins; Plasminogen Activator Inhibitor 1; RNA, Messenger; Tissue Plasminogen Activator, EC 3.4.21.68

In this study the effect of vitamin A status and retinoid treatment on the activity of tissue-type plasminogen activator (TPA) and its inhibitor, plasminogen activator inhibitor-type 1 (PAI-1), in plasma and in several tissues was investigated in BN/BiRij rats. Hypervitaminosis A and retinoic acid treatment increased plasma TPA activity by approximately 50%, but PAI-1 activity was not affected. The effect of retinyl palmitate treatment on plasma TPA activity was already significant after 5 days and continued for at least up to 8 wk. Isotretinoin treatment affected neither plasma TPA nor PAI-1 activity. In plasma of vitamin A-deficient rats, TPA activity was decreased by a factor of three, whereas PAI-1 activity was increased twofold. Modulation of plasma TPA activity by vitamin A status and retinoic acid treatment was associated with similar changes in tissue TPA activity. This suggests that the changes in plasma TPA activities might be the result of changes in tissue TPA synthesis.

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 μM) 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 N(G)-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. Chemicals/CAS: 8 bromo cyclic AMP, 23583-48-4; adenylate cyclase, 9012-42-4; atrial natriuretic factor, 85637-73-6; bradykinin, 58-82-2, 5979-11-3; forskolin, 66575-29-9; guanylate cyclase, 9054-75-5; n(g) nitroarginine, 2149-70-4; nitric oxide, 10102-43-9; nitroprusside sodium, 14402-89-2, 15078-28-1; thrombocyte activating factor, 64176-80-3, 65154-06-5; tissue plasminogen activator, 105913-11-9; von Willebrand factor, 109319-16-6; 8-Bromo Cyclic Adenosine Monophosphate, 23583-48-4; 8-bromocyclic GMP, 31356-94-2; Adenylate Cyclase, EC 4.6.1.1; Atrial Natriuretic Factor, 85637-73-6; Bradykinin, 58-82-2; Cyclic AMP, 60-92-4; Cyclic GMP, 7665-99-8; Forskolin, 66428-89-5; Guanylate Cyclase, EC 4.6.1.2; Nitroprusside, 15078-28-1; Platelet Activating Factor; Tissue Plasminogen Activator, EC 3.4.21.68; von Willebrand Factor

Using a perfused rat hindleg system, release of tissue-type plasminogen activator (t-PA) from endothelial cells could be induced by platelet-activating factor (PAF), bradykinin, substance P, thrombin, carbachol and A23187, while this release was inhibited by mepacrine and by nor-dihydroguaiaretic acid. The PAF-induced release of t-PA was inhibited by the cytochrome P-450 mono-oxygenase inhibitors, metyrapone, ketoconazole and SKF 525A and by eicosatetraynoic acid but not by indomethacin or BW 755C, suggesting the involvement of epoxygenase products. The PAF-induced release of von Willebrand factor (vWF) was also similarly inhibited by the cytochrome P-450 monooxygenase inhibitor, ketoconazole. Phorbol ester and phospholipase C induced the release of both t-PA and vWF, while phospholipase A2 did not. The release induced by PAF and bradykinin was not influenced by pretreatment with pertussis toxin.

Tissue-type plasminogen activator (t-PA) and its inhibitor, plasminogen activator inhibitor 1 (PAI-1), play an important role in regulating the fibrinolytic capacity of plasma. Both t-PA and PAI-1 are synthesized by the endothelium. We report that retinoic acid (vitamin A acid) and other retinoids rather specifically stimulate the production of t-PA by cultured human umbilical vein endothelial cells. Effective retinoids induced a dose-dependent (range: 0.01-50 μM) increase in the production of t-PA of maximally about six-fold, while simultaneously causing no or only a small increase (less than two-fold) of PAI-1. The effects on t-PA synthesis were apparent by 4-8 h, and reached maximal values after about 24-48 h of incubation with retinoid. The retinoid effect on t-PA production was accompanied by increased t-PA mRNA levels, without any parallel change in PAI-1 or GAPDH mRNA concentrations. The study also shows that modifications at the carboxyl group of retinoic acid are associated with a decrease in stimulatory potency. The stimulatory pathway appears to be identical for all retinoids but distinct from a pathway by which another strong inducer, sodium butyrate, induced t-PA synthesis in endothelial cells. The induction of t-PA by retinoids might involve protein kinase C (PKC) as judged by an experiment using a specific PKC inhibitor. The effect of retinoids on the fibrinolytic system in vivo was assessed by feeding rats with a vitamin A deficient diet or a diet with excess of vitamin A or other retinoids. The activity and antigen levels for t-PA in plasma and tissue samples were strongly decreased in vitamin A-starved animals, and enhanced in retinoid-fed animals. The recognition of a class of compounds, the retinoids, that can increase t-PA synthesis in cultured human endothelial cells and in rat tissues in vivo, may be of importance in designing an effective method to enhance plasma fibrinolytic activity, and thereby in preventing thrombotic phenomena. Chemicals/CAS: isotretinoin, 4759-48-2; plasminogen activator, 9039-53-6; retinol, 68-26-8, 82445-97-4; Butyric Acid, 107-92-6; Butyric Acids; DNA Probes; Plasminogen Inactivators; Protein Kinase C, EC 2.7.1.37; Retinoids; Tissue Extracts; Tissue Plasminogen Activator, EC 3.4.21.68

The tissue-specific distribution of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA) and their inhibitor type 1 (PAI-1) was analyzed at mRNA level in five major rat organ tissues. t-PA mRNA was detected in lung, kidney, heart, and liver. u-PA mRNA was detected in kidney and lung. Presence of PA mRNA correlated with the detection of PA activity in extracts of these tissues. PAI-1 mRNA was detected predominantly in heart and lung. Although PAI activity could not be measured directly in tissue extracts, the presence of PAI-1 mRNA correlated with the occurrence of PA·PAI complex in fibrin autography of tissue extracts. Endotoxin injection caused a very large increase in plasma PAI activity. This increase correlated with a marked increase in PAI-1 mRNA in nearly all tissues studied. The increase in PAI-1 mRNA is most pronounced in lung and liver. Endotoxin injection also caused an increased level of t-PA mRNA in heart and kidney, and an increased u-PA mRNA level in kidney. mRNA analysis of freshly isolated and separated subfractionated liver cells showed that the marked increase in PAI-1 mRNA in the liver after endotoxin injection may be due mainly to a strong increase of PAI-1 mRNA in the liver endothelial cells.

Problems involved in studying the effect on fibrinolysis of dietary intervention are briefly discussed, emphasizing the importance of follow-up studies to delineate possible mechanisms involved.