Structure and functions of the endothelium

The vascular endothelium is a dynamic interface between blood and tissues, which releases vasoconstrictors or vasodilators regulating the vascular tone. The endothelium modulates the balance between thrombosis and haemorrhage. Activated endothelium may produce tissue factor which triggers the coagulation cascade. In different tissues, the endothelial cells become specialised and may participate to the immune response and inflammation. Various metabolic or immune stimuli may alter endothelial cell functions, induce leukocyte adhesion through expression of specialised molecules and modify the release of fibrinolytic agents, cytokines, and growth factors.     

Tissue factor expression by endothelial cells in sickle cell anemia

The role of the vascular endothelium in activation of the coagulation system, a fundamental homeostatic mechanism of mammalian biology, is uncertain because there is little evidence indicating that endothelial cells in vivo express tissue factor (TF), the system's triggering mechanism. As a surrogate for vessel wall endothelium, we examined circulating endothelial cells (CEC) from normals and patients with sickle cell anemia, a disease associated with activation of coagulation. We find that sickle CEC abnormally express TF antigen (expressed as percent CEC that are TF-positive), with 66+/-13% positive in sickle patients in steady-state, 83+/-19% positive in sickle patients presenting with acute vasoocclusive episodes, and only 10+/-13% positive in normal controls. Repeated samplings confirmed this impression that TF expression is greater when sickle patients develop acute vasoocclusive episodes. Sickle CEC are also positive for TF mRNA, with excellent concurrence between antigen and mRNA expression. The TF expressed on the antigen-positive CEC is functional, as demonstrated by a binding assay for Factor VIIa and a chromogenic assay sensitive to generation of Factor Xa. By establishing that endothelial cells in vivo can express TF, these data imply that the vast endothelial surface area does provide an important pathophysiologic trigger for coagulation activation.     

Modulation of [3H]dopamine release by neuropeptide Y in rat striatal slices

Polycythemia vera (PV) is associated with a high incidence of thrombosis. The association of apparent and secondary polycythemia with thrombosis is not clear. It was suggested that activation of the coagulation system contributes to thrombus formation in PV. However, the mechanism of activation is unknown. Monocytes generate a potent tissue factor (TF) upon stimulation with various substances, which is involved in thrombus formation in various disorders. Therefore, we studied the possibility that the factor is involved in the activation of coagulation and thrombus formation also in PV. Unstimulated peripheral blood mononuclear cells (PBMC) from each of the different types of polycythemia expressed weak TF activity (2 U) and antigen (41.4 to 52.9 pg/ml), which were similar to normal controls. Following stimulation with endotoxin, PBMC from normal controls and from apparent and secondary polycythemia showed a 3.9- to 4.5-fold increase in TF, while cells from PV showed a 21-fold increase (P<0.001). Similar levels were generated by PBMC after treatment of PV and at the spent phase. TF was generated by monocytes but not by lymphocytes. Plasma prothrombin fragment1+2 (F1+2) levels, assayed at the same time, were significantly higher in PV (2.46 nm) compared to normals and apparent and secondary polycythemia (0.22 to 0.32 nm), and were in a significant correlation with monocyte TF activity and antigen levels (r = 0.77, 0.87). The high levels of F1+2 confirm that the coagulation system is activated in PV. The increased capacity of monocytes to generate TF may be responsible for the activation of the coagulation system and thrombus formation. The hypercoagulability state that is induced by this mechanism suggests that long-life oral anticoagulation should be considered once thrombosis has been developed in PV.     

No evidence for direct incorporation of esterified palmitic acid from plasma into brain lipids of awake adult rat

Thrombin is a multifunctional enzyme involved in coagulation, cell modulation and inflammation. We recently reported a novel membrane-associated prothrombin activator, abbreviated as MAPA, found in cultured fibroblasts and glial cell lines. In this study, we examined the physiological role of this enzyme. MAPA-like activity was detected in the liver, kidney, lung and heart but not in the spleen or brain in normal mice. To examine whether MAPA participates in biological reactions, hepatic and renal injury were induced by administration of CCl4 and HgCl2, respectively. MAPA-like activity was specifically increased in the injured tissues: the activity was elevated by about 100-fold in 48 h in the liver and increased by about 5-fold in 12 h in the kidney. Their enzymatic properties were the same as those of MAPA in 8C feline kidney fibroblast cells. Phospholipids are required for activation of prothrombin by MAPA obtained from both 8C cells and tissues. These results suggest that MAPA activates prothrombin on the cell surface in injured tissue and participates in inflammation and regeneration associated with tissue injury.     

Rapid mechanotransduction in situ at the luminal cell surface of vascular endothelium and its caveolae

The vascular endothelium is uniquely positioned between the blood and tissue compartments to receive directly the fluid forces generated by the blood flowing through the vasculature. These forces invoke specific responses within endothelial cells and serve to modulate their intrinsic structure and function. The mechanisms by which hemodynamic forces are detected and converted by endothelia into a sequence of biological and even pathological responses are presently unknown. By purifying and subfractionating the luminal endothelial cell plasma membrane from tissue, we show, for the first time, that not only does mechanotransduction occur at the endothelial cell surface directly exposed to vascular flow in vivo but also increased flow in situ induces rapid tyrosine phosphorylation of luminal endothelial cell surface proteins located primarily in the plasmalemmal invaginations called caveolae. Increased flow induces the translocation of signaling molecules primarily to caveolae, ultimately activating the Ras-Raf-mitogen-activated protein kinase pathway. This signaling appears to require intact caveolae. Filipin-induced disassembly of caveolae inhibits both proximal signaling events at the cell surface and downstream activation of the mitogen-activated protein kinase pathway. With the molecular machinery required for mediating rapid flow-induced responses as seen in endothelium, caveolae may be flow-sensing organelles converting mechanical stimuli into chemical signals transmitted into the cell.     

Intra-articular glucocorticoid injections in joint diseases

Intra-articular glucocorticosteroid injections are widely used in mono- or oligoarticular flares of patients with rheumatoid arthritis and other aseptic inflammatory joint diseases, as well as in osteoarthritis. Rapid and pronounced, but usually temporary, suppression of local joint inflammation may be achieved with only minor systemic effect. In osteoarthritis the effect is brief and transient. Triamcinolone hexacetonide provides the longest clinical effect, but since this drug may cause local tissue necrosis when injected outside a synovial cavity it should be used only by experienced clinicians. The risk of glucocorticoid-induced cartilage damage is discussed. The risk is probably less than that of untreated joint inflammation. Nevertheless, it is recommended that injections into the same joint are limited, for instance to one injection every six weeks and no more than three or four in one year. Furthermore, indications and contraindications should be carefully considered prior to each injection. Intra-articular glucocorticoid therapy may be of considerable clinical value in the management of aseptic arthritis, if administered on correct indications using a correct technique.     

Role of tissue factor in adhesion of mononuclear phagocytes to and trafficking through endothelium in vitro

An in vitro model consisting of endothelium grown on collagen was used to investigate how mononuclear phagocytes traverse endothelium in the basal-to-apical direction (reverse transmigration), a process that mimics their migration across vascular and/or lymphatic endothelium during atherosclerosis and resolution of inflammation, respectively. Monoclonal antibody (MoAb) VIC7 against tissue factor (TF) inhibited reverse transmigration by 77%. Recombinant tissue factor fragments containing at least six amino acids C-terminal to residue 202 also strongly inhibited reverse transmigration. TF was absent on resting monocytes but was induced on these cells after initial apical-to-basal transendothelial migration. Two additional observations suggest that TF is involved in adhesion between mononuclear phagocytes and endothelium: (1) when monocytes were incubated with lipopolysaccharide (LPS) to stimulate expression of TF before they were added to endothelium, VIC7 or soluble TF modestly inhibited their adhesion to the apical endothelial surface, each by about 35%; and (2) endothelial cells specifically bound to surfaces coated with TF fragments containing amino acids 202-219. This binding was blocked by anti-TF MoAb, suggesting that endothelial cells bear a receptor for TF. These data suggest that mononuclear phagocytes use TF, perhaps as an adhesive protein, to exit sites of inflammation.     

Platelet functions and haemostasis parameters in pigs: absence of side effects of a procedure of general anaesthesia

Pigs are largely used as experimental animal models of thrombosis and for testing the anti thrombotic drug efficacy. Generally experiments are performed on pigs under general anaesthesia and observations can be affected by the anaesthetic drugs used. The effects of a general anaesthetic procedure were checked on pig haemostasis parameters; the pig was pre-anaesthetized with ketamine chloride, then intubated and ventilated with a mixture containing halothane, nitrous oxide and oxygen. Bleeding time, platelet aggregations, coagulation factors, coagulation inhibitors, fibrinolysis parameters and markers of activation of coagulation were determined on 30 Large White pigs before and under this anaesthesia procedure. Compared to human coagulation, pig is characterized by very high levels of factor V, VIII, IX, XI, XII activities, same levels of factor II, fibrinogen, antithrombin III (ATIII), low levels of protein C activities. Thrombin-antithrombin complex (TAT) and tissue plasminogen activator antigen (tPA) values were dispersed. With the reagents used, protein S, prothrombin fragment 1 + 2 (F1 + 2), D Dimers (D-D), plasminogen activator inhibitor (PAi) levels could not be determined. No difference was observed between results obtained before and under anaesthesia, particularly to increase of bleeding time, no modification of platelet aggregations and no activation of coagulation. This anaesthetic procedure does not induce any modification of pig haemostasis and can be used, without side effects, for experimental thrombosis studies in pigs.     

Endothelial cells in culture: an experimental model for the study of vascular dysfunctions

Culture of endothelial cells started two decades ago and is now a useful tool in understanding endothelial physiology and the study of the interaction of endothelial cells with blood cells and various mediators. In vitro proliferation can be measured by [3H]thymidine incorporation in defined conditions and gives reproducible results. Endothelial cells can be activated by several stimuli, including cytokines such as tumor necrosis factor-alpha and interleukin-1. Part of endothelial cell activation is defined by expression or overexpression of leukocyte adhesion molecules. Intracellular adhesion molecule (ICAM), E-selection and vascular adhesion molecule (VCAM) are receptor molecules for leukocyte adhesion. Leukocyte adhesion to endothelium can be measured in static but also in rheologically defined flow conditions. Normal red blood cells (RBCs) do not adhere to endothelium, while RBC from patients with sickle cell anemia, diabetes mellitus, and malaria have an increased adhesion to endothelium which is mediated by specific VCAM, receptor for advanced glycated end-products (RAGE), and ICAM, respectively. Binding of blood cells or activation by cytokine is followed by a series of reactions in endothelial cells associated with the modulation of prostacyclin, nitric oxide, tissue factor, and cytokine production. Modification of endothelial cell functions in culture is correlated to in vivo alteration of vascular wall properties, further supporting these cells in culture as a relevant experimental model.     

Case management: a literature review

There is considerable evidence that the hemostatic system is involved in the growth and spread of malignant disease. There is an increased incidence of thromboembolic disease in patients with cancers and hemostatic abnormalities are extremely common in such patients. Antihemostatic agents have been successfully used to treat a variety of experimental tumors, and several clinical trials in humans have been initiated. Although metastasis is undoubtedly multifactorial, intravascular coagulation activation and peritumor fibrin deposition seem to be important. The mechanisms by which hemostatic activation facilitates the malignant process remain to be completely elucidated. Of central importance may be the presence on malignant cells of tissue factor and urokinase receptor. Recent studies have suggested that these proteins, and others, may be involved at several stages of metastasis, including the key event of neovascularization. Tissue factor, the principal initiator of coagulation, may have additional roles, outside of fibrin formation, that are central to the biology of some solid tumors.     

Quantitative studies of the pathway to acute carrageenan inflammation

A 12-step scheme representing initiation and development of acute carrageenan inflammation in the rat has been devised. Simultaneous quantitative temporal measurement of the number of inflammatory cells mobilized and edema volume produced as carrageenan pleurisy developed helped elucidate several of the early steps, In the pleurisy a 20-min lag phase preceded both the mobilization of neutrophils and edema formation. Temporal histological studies suggested that the lag phase represents the time needed for the neutrophil chemotactic factor to be generated and/or released (steps 2 and 3) in addition to the time required for these cells to move through the walls of the capillaries into the pleural space (steps 4 and 5). The neutrophil chemotactic factor is unknown. The complement system is not involved since cobra venom factor-treated animals produced a normal edematous and cellular response to carrageenan in spite of severely depressed complement levels. The mobilized neutrophils are believed to phagocytize the carrageenan(step6). In this process lysosomal enzymes are released (step 7). Drug inhibition studies suggest that lysosomal enzymes are not the edemagenic agents in carrageenan inflammation. In the scheme these enzymes are responsible for activation of the prostaglandin biosynthetic chain (step 8). Prostaglandin biosynthesis (step 9) leads to the release of an intermediate (step 10), as yet unidentified, which is responsible for increased tissue permeability (step 11) and edema formation (step 12). Histamine, serotonin, bradykinin, arachidonic acid, and prostaglandin's E1 and E2 are not deemagenic in the rat pleural cavity and therefore cannot be responsible for edema formation. Aspirin and indomethacin reduce the edema produced by carrageenan in a dose-related manner without affecting the magnitude or the time course of neutrophil mobilization. These findings led to the concept that edema formation is not the result of inflammatory cell mobilization but is rather a consequence of cellular activity, presumably phagocytosis, after mobilization.     

Embryonal histogenesis of endothelium

Development of the mammalian vascular system is characterized by recapitulation of many morpho-functional signs peculiar to certain ancestral forms, as well as by certain aromorphous reorganizations reflecting in histogenesis of endothelium. Prior to the period of the common blood circulation in the mammalian embryos, the endothelium is developing within the endomesenchyme constructed by cells of a peculiar - endothelial - germ, but not of the endomesenchymal cells, as numerous investigators consider. The endothelium is developing as a capacitance tissue which presents by itself an epitheliomorphous duplex selective membrane, functionally connected with blood, tissue fluid and lymph in order metabolism and other functions of the organism's cells and tissues can be performed. Increase in cell number and their proendothelial derivatives at pretissue, tissue and hemoendothelial levels and successive union of recapitularities with the same name at the formation of the endothelial vessels add certain essential features to histogenesis of the endothelium and to its normal reactions. Polymerization and union of the recapitularities with the same name are the ways of integration; they are important ingredients of hereditarily determined differentiation. The latter is realized in the development of hemoendothelial offsprings which appear after the common blood circulation is started. The endothelial genesis in its capacitant form is complemented with blood cells incorporated, the means which is subjected to certain essential evolutional reorganizations. In its turn, this results in appearance of the double tissue hemoendothelial complex possessing features of an elementary organ.     

The role of the host defence response in the progression and outcome of ARDS: pathophysiological correlations and response to glucocorticoid treatment

The host defence response (HDR) to insults is similar regardless of the tissue involved and consists of an interactive network of simultaneously activated pathways that act in synergy to increase the host's chance of survival. Among this cascade of integrated pathways, three aspects of the HDR, inflammation, coagulation and tissue repair, are analysed separately to explain the histological and physiological changes occurring at the tissue level in unresolving acute respiratory distress syndrome (ARDS). Cellular responses in HDR are regulated by a complex interaction among cytokines, and cytokines have concentration-dependent biological effects. The degree of initial HDR may determine the progression of ARDS. On Day 1 of mechanical ventilation and over time, nonsurvivors of ARDS have significantly higher plasma and bronchoalveolar lavage inflammatory cytokine levels than survivors. In the absence of inhibitory signals, the continued production of HDR mediators prevents effective restoration of lung anatomy and function by sustaining inflammation with tissue injury, intra- and extravascular coagulation and proliferation of mesenchymal cells (fibroproliferation) with deposition of extracellular matrix resulting in fibrosis. Glucocorticoids inhibit the HDR cascade at virtually all levels; their gradual and generalized suppressive influence protects the host from overshooting. In patients with exaggerated HDR, however, cytokine elevation may cause a concentration-dependent resistance to glucocorticoids by reducing glucocorticoid receptor binding affinity. Recent clinical and experimental studies have shown that effective containment of the HDR in unresolving ARDS may be achieved only if glucocorticoid administration is prolonged. A double-blind randomized study is in progress to evaluate the role of prolonged glucocorticoid treatment in unresolving ARDS.     

Complex regulation of granulocyte adhesion to cytokine-activated endothelium

The adhesion of leukocytes to the endothelium is a hallmark in the development of an inflammation. Adhesion is caused by a number of mechanisms that depend upon the activation of the endothelium. The adhesion has to be specific for the different leukocyte types. I review here recent data with respect to polymorphonuclear granulocytes (PMN) adhesion to the endothelium of an inflammatory site. I especially focus on the pivotal role of Interferon gamma in the regulation of PMN adhesion to the endothelium. It modulates the adhesion of PMN caused by activation by other cytokines like IL-1, but does not affect the cell surface expression of the known adhesion molecules. I postulate the existence of a new class of cell surface modulators of adhesion which participate in the multiple step process of cell-cell adhesion.     

Carrier screening for cystic fibrosis: costs and clinical outcomes

Generally, interferon-gamma (IFN-gamma) is considered a critical regulator of T cell mediated inflammation. For this reason, we investigated the pathogenesis of lymphocytic choriomeningitis in mice with a targeted defect of the gene encoding this cytokine. Our results revealed that IFN-gamma is redundant in the afferent phase of the antiviral T cell response as well as a local mediator of this T cell mediated inflammatory disease. However, IFN-gamma may play an indirect role as it is involved in reducing extraneural infection that may compete with CNS for available effector cells. Analysis of the inflammatory exudate disclosed that leucocyte recruitment was unimpaired in the absence of IFN-gamma as was the upregulation of ICAM-1 and VCAM-1 on endothelium at the inflammatory site. However, local macrophage activation (production of tumor necrosis-alpha and NO) was significantly impaired. Notably, a viral peptide could also elicit a T cell mediated inflammatory response in virus-primed IFN-gamma knock-out mice, indicating that redundancy of this cytokine as a proinflammatory mediator is not restricted to inflammatory reactions triggered by an active infection. Thus, T cell mediated inflammation may be induced in the absence of IFN-gamma and local macrophage activation.     

Monocyte procoagulant activity induced by adherence to an artificial surface is reduced by end-point immobilized heparin-coating of the surface

Heparin-coating improves the biocompatibility of blood contacting artificial surfaces. This led us to investigate the impact of heparin-coating (Carmeda AB, Stockholm) of polymetylmetacrylate on the expression of monocyte tissue factor procoagulant activity (TF-PCA) by surface adhesion. Also, the anticoagulant effect of heparin-coating in the presence or absence of adherent procoagulant monocytes was assessed. This is of particular interest, since activation of extrinsic coagulation by adherent monocyte TF-PCA may play a significant role in thrombin generation during extracorporeal circulation. Monocytes exposed to heparin-coated or non-coated polymetylmetacrylate expressed TF-PCA. The heparin coat did not affect the rate of monocyte adhesion. However, heparin-coating reduced the induction of TF-PCA of non-adherent and adherent monocytes by 17 and 33% (p <0.001 and p <0.0003), respectively. Heparin-coating in the absence of monocytes, totally inhibited the clotting of recalcified plasma (p <0.003). In contrast, in the presence of adherent monocytes expressing TF-PCA, surface-bound heparin did not inhibit clotting. However, inclusion of heparin in a plasma concentration of 8.9 IU/ml totally inhibited the activation of coagulation. It is apparent that heparin-coating of an artificial surface is an efficient means to inhibit coagulation of recalcified plasma, but much less so when procoagulant monocytes are adherent to the coated surface. The present findings are of clinical relevance, since monocytes will adhere to blood contacting surfaces of extracorporeal circuits or to implanted vascular prostheses and subsequently express TF-PCA, and this may promote thromboembolism.     

Role of the endothelium in atherosclerosis

Endothelial injury has long been considered as a prerequisite for atherosclerotic plaque formation. Experimental and clinical studies have proven that during lesion development the endothelium is morphologically intact, but its phenotype is changed to an activated state, which favours the chronic inflammatory process leading to atherosclerotic disease. Endothelium is involved in atherosclerosis in several manners including: 1. subendothelial accumulation of atherogenic lipoproteins, through the barrier properties of the endothelium; 2. intimal accumulation of macrophages, through recruitment and adhesion of monocytes, when endothelial cells are activated; 3, vasospasm, when its vasodilatory properties are altered; 4, thrombus formation, when the endothelium is eroded.     

Venocclusive disease of the liver after bone marrow transplantation: the role of hemostasis

Venocclusive disease of the liver is a relatively frequent early complication of bone marrow transplantation related to pre-transplant toxic injury to the liver. Events that lead to toxicity of the liver in the pre-transplant setting are infection, anti-neoplastic and anti-infectious drug administration and radiation. The histological correlates of venocclusive disease are lesions mainly localized to structures in zone 3 of the liver acinus and in the sublobular central venules. At some point in the pathogenesis of venocclusive disease, blood clotting and inflammation occur. The first is characterized by laboratory signs of coagulation activation, by an increase in several procoagulant proteins and by a decrease in naturally occurring anticoagulants, particularly protein C, the latter being a sensitive index of liver injury. Inflammation is mediated by cytokine production, which maintains procoagulant endothelial responses and liver injury. Severe venocclusive disease is associated with multi-organ failure and elevated mortality. Attempts at finding predictive markers of the disease have succeeded in identifying some coagulation and inflammatory proteins which can be useful in predicting the occurrence of VOD in selected patient groups. The role of hemostasis in venocclusive disease is underscored also by the reports on the successful prophylaxis and management of the disease with heparin and thrombolytic agents.     

Stimulation of monocyte tissue factor expression in an in vitro model of bacterial endocarditis

The coagulation system plays a major role in the formation of the infected endocardial vegetation in bacterial endocarditis. Since monocytes can express tissue factor (TF) on their surfaces, they are thought to be responsible for the extrinsic activation of the coagulation cascade during this disease. The present study used an in vitro model in which fibrin plates, isolated adherent monocytes, and Streptococcus sanguis were used as an analog for endocardial vegetations. Adherence to fibrin by itself was found to stimulate TF expression on the monocytes, but stimulation by S. sanguis significantly increased TF expression, which was found to be maximal at a bacterium-to-monocyte ratio of 9 or more.     

Pregnancy induced hypertension

Hypertension remains a leading cause of perinatal morbidity and mortality. Classification of the hypertensive disorders of pregnancy is 1) preeclampsia-eclampsia, 2) chronic hypertension, 3) chronic hypertension with superimposed preeclampsia-eclampsia. Preeclampsia is characterized by the triad of hypertension, proteinuria, and edema but these findings are not specific. Although the etiology and pathogenesis of preeclampsia remain unknown, several factors such as abnormalities in prostaglandin systems, in coagulation process, derangements of the endothelium and so on. Management of preeclampsia is bed rest, aspirin administration, antihypertensive agents (beta-blockers, hydralazine, alpha-methyldopa) would be used for reduction of blood pressure.