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Biomedical subjects

M C Alessi

Publications and source records attributed to M C Alessi.

At least 91 records · Page 5Linked to original sources
PubMed1992

Endothelial cell dysfunction in HIV infection.

We have investigated plasma levels of endothelial cell products playing a role in hemostasis in 125 HIV-positive patients and 30 controls. Antigenic von Willebrand factor increased significantly with disease progression and was closely correlated with CD4+ cell counts and beta 2-microglobulin levels. Tissue-type plasminogen activator was normal in CDC II/III and CDC IVC2 patients and was slightly increased in AIDS patients, whereas plasminogen activator inhibitor was increased in each group, the stage of the disease not having any effect. Mean total protein S levels were lower in HIV-positive patients and, in 27.2% of the cases, were associated with a decrease in free protein S levels. Such abnormalities could be responsible for a hypercoagulable state in these patients and could be explained by endothelial cell damage during HIV infection. Whether this injury is due to HIV itself remains to be further investigated.

Adult↗
PubMed1991

Characterization of epitheloid cells from human omentum: comparison with endothelial cells from umbilical veins.

Capillary cells represent 95% of the vascular bed, and cells from large and micro-vessels do not express identical functions. In order to study the hormonal regulation of plasminogen activator inhibitor 1 (PAI-1) secretion by human capillary cells we used epithelial cells from omental tissue (HOTMEC). As their endothelial origin is subject to controversy, we attempted to determine their characteristics by comparing them to human umbilical vein endothelial cells (HUVEC). Morphological and biological criteria were studied. By phase contrast microscopy HOTMEC elicited a cobblestone pattern similar to HUVEC. Weibel-Palade bodies were not found in the cytoplasm with electron microscopy. Fluorescence microscopy studies indicated that HOTMEC took up acetylated-LDL more intensely than HUVEC, and showed no staining for von Willebrand factor. The phenotype of HOTMEC was studied by flow cytometry using monoclonal antibodies (mo Ab) directed against epitopes either specific for endothelial cells or for mesothelial cells. We showed that in our preparations only 10% of cells reacted with mo Ab specific for endothelial cells. About 60% of the HOTMEC were labelled with an antibody directed against mesothelial cells. HOTMEC expressed fibrinolytic factors. Tissue plasminogen activator (t-PA) levels in HOTMEC conditioned medium were 50 fold higher than those of HUVEC, and the PAI-1 secretions were identical in both cell types. Insulin which is known to increase PAI-1 synthesis by hepatocytes did not enhance the PAI-1 level either in HOTMEC or in HUVEC conditioned media. Our results suggested that morphological and functional methods did not allow discrimination between the cell types present in the omentum tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1991

Molecular forms of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA) in human plasma.

Molecular forms of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA), identified by gel filtration and specific immunoassays, were studied in plasma from subjects with normal and elevated PAI-1 levels before and after in vitro or in vivo addition of t-PA. In normal plasma, PAI-1 occurs in three molecular forms, a Mr greater than 700 KDa inactive form of heterogeneous composition, an active 450 KDa form containing PAI-1/vitronectin complex and an inactive peak at Mr 50 KDa containing free PAI-1. Stimulation of platelets results in a significant increase of the 50 KDA form and a slight increase of the 450 KDa form. Patients with increased PAI activity levels have an increase of both the 450 KDa and the 50 KDa forms, whereas patients with thrombotic thrombocytopenic purpura have an increased 50 KDa form. In normal plasma, collected in the presence or absence of D-Phe-Pro-Arg-CH2Cl, t-PA occurs primarily as a Mr greater than 700 KDa form containing t-PA/PAI-1 complex. Addition of high concentrations of t-PA (70 ng/ml) to plasma in vitro or t-PA injection in vivo, results in t-PA inhibitor complexes, including t-PA/ alpha 2 antiplasmin. It is concluded that in subjects with increased PAI-1 levels in plasma, PAI-1 may occur as high molecular weight complexes with vitronectin of which 450 KDa was the most important part and as a 50 KDa inactive form; t-PA circulates primarily in complex with inhibitors. Thus, some of the molecular interactions between PAI-1, t-PA and vitronectin, previously demonstrated in purified systems in vitro, also occur in plasma.

Amino Acid Sequence↗
PubMed1991

Increased plasma plasminogen activator inhibitor 1 levels. A possible link between insulin resistance and atherothrombosis.

According to recent prospective studies, hypofibrinolysis due to elevated plasma plasminogen activator inhibitor 1 levels appears to be an independent risk factor for myocardial reinfarction in men, and hyperinsulinaemia, a major indicator of insulin resistance is considered as a risk factor for coronary disease. It has recently been shown that insulin resistance is accompanied by an increased plasma plasminogen activator inhibitor 1 concentration: A significant correlation coefficient was demonstrated between plasminogen activator inhibitor 1 and fasting plasma insulin in the normal population, in obese subjects, in Type 2 (non-insulin-dependent) diabetic patients and in angina pectoris. Attempts to decrease insulin resistance such as fasting, diet, or administration of an oral anti-diabetic drug such as Metformin induced a parallel decrease in plasma insulin and plasminogen activator inhibitor 1 levels. This inhibitor is produced by endothelial cells and by hepatocytes in culture. Plasminogen activator inhibitor 1 synthesis by hepatocytes in culture was stimulated by an increasing insulin concentration, or low density lipoproteins, whereas the endothelial cell synthesis was stimulated by very low density lipoproteins especially when they were obtained from hypertriglyceridaemic patients. Therefore, a direct effect of insulin or lipoprotein changes on the cells which synthesize plasminogen activator inhibitor 1 could be responsible for its increased plasma concentration in insulin resistance states. The increase in plasma plasminogen activator inhibitor 1 levels linked to hyperinsulinaemia is a tempting partial explanation for the association between insulin resistance and coronary disease.

Arteriosclerosis↗
PubMed1990

The increased plasma Lp(a): B lipoprotein particle concentration in angina pectoris is not associated with hypofibrinolysis.

The structural homology between plasminogen and apolipoprotein (a), the specific glycoprotein of Lp(a) lipoprotein, raises the possibility of a relationship between this lipoprotein and the plasma fibrinolytic system. The present study examines this proposal by studying 66 patients with angina pectoris. As compared to normal controls, the patients had raised concentrations of Lp(a): B lipoprotein particles. No correlation was found between circulating Lp(a): B and the fibrinolytic system. The pathogenic role of Lp(a): B lipoprotein seems therefore not mediated by its effect on the plasma fibrinolytic system.

Aged↗
PubMed1990

Secretion of tissue-type plasminogen activator and plasminogen activator inhibitor by Rickettsia conorii- and Rickettsia rickettsii-infected cultured endothelial cells.

Hemostasis abnormalities have been described in patients with Mediterranean spotted fever and Rocky Mountain spotted fever. Evidence of the activation of the fibrinolytic system has been obtained in both diseases. After experimental Rocky Mountain spotted fever, an elevated level of fibrinogen was found in parallel with the activation of the fibrinolytic system and transient elevation of the tissue-type plasminogen activator. Later protein is mainly synthesized by endothelial cells. The ability to culture human endothelial cells in vitro provides a unique system to study the secretion of tissue-type plasminogen activator and of plasminogen activator inhibitor after rickettsial infection. Human vascular endothelial cells derived from the umbilical vein, when infected with Rickettsia conorii or Rickettsia rickettsii, secreted as much tissue-type plasminogen activator as control cells. The activity of plasminogen activator inhibitor however, was higher in the supernatants of infected cells than in those of control cells. This rickettsia-induced imbalance of the tissue-type plasminogen activator-inhibitor pair was a very early event after in vitro infection. The involvement of this system during Mediterranean spotted fever and Rocky Mountain spotted fever remains to be demonstrated.

Antibodies, Monoclonal↗
PubMed1990

Modulation of tPA, PAI-1 and PAI-2 antigen and mRNA levels by EGF in the A431 cell line.

It has been reported that EGF treatment enhances uPA but not tPA in the A431 epidermoid carcinoma cell line. To determine whether the absence of tPA modulation by EGF could be due to the action of inhibitors, we assayed tPA, PAI-1, PAI-2 and tPA/PAI-1 complexes by immunological assays and zymography in A431 serum-free medium. We found that, under conditions in which EGF had no effect on tPA activity, tPA antigen increased with a concomitant rise of tPA/PAI-1 complexes, indicating the action of an inhibitor. Both tPA antigen and tPA/PAI-1 complexes were modulated by EGF in a time and concentration dependent manner. tPA/PAI-1 complex levels were lower than tPA levels, suggesting the presence of other inhibitors. Immunological assays detected PAI-2 in addition to PAI-1 and showed a time and dose response to EGF. Modulation of tPA and the anti-activators by the growth factor was confirmed by identification of the corresponding transcripts with cDNA probes. We conclude that the net plasminogen activator activity in A431 cells is the result of a balance between activators and inhibitors.

DNA↗
PubMed1989

Increased plasminogen activator inhibitor activity in non insulin dependent diabetic patients--relationship with plasma insulin.

Type 2 diabetic patients are known to frequently have a high insulin level and were recently described as having high plasminogen activator inhibitor (PAI) activity, compared to normal controls. As we have shown in several clinical conditions (normal subjects, obese patients, angina pectoris patients) that plasma PAI activity was linked with plasma insulin, we have studied in 38 type 2 diabetic patients the relationship between PAI activity, insulin and other parameters. Patients showed higher level of PAI activity, as well as plasma glucose, insulin, triglyceride, cholesterol and Apolipoprotein B levels than normal controls; highest values were observed with diabetic patients also affected by coronary artery disease. A significant correlation was found between PAI activity and insulin (r = 0.60, p less than 0.001), body mass index (r = 0.32, p less than 0.05) and Apolipoprotein B (r = 0.33, p less than 0.05). The two latter correlations disappeared after adjustment for insulin. These results are in agreement with our previous report showing an in vitro effect of insulin on the synthesis of PAI by a hepatocellular cell line. Hyperinsulinemia presented by type 2 diabetic patients may increase the hepatic synthesis of PAI, inducing an hypofibrinolysis, which could play a role in the development of the vascular complications. Attempts to reduce hyperinsulinemia could have a favorable effect by lowering PAI activity.

Adult↗
PubMed1989

Fat distribution and plasminogen activator inhibitor activity in nondiabetic obese women.

Epidemiologic data strongly suggest that upper distribution of body fat and high plasminogen activator inhibitor (PAI) activity are risk factors for cardiovascular disease. Therefore, a link between these two parameters was evaluated by studying 51 menstrually active nondiabetic obese women. In this group positive correlations were observed between body mass index (BMI), waist to hip circumference ratio (W/H ratio, which estimates body fat distribution), plasma insulin, and PAI activity. In addition, plasma triglycerides were related to the W/H ratio and insulin and PAI activity. Partial correlations revealed that BMI was independently and solely related to W/H ratio, which was also independently related to plasma insulin, which in turn related to PAI activity. These results suggest that upper body fat distribution acts as a risk factor of cardiovascular disease through its association with high PAI activity.

Adipose Tissue↗
PubMed1988

Insulin stimulates the synthesis of plasminogen activator inhibitor 1 by the human hepatocellular cell line Hep G2.

Secretion of plasminogen activator inhibitor 1 (PAI-1) by cultures of human umbilical vein endothelial cells and human hepatocellular cell line Hep G2 was evaluated after insulin stimulation. The secretion of PAI-1 antigen and activity was measured in the conditioned medium and the cellular extracts after incubation of confluent cultures with 1% serum medium for 24 hours. Insulin induced a dose dependent increase of the PAI-1 secretion by Hep G2 cell line. At 10(-8) M a two fold increase of PAI-1 antigen and activity were observed whereas alpha 2 antiplasmin and fibrinogen were not significantly modified. No effect of insulin was observed on PAI-1 antigen and PAI activity production by human endothelial cells whereas endotoxin resulted in a two fold increase in PAI-1 secretion. In recent clinical studies we have demonstrated that the level of plasma insulin correlated with that of PAI-1. Thus we hypothesize that hepatocytes represent a physiological source of plasma PAI-1 which is modulated by plasma insulin level.

Animals↗
PubMed1988

Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin).

A binding protein for plasminogen activator inhibitor 1 (PAI-1-BP) was isolated from human plasma by a four-step procedure. 1) The 7 S globulin fraction of plasma was isolated by gel filtration on Sephacryl S-300. 2) Human endothelial cell-type plasminogen activator inhibitor (PAI-1), pretreated with 12 M urea, was added to this fraction (22 micrograms of PAI-1/ml of plasma), and a PAI-1 antigen peak with apparent mass 450 kDa (representing 65% of PAI-1 antigen and 85% of PAI activity) was isolated by gel filtration of this mixture. 3) The PAI-1.PAI-1-BP complex was further purified by immunoadsorption on an immobilized murine monoclonal antibody directed against PAI-1 (MA-7D4) and by elution with 4 M KSCN. 4) The complex was then dissociated by addition of excess human tissue-type plasminogen activator (t-PA), and t-PA and PAI-1 antigen (t-PA.PAI-1 complexes and free t-PA and PAI-1) were removed by immunoadsorption on monoclonal antibodies directed against t-PA (MA-62E8) and against PAI-1 (MA-7D4 and MA-12A4). Sodium dodecyl sulfate-gel electrophoresis of the purified material under nonreducing conditions revealed two bands with apparent mass approximately equal to 150 kDa and two bands with mass 74 and 68 kDa. Reduced sodium dodecyl sulfate-gel electrophoresis displayed two main bands with apparent masses of 73 and 64 kDa. The PAI-1-BP reacts with urea-treated, but not with inactive PAI-1. t-PA dissociates the complex between PAI-1 and PAI-1-BP. PAI-1 in complex with PAI-1-BP is 2-3-fold more stable at 37 degrees C than purified PAI-1, suggesting that PAI-1-BP may stabilize PAI-1 in blood. The concentration of PAI-1-BP in plasma determined by titration with PAI-1 is approximately 130 mg/liter. The isolated PAI-1-BP was shown to be identical to S protein (vitronectin) both by cross-reactivity with monospecific rabbit antisera and by NH2-terminal amino acid sequence analysis. The gel filtration behavior, mobility on sodium dodecyl sulfate-gel electrophoresis, and concentration in plasma suggest that PAI-1-BP is a multimer (presumably a dimer) of S protein accounting for approximately 35% of the S protein in plasma.

Amino Acids↗
PubMed1988

Purification and characterization of natural and recombinant human plasminogen activator inhibitor-1 (PAI-1).

Human plasminogen activator inhibitor-1 (PAI-1) was purified from the conditioned medium of endotoxin-stimulated umbilical vein endothelial cell cultures by combinations of zinc-chelate-Sepharose chromatography, gel filtration on Sephacryl S-300 and immunoadsorption on an insolubilized murine monoclonal antibody (MA-7D4). The final product was obtained with a recovery of approximately 20% from conditioned medium containing about 3 micrograms/ml PAI-1. The yield of PAI-1 was 15-100 micrograms/umbilical cord, depending on the culture and harvest conditions. SDS gel electrophoresis revealed a main band with Mr = 46,000 both under reducing and non-reducing conditions. On gel filtration on Sephacryl S-300, however, the material was separated in two fractions, one eluting at the void volume, which contains active PAI-1, and one with Mr = 46,000 containing inactive material that could be reactivated with 12 M urea. SDS gel electrophoresis of the isolated high-Mr fraction revealed several bands including a main 46,000-Mr component, which reacted with anti-(PAI-1) antibodies on immunoblotting and neutralized tissue-type plasminogen activator (t-PA). The active high-Mr fraction and the reactivated low-Mr fraction of PAI-1 inhibited t-PA very rapidly with an apparent second-order rate constant of (1.5-4) x 10(7) M-1 s-1. The cDNA of endothelial cell PAI-1 was cloned and expressed in Chinese hamster ovary cells. The translation product, purified from conditioned medium of transfected cells, also revealed a high-Mr and a low-Mr fraction on gel filtration, which were indistinguishable from the natural proteins by physicochemical, immunochemical and functional analysis. On reduced SDS gel electrophoresis, the high-Mr fraction was separated into the Mr-46,000 low-Mr PAI-1 and two other components with Mr 65,000 and one barely entering the gel. When reactivated low-Mr PAI-1 was added to plasma, PAI activity and PAI-1 antigen eluted with an apparent Mr greater than or equal to 300,000 on gel filtration, indicating that active PAI-1 complexes with one or more binding proteins in plasma.

Cells, Cultured↗