Mireille Henry

Production of Plasminogen Activator Inhibitor 1 by Human Adipose Tissue: Possible Link Between Visceral Fat Accumulation and Vascular Disease

Plasminogen activator inhibitor type 1 (PAI-1) contributes to the pathogenesis of atherothrombosis. Its plasma level is strongly correlated with parameters that define the insulin resistance syndrome, in particular with BMI and visceral accumulation of body fat, suggesting that PAI-1 may be an adipose tissue–derived circulating peptide. The present study was designed to investigate PAI-1 expression by human adipose tissue and its different cellular fractions. Special interest has been paid to the amount of PAI-1 antigen produced by omental versus subcutaneous fat. PAI-1 protein detected by immunolocalization was present at the stromal and adipocyte levels. PAI-1 mRNA was detected in stromal vascular cells freshly isolated and under culture conditions. It was also detected in whole adipose tissue and adipocyte fraction under culture conditions. The mRNA signal from the adipocyte fraction was detected as early as 2 h of incubation. The increase in PAI-1 mRNA was followed by an increase in PAI-1 antigen in the conditioned medium that was suppressed by treatment with cycloheximide. Transforming growth factor-β1 significantly increased PAI-1 antigen production by the adipocyte fraction, whereas tumor necrosis factor-α did not have any effect. Interestingly, after 5 h of incubation, omental tissue explants produced significantly more PAI-1 antigen than did subcutaneous tissue from the same individual, whereas similar production of leptin by the two territories was observed. These results strongly suggest that human adipose tissue, in particular visceral tissue, can be an important contributor to the elevated plasma PAI-1 levels observed in central obesity.

The A −844G Polymorphism in the PAI-1 Gene Is Associated With a Higher Risk of Venous Thrombosis in Factor V Leiden Carriers

Identification of combined genetic factors in factor V Leiden carriers is important for a more accurate risk assessment for venous thrombosis (VT). Among these individuals, we evaluated the role of polymorphisms of the plasminogen activator inhibitor-1 (PAI-1) gene in the thrombophilic phenotype. A total of 382 factor V Leiden carriers were included in the study. This population was divided into 3 groups. Group 1 (n=168) included individuals with a personal history of VT; group 2 (n=140) included individuals without personal VT but with a familial history of VT; and group 3 (n=74) included individuals without VT and with a fortuitous discovery of the factor V Leiden mutation. We compared the genotype distribution of 2 polymorphisms, A -844G and -675 4G/5G, located in the promoter region of the PAI-1 gene among these 3 groups of individuals. The A -844G allele frequency differed significantly among the 3 groups (P=0.048), the A allele being more frequent in patients who suffered from VT (61%) than in subjects without VT (52%, P=0.015), whereas no difference was observed between the 2 groups of asymptomatic individuals. The prevalence of genotype AA carriers was higher in patients with VT (38%) than in asymptomatic individuals (21%, P=0. 015), leading to an odds ratio of 1.74 (95% confidence interval, 1.3 to 3.8). Carrying the AA genotype conferred a risk of deep VT of 2. 08 (95% confidence interval, 1.28 to 3.40), whereas it did not seem to significantly influence the risk of pulmonary embolism. Concerning the -675 4G/5G polymorphism, no significant difference was observed among the 3 groups, the 4G allele frequency being 0.54 (in group 1), 0.49 (in group 2), and 0.45 (in group 3). These data suggest a role for the -A844G PAI-1 gene polymorphism in the thrombophilic phenotype of factor V Leiden carriers.

Use of recombinant epitopes to study the heterogeneous nature of the autoantibodies against thyroid peroxidase in autoimmune thyroid disease

Microsomal antigen is often recognized by the sera from patients with autoimmune thyroid disease (AITD). Human thyroid peroxidase (hTPO) is the main component of this antigen. In a previous study, we expressed hTPO cDNA as fusion proteins in prokaryotic vector; we thereby defined seven antigenic peptides by using two rabbit polyclonal anti‐hTPO antibodies. In the present study we used the seven epitopes and three widened peptides to define the reactivity pattern of 61 sera from patients with AITD. Thirty‐eight of them reacted against at least one of the seven hTPO‐restricted epitopes; 14 were negative against the seven determinants but recognized one or two of the extended peptides. Thus, the antibody response against hTPO appeared to be highly heterogeneous in AITD patient sera. Moreover, we demonstrated that the immunodetection of the hTPO on Western blotting with deoxycholate solubilized microsomes can be perfectly correlated with the recognition of one of the epitopes in the region 554–735.

Intracellular Calcium Mobilization Suppresses the TNF-α–Stimulated Synthesis of PAI-1 in Human Endothelial Cells Indications That Calcium Acts at a Translational Level

We investigated in human umbilical vein endothelial cells (HUVECs) the interaction between the signaling pathways triggered by calcium mobilization and those affected by human recombinant tumor necrosis factor-alpha (TNF) on the expression of type-1 plasminogen activator inhibitor (PAI-1). Calcium ionophore A23187 alone exerted a modest increase (50%) on PAI-1 synthesis. TNF alone increased PAI-1 accumulation in the culture medium in a time- and dose-dependent fashion, but this increase was abolished when A23187 was added simultaneously with TNF. The downregulating effect of A23187 was not the result of impaired protein secretion, proteolysis, cytotoxicity, or an apoptotic process. A23187 did not decrease the TNF-enhanced PAI-1 mRNA level but did provoke a significant shift in the distribution pattern of PAI-1 transcripts by increasing the 2.3-kb relative to the 3.2-kb form. Comparable inhibitory effects on PAI-1 protein synthesis were observed when A23187 was added 7 hours after the onset of TNF stimulation, strongly suggesting a posttranscriptional inhibitory action of calcium signaling on TNF-stimulated PAI-1 synthesis. However, treatment with actinomycin D showed that PAI-1 mRNA stability was not altered by the various treatments. Chelation of extracellular calcium by EGTA did not prevent the A23187-induced inhibition of TNF-stimulated PAI-1 protein synthesis, emphasizing the role of internal calcium stores in the inhibition of PAI-1 synthesis. Sucrose gradient fractionation of cell lysates revealed that regardless of which treatment was used, both PAI-1 mRNA transcripts exhibited similar sedimentation profiles in the actively translating polysomal pool, suggesting that the A23187-induced shift had no functional consequence on translation. However, in TNF-stimulated cells, A23187 induced a higher proportion of PAI-1 mRNAs that sedimented in fractions corresponding to less dense polysomes, a phenomenon that usually reflects a slower initiation rate during mRNA translation. A23187 also abolished the increase in PAI-1 synthesis induced by recombinant human interleukin 1 beta, and thapsigargin exerted effects comparable to those of A23187 on PAI-1 synthesis in TNF-stimulated cells. It is proposed that in HUVECs, the A23187-induced release of calcium from endoplasmic stores suppresses at the translational level the increase in PAI-1 synthesis triggered by proinflammatory cytokines.

Up-regulation of PAI-1 Synthesis by Insulin and Proinsulin in HEP G2 Cells but not in Endothelial Cells

Summary Increased plasminogen activator inhibitor 1 (PAI-1) plasma levels have been described in patients with ischemic heart disease. Although the regulation of PAI-1 production is still not completely understood, the important role of metabolic factors has been suggested. Clinical and biochemical changes found in the ‘syndrome X’ are significantly correlated to plasma PAI-1 levels. Intact proinsulin and its derivates have been observed in plasma and shown to possess immunological reactivity similar to that of insulin in conventional radioimmunoassay. PAI activity has been shown in diabetic patients to better correlate with plasma levels of proinsulin and split products than with insulin. These data question the existing relationship between insulin, proinsulin, and cellular PAI-1 production. We have compared the effect of insulin and proinsulin on PAI-1 synthesis by Hep G2 cells, and endothelial cells from human umbilical vein and porcine aorta. Our results indicated that proinsulin and insulin increased PAI-1 synthesis by Hep G2 cells. Nevertheless the proinsulin-induced up-regulation always represented 2 to 4% of the insulins effect, on a molar basis. Conversely to previous reports we cannot demonstrate an effect of proinsulin or insulin on PAI-1 synthesis by endothelial cells irrespective of their origin. Therefore, in so far as in vitro results can be extrapolated to the in vivo situation in insulin resistant patients, and in so far as insulin and precursors play a role in PAI-1 synthesis regulation in these patients, elevated PAI-1 levels observed in insulin resistant and/or coronary heart disease patients might be the result of increased synthesis of PAI-1 localized at the hepatocyte level.

EPITOPE MAPPING OF HUMAN THYROGLOBULIN REVEALS A CENTRAL IMMUNODOMINANT REGION

Thyroglobulin is the thyroid hormone precursor and the major antigen frequently involved in autoimmune diseases. The primary structure of human thyroglobulin is known but the spatial structure remains largely undetermined. By using fusion protein produced in prokaryotic system we have characterized seven short immunoreactive properties carrying at least one epitope. None of them includes hormonogenic sites, but five are concentrated in the central part of the monomeric molecule, which thus emerges as the major immunogenic region of this protein.

Anti-human thyroid peroxidase and anti-human thyroglobulin antibodies present no cross-reactivity on recombinant peptides.

Thyroglobulin (Tg) and thyroid peroxidase (TPO) are two antigens largely recognized by the sera from patients with autoimmune thyroid disease (AITD). Recently, the complete mapping of both antigens was established with rabbit polyclonal antibodies by the use of recombinant proteins expressed in prokaryotic vector. Several investigators have argued for the existence of a crossreactivity of some hetero‐ and autologous antibodies versus these two proteins. In the present study, using rabbit polyclonal antibody, mouse polyclonal antibody and autoimmune antibody (aAb). we observed no common epitope on human Tg (hTg) and human TPO (hTPO).