Plasminogen Activator Inhibitor-1 Synthesis Research Articles

SERPINE-1 gene polymorphism in patients with cardiovascular diseases

Currently, the issues of recurrent course of cardiovascular diseases are given great importance. Today, there is a search for more and more new factors and causes, including genetic ones, that contribute to the increase in the incidence of circulatory system diseases. The study of polymorphic variants of hemostasis system genes made it possible to study the molecular mechanisms underlying the causes of cardiovascular complications. Polymorphism of theSERPINE-1gene, encoding plasminogen activator inhibitor-1, is associated with the occurrence of cardiovascular diseases. This literature review examines the influence ofSERPINE-1gene polymorphism and the concentration of the plasminogen activator inhibitor-1 it encodes on the development and severity of circulatory system diseases; as well as the role of plasminogen activator inhibitor-1 as one of the indicators reflecting the antifibrinolytic potential of the blood. Taking into account the opinion of most authors, we can conclude that the polymorphism of theSERPINE-1gene and its homozygous variant 4G/4G, due to which the synthesis of plasminogen activator inhibitor-1 is increased, is an unfavorable predictor of many pathological processes. However, most of the data have been obtained on the association of theSERPINE-1gene polymorphism with cardiovascular diseases, where, according to most authors, the 4G/4G genotype is a prognostically negative variant. However, a number of researchers believe that the heterozygous 5G/4G variant is likely associated with the occurrence of cerebral ischemia. The inconsistency of the data obtained, ofcourse, requires further study of the characteristics of theSERPINE-1gene polymorphism in various pathological conditions, which is an important prerequisite for understanding the mechanisms of a number of diseases. To prepare the review, a literature search method in PubMed databases for the period 2013–2023 was used.

Protective role of N-acetylcysteine and Sulodexide on endothelial cells exposed on patients' serum after SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 causes hyperinflammation and activation of coagulation cascade and, as a result, aggravates endothelial cell dysfunction. N-acetylcysteine and Sulodexide have been found to mitigate endothelial damage. The influence on coronary artery endothelial cells of serum collected after 4 ± 1 months from coronavirus infection was studied. The concentrations of serum samples of interleukin 6, von Willebrand Factor, tissue Plasminogen Activator, and Plasminogen Activator Inhibitor-1 were studied. The cultures with serum of patients after coronavirus infection were incubated with N-acetylcysteine and Sulodexide to estimate their potential protective role. The blood inflammatory parameters were increased in the group of cultures incubated with serum from patients after coronavirus infection. Supplementation of the serum from patients after coronavirus infection with N-acetylcysteine or Sulodexide reduced the synthesis of interleukin 6 and von Willebrand Factor. No changes in the synthesis of tissue Plasminogen Activator were observed. N-acetylcysteine reduced the synthesis of Plasminogen Activator Inhibitor-1. N-acetylcysteine and Sulodexide increased the tPA/PAI-1 ratio. N-acetylcysteine may have a role in reducing the myocardial injury occurring in the post-COVID-19 syndrome. Sulodexide can also play a protective role in post-COVID-19 patients.

Secretory activity of the coronary artery endothelial cells in conditions of the peritoneal dialysis

Introduction Endothelial dysfunction is frequent in patients treated with peritoneal dialysis and may lead to cardiac complications. We evaluated the effect of effluent dialysates and serum on the function of coronary artery endothelial cells (CAEC). Methods Human CAEC in in vitro culture were exposed to serum and dialysates from 24 patients treated with continuous ambulatory peritoneal dialysis (CAPD) and secretion of interleukin-6 (IL6), von Willebrand factor (vWF), tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were measured. Modulation of the secretory activity of CAEC by Sulodexide, mixture of glycosaminoglycans: heparin sulfate and dermatan sulfate, was studied. Results Serum from CAPD patients stimulated synthesis of IL6 (+93%), vWF (+18%), and PAI-1 (+20%) and did not change t-PA secretion in CAEC. Dialysates stimulated secretion of IL6 (+89%), vWF (+29%), and PAI-1 (+31%) and did not change t-PA synthesis. Dialysates collected in 12 patients after 6 months more strongly stimulated synthesis of IL6 (+37%) and PAI-1 (+7%). Sulodexide suppressed the secretory activity of CAEC stimulated by the studied sera: IL6 (–38%), vWF (–19%), t-PA (–13%), and PAI-1 (–12%). Conclusions Serum and the dialysate from CAPD patients induce inflammatory and prothrombotic reaction in coronary arterial endothelial cells. The general pattern of the observed effects for serum and dialysates was similar but the intensity of the effects was not identical. Sulodexide reduced these effects.

Features of plasminogen activator inhibitor-1 synthesis by local fat depots of different localization in cardiovascular diseases

Aim. To determine the features of expression and secretion of plasminogen activator inhibitor-1 (PAI-1) by subcutaneous, epicardial and perivascular adipocytes depending on the complexity of coronary artery disease (CAD).Material and methods. The study included 86 patients with CAD, including 35 with moderate atherosclerotic lesions of the coronary arteries (CA) (SYNTAX Score ≤22), 22 with severe (SYNTAX Score of 23-31 pints), 29 with extremely severe (SYNTAX Score ≥32). As a comparison group, 52 patients with heart defects were examined. During an elective surgical intervention, adipose tissue (AT) samples were obtained for subsequent cultivation and determination of PAI-1 gene expression and PAI-1 secretion in AT supernatants of various localizations. Statistical analysis was performed using Statistica 10.0.Results. In CAD, PAI-1 production in all three types of AT and plasma PAI-1 concentration were increased compared with patients with heart defects. Epicardial AT (EAT) in CAD was characterized by the maximum levels of expression and secretion of PAI-1 relative to patients with heart defects and subcutaneous and perivascular (PVAT) fat depots. Moderate coronary involvement (adipose tissue <22) is characterized by the lowest expression and secretion of PAI-1 in all types of AT and plasma concentration of PAI-1. Direct correlations of PAI-1 expression and secretion in EAT and PVAT and plasma PAI-1 level with its secretion in EAT in patients with CAD and heart defects were revealed. A direct correlation between the plasma level of PAI-1 and its secretion in PVAT was found only in CAD.Conclusion. In CAD, there is not only an increase in the plasma PAI-1 level, but also an increase in the expression of PAI-1 gene and the secretion of PAI-1 in all three types of AT compared with patients with heart defects. PAI-1 expression and secretion in EAT, PAI-1 plasma level, and age are predictors of severe/extremely severe coronary involvement in CAD.

Features of plasminogen activator inhibitor-1 synthesis by local fat depots of different localization in cardiovascular diseases

Aim. To determine the features of expression and secretion of plasminogen activator inhibitor-1 (PAI-1) by subcutaneous, epicardial and perivascular adipocytes depending on the complexity of coronary artery disease (CAD).Material and methods. The study included 86 patients with CAD, including 35 with moderate atherosclerotic lesions of the coronary arteries (CA) (SYNTAX Score ≤22), 22 with severe (SYNTAX Score of 23-31 pints), 29 with extremely severe (SYNTAX Score ≥32). As a comparison group, 52 patients with heart defects were examined. During an elective surgical intervention, adipose tissue (AT) samples were obtained for subsequent cultivation and determination of PAI-1 gene expression and PAI-1 secretion in AT supernatants of various localizations. Statistical analysis was performed using Statistica 10.0.Results. In CAD, PAI-1 production in all three types of AT and plasma PAI-1 concentration were increased compared with patients with heart defects. Epicardial AT (EAT) in CAD was characterized by the maximum levels of expression and secretion of PAI-1 relative to patients with heart defects and subcutaneous and perivascular (PVAT) fat depots. Moderate coronary involvement (adipose tissue <22) is characterized by the lowest expression and secretion of PAI-1 in all types of AT and plasma concentration of PAI-1. Direct correlations of PAI-1 expression and secretion in EAT and PVAT and plasma PAI-1 level with its secretion in EAT in patients with CAD and heart defects were revealed. A direct correlation between the plasma level of PAI-1 and its secretion in PVAT was found only in CAD.Conclusion. In CAD, there is not only an increase in the plasma PAI-1 level, but also an increase in the expression of PAI-1 gene and the secretion of PAI-1 in all three types of AT compared with patients with heart defects. PAI-1 expression and secretion in EAT, PAI-1 plasma level, and age are predictors of severe/extremely severe coronary involvement in CAD.

HDAC Inhibitor Abrogates LTA-Induced PAI-1 Expression in Pleural Mesothelial Cells and Attenuates Experimental Pleural Fibrosis.

Lipoteichoic acid (LTA) stimulates pleural mesothelial cell (PMC) to overproduce plasminogen activator inhibitor-1 (PAI-1), and thus may promote pleural fibrosis in Gram-positive bacteria (GPB) parapneumonic effusion (PPE). Histone deacetylase inhibitor (HDACi) was found to possess anti-fibrotic properties. However, the effects of HDACi on pleural fibrosis remain unclear. The effusion PAI-1 was measured among 64 patients with GPB PPE. Pleural fibrosis was measured as radiographical residual pleural thickening (RPT) and opacity at a 12-month follow-up. The LTA−stimulated human PMCs and intrapleural doxycycline−injected rats were pretreated with or without the pan-HDACi, m-carboxycinnamic acid bis-hydroxamide (CBHA), then PAI-1 and collagen expression and activated signalings in PMCs, and morphologic pleural changes in rats were measured. Effusion PAI-1 levels were significantly higher in GPB PPE patients with RPT > 10 mm (n = 26) than those without (n = 38), and had positive correlation with pleural fibrosis shadowing. CBHA significantly reduced LTA−induced PAI-1 and collagen expression via inhibition of JNK, and decreased PAI-1 promoter activity and mRNA levels in PMCs. Furthermore, in doxycycline−treated rats, CBHA substantially repressed PAI-1 and collagen synthesis in pleural mesothelium and minimized pleural fibrosis. Conclusively, CBHA abrogates LTA−induced PAI-1 and collagen expression in PMCs and attenuates experimental pleural fibrosis. PAI-1 inhibition by HDACi may confer potential therapy for pleural fibrosis.

A Serpin With a Finger in Many PAIs: PAI-1's Central Function in Thromboinflammation and Cardiovascular Disease.

Plasminogen activator inhibitor 1 (PAI-1) is a member of the serine protease inhibitor (serpin) superfamily. PAI-1 is the principal inhibitor of the plasminogen activators, tissue plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). Turbulence in the levels of PAI-1 tilts the balance of the hemostatic system resulting in bleeding or thrombotic complications. Not surprisingly, there is strong evidence that documents the role of PAI-1 in cardiovascular disease. The more recent uncovering of the coalition between the hemostatic and inflammatory pathways has exposed a distinct role for PAI-1. The storm of proinflammatory cytokines liberated during inflammation, including IL-6 and TNF-α, directly influence PAI-1 synthesis and increase circulating levels of this serpin. Consequently, elevated levels of PAI-1 are commonplace during infection and are frequently associated with a hypofibrinolytic state and thrombotic complications. Elevated PAI-1 levels are also a feature of metabolic syndrome, which is defined by a cluster of abnormalities including obesity, type 2 diabetes, hypertension, and elevated triglyceride. Metabolic syndrome is in itself defined as a proinflammatory state associated with elevated levels of cytokines. In addition, insulin has a direct impact on PAI-1 synthesis bridging these pathways. This review describes the key physiological functions of PAI-1 and how these become perturbed during disease processes. We focus on the direct relationship between PAI-1 and inflammation and the repercussion in terms of an ensuing hypofibrinolytic state and thromboembolic complications. Collectively, these observations strengthen the utility of PAI-1 as a viable drug target for the treatment of various diseases.

Platelet HIF-2α promotes thrombogenicity through PAI-1 synthesis and extracellular vesicle release.

Oxygen-compromised environments, such as high altitude, are associated with platelet hyperactivity. Platelets confined within the relatively impervious core of an aggregate/thrombus have restricted access to oxygen, yet they continue to perform energy-intensive procoagulant activities that sustain the thrombus. Studying platelet signaling under hypoxia is, therefore, critical to our understanding of the mechanistic basis of thrombus stability. We report here that hypoxia-inducible factor (HIF)-2α is translated from pre-existing mRNA and stabilized against proteolytic degradation in enucleate platelets exposed to hypoxia. Hypoxic stress, too, stimulates platelets to synthesize plasminogen-activator inhibitor-1 (PAI-1) and shed extracellular vesicles, both of which potentially contribute to the prothrombotic phenotype associated with hypoxia. Stabilization of HIF-α by administering hypoxia-mimetics to mice accelerates thrombus formation in mesenteric arterioles. In agreement, platelets from patients with chronic obstructive pulmonary disease and high altitude residents exhibiting thrombogenic attributes have abundant expression of HIF-2α and PAI- 1. Thus, targeting platelet hypoxia signaling could be an effective anti-thrombotic strategy.

MiR145-5p inhibits proliferation of PMVECs via PAI-1 in experimental hepatopulmonary syndrome rat pulmonary microvascular hyperplasia.

ABSTRACTHepatopulmonary syndrome (HPS) is a triad of advanced liver disease, intrapulmonary vasodilatation and arterial hypoxemia. Increasing evidence shows that HPS is associated with pulmonary microvascular hyperplasia. The aim of this work was to investigate the underlying mechanism of miR-145 in regulating the proliferation of pulmonary microvascular endothelial cells (PMVECs) and angiogenesis in HPS via plasminogen activator inhibitor-1 (PAI-1). To test this, morphology score and number of pulmonary microvascular were assessed in lung tissues from rats with HPS by Hematoxylin and Eosin (H&E) staining. Expression levels of PAI-1 were assessed in lung tissues from HPS rats, as well as in PMVECs treated with HPS rat serum. We also selected the putative microRNA binding site on PAI-1 by bioinformatics analysis. Then, miR145-3p and miR145-5p expression levels in the lungs and PMVECs of rats were detected by qRT-PCR because miR145-5p is a microRNA binding site on PAI-1. In addition, the effects of miR-145-5p regulation on PAI-1 were examined by upregulation and downregulation of miR-145-5p and specific lentivirus transfection was used to overexpress and knockdown PAI-1 to assess PAI-1 function on PMVECs proliferation. Our data showed that levels of PAI-1 expression in lung tissue of rats increased significantly when rats were treated with common bile duct ligation. We found that levels of miR-145-5p were frequently downregulated in HPS tissues and cell lines, and overexpression of miR-145-5p dramatically inhibited PMVECs proliferation. We further verified PAI-1 as a novel and direct target of miR-145-5p in HPS. MiR-145-5p inhibits PAI-1 synthesis and the expression changes of PAI-1 directly affect the proliferation of PMVECs. We concluded that miR-145-5p negatively regulates PMVEC proliferation through PAI-1 expression. In addition, overexpression of miR-145-5p may prove beneficial as a therapeutic strategy for HPS treatment.

Regulatory effects of estetrol on the endothelial plasminogen pathway and endothelial cell migration

BackgroundEstetrol (E4) is a natural estrogen produced solely during human pregnancy. E4 is suitable for clinical use since it acts as a selective estrogen receptor modulator. In clinical trials E4 has been seen to have little or no effect on coagulation. Hence, it is interesting to investigate whether E4 alters endothelial-dependent fibrinolysis. ObjectivesWe studied the effects of E4 on the fibrinolytic system and whether this could influence the ability of endothelial cells to migrate. In addition, we compared the effects of E4 with those of 17β-estradiol (E2). Study designHuman umbilical vein endothelial cells (HUVEC) were obtained from healthy women. Expression of plasminogen-activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator (u-PA) and tissue plasminogen activator (t-PA) proteins was evaluated by Western blot analysis. Endothelial cell migration was studied by razor-scrape horizontal and multiwell insert systems assays. ResultsE4 increased the expression of t-PA, u-PA and PAI-1 in HUVEC, but less so than did equimolar amounts of E2. The effects of E4 on t-PA, u-PA and PAI-1 were mediated by the induction of the early-immediate genes c-Jun and c-Fos. E4 in combination with E2 antagonized the effects induced by pregnancy-like E2 concentrations but did not impair the effects of postmenopausal-like E2 levels. We also found that the increased synthesis of PAI-1, u-PA and t-PA induced by E2 and E4 is important for horizontal and three-dimensional migration of HUVEC. ConclusionsThese results support the hypothesis that E4 acts as an endogenous selective estrogen receptor modulator (SERM), controlling the fibrinolytic system and endothelial cell migration.

Dissociation of SERPINE1 mRNA from the translational repressor proteins Ago2 and TIA-1 upon platelet activation.

Platelets play an important role in haemostasis, as well as in thrombosis and coagulation processes. They harbour a wide variety of messenger RNAs (mRNAs), that can template de novo protein synthesis, and an abundant array of microRNAs, which are known to mediate mRNA translational repression through proteins of the Argonaute (Ago) family. The relationship between platelet microRNAs and proteins capable of mediating translational repression, however, remains unclear. Here, we report that half of platelet microRNAs is associated to mRNA-regulatory Ago2 protein complexes, in various proportions. Associated to these Ago2 complexes are platelet mRNAs known to support de novo protein synthesis. Reporter gene activity assays confirmed the capacity of the platelet microRNAs, found to be associated to Ago2 complexes, to regulate translation of these platelet mRNAs through their 3'UTR. Neither the microRNA repertoire nor the microRNA composition of Ago2 complexes of human platelets changed upon activation with thrombin. However, under conditions favoring de novo synthesis of Plasminogen Activator Inhibitor-1 (PAI-1) protein, we documented a rapid dissociation of the encoding platelet SERPINE1 mRNA from Ago2 protein complexes as well as from the translational repressor protein T-cell-restricted intracellular antigen-1 (TIA-1). These findings are consistent with a scenario by which lifting of the repressive effects of Ago2 and TIA-1 protein complexes, involving a rearrangement of proteinmRNA complexes rather than disassembly of Ago2microRNA complexes, would allow translation of SERPINE1 mRNA into PAI-1 in response to platelet activation.

Bone geometry of the hip is associated with obesity and early structural damage – A 3.0T MRI study of community-based adults

Bone geometry of the hip is associated with obesity and early structural damage – A 3.0T MRI study of community-based adults

Mental Disorders and Thrombotic Risk

Patients with psychosis, severe depression, or chronic stress are at increased risk for thromboembolism. Evidence suggests that tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) imbalance may play an important role in pathophysiology of mental and thromboembolic disorders. tPA facilitates clot dissolution and participates in several brain functions, including response to stress, learning, and memory. Depression is characterized by high PAI-1 levels, and conditions related to a hypofibrinolytic status, such as polycystic ovary syndrome and metabolic syndrome, are associated with an increased risk for both depression and cardiovascular events. Depression/psychosis may occur when estrogen and progesterone levels decrease. Estrogen inhibits synthesis of transforming growth factor-β (TGF-β), a protein increasing PAI-1 synthesis, whereas progesterone induces brain-derived neurotrophic factor release, stimulating tPA expression in neurons. Synthetic progestogens (progestins) may cause severe depression. Notably, progestin metabolites, which may prevent synthesis of natural progesterone by feedback, do not cross the blood-brain barrier. Intense suprapubic menstrual cramp, which may relate to difficulty to dissolve clots, correlates with severe premenstrual tension. Exercise, which increases tPA synthesis, has antidepressive and antithrombotic effects. We suggest that thromboembolism and some mental disorders are mechanistically related: tPA-PAI-1 imbalance seems to be a common denominator.

Abstract 547: Excess Glucose Dowregulates Factors for Invasion and Induces an Anti-angiogenic Milieu in First Trimester Cytotrophoblast Cell

Objective: Diabetes mellitus is a known risk factor for the development of preeclampsia (preE). First trimester cytotrophoblastic (CTB) cells invade the decidua using the serine protease plasmin. The enzymatic conversion from the inactive plasminogen to the active form plasmin, is performed by urokinase plasminogen activator (uPA), which is regulated by plasminogen activator inhibitor 1 (PAI-1) in CTBs. Disruption of placental development, setting the stage for preE development is believed, in part, to be caused by a dysfunction of the invasive nature of CTBs. The aim of this study is to determine whether excess glucose induces anti-invasive and anti-angiogenic effects in CTBs in vitro . Methods: Human CTB cells (Sw. 71) were derived from first trimester placenta. The CTB cells were treated with 45, 135, 225, 495 or 945 mg/dL glucose for 72h. Thereafter, cell lysates were utilized to measure uPA and PAI-1 protein expression and phosphorylation of p38 MAPK by western blot. The mRNA expression of uPA and PAI-1 in glucose-induced CTB cell lysates was measure by qPCR. Levels of angiogenic and anti-angiogenic factors (sFLT-1, sENG, VEGF165, PlGF) and the level of inflammatory cytokine IL-6 were measured in the culture media by the commercially available kits. Statistical comparisons were performed using analysis of variance with Dunnett's post hoc tests. Results: Both uPA and PAI-1 protein and mRNA expression were downregulated (p<0.05) in CTB cells treated with >135 mg/dL glucose as compared to those in basal (45 mg/dL). The anti-angiogenic factors (sENG and sFLT-1) were upregulated, while the angiogenic factors (VEGF165 and PlGF) were downregulated in the presence of >135 mg/dL glucose. The level of IL-6 was higher (p<0.05) in the presence of glucose >135 mg/dL compared to basal (45 mg/dL) conditions. The p38 MAPK phosphorylation was upregulated (p<0.05) in excess glucose-treated CTBs. Conclusions: Exposure of CTB cells to glucose at levels associated with diabetes causes a disruption in the invasive profile of CTB cells by decreasing the synthesis of uPA and PAI-1 (both protein and mRNA expression); by downregulating expression of VEGF165 and PlGF; and by upregulating expression of sENG, sFLT-1, and IL-6. Moreover, glucose induced a stress signaling in CTBs.

The role of thrombin receptors PAR1 and PAR4 for PAI-1 storage, synthesis and secretion by human platelets

IntroductionArterial thrombi contain more platelets than venous thrombi and are more resistant to fibrinolysis. This resistance could partly be due to plasminogen activator inhibitor 1 (PAI-1) secreted by platelets. The aim of this study was to elucidate differences between thrombin receptors protease-activated receptor (PAR) 1 and 4 and platelet storage, secretion and synthesis of platelet PAI-1, as compared to other platelet α-granule proteins such as VEGF and endostatin. Materials and methodsHuman isolated platelets were incubated with thrombin (0.5U/ml), PAR1-activating peptide (AP) (0.4-30μM) or PAR4-AP (1.5-300μM) for up to 24hours. ELISA, western blot and fluorescence microscopy were used to measure secretion, contents and localization of PAI-1, VEGF and endostatin. ResultsOur results show that PAI-1 and VEGF might be co-localized and that endostatin does not co-localize with either PAI-1 or VEGF. PAI-1 and VEGF show a similar secretion pattern, being more sensitive to low grade PAR1 activation, but secretion was also observed with higher concentrations of PAR4-APs. PAI-1 is secreted in an active form. PAI-1 mRNA was found in platelets, and elevated levels of PAI-1 were detected after 24hours incubation of platelets. ConclusionsPAI-1 and VEGF, but not endostatin, might be stored in the same α-granule in human platelets. PAI-1 and VEGF also show a similar secretion pattern, being more sensitive to PAR1 than to PAR4 activation, but the secretion is not exclusively selective. Our results also show that platelet PAI-1 is increased if incubated for 24hours, both with addition of PAR1-activating peptide and without activation, which could indicate de novo synthesis.

Blockade of Cannabinoid Receptor 1 Improves Insulin Resistance, Lipid Metabolism, and Diabetic Nephropathy in db/db Mice

The endocannabinoid system is important in the pathogenesis of obesity-related metabolic disorders. However, the effect of inhibiting the endocannabinoid system in type 2 diabetic nephropathy is unclear. Therefore, we examined the effect of the cannabinoid (CB)1 receptor antagonist, SR141716, on insulin resistance and diabetic nephropathy in db/db mice. Six-week-old db/db mice were treated with the CB1-specific antagonist SR141716 (10 mg/kg · d) for 3 months. Treatment with SR141716 significantly improved insulin resistance and lipid abnormalities. Concomitantly, CB1 antagonism improved cardiac functional and morphological abnormality, hepatic steatosis, and phenotypic changes of adipocytes into small differentiated forms, associated with increased adiponectin expression and decreased lipid hydroperoxide levels. CB1 receptor was overexpressed in diabetic kidneys, especially in podocytes. Treatment with the SR141716 markedly decreased urinary albumin excretion and mesangial expansion and suppressed profibrotic and proinflammatory cytokine synthesis. Furthermore, SR141716 improved renal lipid metabolism and decreased urinary 8-isoprostane levels, renal lipid hydroperoxide content, and renal lipid content. In cultured podocytes, high-glucose stimulation increased CB1 receptor expression, and SR141716 treatment abolished high-glucose-induced up-regulation of collagen and plasminogen activator inhibitor-1 synthesis. Additionally, knockdown of CB1 receptor expression by stealth small interfering RNA abolished high-glucose-induced sterol-regulatory element-binding protein-1 expression in podocytes. These findings suggest that CB1 blockade improves insulin resistance and protect against renal injury through both metabolic and antifibrotic effects in type 2 diabetic nephropathy. Targeting CB1 blockade could therefore provide a new therapeutic target to prevent type 2 diabetic nephropathy.

Stimulated in vivo synthesis of plasminogen activator inhibitor-1 in human adipose tissue

Plasminogen activator inhibitor type-1 (PAI-1) is one of the most important inhibitors of endogenous fibrinolysis. Adipose tissue is a suggested source of the elevated plasma levels of PAI-1 in obesity. The relation between PAI-1 and inflammation is of particular interest, but current knowledge regarding regulation of PAI-1 in adipose tissue is mainly based on animal studies or ex vivo experiments on human cultured adipocytes. So far, no study has described stimulated gene expression and protein synthesis of PAI-1 in vivo in human adipose tissue. We used open heart surgery as a model of acute systemic inflammation. Twenty-two male patients underwent blood sampling and omental and subcutaneous adipose tissue biopsies for gene expression studies before and after surgery. Expression and localisation of PAI-1 antigen was evaluated by immunohistochemistry. After surgery gene expression of PAI-1 increased 27-fold in omental adipose tissue and three-fold in subcutaneous adipose tissue, but no differences were found in tissue-type plasminogen activator (t-PA) mRNA. PAI-1 antigen was localised within endothelial cells and in the adipose tissue interstitium close to vessels. The upregulated gene expression and protein synthesis in adipose tissue was followed by increased concentrations of PAI-1 antigen in plasma. In conclusion, we present for the first time that an acute systemic inflammation in humans increased gene expression and protein synthesis of PAI-1 in adipose tissue and that this increase was most prominent in omental adipose tissue. PAI-1 synthesis in adipose tissue due to acute systemic inflammation may be a link between inflammation and impaired endogenous fibrinolysis.

HDL3, but not HDL2, stimulates plasminogen activator inhibitor-1 release from adipocytes: the role of sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that regulates numerous key cardiovascular functions. High-density lipoproteins (HDLs) are the major plasma lipoprotein carriers of S1P. Fibrinolysis is a physiological process that allows fibrin clot dissolution, and decreased fibrinolytic capacity may result from increased circulating levels of plasminogen activator inhibitor-1 (PAI-1). We examined the effect of S1P associated with HDL subfractions on PAI-1 secretion from 3T3 adipocytes. S1P concentration in HDL3 averaged twice that in HDL2. Incubation of adipocytes with increasing concentrations of S1P in HDL3, but not HDL2, or with S1P complexed to albumin stimulated PAI-I secretion in a concentration-dependent manner. Quantitative RT-PCR revealed that S1P(1-3) are expressed in 3T3 adipocytes, with S1P(2) expressed in the greatest amount. Treatment of adipocytes with the S1P(1) and S1P(3) antagonist VPC23019 did not block PAI-1 secretion. Inhibiting S1P(2) with JTE-013 or reducing the expression of the gene coding for S1P(2) using silencing RNA (siRNA) technology blocked PAI-1 secretion, suggesting that the S1P(2) receptor mediates PAI-1 secretion from adipocytes exposed to HDL3 or S1P. Treatment with the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor RO-318425, or the Rho-associated protein kinase (ROCK) inhibitor Y27632 all significantly inhibited HDL3- and S1P-mediated PAI-1 release, suggesting that HDL3- and/or S1P-stimulated PAI-1 secretion from 3T3 cells is mediated by activation of multiple, downstream signaling pathways of S1P(2).

ChemInform Abstract: Toxicity of Cadmium and Lead on Vascular Cells that Regulate Fibrinolysis

Cadmium and lead are heavy metals that cause vascular lesions such as atherosclerosis and hypertension. Toxicity of cadmium and lead on the regulation of fibrinolysis by vascular-composing cells was investigated using a cell culture system. It was found that cadmium promotes the synthesis of plasminogen activator inhibitor-1 (PAI-1) whereas lead inhibits the synthesis of tissue plasminogen activator (t-PA) in vascular endothelial cells; consequently, both heavy metals reduced fibrinolytic activity in the liquid phase. The responses of endothelial cells to cadmium and lead were different from those to other heavy metals and the release of the fibrinolytic proteins from vascular smooth muscle cells and fibroblasts was perturbed by cadmium and lead in different manners. In conclusion, the present study showed that cadmium and lead exhibit their toxicity on fibrinolysis regulated by vascular cells in different manners among cell types and the individual cell types respond to cadmium and lead in different manners with respect to the release of fibrinolytic proteins.

Blockade of endogenous tissue kallikrein aggravates renal injury by enhancing oxidative stress and inhibiting matrix degradation.

Levels of tissue kallikrein (TK) are significantly lower in the urine of patients with kidney failure, and TK expression is specifically diminished in rat kidney after recovery from ischemia-reperfusion injury. In this study, we investigated the functional consequence of blocking endogenous TK activity in a rat model of chronic kidney disease. Inhibition of endogenous TK levels for 10 days by neutralizing TK antibody injection in DOCA-salt rats caused a significant increase in blood urea nitrogen and urinary protein levels, and a decrease in creatinine clearance. Kidney sections from anti-TK antibody-treated rats displayed a marked rise in tubular dilation and protein cast accumulation as well as glomerular sclerosis and size. TK blockade also increased inflammatory cell infiltration, myofibroblast and collagen accumulation, and collagen fraction volume. Elevated renal inflammation and fibrosis by anti-TK antibody were associated with increased expression of tumor necrosis factor-alpha, intercellular adhesion molecule-1, tissue inhibitor of metalloproteinase-2 (TIMP-2), and plasminogen activator inhibitor-1 (PAI-1). Moreover, the detrimental effect of TK blockade resulted in reduced nitric oxide (NO) levels as well as increased serum lipid peroxidation, renal NADH oxidase activity, and superoxide formation. In cultured proximal tubular cells, TK inhibited angiotensin II-induced superoxide production and NADH oxidase activity via NO formation. In addition, TK markedly increased matrix metalloproteinase-2 activity with a parallel reduction of TIMP-2 and PAI-1 synthesis. These findings indicate that endogenous TK has the propensity to preserve kidney structure and function in rats with chronic renal disease by inhibiting oxidative stress and activating matrix degradation pathways.