Plasminogen Activator Inhibitor-1 Gene Expression Research Articles

Effect of aminoguanidine on plasminogen activator inhibitor-1 and receptor of advanced glycation endproduct in the liver of streptozotocin-induced diabetic rats

Objectives: Advanced glycation end products (AGEs) play an important role in the development and progression of diabetic complications. The receptor for AGE (RAGE) is the ligand-binding site of AGE that initiates and accelerates the atherosclerotic process. Plasminogen activator inhibitor-1 (PAI-1) is a prothrombotic factor that has been proposed as a biological marker for prognostic assessment, monitoring of microvascular and macrovascular complications in diabetes. The purpose of this study is to investigate the effects of aminoguanidine on RAGE and PAI-1 expression levels in the liver of streptozotocin-induced diabetic rats.
 Methods: Diabetes was induced in rats by intraperitoneal injection of streptozocin (STZ, 50 mg/kg). On day 3, diabetic rats were administered 50, 100, and 200 mg/kg/day of aminoguanidine. The expression of PAI-1 and RAGE in the liver tissue was evaluated using real-time PCR.
 Results: PAI-1 and RAGE gene expression levels were higher in the liver of the diabetic rats compared to the control group. Aminoguanidine at 50, 100, and 200 mg/kg decreased PAI-1 and RAGE gene expression in the liver (p<0.001 at all doses). However, these genes were downregulated only at a dose of 200 mg/kg in healthy rats (p<0.0001). In addition, hepatic AGE protein levels were significantly decreased following treatment of the diabetic rats with aminoguanidine (p<0.001). There was also a significant correlation between AGE protein concentration and the expression of PAI-1 and RAGE.
 Conclusion: In summary, the data of the present study suggest that aminoguanidine reduced the expression of PAI-1 and RAGE in the liver of the diabetic rats.

Abstract TP222: Aging-associated Medin Induces Human Brain Microvascular Endothelial Cell Pro-inflammatory And Pro-thrombotic Activation Partly Via NFκB

Background: Vascular aging is characterized by endothelial dysfunction and proinflammatory & prothrombotic changes. It is responsible for aging-associated pathologies including stroke, vascular dementia (VaD) and Alzheimer’s disease (AD). The underlying mechanisms of vascular aging are poorly understood. Medin is an amyloidogenic protein that accumulates in aging vasculature, is associated with VaD and AD, and is a candidate mediator of vascular aging through unknown mechanisms. Our aim is to test the hypothesis that medin induces proinflammatory and prothrombotic changes in human brain microvascular endothelial cells (HBMVECs) through nuclear factor kappa B (NFκB). Methods: HBMVECs were exposed to vehicle, recombinant medin (5 μM, a physiologically relevant dose) without or with RO106-9920 (a small molecule selective NFκB inhibitor, 10 μM) or RO106-9920 alone for 20 hours (N=3-17/treatment). Cell lysates were measured for gene expression of inflammatory cytokines/chemokines (IL-8, IL-6, ICAM-1 and VCAM-1) and thrombotic factors (thrombomodulin (TM), a transmembrane glycoprotein and potent inhibitor of coagulation, plasminogen activator inhibitor-1 (PAI-1) which promotes thrombosis and tissue factor (TF) which initiates the coagulation cascade), using real time PCR. Results: See Figure. Medin increased gene expression of IL-8, IL-6, ICAM-1, VCAM-1 and PAI-1, reduced TM with no change in TF. Co-treatment of medin with RO106-9920 attenuated the increases in IL-8, IL-6, ICAM-1, VCAM-1 and the reduction in TM, with no change in PAI-1. Conclusions: Medin caused increased pro-inflammatory gene expression in HBMVECs. It also caused reduced TM and increased PAI-1 gene expression, signifying prothrombotic activation. Co-treatment with NFκB inhibitor prevented medin’s proinflammatory effect, and effect on TM, but not PAI-1. The effect of inducing endothelial cell activation supports medin as a potential novel target to treat vascular aging.

The role of the platelet pool of Plasminogen Activator Inhibitor-1 in well-controlled type 2 diabetes patients.

BackgroundThe main inhibitor of the fibrinolytic system, Plasminogen Activator Inhibitor -1 (PAI-1), irreversibly binds tissue-type Plasminogen Activator (t-PA) and thereby inhibits the protective action of tPA against thrombus formation. Elevated levels of plasma PAI-1 are associated with an increased risk of cardiovascular events and are observed in subjects with type 2 diabetes (T2D) and obesity. Platelets contain the majority of PAI-1 present in blood and exhibit the ability to synthesis active PAI-1. Diabetic platelets are known to be hyper-reactive and larger in size; however, whether these features affect their contribution to the elevated levels of plasma PAI-1 in T2D is not established.ObjectivesTo characterize the PAI-1 antigen content and the mRNA expression in platelets from T2D subjects compared to obese and lean control subjects, in order to elucidate the role of platelet PAI-1 in T2D.MethodsNine subjects with T2D and obesity were recruited from Primary Care Centers together with 15 healthy control subjects (8 lean subjects and 7 with obesity). PAI-1 antigen levels in plasma, serum and platelets were determined by ELISA, and PAI-1 mRNA expression was analyzed by qPCR.ResultsThere was no significant difference in PAI-1 mRNA expression or PAI-1 antigen in platelets in T2D subject in comparison to obese and lean control subjects. An elevated level of plasma PAI-1 was seen in both T2D and obese subjects. PAI-1 gene expression was significantly higher in both obese groups compared to lean.ConclusionSimilar levels of protein and mRNA expression of PAI-1 in platelets from T2D, obese and lean subjects indicate a limited role of platelets for the elevated plasma PAI-1 levels. However, an increased synthesis rate of mRNA transcripts in platelets from T2D and an increased release of PAI-1 could also result in similar mRNA and protein levels. Hence, synthesis and release rates of PAI-1 from platelets in T2D and obesity need to be investigated to further elucidate the role of platelets in obesity and T2D.

Vitronectin and Its Interaction with PAI-1 Suggests a Functional Link to Vascular Changes in AMD Pathobiology.

The pathogenesis of age-related macular degeneration (AMD), a frequent disorder of the central retina, is incompletely understood. Genome-wide association studies (GWAS) suggest a strong contribution of genomic variation in AMD susceptibility. Nevertheless, little is known about biological mechanisms of the disease. We reported previously that the AMD-associated polymorphism rs704C > T in the vitronectin (VTN) gene influences protein expression and functional aspects of encoded vitronectin, a human blood and extracellular matrix (ECM) protein. Here, we refined the association of rs704 with AMD in 16,144 cases and 17,832 controls and noted that rs704 is carried exclusively by the neovascular AMD subtype. Interaction studies demonstrate that rs704 affects the ability of vitronectin to bind the angiogenic regulator plasminogen activator inhibitor 1 (PAI-1) but has no influence on stabilizing its active state. Western blot analysis and confocal imaging reveal a strong enrichment of PAI-1 in the ECM of cultured endothelial cells and RPE cell line ARPE-19 exposed to vitronectin. Large-scale gene expression of VTN and PAI-1 showed positive correlations and a statistically significant increase in human retinal and blood tissues aged 60 years and older. Our results suggest a mechanism by which the AMD-associated rs704 variant in combination with ageing may contribute to the vascular complications in AMD.

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.

Visceral fat-specific regulation of plasminogen activator inhibitor-1 in aged septic mice.

Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) are documented in patients with sepsis and levels positively correlate with disease severity and mortality. Our previous work demonstrated that visceral adipose tissues (VAT) are a major source of PAI-1, especially in the aged (murine endotoxemia), that circulating PAI-1 protein levels match the trajectory of PAI-1 transcript levels in VAT (clinical sepsis), and that PAI-1 in both VAT and plasma are positively associated with acute kidney injury (AKI) in septic patients. In the current study utilizing preclinical sepsis models, PAI-1 tissue distribution was examined and cellular sources, as well as mechanisms mediating PAI-1 induction in VAT, were identified. In aged mice with sepsis, PAI-1 gene expression was significantly higher in VAT than in other major organs. VAT PAI-1 gene expression correlated with PAI-1 protein levels in both VAT and plasma. Moreover, VAT and plasma levels of PAI-1 were positively associated with AKI markers, modeling our previous clinical data. Using explant cultures of VAT, we determined that PAI-1 is secreted robustly in response to recombinant transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα) treatment; however, neutralization was effective only for TNFαindicating that TGFβ is not an endogenous modulator of PAI-1. Within VAT, TNFα was localized to neutrophils and macrophages. PAI-1 protein levels were fourfold higher in stromal vascular fraction (SVF) cells compared with mature adipocytes, and among SVF cells, both immune and nonimmune compartments expressed PAI-1 in a similar fashion. PAI-1 was localized predominantly to macrophages within the immune compartment and preadipocytes and endothelial cells within the nonimmune compartment. Collectively, these results indicate that induction and secretion of PAI-1 from VAT is facilitated by a complex interaction among immune and nonimmune cells. As circulating PAI-1 contributes to AKI in sepsis, understanding PAI-1 regulation in VAT could yield novel strategies for reducing systemic consequences of PAI-1 overproduction.

Elevated fibroblast growth factor-inducible 14 expression transforms proneural-like gliomas into more aggressive and lethal brain cancer.

High-grade gliomas (HGGs) are aggressive, treatment-resistant, and often fatal human brain cancers. The TNF-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) signaling axis is involved in tissue repair after injury and constitutive signaling has been implicated in the pathogenesis of numerous solid cancers. The Fn14 gene is expressed at low levels in the normal, uninjured brain but is highly expressed in primary isocitrate dehydrogenase wild-type and recurrent HGGs. Fn14 signaling is implicated in numerous aspects of glioma biology including brain invasion and chemotherapy resistance, but whether Fn14 overexpression can directly promote tumor malignancy has not been reported. Here, we used the replication-competent avian sarcoma-leukosis virus/tumor virus A system to examine the impact of Fn14 expression on glioma development and pathobiology. We found that the sole addition of Fn14 to an established oncogenic cocktail previously shown to generate proneural-like gliomas led to the development of highly invasive and lethal brain cancer with striking biological features including extensive pseudopalisading necrosis, constitutive canonical and noncanonical NF-κB pathway signaling, and high plasminogen activator inhibitor-1 (PAI-1) expression. Analyses of HGG patient datasets revealed that high human PAI-1 gene (SERPINE1) expression correlates with shorter patient survival, and that the SERPINE1 and Fn14 (TNFRSF12A) genes are frequently co-expressed in bulk tumor tissues, in tumor subregions, and in malignant cells residing in the tumor microenvironment. These findings provide new insights into the potential importance of Fn14 in human HGG pathobiology and designate both the NF-κB signaling node and PAI-1 as potential targets for therapeutic intervention. MAIN POINTS: This work demonstrates that elevated levels of the TWEAK receptor Fn14 in tumor-initiating, neural progenitor cells leads to the transformation of proneural-like gliomas into more aggressive and lethal tumors that exhibit constitutive NF-κB pathway activation and plasminogen activator inhibitor-1 overexpression.

Urokinase receptor and tissue plasminogen activator as immediate-early genes in pentylenetetrazole-induced seizures in the mouse brain.

Epileptogenesis progressively leads to the rearrangement of normal neuronal networks into more excitable ones and can be viewed as a form of neuroplasticity, the molecular mechanisms of which still remain obscure. Here, we studied pentylenetetrazole seizure-induced regulation of genes for plasminogen activator system in the mouse brain. We found that expression of tissue plasminogen activator (tPA) and urokinase receptor (uPAR) mRNA was strongly increased in the mouse cerebral cortex, hippocampus, striatum and amygdala as early as 3hr after pentylenetetrazole seizures. Such early activity-induced expression of uPAR in the central nervous system has not been demonstrated before. uPAR mRNA accumulation was followed by elevation of uPAR protein, indicating a complete transcription-translation process. Both tPA gene induction and uPAR gene induction were independent of the protein synthesis, suggesting that they are regulated by neural activity as immediate-early genes. In contrast to tPA and uPAR genes, the expression of which returned to the basal level 6hr following seizures, urokinase and plasminogen activator inhibitor-1 gene expression showed a delayed activation only at 3days after seizures. In conclusion, our results suggest an important sensitivity of the brain plasminogen activator system to seizure activity which raises the question of its role in activity-dependent neural tissue remodeling in pathological and normal conditions.

Abstract TP557: Endothelial Cell Proinflammatory Priming by Amyloidogenic Medin via NFκB and Inflammasome Pathways and Reversal by Monosialoganglioside Nanoliposomes

Medin is a common amyloid protein that accumulates in cerebral vessels with aging and cause endothelial dysfunction in human cerebral arterioles. Studying its role in cerebrovascular disease could provide insights on cerebrovascular aging. We aim to test whether medin induces pro-inflammatory priming of endothelial cells (ECs) and probe mechanistic bases, and determine whether monosialoganglioside-containing nanoliposomes (NLGM1), previously shown to protect against AL amyloid vascular dysfunction, would abrogate this response. Methods: Human umbilical vein ECs were treated (20 hours) with physiologic dose of medin (5 μM) or vehicle, and proinflammatory markers IL-1β, caspase-1, IL-8, IL-6, ICAM-1, phospho-NFκB and pro-thrombotic marker plasminogen activator inhibitor-1 (PAI-1) gene and/or protein expressions were measured. In some replicates, medin was co-treated with either specific NFκB small molecule inhibitor RO106-9920 100 μM or NLGM1 (70:20:5% phosphatidylcholine, cholesterol, monosialoganglioside, 1:10 medin:NLGM1 mass ratio). Results: Medin increased gene expression of IL-1β (104.0±30.2x vs. control, p=0.002), IL-8 (5560±1864x, p<0.001), IL-6 (80.4±48x, p<0.001), ICAM-1 (72.0±8.5x, p<0.001) and PAI-1 (5.4±1x, p<0.001) (n=13-44). There were corresponding increases in protein expression as well as increased phospho-NFκB and caspase-1 (Figure). RO106-9920 co-treatment reduced gene expressions of IL-8, IL-6, ICAM-1 and PAI-1 but not IL-1β (versus medin treatment) whereas NLGM1 normalized gene expressions of all cytokines including IL-1β. Conclusions: Medin induced profound proinflammatory and prothrombotic responses in endothelial cells. Both NFκB and inflammasome-mediated mechanisms mediate medin’s vascular priming effects. Medin-induced pathology is a potential new mechanism of aging-related cerebrovascular inflammation. NLGM1 protects against medin’s effects and may represent a new therapeutic approach.

Upregulation of Plasminogen Activator Inhibitor-1 in Irradiated Recipient Arteries and Veins from Free Tissue Transfer Reconstruction in Cancer Patients.

Background Clinical studies have shown that radiotherapy can induce vascular disease at the site of exposure but is usually not clinically evident until years after treatment. We have studied irradiated human arteries and veins to better understand the underlying biology in search of future treatments. The aim was to investigate whether radiotherapy contributed to a sustained expression of plasminogen activator inhibitor-1 (PAI-1) in human arteries and veins. Methods Irradiated arteries and veins were harvested, together with unirradiated control vessels, from patients undergoing free tissue transfer reconstruction at a median time of 90 weeks [5–650] following radiation exposure. Differential gene expression of PAI-1 was analysed, together with immunohistochemistry (IHC) and immunofluorescence (IF). Results PAI-1 gene expression was increased in both arteries (p = 0.012) and veins (p < 0.001) in irradiated compared to unirradiated control vessels. IHC and IF indicated that cells expressing PAI-1 were located in the adventitia of both arteries and veins and colocalized with cells positive for CD68, CD45, and α-SMA in arteries and with CD45 and α-SMA in veins. Conclusion The current study shows a sustained upregulation of PAI-1 in both arteries and veins after exposure to ionizing radiation, indicating a chronic inflammation mainly in the adventitia. We believe that the results contribute to further understanding of radiation-induced vascular disease, where targeting PAI-1 may be a potential treatment.

Reactive oxygen species modulate HIF-1 mediated PAI-1 expression: involvement of the GTPase Rac1.

The hypoxia-inducible transcription factor HIF-1 mediates upregulation of plasminogen activator inhibitor-1 (PAI-1) expression under hypoxia. Reactive oxygen species (ROS) have also been implicated in PAI-1 gene expression. However, the role of ROS in HIF-1-mediated regulation of PAI-1 is not clear. We therefore investigated the role of the GTPase Rac1 which modulates ROS production in the pathway leading to HIF-1 and PAI-1 induction. Overexpression of constitutively activated (RacG12V) or dominant-negative (RacT17N) Rac1 increased or decreased, respectively, ROS production. In RacG12V-expressing cells, PAI-1 mRNA levels as well as HIF-alpha nuclear presence were reduced under normoxia and hypoxia whereas expression of RacT17N resulted in opposite effects. Treatment with the antioxidant pyr-rolidinedithiocarbamate or coexpression of the redox factor-1 restored HIF-1 and PAI-1 promoter activity in RacG12V-cells. In contrast, NFkappaB activation was enhanced in RacG12V-cells, but abolished by RacT17N. Thus, these findings suggest a mechanism explaining modified fibrinolysis and tissue remodeling in an oxidized environment.

Plasminogen Activator Inhibitor Type-1 as a Regulator of Fibrosis.

Fibrosis is known as a frequent and irreversible pathological condition which is associated with organ failure. Tissue fibrosis is a central process in a variety of chronic progressive diseases such as diabetes, hypertension, and persistent inflammation. This state could contribute to chronic injury and the initiation of tissue repair. Fibrotic disorders represent abnormal wound healing with defective matrix turnover and clearance that lead to excessive accumulation of extracellular matrix components. A variety of identified growth factors, cytokines, and persistently activated myofibroblasts have critical roles in the pathogenesis of fibrosis. Irrespective of etiology, the transforming growth factor-β pathway is the major driver of fibrotic response. Plasminogen activator inhibitor-1 (PAI-1) is a crucial downstream target of this pathway. Transforming growth factor-β positively regulates PAI-1 gene expression via two main pathways including Smad-mediated canonical and non-canonical pathways. Overexpression of PAI-1 reduces extracellular matrix degradation via perturbing the plasminogen activation system. Indeed, elevated PAI-1 levels inhibit proteolytic activity of tissue plasminogen activator and urokinase plasminogen activator which could contribute to a variety of inflammatory elements in the injury site and to excessive matrix deposition. This review summarizes the current knowledge of critical pathways that regulate PAI-1 gene expression and suggests effective approaches for the treatment of fibrotic disease. J. Cell. Biochem. 119: 17-27, 2018. © 2017 Wiley Periodicals, Inc.

Enhanced mRNA expression of plasminogen activator inhibitor-1 in livedoid vasculopathy lesions.

Thrombosis and inflammation play an important role in pathophysiology of livedoid vasculopathy (LV). Plasminogen activator inhibitor-1 (PAI-1) is the main physiological inhibitor of fibrinolysis and is a pivotal modulator in a broad range of biological processes. The study specimens were retrospectively selected from archives of pathology department. We investigated PAI-1 mRNA expression in the paraffin blocks of patients with biopsy-proven LV and controls. We analyzed the presence of thrombus, fibrinoid necrosis, ulcer, and epidermal atrophy in study samples. The correlation between histologic findings and PAI-1 expression was investigated. Analyses were performed in 14 LV patients (mean age 31±20, 79% female) and 4 controls (mean age 64±19, 50% female). PAI-1 gene expression was significantly higher in LV compared to the control group (median 7.74 (Iqr:13.94) vs 1.0 (0.31)), P=.011. Histological analysis displayed that fibrinoid necrosis was present on all patients with LV, 61.5% displayed thrombus, 46.2% displayed ulcer, and 15.4% displayed epidermal atrophy. Overall, we did not observe any discerning difference in PAI-1 expression between the LV blocks with or without thrombus, fibrinoid necrosis, or epidermal atrophy, yet the LV specimens that displayed ulcer histologically had higher PAI-1 mRNA expression compared to those without ulcer (median 13.98 (Iqr:19.21) vs 2.86 (5.59)), (P=.046). PAI-1 mRNA expression is significantly increased in cutaneous lesions of patients with LV. Histological finding of ulcer is associated with increased PAI-1 expression in LV specimen. In the current era of PAI-1 inhibitors, enhanced local PAI-1 expression can form a novel and local therapeutic target in LV.

A 2-year Double-Blind RCT Follow-up Study with Fermented Papaya Preparation (FPP) Modulating Key Markers in Middle-Age Subjects with Clustered Neurodegenerative Disease-Risk Factors.

In recent years a number of studies have reported the significant relationship between metabolic syndrome and neurodegenerative disease. There is accumulating evidence that the interplay of combined genetic and environmental risk factors (from diet to life style to pollutants) to intrinsic age-related oxi-inflammatory changes may be advocated for to explain the pandemic of neurodegenerative diseases. In recent years a specific Fermented Papaya Preparation (FPP) has been shown to significantly affect a number of redox signalling abnormalities in a variety of chronic diseases and as well in aging mechanisms either on experimental and on clinical ground. The aim of the present study was to evaluate FPP use in impending metabolic disease patients with potentially neurodegenerative disease clustered risk factors. The study population consisted of 90 patients aged 45–65 years old, with impending metabolic syndrome and previously selected as to be ApoE4 genotype negative. By applying a RCT, double-blind method, one group received FPP 4.5 g twice a day (the most common dosage utilized in prior clinical studies) while the other received an oral antioxidant cocktail (trans-resveratrol, selenium, vitamin E, vitamin C). Then, after 21 month treatment period, a selected heavy metal chelator was added at the dosage of 3 g/nocte for the final 3 months study treatment. The parameters tested were: routine tests oxidized LDL-cholesterol, anti-oxidised LDL, Cyclophilin-A (CyPA), plasminogen activator inhibitor-1 and CyPA gene expression. From this study it would appear that FPP, unlike the control antioxidant, significantly decreased oxidized-LDL and near normalizing the anti-Ox-LDL/Ox-LDL ratio (p<0.001) although unaffecting the lipid profile per sè. Moreover, only FPP decreased cyclophilin-A plasma level and plasminogen activator-inhibitor (p<0.01) together with downregulating cyclophilin-A gene expression (p<0.01). Insulin resistance was only mildly improved. Heavy metals gut clearance proved to be effectively enhanced by the chelator (p<0.01) and this was not affected by any of the nutraceuticals, nor it added any further benefit to the biological action of FPP.

MnTE-2-PyP modulates thiol oxidation in a hydrogen peroxide-mediated manner in a human prostate cancer cell

To improve the treatment of advanced prostate cancer, the development of effective and innovative antitumor agents is needed. Our previous work demonstrated that the ROS (reactive oxygen species) scavenger, MnTE-2-PyP, inhibited human prostate cancer growth and also inhibited prostate cancer migration and invasion. We showed that MnTE-2-PyP treatment altered the affinity of the histone acetyltransferase enzyme, p300, to bind to DNA. We speculate that this may be one mechanism by which MnTE-2-PyP inhibits prostate cancer progression. Specifically, MnTE-2-PyP decreased p300/HIF-1/CREB complex (p300/hypoxia-inducible factor-1/cAMP response element-binding protein) binding to a specific hypoxia-response element (HRE) motif within the plasminogen activator inhibitor-1 (PAI-1) gene promoter region, and consequently, repressed PAI-1 expression. However, it remains unclear how MnTE-2-PyP reduces p300 complex binding affinity to the promoter region of specific genes. In this study, we found that overexpression of Cu/ZnSOD (superoxide dismutase 1, SOD1) significantly suppressed PAI-1 gene expression and p300 complex binding to the promoter region of PAI-1 gene, just as was observed in cells treated with MnTE-2-PyP. Furthermore, catalase (CAT) overexpression rescued the inhibition of PAI-1 expression and p300 binding by MnTE-2-PyP. Taken together, the above findings suggest that hydrogen peroxide (H2O2) is likely the mediator through which MnTE-2-PyP inhibits the PAI-1 expression and p300 complex binding in PC3 cells. To confirm this, we measured the production of H2O2 following overexpression of SOD1 or catalase with MnTE-2-PyP treatment in the presence or absence of radiation. We found that MnTE-2-PyP increased the intracellular steady-state levels of H2O2 and increased nuclear H2O2 levels. As expected, catalase overexpression significantly decreased the levels of intracellular H2O2 induced by MnTE-2-PyP. We then determined if this increased H2O2 production could result in oxidized protein thiol groups. In the presence of MnTE-2-PyP, there was a significant increase in oxidized thiols in PC3 cell lysates and this was reversed with catalase overexpression. Specifically, we showed that p300 was oxidized after MnTE-2-PyP treatment, indicating that MnTE-2-PyP is creating a more oxidizing environment and this is altering the oxidation state of p300 thiol residues. Our data provide an in depth mechanism by which MnTE-2-PyP regulates gene transcription through induced H2O2 mediated oxidation of particular proteins, supporting an important role for MnTE-2-PyP as an effective and innovative antitumor agent to enhance treatment outcomes in prostate cancer radiotherapy.

Protective Role of PEDF-Derived Synthetic Peptide Against Experimental Diabetic Nephropathy.

Pigment epithelium-derived factor (PEDF) is a glycoprotein with complex neuroprotective, anti-angiogenic, and anti-inflammatory properties, all of which could potentially be exploited as a therapeutic option for vascular complications in diabetes. We have previously shown that PEDF-derived synthetic peptide, P5-3 (FIFVLRD) has a comparable ability with full PEDF protein to inhibit rat corneal neovascularization induced by chemical cauterization. However, the effects of PEDF peptide on experimental diabetic nephropathy remain unknown. To address the issue, we modified P5-3 to stabilize and administered the modified peptide (d-Lys-d-Lys-d-Lys-Gln-d-Pro-P5-3-Cys-amide, 0.2 nmol/day) or vehicle to streptozotocin-induced diabetic rats (STZ-rats) intraperitoneally by an osmotic mini pump for 2 weeks. We further examined the effects of modified peptide on human proximal tubular cells. Renal PEDF expression was decreased in STZ-rats. Although the peptide administration did not affect blood glucose or blood pressure, it decreased urinary excretion levels of 8-hydroxy-2'-deoxyguanosine, an oxidative stress marker, and reduced plasminogen activator inhibitor-1 (PAI-1) gene expression, and suppressed glomerular expansion in the diabetic kidneys. High glucose or advanced glycation end products stimulated oxidative stress generation and PAI-1 gene expression in tubular cells, all of which were significantly suppressed by 10 nM modified P5-3 peptide. Our present study suggests that PEDF-derived synthetic modified peptide could protect against experimental diabetic nephropathy and inhibit tubular cell damage under diabetes-like conditions through its anti-oxidative properties. Supplementation of modified P5-3 peptide may be a novel therapeutic strategy for diabetic nephropathy.

Activation of cardiac renin–angiotensin system and plasminogen activator inhibitor-1 gene expressions in oral contraceptive-induced cardiometabolic disorder

Context: Clinical studies have shown that combined oral contraceptive (COC) use is associated with cardiometabolic disturbances. Elevated renin–angiotensin system (RAS) and plasminogen activator inhibitor-1 (PAI-1) have also been implicated in the development of cardiometabolic events. Objective: To determine the effect of COC treatment on cardiac RAS and PAI-1 gene expressions, and whether the effect is circulating aldosterone or corticosterone dependent. Methods: Female rats were treated (p.o.) with olive oil (vehicle) or COC (1.0 µg ethinylestradiol and 10.0 µg norgestrel) daily for six weeks. Results: COC treatment led to increases in blood pressure, HOMA-IR, Ace1 mRNA, Atr1 mRNA, Pai1 mRNA, cardiac PAI-1, plasma PAI-1, C-reactive protein, uric acid, insulin and corticosterone. COC treatment also led to dyslipidemia, decreased glucose tolerance and plasma 17β-estradiol. Conclusion: These results demonstrates that hypertension and insulin resistance induced by COC is associated with increased cardiac RAS and PAI-1 gene expression, which is likely to be through corticosterone-dependent but not aldosterone-dependent mechanism.

Role of COX-2-derived PGE2 on vascular stiffness and function in hypertension.

Prostanoids derived from COX-2 and EP receptors are involved in vascular remodelling in different cardiovascular pathologies. This study evaluates the contribution of COX-2 and EP1 receptors to vascular remodelling and function in hypertension. Spontaneously hypertensive rats (SHR) and angiotensin II (AngII)-infused (1.44 mg · kg(-1) · day(-1), 2 weeks) mice were treated with the COX-2 inhibitor celecoxib (25 mg · kg(-1) · day(-1) i.p) or with the EP1 receptor antagonist SC19220 (10 mg · kg(-1) · day(-1) i.p.). COX-2(-/-) mice with or without AngII infusion were also used. Celecoxib and SC19220 treatment did not modify the altered lumen diameter and wall : lumen ratio in mesenteric resistance arteries from SHR-infused and/or AngII-infused animals. However, both treatments and COX-2 deficiency decreased the augmented vascular stiffness in vessels from hypertensive animals. This was accompanied by diminished vascular collagen deposition, normalization of altered elastin structure and decreased connective tissue growth factor and plasminogen activator inhibitor-1 gene expression. COX-2 deficiency and SC19220 treatment diminished the increased vasoconstrictor responses and endothelial dysfunction induced by AngII infusion. Hypertensive animals showed increased mPGES-1 expression and PGE2 production in vascular tissue, normalized by celecoxib. Celecoxib treatment also decreased AngII-induced macrophage infiltration and TNF-α expression. Macrophage conditioned media (MCM) increased COX-2 and collagen type I expression in vascular smooth muscle cells; the latter was reduced by celecoxib treatment. COX-2 and EP1 receptors participate in the increased extracellular matrix deposition and vascular stiffness, the impaired vascular function and inflammation in hypertension. Targeting PGE2 receptors might have benefits in hypertension-associated vascular damage.

Diet-Induced Podocyte Dysfunction in Drosophila and Mammals

Diabetic nephropathy is a major cause of end-stage kidney disease. Characterized by progressive microvascular disease, most efforts have focused on injury to the glomerular endothelium. Recent work has suggested a role for the podocyte, a highly specialized component of the glomerular filtration barrier. Here, we demonstrate that the Drosophila nephrocyte, a cell analogous to the mammalian podocyte, displays defects that phenocopy aspects of diabetic nephropathy in animals fed chronic high dietary sucrose. Through functional studies, we identify an OGT-Polycomb-Knot-Sns pathway that links dietary sucrose to loss of the Nephrin ortholog Sns. Reducing OGT through genetic or drug means is sufficient to rescue loss of Sns, leading to overall extension of lifespan. We demonstrate upregulation of the Knot ortholog EBF2 in glomeruli of human diabetic nephropathy patients and a mouse ob/ob diabetes model. Furthermore, we demonstrate rescue of Nephrin expression and cell viability in ebf2(-/-) primary podocytes cultured in high glucose.