Ultrasmall superparamagnetic iron oxide nanoparticles acutely promote thrombosis and cardiac oxidative stress and DNA damage in mice.

BACKGROUND: Ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) are being developed for several biomedical applications including drug delivery and imaging. However, little is known about their possible adverse effects on thrombosis and cardiac oxidative and DNA damage. METHODS: Presently, we investigated the acute (1 h) effect of intravenously (i.v.) administered USPIO in mice (0.4, 2 and 10 µg/kg). Diesel exhaust particles (DEP; 400 µg/kg) were used as positive control. RESULTS: USPIO induced a prothrombotic effect in pial arterioles and venules in vivo and increased the plasma plasminogen activator inhibitor-1 (PAI-1). Both thrombogenicity and PAI-1 concentration were increased by DEP. The direct addition of USPIO (0.008, 0.04 and 0.2 µg/ml) to untreated mouse blood dose-dependently induced in vitro platelet aggregation. USPIO caused a shortening of activated partial thromboplastin time (aPTT) and prothrombin time (PT). Similarly, DEP administration (1 µg/ml) triggered platelet aggregation in vitro in whole blood. DEP also reduced PT and aPTT. The plasma levels of creatine phosphokinase-MB isoenzyme (CK-MB), lactate dehydrogenase (LDH) and troponin-I were increased by USPIO. DEP induced a significant increase of CK-MB, LDH and troponin I levels in plasma. The cardiac levels of markers of oxidative stress including lipid peroxidation, reactive oxygen species and superoxide dismutase activity were increased by USPIO. Moreover, USPIO caused DNA damage in the heart. Likewise, DEP increased the markers of oxidative stress and induced DNA damage in the heart. CONCLUSION: We conclude that acute i.v. administration of USPIO caused thrombosis and cardiac oxidative stress and DNA damage. These findings provide novel insight into the pathophysiological effects of USPIO on cardiovascular homeostasis, and highlight the need for a thorough evaluation of their toxicity.

Functional relevance of D,L-sulforaphane-mediated induction of vimentin and plasminogen activator inhibitor-1 in human prostate cancer cells.

PURPOSE: D,L-Sulforaphane (SFN) is a promising chemopreventive agent with in vivo efficacy against prostate cancer in experimental rodents. This study was undertaken to determine the role of vimentin and plasminogen activator inhibitor-1 (PAI-1) in anticancer effects of SFN. METHODS: Effect of SFN on levels of different proteins was determined by Western blotting or immunofluorescence microscopy. RNA interference of vimentin and PAI-1 was achieved by transient transfection. Apoptosis was quantified by flow cytometry. Transwell chambers were used to determine cell migration. RESULTS: Exposure of PC-3 and DU145 human prostate cancer cells to SFN resulted in induction of vimentin protein, which was accompanied by down-regulation of E-cadherin protein expression. The SFN-mediated induction of vimentin was also observed in a normal human prostate epithelial cell line. RNA interference of vimentin did not have any appreciable effect on early or late apoptosis resulting from SFN exposure. On the other hand, SFN-mediated inhibition of PC-3 and DU145 cell migration was significantly augmented by knockdown of the vimentin protein. Knockdown of vimentin itself was inhibitory against cell migration. The SFN-treated cells also exhibited induction of PAI-1, which is an endogenous inhibitor of urokinase-type plasminogen activator system. Similar to vimentin, PAI-1 knockdown resulted in a modest augmentation of PC-3 cell migration inhibition by SFN. Tumors from SFN-treated transgenic adenocarcinoma of mouse prostate mice showed a 1.7-fold increase in vimentin protein level compared with control tumors. CONCLUSION: The present study indicates that vimentin and PAI-1 inductions confer modest protection against SFN-mediated inhibition of prostate cancer cell migration.

Impact of lysophosphatidylcholine on the plasminogen activator system in cultured vascular smooth muscle cells.

The balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolysis. PAI-1 expression increases in atherosclerotic arteries and vascular smooth muscle cells (VSMCs) are one of major constituents of atheroma. We investigated the impact of lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, on the plasminogen activator system of the rat VSMCs. The lysoPC stimulated the protein and gene expressions of PAI-1 but did not affect the protein expression of t-PA. Fibrin overlay zymography revealed that lysoPC increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA. Vitamin E inhibited the lysoPC-induced PAI-1 expression. Further, lysoPC increased the intracellular reactive oxygen species (ROS) formation. Caffeic acid phenethyl ester, an inhibitor of NF-κB, blocked this lysoPC effect. Indeed, lysoPC induced the NF-κB-mediated transcriptional activity as measured by luciferase reporter assay. In addition, genistein, an inhibitor of protein-tyrosine kinase (PTK), diminished the lysoPC effect, while 7,12-dimethylbenz[a]anthracene, a stimulator of PTK, stimulated PAI-1 production. In conclusion, lysoPC does not affect t-PA expression but induces PAI-1 expression in the VSMC by mediating NF-κB and the genistein-sensitive PTK signaling pathways via oxidative stress. Importantly, lysoPC stimulates the enzyme activity of PAI-1 and suppresses that of t-PA.

Regulation of tissue plasminogen activator/plasminogen activator inhibitor-1 by hydrocortisone in rat primary astrocytes.

Although originally known as a plasma serine protease involved in clot dissolution, tPA and its primary inhibitor, PAI-1, play crucial roles in synaptic reorganization and plasticity in the central nervous system. In contrast to the wide array of work conducted in neural cells, relatively little is known about the regulatory mechanism governing tPA/PAI-1 expression in astrocytes. Glucocorticoids (GCs) such as hydrocortisone regulate the expression of tPA/PAI-1 in various biological systems in a tissue-specific manner. However, little is known about GC-mediated regulation of tPA/PAI-1 system in CNS. The aims of the present study were to investigate whether tPA/PAI-1 expression is regulated by hydrocortisone in rat primary astrocytes. Enzyme activity of tPA was decreased in a concentration-dependent manner by hydrocortisone treatment, and the activity of PAI-1 was increased by hydrocortisone. Hydrocortisone did not affect the level of tPA mRNA, which suggests that transcriptional down-regulation of tPA mRNA is not involved in the down-regulation of tPA enzyme activity in astrocytes. However, the level of PAI-1 mRNA and protein was increased. Both hydrocortisone and a tPA-Stop treatment prevented glutamate-induced neurotoxicity in rat cortical primary mixed astrocyte-neuron culture, which suggests a neurotoxic role for tPA in our culture system. Interestingly, hydrocortisone further increased LPS-induced up-regulation of PAI-1 while inhibiting the up-regulation of iNOS and COX-2 expression. Our data show that hydrocortisone up-regulated PAI-1 expression along with down-regulation of tPA activity in both normal and inflammatory conditions.

Plasminogen activation is required for the development of radiation-induced dermatitis.

Skin damage caused by radiation therapy (radiodermatitis) is a severe side effect of radiotherapy in cancer patients, and there is currently a lack of effective strategies to prevent or treat such skin damage. In this work, we show with several lines of evidence that plasminogen, a pro-inflammatory factor, is key for the development of radiodermatitis. After skin irradiation in wild-type (plg+/+) mice, the plasminogen level increased in the irradiated area, leading to severe skin damage such as ulcer formation. However, plasminogen-deficient (plg-/-) mice and mice lacking plasminogen activators were mostly resistant to radiodermatitis. Moreover, treatment with a plasminogen inhibitor, tranexamic acid, decreased radiodermatitis in plg+/+ mice and prevented radiodermatitis in plg+/- mice. Together with studies at the molecular level, we report that plasmin is required for the induction of inflammation after irradiation that leads to radiodermatitis, and we propose that inhibition of plasminogen activation can be a novel treatment strategy to reduce and prevent the occurrence of radiodermatitis in patients.

Regulatory roles of miR-155 and let-7b on the expression of inflammation-related genes in THP-1 cells: effects of fatty acids.

The main aim of this investigation was to study the regulatory roles of let-7b and miR-155-3p on the expression of inflammation-associated genes in monocytes, macrophages, and lipopolysaccharide (LPS)-activated macrophages (AcM). A second goal was to analyze the potential modulatory roles of different fatty acids, including oleic, palmitic, eicosapentaenoic (EPA), and docosahexaenoic (DHA), on the expression of these miRNAs in the three cell types. This hypothesis was tested in human acute monocytic leukemia cells (THP-1), which were differentiated into macrophages with 2-O-tetradecanoylphorbol-13-acetate (TPA) and further activated with LPS for 24 h. Monocytes, macrophages, and AcM were transfected with a negative control, or mimics for miR-155-3p and miR-let-7b-5p. The expression of both miRNAs and some proinflammatory genes was analyzed by qRT-PCR. Interestingly, let-7b mimic reduced the expression of IL6 and TNF in monocytes, and SERPINE1 expression in LPS-activated macrophages. However, IL6, TNF, and SERPINE1 were upregulated in macrophages by let-7b mimic. IL6 expression was higher in the three types of cells after transfecting with miR-155-3p mimic. Similarly, expression of SERPINE1 was increased by miR-155-3p mimic in monocytes and macrophages. However, TLR4 was downregulated by miR-155-3p in monocytes and macrophages. Regarding the effects of the different fatty acids, oleic acid increased the expression of let-7b in macrophages and AcM and also increased the expression of miR-155 in monocytes when compared with DHA but not when compared with non-treated cells. Overall, these results suggest anti- and proinflammatory roles of let-7b and miR-155-3p in THP-1 cells, respectively, although these outcomes are strongly dependent on the cell type. Noteworthy, oleic acid might exert beneficial anti-inflammatory effects in immune cells (i.e., non-activated and LPS-activated macrophages) by upregulating the expression of let-7b.

High glucose induces inflammatory responses in HepG2 cells via the oxidative stress-mediated activation of NF-κB, and MAPK pathways in HepG2 cells.

OBJECTIVE: The aim of this study was to investigate the effects of high glucose (HG) on inflammation in HepG2 cells. METHODS: The molecular mechanisms linking HG to inflammation was assessed in HepG2 cells exposed to HG (33 mM). RESULTS: The results showed that HG significantly enhanced TNF-α, IL-6 and PAI-1 expression in HepG2 cells. Increased expression of cytokines was accompanied by enhanced phosphorylation of JNK, P38, ERK and IKKα/IKKß. In addition, JNK, ERK, P38 and NF-kB inhibitors could significantly attenuate HG-induced expression of TNF-α, IL-6 and PAI-1. Furthermore, HG could promote the generation of reactive oxygen species (ROS), while N-acetyl cysteine, a ROS scavenger, had an inhibitory effect on the expression of TNF-α, IL-6 and PAI-1 in HG-treated cells. CONCLUSIONS: Our results indicated that HG-induced inflammation is mediated through the generation of ROS and activation of the MAPKs and NF-kB signalling pathways in HepG2 cells.

PAI-1 is a critical regulator of FGF23 homeostasis.

Elevated levels of fibroblast growth factor 23 (FGF23), a bone-derived phosphaturic hormone, are associated with a number of pathologic conditions including chronic kidney disease, cardiac hypertrophy, and congestive heart failure. Currently, there are no specific treatments available to lower plasma FGF23 levels. We have recently reported that genetic plasminogen activator inhibitor-1 (PAI-1) deficiency provided a significant reduction in circulating FGF23 levels while simultaneously prolonging the life span of Klotho-deficient mice. We extend our investigations into the effect of PAI-1 on FGF23 homeostasis. Transgenic overexpression of PAI-1 resulted in threefold increase in FGF23 levels compared to wild-type littermates. Moreover, pharmacological modulation of PAI-1 activity with the small-molecule PAI-1 antagonist TM5441 significantly reduced FGF23 levels in PAI-1 transgenic and Klotho-deficient mice. In addition, TM5441 treatment or PAI-1 deficiency significantly accelerated the clearance of endogenous FGF23 and recombinant human FGF23 from circulation in mice with acute kidney injury. On the basis of these observations, we studied the effects of plasminogen activators (PAs), tissue-type PA (tPA) and urokinase-type PA (uPA), on FGF23. We demonstrate that both PAs directly cleave FGF23; however, it is not known whether the PA-generated FGF23 peptides retain or acquire functions that affect binding and/or signaling properties of intact FGF23. PAI-1 inhibits the PA-dependent cleavage of FGF23, and TM5441 inhibition of PAI-1 restores the proteolysis of FGF23. Furthermore, top-down proteomic analysis indicates that tPA cleaves FGF23 at multiple arginines including the proconvertase sensitive site R176. In summary, our results indicate that PAI-1 prevents the PA-driven proteolysis of FGF23 and PAI-1 inhibition provides a novel therapeutic approach to prevent the pathologic consequences of increased FGF23.

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 17ß-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.

The mechanism and significance of synergistic induction of the expression of plasminogen activator inhibitor-1 by glucocorticoid and transforming growth factor beta in human ovarian cancer cells.

Plasminogen activator inhibitor-1 (PAI-1) plays a key role in tissue remodeling and tumor development by suppression of plasminogen activator function. Glucocorticoids (GCs) and transforming growth factor beta (TGF-ß) signal pathways cross-talk to regulate gene expression, but the mechanism is poorly understood. Here we investigated the mechanism and significance of co-regulation of PAI-1 by TGF-ß and dexamethasone (DEX), a synthetic glucocorticoid in ovarian cancer cells. We found that TGF-ß and DEX showed rapidly synergistic induction of PAI-1 expression, which contributed to the early pro-adhesion effects. The synergistic induction effect was accomplished by several signal pathways, including GC receptor (GR) pathway and TGF-ß-activated p38MAPK, ERK and Smad3 pathways. TGF-ß-activated p38MAPK and ERK pathways cross-talked with GR pathway to augment the expression of PAI-1 through enhancing DEX-induced GR phosphorylation at Ser211 in ovarian cancer cells. These findings reveal possible novel mechanisms by which TGF-ß pathways cooperatively cross-talk with GR pathway to regulate gene expression.

Relation of fibrinolytic potentiation by estrogen to coagulation pathway activation in postmenopausal women.

To determine whether enhanced fibrinolysis is a primary effect of estrogen or is secondary to activation of coagulation, we measured hemostatic factors before and after conjugated equine estrogen 0.625 mg/day for 1 month in 9 postmenopausal women. We found that potentiation of fibrinolysis is a primary effect of conjugated equine estrogens at this commonly used dosage in healthy postmenopausal women.

Synergistic augmentation of expression of plasminogen activator inhibitor type-1 induced by insulin, very-low-density lipoproteins, and fatty acids.

BACKGROUND: Plasminogen activator inhibitor type-1 (PAI-1) implicated as a determinant of thrombosis, and possibly of atherosclerosis, is increased approximately four-fold in the blood of subjects with type II diabetes, and the increase is closely correlated with concentrations of insulin. Insulin can stimulate expression of PAI-1 in vitro, but infusions of insulin in human subjects have not increased PAI-1 in blood in acute, euglycemic clamp studies. OBJECTIVE: To determine whether the dichotomy reflects interactions between insulin and both very-low-density lipoprotein (VLDL)-bound triglycerides (VLDL-TG) and albumin-bound nonesterified (free) fatty acids (FFA) affecting PAI-1 elaboration. METHODS: HepG2 cells (human hepatoma cell line) were exposed to insulin, VLDL, and FFA alone and in selected combinations for 24 h. RESULTS: Striking synergistic effects were observed. Thus, compared with control, pathophysiologic concentrations of insulin increased PAI-1 accumulation in conditioned media modestly, as did VLDL, but the combination elicited a marked nine-fold increase (control PAI-130 +/- 2 ng/ml, PAI-1 with 400 mg/dl VLDL-TG 97 +/- 6 ng/ml; with 4 nmol/1 insulin 45 +/- 6 ng/ml, with 400 mg/dl VLDL-TG plus 4 nmol/1 insulin 276 +/- 47 ng/ml; P < 0.01 for the combination compared with control or with either agent alone). Similarly, a modest increase was found either with insulin or with FFA alone, but a synergistic increase was evident when they were combined (control 27 +/- 3 ng/ml; 1 mmol/l FFA 36 +/- 2 ng/ml; 10 nmol/l insulin 59 +/- 6 ng/ml; 1 mmol/l FFA plus 10 nmol/l insulin 82 +/- 1 ng/ml; P < 0.01 for the combination compared with control or with either agent alone). CONCLUSION: The combination of increased insulin with elevated VLDL-TG and increased FFA appears to cause the increase in PAI-1 in blood of subjects with type II diabetes mellitus and other insulin-resistant states, providing novel and promising targets for normalizing altered fibrinolytic system potential and thereby ameliorating the associated predisposition to persistent thrombosis and possibly atherosclerosis.

Dietary trans fats enhance doxorubicin-induced cardiotoxicity in mice.

This study investigated the combined effects of trans fat diet (TFD) and doxorubicin upon cardiac oxidative, inflammatory, and coagulatory stress. TFD increased trans fatty acid deposit in heart (P < 0.05), and decreased protein C and antithrombin-III activities in circulation (P < 0.05). TFD plus doxorubicin treatment elevated activities of plasminogen activator inhibitor-1, lactate dehydrogenase, and creatine phosphokinase (P < 0.05). This combination also raised xanthine oxidase activity, and enhanced cardiac levels of reactive oxygen species, interleukin (IL)-6, IL-10, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 than TFD or doxorubicin treatment alone (P < 0.05). TFD alone increased cardiac nuclear factor kappa B (NF-κB) activity (P < 0.05), but failed to affect expression of NF-κB and mitogen-activated protein kinase (MAPK) (P > 0.05). Doxorubicin treatment alone augmented cardiac activity, mRNA expression, and protein production of NF-κB and MAPK (P < 0.05). TFD plus doxorubicin treatment further upregulated cardiac expression of NF-κB p65, p-p38, and p-ERK1/2 (P < 0.05). These findings suggest that TFD exacerbates doxorubicin-induced cardiotoxicity.

Combined TGE-SGE expression of novel PAI-1-resistant t-PA in CHO DG44 cells using orbitally shaking disposable bioreactors.

An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncatedmutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncatedmutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.

C5a stimulates production of plasminogen activator inhibitor-1 in human mast cells and basophils.

We have recently shown that resting human mast cells (MCs) produce tissue-type plasminogen activator (t-PA) without simultaneously expressing plasminogen activator inhibitor 1 (PAI-1). In the present study we have identified the anaphylatoxin rhC5a as a potent inducer of PAI-1 expression in human MCs and basophils. In primary human skin MCs and primary blood basophils, exposure to rhC5a was followed by an increase from undetectable to significant levels of PAI-1. In addition, rhC5a induced a concentration- and time-dependent increase in PAI-1 antigen in the MC line HMC-1 and the basophil cell line KU-812 and increased the expression of PAI-1 mRNA in HMC-1. In conditioned media of HMC-1 treated with rhC5a, active PAI-1 could be detected. A simultaneous loss of t-PA activity in conditioned media from the same cells indicated that rhC5a-induced PAI-1 was capable of inhibiting the enzymatic activity of coproduced t-PA. Correspondingly, the levels of t-PA-PAI-1 complexes increased in rhC5a-treated cells. When HMC-1 cells were incubated with pertussis toxin or anti-C5a receptor antibodies, the effect of rhC5a on PAI-1 production was completely abolished. Treatment of C5a with plasmin resulted in loss of its ability to induce PAI-1 production in MCs. Considering the suggested role for MCs and components of the complement system in the development of cardiovascular diseases, we hypothesize that MCs, by producing t-PA in a resting state and by expressing PAI-1 when activated by C5a, might participate in the modulation of the balance between proteases and protease inhibitors regulating tissue injury and repair in such disease processes.

Effects of steroid hormones on fibrinolytic system in cultured human endometrial cells.

In a primary human endometrial cell culture, the addition of progesterone resulted in an approximately 2-fold increase in the amount of tissue-type plasminogen activator (t-PA) released into the culture media, with the minimal effective dose being 10(-7) M. In contrast, progesterone significantly reduced the release of urokinase-type PA (u-PA). Endometrial cells are known to release a major PA inhibitor, PAI-1. Progesterone stimulated the release of PAI-1. These observed effects of progesterone seem to be mediated through the progestin receptor in that R5020, a specific ligand for progestin receptor, mimicked the effects of progesterone, and RU486, an antagonist of progesterone, completely eliminated the effects of progesterone. It is notable that estradiol, when added alone or in combination with progesterone, caused no discernible effect on the release of PAs and PAI-1. These results suggest that progesterone is a key hormone in regulating the PA/plasmin system in the human endometrium, thereby playing a pivotal role in implantation and ensuing embryonal development.