Synthesis Of Plasminogen Activator Research Articles

Effects of Ghrelin on Plasminogen Activator Activity in Human Umbilical Vein Endothelial Cells

Ghrelin and its growth hormone secretagogue receptor (GHSR) have been found in the placenta, both in endothelial and trophoblast cells. Ghrelin has been shown to decrease blood pressure in several systems and improve endothelial function by stimulating VEGF production. Because locally increased Ghrelin was detected in the preeclamptic fetoplacental unit, we hypothesized its involvement in the fibrinolysis and vascular tone typically observed in preeclamptic patients. This study aimed to evaluate the synthesis of plasminogen activators (PAs), PA inhibitor-1 (PAI-1), and urokinase-type PA (uPA) receptor (uPAR) in human umbilical vein endothelial cells (HUVECs) since the components of the PA/plasmin system are vital players in the extracellular matrix remodeling process necessary for angiogenesis. HUVECs were treated for 24 h with increasing concentrations of Ghrelin (10−11–10−7 M) or IL-1β (0.1 ng/mL). PAs, PAI-1, and uPAR mRNAs were determined by real-time PCR and PA activity was determined by casein underlay. We demonstrated an increase in uPA, tissue-type PA (tPA), and uPAR mRNA; a reduction in PAI-1 mRNA in HUVECs treated with Ghrelin; and an increase in total uPA activity. In conclusion, our results suggest a potential compensatory physiological mechanism for Ghrelin in response to the maternal endothelial dysfunction observed in the preeclamptic fetoplacental unit.

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.

Aging Is Associated with a Circulating Endothelial Cell-Derived Microvesicle Phenotype that Impairs Cerebral Fibrinolytic Capacity

Aging is associated with increased risk and prevalence of ischemic stroke. Endothelial cell dysfunction is considered to be a central mechanism underlying the age-related increase in cerebrovascular accidents (CVA). However, the direct, and indirect, effects of aging on endothelial cell biology are diverse and not completely understood. Reduced endogenous fibrinolytic activity is a central etiologic factor underlying ischemic stroke. Endothelial cells are the primary site of synthesis and release of tissue-type plasminogen activator (t-PA), the main plasminogen activator in fibrinolysis and central regulator. Clinical interest in circulating extracellular vesicles, particularly endothelial cell-derived microvesicles (EMVs), has intensified due to their involvement in the development and progression of endothelial dysfunction and CVA. The aim of this study was to determine, in vitro, the effect of circulating EMVs isolated from young and older adults on brain endothelial cell fibrinolytic capacity. We hypothesized that EMVs from older adults would reduce brain endothelial cell t-PA production and release and increase PAI-1 production compared with EMVs from young adults. Twenty-two healthy, non-obese, normotensive, sedentary adults were studied: 10 young (6M/4F; age: 25±1 yr) and 12 older (7M/5F; 64±2 yr). EMV identification (CD144 + ) and isolation from peripheral blood were performed by flow cytometry. Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject. Cultured hCMECs were treated with EMVs in the absence and presence of thrombin (1 unit/mL) for 24 hours. Basal expression of t-PA (25.2±1.5 vs 39.8±1.8 AU) and the t-PA response to thrombin (32.5±1.5 vs 57.2±2.2 AU) were significantly lower (~35%) in hCMECs treated with EMVs from older compared with young adults. Concordant with changes in cell protein expression, basal t-PA production (55.2±3.5 vs 70.1±3.7 ng/mL) and thrombin-stimulated t-PA production (61.9±3.7 vs 88.4±3.8 ng/mL) was significantly lower in cells treated with EMVs from older adults. In conclusion, circulating EMVs may contribute to the increased risk of ischemic stroke with aging by impairing brain endothelial cell fibrinolytic capacity. Diminished capacity of the endothelium to release t-PA is a key mechanistic factor underlying cerebral thrombosis. Nothing to Disclose This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of polyamines in the regenerative process of skin aerobic-purulent wounds

Aliphatic polyamines (putrescine, spermidine, spermine) are organic polycations that play an important role in wound healing by stimulating several cellular mechanisms. In a human skin wound sample, the activity of the enzyme ornithine decarboxylase, which regulates the rate of polyamine synthesis, rapidly increases along the wound edges and leads to the activation of the polyamine synthesis cascade. Under the influence of polyamines, some signaling systems are also activated in wounds, which are the main pathways for the release of cellular mechanisms, and thanks to them, the healing process begins in wounds. For example, spermine induces the synthesis of urokinase-type plasminogen activator, the binding of which to the corresponding receptor at the wound margins executes the urokinase-type plasminogen activator and its receptor signaling system, which is the main driver of keratinocyte migration. Eukaryotic cell proliferation depends on precise modification of the eukaryotic initiation factor 5A1, in which spermidine plays an indispensable role. However, in addition to the significant functions performed by polyamines in the human body, polyamines are also necessary for the normal growth and development of fungi and bacteria. Small amounts of some microorganisms have a positive effect on the healing of wounds, but their increase, on the contrary, leads to the impairment of the normal course of wound healing due to their enhanced synthesis of polyamines. On the other hand, many studies show that excess ornithine decarboxylase and polyamines increase the risk of skin cancer. Suppression of polyamine synthesis by pathogenic microflora during wound healing can contribute to both rapid healing and the prevention of skin cancer. In our study, we offer a way of inhibition of polyamine synthesis by wound microflora for rapid wound healing and prevention of subsequent cancer. The medicinal mixture “Armenicum/Eflornithine” is a mixture of the drug “Armenicum” and α-difluoromethylornithine.

Uremic serum induces prothrombotic changes in venous endothelial cells and inflammatory changes in aortic endothelial cells

Background Uremia induces various pathologic changes in the endothelium. However, there is limited information about the differences of these effects in endothelial cells originating from different parts of the vascular tree. Methods The effect of uremic serum obtained from patients with end stage renal failure on the gene expression and secretory activity of venous endothelial cells (VEC) and aortic endothelial cells (AEC) was studied in in vitro culture. Results In VEC, the expression of genes regulating the synthesis of von Willebrand factor (vWF) was increased by 254% (p<.005), vascular endothelial growth factor (VEGF) synthesis by 150% (p<.001), tissue plasminogen activator (t-PA) synthesis by 62% (p<.005), platelet endothelial cell adhesion molecule by 89% (p<.005), and the expression of gene regulating interleukin-6 (IL-6) synthesis was reduced. In AEC, the expression of the gene regulating synthesis of IL-6 was increased by 174% (p<.001), and the expression of the other genes was reduced. The secretion of IL-6 was reduced in VEC by 38% (p<.01) and increased in AEC by 55% (p<.005). In VEC, increased synthesis of VEGF 64% (p<.001) vWF (+34%, p<.01), and t-PA (+53%, p<.002) was observed, and in AEC it was reduced. Conclusions VEC and AEC respond in different ways after exposure to uremic serum. VEC acquires the prothrombotic phenotype, whereas in AEC the inflammatory phenotype appears.

N-acetylcysteine modulates effect of the iron isomaltoside on peritoneal mesothelial cells.

Intravenous (i.v.) iron supplementation is used in patients on chronic peritoneal dialysis (pd). Iron induced intraperitoneal inflammation observed in our previous studies with iron sucrose may deteriorate the function of the peritoneum as the dialysis membrane. We evaluated effect iron compound, iron-isomaltoside-100 (IIS) on the peritoneal mesothelial cells (MC). We studied the effect of iv treatment with IIS ± N-acetylcysteine (NAC) on the dialysate parameters and function of MC. In 7 uremic pd patients IIS 200 mg was infused i.v. ± NAC 600 mg. Afterward, a 4 hours exchange was performed with Dianeal 1.5%. As a control dialysate exchange preceding IIS treatment was used. Inflammatory parameters of the drained dialysates as well as the dialysates and IIS effects on MC were evaluated in ex vivo experiments. Intravenous infusion of IIS resulted in an increase of the dialysate Fe (+147%, P < 0.01). Concentrations of the dialysates inflammatory mediators were increased: interleukin-6 (IL-6) +39%, P < 0.02, monocyte chemoattractant protein-1(MCP1) +50%, P < 0.02, and hyaluronan (HA) +64%, P < 0.02. Simultaneous i.v. infusion of NAC prevented increase of the dialysate inflammatory mediators. Dialysates collected after IIS treatment induced oxidative stress in MC (+29%, P < 0.05) and stimulated IL-6 synthesis (+64%, P < 0.05) in MC; no such effect was seen in dialysates obtained after simultaneous IIS and NAC i.v. treatment. IIS used as the additive to culture medium stimulated synthesis in MC of IL6 (+76%, P < 0.001) and plasminogen activator inhibitor-1 (PAI-1) (28%, P < 0.001) whereas synthesis of tissue plasminogen activator (t-PA) was reduced (-16%, P < 0.001). These changes were prevented in the presence of NAC 1 mmol/L. Intravenous administration of IIS results in the mild stimulation of intraperitoneal inflammation. IIS changes MC phenotype to the inflammatory one with reduced fibrinolytic activity. These effects are prevented by NAC.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a negative regulator of tissue plasminogen activator synthesis in cultured human vascular endothelial EA.hy926 cells.

Blood coagulation and the fibrinolytic system contribute to vascular lesions. Fibrinolysis in normal circulating blood strongly depends on the balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) secreted from vascular endothelial cells; however, the mechanisms by which endothelial fibrinolysis is regulated remain to be fully understood. In the present study, human vascular endothelial EA.hy926 cells were transfected with small interfering RNA for nuclear factor erythroid 2-related factor 2 (NRF2) and the expression of t-PA and PAI-1 and fibrinolytic activity in the conditioned medium were examined. EA.hy926 cells were also treated with sulforaphane, an NRF2 activator, and fibrinolytic activity was examined to confirm the NRF2 signaling pathway's effect. Enhanced fibrinolytic activity in the conditioned medium was observed in association with increased expression and secretion levels of t-PA in NRF2 knockdown EA.hy926 cells. However, sulforaphane inhibited fibrinolytic activity and t-PA synthesis in EA.hy926 cells without any cell damage. The expression level of PAI-1 did not change in either NRF2 knockdown or sulforaphane treated cells. These results suggest that transcription factor NRF2 may play a role in down-regulating endothelial t-PA synthesis and fibrinolytic activity.

Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator

Stroke is the leading cause of death with over 5 million deaths worldwide each year. About 80% of strokes are ischemic strokes caused by blood clots. Tissue plasminogen activator (tPa) is the only FDA-approved drug to treat ischemic stroke with a wholesale price over $6000. tPa is now off patent although no biosimilar has been developed. The production of tPa is complicated by the 17 disulfide bonds that exist in correctly folded tPA. Here, we present an Escherichia coli-based cell-free protein synthesis platform for tPa expression and report conditions which resulted in the production of active tPa. While the activity is below that of commercially available tPa, this work demonstrates the potential of cell-free expression systems toward the production of future biosimilars. The E. coli-based cell-free system is increasingly becoming an attractive platform for low-cost biosimilar production due to recent developments which enable production from shelf-stable lyophilized reagents, the removal of endotoxins from the reagents to prevent the risk of endotoxic shock, and rapid on-demand production in hours.

Synthesis and Characterization of Tissue Plasminogen Activator-Functionalized Superparamagnetic Iron Oxide Nanoparticles for Targeted Fibrin Clot Dissolution.

Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted great attention in many biomedical fields and are used in preclinical/experimental drug delivery, hyperthermia and medical imaging. In this study, biocompatible magnetite drug carriers, stabilized by a dextran shell, were developed to carry tissue plasminogen activator (tPA) for targeted thrombolysis under an external magnetic field. Different concentrations of active tPA were immobilized on carboxylated nanoparticles through carbodiimide-mediated amide bond formation. Evidence for successful functionalization of SPIONs with carboxyl groups was shown by Fourier transform infrared spectroscopy. Surface properties after tPA immobilization were altered as demonstrated by dynamic light scattering and ζ potential measurements. The enzyme activity of SPION-bound tPA was determined by digestion of fibrin-containing agarose gels and corresponded to about 74% of free tPA activity. Particles were stored for three weeks before a slight decrease in activity was observed. tPA-loaded SPIONs were navigated into thrombus-mimicking gels by external magnets, proving effective drug targeting without losing the protein. Furthermore, all synthesized types of nanoparticles were well tolerated in cell culture experiments with human umbilical vein endothelial cells, indicating their potential utility for future therapeutic applications in thromboembolic diseases.

Copper diethyldithiocarbamate as an inhibitor of tissue plasminogen activator synthesis in cultured human coronary endothelial cells.

Recent developments have shown that organic-inorganic hybrid molecules have the potential to provide useful tools for analyzing biological systems. In the case of fibrinolysis, which is the phenomenon whereby fibrin is degraded by plasmin that has been converted from plasminogen via tissue plasminogen activator (t-PA) secreted from vascular endothelial cells, we hypothesized that there may be organic-inorganic hybrid molecules that could be used to analyze the mechanisms by which endothelial fibrinolysis is regulated. In our present study, we found that a copper complex - copper diethyldithiocarbamate (Cu10) - reduces t-PA activity in a conditioned medium of cultured human coronary endothelial cells by inhibiting the t-PA synthesis without changing the synthesis of plasminogen activator inhibitor type 1, which is a t-PA inhibitor. Copper sulfate, the Cu10 ligand, and zinc/iron complexes with the same Cu10 ligand, did not exhibit such biological activity. These results indicate that Cu10 has the potential to provide a useful tool for finding alternative pathways that downregulate endothelial t-PA synthesis.

Abstract 570: Long-term efficacy and downstream mechanism of anti-Annexin A2 monoclonal antibody in a pre-clinical model of aggressive human breast cancer

Abstract Abstract: There are considerable direct evidences to support that calcium binding protein ANX A2 is a potential target for aggressive breast cancer. The most compelling evidence is based on the fact that ANX A2 overexpress specifically in aggressive triple negative human breast cancer (TNBC) cell lines as well as in human breast cancer tissues. Previously, we and others have demonstrated a unique role of ANX A2 in cancer invasion including breast cancer. Moreover we demonstrated that anti-ANX A2 monoclonal antibody (anti-ANX A2mAb)-mediated immunoneutralization of ANX A2 inhibited invasive human breast growth in a xenograft model. We further evaluated long-term effect of multiple treatments of anti-ANX A2mAb and its mechanism of inhibition of human breast tumor growth. We now show that three treatments of anti-ANX A2mAb showed significant inhibition of breast tumor growth in immunodeficient mice and anti-tumor response was started from day 94. After treatments, we followed tumor growth for 172 days and our results demonstrated 67% inhibition of tumor growth without detectable adverse effects. Biochemical analysis demonstrated anti-ANX A2mAb treatment caused significant inhibition of tissue plasminogen activator (tPA) synthesis in the tumor microenvironment. This led to disruption of plasmin generation that consequently inhibited activation of MMP-9 and MMP-2. These results suggest that ANX A2 plays an important role in aggressive breast tumor growth by regulating proteolytic pathway in the tumor microenvironment. ANX A2 may represent a new target for the development of therapeutics for the treatment of aggressive breast cancer. Citation Format: Mahesh C. Sharma, George P. Tuszynski, Marc R. Blackman, Meena Sharma. Long-term efficacy and downstream mechanism of anti-Annexin A2 monoclonal antibody in a pre-clinical model of aggressive human breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 570.

Endogenous plasminogen activators mediate progressive intracerebral hemorrhage after traumatic brain injury in mice

AbstractPersistent intracerebral hemorrhage (ICH) is a major cause of death and disability after traumatic brain injury (TBI) for which no medical treatment is available. Delayed bleeding is often ascribed to consumptive coagulopathy initiated by exposed brain tissue factor. We examined an alternative hypothesis, namely, that marked release of tissue-type plasminogen activator (tPA) followed by delayed synthesis and release of urokinase plasminogen activator (uPA) from injured brain leads to posttraumatic bleeding by causing premature clot lysis. Using a murine model of severe TBI, we found that ICH is reduced in tPA−/− and uPA−/− mice but increased in PAI-1−/− mice compared with wild-type (WT) mice. tPA−/−, but not uPA−/−, mice developed a systemic coagulopathy post-TBI. Tranexamic acid inhibited ICH expansion in uPA−/−mice but not in tPA−/− mice. Catalytically inactive tPA-S481A inhibited plasminogen activation by tPA and uPA, attenuated ICH, lowered plasma d-dimers, lessened thrombocytopenia, and improved neurologic outcome in WT, tPA−/−, and uPA−/− mice. ICH expansion was also inhibited by tPA-S481A in WT mice anticoagulated with warfarin. These data demonstrate that protracted endogenous fibrinolysis induced by TBI is primarily responsible for persistent ICH and post-TBI coagulopathy in this model and offer a novel approach to interrupt bleeding.

THE EFFECTS OF DIPICOLINIC ACID ON THE THROMBOLYTIC ACTIVITY OF HUMAN CELLS

Dipicolinic acid [2,6-pyridinedicarboxylic acid (DPA)] is present in the spores of the bacteria belonging to the Bacillus genus. Relatively large amounts of DPA are present in natto, a traditional Japanese food consisting of fermented soybeans. The daily average intake of DPA derived from natto is 0.6–4.0 mg in the Japanese people. We have already reported that one of the physiological properties of DPA is that it inhibits platelet aggregation. In the current study, we confirmed that DPA promotes the synthesis of tissue-type plasminogen activator (t-PA), the thrombolytic enzyme secreted by human cells. Incubation of cells with 5 mM DPA resulted in a peak increase in the amount of t-PA secretion to a level that was 46.7 times that of the control. Experiments conducted using eight varieties of DPA derivatives showed that such functions were specific to DPA alone. PRACTICAL APPLICATIONS It is known that nattokinase produced by Bacillus subtilis natto directly dissolves fibrin and is used for thrombosis prevention. It is thought that the thrombus-dissolution activity of the B. subtilis natto is because they contain nattokinase. DPA is present in higher quantities in the traditional fermented food known as “natto” in Japan. We have earlier reported that DPA inhibits platelet aggregation and influences the activity of the blood coagulation-fibrinolysis system. In this study, we confirmed an additional physiological role of DPA, namely, to enhance t-PA synthesis. DPA has a low molecular weight and heat resistance, which is origin of the food microorganism with high safety. It is believed that in future, DPA might be used for thrombosis prevention like a aspirin. The market of natto and B. subtilis natto will expand all over the world as a functional food.

Epigenetic control of tissue-type plasminogen activator synthesis in human endothelial cells

Tissue-type plasminogen activator (t-PA) is produced by endothelial cells (EC) and is responsible for the removal of intravascular fibrin deposits. We investigated whether expression of t-PA by EC is under epigenetic control. Methylation analysis of the proximal t-PA promoter revealed a stretch of unmethylated CpG dinucleotides from position -121 to +59, while upstream CpG dinucleotides were all methylated. In contrast, in human primary hepatocytes, which express t-PA at much lower levels than EC, the proximal promoter was partially methylated. Treatment of EC with the non-specific histone deacetylase (HDAC) inhibitors butyrate and trichostatin and with MS275, a specific inhibitor of class I HDAC, resulted in a time- and dose-dependent increase in t-PA expression. Garcinol and anacardic acid, inhibitors of the histone acetyl transferases CBP/p300 and PCAF, reduced basal and HDAC inhibitor-induced t-PA expression, whereas curcumin, an inhibitor of CBP/p300 only, had no effect. We performed chromosome immunoprecipitation analysis of the t-PA promoter using antibodies specific for acetylated histone H3 or H4 and observed an increase in H3 acetylation of 10 ± 3 and 44 ± 14-fold in EC treated with trichostatin or MS275, respectively, and in H4 acetylation of 7.7 ± 1.4 and 16 ± 3-fold, respectively. The proximal t-PA promoter is unmethylated in human EC and partially methylated in human primary hepatocytes. Expression of t-PA by EC is repressed by HDACs in a mechanism that involves de-acetylation of histone H3 and H4.

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.

Viral pathogens as novel activators of procoagulant signalling

Thromb Haemost 2009; 101: 993–994 Toll-like receptors (TLR) constitute a protein family of 13 different mammalian subtypes of heterodimeric immune receptors which are activated by a diverse range of molecules specific to certain microbial hazards. Among them, TLR subtype receptor 3 (TLR3) is unique because it is activated by double-stranded RNA (dsRNA) of viral pathogens. Polyinosinic:polycytidylic acid (poly I:C) serves as a well-known activator of TLR3 in cell culture studies to mimic the agonistic effect of dsRNAs (1). In general, TLRs are expressed on immune cells of monoblastic or myeloblastic origin, such as dendritic cells, lymphocytes or monocytes and play a pivotal role in innate immunity. Moreover, certain TLRs have been described on certain epithelial cell types and in particular TLR3 was found to be present on epithelial and neuronal cells, in the kidney and also on mesothelial cells (2–5). In contrast to most TLRs, signalling of TLR3 is independent of the common TLR adaptor protein MyD88 but involves the adaptor protein Trif (TIR-domain-containing adapter-inducing interferon-β, TICAM-1), which ultimately induces transcriptional activation of interferon-α and -β, both of which play a substantial role in the inflammatory immune response following viral infections. In this issue of Thrombosis and Haemostasis, Wornle et al. describe a novel link between viral infection of serosal cavities and regulation of fibrin generation and fibrinolysis (6). In a cell culture model of mesothelial cells isolated from explanted human omental tissue, they show that stimulation with poly I:C leads to both a concentration-dependent increase of plasminogen activator inhibitor 1 (PAI-1) synthesis and a concentration-dependent decrease of tissue plasminogen activator (t-PA) synthesis. Aside from TLR3, mesothelial cells also express the helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5),which may also act as sensors of viral infections through recognition of viral dsRNA and may up-regulate type I interferons. The specifity of the involvement of TLR3, RIG-I and MDA5 in these observations is confirmed by the use of RNA interference technology. These results indicate that the effect of poly I:C on increased PAI-1 but decreased t-PA synthesis is mainly mediated by TLR3. The authors interpret their findings to be of potential importance for the pathogenesis of viral diseases in serous tissue such as the mesothelium, pleura or pericardium because fibrin deposition at these sites could be promoted through the described mechanism. This could ultimately determine the course of the infectious disease e.g. by leading to pleural or pericardial fibrosis. In the mesothelium, similar mechanisms may be responsible for the failure of the peritoneal space to contribute to permeability changes. Hence, in this specific context, activation of mesothelial viral receptors followed by disturbance of the fibrinolytic system could explain why patients undergoing chronic abdominal peritoneal dialysis only profit from this type of treatment for a limited time span. From the data obtained by Wornle et al., such conclusions seem straightforward and comprehensible, yet another implication of the findings presented in this work could be related to the change in gene expression of adjacent vascular tissue. Since under guiescent conditions in vivo the ratio in gene expression between t-PA and PAI-1 in vascular endothelium is very high, poly I:C could dramatically shift this value resulting in the upregulation of PAI-1. This would lead to a more prothrombotic cellular phenotype as recently documented by the poly I:C induced up-regulation of tissue factor and down-regulation of thrombomodulin in the vessel wall (7). Indeed, increased levels of t-PA and PAI-1 have long been known to be present in the early stages of atherosclerosis and to be independent risk predictors for myocardial infarction (8, 9). However, according to present data, they seem to be markers of increased endothelial activation rather than playing an active role in promoting atherosclerosis (10). Intriguingly, besides being expressed in the above mentioned tissues, TLR3 also appears to be expressed on cells of the vascular endothelium, where it most likely acts as a receptor for viral dsRNAs. Thus, activation of endothelial innate immune receptors such as TLR3 could actually represent an active promoting mechanism in atherosclerosis and its thrombotic complications because it might not only result in dysregulation of the t-PA-PAI-1 system, but could also activate other signalling pathways within endothelial cells that directly promote endothelial dysfunction. Indeed, in a very recent report by Fischer et al., the authors describe endothelial activation through extracellular RNA (but not DNA) leading to binding of vascular endothelial growth factor (VEGF) to neuropilin-1, © 2009 Schattauer GmbH, Stuttgart

Effect of Norgestomet Treatment on Plasminogen Activator Activity in the Cervical Mucus and the Endometrium in Dairy Cows

The aim of this study was to determine the effect of norgestomet treatment, in the absence or the presence of a functional corpus luteum (CL), on plasminogen activators activity (PAA) in the cervical mucus and the endometrium in dairy cows. Eleven days after oestrus (Day 0 = oestrus), 38 cows were randomly assigned to one untreated control group (n = 9) and three treatment groups (S(1), S(2) and S(3)). Animals of S(1) group (n = 9) received an implantation of norgestomet on the outer surface of the ear for 8 days, simultaneous injection of oestradiol valerate 5 mg and norgestomet 3 mg, i.m., and on Day 19 an injection of ECG 500 IU, i.m. Animals of S(2) group (n = 11) received the treatment of S(1) group, plus an administration of PGF(2)alpha on Day 10 for the regression of CL. Animals of S(3) group received the treatment of S(2) group, plus two additional norgestomet implants inserted on Day 16 for 36 h. Both types of plasminogen activators [the tissue-type (t-PA) and the urokinase-type (u-PA)] were detected in the cervical mucus and the endometrium of the cows. Plasminogen activators activity in the cervical mucus was higher in control group than in S(1), S(2) and S(3) groups (P < 0.001). In contrast, endometrial PAA did not differ among groups (P > 0.05). Oestradiol-17beta concentrations on Day 21 were higher in S(2) group than in control group (P < 0.01) and S(3) group (P < 0.05). Progesterone concentrations did not differ among groups (P > 0.05). Oestradiol-17beta concentrations could positively affect cervical mucus PAA in control group (P < 0.1), but not in other groups (P > 0.05). These findings suggest that control of estrous cycle by norgestomet administration, in dairy cows, exerts a suppressive effect on plasminogen activators synthesis and/or secretion in the cervical mucus, regardless of the absence or the presence of the CL. On the contrary, endometrial PAA is not affected by norgestomet treatment.

A Critical Role for System A Amino Acid Transport in the Regulation of Dendritic Development by Brain-derived Neurotrophic Factor (BDNF)

Dendritic development is essential for the establishment of a functional nervous system. Among factors that control dendritic development, brain-derived neurotrophic factor (BDNF) has been shown to regulate dendritic length and complexity of cortical neurons. However, the cellular and molecular mechanisms that underlie these effects remain poorly understood. In this study, we examined the role of amino acid transport in mediating the effects of BDNF on dendritic development. We show that BDNF increases System A amino acid transport in cortical neurons by selective up-regulation of the sodium-coupled neutral amino acid transporter (SNAT)1. Up-regulation of SNAT1 expression and System A activity is required for the effects of BDNF on dendritic growth and branching of cortical neurons. Further analysis revealed that induction of SNAT1 expression and System A activity by BDNF is necessary in particular to enhance synthesis of tissue-type plasminogen activator, a protein that we demonstrate to be essential for the effects of BDNF on cortical dendritic morphology. Together, these data reveal that stimulation of neuronal differentiation by BDNF requires the up-regulation of SNAT1 expression and System A amino acid transport to meet the increased metabolic demand associated with the enhancement of dendritic growth and branching.

Urokinase (u‐PA) is produced by collecting duct principal cells and is post‐transcriptionally regulated by SV40 large‐T, arginine vasopressin, and epidermal growth factor

We have analyzed the expression and regulation of plasminogen activators (PA) in principal cells of the renal collecting duct. We used a rabbit principal cell line (RC.SVtsA58) infected with the temperature-sensitive SV40 strain tsA58. Transformed cells cultured at permissive temperature (33 degrees C) produced only tissue-type plasminogen activator (t-PA). Shifting the cells to nonpermissive temperature (39.5 degrees C) induced their differentiation and a marked increase in total fibrinolytic activity due to the induction of urokinase-type plasminogen activator (u-PA) synthesis and secretion. The effect on u-PA was post-transcriptional and it could be attributed to large-T inactivation at 39.5 degrees C since it was abolished by re-infecting the cells with wild-type SV40. Run-on assay and real-time RT-PCR of u-PA transcripts indicated that large-T altered post-transcriptional regulation. u-PA was also produced by primary cultures of collecting duct cells and was present in the rabbit urine. In the kidney, u-PA and its receptor (u-PAR) were almost exclusively expressed at the apex of collecting duct cells. We then analyzed the regulation of u-PA by arginine vasopressin (AVP) and epidermal growth factor (EGF), two key regulators of principal cell functions. We found that AVP and EGF, which have opposite hydro-osmotic effects in the collecting duct, also exhibited contrasted effects on u-PA synthesis in differentiated RC.SVtsA58 cells. EGF increased but AVP suppressed u-PA activity and protein, and these regulations occurred at post-transcriptional level. These results point to a physiological role of u-PA in principal cells of the renal collecting duct.

Atorvastatin Affects Several Angiogenic Mediators in Human Endothelial Cells

The pleiotropic effects of statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, have been recently extended to the modulation of angiogenesis. Here, to get more insight into the statins action, the authors have investigated the effect of atorvastatin on the expression of several angiogenic and inflammatory genes in human umbilical endothelial cells (HUVECs). Atorvastatin was proangiogenic at the dose of 10 nM, and antiangiogenic at the concentrations of 1 to 10 micro M. Moreover, these higher concentrations inhibited also the proliferation of HUVECs induced by vascular endothelial growth factor (VEGF). Lower doses of atorvastatin did not influence endothelial cell proliferation. Importantly, atorvastatin at the micromolar concentrations diminished the production of interleukin (IL)-8, a proinflammatory and proangiogenic chemokine, and inhibited the synthesis of urokinase plasminogen activator (uPA), a potent proinflammatory mediator. However, it decreased also the expression of plasminogen activator inhibitor-1 (PAI-1) and thrombospondin-1 (TSP-1), the inhibitors of angiogenesis. Atorvastatin stimulated the expression of angiopoietin (Ang)-2 and moderately enhanced the expression of endothelial nitric oxide synthase (eNOS), whereas heme oxygenase-1 (HO-1) was not significantly affected. In conclusion, the present findings points to other angiogenesis-related effects of atorvastatin, which may be of relevance to the beneficial influence of statins in cardiovascular system.